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Find below our yearly gardening update on our previous growing season. All the best to you this growing season!
Dear Fellow-Gardeners,
Cool wet springs seem to be the rule here now. In 2013 summer didn't begin until August but then we had nearly 3 months of it. The pea seed crop was quite good but we had quite a bit of deer damage. We had a wonderful crop of beets and carrots so we are looking forward to a good seed crop next year as these crops are biennial. We had a good lettuce seed harvest and also lots to eat but we have to get in the habit of seeding a crop every 10 days or so. The tomatoes did well, both indoors and out, with no late blight damage. See some new tall cultivars, with interesting colours, flavours, sizes and shapes in our tomato section. We ate all the fresh beans we wanted and had a bumper seed crop. Most of the vine crops did well but, even though the melons were very productive, they ripened badly.
In our grain patch we grew a selection of 29 cereals from our collection, including 15 samples of einkorn wheat. They all did well and we will be continuing these trials next year to try and identify which einkorns grow best here. Many people appear to have health problems related to consumption of modern wheats so we are going back to einkorn, the first domesticated wheat from about 10,000 years ago.
Judy (sister/aunt) Ternier, grew much of the seed on the Cochin farm again this year. Rachelle and Jim gardened at the Abbey all summer producing seed and a bountiful harvest of food. If you ever come to experience the hospitality of St. Peter's Abbey you will be eating some of the fruits of our labour. A special thanks to the WWOOFer volunteers who came to work with us this past summer.
Frank Morton, of Wild Garden Seeds, is doing wonderful work in Oregon breeding and selecting seed, mostly for leaf crops. He emphasizes organic growing and he breeds for taste and disease resistance. He promotes biodiversity and offers a wide range of colours and forms, often in mixes. See our lettuce section as well as other leaf crops and a few root crops.
We are always interested in the history of seeds so we indicate a date of introduction for many varieties, and something of their origins whenever we can. We also note All American Selections (AAS) awards. Each year since 1933 seed breeders and companies have been submitting new varieties to be tested by seed companies and academic and research institutions all over North America. Those which appear to be a significant improvement over previous varieties are given an AAS award.
We continue to garden without agricultural chemicals. A number of friends and relatives, and small growers, are providing us with some seed, and we are always ready to talk to new growers. A few of our seeds are from commercial sources. These commercial seeds are all open-pollinated and none of the seeds are treated, but they may have been grown using agricultural chemicals. We have indicated this by placing a C (for commercial seed) after the variety name.
PLEASE NOTE: During the peak season (February-May) it will probably take six weeks to process an order. We encourage you to group orders whenever you can. On orders sent in one package to one address there is only one handling charge, and there is a 10% discount on orders over $100. We hope you will forgive us if we occasionally group orders when it seems appropriate. If you are dissatisfied with any of our seeds we will replace them, refund you your money or give you a credit.
If anyone is interested in older varieties or ones not readily available commercially, we invite them to become members of Seeds of Diversity Canada (SoDC, formerly known as Heritage Seed Program), Box 36, Station Q, Toronto, ON, M4T 2L7. One of the activities promoted by SoDC is Seedy Saturdays (and Sundays), held in various locations throughout Canada in the winter and early spring. Each one is a day of buying, selling and swapping seeds as well as talks, displays and other educational events. We attend events every year in Alberta, Saskatchewan and Manitoba. Please check our events page for more details about the Seedy Saturday/Sunday events we attend. We would be happy to meet any customers who come to these events, and will bring seed orders with us. See a listing of all Seedy Saturday/Sunday events throughout Canada here.
SoDC offers a 60-page booklet entitled How to Save Your Own Seeds and La conservation des semences. It is available through their website.
All seeds are open pollinated,
untreated and not genetically engineered.
All seeds are grown without agricultural chemicals
except those marked (C) (commercial seed).
Prairie Garden Seeds has added their voice with others on the Safe Seed Pledge (put together by the Council for Responsible Genetics). The SAFE SEED INITIATIVE has joined with a number of seed companies to create the Safe Seed Pledge which states:
"Agriculture and seeds provide the basis upon which our lives depend. We must protect this foundation as a safe and genetically stable source for future generations. For the benefit of all farmers, gardeners and consumers who want an alternative, we pledge that we do not knowingly buy, sell or trade genetically engineered seeds or plants.
The mechanical transfer of genetic material outside of natural reproductive methods and between genera, families or kingdoms, poses great biological risks as well as economic, political, and cultural threats. We feel that genetically engineered varieties have been insufficiently tested prior to public release. More research and testing is necessary to further assess the potential risks of genetically engineered seeds. Further, we wish to support agricultural progress that leads to healthier soils, genetically diverse agricultural ecosystems and ultimately healthy people and communities."
We try to thank each person who sends us seed samples and used stamps (yes, Jim collects stamps as well as seeds!), but in case we forgot: THANK YOU. Every year a few new seeds find their way into our seed catalogue, many of which have been gifts from you who are reading this.
A big thank you is due to Betty Ternier Daniels and Judy Ternier (Jim's sisters). They have been very generous in providing seeds for the catalogue from their gardens. As we already mentioned, Judy grows a lot of the seeds found in this listing. A final thank you goes to Marie-Louise (wife/mom) for all the work she has done and for supporting and loving the farmer for the past 34 years.
Do you have seeds you would like to trade, or is there anything you would like us to offer in future years? If you do not see a seed you would like in our listing, ask and we might have a bit on hand. We are happy to have your comments on our seeds, or on any aspect of gardening on the prairies.
Rachelle and Jim


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25thframe.co.uk > Action | Thriller > Into The Storm
This film has not been scored.
What's your score?
The biggest selling video of the year is staying put at the top of the video chart this week, for a second week, although it is it's fourth time there.
It's also having a resurgence at the box office in the sing along version, hundreds of kids belting Let It Go at the top of their voices, a must for any parent!
Highest new film of the week is right down at Seven, Sin City: A Dame to Kill For makes a soft entry on it's debut week.
The sequel to the 2005 film, Sin City, was released on 29th August this year and had a very soft entry at 4 with just over £1 million in weekend takings.
The film fell to 12 the following week and then left the box office after just 2 weeks of release, it took only £1.6 million on it;s whole run.
With Christmas just round the corner no big new releases to mention except for Into The Storm which enters at number 20.
At the UK box office this week Luc Besson's directed Lucy about a woman using 100% of her brain takes over at the top.
Starring Scarlett Johansson Lucy tops the box office on it's debut week of release with a weekend gross of £3.07 million.
Compared to Luc Bessons previous films this is by far the best opening he has had, Arthur and the Invisibles is his highest grossing British release which took just over a million on it's release in 2007.
Scarlett Johansson is part of the Marvel Avengers universe so has seen some astronomical releases over the past few years and so it's a little unfair to compare!
Falling into second place is last weeks top film The Inbetweeners 2 which has now grossed £27.6 million after 3 weeks of release.
There is a few other new entries this week which are
Into the Storm at 4 Deliver Us From Evil at 5
What if at 6
Doctor Who: Deep Breath at 8, from a single day special showing on Saturday
Two Days, One Night at 14
Last year Matt Damon was starring in Elysium which entered at the top of the box office knocking Kick-Ass 2 right down to 7.
Five years ago Inglourious Basterds was the new top film in the UK knocking The Time Travers Wife down to the runner up spot.
Ten years ago M. Night Shyamalan released The Village which hit the top spot on it's debut with The Bourne Supremacy falling a single place to number 2.
Fifteen years ago Star Wars Episode I: The Phantom Menace reclaimed the top spot knocking Austin Powers The Spy Who Shagged me to number 2, highest new film was Micky Blue Eys at 3.
With very little in the way of new films this weekend the Daniel Radcliffe starring What If seems to have the biggest potential with Lucy and Into The Storm also on release. The former Harry Potter star has really built up a following since his days as the boy wizard and he has had big success outside the series with The Woman in Black.
A fairly simple love story What If should see a top 5 entry, especially for the Potter fans wanting to see how Radcliffe does as a romantic lead.
Possibly out to spoil What If's party is Lucy starring Scarlett Johansson and directed by Luc Besson, the French director who brought us Le Femme Nikita and Leon.
With a top hollywood star, Morgan Freeman in for good measure and good pre-word this could easily out perform What If this weekend.
Into The Storm is also out, but no star appeal and a tried and tested them of weather elements gone wrong this should limp into the top 10.
Also worth a mention is Doctor Who, a special event screening of the first episode of the new series is happening on Saturday night, the previous Doctor Who event screen took Day of the Doctor to number 3 with £1.8 million.
The top 2 films of The Inbetweeners 2 and Guardians of the Galaxy should stay in place but maybe there will be a swap around with Guardians going back to the top?
Come back next week to find out the final weekend results.
14 years after their first appearance on the big screen the Teenage Mutant Ninja Turtles are back and dominating the box office.
In a surprise move the Turtles stormed the box office beating expectation and knocking last weeks top film Guardians of the Galaxy from the top spot.
The Turtles took a $65 million (including Thursday night previews) over it's debut weekend, making it one of the biggest August debuts, it's a boon a good year for August debuts with Guardians breaking the record last week.
Guardians of the Galaxy fell to number 2 this week, taking $42 million for the weekend it has not made $176 million in the US after 2 weeks of release.
Also hitting the US box office are:
Into The Storm - number 3 with $18 million
The Hundred-Foot Journey - at number 4 with $11 million
Step up 5 All In - number 6 with $6.5 million
UK release date: 22nd August 2014
Highest chart position: 4
Weeks on box office: 3
Total UK gross £2,632,817 and the 1770th top grossing film in the UK
UK BBFC Certificate: 12AGenres:

subset: commoncrawl


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Hi I have recently been diagnosed with pyelonephritis (kidne
Patient : Hi. I have recently been diagnosed with pyelonephritis (kidney infection). Because of genetics I am very susceptible to UTIs and have had them frequently. My symptoms started 55 hours ago, and I started antibiotics 39 hours ago. My fever has subsided, but I still have tenderness in my back and achiness in the rest of my body. Is this normal timing for the healing process of a kidney infection? I am a 20 y/o female, and some input would be much appreciated!
Doctor : This question is currently in queue and will be answered shortly by one of our doctors. In the meantime you may browse our library of answered questions by clicking here.
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14th-21st May 2017
We will be heading out to Greece again in May to train EP and CP students. The aim of this trip is to get as much training as we can within a week by using the fantastic training hills and mountains and by taking advantage of the more predictable weather.
The course is run from Sunday to Sunday and includes all airport transfers, Bed and Breakfast, all flying equipment.
You will be trained by two BHPA senior instructors who have over 40 years of teaching Paragliding. Whilst we are in Greece we will also be guided by one of the most experienced local pilots in the area ensuring we are always on the right hill at the right time.
Unlike other schools we do not charge separately for EP and CP we only charge for the week. We aim to get as much training as possible during that time but a typical week should see you complete EP and most of CP. The cost is Ł750 click here to pay for the week.
If you take advantage of our package deal the trip to Greece is Free!!!!!!
Please use the online diary below to secure your place which is limited to 6 students.
Once you have confirmed your place and we have filled the course flight details will be issued.
If you require any additional information, please call Lee on 07795632710


