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November 25, 2015
ST. PETERSBURG, Fla. (AP) - Will Power has won the rain-delayed
Honda Grand Prix, his second IndyCar victory in as many races this
Power and Justin Wilson ran away from the field late, but Wilson
could never make a great run at the lead in the final laps. Power
prevailed by nearly a full second Monday, with Ryan Briscoe third and Helio Castroneves one spot behind him - that duo giving Penske Racing three of the top four spots.
Power was the polesitter after qualifying Saturday, and was the fastest driver in every practice session but one all weekend.
The race was scheduled for Sunday before thunderstorms put much
of the 1.8-mile street circuit under water. A total of 4.03 inches of rain fell in a span of about 19 hours, but the course was fairly dry when the green flag finally fell Monday morning.
(Copyright 2010 by The Associated Press. All Rights Reserved.)
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he may have become may favorite NFL player after seeing this video
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Eric Berry is scared...
Started by CockyinCola, Nov 13 2012 04:59 PM
Posted 13 November 2012 - 04:59 PM
Posted 13 November 2012 - 05:07 PM
Posted 13 November 2012 - 05:11 PM
lololol....too funny
"Whenever I see those crimson jerseys and crimson helmets, I feel humbled to have played football for Alabama. Other players in the NFL talk to me about their schools and their traditions. I just smile knowing the immense love Alabama fans have for our school and its football program. I'm proud to be a part of that Crimson Tide heritage."
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Art directors to honour animation guru Lasseter
Oscar-winning animation wunderkindJohn Lasseter, whose directing credits include Toy Story, A Bug's Life and Toy Story 2, will receive the Contribution to Cinematic Imagery Award at the 8thAnnual Art Directors Guild Awards in Los Angeles on Feb 14.
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Michigan State head coach Mark Dantonio watches from the sidelines Oct. 20, 2012.
EAST LANSING -- Michigan State has yet to make official the hiring of Jim Bollman for its football coaching staff, although that could come today.
Purdue coach Darrell Hazell confirmed Friday that Bollman was leaving his staff to become MSU's offensive coordinator. Bollman's move was first reported Wednesday by Coachingsearch.com.
MSU has yet to comment on the longtime Ohio State offensive coordinator and offensive line coach, who won the 2002 national championship at OSU under Jim Tressel. MSU coach Mark Dantonio was that team's defensive coordinator. ...


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When The Beauty Box—the popular apothecary with a curated selection of niche and well-known beauty products on Ventura Boulevard in Studio City, California—launched in 2005, it was an instant success with makeup artists, hairstylists, celebrities and consumers.
Luxury-beauty-products and spa-retail chain bluemercury has recently opened three locations, with more planned for 2012.
Woodley & Bunny now has a second brick and mortar—a West Hollywood, California, beauty apothecary—in the Los Angeles area.
Six years ago, co-founders Misha and Erin Anderson opened their Brooklyn, New York-based, hybrid apothecary and full-service salon.


text:
Jesper de Jong wrote:The int compareTo(Object other) method is indeed from the Comparable interface. The equals() method comes from class Object, the superclass of all classes in Java. Note that because equals() is in the Object class, all objects in Java always have an equals() method (if you don't declare one yourself in your class, it will inherit it from class Object). But not all objects have a compareTo() method; they will only have that if the class (or a superclass) implements the Comparable interface.
Type casting is a way to tell the compiler "look, I have an object of type X here, but I want you to treat it as type Y. Don't check it, just trust me!". What it essentially does is deferring type checking from compile time to runtime - when, while you run the program, it turns out that the object that you cast cannot be treated as type Y, you will get a ClassCastException.


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Cape Girardeau, MO – Americans for Prosperity-Missouri, the state's leading advocate for fiscal responsibility and prosperity, earned media more media coverage on its Economic Prosperity Report Card for the 2013 Missouri legislative session on KZIM 960/KSIM 1400 Radio.
The Missouri Economic Prosperity Report card continues to make its way throughout the state's media outlets as a tool for Missourians to find out how their elected officials voted on key issues of economic prosperity.
These scores were based on their votes opposing Medicaid expansion, Common Core education standards, a transportation sales tax proposal and a new utility surcharges, and supported measures for paycheck protection, lifting prevailing wage requirements and imposing spending caps.
AFP – Missouri State Director, Patrick Werner said, "The score card was based on the key issues from this session that impacted our mission of smaller government, lower taxes, and policies that produce economic freedom. The score card gives an A, B, C, D, or F grade and percentage to each member of the Missouri House & Senate based on how each member voted on legislation impacting our 2013 legislative agenda."
Werner added, "The purpose of creating a legislative score card is to provide our members and all Missouri taxpayers with a general overview of how their elected officials voted on key bills that impact our economic freedoms and liberties. AFP – Missouri is dedicated to free market policies that will improve the lives of Missouri families and businesses. Report cards shed light on who is standing up for limited government, lower taxes and regulatory
- The KZIM 960/KSIM 1400 article can be read here.
- To read the complete Missouri Torch article, please click here.
- For the KDKD AM article, please click here.
- The Joplin Globe article can be found here.
For a complete 2013 Economic Prosperity Report Card, visit www.MOAFP.org.
For further information or an interview, please contact Patrick Werner at firstname.lastname@example.com or 314-780-7687.
Americans for Prosperity (AFP) is a nationwide organization of citizen-leaders committed to advancing every individual's right to economic freedom and opportunity. AFP believes reducing the size and intrusiveness of government is the best way to promote individual productivity and prosperity for all Americans. For more information, visit www.americansforprosperity.org


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Archive for Thursday, October 6, 2005
Tuesday was another night of wins for the Eudora Cardinal volleyball team at Central Heights.
The team swept a league quadrangular featuring Anderson County, Spring Hill and the host Vikings, but it wasn't the fire and brimstone dominance that has been on display in recent Cardinal outings.
"We didn't come ready to play tonight," coach Jill Stutler said. "We didn't have any enthusiasm, and we didn't have focus during our rotations, and honestly we were lucky to come away with the wins."
The Cardinals began the night against Anderson County and came away with a relatively easy win in the first game 25-17.
But it was in game two that things began to get away from the Cardinals.
Eudora had built a seemingly insurmountable 17-4 lead and were poised to bury the Bulldogs and move on.
But a sudden lack of poise and focus descended over the court as the Bulldogs ripped off 14 of the next 19 points to close the gap to 22-18.
The Cardinals looked confused for most of that stretch, before finally regrouping with some strong net play from sophomore Nikki Snider, grabbing a 25-19 win.
"That's the first time that we've allowed a team to come from that far behind," Stutler said.
The ability to put teams away has been a strong point of the Eudora squad throughout the year, but Tuesday's contest against Anderson County affected the night's other matches.
The Cardinals took to the court in their second match against Spring Hill.
Eudora had little problem with the Broncos overall, winning 25-17, 25-14. But as the game went on the Cardinals lacked a little bit of the fire.
With the exception of a devastating Erin Kracl kill late in the first match, most of the Cardinal points came from the net in the form of tips and blocks.
In the third match the Cardinals took on the Central Heights Vikings. The host team was winless throughout the night, and Eudora kept it that way with a 25-11, 25-15 victory.
The Cardinals regained a little bit of their fire in the early stages of game one, building a staggering 10-1 lead.
The stretch included strong play from Kracl, who had two of her 20 kills in the run, and more consistent play from senior setter Kelsey Epperson.
Epperson finished the night with 49 assists.
The Vikings never recovered from that opening blitz and never got as close as 11 points.
Game two was more of the same for the Cardinals as the team kept the home squad at arm's length for the entire match.
Eudora led by as many as nine throughout the game before finally getting the 10-point win.
But the seeming return to dominance against the Vikings still did not wash away the taste of the earlier struggles.
"We need to work on our work ethic and defense," Stutler said. "Our defense is not where it should be if we're going to go as far as we think we can."
Eudora racked up six more wins Saturday, cruising through the Wellsville tournament undefeated and unchallenged.
The Cardinals knocked off Pomona, Baldwin, Waverly, Williamsburg and Kansas City Metro Academy in ridiculous fashion, never surrendering more than 13 points in any game. They beat Baldwin 25-11 and 25-1, then handled Waverly 25-4 and 25-6.
Eudora beat Wellsville for the championship, knocking off the home team in three games, 25-12, 21-25 and 25-19.
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I had the pleasure of viewing this vehicle in RIGHT HAND DRIVE, pre production in Australia this morning. It will be a direct competitor for the model E. Let me say that this is a very impressive car, albeit with funky looks. Yes, it is a commuter car, but BMW with its ICE package loaner for those rare occasions when one wants to travel long distance make it a compelling and realistic alternative for those of us waiting and waiting on the list (now for 3 plus years), with no store or sales staff in Australia to really give us realistic hope that the car really will be delivered here in the first half of 2014.
