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- Please select a date to see the proper prices!
Description of the accommodation:
Rooms, services Guests looking for accommodation in Pingtung City are welcome to book 180 nicely furnished rooms at Uni Resort-Kenting Pingtung City. Should you be arriving by car, parking will be available nearby. The hotel is offering wireless Internet connection (WiFi) without charging extra fees, so you may go online anytime with your laptop during your stay. To learn more about the exact range of services of Uni Resort-Kenting Pingtung City (including services for an extra fee) and the exact equipment of the rooms, please check the information sheet below.
Payment Credit card guarantee or advance bank card payment may be required for the reservation, depending on the chosen room, date and price.
Arrival, opening hours The earliest time for arrival and check-in is 15:00, the latest time for departure and check-out is 12:00. The personnel speaks Chinese - China and English.
Uni Resort-Kenting Pingtung City rooms Rooms
- Please select a date to see the proper prices!
Accommodation services
Free WiFi in shared places
- Pool (Outdoor - seasonally open)


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Jo Rees
Current Location:
Montgomery, Powys
My Profile
Hello, thank you for visiting my site!
My name is Jo Rees and I am moving to Ontario, Canada to volunteer for Young Life, a not for profit organisation who works with teens. My role will be to go into the world of teens, building relationships with them through organised events and meeting up with them on a one-to-one basis. As I will be volunteering I need to raise my funds in order to fully support myself and the work I do in Canada.
Thank you so much for your support and donations, I really appreciate it!
Friend Request
has been sent a request to be your friend.
Total money raised so far: £0.00
My Friends
Become the first Charity Choice friend of Jo.

subset: commoncrawl


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The ATEE takes part in a board network of national and international institutions, associations and governmental bodies in the area of teacher quality and teacher education.
Through its memberships, the ATEE has a broad network of pre-service and in-service teacher education institutions in a wide variety of countries, covering the most of Europa and countries from outside Europe (including the United States, Australia and African countries). Next to these institutional network, the ATEE has through its members relations wiith national associations and organisations in the area of teacher education (e.g. the MOFET Institute in Israel, the VELON in The Netherlands, etc.).
To strengthen the voice of teacher education in the world and to stimulate exchange in the worldwide community of teacher education, the ATEE participates in the World Federation of Associations for Teacher Education, where the ATEE cooperates with the Association of Teacher Educators ATE in the United States, the Australian Teacher Education Association ATEA and the African Association for Teacher Education.
On a regular basis the ATEE is invited by the European Commission to give the association's view on policies concerning teacher quality and teacher education.


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The springs available from MALOSSI for the converter unit (driven pulley) are constructed using a silicium steel alloy which guarantees extra stability during use under extreme conditions and operating temperatures. This is reached through a high proportion of carbon in the steel alloy and a special tempering process for the outer surface. In addition MALOSSI variator springs are dynamically balanced and specifically developed for individual demands.
They are available in three categories:
SIP TIP : The MALOSSI converter grease was specifically developed for use at higher motor RPM. It is available in two versions : MRG grease and Racing grease.
OEM Numbers (for the purpose of comparison)


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Please disable your Ad Blocker to better interact with this website.
Josephine Dumont – is an old school conservative on a quest to find her people. Committed to common sense journalism, Dumont hopes to distinguish herself by stepping out of the debacle of modern political propaganda to ask tough questions of all sides. Dumont feels that the biggest threat to America at present is Lincoln's "enemy within," a moral social cancer that threatens to corrupt the courage and character of the Republican Party. Standing against fear and fury, Dumont desires to urge conservative readers to embrace compassion, integrity, and faith on their quest to reclaim their homeland.


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Rep. Nancy Pelosi (D-Calif.) issued a stern warning to her colleagues yesterday during a closed-door meeting, telling them not to buck their leadership on a pivotal lobbying reform bill.
"They want to use you to defeat us," she warned. "Trust us on this. It has absolutely no downside to your district," she said, as she urged Democrats to vote against the House GOP leadership's lobbying and ethics reform bill.
Democrats have pinned their election-year hopes on painting an image of the GOP as a corrupt, entrenched majority that voters should reject in November. Pelosi and other leaders argued that Democrats would hand Republicans a victory if they voted for a measure that Democrats and others have denounced as a sham.
Pelosi recalled that some members who had defied leadership on other important votes had come to her later and said they regretted the move. She took issue with those Democrats in particular: "You need to reach your wisdom on this before the vote rather than after."
Pelosi made the comments before a record crowd at the weekly House Democratic Caucus meeting. The gathering attracted 102 House Democrats, nine more than the previous attendance record.
A Democratic aide hinted that there could be consequences for members who backed the lobbying bill. Pelosi has previously threatened to pull coveted committee assignments of members who are disloyal to leaders. During a vote on the Central America Free Trade Agreement last summer, Pelosi told the members that siding with Republicans could jeopardize their committee assignments.
"There should be no reason for anyone to register a yes vote on the lobbying reform bill," the aide said. "If the Republicans win with Democratic votes, that is a slap in the face to the caucus."
Pelosi, Democratic Whip Steny Hoyer (Md.) and Rep. Rahm Emanuel (D-Ill.), head of the House Democrats' campaign arm, were whipping the bill feverishly yesterday as The Hill went to press, and it appeared that a few Democratic rebels could help secure a GOP win.
Eight Democrats voted for the lobbying reform bill: Reps. John BarrowJohn Jenkins BarrowOur democracy can't afford to cut legal aid services from the budget Dem files Ethics complaint on Benghazi panel Barrow thanks staff in farewell speech MORE (Ga.), Dan Boren (Okla.), Leonard Boswell (Iowa), Henry Cuellar (Texas), Jim Marshall (Ga.), Jim MathesonJim MathesonTrump's budget targets affordable, reliable power Work begins on T infrastructure plan New president, new Congress, new opportunity MORE (Utah), Charlie Melancon (La.) and Gene Taylor (Miss.). All but two are among the Democrats' 10 most vulnerable members.
Pelosi's comments to the caucus struck a stern note just hours after she and Emanuel had led an upbeat political pep rally Tuesday night for House Democrats.
During that gathering, Pelosi and Emanuel energized members with poll numbers, anecdotal evidence of changing public opinion and comments from the punditry that painted a picture of a nation poised to turn House seats over to Democrats.
Both Pelosi and Emanuel noted their belief that "the intensity" in the 2006 election was on the side of Democrats, one attendee said.
Emanuel showed a PowerPoint presentation and a videotape documenting the Democrats' chief political fundraising programs.
"It set a very positive tone in terms of Democratic prospects," said Rep. Al Wynn (D-Md.), who attended the meeting along with more than 100 other Democrats. "It reinforced the call to unity and the idea that we should roll up our sleeves."
Pelosi again told Democrats that they need to remain unified to create a clear distinction between the agendas of the two parties.
Given the upbeat nature of the dinner and the belief of many Democrats that such unity is key to their election prospects, some members were upset that some Democrats were considering voting with Republicans on the lobbying bill yesterday.
"You have to ask yourself: Why provide health and welfare to a majority that hasn't called, hasn't written and hasn't sent flowers?" said Rep. Ellen Tauscher (D-Calif.).
Rep. John Larson (D-Conn.), vice chairman of the Democratic Caucus, said members knew there would be consequences to bucking leadership.
"There are always repercussions," he said. "Those factors are taken into account by leadership. … You can't always make them vote with you, but you can make them wish they did."


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Former surgeon on trial for leaving woman permanently disabled
Former neurosurgeon Christopher Duntsch, who was stripped of his medical license, is on trial in Dallas and charged for a botched surgery. But prosecutors say he's connected to two deaths and 34 seriously injured patients.
Duntsch was indicted in several cases where patients were left seriously injured, paralyzed and even dead.
In a somewhat unusual move, prosecutors will be allowed to call other patients who will testify their lives are forever changed by the gross malpractice of the former neurosurgeon.
79-year-old great grandmother Mary Efurd told the jury she'd gone to Dr. Christopher Duntsch for back surgery in 2012 to eliminate chronic pain but left barely able to stand or walk.
"I had never had pain like that before. It was a 10 + if you know what that means," she said.
Prosecutors say Duntsch not only severely bungled her spine surgery but amputated part of her nerve root.
Dr. Robert Henderson testified he was called in to try to correct Duntsch's surgery and called Efurd's case an "atrocity" and questioned his qualifications.
"You asked how egregious it was. It was as egregious as you can imagine," Henderson said. "I became concerned whether or not he was a physician and a surgeon."
FOX4 first covered Duntsch in 2013. Patients were showing up to his Plano office trying to get records after his medical license was suspended, then finally revoked. In 2015, he was arrested for shoplifting and aggravated assault. There were allegations of drug and alcohol abuse, including a DWI conviction out of Colorado.
Duntsch's defense attorney Robbie McClung says this case is even more unusual because the jury will be allowed to hear testimony from other patients from other indictments involving her client.
"It's just important that they not let emotion not take control and they stick to the facts since that's what guilt-innocence is supposed to be about," the attorney said.
The trial will start back up Friday morning at 9 a.m.


