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10 Questions With: Actor & Entrepreneur Daniel Franzese
To a lot of people, actor and entrepreneur Daniel Franzese is best known for his roles in Mean Girls ("She doesn't even go here!"), HBO's Looking, and countless other film and TV credits. But to fashion-conscious dudes like me, it's his work in the body positive community and as the Creative Director of the super-cool big & tall clothing subscription company The Winston Box that makes us fan girl.
I sat down with him to ask him my 10 hard-hitting questions about life (and style) as a big guy. Like this series? Check out my other interviews here.
What is your favorite part of being big and/or tall?
While there are a lot of challenges to being an above-average-sized guy in an average world, there are some great things about it. I'm least likely to be challenged to fight in a bar and I always feel strong. People feel safe around me and it's nice to easily reach cereal boxes off the top shelf for old ladies at the supermarket
What is the hardest part of being big and/or tall?
This world is not made for us. Doorways, plane/theater/car seats, even chairs that are wide enough are still not tall enough. Bending our legs in ways that cause knee and back problems. We are living in a small person's world.
If you could only wear one outfit for the rest of your life, what would it be?
My Winston stretch denim jogger and black Pima cotton scoop shirt. It's hard to get me to wear anything else now even though I have so many choices.
What's one fashion item every guy should own?
A well-fitting black T shirt. It's so versatile. Perfect for the gym or put a blazer over it and you can wear it on the town.
What are your go-to stores or sites for clothes?
There aren't that many options for bigger guys but I love shopping vintage. I can find some really incredible pieces and then spend the money on a piece to go with it. I love a good thrift find!
What do you wish retailers would do differently?
I wish there was more consistency in sizing. There should be some kind of regulation. Vanity sizing and just plain fashion sizing is so off nowadays. You can't call a store and hour away and ask if they have your size. It's a guessing game and I think it actually does mental damage to a lot of young people.
What does being "body positive" mean to you?
It means treating yourself well. Apologizing to your body and caring for it. Being proud of who you are at the stage that you are at in your journey and not letting anyone or anything stop you from enjoying your body to its fullest. Be shirtless in the sun, wear something form-fitting just celebrate that any body can be a sexy body.
What is your biggest style regret?
The way I treated my hair in high school. If I still had that much hair I wouldn't torture it the way I did.
Who are your style icons?
David Hockney, Marcello Mastroianni, Andre 3000, Winston Churchill, every 80s movie professor, and the Gorton's fisherman.
Who do you nominate to answer these questions next?
Winston model Shawn Jesus Davis.
You can find Daniel Franzese @whatsupdanny on Twitter, instagram, Snapchat and Tumblr .
Sign up for your own box at TheWinstonBox.com (use code #danfranfans to save $15 on your first Winston Box!).


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Nicker Kisses Horse Treats is committed to producing a quality product for your horse. Our treats are handmade, baked fresh with the finest ingredients and made with love. They are naturally preserved through the baking process, no artificial flavorings or preservatives have been added.
All ingredients used in Nicker Kisses are USEF and FEI compliant.
Ingredients include:
- Pelleted grains – oats, wheat, soy, stabilized rice bran
- Molasses
- Flax Seeds
- Stone Ground Wheat Flour
- Natural Dried Apples, Peppermint Candies
- Prebiotic Herbal Blend
*Check back for our seasonal, limited batch flavors!
Fill out the form below to place your order.
Each package contains Original, Apple, and Peppermint flavors. Should you want only 1 or 2 flavors, please specify in the "Special Instructions" box during ordering.

subset: commoncrawl


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B of the Bang – Southsea Fest will be our last ever show
B of the Bang are a fantastic Portsmouth band, they create beautiful melodies and addictive music, which in turn creates records that have longevity.
We first discovered them at Southsea Fest a few years ago, they were playing in one of the many venues that takes part in the day long festival. That day was spent trying to catch as many bands as possible so we didn't get to see too much unfortunately.
Then last yet we caught the end of their set at Victorious, it was brilliant and captivating, because stage presence is another thing they can add to their list.
So it is with a great amount of pleasure that we can post an interview with Wit, who sings and plays the guitar, from the band. However, it does come with bad news.
Musical Nourishment (MN): Most famous person you've ever met?
Wit (W): I'll have to go with two here. The first is Liam Gallagher who I walked straight into whilst leaving the gents toilets at an airport when I was about 18. I looked up and said 'sorry mate' before realising who it was and he said 'nice one our kid' exactly as you'd expect. I got my then girlfriend to go and get his autograph afterwards as I was too embarrassed and he glanced over, saw it was actually for me, shook his head and laughed.
The second is Peaches Geldof (RIP). The B of the Bang (TBOTB) supported The Horrors early on in their career when the frontman, Faris, was going out with her. I didn't know who she was at the time and when he introduced me to her I misheard and thought he said her name was 'Beatrice'. I then spent the whole evening asking 'Beatrice' if she was OK and whether she wanted a drink etc…as we were all together back stage. She seemed pretty annoyed with me. It was only when someone pointed it out at the end of the evening that I realised. Oh dear.
MN: If someone wrote a biography about you what would it be called and why?
W: A Lifetime of Underachieving. I'm essentially a very lazy person. So much potential, so many sofas to lay on.
MN: Tell me a secret.
W: When I was young I had a weird, rare illness called Henoch-Schönlein purpura. It was one of Hugh Laurie's amazing diagnoses on 'House MD' once and I felt famous.
MN: What is the weirdest experience you've ever had on tour?
W: Too many to mention here without writing an essay. You constantly find yourself in bizarre situations. Naked in hotel hallways, swallowing coins, broken ribs in A&E, naked in kebab shops, waking up on a boat…all these things and many more have occurred in TBOTB…
MN: When did you first realise music is what you wanted to do and why?
W: My older brother had albums by bands like Guns n Roses and Aerosmith which I started listening to around 11 I guess. It was only upon discovering Nirvana's In Utero that it really hit me though. We had a battered old classical acoustic guitar in the house and before I'd even learnt a chord I started writing songs. Terrible ones, mind. First year of senior school…grow long hair…start writing miserable music. Nothing's really changed since.
MN: Anything to talk about as a band?
W: Unfortunately we are splitting up so not many plans for the future now! We're going to play Bestival, and the Kings Theatre at Southsea Fest will be our last ever show. It will be emotional!
We're very upset to hear the news that B of The Bang are splitting up, they're a fantastic group and Desire Lines is one of our favourite songs. They are all exceptionally talented, so thank you for the music you have given us, it's been fantastic!
With Bestival now over it's not long to go until Southsea Fest. If you've got tickets make sure you go and watch The B of The Bang's last show at the King's Theatre.


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Poor beets. Beets get a bad rap. Most people I talk to say, "Ew, beets. They taste like dirt!"
I don't think they taste like dirt. I think they are smooth and creamy and yummy. They do require a bit of scrubbing to remove the dirt, but once that is done, any taste of dirt should be eliminated.
My absolute favorite way to eat beets is roasted with olive oil, sea salt, and freshly ground pepper. Actually, I think all vegetables are better this way. Add some Parmesan cheese and crusty bread, and it would make a lovely lunch.
Oh, and if you haven't tried sea salt yet, give it a shot. I thought it was all snooty hype, but I find myself reaching for the sea salt more than any other variety. I just love getting the occasional crunchy burst of salt from the large grains.
Back to roasting beets. It's simple.
- Scrub your beets. (I used two golden beets and one traditional red beet.) Scrub them until your arms are sore. Every centimeter. Really. Don't skimp here.
- Place them on a sheet of foil and drizzle with olive oil.
- Wrap up the beets, seal the foil, and place on a baking sheet.
- Roast at 400° for a while. Exact time depends on the size of your beets. Small beets could be done in 20 minutes, larger ones may take 40 minutes or even more. They should be softened, but still fairly firm. I didn't try this, but I would guess that a knife would slip in with little resistance when the beets are fully cooked.
- Unwrap and allow the beets to cool a bit.
- Once the beets are cool enough to hold, trim off the ends and peel off the skin. The skin may come off easily, or you may need to use a vegetable peeler. (There are some great photos and instructions here and here.)
- Once peeled, slice into wedges, drizzle with more olive oil and sprinkle with salt and pepper. Enjoy!
How do you feel about beets?


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NBAA and the General Aviation Manufacturers Association (GAMA) have launched a joint advocacy campaign to reinforce the value of business aviation to citizens, companies and communities across the country. The program takes full advantage of new communication technologies while building on proven advocacy techniques. A dedicated web site, thorough research, paid advertising, news media interviews, online education offerings, YouTube placements, podcasts and more are part of the initiative. Below are some featured No Plane No Gain headlines and articles.
Visit the No Plane No Gain web site at www.noplanenogain.org.


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Dirt Merch PTY LD
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Customer enquiries, email@example.com or 0499999684
Office hours 9am - 4pm QLD time.
God dam we are dissapointed to not be able to attend interbike this year …
Scotland brought on the weather and the racing was fast! If you couldn't stay …
Fort Bill qualy has gone down with Bergamont rider Eddie Masters getting his …
All prices are in AUD. © 2019 Dirt Merch.
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I had tasted this dish in a restaurant in Kundapur and fell in love with it immediately so much that I asked the waiter for the recipe :) Of course, I did not remember the recipe word to word and cooked my own version.
We dont get as big Kaane fish ( also called Nogli ) here so managed with smaller ones. This fish is probably called Lady fish in English (not sure though - Google came up with some results which said that)
1. Clean the fish, cut off the head and the tail. Make slits in the fish for masala to sink in. Marinate the entire fish with salt, turmeric and chilli powder.
2. Grind together 1/2 onion sliced, 1 tsp coriander seeds, 3 red chilli, 5-6 garlic flakes, 1 marble sized tamarind, a pinch of asafoetida, 1 tsp turmeric and 1 inch ginger.
3. Add this masala to the fish and keep it aside for 1 hour.
4. Take a pan and add 2 tbsp coconut oil. Add 1 small onion finely clopped and saute till translucent. Add 1 twig of curry leaves. Now add the fish along with the paste and let it cook on low flame. Dont stir too much. Let the fish cook in the masala. It takes around half an hour for the raw taste of the masala to go away.