The car comes either in full electric or with range extender motorcycle 650cc engine, which gives it an extra 100 km (60miles) of range. Range with battery only is 130 - 160 km and with range extender quoted as 240 to 300 km.
Tesla really need to come forward with pricing for this market, since if they come in at say $150000 to $160000, they are going to lose sales, that to me included.
Yes, i know the advantages of the Tesla only too well. I have been to the factory in 2011 and had the test ride then, and have been assiduously following the forums ever since. A startup company in Australia is different to the USA especially since the factory is there. BMW has the advantage of gravitas.
In truth, the modelS is probably too large a vehicle for my needs now, but i am sold on the electric vehicle concept. The Volt does not appeal, mainly because it is a GM product, and i have had bad experiences with GM products previously.
Lots of wanna bee low end short range cars coming into the market. I bought Nissan Leafs to drive while I waited for a "real" EV. Tesla is the only true EV that can replace an existing ICE car.
You may have to learn EV on a shorter range less capable car while you wait for Tesla to deliver there. I had to do it for a few years while I waited for the Tesla to be available. The limited range and limited performance cars being rushed out by all the legacy automakers will just make you appreciate the Tesla more when you can finally get one.
The major car companies are all market cowards. They are trying to get into EV with econo box low end electric that are pricy for normal low end econo box buyers. Tesla was brilliant to start at the high end with a higher end high performance product. That is the market segment that can afford what it costs to go full EV with performance and quality. Everyone else is cautiously dipping their toe into the market with short range econo boxes.
Not a big fan of plug in hybrids liked the Volt. You end up wit the least of both worlds, a crappy ICE married to a limited short range EV with all the maintenance of both car types rolled into one. Go big or go home.
Right now Tesla is the only company that has gone big.
I don't think i3 is any competition for Tesla. Not even close. Just like Leaf is not a competition nor Chevy Volt. Tesla is in a different class. If BMW wants to compete with Tesla they need to first hire someone who can design a better car.
The I3 looks like a high speed collision between a Leaf and a Smart car. Would have to drive it with a bag over my head. That from someone that has owned and driven lots of BMW products over the years.
Good thing you liked the i3 since the Model S doesn't seem to be an option--good luck with that.
Nissan has been offering loaners to LEAF buyers for years.
What currency are you using $150-160k for the GenIII? Surely, that can't be AUD as the exchange ratio is close to 1:1. Everything so far indicates it will be around $35-40k USD, which will be less than the base model for the i3, much less with the range extender. Note that even with the range extender (which they don't want you to use in normal situations), you don't have as much combined range as the 85kWh Model S.
I think the I3 is more evidence that other EV manufacturers (with the exception of Ford) are trying to out ugly each other!
The model S may come in here at 150 to 160 Australian dolls even at parity with the US dollar. If so, that will be too rich for me. 125 to 130 then yes, i will get the Model S.
What are your customs and taxes like? What are you basing your price estimate on? Also, do you have any incentives for EVs? While a Volt isn't in the same league as a Tesla by any means, it could help you wait for it (maybe a used one or smth) but I think it is easiest if you email Tesla and ask them when they expect to be operational in Australia and have steering wheels on the wrong side :) Do you plan on placing a roadkill bumper on it? :)
Skip the i3 but you may consider a Fiat 500 electric in between since it is a fun little bastard but I don't think they sell them in Australia.
dborn, not sure what your time frame is, but it sounds like you've been waiting anxiously for quite some time (3 plus years).
Is there any great urgency to buy a car right now? If not, is it possible to wait, continue to save and make up the difference in the next 2 years through saving?
I had a similar experience about 3-4 years ago (with model S). I was in the market for a second car (one for wife and me). instead of buying something we held off for 4 years, and we basically saved and saved and saved. In the end, was able to afford the Model S because we planned and put aside money each month. Our main car was a honda civic hybrid (and you can imagine that the MS was an upgrade for us).
When that time rolls around, you can see what the differences will be between the GenIII and model S. If you opt for BMW i3 or Gen III, keep the 100K AUD and invest it. If you opt for the Model S, congrats and enjoy it ... you've earned it.
I guess the key is ... do you NEED a new car right now, or can you hold off?
I saw prototype i3 and i8 at BMW Welt during a recent trip to Munich. Both of them are very credible competitors to the MS. In terms of built-quality and fit / finishing and comfort / conv features, BMW is probably miles ahead of the MS. Though I'm anxiously waiting for my MS, but currently drive both BMW and Mini.
Having said that, I just don't get that spark within me to desire a i3 or i8 when comparing to the MS when I looked at them. If you're a rational person, you should go for i3 or i8 with BMW behind them; but if you like your car or the drive, this is going to be an emotional decision. I placed my order for the MS (unsight / un-test drive) because it looks cool and it's an awesome product concept.
Shame about Australian pricing for the MS (as well as i3 and i8), Australia is an unlucky country when it comes to buying a new car... hope this changes with Tesla. I was told for Asia-Pac markets, the base price will be similar to "Other European Markets", plus local applicable taxes.
If you can afford to wait for 2-3 more years, I believe Gen III and BMW i5 should be out by 2015 with similar and lower pricings. Hopefully you can decide with your heart then!
Always happy to hear when ICE manufacturers are (often against their will) begrudgingly taking the plunge into EVs. Especially BMW. But, it's frustrating that the range of the i3 is that of the Leaf for twice the price... and that they offer a side-by-side lawnmower engine - as if an EV drivetrain is still not enough for your driving needs.
There is one excellent analysis of the BMW i3 that really captures the essence of this:
I don't get it how i3 is competitor of MS? is like saying that BMW 3 series is competitor of Mercedes S class. i3 is competitor of Volt and the Leaf 44 Kw/h will be competitor of i3. Now that you love i3 "I'm BMW lover" tell me how the small ICE motor sound while charging like a blender?
Tesla gets glowing testimonials from those in the racing industry (Leilani Munter), those in the automotive media (Car and Driver, Motortrend), and Consumer Reports. Who does BMW have to say good things about the i3? Hold on to your pants, people...
Didn't click--are they using it as a golf cart?
Exactly. This video is posted at the BMW i3 web site. It's almost absurd to watch pro golfers drive an i3 and proclaim how wonderful it is. Maybe it's an inside joke at BMW, but why promote your flagship hybrid-in-EV-clothing as a golf cart?
Now, that is funny. Although didn't click.
Well, golfers do like carbon fiber.
Is there any automaker (besides Tesla) that has a pure EV in development that gets over 150 miles of range on a single charge? I don't count that fake GM announcement. Is there any other EV with a name on it?
This has always been the issue with the "traditional" car manufacturers. They don't want to cannibalize their mainstream money makers. Billions of R&D dollars were invested into their internal combustion engines and all related parts (transmission, exhaust system, etc.). Their supply management is fine-tuned to deliver the best pricing to support building an ICE vehicle. Their dealership networks are working in concert to squeeze as much money out of you in acquisition and ongoing maintenance. For them, it make little business sense to switch gears (pun intended) to build an EV that a consumer will want to drive.
"why promote your flagship hybrid-in-EV-clothing as a golf cart?"
Maybe they don't want to promote it at all.
Folks, I am a Tesla fan. However, don't get too blinded. This car makes a great deal of sense for those living in crowded cities such as New York, Chicago or San Fran. It does not make a lot of sense in other areas of the USA . It makes a great deal of sense in most of Europe. If you rarely drive more than 30 or 40 miles a day, then this car could well work for you. It's displays are hi res and the main screen is 10 odd inches. If only two people are likely to use it most of the time such as retirees, it is a really good option. It has a high level of equipment and safety features and all the latest technology. Yes the appearance is funky, but google the reviews, they are universally positive with some detractions such as looks especially from the rear, but from a technical standpoint the reviews are basically all favorable.
The first clear demonstration that the BMW grill is mean to subliminally suggest a bust! Sex sells.
mean meant
No kidney, BrianH? ;-)
I always think of this when I read the word "bust":
Is that the first archeological evidence of an afro hairdo?
Another Roman invention? Who would have known!
I would not expect much love for the i3 on a Tesla forum.
For the record, I have a reservation for the Model X. I firmly believe that my Model X is about two years away since I am number 6336 and will not order a loaded X for cost reasons.
So far, I really like what I see in the i3. It is a quirky little city car with decent performance, luxury and range for it's cost. I actually like the looks. I really like the interior. It will have a higher ride height and seating position for people like me (and my wife) with bad backs. This is why I did not order a Model S. We are going to order one next month with delivery in the spring of 2014. We are going to order the REX version as we will probably make a trip or two each month just over the 80-90 mile range of the pure BEV model.
I realize that the models S is better in almost every way, but it is also at least $28,000 more expensive. I still think i3 will be a comfortable and fun little car to drive around town. The 0-60 in 7-8 sec performance is not exactly a "golf cart" as a few of you have stated. I also own a converted EV with a similar range (and even less performance) and find it extremely fun to drive. Admittedly it is mostly a toy.
