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Cowboys 16, Chargers 14: My Top 10 Observations
10. I'm out of excuses/reasons/explanations. Any theories as to why - still - quarterback Tony Romo and receiver Roy Williams don't seem to be on the same page? Sometimes it's Romo overthrowing him and sometimes it's Williams falling down. Whatever, they just don't click.
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7. You can complain about offensive coordinator Jason Garrett's third-and-one call in the first quarter - the rollout pass that Miles Austin dropped - but remember this: The Cowboys aren't yet game-planning. Their goal isn't to win games. It's to work on plays that may help them once the real games start. Geez. Some people.
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5. One thing's for certain, the Cowboys have found themselves a fourth cornerback. His name is Cletis Gordon. The former Charger joins Keith Brooking and Igor Olshansky as former Wade Phillips' players who can still play in Dallas. Maybe Wade knows talent after all.
4. I know it's early, but rookie linebacker Sean Lee looks like a player. Yes?
3. Couple thoughts on the 80-yard fumble return in the second quarter. Did Barry Church really run out of gas? Seriously? He's a safety, not a nose tackle. Looked like he had just run 800 yards, not 80. And what the what was Anthony Spencer doing? A shot to cleanly plow over San Diego's Pro Bowl quarterback and he just sorta jogs by doing, um, nothing.
2. When Martellus Bennett cherishes football over fame, the results can be scary. More tight end. Less "Kung Fu Astronaut."
1. Main negative to the preseason isn't the lackluster offense, but more so the injuries. With the addition of starting strong safety Gerald Sensabaugh (dislocated left shoulder), that's now four starters - along with Marcus Spears, Marc Colombo and Kyle Kosier - who might not be ready for the September 12 opener at Washington. And I almost forgot about Dez Bryant.
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Does what it is supposed to do.
By RiderRick
from Dayton, OH
About Me Avid Cyclist
See all my reviews
Comments about Headsweats CoolMax Shorty Skull Cap - Discontinued Style:
Cycling friends of mine use this product. I am a heavy sweater, so I thought I would give it a try. It really does help keep the sweat out of my eyes and off of my sunglasses. It seems to funnel a lot of the sweat back to the tail and down my back, help keeping your upper body cool as well. It is not 100% effective. Some sweat on hard rides can still come off the front of my head, but it significantly reduces the sweat going into my eyes and onto the front of my bike.
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Comment on this review
Good service. Good price. Keeps most of sweat out of eyes.
By Ron
from Loxley, al
Good at keeping eyes clear. Good price. Headband could be a tad thicker. No complaints though. Would recommend the product
Everyone active should have oneof these
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from Canfield,ohio
This product really works for me. It has helped me be able to wear a bike helmet without getting bugs and flies in my scalp as well as absorb some sweat along with not being unable to wear a helmet or hat without it falling off. I am also going to use it small game hunting in Minnesota next week!!
Bottom Line Yes, I would recommend this to a friend
Cool Style
By Ricko
from Ames Iowa
Perfect under your helmet ... should not have been discontinued.
Works well
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from Wausau WI
Works well to keep the sweat out of my eyes and from keeping bugs out of the little hair I have left when they get through vents in the helmet. Just rinse it out and let it dry after a hard ride, and wash it occasionally.
keeps the sweat out of my eyes.
By ibgready
from Baldwin County Georgia
This is the first sweat band skull cap that I ever bought. It works and it looks ok. I think the sweat band part of it could be tacked in place a bit better and a thicker terry cloth on the band would be better. I imagine you would have to pay more for these features this was a cheap deal so I am happy
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from O'Fallon, IL
In this humid mid-west weather, I needed something to keep the sweat out of my eyes. I wear contact lenses and, after about 20 minutes on my bike, sweat starts to drip between my lens and my eye. What a sting! Sometimes it's so bad I have to stop and wipe down. This lightweight sweatband solves the problem. I still get a little sweat that leaks through, but what an improvement! Love it.
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It is light as air and does a good job of keeping the sweat out of my eyes, fits under the helmet nicely, ventilates well when warm.
Great product
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Keeps sweat out of my eyes
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Counting down the hours
Credits & Info
- Uploaded
- Sep 1, 2010 | 11:28 PM EDT
- File Info
- Song
- 3 min 10 sec
- Score
- 4.41 / 5.00
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Heard in 6 Entries!
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Licensing Terms
You may not use this work for commercial purposes without making specific arrangements with the artist.
- Score:
- Rated 4.41 / 5 stars
- Plays & Downloads:
- 22,768 Plays | 2,683 Downloads
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- Genres:
- Easy Listening - Classical
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Author Comments
Rated 5 / 5 stars
Easily the best track in K.O.L.M., full of emotional value.
Counting down the hours spent pursuing your dreams, facing obstacles, never giving up, and learning from mistakes.
Well done mate,
Rated 5 / 5 stars
Beautiful, mate.
Really don't know what to say, past that you should honestly consider pursuing a career in film or video-game scoring.
I fell in love with this song playing K.O.L.M., and I'll probably leave it looped while I'm working on my novel.
I am, so this is very good to hear :)
Thanks very much and good luck with your novel
Rated 5 / 5 stars
Tear Jerking.
I Love This Music.It's So Sad And You Can Feel Someone Lost Alone And Crying I Myself Cryed It's Just So Moving.
Rated 5 / 5 stars
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Rated 4.5 / 5 stars
Wow dude
This is a pretty cool song, very sad but i guess thats what u were going for and u nailed it, yo sonds kinda like a movie soundtrack, sry 8/10 for the sadness but realy well thought out. well played my friend


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Average Cost is More Than $90,000 Among Midsized Firms Reporting Noncompliance with Payroll Tax Laws, Other Government Regulations
ROSELAND, N.J. – December 19, 2013 – ADP®, a leading global provider of Human Capital Management (HCM) solutions, reveals a disconnect between midsized business leaders' confidence in their ability to manage regulatory compliance compared to the reality of keeping pace with evolving requirements. According to a recent ADP Research Institute® study, Top Concerns and Challenges of Midsized Business Leaders in 2013, 30% of midsized companies reported unintended expenses, including fines, penalties or lawsuits, in the past 12 months due to noncompliance with government workforce and payroll regulations.
The study found that 83% of midsized business owners are confident their firms are compliant with payroll tax laws and regulations. Similarly, 80% of respondents felt the same about government workforce regulations, such as those related to 401(k) plan management and workers' compensation. ADP's study also found that nearly one-third of midsized firms fined or penalized in the past year reported an average of six fines or penalties with an average total cost of $91,300. Interestingly, midsized organizations processing payroll in-house received almost twice as many fines or penalties as those that outsource processing to a third party.