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RRP: 208.1800 Save:
Found this item cheaper elsewhere? Let us know
This Atomos Sunhood is specially designed for Shogun Flame, Samurai Blade and the Ninja Flame.
It is designed to secure to the 1/4"-20 threaded hole on the top of the monitor using an included thumbscrew. A viewing cover is also included that magnetically snaps to the sunhood to provide an even more concentrated viewing window.
Copyright 2019 Lemac Films Australia PTY LTD


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Explore a different world and a different reality, Tomorrowland is a wonderful experience. An experience that will change your life and transforms whatever you worked hard on as a heaven like experience. We introduce to you the Tomorrowland Experience.
Once upon a time, the beginning of Tomorrowland's fairy tale was witnessed with the total of 10,000 people and a single stage. How crazy is it to imagine that Tomorrowland is officially the world's largest music festival. Let's take a trip down a memory lane and reveal the history of all Tomorrowland themes ever since
The Story of Planaxis is finally revealed. "Be part of this fascinating new adventure and discover a magnificent new world with The Story of Planaxis"- Tomorrowland 2018 The people of Tomorrow are you all ready to experience the madness of Tomorrowland 2018? Tomorrowland, the biggest music dance festival in the history, has finally revealed the
Beirut, Lebanon's Capital City is definitely one of the destinations you should mark down to visit. Not many leave the city without an eye opening and a memorable experience! If you are still doubting wether you should visit Beirut, here I will be writing down why it is an opportunity not to miss! Let's talk


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Finished* my pieces for the ReStore Art Challenge– Huzzah!What's that? These photos don't give a complete and accurate picture of the finished pieces? CORRECT.Guess you'll have to stop by the opening:
Where: Delaware City Art Center 22 E. Winter Street, Delaware, Ohio
When: April 6 – 28, 201
See you there!
*well, okay, there are a few tiny touches to finish, but for the most part, they're done.


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25 March 2016
Renew! Renew!
Got a shiny new Concealed Weapon or Firearm License from the "local" regional office of the Florida Department of Agriculture and Consumer Services today.
If you go to the regional office, the photo and notarization are included in your renewal fee. You lose travel time and gas instead.
I've had worse drives... ;)
It honestly felt strange getting my card the same day I wrote the check.
5 comments:
OMG! Thanks so much for this post! I'm leaving for Florida tomorrow, and I just got my Renewal packet!ReplyDelete
I'll have to make some time to renew whilst I'm down there. I haven't done much research, is there a website that can help me locate the nearest office to where I'll be staying?
Klickst Du hierReplyDelete
That link gives the list of regional offices. You MUST have your unsigned renewal packet with you!
In addition to those, lots of the driver's license stations are doing renewals. More fees, but prolly closer.Replies
I'll be here until Thursday, I'll let you know when I settle in...but most of the time i'll be chasing a small tornado masquerading as a small girl.ReplyDelete
You are a guest here when you comment. Inappropriate comments will be deleted without mention. Amnesty period is expired.
If you can't comprehend this, don't comment.


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I went shopping again today and picked up some new stuff, including, finally, a couple of Essie polishes! I thought that because I've been wanting to try Essie for so long, I'd do some mini reviews rather than a big post about the stuff I bought:) The rest of non-Essie stuff will have it's own post too, so don't worry;)
Lapiz of Luxury
Colour- I am absolutely completely loving this colour. It's so beautiful, it's like a dark cornflower blue, and it's gorgeous. It's just become one of my favourite nail polish colour, and it would be gorgeous as a background for the dandelion nail art I keep seeing everywhere and still want to try.
Price- £7.99 from Boots or Superdrug, well worth the price.
Quality&Application- The quality is brilliant, I have two coats on in the pictures above, and looks surprisingly shiny. It dries fairly quickly too. The application was really easy, the brush is brilliant and really wide, so you only have to do two strokes across your nail.
Packaging- I love it! It's a really gorgeous 13.5ml bottle, I like the white polish top, most of mine are black so it really stands out.
Lasts for- I only tend to wear nail polishes for a day and then I change the colour. I know that's really weird;) but I only get two days a week to be able to wear nail polish, so I like to use as many colours as possible. It's looking good so far though:)
Overall- Totally loving Essie, and this colour. Will definitely be trying more Essie polishes, but I've got another two parts to this post so you'll be able to see some more of these soon!:) Recommend me some colours to try k?;)


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3 Reasons to Buy $600 Shoes
If you read the last post on shoes you know I'm a believer in buying quality over quantity. The Allen Edmonds shoes I mentioned will last you about 10 years. But what if you really don't like shopping?
The answer: Shell Cordovan.
- They require next no maintenance – Rich in natural fats, the shoes are 'self lubricating' and simply need to be brushed on occasion to raise them to a high shine.
- Non-porous – Cordovan lacks the pores of calf skin leather and is waterproof.
- Strong – These shoes will last upwards of 50 years in normal rotation.
Most dress shoes you'll find in department stores are made from some variation of calf skin—this is common and probably not groundbreaking. Calf skin (leather from a cow) is porous and strong and a natural first choice for making quality shoes…and a really well made pair like the AEs will give you a decade of wear.
Shell cordovan is a step above. While most people perceive cordovan as a color, it is first and foremost equine leather from hindquarters covering the rump. In layman's terms, that's the covering of a horse's ass. Because each horse produces only enough shell cordovan for a single pair of shoes, because there is a ban on raising horses exclusively for leather, and because there is only one production company worth their salt (Horween) the material cost is high.
Most women would balk at having the same pair of shoes for 50 years, but in a men's store "brown or black" can be an appropriate adage.
I recommend the Allen Edmonds cordovan collection as a great place to start. You're paying for the quality of the craftsmanship and the level of customer service over the name. If you're in the Atlanta area, check out the Dunwoody store and ask for Lupe or Cody.
For a step up the fashion ladder, Alden makes an impeccable shoe with a New England flair lower on the customer service totem pole but higher on quality. If you're in the market for Aldens, contact Brand at H. Stockton Lenox or shop online at TheShoeMart.com.


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Navigating Cancer Care at the Hospital with Moral Support
When a person is diagnosed with cancer, taking the next steps is overwhelming in the midst of physical and emotional pain. That's where the role of the Cancer Patient Navigator comes in, said Carolyn Henry, Care Manager for the Bow Valley Cancer Centre.
"You have the role of being a case manager for them," said Henry, an RN and community care nurse who previously worked as Cancer Patient Navigator.
The navigator is "really being the person that they call" for all aspects of a patient's experience, including "emotional and spiritual support," said Henry.
Henry, who has worked at the Canmore General Hospital since 2013, said the team has grown to include two nurses devoted part-time to the unit, as well as three casual nurses and a unit clerk.
The Canmore and Area Health Care Foundation has supported cancer care at the Canmore General Hospital for years. Beyond the expansion of and improvements to the cancer care unit, the foundation provides ongoing funding to programs for cancer patients and their families, including yoga, mindfulness and support groups. For patients going through treatment, Henry said it's hard for them to stop thinking about their diagnosis.
"Those programs allow them to have time to learn how to have a break from their cancer with people who understand what they're going through," said Henry. "I think those activities bring people together in the community who are sharing the same thing."
When patients or family members ask the hospital staff how they can contribute to continued programming for cancer patients in the Bow Valley, Henry tells them about the foundation. For her part, Henry is a devoted champion of the CAHCF, donating money from each pay cheque directly to the foundation.
"It's really great to see that they are really working hard to make a difference in the cancer client's journey," said Henry of the foundation's support. "It does make a difference. Without them, we wouldn't be able to provide those amazing programs that help a lot."
Beyond programming, the CAHCF pays for medical staff – including nurses, physicians and pharmacists – to attend national and international conferences to stay up-to-date on cancer treatments and medications. This, said Henry, is an important part of staff providing the best and most current care for cancer patients.
Carolyn Henry, an RN and community care nurse, manages the cancer unit staff at the Canmore General Hospital. The CAHCF plays a large role in ongoing training and paying conference fees for staff to stay up-to-date on treatments and medications.


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In the past week, a couple leaked images of Vision appeared on the internet. The fact that Marvel has been adamantly hunting them to remove them based on copyrights means that they are probably legit. They don't show us much, but they're still pretty cool to confirm what we know.
So without further adieu, here is Vision from Age of Ultron:
These images look almost exactly like the Vision from the comics. There is no close up on the face, which is what I am very interested in seeing, but they confirm that the costume will be nearly exactly the same from the comics. Granted, comic Vision has a rather fantastic collar that seems to be dropped, but I am personally impressed with how similar they are keeping it.
Paul Bettany is a very interesting choice for Vision. His interpretation is one of the biggest things I am looking forward to in Age of Ultron.
Questions still circulate about the Jarvis-Vision connection, which undoubtedly falls back to the Stark-Ultron question. For now, only time will tell.


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About me
I started The Social Walker Agency to help other business owners achieve their business dreams. Along the way, I decided I wanted to combine what I was learning in my personal life along with my business tips and goals. My heart is to work with business owners and


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Today's Right-Now Economy demands continuous engagement through instantly responsive, always available applications. DSE is the always-on, distributed cloud database that allows you to build real-time applications at scale while providing the data autonomy you need to thrive in an increasingly hybrid-cloud world.
DSE is the only database that can power Contextual, Always-on, Real-time, Distributed, and Scalable applications.
Retain control of data and manage it anywhere (ie, public cloud, private cloud, on-premises, hybrid cloud, or multi-cloud).
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Supports tabular, key-value, JSON, and graph (by the team that created Apache Tinkerpop™).
Provides a unified layer of database, search, and analytics that can be fully managed by DataStax or self-managed.
DataStax Enterprise, a unified data layer of database, search, and analytics, is the only hybrid cloud data management layer that allows enterprises to meet the contextual, always-on, real-time, distributed, and scalable requirements of Right-Now Economy applications.Try DataStax
DSE offers advanced security, self-driving operations, and twice the performance of open source Apache Cassandra. With DSE, enterprises get a comprehensive, always-on, secure, easy-to-use data layer backed by expert support and services that helps them WIN in the Right-Now Economy.
DSE provides a uniform security model across the entire data layer (database – including graph, search, and analytics) and includes advanced features such as unified authentication, authorization, profile access control, row-level access control, encryption, data auditing, and more to prevent unauthorized access.
DSE can handle transactional, search, and operational analytics workloads all running on the same cluster and possesses built-in workload isolation and replication capabilities that keep these workloads from competing for resources.
DSE reduces manual repair operations significantly, eliminates cluster outages attributed to manual repair operations, and provides visual management and monitoring of DSE clusters across cloud, on-premises, or in hybrid environments, allowing even novice DBAs and DevOps personnel to manage operations like seasoned professionals.
DSE packs twice the performance of Apache Cassandra and three times the performance for reads against open source Apache Spark™, allowing for double the workload or users for the same amount hardware. And DataStax Bulk Loader accelerates the loading and unloading of data by 4x.
DSE offers an array of options for developers to quickly and easily build applications, including CQL, which is quite similar to SQL and designed specifically for DSE's powerful capabilities, or Spark SQL for analytic requirements.
Learn about DataStax Enterprise and how it can transform your company.View Products