You can see it at www.EVThing.me if you like.
The wife likes my little car too, but wants a modern EV with all of the creature comforts. The i3 is a bit of a compromise, but if her i3 performs well over the next year I would be very tempted to cancel the Model X and just get an REX i3 for myself. If the i3 does everything I need and it is fun to drive, I would have a hard time justifying the extra $30,000 to $40,000 to get the Model X.
I know I would prefer a Tesla Model ___ (GEN III), but that is at least 4-5 years away and I do not want to wait that long so the Model X is the only real alternative for me...
I like you reasoning but its seems odd that you would have such a wide range of what you want out of an EV. The Model X and the i3 are so different. Why even consider an EV like the Model X if you don't need or really want it?
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text: Snooker: The Masters 2014 Fourth Quarter-Final Facebook Twitter Google Tumblr Pinterest Blogger Reddit VKontakt Print Email About this programme Neil Robertson v Stephen Maguire. Hazel Irvine presents highlights of the fourth quarter-final match in the non-ranking tournament at Alexandra Palace in London. The winner of this contest would set up a semi-final against the winner of the day's early game between Ricky Walden and Ronnie O'Sullivan. With commentary by Dennis Taylor, Ken Doherty, Stephen Hendry and John Virgo, and analysis by Steve Davis and John Parrott. Cast and crew Cast Guest Steve Davis Guest John Parrott Commentator Dennis Taylor Commentator Ken Doherty Commentator Stephen Hendry Commentator John Virgo Presenter Hazel Irvine Crew Director Steve Docherty Director Simon Wheeler Executive Producer Alison Witkover Categories Sport Facebook Twitter Google Tumblr Pinterest Blogger Reddit VKontakt Print Email


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Having a great display and unique items can make your craft fair experience a success. This guide is about selling crafts at craft shows.
Read and rate the best solutions below by giving them a "thumbs up".
By Linn from Dartmouth, Nova Scotia
Need ideas on displaying your items at craft shows or in your booth?
The number one thing in displaying your crafts is make sure all of your items are priced and the tag can be easily seen. 9 times out of 10 those few items that are not priced will be walked away from because most shoppers are just simply too "shy" to ask.
Even if your not pricing your items but are using the "Here's your Sign" tactic by displaying a large sign stating something like 1 for 2.99 buy 2 for $5.00, I suggest to still price everything! Shoppers are so excited and distracted from looking through all the products and discussing the oohh's and aahh's with their shopping friends that the signs are sometimes missed especially in large craft shows where they get crowded.
Make your display booths unique and interesting to draw in attention. Using a folding table with a country plaid tablecloth are great, but why not really spice it up and use something out of the ordinary to grab the shopper's eye from across the room. These are depending on your space size of course, or could be scaled to fit.
Some other ways to display items are using baskets, ladders, stacking suitcases, crates, crocks, coat racks, stacking various sized tables, cutting barrels in half and adding shelving inside, Christmas trees, old galvanized wash tubs, dress mannequins, wood ironing boards, quilt racks, shutters, or fencing.
And one last craft display tip. Don't forget the kids! Most shoppers will be bringing along young children. Have items to catch a child's eye down low or invest in a bubble gum machine and fill it with sweets. A few turns on the irresistible machine will make you some profits as well.
Give a "thumbs up" to the solution that worked the best! Do you have a better solution? Click here to share it!
Here are questions related to Selling Crafts at Craft Shows.
What crafts are selling best at craft shows?
Jean from Columbus, Ohio
AnswerWas this interesting? Yes No
By Cyinda [214] 03/06/2008
I do craft show for a living (I sell hand painted pottery) and the one thing that always sells and makes money is garden crafts. It may be actual plants, or seeds or live floral wreaths or flowers planted in unusual things like in boots or old chairs or tires. Wooden window boxes always sell too, or even garden art or yard furniture or bird baths and stepping stones, but let me tell you, anything for the yard or garden sells like crazy!
You gotta have 2 price scales; the pricey stuff and also something to sell that's under 20 or 30 dollars. The lower priced stuff is what people will buy without thinking to hard about it.
Craft fairs aren't bringing in the money they used to (Believe me!), with the economy these days being so bad you have to have something either people need and is useful or is super cheap that they can buy for a gift. I'm making less than half what I made before 2000. Really! Even though I have a great reputation and people know me better and better each year). When the economy sucks, so do sales at craft fairs (unless you're selling food items). I sometimes drive 180 miles (round trip) to sell my stuff "where the money is". Try to sell your stuff at a fair where the people with money live. Get my drift!?
In my community, craft fair season is fast approaching and I would like to try selling my own handmade items. Since I have never done this before, any suggestions would be helpful. What sells well? How do I determine how much to charge for each item? (For example, I like to rag quilt, so do baby quilts sell better than twin or full size? I'd rather make 3 baby quilts that sell than 2 twin size that don't.)
Do I need to claim my earnings on my taxes and do I obtain a tax-exempt number to buy my materials? If so, how? I have been told that I can't sell fabric items with licensed characters on the fabric, why? Thanks for the help?
By Marie from Idaho Falls, ID
AnswerWas this interesting? Yes No
By Ivalou 05/11/2013
Here in Lawton, Oklahoma our craft club has one large craft show a year. We mark off 211 10X10 ft booths. We rent these booth to mostly crafters. If we do not fill all booth we will allow select comercial booths. Items have to be sold not party set up. We have great sucess each year. We are always looking for new crafters.
Many of our crafters have campers and stay on the grounds where the show is happening. There are hook ups for campers. We provide security inside the building each day and over night to protect our booths. We have some members who live out of state. Many of our crafters who are not members come back year after year. Contact ivazimmerman AT aol.com for more information.
I'm looking for craft fairs that will give you a booth for low cost or free. I'm a first timer and don't want to spend a bunch of money for a booth right now. Are there any craft fairs in Minnesota that have really cheap or free available booths for selling?
By Danica
AnswerWas this interesting? Yes No
By Marla Ball 01/16/2013
You're so right in thinking of doing the lower dollar craft shows. These shows are so. Participating in such shows can be so iffy. You just never know how well things will go. Keeping the expense of participating down betters your opportunities of doing well. Googling "Craft Shows" is a great way to get links to various sights that list shows around the country. I know there is a Church there in MN that does a Summer event each yr. I've donated a gift basket to them for their event before. Best of luck!
Does anyone know of a website or place that lists all upcoming craft shows, fairs, and markets for a certain area? I am in Chicago and I'm interested in selling handmade purses and pillowcases. I am having trouble finding places other than online. I have never sold anything, but now I'm ready with enough product. Do I really have to wait until summer? Don't these things happen indoors throughout the winter?
By down n dirty from Chicago, IL
AnswerWas this interesting? Yes No
By Georgetta Ruth [72] 01/01/2011
Here is a link that I have used in the past.
http://www.eventlister.com/. You can also check with local churches, schools andevent planners. Where at in Chicago are you located. I use to live there and I did a number shows in the area.
Please help. I would like to sell my handmade patchwork tote bags (made out of old jeans and new fabric) at a local farmer's market this summer, but I don't know how much to charge. I know how much the jeans and fabric cost, but I don't know how much to charge for labor, thread, use of my machine, etc. I would also need to invest in a canopy and tables for my booth. The bags are an easy technique, but it takes 2-3 days to make a bag, if I don't have any other wife/mom things to do. Plus, I live in a community with a lot of crafters who could make the items they see at a craft fair and people who like to negotiate for a good price. Thus, I need to find a fair price that will not scare away potential buyers, but will give me room to negotiate and still make a decent profit. How do I make my craft fair dreams happen?
By Marie V.O. from Idaho Falls, ID
Thrifty Fun has been around so long that many of our pages have been reset several times. Archives are older versions of the page and the comments that were provided then.
My daughter Katie and I would like to make crafts items to sell at local craft shows. We have some ideas but would like more. Does anyone know of some good websites for free craft patterns and ideas? Thanks! Sharon Young from Benton, AR
By bjptl
By CaseyT6
By Mr. Thrifty
By Amber
I live in Essex in the UK and along with a friend, we would like to start making and selling at craft fairs to enable us to continue to stay home with our children.
I thought I would ask for suggestions on here as being an american site, you would probably come up with suggestions that we don't have over here.
Thanks in advance,
Tracey from Essex
Make sure your displays are really good. If you sell earrings, look for earring racks or some unique way to display your earrings. If you sell dolls, make sure they can sit or stand to display them best. Whatever you are selling, make sure that it is easy to see from a distance what you have. Use nice table coverings that match and tie your booth, table or displays together.
I've bought and sold crafts for years and just like the big merchandisers how you present yourself and your products really helps make sales. It doesn't have to cost a lot but always stand back and look at your displays before starting your sale. Anything that distracts you from the "goods" should be avoided (i.e. a rusty table leg, a stain, dirty mirror, papers sitting around, loose threads).
