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I'm ambivalent about heritage turkeys, which is to say I feel strongly and not so strongly about them all at once. Not so strongly because I've managed to convince my family to partake in the ridiculously complex (but enjoyable) ritual of brining-roasting-poaching-steaming the Thanksgiving turkey to get good flavor. That's not necessary with the heritage breeds; in fact, brining them is exactly what you shouldn't do because it masks the flavor. So now that I'm sourcing heritage breeds each year, I may be increasing the diversity of our farm (which I feel strongly about), but I'm also aiding in the disappearance of a family tradition — the ten-step turkey roast dinner.
Fortunately the turkey operation at Stone Barns accommodates both camps. Livestock manager Craig Haney raises two breeds: Broadbreasted Whites and Bourbon Reds.
While their rapid growth rate and large breasts make Broadbreasted Whites an attractive candidate for the farmer (and the white meat crowd), if you're thinking of flavor alone, or the preservation of rare breeds, Bourbon Reds are the natural choice. Unchanged by the homogenizing forces of the mass market, this heirloom breed has managed to maintain its genetic legacy of rich, dark meat. It's turkey that actually tastes like turkey, which is why I prefer the Reds in the end.
As far as the Thanksgiving table goes, though, I'm still undecided.
Turkey pot pie
- 2 pounds Yukon gold potatoes
- 1½ cups milk
- 3 tablespoons butter
- 6 cups shredded roast turkey meat
- 1 cup turkey gravy
- 2 tablespoons sherry wine vinegar
- 5 tablespoons herbs (such as parsley,
- chives, and tarragon), chopped
- 1 tablespoon shallots, chopped
- ½ cup fine bread crumbs
- Salt and pepper, to taste
1. Preheat oven to 375°F.
2. Boil potatoes in well-salted water until fork tender. Drain and peel when cool enough to handle.
3. Heat milk and butter until butter melts.
4. Pass potatoes through a food mill or ricer; gently fold in hot milk mixture until well incorporated. Season well with salt and pepper.
5. In a large bowl, mix together the turkey, sauce, vinegar, herbs and shallots; season well with salt and pepper.
6. Divide turkey among 12 ramekins; top with potato puree and smooth with a spatula.
7. Heat for 20 minutes.
8. Remove from oven and sprinkle with bread crumbs. Place under a broiler for about 1 minute, until the bread crumbs are golden brown. Serve with glazed carrots or other roasted vegetables for a hearty fall meal.
Story by Dan Barber. Dan Barber is the executive chef and co-owner of Blue Hill restaurant in new york City and blue hill at Stone barns, located within Stone barns Center for Food and agriculture, a pioneering farm and education facility in pocantico hills, new york (bluehillnyc.com). This article originally appeared in Plenty in November 2008. The story was added to MNN.com.


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Model in Focus: Toyota AvanzaBy marian on July 31st, 2015
Like the Toyota Innova in the Philippines, the Toyota Avanza is one of the best SUV's to have in the country. Not only is it a good choice for private car owners, but it's also an ideal vehicle for public transportations such as taxi drivers. So what makes people so attracted to this ride? Find out below!
Family Friendly
One of the most important tenets of Filipino culture is family and friendship. We spend quality time with them at malls, restaurants, parks, and out-of-town destinations. And when we do, it's usually with the entire family or gang—leaving even just one member behind is never the way to go. Thankfully, with the Avanza, you will never have to!
The seating space inside the Toyota Avanza is more than enough to fit in six people comfortably with more than enough leg space. And if you plan to either bring with or bring home a few things, then you don't have to worry, as there is more than enough cargo space, especially if you fold the rear seats.
Smooth and Efficient
While a smooth and comfortable riding experience doesn't necessarily mean fast, an unbelievably sluggish one is definitely not a pleasant one. We here at Toyota know that, so we equipped the Toyota Avanza with the DOHC 16-Valve EFI VVT-I engine. It's powerful, fast, and fuel-efficient, allowing you to get you and your family and friends anywhere you want to go.
Steady Roll
Nobody likes a rough and bumpy ride, especially when there are a lot of you sharing the same car. Mostly it's because of the bumpy road, but sometimes it's the car's fault, as it can have an inefficient suspension system. With Toyota Avanza, however, you won't have to experience the latter. In fact, it's very effective in providing both the driver and the passengers a smooth ride.
Outside Appeal
No matter how productive, fast, or fuel-efficient a car may be, you wouldn't drive it at all, let alone get your family and friends to ride with you, if it looks too bizarre, uncool, or out of style. Thankfully, the Toyota Avanza's exterior is none of those things.
The Toyota Avanza is an MPV that has rounded and sleek edges, making it visually smooth yet functionally aerodynamic. Gone are the days when SUV's look and feel uncool! And if you're not exactly a fan of the standard silver, you have five other colors to choose from: light blue metallic, champagne metallic, gray metallic, white, and dark steel mica. Now you don't have to worry about being exactly identical with other Avanzas on the road!
Safe and Sound
Last but definitely not least is safety, an important factor some car manufacturers and buyers neglect. You can rest assured that Toyota is not one of these blokes; in fact, their great dedication towards this aspect is shown in the Avanza's safety systems, which offers an extra shell of protection and airbags.
Cool, comfortable, and convenient—that's just some of the things the Toyota Avanza is. So what are you waiting for? Go to the nearest Toyota outlet now and get one so you, your family, and your friends can enjoy many memorable rides together!

subset: commoncrawl


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Wyckoffians find Relative Strength analysis to be very useful and illuminating. Relative Strength studies can be like having X-ray vision. With Relative Strength tools, one can see important hidden secrets about the health of a stock or industry group. Relative Strength analysis is simple and straightforward to apply and is one of the most useful tools in the Wyckoff toolkit.
(click on chart for active version)
Relative Strength gauges leadership, or the lack of it. My preferred method of market study is to drill down from Sector studies, to Industry Groups, to stocks. Always seeking the leading stock, in the leading industry group, in the leading sector. Evaluating weekly data puts us in synch with the major trends and their rotation in and out of favor. Once a price trend forms it tends to persist for a long time. This is also true of Relative Strength. Once a stock or an Industry Group emerges into a Relative Strength trend, it can go on for months and years. Big trends and how to trade those trends is our Wyckoffian edge.
During a healthy long term uptrend, price and Relative Strength will mostly be in gear with each other. They do this by making higher highs and higher lows in unison (in downtrends the opposite condition is seen). When price and RS fall out of synch the existing trend becomes suspect. In the above case study, note the US Aerospace Index ($DJUSAS) price pushes to a new high in early 2015 while RS lags and makes a lower high. This lower peak has us drawing a downtrend line. Here is where the x-ray vision comes in. If a healthy uptrend in price is accompanied by a higher peak in RS, we would consider them 'in gear up'. A lower high in RS while price pushes to a new high is an 'Upside Non-Confirmation (UNC). This condition gives us cause for concern that price will follow RS lower. Which it does. The veracity of the decline that follows the Upthrust in price is surprising unless compared to the Relative Strength which is issuing a large warning.
At the beginning of 2016, $DJUSAS and RS made an important low at the same time. Thereafter the price and RS began uptrending (note the rise above the downtrend line). Now price and RS are 'in gear up' and $DJUSAS is making a new high.
Our goal is to make positive returns year-in and year-out. And to have relative outperformance to our benchmarks (here we use the S&P 500 for Relative Strength comparison purposes as many institutions use the same). When both the price and the RS are in gear upward these goals are being met. If either condition is out of gear, this goal will most likely not be achieved.
(click on chart for active version)
The DJ US Commodity Chemicals Index ($DJUSCC) entered a multi-year Reaccumulation in 2014. Note how severe the break in the Relative Strength was after the Buying Climax (BCLX). Though the industry group recovered into 2015, we can see from the badly lagging RS that this condition was Distribution. At the Secondary Test (ST) in price another stiff decline begins. This deep decline is signaled and explained by the badly lagging RS which is well below the prior high and the long term moving average. A Downside Non-Confirmation (DNC) arrives at the Test of the Shakeout low. This is a subtle, but valuable, clue of a change of character in Commodity Chemicals. An uptrend is beginning where higher highs in price and RS are developing. Our view is that such an in gear uptrend will remain in force for potentially years to come.
In future posts we will dig deeper into this very useful concept. In the meantime practice drilling down from Sector, to Industry Group, to Stocks to find the best leadership themes. Stockcharts.com has created outstanding tools to leverage your analysis time finding these leadership themes.
All the Best,


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Category Archives for "Losernomics"
Charles Ramsey has become an overnight sensation on the internet. The reportedly lower-income black person with a vigorous, animated speaking style and quick with his own brand of witticisms has become an internet phenomenon and according to some, "that ain't right". Exactly what's wrong with this is a mystery to me. The Atlantic Wire is […]Continue reading
It's impossible that this wouldn't go viral across the internet, but some guy in New Hampshire lost his life savings ($2,900) at a carnival trying to win an Xbox Kinect. Henry Gribbohm, pictured here with a life-sized dreadlocked banana he did win, went to a carnival and after winning said banana, pressed his luck going […]Continue reading


text: "I had a 60 minute massage with Jo last Saturday and what a lovely treatment it was. Jo's approach is very warm and the treatment felt very individualised for my needs. She is a wonderful therapist and I can't recommend her enough. Jo has an innate ability to find the right level of chat, uses beautiful music and is so skilled at making the massage truly feel like time out of my extremely busy week. Thanks Jo!"


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Available on Kobo, "The Asset" (A Wounded Warrior Novel # 1) by Anna del Mar
#AromasAuthors #AnnadelMar @anna_del_mar
Anna del Mar's explosive, sexy debut novel in the Wounded Warrior series, perfect for fans of Lisa Marie Rice and Lora Leigh—the story of a woman desperate to escape her dangerous past and the navy SEAL who would lay down his life to save her
Ash Hunter knows what it is to run. A SEAL gravely injured in Afghanistan, he's gone AWOL from the military hospital. Physically and mentally scarred, he returns home to his grandmother's isolated cottage—and finds a beautiful, haunted stranger inside.
Like recognizes like.
Lia Stewart's in hiding from the cartel she barely escaped alive, holed up in this small Rocky Mountain town. Surviving, but only just. Helping the wounded warrior on her doorstep is the right thing to do…it's loving him that might get them both killed.
Soon, Ash realizes he's not the only one tormented by the past. Pushing the limits of his broken body, testing the boundaries of her shattered soul, he'll protect Lia until his last breath.
To add this book to your library simply click on, Save Preview 😉
Thank you!
Have a great day!