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St Kentigern's Church
|860 (Census 2011)
|OS grid reference
|Civil parish
|Shire county
|Sovereign state
|United Kingdom
|Post town
|Postcode district
|Dialling code
|North West
|EU Parliament
|North West England
|UK Parliament
Irthington is a village and civil parish within the City of Carlisle district in Cumbria, England, situated to the north-east of Carlisle Lake District Airport. The population in 2011 was 860 according to the 2011 census.[1]
The name Irthington derives from Old English and means farmstead or village on the river Irthing.[2]
The most important period in Irthington's history was during the Romano-British era. Irthington lies on top of the line of the Roman Stanegate road which preceded Hadrian's Wall and ran from Corbridge (Coria) west to Carlisle (Luguvalium). The village was described in 1884 as being "intersected by the site of the great Roman wall, and also by the military road [ie the Stanegate] from Newcastle to Carlisle".[3] Some of the building stone used in the south wall of the church of St Kentigern's chancel is thought to come from Hadrian's Wall, which ran less than 1 kilometre (0.62 mi) to the northwest of the village, though its stone locally has been robbed. St Kentigern's church is dated as far back as Norman times. Restoration took place in 1849 in the gothic tradition. Since then, more restoration has taken place including the addition of a five-bell clock tower. The historical remains of the arcades of round arches set on round piers remain by the church, and are dated to the 1170s.[4]
There is also a medieval motte in Irthington, a hundred yards or so south of the church.
In the 1870s Irthington was described as:
- " a village, a township, and a parish, in Brampton district, Cumberland. The township comprises 947 acres"[5]
In the 2011 census the population of the village was 860. However, as early as 1831 it stood at 1049 and has since been variable. This number has not changed significantly over the last 200 years apart from a sharp increase as shown in the population time series in 1951 due to a change in area boundary.[6] The male to female ratio has rarely been more than marginally different. In 1801, there was 430 men to 440 women while in 2011 there were 416 men to 444 women, only a small increase from 10 to 28. Another aspect of the population is that as of 2011, 840 of the 860 residents were born within the UK. This gives a total percentage of UK born residents of 98%.[7]
Occupation data[edit]
The data taken from 1881 shows that 197 people worked within the Agriculture or Farming occupation. This means that 23%, almost a quarter of the 853 people living in Irthington during this census were under this occupation, the largest in the village. The next largest occupation is of people without a specified occupation. 102 people during this census come under this occupation, 12% of the population. The fact that second largest occupation is almost half of the highest occupation shows the importance that agriculture and farming played. The third highest populated occupation in 1881 was "domestic services and offices". 57 people, 7% of the population were employed or engaged in this area of work. These top three occupations accounted for 42% of the occupation of the entire population of the village.[8] More recently, the occupations of the population have changed. Of the 860 residents, 118 people work within the skilled trades occupation which equates to 14% which is above the national average. The second most populated occupation is that of professional occupations where 80 people work within, which accounts for 9%, almost half the national average. The third highest occupation within Irthington is of Managers, directors and senior officials. This accounts for 6% of the total employment and is also below the national average for the same occupation. The occupational transition has seen a large shift away from reliance on the agricultural sector. It has also seen a reliance a limited number of occupations with the top 3 occupations accounting for 29%, down 11% in 1881.[9]
Irthington offers a range of holiday accommodation with eight hotels/inns available in the area.[10] The airport located nearby should be useful for anyone who is a tourist to Irthington once passenger services as well as freight are provided. The main element of tourism in Irthington is the close proximity to the Hadrian's Wall trail which attracts people to stop in the village. However, tourism does not play a major part in the local economy.[11]
Irthington is a rather remote village. In terms of rail link the closest train station is Carlisle railway station which is 9 miles (14 km) away from Irthington, which equates to approximately 20 minutes away by car.[12] The nearest aviation transport is Carlisle Lake District Airport, which is located just over 1 mile (1.6 km) away from Irthington.[13] The airport is not currently offering commercial flights, but redevelopment which started in December 2014 was predicted to see flights available to London and Dublin by 2016. This development has also reportedly safeguarded the employment of 800 people.[14]
See also[edit]
- ^ "Irthington (Parish): Key figures for 2011 Census: Key statistics". Neighbourhood statistics. Office for national statistics. Retrieved 27 January 2015.
- ^ "History of Irthlington" (PDF). Archived from the original (PDF) on 2 April 2015. Retrieved 29 March 2016. Cite uses deprecated parameter
- ^ Bulmer, T. (1884). History, Topography and directory of East Cumberland.
- ^ "Irthington". The Cumbria Directory. Retrieved 27 March 2015.
- ^ Marius Wilson, John (1870). Imperial Gazetteer of England and Wales.
- ^ "Irthington AC/CP through time". A vision of Britain. GB Historical GIS/ University of Portsmouth. Retrieved 27 March 2015.
- ^ "Irthington: Key figures for 2011 Census: Key statistics". Office for National Statistics. Retrieved 27 March 2015.
- ^ "Irthington Occupational Data". A vision of Britain. GB Historical GIS/ University of Portsmouth. Retrieved 27 March 2015.
- ^ "Occupational data for Irthington". Office of National Statistics. Retrieved 27 March 2015.
- ^ "Irthlington: Tourist Information". Archived from the original on 4 March 2016. Retrieved 29 March 2016. Cite uses deprecated parameter
- ^ "Carlisle District: Rural masterplannng" (PDF). Carlisle City Council. Carlisle City Council. Archived from the original (PDF) on 27 April 2015. Retrieved 20 April 2015. Cite uses deprecated parameter
- ^ "Irthington to Carlisle Railway Station". Retrieved 29 March 2016.
- ^ "Irthington to Carlisle Airport". Retrieved 20 March 2016.
- ^ "Work begins on £12m Carlisle Airport Revamp". BBC. 3 December 2014. Retrieved 20 April 2015.
External links[edit]
Media related to Irthington at Wikimedia Commons


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Search Jobs
Building | Assembly
Do you have construction experience? Are you looking for a new opportunity in the Morristown area? Luttrell Staffing Group is currently seeking applicants for Building | Assembly positions!
Pay rate: $12 / hr
Schedule: 4 a.m. - 12:45 p.m. or 7 a.m. - 3:45 p.m. (40+ hrs / week)
Building | Assembly details:
- Assembling sound isolation enclosures
- Using hand tools, air tolos, tape measure
- Fast paced work
- Heavy lifting (up to 80 lbs) required
- At least two years of related construction experience required
Building | Assembly benefits:
- Long-Term Temporary position
- Eligible for Health, Vision, Dental, and Term-Life Insurance
- Vacation and holiday pay
- Paid Weekly
- Direct-Deposit Available
Interested? Click the link to apply then call our office at 423-586-5008 to schedule your interview TODAY!
Our office is located at 2629 W. Andrew Johnson Hwy., Suite 130 | Morristown, TN 37814
*For more information on this or other opportunities, text Luttrell to 25000!*
Louisa Jones
Staffing Coordinator
Louisa is a Staffing Coordinator for Luttrell Staffing Group-Morristown. Call her to find a great job!


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Charges are a bit decrease right here in the US, nonetheless, typically it makes extra sense to lock within the fee in case you plan to personal the property over the long term. When you're confident your turning the property over in the next year or two, then you can make an argument for the variable rate. The U.S. Division of Labor issued its final Conflict of Curiosity" (C..I.") regulation in April 2016, with the effective date of its core provisions on April 10, 2017. Underneath the C..I. regulation, fiduciary standing is imposed on nearly everyone offering funding suggestions to ERISA-covered plan sponsors and plan contributors, as well as to owners of IRA accounts, Keogh plan accounts and well being savings accounts. While prohibited transaction exemptions (PTEs) (together with the B.I.C.E. permit the receipt of third-celebration compensation, as a sensible matter corporations – and their advisors – should transition to price-based accounts. Something less will result in important reputational threat to advisors, in addition to considerably elevated litigation threat to both companies and advisors.
A two-year degree option, the associate degree provides college students primary data in regards to the world of finance, getting ready graduates for entry-stage roles or for switch to a bachelor's diploma program. Along with introductory programs in finance, college students additionally discover foundational enterprise topics and full common training in areas reminiscent of English composition, math, humanities, and social and behavioral sciences.
Two wheeler loans are a quick way of raising finance for the purchase of your dream journey. It comes with flexible phrases of compensation and has a fast approval process. The mortgage application is often accredited inside a period of 48 to 72 hours and you can be riding your new ride residence in no time. For any type of finance, analysis is the key. Before making any decisions, research the market and the prevailing norms of credit to ensure that you're taking the proper step.
This technique, in fact, has its personal problems. For one, you should be talented at managing, mentoring, and growing younger expertise. This is a multi-yr process that requires a large e-book to assist. Likewise, it's important to perceive youthful advisors. Generational differences in how and the place they work, what tools they use, and how they interact with purchasers needs to be appreciated, not snuffed out. These are variations in type, not substance. On high of that, you are paying them and they'll likely not be a profit-middle for your small business. In different phrases, you are investing upfront for a better transition with more favorable phrases.
In case you are good on this, companies, blogger and Web marketer will all be looking for you and you'll make lots of cash. We work onerous to earn money and therefore are very scared to lose it. We let the money lie idle in the financial savings checking account thinking that we might make bad funding selections which can lead us to lose cash. Affiliate marketing is the method of merchandising the products and providers of one other organization on their behalf. Here you're employed as the affiliate of that group.