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A few thoughts have come to mind since my last post (and I've heard from some other folks on what you should try to avoid when applying for positions). So a couple more things...
-I can't tell you how many times I've received a cover letter that starts "Dear Sir". OK, maybe everyone isn't familiar with the name Heather and it's implied female-ness. I guess I would say if you are not reasonably sure of the gender of the recipient of the e-mail, you should use a gender neutral greeting. "Hello" always works for me (cheerful, not too familiar, etc.). Even "Dear Recruiter" is OK (although I always wonder why we use "dear" in business communications). In case anyone out there is going to send me a resume...."Hi Heather" is just fine!
-Follow the instructions of the posting. I was reading someone else's blog about this subject yesterday and it struck me how often applicants don't follow the instructions and how much of my time is taken up because of that. There are a couple reasons why it is important (at least to me). First, recruiters tend to recruit on number of positions at the same time (for me, it has been anywhere from 25 to 80). So when we post jobs, we use job codes to help us identify where you saw the posting and which job you were interested in. When I post positions, I ask candidates to include that info in the subject line. This way, I can forward their resume to our database (which tracks where the candidates come from and what job they are interested in) and also immediately start comparing their background to the requirements of the position of interest. If I receive a resume submission without this info, I have to find the info and add it to the subject line to get it into our database. That's probably more detail than you needed about how we track sources, but I just wanted to illustrate that applicants not following the submission instructions takes up a lot of time. Secondly, recruiters tend to use inbox rules to route incoming resumes to folders. In my case, some of those rules are often based on what is in the subject line. If the applicant doesn't put the appropriate info in the subject line, the resume doesn't get into the right folder. So when I go to review applicants for a specific job, their resume could still be sitting in my inbox. Finally, not following the instructions on the posting may represent a lack of attention to detail. Trust me, we try to make the process as simple for the candidate as possible because we want to see your resume. When the instructions aren't followed, it makes me wonder whether the person took the time to read the full job description, disregarded the instructions, etc. Or just firing pout a bunch of resumes indiscriminately. OK, enough about that.
-As I mentioned above, our database tracks which positions people apply to. And we do get people that seem to apply to just about all of them (that might be an exaggeration, but some have applied to hundreds). We have upwards of 2500 positions open at any time. Does the applicant really think they are a fit for all of them? I would recommend limiting the number of positions you apply to...focus only on your top 5 to 10. Recruiters will still pick up your resume for other roles through keyword searches. But this way, it shows that you took the time to do a thoughtful search, you understand the requirements listed and your own skills and you are really looking for the right position (versus any position at Microsoft).
-Also, read the requirements of the position. If we are looking for a Director of Product Management, which requires X number of years in product management, someone making a career change from another field is not going to fit. We try to differentiate what is "required" versus "preferred" and yes, I do think equivalent experience can substitute for degree requirements. So use your best judgment here. You need to have most of the requirements to fit. If you aren't finding positions that fit your background, but still want to get them into our system, send them to email@example.com and in the subject line include "DS-internet research-heathham blog".
-I've also seen some folks talking about e-mail addresses and which one you use. Some people think it's OK to use your current work addresses, some don't. Some like hotmail addresses, some don't. My opinion is that you should not use your current work address on your resume. To me using your current work address says 1) you are not loyal to your current employer and 2) you use your time at work to do personal stuff. If I see you doing that kind of thing at your current employer, who is to say you wouldn't do that same thing here. I know that we all do a certain amount of personal things at work, but using that time to look for a new job is a little too much. I think hotmail addresses are OK if they aren't too goofy. A name and number combination is better than a nickname. I am very fond of the university alumni org e-mail addresses that many people are using. They follow you wherever you go.
-The other thing I wanted to re-emphasize is proof-reading. Spell check picks up blatant spelling errors but not misuse of words. My peeves: to/two/too, there/their, your/you're mistakes. It's not that we are looking for national grammar contest winners (if are such people). This is about attention to detail. Employers want to know that you are taking your search and your interest in their company seriously. Take the time to make sure your resume and cover letter (if you create one) are great.


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the sky let loose the rain it had cradled in its arms
all morning. A yellow dog pranced ahead of a man balancing
his coffee cup and folded newspaper umbrella. Spontaneous,
you'll say later when I ask as though out of the blue, lips were
close enough for other lips to touch. Soft pink blossoms
crowd the tiny tips of tree branches. Shoulder to shoulder,
ducks peck at the last bits of stale bread. Impractical white
feathers. Pale petals on wet streets. Even the wind can't lift them
clinging as they do.
-refers from the phrase "Take up indefinite words, cover" from 2, From Catalogue of Utilities by Laressa Dickey
Carolee is a painter, mixed media artist and poet living in downtown Albany. She has roots in Northern Maine, where she was born and raised, and in West Virginia, the college "home among the hills" where she received an undergraduate degree in journalism. Her poetry is published locally and nationally and has been nominated for a Pushcart Prize. In 2010, she was the "almost" Poet Laureate of Smitty's Tavern in Voorheesville, New York, placing as first runner-up in the inaugural contest.


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Пользователь обратился в сервис 911, указав на следующие проблемы в работе его компьютера:
I'm getting memory dump continuously and system gets shut down by itself, and a new window is getting popped up continuously when I run my machine in safe mode saying perform security repair, window alert. In IE the sites go to some error page by itself. These are all some Trojan viruses as I keep seeing the names in the pop up list.
I would be very much thankful if you resolve the infections before something happens to my machine. I got lot of secured data in my laptop.
Surendra Reddy.D.V.
Дата обращения: 08.06.2011 6:54:19
Номер заявки: 77237


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Results 1 to 2 of 2
Thread: View Date/Time of Update
12-11-04, 20:40 #1Registered User
- Join Date
- Dec 2004
- Posts
- 42
Unanswered: View Date/Time of Update
Hey there,
I'd like to show, on a form in Access, the date and time of the last update to the tables ....
I usually update the tables and a regular basis, but it would help tremendously if the users know exactly the last time they were updated. I've tried using a macro and update query to update a seperate table, and I've reviewed previous posts, but all to no avail.
Any help appreciated!
12-12-04, 06:48 #2Registered User
- Join Date
- Dec 2003
- Location
- Dallas, TX
- Posts
- 1,004
Hi Reg and "Welcome to the Forum"
What you are wanting I have done myself, though may not be the best of ways, but the end result is that IT WORKS.
First, in your Table make a field named something like LastModified and set it as Date/Time.
Next, go to that Form in DesignView and pull that field onto the Form wherever you like.
Now, create a new Module for that form and place the below Function in it:
'------------------------------------------------------------ ' modGlobal_Form_BeforeUpdate created by "BUD" 12-12-2004. 'This was created to Update the Form to show the last time it was modified. By modified meaning is when something was actually changed and saved 'to the form. 'This was part of a larger GlobalForm module I was using. '------------------------------------------------------------ Function modGlobal_Form_BeforeUpdate() On Error GoTo modGlobal_Form_BeforeUpdate_Err With CodeContextObject ' Updates the control DateModified to show date record was last updated. .DateModified = Now() End With modGlobal_Form_BeforeUpdate_Exit: Exit Function modGlobal_Form_BeforeUpdate_Err: MsgBox Error$ Resume modGlobal_Form_BeforeUpdate_Exit End Function
Hope this helps you with what you want. Wanted to give it a try anyway.
have a nice one,
Last edited by Bud; 12-12-04 at 06:51. Reason: Control the long text line, shortened it for readability


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Discussion in 'Sexy & Naked Women Pictures' started by Angiebaby8, Jan 3, 2016.
She would look hot with a big black cock I'n her
I want to fuck that pussy.
Yes she would
Very sexy! I would love to play with you
Eatable, lickable, fuckable.
Where in Houston are u?
Near the galleria area. What about you?
Very nice in dead
Separate names with a comma.


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http://www.promonsale.co.uk/discount-prom-dresses-77/ provides a range of gorgeous long prom dresses and evening dresses. You can search your love gown there and share it with your friends.
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.