Once you are selling, figure out what sells the best and concentrate on those items. Also pay attention to where the items were placed as that can also make a difference. Make sure all of your items are priced.
If there are two of you, avoid both of you sitting staring out waiting for people to come. Have one of you stand up, walk out and walk up to the booth, people tend to go to tables where other people are. It's funny but it works. (01/20/2007)
My friend and I have been setting up at craft shows for about 8 years. We have gone with all homemade items and we have made items that are on the normal persons budget. Most of our items are $5.00 and under. We make crochet towels, crocheted slippers, mittens, hats, dish cloths, scrubbies out of nylon net, barbie cloths, and we also make many gag gifts that we sell for 50 cents a piece. We are busy from the time we get set up to the end of the day. People love to be able to buy something even if it just a little item. We do have a few higher priced item at different shows but keep your booth friendly and bright. We sometimes have a tape player playing very soft music, helps make people feel welcome. Good luck! (01/22/2007)
By dameemag
I am a stay at home mom that wishes to earn extra income selling crafts at craft fairs/flea markets. Any ideas or suggestions of crafts that sell well?
I am going to start selling at a local market day in my town and I was hoping for some advice. I like to sew and so far I have decided to sell children's aprons and chef hats.
Is it necessary to disclose the origin of fabric at a craft fair? I sew dog dresses to sell at craft fairs and although I do buy new fabric from the store, I also get fabric from my local Goodwill.
Does anyone know how I would find out who to contact about selling my crafts at craft shows or festivals in my local area?


text:
For Barack Obama, The Party's Never Really Getting Started
- For those of you expecting a week-long party to start off the Obama Administration, prepare to be disappointed. There will be one public event welcoming you to D.C. on Sunday afternoon. Monday will be a day of community service, and the Inauguration will occur on Tuesday out in the cold, followed by formal balls… »


text:
Proteases regulate pathogenesis in apicomplexan parasites but investigations of proteases have been largely confined to the asexual stages of Plasmodium falciparum and Toxoplasma gondii. Thus, little is known about proteases in other Apicomplexa, particularly in the sexual stages. We screened the Eimeria tenella genome database for proteases, classified these into families and determined their stage specific expression.
Over forty protease genes were identified in the E. tenella genome. These were distributed across aspartic (three genes), cysteine (sixteen), metallo (fourteen) and serine (twelve) proteases. Expression of at least fifteen protease genes was upregulated in merozoites including homologs of genes known to be important in host cell invasion, remodelling and egress in P. falciparum and/or T. gondii. Thirteen protease genes were specifically expressed or upregulated in gametocytes; five of these were in two families of serine proteases (S1 and S8) that are over-represented in the coccidian parasites, E. tenella and T. gondii, distinctive within the Apicomplexa because of their hard-walled oocysts. Serine protease inhibitors prevented processing of EtGAM56, a protein from E. tenella gametocytes that gives rise to tyrosine-rich peptides that are incorporated into the oocyst wall.
Eimeria tenella possesses a large number of protease genes. Expression of many of these genes is upregulated in asexual stages. However, expression of almost one-third of protease genes is upregulated in, or confined to gametocytes; some of these appear to be unique to the Coccidia and may play key roles in the formation of the oocyst wall, a defining feature of this group of parasites.
Keywords:Eimeria; Apicomplexa; Protease; Protease inhibitors; Gametocyte; Oocyst wall
Proteases are essential regulators of pathogenesis in the Apicomplexa, a phylum that includes obligate, intracellular protozoan parasites of great human health (e.g., Plasmodium species, causing malaria, Toxoplasma gondii, causing toxoplasmosis, and Cryptosporidium, causing cryptosporidiosis) and agricultural and economic significance (e.g., Neospora caninum, the cause of foetal abortion in cattle, and Eimeria species, the causative agents of coccidiosis in poultry, cattle, sheep and rabbits). Extensive study of Plasmodium species and T. gondii has established that proteases help to coordinate and regulate the lifecycles of these parasites, playing key roles in host cell invasion, general catabolism, host cell remodelling and egress from host cells [1]. These processes are all associated with the asexual stages of apicomplexan parasites. By contrast, relatively little is known about what roles proteases may play in the sexual phase of the apicomplexan lifecycle though it is known that a subtilisin 2 is detected specifically in the gametocyte proteome [2] and expression of falcipain 1 is upregulated in gametocytes [3] of P. falciparum. Moreover, it has been demonstrated that the cysteine protease inhibitor, E64d, or the targeted genetic disruption of falcipain 1 can inhibit oocyst production in P. falciparum[3,4]. Likewise, the proteosome inhibitors, epoxomicin and thiostrepin, exhibit gametocytocidal activity [5,6].
In comparison to P. falciparum and T. gondii, proteases from Eimeria species have been studied far less intensively, despite the economic importance of this genus of parasites. Thus, homologs or orthologs of several classes of proteases found in P. falciparum and/or T. gondii have also been identified in Eimeria species including an aspartyl protease [7-10], an aminopeptidase [11], a rhomboid protease [12,13], a subtilisin 2-like protease [10,13,14], three cathepsin Cs [15], a cathepsin L [15] and an orthologue of toxopain, a cathepsin B cysteine protease [14,15]. As for P. falciparum and T. gondii, these proteases have been found in the asexual stages of Eimeria and are mostly predicted to play roles in host cell invasion, though expression of some of these enzymes is associated with the sporulation of the developing oocyst [11,13,15]. However, it is hypothesized that proteolytic processing of two proteins from the wall forming bodies of the macrogametocytes of Eimeria – GAM56 and GAM82 – is essential for the subsequent incorporation of tyrosine-rich peptides into the oocyst wall [16].
In this study, we screened the E. tenella genomic database for genes encoding proteases, classified these into clans and families and designed PCR probes for them. Using cDNA produced from E. tenella stage specific mRNA, we carried out semi-quantitative PCR to determine the stage specificity of expression of the protease genes, especially to identify protease mRNAs that were upregulated in gametocytes. In order to further resolve which of these may be involved in oocyst wall formation, we carried out a processing assay using gametocyte extracts of E. tenella, whereby a variety of specific protease inhibitors were tested for their ability to inhibit the processing of GAM56 into smaller, putative oocyst wall proteins.
Identification of potential protease genes in Eimeria tenella
The genome of E. tenella (Houghton strain) was sequenced by the Parasite Genomics Group at the Wellcome Trust Sanger Institute and provided pre-publication for the current analysis. The Parasite Genomics Group plan to publish the annotated sequence in a peer-reviewed journal in the coming future. The E. tenella genome database (http://www.genedb.org/Homepage/Etenella webcite) was explored to identify genes that were automatically predicted to code for aspartic, cysteine, metallo and serine proteases. Database mining revealed over 60 gene sequences whose predicted open reading frames were associated with potential peptidase activity. Manual annotation of the genes was performed by BLAST search of apicomplexan genome databases to identify phylogenetically closely related nucleotide sequences and by BLAST search of various protein databases to identify the most closely related, experimentally characterized homologs available (Table 1). Additionally, the predicted proteins were analysed for conserved motifs and domains to further validate protein function (Table 1). Each predicted protein was then assigned a five-tiered level of confidence for function using an Evidence Rating (ER) system (Table 1). The evidence rating system, described previously [17], allocates genes an overall score (ER1-5), indicating how compelling the bioinformatic and experimental evidence is for protein function. An ER1 rating signifies extremely reliable experimental data to support protein function in the particular species being investigated, in this case Eimeria, whereas ER5 indicates no experimental or bioinformatic evidence for gene function. Genes with an ER5 were eliminated from further investigation. After this validation process was performed, 45 putative protease genes remained and these could be classified into clans and families of aspartic, cysteine, metallo and serine proteases (Table 1), including: three aspartic proteases, all within family A1 in clan AA; 16 cysteine proteases, the vast majority (15) of which were in clan CA, five being cathepsins (family C1), one calpain (family C2), eight ubiquitinyl hydrolases (family C19) and one OTU protease (family C88), as well as a single clan CF pyroglutamyl peptidase (family C15); 14 metallo proteases, distributed over five clans (MA (6), ME (5), MF (1), MK (1) and MM (1)) and seven families (M1 (2), M41 (3), M48 (1), M16 (5), M17 (1), M22 (1) and M50 (1)); and 12 serine proteases in clan PA (three trypsin-like proteases in family S1), clan SB (six subtilisin-like proteases in family S8), clan SC (one prolyl endopeptidase in family S33), clan SK (one Clp protease in family S14) and clan ST (a rhomboid protease – rhomboid protease 1 – in family S54). Three additional rhomboid proteases were identified in the E. tenella genome database by using BLASTP to search the database using, as queries, homologs described in T. gondii: rhomboid protease 3 (ETH_00032220, Supercontig_69: 140161–141340; 4.0e52); rhomboid protease 4 (ETH_00009820, Supercontig_44: 17996–24858; 9.8e-164); and rhomboid protease 5 (ETH_00040480, contig NODE_916_length_3953_cov_17.775614: 53–3466; 7.2e-65). However, we were unable to confirm coding sequences or stage-specific expression for any of these three genes.
