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ELOY — Pinal County's employment situation is on the upswing, but there's still plenty of work to be done.
That was the primary message from the leaders of central Arizona economic development group Access Arizona during its annual investor luncheon Thursday at Robson Ranch's Hermosa Ballroom.
Access Arizona Executive Director Jim Dinkle said Pinal County's unemployment rate has dropped from 8.3 percent to 7.1 percent in the last year, which is more in line with the statewide unemployment rate of 6.7 percent. Representatives from PhoenixMart, Tractor Supply Co. and Alliance Lumber highlighted the number of jobs and revenue their businesses have generated in Pinal County or are expected to generate in the future.
But Luke Jackson, Access Arizona's director of economic development, said the organization needs to do more in the area of workforce development, noting complaints he's heard from local business owners about the rate of employee turnover.
"We have to get more involved and partner with them to really create results, a better workforce, a more stable workforce, a more educated workforce," he said.
Jackson said the best way to attract new businesses isn't just with lower taxes and property costs, but with a higher-skilled workforce.
"We need more people working in workforce development, more people supporting them, and I hate to say this, but more money for those people, as well, if we're truly going to be effective," he said. "But we have the right pieces in place."
One of those pieces is Banner Health, a nonprofit hospital network based in Phoenix and one of Access Arizona's member investors. In June, Banner acquired Casa Grande Regional Medical Center, the culmination of a process that began in December 2013.
"There is no other organization in the entire state of Arizona that can stand before you and say" they have cut costs as dramatically as Banner has, said Rebecca Kuhn, president, Arizona east region for Banner Health and keynote speaker at Thursday's luncheon.
Through the acquisition, Banner retained CGRMC CEO Rona Curphy, who introduced Kuhn on Thursday.
"Basically we saw an opportunity to assist and support Rona and her team in this community in terms of delivering value-based care," Kuhn said.
"It represents a growth opportunity for us. Our goal is to expand, be a statewide network, because we think we can bring value to the people in multiple communities."
Kuhn said Banner is enhancing services to women and infants at its Casa Grande location and is improving emergency, trauma and surgical services. It has also introduced telehealth, which helps critical-care nurses more accurately detect symptoms and warning signs in patients.
"We are saving lives through this technology, and this is a technology that we've been able to bring to your facility here in Casa Grande."
Since the acquisition, Kuhn said Banner Casa Grande has provided a community benefit of $44 million, including $12 million in unpaid Medicare and AHCCCS costs and $3 million in charitable contributions. She said the hospital employs 780 people paying a total of $54 million in salaries, wages and benefits.
"As business leaders, we know what a burden that is for you, the cost of health care," she said. "We're doing a lot of great things in terms of providing care and managing care. We are also absolutely committed to improving outcomes and reducing costs."
Access Arizona similarly reaffirmed its commitment Thursday to improving economic conditions in Pinal County. Its leaders celebrated the county's recent job growth but acknowledged there's much work ahead.
"We're at a crossroads because we want to be competitive in the future and build on the successes that we have and that we announced," Jackson said. "We're going to have to increase the ability, the size and the capacity of the workforce or we'll just cease to be competitive."


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Sunday, May 24, 2015
New 'Teen Titans Go!' to Fly on DC Nation
Robin, Starfire, Raven, Beast Boy and Cyborg are coming back to Cartoon Network in 2013. A new generation of Teen Titans will make their debut on the cable network's DC Nation block ... Continue Reading →
WB TV Reveals Comic-Con Line Up
With just over a month remaining until the pop culture fandom hordes invade San Diego for Comic-Con International (July 12-15), Warner Bros. Television Group has announced their bang-up ... Continue Reading →
Cartoon Network Schedules Father's Day Marathon
After taking dad to brunch this upcoming Father's Day, you can catch some favorite funny dad-themed episodes of Regular Show, Johnny Test and The Amazing World of Gumball on Cartoon ... Continue Reading →
'Aqua Something' Premieres June 24 on Adult Swim
If you've got a hankering for new adventures starring those tasty toonsters Frylock, Master Shake and Meatwad, we've got good news for you: Starting June 24 at midnight, ... Continue Reading →
Call for Submissions: MIA Animation Festival
The MIA Animation Conference & Festival, presented by Miami International University of Art & Design, is seeking submissions for its 2012 event which is set to take place October ... Continue Reading →
June 21 WIA Panel to Explore the Green Light Process
Women in Animation's next general meeting will include a special panel discussion titled "The Green Light: Who Says 'Yes' and Why?" The panel will include ... Continue Reading →
Tales from the Weird Side
Gravity Falls creator Alex Hirsch taps his childhood memories to create the offbeat world of the hot new Disney Channel cartoon. The fun tagline for Disney Channel's new show Gravity ... Continue Reading →
CN Ups Curtis Lelash to VP of Comedy Animation
Curtis Lelash has been promoted to vice president of comedy animation for Cartoon Network. Lelash will oversee the development of Cartoon Network's animated comedy series, which ... Continue Reading →
Pitch Party '12: Adult Swim's Manning Joins Judges' Table
Animation Magazine is pleased to announce that Adult Swim programming honcho Kim Manning has joined our judges line-up for the 2012 Pitch Party competition! Entries to our annual animation ... Continue Reading →
'LEGO Ninjago Masters' DVD to Attack in June
The season of Cartoon Network's Lego Ninjago: Masters of Spinjitzu is coming to DVD on June 26. The two-disc DVD set will include all 13 episodes of the first season, which chronicles ... Continue Reading →

subset: commoncrawl


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Winter!: Prepare for holiday gaming!
Sand Giant down! After weeks of leveling, the Sand Giants in Ro are no longer as dangerous as they once were. I was passing through the zone on my way to Cazic Thule and arrived at this battle just after it had finished. Otherwise I would have clicked a shot of the giant in mid fall, hehe. Although Taznarrok does add some insult to injury payback.
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Leave a Review
I made this dish, in addition to potato and cheese soup, and a shrimp/asparagus salad. the pasta came out too dry. i think i should have added another egg? the flavors of the cheese was too sharp for my friend. he put the noodles into a little bit of the soup broth and ate it like that. he ate a lot of it like that though, so i guess that combination works well
Only substitution was that I only had 2 eggs, but it worked out fine. My Roman boyfriend (meaning a picky eater --like a 5 year old) said it was the best carbonara he'd ever eaten, then gave the reciepe to his mom! Now I'm afraid to visit her. :-)
This is the way to make carbonara. Period, full stop.
That's ~1 oz of pancetta, 1/2 oz of cheese and less than 1 egg per person. You're right, might as well just eat the tub of lard! that's what I really wanted anyway.
Sort of low fat? LOL. 2 oz of cheese, pancetta and 3 eggs is low fat? Yeah, I guess compared to eating spoonsful of lard straight out of the crock, it's low fat.
I like the fact that this recipe was sort of low fat. Next time I would add some garlic and less onions.
Been to Italy, come from an Italian family and loved it! I thought it was delicious. No snobby authentic comments here. In a completly heinous move, I had to use salt pork because my local market was out of pancetta. It was STILL good. Pleased that it had no cream in it. I do recommend free range or organic eggs though as the yolks are much more flavourful and that is a big part of this dish.
I used the ready crisp bacon from Costco. It was probably a bad idea.
This was a fast, delicious weeknight supper. I used good quality pancetta and cheese, and served it with a simple salad and baked fruit for dessert.
I have been looking for a good carbonara recipe for ages - strange that there is so many different recipes for such a simple dish. This is a great one - and the one I'm going to stick to. What works so well is the addition of wine and the absence of cream. I shouldn't eat it as I'm pregnant and our UK eggs can be a bit dodgy - but cravings must be obeyed!


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PIQUA — The Miami County Foundation will offer a brief instructional session to explain its grant application process. The session will be held from 9-10 a.m. July 30, in room 511 at Edison Community College, 1973 Edison Drive, Piqua. Advance registration is required by phoning the foundation office at 773-9012 or email firstname.lastname@example.com.
To be eligible for Miami County Foundation grant submission the organization must provide services directly to the citizens of Miami County, must be certified federally tax-exempt by the IRS as a 501(c) or equivalent organization, preferably 501(c)(3). Grants will not be considered for organizations that limit their services to members of any one religious group, political organizations or those whose primary purpose is to influence legislation or political view or general operating funds. Grant deadlines are the last day of February and August each year.
The purpose of the Miami County Foundation is to solicit, receive and administer assets exclusively for charitable purposes that would most effectively assist, encourage and promote the health, education and welfare of the residents of Miami County. For more information visit www.miamicountyfoundation.org.
At Edison Community College on the same day, July 30, there will be a free workshop to demonstrate the Foundation Directory Online for representatives of not-for-profit organizations. The college is a cooperating collection sight for the Foundation Center. The college library houses free grant information, core collections of Foundation Center publications and a variety of supplementary materials and services in areas useful to grant seekers. This online directory is the Foundation Center's top-tier searchable database providing information on more than 100,000 grant makers and more than a million grant records. Participants will learn grant seeking basics for not-for-profit organizations using the online database. This introductory session is ideal for those seeking grants from foundations, corporations and grant making public charities.
Training for the online grant directory will be from 10-11 a.m. July 30 in room 511 at Edison. Space is limited so pre-registration for this free workshop is required by phoning 773-9012 or email email@example.com.