View infographic
"Our research reveals a 'compliance overconfidence' in the midmarket that's creating costs these firms can ill afford," said Regina Lee, President, Major Account Services and ADP Canada. "With more than 60 years of experience helping companies adapt to a changing regulatory landscape, ADP's dedicated team of compliance experts and integrated HCM platforms empower organizations to reduce compliance risk and unintended expenses."
Complex legislation, such as the Affordable Care Act (ACA), and growing pressure from global competition make regulatory compliance even more challenging for companies of all sizes. The same ADP study found that 70% of midsized business leaders expressed confidence that their firms would be in "complete compliance" with ACA, yet less than half expressed confidence in their firms' understanding of all the new ACA regulations. As midsized companies expand operations into multiple countries, they need integrated HCM tools to simplify compliance with diverse payroll laws and streamline reporting.
Recent enhancements to the ADP Workforce Now® platform directly address midsized companies' top compliance challenges, such as new features to support ACA provisions stemming from the law's employer "shared responsibility" requirements. This all-in-one HCM platform also helps midsized businesses quickly implement ongoing changes in payroll and employment tax laws, such as those mandated by the American Taxpayer Relief Act of 2012.
More information about how ADP Workforce Now helps midsized companies overcome compliance challenges is available at www.adp.com/workforcenow.
About the ADP Research Institute®
The ADP Research Institute is a specialized group within ADP that provides insights to leaders in both the private and public sectors around issues of human capital management, employment trends and workforce strategy. For more information about the ADP Research Institute, visit:www.adp.com/research.
About ADP
With more than $11 billion in revenues and more than 60 years of experience, ADP® (NASDAQ: ADP) serves approximately 620,000 clients in more than 125 countries. As one of the world's largest providers of business outsourcing and human capital management solutions, ADP offers a wide range of human resource, payroll, talent management, tax and benefits administration solutions from a single source, and helps clients comply with regulatory and legislative changes, such as the Affordable Care Act (ACA). ADP's easy-to-use solutions for employers provide superior value to companies of all types and sizes. ADP is also a leading provider of integrated computing solutions to auto, truck, motorcycle, marine, recreational vehicle, and heavy equipment dealers throughout the world. For more information about ADP, visit the company's Web site at www.adp.com.
The ADP Logo, ADP, ADP Workforce Now and ADP Research Institute are registered trademarks of ADP, Inc. All other marks are the property of their respective owners.
Copyright © 2013 ADP, Inc.
Media Contact:
Michael Schneider
(973) 404-4241


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Delon misses Opera Ball over health concerns
The Local/AFP · 4 Feb 2016, 15:02
Published: 04 Feb 2016 15:02 GMT+01:00
- Hundreds of police to guard Opera Ball guests (03 Feb 16)
- Canalis' breast slips out at Opera Ball (13 Feb 15)
"Alain Delon's cardiologist said he wasn't fit to travel," German fashion designer Florian Wess, who had invited the one-time heartthrob star to the event, said on Thursday.
"It's a real shame that Mr Delon can't come. But we understand the situation, he's 80 after all and you've got to be careful at that age," he told the APA news agency.
Instead, Delon's son Anthony, also an actor, will attend the opulent shindig at the State Opera in the Austrian capital, Wess revealed.
The ball is the highlight of Vienna's social calendar and draws celebrities from around the world.
The event's 60th edition is set to open on Thursday evening with a performance by renowned tenor Placido Domingo.
The celebrity guest list features American actress Brooke Shields and former Playboy model Pamela Anderson, who will rub shoulders with Austria's political elite, including Chancellor Werner Faymann.
For more news from Austria, join us on Facebook and Twitter.


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3rd May 2016
Exhibitions and Trade Shows are a great way of promoting your business within your industry sector, be it at the NEC, Excel Arena, Ricoh or events like Agricultural shows where you just set up in a...… more
25th April 2016
GL events UK recently announced the Operations Director of its UK brands, Darren Woodhouse in a series of new recruits that the company sees as the stimulus for further growth within its business.… more
18th April 2016
Teely Pearson Webb is an award winning PA who will be speaking at the Conference and Hospitality Show, Leeds on Tuesday 26th April.… more
12th April 2016
By creating two new event spaces which can cater for up to 100 people, Lord's Cricket Ground has invested £3.8m at the famous venue and refurbished a further space at the venue.… more
11th April 2016
The Conference and Hospitality Show (CHS) is now in its seventh year, and is the leading event in the North for event... more
7th April 2016
Nominations for Event Technology Awards 2016 are now open and will close on 31st May 2016.… more
29th February 2016
Bristol Live Events Firm teamed up with its customer and garnered an award winning stand design at the Wedding Journal Show 2016. The Wedding Shop with its collaborative effort with Bristol based...… more
25th February 2016
Come and meet the Exhibitions team at International Confex, 2nd – 3rd March 2016 at Olympia, London.… more
22nd February 2016
This year's International Confex, Lily Cole will keynote the educational programme. The actor and social entrepreneur is also a renowned and engaging speaker and will take to the stage on day two of...… more
19th February 2016
Join Studio Souffle for a drink, talk inflatable events and play with their Baby Robot "Robob". They will be exhibiting at the Event Production Show in March. Pop round and see them if you're...… more
16th February 2016
In partnership with International Confex, The AEO will once again be running its Sales Conference as part of the show's educational programme. Last year, the conference debuted at the show and has...… more
10th February 2016
Where can I find an events venue that is going to meet my needs and the needs of my organisation, my delegates and my budget?… more
8th February 2016
During UK Events Week, Jez Jowett, Havas Media Group's Global Head or Creative Technology will join Henry Stuart Found and CEO of virtual reality production company Visualise and Scott Hope,...… more
4th February 2016
International Confex will be dominated by some of the biggest and most prestigious hotel, destination and venue brands in the world when the show opens on 2-3 March 16 at Olympia London.… more
2nd February 2016
2nd February 16, UK: Nick de Bois and Kevin Jackson, will open UK Events Week with two exclusive sessions, which will look at the future of the events industry amongst major corporate businesses,...… more
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With bowl game berths and conference championships on the line, Week 14's slate of games produced moments that college football players around the nation will never forget.
The SEC championship game was the marquee matchup on the weekend, and it undoubtedly lived up to its billing. Alabama won in an incredible game, and while the Big Ten, Big 12 and ACC championship games did not offer as much excitement, Wisconsin, Kansas State and Florida Sate fans all enjoyed themselves.
But the action started Thursday when Louisville bested Rutgers to secure a sport in a BCS bowl game, and the thrills continued into Friday night as the Pac-12 and MAC championships produced thrilling contests.
Stanford is Rose Bowl bound while Kent State has fallen short of a shocking BCS bid.
Here are the best images from all of these fantastic games.