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It's time to Fix I-35!
The Plan
The Texas Department of Transportation (TxDOT) is refining its Unified Transportation Program (UTP) which will serve as a state guide for the next ten-years of new road construction and infrastructure projects. In order to give Texans a chance to voice their priorities about future projects, TxDOT has opened up a public comment period, but only through August 20. It's time for you to weigh in!
Demand Change on I-35
It's time to voice your opinion about how bad Texas traffic is and demand relief on I-35!
Congestion on state highways, particularly on I-35, has gotten out of hand. Politics is getting in the way of moving forward with the I-35 expansion project and every day we fail to act is another day our state falls behind. In order to create real change and let our politicians know you want action, we need you to voice your opinion about how bad traffic has gotten in Central Texas and specifically on I-35.
You only have until August 20 to voice your opinion about traffic relief!
Suggested Comments
Take a moment to submit your own comment, or select one of the prewritten ones below:
- Central Texas has experienced a huge population boom that has led to major overcrowding on I-35. It's time for a dedicated plan that gives drivers on I-35 traffic relief.
- The portion of I-35 between SH 71 and US 290 is one of the most congested roads in the state. If we can add new managed lanes and maintain free lanes to this road without raising taxes, it would be a win-win situation for all who travel on this thoroughfare and Texas taxpayers.
- The I-35 expansion project would provide relief to hundreds of thousands of commuters by reducing traffic congestion, improving safety, and giving commuters a choice when it comes to using managed lanes.
- Texas is an economic powerhouse. However, with traffic levels continuing to rise, Central Texas commuters are faced with impending gridlock and our economy is at risk if drastic changes aren't made.


text: Julianne Kanzaki May 13, 2017 Simple Truths. Julianne Kanzaki May 13, 2017 Jedidiah Smith Redwoods State Park When I am among the trees, especially the willows and the honey locust,equally the beech, the oaks, and the pines, they give off such hints of gladness.I would almost say that they save me, and daily.I am so distant from the hope of myself,in which I have goodness, and discernment, and never hurry through the world but walk slowly, and bow often. Around me the trees stir in their leavesand call out, "Stay awhile."The light flows from their branches.And they call again, "It's simple,"they say, "and you, too, have comeinto the world to do this, to go easy,to be filled with light, and to shine. — Mary Oliver, "When I Am Among Trees"


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Should ethnicity be used to interpret relations between the Deaf community and the hearing people? Recent scholarship questioning the merits of Deaf ethnicity suggests a need to reexamine the use of ethnicity when describing Deaf identity and culture. This article provides an overview of key contributions to race and ethnicity discourse in the 20th century, identifies epistemological and ontological errors to avoid, suggests adherence to the classical Greek concept of ethnos as an alternative to ethnie, and argues for the continuing significance of Deaf ethnicity. Specifically, I propose that Deaf ethnicity is a triadic relational nexus that approximates communities of origin, language, and religion. This is expressed as Deafnicity ≈ D/deaf (Hómaemon • Homóglosson • Homóthreskon).Eckert goes on to comment that -
Deafnicity offers a promising alternative for examining relations between Deaf and hearing communities, exploring variance between nationalized Deaf communities, and expanding our understanding of audism
What is ethnicity? Sociologists have been defining ethnicity for almost a century. Ethnicity is an expression of self and community (Weber 1922/1978). Ethnicity is a negotiated product of dialectical tensions between internal self-identification and external ascriptions, who we say we are in a set of relations with who others say we are (Nagel 1996). It is an emergent process (Yancey, Ericksen & Juliani 1976) and situational (Gans 1979). Ethnicity is a strategic and adaptive process of reorganizing identity boundaries and the cultural content within those boundaries (Nagel & Snipp 1993). Ethnicity is a rationale choice (Nagel 1996). Should the concept of Deaf ethnicity be used to describe the set of relations between the Deaf community and those who are hearing?1
In this article, I retrace the social scientific understanding of ethnicity. I attempt to expose some of the misunderstandings that accompany recent discussions about Deaf ethnicity. I call for a return to how the ancient Greeks applied the concept of ethnos. In ancient Greece, ethnos included a wide range of concepts involving identity boundaries (Smith 1986, p21). I argue that if Deaf ethnos is viewed as a triadic relational nexus of Hómaemon (community of common origin), Homóglosson (linguistic community), Homóthreskon (community of religion), it has greater explanatory power than the modern concept ethnie (collective name, myth of common descent, a shared history, a shared culture, an association with a specific territory, and a sense of solidarity; Smith 1986, pp22–31).
Stokoe's (1960/2005) recognition of sign language, Woodward's (1972), distinguishing cultural constructions (Deaf) from medical circumstance (deaf), and Humphries' (1977) coinage of the term "audism" imply or infer Deaf ethnicity. However, recognition of the ethnic processes of constructing, maintaining, and reorganizing Deaf identity boundaries is relatively recent (Erting 1978; Markowicz & Woodward 1982). Lane, Pillard, & Hedberg (in press), Lane (2005), and Eckert (2005) argue the merits of Deaf ethnicity using modified versions of Smith's (1986) dimensions of ethnie. Lane et al (in press) and Lane (2005) rely on historical comparative data to demonstrate the presence of all six dimensions in the Deaf community. Eckert (2005) hypothesized a nationalized concept of Deaf American ethnicity, or what he called Deafnicity, as a counter hegemonic response to audism.
Lane's (2005) and Eckert's (2005) usage of Smith's (1986) dimensions of ethnie illuminates a few problems with the model when applied to the Deaf community. First, Smith (p27) deemphasizes the relevance of language. Smith writes "Examples could be multiplied to show that language, long held to be the main, if not sole, mark of ethnicity, is often irrelevant or divisive in the sense of ethnic community" (p27). Sign language is a critical component of Deaf identity and culture. Lane et al (in press), Lane (2005), and Eckert (2005) do emphasize the importance of sign language in their modifications to ethnie.
Second, the Smith model of ethnie lacks precision when defining the myth of common descent. On the one hand, there is a "self-same ancestor" (Smith 1986, p24). On the other hand, the sense of tribal belonging is something based on "common family ties, rather than any sense of genetic and blood ties" (Smith 1986, p24). Although Smith does not appear to treat ethnie as biologically determined, there is a need to amend the ethnie model in a way that clearly identifies kinship ties as an effort to organize human interdependence through rules of relatedness (see Macintyre 1993). Fictive kinship, as understood by anthropologists, provides an avenue to go beyond the idea of Deaf identity being one generation thick as argued by Davis (2008). Eckert (2005, pp108–110) asked Deaf respondents to compare meeting a Deaf person for the first time with meeting a hearing person for the first time. Respondents described Deaf people as being those they felt instantly connected to and could be their real self around. The lack of connection to hearing people was also highlighted. One respondent (Jim) said, "It is different because the Deaf person feels like family" (Eckert 2005, p110).8 The Deaf self-same ancestor, sometimes signed as "DEAF-SAME," is not a matter of genetics.
The Smith model of ethnie also fails to account for what Nagel (2003) calls the sexualized boundaries of ethnicity or ethnosexual frontiers. These "are the borderlands on either side of ethnic divides; they skirt the edges of ethnic communities; they constitute symbolic and physical sensual spaces where sexual imaginings and sexual contact occur between members of different racial, ethnic, and national groups" (Nagel 2003, p14). Nagel (2003) defines ethnosexuality as "the intersection between ethnicity and sexuality and the ways in which each defines and depends on the other for its meaning and power" (p10). Padden and Humphries (2005) describe a set of power relations that includes sexual domination of the Deaf community in America from as far back as the early 1800s. Padden and Humphries (2005, ch 1) description of "silenced bodies" illuminates the need to consider the ethnosexualized boundaries of Deaf identity and culture. Stereotypes used to describe the Deaf population offer some of the more extreme examples of sexualizing Deaf identity and culture. The Peoples Common Sense Medical Advisor published in 1890 provides a list of causes for the "paralysis of the auditory nerve" (Pierce 1890, p681). The list includes masturbation, excessive sexual excitement, and debauchery (Pierce 1890, p681).
Smith's ethnie does not account for sexualized stereotypes nor does it include important cultural aspects of selective mating habits, sexualized stereotypes, abuse, and exploitation. Lane et al. (in press) includes a wealth of information about Deaf ancestry that implies selective mating habits. Eckert (2005) provides narrative descriptions told by Deaf previously married to hearing individuals. However, neither Eckert (2005) nor Lane et al. (in press) provide an analysis that includes ethnosexual settlers, sojourners, adventurers, invaders (Nagel 2003) in the context of the sexualized relations between the dominant hearing majority and the Deaf community that Padden and Humphries (2005) highlight.
Finally, Smith's idea of ethnie conflates a number of Greek definitions of ethnos. The synthesis of different types of ethnos is a useful starting point. Though, without language, a clear distinction of kinship, or recognition of the sexualized borders of ethnicity, the explanatory powers of the Smith model are inadequate when addressing the continuing significance of Deaf ethnicity in contemporary society. As such, one is compelled to ask if the classical Greek concept of ethnos has greater explanatory powers than Smith's (1986) model of ethnie. Does ethnos include language? Does ethnos clarify the role of fictive kinship? Does it account for the sexualized borders of ethnicity?
Critics of Deaf ethnicity include Davis (2008), Sabatello (2005) and Tucker (2004). Davis' objections to the idea of Deaf ethnicity appear to be with both the broader concept of ethnicity and the narrower dimensions of ethnie. Davis (2008) seeks to replace Deaf ethnicity with what he calls postdeafness. For example, Davis (2008) asks "Why use, outdated, outmoded, and potentially dangerous categories of ethnicity, minority status, nationhood (including 'world' and 'culture'), when one might do better to use the category of 'one-generation' identities to redefine the nature of social identity?" (p323).
Sabatello (2005) argues "The Deaf community is arguably a linguistic minority based on fluency in sign language that is different from the majority language, or alternatively, an ethnic minority based predominantly on common history and culture" (p1036). Sabatello asserts a need to differentiate "between those who are legally recognized as ethnic, linguistic, and religious communities, and other sorts of 'life-styles cleavages, social movements and voluntary associations' notwithstanding their internal claims for shared linguistic and cultural systems" (pp1048–1049). The arguments of Sabatello highlight the importance of establishing the Deaf community as an ethnic community.
Tucker (2004) argues that Deaf identity, in the context of a deviant medical minority, needs to be rehabilitated and conform to the cultural hegemonic goals of the dominant hearing majority. Tucker argues "deaf people with cochlear implants, particularly children, have a wealth of opportunities and potential life experiences available to them" (p186). Tucker goes on to say, "To deny such opportunities based on theories of segregation is indeed illogical" (p186). Tucker's argument is framed first by identifying Deaf people as second-class citizens using a Washington Post article as proof of second-class citizenship and then claiming that cochlear implant technology will 'alleviate the ramifications of deafness" (p186).
Sabatello (2005) and Tucker (2004) each suggest that the Deaf community is attempting to impose a Deaf centric view on families with Deaf children and in the process deny Deaf children the ability to successfully assimilate into the dominant hearing majority. Sabatello argues "Thus, rejecting cochlear implants for a deaf child, as advocated by members of the Deaf community, coerces the Deaf culture on the child" (p1033).
Davis, Sabatello, and Tucker each express major misunderstandings of Deaf identity and culture. As such, demystification of ethnicity must accompany discussion of whether Deaf Americans are an ethnic population. The larger problem with the assertions of Davis (2008) and Sabatello (2005) is their pretension of recognizing Deaf human identity while perpetuating negative stereotypes that challenge that humanity. For Tucker, assimilation provides human identity. This differs greatly from those advocating Deaf ethnicity who do not view assimilation as a prerequisite of human identity.