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How to Get more users to like your facebook page
Special Ways to Get 1000s of new likes easily in almost no time
Advertising on Facebook is really effective, Facebook advertisements are more effective than Adwords if your product/service is somehow linked to social media. Facebook advertising costs you very less and gets you a lot of views and likes.
Advertising is an initial or momentary boost, not an alternative to organic likes.
Organic Search
We all know how to advertise, but organic search or non-paid likes are the ones we look for. How to get organic likes? A little is known about facebook search algorithm, but here are some useful actions to take
- Include Keyword in Page Name – If you are not representing a big brand which is already very familiar, you need to include a keyword in your page name. Your visitors will drastically increase if you include the most relevant keyword to your title. You might need to change it later, it can be done but not too much.
- Be active – This is no news to regular FB users, active people show up on the top of search results, most popular and most active pages are placed above all others.
- Get a few Likes – Get a few likes somehow, whether its through advertising or otherwise. Likes increase your click through rate, more people will visit your page if it comes up in search results. Likes build trust among visitors.
- Post in groups – There are many groups dedicated to share pages, post your page link. Just be sure to not overdo it, as it might lead to temporary or permanent account restrictions.
- Share posts to Groups and invite people to like page – Share posts to relevant groups, when a user likes your post, it will give you a chance to invite him to like your page.
Social Methods
Building a strong account automatically increases followers and likes on your pages. You can build up a strong account in a number of ways. Instead of adding just your friends and family, get more people to follow you.
- Look for people with similar interest and befriend or follow them
- Share interesting things randomly to groups and talk to people
- Chat and request people to like your page
- Join add friend groups
- Invite friends to like interesting things, but not too much it creates an ignorance attitude towards you
- Join discussion groups and comment on it, be biased and make provocative comments that makes you stand out, making enemies are just as good as making friends online, all you need is a reaction


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TBS Trailer (2008)
"Not everything is love" 31 January 2008 Thriller, Crime, Foreign 90 mins
Johan has been locked up - wrongly in his opinion - for the murder of his father and little sister. His mother is the only one who can prove his innocence, but he hasn't been able to contact her for years. When he is transferred within the psychiatric prison to the long-stay department, it's the last straw. He decides to escape and to go looking for his mother.
Watch the official TBS 2008 trailer in HD below or find more TBS videos on Vidimovie.