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Mr Scott Alberts
Scott Alberts has more than 25 years of industry experience including solid state lighting and semiconductors/embedded systems. His areas of expertise include strategic marketing and business development, along with finance, mergers and acquisitions, as well as Wall Street investor relations. Scott has held senior management positions and consulted with multinational corporations, start-ups and venture firms. As a Venture Partner, Scott focuses on Cleantech and Component investment opportunities and helps DCM portfolio companies with product strategies, business development and strategic alliances. His areas of interest include digital video, solid state lighting and energy management. Prior to joining DCM, Scott provided strategic consulting to companies such as Agilent, Broadcom, Fortinet and Sand Video. Previously, he was co-founder of WebPartner, a venture-backed start-up providing web performance management software. Earlier, Scott spent ten years with Oak Technology, joining the company as its first marketing manager and playing a key role in growing the business from a start-up to $250 million in revenue and a successful IPO and secondary. He created Oak's business development program, executing several acquisitions, including Pixel Magic, which firmly established Oak as a leader in the digital imaging market. Scott started his career at Hewlett Packard's Components Group (now Avago Technologies & Philips-Lumileds) where he led the LED automotive marketing efforts and later established a successful Japan market development program.
Venture capital firm.
Add a job
Add education


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school partnership programme -TWINNING of schools- Implementation guidelines-Rc.26Dt.27/9/17
The "Partnership between School / Twinning of Schools" Programme is aimed to link schools located in rural areas with Private, Aided or Government Schools in urban or semi-urban areas to enable interaction amongst the students and sharing of experiences.
"Well functioning Private or Government Schools in urban or semi-urban areas may be linked with schools located in rural areas for interaction and exchange of experiences. Under the exchange programme the students of each section from higher classes from rural schools may be brought to schools in urban areas for one week and vice-a-versa for greater exposure".
With reference to the letters dated 9th May 2016, 30th July 2015, and 12th January 2016 from MHRD (D.O No. 2-8/2015-EE.3) to SSA, Andhra Pradesh regarding designing the "Partnership between School / Twinning of Schools" programme.
The above said programme is designing for the Schools the State of Andhra Pradesh on pilot basis in all the Districts. The number of Schools, Teachers and Students will be decided based on the availability of facilities, infrastructure, teaching aids, etc. While implementing the programme, core teams has to be constituted at District level and State level. Also certain guidelines need to be framed for Students, Teachers, Headmasters, Complex School Headmasters, Parents and team's viz., administration, monitoring teams. The overall programme has to be documented for every quarterly by the concerned School team which will be further coordinated by District Authorities.
Targeted Age Group, Classes and Schools:
- Target age group: every child of 6 to 18 years age.
- Target classes: Children from classes I-XII
- Target Institutions: State Government and Local body schools, Kendriya Vidyalayas, Navodaya Vidyalayas and other Central Government and Private unaided schools.
List of Programmes: The following list of activities will be shortlist by the district level team with the support of shortlisted school headmasters and teachers.
- List of Sports
- Citizenship
- Art and Craft
- List of local places to visit
- List of local festivals
- List of Local art, craft and music
- List of life skills
- List out the local livelihoods (Agriculture, Industry or Services)
- List out Best practices of selected schools.
Proposed Team Structure: The core teams in various levels have to be constituted for efficient implementation of the programme. The teams are broadly categorized into 3 core teams (viz., Administration and Monitoring teams) at State, District level and local support team.
I. State level:
1. Administration: the SSA State level officers will provide budgetary support for the programme.
· Team: SSA State Officers
2. Monitoring: Monitoring team will monitor and coordinate the entire programme and collect the reports from all the Districts and consolidate the report. The detailed report will be submitted to the Commissioner of School Education and the State Project Director, Sarva Siksha Abhiyan.
· Team: SAMO, SSA.
II. District level:
1. Administration: the District level administration will administer the entire programme in their District. The administrative team will collect the reports from all the twinned Schools of their District and consolidate the reports including photos and data and send to the State Monitoring Team.
2. Monitoring: AMO will monitor the District level activity and MEO will monitor the Block/Mandla level activity.
· Team: SSA PO, AMO, MEO
III. Local Support level
1. Monitoring: This team is meant for monitoring the activity at Block/Mandal level and School level.
• Team: MEO, School Complex Headmaster, Headmaster
2. Small Group for Mentoring: The group is meant for mentoring the Block/Mandal and School level programme (Partnership of Schools).
· MEO, School Complex Headmaster, Mandal Resource Coordinator – MRC.
1. Students:
· Student must be in discipline in the class and while presenting the projects of other students at host school.
· Students are to bring all required materials to class / venue each day.
· Students are to remain in their seats unless given permission to leave the class room / workshop).
· After entering the room, students may not leave without permission.
· Students may not leave books and materials outside the classroom for any reason.
· Students are responsible for cleaning the area around their desks before leaving the room.
· In the event of a student's absence from school / went to home, while mid of the programme, parents must provide an explanation for the student's absence, below. (Valid for only up to two days of absence. Beyond that, an application duly signed by parent along with Medical Certificate and prescription is a must.).
· Students are expected to maintain proper decorum in the classroom.
· Students must adhere to the rules set forth by the instructor / teacher.
· The classroom should be a learning centered environment in which teachers and students are unhindered by disruptive behavior.
· Failure to comply with classroom rules, instructor orders or teacher's orders may result in dismissal from the class and/or the school.
2. Teachers:
· The classroom (host school) should be a learning centered environment in which teachers and students are unhindered by disruptive behavior.
· Teachers have the authority to manage their classrooms (Source schools) to ensure an environment conducive to learning.
· Teachers have to support the students and provide guidance, while preparing the projects and Science fairs.
· Teachers should take care of the students in host school.
· Teachers should update the activity information to the District core team and local support team timely.
· Teachers must share the student activity with their parents timely.
· Teachers should prepare the requirements viz., materials, budget, local logistics viz., accommodation, food, etc. for students, before commencing the partnership programme.
· Teachers should collect the information on local places of importance and make action plan.
· Teacher must prepare an activity wise report on daily basis and weekly basis and submit it to the Local Support Team and District Level Teams.
· Teachers must capture the photos covering all the activities of the entire programme and provide the information with data evidence, while performing field visits, student demonstrations, exhibitions etc. Also he/she must prepare student assessment report activity wise.
3. Parents:
· Parents permit the child to participate in the partnership school programme.
· Parents should interact their child and teacher for taking the programme review and status of their child performance.
· Parent Teacher Association (PTA) members must visit the host school and review the programme and provide their feedback.
4. Management: Host School Management / Headmaster / Instructor / Principal will make all arrangements in the School for students and teachers of other school including local stay, food, medical facilities, etc. Monitor the programme in the school and provide necessary infrastructure, support timely.
5. Administration and Logistics: The District Administration is responsible for smooth functioning of the partnership programme. For the purpose, the District officials should collect the schools information, then scrutiny the schools for piloting the programme. Further, they can issue the official orders to the respective schools by marking / copy to all concern Departments of the State and District. Apart from that, the District Project Office can coordinate the budget provided by the State Office for the programme and also provide necessary logistics support.
School selection criteria: The initiative needs 2 schools from rural and urban areas. While piloting the programme, we may need few schools from each District for pairing and bridge the activity in both schools under student exchange.
· Schools should have good infrastructure facilities viz., good classrooms, teaching learning materials, auditorium, play ground, computer lab with internet connection, digital classroom, science labs and good sanitation.
· Well qualified teachers and supporting staff.
· Campus should have greenery.
· Campus should be located at proper access of transport facilities.
· Campuses are preferred which are having student hostel facilities.
· The School having achievements in curricular, extra-curricular, other curricular activities and sports and games.
· The schools which are having innovative practices in teaching and learning.
Student's selection criteria:
- The student must be energetic, talented, brilliant and enthusiastic in any one of the themes provide in the draft.
- The student is willingness to stay out of his/her home or residence and school.
- Meritorious students will be selected for the programme in any one of the themes provide in the draft.
- Preference will be given for Socially, Economically poor and backward students in the school.
- Students those who are shine in Sports, Games, and Cultural & Literary activities in local / regional / state / national levels.
Assessment Criteria for Student outcomes: The assessment of the student will be done by the respective teacher, who is involving / leading the programme. In-charge Headmaster of the host and source school will fix the assessment format of students. Final assessment format should provide to the State project office SSA and district project office SSA, 2 weeks before of the school twinning pilot project.


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Elite track and field in-state recruit Anna Holdiman has received a release from her letter of intent to attend Iowa State and will now attend Florida State, she said Friday.
Elite track and field in-state recruit Anna Holdiman has received a release from her letter of intent to attend Iowa State and will now attend Florida State, she said Friday.
Holdiman�s decision stemmed from the coaching change at ISU that saw Corey Ihmels leave for Boise State and Martin Smith come in with an entirely new staff. A distance runner who would�ve arrived in Ames as a freshman this August, Holdiman had selected the Cyclones primarily because of her relationship with Ihmels and his staff.
�I chose Iowa State for coaching, and I just kind of wanted to go into a program where I knew what I was signing up for and knew what I was getting into,� Holdiman said. �The way I looked at it was you only have four years to make the most of where you want to go to school, and I just wanted to explore my options while they hired a new coach just because I had no idea the direction the program was going.�
Holdiman asked for and was granted her release by ISU athletic director Jamie Pollard in-mid June, she said. Ihmels announced on June 14 that he was heading west. At that point, Holdiman reopened her recruitment and recently decided to attend Florida State, she said.
Smith, Pollard and potential Cyclone teammates made clear they still wanted Holdiman at ISU and were �gracious� and �first class� throughout the process, she said, but she was unfamiliar with the new staff. Asked if there was anything ISU could�ve done to keep her once Ihmels left, Holdiman said, �No � they honestly couldn�t have done anything more.�
�It was hard to see them go,� Holdiman said of Ihmels and his staff. �But everyone moves on in different parts of their career.�
Holdiman, a Waverly Shell-Rock graduate, was arguably the top incoming freshman for the Cyclones. She took second in the women�s junior 3,000 meters at the U.S. Outdoor Track and Field Championships in June in Des Moines. She was also named the Gatorade runner of the year in Iowa for cross-country this past season after winning a Class 3A state title.
Holdiman was sold on Florida State after a visit there, she said.
�It wasn�t just an athletic choice,� Holdiman said. �As far as academics � I�m interested in the health profession, and there�s a couple majors I�m interested in that Iowa State didn�t offer. So it�s also a chance to further my education � I liked the girls on the team a lot and that was really important to me, and I really liked the coaches there.�


text: Melon with Basil-Lime Granita is a perfect summer dessert, light and refreshing. Blend a simple sugar syrup, basil, lime juice, and freeze, scraping every 30 minutes to get a slushy ice texture for the granita. Then serve it on top of canteloupe and honeydew macerated in a little bit of sugar. To go over the top, serve with a delicious, not-too-sweet cornmeal cookie. A definite hit dessert. Recipes from the dearly departed Gourmet magazine.