Table 1. Protease genes identified in the Eimeria tenella genome database
Stage-specific protease gene expression
To assess the stage specific gene expression of putative proteases identified in the E. tenella database, different stages of the parasite lifecycle were isolated and total RNA purified. These stages included merozoites, 134 h gametocytes, unsporulated oocysts, sporulated oocysts as well as uninfected caeca control tissue. RT-PCR was performed and the stage-specific cDNA samples were subjected to control PCRs to determine purity (Figure 1). Purification of merozoite and gametocyte lifecycle stages inevitably results in co-purification of host tissue, hence, the E. tenella β-actin structural gene was amplified to optimize relative amounts of parasite starting material as described previously [18]. The E. tenella β-actin gene was amplified from each of the parasite lifecycle cDNA samples and quantification of bands visualized by agarose gel electrophoresis allowed the specific E. tenella cDNA to be standardized to each other accordingly. The E. tenella gam56 gene product, which is predominantly expressed in gametocytes but largely down-regulated in unsporulated oocysts, confirmed the quality of gametocyte cDNA and served as a gametocyte-specific positive control, establishing the lack of gametocytes in merozoite and oocyst samples. The amplification of the tfp250 gene, specifically expressed in the asexual stages [19], indicated contaminating merozoite cDNA in the gametocyte cDNA sample, as anticipated, at the 134 h time point. Furthermore, amplification of a chicken host-specific lysozyme gene indicated host cDNA was present in both merozoite and gametocyte preparations, also as anticipated.
Figure 1. RT-PCR assessment of purity of Eimeria tenella merozoites, gametocytes and oocysts. Total RNA was extracted from E. tenella merozoite (M), gametocyte (G), unsporulated oocyst (UO) and sporulated oocyst (SO) preparations. cDNA was synthesized and stage-specific parasite genes (GAM56 for gametocytes; TFP250 for merozoites and sporozoites) was amplified by RT-PCR. PCR samples were run alongside chicken host cDNA (H), E. tenella genomic DNA (gD) and a negative control sample with no template (−) on a 1% agarose gel. The E. tenella β-actin gene product was used to standardize loading of parasite cDNA samples.
After optimisation of parasite lifecycle stage cDNA samples, primer pairs were designed to generate PCR products from exons of less than 1 kb in size, where possible. PCRs were performed at optimal annealing temperatures specific for the individual primer pairs and annealing times optimal for predicted cDNA sized products. PCRs were performed at least twice (and normally three times) for each gene product, by different researchers each time. In the case of failed PCRs, primer pairs were redesigned and retested. Results of PCR on the different lifecycle stages of E. tenella indicated that 40 of the 45 protease genes could be amplified from parasite cDNA (Table 2). The five PCRs that failed to amplify a product from cDNA were for three of the eight ubiquitinyl hydrolases, the single OTU protease and one of the six subtilisins. However, it was possible to amplify PCR products from gDNA for all five of these proteases that, when sequenced, confirmed primer specificity (data not shown). The failure to amplify a product from cDNA for these genes may be due to genome annotation problems; possibly the sequence targeted by our primers is not transcribed or falls in unpredicted intronic regions. Alternatively, a low abundance of these transcripts may have contributed to the failure to detect cDNA amplification products. Further work will be required to characterize these genes. All other PCR products from cDNA from the four E. tenella lifecycle stages were directly sequenced to confirm the correct coding sequence. Expected and actual cDNA amplicon sizes and their corresponding sequence accession numbers are shown in Table 2.
Table 2. Expression of protease genes in merozoites, gametocytes, unsporulated oocysts and sporulated oocysts of Eimeria tenella
The majority of the protease genes were expressed in more than one of the four parasite stages investigated (Table 2). However, stage-specific up- or downregulation of protease gene expression was evident. Thus, taking into account that merozoite cDNA contaminates the gametocyte samples, it is safe to conclude that there were a large number (at least 15, probably 17) of protease genes whose expression was upregulated in merozoites including eimepsin 3, cathepsin C1, calpain, several of the ubiquitinyl hydrolases, an ATP-dependent Zn protease, the CAAX prenyl protease, three of the five insulysins, the leucyl aminopeptidase, the O-sialoglycoprotease, one of the trypsins, a subtilisin, the Clp protease and rhomboid protease 1. Aminopeptidase N1 appeared to be downregulated specifically in merozoites. Gametocyte-specific or gametocyte-upregulated proteases were also common, with thirteen in all, also distributed across the four groups of proteases, including eimepsin 2, cathepsin C2, ubiquitinyl hydrolase 2 and 5, the pyroglutamyl peptidase, aminopeptidase N2, insulysin 4, the S2P-like metalloprotease, two trypsin-like proteases and three of the subtilisins. Additionally, two other proteases were upregulated or specific for the sexual phase of the lifecycle (i.e., gametocytes and unsporulated oocysts), namely, cathepsin C3 and subtilisin 4. Cathepsin L appeared to be downregulated specifically in gametocytes. Only two protease genes, a pepsin-like protease with high homology to eimepsin (eimepsin 1) and an insulysin, were switched on exclusively in oocyst lifecycle stages. In contrast, numerous protease genes appeared to be downregulated in sporulated oocysts (Table 2).
Protease processing of GAM56
Gametocytes from E. tenella-infected caeca were lysed and immediately incubated with or without protease inhibitors for various lengths of time, and the native GAM56 protein analysed by SDS-PAGE and western blotting with anti-GAM56 antibodies, as described previously [20,21], to track the disappearance of the protein to determine whether any inhibitors could prevent the degradation observed in the presence of native gametocyte proteases. The precise epitopes recognised by the anti-GAM56 polyclonal antibodies are not known for E. tenella though there is some evidence, from work with E. maxima[21], that they are located in the conserved amino-terminus of the protein. The anti-GAM56 antibodies are, thus, very useful for sensitive and specific tracking of the degradation of GAM56. No disappearance of GAM56 was apparent after 2, 4, 6, 8, 10, 12 or 16 h (data not shown) but was obvious at 24h (Figure 2). The 24 h assay was therefore repeated three times with a comprehensive range of protease inhibitors (Table 3) targeting the four protease families identified in the genome. The aspartyl protease inhibitor, pepstatin A, had no effect on GAM56 disappearance (Figure 2). None of three cysteine protease inhibitors investigated, Z-Phe-Ala-diazomethylketone (data not shown), N-ethylmalemide (data not shown) or E64 (Figure 2) inhibited GAM56 disappearance. The serine/cysteine protease inhibitor, chymostatin (data not shown) and leupeptin (Figure 2), inhibited GAM56 disappearance but another inhibitor with the same specificity, antipain, did not (data not shown). The serine protease specific inhibitors, benzamidine HCL (data not shown), soybean trypsin inhibitor (data not shown) and aprotinin (Figure 2) all inhibited the disappearance of GAM56 but AEBSF did not (Figure 2). The metal chelating agent, EDTA, also inhibited the disappearance of GAM56 but more specific metalloprotease inhibitors, bestatin and phosphoramidon, did not (Figure 2).
Figure 2. The effect of protease inhibitors on degradation of GAM56 in Eimeria tenella gametocytes. A sample of purified E. tenella gametocytes was lysed and equal volumes of lysate added to a range of protease inhibitors (see Table 3 for details on concentrations and specificity) or PBS. A zero time point sample was taken immediately. Other samples were incubated for 24 h at 37°C, after which the assay was halted by addition to Laemmli buffer and the samples subjected to SDS-PAGE and immunoblotting as described previously [20,21] to assess the disappearance of GAM56. Lane 1, time = zero sample; Lane 2, pepstatin A; Lane 3, E64; Lane 4, aprotonin; Lane 5, AEBSF; Lane 6, bestatin; Lane 7, leupeptin; Lane 8, phosphoramidon; Lane 9, EDTA; Lane 10, a cocktail of pepstatin A, E64, aprotonin, AEBSF, bestatin, leupeptin, phosphoramidon and EDTA; Lane 11, control sample containing no protease inhibitors.
