text:
RAISE YOUR VOICE: Share your own opinion online at dallasnews.com/sendletters. Sign up for Sounding Off or submit a guest column (and include your full name and contact information) by visiting dallasnews.com/voices.
How aware of your surroundings are you while you are at the gas pump? What precautions do you take to keep your valuables safe?
Jimmie Wilson, Richardson: I only have the door unlocked on the driver's side and I pump gas on that side, so I am very aware of my surroundings. Maybe I should start taking my dog with me, as she will bite anyone who tries to get in the car.
Tom Naylor, Crowley Park in Richardson: While I do believe that I have "situational awareness" while I am pumping, I can't say that I don't sometimes let my mind wander a bit, usually when I am pumping on a very cold day and I am just wishing I were somewhere warm. The main thing that I do is to take my keys with me out of the car so the car can't be stolen. Most importantly, I don't leave valuables in my car. My wallet is always in my pocket. If I did have something valuable in the car, it would probably be in the trunk, but it is only rarely that I would have something of value in the car. A thief sliding into my car could not make a living from anything that might be there.
Suzanne Harned, Lake Highlands: Wow, I was ready to be all self-righteous and write that I always take my keys and stand by my car while pumping gas. Then I viewed the link you provided showing a man coming in from the passenger side to snatch what he could. Well, I certainly could have been a victim many times as I never think to lock my car while I'm right beside it. You can bet that I will from now on. Thanks for showing me the error of my ways.
Jack Orr, Far North Dallas: This problem just shows that the crooks are always thinking, and that honest folks need to use their heads and be alert when out amongst them.
Since most modern cars these days have a lock console on the left door, it would be good practice to lock all doors, including the driver side, when exiting the vehicle, and you are able to have the key fob with you.
It might help to get gas in neighborhoods where criminals are less likely to be found.
Jason Nancarrow, Lake Highlands: I thought "sliders" we're delicious miniature hamburgers from my childhood; criminals take everything. Here are some changes we can make.
First, close down the gas stations that tolerate this kind of behavior. I'm a big believer in the broken-window theory. Crime festers when people don't care.
Second, we need to legalize vigilantism. It seems to be working in Mexico. And if characters like Batman can pull it off, why can't people pull it off in real life?
Kathy Minde, Arapaho Heights in Richardson: My car has the option to only unlock the driver's door when the key is turned off. Plus, my gas tank is on the driver's side. I stand in the doorway to my car while filling with gas so no one can get to my purse. I also make a point to pull my keys and have them with me. One day while filling with gas, I accidentally hit the door lock button as I got out and the wind blew the door shut with keys inside. Slightly off topic, but a good habit to get into, if you do need to move away from the car, you can close the door and lock it immediately.
Marna Irby, Richardson: I do not pump the gas, but my thought here is that since we now have identified "sliders," what next? Is there no end to what people will do to steal from others? We can't police everything 24 hours a day.
No one seems to talk about how we became a society where anything goes if you don't get caught. Maybe we need a think tank to solve that. Getting to the root of our immorality, and acting on it, is the only way to find a solution
Anne Healy, Richardson: Sad to say I've always been apprehensive when filling my gas tank. I take my keys and wallet with me and leave my purse by the driver's side door on the floor. As the pump is filling the tank I stand by that door. I never get back into the car, not even on the windiest or coldest of days, because of the possibility of static electricity doing a number on me and/or the car.
Patricia Allbee, Richardson: I always auto-lock my car when I'm pumping gas, because my gas tank is on the driver's side, and I've always thought it would be easy for someone to quickly open the passenger side door and take whatever is on the front seat while my back is turned and I'm going through all the prompts on the credit card screen ... to be really safe, I probably should put my purse somewhere out of reach, as a "smash and grab" is not out of the realm of possibility even if the door is locked! In addition, I usually pump gas using the trigger lock so I can stay inside my car while the tank is filling up, which I think is safer, not to mention warmer!
Eric Strull, Far North Dallas: I pump gas facing the pump with the intention of being aware of wha's going on around me. Since I'm a guy, my wallet is in my pocket. For women it would be best if they hold on to their purse while pumping gas. I have no valuables in the car so I don't lock it so a thief would not find anything of value in my car. Should a woman not want to hold her purse, then the doors must be locked. Just be mindful of not locking your keys in the car.
LaRuth Morrow, The Reservation in Richardson: I am extremely aware of my surroundings when filling my car's gas tank. First, I only go when there are others at the self-serve pumps. I get my credit card out and have it on me while outside at the pump; turn off the ignition and take the car keys out and then lock the car. I pay at the pump and only after I have my receipt in hand, I unlock the car and get back in. When I turn on the ignition, the car doors are automatically locked. So far this plan is working well for me.
Mike Lysell, Richardson: As the male in the family, I'm the person who gets stuck filling the gas tank. I say stuck because sometimes it's pretty cold, sometimes pretty hot and it always seems windy! The station we usually use and the time of day we fill the tank means there are a lot of other motorists around us. I always take the key and my billfold is in my pocket. Any valuables are either in the trunk or out of plain sight. There have been times when I've been there later in the evening when no one else is around. The station is well lit and on a busy street, but I always keep an eye peeled for anybody that looks out of place. I am more concerned about someone trying to carjack my vehicle than I am about the "slider" thieves.
Tami Cannizzaro, Lake Highlands: I like to think that I am hyper-aware of my surroundings. It never hurts to take a friend when going to the gas station, but that's not always possible. What is possible and many women don't like to hear this is to not carry your handbag with you. I'll take my driver's license, insurance card and credit card or cash in my front pocket. If you have your handbag with, then try to hide it or put in on the floor before arriving at the gas station. Try to pick a pump that is front and center of the store. Never leave your car unlocked! My car has a locking gas cap which means it must be unlocked to remove the cap, so before I start my transaction I take the gas cap off then quickly lock the car.
While the gas is pumping I am watching in all directions. I'm aware of the cars and people on all sides, making eye contact with them. When I'm done, I quickly put the gas cap on, get in the car and lock the doors again. If I happen to have my handbag with me, I don't sit there rearranging my handbag and wallet. I can do that when I get home or at my next destination. It's a shame that we have to be so careful, but we do.
Carole Crawford, College Park in Richardson: I try to stay very aware of my surroundings whatever I'm doing. At gas stations, I keep my wallet, which contains my credit cards and cash, with me, and I leave my doors locked. It is ridiculous to leave valuables in an unlocked car or leave a car running while you "run in to grab something." I've had excellent luck. I left my purse in my grocery cart one day, but by the time I got back to the store, my purse had been turned in completely intact, and the store had called me at home to alert me that they had my purse. That's how neighbors take care of each other!
sounding off


text: Encompassing over six million acres, Denali National Park is home to diverse wildlife including grizzly bears, caribou, wolves, Dall sheep, and moose. The glaciated landscape offers ample opportunity for sightseeing, backpacking, and mountaineering. Mount McKinley, North America's tallest mountain at 20,320 feet (6,194 meters), helps make Denali one of the state's most visited National Parks.Hide

subset: commoncrawl


text:
Chicken Recipes..
By Maya Inc.
This app is only available on the App Store for iOS devices.
It's easy to prepare, budget-friendly and great-tasting, so what's not to love about chicken! Chicken is a family favourite ingredient; yet we often stick to the tried and tested chicken recipes we know by heart. Why not try some new chicken recipe ideas, add a bit of spice to mid-week chicken breast recipes, and really make the most of this versatile food. We've got chicken pastas, chicken bakes, barbecued chicken, chicken soup, chicken stir fries, chicken sandwiches and wraps, chicken slow cooker.
Great chicken recipes like these are perfect for pleasing picky eaters and the cooks who like a good, easy meal.
Plain chicken has been dressed up in a variety of different recipes with delicious spices and sauces; then served as chicken casseroles, fried chicken, chicken soup, chicken tenders, and more.
You'll find recipes that have been passed from one family's recipe box to another, the home cooked favorites that folks come to the table and linger a bit longer for.
The chicken recipes included are tested and approved by generations of families and friends. Enjoy them with those who gather at your table.
Some of the Chicken Recipes included in the app are:
Chicken Appetizers
Chicken Breasts
Chicken Thighs and Drumsticks
Baked Chicken Wings
Baked and Roasted
Barbeque and Grilling
Grilled Salads
Chicken Casserole
Chicken and Broccoli
Chicken and Pasta
Chicken and Rice
Chicken and Stuffing
Pasta Dishes
Chicken Alfredo
Sandwiches and Wraps
Soups and Stews
Slow Cooker
✔Popular Casseroles
✔Beef Casseroles
✔Breakfast Casseroles
✔Broccoli Casseroles
✔Noodle Casseroles
✔Potato Casseroles
✔Rice Casseroles
✔Tuna Casseroles
What's New in Version 1.15
Recipe sharing facilities are added (Facebook, Email, Sms),
New Recipes categories are added,
Favorite tab is included for favorite recipes.
Customer Reviews
Very disappointed!!
Yes it does include some recipes HOWEVER the majority of the recipes must be purchased & are separate apps. I didn't purchase an app about purchasing apps. I purchased an app for recipes. This is just underhanded trickery
- Free
- Category: Health & Fitness
- Updated: Jun 26, 2012
- Version: 1.15
- Size: 29.3 MB
- Language: English
- Seller: Atia Feroz
- © 2012 Maya Inc.
Compatibility: Requires iOS 5.0 or later. Compatible with iPhone, iPad, and iPod touch.
Top In-App Purchases
- Full Database of Beef Casseroles$0.99
- Full set of Slow Cooker Chicken Recipes with full app features$0.99
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- Full set of Baked Chicken with full app features$0.99
- Full Database of Rice Casseroles$2.99
- Full set of Chiecken Appetizers with full app features$0.99