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Vietnam based carrier Vietjet Air announces its interline partnership with Qatar Airways, an agreement which will allow Qatar Airways passengers to travel to and from points in Vietnam not served directly by Qatar Airways using a single reservation across both airlines' networks.
The new partnership will allow passengers traveling to and from eight new domestic points within Vietnam to connect via Qatar Airways' existing gateways in Vietnam – Ho Chi Minh City (SGN) and Hanoi (HAN) – providing them with a more seamless travel experience. The agreement includes the popular coastal city Da Nang (DAD), as well as Ban Me Thuot (BMV), Dalat (DLI), Hai Phong (HPH), Nha Trang (CXR), Phu Quoc (PQC), Pleiku (PXU), Vinh City (VII) and one international point, Taipei (TPE).
Qatar Airways first commenced operations to Ho Chi Minh City on March 25, 2007. The airline was also the first Gulf carrier to offer non-stop service to Vietnam's largest city. In addition, Hanoi was added to the airline's fast-growing global network in November 2010. Passengers will benefit from frequency increases to both cities starting from January 01, 2018, with Qatar Airways flying double-daily to Hanoi and providing 10 flights a week to Ho Chi Minh City.


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As our patient volume grew, it became more difficult to find the time to put together a professional-looking newsletter. Our clinic contracted a professional newsletter company to do that work. After six months, we reviewed our public relations/marketing budget and realized that we were spending almost one-third of our monthly budget on reactivation that seldom happened. (In the last six months, we only had two reactivations from the newsletters.) We stopped including newsletters in our PR budget, at least temporarily.
Newsletters can be a useful educational and marketing tool, but it has been my professional experience that using newsletters solely as a reactivation tool is not the best use of a clinic's PR budget. The key word here is solely.
Newsletters can be so much more to a chiropractic clinic. First, CAs, your time is much too valuable to spend three days a month doing all it takes to put together a great newsletter. On the other hand, I feel that using one-fourth to one-third of the PR budget on an outside service is just plain poor business management.
The first thing you need to do is look for a publication service that meets your clinic's needs. I know of one service that creates a two-page weekly handout that comes to the clinic ready for printing (please contact me for that information). Copy as many newsletters as you need throughout the week. I suggest using colored paper. (I like bright colors over pastel colors. I feel they are more eye-catching.) With this publication, 52 issues a year cost less than one month's worth from many other newsletter companies that only print one issue a month.
The second thing you need is a plan of action to put your newsletters to work to get the maximum from your PR dollars. Here are some suggestions that will educate your active patients and pass along invaluable information to your community.
You can easily laminate "flyer-type" newsletter (one page, with information on both sides). (I told you in my last article in the January 1 issue that we would get our doctor's money's worth out of that laminating machine.) I suggest laminating between 10-12 of each week's newsletter. Put one in every seat in either the reception room, adjustment suite, or on the clinic's "hot seats."
A CA needs to be in charge of making sure that the newsletters are put back into the chairs between patients. You definitely want your patients to have to pick up the flyer before they sit down. Patients are more likely to read it if they have to pick it up before sitting down. Putting flyers in the magazine racks just doesn't cut it. Also, have a dozen or more newsletters at the front desk (not laminated) so the front-desk assistant can offer them to patients to take with them, to give to their friends, family members or co-workers.
How about this one? If people walk by your clinic in a downtown location or in a strip mall area, it's likely people passing by may look into your windows during your off hours. If you have an attractive window at the front door of your practice, display a laminated copy of that week's newsletter in the window. Take this opportunity to educate them about the benefits of chiropractic care while they become familiar with your location.
Now that we have all of these laminated newsletters from last week, it would be a real waste to throw them away. You and your doctor(s) can choose 10 or more of your patients who you believe would be willing to be helpful ambassadors to your chiropractic cause. These patients should be people who own, manage or work in a service environment; who have reception rooms for their clients; and who you believe would be willing to have your clinic's newsletter in their lobby or reception area. Asks these chosen few to be the clinic's "ambassador to health" by having the weekly laminated newsletter available for their clients to read. CAs, be sure that if the ambassador does not come into the office weekly, make arrangements to drop a copy by their business location each week.
Here are some other ideas for getting your doctor's money's worth out of the flyer-type newsletter. Recently, in one of my clinics, I started saving one of the laminated newsletters each week for the clinic. Why not make a binder with a collection of your favorite newsletter?
Think about the possibilities of this collection. Keep it in your reception room as an educational reference. Additional copies can always be made from the collection later for a potential patient wanting information on a certain condition or health concern. Why not refer to the information in the collection when you or your doctor are scheduled to speak to a civic group, organization and/or industry in your community? Make more copies and use them at the talk as a reference to the subject you spoke on to take home with them.
Getting known in your community is an ongoing project. When a chiropractic clinic thinks that everyone knows their doctor or location or understands what services your clinic has to offer and stops promoting, watch your statistics. In about three to six months, your numbers will begin to slide in the wrong direction.
Even megabusinesses like McDonald's and Disney constantly market themselves. They keep themselves in sight and in mind every day, and your clinic should do the same. CAs, keep your clinic's name visible and remember that even little things like cost-effective newsletters can be used for a lot of mileage if marketed effectively. Until next time, go out and make a difference.
Click here for previous articles by Rose Jacobs, CA.