Table 3. Protease inhibitors used in the Eimeria tenella GAM56 processing assay
Mining of the E. tenella genome database has revealed over 40 protease transcripts distributed over 13 clans and 18 families of aspartic, cysteine, metallo and serine proteases. Such diversity of proteases is not unusual, indeed it may be an underestimate of the true number of protease genes in this parasite since other apicomplexan parasites are known to possess substantially more protease genes (Table 4); thus, for example, there are at least 70 in Cryposporidium parvum, more than 80 in P. falciparum and over 90 in T. gondii, though other apicomplexan parasites possess similar numbers of protease genes as E. tenella. Eimeria tenella also has lower numbers of protease genes than protozoan parasites like Leishmania, Trypanosoma and Trichomonas (though the latter is known to have an unusually expanded genome in general [22] and, apparently, in C1 and C19 cysteine proteases and M8 metalloproteases, in particular; Table 4). But, again, E. tenella has a broadly similar total number of protease genes to Entamoeba dispar and Giardia intestinalis, which are also intestinal parasites. However, the fact that our dataset for E. tenella lacks protease genes for several families, across all four types of proteases, that are represented in all other Apicomplexa and most other protozoan parasites, including A28, A22, C12, C85, C86, C13, C14, C50, C48, M24, M18, M67, S9, S26 and S16, provides reason to believe that some E. tenella protease genes remain unannotated.
Table 4. Distribution of protease clans and families in Eimeria tenella, its host and other protozoan parasites
The apparent stage-specific regulation of protease genes in E. tenella is striking and intriguing. Most investigations of parasitic protozoan proteases have focused on the asexual stages of the apicomplexan parasites, T. gondii and P. falciparum, establishing crucial roles for proteases in host cell invasion, remodelling and egress by the asexual stages of these parasites [1]. Our finding that expression of up to 17 of 40 protease genes examined in E. tenella is upregulated in merozoites further underscores the importance of proteases in the biology of the asexual stages of apicomplexan parasites. Not surprisingly, therefore, an eimepsin, several cathepsins, a calpain, a trypsin-like protease, subtilisins, Clp and a rhomboid protease are upregulated in the asexual stages of E. tenella (Table 2). Likewise, eimepsin1 and insulysin 3 are expressed specifically in oocysts and may play an important role in the first steps of the parasite lifecycle, such as host cell invasion; they are, therefore, worthy of further research. The downregulation of several proteases (including cathepsin B, ATP-depenedent ZN proteases 1 and 3, and a prolyl endopeptidase) in sporulated oocysts may be, in part, attributed to the dormancy of this lifecycle stage, yet still warrants further investigation.
Perhaps the most significant finding of our stage-specific expression study was the relatively large number of protease genes whose expression is upregulated specifically in the gametocytes stage – a total of at least 13 genes, including six that are only expressed in gametocyte (Table 2). This observation becomes even more intriguing when examined in the context of the distribution of different families of proteases across parasitic protozoa (Table 4). Four classes of proteases stand out amongst the protozoa because they are only found, or are "over-represented" in the two Coccidian parasites, E. tenella and T. gondii – families C15, M50, S1 and S8. Eimeria tenella contains a total of eleven protease genes distributed unevenly across these families, with only one in C15 and M50 and three and six in the serine protease families, S1 and S8, respectively. But, even more significantly, all but three of these unique protease genes are upregulated or confined in expression to the gametocyte stage of the parasite. Thus, expression of a pyroglutamyl peptidase, a trypsin-like protease and subtilisin 4 is upregulated in gametocytes whilst expression of an SP2-like protease, a trypsin 1-like protease and three subtilisins is entirely gametocyte specific.
One of the defining features of the Coccidia is the possession of a hard-walled oocyst that originates from specialized organelles (wall forming bodies) in macrogametocytes. It is hypothesized [16] that degradation of two proteins found in the wall forming bodies of macrogametocytes of Eimeria, namely GAM56 and GAM82, is integral to oocyst wall formation; tyrosine-rich peptides formed by the degradation of these two proteins are believed to be subsequently cross-linked via dityrosine bonds [23], giving the oocyst wall its renowned strength and resistance to environmental and chemical insults [24]. To test this hypothesis, we designed an assay to follow the degradation of GAM56 in freshly harvested gametocytes (Figure 2). This assay has certain inherent limitations: first, it relies on sensitive antibodies for detection of specific degradation of GAM56 and, unfortunately, the lack of suitable antibodies for detection of GAM82 in E. tenella[21] meant that we were unable to run confirmatory experiments with this protein; and, second, the only controls possible are a zero time point and a cocktail of protease inhibitors designed to prevent all proteolytic activity. These limitations require us to be cautious in our interpretations; none-the-less, the inhibition of degradation of native GAM56 by a very specific group of protease inhibitors reveals that this function may be carried out by subtilisin-like proteases. Thus, degradation of GAM56 was inhibited by the serine/cysteine protease inhibitors, chymostatin and leupeptin, and the serine protease specific inhibitors, benzamidine HCL, soybean trypsin inhibitor and aprotinin but not by AEBSF (Figure 2). Intriguingly, the metal chelating agent, EDTA, also inhibited degradation of GAM56. This profile indicates that serine proteases are critical for degradation of GAM56 but it seems to rule out participation of rhomboid proteases, which are unaffected by EDTA, aprotonin, leupeptin and chymostatin [25]. Trypsin-like proteases can, perhaps, not be completely ruled out of this process but the inhibitory profile, particularly the lack of inhibition by AEBSF coupled with the inhibitory effect of EDTA, points to a subtilisin or subtilisins as P. falciparum subtilisin 1 is inhibited in exactly the same fashion [26].
Subtilisins are further implicated in the formation of the oocyst wall of Eimeria through analogy with their known role in the formation of the cuticle of nematodes. Thus, the assembly of collagens to form the cuticle involves a number of molecular events that strikingly resemble our model of oocyst wall formation pathways: first, collagens are the result of degradation of proproteins by a subtilisin-like protease [27-30]; and, second, these collagens are subsequently bonded together by di- and tri-tyrosine crosslinks [31]. A failure in either of these steps, results in a malformed cuticle and parasite death [31]. Subtilisins are currently being further investigated as potential candidates in the catalytic cleavage of the oocyst wall precursor proteins.
Eimeria tenella possesses a large number of genes coding for proteolytic enzymes, which display a remarkable pattern of stage specific expression. As in other apicomplexan parasites such as P. falciparum and T. gondii, expression of many of these genes is upregulated in the asexual, invasive stages, possibly indicating important roles in host cell invasion, remodelling and egress. However, expression of almost one-third of the protease genes identified in the E. tenella genome is upregulated or confined to the sexual gametocyte stage of this parasite's lifecycle; some of these appear to be unique to Coccidia and may play key roles in the formation of the resilient oocyst wall, a defining feature of this group of important parasites.
Data-base mining
Eimeria tenella genome sequences and gene models were downloaded from GeneDB (http://www.genedb.org/Homepage/Etenella webcite). The genome of E. tenella (Houghton strain) was produced by the Parasite Genomics Group at the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/research/projects/parasitegenomics/ webcite) and has been provided prepublication. The E. tenella genome database was searched for genes predicted to code for proteins with peptidase activity. All auto-annotated peptidase genes identified were manually curated by performing BLAST analysis against apicomplexan genome sequence databases and against various protein databases [32] such as the protein data bank (PDB), Swiss-Prot and non-redundant (NR) protein sequence databases. In addition, signature protein motifs for the protein sequence of each gene were identified through Pfam (http://pfam.sanger.ac.uk/search webcite; [33]), InterproScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/ webcite) and the MEROPS databases (http://merops.sanger.ac.uk/ webcite; [34]). Further gene sequence manipulations, such as translation into amino acid sequences and ClustalW alignments, were performed using the DNASTAR Lasergene™ 9 Core Suite. After the bioinformatic information was collated, genes were assigned a five-tiered level of confidence for gene function using an Evidence Rating (ER) system giving an overall score of ER1-5, where ER1 indicates extremely reliable experimental data to support function and ER5 indicates no evidence for gene function [17].
Animals and parasites
One day old chicks (Australorp; Barter and Sons Hatchery, Luddenham, Australia) were housed at the Ernst Facility Animal House (University of Technology, Sydney), under heat lamps for the first 2 weeks of their life and, thereafter, at 21°C with a 12 hour light/dark cycle with free access to food and water. Chickens were infected orally at 4.5 weeks of age with 2.5 × 103 sporulated oocysts of Eimeria tenella (Houghton strain originally provided by Janene Bumstead, Institute for Animal Health, Compton, UK). Fresh E. tenella oocysts were harvested 7 days post infection from the caeca following protocols published previously [35]. Sporulation of oocysts was carried out at 28°C for 72–120 hours using a low-pressure aquarium pump to aerate the suspension. Sporulated oocysts were then treated with 2.8 M NaCl and 2% sodium hypochlorite (Milton solution) and stored in 2% potassium dichromate at 4°C until required. Unsporulated oocysts were also treated with Milton solution and stored at −80°C. Merozoites (112 h p.i.) and gametocytes (134 h p.i.) were isolated from infected chicken caecae following techniques published previously [36,37]. Aliquots of parasites were either frozen at −80°C as pellets or were stored in TRIzol® reagent (Invitrogen) at −80°C for further use in RNA purification.