text:
Last weekend, I had a chance to corner Julius Randle (Dallas/Prestonwood Academy), No. 3 overall in the 2013 class, and talk to him about hoops and his recruitment. Shortly afterward, he went and won the Elite 24 slam dunk competition and was an MVP in the game.
Two days later, Randle said he was considering 10 schools (notice I didn't say "trim" because 10 schools is a lot to have on a list in September). The 6-foot-9 power forward will decide amongst Florida, Baylor, Duke, Kansas, Kentucky, North Carolina, NC State, Oklahoma, Oklahoma State and Texas.
Here is the Q&A I had with him:
Where does the intensity come from?
"I just want to be the best player on the court. It comes from just watching great players like Kobe, LeBron and KD; you see what they do. Me as a competitor watching them it motivates me and I'm a self motivator."
There's not much intensity out here, so what's the Elite 24 like?
"This is more fun, it's not too much of a reality of what basketball is. You aren't going to face too much adversity out there. It's more of an honor and you just want to enjoy yourself."
Over the years, who gave you the most trouble?
"Aquille Carr is always hard to play against. Andrew Wiggins, he's tough. I'd say the player that I least expected to be that hard when we played against him was Tyus Jones. How he plays, he's kind of like Chris Paul, he makes no mistakes on the court and it seems like he plays a perfect game. I really enjoyed playing against him."
When you look at colleges, what are the pieces that have to be there for you?
"Point guard, shooters and another big man. That's pretty much it. I want guys who are going to play hard with me."
How much has Matt Jones (Duke commit) leaned on you?
"He's leaned on me a little bit. Matt's a good person. He doesn't care where I go. He's a good person and a good friend. He wants me to come with him but down inside I know he wants what's best for me."
Do you have a place in mind that you think is going to be hard to beat?
"It's been like that at times but it's changed from place to place. Then I say I want to go here and then six months later I want to go here. Sometimes I do, but my mind changes a lot. I just have to sit down and study the system."
What happens when you switch back and forth on a favorite?
"Maybe you see a player that's gone there and done well or it's the coach who is the hottest. You may go take a visit and say this is the place for me. Then once you sit down and think about it you have to take the emotional stuff out of it and think about a business decision."
You recently snuck up to Oklahoma State. Are they in there?
"For sure. I played with Marcus Smart on the USA Team and their coaches talk to me a lot. They kept texting Jeff Webster and he brought me up there. They have the best locker room, it's a two-story locker room and it's the best one I've ever seen."
Is the plan to wait until the spring?
Probably in the spring to see what players are going there, see what coaches leave and it gives me a last time to really study the system and see what kind of offense they run. It gives me time to look. I'll probably save some of my visits; I want to go to some basketball games."
What's been the best experience?
"USA Basketball. It was the longest time I was away from home. A lot of those guys are going to be freshman in college. I played for college coaches and it was the first time I went overseas. We bonded with other players and people I don't usually hang out with. It helped me mature."
What's the process been like?
"I've had fun with all of it. I don't think I've stepped on too many toes. You don't want everybody to think you don't like them. You want to make everyone happy. I don't want to be a jerk about it. I just want to be as civil as I can."


text:
Genre: Action, Adventure, Sci-Fi
In delivering PG-13-rated excitement, Alien vs. Predator is an acceptably average science-fiction action thriller with some noteworthy highlights, even if it squanders its opportunity to intelligently combine two popular and R-rated franchises. Rabi… more »
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text:
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text: [Federal Register Volume 76, Number 73 (Friday, April 15, 2011)] [Notices] [Pages 21411-21413] From the Federal Register Online via the Government Publishing Office [www.gpo.gov] [FR Doc No: 2011-9202] ======================================================================= ----------------------------------------------------------------------- OFFICE OF PERSONNEL MANAGEMENT Excepted Service AGENCY: U.S. Office of Personnel Management (OPM). ACTION: Notice. ----------------------------------------------------------------------- SUMMARY: This gives notice of OPM decisions granting authority to make appointments under Schedules A, B, and C in the excepted service as required by 5 CFR 213.103. FOR FURTHER INFORMATION CONTACT: Roland Edwards, Senior Executive Resource Services, Executive Resources and Employee Development, Employee Services, 202-606-2246. SUPPLEMENTARY INFORMATION: Appearing in the listing below are the individual authorities established under Schedules A, B, and C between February 1, 2011, and February 28, 2011. These notices are published monthly in the Federal Register at http://www.federalregister.gov/. A consolidated listing of all authorities as of June 30 is also published each year. The following Schedules are not codified in the Code of Federal Regulations. These are agency-specific exceptions. Schedule A No Schedule A authorities to report during February 2011. Schedule B No Schedule B authorities to report during February 2011. Schedule C The following Schedule C appointments were approved during February 2011. ---------------------------------------------------------------------------------------------------------------- Effective Agency name Organization name Position title Authorization No. date ---------------------------------------------------------------------------------------------------------------- DEPARTMENT OF COMMERCE........... Office of Director of DC110032 2/2/2011 Legislative and Legislative Affairs. Intergovernmental Affairs. Office of the Under Speechwriter........ DC110034 2/4/2011 Secretary. Office of the Chief Director, National DC110027 2/4/2011 of Staff. Export Events. Office of Assistant Associate Director DC110038 2/4/2011 Secretary for of Legislative and Legislative and Intergovernmental Intergovernmental Affairs. Affairs. Office of Assistant Associate Director DC110037 2/9/2011 Secretary for of Legislative Legislative and Affairs. Intergovernmental Affairs. COMMISSION ON CIVIL RIGHTS....... Staff Members....... Special Assistant... CC110005 2/7/2011 DEPARTMENT OF DEFENSE............ Office of Assistant Special Assistant... DD110041 2/28/2011 Secretary of Defense (Legislative Affairs). Office of the Under Special Assistant DD110042 2/28/2011 Secretary of for Reserve Affairs. Defense (Personnel and Readiness). DEPARTMENT OF THE NAVY........... Office of the Under Special Assistant... DN110012 2/8/2011 Secretary of the Navy. [[Page 21412]] DEPARTMENT OF EDUCATION.......... Office of English Deputy Director..... DB110025 2/4/2011 Language Acquisition, Language Enhancement, and Academic Achievement for Limited English Proficient Students. Office of Special Confidential DB110015 2/8/2011 Education and Assistant. Rehabilitative Services. Department of Deputy Director DB110021 2/8/2011 Education. Program Mgmt and Performance Unit. Office of the Special Assistant... DB110019 2/10/2011 Secretary. Office of Planning, Confidential DB110031 2/11/2011 Evaluation and Assistant. Policy Development. Office of Confidential DB110030 2/11/2011 Legislation and Assistant. Congressional Affairs. Office of Safe and Special Assistant... DB110020 2/11/2011 Drug-Free Schools. Office of Special Assistant... DB110029 2/11/2011 Legislation and Congressional Affairs. Office of Elementary Confidential DB110032 2/23/2011 and Secondary Assistant. Education. DEPARTMENT OF ENERGY............. National Nuclear Deputy Director of DE110041 2/23/2011 Security Public Affairs. Administration. Loan Programs Office Assistant Director DE110036 2/23/2011 for External Relations. National Nuclear Director of DE110047 2/23/2011 Security Congressional Administration. Affairs. ENVIRONMENTAL PROTECTION AGENCY.. Office of the Senior Advisor...... EP110018 2/18/2011 Associate Administrator for Congressional and Intergovernmental Relations. GENERAL SERVICES ADMINISTRATION.. Mid-Atlantic Region. Special Assistant... GS110024 2/18/2011 Office of Deputy Associate GS110026 2/18/2011 Congressional and Administrator for Intergovernmental Legislative Affairs. Affairs. Office of Deputy Associate GS110025 2/18/2011 Congressional and Administrator for Intergovernmental Policy. Affairs. DEPARTMENT OF HEALTH AND HUMAN Office of Public Senior Advisor, DH110053 2/28/2011 SERVICES. Affairs. Office of Public Affairs, Administration for Children and Families. Center for Consumer Senior Advisor...... DH110057 2/28/2011 Information and Insurance Oversight. DEPARTMENT OF HOMELAND SECURITY.. Office of the Special Assistant... DM110033 2/2/2011 Assistant Secretary for Policy. Office of the Chief White House Liaison. DM110075 2/11/2011 of Staff. DEPARTMENT OF HOUSING AND URBAN Office of Senior Counsel...... DU110016 2/18/2011 DEVELOPMENT. Sustainable Housing and Communities. DEPARTMENT OF THE INTERIOR....... Office of the Attorney-Advisor.... DI110032 2/4/2011 Solicitor. Assistant Secretary-- Deputy Chief of DI110033 2/17/2011 Indian Affairs. Staff. Office of Special Assistant... DI110037 2/23/2011 Congressional and Legislative Affairs. DEPARTMENT OF JUSTICE............ Office of Public Deputy Speechwriter. DJ110054 2/18/2011 Affairs. DEPARTMENT OF LABOR.............. Wage and Hour Senior Advisor...... DL110012 2/17/2011 Division. Bureau of Deputy Chief of DL110009 2/25/2011 International Labor Staff. Affairs. OFFICE OF MANAGEMENT AND BUDGET.. Office of the Confidential BO110012 2/22/2011 Director. Assistant, Government Reorganization Initiative. Office of the Confidential BO110010 2/23/2011 Director. Assistant. OFFICE OF THE UNITED STATES TRADE Office of the Special Assistant TN110003 2/2/2011 REPRESENTATIVE. Ambassador. for Scheduling. SECURITIES AND EXCHANGE Office of the Confidential SE110002 2/17/2011 COMMISSION. Chairman. Assistant. SMALL BUSINESS ADMINISTRATION.... Office of the Special Assistant... SB110018 2/17/2011 Administrator. Office of Management Senior Advisor for SB110019 2/23/2011 and Administration. Management and Administration. Office of the Special Assistant SB110021 2/28/2011 Administrator. and Scheduler. DEPARTMENT OF STATE.............. Office of the Special Assistant... DS110046 2/23/2011 Counselor. UNITED STATES INTERNATIONAL TRADE Office of the Staff Assistant..... TC110003 2/25/2011 COMMISSION. Chairman. DEPARTMENT OF VETERANS AFFAIRS... Office of the Special Assistant... DV110004 2/7/2011 Assistant Secretary for Public and Intergovernmental Affairs. Office of the Special Assistant, DV110017 2/23/2011 Secretary and White House Liaison. Deputy. ---------------------------------------------------------------------------------------------------------------- [[Page 21413]] Authority: 5 U.S.C. 3301 and 3302; E.O. 10577, 3 CFR 1954-1958 Comp., p. 218. John Berry, Director, U.S. Office of Personnel Management. [FR Doc. 2011-9202 Filed 4-14-11; 8:45 am] BILLING CODE 6325-39-P