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Crucial to glucose homoeostasis in humans, the hPDC (human pyruvate dehydrogenase complex) is a massive molecular machine comprising multiple copies of three distinct enzymes (E1–E3) and an accessory subunit, E3BP (E3-binding protein). Its icosahedral E2/E3BP 60-meric 'core' provides the central structural and mechanistic framework ensuring favourable E1 and E3 positioning and enzyme co-operativity. Current core models indicate either a 48E2+12E3BP or a 40E2+20E3BP subunit composition. In the present study, we demonstrate clear differences in subunit content and organization between the recombinant hPDC core (rhPDC; 40E2+20E3BP), generated under defined conditions where E3BP is produced in excess, and its native bovine (48E2+12E3BP) counterpart. The results of the present study provide a rational basis for resolving apparent differences between previous models, both obtained using rhE2/E3BP core assemblies where no account was taken of relative E2 and E3BP expression levels. Mathematical modelling predicts that an 'average' 48E2+12E3BP core arrangement allows maximum flexibility in assembly, while providing the appropriate balance of bound E1 and E3 enzymes for optimal catalytic efficiency and regulatory fine-tuning. We also show that the rhE2/E3BP and bovine E2/E3BP cores bind E3s with a 2:1 stoichiometry, and propose that mammalian PDC comprises a heterogeneous population of assemblies incorporating a network of E3 (and possibly E1) cross-bridges above the core surface.
The hPDC [human mitochondrial PDC (pyruvate dehydrogenase complex)] is a large macromolecular machine (Mr, ~9.5 × 106), responsible for the conversion of pyruvate into acetyl-CoA and NADH, the key committed step in carbohydrate utilization in humans. It is assembled from multiple copies of four components: pyruvate decarboxylase (E1), dihydrolipoamide acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and E3BP (E3-binding protein). Its E2 and E3BP subunits form a 60-meric pentagonal dodecahedral core to which 20–30 α2β2 E1 tetramers and 6–12 E3 homodimers bind tightly but non-covalently. Human E2 and E3BP have similar modular domain organizations comprising outwardly extended N-terminal LDs (lipoyl domains) (two on E2 and one on E3BP), an SBD (subunit-binding domain) and a large CTD (C-terminal domain) that mediates core assembly. Individual domains are joined by flexible linkers rich in alanine and proline allowing the peripherally located LDs to act as 'swinging arms', such that their lipoamide cofactors can visit all three active sites in turn during the catalytic cycle. PDC deficiencies have been implicated in a broad range of genetic, metabolic, autoimmune and neurodegenerative disorders [1–7].
PDCs from prokaryotes lack E3BP, and the crystal structure of the Bacillus stearothermophilus (now Geobacillus stearothermophilus) (PDB code 1B5S) E2-CTD core provides clear insights into its subunit organization, with the basic building blocks, namely E2 trimers, forming the 20 vertices of the dodecahedron [8]. The mammalian E2/E3BP core is also assembled from 20 trimeric units [9]. Interaction of E3BP with E2 occurs co-translationally and is mediated by their respective CTDs.
The structure and subunit composition of E2/E3BP is critical to the efficient functioning of the PDC. Initially it was reported that 12 E3BPs were situated on the 12 pentagonal faces of the E2 assembly in mammalian and yeast PDCs – the so-called 'addition' model of core organization (60E2+12E3BP) [10,11]. However, in subsequent 48E2+12E3BP and 40E2+20E3BP 'substitution' models, 12 or 20 E3BPs were proposed to replace an equivalent number of E2s within the 60-meric core structure [12,13]. AUC (analytical ultracentrifugation) studies, SAXS (small-angle X-ray scattering) and SANS (small-angle neutron scattering) solution structures combined with cryo-EM (cryo-electron microscopy) reconstructions of rhE2/E3BP (recombinant human E2/E3BP) assemblies reveal the presence of open pentagonal faces, strongly supporting the substitution model [12–14].
Current substitution models vary in the trimer population forming the 60-meric core assembly. Thus the 48E2+12E3BP version would be assembled from a mixture of eight E2 homotrimers and twelve 2E2–1E3BP heterotrimers, whereas the 40E2+20E3BP core would be formed exclusively from the latter. The number and distribution of E3BPs within the core is a key issue, as overall geometry and subunit composition will influence core stability, catalytic efficiency and PDK (pyruvate dehydrogenase kinase)-mediated regulation [14].
The stoichiometry of E1 and E3 interactions with the central core has also proved controversial. In B. stearothermophilus PDC, E1 and E3 bind to E2-SBDs with 1:1 stoichiometries in a mutually exclusive fashion [15–17], whereas, in eukaryotes, E1 binds to a specific E2-SBD and E3 binds exclusively to an equivalent E3BP-SBD in normal circumstances. Crystal structures and solution studies of bacterial E1–E2-SBD [18] and E3–E2-SBD [19] sub-complexes show that association of a second E2-SBD to either E1 or E3 is prevented by steric hindrance. The crystal structures of rhE3 (recombinant human E3) bound to E3BP-SBD [20,21] as well as ITC (isothermal titration calorimetry) studies of rhE2/E3BP–rhE3 and rhE3BP-SBD–rhE3 interactions [13] suggest a 1:1 stoichiometry [20]. In contrast, a SAXS solution structure of rhE3 complexed with an E3BP-didomain (E3BP-DD, LD plus SBD) indicates a 2:1 (E3BP-DD–E3) relationship [22].
Although considerable structural and biochemical information is available for the individual mammalian PDC components, relatively little is known about the arrangement of its icosahedral E2/E3BP core, including its precise subunit composition, overall organization and stoichiometry of binding to E1 and E3. In the present study, we use a range of biochemical and biophysical approaches to demonstrate clear differences in the subunit compositions of native bovine and rhE2/E3BP cores, and propose a new variable substitution model. Structural and geometric constraints that govern core organization and assembly have also been explored by mathematical modelling, revealing a plausible basis for favouring the 48E2+12E3BP model in situ. Moreover, we show that, at maximal occupancy, the number of E3 homodimers tethered to the assembled E2/E3BP 60-mer is equivalent to 50% of the E3BP subunits present, indicative of a 2E3BP–1E3 stoichiometry and suggesting the presence of a novel architectural feature, namely a network of E3 cross-bridges linking pairs of E3BPs above the core surface.
Part of this work was first presented at the 16th European/UK Analytical Ultracentrifuge Meeting 'AUC and Hydro' held at the Newcastle University, Newcastle upon Tyne, U.K., in September 2008.
Protein expression and purification
rhE3 (in pET14b) and rhE2/E3BP (in pET11/pET28b) were overexpressed in Escherichia coli BL21 star (DE3) cells (Invitrogen), grown in LB (Luria–Bertani) medium supplemented with the appropriate antibiotics. Cells were induced at an A600 of 0.6–0.8 with 1 mM IPTG (isopropyl β-D-thiogalactopyranoside) for 4 h at 30 °C (rhE3) or 16 h at 15 °C (rhE2/E3BP). rhE2/E3BP cultures were supplemented with 100 μg/ml lipoic acid. Cells were harvested (10000 g for 15 min at 4 °C) and overexpression was assessed by SDS/PAGE. Cell pellets [in 0.1 M NaCl, 0.01 M imidazole and 50 mM potassium phosphate buffer (pH 8.0) supplemented with EDTA-free protease inhibitors (Roche), DNase I (Sigma) and Halt protease inhibitor cocktail (Thermo Scientific)] were lysed by French press treatment and clarified by centrifugation (10000 g for 15 min at 4 °C). Proteins were purified by zinc-chelate chromatography on a BioCAD 700E Workstation by virtue of their N-terminal His tags. In the case of the purified rhE2/E3BP assembly, only E3BP-contained a His tag, so isolation of intact rhE2/E3BP core required co-integration of E2 and E3BP. Further purification involved either anion exchange on a high-capacity 20HQ column (rhE2/E3BP) for selective DNA removal (Applied Biosystems) and/or gel filtration (rhE3, rhE2/E3BP) on a Sephacryl S-300 column (Amersham). Excess unincorporated, monomeric E3BP was removed from the high Mr E2/E3BP core during the gel-filtration step. Purified enzymes in PEBS100 buffer [2 mM EDTA, 0.01% sodium azide, 0.1 M NaCl and 50 mM potassium phosphate (pH 7.5)] were analysed by SDS/PAGE. A molar absorption coefficient of 11300 M−1·cm−1 at 450 nm was used for FAD and 1951320 M−1·cm−1 at 280 nm calculated for the rhE2/E3BP assembly using ProtParam (http://www.expasy.ch/tools/protparam.html).
dE3 (deuterated E3) was overexpressed as follows: plasmid encoding rhE3 was transformed into BL21 (DE3) cells and overexpression was carried out in Enfors minimal medium (containing 85% 2H2O and hydrogenated glycerol as the carbon source) at 30 °C to an A600 of 15 using an Enfors fermentation system. dE3 was purified as described above for hydrogenated rhE3 and purity assessed by SDS/PAGE.
Bovine heart PDC was purified as described previously [23] with slight modifications. PDC was kept at 4 °C in 2 mM EDTA, 0.01% sodium azide, 0.5% Triton X-100 and 50 mM potassium phosphate buffer (pH 7.4). Subsequently, bovine E2/E3BP core (termed bE2/E3BP) was purified on discontinuous sucrose gradients [24] and its concentration measured using the biuret method.
Purified enzymes were exchanged into PEBS100 buffer (or PEBS100 with 0.5% Triton X-100) and used within 1–2 days.
SV (sedimentation velocity) AUC
SV experiments were performed using an Optima XL-I analytical ultracentrifuge (Beckman Coulter) and an An-50 Ti rotor. rhE2/E3BP complexes were maintained at 191.5 nM and the rhE3 concentration varied to achieve E3BP to E3 ratios from 4:1 (48E2/12E3BP to 3E3) to 1:4 (48E2/12E3BP to 48E3). Samples (360 μl) in PEBS100 buffer were loaded into 12 mm double-sector centrepieces and centrifuged at 20000 rev./min at 4 °C. Sedimentation profiles acquired at 1 min intervals with Rayleigh interference optics were analysed using c(s) analysis in SEDFIT [25]. The partial specific volumes of rhE2/E3BP and rhE3, calculated using SEDNTERP [26], were 0.744 and 0.739 ml/g respectively at 20 °C.
GFC (gel-filtration chromatography)
rhE2/E3BP–rhE3 samples were prepared by maintaining rhE2/E3BP at 0.42 mM and varying rhE3 levels to satisfy rhE2/E3BP to rhE3 ratios ranging from 3:1 (48E2/12E3BP to 4E3) to 1:3 (48E2/12E3BP to 36E3). Samples were analysed by Sephacryl S-300 gel filtration with uncomplexed rhE2/E3BP and rhE3 included as controls.
SANS was conducted on beamline D22 of the ILL (Institut Laue-Langevin), Grenoble, at detector distances of 4 and 14 m. Transmission and scattering data (buffer and sample) were recorded for 4 min and 15 min respectively at 4 °C in 1 mm quartz cuvettes, covering an overall momentum transfer (Q) range 0.0034<Q<0.143 Å−1 (1 Å=0.1 nm). The response of the two-dimensional area gas detector was calibrated against water. Raw data were analysed using the program GRASansP written by Charles Dewhurst, ILL (http://www.ill.eu/instruments-support/instruments-groups/groups/lss/grasp/home/). The p(r) distance distribution, maximum dimension (Dmax) and radius of gyration (Rg) were obtained using the programs PRIMUS [27] and GNOM [28,29].
N-ethyl-1-[14C]maleimide) radiolabelling and counting
Purified PDC (40, 60 and 120 μg) and rhE2/E3BP (20 and 40 μg) complexed with rE3 (at stoichiometries of 2:1, 10:1, 20:1 and 40:1) suspended in PEBS100 buffer were radiolabelled with 0.65 mM [14C]NEM (33.2 mCi/mmol, PerkinElmer) and incubated for 30 min at room temperature (18 °C). All samples were subsequently mixed with NAD+ (0.5 mM) or NADH (1 mM) followed by incubation for 10 min at room temperature. Reactions were terminated by the addition of 0.05 M DTT (dithiothreitol) after which SDS-loading dye was added. Labelled samples were assessed by SDS/PAGE on 8% (w/v) slab gels (17 cm×15 cm×2 mm) at 400 V, 65 mA. Gels were stained and destained, and the radioactive bands were excised and dissolved overnight in SOLVABLE (PerkinElmer). Gel pieces from blank tracks were used to estimate background radioactivity. Solubilized samples were mixed with EcoScint A (National Diagnostics) and counted (10 min/sample) in a Beckman LS 6500 scintillation counter. Four bovine PDC and four rhE2/E3BP:E3 preparations were separately radiolabelled. Counts for each were determined in triplicate. Data were analysed statistically as follows [30]:
where CE2 and CE3BP, and σE2 and σE3BP represent the [14C] counts and associated errors determined for radiolabelled rhE2 and rhE3BP respectively.
To eliminate background labelling of accessible cysteine residues as a possible source of error in the [14C]NEM radiolabelling study, fluorographic analysis of rhE2/E3BP–rhE3 was undertaken. Samples of radiolabelled rhE2/E3BP (6 μg) complexed with rhE3 at a stoichiometric ratio of 10:1 were run on SDS/PAGE (8% gels), then stained and fixed overnight. Fluorography was performed as described by Chamberlain [31]. Fixed gels were immersed in Amplify (GE Healthcare) and incubated for 30 min prior to drying under vacuum at 80 °C for 2 h. Fluorographs were stored at −80 °C and developed on X-ray film (Kodak) after 4–7 days.
Mathematical modelling
The algorithm for modelling the 60-meric core organization was based on its assembly from 20 trimeric units employing two important constraints: only one E3BP was allowed per trimer and E3BP was allowed to interact only with itself. On this basis, core subunit arrangements ranging from 60E2+0E3BP to a maximum of 40E2+20E3BP can be theoretically envisaged. Dodecahedral faces were parameterized by [X,Y,Z] (the number of pentagonal dodecahedral core faces that incorporate 3, 4 or 5 pairs of E2s respectively). The resulting set of simultaneous linear constraint equations was solved to obtain a list of solutions for the number of 3-, 4- and 5-E2 pair faces at each E2+E3BP subunit stoichiometry. The equations were implemented as a finite domain constraint logic program in Sicstus Prolog (http://www.sics.se/sicstus), using its in-built constraint solver to generate the solutions.
Purified proteins were dialysed at 4 °C in PEBS100 buffer (rhE2/E3BP and rhE3) or PEBS100 plus 0.5% Triton X-100 (bE2/E3BP and rhE3). ITC measurements were conducted on a MicroCal VP-ITC titration microcalorimeter at 20 °C [32]. Analysis and curve fitting to the binding isotherms using MicroCal ORIGIN v7.0 gave values for the association constant (Ka), the molar ratio (n) and the change in enthalpy (ΔH) for protein–protein interactions.
Titration of the rhE2/E3BP core assembly with increasing amounts of rhE3 indicates saturation at a 2:1 stoichiometry
Initial GFC and AUC studies were conducted to assess the number of E3 dimers tethered to the rhE2/E3BP core at maximal occupancy as a means of assessing binding stoichiometry and E3BP content. The GFC profiles of a series of rhE2/E3BP–rhE3 mixtures (based on the 48E2+12E3BP model) covering ratios from 48E2/12E3BP to 4E3 (3:1) to 48E2/12E3BP to 36E3 (1:3) are shown in Figure 1(A). The Kd values for rhE3BP–rhE3 and E3BP-DD–E3 sub-complex formation are reported to be 6.3 nM and 35.7 nM respectively [24,33]. E3 and E3BP concentrations used in the present study were in the low micromolar range, ensuring tight and complete binding.
Binding stoichiometry of rhE3 to the rhE2/E3BP core assembly determined by GFC and AUC
Void volume peaks (38 ml) for the high Mr rhE2/E3BP core uncomplexed and complexed with rhE3 were apparent at all ratios, whereas free rhE3 (Ve of 65 ml) was evident only in the range from 1:1 to 1:3, i.e. 12–36 E3 dimers per 48E2/12E3BP core. SDS/PAGE analysis confirmed the presence of free E3 at these ratios (Supplementary Figure S1 at http://www.BiochemJ.org/bj/437/bj4370565add.htm). These estimates of maximal E3 binding to assembled E2/E3BP core indicated that saturation was achieved prior to addition of 12 E3BPs per core and supported our previous data indicating 2:1 binding between a free (monomeric) truncated version of E3BP (LD plus adjacent SBD) and E3 [22].
SV AUC analysis was also conducted for a similar series of rhE2/E3BP–rhE3 mixtures, and resultant profiles were analysed using the c(s) model in SEDFIT [25] (Figure 1B). s20,w0 for free rhE3 was 5.8 S, in agreement with previous data [22]. An increase in sedimentation coefficient for the rhE2/E3BP core from 29.3 to 35.3 S was observed on rhE3 binding. Moreover, uncomplexed rhE3 became detectable at a 1:1 stoichiometry (12 E3 dimers per 48E2/12E3BP core), again consistent with 2:1 binding. GFC and AUC studies were designed on the basis of the 48E2+12E3BP substitution model; however, the presence of excess rhE3 at 12 E3 dimers per core is also consistent with a 40E2+20E3BP model with rhE3 saturation occurring at 10 E3s for a 2:1 stoichiometry in this case.
Ten rhE3s bind to the rhE2/E3BP core
To establish the binding stoichiometry and assess the rhE2/E3BP core composition more precisely, rhE2/E3BP was reconstituted with increasing amounts of dE3 and complex formation was monitored by SANS. Given the high molecular mass of the rhE2/E3BP core assembly (~3.5 MDa) compared with E3 (106 kDa), accurate titration measurements of hydrogenated E3 binding using SAXS would prove difficult. By exploiting neutron scattering, it was possible to increase the E3 signal by deuteration, markedly enhancing the sensitivity of stoichiometry measurements. Scattering curves for rhE2/E3BP and rhE2/E3BP–dE3 obtained at various levels of dE3 saturation are shown in Figure 2(A). Intensity increases, particularly in the low angle region, were clearly evident on dE3 addition, over a stoichiometry range from 0 (uncomplexed rhE2/E3BP) to 18 dE3 dimers per core. As expected, dE3 attachment to rhE2/E3BP resulted in an increased radius of gyration, Rg, and scattering intensity at zero angle, I(0) (Table 1). Moreover, dE3 addition yielded a maximum Rg of 162 Å (1 Å=0.1 nm) for the E2/E3BP–E3 sub-complex, in agreement with a previous report [12]. The I(0) value, normalized for concentration and plotted against the number of dE3s present per core is shown in Figure 2(B). Importantly, at ten dE3 dimers bound per core, the gradient of the line changes markedly, indicating saturation.
Binding stoichiometry of dE3 to rhE2/E3BP measured by SANS
|Subunit stoichiometry rhE2/E3BP–dE3 .
|Radius of gyration Rg (Å) .
|Intensity I(0) .
|Subunit stoichiometry rhE2/E3BP–dE3 .
|Radius of gyration Rg (Å) .
|Intensity I(0) .
The observed saturation value is close to the published value of 12:1 [12] and could be interpreted as evidence for 1:1 binding assuming the 48E2+12E3BP substitution model. On repeated measurement, however, saturation was always obtained at exactly ten dE3 dimers per rhE2/E3BP core. Furthermore, possible dE3 aggregation was eliminated as a source of error. Additionally, the Rg (34 Å) and Dmax (130 Å) values for dE3 (Supplementary Figure S2 at http://www.BiochemJ.org/bj/437/bj4370565add.htm) compared well with published data for hydrogenated E3 (38 Å and 130 Å), indicating no major structural changes on E3 deuteration [22]. A marked increase in particle diameter, Dmax, from 462 Å to 520 Å, was also evident from the rhE2/E3BP p(r) distribution function on dE3 saturation (Supplementary Figure S3 at http://www.BiochemJ.org/bj/437/bj4370565add.htm).
Since E3 interactions with the intact E2/E3BP scaffold will be governed by core geometry, such stoichiometry measurements are more physiologically relevant than previous analyses of E3 binding to monomeric E3BP-SBD or E3-DD constructs [13,19–22]. However, a 1:1 stoichiometry is observed in crystal structures of E3-DD and E3-SBD sub-complexes [19–21] where steric hindrance prevents access to a putative second binding site located near the two-fold axis of symmetry. It is possible that crystal packing could account for this key difference, implying that the E3 crystal structure differs significantly from its structure in solution. In this regard, the Dmax for protonated or deuterated E3 (determined by SAXS and SANS) is 130 Å [22] (Supplementary Figure S2) compared with 100 Å in the crystal structure, indicating that E3 adopts a more open and dynamic conformation in solution. Thus a plausible explanation for the SANS-based estimate of ten E3-binding sites per core is that it represents 2:1 binding to a 40E2+20E3BP assembly.