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GraphicRiver Modern Geometrical Art Background Templates 5603961
Stock Vector \ Conceptual \ Business \ Backgrounds
Vector \ Design \ Illustration \ Included \ Wallpaper \ WhiteVector \ Editable
Stock Vector - Modern Geometrical Art Background Templates 5603961 by antishock @ GraphicRiver
Stock Vector \ Conceptual \ Business \ Backgrounds
Vector illustration. Fully editable vector. All design elements included in EPS file (use of Adobe Illustrator or other vector graphics editors is preferred)
Layered: No; Graphics Files Included: JPG Image, Vector EPS; Minimum Adobe CS Version: CS.
Triangle Geometric Concept; Triangle Geometric Concept; Merry Christmas modern cards - abstract baubles; Christmas Background; Christmas Background; Happy New Year Concept; Geometric Christmas Tree.
Keywords: abstract, art, backdrop, background, card, circle, cover, decor, decoration, decorative, design, element, geometric, graphic, illustration, line, modern, pattern, set, shape, square, stripe, style, techno, template, texture, triangle, vector, wallpaper, white.


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Over on The Post Money Value Rick Segal writes that WiFi is Secretly Free Everywhere. Of course. Most hotels (at least the ones I stay at) will comp you the WiFi if you ask. Most people just don't ask.
Show me the WiFi!
{ 0 comments… add one }
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Donald Trump's taxes are a big deal
Hillary Clinton is getting 98% of the African-American vote in the state, and she has seen her support among women grow from 53% to 61%. Hear his misogynistic commentary in the clip below. Trump sees it as smart decision for individuals to take any legitimate and legal deductions in an effort to reduce their individual tax burdens or those of their small business.
NC radio station drops football game over anthem protest
I'm proud of our country and I'm proud of our soldiers.especially our soldiers from FORT BRAGG. fighting for our country so I've decided that ESPN FAYETTEVILLE will 'protest the protest.' I talked with some of our EAST CAROLINA football sponsors yesterday and will be talking with more of them today.
Ravens release seldom-used RB Forsett, who asked to leave
In three starts, Forsett carried the ball 31 times for 98 yards (long run of 11) and caught 11 passes for 36 yards (long of 10) before being benched on Sunday in a loss to the Oakland Raiders. In other words the team cut him. Rookie Kenneth Dixon, who led the Ravens in rushing in the preseason before suffering a knee injury, returned to practice last week and is expected to be ready to make his regular season debut.
Wenger hails Arsenal after win over Chelsea
The midfield maestro put up a picture of himself along with his teammates Granit Xhaka, Shkodran Mustafi and Lucas Perez in the dressing room, each one of them beaming following the dominant performance they just put on. " All the more, we have proved now that this wasn't a one-off and we'll have to perform equally as well against Basel ". Two at the back, three at the back, four at the back, I don't care.
Low-key start for Danny Willett despite brother's comments
Sign up to our daily newsletter for up to date global news and features. The Masters champion initially apologised for the comments, before going on to have a poor Ryder Cup debut, failing to win a point as Europe lost 17-11 to the States.
Mickelson starts war of words with former Cup captain Sutton
Jamie Weir and Rob Lee previews this weekend's Ryder Cup with a look at the potential pairings. - Make sure the pingpong table in the team room has extra balls. Love said he is better able to cope with any situation than he was in 2012, when Europe built momentum in the last-day singles while the U.S. team struggled to the finish.


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Shy People Trailer (1987)
A New York journalist visits her distant cousin for the first time to write an article about her hard life in the bayous of Louisiana. The journalist's wild, drug-addicted daughter adds tensions between the two relatives' cultures.
Watch the official Shy People 1987 trailer in HD below or find more Shy People videos on Vidimovie.


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Registration for the next session of Swim Lessons begins Monday, August 28th for members and Wednesday, August 30th for non-members.
Please call the YMCA at 224-1683 or email the Aquatics Director firstname.lastname@example.com for information on instructors.
AGE REQUIREMENTS: Participants in our Parent/Child, which is our infant/toddler program, must be at least 6 months of age, up until 5 years of age.
Participants in our Pre-School Aquatics program must be about 3 years old, up until 5 years of age. We offer 4 levels of pre-school levels.
Participants in our Youth Swim Lesson program, should be 6 years of age and older. We offer 6 levels of Youth Swim Lessons. Levels are grouped by ability.
PARENT OBSERVATION: Unless involved in the parent/child class, parents are not allowed to be on the YMCA pool deck. This is to provide your child with the best possible learning experience. It is our ambition to help your child grow in their confidence and to progress with their skill level. Parents and guardians are to watch swimming lessons from the observation room located above the YMCA Pool.
PASSING CLASSES: It is not uncommon for a child to stay in a particular level several times before successful completion. It is natural for participants to develop motor skills and become comfortable with water at a different pace. Practicing skills during open and family swim is strongly recommended to improve swim skills.
MISSED CLASSES:Sorry, missed classes cannot be made up; nor are they refundable.
(6 months to 5 years old)
An adult must participate in the water with the child. The adult need not be a parent. This program builds swimming readiness by emphasizing fun in the water. Elementary skills are covered such as water entry, bubbles, front kicking, back floating, and underwater exploration.
If your child is between 3 and 5 years old look for Pike, Eel, Ray and Starfish. These have similar skills to the school aged classes but are geared for younger swimmers. If your child is 6 years old and older sign up for Polliwog, Guppy, Minnow, Fish Flying Fish, and Shark.
PIKE- Beginning Swimmer (Preschool 1)
Little swimmers develop safe pool habits, adjust to the water, and basic paddle stroke (front crawl). To progress, child must feel comfortable holding face in water while blowing bubbles, and must be able to float on their back.
EEL (Preschool 2)
Child is comfortable in the water. Swimmer is taught to kick, dive, float and paddle stroke (front crawl). To progress, child must swim across the pool without assistance with their face in the water.
RAY (Preschool 3)
Child can briefly swim independently without a floatation device.
STARFISH (Advanced Preschool)
Child improves stroke skills, builds endurance, and treads water. Child can swim on their front and back without assistance.
*If students have completed the RAY or Starfish level they skip Polliwog and move immediately to the GUPPY level.
POLLIWOG - Beginner (Level I)
Students become acquainted with the water, floatation devices, and pool. Students will learn paddle stroke, side and back paddle. To move to the next level they will need to swim across the pool with assistance with their face in the water.
GUPPY (Level 2)
Students are introduced to lead-up strokes: Front and back crawl, sidestroke, breaststroke, and elementary backstroke. Before moving on students must swim the length of the pool without assistance.
MINNOW (Level 3)
Students further refine the lead-up strokes: Front and back crawl, sidestroke, breaststroke, and elementary backstroke. To move to the next level they need to be comfortable swimming all strokes 25 yards.
FISH (Level 4)
Students work to perform the crawl stroke, elementary backstroke, back crawl stroke, and side stroke with turns. They are introduced to butterfly stroke. To advance, students must be able to swim all strokes 50 yards without stopping.
FLYING FISH (Level 5)
Perform the front crawl, back stroke, elementary backstroke, sidestroke, & breast-stroke; to advance student needs tread water for three minutes with combination kicks plus swim all strokes 100 yards.
SHARK (Level 6)
Swimmers focus on perfecting strokes and increasing endurance with open and flip turns. Students also learn advanced skills in boating, safety, and rescues. Perform front crawl, breaststroke, elementary backstroke, & sidestroke with open turns; perform the butterfly.
Conquer Your Fear | Monday 7:15-8pm
Are you afraid to put your face in the water? Have you had lessons but still don't think you are a swimmer? Can you swim but never got the hang of the breathing? Do you think you will sink like a rock? Come and try a whole new way of learning to swim. Guaranteed to conquer your fear of water at your own pace. Contact Holly at 224-1683 or email@example.com more information.
Member $19 Non-Member $38
Are you not able to make the scheduled swim lesson times? Have you always wanted to learn to swim but wanted to work at your own pace? Private lessons may be right for you. We offer private lessons for ALL ages to accommodate to your (and the instructors') schedule. Punch cards are for sale at the front desk which contain four 30 minute lessons.
Prices range from$56-$64 depending on the instructor. Contact Holly at 224-1683 or at firstname.lastname@example.com for more information.
Swimming lessons, water safety, and water recreation for home-schooled students age 6 and up. A great way to fulfill P.E. requirements. Beginners will get comfortable in the water and learn basic skills. Intermediate will add more skills and strokes and refine basic skills. Advanced will refine technique and work on fitness principles. Water Safety and recreation explored at all levels.
Financial assistance available for those who qualify
For more info on YMCA swim lessons contact Holly Hardy, Aquatic Director at 605-224-1683 or firstname.lastname@example.com


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Creative Web Design Company
We are a creative web design Company and we prefer to deliver products, with minimum cost before time with best quality to our Clients. We customise your happiness with alluring results. Web Designing is a combination of creativity and beauty. A perfect blend of these combination happens only when there is teamwork.
We focused being right in everything. Our website solution gives deeper insights about consumers interests, needs and behaviors. We don't have any hidden charges for making the websites. Our customer service goal is to provide support to the clients and to understand technical terms.


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Results 1 to 2 of 2
05-02-14, 15:48 #1Registered User
- Join Date
- May 2014
- Posts
- 1
New Member Question: Online interactive exercises on ERD and Normalization?
Im not sure where to put this question so I figured id post here and if mods can move it to appropriate section if necessary.
Ok well im a Computer Information Systems major and currently at the finish line for my Database Management class. Portion of the final will be multiple choice and T/F and the rest will be questions to work out such as SQL, ERD and Normalization.
I wanna be able to practice building a ERD from 10-15 entites and be able to normalize from unnormailzied to 1st - 3rd and maybe 4th. Ive practiced questions ive been given and want more different problems to work on to get more practice and grasp of it.
Question: Can someone suggest a place online that gives out ERD or Normalization exercise problems thats possibly interactive or atleast shows the answers with steps on how to do it at the end?
05-02-14, 16:24 #2Resident Curmudgeon
- Join Date
- Feb 2004
- Location
- In front of the computer
- Posts
- 15,579
I'd like to hear about this too if anyone has any recommendations. I know of a few paid sites that do this as part of their curriculum, but I've never seen a free site that was of any value to me.
-PatPIn theory, theory and practice are identical. In practice, theory and practice are unrelated.