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Prambanan is a 9th-century Hindu temple compound in Central Java, Indonesia, dedicated to the Trimurti, the expression of God as the Creator (Brahma), the Preserver (Vishnu) and the Destroyer (Shiva). The temple compound is located approximately 20 kilometres northeast of the city of Yogyakarta on the boundary between Central Java and Yogyakarta provinces.
The temple compound, a UNESCO World Heritage Site, is the largest Hindu temple site in Indonesia, and one of the biggest in Southeast Asia. It is characterized by its tall and pointed architecture, typical of Hindu temple architecture, and by the towering 47-metre-high central building inside a large complex of individual temples. The temple was damaged during many severe eartquakes but still attracts many visitors from across the world.


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Well, my damn awesome plan to do nothing but write today has fallen by the wayside. I mean, crap! When it get to be nighttime? I find this strange and confusing and probably all part of an alien plot to destroy my sense of space and time and take over the world. Because I am OBVIOUSLY the linchpin that holds the motherfucking world together and keeps it from drifting endlessly through the cosmos.
However, seeing as it's only 9-ish, I technically have 5 or 6 hours of awake time before tiredness hits me and I'm forced to go to bed. I could write my planned quota for the day. I could. I might even still have some coffee stashed away downstairs. That could give me another hour or two on top of that. Dudes! Why sleep at all? I could just stay up until tomorrow morning and then go to bed. Cole can totally fend for himself. I mean, I'm fairly certain that he can. You know, in a "he probably won't burn the house down while trying to make himself breakfast" sort of way. Pretty sure. Maybe this isn't the best plan.
Anyway. I have 2 more reviews to write and then I'm caught up to my current books I'm reading. Unless I count the short stories I've been sinking my teeth into recently. Uggh. Too much. Brain going into terminal overload. Maybe I'll skip those short stories. Maybe.
Book #157 - Derrick (Circe's Recruits #3) by Marie Harte - Picking up practically from where Book #2 left off, Sabrina's on the run from the PPA and Derrick can't get her out of his head. My biggest question with this book was why Roane had to claim Sabrina and mark her as one of the Pack when he didn't have to claim Kelly in Book #2. As far as I can figure that was entirely handwaved. I really don't remember any actual explanation for it. Other than that, this was a fun little read that moved the overall series arc toward the final book.
Book #158 - Hale (Circe's Recruits #4) by Marie Harte - I adored this book. Maybe not quite as much as Zack and Ace's story but there was still plenty of adoration. I liked how the storyline was tied up and the introduction of the SEAL team (heroes of the Dawn Endeavor series). I thought McKinley was a growly, sexy, scary character and somehow his relationship with Hale and Paige worked perfectly for me. As the overall arc winds down, the Recruits are desperately trying to find out who's running Pearson Labs while Hale is hunting down Paige, the daughter of the mad scientist who helped create the Circs, to keep her safe. As if finding his mate isn't enough, Hale also realizes that he's drawn to McKinley, the Circ who helped them from within Pearson Labs. I really enjoyed the entire series and was happy with the way things progressed from book to book and the way the final resolution played out. Now I'm anxious to get started on the Dawn Endeavor series.
Writing now? Okay.
( 2 comments — Leave a comment )
i was just thinking, i was only a little bit older than cole (i was almost 12) when i was in charge of my little brothers while my mom and stepdad worked. and taylor was just an infant at the time. that would be like cole watching ryan full time, plus a four year old. weird.
I canNOT imagine leaving Cole in charge of anyone. You were obviously an exceptional child.


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|Floating Treatment Wetland planted with bulrushes
My Aunt Shelagh and I have been playing with the idea for years. Last summer we made several models for testing and this winter we acquired several rotten wharfs from around the lake so we could start planting.
|The first Floating Treatment Wetland I built in August 2013. It was a mesh of willow branches that rooted into the lake.
Our first constructed wetland was a happy accident. It was an old styrofoam box that floated a wharf. Someone had filled it with willow cuttings and let it free on the lake. We moored it up, not thinking much at the time and suddenly in spring it was sprouting roots and shoots.
|First constructed Floating Wetland at sunset. We put solar lights on it so it would not be a hazard floating around.
|Prototype Floating Treatment Wetland with potting soil.
We rearranged the willow and planted some bulrushes, mosses and ferns on to. It was a bit unstable; the dogs would step on it and both would flip into the lake and I would be diving for the plants and moss.
When we rebuilt our wharf we had four boxes filled with syrofoam left over. I quickly planted two with ferns, moss, grass and wildflowers. As an experiment I used potting soil on one of them and a rope to wick water up. On the other one I only laid down moss and planted into the styrofoam.
This was in early October 2013 so winter was coming. Luckily our local nursery, Dinter Nursery, had poly hoop houses that were the exact same width as the boxes. I used electrical staples to secure the metal rods to the wood so nothing would blow away in the winter storms.
|Some days it was tropical inside the hoop house.
The hoop houses kept everything warm over the winter, including one major snowfall. The sheltered environment allowed some wildflowers to bloom in January! They died back during the spring snowfall but it was impressive.
The gardens thrived over winter and I took off the hoop houses in April. The vegetation was lush and had a lot of species. The roots from the flowers are floating in the lake. The roots provide a home for bacteria that break down toxins in the water and consume excess nutrients.
|Root system taking up nutrients and water from the lake.
|Flowers sprouting out of moss.
In May 2014 we took a Permaculture Design Course at OUR Ecovillage and one of our instructors, Javan Kerby Bernakevitch issued the class a challenge to create a Permaculture project in no more than 10 days, with no more than 10 hours of work, and less than $100. We did ours in 8 hours with lots of cider breaks. We got some gas to go out to the ditch and dig bullrushes but besides that we used materials we had on hand. Another cost that could be factored in is $8.98 plus taxes on a roll of garden felt. I bought it for another project but used it for this instead so it was brand new.
We covered the styrofoam and boards with the felt. The previous owner of the wharf had already removed the deck so we had to balance along 2x4 boards or fall in the lake. After we stapled the felt in place we used a utility knife to cut out slits for our plants.Shelagh took a length of PVC pipe and a small sledge hammer to punch holes through the styrofoam. The pipe worked really well and contained nearly all the bits of foam inside the pipe.
|Shelagh makes holes in the styrofoam
|Various sized holes in the styrofoam
|Planting the first bulrushes.Note the myceliated cardboard on the mulch.
After about an hour of leisurely work we had planted the whole thing and mulched it as well. Our mulch had been sitting for a year and was completely infiltrated with mycelium. Hopefully we will have a strong mycelial mat form in the mulch. Mycelium are excellent water cleaners. The saprophytic fungi can break down hydrocarbons like motor oil which would be wonderful when all the power boats come out to play in summer time.


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Professional Profile
Melisa C. Zwilling is a shareholder with Carr Allison and is a resident in its Birmingham, Alabama office. Her practice focuses exclusively on Medicare Compliance and she chairs the firm's Medicare practice group. She has been serving clients in this area for over 18 years and is considered a national authority on Medicare Secondary Payer issues. Melisa has published numerous articles in major journals nationwide and has been a guest speaker at countless seminars and conferences across the country. She is admitted to practice before the United States Supreme Court and has submitted a brief to the Court concerning Medicare Secondary Payer issues.
Melisa earned a rating of AV, the highest available, with the national lawyer ranking service of Martindale-Hubbell. Additionally, in 2014 and again in 2015, she was selected as one of Fortune Magazine's Women Leaders in the Law. In 2013, she was named a Top Rated Lawyer in Insurance Law. She has also been recognized by Alabama Super Lawyers® and as one of Birmingham's Top Rated Lawyers. In 2008, she was invited to Time Magazine's list of Most Influential Women in America and in 2007, to Fortune's List of Premier Lawyers.
Melisa received two Bachelor of Science degrees, magna cum laude, from Athens State University and her law degree from the University of Alabama School of Law, cum laude.
Having been raised in the country, Melisa spends most of her free time outdoors with her 13-year-old daughter and 8-year-old son. She can also be found on the back of one of her favorite horses or fishing as often as possible.
Although never shying from a challenge, Melisa freely admits that she is unable to swim, whistle or, unfortunately, cook. What she lacks in those areas, though, she makes up for with an astounding ability to organize things, as well as people, and "get things done."
After having a stroke at the age of 35, Melisa has spent the past several years as a volunteer and advocate for the American Heart and Stroke Associations. She tries to live every day to the fullest and takes advantage of opportunities to make a positive difference in the lives of others.
Visit the Medicare Compliance website
Visit the Medicare Compliance blog


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The industries we serve are industries we know
Financial Services
Medical Devices
Life Sciences
Real Estate
Happy Clients in their own words
Solugenix takes the time to listen to our needs and then delivers exactly what we need. Orion Pacific highly recommends Solugenix for all your IT needs.
I am thoroughly impressed with the Solugenix team. The combination of sound process design, capable employees and rigorous oversight truly set them apart.
Solugenix is a great company to work with. They are quick to respond, knowledgeable and provide excellent service. Their personnel go out of their way to be helpful and have made our company more efficient. We are lucky to have them!
Get the Right Professionals Now
Our strategic IT staffing solutions bring a deep and wide understanding of these industries. As such, we can provide IT professionals who speak the language of your industry as well as IT so that they can support your business goals.
To learn how Solugenix can help strengthen your business as your long-term IT staffing partner, please give us a call today at 1-866-749-7658, or contact us online for more information.