Distinctive subunit organization of the rhE2/E3BP and bE2/E3BP cores
As already indicated, there are currently two conflicting 48E2+12E3BP and 40E2+20E3BP substitution models for rhPDC core subunit content and organization in which 12 or 20 E3BPs are proposed to replace an equivalent number of E2s within the 60-meric assembly [12,13]. Importantly, evidence for both of these models was obtained with rhE2/E3BP cores produced using similar recombinant vectors, overexpression systems and protein purification regimes. However, no results were provided on the relative levels of E2 and E3BP expression in either case. Our present study relies on the presence of a His-tag on E3BP for the isolation of the integrated rhE2/E3BP core. Excess monomeric E3BP is always present after the initial purification step prior to its removal by gel filtration, ensuring maximal E3BP incorporation. Interestingly, however, previous studies on native bovine heart and yeast PDCs all report the presence of only 12 E3BP subunits per core (i.e. 48E2+12E3BP) [10,11]. Importantly, both models yield predictable and distinct numbers of bound E3s at saturation (Supplementary Table S1 at http://www.BiochemJ.org/bj/437/bj4370565add.htm).
In view of these conflicting reports, NADH-induced E2 and E3BP lipoamide group radiolabelling was performed to assess possible differences between native bovine (bE2/E3BP) and human (rhE2/E3BP) cores. Specific [14C]NEM modification of reduced E2 and E3BP lipoamide thiols is achieved in the presence of NADH and catalytic amounts of rhE3. The ratio of [14C]NEM incorporated into E2 (two LDs) and E3BP (one LD) subunits alters significantly depending on the model in question (Table 2). In particular, a marked difference in ratios is predicted for the 48E2+12E3BP (8:1) [12] and 40E2+20E3BP substitution models (4:1) [13], whereas potential variations in ratio for E2+E3BP core compositions in the range 44E2+16E3BP (5.5:1) to 40E2+20E3BP (4:1) are relatively minor (Table 2). Radiolabelling was conducted for triplicate samples of four bovine heart PDC and rhE2/E3BP–rhE3 preparations, to prevent uncertainties owing to batch variation or sample degradation.
|Model .
|Stoichiometry .
|Ratio of E2 to E3BP LDs .
|Theoretical radiolabelled E2 to E3BP ratio .
|Variable substitution
|Model .
|Stoichiometry .
|Ratio of E2 to E3BP LDs .
|Theoretical radiolabelled E2 to E3BP ratio .
|Variable substitution
Ratio analysis of radiolabelled subunits in rhE2/E3BP and native bE2/E3BP cores is shown in Table 3. Values for the bE2/E3BP core supported the 48E2+12E3BP model (8:1, Table 2), as reported previously [12], whereas rhE2/E3BP cores generally gave values (4:1) consistent with the 40E2+20E3BP model [13]. Occasional higher values were obtained in the latter case with 5.63, the highest ratio observed, equivalent to a 44E2+16E3BP composition. In summary, radiolabelling data suggested distinctive core models with average compositions of 48E2+12E3BP for bE2/E3BP, and approaching 40E2+20E3BP for rhE2/E3BP.
|14C counts .
|Samples .
|Ratio of E2 to E3BP .
|14C counts .
|Samples .
|Ratio of E2 to E3BP .
To eliminate background labelling of accessible cysteine residues as a possible source of error, fluorographic analysis of rhE2/E3BP–rhE3 (Supplementary Figure S4 at http://www.BiochemJ.org/bj/437/bj4370565add.htm) was conducted after modification with [14C]NEM in the presence of NAD+ or NADH to maintain the E2 and E3BP-linked lipoamide moieties in their oxidized or reduced states. Specific NADH-induced modification of E2 and E3BP was observed with no discernable labelling of complexed rhE3. However, radiolabelling of accessible E3 cysteine residues, possibly the active-site disulfide pair, was detectable with non-complexed E3 (Supplementary Figure S4).
Previous studies have established specific modification of E2 and E3BP-reduced lipoamide moieties in bovine PDC [34]. Under these conditions, estimates of the rhE2/E3BP core composition obtained by specific NADH-induced [14C]NEM labelling of E2 and E3BP lipoamide thiols are in agreement with the 40E2+20E3BP model. Brautigam et al. [13] provided several lines of evidence for a 40E2:20E3BP core stoichiometry using quantitative Coomassie Blue staining, AUC, native gel electrophoresis and ITC measurements. They also concluded that previous AUC analyses of maximal E1 and E3 binding to the high Mr E2/E3BP core leading to the development of the original 48E2+12 E3BP model by Hiromasa et al. [12] were technically flawed. However, neither group took account of the relative levels of E2 and E3BP expression in their systems. This is likely to prove a key factor in determining the final core composition, particularly as E2 itself is capable of self-assembly into a 60-meric icosahedron, whereas E3BP is largely monomeric (see Figure 4) and requires to be co-expressed with E2 to achieve core integration. In this context, it is apparent that generation of recombinant cores with various E2/E3BP subunit contents is a reflection of the relative E2 and E3BP expression levels.
A clear difference in subunit organization and composition between recombinant human and native bovine cores was also confirmed by ITC. Binding isotherms were generated by titrating rhE3 into the reaction cell containing rhE2/E3BP (Figure 3A) or bE2/E3BP (Figure 3B). On curve fitting, molar ratios (n, E3BP to E3) of 9.68±0.18 and 5.70±0.09 were obtained for rhE2/E3BP to rhE3 and bE2/E3BP to rhE3 respectively. The E3 titration profile for rhE2/E3BP and bE2/E3BP yielded Kd values with an upper limit of 7 nM and 10±5 nM respectively, consistent with previously reported values [24,33], although binding was too tight for accurate measurement. Three independent binding isotherms for rhE2/E3BP–rhE3 and bE2/E3BP–rhE3 complex formation yielded n values within 10% of 9.68 and 5.70 respectively, consistent with 2:1 E3BP to E3 binding molar ratios predicted (Supplementary Table S1 at http://www.BiochemJ.org/bj/437/bj4370565add.htm) for the recombinant 40E2+20E3BP and bovine 48E2+12E3BP models respectively. The recombinant E3–E2/E3BP complex molar ratio (9.68) was also consistent with our SANS data (ten E3 dimers per core).
Stoichiometry of binding of rhE3 to rhE2/E3BP and bE2/E3BP core assemblies determined by ITC
A problematic issue is that our present ITC and previous biochemical and biophysical analyses [22], all indicating a 2:1 stoichiometry of E3 binding to E3BP using either truncated E3BP constructs or the fully assembled E2/E3BP core, are in direct contrast with the findings of Brautigam et al. [13]. In our case, ITC evaluation of E3 binding to rhE2/E3BP shows saturation at n=10 E3 dimers per core, with a maximal Kd of 7 nM. In contrast, Brautigam et al. [13] report a much higher Kd (102 nM) that may account for their high n value of 18.8, implying 1:1 E3 binding to a 40E2+20E3BP assembly. In this context, it is noteworthy that PDC-deficient patients totally lacking E3BP retain partial PDC activity (10–20% of controls), suggesting that E3 may still interact with the E2 core to a limited extent in the absence of its normal binding component. In support of this idea, bovine PDC reconstituted in vitro with a homogeneous 60-meric E2 core is virtually inactive at stoichiometric E3 levels, whereas overall complex activity (approaching 40–50% of wild-type) can be restored with a 200-fold E3 excess, suggesting that E3 can interact weakly with the normal E1-binding site on E2 under these conditions [35]. Recent surface plasmon resonance data from our laboratory (G. Singh, A. E. Brown, S. D. Richman, Z. Cao, H. Al-Amodi, S. M. Kelly, D. McGow, O. Byron, G. K. Brown and J. G. Lindsay, unpublished work) confirm that E3 has a low-affinity interaction with a secondary binding site on E2 (Kd≈410 nM) that can lead to partial E1 displacement at markedly elevated E3 concentrations. This Kd value is only 4-fold higher than the Kd of 102 nM for E3–E3BP complex formation [13], and may account for the additional E3 binding required to achieve saturation in their experiments leading to an erroneously high estimate of E3 binding stoichiometry. In our case, E3 interacts with its primary E3BP-binding site with a much lower Kd (7 nM or less), i.e. with at least a 60-fold greater affinity than its weak secondary interaction with E2. It is difficult to assess the reasons underlying these marked differences in E3-binding affinity for the rhE2/E3BP core without a detailed knowledge of the sequences of the E2, E3BP and E3 constructs and recombinant expression system used by Brautigam et al. [13]. Inaccurate protein measurement is another possible source of error in these experiments. In our case, the concentration of purified E2/E3BP and E3 samples was estimated by two independent methods. For E2/E3BP, this involved A280 measurement based on the calculated molar absorption coefficient of the core assembly and biuret analysis. Possible DNA contamination of purified rhE2/E3BP was eliminated by use of an ion-exchange step. The E3 concentration was also estimated by the biuret method and by A450 measurement of FAD content after ensuring that the protein contained a full complement of cofactor.
E2/E3BP core composition: investigation by mathematical modelling
Mathematical modelling of possible E2/E3BP core arrangements was conducted by systematic variation of E2 homotrimer and 2E2–1E3BP heterotrimer populations that form the 60-meric icosahedron (Supplementary Table S2 at http://www.BiochemJ.org/bj/437/bj4370565add.htm). Two fundamental constraints were applied to the modelling process: first, only one E3BP was allowed per heterotrimer, and secondly, E3BPs of neighbouring heterotrimers could interact only with each other. The first constraint recognizes that E3BP is itself incapable of core formation with no evidence for the existence of 1E2–2E3BP heterotrimers. Thus, despite high rhE3BP expression relative to E2, purified rhE2/E3BP always contains E2 as the major species, as judged by SDS/PAGE and radiolabelling (Supplementary Figures S1 and S4). Justification for the second constraint is that E3BP lacks key residues involved in the hydrophobic 'ball and socket' connection that mediates inter-trimer contacts within the core [12]. It has been suggested that E3BP could self-associate on the core surface [12], and in our earlier AUC studies [14], purified rhE3BP showed little or no capacity to form higher oligomeric states. As shown in Figure 4, non-specific cross-linking of purified rhE3BP with glutaraldehyde revealed a weak tendency to form dimers, although the majority of rhE3BP remained monomeric. Successful cross-linking of rhE3, a naturally occurring homodimer, served as a positive control.
Glutaraldehyde cross-linking of E3BP
Several solutions of core organization for each model were obtained by mathematical modelling (Table 4). Each represents an E2 and E3BP arrangement that satisfies a particular core composition ranging from 60E2+0E3BP to 40E2+20E3BP. Final states were parameterized by [X,Y,Z] (the number of pentagonal dodecahedral core faces that incorporate 3, 4 or 5 pairs of E2s respectively). The number of possible final states follows a near Gaussian distribution (Figure 5A). Importantly, whereas the 40E2+20E3BP model has only two final states (one symmetric and one asymmetric; Figure 5B), the number of possible states peaks at seven for the 48E2+12E3BP core (Figure 5C), with each state showing a distinctive distribution of E3BPs around the core surface. Thus a 48E2+12E3BP subunit stoichiometry may be advantageous in that it ensures the greatest number of routes to, and final arrangements of, the core structure, enabling rapid and efficient production in vivo. Moreover, the relative abundance and distribution of E2 and E3BP subunits will have a direct effect on the number and location of associated E1 and E3 enzymes. Thus, from a functional and regulatory standpoint, a 48E2+12E3BP core composition may be favoured in that it recruits the optimal balance of cognate E1s and E3s for achieving optimal catalytic efficiency and regulatory fine-tuning. E1 normally catalyses the rate-limiting step in the reaction sequence, so alterations in bound E1 levels will directly affect the overall catalytic rate in most circumstances. Interestingly, a reconstituted bE2/E3BP core containing only 35% of the normal E3BP complement supports a correspondingly reduced level of PDC activity, indicating that the ratio of bound E1s and E3s (as determined by the E2/E3BP core composition) is also important in regulating the overall rate of catalysis [35].
|Core organization .
|Number of homotrimers .
|Number of heterotrimers .
|Number of final states/solutions .
|Core organization .
|Number of homotrimers .
|Number of heterotrimers .
|Number of final states/solutions .
Mathematical modelling of E2/E3BP core organization
Variable subunit composition of the rhE2/E3BP core assembly
Based on current evidence, it is apparent that there is considerable potential for variation in both overall E2/E3BP core organization and subunit stoichiometry. In our case, rhE2 and E3BP co-expression is carried out under conditions where E3BP is produced in excess leading to formation of a 40E2+20E3BP core in support of the hypothesis that only one E3BP subunit can be incorporated per trimer. We also demonstrate for the first time that, in contrast with E2, rhE3BP was incapable of independent self-assembly into a high Mr 60-meric complex. Indeed, it is largely monomeric with a weak tendency to form dimers (Figure 4). In this context, we have previously reported that the stability of the rhE2/E3BP core was reduced in comparison with a homogeneous 60-meric E2 core, indicating that the E3BP content was likely to influence core 'breathing' and protein dynamics [14,37].
Advantages in maintaining a standard 48E2+12E3BP core structure in native eukaryotic PDCs
Although current evidence lends support to the 40E2+20E3BP model for the rhE2/E3BP core when E3BP is present in excess, we also show that the native bovine assembly has a distinctive 48E2+12E3BP subunit stoichiometry. Interestingly, in vivo, E3BP subunit levels per core have always been estimated at 12 in mammalian and yeast PDCs [10,11], so it will be essential in future to determine whether the 48E2+12E3BP core represents the standard archetypal assembly that has been widely adopted in eukaryotes or whether there are characteristic tissue- or species-specific alterations.
As a central 48E2+12E3BP core is composed of two types of trimeric units, namely E2 homotrimers and 2E2/1E3BP heterotrimers, E2 and E3BP synthesis must be tightly co-ordinated in vivo, with E3BP being produced in limiting amounts leading to formation of non-uniform cores averaging 12E3BPs per icosahedron. Electron microscopy studies of PDC have highlighted its inherent heterogeneity [37–39], so it is also probable that a mixed population of E2/E3BP assemblies is generated in vivo such that the 48E2+12E3BP model represents an 'average' structure. As illustrated in Figure 5, even assuming that all cores have a uniform 48E2+12E3BP subunit composition, they can still be arranged in seven possible final states differing in the distribution of E2 and E3BP subunits around the core surface and consequently the spatial organization of its interacting E1 and E3 partners.
In summary, on the basis of limited stoichiometry data available to date, the 48E2+12E3BP model appears to represent the standard archetypal PDC core assembly found in eukaryotes, although there is undoubtedly potential for significant variation in the subunit content of this central structural framework. Mathematical modelling and biochemical data on E3BP-deficient core assemblies also give important insights into potential advantages of deploying a 48E2+12E3BP core composition. In future, it will be essential to determine whether there are species- or tissue-specific variations in the native E2/E3BP subunit content which has the potential for modulating overall PDC composition leading to subtle alterations in catalytic efficiency and regulatory fine-tuning.
Stoichiometry of E3 interaction with the recombinant human and native bovine E2/E3BP assemblies
Our current results using GFC, AUC, SANS and ITC confirm that ten and six E3s respectively bind to individual recombinant human and native bovine E2/E3BP cores at maximal occupancy. These data highlight their distinctive 40E2+20E3BP and 48E2+12E3BP compositions while confirming the 2:1 stoichiometry of E3BP–E3 binding to the surface of these large pentagonal dodecahedral assemblies. It should be noted, however, that final rhPDC core composition is governed by the relative levels of E2 and E3BP expression. Thus the production of a 40E2+20E3BP assembly in recombinant human systems merely reflects one possible scenario, namely where there is excess production of the E3BP subunit (which is never detected in situ as an independent non-integrated species) thereby ensuring its maximal incorporation into the assembling E2 core.
A schematic illustration of an archetypal 48E2+12E3BP core assembly is shown in Figure 6 with representative E2 and E3BP N-terminal segments, consisting of LDs and SBDs joined by flexible linkers, extending outwards from the core. E3BP content and distribution may vary considerably, so the core would be more accurately depicted as a heterogeneous population of closely related assemblies with an average 48E2+12E3BP composition. Representative E3 cross-bridges linking adjacent pairs of E3BPs are also illustrated. There is an additional possibility of E1 heterotetramers linking neighbouring E2-SBDs highlighting the existence of a network of cross-bridges on the core surface (Figure 6 and Supplementary Movie S1 at http://www.BiochemJ.org/bj/437/bj4370565add.htm). This architectural feature (i) has the potential to moderate the flexibility of the E2 and E3BP lipoyl 'swinging arms' and (ii) may facilitate PDK movements around the core. Interestingly, cryo-EM images of rhE2/E3BP reveal the appearance of prominent E3-induced 'spikes' located just above the surface of the CTD. The increased order in this region permits it to be properly resolved on cryo-EM reconstruction, and is consistent with E3 cross-bridge formation restricting movement of the flexible lipoyl 'swinging arms' in this region [14].
Cross-bridge formation in human PDC
analytical ultracentrifugation
- bE2/E3BP
- bovine
- cryo-EM
cryo-electron microscopy
C-terminal domain
- dE3
deuterated E3
E3-binding protein
gel-filtration chromatography
isothermal titration calorimetry
lipoyl domain
pyruvate dehydrogenase complex
- hPDC
human PDC
pyruvate dehydrogenase kinase
- rhE2/E3BP
recombinant human E2/E3BP
- rhE3
recombinant human E3
small-angle neutron scattering
small-angle X-ray scattering
subunit-binding domain
sedimentation velocity
Swetha Vijayakrishnan designed and performed the experiments, analysed the data and wrote the manuscript. Philip Callow carried out the SANS experiment and analysis. Matthew Nutley performed the ITC experiment and analysis. Donna McGow provided technical support. Mathematical modelling and data analysis was carried out by David Gilbert and Peter Kropholler. Alan Cooper provided conceptual advice on the ITC. Olwyn Byron and Gordon Lindsay supervised the project, designed the experiments and edited the manuscript before submission.
We thank the staff of the ILL-EMBL Deuteration Laboratory for their assistance during sample preparation, the ILL for use of the D22 diffractometer and the EPSRC (Engineering and Physical Sciences Research Council). We thank Michael Haertlein, Peter Timmins and Trevor Forsyth for advice, support and encouragement.
This work was partly supported by the Engineering and Physical Sciences Research Council (EPSRC) [grant numbers GR/R99393/01, EP/C015452/1]. S.V. was the recipient of a Wellcome Trust-funded Ph.D. studentship and a Wellcome Trust VIP award. J.G.L. acknowledges continued financial support from BBSRC (Biotechnology and Biological Sciences Research Council), U.K. and the UMDF (United Mitochondrial Disease Foundation), U.S.A.. This research has also benefited from the activities of the DLAB consortium funded by the EU under contracts HPRI-2001-50065 and RII3-CT-2003-505925.