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Severe Hypoglycemia From Evidence to Practice: Guidelines and Policy Development. Alexis Farrington NUR 4745 Practice Pathways III Florida State College at Jacksonville Dr. Susan Shultz, DNP, CCRN, CNE. From Evidence to Practice: Preceptor. Tina Debile , BSN, PCCN (904)308-7300
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Alexis Farrington
NUR 4745 Practice Pathways III
Florida State College
at Jacksonville
Dr. Susan Shultz, DNP, CCRN, CNE
Is nurse-friendly
Limits additional documentation
Manages and controls symptoms
Promotes early identification of symptoms
Prevents adverse events
Functions within facility policy
ADA & AACE Practice Guidelines
National Institute for Health and Clinical Excellence (NICE)
St. Vincent's Intranet (2011). Policies and Procedures.
The Evidence
SVMC Policy
Minimum baseline for treatment of hypoglycemia and subsequent symptoms at =/< 70.
Allows more time for recognition of signs, symptoms and treatment targeted at prevention of an episode versus treatment of an event.
Variances in outcomes (previous policy).
(Reider, J. D., 2009)
Data collected from ten patients with varying diagnoses
for staff-friendly protocol.
support staff.
protocol implemented.
Study time and duration limited to a few days
Limited data to plot more specific event and evaluation over time
Multiple co-morbidities associated with the cause of alterations in therapeutic levels
- Result in successful outcomes for our patients
- Promote patient and family satisfaction of care
- Maintain the preservation of confidence in our facility's
clinical performance
- Reinforce competencies of our bedside nurses.
A Comparative Study. Diabetes Technology & Therapeutics, 635-639.
Algorithms and Insulin Pump Suspension. Diabetes Care, 1013-1017.
practice. St. Vincent's Health System Intranet Policies and Procedures. Retrieved
March 10, 2011from Jaxhealth.com: http://www.jaxhealth.com
Subcutaneous Insulin Infusion: Is It Associated with Poor Glycemic Control. Diabetes
Technology Therapeutics, 93-98.
control. American Association of Clinical Endocrinologists, 801-808.
Blood Glucose
(If unable to swallow or NPO)


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How do I get more involved in the SFA?
Please join us as an SFA member. We also encourage you to like us on Facebook, follow us on Twitter, and check our calendar to find out about SFA events in your area.
How do I submit an article to Gravy?
Gravy accepts unsolicited submissions under 1,000 words. Please send your submission to Sara Camp Arnold. Please allow 3–4 weeks for a response.
Where do I buy a "Make Cornbread, Not War" hat?
Our dear friends at Billy Reid make those rad hats. And other SFA-inspired stuff, too. You can shop in their stores or online.
What are the benefits of joining the SFA?
So glad you asked! As a member, your dollars are supporting the mission and vision of the SFA. You help us do what we do. In addition, other benefits include:
* a subscription to our Gravy quarterly
* advance notice of SFA events, including the ability to register for the Symposium and Summer Symposium
* access to the SFA Member Directory
I'm a student. How do I become an intern with the SFA?
If you are not a student at the University of Mississippi, the best way to get involved with the SFA is to come volunteer at our annual symposium, held in Oxford every October. Contact Mary Beth Lasseter if you are interested in becoming a volunteer.
How do I host an event for the SFA?
Wow, thanks! Please click here to read our guidelines for hosting a Piggy Bank Dinner. And if you've never been to an SFA event before, we invite you to attend one (or two, or more!) before becoming a host.
Do you take guest bloggers?
In general, we invite guest bloggers to write for us. If you've got an idea for a series of 4–6 blog posts that you think we'll love, please e-mail Sara Camp Milam
I want to visit SFA World Headquarters. Where are you?
We LOVE visitors here at the SFA World Headquarters! You can visit our funky office any time (or at least, Monday-Friday, 8:00am-5:00pm), in Barnard Observatory on the campus of the University of Mississippi. Click here for a map. We might even have pie.
I'd like to invite an SFA staffer to talk to my organization. How do I make arrangements?
We are pleased to speak with your group via Skype or in person whenever it's feasible. To make a request and help identify the right person for your needs, please begin by contacting Melissa Hall. We make it a point to pay for speakers' travel to our own events, and we hope you'll understand that we will expect similar consideration from your organization.
Where can I find a copy of Gravy quarterly?
The best way to make sure you receive every copy of Gravy is to become an SFA member! You can also find Gravy at Square Books in Oxford, MS; A Capella Books in Atlanta, GA; Star Provisions in Atlanta, GA; The Stacks in New Orleans, LA; Cochon Butcher in New Orleans, LA; the Alabama Chanin Factory in Florence, AL; and Parnassus Books in Nashville, TN.
If you would like to carry Gravy in your bookstore or other establishment, please contact Mary Beth Lasseter.
How do I obtain an SFA film to screen at my event?
We most often screen SFA films at events that are sponsored by the SFA or put on by others as a fundraiser in our benefit. Melissa Hall is happy to chat with you about other film requests on a case-by-case basis.
How do I check on the status of my SFA membership?
Our membership directory is coming soon! Until then, just e-mail us. We'll check for you!
What is your relationship to the University of Mississippi?
SFA is an institute of the Center for the Study of Southern Culture at the University of Mississippi. Our World Headquarters (SFAWHQ) are inside Barnard Observatory on the university campus.
Do you offer grants to documentarians or film producers?
We do offer documentary film grants on a limited basis through our Greenhouse Films initiative. Stay tuned to our website and social media, where we will send out a call for film proposals approximately twice per year. We also encourage you to visit our Vimeo page to get an idea of the projects we commission and/or fund. If you have more questions, email Sara Camp Milam.
Where should I direct media inquiries?
Please e-mail Melany Robinson at Polished Pig Media.
How do I keep up with SFA content, events, and news?
There's always something fresh and new on our website, and we don't want you to miss any of it. Add southernfoodways.org to your RSS feed or follow us on social media to get all the latest updates.
Facebook: facebook.com/SouthernFoodwaysAlliance
Twitter: twitter.com/potlikker (@potlikker)
Instagram: instagram.com/southernfoodways (@southernfoodways)
Pinterest: pinterest.com/sfapotlikker (Southern Foodways Alliance)
I'm interested in collaborating with the SFA on a project. What's the best way to learn about the culture of the organization?
The SFA created this online primer for prospective collaborators. Click here to download a copy.
How does the SFA operate? What is it like in the SFA offices?
Read through our mission, vision, and values, and you might get a better sense of how we work. If you want more specifics, click here to view a more detailed presentation of the SFA.


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SuperIcon (Life of Pixel) is hard at work on Vector Wars, developed in Unity, it is confirmed for the Vita, among other formats. It seems to offer a great city to explore and engage in combat around with bags of detail and effects that bring the old vectors to life, looking a little similar to Vektropolis, which seems to have fallen off the radar.
Looks pretty sweet. If it's priced reasonably I'll pick it up.


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The major principle of a newsgroup and online conferencing is the counterpart. Since they facilitate numerous people to correspond with each other. On the other hand, they vary in their tasks. Newsgroup is an online community where people can exchange their visions about different matters. While, online conferencing is a live assembly performed for staging or preparation. The newsgroups really facilitate people recognize dissimilar customs, conventions, ideas and outlooks of people. Somewhat fascinatingly, religion is one of the most talked about issues on the newsgroups. By Christians, Muslims, Jews and devotees of other religions striving to appreciate each other and representing their personal viewpoint. Newsgroups can also be used for instructive functions.
For example, as a teacher can correspond to a broad variety of students handily through this means. These groups took part in an essential task in the latest Arab Spring which was able to get rid of an amount of dictatorial administrations. To systematize the affairs, social media develop to be a significant instrument. Nevertheless, multi-nationals frequently encounter a circumstance where they have to carry out an essential assembly. Yet all partakers are in numerous districts of the world. Online conferencing has deciphered this difficulty of numerous groups as they are now competent to correspond to each other via the newest technology.
Online conferencing is lucrative and saves plenty of time and disturbances. Likewise, it can be successfully utilized for any sort of working out or learning. There are a few websites and other applications which recommend this facility to their clients. Newsgroup is an online population, which is committed to talk about or contribute information and views about a certain subject of concern. The conversations on the newsgroups are not restricted to existing issues merely and people may contribute to their outlooks on any matter, which is opposed to 'news' in the name. However, online conferencing is a live convention, which is performed online for managements or preparation, with the assistance of various applications and facilities. It could simply be an audio conference yet most of the associations utilize web cams to develop into a video conference.