text:
U-14 softball clinches No. 2 playoff seed with sweep of Moorhead
DL's U-14 team swept Moorhead Tuesday night, 14-1 and 25-1, improving their season record to 10-2 and clinching the No. 2 seed in the playoffs, which start next Tuesday in Hawley.
In Game 1, Emma Maguire picked up the win, striking out 5.
Macy Kirchner was 2-3 with a triple and 2 RBI. Sydney Prussia was 2-2 with an RBI.
In Game 2, Emma Maguire and Madi Kirchner combined on the mound to give up just one hit, with Maguire getting her second win of the day.
Sydney Prussia had three hits and five RBI. Kirchner, Dallmann and Emily Schiltz each collected a pair of hits in the victory as well.
DL also picked up the sweep against Hawley in a July 2 doubleheader.
In Game 1, DL won 15-3, with Maguire earning the win as pitcher once again.
Dallmann went 1-2, with a double and three RBIs. Amber Lyman also went 2-1, including a triple, while Madi Ramsey finished 2-4 with a triple and four RBIs.
In Game 2 DL won even more convincingly, 17-0, with Maguire once again being the winning pitcher.
She also went 1-1 at the plate, with a triple and two RBI.
Macy Kirchner went 1-1 on the day, with a triple and 4 RBIs, while Madi Ramsey collected two more hits and a pair of RBIs.
Betty Schouviller went 1-2 with a triple and two RBI.
Detroit Lakes is the defending Class D state champion, and the team hopes to defend their title by qualifying for the state tournament next week in Hawley. Games start at 9 a.m.
U-12 softball sweeps doubleheader vs. Hawley
Detroit Lakes' U-12 softball improved to 10-1 on the season with a 9-2 win over Hawley in game 1 Monday.
Madi Kirchner was the winning pitcher, striking out five in three innings of work. Kirchner also helped herself by going a perfect 3-3 with a single, triple, and three-run homerun in the effort. Brianna Clem homered for Detroit Lakes as well.
Lauryn Manning and Ava Steimentz also collected hits in the win.
In game two, Madi Anderson picked up the win in a 7-0 shutout. Mattea Borah worked a scoreless inning in relief.
Rachel Perkins, Madi Anderson, and Caitlin Dallmann all collected safeties in the win.
Detroit Lakes finishes its regular season on Thursday versus Moorhead at Snappy Park, with games starting at 4:30 and 5:30 p.m.
The season ending tournament is scheduled for Thursday, July 18 in Hawley. The top two teams will advance to state tournament action July 27-28 in Buffalo, Minn.
U-12 White splits with FF
Detroit Lakes' U-12 White team split with Fergus Falls, losing the first game 10-9 and winning game two 10-7.
Mirella Grimm tripled and singled in the winning effort.
Danielle Bishop made her pitching debut, striking out four to pick up the win.
U-18 team earns split with Hawley
The DL U-18 team split its doubleheader with Hawley last week, winning game one 5-4.
Chaz Butz was the winning pitcher, while Kathryn Kinney finished with two hits and a homerun. Kyra Vagle also had three hits in the win.
In game two, DL lost a close one, 3-0. Kyra Vagle collected two more hits in the effort.
DL's U-18 team is now 7-5 on the year.


text:
The 5th Album
|CD, Released on 2004
The 5th Album
I i could put 10000 stars i would because i live for Infected Mushroom i love them so much every single CD and track in unique and totally BLISS. im only 18 and i dont think i will EVER hear anything like Infected Mushroom and i will always be listening to them in my sleep and aaaalll day the have definatly infected me. xxxx Steff Stephanie Hunter


text:
Lizzie Bright and the Buckminster Boy by Gary D. Schmidt
My review
rating: 5 of 5 stars
I ordered "Lizzie Bright and the Buckminster Boy" from PaperbackSwap.com, as an afterthought. I had never heard of it, but it had the Newbery, so I thought I'd try it out.
Oh, my. I can't recommend this book enough. The writing itself leaves me speechless. The characterizations, the excellent balance of action and introspection, the dialogue are all completely flawless.
Then there's the story - a tale of a small Maine town at the turn of the 20th century, and the 13-yr-old minister's son who has just relocated there. Although he wants to move back home to Boston, he meets a girl just his age named Lizzie. Their friendship defies everyone's expectations, and changes his entire life.
The backstory of the race relations is true; the rest is fiction, but it seems like the quiet, deft tale handed down age to age, hand to hand. It's truly a brilliant, lovely piece of literature.
View all my reviews.


text:
High Precision 03 20 Series Zys Tapered Roller Bearings
|FOB Price:
|US $1-855.5 / Piece
|Min. Order:
|1 Piece
|Min. Order
|FOB Price
|1 Piece
|US $1-855.5/ Piece
|Production Capacity:
|10000 Per Month
|Transport Package:
|Cases and Pallets or as Customer′s Requirment
|Payment Terms:
Request a custom order and have something just for you!Send Customized Request
- Recommended Products
- 7 Products
- Content
- Product Image
- Product Name
- FOB Price
- Port
- Model NO.: 03 20 series
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- Material: Bearing Steel
- Separated: Separated
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Packing :
Bearing workshop :
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Address: No.1 Jilin Road, Luoyang City, Henan, Province, China.
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kype: zysdongoffice
|High precision angular contact ball bearing Precision spindle bearing
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|Material: GCr15,9Cr18,G20CrMo or others
|Certificate: GB/T19001-2000(ISO9001:2000), GJB/Z9001A-2001, GJB9001A-2001