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Filed under: North Star Sermons-Daniel | Leave a comment »
Daniel 1:1-8 "Counter-culture Faith"
Veteran's Day 2017
For the week leading up to Veteran's Day I wanted to post a few pictures from the old days, and pictures of old men. I was a lot younger then. On the left bottom is Gary "Fert" Grow, Ghost Rider 58, my flight school roommate (passed on). I wish I had a digital picture of my brother Mike, he was a Soc Trang T-Bird, gunship pilot, and my brother, Don. He was on Group Staff for 23rd Arty. Three of the four "Brown boys" were in country at the same time. The pictures of the Wall are from our unit reunions, and when we gathered to lay a Ghost Rider to rest among our brothers at Arlington. The Rock Pile was a bad area along the DMZ and east of Khe Sanh. I have placed one skid on the top while grunts unloaded. The picture from Life Magazine was taken during Lam Son 719. THe Army Times picture was south of the Rock Pile after Lam Son 719. The picture of the hooch includes, Toad Rivet, Slim Pickens (classmate/passed on), my favorite "Peter Pilot" Blackbird, Jim Starling , and Gov Wallace. It wasn't much (and I watched it get blown away on TV when the NVA rolled down Hwy 1), but it was home. Out of the five flight school classmates in the Ghost Riders only two of us are left; John "Cowboy" Lucas and me. All of us survived our tours, but Stover died as a bush pilot in Alaska. His crash site was not found for about 20 years. David P. Soyland was a classmate and in our sister company. He was Phoenix 22. I believe every pilot who had the call sign did not complete their tour. David was shot down near the Rock Pile on a CCN (special ops) mission extracting a team under heavy fire. He spent several days evading the NVA and then was probably killed after capture. They did not like helicopter pilots. The picture of the man standing in front of his wheelchair, I saw one time, well before the current NFL fiasco. There is unspeakable worth to all our flag represents. We have never been a perfect nation, but we have been willing to pay the price to strive to be "perfect". Veterans understand the cost and respect the symbol of our freedoms. To my fellow Vets and Ghost Riders, have a blessed week.
Filed under: Uncategorized | Leave a comment »
Four Lessons I Learned From the Army, Outdoors, and My Faith. Pt.1
Recently a friend asked me, "What did you learn from your time in the Army and the outdoors and how does it apply to your Christian life?" So, after thinking about it, I have come up with four lessons.
Mentoring/Discipleship/Shared Life
I celebrated my 18th birthday at the US Army Rotary Wing Aviation Course and my 20th birthday after returning from Viet Nam with over 1,000 hours of combat flying. I served as an Army helicopter pilot in the 101st Airborne. Our company call sign was Ghost Riders. I was/am Ghost Rider 54. Our company was a highly decorated unit. We flew in I Corps, which was in the mountains along the Demilitarized Zone and Laos. During its time in Viet Nam the company earned two Presidential Unit Citations (for Ripcord and Lam Son 719). At last year's reunion in D.C. a Pentagon representative said there were only 14 given during the war. Off hand, I can think of four or five men who earned or were recommended for Silver Stars. Most of the pilots earned Distinguished Flying Crosses. The unit flew in support of troops at Hamburger Hill, the A Shau Valley, Ripcord, and Lam Son 719. All of the major combat events in I Corps during the units time in country. Through all of these actions we had a low casualty rate. This was not by accident. Helicopter pilots arrived in Viet Nam with about 200 hours. We could fly the aircraft but we were a long way from being real pilots. The first 350-500 hours of flying in-country were spent as a Peter Pilot. Sitting in the right seat we had to learn the skills to hover with one skid on a ridge line while heavily loaded troops would climb on or off the aircraft. We were expected to hover down 150 feet into jungle landing zones with just a few feet clearance on each side. The pilot would focus on the trees a few feet from the rotor blades while moving the tailboom a foot or two left or right depending on the directions of the crewchief and doorgunner. We were expected to be able to operate in the mountains with maximum loads under all sorts of conditions. All of this was to be done while taking enemy fire. An aircraft commander could drop through an opening in the clouds and know where he was by looking at a river valley or hillside. That did not happen by accident. Aircraft commanders were expected to pass on all of their skills and knowledge to the new guys. Some pilots never made AC because of lack of skills or mental toughness. We all knew that one day we would go home and we would turn our aircraft and crew over to one of the new guys. I wanted my crewchief and gunner to come home safe. That meant I needed to make sure my replacement was well trained. We understood the difference between a guy with wings and a real combat pilot required mentoring. Pilots would enter the company and spend months flying with the experienced AC's. When the old guys DEROSed (went home) the new "old guys" would pass on the unit traditions, knowledge, and skills to the new "new guys". I knew the names of the aircraft commanders who had taught my aircraft commanders. It was an unbroken chain of mentoring. It was more than a transference of information. Ghost Riders believed in a shared live. We did everything together. That is why we are still a part of each others' lives forty plus years later.
I also learned the importance of this when I was commercial fishing in SE Alaska. I was a good sport fisherman, but then I began long-lining for halibut and trolling for salmon. I had an Oregon dory with a small cabin and I would go out for three to five days at a time. I was OK, but knew I did not know all I needed. One of the top handtrollers in Southeast Alaska was a guy named Mick. I don't know why Mick took pity on me. I think it was because I was a hard worker. I was usually right in front or behind him pulling out of the anchorage each morning and was one of the last boats to come in to anchor at night. I usually put in 20 hour days. One night Mick invited me over to his boat to mug up. (You grab your coffee, or hot chocolate, mug and sit around the galley stove.) From that night on, I would anchor up, clean my gear, and then sit with Mick as he taught me to rig gear, how to fish, when to fish, everything I needed to know as a fisherman. This was unusual because commercial fishermen are secretive. Mentoring was not a commonly used word at that time, but that was what Mick did.
Jesus' ministry was one of mentoring. The Bible calls this discipleship. Everyday Peter, James, John, and the others watched Jesus minister and teach. They asked him questions. They watched him serve people. So many of the passages in the Gospels could be described as serendipitous. A great example was when Jesus was walking through the wheat field on a Sabbath (Matthew 12). He "harvested" and milled the wheat causing the Pharisees to accuse Jesus and the disciples of breaking the Sabbath. Jesus then taught about the purpose of the Sabbath. When you share life, the teacher does not present a canned lesson, he answers the student's questions as they arise from day to day life. After Jesus ascended into heaven his enemies commented that it was clear His disciples were just like Jesus. I have been blessed over the years since Viet Nam to be discipled/mentored by older Christians. I learned about being a father, husband, and man through them. In turn I like to think my three sons are better Christian men, husbands and fathers because of their time with me.
I have long said after Viet Nam I knew more about how to be a "real helicopter pilot" than living the Christian life. On one hand I was mentored; on the other I was not. In truth, both settings are war zones. Survival and victory depend on each generation passing on the knowledge and skills to the next.
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Dr. Paige Patterson on 2016 Election
I would highly recommend reading the article at this link. Dr. Patterson has always had a gift for using examples from history to shed light on present situations.
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CW4 Duane "Muddy" Watters
"In great deeds, something abides. On great fields, something stays. Forms change and pass; bodies disappear; but spirits linger, to consecrate ground for the vision-place of souls…" General Joshua Chamberlain, Congressional Medal of Honor. He held the Union left flank at Little Round Top, Gettysburg.
Last night I
was called and informed that Duane "Muddy" Watters had recently died from complications related to lung cancer. Two of the most important days of my flying career (March 19 & 20) were spent flying right seat for Muddy. Muddy was one of the old guys (24). He was an excellent pilot, calm under fire, and committed to his fellow Ghost Riders. He would not leave a man behind, no matter what. He retired a CW4. Muddy said he wanted to be remembered by the words: "Duty, honor and country." He lived it. Muddy, Itty Bitty, and others "consecrated" the air and landing zones in the action known as Lam Son 719. I was hoping to see him at the October reunion. He is survived by his wife, Candice and family in Fairbanks, AK. He is missed. He is remembered. And, we are growing old.
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The Intellectual And Spiritual Wanderings Of American, Conservative, Evangelical Christians: Or, Why I Do Not Listen To Glenn Beck.
To begin, I have several confessions to make. First of all, I listen to talk radio. Not every day, but several times a week. I like Rush Limbaugh. Maybe because I understand his humor. I do not hang on every word, but I appreciate another perspective than network news. I listen to several other radio talk show hosts depending on when I am driving in the truck. That said, I do not listen to Glenn Beck. I use to, but not now.
I think Glenn Beck is an expert on American history, and insightful in American politics. I agree with many things Beck says. I also think he and his radio show crew have a great time. Sometimes they are hilarious. But, except for occasional short segments transitioning to or from other shows, I do not listen. My problem is a problem of Christology and eschatology.
First the Christology; on Glenn Beck's Facebook page he states:
I am a born again LDS Christian who teaches Gospel Doctrine in my church, speaks about God and Jesus more than any other main stream radio or television host in the last 70 years, who also studies the Torah with rabbis on a regular basis and met today with the Dalai Lama. [smile emoticon.]
Glenn Beck does sound more like a televangelist than a radio talk show host. And, the times I have listened to his program he is constantly calling for prayer, revival, and for Christian unity. In the above quote he is obviously proud of his ecumenical spirit. He often invokes Christ's name in his pleas. He is quiet passionate about God and Jesus.
The conundrum is his adherence to the "Gospel Doctrine in my church." The LDS Christ was once a man, just a man, not the 100% God/100% man of the Bible and the historic creeds of the Christian faith. The gospel according to Glenn Beck's LDS teachings is not the redemptive work of God through Christ and the cross. The LDS gospel teaches salvation through obedience to the teachings of the LDS church. The LDS Jesus was a man as we are; and if we avail ourselves of baptism in a Mormon Temple, marry in a Mormon Temple, spend two years as a self-supporting LDS missionary, earn and maintain a "Temple Recommend", and in general work your way through life you will experience exaltation. Your eternity will consist of becoming a god, like Jesus is now, and becoming a god, along with your wife (wives) to another world. I am sure some Mormons may say I don't have it exactly right, but I am correct in the fact the LDS does not use the word "gospel" as the Christian Church does. The Christ of the LDS is not the Christ I worship and serve. Therefore, to hear Glenn Beck evoke Christ and claim a unity on spiritual grounds is unacceptable.
Glenn Beck is well informed concerning Shiite eschatology. This branch of Islam contents the twelfth and final Imam will come for the final reckoning. As Beck has made clear, the danger of a nuclear Iran is the fact that Iran's leadership believes they can hasten the twelfth Imam's return by starting a nuclear war. Just as the hijackers did not hesitate to fly planes into buildings on 9/11, Iran's leadership will not hesitate to start Armageddon. Their eschatology (doctrine of end times) drives their thinking. Securing the return of the twelfth Imam and earning their place in Paradise is worth it all.
In the same way we need to understand LDS eschatology. The LDS church is similar to historical Christianity in regards to the return of Christ. However, Mormons place a greater emphasis on the end times, the cataclysmic events preceding His return, and the need to prepare for those preliminary events. You might say, Christians believe the same. I am basically a Pre-Tribulation supporter. I am not rigid and can even go for a Mid-Tribulation Rapture. However, if I am wrong, that is OK. I do not spend a lot of time preaching about the events of Revelation. I think it is largely written to the people who will experience it. I do not stockpile large supplies of food as LDS encourages.
It might be wise to stockpile food supplies and doing so is not a bad thing. However, that eschatology is apparent in the Glenn Beck Show's tone. I get depressed when I listen to Beck for any length of time. Beck expects the worse. Every show I have heard is a litany of coming disasters. I do believe America will be judged and receive the consequences as we continue to move farther from our Founder's moral base. However, I am also a student of revivals and believe God will have another great harvest before He shuts this all down. Therefore, in the midst of all the crazy (and sick) things occurring in our world, God is still in control.
Can I, as a conservative Christian, work with people of like morals and values? Yes. I will be happy to work with people of all faiths to stop abortion, sex trafficking, and other societal sins. I have had, and still have, friends of various beliefs, including LDS. However, there is a line that cannot be crossed. After 9/11 there was a ecumenical "worship" service in New York led by Oprah Winfrey. I would not have participate in that service if invited. Would I attend a community gathering honoring the victims? Yes. Actually, I have less problems with Oprah because she doesn't present herself as a Christian, and that is the rub. Beck thinks he is.
In the last year Glenn Beck has promoted various events with a spiritual emphasis. He is calling "Christians" to unite. He has spoken from the podiums of various Christian churches. He presents himself as a "Born-again Mormon." Unfortunately, many Christians now see Glenn Beck as a prophetic voice like a modern day Dwight L. Moody. They are not only trusting him for political advice, but spiritual direction. When I invite anyone to speak in the church I pastor, or attend a spiritually focused event, I best consider who is leading the movement.
Can I accept Glenn Beck, or any other Born-again Mormon as a brother in Christ? Yes, on one condition. To accept Christ as Lord and Savior requires me to reject all other ways of salvation. I pray for the day Glenn Beck denounces the "Gospel Doctrine [ of the LDS] church" as a false gospel and Jesus Christ and His substitutionary, propitiatory sacrifice as the only means of salvation. No one can accept the gospel piecemeal. What it means to be a Christian is not determined by other religious groups or someone desiring to adapt elements of Christian doctrine within his own religious framework.
I am in agreement, we probably are in "the last days". Therefore, I need to remember 2 Corinthians 11:13-15, 2 Peter 2:1, and other passages in which I am warned of the danger of false teachers. Do not expect them to identify themselves as anything other than Christians. I also consider the warning of Matthew 7:21-23 (NKJV)
Matthew 7:15-20 (NKJV)
15 "Beware of false prophets, who come to you in sheep's clothing, but inwardly they are ravenous wolves. 16 You will know them by their fruits. Do men gather grapes from thornbushes or figs from thistles? 17 Even so, every good tree bears good fruit, but a bad tree bears bad fruit. 18 A good tree cannot bear bad fruit, nor can a bad tree bear good fruit. 19 Every tree that does not bear good fruit is cut down and thrown into the fire. 20 Therefore by their fruits you will know them. 21 "Not everyone who says to Me, 'Lord, Lord,' shall enter the kingdom of heaven, but he who does the will of My Father in heaven. 22 Many will say to Me in that day, 'Lord, Lord, have we not prophesied in Your name, cast out demons in Your name, and done many wonders in Your name?' 23 And then I will declare to them, 'I never knew you; depart from Me, you who practice lawlessness!'