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Bonding is a popular method to enhance the aesthetics of your smile. Bonding can be used to correct cracks or gaps in teeth, as a filling after a cavity has been removed, or to cover up stains or discolored teeth.
A composite resin is used on the affected tooth or teeth. It is molded and sculpted over an adhesive gel that is placed on the tooth. After the resin has been applied an ultraviolet light is used to harden the resin, which is then polished to give you a fresh, new smile.
Bonding is an obvious improvement over unsightly silver amalgam fillings. With the advancements in dental technology, bonding usually lasts for over 10 years. It is a safe, affordable, and attractive solution.
If you have crooked teeth that you've always wanted to straighten out for a more attractive, youthful smile, you have multiple choices. Whether traditional metal or enamel-colored ceramic braces that are affixed to the teeth or removable clear braces (such as Invisalign), this is the most conservative way to get your smile in line.
Lumineers are very thinly shaped porcelain that is bonded to your tooth. The fact that they are very thin allows us to minimize the preparation of the tooth to create beautiful transformations of your teeth.
Due to the fact that there is minimal preparation, there is often little to no discomfort in this procedure. The procedure often requires 1-2 visits.
Give us a call today to see if Lumineers will work for your smile!
Veneers are an excellent way to redesign and reshape your entire mouth. Veneers are a very thin ceramic shell that covers your existing tooth structure. In placing these new veneers on your teeth, you are able to fix the look of issues such as staining, cracked teeth, spaces, misaligned teeth, chipped teeth and many other issues.
In terms of the process to place veneers, it is a process that takes approximately two to three visits. In the first visit, we will discuss the new shape and look of what we are going to accomplish by placing the veneers. Once we have put a plan in place, we will carefully prepare the teeth for the placement of the new veneers. As we complete this piece of the process, the next step is to take an impression of the newly prepared teeth. This impression will be sent to our laboratory for the final product to be fabricated. We only utilize the highest standard laboratory for fabrication of all of our work. This process at the laboratory takes between 10-14 days. While the new veneers are being fabricated at the lab, we will provide you with a beautiful temporary solution to wear for this time.
As the veneers are finalized, we will make every effort to make sure that your new veneers will feel just like your natural teeth. We will bond them into place ensuring that you can enjoy your new smile for a very long time!
Keeping our teeth their whitest is a lot harder than it sounds. With all the coffee, wine, smoking and other foods that have the ability to stain our teeth on a daily basis, even proper maintenance sometimes leaves them a little lackluster.
Teeth whitening is an excellent way to restore the natural color of your teeth or even make them whiter than your natural color if you would like.
To accomplish the whitening of your teeth, we utilize a tray whitening system. This tray whitening system is completed in 2 steps. The first step is to make an impression of your teeth. With this impression, we'll craft you custom whitening trays that you can use over and over. Finally, you will take the whitening gel and put it in the gel for a short period of time over a period of a few days. This often results in a whiter smile of 4 – 8 shades!
Please contact us today to see if you are a candidate for this type of whitening.
To accomplish the whitening of your teeth, we utilize a product called ZOOM!. ZOOM! is a chairside whitening procedure that uses a safe whitening gel and a low intensity light to activate the gel. This combination allows us to provide you with teeth that can be 6-10 shades whiter in about an hour!
Service areas include: Greenwood, Indianapolis, Martinsville, New Whiteland, Beech Grove, Southport, Bargersville & Franklin, Indiana.
Dr. Walton is worth the short drive and has many patients from Mooresville, Carmel, Westfield, Plainfield, Zionsville, Center Valley & Speedway, Indiana.


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Lisa & Tom's Yeppoon wedding
Oh how I love a back yard wedding! Lisa and Tom's ceremony was in the back yard of Tom's family home in Yeppoon. Loved ones gathered, champagne flowed and vows were excitedly exchanged. They celebrated being just married not with a kiss, but by champagne sabrage. With two swords they kicked off the party in serious style. Then they kissed.
Lisa and Tom's wedding is a personal fave of mine. I can't help it, the whole day was packed full of the most genuine, warm and fun loving people. Plus one gorgeous cat.
Here is their story...


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St. Luke's Community House creates a community where children, families, and seniors from different backgrounds can easily access the resources needed to live fulfilling lives.
Deborah Wallace's team of Community Association Managers volunteered on December 7th, helping families shop for gifts at their annual Christmas House Toy Store. This was a fun opportunity to give back to the community and grow as a team.
For decades, the St. Luke's Christmas programs helped deserving families and seniors have presents under the tree. Instead of accepting free gifts someone else chose for their children, parents can now purchase new toys for a very small amount they can afford.