text:
This product is no longer available.
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Genetics Research International
Volume 2011 (2011), Article ID 572689, 11 pages
Comparative Study of Cyanobacterial and E. coli RNA Polymerases: Misincorporation, Abortive Transcription, and Dependence on Divalent Cations
1Structural Biology Center, National Institute of Genetics, and Department of Genetics, The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan
2Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute at Frederick, MD 21702, USA
3Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo-shi, Chiba 271-8518, Japan
4Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kita-Ku, Kyoto 603-8555, Japan
Received 7 June 2011; Accepted 31 July 2011
Academic Editor: Sebastián Chávez
Copyright © 2011 Masahiko Imashimizu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
If Mg2+ ion is replaced by Mn2+ ion, RNA polymerase tends to misincorporate noncognate nucleotide, which is thought to be one of the reasons for the toxicity of Mn2+ ion. Therefore, most cells have Mn2+ ion at low intracellular concentrations, but cyanobacteria need the ion at a millimolar concentration to maintain photosynthetic machinery. To analyse the mechanism for resistance against the abundant Mn2+ ion, we compared the properties of cyanobacterial and E. coli RNA polymerases. The cyanobacterial enzyme showed a lower level of abortive transcription and less misincorporation than the E. coli enzyme. Moreover, the cyanobacterial enzyme showed a slower rate of the whole elongation by an order of magnitude, paused more frequently, and cleaved its transcript faster in the absence of NTPs. In conclusion, cyanobacterial RNA polymerase maintains the fidelity of transcription against Mn2+ ion by deliberate incorporation of a nucleotide at the cost of the elongation rate. The cyanobacterial and the E. coli enzymes showed different sensitivities to Mg2+ ion, and the physiological role of the difference is also discussed.
1. Introduction
A DNA-dependent RNA polymerase (RNAP) has a catalytic center chelating Mg2+ ions to form phosphodiester bonds [1, 2]. Most other divalent cations inhibit the activity, but Mn2+, which has a similar but only slightly larger ionic radius than Mg2+, supports the polymerizations. In the case of Escherichia coli (Esc) RNAP, Mn2+ causes the enzyme to misincorporate deoxyribonucleoside triphosphate (dNTP) instead of ribonucleoside triphosphate (NTP) [3] as well as noncognate NTPs [4–6]. Therefore, the growth of E. coli is inhibited in the presence of Mn2+, and the inhibition is mitigated by GreA and GreB which bind to RNAP [7].
In cyanobacteria, the intracellular concentration of Mn2+ is considered to be higher by two orders of magnitude than in E. coli [8], because Mn2+ is required at higher concentrations to assemble the photosynthetic reaction center carrying Mn2+ cluster, and/or to scavenge reactive oxygen species generated from photosynthesis [9, 10]. Since cyanobacteria have no genes orthologous to greA or greB, cyanobacteria should have the mechanism of detoxifying Mn2+.
We thus made two purified reconstituted transcription systems from a mesophilic cyanobacterial species, Synechococcus sp. PCC 7942 (Syn), and from a moderately thermophilic species, Thermosynechococcus elongatus BP-1 (The). By comparing these systems with the system of E. coli, we here examine the possibility that the mechanism of detoxifying Mn2+ is installed in the poisoning target, RNAP, and ask whether or not the cyanobacterial RNAP has a sensitivity to Mg2+ different from that of E. coli RNAP.
2. Materials and Methods
2.1. Materials
All of the oligo DNAs and RNA, NTPs, and [γ-32P]ATP were obtained from Hokkaido System Science, Yamasa and Perkin Elmer, respectively. Restriction enzymes were purchased from New England Biolabs and Takara. Primestar HS and Primestar Max DNA polymerases used for PCR were purchased from Takara. Syn cells used for RNAP purification were partially gifted from Dr. Mitsumasa Hanaoka. The culture condition of the gifted cells is the same as shown in the next section.
2.2. Protein Purification
Cells of Syn and The were cultivated and harvested as described [15, 16]. About 10 g of the wet-cell paste was suspended in 30 mL of TGED buffer [10 mM Tris-HCl (pH 8.0), 5% (v/v) glycerol, 0.1 mM EDTA, and 1 mM DTT] containing 1 mM phenylmethylsulfonyl fluoride and 0.2 M NaCl, and then, cells in suspension were lysed by sonication. A 35%- saturated solution of ammonium sulfate was added to a soluble fraction of the cell lysate which was obtained by centrifuging for 30 min at 20,000 × g. The obtained mixture was added to 10 mL of phenyl sepharose resin (GE Healthcare), pre-equilibrated with the TGED buffer containing 0.2 M NaCl and 35% saturated ammonium sulfate, and the mixture was washed by the same buffer, eluted with the TGED buffer. The eluate was applied to a column containing 10 mL of DEAE resin (TOSOH) pre-equilibrated with the TGED buffer, and the column was washed with TGED buffer containing 0.2 M NaCl, eluted with the TGED buffer containing 0.5 M NaCl. The eluted fraction was further chromatographically purified by using Hi-trap heparin affinity column (GE Healthcare) and MonoQ anion exchange column (GE Healthcare) as described [16]. The purified core enzyme was dialyzed against the TGED buffer containing 0.2 M NaCl and 50% glycerol and stored at −80°C. The core enzyme of E. coli was prepared according to [17], and σ70 of E. coli and σAs of Syn and The were obtained as described in [15, 16].
2.3. In Vitro Transcription Assays
The liner DNA template containing the T7A1 promoter from −147 to +87, when +1 is the transcription start site, was prepared by PCR using the plasmid pAR1435 [18] and the following digestion by HaeIII. The template containing the psbA2 promoter from −127 to +101 was prepared by PCR using a genomic DNA of a cyanobacterium Synechocystis sp. PCC 6803. These DNA templates were purified by PAGE. The holoenzyme was reconstituted by incubating core RNAP mixed with a 3-fold molar excess of the primary σ factor for 10 min at 37°C. We used twice more Syn and The RNAPs, because they have smaller affinities for the psbA2 promoter. The reconstituted holoenzyme (50 nM for E. coli or 100 nM for Syn and The) and 20 nM DNA template was preincubated for 10 min at 37°C in 8 μL of T-buffer [50 mM Tris-HCl (pH 7.9), 100 mM KCl, 10 mM MgCl2 (see figure legends and the main text in the cases that the concentration was changed or MgCl2 was changed to MnCl2), 1 mM DTT, and 150 μg mL−1 partially hydrolysed casein]. Reaction was started by adding 2 μL of prewarmed substrate mixture: 5 μM [γ-32P]ATP (40 Ci mmol−1) as well as 100 μM each of GTP, CTP, and UTP. In a single-round reaction, heparin (100 μg mL−1) was added together with the substrates to eliminate enzyme turnover. After incubation for 20 min at 37°C, the reaction was stopped by phenol/chloroform/isoamyl alcohol (25 : 24 : 1). Transcripts were analyzed by PAGE using a 20% gel containing 7 M urea. The sequences of abortive transcripts from the T7A1 promoter were assigned according to [19], and those from the psbA2 promoter were determined using 5′ end-labeled RNA marker 5′-AGUCAGUU-3′ and a chemical cleavage assay (Figure S2 which is available online at doi:10.4061/2012/572689). The assignment of abortive transcripts are often confused by the presence of cleaved fragments of the run-off transcript. The cleavage occurs during the extraction with oxidized phenol but the fragments are distinguished by the retention of 3′-phosphate as described in Figure S2 which is available online at doi:10.4061/2012/572689.
All the experiments with presented results in this study were repeated two or more times and the represented ones are shown.
2.4. TEC Formation
In order to stop elongation at Position +18 by a lack of the cognate CTP for Position +19, the intrinsic cytosine residues (nontemplate strand) at Positions +4, +9, +10, and +14 of the psbA2 promoter were replaced by thymine by PCR-based mutagenesis, following cloning of the DNA template from −127 to +101 into a plasmid pUC19 (see Figure 4(a)). For the TEC9 formation, the intrinsic cytosine residues at Positions +10 and +14 were retained. The binary complex was formed by mixing the holoenzyme (50 nM for E. coli or 100 nM for Syn) and 40 nM DNA template containing the psbA2 promoter as described above. The ternary complex was formed by incubating 5 min at 37°C with 5 μM [γ-32P]ATP (40 Ci mmol−1) as well as 50 μM each of GTP and UTP in T-buffer containing 10 mM MgCl2 or 1 mM MnCl2. The TEC was isolated from the substrates including [γ-32P]ATP as well as released abortive transcripts by triplicated passing through a MicroSpin G50 column (GE Healthcare) equilibrated with T2-buffer [50 mM Tris-HCl (pH 7.9), 100 mM KCl, 10 mM MgCl2 or 1 mM MnCl2, 1 mM DTT, and 5% glycerol] at room temperature. After this treatment, no elongations by the TEC9s and TEC18s of both Syn and E. coli in the T2 buffer were detected for 10 min at 37°C, without adding substrates.
2.5. Single-Step Elongation Assay
Reactions were started by adding cognate or noncognate NTP at the final concentration of 0.5 mM to 9 μL solution containing the TEC and the T2-buffer. The obtained mixture was incubated at 37°C for the indicated time and the reaction was stopped by adding an equal volume of gel-loading solution, containing 95% (v/v) deionized formamide, 20 mM EDTA (pH 8.0), bromophenol blue and xylene cyanol, 0.05% (w/v) each, and analyzed by PAGE using a 20% gel containing 7 M urea.
3. Results
3.1. Cyanobacterial Transcription System Reconstituted from Purified Components
For the comparative study, it is essential to purify cyanobacterial RNAPs. The tedious step of the purification is to remove thylakoid-membrane fragments and the associated proteins [20, 21]. We thus invented a batch-wise removal with a hydrophobic resin (see Materials and methods). The Syn and The core enzymes were purified by the improved procedure, resulting in a preparation which is more than 95% pure, judged by CBB stain, within a day (Figure 1(a)). The core enzyme of cyanobacteria is composed of α2ββ′ω as E. coli enzyme, but its β′ subunit is composed of two polypeptides [20]. A large nonconserved domain is inserted in the G region as chloroplast RNAP (Figure 1(b)) [21].
We examined the transcription by the holoenzymes of Syn, The, and E. coli retaining their primary factors (σs and σ70) at the T7A1 promoter (Figure 2(a)), which is the strongest among the standard promoters for the E. coli enzyme [22]. The Syn enzyme was active on this transcription unit but much weaker than the E. coli enzyme: the runoff transcript was observed only at the multiround condition, and its amount was lower by an order of magnitude (Figure 2(b)). Moreover, the The enzyme did not synthesize any detectable transcripts in all conditions (data not shown).
We thus searched for a promoter driving transcription by both cyanobacteria and E. coli holoenzymes without adding specific activators. The cyanobacterial psbA2 gene encoding D1 protein of the photosystem II is highly transcribed in the daytime [23] and the sequence of its promoter shares the −10 and −35 elements recognized by the E. coliσ70 holoenzyme (Figure 2(a)) [24]. As expected, the E. coli enzyme was active on this promoter and produced similar amounts of runoff transcripts from the psbA2 promoter in the single-round condition (Figure 2(c)). We thus used this promoter in comparison between these enzymes.
3.2. Cyanobacterial RNAPs Are Less Abortive due to Their Core Parts
In contrast to the similar amounts of runoff transcript, Syn RNAP produced a much lower level of 2–11 nt long transcripts (Figure 2(c)), which is known as abortive transcription, an iterative synthesis and release of oligo-RNA in initiation [25, 26]. The ratio of abortive synthesis to runoff synthesis is smaller than that in E. coli by two orders of magnitude. The ratio for The RNAP could be determined only in a multiround transcription condition and was as low as that of Syn RNAP (Figure 2(c)).
The observed small ratios were not specific to the psbA2 promoter, because in multiround transcriptions from the T7A1 promoter, the ratio for Syn RNAP was also two orders of magnitude smaller than that for E. coli (lanes 4 and 6 in Figure 2). In this experiment, we used the holoenzymes reconstituted from the σAs purified from the overproducing E. coli strains because of low yields of holoenzymes. The observed low activities of abortive transcription are not due to the artifacts of the reconstitution. We independently purified the histidine-tagged holoenzyme of a Synechocystis sp. PCC 6803 and it also produced abortive transcripts as low as the Syn and The RNAP (Figure S1A). In conclusion, cyanobacterial RNAPs are generally much less abortive than E. coli RNAP.
The level of abortive transcription is known to depend on mutations of both core enzyme and σ factor [27–29], indicating that both the components are responsible. We addressed the question of which component is more responsible by constructing chimeric holoenzymes. Taking into account the large difference between the levels of cyanobacterial and E. coli RNAPs, we found that the large difference was associated with the core part of RNAP but not σ (lanes 2, 5, 8, 11, and 12 in Figure 2). In spite of the absence of detectable transcripts by The enzyme in Figure 2, the above conclusion is also applicable to the enzyme because of the results of transcripts labeled with [α-32P] UTP (Figure S1B).
Among the abortive transcripts at the psbA2 promoter, the 10 nt- and 11 nt-transcripts of E. coli RNAP accompanied the bands with decreased migration (Figure 2(c)). These transcripts were shown to contain a purine nucleotide instead of the cognate cytosine at Position +10, a misincorporation, by a chemical cleavage assay (Figure S2). In contrast to abortive transcripts, the runoff transcript did not contain any detectable misincorporation. The chimeric enzymes containing E. coli core enzyme also showed the misincorporation (lanes 7, 8, 10–12 in Figure 2). Such a misincorporation by the E. coli enzyme had already been known for several promoters [19], and thus, this result suggests that the core enzyme is responsible for the misincorporation.
When the enzymes of two different species with different typical growing temperatures are compared, there are no absolute choice of the temperature. Since we focused to the difference in the catalytic properties of RNAPs rather than the difference in its role in their growth, we selected the same 37°C for in vitro assays. In this way, we can avoid the effect of the different stabilities of DNA duplex in the promoter. The both cyanobacteria grow and survive at 37°C although the optimal temperature of The is 55°C. Another lines of circumstantial evidence for our choice is that the levels of the runoff RNA synthesis are similar for the The and E. coli enzymes for the Syn rrnA promoter (Panel B in Figure S1).
3.3. Syn RNAP Is Likely to Form Moribund Complex which Produces Only Abortive Transcripts
For E. coli RNAP, only a part of the promoter complex synthesizes the full-length RNA, while the rest produces the majority of abortive transcripts at the promoter (Figure S3), the latter being named moribund complex [7, 30, 31]. To test whether or not the Syn RNAP has a similar property, we carried out the most sensitive assay for detecting the moribund complex: inverse pulse-labeling which monitors the fates of promoter-RNAP complex [30]. In this assay, a single-round transcription from the psbA2 promoter was started with unlabelled 4NTPs (ATP, CTP, GTP, and UTP) at time zero. The γ-32P-labeled initiating ATP was then added at various time points, followed by incubating for a further 20 min to complete the round (Figure 3(a)). Since the [γ-32P]-labeled runoff and abortive transcripts are both produced only by the RNAP that still survive at the promoter at the time point of adding [γ-32P]ATP, the ratio of the two kinds of labeled transcripts should reflect the preference of producing abortive transcripts.
As shown in Figures 3(b) and 3(c), the preference becomes stronger at the later time points, showing that the RNAP bound at the promoter at time zero is not homogeneous. Although there is no evidence that cyanobacterial RNAP forms the same moribund complex as E. coli RNAP, which produces only abortive products, a fraction of the Syn enzyme produces abortive transcripts preferentially over others, and the fraction is enriched at the later time points.
3.4. Syn RNAP Pauses More Frequently and Elongates Transcript More Slowly Than the E. coli Enzyme
We next compared the elongation by Syn with that by E. coli RNAPs. To isolate elongation from initiation, we used a ternary elongation complex retaining 9-mer transcripts, TEC9 (Figure 4(a)). It was elongated with 4NTPs in the presence of 10 mM Mg2+. The E. coli TEC9 smoothly elongated its transcript, and the elongation was completed at 40 s (Figure 4(b)). The 6- and 7-mer transcripts existed at time zero, and their amounts did not change, demonstrating that they had been contaminating TEC9. The 9-mer transcript, as well as a part of the 8-mer transcript, was elongated. Therefore, there is no significant pausing of the E. coli enzyme on this template.
In contrast, the elongation of the Syn TEC9 was paused at a number of lengths and was completed at later than 200 s (Figure 4(b)). When the time courses of syntheses of the run-off transcripts are approximated to single-exponential curves, the elongation of the Syn TEC9 has a time constant of 6 times longer than that of the E. coli TEC9, that is, time constants of 164 s compared with 26 s (Figure 4(c)).
The Syn TEC9 contains more amounts of 7-mer and 8-mer transcripts than the E. coli TEC9 at time zero. Since no such short transcripts were found prior to the step of removing NTPs in the preparation (data not shown), the shorter transcript must be generated in the absence of NTP by hydrolysis and/or pyrophosphorolysis of TEC9's, suggesting that the Syn TEC9 had a higher activity of shortening a transcript than E. coli had. In addition, the E. coli TEC9 is contaminated by a 13-mer transcript. This might be due to the slippage synthesis [32–34] on the psbA2 promoter, where the segment of AGUU transcribed from +1 to +4 slips back to the agtt template sequence from −4 to −1, resulting in an addition of 4 base extra sequence at the 5′-end (Figure 4(a)). Again, the Syn enzyme does not catalyze such a misincorporation.
3.5. Sensitivity to Mg2+ Ion of Syn RNAP
The Mg2+ concentration in chloroplast is observed to be changed at dark and at light [35]. In cyanobacteria, the gene expressions are globally repressed at dark, including the psbA2 expression, [36]. To test the possibility that transcription is directly controlled by Mg2+ concentration, we examined the sensitivity of Syn RNAP to the concentration (Figure 5). According to the increasing Mg2+ concentration from 0.5 to 2.5 mM, the level of runoff synthesis was increased by 25-fold, while the ratio of abortive synthesis to runoff synthesis was decreased to 1/15. In other words, it is possible to control RNAP to produce mainly abortive transcripts at a concentration and mainly mature transcripts in another concentration. However, such control would satisfy several quantitative conditions on the Mg2+ concentration.
3.6. Syn RNAP Shows Higher Fidelity in Elongation Than the E. coli Enzyme
The results above described show that the Syn RNAP is slower and more accurate in the whole elongation than the E. coli RNAP. We then examined the elongation at the resolution of one nucleotide and arbitrarily selected the elongation at +18. By using a psbA2 DNA with cytosine replaced as shown in Figure 6(a), we have prepared TEC18s by the Syn and E. coli enzymes. The elongations of Syn and E. coli TEC18s were carried out for 10 min with CTP, the cognate substrate, or ATP, a noncognate substrate, in the presence of 10 mM Mg2+ or 1 mM Mn2+ (Figure 6(b)). Similarly to TEC9s, a part of the transcript of the Syn TEC18 was shortened during the preparation, as shown in the lanes at time zero in Figure 6(b), probably due to hydrolysis or pyrophosphorolysis in the absence of NTPs. However, in contrast to TEC9, the shortening was not distinct for the E. coli TEC18, demonstrating that the Syn enzyme tends to shorten the retaining transcript more than the E. coli enzyme at Position +18.
In the presence of the cognate CTP, the Syn and E. coli TEC18s incorporated CMP at Position +19 within 1 min and then slowly misincorporated CMP at Position +20 (Figure 6(b)). The level of the CMP misincorporation at 10 min was lower for the Syn TEC18 than for the E. coli, being consistent with the more accurate whole elongation by the Syn enzyme than by the E. coli enzyme. In the presence of the noncognate ATP, both the TEC18s incorporated two AMP molecules, the misincorporation at Position +19 and +20, and again the Syn enzyme was more accurate. The level of the successive two AMP incorporations was by an order of magnitude higher for the E. coli TEC18 than for the Syn one (Figure 6(c)). The Syn TEC18 showed higher fidelity than the E. coli TEC irrespective of the coexisting divalent cations, also for misincorporating UMP and GMP (Figure 5(c)). Because this misincorporation may be interpreted as the misincorporation at +19 with the substrate at +20, this misincorporation could be due to the misalignment mechanism [37, 38].
In conclusion, the Syn enzyme showed higher fidelity than the E. coli one, both in initiation and in elongation, higher shortening activity, and slower rates of elongation. For most sets of substrates and enzymes, misincorporations were enhanced by the replacement of Mg2+ with Mn2+ except for the misincorporation by the E. coli TEC of AMP at +20 (Figure 6(c)).
4. Discussion
As shown in Figure 1(b), cyanobacterial RNAPs have large inserted domains in the G region of the β′ subunit [14]. In yeast RNAP II, the lack of Rpb9, which can be considered to be the counterpart of a part of the inserted domain of the bacterial β′ subunit, makes elongation faster and fidelity lower [4]. Our results share a similar feature: E. coli RNAP bearing smaller insertion elongates faster with lower fidelity than the cyanobacterial enzyme bearing a larger insertion. Therefore, the size of the inserted domain could be a determinant of the rate of elongation and the fidelity of an RNAP.
The holoenzyme containing the primary σ factor occupies less than 0.2% of the soluble proteins in cyanobacteria, and the content is at least 3 to 10-fold smaller than that in E. coli [24, 39, 40]. This is also consistent with the slower rates of elongation by cyanobacterial RNAP. As proved in E. coli, the release of sigma is a time-dependent event costing several seconds [41, 42], but elongation of several kilo base takes several minutes at least. Therefore, slower elongation requires less σ factor. In addition, the less formation of moribund complex also decreases the requirement of σ factor, because the complex contains the factor.
In E. coli, moribund complex blocks a promoter at the cost of abortive transcripts which are elongated at a rate of orders of magnitude smaller than the productive transcripts [30]. Therefore, repression by forming moribund complex at a promoter is considered to be less wasteful than abortion of long transcripts by elongation pause or immature termination (Figure S3). The Gre factors enable the conversion of moribund complex into productive complex [31] to eliminate the repression by moribund complex [43]. The factors finely tune the levels of some proteins which are important to the growth in rich nutrient conditions [7, 44] although the factors are not essential. For example, the transcriptional level of atp operon encoding FoF1ATPase, the main generator of ATP, is reduced to one fourth in the disruptant of the gre genes [7]. Therefore, the Gre factors allow E. coli to make a quick uptake of nutrients at the moderate cost of abortive synthesis.
The two cyanobacteria examined in this study, as well as most cyanobacteria, grow in poorer nutrient conditions than E. coli. They may not be allowed to use the moderate cost as E. coli does with its Gre factors, and this might be the reason why they exceptionally lack the genes orthologous to greA which is widely conserved in eubacteria. Consistently, their RNAPs elongate transcripts slower and form less moribund complex, making the Gre factors insignificant.
If the discussion above is the case, why is the branched pathway preserved in the cyanobacterial RNAP? There could be two answers. The first is that the mechanism is inevitably accompanied with the Brownian ratchet mechanism of RNAP elongation [45]. The other is that the cyanobacterial RNAP may use moribund complex only in limited conditions such as low Mg2+ concentrations. The ratio of abortive to runoff transcripts was 50 or more at a Mg2+ concentration lower than 0.5 mM (Figure 5), which is large enough for the repression by moribund complex to be significant.
The concentration of Mg2+ in a stroma of chloroplast has been measured to change from 0.5 mM at dark to 2 mM at light [35]. This increase by illumination is due to the uptake of Mg2+ from the inner region of the thylakoid membrane, and electrically compensated by the antitransport of proton from a stroma due to photosynthesis [46, 47]. The cytosolic pH in cyanobacteria has been measured to change as in the stroma between light and dark [48, 49]. In contrast to Mg2+, little is known about the variation of Mn2+ concentration in cyanobacterial cells. Although a measured value of the concentration of Mg2+ or Mn2+ in vivo cannot be directly correlated to its concentration effect on in vitro transcription (Figure S4), it is an important clue in comparative studies of RNAPs.
This work was supported by an NIG scholarship and Leaveanest Grant to M. Imashimizu. The authors thank Ms. Harriet Sallach for reading the paper.
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text:
Squartatore di New York, Lo1982
Chief of Police:
Fred, have you flipped out, or are you trying to give me an ulcer? A smart-ass coroner comes out with a little verbal diarrhea, and you immediately go around declaring there's a maniac loose in the city.
Lt. Fred Williams:
Yeah, so what do you want me to say? A boy scout's been wildling on girls with his knife?
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text: Ivan Lazar Miljenovic wrote: > Stephen Tetley <stephen.tetley at gmail.com> writes: > >> On 3 July 2010 14:00, Ivan Lazar Miljenovic <ivan.miljenovic at gmail.com> wrote: >> >>> So this argument isn't valid ;-) >> I think it was Hugs compliant as least for some revisions - I seem to >> remember looking at it before I switched to GHC. > > Actually, how would you call it in Hugs? Just start it explicitly with > the -98 flag? For most extensions you use the -98 flag, for OverlappingInstances you use +o, and for CPP you use -F'cpp -P -traditional -D__Hugs__' (or similar). If FFI is required then call the program with ffihugs first. E.g. $> ffihugs -98 +o -F'cpp -P -traditional' Bar.hs $> hugs -98 +o -F'cpp -P -traditional' Bar.hs Hugs does not know about LANGUAGE pragmas, as those were added in GHC 6.8. And Cabal does not infer the need for -98 or +o based on the Extensions: field, so you'll have to add them to Hugs-Options: manually. Also, there are some bugs in Cabal preventing the mixture of CPP and FFI, even though this is fine in Hugs. See http://community.haskell.org/~wren/cabal-ffihugstest for more details. -- Live well, ~wren