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|Property type
|Minimum Stay
|5 - 7 nights
Our villa is brick built, fully air conditioned throughout and has a fully equipped kitchen with all modern appliances. Both the pool and Jacuzzi can be heated if required during the cooler months, and for those that like the outdoors with some shade then our pool deck alcove would be a great place to relax. All furniture is brand new and we have TV's throughout including all of the bedrooms complete with HD Cable Channels along with free WI FI. The Family Room and Games/Entertainment Lounge both have 46" LED TV's with Blu-Ray Players and the Entertainment Lounge also offers a Pool table and XBOX 360 with internet connection.
We are only 10 minutes away from Disney's Main Gate, Universal and SeaWorld are also close by being a short drive further up I-4. Shops, restaurants, banks and Petrol/fuel stations are all within 3 minute drive away from our villa. Cypress Pointe has easy access to I-4 for all the Theme Parks. Daytona and Tampa Bay are both about 1 hours drive away.
•5 Bedrooms, 1 King Bed, 2 Queen Beds all with en-suites, 1 further Queen Bed and also a Twin Bedroom (these two bedrooms share a bathroom)
•South Facing Pool "Have a pool that will give you the sun all day long"
•Large Pool and Jacuzzi, also with a covered area for those that want some shade.
•Pool overlooks Nature Conversation Area, peaceful, quiet, and just perfect for relaxing.
•Sunset Villa is on a small gated community, yet only 3 minutes' drive to Publix (supermarket), Restaurants and many other shops.
•Easy Access to all Parks, Disney Area will take you less than 10 minutes, SeaWorld 20 minutes and Universal about 30 minutes.
•FREE Wi-Fi and local calls, just bring your laptop!
•TV's in all Bedrooms (hairdryers in some of the bedrooms).
•We have 46" LED TV's/Blu-Ray Players in the Family and Games/Entertainment Lounge which can play DVD's as well as CD's.
•The Games room has a 7ft Pool Table, selection of DVD's, XBOX 360 with Wi Fi with some games. We also have other board games, everything to keep the whole family entertained.
•Cots and Highchairs can be provided if requested.
•FREE use of safe.
•If you enjoy Golf then we have 5 Championship courses all within less than 15 minutes drive away, the nearest being Reunion only 4 minutes away.
The villa is fully air conditioned throughout along with ceiling fans in most rooms. All linen, pots, pans and silverware and towels (both bath and pool) are provided. The property has a management company that will offer first class service should you have any problems during your stay and would be more than willing offer any advice that you require.
The kitchen comes fully equipped with everything you need, all modern appliances, dishwasher, larger cooker, microwave, Fridge/freezer with chilled water along with crushed/cubed ice, coffee maker, toaster and for the British a electric kettle for that all important cup of tea. Breakfast bar with four high stools.
Dining Area
The dining area is the place for formal meals, whether you prepare something yourself in the fully equipped kitchen or perhaps it is something delivered or brought home after your day out.
Family Room
The family room is furnished to a high standard and has an office desk, 3 seater settee, 2 seater settee, arm chair, 2 small tables, 1 coffee table with 4 foot stools, console table, lamps, 46" LED TV with HD cable channels, Blu-ray player, TV stand, ceiling fan. Free Wi-Fi throughout the villa.
Master Suite
The Master suite has a King size bed, 2 bedside tables with lamps, large dressing table with drawers, alarm clock, TV with HD cable, en-suite with double sink unit, large walk-in shower, toilet and cupboard for fresh towels. Walk-in wardrobe, safe (for guests use), hair dryer, ceiling fan.
Queen Suite
There is a Queen size bed, 2 bedside tables with lamps, large dressing table with mirror, alarm clock, TV with HD cable, en-suite with single sink unit, walk in shower, toilet, ceiling fan, built in wardrobe.
Queen Suite (2)
In the second Queen suite there is a Queen size bed, 2 bedside tables with lamps, chest of drawers, large wall mirror, alarm clock, TV with HD cable, en-suite with single sink unit, bath with shower over, toilet, hair dryer, ceiling fan, built in wardrobe.
Queen Bedroom
Yet another luxuriously furnished Queen bedroom with Queen bed, 2 bedside tables with lamps, chest of drawers, TV with HD cable, built in wardrobe, ceiling fan. The room shares a bathroom with the twin bedroom.
Twin Bedroom
This tastefully furnished twin bedroom has 2 twin beds, bedside table with lamp, chest of drawers, TV with HD cable, built in wardrobe, ceiling fan and shares a bathroom with the Queen bedroom.
Games & Entertainment Room
In this purpose built game and entertainment room there is a 7ft Pool Table, selection of board games, 2 seater settee, 2 bean bags, 4 stools, TV stand, 46" LED TV with HD Cable, Blu-ray player, over 30 DVD's, Xbox 360 with internet connection, selection of Xbox games, ceiling fan.
Pool Deck
What better way to cool down than in our large pool. This will be a real favourite with the kids! The main features of this area are the large swimming pool & Jacuzzi, pool and Jacuzzi lights for night bathing, Ideal to relax and unwind after a hectic day. There are 5 Sun beds. The pool is South facing (sun all day long) over looking nature conservation area. Inflatables for use in the swimming pool.
What better way to relax and leave behind all the everyday pressures of work than to have a soak in the spa, perhaps with your favourite drink and recalling the days experiences. It doesn't get much better than this.
Dear Kevin & Sue
I am writing following our recent return to the United Kingdom following our visit to Florida during the period 10 April to 24 April 2012. The family and I had a fantastic time and central to this was our stay at your beautiful Villa with its fantastic facilities.
All the rooms were beautifully decorated whilst the living areas were spacious and luxuriously appointed. The large pool area and Jacuzzi were also greatly appreciated after our long days at the Disney and Universal Parks which were all within 15 to 25 minutes from the Villa and avoided the dreaded Highway 192. Please also pass on my thanks to Richard at your local Property Management Company who was always very helpful and courteous on those odd occasions when we sought advice.
I would certainly recommend your Villa to any family planning a holiday in Florida. For our part we hope to be able to return to Florida in the near future and hope that your Villa will be available at that time. Thank you once again from all of us.
Kind Regards
Michael, Sarah, Matthew & Molly Young
|Rate Period
|Weekend Night
Low Season
Apr 11 2015 - Jun 14 2015
5 night minimum stay
Jun 15 2015 - Aug 19 2015
7 night minimum stay
Low Season
Aug 20 2015 - Sep 30 2015
5 night minimum stay
Oct 1 2015 - Oct 31 2015
5 night minimum stay
Low Season
Nov 1 2015 - Dec 18 2015
5 night minimum stay
Dec 19 2015 - Jan 4 2016
7 night minimum stay
Low Season
Jan 5 2016 - Jan 20 2016
5 night minimum stay
Payment is usually accepted in the quoted currency (USD) unless the currency and the amount is specifically agreed in advance with the owner / advertiser.


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Space News Archive - May 05, 2011
NASA's Gravity Probe B (GP-B) has produced astonishing new confirmation of two key predictions derived from Albert Einstein's general theory of relativity, which the spacecraft was designed to assess.
Early Wednesday in the skies over Mojave Air and Spaceport CA, the world's first commercial spaceship, SpaceShipTwo, made a milestone glide-test flight and demonstrated its unique reentry 'feather' configuration for the first time.
Whenever someone proposes to do something that has never been done before, there will always be skeptics.
The team exploring Mars via NASA's Opportunity rover for the past seven years has informally named a Martian crater for the Mercury spacecraft that astronaut Alan Shepard christened Freedom 7.
You may have heard the news: Comet Elenin is coming to the inner-solar system this fall.
NASA Administrator Charles Bolden issued this statement Thursday, May 5, about the 50th anniversary of United States human spaceflight.
NASA has selected three science investigations for a potential 2016 mission that would look at Mars' interior for the first time; study an extraterrestrial ocean on one of Saturn's moons; and study in unprecedented detail the surface of a comet's nucleus.
- The horn of a unicorn considered as a medical or pharmacological ingredient.
- A winged horse with a single horn on its head; a winged unicorn.


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FRIDAY NIGHT VIDEO: The Soloing Netby Mick Ryan - UKClimbing.com Apr/2008
This news story has been read 27,528 times
With pads getting bigger and bigger, and stacked higher and higher, and with the development of inflatable Air pads, climbing short routes ropeless can be made relatively safe.
But does the below count as a solo? Some may have seen this before. Looks like the future is here and no doubt some enterprising company will be developing circus safety nets specifically for climbing.
Have a good weekend.


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Hi ,My Family name is BASSANI, I was born and live in Australia and my fathers people immigrated from Italy after the ww2. My fathers name was Guiseppe Bassani, His Fathers name was Amedeo Bassani(B1909), his fathers name was Guiseppe Bassani (B1866) and the furtherest we can go back is to Alessio Bassani all from the Vicenza Area of Northern Italy.
I am keen to learn more as I believe I have blood relatives in the USA,Brazil and Argentina. If anybody can help with this it would be appreciated. I use PAF5 . BASSANI.