RNA purification, cDNA synthesis and cDNA standardisation
To isolate total RNA, purified merozoites (1 × 107) and gametocytes (1 × 106) were resuspended in 1 ml TRIzol® Reagent and homogenized by pipetting. Unsporulated oocysts (2 × 105) and sporulated oocysts (5 × 105) were resuspended in 1 ml TRIzol® Reagent and one volume of glass beads were added to the sample, which were then vortexed for 1 min intervals until disruption of oocyst was confirmed by bright field microscopy. All TRIzol® treated samples were left at room temperature for 10 min and total RNA isolated by chloroform extraction and isopropanol precipitation. RNA was quantified using a NanoDrop™ ND-1000 Spectrophotometer and cDNA was synthesized using SuperScript™ III Reverse Transcriptase (Invitrogen) according to manufacturer's instructions.
Parasite cDNA samples were standardized by relative quantification of an E. tenella β-actin PCR product. β-actin forward primer E0043 (5′ ggaattcgttggccgcccaagaatcc 3′) and reverse primer E0044 (5′ gctctagattagctcggcccagactcatc 3′) were used to generate the 1020 bp β-actin cDNA PCR product. Each PCR reaction contained 50 ng of parasite stage specific cDNA, 0.2 μM forward primer, 0.2 μM reverse primer, 1 × AccuPrime™ reaction mix, and AccuPrime™ Pfx DNA polymerase (Invitrogen). The PCR reaction was carried out as follows: initial denaturation 95°C for 3 min; 95°C for 30 s; 61°C for 1 min; 68°C for 1.5 min, for 25 cycles with a final extension at 68°C for 10 min. All products were electrophoresed on a 1% agarose gel and visualized using Gel Red™ (Biotium). The net intensity of each band was determined using the Kodak EDAS 290 Electrophoresis Documentation and Analysis System and serial dilutions performed until relative intensity of PCR products were equal.
In addition, three control genes were amplified to determine the purity of parasite lifecycle stages. The GAM56 gene was used as a gametocyte specific gene. GAM56 forward primer E0030 (5′ catatggtggagaacacggtgcac 3′) and reverse primer E0031 (5′ ctcgagttagtaccagctggaggagta 3′) were designed to amplify a 906 bp gametocyte cDNA product at an annealing temperature of 61°C. The EtTFP250 gene, a homolog of an E. maxima gene encoding a microneme protein, was used as an asexual stage control. The EtTFP250 forward primer Et250F (5′ gcaaggacgttgacgagtgtg 3′) and Et250RV1 (5′ gttctctccgcaatcgtcagc 3′) were designed to amplify an 805 bp cDNA product, at an annealing temperature of 60°C. The chicken lysozyme gene was used to determine relative quantities of contaminating host cDNA. The forward primer RW3F (5′ acaaagggaaaacgttcacgattggc 3′) and reverse primer RW4R (5′ tgcgttgttcacaccctgcatatgcc 3′) were designed to amplify a 280 bp host cDNA product at an annealing temperature of 60°C.
Semi-quantitative PCR
The predicted coding regions of each protease gene were examined for potential primer sites within 1 kb of each other where possible. Primers were designed as detailed in Table 5.
Table 5. Primers for Eimeria tenella protease genes for the amplified coding sequences
PCRs were conducted on cDNA samples from E. tenella merozoites, gametocytes, unsporulated and sporulated oocysts. PCR were optimized to produce cDNA sized products. Negative controls of no DNA template and host cDNA were run alongside a positive genomic DNA control. When genomic DNA products were not amplified, a repeat PCR was performed at longer annealing times to produce the often much larger genomic DNA product. A typical PCR was as follows: 1μL of standardized cDNA sample, 0.2 μM forward primer, 0.2 μM reverse primer, 1 × AccuPrime™ reaction mix, and AccuPrime™ Pfx DNA polymerase (Invitrogen). Cycling conditions typically involved an initial denaturation at 95°C for 3 min, followed by 25 cycles of denaturation 95°C for 30 s; annealing at Tm-5 for 1 min; extension at 68°C for 1.5 min. When products were to be sequenced, a final extension at 68°C for 10 min was performed at the end of the PCR reaction. PCRs were performed at least twice and, generally, three times for each gene product by a different researcher each time.
All amplified products were gel purified using a QIAquick® Gel Extraction Kit (QIAGEN) according to the manufacturer's instructions and sequenced (Australian Genome Research Facility, Queensland). When cDNA products were amplified from different parasite stages, these were pooled and used in sequencing reactions. When cDNA products were not obtained, additional primers were designed and used. If a cDNA product was still unable to be amplified with the second primer pair, genomic DNA products were sequenced to confirm primer specificity. Sequences were analysed using DNASTAR Lasergene™ 9 Core suite.
GAM56 processing assay
A frozen sample of purified E. tenella gametocytes (1 × 106 cells in 100 μL) was resuspended in PBS (145 mM NaCl, 7.5 mM Na2HPO4, 2.5 mM NaH2PO4.2H2O, pH 7.4) to a final volume of 500 μL. Glass beads (250 μL of 710-1180 μm, Sigma) were added to the suspension and vortexed at full speed for three 1 min pulses with a 1 min pause on ice between each pulse. After three vortex cycles, the sample was centrifuged and the lysate transferred to a clean tube. Equal aliquots of the gametocyte extract (18 μL) were immediately added to either 2 μL of 10× protease inhibitor (Table 3) or PBS. A zero time sample was taken from the PBS control and immediately added to Laemmli sample buffer [38] and frozen. The assay tubes were incubated at 37°C for 2, 4, 6, 8, 10, 12, 16 or 24 h, after which Laemmli sample buffer was added and samples stored at −20°C for further assessment.
SDS-PAGE and immunoblotting were carried out as described previously [20,21]. Briefly, gametocyte assay samples, resuspended in Laemmli sample buffer (5 μL), were boiled for 5 min at 100°C prior to SDS-PAGE on a NuPAGE® Novex 4-12% Bis-Tris Gel (Invitrogen). SeeBlue® Plus2 Pre-Stained Standards (Invitrogen) were used as a marker. Proteins separated by SDS-PAGE were transferred electrophoretically (100 V, 1 hour, 4°C) to Immobilon-P membrane (Millipore) in transfer buffer (25 mM Tris–HCl, 192 mM glycine, 10% methanol). Membranes were rinsed in methanol and water then soaked for 10 min in transfer buffer prior to transfer. Gels were pre-soaked for 15 min in transfer buffer. After transfer, membranes were incubated overnight in blocking solution (5% w/v skim milk powder in PBS) at 4°C and then incubated with primary antibody (1:1000 rabbit anti-EmGAM56) for 2 h at room temperature. Each membrane was washed twice for 5 min and twice for 10 min in 0.05% Tween 20 in PBS then incubated with secondary antibody (1:1000 anti-rabbit IgG conjugated to alkaline phosphatase; Sigma) for 2 h at RT. Membranes were washed as above and bands visualized with SIGMA FAST™ BCIP/NBT buffered substrate (Sigma).
Competing interests
The authors declare they have no competing interests.
Authors' contributions
NCS, MK and SIB conceived the study. MK coordinated the study and performed data analysis, along with FB, FMT and NCS. MK, RJI, MR, IS, PAS and RAW carried out the laboratory work. NCS, MK, FMT and FB drafted the manuscript. All authors read and approved the final manuscript.
This work was supported by an Australian Research Council Discovery Project grant (DP0664013) and a Department of Innovation, Industry, Science and Research International Science Linkage grant (FR110089). Neither funding body played any role in: the design, collection, analysis, or interpretation of data; the writing of the manuscript; or the decision to submit the manuscript for publication. The authors gratefully acknowledge Professors John Dalton and Michael Wallach (University of Technology, Sydney) for helpful discussions, the staff of the Ernst Facility at the University of Technology, Sydney for their excellent care of the chickens used in this study, and the Parasite Genomics Group at the Wellcome Trust Sanger Institute for providing us pre-publication access to the E. tenella genome database.
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Facing a friend in competition can be difficult to handle, especially if the friend is a teammate and the competition is the state singles final. And when top-seeded Christina Sisti faced second-seeded Sabrina Barisano on Monday, the winner of the third set would become Ursuline's first state tennis champion.
Sisti folowed up her Section 1 championship with a 6-4, 2-6, 6-0 win over Barisano at Tri-City Tennis Courts.
"Every time I play her, I know I have to really work hard," Sisti said of Barisano. "I have a lot of respect for her. She's a really nice person and a great competitor." ...


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District notebook: Three former soccer stars set to begin pro careers
College Football Videos
Three former players with ties to local colleges were among the 55 players allocated to the National Women's Soccer League.
The trio of Canadians — goalkeeper Erin McLeod (Penn State), defender Carmelina Moscato (Penn State) and outside back Bry McCarthy (West Virginia) — are slated to play in the latest attempt at U.S. professional women's soccer.