Bama Ready to Go Back-to-Back
The showdown between No. 2 Alabama and No. 3 Georgia will not be forgotten anytime soon, and the Crimson Tide came out on top in a wild game. The most talked-about play from the first half came when Alabama's Quentin Dial blindsided Georgia quarterback Aaron Murray and somehow was not penalized for this hit.
Seminoles Squeak By Yellow Jackets
No. 13 Florida State won the ACC as expected, but Georgia Tech made sure the Seminoles had to work for a spot in the Orange Bowl. FSU's James Wilder Jr. had two second-quarter touchdowns and helped build a big lead.
Big Ten Blowout
Wisconsin started fast against Nebraska and did not let up until they had roses in their teeth. Montee Ball had three of the Badgers' 10 touchdowns in the 70-31 rout.
Wildcats Coast Into Fiesta Bowl
The Big 12 has no conference championship game, but the conference's teams still had to finish up their schedule. The marquee match was No. 18 Texas taking on No. 6 Kansas State, and Collin Klein made one final Heisman push by accounting for three touchdowns.
Roses are
Red Cardinal
UCLA faced off with Stanford for the second time in six days, and once again, the Cardinal came out on top. This time, it was a much closer contest with significantly higher stakes.
The Bruins looked to be the better team early and were driving towards a two-touchdown lead, but an Ed Reynolds interception and long return flipped this game on its head.
Headed to the Rose Bowl: usat.ly/Swe3up //twitpic.com/bhzhm1— USA TODAY Sports (@USATODAYsports) December 1, 2012
Battle for the Big East
While many college football fans would argue that no Big East team deserves to be in a BCS bowl, supporters at this game cared little and were completely aware of what was on the line.
Kent State Can't Cut It
Kent State was on the verge of cracking the Top 16 in the BCS standings, and that would have booked them a spot in a BCS bowl. Northern Illinois now has a chance to break that threshold, and the players held out hope after their 44-37 victory.


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Does anyone have any information on cleaning trumpets and hard water (well water without softener) issues?
Was just wondering if it contributed to scale on the inside of the instrument or not.
Does anyone have any information on cleaning trumpets and hard water (well water without softener) issues?
Was just wondering if it contributed to scale on the inside of the instrument or not.
Little Horns
Jupiter 416 Pocket Trumpet
2013 Corneta Traspositor C/Db
'32 Conn 40B Vocabell
'32 Selmer Louis Armstrong Special
'50 Blessing Super Artist
Bigger Horns
2009 Phaeton Flugelhorn
Hi John,
I would have thought that hard water would still be OK to clean, it just will not suds up with detergent as much.
If it was boiled then the deposits would get left behind, (we do not use boiling water) but the small amount of moisture left in the horn after flushing and cleaning would not affect it IMO. But happy to hear from others on this.
Why do I like a bell that Points UP ?
- because the spit does not run back into my mouth!
In many ways, hard water is better since the high calcium content keeps the chemistry on the alkaline side of neutral. Soft water can easily pick up a bit of carbon dioxide and stray into the acidic side.
If your tap water is prone to significant scaling, you'll see either a white (calcium) or brown (iron) crustiness around your hot water tap nozzles, and the sink/bath below. They won't harm the instrument and are quite easy to shift.
Bb Trumpets: Yamaha YTR-6335HSII - Flip Oakes "Wild Thing" - 1972 Getzen Eterna "Severinsen" - 1980 Boosey & Hawkes Sovereign Studio - B&S 3005 WTR-L - 1963 Besson 10-10 - Monke Mystery Horn - Spiri Vario
C Trumpet: Inderbinen Alpha 200
Bb Bass: 1961 Holton #58 "Symphony"
Wyrd oft nereð unfågne eorl, þonne his ellen dëah.
"Pypes, trompes, nakers, clariounes, that in bataille blowen blody sounes"
I live in the land of crunchy water and I can tell you that it makes absolutely no difference.
Buescher Lightweight 400
Other Buescher horns 1939--1955
Al Cass 1-28 mouthpiece
Humes and Berg mutes
I'll second that. Any minerals in the tap water is nothing compared to the minerals that can accumulate in a horn just from playing it one time.
Stop acting like someone shot your dog.
Alka Seltzer has some aspirin, sodium citrate, and sodium bicarbonate in it so will buffer and clean and tie up tramp metals like calcium and magnesium from hard water. It also takes many of the minerals as gzent mentions. The aspirin does nothing unless your horn gets migraines.... but the other ingredients are the most easy way to get properly buffered and compounded sodium citrate if you are not a chemist and into mixing your own stuff. You can also mix most dishwater detergents like Dawn etc into the warm Alka Seltzer treated solution. Just be sure to rinse everything out well at the end of this with regular water when you are done.
Not comfortable with this? Then test this procedure and see how it performs with you and your local water supply (they vary a lot across the US) on a student horn before using it on a prized 1946 LB Martin Committee or Olds Super Recording. That's what I did myself....my old 1933 Liberty really cleaned up nicely inside with this and some snake action.
Getzen Custom 3051 (.462) RL
French Besson Marvin Stamm 94 BB (.464) (Kanstul)
Kanstul Chicago 1001 MLP (.464)
Lawler C7 Step bore (.453/.460/.462) 1A
Unless you plan to use your trumpet as a nozzle for a garden hose, hard water will make no contribution to the use of your horn. If you still worry about this after all the above posts validating the safe use of hard water, perhaps hard liqueur will help.
Unless you plan to run water through the horn 24 hours a day, 365 days a year, and do this for a number of years, there is no problem. Point being, you will have absolutely NO buildup washing your horn once a month. now, for those who use the salt and aluminum foil method to clean the sliver on their trumpet, I have an idea hard water likely keeps this process from working as efficiently (any of you chemistry folks are welcome to confirm or correct me).
Put the horn in hot water for silver plate, mild temperature if an older lacquered horn, all in a tub (plastic preferably or put a towel on the bottom to keep down scratches if porcelain etc.), add several good squirts of Dawn (hey they use it to clean water fowl soaked with oil from oil spills), let soak, then scrub well with your bristled brush and snake.