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Cheap LWT to King Cove flights
Popular Flights from Lewistown, Montana
Top Lewistown Flights to US Destinations
Popular Flights from King Cove, Alaska
Top King Cove Flights to US Destinations
LWT to KVC Route Map
Lewistown to King Cove Flights, Stats and Airplanes
Route information for flights from Lewistown Municipal to King Cove AK.
2,299.10 miles
KVC - King Cove AK Airlines
Browse the most popular airlines servicing the King Cove (KVC) airport. Visit their flights pages to learn more about the most popular routes these carriers fly.


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Unless indicated, documents on this Web site are Adobe Acrobat files, and require the free
reader software. Further assistance can be found on the
DARM Support Desk page or by contacting the
Web Administrator.
View information related to Fast Track Adoption here. For further information regarding this rulemaking project please send your request to Marnie Brynes
62-204.800: Federal Regulations Adopted by Reference
Rule as Certified, Effective November 18,
Notice of Proposed Rule, October 16, 2009
[PDF - 47kb]
Notice of Withdrawal, October 16, 2009
[PDF - 14kb]
Notice of Proposed Rule, September 11,
2009[PDF - 500kb]
Last updated:
August 04, 2014
Email Your Questions or Comments
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Division of Air Resource Management2600 Blair Stone Road MS 5500Tallahassee, Florida 32399-2400


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Recommended Just For You
Customer Raves & Reviews
I needed my logo to stand out more & be slightly larger. All I had to do was give them the specifics, and they did it!
nice colors
A good product that I have used for years. The colors are pleasant, however, I think that the pastel colors over the month's headings are not clear enough, i.e. November.


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I photograph people because they fascinate me.
Portraits, to make them look great, and to stop time. So they and other people can look at then down the road and to see them as they were.
Photojournalism, to make a statement, either their statement or mine.
I rarely do scenics but I love to look at old pictures of places that are no longer there or have changed considerably.
I think I have a melancholy approach to photography, and perhaps to life. I think I'm trying to stop time.


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Hundreds at risk of losing their homes
Published at 07:38, Wednesday, 27 August 2014
NEARLY 600 households in England are at risk of losing their homes every day, new research has revealed.
The findings, which come from Ministry of Justice statistics, reveal that sky-high housing costs are pushing more and more families to the brink.
With finances stretched to breaking point, just one thing, like a sudden illness or job loss, can leave a family facing homelessness.
With cuts to the safety net hitting hard, Shelter's helpline is fighting to meet the demand.
Since 2011, calls from people struggling with rent arrears have almost doubled.
Campbell Robb, Shelter's chief executive, said that 582 households were at risk of losing their home every day, which was 582 too many.
"Each one of these will have had their lives turned upside down by this experience.
"Tragically, we are seeing more and more people coming to us for help – people who were struggling to make ends meet, and then just one change of circumstances sent them spiralling towards homelessness.
"We urgently need people's financial support so we can help more people avoid the nightmare of losing their home."
Published by http://www.hexhamcourant.co.uk