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Cholesterol synthesis occurs in the ER (endoplasmic reticulum), where most of the cholesterogenic machinery resides. As membrane-bound proteins, their topology is difficult to determine, and thus their structures are largely unknown. To help resolve this, we focused on the final enzyme in cholesterol synthesis, DHCR24 (3β-hydroxysterol Δ24-reductase). Prediction programmes and previous studies have shown conflicting results regarding which regions of DHCR24 are associated with the membrane, although there was general agreement that this was limited to only the N-terminal portion. Here, we present biochemical evidence that in fact the majority of the enzyme is associated with the ER membrane. This has important consequences for the many functions attributed to DHCR24. In particular, those that suggest DHCR24 alters its localization within the cell should be reassessed in light of this new information. Moreover, we propose that the expanding database of post-translational modifications will be a valuable resource for mapping the topology of membrane-associated proteins, such as DHCR24, that is, flagging cytosolic residues accessible to modifying enzymes such as kinases and ubiquitin ligases.
- cholesterol
- membrane
- peduncle
- topology
Cholesterol synthesis enzymes are typically localized to the ER (endoplasmic reticulum), and as membrane-associated proteins, little has been done to determine their exact topology and structure. We are particularly interested in DHCR24 (3β-hydroxysterol Δ24-reductase), the final enzyme in cholesterol synthesis, as it is a multifunctional protein, which is intricately regulated (reviewed in [1]). Initial studies characterizing DHCR24 by Greeve et al. [2], and subsequently confirmed by Wu et al. [3], found that it was targeted to the ER, and to a lesser degree, to the Golgi.
Since the discovery of DHCR24, numerous binding partners and cofactors have been identified and characterized. DHCR24 contains a highly conserved FAD (flavin adenine dinucleotide)-binding domain [4–6], and reduction of desmosterol to cholesterol is dependent on FAD [7], suggesting functionality of the conserved domain. In addition, DHCR24 contains conserved binding domains for both p53 and Mdm2, required for mediating cellular responses to oncogenic and oxidative stress [3]. DHCR24 is reported to be proteolytically cleaved during apoptosis, at two highly conserved caspase recognition motifs located in the FAD- and p53-binding domains, presumably destroying them, producing a soluble, 40 kDa peptide [2]. How DHCR24 interacts with the membrane, however, is less well known.
Using simulated DHCR24 membrane models with and without substrate and cofactors, Pedretti et al. [5] predicted DHCR24 as a monotopic membrane protein; with the N-terminus partially embedded in the membrane as a stem or 'peduncle' (meaning 'little foot'), rather than traversing the membrane bilayer (bitopic). This 'peduncle' firmly anchors the protein to the membrane, with the C-terminus protruding into the cytoplasm, allowing access to substrates and cofactors [5]. The DHCR24 homologue, DWF1 in Arabidopsis thaliana, has a similar predicted structure, based on hydropathy predictions: strong membrane association and a cytoplasmic C-terminus [8].
However, Lu et al. [9] published the experimental work in support of a bitopic, single TMD (transmembrane domain) model of DHCR24: a lumenal N-terminus, followed by a single TMD (also at the N-terminus), and cytoplasmic C-terminus. The N-terminus was essential for DHCR24 membrane association, as deletion of this region (1–58, Δ58 DHCR24) translocated DHCR24 to the cytoplasm. The orientation of the N- and C-termini were deduced from protease protection assays of fluorescent fusion constructs; an N-terminal Ds-Red fluorescent fusion protein was preserved, indicating that it was lumenal, whereas a C-terminal green fluorescent protein was degraded by trypsin, indicating cytoplasmic localization. By contrast, Pedretti et al.'s 'peduncle' model [5] predicts that the N-terminus is buried in the membrane, and the C-terminus is cytoplasmic. However, fusion of the large (~28 kDa), soluble, Ds-Red fluorescent protein to the N-terminus could feasibly draw the membrane-embedded N-terminus into the lumen, that is, interfere with the native membrane topology of DHCR24. Therefore we aimed to determine the validity of the two different published models [5,9], investigating how DHCR24 interacts with the ER membrane, and its membrane topology.
CHO (Chinese-hamster ovary)-7 cells, the Insig-myc plasmid and the antibody against the trypsin-protected SREBP-cleavage activating protein (Scap, IgG-R139) fragment were generous gifts from Drs Michael S. Brown and Joseph L. Goldstein (UT Southwestern Medical Center, Dallas, TX, USA). IgG-R139 is a rabbit polyclonal antibody against hamster Scap (residues 54–277 and 540–707 [10]). Trypsin was from Sigma-Aldrich. Lipofectamine LTX Reagent was from Life Technologies. Plasmids expressing DHCR24 with a V5 epitope tag at the N- or C-terminus were created in pcDNA3.1 (Life Technologies). Truncations of the DHCR24-V5 plasmid were made using PIPE (polymerase incomplete primer extension) [11] or by two stage mutagenesis [12] for Δ56 DHCR24-V5.
Cell culture and transfection
CHO-7 cells were cultured in 5% (v/v) LPDS (lipoprotein-deficient serum)/Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12). Cells were transfected using Lipofectamine LTX Reagent according to the manufacturer's instructions.
Cell fractionation
The cell fractionation protocol was adapted from Feramisco et al. [13]. Cells were washed and scraped into PBS on ice and centrifuged at 1000 g. The cellular pellet was resuspended in 400 μl Buffer A [10 mM Hepes–KOH (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 5 mM sodium EDTA, 5 mM sodium EGTA and 250 mM sucrose] and passed through an 18 gauge needle 50 times. Protein concentration of cell lysate was determined using the BCA assay. Cell lysate was centrifuged at 1000 g for 5 min at 4°C. The 1000 g pellet was resuspended in 100 μl Buffer B [20 mM Hepes–KOH (pH 7.6), 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 5 mM sodium EDTA, 5 mM sodium EGTA], and incubated at 4°C for 1 h with end over end mixing, and centrifuged at 100000 g for 30 min at 4°C. The supernatant from this centrifugation was collected and designated the nuclear fraction.
The supernatant from the 1000 g centrifugation was centrifuged at 100000 g for 30 min at 4°C. The supernatant was collected and designated the cytosolic fraction. The pellet was resuspended in 100 μl Buffer C [10 mM Tris–HC1 (pH 7.4), 100 mM NaC1, 1% (w/v) SDS], and designated the membrane fraction.
Membrane isolation
To prepare membranes for differential solubilization and protease protection assays, cells were washed and scraped in PBS and centrifuged at 1000 g for 5 min at 4°C. The cellular pellet was resuspended in Buffer A or D (Buffer A containing 100 mM NaCl; as indicated in figure legends), passed through an 18 gauge needle 50 times, and the cell lysate was centrifuged at 1000 g for 5 min at 4°C. The supernatant was then centrifuged at 20000 g for 15 min at 4°C, and the resulting membrane pellet was resuspended in 65 μl Buffer A or D. Membrane protein concentration was determined using the BCA assay.
Differential solubilization
To determine the strength of the protein–membrane interaction, equivalent amounts of membrane were treated with 200 μl 1% SDS, Buffer A, 0.1 M Na2CO3 (pH 11.5), or 1 M NaCl, and incubated at 4°C for 30 min with end over end mixing. Treated membranes were centrifuged at 100000 g for 30 min at 4°C; the supernatant was designated the soluble cytoplasmic fraction (C), and the membrane pellet was resuspended in 200 μl Buffer D and designated the insoluble membrane fraction (M).
Protease protection assay
To determine the membrane orientation of the N- and C-termini, equivalent amounts of membrane were treated with trypsin as described [13]. Briefly, membranes were treated with the indicated amount of trypsin in the presence or absence of Triton X-100 in Buffer A, for 30 min at 30°C. Reactions were stopped by the addition of loading buffer and heat inactivation at 95°C for 10 min.
Immunofluorescence microscopy
To determine intracellular localization, cells grown on coverslips were cotransfected with a DHCR24 expression construct and an ER marker plasmid (pDsRed-ER, Clontech). Cells were then fixed with 3% (v/v) formaldehyde/PBS for 10 min. Cells were rinsed with PBS (three times for 5 min) and then permeabilized with 0.3% (v/v) Triton X-100/PBS for 5 min. Cells were washed with PBS, then incubated with 10% (v/v) FCS/PBS for 1 h. Cells were then incubated with 1:2000 V5 antibody in 10% FCS/PBS including 0.1% (w/v) saponin for 16 h at 4°C. After washing with 10% FCS/PBS (three times for 5 min), cells were incubated with 1:1,000 Alexa Fluor-488-conjugated secondary antibody (Molecular Probes) in 10% FCS/PBS for 1 h. Cells were washed with 10% FCS/PBS (three times for 5 min) and then mounted on glass slides with ProLong Gold AntiFade Reagent with DAPI (4′,6-diamidino-2-phenylindole; Life Technologies). Images were obtained using a Nikon C1 confocal microscope with laser excitation at 408 nm (DAPI), 488 nm (DHCR24-V5) and 561 nm (ER marker).
Bioinformatics tools
For in silico analysis, the complete human amino acid sequence of DHCR24 (Q15392) was used. An SP (signal peptide) was predicted using SignalP v4.1 [14] and a hydrophobicity profile was returned by ProtScale [15]. TMDs and membrane topology were predicted using TMHMM (TM hidden Markov models) 2.0 [16], TOPCONS [16] and ΔG predictor server v1.0 [17]. Myristoylator [18], NMT [19], big-PI predictor [20] PrePS [21], CSS-Palm 2.0 [22] were used to predict PTMs (post-translational modifications) involved in membrane attachment. To model the membrane topology, the LaTeX package TeXtopo [23] was employed, with a minor modification to the source code to enable a 'half-loop' to extend beyond 14 amino acids.
DHCR24 has an extremely hydrophobic N-terminus, suggesting a candidate region for TMDs
To investigate the topology of DHCR24, we first used an in silico approach. TM (transmembrane) proteins are hydrophobic by nature, and can therefore be predicted based on this characteristic. The human DHCR24 sequence from UniProt (Q15392) was analysed for putative TMDs based on hydrophobicity by the method of Kyte and Doolittle (Figure 1A) [15]. Two potential TMDs were identified at peak regions of hydrophobicity at the extreme N-terminus of DHCR24 (A, B; Figure 1A), with scores above the recommended threshold by Kyte and Doolittle (dotted red line, 1.6) [15]. As DHCR24 is not an extremely hydrophobic protein overall, having a GRAVY (grand average of hydropathicity) score of −0.061 (blue line), other hydrophobic peaks are discernible above this value, but do not reach the Kyte and Doolittle threshold of 1.6.
A more specific prediction of regions that lie within the ER membrane is the free energy calculation for membrane insertion by means of the Sec61 translocon (ΔGmi): negative ΔGmi values are indicative of potential TMDs of strong hydrophobicity and sufficient length; positive ΔGmi values are less likely to be TMDs [24]. Both potential TMDs identified by the method of Kyte and Doolittle have a negative ΔGmi, further indicating them as putative TMDs (A, B; Figure 1B). Three additional regions were identified as putative TMDs based on their ΔGmi values (c, d, e; Figures 1A and 1B); however, their ΔGmi values were positive, and these regions did not reach the threshold for detection of TMDs in the hydropathy plot. Therefore based on their hydrophobicity and their ΔGmi values, they are less likely to form TMDs.
DHCR24 is not predicted to associate with the membrane via post-translational modifications
Hydrophobic PTMs (post-translational modifications) can facilitate membrane attachment. Common groups include the irreversible attachment of various lipid groups, such as prenyl, myristoyl or GPI (glycophosphatidylinositol) groups or the reversible attachment of a palmitoyl group. The prediction programs Myristoylator [18], NMT [19], big-PI predictor [20], PrePS [21] and CSS-Palm 2.0 [22] predict that DHCR24 is unlikely to associate with the ER membrane via PTMs; therefore, these means of membrane attachment were not considered for DHCR24.
Predicted membrane topology of DHCR24 consists of N-terminal TMDs
For advanced DHCR24 TMD prediction, TOPCONS and TMHMM membrane topology prediction programs were employed. Both of these programs use HMM and/or neural network algorithms to integrate sequence information, and MSAs (multiple sequence alignments). TOPCONS uses multiple TMD prediction programs: SCAMPI-seq and SCAMPI-msa [25], PRODIV and PRO [26] and OCTOPUS [27]. The TOPCONS global prediction (TOPCONS consensus) [25] is a consensus of these five predictions, as well as the ΔGmi [24] and ZPRED [28] algorithms. Both regions identified by their hydrophobicity and ΔGmi as putative TMDs (A, B; Figure 1A) and the three regions with positive ΔGmi values (c, d, e; Figure 1A) were predicted by at least one of the TOPCONS prediction algorithms (Figure 1C); however, only the two N-terminal hydrophobic regions (A, B) were uniformly predicted by all five of the individual algorithms, and the global TOPCONS prediction (TOPCONS consensus; Figure 1C). Furthermore, the self-assessed reliability of the TOPCONS consensus is lowest in the region of the three other putative TMDs (c, d, e).
The TMHMM prediction [16], which also includes information on evolutionary conservation from MSAs, agreed with the TOPCONS consensus, predicting two N-terminal TMDs with high probability (Figure 1D). However, TOPCONS predicted a cytosolic C-terminus, whereas TMHMM predicted it to be lumenal.
A secretory SP is predicted for DHCR24
DHCR24 contains a putative TMD at its extreme N-terminus (A; Figure 1), which is usually the location of a secretory sequence. Signal sequences allow translocation of a protein to, or across, the ER membrane, which is cleaved for targeting beyond the ER membrane (SP), or retained within the membrane for ER membrane localization [SA (signal anchor)] [29]. Both SPs and SAs contain a stretch of hydrophobic residues, and therefore can be incorrectly predicted as TMDs by many prediction algorithms [30]. Most TMD prediction programs, such as TMHMM cannot discriminate between N-terminal TMDs and SPs and SAs [30]. Prediction algorithms, such as SignalP, use a neural network to distinguish N-terminal TMDs from SPs based on a combined scoring system for each position at the N-terminus [31]. The most N-terminal putative TMD for DHCR24 (A; Figure 1A) was predicted to be an SP (Supplementary Figure S1; available at http://www.bioscirep.org/bsr/034/bsr034e098add.htm), with the cleavage site at 22–23 based on all scoring systems: predicted cleavage site (C-score), SP length (S-score), and the combined cleavage site score (Y-score; a combination of the C-score and the slope of the S-score), which is a better cleavage site prediction than the raw C-score alone.