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Everyone loves a good fact, don't they? Well, I do, anyway. A couple of months ago, on a Friday night, my mate told me a fact that I found totally fascinating (I didn't believe him at first, but good old Google verified it. Yes, he was smug as fuck) and because it was a Friday we decided that Friday's would now become Fact Friday's, so I thought I'd write a post every Friday with some cool facts that I find online. Now, I haven't verified any of these, so don't come crying to me if they turn out to be bullshit.
First, I'll give you the fact that my friend told me……..
* When someone yawns in your vicinity, it can often make you yawn too, as though the yawn is somehow 'catching'. (You all know what I mean, right?). Psychopaths are immune to 'catching' yawns.
Now, come on, that's a pretty sweet fact by anybody's standards. You know you're going to be on alert now anytime someone yawns, to see who the psychos are. Here are a few others that I think are pretty cool……
* The name for the shape of a Pringle is a 'Hyperbolic Paraboloid'.
* Albert Einstein's last words were spoken in German to a nurse who couldn't speak the language and are lost forever.
* No one who was born blind has ever developed schizophrenia.
* The higher your IQ, the more forgetful you will be.
Hang on a minute, what was I doing again? Oh yeah, I remember now. Haha.
* More than 50% of koalas have got Chlamydia.
* The woolly mammoth was still alive when the pyramids were being built.
* Internet scams are among the top 5 industries in Nigeria.
Some pretty neat ones in there, I reckon. I'll give you another dose (like a Chlamydia ridden koala) next Friday.