Olds Supers, LA (1953), Ful. (1962)
Olds Recording, LA (1952)
Olds Studio, LA (1953)
Olds Special, Ful. (1964)
Olds Ambassador, LA (1954)
Olds Ambassador, Ful. (1973)
Bach Strads 37-(1967, 1970, 1974, 1982)
Bach Strad 72 MLV (1973), 72* (1982)
Kanstul 1500 (2002), 1502 (2008), 1503 (2002)
Kanstul 1537 (2007)
Kanstul Chicago (2000)
Kanstul 1510 C
King Liberty (1929,1929)
King Liberty 2 (1938, 1944)
King Liberty 2b (1950)
J.H. Darby 45 USA
Holton (Revelation) 1924
Kanstul 1525 Flugelhorn
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Welcome to City Physiotherapy & Sports Injury Clinic Adelaide
At City Physiotherapy & Sports Injury Clinic Adelaide our staff are experienced and professional and are committed to providing you with hands on physio solutions to your musculoskeletal aches, pains and strains. We promise to listen to your needs and to provide you with hands on individual solutions in a friendly and caring environment. We are here to minimise your pain so you can get on with enjoying your life. We value our partnership with you in your wellness and health journey. Value and invest in your wellbeing and health and let us help you to feel better now.
Why choose City Physiotherapy?
Why choose City Physio? Connection and caring is what it's all about. Our physiotherapists are skilled and over the years we've learnt countless new physio treatment techniques and keep up with the latest research- you can rest assured you will get the results and help you're looking for with City Physiotherapy. However, good outcome orientated therapeutic physiotherapy treatment aside one of our best qualities is that we connect with each patient and we understand the meaning of caring treatment to the highest level. Our patients keep coming back to us because they trust us to partner with them on their journey to improve their health, to minimise their pain and so they can get on with living and being active. We love getting to know the people we treat and we know that City Physiotherapy can offer you the care and support that you deserve through all the stages of your life.
If you'd like to be kept informed of friendly and caring Adelaide City Physiotherapy, health and wellbeing tips then click on our FACEBOOK page and like us.
Convenient CBD Location & Opening Hours
City Physiotherapy & Sports Injury Clinic is well located in the Adelaide CBD. You can find us near the corner of King William and Pirie St opposite the Adelaide Town Hall on Level 6/108 King William Street. Public Transportation Tram and Bus stops are located right out the front making access easy for everyone, whether you work in the City of Adelaide or are coming from further afield. With opening times Monday to Friday 8am to 6pm we aim to have you back home feeling better so you can enjoy your valuable leisure time with family and friends. We also have a second clinic within the Adelaide City Goodlife Gymnasium located at 81 King William Street. You can ask for a one on one gymnasium physiotherapy session with your physiotherapist at City Physiotherapy and Sports Injury Clinic for those needing hands on rehabilitation or first time gym users, we are there to help you.
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At City Physiotherapy we understand that you need us to run our appointments on time so you can get on with the rest of your day. We aim to have you in our waiting room for the least possible time wherever we can. If you do get the opportunity to sit for a short time in our waiting room, we have a fabulous range of latest magazines for you to read. Sometimes our patients have been known to deliberately come 15 minutes before their appointment just to catch up on reading the magazines! Our friendly administration staff will do their best to attend to your questions on products and services, claiming and appointments and offer you all this with a smile.
Established over 22 years
City Physiotherapy & Sports Injury Clinic has provided Physiotherapy services to the People of Adelaide for over 22 years. With established roots in Adelaide and a wealth of experience we are committed to providing a lasting relationship with you and will do all we can to provide the best possible outcomes for you, your family and colleagues.
May 2016 Newsletter. (PDF file, 386KB)
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realmagick.com The shrine of knowledge.
Drilling Mud Purpose
A selection of articles related to drilling mud purpose.
Original articles from our library related to the Drilling Mud Purpose. See Table of Contents for further available material (downloadable resources) on Drilling Mud Purpose.
- Basics of Magick: The Use of Color in Magick
- White | Silver | Grey | Pink | Red | Purple | Brown | Blue | Green | Yellow | Gold | Black In magick, colors represent certain energies, goals, people and non-physical beings, such as deities or spiritual forces. For this reason, you should include candles,...
Symbology >> Colorology
- Survivalists' Guide for the New Millennium: Chapter 2
- WORKING TO LIVE RATHER THAN LIVING TO WORK Before the dawn of recorded history, a major period of transition occurred. Nomadic hunter-gatherer clans transformed themselves into larger communities who farmed the land. This they did by planting their own...
Philosophy >> Survivalists Guide for the New Millennium
- The Earthmind Material
- Part 1 | Part 2 Author's Note All references made in the first person(s) herein, are grammatical conveniences created for the sake of the reader. Such words as "I", "OUR", and "I/WE" are therefore not meant to be taken as literal...
Mind >> World Mind
- The Perpetual Raising: Part 3
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Mind >> World Mind
- Survivalists' Guide for the New Millennium: Chapter 5
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Philosophy >> Survivalists Guide for the New Millennium
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Mystic Sciences >> Mind
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Pokemon Nostalgia
Maybe a thread has already been made before but... I can't take it anymore. :py06:
These past 2 months, getting nostalgia from the old pokemon games. Being Red/Blue and Gold/Silver. The graphics, the music, the memories... all the time spent on there back when I was younger. Pokemon make me more nostalgic than anything else! :py11:
Anyone else feel nostalgic when thinking about the old ones for Gameboy? Create that darn feeling of warm, happy and yet depressed in a way? So tough to handle... :py64:
( On a side note, nostalgia probably protects games that even today people say it's good but probably not so much anymore and seems overrated. Not the pokemon games though. Thinking of other games. )
Anyway just wanted to let that out and let other share at their feelings of pokemon.
I miss my first pokemone game @.@. I used to play Silver like freaking crazy and I had a great team even though I didn't even level them up or play them as most players nowadays would say is "correct".
It was also funny because I would keep leveling up my Sudowudo thinking he would level xD. I got so disappointed when it never did D:
I really wish they'd rerelease it again but that won't happen. It's not the same with using an Emulator.
i like pokemon yellow. Didn't have to hunt in the forest for hours for a pikachu.
Pokemon Yellow and Silver ;_;
....those were the days.
I actually tried to restart my gold version for kicks yesterday, but I dishearteninlgy found out that my save battery is DEAD so it can't hold a save anymore.
g/s remake for ds faflksdjfkjsdlf yes plz
red/blue > *
Trades it over to blue.
"Pikachu evolved into Raichu!"
Trades back. Flips off Yellow cartridge.
Notice on GameFAQs how seems only Gold and Silver has reached that 9.0 score. All others are 8.9 or below. Besides Platinum being a 9 at the moment, it might go below a 9 once it comes out and more people rate it.
Also notice the dates Red/Blue were released and ones to follow. G/S came out about 2 years later. R/S came out was about a 3 year difference. Then to D/P was about a 4 year difference. So we can pretty much expect a new pokemon game in about 2-4 years. Those time differences seem so short yet so long.
But yes, nothing can bring back such a feeling. Even if I were to play the old ones it does not feel the same. That is how nostalgia is after all. :py06:
My first pokemon game was...eh...Pokemon FireRed/LeafGreen...then I got Pokemon XD: Gale of Darkness....then my friend gave me his Pokemon: Yellow....Then I got pokemon D/P and I traded in FR/LG
...To keep my mom from pawning my Yellow I hid it behind my gamecube LOL.