To test whether the putative SP is required for ER membrane localization, truncated DHCR24 (Δ23 DHCR24) was transfected into CHO-7 cells, and the cellular localization examined. Through cell fractionation, Δ23 DHCR24 was smaller than WT DHCR24 and, like WT DHCR24, Δ23 DHCR24 localized primarily to the membrane fraction (results not shown). This demonstrates that the putative SP is not cleaved off in WT DHCR24, and is not necessary for membrane retention.
The discounted SP and TMD lie in one of the least conserved regions of DHCR24. The C-terminal region, however, is highly conserved and contains multiple protein-binding/recognition sites for FAD [4–6], caspase 3 [2], p53 and Mdm2 [3]. The three predicted TMDs that did not have negative ΔGmi values all reside close to functional domains of DHCR24 (136–156, FAD; 210–230, Mdm2; 349–369, p53), that by definition should be cytosolic. Furthermore, a single TMD model is fitting, as it would allow the C-terminus to reside in the cytoplasm, making the multiple binding sites in DHCR24 accessible to soluble-binding partners.
Membrane orientation of the N and C termini of DHCR24
To determine the localization of the N- and C-termini of DHCR24 with respect to the ER membrane, CHO-7 cells were transfected with DHCR24 with a V5 epitope tag at either the N-terminus (V5-DHCR24) or C-terminus (DHCR24-V5), and co-transfected with Insig, an integral membrane protein with a myc epitope on the cytoplasmic C-terminus (Insig-myc) [13] (Figure 2A). Intact ER membrane vesicles were isolated and treated with trypsin in the presence or absence of Triton X-100, subjected to SDS–PAGE and immunoblotted for V5 (Figure 2A). As controls, samples were also immunoblotted with myc for Insig, and R139 for Scap, which recognizes a lumenal loop (residues 540–707) in hamster Scap [32] (Figure 2A). Therefore when trypsin was added to microsomes, the myc epitope on Insig was digested, and the R139 epitope of Scap remained intact, as it is located within the lumen (Figure 2A, lanes 1–4). Similar to Insig, DHCR24-V5 was digested. However, V5-DHCR24 remained stable (Figure 2A, lanes 1–4). When Triton X-100 was added to partially solubilize the membrane, allowing trypsin access to lumenal peptides, the R139 epitope of Scap was digested. However, V5-DHCR24 remained relatively stable (Figure 2A, lanes 5–8), indicating that the N-terminus was protected from trypsin digestion by the membrane.
Although there was no obvious change in the size of the V5-DHCR24 band with trypsin and Triton X-100 treatment, the band intensity slightly decreased (Figure 2A, lanes 5–8), and a small fragment was discernible with increasing amounts of Triton X-100 (~11 kDa, Figure 2B, lanes 6–9). This smaller band was not visible in non-treated conditions (lanes 1–4) or when trypsin was inactivated with SDS (lane 10). Furthermore, the band appeared with or without Triton X-100 treatment, indicating a partially accessible cytoplasmic loop following a membrane embedded N-terminus. The calculated molecular weight of this digested fragment indicates that the loop is located between residues 70–100, which contains six possible arginines/lysines that could be cleaved by trypsin (dotted red line; Figure 2B). Altogether, these data are indicative of a cytoplasmic C-terminus and a non-accessible N-terminus, which is either embedded within the membrane, or lumenal but inaccessible, due to strong interactions with the ER membrane (Figure 2).
The putative TMD is not essential for DHCR24 membrane association
To determine if the hydrophobic N-terminus containing the putative TMD of DHCR24 is required for ER membrane attachment, truncated DHCR24 (Δ56) was transfected into CHO-7 cells, and the cellular localization examined. DHCR24-V5, like Insig-myc, localized only to membranes (Figure 3A). The truncation lacking the putative TMD, Δ56 DHCR24-V5, also localized specifically to membranes (Figure 3A). Therefore contrary to the predictions in Figure 1, these very hydrophobic regions are not necessary for DHCR24 membrane association.
DHCR24 associates strongly with membranes
To determine if DHCR24 is an integral membrane protein, differential solubilization was performed on membranes to determine the manner in which DHCR24 associates with it. The ability of buffers (detergent, high pH, high salt) to dissociate DHCR24 from the membrane (M) into the cytoplasm (C) indicates the strength of its association with the membrane (ionic or hydrophobic).
When exposed to aqueous buffer, DHCR24 was located in the membrane fraction, but was released into the cytoplasmic fraction upon treatment with 1% (w/v) SDS (Figure 3B). Treatment with 0.1 M Na2CO3 or 1 M NaCl, reagents known to disrupt peripheral associations by altering pH and electrostatic interactions, respectively, were not able to solubilize DHCR24, and it remained in the membrane fraction (Figure 3B). For all treatments, Insig-1 displayed similar results, indicating DHCR24 is associated with membranes, and that it is an integral membrane protein (Figure 3B). Similarly, Δ56 DHCR24-V5 behaved like an integral membrane protein (Figure 3B). Although Δ56 DHCR24-V5 remained in the membrane fraction after treatment with 0.1 M Na2CO3, the presence of a weaker signal in the cytoplasmic fraction indicated that Δ56 might be solubilized with this treatment. However, performing the treatment and centrifugation process a second time showed that Δ56 DHCR24-V5 remained membrane associated (Supplementary Figure S2; available at http://www.bioscirep.org/bsr/034/bsr034e098add.htm), suggesting that regions beyond the first 56 residues are integral for strong membrane association.
Other candidate TMDs are not essential for DHCR24 membrane association
Having established that the most likely TMDs (A and B, Figure 1) was not essential for membrane localization, we next examined the other predicted TMDs. Based on the ΔGmi values, and the TMHMM probability score (Figure 1), the two next best TMD candidates were examined (c, 136–156; d, 210–230). Truncated versions of DHCR24 (Δ160, Δ240; Figure 4A) were transfected into CHO-7 cells, and the cellular localization examined. Similar to WT DHCR24–V5, both truncations were found primarily in the membrane fraction (Figure 4B), demonstrating that the first 240 residues are also not necessary for membrane attachment. The appearance of secondary bands (~22 and ~32 kDa) in the Δ240 DHCR24 membrane fraction is indicative of proteolytic fragments, which are also associated with membranes, and would encompass the untested putative TMD (e). This further suggests that there are more membrane-binding site(s) closer to the C-terminus. To confirm that the truncations are not mislocalized or aggregating, immunofluorescence microscopy was performed. WT and Δ240 DHCR24 both localized predominantly to the ER (Figure 5), indicating that DHCR24 remains ER membrane associated despite N-terminal deletions.
To further elucidate the membrane topology of DHCR24, we predicted how it interacts with the ER membrane through the use of numerous membrane insertion algorithms, as well as considering the current literature on DHCR24 topology. We then characterized this interaction using biochemical methods.
To create a DHCR24 topology model, we first characterized DHCR24 based on its hydropathy using the method of Kyte and Doolittle [15]. Two regions of peak hydrophobicity were identified as candidates for TMDs (Figure 1A), which were confirmed by numerous TMD prediction algorithms (ΔGmi; Figure 1B) and programs (TOPCONS and TMHMM; Figures 1C and 1D). DHCR24 was predicted to contain an SP (Supplementary Figure S1), which generally targets proteins to or across the ER membrane, and this was located at the first putative TMD. The efficacy of a DHCR24 SP was tested by the creation of a truncation construct, which did not contain the putative SP. This was smaller than its WT DHCR24 counterpart, and still associated with membranes. Secondly, the N-terminal V5 tagged DHCR24 construct (V5-DHCR24) was the same size as C-terminal DHCR24-V5 (V5-tagged DHCR24), indicating no cleavage of the N-terminus, which would be predicted for a true SP. These data show that the N-terminus is not cleaved, nor is it required for membrane targeting, demonstrating that DHCR24 does not contain an N-terminal SP. This is similar to cytochrome P450, which is also an integral ER membrane protein that does not require an N-terminal SP for ER targeting, but contains complex N- and C-terminal retention signals [33].
Upon deletion of the N-terminus (including the putative TMDs; Δ56), DHCR24 still associated with the membrane (Figure 3A), and could not be solubilized by various treatments (Figure 3B). Again, this is similar to cytochrome P450, which contains a hydrophobic N-terminus predicted to associate with the ER membrane, which retained strong membrane attachment after truncation experiments and differential solubilization [34,35]. Further N-terminal truncations in DHCR24 that deleted lower scoring TMD candidates also associated exclusively with the membrane (Figure 4). These data discount the findings of Lu et al. [9] that the N-terminus is essential for membrane attachment, and that deletion of the N-terminus (Δ58) results in altered cellular localization of DHCR24. In contrast, our findings demonstrate that the membrane association of DHCR24 is not reliant on TMDs at the N-terminus, but contains membrane associated region(s) beyond the hydrophobic N-terminus.
We also elucidated the membrane orientation of the N- and C-termini of DHCR24, for which the TMD prediction programs gave different results. Using protease protection assays, we determined that the C-terminus was cytoplasmic, due to its accessibility to trypsin. The N-terminus, however, was strongly protected, even when trypsin was accessible to the lumen by partial solubilization of the membrane (Figure 2A). This suggests that the N-terminus is 'hidden' from trypsin; either due to being embedded within the ER membrane, which would support (in part) Pedretti et al.'s 'peduncle' model [5], or due to peripheral interactions with the ER membrane. Both of these possible models disagree with the findings of Lu et al., that the N-terminus is lumenal, which is most likely due to the difference in epitopes used in the protease protection assay [9]. As mentioned, Lu et al. [9] used a large fusion protein (~28 kDa), whereas we used a small V5 epitope (~1 kDa). Testing the membrane association of a C-terminal truncation using a differential solubilization assay would determine the type of interaction the N-terminus has with the membrane (integral or peripheral). Finally, the identification of a digested N-terminal ~11 kDa fragment suggests that the membrane associated N-terminus is followed by a sterically hindered cytosolic loop at position ~70–100 (Figure 2B).
Overall, our findings demonstrate that DHCR24 has a strong affinity with the ER membrane, due to multiple membrane-associated regions, including beyond the N-terminus (Δ240; Figure 4). The extreme hydrophobic nature of the N-terminus suggests that it is also membrane associated, but this needs to be confirmed experimentally by C-terminal truncations. We hypothesize that there are multiple membrane associated regions throughout the protein, or re-entrant loops that associate with only one leaflet of the membrane. Re-entrant loops are typically rich in small residues such as glycines and alanines [36], and one of the lower scoring putative TMDs (d, Figure 1) is rich in these amino acids, and therefore a prime candidate for a re-entrant loop. Our proposed model may also contain classical TMD(s), as although this study ruled out the most likely putative TMD, one low-scoring putative TMD was untested (e, Figure 1). As this region is beyond the first 240 residues, it is likely to explain the strong membrane localization of Δ240 DHCR24 (Figure 4). Our data strongly suggest that relocation of DHCR24 to other cellular compartments, proposed to occur in response to stress signals, is highly unlikely [3]. This conclusion should be re-examined in light of our results.
Altogether, our findings are at odds with prediction algorithms and published topology models (Figure 6, [5,9]). We demonstrated that DHCR24 does not conform to either published topology model; being neither monotopic, like Pedretti et al. suggest, nor bitopic, as Lu et al. propose, but is polytopic, with multiple hydrophobic domains passing through and/or anchoring DHCR24 to the hydrophobic core of the lipid bilayer of the ER.
To create a hypothetical membrane topology model of DHCR24, we have also utilized in addition to our experimental findings our knowledge of known PTMs on DHCR24 [37]. Phosphorylation and ubiquitination sites must be accessible to cytosolic kinases and ubiquitin ligases respectively, and therefore we have used these data to refine our topology model of DHCR24 (Figure 7). To the best of our knowledge, we are the first to propose that the expanding databases of PTMs provide an unlikely but invaluable resource for membrane topology mapping, including for other atypical cholesterogenic membrane proteins [38]. After all, this is not the first example of a cholesterol biosynthetic enzyme that has an atypical membrane topology. Lanosterol synthase [also known as OSC (oxidosqualene cyclase)] is a monotopic ER membrane protein, containing a membrane associated region, which resides in one leaflet of the membrane, and therefore does not span the bilayer [39]. This hydrophobic catalytic domain resides in the membrane, which allows access to the hydrophobic substrate, oxidosqualene. As the substrate-binding domain of DHCR24 is as yet uncharacterized, and desmosterol is also hydrophobic, a membrane associated catalytic site is likely.
To further refine our membrane topology model of DHCR24 (Figure 7), additional experimentation is required to precisely define membrane, cytoplasmic, and lumenal regions, including whether the final putative membrane region (e) is bona fide. This commonly requires modification of the specific residues or introduction of epitopes and recognition sites. Cysteine derivitization and glycosylation site mapping have previously been successful in elucidating the membrane topology of membrane bound proteins, such as Insig [13], and these techniques could similarly be applied here.
Eser Zerenturk, Laura Sharpe and Andrew Brown designed the experiments. Eser Zerenturk and Laura Sharpe performed the experiments. Eser Zerenturk, Laura Sharpe and Andrew Brown analysed the data. Eser Zerenturk, Laura Sharpe and Andrew Brown wrote the paper.
AJB's laboratory is supported by grants from the National Health and Medical Research Council of Australia [grant number 1008081] and the National Heart Foundation of Australia [grant number G11S5757].
We thank Julian Stevenson for his involvement in the early stages of this project and Timothy Amos for his assistance with the topology model.
Abbreviations: CHO, Chinese-hamster ovary; DAPI 4′, 6-diamidino-2-phenylindole; DHCR24, 3β-hydroxysterol Δ24-reductase; ER, endoplasmic reticulum; FAD, flavin adenine dinucleotide; GRAVY, grand average of hydropathicity; HMM, hidden Markov model; MSA, multiple sequence alignment; PTM, post-translational modification; SA, signal anchor; SP, signal peptide; TM, transmembrane; TMD, transmembrane domain
- © 2014 The author(s)


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