text:
"THE Scottish economy is now enjoying a strong recovery ..."
"THE Scottish economy is now enjoying a strong recovery ..."
This is the statement with which Professor Brian Ashcroft kicks off the latest economic commentary from Strathclyde University's Fraser of Allander Institute. So far, so good.
Loading article content
However, he does not even get to the end of the opening sentence before the caveats come thick and fast.
He cites the risks of a continuing lack of balance in the economic recovery in Scotland and the UK as a whole, falling real wages, booming prices in the London housing market, and deflation in the eurozone.
Mr Ashcroft also highlights the fact that the recovery continues to be considerably weaker than any since the 1930s.
He also flags the fact that household debt levels, as a percentage of income, remain high.
And Fraser of Allander, while noting the fall in the unemployment rate in Scotland to 6.6 per cent on the International Labour Organisation measure, observes many people are having to take part-time positions because they cannot find full-time work.
The same point has been made by others, including Bank of England Governor Mark Carney.
We should perhaps afford ourselves the luxury of a sigh of relief on reading the first part of Mr Ashcroft's sentence, that recovery has at long last got under way. After all, it has been a long haul, with the scale and mix of the Coalition Government's austerity programme having almost certainly impeded progress.
Having said that, no-one should under-estimate the reality or scale of the risks cited by Mr Ashcroft.