I played:
Red [emulator/rom]
Blue [emulator/rom]
Yellow [emulator/rom]
Green [emulator/rom]
Gold [emulator/rom]
Silver [emulator/rom]
Crystal [emulator/rom]
FireRed [owned]
Diamond [owned]
my first pokemon game was silver
i wanted to play it again a couple of moths ago but it's broken now ;_;
I first started the Pokemon series with Blue. And it was really great. I then moved to Gold, which was my favorite game. With all that beautiful music and stuff. May have been the easiest version, but man was it hella fun. Too bad it's save battery is broken too. XD
I really do want a Gold remake though. That'd be so cool. I personally liked how the trainer looked. He wasn't so generic imo. He actually wore clothes that people would wear. Now in Pokemon games it's all like, "WHO WOULD OWN THIS STUFF??". Oh well. /waits for "White Gold".
@Jono: Shoddy only covers D/P. We'd have to play NetBattle and play in the R/B/Y or G/S/C.
I liked pokemon games with a buttload of secrets. I remember being the envy of my whole class for catching Entei in Gold when the 3rd movie came out. Such awesomeness..
Silver and Gold were my favorite, I had gold and my bro play silver, so that made us a good combination xD
There were really a lot of memory, such as my bro at one point got stuck on the ship (forgot name), and didn't know that he has to go to room to sleep to continue (at the end he gave up and re-start the whole game lol xD);
and in Gold I leveled this Sandslash to lvl 89 (I gave up on 4X% on way to 90, it was just too hard...), and I still remembered all 4 techniques it used...lol he was unbeatable beside water type pokemon, almost all pokemon get 1hko from ANY of my techniques lol
Sadly, neither of our 2 games are around now, I lost my Gold somewhere in the house, as for Silver...the out of internal battery thingy erased my bro's save and he can't play it anymore...then it got lost too ;-;
P.S. if they remake gold and silver, I'd really prefer they just make it in the quality of GBA version (like Ruby and Emerald), I'm not really into the 3D effect in Pearl and Diamond
I personally don't care. I think the 3D would compliment it. But either way I'd be satisfied. I'd also like if you couldn't EV train in it, just like the old days. But I know that won't happen. xD
I just liked how Pokemon was Pokemon back then. If you fell asleep you were screwed. Wrap was "strong" with a fast Pokemon. And the music, was beautiful.
If they remake Gold and Silver, it would most likely be in 3D which I am fine with either way. Because no matter the graphics in the remake, it won't feel the same, but would still be nice anyway. No game feels the same when you play it even out of nostalgia, after all.
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Clostridium perfringens type A isolates carrying a chromosomal copy of the enterotoxin (cpe) gene are involved in the majority of food poisoning (FP) outbreaks, while type A isolates carrying a plasmid-borne cpe gene are involved in C. perfringens-associated non-food-borne (NFB) gastrointestinal diseases. To cause diseases, C. perfringens spores must germinate and return to active growth. Previously, we showed that only spores of FP isolates were able to germinate with K+ ions. We now found that the spores of the majority of FP isolates, but none of the NFB isolates, germinated with the cogerminants Na+ and inorganic phosphate (NaPi) at a pH of ~6.0. Spores of gerKA-KC and gerAA mutants germinated to a lesser extent and released less dipicolinic acid (DPA) than did wild-type spores with NaPi. Although gerKB spores germinated to a similar extent as wild-type spores with NaPi, their rate of germination was lower. Similarly, gerO and gerO gerQ mutant spores germinated slower and released less DPA than did wild-type spores with NaPi. In contrast, gerQ spores germinated to a slightly lesser extent than wild-type spores but released all of their DPA during NaPi germination. In sum, this study identified NaPi as a novel nutrient germinant for spores of most FP isolates and provided evidence that proteins encoded by the gerKA-KC operon, gerAA, and gerO are required for NaPi-induced spore germination.
Clostridium perfringens is a gram-positive, anaerobic, spore-forming, pathogenic bacterium that causes a wide array of gastrointestinal (GI) diseases in both animals and humans (14, 15). However, Clostridium perfringens type A food poisoning (FP) is the most common C. perfringens-associated illness among humans and is currently ranked as the third most commonly reported food-borne disease (14). Mostly type A isolates that produce the C. perfringens enterotoxin have been associated with C. perfringens-related GI illnesses (14). C. perfringens cpe-positive isolates can carry the cpe gene on either the chromosome or a plasmid (3, 4). Interestingly, the majority of C. perfringens type A FP isolates carry a chromosomal copy of the cpe gene, while all non-food-borne (NFB) GI disease isolates carry a plasmid copy of cpe (3, 4, 11, 29). The genetic differences involved in the pathogenesis differences between C. perfringens FP and NFB isolates seem to involve more factors than the simple location of the cpe gene. For example, spores of FP isolates are strikingly more resistant than spores of NFB isolates to heat (100°C) (27), cold (4°C), and freezing (−20°C) temperatures (12) and to chemicals used in food industry settings (13), making FP spores more suited for FP environments. Under favorable environmental conditions, these dormant spores germinate to return to active growth, proliferate to high numbers, and then produce toxins to cause disease (14).
Bacterial spores germinate when they sense the presence of nutrients (termed germinants) in the environment through their cognate receptors located in the spore inner membrane (18). For C. perfringens, some nutrients that initiate germination include l-asparagine, KCl, a mixture of l-asparagine and KCl, and a 1:1 chelate of Ca2+ and dipicolinic acid (DPA) (Ca-DPA) (20). The main receptor(s) involved in sensing these compounds is the GerKA and/or GerKC receptor(s), which is required for l-asparagine and Ca-DPA and only partially required for KCl and an l-asparagine-KCl mixture (20, 21). Upon binding of the germinant to its cognate receptor, a variety of biophysical events take place, including the release of monovalent ions (i.e., Na+, K+, and Li+) followed by the release of the spore's large depot of Ca-DPA (28). In Bacillus subtilis, release of Ca-DPA acts as a signal for activation of the cortex-lytic enzyme CwlJ (17). In contrast, Ca-DPA release from the spore core has no role in triggering cortex hydrolysis during C. perfringens spore germination (19, 22, 23); instead, Ca-DPA induces germination via the GerKA and/or GerKC receptor(s) (20, 21). Degradation of the cortex in both species leads to hydration of the spore core up to levels found in growing bacteria, allowing resumption of enzymatic activity and metabolism, and consequently outgrowth (22, 28).