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Sorry, page not found
The page you are looking for could not be found. Go back to the home page or use the navigation above.
FRP Development Corp / Florida Rock Properties, Inc.
The page you are looking for could not be found. Go back to the home page or use the navigation above.
FRP's Land Development team purchases and develops properties to fuel our development pipeline while enhancing the value of the remaining acreage. Our land development platform works alongside local, state, and federal jurisdictions in order to bring undeveloped properties through the permit and design phase.
FRP develops, owns and manages its properties, meaning we have a vested interest in the quality and longevity of our buildings. Our knowledgeable development team understands the high level of construction that is expected from them, and each of our projects reflects this standard.
FRP's Interiors division understands your requirements, and has the experience and dedication needed to deliver results beyond your expectations.


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There are very few people who could pull off a head-to-toe yellow look like this. But Gigi Hadid is definitely one of them. And she killed it this week in this canary Ralph Lauren Collection minidress and matching oversized coat. She complimented her bright, bold ensemble with glitter pumps, no less. A winner in my style book. How about yours?
Beauty April 24th, 2020


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In addition to delicious noodle bowls and fine cuisine we offer an array of noodles, savory sauces, condiments and cooking ingredients to purchase in our NK – Noodle Kitchen and Market South Tampa location.
Top off your noodles and bring home the tastes and delicious flavors that we have created though years of culinary experience and customer satisfaction. We have numerous flavors to sample and experience on your noodles and to enhance your home cooking.
NK brand sauce flavors include:


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A Texas Business Insurance Update
Most businesses purchase general liability insurance to protect the business against certain kinds of claims. These claims could include bodily injury and property damage. Professional Liability insurance, also called Errors and Omissions insurance, protects against a claim from a third party who suffers a financial loss because of services or actions. Any individual or business in a profession that provides advice, expertise, recommendations, or a professional service to customers, clients, or another party, would benefit from Professional Liability insurance.
Typically, professionals such as doctors, accountants, nurses, insurance agents, lawyers, and architects purchase Professional Liability insurance. However, if you have clients who are in the business of providing advice, opinions, or services, they should consider Professional Liability insurance. Professionals who provide advice are expected to have knowledge or training in their particular area of expertise. They are also expected to perform the services for which they were hired according to the standards of conduct in their profession. Businesses and individuals can be held legally responsible for any financial loss caused.
It's important to note that almost all Professional Liability insurance policies are sold on a "claims-made" basis. This means that the insurance only covers work performed while the policy is in force, and for claims actually filed during the term of the policy. Professional Liability insurance is offered in many different forms that protect many kinds of firms.
If someone feels he or she has suffered loss or damages due to a professional service, often their next action is a lawsuit. These kinds of actions can be costly and severely reduce a firm's resources. Professional Liability insurance can help preserve assets and more importantly help preserve the firm's reputation.
As an Independent Insurance Agency we represent many insurance companies to fit your businesses dynamic needs. Whether you're a new business or a small business, we can help. Being an Independent Agent means we are not only your agent, but we are your business insurance broker as well. We understand every business and industry is unique, so we will work with you to find the best business insurance coverage at the lowest rate. Contact us today for a free quote.
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April 2020


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The Mummy is a strange contradiction. It was a flop in North America but proved to be the biggest international opening of Tom Cruise's career. The guys break it down and what this means for the future of the Dark Universe and cinema.
Check us out at Get2thePodcast.com and follow @get2thepodcast on Twitter, Facebook and Instagram!


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Arsenal Jersey Home & Away 2015/16 by Puma Newest Arrival
Vancouver soccer store North America Sports has received the new Arsenal jersey in home & away for 2015/16 by Puma.
Adult and youth sizes are currently available in the Arsenal home jersey.
The new Arsenal away jersey 2015/16 by Puma is now in stock in adult sizes.
Get the new Arsenal jersey 2015/16 in Home & Away by Puma at North America Sports the Soccer Shop in Vancouver BC Canada or call 604-299-1721.
Labels: arsenal jersey 2015 2016 puma vancouver bc canada, arsenal jersey puma 2016 vancouver bc, arsenal jersey vancouver


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« Students With Disabilities in Florida District Outpace Peers on NAEP | Main | FBI Probe Finds No Misuse of Title I Funds in Providence, R.I. District »
Special Educator Interest in Common Core Heated Up in 2013
2013 was a tough time to eke out any news on special education at the federal level. Sure, there were the effects of the sequester cuts (and the prospect that those cuts may soon be alleviated), but Washington was not where special educators were looking in the past 12 months.
Instead, they were intensely interested in any news relating to the Common Core State Standards, judging by a look at the most-read blog entries for On Special Educaton Blog in 2013. A post noting that the Partnership for the Assessment of Readiness for College and Careers, one of the test-writing consortia, was seeking comment on proposed accommodations drew high readership, as did follow-up blog posts noting that PARCC had released a proposed accommodations and accessibility manual for students with disabilities, and that the organization adopted a final version of the manual in June.
A post on PowerUp WHAT WORKS, a federally-funded collaboration that offers common-core themed lesson plans for special education teachers, also drew attention, as did an entry on common-core resources compiled by PARCC.
Readers were also interested in classroom research: a study that found high-quality teaching for young students with autism was more important than a particular program got attention. So did a study from the What Works Clearinghouse that found that most postsecondary programs for students with disabilities have not been studied rigorously for effectiveness, and a report spelling out the poor graduation rates of students with learning disabilities.
Response to intervention also made its way onto readers' radar, with a post on a University of Kentucky student's doctoral research that outlined inconsistencies in RTI implementation.
The Education Department did make news that garnered reader attention this year; for example, when it released guidance saying that schools must provide equal access to athletic opportunities for students with disabilities, and when it asked for comment on a proposal to revise its monitoring focus to pay more attention to outcomes, not just procedural compliance.
Finally, this year marked the long-awaited fifth revision to the Diagnostic and Statistical Manual of Psychiatric Disorders, which, among its changes, eliminated some subsets of autism such as Asperger's Syndrome. And the American Association of School Administrators tried to make sure no one forgot the Individuals With Disabilities Act, through a proposal that would dramatically change due-process procedures. Will we see those changes come to pass in 2014? Stay tuned!