The people of this passage will be stunned when they are cast out. They thought they were Christians. I am sure others also thought the religious workers were Christians.
In closing I might as well kick the dog on my way out. I would have made some comments about the way Christians are jumping on the political bandwagon of people like Donald Trump because they like his bluntness, but I have said enough for now.
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The Cross and Political Correctness
In the last few weeks the national news has been focusing on the legislation in Indiana and Arkansas dealing with protecting religious liberty. We are in the midst of a collision of biblical and non-biblical values. Before dealing with the contemporary events I would like to go back to the Roman Empire and its use of crucifixion.
The Romans were not inventive. They were innovative. They had the practice of using other cultures' ideas and improving them. Crucifixion was used by the Persians and the Macedonians, but the Romans took the practice to a new level. Crucifixion was not about killing the victim. There were much more efficient means of execution, and the Romans loved efficiency. The main purpose of crucifixion was silencing opposition. If a non-citizen was determined a threat to Roman rule he was crucified. You may have been unhappy under Roman rule, but you kept your thoughts to yourself, because the cross was always looming. Crassus crucified 6,000 slaves after crushing their uprising. That was the last revolt by the slaves. Crucifixion silenced the opposition.
We do not literally crucify people today. We are more civilized in silencing opposition than that. The Indiana Freedom of Religion bill was in line with federal legislation signed by President Bill Clinton. The purpose was to support the First Amendment protections for Indiana residents. The news media "carried the water" for the homosexual community portraying the law as promoting businesses who wish to discriminate against homosexuals. Like numerous other recent events, the facts of the case were never presented. America has become a society that does not desire truth. Our culture does not want an exchange of ideas. Instead of presenting the various positions in the public arena and trusting that the truth and the superior position will win the day, divergent positions are silenced by "crucifixion", that horrible death.
Crucifixion was designed to humiliate and intimidate. The victim was scourged with lashes tipped with metal and bone. They were disfigured with pieces of flesh hanging from the open wounds exposing bones. They were stripped of their clothes. They were hung along the public roadways. They were made less than human. American "crucifixion" involves a social media blitz bombarding the enemy with hate mail and threats. The guilty party's livelihood or business is threatened. The person's integrity, character, and morals are maligned. In some cases the federal government becomes involved with threats of IRS audits or justice department investigations. The message to the observing masses is clear, do not express your views, especially do not take a stand for a biblical worldview, go with the flow. It is not surprising that our politicians run for cover, who wants that kind of attention. But, we are not politicians. We are followers of Christ.
As His followers, we are to love all people while standing on His word. As Americans we have a unique place in history. As an army officer, I took an oath to uphold and protect the Constitution. We have been given a priceless gift of the First Amendment. The freedom of religion is the foundational right. When it falls, all others will follow. As a Christian, I am a bondservant of Christ. I am to be faithful to my Lord even if it conflicts with the government. We have a moral obligation to protect our constitutional rights for future generations. In the recent collisions of values there is a clearly stated constitutional right, the freedom of religion, versus a lifestyle choice. Christianity is not a religion of coercion. You must come to Christ of your own free will. Whether you come or not, you are to be loved as a human being. The sanctity of human life is true for all. I reject all lifestyles that do not conform to God's word, but respect the person's right to choose their own life. In turn, I expect others to respect my choice. Do not try to force me affirm yours. The homosexual social agenda is the affirmation of their worldview while stripping all others of theirs, and one of their most powerful tools is "crucifying" anyone brave, or foolish, enough to speak in opposition. Christians and others can hold their private views, but cannot express those views as others do. We are being forced out of the public arena. Christian businesses daily serve people of other faiths and lifestyles, but that is not enough. Now those businesses must participate in practices contrary to Christian beliefs. There is a difference between others practicing their lifestyles (i.e., homosexual marriages) and being required to approve of the practice by catering the event, or providing flowers. You know it is political correctness when a Muslim business does not have to serve me pork barbeque, or the equivalent for businesses of a liberal viewpoint.
The Jewish religious leaders and the Roman government thought they had silenced the teacher from Nazareth. By all appearances they had won. The followers of Jesus were scattered, in hiding, and terrified. That was the usual effect of crucifixion. But, they were wrong. The Resurrection changed everything. The world was "turned right side up" by His followers. (Remember, it was the lost who said the disciples were turning the world upside down.) The Holy Spirit transformed the disciples into men who were more in awe of God than afraid of the cross. Tradition tells us that the disciples died for the faith. Thousands of Christians died under Roman persecution and the Church's response to the hate was love.
Political correctness is effective as long as people are intimidated. As American Christians we have the right to hold our moral positions and exercise them in the political process. That said, our greatest concern is not the political ramifications of our silence. Our faith is not lived out in isolation. We cannot be not salt and light in selected arenas. If we are followers of Christ, He is Lord of all the arenas of our lives. We must speak truth in love and with boldness. Where would we be if the first century disciples were silenced by the political correctness of their time? Where will our grandchildren be if we are not faithful? No one wants to be vilified, but Jesus was not excited about his looming crucifixion.
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Spring and preparing for the harvest.
I love this time of year. This past winter has been cool and wet enough that it was hard to get enthused about riding my bike enough to stay in shape. Winter is a time of tarped boats and occasional rides. When I lived in Alaska it was a time to ski and ice climb. This part of North Carolina is too cold for summer sports and too warm for winter sports. So, winter is a time to rest a bit and recharge. Then comes spring. When I commercial fished in Alaska spring was filled with cleaning and rigging the boat, checking and tying new gear, and counting down the days until the king salmon showed up in numbers. Spring was a time of anticipation, and preparation.
We need times of refreshing. Ecclesiastes 3 declares there is a season for everything. There are times for sabbaticals, but a never ending sabbatical is called laziness. I have found it hard to shift from resting to activity. After sitting in neutral it is just hard for me to shift back into high gear. You may have the same problem. If so, then perhaps, the following motivational tools I use could benefit you.
Set a goal with a "price". During the winter I ride a couple of times a week if the weather is good. It is hard to shift into a daily ride mode, which I desperately need to shed my winter weight gain. In January I register (and pay) to participate in a major ride. Usually I ride a century (100 miles in one day) at the end of April. This year I am riding a 75 mile ride from Emerald Isle to Harker's Island and back. These rides are fun, but require preparation. I know it will be agony to ride the mileage if I have not prepared, and I won't back out because I paid to enter. I have a ten week training program which adds mileage each week. At the end of February my lazy side does not want to go for a ride, but I know the "price" of not going. I have a goal with a timeline that will not change. On May 5 I will either enjoy a great ride with a friend, or I will suffer for five hours, or forfeit the fee.
Find a partner. Last spring my middle son committed to riding a century with me the end of April. He was living in Macon so we could not train together, but we would often compare training rides using a smartphone app. This year a former colleague, John, approached me about "Ride the Bank." John and I were professors together a few years ago. At that time neither of us rode. Since then John began doing triathlons and I started riding centuries. John wanted to try his hand at longer rides (he is used to 18-20 milers), and had heard I was riding. So John and I meet two or three times a week to train together. We have busy schedules so we can't ride everyday at the same time. We are riding on our own, but the common training sessions help us stay accountable. For a couple of old guys we are doing pretty good.
Related to both of the above is the motivation of making it public. Before my first long ride I made sure my family knew, so I could not back out. (It was nice to hear the grand-kids express their pride when I completed it.) Recently, I used this tool in my small group. For several years I have done little scripture memorization work. In the past I memorized Colossians and Titus, but then stopped. I took a sabbatical from memorizing new material. It was comfortable just repeating those books on a regular basis. Some days I would not quote any scriptures. Last month, two of our small group members talked about the book of James. Their comments were not about memorization, just about how James blessed them. I was convicted that I could have memorized the book if I had not taken a "sabbatical" from memorization. Actually, sabbatical is just a term to make me feel better. I had become lazy in regards to committing God's word to memory. I was satisfied with maintaining the status quo. That night I made a public commitment to memorize James. Now, I am making a commitment to not stop with James. The days I start to skip my memory work I think of my small group members and my commitment to them.
I enjoy cycling. There is a sense of accomplishment when I finish a hard ride. I started riding as a means of exercising. I cannot stand working out in a gym or running. Riding a bike is like being a kid again. I determined that I want to be active when I am in my eighties. I also realized for that to happen, I could not wait until I am eighty to begin exercising. I am investing now for a goal years away. In the same fashion, I want to be a godly man who knows God's word. I have always admired those elderly saints that exude God's love and have His word hidden in their hearts. That will not take place unless I prepare now.
God rested on the seventh day to demonstrate our need for rest. God did not create everything in one day, though He could have, and then take six days of rest. We will have times that we need to take a break, but then we need to return to work. Jesus looked upon the multitude and encouraged his disciples to pray to the Lord of the Harvest to send forth laborers into the harvest. (Matt 9:37-38) Winter is over. Spring is here. Summer is coming, and then the harvest. Will we be ready? Set the goal. Pay a price. Make it public. Find that yoke fellow who will challenge you and hold you accountable. Everything we invest now will come back as a blessing when we stand before the Lord of the Harvest. The fields are white unto harvest.
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Toffees come unstuck at Old Trafford; 10-man Stoke see off Man City; Arsenal & Villa fire blanks; Bolton strike a late blow to down Spurs
Manchester United 1 – Everton 0 (Premier League, January 31, 2009) An early wobble in the Everton defence produced a nervous moment from a dangerous Park cross. Tim Howard was then forced to turn over Carlos Tevez's shot from inside the six-yard box before Cristiano Ronaldo came within inches of scoring, seeing his shot rebound off the inside of the post. The barrage continued, Howard tipped over again from Ronaldo, and just before the break to pressure paid off as Ronaldo slotted home a penalty after Carrick was tripped in the box. (First half highlights here.) Carlos Tevez skied a good opportunity inside the box at the start of the second half, and later in the half a deflected Tevez free-kick forced Howard to tip-over yet another effort on his goal.
Stoke 1 – Manchester City 0 (Premier League, January 31, 2009) Stoke subjected City to their set-piece barrage early on, but their hopes for a crucial win took a blow when Rory Delap was sent off for a foul on SWP which was compounded by kicking the ball into the former Chelsea winger. Deep into lengthy first-half injury time, Stoke's January signings Matthew Etherington and James Beattie combined to put the ten men into the lead.
Arsenal 0 – West Ham 0 (Premier League, January 31, 2009) (Longer highlights here.) (MOTD highlights here.)Â Abou Diaby headed against the bar off from Nasri's inswinging corner, while at the other end Gael Clichy cleared James Collins' effort off the line in West Ham's only chance of note during a drab first half (highlights here) at the Emirates. After the break Emmanuel Adebayor forced Green into saving his point-blank header. (Second half highlights here.)
Aston Villa 0 – Wigan 0 (Premier League, January 31, 2009) (MOTD highlights here.)Â Villa came within a whisker of scoring against Wigan, the Latics were fortunate to see Zat Knight's header hit the post and stay out. (First half highlights here.)
Hull 2 – West Brom 2 (Premier League, January 31, 2009) Hull took the lead on the stroke of halftime as Bernard Mendy took the ball round Scott Carson to score. The Baggies levelled after the break, Jay Simpson scoring his first Premier League goal. Mendy turned provider for Craig Fagan to put Tigers back in front in the 69th minute but a Chris Brunt penalty four minutes later earned the Baggies a point.
Bolton 3 – Tottenham 2 (Premier League, January 31, 2009) (MOTD highlights here.)Â Sebastien Puygrenier put the Trotters in front heading in Davies's free-kick. Bolton doubled their lead in the 64th minute when striker Kevin Davies shot into the far corner from a Cahill flick-on. Chimbonda's cut-back found Darren Bent minutes after his introduction from the bench, and two minutes later Bent netted again when Palacios's shot was parried into his path. But Bolton struck late on, Kevin Davies scoring his second heading in a Taylor corner.
Fulham 3 – Portsmouth 1 (Premier League, January 31, 2009) Andrew Johnson seized upon a fine Clint Dempsey pass to open the scoring for Fulham in the 14th minute. Erik Nevland put Fulham two up and added another to secure the win with David Nugent adding a late consolation for Portsmouth.
Middlesbrough 0 – Blackburn 0 (Premier League, January 31, 2009) Sam Allardyce extended his promising sequence of results to eight without defeat, six in the Premier League, to take a 10th point from a possible 18.
Sheffield Wednesday 1 – Birmingham 1 (Championship, January 31, 2009) Lewis Buxton broke the deadlock for the Owls with his first goal for the club in the 56th minute. In the 93rd minute, Kevin Phillips stole in at the far post to head home Scott Sinclair's fine cross and salvage a point from a game they appeared destined to lose.
Southampton 2 – Swansea 2 (Championship, January 31, 2009) Marek Saganowski gave the home side the lead after 17 minutes when he took full advantage of Monk's poor backpass, only for Jordi Gomez's to level matters before halftime. Saints' Lee Molyneux saw red shortly after the interval and Gorka Pintado's close-range finish looked like making it six consecutive wins for the Swans. Saganowski had other ideas though, and he converted Surman's cross after 76 minutes to earn a share of the spoils.