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Jimmy D's Car Care Center
Review for Jimmy D's Car Care Center
ROGER F. on 11/19/2015
"Great and honest mechanics"
I took my car in for a long-overdue tuneup and oil change. The job was done quickly and at a reasonable price. Jimmy D's it the only auto shop I go to.
Vehicle: Dodge Caravan
Service Date: 11/17/2015
Review Created: 11/19/2015 01:42 PM


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- Home
- Commissioner Biographies
- Commissioner Login
- Contact Us
- About the Commission
- Latest News
Rt Revd Nigel McCulloch (Bishop of Manchester)
Nigel McCulloch has been Bishop of Manchester since 2002. He began his ministry in the 1960s at the other end of the Ship Canal, working primarily on an overspill estate for ex-Liverpool residents, some of whom were experiencing serious poverty. Prior to coming to Manchester, he was Bishop of Wakefield - where he was greatly involved in supporting mining communities in South and West Yorkshire during and after the pit closures.
A member of the House of Lords since 1997, he has served on two Select Committees – BBC Charter Renewal, and Communications. Currently he chairs the Church of England's Legislative Committee for the appointment of Women Bishops; and the national Council of Christians and Jews. He is deputy Chair of Governors at Bolton University.
The first Bishop of Manchester to have Mancunian blood (his great-grandfather was born and brought up in the 1830s in Bengal Street, Ancoats – an area of enormous poverty and disease at that time), he has, during his time as Bishop here visited, at least three times, every one of his parishes across Greater Manchester (a significant number of which are coterminous with areas of deep deprivation).
He chairs the Greater Manchester Faith Community Leaders, and is a member (with Councillor Murphy) of the Manchester Board.
He accepted the invitation from local MPs to chair the Commission because of his known commitment to supporting the marginalised, and his long-held belief that society has a responsibility to help people out of poverty.


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Gardenia plays jazz at Gibbs
Washington — The Gardenia jazz trio will perform classic jazz and popular standards Saturday, Jan. 14, at 7 p.m. in the Bryant Room of Gibbs Library, 40 Old Union Road.
Gardenia features Washington vocalist Cindy Millar, Spike Hyssong on standup bass and Dick White on guitar. Coffee and other beverages and delicious desserts will be served. A $5 per person donation is suggested for the Jazz Café, which will benefit the library.
Courier Publications' A&E Editor Dagney C. Ernest can be reached at (207) 594-4401, ext. 115; or firstname.lastname@example.com.