The ability of bacterial spores to sense different nutrients appears to be tightly regulated by their adaptation to different environmental niches. For example, spores of FP isolates, but not NFB isolates, are capable of germinating with KCl (20), an intrinsic mineral of meats that are most commonly associated with FP, suggesting an adaptation of FP isolates to FP environments. In addition, the level of inorganic phosphate (Pi) is also significant in meat products (42 to 60 mM) (USDA [http://fnic.nal.usda.gov/nal_display/index.php?info_center=4&tax_level=1&tax_subject=242]). Similarly, sodium ions are also present in meats (~30 mM), especially in processed meat products (~300 to 400 mM) (USDA). Consequently, in this study we found that Na+ and Pi at ~100 mM and pH 6.0 are unique cogerminants for spores of C. perfringens type A FP isolates, act through the GerKA and/or GerKC and GerAA receptors, and also require the presence of the putative Na+/K+-H+ antiporter, GerO, for normal germination.
C. perfringens isolates used in this study included the following: six FP isolates carrying a chromosomal cpe gene, namely, SM101 (electroporatable derivative of an FP type A isolate; NCTC8798) (30), NCTC8798 (6), NCTC10239, E13, FD1041 (3), and 6263 (8); three NFB isolates carrying a plasmid copy of the cpe gene, namely, F4969, NB16, and B40 (3); one type C cpe-negative isolate, JGS1495 (from a diarrheic pig) (7); C. perfringens SM101 ger mutants DPS101 (gerKA-KC), DPS103 (gerAA) (20), and DPS108 (gerKB) (21); and C. perfringens SM101 antiporter mutants DPS113 (gerQ), DPS116 (gerO), and DPS115 (gerO gerQ) (24).
Starter cultures (10 ml) of C. perfringens isolates were prepared by overnight growth at 37°C in fluid thioglycolate broth (Difco) as described previously (10). Sporulating cultures of C. perfringens were prepared by inoculating 0.2 ml of a fluid thioglycolate starter culture into 10 ml of Duncan-Strong sporulating medium (5); this culture was incubated for 24 h at 37°C to form spores, as confirmed by phase-contrast microscopy. Spore preparations were prepared by scaling up the latter procedure. Spore preparations were cleaned by repeated centrifugation and washing with sterile distilled water until spores were >99% free of sporulating cells, cell debris, and germinated spores and then suspended in distilled water at a final optical density at 600 nm (OD600) of ~6 and stored at −20°C (20).
Spores at an OD600 of ~1.0 were routinely heat shocked (80°C; 10 min), cooled in water at ambient temperature, and germinated at 40°C and pH 6.0 with 100 mM sodium phosphate (NaPi), citric acid, sodium citrate, 3-morpholinopropanesulfonic acid (MOPS), maleic acid, acetic acid, or sodium acetate as previously described (20). Spore germination was routinely measured by monitoring the OD600 of spore cultures (Smartspec 3000 spectrophotometer; Bio-Rad Laboratories, Hercules, CA), which falls ~60% upon complete spore germination, and levels of germination were confirmed by phase-contrast microscopy. To measure the rate at which the spore population decreased its OD, the maximum rates of spore germination were determined by measuring the OD600 of germinating cultures every 2.5 min, the maximum slopes were calculated, and maximum rates were expressed as the maximum rate of loss in OD600 of the spore suspension relative to the initial OD600 of the culture.
DPA release during NaPi germination was measured by incubating a heat-activated spore suspension at an OD600 of 1.0 at 40°C with 250 mM NaPi (pH 6.0). Aliquots (1 ml) of germinating cultures were centrifuged for 3 min in a microcentrifuge, and DPA in the supernatant fluid was measured by monitoring the OD270 as described previously (2, 20). To measure the total spore DPA content, a 1-ml aliquot of the germination culture was boiled for 60 min and centrifuged for 5 min in a microcentrifuge, and the OD270 of the supernatant fluid was measured (2, 20). In C. perfringens, ~90% of the material absorbing at 270 nm comprises DPA (22).
Student's t test was used for specific comparisons. All experiments were repeated four times with two independent spore preparations.
In our previous study, we observed that 25 mM NaPi (pH 7.0) was able to induce little, but significant, germination of C. perfringens spores (20). To evaluate if NaPi could indeed induce spore germination, we assayed the germination of spores of SM101 (an FP isolate) and F4969 (an NFB isolate) with various concentrations of NaPi at various pHs. Surprisingly, NaPi (100 mM) was able to induce germination of SM101 spores at a pH range of 5.0 to 6.5, but not below pH 5.0 or above pH 7.0 (Fig. (Fig.1A).1A). Although the rates of germination at pH 5.0 and 6.5 were much lower than that at pH 6.0, SM101 spores were able to germinate to similar extents at a pH range of 5.0 to 6.5 (Fig. (Fig.1B).1B). Indeed, phase-contrast microscopy indicated that >90% of SM101 spores had become phase dark when germinated with NaPi at a pH range of 5.0 to 6.5 (data not shown). However, NaPi was unable to induce germination of F4969 spores at all tested pHs (Fig. 1A and B). Phase-contrast microscopy showed that 100% of F4969 spores incubated with 100 mM NaPi (at various pHs) remained phase bright, indicating no germination (data not shown).
SM101 spores exhibited a maximum rate of germination with 250 mM NaPi at pH 6.0 (Fig. (Fig.1C).1C). Interestingly, SM101 spores were able to germinate significantly after 60 min with NaPi concentrations as low as 20 mM (Fig. (Fig.1D),1D), as phase-contrast microscopy showed that ~85% of the spores had become phase dark. In contrast, F4969 spores showed no significant increase in their rate of germination (Fig. (Fig.1C)1C) and no significant extent of germination, with the exception of a small but significant decrease in OD600 at 400 mM NaPi (Fig. (Fig.1D).1D). Phase-contrast microscopy of F4969 spores with NaPi concentrations of <250 mM showed that >99% of spores remained phase bright, but with a 400 mM concentration ~10% and ~30% of the spores became phase dark and gray, respectively, after 60 min of incubation. Collectively, these results suggest that NaPi at pH 6.0 is a unique germinant for spores of SM101 but not for those of F4969.