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President - Engineering and India Site Leader for Cisco
Amit Phadnis is the President – Engineering and India Site Leader for Cisco. Amit heads Cisco's Core Software Group (CSG) India, a team responsible for $ 11 Bn business portfolio for Cisco. As a CSG head, Amit leads a team of 2500+ engineers to drive innovations and product developments, architectural alignment and integration with GTM (go-to-market) strategies in the routing, switching and wireless area. Amit has over 20 US PTO patents in his name.
Before joining Cisco, Amit was the Country Manager/CEO and Vice President of Engineering at Symbol-Motorola. In his role with Symbol, Amit was involved in building a strong product organization as a country manager. He contributed to technologies related to handheld computing, wireless and RFID. Amit has a Masters Degree in Electronics and Communication from the Indian Institute of Science (IISc), Bangalore.


text: WOW!! I love this board!!!! It's new I think...haven't seen it before! Anyway...DH and I were at WDW for our honeymoon but plan on renewing our vows there in a small romantic ceremony....I am wondering if anyone has any ideas!!! This is place to find them I know! Our wedding was so stressful and large that I want this to be the exact opposite!!


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Drake celebrates his third week at No. 1 by releasing a new remix for "Hype" featuring Lil Wayne.
Drake remains top dog on the Billboard 200 and the Hot 100 charts, so to help ensure he stays there, the Toronto rapper dropped a new re-working of Views track "Hype" featuring Weezy. Following YG's premiere of his Drake-assisted single "Why You Always Hatin?" on OVOSOUND Radio on Saturday, Drizzy decided to share his new tunes too.
Weezy fires up the track with a Prince mention ("I pull up on ya in that purple thang, bumpin' 'Purple Rain'") as well as his ex Christina Milian ("You don't see 'Tina right next to me").
Listen here:
Thoughts? Let us know in Exhale.
And while you're at it… leave a comment and follow us on Facebook, Twitter & Instagram.


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A Dangerously Confused AUMF Discussion in Congress as Escalation Looms
A Dangerously Confused AUMF Discussion in Congress as Escalation Looms March 13, 2015 Earlier this week, top officials appeared before the Senate Foreign Relations Committee to discuss the Obama Administration's proposed authorization for use of military force (AUMF) against the Islamic State. Secretary of Defense Ashton Carter, Secretary of State John Kerry, and Chairman of the Joint Chiefs of Staff Gen. Martin Dempsey took part in a conversation that highlighted several areas of concern for which additional focus is warranted going forward. In particular, the hearing added confusion to the proposed definition of "associated forces" and how it is currently interpreted or could be interpreted in the future. The hearing also confirmed the proposed language that would prohibit "enduring offensive ground combat operations" lacks significant meaning.… Read More ›