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Pseudomonas aeruginosa, a pathogen infecting those with cystic fibrosis, encounters toxicity from phagocyte-derived reactive oxidants including hydrogen peroxide during active infection. P. aeruginosa responds with adaptive and protective strategies against these toxic species to effectively infect humans. Despite advances in our understanding of the responses to oxidative stress in many specific cases, the connectivity between targeted protective genes and the rest of cell metabolism remains obscure.
Herein, we performed a genome-wide transcriptome analysis of the cellular responses to hydrogen peroxide in order to determine a more complete picture of how oxidative stress-induced genes are related and regulated.
Our data reinforce the previous conclusion that DNA repair proteins and catalases may be among the most vital antioxidant defense systems of P. aeruginosa. Our results also suggest that sublethal oxidative damage reduces active and/or facilitated transport and that intracellular iron might be a key factor for a relationship between oxidative stress and iron regulation. Perhaps most intriguingly, we revealed that the transcription of all F-, R-, and S-type pyocins was upregulated by oxidative stress and at the same time, a cell immunity protein (pyocin S2 immunity protein) was downregulated, possibly leading to self-killing activity.
This finding proposes that pyocin production might be another novel defensive scheme against oxidative attack by host cells.
Many microorganisms continuously face a range of reactive oxygen species (ROS) including hydrogen peroxide, superoxide, and the hydroxyl radical derived from many sources. During the process of active infection, pathogenic bacteria are exposed to exogenous oxidative stress that phagocytes utilize as a host defense mechanism [1]. Even normal cellular metabolism produces cytotoxicity arising from its partially-reduced intermediates [2]. For instance, by reacting with intracellular iron, hydrogen peroxide can form the hydroxyl radical through the Fenton reaction, which damages various cellular molecules including lipids, proteins, and DNA [1,3]. Superoxide is also capable of promoting oxidative damage by increasing the concentration of intracellular iron [2,4,5]. Because of the vast array of stimuli and their sources, it is intuitive to anticipate that organisms have developed complex antioxidant strategies that serve to neutralize and repair oxidative damage.
Pseudomonas aeruginosa PA01 (P. aeruginosa), a Gram-negative pathogen responsible for respiratory infections in individuals with cystic fibrosis and cancer, is also known to possess a multifaceted defense system against reactive oxidants that includes such enzymes as catalase and superoxide dismutase [6-8]. There are many specific defense genes that have been identified and regulatory aspects of their activities have been elucidated in many cases [1,5]. Despite this marked progress, cystic fibrosis remains problematic and our knowledge of P. aeruginosa pathogenicity remains incomplete. A more thorough understanding of this bacterium's defense system might serve to enhance the development and efficacy of therapeutic agents for this disease. In particular, an understanding of the linkage between the cell's ROS defense mechanism and the remainder of the cell's metabolism can lead to more innovative methods for combating this pathogen. For example, better elucidation of the molecular events responsible for establishing and maintaining pathogenicity might improve optimal drug and vaccine design [9]. That is, by using microarray analysis that enables us to simultaneously and globally examine the complete transcriptome during cellular responses, we might reinforce known relationships between genes with previously identified functions, and also reveal new target genes that give us more insight into P. aeruginosa-host interactions.
To provide a more complete linkage between cell physiology and the well-characterized defense response, we investigated genome-wide changes in P. aeruginosa gene transcription upon exposure to hydrogen peroxide using Affymetrix P. aeruginosa GeneChip arrays. Notably, we made a significant finding that hydrogen peroxide induced the transcription of each and every pyocin (bacteriocins) reported in P. aeruginosa. Moreover, we found that a pyocin immunity gene, which prevents bacterial cell death during pyocin synthesis, was downregulated, possibly leading to self-killing activity. Finally, we've corroborated an anticipated result regarding iron uptake; that oxidative stress in our experimental conditions lead to the repression of iron uptake genes.
Results and discussion
To investigate the effect of sublethal oxidative stress on P. aeruginosa, we performed a transcriptome analysis with microarrays upon 20 min exposure to 1 mM hydrogen peroxide. This concentration successfully induces sublethal oxidative damage in Escherichia coli and P. aeruginosa [10-12]. Besides providing requisite quantities of mRNA for microarray analyses, sublethal doses of antibiotics are receiving increased interest because of their potential for attenuating pathogenicity but with concern for increased rates of mutation and resistance [13,14]. We confirmed that 1 mM hydrogen peroxide caused strong growth inhibition but not cell death for the first 60 min post-treatment (data not shown).
To determine genome-wide transcriptional changes in response to hydrogen peroxide, we conducted four and five independent microarray experiments in the absence (control) and the presence (experimental) of hydrogen peroxide, respectively. Transcriptome analysis with Affymetrix P. aeruginosa GeneChip arrays suggested that mRNA levels of 805 and 827 out of a total of 5,570 genes were increased and decreased (IR <> 1), respectively, after 20 min treatment. We refer to "statistically marked" changes in transcript level for those genes that meet the following criteria: (i) a p-value for a Mann-Whitney test should be less than 0.05, (ii) an absolute fold change in transcript level should be equal to or greater than 2, and (iii) a gene should have a presence or marginal call [15] from 50% or more replicates on both the experimental and control replicate sets. Based on these criteria, we pared the set to 116 and 107 genes (a total of 223) that had statistically marked increases and decreases in transcript level, respectively. This result suggests that these genes might be associated with the defensive response of P. aeruginosa to hydrogen peroxide-induced oxidative stress. Interestingly, this number is comparable to that found in a study of E. coli cells treated with 1 mM hydrogen peroxide (140 genes, = 4-fold) [16], and significantly less than those of a comparable P. aeruginosa study (1,854 genes, ≥ 2-fold) [12]. In Table 1, from among the 223 genes, we list 30 most strongly induced and repressed genes as well as their putative functions, many of which are discussed below. Additionally, Supplementary Table 1 shows the average signals, p-values, and fold changes of all 5,570 predicted P. aeruginosa open reading frames. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus [17] and are accessible through GEO Series accession number GSE3090.
Table 1. List of 30 P. aeruginosa genes most strongly induced and repressed in response to hydrogen peroxide
To validate the relative transcript levels obtained by the array analysis, we employed quantitative real-time PCR analysis on PA4613 (katB), PA2850 (ohr), PA4763 (recN), and PA5530. These genes were selected since they displayed a range of mRNA level changes (-1- to 24-fold). Moreover, PA0576 (rpoD) was used as a control gene for the relative mRNA level calculation due to the fact that rpoD exhibits stable expression level [18]. As shown in Table 2, our microarray results were corroborated with quantitative real-time PCR analyses of the selected genes.
Table 2. Transcript level comparison of P. aeruginosa genes between real-time PCR analysis and microarray analysis
Functional classification
To examine how the genes are distributed with regard to their functions, we classified these 223 genes according to the categories described in the Pseudomonas aeruginosa Community Annotation Project [19] (see, Figure 1). Both Figure 1 and Table 1 indicate that the most distinctive feature was the completely uniform upregulation of genes involved in DNA modulation (Phage, transposon, or plasmid and DNA replication and repair); this is discussed further below. It also appeared that genes related to various membrane functions including "transport of small molecules", "secreted factors", and "membrane proteins" were noticeably downregulated, suggesting that hydrogen peroxide alters the regulation of membrane proteins. Even among the 30 most downregulated genes presented in Table 1, 17 genes belonged to these functional classes (PA2398 (fpvA), PA4156, PA4230 (pchB), PA2409, PA5235 (glpT), PA4225 (pchF), PA2403, PA5479 (gltP), PA2404, PA3610 (potD), PA0280 (cysA), PA2408, PA4231 (pchA), PA0281 (cysW), PA4221 (fptA), PA4229 (pchC), and PA4218). This result may reflect attenuation of active and/or facilitated transport through the cell membrane. Notably, many genes associated with these classes were found linked with iron regulation (discussed below). Also, consistent with the sublethal but significant applied stress, as shown in Table 3, several genes involved in primary metabolism exhibited decreased mRNA levels: (i) energy metabolism-related genes, PA1317 (cyoA), PA1319 (cyoC), and PA3621 (fdxA), and PA4133, (ii) polyamine synthesis and uptake genes (related to amino acid synthesis and metabolism), PA0654 (speD), PA1687 (speE), PA4839 (speA), and PA3607-3610 (potABCD), and (iii) ribosomal protein genes, PA4432 (rpsL), PA4563 (rpsT), PA5049 (rpmE), and PA5315 (rpmG). It is also interesting that putative cell division inhibitors such as PA0671 and PA3008, which are similar to E. coli sulA [20], showed increased transcript levels upon exposure to hydrogen peroxide, suggesting that these genes might promote the repression of primary metabolism.
Figure 1. Functional classification of genes with statistically significant increase and decrease in mRNA level (a total of 223 genes). The number in parenthesis represents the percentage of the total number of genes within the genome in each functional class.
Table 3. List of P. aeruginosa genes that were discussed in this report, categorized by their related functions.
Genes related to cellular protective mechanisms
Figure 1 and Table 3 show that a number of genes primarily in the classes of "adaptation, protection" and "DNA replication, recombination, modification and repair", which are known to be involved in cellular protective mechanisms, were induced in response to hydrogen peroxide. First, two catalase genes, PA4236 (katA), and PA4613 (katB) showed significant increases in mRNA levels. It was reported that the KatB enzyme is essential for optimal resistance to hydrogen peroxide [6,21]. The mere 2-fold upregulation of katA may support a prior hypothesis that katA serves as a primary housekeeping catalase; expressed constitutively throughout the growth cycle [7,21]; as a result, dramatic transcript increases might not occur. Surprisingly, superoxide dismutase genes, PA4366 (sodB) and PA4468 (sodM), did not show significant increases in mRNA levels. However, sodB was among genes that had the highest signal intensities on both control and experimental replicates, whereas sodM exhibited low mRNA levels under both conditions. Again, this result may correspond well to a previous report suggesting that SodB expression may be important even during normal aerobic growth while SodM activity is increased only in response to iron deprivation, which is not the case in our study (discussed below) [22].
Second, DNA repair-related genes were highly induced in the presence of hydrogen peroxide. Indeed, it is known that hydrogen peroxide causes oxidative DNA damage by generating hydroxyl or ferryl radicals [1,2,23]. Palma et al. demonstrated that genes of P. aeruginosa SOS regulon exhibit increases in mRNA level upon exposure to 1 mM hydrogen peroxide [12]. Reportedly, RecA stimulates autodigestion of repressor protein LexA for the activation of the SOS regulon in E. coli [24]. Our study also revealed increased expression of PA3007 (lexA) and PA3617 (recA) (Table 3). Furthermore, PA3008, adjacent to lexA, showed high increase in transcript level; notably, this gene is similar to E. coli sulA, which inhibits cell division until DNA repair is finished [12,25]. Besides, PA3616 (probable recX), a downstream gene of recA, is reportedly involved in regulation of RecA expression [26,27]. Other interesting genes possibly relevant to DNA repair-related functions included PA0669 (a DNA polymerase gene), PA3413-PA3414, and PA4763 (recN, a DNA repair gene). To be specific, a protein encoded by PA3414 exhibited a high homology to YbeG in E. coli, which is inducible by DNA damage [28,29]. Moreover, the fact that DnaE, similar to a product encoded by PA0669, is involved in DNA mismatch correction or DNA repair in Bacillus subtilis suggests that the PA0669 protein might also participate in repairing DNA damage caused by hydrogen peroxide in our study [30,31].
As shown in Figure 2, the complete profile of our response is compared directly to that of Palma et al. [12]. Our results were generally consistent and, overall, this portion of our analysis corroborates previous studies that have associated oxidative stress response genes with hydrogen peroxide and other ROS insults, and reinforces the conclusion that DNA repair proteins and catalases may be among the most central mechanisms that P. aeruginosa employs to counteract lethal effects of reactive oxygen intermediates.
Figure 2. Comparison of 223 genes with statistically significant mRNA level changes in this study with those of Palma et al. [12]. The fold change is a positive number when the expression level in the experiment increased compared to the control and is a negative number when the expression level in the experiment declined.
Genes related to iron regulation
As mentioned above, however, one of the aims of this study was to examine the responses of ROS stress genes to the rest of cellular metabolism, in order to provide new insight on P. aeruginosa regulation in response to varied oxygen conditions as is expected in niche environments of the human lung. Among the more interesting findings was an apparent discrepancy between previous reports of iron-regulated genes. These genes are highlighted in Figure 2. Iron metabolism is important for many reasons: there are many iron catalyzed oxygen reactions that can damage cells; there are many sources of iron, particularly localized in the human lung; and iron-related genes are often coordinately regulated with oxidative stress defenses in E. coli [32]. Notably, this study also revealed that hydrogen peroxide treatment drastically altered iron regulation in P. aeruginosa. In particular, the following genes, all of which are known to be regulated by the ferric uptake regulator (Fur, encoded by PA4764), were repressed in response to hydrogen peroxide (Table 3): (i) iron starvation sigma factor, PA2426 (pvdS), (ii) ferri-siderophore (iron-chelating compound) receptor genes, PA2398 (fpvA) and PA4221 (fptA), (iii) siderophore (pyochelin) biosynthesis genes, PA4225-PA4226 (pchFE) and PA4228-PA4231 (pchDCBA), and (iv) siderophore (pyoverdin) system-related genes, PAPA2403-PA2410 and PA4156 [33,34].
As is widely accepted, in order to overcome a limited supply of iron, P. aeruginosa produces two major siderophores, pyoverdin and pyochelin [34-36] and upon iron deprivation, these siderophores are excreted from the cells, chelate iron and transport it back to the cells through outer membrane receptors, FptA and FpvA, which are specific for the iron-siderophore complexes [37,38]. Moreover, in the absence of iron, PchR, encoded by PA4227, seems to become a repressor of the receptor gene, fptA, and of its own gene, pchR. On the other hand, excess iron represses the biosynthesis of pyoverdin and pyochelin [35,39]; that is, in the presence of abundant iron, the Fur protein, a repressor, binds to the promoters of genes encoding PvdS and PchR proteins, which positively regulate the biosynthesis of pyoverdin and pyochelin, respectively [38]. These phenomena seem congruent with the transcript levels of iron-related genes in this study.
It has been documented that the intracellular iron level could be affected by oxidative stress [3,5,32]. In particular, superoxide, generated during the reduction process of oxygen, releases free iron from iron-sulfur proteins, thus increasing the levels of intracellular iron [2-4]. Consequently, the result that the iron regulation-related genes such as pvdS, fpvA, fptA, and pchABCDEF were repressed in our study could be accounted for by prior reports suggesting that the amount of intracellular iron might be increased as a result of exposure to reactive oxidants [4]. At this point, it should be pointed out that "free iron" was intended to suggest not that the iron is hexa-aqueous, but that the iron is not integrated into enzymes. Iron vigorously binds to most biomolecules so that iron atoms free inside the cell likely exist in a form associated with the surfaces of cellular molecules. As shown in Figure 2, Palma et al. previously reported induction of several iron starvation-inducible genes such as PA0471, PA0472, PA2426 (pvdS), PA2686 (pfeR), PA4221 (fptA), PA4227 (pchR), PA4228 (pchD), PA4230 (pchB), and PA5531 (tonB) upon exposure to hydrogen peroxide, suggesting that cells experience iron starvation and/or a transient loss of Fur repressor function [12]. The reason for this discrepancy is not understood at present; however, it should not be excluded that different growth mediums, exposure times, and/or growth phase (tryptic soy broth, 10 min, optical density 0.5, respectively) as well as a different P. aeruginosa isolates were used in the two studies (discussed in more detail later).
Lastly, it should be pointed out that iron regulation system might be an attractive therapeutic target because it appears to be closely related to the antioxidant mechanism of P. aeruginosa. For instance, siderophore malfunction by potent drugs could lead to inhibition and/or imbalance of iron regulation and thus, could increase the cell's susceptibility to exogenous oxidative stress that phagocytes utilize during active infection.
Genes related to pyocin system
The most striking result of our study, which has not been shown previously in any oxygen-insult studies of any bacterium, was that F-, R- and S- type pyocins, bacteriocins of P. aeruginosa, were strongly induced in response to hydrogen peroxide. As shown in Figure 2, these 41 genes were uniformly upregulated. Many strains are known to adapt bacteriocins so as to preserve the initial predominance of bacteriocinogenic bacteria [40]; however, bacteriocins are effective only against the same or closely related species [41-43]. Surprisingly, our study revealed that PA0985 (probable pyocin S5 gene), PA1150 (pys2, pyocin S2 gene), and PA3866 (probable pyocin S3 gene) were induced upon exposure to hydrogen peroxide (Table 3). Further, as shown in Figure 3 and Table 3, all the genes of a R2/F2 pyocin gene locus (PA0612-PA0648) exhibited increased mRNA levels (Figure 1, the "phage, transposon, and plasmid" class). Thus, R2 pyocin (PA0615 – PA0628), F2 pyocin (PA0633 – PA0648), and lysis enzymes (PA0614, PA0629 – PA0631) were likely expressed with the hydrogen peroxide treatment (Figure 3). Pyocins cause cell death by DNA breakdown and the inhibition of lipid synthesis; their production is inducible by mutagenic agents such as UV and mitomycin C [40,44]. RecA (a DNA repair protein), PrtR (a repressor protein) and PrtN (an activator protein) co-regulate activity of many pyocin genes. As noted above, DNA-damaging agents increase production of RecA, which in turn, cleaves PrtR and releases PrtN [45]. Hence, since DNA repair-related genes including recA were upregulated in our study and regulatory genes such as recA, prtN, and prtR are probably shared by S-, F-, and R-type pyocins [41], we may conclude that DNA damage might also be a reason for the induction of these pyocins, S2, S3, S5, R2, and F2. Moreover, the induction of lysis enzymes (PA0614, PA0629, PA0630, and PA0631), which are likely shared by S-, F-, and R-type pyocins, indicates that these pyocins might have been released from the cells [41].
Figure 3. Genetic organization of the R2/F2 pyocin gene locus according to Michel-Briand and Baysse [40] and Nakayama et al. [41]. Each box represents a gene between PA0609 (trpE) and PA0649 (trpG). Genes inside the dotted line were upregulated upon exposure to hydrogen peroxide (see, Table 3).
These results suggest that host cells are exposed to pyocins that are induced by oxidative stress during active infection. As indicated above, pyocins are known to kill species closely related to P. aeruginosa (e.g. some of Gram-negative bacteria). Interestingly, pyocins display cytotoxic effects on human cancer cells, triggering lethal events [46,47]. At present, it is unclear whether pyocins are causative toxic agents in patients with defective immune systems; however, it should not be excluded that pyocin production might be another defense mechanism that P. aeruginosa employs against oxidative attack by host cells.