McCarthy, the Big 12 Defensive player of the Year, will play for the Canadian national team next week in the Yongchuan Cup, a four-nation women's tournament in China. Meghan Klingenberg, a Pine-Richland graduate and a reserve on last summer's U.S. Olympic team, opted to play this season in Sweden.
• Penn State senior Christine Nairn is a finalist for the Hermann Trophy, presented to the nation's outstanding soccer player. Crystal Dunn of North Carolina and Caroline Miller of Virginia also are finalists for the award, which will be presented by the Missouri Athletic Club on Friday.
Men's basketball
• Slippery Rock (8-2, 5-1) assumed sole possession of first place in the PSAC Western Division with a 69-64 win Friday over Millersville.
Women's basketball
• No. 6 Gannon (13-1, 7-0 PSAC) won its 12th consecutive game Saturday, beating Mansfield, 80-49. It's the team's longest winning streak since the 2009-10 team won 37 in a row.
• Thiel senior guard Kelly Muffley scored 10 points in an 80-54 loss to Thomas More on Saturday to become the 13th player in school history to reach 1,000 points.
• California (Pa.) tight end Blake Williamson will play in the Casino Del Sol College All-Star Game at 9 p.m. Friday in Tucson, Ariz. Williamson (Chartiers-Houston) will be joined by Penn State tackle Mike Farrell (Shady Side Academy), Pitt guard Chris Jacobson (Keystone Oaks) and receiver Mike Shanahan (Norwin), and West Virginia linebacker Terence Garvin and fullback Ryan Clarke.
Women's ice hockey
• Penn State director of operations and video coach Mo Stroemel will coach the U.S. women's national university team at the World University Games in December in Italy.
Men's track and field
• Penn State junior Jon Hendershot took first place in the high jump at the Penn State Relays over the weekend. His jump of 2.15 meters is tied for fourth best in the country.
Women's track and field
• Penn State senior Erika Morgan won the high jump at the Penn State Relays. Her jump of 1.79 meters is tops in the Big Ten and fifth in the country. Junior Mahagony Jones ran 60 meters in 7.44 seconds, good for ninth in the nation.
• Penn State sophomore brothers Andrew (149 pounds) and Dylan Alton (157) each went 5-0 to win championships Saturday at the Franklin & Marshall Open. Pitt redshirt junior P.J. Tasser (Belle Vernon) won the title at 174 pounds.
• Waynesburg junior Sam Lombardo (Canon-McMillan) improved to 14-2 as he won the 157-pound crown Saturday at the Waynesburg Invitational.
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text: So, you wanna be a Hilton? Well my friend, you've come to the right place. VH1 has compiled the definitive how-to guide that will transform any average Joe into America's favorite tabloid darlings. This lighthearted half-hour program will include lessons in "red carpet mastery," "how to deal with... Read Full Summary »


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Teaching Heterodox Economics
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Congratulations to the organizers. I will participate and hope that many others will take part also.
Yes, I agree. This is a great
Yes, I agree. This is a great help to each and every school. Heterodox economics is an excellent move to gain more knowledge.
As a former director af an
As a former director af an economic education program, I want to commend you for developing this vital enterprise. I cannot fully express my gratitude for your initiative.
Bill Luker
Dear Bill and Haider
Thanks for the comments. Haider, please send me (firstname.lastname@example.com) your email address for a login to enable you to participate.
Greater Open-Mindedness in Academia....................
There is a growing need for greater appreciation in education for the pluralism of ideas in economics. Furthermore, the subject is continually becoming more complex with its many "branches," and it is important for anyone to gain easier access to authorative, and obscure areas of economic data via the internet, and living experts. This is essential for society, and ofcourse, academia.
One gripe one has is that radical monetary reforms, and radical monetary concepts are ignored by the mainstream, and ofcourse by universities in the main. This is wrong, and greater open-mindedness is required.
I, myself, am involved in developing a project known as Transfinancial Economics. It is essentially a very advanced kind of monetary reform...
Bravo for this intiative.
Economic thought as its teaching needs more open approaches, especially non-sectarian and more objective.
I hope this project will create a new breath.
Faruk and Robert - thanks for
Faruk and Robert - thanks for the comments.
Congratulations to the organizers, these materials are helpful. I'll translate some of them since I'm from Ecuador and we speak spanish. Long life to TRUE project!
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Welcome to Heterodox Economics education! Our aims are:
- To encourage and support pluralism in the teaching and learning of economics in UK universities.
- To encourage and support the teaching and learning of heterodox economics in UK universities.
- To create a community of pluralist economics teachers willing to share ideas, resources and expertise in economics teaching.
- To promote alternative economic paradigms to potential students.
Heterodox Economics Education is led by Dr Andy Denis (firstname.lastname@example.com), Senior Lecturer in Political Economy at City University London.
This page, and the pages linked from it via the navigation bar on the left, are part of the TRUE project, aiming to make teaching resources in economics freely available online.
Pages for each of the 14 TRUE specialist areas are Wikis: to edit them, all you have to do is to ask for a login and password. The same login details will work for all of the specialist areas, so you only need to do this once. If you teach degree-level economics, please consider signing up, by sending me an email (at firstname.lastname@example.com), so that you can upload your teaching materials and edit the pages in other ways.
If you teach a module with any heterodox or pluralistic content, please contribute your teaching materials - syllabus, reading list, lecture notes and PowerPoints, handouts and exam papers, etc. - by uploading them to one of the pages you can see linked to on the navigation bar.
For the other 13 specialist areas, the emphasis is on second and third year options, as it was felt by project leaders that core micro, macro and quants at all levels are relatively well provided for by textbooks and open content through the web, but there was much less open availability of resources in specialist areas. This, however, does not apply to heterodox and pluralistic teaching, where there is a need for resources in core and first year modules quite as much as in optional modules taken in the second and third years of the undergraduate programme.
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AltaSim Knows How to Use COMSOL Multiphysics, You Can Too
Fanny Littmarck | May 17, 2012
Our Certified Consultants, AltaSim Technologies, are excellent at using COMSOL Multiphysics. If you read one of Phil's blog posts back in April, you would already be familiar with their modeling of Surface Plasmon Resonance. Also mentioned, in passing, are the training courses AltaSim runs.
Their 4-day course on "Solving COMSOL Multiphysics Problems" held this month turned out to be very popular and filled up quickly. The training is centered on the practical aspects of using COMSOL Multiphysics and learning how to use our simulation tool effectively. AltaSim's instructors combine presentations, workshops, demonstrations, individual problems, and one-on-one instruction to guide students through the training. Got a specific problem you need help with? No problem – AltaSim's experienced engineers are there to help you set up these problems.
We are proud to attach our "Certified Consultants" badge to the AltaSim name, and are happy to point those with little or no previous experience using COMSOL to AltaSim's training events. These guys use our software on a daily basis and are sure to be able to share some tips and tricks with you. If you were one of the unlucky ones who were unable to register for the course in May, you'll be happy to know that they have another "Solving COMSOL Multiphysics Problems" course scheduled for June 19-22. The upcoming course is still open for registrants.
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Research firm YouGov says it will save £100,000 a year after migrating its IT infrastructure from its Berlin datacentre, which was managed by Interoute, to a new datacentre in London.
Nicholas Carter, director of global systems operations at YouGov, told Computing that the organisation has saved significant sums by switching to its own datacentre.
"The cost savings come from the additional capacity that we wanted to add. If we were trying to implement the current system in our previous environment, we would have had to pay an additional £10,000 to £20,000, if not more," he said.
"In addition, we would have had to pay £3,000 to £4,000 per month to host it."
The firm used virtual infrastructure and data protection provider Veeam Software's backup and replication tool, Backup 5, to migrate its core IT infrastructure. It made the move after having several problems with its datacentre in Berlin.
"We had a managed service in Berlin. YouGov owned the hardware, but the hardware was managed by datacentre provider Interoute and we had a lot of issues with that," explained Carter.
For example, he claimed the cost of adding new hardware was inflated because YouGov had to buy the equipment through Interoute.
"We had the required skillset within the IT team in London, so we decided to move our datacentre there," he said.
The migration process took place after YouGov had built its own datacentre, purchasing all the required hardware itself.
"We used Veeam to do a lot of the complex migration. The physical systems were different. We had NetApp in Berlin and we had a Dell Compellent Storage Array in London, which made it difficult moving data across. This is why we virtualised our environments and used Veeam to migrate the data," said Carter.
YouGov transferred about 30TB of data from Berlin to London from the end of May to 28 July last year.
"A single virtual machine took only eight to nine hours to replicate and, using Veeam, we replicated 40 of them," said Carter.
YouGov was also able to move its applications, such as Microsoft SharePoint and Symantec Enterprise Vault, onto the new infrastructure.
Carter said that YouGov chose Veeam to help it migrate its IT infrastructure because it was looking into backup software for its virtual machines, for which Veeam has a tool. It was also satisfied by the company's reputation.