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Elton John AIDS Foundation writes:
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Enjoy this gift of Lambertz Chocolates.
Enjoy your break and we look forward to seeing you in the New Year.
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I have been wanting new silverware for a long time. A friend gave me twelve 5 piece settings that were brand new. She bought them when they were 75% off the 189.99 price. They were sitting in her attic not being used. I used them for a special event…but now I am considering using them as my daily silverware.
Check out my Thrifty Shopping articles on examiner.com.
Thanks for stopping by Cottage Lifestyle and examiner.com.
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"Admiral's Early Bond Releases" - Veritas Report - November 5, 2016
It's only been a few short weeks since I began sharing my thoughts with you. For years, I simply used this resource as an information source, something to read late at night after the long day's work.
Why did I suddenly come forward and begin to voice my information?
It is a position of vulnerability, surely. One opens to all types of criticism and comments, something I tend to avoid, preferring to stay in the background quietly.
Over the years, my own contacts have grown to be such that I lost any doubt as to what is happening and what may happen in our country. I came to look outward, to other countries for business investment and the future. I lost hope that it could get better here.
To be honest, I felt that the corruption here was so immense, so deep and all-encompassing, that it could never be overcome. I gave up on the country that I was raised to love. My family history has deep and visible roots in the very beginnings when America became America. I never imagined that there would be a time I would harbor such doubts, yet there I was.
That is why I began writing. I saw a despair, even an anger, in many as time went along and it was the "next week" phrase, the eternal "almost there" that had driven me so crazy before I met and interacted daily with so many individuals actually fighting this fight, so to speak. I couldn't remain silent any longer.
Tonight I sit here very soberly, watching once again with a hope for the United States.
It is not the time for despair but for real hope.
It is not the time for discouragement but for action.
It is not the time for hate but for love.
Not since the beginning of our country has there truly been such a need for the intelligent, thoughtful, loyal and determined citizens to be involved. As a currency exchange participant, you should truly feel good because that action alone sets you apart; makes you actively involved in changing how things will happen from now on.
Perhaps in the beginning it was a move to get ahead; a way to catch up; a way to multiply your money and change your financial position. I would solidly bet that it isn't just about that for you now.
I'll bet that, like me, you have regained a hope that we can fix our country.
That thought is the one I want to flame tonight, particularly if you feel uncertain or are faltering under this enormous deluge of information and political slime.
Don't give up.
This is not a fight over political parties. This is a war to the death of good vs. evil. It is the ultimate battle of truth over use of ignorance to manipulate and control. It is the choice of self-decisive life over mindless submission in order to avoid taking responsibility.
This is why I respect those fighting behind the scenes, risking and sometimes losing their lives for us in ways we would never imagine. If only I could share fully all that I now know and have seen and witnessed! I can only share with you the conviction I have gained from this education. It is essential to leave our naivety behind and to wake up to all that is involved; even to assist directly - even if it is merely our positive, energetic and powerful thinking that we throw into the mix.
Your thoughts and actions are so very important. This moment in time is a turning point for all of us, one individual at a time. Together, we are a vast army of intelligent, determined people who simply won't take it any more.
I realize I go on, but you asked and I feel impassioned to answer straight from my being.
Don't give up.
Where are we?
First let me share the status of the Admiral's transactions I mentioned in my last post that the Admiral's transactions had released and that we had a few individuals on our team that have received and can spend.
When the flood of obvious total releases did not occur, I did a detailed back trail research into those that had. Each was traced back to early historical bond releases, the very few that were allowed to already go out from Reno.
Each had a small portion as an up-front deposit with the remainder to be released via traveling to Hong Kong. I should have investigated to this level prior to stating what I did, however, I share with you now the final results.
So, yes, some were received and released; the majority are still stuck in the vast morass of "Paymaster" where they are held for final release pending the great "go" for which we all wait. Now you know that I am infallible. I will be more thorough in the future.
You ask when the 800 registration number will release. I will share what I can.
Sometimes I feel like we are all standing at a vortex of immense proportions and complexity. There are a hundred different ways that it can go, it seems.
I love how an Admiral friend put it the other day, "There is not a scenario that isn't already fully planned out. It is coming down and in full control." It looks anything but, doesn't it?
I do think that everything is going to be as we would like to see it.
I frankly thought that the full release of the Admiral's should have already occurred. That is partly why I didn't write today, just sitting here in anticipation of it and not needing to see what else might be up. Obviously that didn't happen. I just know that it will.
At this point I can only share my opinion, but based on all the exposure that I have.
I know that everything is totally prepared. All funds are in final accounts, fully primed, just waiting authorization for use.
I know that everyone that needs to be in Reno to make this happen is there and ready to go.
I know that behind the scenes, the most unbelievable military actions are taking place, not because they are anticipated to be needed, but because this is all driven from the military and face it, they prepare for anything
I have personally witnessed several locations and the activity I mention. When I inquire if we are going to see some action, the military advises "NESARA" as the single reason. I think of the vast resources being displayed and I realize how deeply the military have prepared financially to have the ability to sustain such a massive preparation and readiness.
I know that I am in a state of readiness to receive the 800 registration number as requested by those informing me and who move my own funds. In all the years of waiting, it simply has never been like this. There isn't anything else that needs to happen. They all know it.
They all are ready 24/7 for the final "go".
I believe that at any point in time we will see a quick sequence of events that finish this.
Even at the top there is such a discussion about what will happen - will the 800 registration number release prior to a NESARA announcement -
Will there be a public NESARA announcement or will it go behind the scenes - will they use NESARA to stop the election or will they ride it out and handle going forward after the election with an easy way forward through Trump to accomplish what needs to happen or an indictment and/or impeachment of Clinton if voting results are forced to her favor - to will the U.S. be included or excluded due to all of these difficulties.
I consistently receive information from the highest sources that it is all going and reassurance that all the scenarios I worry about I should stop worrying about. It is happening and soon.
I certainly believe that this will be over the next few days. Again, some indicate prior to the election and over the weekend. That is the majority. One indicates a belief that they will wait to see how the election goes. When there are gag orders, the higher-ups speculate as you do.
So I sit here now with you - waiting and wondering when the exact moment will be.
It is really aggravating. I meditate, I hit the gym, I do something else.
I'm just glad I don't hit the Red Bull like my cohorts. They drop like flies with premature heart attacks. Just sad.
The last word I have is that it may hit during the night, so once again I am up. I am certainly used to doing the night stint. If it isn't tonight, then I still know that they are ready to hit the button and get this finished. Since the night is effectively over, I hope for action today. I know that only the NPTB will know the moment they feel is perfect to hit this button.
As I get definitive information, I will share all that I can here.
I'm glad that I have those like you to keep me company.
I want to thank you for listening to my words. To me, they are little parts of myself, shining outward like the stars of the Milky Way and spreading across a vast sky of thought. I didn't imagine that they could really make a difference.
Copyright 2016 All rights reserved VERITAS WITTICISMS
Saturday UPDATE for November 5, 2016 -
It was reported to me late Friday evening that the 2nd reading of the budget is scheduled for Saturday then on to a final vote next week, hopefully.
Mosul is being reported as "clear" and they are still awaiting PM Abadi to plant the "official" flag. Time Will Tell!! smile
PS - Looks like two contract rates will still be available at exchange time.....based on member reports.
AJTexas: Outstanding! Iraq is also meeting with the IMF on Monday regarding the $2.4 billion loan.
Dinera: I always thought it would happen Saturday night while I'm asleep and wake up Sunday morning to everything different! Is that wishful thinking?
Dinera: Someone posted this earlier but I was just able to watch it. What keeps you up at night and mentions currencies. It goes along with my thinking of happening on a Saturday night and waking up on Sunday to it being different.
I think that everything has to line up at the same time which hasn't worked out yet for everyone to agree upon. http://www.bloomberg.com/news/videos/b/ba9798f7-258a-4cb3-9ffb-b651a9e5c291
Dinara: It might be wishful thinking but that's ok…..
Jacksmits: i did like the part you refer too where they said the change/announcement would come in the middle of the night on a weekend or such...makes sense
JackSmits: honestly, i think the whole thing with the RV is all about progress in Iraq, particularly security and stability in the country...when they feel like they have that....ie ISIS in check, we will see what we are looking for
IdahoUSA: DAYLIGHT SAVINGS TIME ENDS ON SUNDAY NOVEMBER 6TH, 2016. Set your clocks back one hour on November 6, 2016 at 2am (which becomes 1am) REMEMBER TO ALSO REPLACE BATTERIES IN SMOKE AND CARBON MONOXIDE DETECTORS.
MagLiteParty: BOOM !!!!! This article should be retitled..."if u own dinar GET READY TO RETIRE IN 2017"...
Militia: Lol.. Might be ready now to retire, as election days is coming.....exciting times, tonight is! The scratch of grains and the metal so soft, is at hand for us all. If the 2016 data from the IMF is complete and implementation effectively imminent, IMO it would be a good idea to be ready..
I felt awhile ago that the country of Iraq has been taking on huge responsibility with the IMF and with the way the articles of late read they fully intend and are accepting thier obligations in regard to reforms. Thus, our time is near for the project to delete the zeros is finished, and for all to see. IMO the ccs are filled with reason that the beginning of the end is upon us. MMTC cheers
Cole: When Kuwait RV'd their currency, didn't they RV days after an article like this one came out? IMO
Don961: Learn the benefits of removing zeros from the Iraqi currency
04/11/2016 World News , Breaking News
Central Bank: the deletion of zeros in early 2017
1.adraj Iraqi dinar within the global currency basket and handle it officially the world 's stock exchanges and financial markets.
2.amkanah held economic agreements to handle the national currency as Iraq could hold Order dollar instead dinar when the contract to build two production lines deals agreements or transactions Stirad goods this will cover a very large part of the foreign remittances that are squandering of national wealth from foreign exchange.
3.illt trading allows dealing in Iraqi dinar in the Iraqi market and not rely on foreign and squandered currencies and Bosrtha transfer abroad by the private and public sector, which is the sovereign wealth dissipated abroad a small, non - essential positions and luxury in Iraq at the time need this wealth of storage as a reserve.
4.seetm rid of manipulating market currency exchange and finish the so - called auction sell the currency , "" burning oven dollar ""
5.satoagaf work corrupt suspicious of banks and their agents and Smaserthm "" contractor and investor cartoons "