Another notable finding was that PA1151 (imm2) encoding the pyocin S2 immunity protein exhibited a significant decrease in transcript level (Table 3). That is, secreted bacteriocins pose a survival problem to host bacteria, so cells produce immunity enzymes, high affinity inhibitors of bacteriocins, during bacteriocin synthesis [42]. P. aeruginosa possesses several pyocin immunity proteins that provide a self-protection function against S-type pyocins [26,40], by binding to the pyocin nuclease domain and neutralizing its activity [40,42]. Thus, our result that the pyocin S2 immunity protein was repressed during pyocin production could render P. aeruginosa vulnerable to this bacteriocin, which might even lead to its own death during pyocin synthesis.
Coincident with this, it was earlier observed that strains of P. aeruginosa produce pyocins active against themselves [48]. The conditions leading to their production were not investigated. The potential for programmed cell death in this system is an intriguing speculation. It may be that self-killing activity by part of a P. aeruginosa population benefits the remainder. For example, microbial population may dissipate damaging effects of extracellular stress such as pH change and oxidative stress by lysing some of the cells, which might be particularly effective for P. aeruginosa biofilms where layers of cells exist. Besides, given that DNA damage might elicit pyocin production, cells with damaged DNA may be the most appropriate targets for cell death, enabling preservation of the remainder of the population.
These kinds of activities apparently require cell-to-cell communication; therefore, it is interesting to question whether the quorum sensing system might be involved in pyocin synthesis. Based on quorum sensing-regulated genes that have been previously reported (163 genes from Hentzer et al. [49]; 315 genes from Schuster et al. [50]; 388 genes from Wagner et al. [51]), we found that 62, 69 and 56 genes, respectively, also showed transcription level changes in response to hydrogen peroxide in our study. While coincident up- or down-regulation does not establish a connection between quorum sensing and pyocin systems, this result might indicate that some genes involved in the quorum sensing system were also activated during cellular response to oxidants. In this light, it is noteworthy that degradation of secreted peptide Staphylococcus aureus autoinducers has been linked to the antioxidant response [14].
The last and perhaps most interesting result is that S-type pyocins become more lethal under iron-limited conditions because they presumably enter cells through siderophore receptors [52]; thus, the repression of siderophore receptors in our study (discussed above) may increase the possibility that the activation of S-type pyocins had larger impact on producer cells. However, in contrast, it might also be possible that the induction of pyocin production might contribute to the repression of iron uptake-related genes of the other population. Further investigation is required to verify the role of the immunity protein repression and the relationship between pyocins and iron-uptake genes.
Co-transcription of genes in operons
In our attempt to further evaluate the transcription levels of genes discussed above, we investigated the co-transcription of their operons. That is, since genes within operons are largely co-regulated, we expected a considerable correlation between these genes and their neighboring genes within the same operon. To test this, we first selected 16 putative operons of the genes described in this report using the "Predicting operons in microbial genome" resource of the Institute for Genomic Research http://www.tigr.org/tigr-scripts/operons/operons.cgi webcite. We then calculated the Pearson's correlation coefficient (R) from the transcript levels of 55 pairs of neighboring genes in these 16 operons. For baseline comparison, we also analyzed 1000 pairs of randomly selected neighboring genes across the genome. Note that each pair has nine (four control and five experimental) array datasets. In Table 4, we observed that the neighboring genes sharing the same operon exhibited significantly high co-transcription levels (the mean and median R values) in this study. This result also provides a high degree of confidence in the transcriptome data we presented herein.
Table 4. Descriptive statistics for the co-transcription level of neighboring gene pairs
Comparison of microarray data to other data in the literature
Lastly, in order to provide another assessment on our results, we compared our genes with statistically marked transcript level changes with those published in Palma et al. [12]. As indicated above, our study examined the transcriptional response upon 20 min exposure to 1 mM hydrogen peroxide in Luria-bertani broth, whereas Palma et al. [12] utilized 10 min exposure time in tryptic soy broth. Since the Mann-Whitney test with the cutoff p and fold change values of 0.05 and 2, respectively, was applied to both studies, we performed a comparative analysis between the two transcriptome profiles. Supplementary Table 2 shows that of 223 genes that we previously considered statistically marked in our report, 41 and 46 genes were upregulated and downregulated, respectively, in both studies. Further, 4 genes induced in this report were repressed in Palma et al. [12], while 11 genes displayed the opposite expression pattern. In Supplementary Table 2, 70 and 46 genes that were upregulated and downregulated, respectively, in this study were not among genes with statistically significant mRNA level changes in Palma's report. Interestingly, the fold changes were generally lower under our experimental conditions. This result demonstrates that about 40% of our statistically marked genes were regulated in the same direction in Palma et al. [12].
Next, we focused on genes that we earlier discussed in this report, which were originally listed in Table 3. Supplementary Table 3 shows that most genes in the categories of "primary metabolism-related" and "cellular protective mechanism-related" were concurrently regulated in the two reports. To be particular, genes encoding enzymes that play integral roles in oxidative response, such as catalases and DNA repair proteins, were consistently upregulated. This result reinforces our prior conclusion that the early transcriptional responses of P. aeruginosa to hydrogen peroxide include the repression of primary metabolism and the induction of protective mechanisms. On the other hand, most genes related to iron regulation and pyocin system were discordantly regulated in the two studies (Supplementary Table 3). As previously discussed, while we cannot fully elucidate this disagreement, we should not exclude the possibility that different growth mediums, strain backgrounds, and exposure times account for the divergent microarray data. Indeed, several reports are in line with this speculation: (i) Vasil proposed that dissimilar experimental conditions including strain history and growth mediums bring about discrepancies in the quorum-sensing transcriptional profiles [53], (ii) Spiegeleer et al. demonstrated that oxidative stress resistance of E. coli is dependent on the growth medium [54], (iii) Kang et al. suggested that strain background is one of the factors affecting a microarray-based transcriptome analysis [55], and (iv) our laboratory observed a large difference in the transcriptional profiles of Staphylococcus aureus response to oxidative stress between 10 min and 20 min exposures (unpublished data). Therefore, this result suggests that further investigation on iron regulation and pyocin system in P. aeruginosa under oxidative stress requires careful evaluation of transcriptional effects by experimental variances.
In summary, we demonstrated how oxidative stress-induced genes were related and regulated in P. aeruginosa by utilizing whole-genome microarrays. Our results suggest that DNA repair proteins and catalases be among the most vital antioxidant defense systems of P. aeruginosa for preventing lethal effects of reactive oxygen intermediates. Second, a slowdown of membrane function-related genes was observed, implying that sublethal oxidative damage reduced active and/or facilitated transport through the cell membrane. Third, it was confirmed that oxidative stress could affect iron metabolism in that many of Fur-regulated genes were repressed upon exposure to hydrogen peroxide. In addition, this finding, together with other reports [5,32] suggests that intracellular iron level might be a key factor for the relationship. Most importantly, we provided evidence that reactive oxidant treatment results in the induction of all three types of pyocin genes in P. aeruginosa. Furthermore, it was demonstrated that a pyocin immunity protein was repressed, which if coincident events occur might lead to self-killing activity. To our knowledge, this is the first study reporting pyocin upregulation in response to oxidative stress. Hence, we are currently exploring whether the repression of an immunity protein increases the sensitivity of P. aeruginosa to pyocins and pyocin production benefits its population against toxic oxidants.
Bacterial strains and growth conditions
In this study, we used Pseudomonas aeruginosa PA01 obtained from Dr. E. Peter Greenberg's laboratory at the University of Iowa. To maintain homogeneous culture samples throughout our experiments, we employed the following three steps: (i) we initiated P. aeruginosa cultures at 37°C with shaking at 250 rpm using sterilized Luria-Bertani (LB) broth (10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride per liter), (ii) after 17 hours, we diluted the overnight cultures 1:100 in pre-warmed LB broth and incubated at 37°C with shaking at 250 rpm until an optical density at 600 nm (OD600) reached the early logarithmic phase (~ 0.8), and (iii) we re-diluted the cells 1:10 in pre-warmed LB broth and incubated at 37°C with shaking at 250 rpm [10,56]. Then, we added 1 mM hydrogen peroxide (Aldrich Chemical Co., St. Louis, MO) immediately after OD600 reached 0.8. Note that culture volumes for all growth conditions were adjusted to be less than 1/10 the total flask volume to maximize aeration.
Affymetrix P. aeruginosa GeneChip arrays
Total RNA was isolated after 20 min incubation using RNeasy Mini kit (Qiagen, Inc., Valencia, CA) according to the manufacturer's protocol. As described previously [56], we added 2 mL of RNAprotect Bacteria Reagent (Qiagen, Inc., Valencia, CA) into 1 mL of the culture before the isolation to stabilize our RNA. RNA quality was determined using both Lambda 25 spectrophotometer (PerkinElmer, Inc., MA) and RNA 6000 Nano LabChip with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Next, we used 12 μg of total RNA with random primers and SuperScript II (both from Invitrogen Corp., Carlsbad, CA) for cDNA synthesis, cDNA fragmentation, labeling, hybridization, staining and washing steps were performed according to the manufacturer's protocol for the Affymetrix P. aeruginosa GeneChip arrays (Affymetrix, Inc., Santa Clara, CA). Finally, the arrays were scanned with the Affymetrix GeneChip Scanner 3000.
Data analysis and real-time PCR
To analyze the array data, we utilized Affymetrix GeneChip Operating Software (GCOS) v. 1.0 and Data Mining Tool (DMT) v. 3.1 (Affymetrix, Inc., Santa Clara, CA) with the following parameters: alpha 1, 0.04; alpha 2, 0.06; tau, 0.015; target signal, 150. Alpha 1 and 2 are significance levels that define detection calls (see below), while tau determines analysis sensitivity [15]. Further, the average intensity of arrays was scaled to a target signal. We calculated fold change as the ratio between the signal averages of four untreated (control) and five hydrogen peroxide-treated (experimental) cultures. Gene expression fold changes were identified with statistical significance by the Mann-Whitney test (cutoff p-value, 0.05). The GCOS detection calls of "Present", "Marginal", and "Absent" are determined based on the Affymetrix detection algorithm [15]. This call indicates whether a transcript is reliably detected (present) or not detected (absent). Genes that received absent calls from 50% or more of the replicates in GCOS were excluded from the final list.
Lastly, to determine the validity of the array data, transcript level changes obtained with the microarray analysis were compared with those from quantitative real-time PCR. Genes and primer sequences employed for the real-time PCR analysis are listed in Table 2. We performed the real-time PCR by utilizing iCycler iQ Real-Time PCR Detection System with iScript cDNA Synthesis Kit and One-Step RT-PCR Kit with SYBR Green (BioRad Laboratories, Inc., Hercules, CA). As stated above, RNA samples were treated with DNase I (Qiagen, Inc., Valencia, CA) to preclude DNA contamination, which was confirmed with Agilent 2100 Bioanalyzer LabChip and gel electrophoresis. In this report, relative quantification based on the relative expression of a target gene versus a reference gene was utilized to determine transcript level changes [57]. For each gene, five biological replicates with three technical replicates each were employed. PCR efficiencies were also derived from standard curve slopes in the iCycler software v. 3.1 (BioRad Laboratories, Inc., Hercules, CA). Finally, melt-curve analysis was performed to evaluate PCR specificity and resulted in single primer-specific melting temperatures.
Authors' contributions
WC performed microarray experiments, and data analysis, and drafted the manuscript. DAS performed microarray experiments. FT initiated and supervised the study, and reviewed the manuscript. WEB supervised the study and reviewed the manuscript.
Additional File 1. Probe set data (average signals, p-values, and fold changes) for experimental and control samples.
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Additional File 2. Comparison of the fold changes and their directions of the significantly marked genes in this study with those in Palma et al. [12](a total of 223 genes). The Mann-Whitney test with the cutoff p and fold change values of 0.05 and 2, respectively, was applied to both studies,
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Additional File 3. Comparison of the expression change directions of genes that we discussed in this study with those in Palma et al. [12]. The genes were classified into the categories of "primary metabolism-related", "cellular protective mechanisms-related", "iron regulation-related", and "pyocin system-related", as presented in Table 3.
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We thank Hyunmin Yi for thoughtful comments on the manuscript. This research is supported by the United States Environmental Protection Agency Grants T- 83100801-0, IPA HQ-514-04-06, and IPA HQ-515-04-06. Although the research described in this paper has been funded wholly by the United States Environmental Protection Agency, it has not been subjected to the Agency's peer and administrative review and therefore may not necessarily reflect the views of the EPA; nor does the mention of trade names or commercial products constitute endorsement of recommendation of use.
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Serino L, Reimmann C, Visca P, Beyeler M, Chiesa VD, Haas D: Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa.
J Bacteriol 1997, 179:248-257. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Michel-Briand Y, Baysse C: The pyocins of Pseudomonas aeruginosa.
Biochimie 2002, 84:499-510. PubMed Abstract | Publisher Full Text
Nakayama K, Takashima K, Ishihara H, Shinomiya T, Kageyama M, Kanaya S, Ohnishi M, Murata T, Mori H, Hayashi T: The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage.
Mol Microbiol 2000, 38:213-231. PubMed Abstract | Publisher Full Text
Kleanthous C, Walker D: Immunity proteins: enzyme inhibitors that avoid the active site.
Trends Biochem Sci 2001, 26:624-631. PubMed Abstract | Publisher Full Text
de Zamaroczy M, Buckingham RH: Importation of nuclease colicins into E. coli cells: endoproteolytic cleavage and its prevention by the immunity protein.
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Sano Y, Matsui H, Kobayashi M, Kageyama M: Molecular structures and functions of pyocins S1 and S2 in Pseudomonas aeruginosa.
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Matsui H, Sano Y, Ishihara H, Shinomiya T: Regulation of pyocin genes in Pseudomonas aeruginosa by positive (prtN) and negative (prtR) regulatory genes.
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Farkas-Himsley H, Hill R, Rosen B, Arab S, Lingwood CA: The bacterial colicin active against tumor cells in vitro and in vivo is verotoxin 1.
Proc Natl Acad Sci 1995, 92:6996-7000. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Abdi-Ali A, Worobec EA, Deezagi A, Malekzadeh F: Cytotoxic effects of pyocin S2 produced by Pseudomonas aeruginosa on the growth of three human cell lines.
Can J Microbiol 2004, 50:375-381. PubMed Abstract | Publisher Full Text
Goodwin K, Levin RE, Doggett RG: Autosensitivity of Pseudomonas aeruginosa to its own pyocin.
Infect Immun 1972, 6:889-892. PubMed Abstract | PubMed Central Full Text
Hentzer M, Wu H, Andersen J, Riedel K, Rasmussen T, Bagge N, Kumar N, Schembri M, Song Z, Kristoffersen P, Manefield M, Costerton J, Molin S, Eberl L, Steinberg P, Kjelleberg S, Hoiby N, Givskov M: Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors.
EMBO J 2003, 22:3803-3815. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Schuster M, Lostroh C, Ogi T, Greenberg E: Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis.
J Bacteriol 2003, 185:2066-2079. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Wagner V, Bushnell D, Passador L, Brooks A, Iglewski B: Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment.
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Baysse C, Meyer JM, Plesiat P, Geoffroy V, Michel-Briand Y, Cornelis P: Uptake of pyocin S3 occurs through the outer membrane ferripyoverdine type II receptor of Pseudomonas aeruginosa.
J Bacteriol 1999, 181:3849-3851. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Vasil ML: DNA microarrays in analysis of quorum sensing: strengths and limitations.
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de Spiegeleer P, Sermon J, Lietaert A, Aertsen A, Michiels CW: Source of tryptone in growth medium affects oxidative stress resistance in Escherichia coli.
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J Bacteriol 2005, 187:1135-1160. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Chang W, Small DA, Toghrol F, Bentley WE: Microarray analysis of toxicogenomic effects of peracetic acid on Pseudomonas aeruginosa.
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|Originally posted by Alexander
[Reality check]
These are "instant gratification pictures" right?
OK. Maybe you'll put them in the album.
Too bent? Get reprints.
[/Reality check]
|I also think it helps to remember to keep my feeling seperate from his. Just because he is upset I don't have to be. I had to remove the pictures to keep them whole. I don't have to be upset that this makes him very angry. And not worry about what others think
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text:
Research, generally speaking, is the foundation of the social-issue documentary, and the more exhaustive, the better. It behooves documentarians to conduct a rigorous study, whatever their subject, and the depth of such films is, to a large extent, determined by the diligence and assiduity of what is, in essence, reportage. Without Shepherds, a new documentary about life in contemporary Pakistan, is the product of a tremendous investment of time and effort: Its apparently indefatigable co-directors, Cary McClelland and Imran Babur, gathered more than 900 hours of interview footage with participants across the country, shot, edited, transcribed, and translated painstakingly over two years. (McClelland and Babur plan to compile the remaining transcriptions into a kind of "spin-off" volume to accompany the film's home-video release.)
The problem is one of disorder. Pure research, while certainly valuable, must be given editorial shape and direction; this kind of raw material needs to be sculpted into something cogent, streamlined, and shorn of excess. Without Shepherds is all sprawl, a loose mélange of talking heads and landscape b-roll. Political rhetoric is invoked but not elaborated. The state of modern Pakistan is bemoaned at length, though rarely compellingly. And so McClelland and Babur are left to meander between their half dozen principal subjects without any sense of design or momentum. The press materials -- as well as an echo chamber of early reviews -- describe the result as something of a "tapestry," but this hardly seems accurate. A tapestry weaves its component parts together with purpose and intricacy. Without Shepherds does neither.
Find capsule reviews, showtimes & tickets for all films in town.