text:
Meet the Humble THQ Bundle; huge deals on great games that helps worthy charities
So, THQ is offering a number of games through the Humble Bundle website. At this point, some of you are probably a little confused. After all, doesn't it seem like the Humble Indie Bundle has kind of skimped on the "indie" in this case.
Let's be honest: they have, but that's alright. It's still a bloody excellent deal for anyone who doesn't have any of the titles they're offering. Besides, THQ could use the help.
Plus, a portion of the proceeds (you get to choose how much) goes to charity, and if you pay more than the average, you even get Saints Row: The Third in addition to all the other titles and the soundtrack for each game. The bundle includes the Company of Heroes series, Metro 2033, Darksiders, and Red Faction: Armageddon.
Video Permalink: http://youtu.be/8vX4jd3yrsM
Of course, if you've even one game on the list, don't expect to receive a gift copy. You don't get a code for each individual game: you just get a bundle code.
Still, it's a great deal if you don't have the titles, and could make an awesome Christmas gift, to boot.
Source: Humble Bundle
Via: Gamespy
|Nicholas Greene
A gamer at heart, Nick started writing when he was a child. He holds a BA in English, works as a freelancer, and loves every minute of it. One day, he hopes to net himself a career in game design - but that's something for the future.
[Other Gaming News +]