6.seetm capping the highest amounts transferred abroad of currency the dollar and the euro and the yen sterling and the yuan exchange - active and other pound to ensure that no smuggling the vast majority of trades of currency smuggling.
and the Iraqi Central Bank Governor said the project to delete the zeros will be implemented in the beginning of 2017, indicating that the bank began to develop the first steps of the project.
and he said: " the purpose of deleting three zeros from the Iraqi currency is to support the Iraqi dinar and make it a competitor of foreign currency", returned him as "this strategic project will serve the country 's economy if will be inclusion of the Iraqi dinar in the global basket of currencies in the vital world stock markets. "
He added that" the Bank is working on mechanisms for the implementation of this project is expected to start at the beginning of 2017 , "noting that" the central bank began the first steps of the project through the selection of the designs , companies and the country that will be the currency printing. "
He pointed out that" the new currency entry does not mean the destruction of ancient coins, but will continue to trade for about ten years , "pointing out that" this period will enable the central bank to withdraw the old currency in accordance with the dynamics and mechanics in order to preserve liquidity In the market
Don961: As Frank has mentioned on the CC , articles like this pop up in the most curious places .... this headline appeared on a scrolling news wire ... amidst a plethora of news about Mosul ... and the different battles going on ... IMO
Militia: Thanks Don. Do we really believe the early 2017 part or is it misdirection and will POP anytime now?? That is the question for the day. Sure will be nice to live in a world without lying media and deceitful governments....
PappaJ: IMO 2017 IS NOT A MISDIRECTION!!! IRAQ IS BEING HELD IN CHECK FOR THE RELEASE TO THE INTERNATIONAL WORLD OF THE IQD. THEY MAY VERY WELL BE A VALUE CHANGE INSIDE OF THE COUNTRY TO GIVE THE CITIZENS POWER OF PURCHASE AND YES THEY CAN DO IT --HOW?
PJ just my thoughts time will tell------- PATIENCE FOR WE ARE ALL PADUWANS IN THIS INVESTMENT!
Don961: Nice PJ .....The article speaks of deleting the zeroes from currency notes themselves ... "the central bank began the first steps of the project through the selection of designs, companies and the country in which the currency will be printed." ... the lower denoms .... perhaps there will be an announcement for citizens to go to exchange centers and/or banks to exchange their old currencies for new .... just like we will exchange these dirty old dinar notes for fresh new money we can use .... electronically deleting doesn't have the burden of dealing with exchanging physical notes ... IMO
11/5/2016 12:05:26 pm
DEAR VERITAS --- WHAT A TREMENDOUS AND REFRESHING POST!!! You are stunningly well-written and have risen to the short list of "must read" for me and, believe me, it is a VERY SHORT list at this juncture.
11/5/2016 04:28:41 pm
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Pulmonary host defense employs a combination of biochemical and biophysical activities to recognize, inactivate, and mediate clearance of environmental agents as well as modulate the overall response to such challenge. Dysregulation of the inflammatory arm of this response is associated with chronic lung diseases (CLD) including cystic fibrosis and chronic obstructive lung disease. Although mechanisms mediating immunoregulation are incompletely characterized, decrements in levels of the nonciliated secretory cell product Clara cell secretory protein (CCSP) in numerous CLD and identification of proinflammatory state in mice homozygous for a null allele of the CCSP gene (CCSP−/−) suggest a central role for the nonciliated secretory cell in this process. In an effort to determine the molecular basis for immunoregulatory defects associated with CCSP deficiency, we utilized difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight to compare the proteomes of wild-type and CCSP−/− mice. We demonstrate a shift in the isoelectric point of the immunomodulatory protein annexin A1 (ANXA1) to more acidic isoforms in CCSP−/− mice. Similar ANXA1 mRNA and protein abundance in wild-type and CCSP−/− tissue and identical localization of ANXA1 protein to alveolar macrophages and the ciliary bed of ciliated cells demonstrated that CCSP deficiency was associated exclusively with altered posttranslational modification of ANXA1. These results suggest that both long- and short-range paracrine signaling between nonciliated secretory cells and cells of the immune system and epithelium impact modification of cell type-specific proteins and implicate nonciliated secretory cells in a regulatory axis that might integrate critical aspects of host defense.
- Cara cell secretory protein
- chronic lung disease
efficacy of mucosal defense mechanisms is dependent on selectivity of the system for pathogenic agents and appropriate regulation of the magnitude and duration of the response. In the gut, identification of anti-inflammatory molecules derived from various tissue compartments as well as leukocytes has led to the concept that cross talk between epithelial, vascular, mesenchymal, and inflammatory system cells creates an "anti-inflammatory network" that is critical to balanced activation and termination of the host defense response (9, 12, 16). Dysregulation of feedback mechanisms that are an essential component of the anti-inflammatory network are postulated to contribute to hyperinflammatory states associated with inflammatory diseases. Thus disruption of regulatory cascades has the potential to transform a beneficial response into a pathological situation that contributes to initiation and exacerbation of chronic disease.
Continual exposure of the pulmonary mucosa to inhaled microorganisms, particles, and gases has necessitated development of host defense mechanisms that are highly responsive to infection or injury but are effectively attenuated following clearance of the pathogen, withdrawal of the irritant, or repair of the lesion. Multiple tissues and cell types contribute to the waxing and waning process that constitutes the innate response to environmental agents in the lung. Traditional roles for the airway epithelium in host defense include function as a dynamic barrier to inhaled agents and contribution to the biochemical and mechanical aspects of mucociliary clearance. In addition, epithelial irritation and injury results in rapid alterations in the spectrum of proteins secreted into the airway lumen. Epithelial-derived host defense proteins include antioxidant, bacteriostatic, and bacteriolytic proteins as well as chemokines and cytokines that recruit immune system cells and modulate the immune response (20). The vital role for appropriately regulated epithelial responses in mucosal defense is highlighted by the potentially life-threatening sequelae of chronic inflammatory diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) (19, 25). In such individuals, deviations in the normal inflammatory response have been correlated with a decline in the most abundant secreted product of nonciliated secretory cells, Clara cell secretory protein (CCSP/CC16), and increased abundance of annexin A1 (ANXA1)/lipocortin1 (2, 10, 27, 30, 38). Both of these proteins have been proposed to exhibit immunomodulatory properties, yet their role in modulation of airway inflammation has not been determined.
ANXA1 is a 37-kDa protein that is abundantly expressed in cells critical to the innate immune response, notably blood leukocytes, macrophages, and epithelial cells (1). Putative roles for ANXA1 in modulation of inflammation stem from the finding that ANXA1 antagonizes neutrophil extravasation (8). In lipopolysaccharide-exposed macrophages, the anti-inflammatory effects of ANXA1 may be mediated by ANXA1-dependent alterations in the activity of proinflammatory enzymes including inducible nitric acid synthetase, cPLA2, and potentially in cyclooxygenase-2 (33). In these cell types, interactions between ANXA1 and target proteins are regulated through receptor tyrosine kinase or PKC-mediated phosphorylation of specific serine, tyrosine, and threonine residues (9). Alternative posttranslational modifications to ANXA1 result in generation of isoforms with distinct isoelectric point (pI). Interestingly, lymphocytes from individuals with Fragile X syndrome express multiple pH isoforms of ANXA1 that are not phosphorylation intermediates and may be a consequence of hyperacetylation of the NH2-terminal domain (24, 37). Within the lung epithelium, ANXA1 is expressed specifically in ciliated cells of the conducting airway (26), colocalizes with the chloride transporter, CF transmembrane conductance regulator (CFTR), and is severely downregulated in the context of CF disease in humans and in CFTR knockout mice (2).
To define roles for CCSP in airway homeostasis, mice have been established that are homozygous for a null allele of the CCSP gene (CCSP−/−). Even though CCSP−/− mice have no overt lung phenotype in the steady state (35), challenge studies have revealed elevated epithelial cell susceptibility and exaggerated inflammatory responses to inhaled oxidant pollutants (21, 22), an elevated inflammatory response to challenge with either respiratory viruses or live Pseudomonas aeruginosa (17, 42), and enhanced epithelial remodeling in an ovalbumin model of allergic inflammation (43). Moreover, CCSP deficiency in CCSP−/− mice is associated with changes in nonciliated secretory cell secretory function and the steady-state abundance of specific mRNA species within the lung including those coding for secretory proteins (SCGB3A2) and immunoglobulin A (IgA) (11, 36, 45). These reports suggest that CCSP−/− mice phenocopy aspects of chronic lung diseases seen in human patients with COPD, asthma, and CF and support a role for CCSP and/or altered secretory cell function in the pathophysiology of these diseases.
The elevation of IgA and the greater inflammatory response of CCSP−/− mice to injury suggested that CCSP deficiency results in a proinflammatory state. The present study uses difference gel electrophoresis (DIGE) (39) to further explore the molecular basis for immunoregulatory defects associated with CCSP deficiency. Our initial comparison of the total lung proteomes of wild-type and knockout mice by DIGE revealed one consistent, significant difference. ANXA1 undergoes posttranslational modifications in knockout mice that shift the pI of ANXA1 from a single protein found at ca. pH 7.0 to multiple more acidic forms separated by ∼0.2–0.4 pH units. We demonstrate that CCSP-dependent modifications to ANXA1 are common to airway ciliated cells and macrophage populations from lung and the peritoneum. These findings suggest that CCSP functions as a biochemical link between functionally distinct airway epithelial cells and among cells of the innate immunity network. Disruption of this paracrine signaling network in the context of CCSP deficiency coupled with secondary effects on ANXA1 function may contribute to hyperinflammation in chronic lung disease.
Wild-type 129 SvEv (Taconic, Germantown, NY) and C57Bl/6 (Jackson Laboratories, Bar Harbor, ME) mice were maintained as inhouse breeding colonies within the University of Pittsburgh barrier facility. Two lines of mice harboring a null allele of the Scgb1A1 gene (formerly termed CCSP), 129P2/OlaHSD*129P3/J-Scgb1A1tmBrst (129 CCSP−/−, Mouse Genome Informatics no. 2684333), and B6.129OlaHSD*129P3/J-Scgb1A1tmbrst (B6 congenic CCSP−/−), were maintained in a homozygous condition through interbreeding in the same facility. All animals were maintained on a 12-h light/dark cycle and were allowed food and water ad libitum. Animals were monitored quarterly for known mouse pathogens and entero- and ectoparasites and were consistently disease-free. Female mice between 2 and 4 mo were used for all experiments. All procedures involving animal use were reviewed and approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
Tissue homogenate.
Wild-type and CCSP−/− mice of either the 129 or B6 congenic background were anesthetized with 15 ml of 2.5% avertin/kg body wt and exsanguinated. Left lung lobes were homogenized with a Polytron homogenizer in 0.5 ml of denaturing lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 10 mM DTT, 10 mM HEPES, pH 8.0) on ice. Particulate matter was removed by centrifugation (12,000 g, 15 min, 4°C). Supernatants were aliquoted, frozen in liquid nitrogen, and stored at −80°C.
Bronchoalveolar and peritoneal lavage.
Groups of five C57Bl/6 wild-type or B6 congenic CCSP−/− mice were used for analysis of alveolar and peritoneal macrophage proteins. Mice were anesthetized as detailed above, and lungs were lavaged via a tracheal cannula with 2 × 1 ml of phosphate-buffered saline. The peritoneum was then lavaged by injecting 5 ml of phosphate-buffered saline, massaging the abdominal cavity, and recovering the fluid. Either peritoneal or bronchoalveolar lavage from animals of like genotype was pooled, and cells were recovered by centrifugation at 300 g, for 5 min, at 4°C. Cells were washed once with saline, and the pellet was aspirated to dryness. Cells were lysed in 100 μl of denaturing lysis buffer as described above. Debris was removed by centrifugation as described above.