text:
Buenos Aires City Guide by TripAdvisor
50,000 - 100,000 downloads
- Downloadable to be used totally offline
- Will save you in roaming charges
- Includes all the extensive restaurant, hotel, and attraction info on the TripAdvisor site
- Huge variety of cities available
- None
+ By TripAdvisor
"Pack your bags"
Reviewed by Janel Torkington / Apr 25, 2014
Travel guides freed from the twin chains of 3G and wifi
No free wifi: the traveler's nightmare. Trusted review and advice site TripAdvisor presents the solution on a silver platter: downloadable Offline City Guides, including all the hotel, attraction, and restaurant information you already use from the website. Data roaming, begone!
Yup, this series of guides will work totally offline if you take a moment to download the one(s) that interests you onto your device. Select from a huge offering of internationally fascinating cities, from New York to Paris to Tokyo to Marrakech. Taking the time to download the guide when you do have a wifi connection means that it works ultra-fast when you're out actually roaming the mean streets of Rome. You can actually update the app with your own reviews as well, just like on the website - they'll upload when you finally do hook up to a sweet source of wifi goodness.
The guides include self-guided tours, which include both famous landmarks and local secrets. Using the GPS in your phone won't cost you a dime, so you can follow along the maps you've downloaded without a hitch and read extra info about la Sagrada Familia as you ogle its towers.
TripAdvisor Offline City Guides are simple and intuitive to use, it's just as interactive as you need it to be, and it can potentially save you a heap of cash in roaming charges. They're also completely and totally FREE. Highly, highly recommended if you're headed for adventure!Developer's original description available here
Key reasons millions of travelers love this app:
Need we say more? Download the app now, there's absolutely no risk, and we're sure you'll love it!
There is no need for a live data connection while using this app, as everything is stored on your phone, after you do an initial update. This means you won't be charged for expensive data roaming, and the app is super fast to use. You can update user reviews periodically when you are on WiFi.
All places that are listed on TripAdvisor.com in this city are included in the app.
TripAdvisor's community of millions of worldwide travelers ensures that content is fresh, plentiful and honest. With over 50 million reviews and thousands more added daily, City Guides offer the best and most recent content.
TripAdvisor members and destination experts have handpicked the best itineraries in the city for you, enabling you to explore interesting neighborhoods, famous landmarks and hidden gems. Using the phone's GPS, you can follow along and read about the interesting sights along the route.
Want to remember the places you visit? It is now easy to do this with TripAdvisor City Guides. Simply Check In, take a photo and add a note for each location, and your journal and travel album will be created for you automatically. You can share it with friends and family on Facebook, or just use it to remember your trip. And, you can even Check In while you are offline and share later when you are online.
Searching for pizza around Times Square? Coffee near the Eiffel Tower? Or do you just want to find a highly recommended place near you? With this app you can easily search any way you want, and find exactly what's best for you and your travel companions.
Once you have selected a restaurant or attraction, this app will guide you there using the fun & useful "Point Me There" screen, which uses the phone's GPS and Compass.
Written by TripAdvisor members and edited by professionals, City Facts articles provide helpful information for when you plan your trip, such as when to go, how to get around in the city, what neighborhoods to visit, etc.
This app leverages the GPS in the phone to show you the best restaurant, attractions etc nearby. The app also uses the compass to guide you to a place once you selected it
- Bug fixes and stability improvements
Trip advisor is the best app for traveling. Whether it be a beginner or an avid traveler.
You are crazy to not have this app! You will never be homeless, hungry or lost with this stroke of genius. I used the map constantly and it's excellent for the non español speaking as you can just search your hotel/restaurant etc, press contact and show the exact address to your taxi driver with zero hassle. Teamed with the Word Lens translation app, this has made my daunting trip an absolute bree
Have used TripAdvisor already before, and again it showed great. That's the only app I could find to offer not only great recommendations but also an offline map of the city - never had to ask for directions.
must have in all travels
Helpful maps, and historic information about Buenos Aires
This app has helped us plan our days while here in BA. It's been very useful
Sooo helpful! It's perfect! I've been living in Buenos Aires for a while but just now I've started to know it since I'm using this app! Ty!