text:
The Dumbell Apartment
Name does not inspire trust, residents say. If you live in New York, you know these buildings and probably lived in one at some point (or still do...). These are the ones that have the well worn stairs, the central staircase, the old toilet doors in the hallway and NO CLOSETS anywhere. This the the "dumbell" design that won a design competition sponsored by the magazine, The Plumber and Sanitary Engineer, in 1879 and fueled a housing boom thereafter....Using the great Google we just found out all about it.... at The Living City. MGR


text:
You won't get hungry on Qantas anymore
- Published by Ozgur Tore
Qantas today unveiled details of its new inflight dining offering, which is set to re-define the travel experience for international Economy customers when it rolls out across the network from 25 November.
Customers will enjoy a wider choice of meals, fifty per cent larger servings and faster service.
Qantas Group Chief Executive Officer Alan Joyce said the dining experience will set a new standard for the five million Economy customers travelling across the airline's international network each year.
"We have worked closely with Australia's leading producers and suppliers to deliver what we believe to be the best inflight economy dining experience offered by any international carrier," said Mr Joyce.
"The new menu offers more generous meals and an increase from two to four meal options. We also have new meal categories, including a healthy option, comfort food and a meal inspired by the route itself, like Smoky Barbeque Beef on our US flights.
"We have extended our Select on Q Eat service to Economy passengers enabling them to pre-order their meal online with access to an exclusive dish as the fourth dish.
"Together with new serving plates, which remove the need for trays, our crew will be able to serve and clear meals up to 30 minutes faster giving them more time to interact and assist passengers," added Mr Joyce.
Some of the new dishes on the menu include smoky barbecue beef with roasted sweet potato, broccoli and corn; honey roasted chicken farro salad and seasonal vegetables with pumpkin and sesame seeds; scrambled eggs with chicken sausage, tomato, hash brown and baked beans; and Ruby & Roy's traditional Greek yoghurt with granola.
Pulled beef sliders with capsicum and tomato relish, Weis ice-cream bars, Maltesers and fresh fruit are among the self-serve grazing options. Shortly after take-off passengers will be served a welcome drink from Bickford's signature range, featuring flavours exclusive to Qantas such as Lemon & Elderflower, and Pink Grapefruit.
A team of Qantas food and beverage experts spent 12 months researching and developing the new menu and service, speaking with customers, visiting local producers and suppliers and researching food and dining trends. Trials were run on a number of international flights and customer satisfaction on those flights almost doubled, hitting record highs.
The new Economy dining experience will be available on the following dates:
· 25th November 2014: Melbourne-Dubai-London
· 26th November 2014: Sydney-Dubai-London
· 28th November, 2014: Tasman
· 5th December, 2014: Perth-Auckland seasonal service
· 9th December, 2014: US services; Los Angeles, New York and Dallas/Fort Worth
· 3rd January 2015: Vancouver seasonal service
· 13th January, 2015: Singapore
· 10th February, 2015: Hong Kong
· 1st March, 2015: Santiago, Johannesburg, Honolulu, Bangkok, Shanghai, Narita, Manila, Jakarta and Noumea


text:
A New Jersey bloggger and Rooer, Ellie Delancey, has posted a delightful twist on the Ben & Jerry's Bonnaroo Buzz flavor...with cupcakes! However trendy cupcakes have become the past few years, from Sex & the City's Magnolia Bakery plug (video above) to Brooklyn's Butch Bakery cupcakes for men, these Bonnaroo Cupcakes mix the malt and whiskey signature base with chocolate. She's got step-by-step and photo-by-photo instructions on how to create the jammin' cakes in your home. These just might be perfect road trip desserts for Bonnaroo 2012! :)
Bonnaroo and Woodstock Parallels
Music festival community and adventure are paralleled by blogger Sara Melvin's latest post. Check out the montage of Woodstock and Bonnaroo photos. The cross-generational parallels are quite amusing!
Bonnaroo News
Look for the latest music festival news here!
May 2012


text:
Farhana Pervin
Farhana Pervin has been a leading researcher in nanocomposite, traumatic brain injury and thin film for 7 years. She is the pioneering researcher in development of the novel experimental technique to assess the traumatic brain injury under impact loading. Her research in traumatic brain injury has made significant contributions to understand the tissue level mechanics of blast injury. She has published 11 international journal articles and 11 international conference papers and coauthored two book chapters. She is a Member of BSME, ASME, and SEM. She is the first woman from Bangladesh to be the recipient of Zonta International Amelia Earhart fellowship twice. She obtained her Ph.D. degree in 2010 from Purdue University, Ind, USA. Dr. Pervin was a Postdoctoral Research Associate at UIUC, Ill, USA, under the advisement of Professor Nancy Sottos after her graduation from Purdue University. She completed her M.S. degree in mechanical engineering at Tuskegee University, Tuskegee, Ala, USA, in 2006 and B.S. degree in mechanical engineering from Bangladesh University of Engineering and Technology, Dhaka, Bangladesh, in 2002.
Biography Updated on 25 December 2012


text:
As anyone driving through the capital will know, yellow box junctions – areas of the road marked off with do-not-enter-me yellow lines – are a deadly sight.
Yes we're not supposed to stop in them, and yes we should be prepared to receive a fine if we stray into one for more than a few seconds, but what if councils are playing it cheeky: Constructing situations where motorists have no choice but to be fined?
That's the view of the Daily Mail with regards to one particular camera-covered junction, which they – and some motorists – claim has been set up as a "cash cow" for the local council.
The yellow box, which is located on the corner of Bagley Lane and New King's Road in Fulham, is surrounded by a selection of traffic lights that the Mail claims are timed incorrectly.
The "trap", as the paper calls it, involves one set of lights – the closest to the drivers heading north-east along New King's Road – turning green, while the second set – located a few car lengths after the box junction – turn red.
As drivers unwittingly pass through the first set, the second turn red: Thereby creating a queue of traffic and forcing some drivers to sit illegally in the yellow box.
In fact, it's this very junction that caught AOL Cars editor James Baggott back in October. Unlike the drivers reported in the Mail, though, Baggott wasn't quite so quick to point the finger.
"The fact of the matter was – just like when you stray over the speed limit and get caught by a camera – I was in the wrong," he said in an opinion piece back in October.
"I'm glad – even though it was done (almost) completely innocently – that I got slapped with that fine, because for every person that does it by mistake there'll be five more drivers who have done it on purpose. And it's those very few that deserve a £65 slap on the wrist."


text:
We know. Next time search the forums first. Even the google results will link to our site... Or visit the FAQ/Tutorials section. At lot of stuff has already been answered. We even have a game compatibility list ongoing.
U need hermes cIOS 222/223. then change the settings for the IOS in the game load menu.
And stop posting the same topic in different forums. I figured I'd answer u before any more duplicates are made. Take a step back to see what your problem is then choose the right forum to post in. Otherwise your responses won't be so "nice".