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Mitochondria are centers of metabolism and signaling whose content and function must adapt to changing cellular environments. The biological signals that initiate mitochondrial restructuring and the cellular processes that drive this adaptive response are largely obscure. To better define these systems, we performed matched quantitative genomic and proteomic analyses of mouse muscle cells as they performed mitochondrial biogenesis. We find that proteins involved in cellular iron homeostasis are highly coordinated with this process and that depletion of cellular iron results in a rapid, dose-dependent decrease of select mitochondrial protein levels and oxidative capacity. We further show that this process is universal across a broad range of cell types and fully reversed when iron is reintroduced. Collectively, our work reveals that cellular iron is a key regulator of mitochondrial biogenesis, and provides quantitative data sets that can be leveraged to explore posttranscriptional and posttranslational processes that are essential for mitochondrial adaptation.
Mitochondria are ubiquitous organelles that are essential for cellular energy generation and a range of key metabolic pathways. The production of mitochondria—termed mitochondrial biogenesis—is a complex process involving the orchestrated transcription, translation, and import of more than 1,000 proteins encoded by two genomes (Mick et al., 2011; Pagliarini et al., 2008; Scarpulla, 2008; Schmidt et al., 2010). Moreover, these organelles vary considerably in composition across tissues (Mootha et al., 2003a; Pagliarini et al., 2008) and remodel to meet cellular needs (Baltzer et al., 2010; Hock and Kralli, 2009), indicating that the mitochondrial biogenesis program is customizable and responsive to environmental conditions. Defects in this process are associated with a range of human disorders, including mitochondrial encephalomyopathy with ragged red fibers (MERRF), type 2 diabetes, and various cancers (Calvo and Mootha, 2010; DiMauro and Schon, 2003; Lowell and Shulman, 2005; Wallace, 2005).
During the past two decades, major progress has been made in deciphering the transcriptional networks that drive mitochondrial biogenesis. Of particular importance was the identification of peroxisome proliferator-activated receptor γ, coactivator 1 α (PGC-1α) (Puigserver et al., 1998). PGC-1α and related coactivators PGC-1β (Kressler et al., 2002; Lin et al., 2002) and PRC (PGC-1 related coactivator) (Andersson and Scarpulla, 2001) coordinate and activate the various transcription factors required for mitochondrial biogenesis (Scarpulla, 2008). Additionally, PGC-1α is activated by exercise, adaptive thermogenesis, changes in cellular redox state, and the availability of nutrients and growth factors (Hock and Kralli, 2009), helping to explain how mitochondrial content is responsive to changing cellular conditions.
Despite significant advancements in our understanding of PGC-1α and its corresponding transcription factors, important aspects of the cellular control of mitochondrial content remain unclear. These include posttranscriptional processes that control mitochondrial gene expression, mechanisms of active mitochondrial degradation and clearance, and extramitochondrial processes that help coordinate communication between mitochondria and the nucleus (Goldenthal and Marín-García, 2004). Posttranscriptional control mechanisms, such as upstream open reading frames (uORFs) (Calvo et al., 2009), iron-responsive elements (IREs) (Eisenstein and Ross, 2003), and microRNAs (Li et al., 2012), are already known to affect the expression of select mitochondrial genes, and the global discordance between cellular mRNA and protein levels suggests that these mechanisms are likely more widespread (Mootha et al., 2003a). Additionally, macroautophagy (i.e., mitophagy) is emerging as an important mechanism for eliminating damaged mitochondria (Youle and Narendra, 2011). However, identifying additional genes subject to posttranscriptional regulation, and spotlighting cellular processes that help synchronize the mitochondrial biogenesis program, would benefit from matched, cell-wide quantitative data of protein and mRNA abundance, which has largely been lacking.
Here, to produce such a resource, we performed parallel quantitative SILAC (stable isotope labeling by amino acids in cell culture)-based proteomics (Ong and Mann, 2006) and microarray analyses of PGC-1α-induced mitochondrial biogenesis in mouse muscle C2C12 cells. In doing so, we find that key proteins involved in maintaining cellular iron homeostasis are correlated with mitochondrial biogenesis. We further reveal that depriving various cell types of iron through chelation or active transport leads to a rapid and dose-dependent attenuation of mitochondrial transcript and protein levels that is fully reversible within 3–4 days. Together, our work demonstrates that iron deprivation results in an active and coordinated downregulation of mitochondrial gene expression, suggesting that the bioavailability of iron is a key parameter for establishing a set point of cellular mitochondrial activity. Because iron deficiency anemia is the most common nutritional disorder worldwide (McLean et al., 2009), this work has broad implications for understanding mitochondrial dysfunction in human health and disease. Additionally, our data serve as a resource for investigating genes subject to posttranscriptional regulation and for identifying additional auxiliary pathways that might be important for calibrating or modulating the mitochondrial biogenesis program.
We sought to better define the mitochondrial biogenesis program in C2C12 mouse myotubes by performing complementary RNA and protein profiling. To maximize the transcriptional activation of mitochondrial genes, we overexpressed PGC-1α—the predominant transcriptional coactivator that drives mitochondrial biogenesis—more than 200-fold using an adenovirus-mediated delivery system. We chose C2C12 cells as a model because overexpression of PGC-1α in this cell line is sufficient to cause an approximate doubling of mitochondrial mass in 3 days (Wu et al., 1999). This approach allows us to assess the relative contribution of posttranscriptional mechanisms in regulating mitochondrial gene expression and provides a more complete assessment of the cell-wide proteomic changes that accompany PGC-1α-induced mitochondrial production.
Following PGC-1α overexpression, we tracked changes in cellular mRNA and protein levels using microarrays and quantitative SILAC proteomics (Ong and Mann, 2006), respectively (Figure 1A). Consistent with previous studies, our microarray analyses showed that PGC-1α causes a robust increase in the transcript abundance of nuclear-encoded mitochondrial genes, especially those involved in oxidative phosphorylation (OxPhos) (Figure 1B). Our SILAC data revealed similar results for protein levels: of 442 mitochondrial proteins quantified using at least two unique peptides, 263 significantly increased in abundance (Figure 1C). As expected, PGC-1α-induced mRNA and protein fold changes were largely consistent in the direction of change (Figure S1A). However, the protein and mRNA abundances differed by as much as 9-fold for nuclear-encoded mitochondrial genes, and for 97 of these genes, mRNA abundance was increased, whereas the corresponding protein abundance was decreased (Table S1; Figure S1B). This mRNA:protein discordance reveals that regulation of protein stability or translation is likely to be important for titrating the expression level of select genes during PGC-1α-induced mitochondrial biogenesis. More broadly, these observations reveal the utility of our resource for spotlighting mitochondrial proteins whose expression may be subject to multiple levels of regulation.
Our experimental approach also enabled us to investigate peripheral genes and pathways that may be important for the mitochondrial biogenesis program. Interestingly, we noted that proteins involved in regulating cellular iron levels were among the most highly up- and downregulated proteins in our SILAC analyses. The transferrin receptor, which is responsible for transporting transferrin-bound iron into cells, increased more than 4-fold with PGC-1α overexpression (Figure 1C). Reciprocally, the level of ferritin heavy chain, part of the ferritin complex that sequesters cellular iron, was decreased (Figure 1C). These results suggest that iron might be essential for the mitochondrial biogenesis program. Increased cellular iron availability during mitochondrial biogenesis might simply be necessary to accommodate the mitochondrial proteins that contain iron as a cofactor or may occur in anticipation of increased output from the mitochondrial iron-sulfur cluster biogenesis pathway (Lill, 2009; Richardson et al., 2010). However, the magnitude of these changes prompted us to explore whether cellular iron levels might impact the mitochondrial biogenesis program holistically.
To test whether loss of cellular iron is sufficient to induce a restructuring of cellular mitochondrial content, we modified the experimental approach outlined in Figure 1A. Here, in lieu of PGC-1α overexpression, we treated the cells with deferoxamine (DFO), a clinically used cell-permeable iron chelator (Chaston and Richardson, 2003). Strikingly, the microarray results revealed that DFO treatment strongly diminished the abundance of mitochondrial transcripts (Figure 1D). Overall, the magnitude of the DFO effect was as robust as the powerful PGC-1α effect and was also most prominent for genes encoding proteins involved in OxPhos (Figures 1E and S1C). Once again, our mRNA and protein measurements were largely consistent in direction and magnitude (Figure S1D); however, there were notable differences in these measurements among the OxPhos complexes. Complexes I and II, each of which contain multiple iron-sulfur clusters, were most strongly affected, with protein levels more decreased than the corresponding mRNA levels (Figure 1F). Conversely, transcripts encoding complex V subunits were decreased, whereas their corresponding protein levels were increased or unchanged (Figure 1F). A recent, large-scale study of protein dynamics found that mitochondrial proteins, including complex I and complex V, have largely similar turnover rates under normal conditions (Price et al., 2010), suggesting that the differences in complex I and complex V subunit levels following iron deprivation likely involve posttranscriptional regulation.
To assess whether cellular iron levels affect PGC-1α-induced mitochondrial biogenesis, we repeated the experimental approach a third time with PGC-1α adenovirus added to cells simultaneously with DFO. Here, the presence of DFO had a pervasive dampening effect on the induction of mitochondrial transcripts by PGC-1α (Figures S1E and S1F). Notably, for many of the same OxPhos subunits highlighted in Figure 1F, transcript expression was increased under these conditions, whereas the corresponding protein levels were decreased (Figures 1G and S1G). This again suggests that for select genes, iron chelation may lead to an active, posttranscriptional reduction of expression, as opposed to merely thwarting the effects of PGC-1α.
Our large-scale gene expression and proteomic data show that iron chelation has an approximately equal and opposite effect to that of PGC-1α, a powerful inducer of mitochondrial biogenesis. To validate these results, we performed immunoblots and real-time quantitative PCR (qPCR) on the same samples used for our proteomics and microarray analyses. Consistent with our SILAC data, we found that OxPhos proteins were significantly decreased by the DFO treatment (Figure 2A). These results also validated that this effect was predominantly limited to mitochondrial proteins because representative endoplasmic reticulum, nuclear, and cytoplasmic markers were unaffected by this treatment (Figure 2A). For qPCR measurements, we chose two nuclearencoded OxPhos genes (Ndufb5, which is part of iron-containing complex I; and Atp5a1, a subunit of iron-free complex V) and one mtDNA-encoded gene (COX1, which was not represented on the microarray or captured by mass spectrometry). All three genes were significantly upregulated with PGC-1α overexpression and diminished by DFO treatment (Figure 2B). These PCR results reveal that the iron chelation effect is not limited to nuclear-encoded mitochondrial genes or to genes encoding iron-dependent mitochondrial proteins. Furthermore, our microarray and SILAC data demonstrate that the expression of genes encoding non-mitochondrial iron-dependent proteins is not diminished by the iron chelation treatment (Figure S1H). Collectively, our data show that acute iron deprivation has a specific and dose-dependent dampening effect on mitochondrial protein expression that exceeds a mere cell-wide loss of iron-dependent proteins and processes.
The results above suggest that cells might possess the ability to calibrate their mitochondrial protein levels to the concentration of available iron. To further test this hypothesis, we grew myotubes in the presence of increasing concentrations of DFO. The results show that even 20 μM DFO has a marked effect on cytochrome c and complex I levels and that the effect is directly proportional to the concentration of DFO (Figure 2C). To begin to assess the universality of the iron deprivation effect, we treated myotubes alongside their undifferentiated myoblast counterparts. Surprisingly, the response in myoblasts was even more robust: after just 24 hr of treatment with DFO, the 8 kDa-complex I subunit was nearly completely lost, whereas the level of this protein continued to gradually vanish from myotubes up through 72 hr (Figure S2A). This effect was also evident in a panel of cell lines differing in species and tissue of origin (Figure 2D), suggesting that iron availability might be a universal gauge that cells use to calibrate mitochondrial activity.
To further define the effect of DFO on OxPhos protein abundance in undifferentiated myoblasts, we examined individual subunits from each OxPhos complex using standard SDS-PAGE and fully assembled, native OxPhos complexes using blue native PAGE (BN-PAGE). SDS-PAGE revealed that DFO causes a decrease in the abundance of complex I, complex II, and complex IV subunits (Figure 2E). Consistent with our proteomic analysis of differentiated myotubes (Figure 1F), BN-PAGE revealed that DFO most strongly affects the abundance of complex I and complex II (Figure 2F). Moreover, iron chelation also seems to affect supercomplex formation (Figure 2F). A distinguishing feature of complexes I and II among the OxPhos machinery is that they each possess multiple iron-sulfur cluster centers. As such, although many mitochondrial proteins are affected by iron deprivation, this suggests that iron-sulfur clusters might be particularly important for the cellular response to this perturbation.