text:
More Products By Petsavers:
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Lime Reflective Pet Collar
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(5.0 out of 5, based on 2 reviews)
Photosadd photo
Reader's Reviews (2)
posted 6 years ago
Great for early morning walks, camping or evening time....just adds a little more visability
Pros: great at night
Cons: none
0 of 0 people found this review helpful.
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This is a laser at night
posted 6 years ago
We jog in the early morning and this collar really reflects light off of it. I feel safe and secure for my pooch when he is wearing this on our sometimes busy street jog. good and durable too.
Pros: Good for early morning jogs
Cons: none
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November 15, 2014 | By Robyn
July 14, 2013 | By Xiaoxiao111
Pink is my favorite color these are wonderful.I will recommend a good website fou you: www.9footballshirt.org
Freshpet Select Roasted Meals for Cat
June 28, 2013 | By Kittymom314
I introduced this food to my two cats, one is a senior at 13 years and the other is about 7 years. Both females. The 7 year old is a new addition, recently adopted :) Because of their age differenc… more ›


text:
Total Anomalous Pulmonary Venous Return
Hypoplastic Left Heart Syndrome
Complex Heart Problems
There are many complex problems of the heart that require clinical care by a physician or other healthcare professional. Listed in the directory below are some, for which we have provided a brief overview.
If you cannot find the information in which you are interested, please visit the Cardiovascular Disorders Online Resources page in this Web site for an Internet/World Wide Web address that may contain additional information on that topic.
Disclaimer - This content is reviewed periodically and is subject to change as new health information becomes available. The information provided is intended to be informative and educational and is not a replacement for professional evaluation, advice, diagnosis or treatment by a healthcare professional. © 2009 Staywell Custom Communications.