text:
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text:
During RubyConf 2011, concurrency was a really hot topic. This is not a new issue, and the JRuby team has been talking about true concurrency for quite a while . The Global Interpreter Lock has also been in a subject a lot of discussions in the Python community and it's not surprising that the Ruby community experiences the same debates since the evolution of their implementations are somewhat similar. (There might also be some tension between EngineYard hiring the JRuby and Rubinius teams and Heroku which recently hired Matz (Ruby's creator) and Nobu, the #1 C Ruby contributor)
The GIL was probably even more of a hot topic now that Rubinius is about the join JRuby and MacRuby in the realm of GIL-less Ruby implementations.
During my RubyConf talk (slides here), I tried to explain how C Ruby works and why some decisions like having a GIL were made and why the Ruby core team isn't planning on removing this GIL anytime soon. The GIL is something a lot of Rubyists love to hate, but a lot of people don't seem to question why it's here and why Matz doesn't want to remove it. Defending the C Ruby decision isn't quite easy for me since I spend my free time working on an alternative Ruby implementation which doesn't use a GIL (MacRuby). However, I think it's important that people understand why the MRI team (C Ruby team) and some Pythonistas feels so strongly about the GIL.
What is the GIL?
Here is a quote from the Python wiki:
In CPython, the global interpreter lock, or GIL, is a mutex that prevents multiple native threads from executing Python bytecodes at once. This lock is necessary mainly because CPython's memory management is not thread-safe. (However, since the GIL exists, other features have grown to depend on the guarantees that it enforces.) [...] The GIL is controversial because it prevents multithreaded CPython programs from taking full advantage of multiprocessor systems in certain situations. Note that potentially blocking or long-running operations, such as I/O, image processing, and NumPy number crunching, happen outside the GIL. Therefore it is only in multithreaded programs that spend a lot of time inside the GIL, interpreting CPython bytecode, that the GIL becomes a bottleneck.
The same basically applies to C Ruby. To illustrate the quote above, here is a diagram representing two threads being executed by C Ruby:
Such a scheduling isn't a problem at all when you only have 1 cpu, since a cpu can only execute a piece of code at a time and context switching happens all the time to allow the machine to run multiple processes/threads in parallel. The problem is when you have more than 1 CPU because in that case, if you were to only run 1 Ruby process, then you would most of the time only use 1 cpu at a time. If you are running on a 8 cpu box, that's not cool at all! A lot of people stop at this explanation and imagine that their server can only handle one request at a time and they they rush to sign Greenpeace petitions asking Matz to make Ruby greener by optimizing Ruby and saving CPU cycles. Well, the reality is slightly different, I'll get back to that in a minute. Before I explain "ways to achieve true concurrency with CRuby, let me explain why C Ruby uses a GIL and why each implementation has to make an important choice and in this case both CPython and C Ruby chose to keep their GIL.
Why a GIL in the first place?
- It makes developer's lives easier (it's harder to corrupt data)
- It avoids race conditions within C extensions
- It makes C extensions development easier (no write barriers..)
- Most of the C libraries which are wrapped are not thread safe
- Parts of Ruby's implementation aren't threadsafe (Hash for instance)
Should C Ruby remove its GIL?
- No: it potentially makes Ruby code unsafe(r)
- No: it would break existing C extensions
- No: it would make writing C extensions harder
- No: it's a lot of work to change make C Ruby threadsafe
- No: Ruby is fast enough in most cases
- No: Memory optimization and GC is more important to tackle first
- No: C Ruby code would run slower
- Yes: we really need better/real concurrency
- Yes: Rubber boots analogy (Gustavo Niemeyer)
How can true concurrency be achieved using CRuby?
- Run multiple processes (which you probably do if you use Thin, Unicorn or Passenger)
- Use event-driven programming with a process per CPU
- MultiVMs in a process. Koichi presented his plan to run multiple VMs within a process. Each VM would have its own GIL and inter VM communication would be faster than inter process. This approach would solve most of the concurrency issues but at the cost of memory.
Finally, when developing web applications, each thread spend quite a lot of time in IOs which, as mentioned above won't block the thread scheduler. So if you receive two quasi-concurrent requests you might not even be affected by the GIL as illustrated in this diagram from Yehuda Katz:
This is a simplified diagram but you can see that a good chunk of the request life cycle in a Ruby app doesn't require the Ruby thread to be active (CPU Idle blocks) and therefore these 2 requests would be processed almost concurrently.
To boil it down to something simplified, when it comes to the GIL, an implementor has to chose between data safety and memory usage. But it is important to note that context switching between threads is faster than context switching between processes and data safety can and is often achieved in environments without a GIL, but it requires more knowledge and work on the developer side.
Similar Posts


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Coming out to their families is one of the most difficult things gay children can do. So where can they go if their family rejects them?
Green Chimneys is an organization in Gramercy Park that welcomes and fosters Lesbian, Gay, Bisexual, Transgender, and Questioning kids.
Teresa Nolan, who runs the foster home for LGBTQ Youth, says that more often than not, people in the program are usually kicked out or abused by families not accepting their identities.
Jamie, a 17-year-old gay male who benefits from the group, wasn't welcomed in his own home. "I used to get beat up all the time.. my father didn't accept it," he said.
But now, he feels like he belongs: "I'm just around kids that are just like me. We can relate more with each other."
Now, Jamie can stay in the center until he's 21, or until he's adopted.
But Nolan says that the chances LGBTQ youth actually have of being adopted are very slim. And when it does happen, it's celebrated "because it just doesn't happen very often."
Lena Harris, a social worker at Green Chimneys, allowed for that celebration when she adopted Mel, a young lesbian at the center.
"My mom showed me I could be cared about. She showed me a lot of love and stuff. And we decided she was going to adopt me," Mel said grinning from ear to ear.
Occupying less than 80 out of 36,000 beds in the NYC foster system, LGBTQ youth find Green Chimneys one of the few safe havens in town.
Sadly, however, the center is losing funding for one of its programs this year because of city budget cuts.
In order to fill the gap, they're holding the Power of Pride Concert Sunday, June 13, at Pier 66 on West 26th street at noon.
"Even though I'm gay, my biggest desire is having a family. I want my own family," Jamie said.
If you want to help Jamie, and the other 25 LGBTQ youth at Green Chimneys, you can buy tickets at GreenChimneys.org or at the door.


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text:
A recommendation letter for nanny is generally used to get additional information about a job seeker for employment, study or other opportunity.
It should be written in way which provide prospective employers with a general idea about the candidate's performance, experience, personal characteristics, capabilities, and professional strengths.
The recommendation or reference letter for nanny should be simple and to the point. Keep away from using jargon or language that is too common or extroverted. It should not exceed one page in length and consist of three paragraphs.
Following is a sample reference letter for nanny; you might use this template for these parallel job titles;
• Babysitter
• Live-in Caregiver
• Au-Pair
• Parent's Helper
• Child Care Provider
• Foster Parent
Nanny Recommendation Letter Example
October 8, 2017
To Whom It May Concern
I am writing to recommend Claire Smith as a nanny at your agency. Claire looked after my kids for three years and performed diligently throughout her tenure.
She was highly motivated to learn as much as she could and performed extremely well utilizing her best capabilities. She frequently devoted herself for long hours to develop lesson plans for my children and monitor their progress. As an individual who understands her limitations as a nanny, she never disturbed our privacy.
Particularly, she is very skilled in:
• Supervising children and caring for their needs
• Planning, preparing and serving meals to children
• Bathing, dressing and feeding infants and children
• Preparing formulas and changing diapers
• Performing light housekeeping duties
Claire has a number of strengths but her main strength – according to my observation – is her talent to motivate children. She always seemed genuinely interested in children's mental development and presented new ways of learning that captured their attention.
She is very reliable, trustworthy, motivated, and able to make herself tune-up with the requirements of family and children. In summary, Claire possess all qualities that make a nanny successful.
Once again, I highly recommend her for a nanny position. In case of any queries regrading her qualifications and abilities, please feel free to contact me on my phone number or email address given below.
Janet Monroe
[Email Address]


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The investigation into a single-engine biplane that crashed into a cattle pasture Thursday night is ongoing, according to Federal Aviation Administration officials.
The pilot, Phillip Witt of Roanoke, walked away with only minor cuts and bruises from the crash that was caused by his plane losing power after takeoff.
The Meyer Special aircraft lost power shortly after 6 p.m. at Northwest Regional Airport in Roanoke. The plane skidded through two fences before hitting a gully and flipping over in an open pasture, said Trooper Lonny Haschel, a spokesman for the Texas Department of Public Safety.
The plane crash wasn't far from the site of a fatal crash at the airport that occurred Sept. 22, Roanoke Fire Chief Mike Duncan said.
Last weekend, another plane took off from Northwest Regional Airport and crashed in Van Zandt County, killing four people on board.
Lynn Lundsford, FAA spokesman, said all of the crashes are under investigation.
Regarding safety concerns at the airport, which has been in operation for decades, Lundsford said, "The NTSB [National Transportation Safety Board] is in charge of all investigations involving fatalities and regularly makes safety recommendations when it finds something of concern."
The Dallas Morning News contributed to this report.
BJ LEWIS can be reached at 940-566-6875. His e-mail address is email@example.com.