Two-dimensional gel electrophoresis.
Cleared wild-type and CCSP−/− homogenates were thawed on ice, and protein concentrations were determined with the 2-D Quant Kit (Amersham). Aliquots containing 100 μg of wild-type protein were labeled with Cy3, and similar aliquots of CCSP−/− protein were labeled with Cy5 essentially as described by Unlu and colleagues (39). Dyes were obtained from the University of Pittsburgh Proteomics Core, and labeling was reversed to account for differences in dye labeling efficiency. The differentially labeled wild-type and knockout protein samples were combined and loaded onto either 18- or 24-cm Immobiline pH 4–10 Isoelectric Focusing (IEF) strips (Amersham Biosciences) that were preswollen overnight in lysis buffer plus 2% ampholytes (IPG buffer, Amersham). First-dimension gels were focused to 70,000-V hours on an IPGphor power source (Bio-Rad). Second-dimension linear 5–12% SDS-PAGE gels were run at 25 mA per gel for 30 min and then at 50 mA per gel for ∼5 h.
Gel images were acquired on Typhoon scanner (Amersham) and analyzed using Image J (http://rsb.info.nih.gov/ij/). In Image J, the Cy3 and Cy5 images were stacked, and a two-frame movie was evaluated visually for changes in spot intensity. At least three independent comparisons were performed to identify repeatable differences. Gels were poststained with colloidal Coomassie blue (Bio-Rad Bio-Safe), and proteins of interest were excised from the gel as 1.5-mm diameter plugs with a manual spot picker (OneTouch, The Gel).
Protein Identification
Trypsin digestion.
Gel pieces were dehydrated at room temperature with 50% methanol/25 mM NH4HCO3 (2 × 100 μl wash) followed by 100% acetonitrile (1 × 50 μl wash). Dehydrated pieces were dried in a SpeedVac for 15 min at room temperature. Tryptic digests were performed with Promega Trypsin Gold, 20 ng/μl in 25 mM NH4HCO3 (10 μl) for 4 h at 42°C. Protein digests were removed to a new tube, and peptides were eluted twice with 50% acetonitrile/1% trifluoroacetic acid (2 × 50 μl). Recovered peptides were evaporated to dryness in a SpeedVac at room temperature. Dried peptides were dissolved in 50% acetonitrile/0.3% trifluoroacetic acid/1 mM citric acid, mixed with an equal volume of saturated α-cyano-4-hydroxy cinnamic acid. Multiple 0.75-μl aliquots were spotted on a 192-well matrix-assisted laser desorption ionization (MALDI) plate (Applied Biosystems).
Mass spectrometer analysis.
MALDI-time-of-flight (TOF) and MALDI-TOF-TOF analyses were performed on an ABI 4700 mass spectrometer at the University of Pittsburgh Integrated Core Facility. MALDI-TOF analysis was performed in the reflectron mode. An analysis of peak lists from MALDI-TOF and MALDI-TOF-TOF was performed with an inhouse version of Mascot (Matrix Science).
Western blot analysis.
Lung homogenate (20 μg) was resolved by reducing SDS-PAGE and was electrophoretically transferred to PVDF membrane. The membrane was blocked with 5% powdered milk in phosphate-buffered saline (blocking buffer) and probed using a rabbit polyclonal antibody against ANXA1 (1:4,000 in blocking buffer, Zymed Laboratories). Immunoreactive bands were detected with a goat anti-rabbit HRP conjugate (1:4,000 in blocking buffer, Invitrogen), and peroxidase reaction was developed using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). Bands were detected using BioMax film (Kodak, Rochester, NY).
Quantitative PCR analysis.
Left lung lobes from three wild-type or three B6 congenic CCSP−/− mice were homogenized individually, and total cellular RNA was purified by the method of Chomczynski and Sacchi (6). RNA was quantified spectrophotometrically, and 400 ng was reverse transcribed using a First Strand Synthesis Kit (Invitrogen). Aliquots of wild-type, knockout, and reference cDNA were amplified using TaqMan 1000 RXN Gold with Buffer A Pack (Applied Biosystems) and Assays-On-Demand for ANXA1 and β-glucuronidase (Applied Biosystems). Cross threshold (CT) values were determined, and the ΔΔCT was calculated (18). The means ± SE are reported. Differences were compared by the Student's t-test. Differences were accepted at P = 0.05.
Dual immunofluorescence and confocal microscopy.
Lung tissue from wild-type or CCSP−/− mice was fixed by instillation of 10% neutral buffered formalin (NFB) at 10 cmH2O pressure through a tracheal cannula followed by immersion in 10% NFB for 24 h. The right lower lobe was embedded in paraffin, and 5-μm sections were taken through the major airway with the inclusion of minor daughter branches using a rotary microtome (Fisher Scientific, Pittsburgh, PA). Immunostaining was performed using a combination of airway-specific markers, CCSP (goat-anti-CCSP, 1:8,000, generated in this laboratory), and acetylated tubulin (mouse IgG2b-anti-AcT, 1:8,000, Sigma) in combination with ANXA1 (rabbit-anti-ANXA1, 1:200, Zymed Laboratories). Sections were cleared in xylenes and blocked for 1 h with 5% bovine serum albumin and fraction V protease-free in phosphate-buffered saline (5% BSA/PBS) and then incubated overnight at 4°C with primary antibody diluted in 5% BSA/PBS. Sections were washed with phosphate-buffered saline and stained with the appropriate secondary antibodies: donkey anti-goat Alexa 488 or donkey anti-goat Alexa 594 (1:500; Molecular Probes, Eugene, OR), donkey anti-rabbit Alexa 488 or donkey anti-rabbit Alexa 594 (1:500; Molecular Probes), and goat anti-mouse IgG2b Alexa 594 (1:500; Molecular Probes). Sections were mounted using Flouramount G (Southern Biotech, Birmingham, AL) containing 1 g/ml 4,6-diamidino-2-phenylindole dihydrochloride (Sigma). Immunoreactivity was not detected in the absence of primary antibodies. Images were captured using an Olympus Fluoview BX61 confocal microscope and assembled in Adobe Photoshop.
Epithelial Cell Cultures
Air-liquid interface cultures.
Groups of four to six C57BL/6 wild-type and B6 congenic CCSP−/− mice were anesthetized as detailed above, and the trachea was recovered. Tracheal tissue was digested with 0.15% pronase (Roche) in Ham's/F-12 (GIBCO) overnight at 4°C. Cells were recovered and cultured in mouse tracheal epithelial culture medium as detailed by You and colleagues (47) until individual cultures reached a resistance of ∼1,000 mΩ (5–6 days). At this point, the apical culture medium was removed, and the basal culture medium was replaced with 2% Nuserum. Cultures were fed on alternate days and allowed to differentiate for 9 days. Ciliogenesis was followed visually using a phase-contrast microscope and was used as an estimate of differentiation.
Whole mount immunofluorescence analysis.
Groups of three C57Bl/6 wild-type or B6 congenic CCSP−/− air-liquid interface cultures from two independent experiments were analyzed. The apical surface was washed with 200 μl of 1× phosphate-buffered saline, and the apical and basal compartments were filled with 10% neutral buffered saline. Cultures were fixed at 4°C for 30 min, washed with 1× phosphate-buffered saline, and permeabilized with PBS/BSA containing 0.1% Triton X-100 at room temperature for 30 min. Cellular antigens were detected by dual immunofluorescence and visualized by confocal microscopy as described above.
DIGE analysis of epithelial cultures.
Sets of six wild-type or B6 congenic CCSP−/− epithelial cultures were pooled for protein analysis. The apical surface of each culture was washed with 200 μl of phosphate-buffered saline, and the culture was placed on ice. Denaturing lysis buffer (100 μl) was added to the apical compartment, and the cells were disrupted with the broad end of a pipette tip. Lysates from cultures of like genotype were pooled, and debris was removed by centrifugation at 12,000 g for 15 min at 4°C. Supernatants were frozen in liquid nitrogen and stored at −80°C. Protein labeling and gel electrophoresis were preformed as described above.
Altered Posttranslational Modification of ANXA1 in Lung Tissue of CCSP−/− Mice
We previously demonstrated that nonciliated secretory cells of CCSP-deficient mice exhibit significant perturbations to their secretory apparatus (11). In the present study, we employed two-dimensional differential gel electrophoresis to more precisely define changes in protein abundance and distribution that accompany CCSP deficiency. Total protein recovered from lung tissue of wild-type C57Bl/6 mice and B6 congenic CCSP−/− (CCSP−/−) mice were conjugated to Cy3 and Cy5 dyes and protein profiles visualized by two-dimensional DIGE (2D-DIGE) using a linear 4–10 pH gradient in the first dimension followed by separation using linear 5–12% SDS-PAGE. Pairwise comparisons were performed using three independently generated lung protein samples from individual wild-type and CCSP−/− mice. Figure 1 includes a representative image of profiles generated comparing lung proteins from wild-type and CCSP−/− mice. Approximately 500 distinct protein species were resolved per gel. Differences in spot intensity that were consistently observed among gels comparing wild-type and CCSP−/− lung proteins were confirmed by dye substitution (data not shown). Protein identities of both validated and reference spots were determined by in-gel trypsin digestion followed by MALDI-TOF analysis. One series of protein species showing variability between lung protein samples from wild-type and CCSP−/− mice was found to be ANXA1 (Fig. 2, A–C, and Supplemental Data, which is available online at the AJP-Lung web site). The only ANXA1 species present within lung protein from wild-type mice had a pI of ∼7.0 and predicted molecular weight of 37 kDa (Fig. 1, A and C). In contrast, lung protein from CCSP−/− mice lacked the pI 7.0 isoform but harbored more acidic ANXA1 isoforms with pI values of 6.8 and 6.4 (Fig. 1, B and D). Differences between wild-type and CCSP−/− lung ANXA1 isoforms were retained following treatment of total lung homogenates with alkaline phosphatase (data not shown). Further analysis of ANXA1 protein abundance through Western blot analysis of total lung protein (Fig. 3A) and analysis of ANXA1 mRNA abundance through real-time quantitative RT-PCR amplification of total lung RNA (Fig. 3B) revealed no significant differences in ANXA1 gene expression between wild-type and B6 congenic CCSP−/− mice.
To independently verify the identity of protein species exhibiting differences between wild-type and CCSP−/− mice, unlabeled protein samples from homogenized lung tissue of C57Bl/6 WT and B6 congenic CCSP−/− were resolved by 2D gel separation and assessed by Western blot analysis. Consistent with data from MALDI-TOF analysis was the demonstration that blots of total lung protein from CCSP−/− mice included ANXA1-immunoreactive protein species with more acidic pI (Fig. 4B), whereas lung protein from wild-type mice contained the more basic isoform of ANXA1 only (Fig. 4A). To verify that differences in ANXA1 were dependent on CCSP and not due to strain-specific difference in ANXA1 posttranslational modification, unlabeled total lung protein from wild-type and CCSP−/− mice of the 129OlaHsd strain were compared by 2D gel electrophoresis followed by Western blot and immunodetection of ANXA1 isoforms. Even though strain-specific differences were observed in the abundance of ANXA1 within lung tissue samples, the same pattern of CCSP-dependent posttranslational modification of ANXA1 was observed (Fig. 4, C and D).
ANXA1 Localizes to the Apical Domain of Ciliated Cells Within Conducting Airways
Since differential posttranslational modification of ANXA1 has been linked with changes in its subcellular localization (34), we sought to determine the cellular and subcellular distribution of ANXA1 expression within airways of wild-type and CCSP−/− mice. ANXA1 was localized within airways by immunofluorescence microscopy, with colocalization for cell type-specific markers including CCSP to define secretory cells in airways of wild-type mice and acetylated tubulin (AcT) to define ciliated cells within airways of wild-type and CCSP−/− mice. In wild-type airways, ANXA1 immunoreactivity specifically localized to the apical membrane of a subset of airway epithelial cells that were positive for AcT immunoreactivity (Fig. 5, D–F) and was absent in CCSP-immunoreactive cells (Fig. 5, A–C). The cellular and subcellular distribution of ANXA1 in airways of CCSP−/− mice was identical to that observed for wild-type: ANXA1 immunoreactivity colocalized with that of AcT immunoreactivity within the ciliary bed of ciliated cells (Fig. 5, G–K). These data suggest that CCSP deficiency and associated changes to nonciliated secretory cells result in changes in the posttranslational modification but not the cellular pattern or subcellular distribution of ANXA1 within ciliated cells.
CCSP-Dependent Posttranslational Modification of Macrophage-Associated ANXA1