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Happy Easter!
We had a lot of fun painting Easter eggs with out reading buddies!
Food chemistry
In our new science kit, we are studying 4 types of nutrients in foods, how to test for them and why we need them in our diet. We have so far learned how to test for starch and glucose. In this kit we are also learning how to be careful scientists, making sure to avoid cross contamination by putting the correct foods in each testing tray, using multiple toothpick, and the right spoon for each food. We are also learning that sometimes we need to redo a test when our results don't match up with the rest of the class.
Literature Circles
It is always fun to read and discuss a book with a friend. In reading right now the fifth graders are doing just that. We have started literature circles, each member of the group is responsible for one part of the group discussion about their book. For our first literature circle all students are reading the same book Pink and Say by Patricia Polacco. Every Tuesday and Thursdays groups have been meeting to discuss the section they read. There are 4 parts to out group discussions. We have the discussion director who is in charge of leading the discussion, they have to write 6 questions about their section. We have the word wizard who is in charge of finding new vocabulary or interesting words from the sections. The third role is blogger, they have to take the role of one of the characters and write a diary entry about the sections they read. The last job we will be doing as a whole class, visual recorder, we will be making an avatar to look as close as we can to one of the characters to summarize the import events that took place in the story. Students have a list of meeting dates and what role they are responsible for. Students are placing their roles online using a website I created so there is not papers to hand in. They are also grading themselves each meeting, and I am also grading them as I walk around observing them. After this book, we will be starting another group where students will get to select 2 or 3 books they want to read and then I will place them in one of those groups.
We accept the challenge!
We completed our Einstein kit on Motion and Design just before Spring break. The final task was for each group to complete a challenge where they would have to use everything they have learned about how things move to design a car for a specific challenge. Each one had a different challenge with different requirements. It was not easy and some wanted to quit when they couldn't see a way to solving it. I am happy to say with a lot of hard work, trial and error and starting from the beginning all groups did complete their challenge.
Nonfiction magazine project.
For the past two weeks, the students have been working on creating a magazine on a topic of their choice. With in this magazine the students had to include specific nonfiction features and it had to contain an article written in one of the 5 different types on nonfiction writing. This was not an easy task and they all worked very hard in completing this project. We had a variety of animals, sports figures and one on art.
Order of operations
Do you remember PEMDAS? Please Excuse My Dear Aunt Sally. We are working on solving multi operation expressions. Today we listened and danced to the order of operation shuffle. Students know almost all of the steps, we just need to remember to multiply and divide from left to right and then add and subtract from left to right.
Auction From the Heart
It's getting close to our biggest fund raiser of the year. You can see all the items that have been donates by following the Auction facebook page. Each class is also putting together a class basket, if you can please send your donation to school. Today the students made their thumb print plate which will be auctioned off.
The students had a great time on our field trip. Our first stop was at NWTC where students from Michigan Tech came and performed over 60 different science experiments. Students got the chance to go around and check out the different experiments that looked interesting to them, all from different fields of science. Some of the labs students only got to observe what happened, when they were using fire or other materials like liquid nitrogen for safety reason but they were still cool. Of course there were ones that involved food and those where a big hit, like dragon's breath where they got graham crackers that make it look like there where breathing smoke. I think some of the big hits where the human bowling and the oobleck.
At the Wildlife Sanctuary the students got to learn about and meet a fox snake, skunk and a short eared owl.
Motion and Design
The students are just a little over half way done with our second Einstien kit, Motion and Design. In this kits students are exploring how to make a vehical move using differnt types of force. They first used a falling weight system that hooked up to the front of their vehical and the other end hung off the side of a table. They started with one weight and added more on to make it move. They discovered the more weights they added the farther their car moved. They then began experimenting with rubber band power and figured out that winding the band around the moveable axle made the car go forward. They also learned avout potential and kenetic energy, when they wond the band that stored energy and when they let go that released the energy making the vehical go. This kit the students have to really work on team work, there is a lit of work and everyone needs to do their part to make their lab sucseful. We have been keeping track of all the ways they know on how to make the vehical move fast and slow, they will use this information to help them complete a final challenge at the end of the kit.
Nonfiction text features
This week we have been focusing on nonfiction text features. We spent 2 days going over the definitions of each and how they help readers. Students then had to search through nonfiction resources to find example.
May 2017


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Artistic director Michael Feinstein and interim CEO/President Frank Basile unveiled the 2012-13 season at the Center for the Performing Arts – home of The Palladium, The Tarkington and The Studio. "The 2012-13 Season presented by St.Vincent Health" will offer five subscription series: Classics, Songbook, Jazz & Blues, Country & Bluegrass, and Dance. Additional performances, including Pop, Spotlight, Family and Holiday will be included in the 2012-13 season as single ticket events.
"I'm certain subscribers will be delighted with our lineup of iconic musicians and performers," said Artistic Director Michael Feinstein. "The new season was conceived as a showcase for artistic beauty and our guests fully embrace the Center's focus on world-class performances. From the lyrical vocals of Johnny Mathis to the expressive footwork of Savion Glover, I can hardly wait to share the season ahead. Each series is composed of the best and brightest, backed by their impeccable artistic reputations."
Highlights include concerts by BBC Concert Orchestra, China National Symphony Orchestra, Celtic Thunder and B.B. King. The Country series brings Josh Turner, Martina McBride and the Oak Ridge Boys. Dance performances include Savion Glover and MOMIX.
Renewals for current subscribers and new subscription sales begin at 8 a.m. at the Palladium box office on Monday, April 23. Phone orders may be placed beginning at 10 a.m. on Monday, April 23. Special events including Pop, Spotlight, Family and Holiday will complete the season. Subscribers will be able to purchase tickets to these events prior to single ticket sales to the general public. The only way to guarantee seats to these special events is to subscribe.
Subscribe by calling the Center's box office at 317-843-3800 or toll-free at 877-909-2787. The season lineup is outlined on the Center for the Performing Arts' website: www.TheCenterPresents.org.
Subscribing to the 2012-13 Season presented by St.Vincent Health offers multiple benefits, including priority seat selection, savings on single tickets (up to 15%) as well as discounts on additional single tickets for shows presented by the Center for the Performing Arts.


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Granite Range:[mp_list_products order_by="price" order="DESC" category="granite"]
What is Granite?
Granite is a light-colored plutonic rock found throughout the continental crust, most commonly in mountainous areas. It consists of coarse grains of quartz (10-50%), potassium feldspar, and sodium feldspar. These minerals make up more than 80% of the rock. Other common minerals include mica (muscovite and biotite) and hornblende (see amphibole). The chemical composition of granite is typically 70-77% silica, 11-13% alumina, 3-5% potassium oxide, 3-5% soda, 1% lime, 2-3% total iron, and less than 1% magnesia and titania. Volcanic rock of equivalent chemical composition and mineralogy is called rhyolite.
Granite is the signature rock of the continents. More than that, granite is the signature rock of the planet Earth itself. The other rocky planets—Mercury, Venus and Mars—are covered with basalt, as is the ocean floor on Earth. But only Earth has this beautiful and interesting rock type in abundance.
Granite is found in large plutons on the continents, in areas where the Earth's crust has been deeply eroded. This makes sense, because granite must solidify very slowly at deeply buried locations to make such large mineral grains. Plutons smaller than 100 square kilometers in area are called stocks, and larger ones are called batholiths.
How is it formed?
On the grandest scale, granite represents the way the continents maintain themselves. The minerals in granitic rocks break down into clay and sand and are carried to the sea. Plate tectonics returns these materials through seafloor spreading and subduction, sweeping them beneath the edges of the continents. There they are rendered back into feldspar and quartz, ready to rise again to form new granite when and where the conditions are right.
Granites are the most abundant plutonic rocks of mountain belts and continental shield areas. They occur in great batholiths that may occupy thousands of square kilometers and are usually closely associated with quartz monzonite, granodiorite, diorite, and gabbro.
Debate has long centered on whether granite is igneous or metamorphic in origin. Originally granite was thought to form mainly from magmatic differentiation of basaltic magma, but geologists now believe there is simply too much of it for it to have formed this way, except locally. Most granite seems to have formed either by melting, partial melting, or metamorphism of deeply buried shale and sandstone. Granite dikes are clearly igneous, and granite emplaced in the upper few kilometers of the Earth's crust also often shows evidence of forceful intrusion into surrounding rocks, whereas some granites that formed deeper within the crust seem not to have been forcefully emplaced. Evidence of intrusion or great mobility is considered to indicate an igneous origin that stems from melting of sediments; but where no good evidence of either a magma chamber or of fluidity is observed, a metamorphic origin must be considered.
Granites are classified in three or four categories. I-type (igneous) granites appear to arise from the melting of preexisting igneous rocks, S-type (sedimentary) granites from melted sedimentary rocks (or their metamorphic equivalents in both cases). M-type (mantle) granites are rarer and are thought to have evolved directly from deeper melts in the mantle. A-type (anorogenic) granites now appear to be a special variety of I-type granites. The evidence is intricate and subtle, and the experts have been arguing for a long time, but that is the gist of where things stand now.
Almost all granite is igneous (it solidified from a magma) and plutonic (it did so in a large, deeply buried body orpluton). The random arrangement of grains in granite—its lack of fabric—is evidence of its plutonic origin. Rock with the same composition as granite can form through long and intense metamorphism of sedimentary rocks. But that kind of rock has a strong fabric and is usually called granite gneiss.
Granite is only one of the granitoids, a granitoid with quartz content between 20 and 60 percent and a feldspar content in which alkali feldspar rather than plagioclase feldspar predominates. Granite is a strong stone because its mineral grains have grown tightly together during a very slow cooling period. And the quartz and feldspar that compose it are harder than steel. Granite takes a good polish and resists weathering and acid rain.
Lavas erupt all over the Earth, but lava with the same composition as granite (rhyolite) only erupts on the continents. That means that granite must form by the melting of continental rocks. That happens for two reasons: adding heat and adding volatiles (water or carbon dioxide or both). Continents are relatively hot because they contain most of the planet's uranium and potassium, which heat up their surroundings through radioactive decay. Anywhere that the crust is thickened tends to get hot inside (for instance in the Tibetan Plateau).
And the processes of plate tectonics, mainly subduction, can cause basaltic magmas to rise underneath the continents. In addition to heat, these magmas release CO2 and water, which helps rocks of all kinds melt at lower temperatures. It is thought that large amounts of basaltic magma can be plastered to the bottom of a continent in a process called underplating. With the slow release of heat and fluids from that basalt, a large amount of continental crust could turn to granite at the same time.
The immediate cause of granite collecting and rising in huge stocks and batholiths is thought to be the stretching apart, or extension, of a continent during plate tectonics. This explains how such large volumes of granite can enter the upper crust without exploding, shoving or melting their way upward. And it explains why the activity at the edges of plutons appears to be generally gentle and why their cooling is so slow.
What does it look like?
Granite is made of large mineral grains (which is where its name came from) that fit tightly together. It always consists of the minerals quartz and feldspar, with or without a wide variety of other minerals (accessory minerals). The quartz and feldspar generally give granite a light color, ranging from pinkish to white. That light background color is punctuated by the darker accessory minerals.
Thus classic granite has a "salt-and-pepper" look. The most common accessory minerals are the black mica biotite and the black amphibole hornblende.


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