Our data demonstrate that iron deprivation causes a pervasive, but not complete, reduction in the expression of mitochondrial proteins. This result is consistent with a calibrated remodeling of the mitochondrial proteome, as opposed to total mitochondrial turnover. To further test this, we measured changes in mitochondrial mass from C2C12 myoblasts using MitoTracker and nonyl acridine orange (NAO) following treatment with 100 μM DFO. Consistent with a similar recent study by Yoon et al. (2006), our results revealed either no change or a slight increase in mitochondrial mass after DFO treatment (Figure 2G). Additionally, fluorescence microscopy with MitoTracker staining revealed no obvious changes in mitochondrial mass or morphology after administration of DFO (Figure S2B). Together with the observation that select mitochondrial proteins are not diminished with DFO treatment, including VDAC (voltage-dependent anion channel) (Figures 2A and 2C), these results strongly suggest that the iron chelation effect does not involve complete mitochondrial turnover, as would be expected for a mitophagy process.
Through large-scale and targeted measurements of mRNA and protein abundance, we have found that iron chelation causes a powerful downregulation of mitochondrial protein expression. DFO is a well-characterized and clinically used iron chelator (Chaston and Richardson, 2003); nonetheless, to ensure that our observed effects are not an off-target effect of this drug, we deprived cells of iron through two additional mechanisms. First, we depleted cellular iron stores by overexpressing the iron exporter ferroportin (Nemeth et al., 2004) in human embryonic kidney (HEK) 293 cells (one of the lines tested in Figure 2D). Similar to DFO, ferroportin expression resulted in a loss of cytochrome c, the complex IV COXIV subunit, and the complex I 8 kDa subunit (Figure 3A). Both treatments also led to a decrease in ferritin, indicating a reduction in cellular iron levels (Figure S3A). Second, we demonstrated that a comparable effect was achieved with 2,2′-dipyridyl (DP), a structurally and functionally distinct iron chelator (Figure 3H). Moreover, because the iron chelation effect is robust in the postmitotic myotubes used in our microarray and proteomics experiments, it is likely independent of the known effect that DFO has on cell proliferation (Yu et al., 2007).
We next sought to determine whether established regulators of mitochondrial biogenesis could explain this response. First, because of the largely reciprocal effects of DFO and PGC-1α treatments (Figure 1E), we hypothesized that DFO might simply cause a decrease in the expression of PGC-1α or its associated transcription factors. From a detailed time course, we found that the abundance of mitochondrial transcripts (Figures 3B and 3C) and proteins (Figure S3B) begins to decrease around 12 hr and reaches a minimum approximately 24 hr after DFO treatment. As a control, we measured the transcript level of Tfrc, whose expression is regulated by iron levels (Figure S3C). We found that DFO had little effect on the transcript levels of Esrra, Nrf1, or Gabpa (the primary PGC-1α-associated transcription factors) (Figure S3C) and caused a marked increase in the levels of Ppargc1a mRNA (Figure 3D). Furthermore, PGC-1α−/− cells (Uldry et al., 2006) responded comparably to wild-type cells when deprived of iron (Figure 3E), together indicating that iron deprivation does not affect mitochondrial gene expression by opposing the function of this coactivator. Interestingly, the levels of PGC-1β mRNA (Ppargc1b) (Figure 3F) and protein (Figure S3B) were decreased following DFO treatment. However, similar to PGC-1α−/− cells, depletion of PGC-1β expression in primary satellite cells isolated from soleus muscle of PGC-1βf/f/MLC-Cre mice (Zechner et al., 2010) did not cause diminished levels of mitochondrial markers, nor did it alter the cellular response to DFO treatment (Figure 3G). Together, these results suggest that the observed effects of iron deprivation are independent of PGC-1α and PGC-1β.
Iron chelation is also known to activate the hypoxia response by inhibiting the iron-dependent hydroxylases that constitutively target the transcription factor HIF-1α (hypoxia-inducible factor 1α) for degradation (Schofield and Ratcliffe, 2004), and HIF-1α activation has recently been shown to initiate a loss of mitochondrial mass in renal clear cell carcinoma cells (Zhang et al., 2007). To determine if the loss of mitochondrial proteins observed here is also due to HIF-1α stabilization, we treated C2C12 myoblasts with the 2-oxoglutarate analog dimethyloxalylglycine (DMOG). The same HIF-1α hydroxylases that require iron also require 2-oxoglutarate as a cofactor and can therefore be inhibited by 2-oxoglutarate analogs even when iron levels are normal (Jaakkola et al., 2001). As seen in Figure 3H, C2C12 cells treated with the iron chelators DFO or DP responded comparably in their ability to stabilize HIF-1α. Cells treated with DMOG also stabilized HIF-1α but did not affect the OxPhos proteins, suggesting that HIF-1α activation is not sufficient for the iron chelation response. To test whether HIF-1α is necessary for this response, we treated wild-type and HIF-1α−/− cells with DFO for 24 hr. These cells showed the same response as all other cell types tested, regardless of the HIF-1α genotype (Figure 3I). Coupled with the fact that HIF-2α is not expressed in this cell line (data not shown), these experiments reveal that the widespread loss of mitochondrial proteins following iron chelation is a distinct, HIF-independent process. Overall, our data reveal that mere deprivation of iron elicits an effect comparable in magnitude to, but independent of, the most well-established drivers of the mitochondrial biogenesis program.
As noted above, depletion of cellular iron has various known side effects on cellular functions, including inhibition of cell division, initiation of apoptosis, and induction of autophagy (De Domenico et al., 2009; Yu et al., 2007). Therefore, we next sought to determine whether our observed mitochondrial effect represents a true acute metabolic adaptation or merely irreversible cellular damage. To do so, we treated C2C12 mouse myoblasts with or without DFO and, after 24 hr, passaged the cells into fresh, DFO-free media (Figure 4A). We continued to passage the cells every 24 hr and took samples at each time point (Figure 4A). Once again, 24 hr of DFO treatment resulted in the selective loss of mitochondrial markers (Figures 4B and S4A). However, 72–96 hr following the removal of DFO, protein (Figure 4B) and transcript (Figure S4A) levels were comparable to those found in their untreated counterparts. To determine whether the recovery of mitochondrial proteins also represents a recovery of mitochondrial function, we profiled mitochondrial respiration using a Seahorse XF Analyzer (Figure 4C). As shown in Figure 4D (and Figure S4B), iron chelation led to a strong decrease in basal oxygen consumption rate (OCR) and spare respiratory capacity (Figure 4E). Because DFO is known to suppress cell proliferation (Yu et al., 2007), we normalized the basal OCR of untreated and DFO-treated cells to cell number. Remarkably, the basal OCR of DFO-treated cells recovered within 48 hr following their passage into DFO-free media (Figure 4D). Interestingly, after this recovery of basal OCR, the DFO-treated cells continued to recover their spare respiratory capacity, which reached untreated levels by day 5 (Figure 4E). Coupling efficiency remained unchanged in the iron-depleted cells, indicating that DFO likely does not damage the integrity of the mitochondrial inner membrane (Figure 4F). Altogether, our results reveal that iron deprivation initiates a reversible and adaptive cellular response that involves remodeling of the mitochondrial proteome and a reduction in mitochondrial respiratory function.
Mitochondria are vital metabolic organelles that must continually adapt to changing external environments and cellular needs. To better define this adaptive process, we performed extensive, matched microarray and quantitative proteomic analyses of mouse muscle cells under a variety of conditions. In doing so, we created a robust resource that can be mined to discover proteins whose expression levels are affected by posttranscriptional regulation and auxiliary cellular processes that both sense the need for and drive mitochondrial restructuring. We leveraged this resource to find that proteins involved in cellular iron homeostasis are coordinated with mitochondrial biogenesis and have shown that depriving cells of iron through a variety of mechanisms results in a rapid downregulation of mitochondrial protein levels and oxidative capacity. We further demonstrated that this drastic effect occurs in a wide range of cell types and that it is fully reversible within 2–3 days following reintroduction of iron. Last, we have shown that this process is independent of the well-established PGC-1α-, PGC-1β-, and HIF-1α-driven mitochondrial biogenesis programs. Because iron deficiency is the world's number one nutritional deficiency, affecting approximately 25% of the population (McLean et al., 2009), our work could have significant implications for human health and disease. Our extensive, freely available mRNA and protein profiling data sets will serve as a rich resource for further exploring the cellular response to acute metabolic stress and the roles of transcriptional and posttranscriptional processes important for mitochondrial biogenesis.
Brown preadipocytes were maintained in high-glucose DMEM with 20% FBS and 1 × PS (Invitrogen) at 37°C and 5% CO2. Primary soleus muscle satellite cells were maintained on rat tail collagen (Invitrogen)-coated plates in Ham's F-10 media (as detailed in the Extended Experimental Procedures). All other cell lines were maintained in high-glucose DMEM with 10% FBS and 1 × PS. Deferoxamine mesylate salt (DFO), DMOG, and DP were obtained from Sigma-Aldrich. For ferroportin (Fpn-GFP) expression, HEK293FT cells were transfected with 1 μg Fpn-GFP (Nemeth et al., 2004) using Lipofectamine LTX (Invitrogen). Myotube differentiation, metabolic labeling, and adenoviral infection were performed using standard procedures (as detailed in the Extended Experimental Procedures).
RNA purification, cRNA preparation, and hybridization to Affymetrix 430 2.0 arrays were performed according to established methods (Mootha et al., 2003b). Unique Entrez Gene identifiers from the mapped data were selected and sorted by the average GFP probe intensity from the two metabolic-labeling experiments. Data corresponding to these brightest probes that gave present call (P) across all samples were used for analysis (8,313 probes total).
Mass spectrometry was performed using standard procedures (as detailed in the Extended Experimental Procedures). For comparison of mRNA and protein abundance, all data were mapped to Entrez Gene identifiers. Gene products were identified as mitochondrial using the MitoCarta inventory (Pagliarini et al., 2008). OxPhos and iron-containing proteins were manually curated from Gene Ontology annotations.
Total RNA was purified using an RNeasy Kit (QIAGEN). First-strand cDNA was synthesized from 500 ng RNA using SuperScript III (Invitrogen). Real-time qPCR was performed using predesigned TaqMan Assays or SYBR green-based detection (ABI) with Actb (microarray samples) or Rplp0 as the endogenous control (see the Extended Experimental Procedures for primer sequences).
For immunoblot analysis, 15 μg of cleared whole-cell lysate, as determined by BCA assay (Pierce), was separated on a 4%–12% Bis-Tris Mini Gel (Invitrogen), transferred to PVDF, and probed with primary antibodies (listed in Table S2). For BN-PAGE, 12 μg of mitochondrial protein, prepared as previously described by Pello et al. (2008), was separated on a 3%–12% NativePAGE Bis-Tris Mini-Gel (Invitrogen), transferred to PVDF, and probed with an OxPhos Blue Native Antibody Cocktail (MitoSciences). For ferritin measurement, 15–35 μg of cleared whole-cell lysate was analyzed by ELISA (Ramco Laboratories) and normalized to total protein concentration, as determined by BCA assay.
For determination of mitochondrial mass, C2C12 myoblasts were seeded at 4,500 cells/well in 96-well microplates and incubated for 24 hr. Following DFO treatment, mitochondria were stained with 100 nM MitoTracker Green FM or NAO (Invitrogen) for 30 min. The cells were then washed 2× with PBS, and fluorescence was measured using a BioTek Synergy 2 Microplate Reader with a 485/20 excitation, 528/20 emission filter set. Fluorescence was normalized to cell number (as described in the Extended Experimental Procedures).
OCR measurements were performed using a Seahorse Biosciences XF96 Extracellular Flux Analyzer as previously described by Nicholls et al. (2010). Briefly, C2C12 myoblasts were seeded at 12,000 cells/well in XF96 microplates (Seahorse Biosciences). After a 24 hr incubation, the growth media were exchanged for XF Assay Medium (Seahorse Biosciences) supplemented with 25 mM glucose (Sigma-Aldrich). OCR measurements were 5 min periods following 3 min mix periods. Myoblasts were treated by sequential addition of 1 μg/ml oligomycin (Sigma-Aldrich), 300 nM FCCP (Sigma-Aldrich), and 2 μM rotenone (MP Biomedicals). Spare respiratory capacity and coupling efficiency were calculated using Seahorse Bioscience instructions (see Figure S4B for details). Basal OCR was normalized to cell number (as described in the Extended Experimental Procedures).
p Values were calculated by Student's two-tailed t test, one-way ANOVA with Tukey's post hoc analysis, Spearman's rank correlation test, or χ2 contingency test as indicated in the figure legends.
We would like to thank the members of the Eisenstein, Kaplan, V.K.M., and D.J.P. laboratories for helpful discussions and assistance regarding this project. We specifically thank Sarah Calvo and Dan Arlow of the V.K.M. lab for assistance with microarray analyses, Jerry Kaplan and Ivana De Domenico (University of Utah) for providing the ferroportin expression vector, Bruce Spiegelman (Harvard Medical School) for providing the PGC-1α−/− cells, Daniel Kelly (Sanford-Burnham) for providing the PGC-1 bf/f/MLC-Cre cells, Randall Johnson and Alex Weidemann (UCSD) for providing the HIF-1α−/− cells, Eric Shoubridge (McGill) for providing the MCH58 cells, and Kelly Werner of the D.J.P. lab for critical reading of the manuscript. This work was supported by a Searle Scholars Award, a Shaw Scientist Award, and USDA Hatch Award WIS01671 (to D.J.P.), NIH R01GM077465 (to V.K.M.), and NIH Molecular Biosciences Training Grant 5T32GM007215-37 (to J.W.R.).
Accession Numbers: The Gene Expression Omnibus accession number for the microarray data reported in this paper is GSE42299. The processed microarray and quantitative proteomic data are also available for download from http://www.pagliarinilab.org/datasets.
Supplemental Information: Supplemental Information includes Extended Experimental Procedures, four figures, and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2012.11.029.
Licensing Information: This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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