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Average rating from 4 reviews.
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Readers' Reviews add review
1 review
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Works great!
posted 6 years ago
A good preventative for kennel cough. I think my dog is the only one in my experience that has evern snuffed in when receiving this! Usually dogs blow out and it goes everywhere.
The key is blowing in their face usually helps.
The stuff works!
Pros: none
Cons: none
0 of 0 people found this review helpful.
Did you?
Want to review a product, but not finding it? Just add it and review it!
Recent Product Reviews
November 15, 2014 | By Robyn
July 14, 2013 | By Xiaoxiao111
Pink is my favorite color these are wonderful.I will recommend a good website fou you: www.9footballshirt.org
Freshpet Select Roasted Meals for Cat
June 28, 2013 | By Kittymom314
I introduced this food to my two cats, one is a senior at 13 years and the other is about 7 years. Both females. The 7 year old is a new addition, recently adopted :) Because of their age differenc… more ›


text: Publications Search Results Users may wish to consult What's Included in the Bibliography. Results 1 - 1 Results per page: 25 50 100 Sort by: Pub Date (newest) Title Recent Additions Author Clear All Pub. Year Citation 1999 Glick, Jennifer E. Economic support from and to extended kin: A comparison of Mexican Americans and Mexican immigrants. International Migration Review. 33, (3), 745-765. Full Text Options: Original Source Worldcat Google Scholar Export Options: RIS EndNote XML [1]


text:
vegetable in Marathi
translation and definition "vegetable", English-Marathi Dictionary online
Translations into Marathi:
- पालेभाजी(noun )Any of various herbaceous plants having parts that are used as food.
Other meanings:
Any plant.
any plant
a person whose body or brain has been damaged
of or relating to plants
of or relating to vegetables
An organism that is not an animal, especially an organism capable of photosynthesis.
vegetable (food)
A person with severe brain damage or who is in a persistent vegetative state.
some edible part of a plant
Of or relating to vegetables.
a plant raised for some edible part of it
(figuratively) A person whose body or brain has been damaged so that they cannot interact with the surrounding environment.
A plant raised for some edible part of it, such as the leaves, roots, fruit or flowers, but excluding any plant considered to be a fruit, grain, or spice in the culinary sense.
The edible part of such a plant.
Of or relating to plants.


text:
Birds In Spring: This Is Your Brain On Myeloma
Published: Feb 21, 2012 4:06 pm
Say, how's your brain working these days?
That's a question for many cancer patients and survivors, but it is particularly one for those multiple myeloma patients who have had at least one stem cell transplant.
Getting a stem cell transplant is pretty intense.
First you start treatment with a cancer drug regimen, or radiation, to reduce the level of myeloma cells in your body and to get your myeloma into the best remission that can be accomplished.
Then you take drugs to make your bone marrow produce more stem cells and "mobilize" them into the blood for harvesting.
A few days before your transplant, you get a lethal dose (200 mg) of melphalan (Alkeran) – it's called MEL200 among the transplant doctors. I say lethal, not because it could kill you on the spot, but melphalan targets and destroys your bone marrow. By doing so, it also destroys your immune system, making you a sitting duck for infection. Oh, and melphalan also gives you the plucked chicken look, since every single hair on your body falls out.
The stem cell transplant is just to rebuild your bone marrow that was destroyed by the chemotherapy. It is rather anticlimactic and takes maybe 20 minutes. They call it your new birthday, since it's the day your immune system in reborn.
There is ongoing research into "reduced intensity" stem cell transplants, basically giving you a smaller dose of melphalan, but it's not commonly used to treat myeloma yet.
This brings me back to the brain thing.
The question I raise is whether after you've been through all this, do you think, act, and feel differently than before? Do you react to things differently? Are there changes in your values? Are there changes in your mental abilities?
Take the last item:
Can myeloma or its treatment have a negative impact on your cognitive abilities?
No one considered the possible impact of chemotherapy on mental abilities until thousands of breast cancer survivors started speaking out, saying something like, "Hey, I don't really think straight all the time any more."
Thus, "chemo brain" entered our lexicon.
I've read estimates that as many as three-quarters of cancer survivors experience some change in their mental functioning, and that it can last for several years. Most of the research into chemo brain has involved women with breast cancer, so it's a fair thing to be a bit hesitant to extrapolate the findings across all cancers.
But let's talk about treatment for multiple myeloma specifically.
A transplant is up toward the top of the list as a stressful and traumatic event on your body, particularly one that you are doing to yourself on purpose. Unfortunately, the effect of chemotherapy on cognitive function is one of the least explored areas of transplant and high-dose chemotherapy.
I also suspect that all of the challenges you face because of myeloma can cause some significant cognitive and mood disturbances, perhaps even something akin to post-traumatic stress disorder.
I've chatted about this possibility with various myeloma medical team folks. One doctor commented that even though some patients say they are okay, they also say they "feel different."
Can all that one goes through in dealing with myeloma, the disease itself and the treatments, make you a different person – spiritually, intellectually, and psychologically?
I start my conversation on this topic by talking about myself.
After my first transplant, I emerged with a greatly changed perspective about some of the significant things in my life. Some things just aren't as important to me as they were before. Other things take a more important place in my view of the world than they previously did.
In addition, I quite possibly experience some chemo brain, and I am not always able to do things as easily as before.
I've never really experienced the depression- or "feeling down"-aspect of myeloma, but who's to say that will always be the case?
One myeloma patient I know used to be one of the most positive people living with myeloma that I'd come across. But since he's experienced some significant setbacks in treatment, and things aren't working as he expected, he's changed.
There's frustration, depression, and anger there now – an edge to him that wasn't there before. Anxiety and fear can do bad things to you.
Someone gave me another example of how this disease can change you: Take a truck driver in complete remission. He's a "success story." However, he can no longer drive a truck. He's now on disability with income that's maybe one-quarter what he was making before. We can make him a stereotype – maybe his wife leaves him, and he sits around, alone, drinking beer, and getting fat.
He looks in the mirror and doesn't see "success." Instead, he says, "This disease has destroyed me. It's destroyed the person I used to be."
From my perspective, I think that myeloma, treatment, and transplants can cause changes that are psychological, cognitive, attitudinal, and physical.
Are the causes of the changes chemical or biological?
There's a little bit of research going on to answer this question.
For example, Shelli Kesler and her colleagues at Stanford University are trying to better understand what causes chemo brain. Her recent research shows that chemotherapy has a negative impact on cognitive function, but she was also quoted recently as speculating that the cancer itself could cause chemo brain.
"When you get a disease like cancer, it activates your immune system. Sometimes you can have increased inflammation for a really long period of time, and inflammation can affect the brain," she said.
My additional thoughts on the topic:
Unfortunately, though, much of what I've written about here isn't being addressed by the medical community in general.
For those of us who are transplant patients, we often find that we aren't always able to function as well as we used to. Very little has been done in the post-transplant setting to understand cognitive dysfunction, post-trauma implications, stress, depression, and attitudinal and physical changes.
From an overall physical health perspective, the medical teams treating myeloma patients/survivors are doing a great job. The medical community has invested a lot in helping us "get better."
Those things that help us get better, however, may be affecting survivors in ways no one anticipated in terms of stress and dysfunction – in ways that can impair recovery.
It would certainly be great if there could be more research about this, and perhaps some solutions.
Lou Ganim is a multiple myeloma patient and columnist at The Myeloma Beacon.
If you are interested in writing a regular column to be published by The Myeloma Beacon, please contact the Beacon team at .
Related Articles:
- "Chemo Brain" May Affect Half Of Myeloma Patients After Initial Therapy, And Is Worsened By Stem Cell Transplantation
- The Top Myeloma Beacon Patient And Caregiver Columns Of 2012
- The Top Myeloma Beacon News Articles Of 2013
- Clinical Trials Start For A Pre-Transplant Treatment
- Obesity's Impact On Myeloma Stem Cell Transplant Outcomes: Not What You Probably Expected


text:
Teligent's new chair abruptly resigns
Teligent Inc.'s new chair resigned over the weekend after less than a month on the job.
Teligent spokeswoman Tita Thompson could not comment on why Howard Jonas resigned, nor could she say if there was a replacement. The board will replace Jonas over time, she says.
Jonas, who took the Teligent helm April 30, also is chair and CEO of IDT Corp., which purchased Liberty Media's 37 percent share in Teligent last month. Shortly after, former CEO and Chair Alex J. Mandl stepped down and IDT's Jonas took over as chair, while another IDT alum, Yoav Krill, took over as Teligent's CEO.
IDT spokesman Gil Nielsen would not comment on why Jonas left Teligent, but says Jonas will remain chair and CEO of IDT.
Thompson says Teligent is conducting business as usual and moving forward with its restructuring.
Teligent filed for Chapter 11 bankruptcy last week.


text:
A group where people can discuss anime / manga. What more is there to say?
I like the Pirates :)
You are not logged in, your comment will be anonymous unless you join the community today (totally free - or sign in with your social account on the right) which we encourage all contributors to do.
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Only one episode and I am blown away by sheer awesomeness. Cute pirates in space doing cute AND piratey things..!
More info here: Myanimelist.net


text:
Southall murder: Life sentence for man who stabbed friend's boyfriend then hid from police under the bed
A knife attacker discovered by police hiding under a bed after murdering his friend's boyfriend has been jailed for life.
Marmaduke is the lovable Great Dane of comic-book fame, now translated into a real-life doggie whose best friend is Carlos, a Russian Blue cat, and whose human family are moving from the Midwest to Orange County.
20 August 2010
14 July 2010
18 January 2010
28 July 2009
An economic downturn is usually good news for comedy. Most of you probably don't want to think about the Great Depression but if you do you'll quickly find yourself thinking of Charlie Chaplin and Buster Keaton. In Britain during the Eighties, when unemployment was at a frown-making three million, the nation was rolling about every night to The Young Ones, Spitting Image and Blackadder. There's no business like crap business for exposing the collective funny spot of a generation, which might be a way of saying Ben Stiller and his friends are about to take over the known universe.
22 May 2009
13 May 2009
03 March 2009
Hollywood funnyman Owen Wilson looks as though he's back to his old self as he relaxes on a sunny beach with a mystery lady.
03 August 2008
21 May 2008
21 May 2008