To discriminate if the ability of SM101 spores to germinate with NaPi (pH 6.0) was due to NaPi or pH 6.0 in the absence of NaPi, we evaluated various organic and inorganic buffers. As expected, SM101 spores, but not F4969 spores, germinated well with NaPi (pH 6.0) (Fig. (Fig.22 and data not shown); however, little to insignificant germination was observed when SM101 spores were incubated with a 100 mM concentration of other buffers (e.g., MOPS or maleic acid) at pH 6.0 (Fig. (Fig.2).2). In addition, F4969 spores also exhibited no germination with any of the buffers tested (data not shown). Phase-contrast microscopy also indicated that >90% of SM101 spores and >99% of F4969 spores remained phase bright after 60 min of incubation with the tested organic and inorganic buffers without NaPi (data not shown), suggesting that the effect is uniquely due to the presence of sodium and/or Pi. Since a previous study (20) indicated that K+ can trigger germination of C. perfringens spores, it is possible that Na+ acts as a cogerminant with Pi. Indeed, 100 mM sodium citrate (pH 6.0) and 100 mM sodium acetate (pH 6.0) induced slight increases in the extent of germination of SM101 spores compared to those with citric acid and acetic acid, respectively (Fig. (Fig.2).2). To evaluate further the role of Na+ in spore germination, germination assays were conducted with various concentrations of NaCl (in 25 mM NaPi to adjust the pH) at pH 6.0 and 7.0 (Fig. (Fig.3).3). While SM101 spores exhibited little germination at concentrations of <25 mM NaCl at pH 7.0, a significant level of germination was observed when SM101 spores were incubated at concentrations of >50 mM NaCl at pH 7.0. However, when the pH was reduced to 6.0, the addition of as little as 1 mM Na+ from NaCl, in addition to the 25 mM from the NaPi buffer, was able to induce a significant level of germination (Fig. (Fig.3).3). As expected, the extent of germination of SM101 spores increased when Na+ was replaced by K+ (Fig. (Fig.3).3). The K+ ions were able to induce higher extents of germination of SM101 spores at pH 7.0 than at pH 6.0 (Fig. (Fig.3).3). It was somewhat surprising that a 250 mM concentration of either Na+ or K+ induced similar extents of germination of SM101 spores at both pH 6.0 and 7.0 (Fig. (Fig.3).3). However, these results contradict the results from previous studies (1, 20) where the pH of 50 mM NaCl was not reported (1) and the germination assays were carried out at 30°C (20). Indeed, in this study, no significant extent of germination was observed when SM101 spores were incubated with 100 mM NaCl (pH 7.0 [adjusted with 25 mM NaPi]) at 30°C (data not shown). Collectively, these results suggest that Na+ acts as a cogerminant with Pi.
NaPi-induced germination of spores of SM101 was not due to domestication and manipulation of this laboratory strain, as spores of NCTC8798 (the parent strain of SM101) germinated similarly to SM101 spores with NaPi (Fig. (Fig.4),4), suggesting that NaPi (pH 6.0) might be a universal germinant for spores of C. perfringens FP isolates. To examine this hypothesis, germination experiments were extended to include spores of seven additional C. perfringens isolates, including four type A FP isolates (i.e., E13, NCTC10239, FD1041, and 6263), two NFB isolates (NB16 and B40), and one cpe-negative type C isolate (JGS1495). Strikingly, as observed with spores of SM101 (Fig. 1A to C), spores of all FP isolates, with the exception of FD1041, were able to germinate well with NaPi (pH 6.0) (Fig. (Fig.4).4). In contrast, spores of NFB isolates were unable to germinate with NaPi (pH 6.0) when incubated for up to 60 min (Fig. (Fig.4).4). Although NFB spores required a lower heat activation temperature (20), similar results were observed (i.e., no significant germination) for NaPi germination when NFB spores were heat activated at 75°C for 10 min (data not shown). Interestingly, the negative NaPi germination phenotype was not confined to cpe-positive type A NFB human isolates, as spores of strain JGS1495 (a type C cpe-negative animal isolate) were also unable to germinate with NaPi (pH 6.0) (Fig. (Fig.4).4). Collectively, these results clearly indicate that NaPi (pH 6.0) is a unique germinant for spores of most FP isolates.
To gain insight into the mechanism of NaPi germination of C. perfringens spores, the germination of spores of strains carrying mutations in germination receptor genes gerKA-KC, gerKB, and gerAA (20, 21) was assayed using the optimum (250 mM) NaPi concentration (Fig. (Fig.1C).1C). Although gerKB spores exhibited significantly (P < 0.05) less germination during the first 20 min than wild-type spores did, their extents of germination after 60 min were similar (Fig. (Fig.5A).5A). This was confirmed by phase-contrast microscopy, where >95% of wild-type and gerKB spores had become phase dark after 60 min of incubation with NaPi (data not shown). In contrast, gerAA spores exhibited significantly (P < 0.001) less germination than that of wild-type and gerKB spores (Fig. (Fig.5A),5A), as ~50% of gerAA spores had become phase dark after 60 min of incubation (data not shown). The germination defect in gerKA-KC spores was significantly (P < 0.01) more pronounced than that in gerAA spores (Fig. (Fig.5A),5A), with phase-contrast microscopy results showing that ~35% of gerKA-KC spores had become phase dark after 60 min of incubation with NaPi (data not shown). These results suggest that GerKA-KC and GerAA receptors are the major receptors involved in NaPi germination and that GerKB may play an auxiliary role.
The rate of germination was measured to determine the role of the Ger receptors in the initial triggering of germination by NaPi. Interestingly, gerKA-KC spores exhibited a rate of germination that was ~5-fold lower than that of wild-type spores and ~2-fold lower than those of gerKB and gerAA spores germinated with NaPi (Fig. (Fig.5B).5B). However, gerKB and gerAA spores exhibited similar rates of germination, but these were ~2-fold lower than that of wild-type spores (Fig. (Fig.5B).5B). These results suggest that GerKA-KC and GerAA receptors are the major receptors involved in NaPi germination and that GerKB is required for normal NaPi-triggered germination.
For Bacillus and Clostridium spores, binding of germinants to specific receptors located in the spore's inner membrane triggers the release of the spore core's large depot of DPA as a 1:1 chelate to Ca2+ (Ca-DPA) (20, 28). Consequently, to investigate the role of each Ger receptor in DPA release during NaPi-induced germination, we measured DPA release during NaPi germination. Consistent with the similarities observed in the extent of germination between gerKB and wild-type spores, gerKB spores also released a similar level of DPA to that released from wild-type spores (Fig. (Fig.5C).5C). However, although gerAA spores released the majority of their DPA during NaPi germination, the amount of DPA remaining in the core of gerAA spores was still slightly higher than that in wild-type spores, which is statistically significant (P < 0.01) (Fig. (Fig.5C).5C). gerKA-KC spores released significantly (P < 0.001) less DPA than wild-type spores did. The amount of DPA released by gerKA-KC spores was also significantly (P < 0.05) less than that released by gerKB and gerAA spores. These results suggest that GerKA and/or GerKC is mainly involved in DPA release during NaPi-triggered germination, with GerAA and GerKB having an auxiliary role.