Secretory and ciliated cells share close proximity within airways that includes cell-cell contacts and the potential for junctional communication through tight junctions. However, resident myeloid cells and those recruited through local proinflammatory conditions are also known to harbor significant levels of ANXA1 that contribute to the total lung pool. We sought to determine whether myeloid cells maintained the same difference in ANXA1 posttranslational modification as was apparent within total lung protein. Two-dimensional DIGE was performed on total protein prepared from cells recovered in bronchoalveolar lavage from wild-type and CCSP−/− mice. Macrophages represented the principal cell type recovered within bronchoalveolar lavage from both wild-type and CCSP−/− mice, accounting for ∼98% of total lavagable cells in each case. Protein species separated from lavagable cell pellets included high levels of ANXA1 that were clearly detected by 2D-DIGE with subsequent confirmation of protein identity by Western blot analysis (Fig. 6). Lung macrophage-associated ANXA1 from wild-type mice was represented as a single basic isoform with a pI of 7.0 (Fig. 6A). In contrast, lung macrophage-associated ANXA1 from CCSP−/− mice was represented by primarily by the more acidic isoforms with pI of 6.8 and 6.4 (Fig. 6B). This pattern of ANXA1 pI isoforms was similar to that reported for lymphocyte-derived ANXA1 from Fragile X syndrome patients (37). The demonstration that macrophage-associated ANXA1 exhibits the same CCSP-influenced posttranslational modification as that observed within total lung protein implies that these influences are mediated through a paracrine mechanism.
Even though the lung is the principal site of CCSP expression and protein accumulation, CCSP is present at measurable levels within serum and is present within the glomerular filtrate before uptake and catabolism within the epithelium lining the proximal convoluted tubule (5, 14). We next sought to determine whether posttranslational modifications to ANXA1 were found on macrophages located in nonpulmonary sites. Peritoneal macrophages were recovered from peritoneal lavage fluid of wild-type and CCSP−/− mice, and their proteomes were evaluated by 2D-DIGE followed by MALDI-TOF identification of ANXA1 isoforms. As was the case for lung macrophages, peritoneal cells from wild-type mice exhibited a single ANXA1 isoform with a pI of ∼7.0 (data not shown). However, peritoneal cells from CCSP−/− mice displayed the same acidic isoforms of ANXA1 as had been demonstrated within lung tissue and isolated macrophages (data not shown). These data demonstrate that the paracrine influence of CCSP deficiency extends beyond the lung.
Posttranslational Modification of ANXA1 is an Intrinsic Property of the CCSP-Deficient Epithelium
Since ANXA1 isoforms detected in total lung homogenates (Figs. 1, 2, and 4) could reflect ANXA1 derived from ciliated epithelial cells (Fig. 5) or macrophages (Fig. 6), we assessed ANXA1 expression in differentiated airway epithelial cell cultures. Cultures derived from wild-type mice were harvested at the point of maximal ciliogenesis, the time point at which nonciliated secretory cell abundance was ∼10–15% of nucleated cells (47) (data not shown). ANXA1-immunoreactive protein was present at the apical surface of ciliated cells and colocalized with AcT immunoreactivity (Fig. 7, A–C). ANXA1 was not detected in other cell types (data not shown). When comparisons were made between cultures derived from wild-type and CCSP−/− mice, no differences were observed in the abundance of cilia or in the cellular or subcellular distribution of ANXA1 immunoreactivity (data not shown). This analysis indicated that expression of ANXA1 on ciliated cells was an intrinsic property of the airway epithelium and not a secondary consequence of interactions between the epithelium and inflammatory cells.
Two-dimensional DIGE was performed to determine the ANXA1 isoforms expressed on ciliated cells derived from wild-type and CCSP−/− mice. As was the case in total lung protein from wild-type mice, the predominant ANXA1 isoform in protein recovered from wild-type acute lung injury (ALI) cultures was the basic isoform (Fig. 8A). However, small amounts of the acidic isoforms were also detected. Incomplete modification of ANXA1 in wild-type cultures may be related to underrepresentation of CCSP-expressing cells in tracheal epithelial cell cultures (47). Total protein from ALI cultures of CCSP−/− epithelium lacked the basic isoform of ANXA1, yet possessed acidic ANXA1 isoforms previously identified within total lung tissue homogenate from CCSP−/− mice. These data support the conclusion that posttranslational modification of ANXA1 in ciliated cells was regulated in a CCSP-dependent fashion. The observation that the ANXA1 modification on ciliated cells was similar to that observed for macrophage cells suggests that alterations in ANXA1 posttranslational modification in CCSP−/− mice most likely resulted from short-range interactions between modified secretory cells and adjacent ciliated cells and long-range effects on distant inflammatory cell types.
We demonstrate that CCSP deficiency in mice results in changes to the charge characteristics of ANXA1 in ciliated cells and either pulmonary or peritoneal macrophages. Localization of ANXA1 to ciliated cells of the conducting airway epithelium and to resident macrophages suggests that interactions between these cell types and airway secretory cells are impacted directly by CCSP deficiency. This finding provides, until now, unappreciated links between secretory cell function and the molecular regulation of other lung cell types that fulfill roles in lung homeostasis and disease. Moreover, the finding of CCSP-dependent ANXA1 modifications in peritoneal macrophages suggests that airway epithelial remodeling has the potential to impact immunoregulation in other mucosal sites.
ANXA1 has been shown to undergo a number of distinct posttranslational modifications that have potential to alter its subcellular distribution and/or function. Phosphorylation of a tyrosine residue at position 21, NH2-terminal serine residues, and a COOH-terminal threonine residue, have been shown to be mediated through various receptor tyrosine kinases, PKC, or PKA, respectively (31, 41, 44). Modifications to the NH2-terminal domain influence calcium binding of ANXA1 and subsequently its ability to interact with plasma membranes and plasma membrane-associated EGF receptor (29, 44). However, changes in the subcellular distribution of ANXA1 were not observed between ciliated cells of wild-type and CCSP−/− mice. Moreover, our observation of changes in pI independently of changes in apparent molecular weight, and the resistance of these changes to phosphatase treatment, suggests that posttranslational modifications other than phosphorylation account for the observed differences in ANXA1 isoforms represented within lungs of wild-type and CCSP−/− mice. Other posttranslational modifications could include acetylation, sulfation, or glycosylation. NH2-terminal acetylation of ANXA1 has been suggested to regulate its interaction with cytoskeletal components such as the S100 calcium-binding family of proteins (15). Differences in N-linked glycosylation have also been shown to alter the charge characteristics of ANXA1, resulting in the generation of distinct isoforms that can be distinguished according to their pI (4). The potential for posttranslational modifications of ANXA1 to influence its ability to bind calcium, lipids, or other proteins, coupled with its localization at the base of cilia within ciliated cells, raises the possibility that altered Clara cell secretory function that results from CCSP deficiency may lead to altered ciliated cell function as a result of changes in the ligand-binding properties of ANXA1 (32). Further studies are needed to determine whether CCSP deficiency impacts ciliated and macrophage cell activity and whether this is mediated through changes in the posttranslational modification of ANXA1.
CCSP-dependent changes in the pI isoforms of ANXA1 present within airway epithelial cells and macrophages derived from either lung or peritoneal compartments suggest paracrine influences of CCSP deficiency on these cell types. The obvious candidate mediator of such a paracrine effect is CCSP itself. Like ANXA1, CCSP has been shown to bind both calcium and lipophilic compounds such as lipids, progestins, and certain metabolites of polychlorinated biphenyls. As such, CCSP deficiency may alter the bioavailability of these ligands, thus influencing the properties and functions of ANXA1. CCSP has also been shown to interact directly with cubulin, resulting in megalin-dependent internalization within epithelial cells of the proximal convoluted tubule (5). The cubulin/megalin-dependent catabolism of CCSP highlights the systemic distribution of CCSP that may be responsible for the observed posttranslational modifications to ANXA1 in peritoneal macrophages.
The demonstration that CCSP deficiency in CCSP−/− mice is also associated with perturbations in Clara cell secretory function and altered protein composition of airway lining fluid raises the possibility that other soluble factors may mediate these effects on ANXA1 (11, 45). Other candidate secretory products of Clara cells that may mediate these effects include SCGB3A2, another member of the secretoglobin family of proteins whose expression is elevated in lungs of CCSP−/− mice (45). SCGB3A2 has been shown to interact with the class A scavenger receptor, MARCO (3). Interestingly, interactions between scavenger receptors and other ligands, such as oxidized lipoproteins, have been shown to increase intracellular Ca2+ and activate a variety of cytosolic kinases and phospholipases via stimulation of pertussis toxin-sensitive G proteins (7, 28, 40, 46). Moreover, MARCO has been shown to mediate cytoskeletal rearrangements within dendritic cells, and its expression on macrophages is critical for appropriate trafficking of B cells to the marginal zone of the spleen (13, 23). These reports suggest that extracellular pools of secretoglobins such as SCGB3A2 and potentially CCSP can be sensed through interactions with scavenger receptors and that transduction of these signals impacts the functional properties of monocytic cells. Interestingly, we previously demonstrated that CCSP deficiency is associated with altered immunoglobulin production within lung tissue and increases in the abundance of IgA-expressing B cells in the peribronchial region of conducting airways (45). It is unknown whether SCGB3A2-MARCO interactions results in similar activation of signaling within lung macrophages or whether CCSP may exhibit a similar capacity for interaction with scavenger receptors. However, these reports raise the possibility that changes in the expression of secretoglobins, either CCSP or other members of the secretoglobin family such as SCGB3A2, may result in altered posttranslational modifications of ANXA1 within macrophages through a scavenger receptor-dependent pathway. The demonstration that this mode of epithelial-inflammatory cell cross talk is altered within lungs of CCSP−/− mice suggests that similar immunoregulatory changes may result from CCSP deficiency seen with chronic lung disease.
Roles for altered Clara cell secretory function in the setting of chronic lung disease have been suggested from studies demonstrating that the abundance of CCSP is typically reduced in either airway lining fluid or in the systemic circulation of patients with chronic lung disease. The basis for these changes to airways, be it chronic injury and phenotypic changes to Clara cells associated with their role as the principal bronchiolar progenitor cell, and/or altered differentiation of the epithelium leading to a phenotypic change within the secretory cell population, has not been well established. However, these changes to airways are associated with defects in mucociliary clearance and local inflammatory responses. The finding in the present study that CCSP deficiency leads to biochemical changes within ciliated cells of the airway epithelium both in vivo and in vitro provides a link between these disparate yet functionally coordinated processes. These data combined with the finding that decrements in CCSP protein levels are also associated with decreased abundance, degradation, and altered subcellular distribution of ANXA1 in ciliated airway cells of CF patients (2, 38) suggest that CCSP and ANXA1 may regulate critical aspects of mucociliary clearance. The demonstration that changes in ANXA1 modification are not restricted to ciliated cells of the surface epithelium but are also seen within lavagable macrophages of the steady-state lung and peritoneum provides further insight into mechanisms of cross talk between secretory cells and other lung cells. These data, when coupled with our earlier demonstration that CCSP deficiency sensitizes mice to injury and more profound inflammatory responses to inhaled oxidant gasses, suggest that CCSP deficiency leads to a proinflammatory state for which modifications to ANXA1 are a reflection. Further studies are needed to define CCSP-dependent components of the lung inflammatory response and whether changes to ANXA1 are causative.
This study was supported by National Institutes of Health Grants HL-64888, ES-08964, and HL-70575.
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