We recently identified two putative antiporters (GerO and GerQ) involved in germination of C. perfringens spores (24). GerO, a putative Na+/K+-H+ antiporter, is required for normal germination with all known germinants for C. perfringens, while GerQ is required mainly in the absence of GerO. To evaluate their roles in NaPi-triggered germination, spores of antiporter mutants were assayed for germination in the presence of NaPi (pH 6.0). gerQ spores exhibited a slightly slower and lesser extent of germination than that of wild-type spores (Fig. (Fig.6A);6A); gerO spores germinated to even a lesser extent. Indeed, phase-contrast microscopy indicated that only ~75% of gerO spores had become phase dark after 60 min of germination with NaPi (pH 6.0) (data not shown). Interestingly, gerO gerQ spores had significantly (P < 0.01) slower germination than that of wild-type, gerQ, and gerO spores (Fig. (Fig.6A),6A), and phase-contrast microscopy indicated that only ~30% of gerO gerQ spores had become phase dark (data not shown). The gerQ, gerO, and gerO gerQ spores exhibited 30, 70, and 80% lower rates of germination, respectively, than that of wild-type spores (Fig. (Fig.6B).6B). These results clearly suggest that GerO and GerQ, only in the absence of GerO, are essential for normal NaPi-triggered germination.
Interestingly, gerQ spores were able to release similar amounts of DPA to that released by wild-type spores after 60 min of incubation with NaPi (Fig. (Fig.6C),6C), consistent with the majority of the spores completing germination. To our surprise, gerO spores also released similar amounts of DPA to that released by wild-type spores (Fig. (Fig.6C),6C), although it is unclear why the extent of germination was significantly less than that with wild-type spores (Fig. (Fig.6A;6A; see Discussion). However, gerO gerQ spores released significantly (P < 0.001) less DPA than wild-type, gerO, and gerQ spores did (Fig. (Fig.6C).6C). These results indicate that NaPi-induced DPA release is affected only in the absence of both GerO and GerQ antiporters, similar to the case with K+-induced germination (24).
The ability of spores of FP isolates to adapt to FP environments seems to go beyond the fact that spores of FP isolates exhibit ~60-fold more heat resistance than spores of NFB isolates (27). The unique ability of FP isolate spores to germinate with K+ ions (20) might provide a greater advantage over spores of NFB isolates present in meat products in the ability to germinate, outgrow, and proliferate. In this respect, and perhaps offering the major conclusion of this work, Na+ and Pi are novel cogerminants of spores of C. perfringens FP isolates. Na+ and Pi, together with K+, are intrinsic minerals found in meat and processed meat products and might provide spores of FP isolates significant advantages over spores of other C. perfringens isolates in the ability to germinate, outgrow, and proliferate in meat products during inadequate processing or subsequent warming. For example, in nonprocessed meat products, where the Na+ concentration is ~30 mM, Na+ and Pi might act as cogerminants together with the major germinant K+, triggering germination of FP spores during ripening of the meats. Contrarily, in processed meat products, with Na+ concentrations of ~300 to 400 mM, it is likely that Na+ can become a major germinant together with K+, triggering germination of FP spores. Results from this study also suggest that spores of FP isolates might sense these ions through an evolved ionic germination mechanism not present in spores of NFB isolates. This concept has previously been proposed for some strains of Bacillus megaterium and C. perfringens strain NCTC8238 (1, 25). Several studies (9, 26) indicate that C. perfringens FP isolates belong to a different evolutionary lineage from that of all other C. perfringens isolates. Indeed, the differential germination phenotype of FP spores reinforces the theory that FP isolates have efficiently adapted to fit FP niches. However, it is unclear what genetic difference in the germination apparatuses of FP versus NFB isolates might have evolved to produce this difference in K+ and NaPi germination phenotypes, since strain F4969 (an NFB isolate) has intact copies of all four germinant receptor genes (gerAA, gerKB, and gerKA-KC) and shows >95% identity to those of SM101 (16). One possibility that deserves further research is that perhaps key residues within the receptor proteins might be essential for the unique NaPi and K+ germination of FP isolates.
A second major conclusion is that NaPi germination of FP spores requires all four Ger receptors, although to different extents. The main germinant receptor proteins involved in NaPi germination are GerKA and/or GerKC, which are also essential for l-asparagine germination and have a major role in KCl germination (20). However, gerKA-KC spores are still able to germinate significantly with KCl (20) as well as with NaPi (this study), suggesting that other Ger proteins might have a role in DPA release during NaPi germination. Indeed, in contrast to KCl germination (20), GerAA also has a role in NaPi germination, with gerAA spores germinating at a much lower rate and to a lesser extent than wild-type spores (Fig. 5A and B). In contrast, GerKB, which was previously shown to have no role in nutrient germination of FP spores (21), appears to play a role in normal germination of FP spores with NaPi. Clearly, the majority of C. perfringens Ger receptors are required for initial triggering of germination with NaPi, suggesting that all C. perfringens Ger receptors form a receptor complex and that the absence of GerKA-KC, GerAA, or GerKB has a major, medium, or small effect, respectively, on the receptor complex's function. Further biochemical studies on receptor proteins should help to clarify this issue.
A third notable conclusion is that the putative antiporters GerO and GerQ, only in the absence of GerO, are required for normal NaPi germination of C. perfringens spores. Our previous study showed that these antiporters are also essential for normal germination of C. perfringens spores with various germinants and that their role precedes DPA release (24). Interestingly, GerO was also essential for normal NaPi germination; however, in contrast to gerO spores releasing significantly less DPA than wild-type spores during K+ germination (24), here the gerO spores were able to release the majority of their DPA during NaPi germination, and this seems likely to occur because of the presence of GerQ. However, it is possible that the larger amount of DPA released by gerO spores in this study than in the previous study (24) was due to the nature of the novel germinant (NaPi) or to the fact that we used a twofold higher concentration of NaPi, creating a higher ionic strength in the medium that could in some fashion stimulate more DPA release. Although the rate of germination of gerQ spores was lower than that of wild-type spores, indicating reduced initial triggering of germination, they germinated to a similar extent as wild-type spores and also released the majority of their DPA. In addition, GerQ became essential only in gerO gerQ spores, which had significantly less initial triggering of germination and germinated to a lesser extent than gerO, gerQ, and wild-type spores and released about half of their DPA. These results reinforce the role of cation transporters in germination of C. perfringens spores with all known germinants (24), including NaPi (this study). However, it is not yet clear whether the effect of gerO or gerQ mutation is directly on spore germination or during spore formation. Further studies are currently being carried out to help decide between these two scenarios.
This work was supported by a grant from the N. L. Tartar Foundation of Oregon State University, by a grant from the Agricultural Research Foundation of Oregon State University, by a grant from the Army Research Office (all to M.R.S.), and by a fellowship from the Office of the Higher Education Commission (Thailand) to P.U.
Published ahead of print on 7 August 2009.
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