text:
Its been a tale of two teams united by one coach.
Jeff Cruce is his second year as coach of the Berkeley Stags, and will lead his team into the second round of the Class AAA playoffs tonight when the Stags travel to the Grand Strand to battle second-ranked and undefeated Socastee in a 7:30 p.m. showdown.
The Stags are a completely different team than the one that began the season with a 1-6 record and were outscored 181-83. The team thats hitting the road today is 4-0 and has outscored foes 155-54 in its last four games.
Its been a season of life lessons, said Cruce, who posted a 4-7 last fall after replacing the legendary Jerry Brown, who won 175 games and three state championships at the school. We fumbled the ball here and made a mistake there but we never quit. The biggest life lesson is perseverance. When things go bad, you dont give up. Its been a spiritual life lesson.
Cruce admits that coaching in Moncks Corner, where football is king, can be tough when the losses outnumber the wins. Hes heard the critics in the stands and on the telephone and has received their emails. But he never wavered in his faith in himself.
Moncks Corner is a very unique place, Cruce said. I dare say but it is one of the top five communities in the state as far as high school football is concerned. Berkeley High School and its football team is the centerpiece of the community.
If you listen (to some fans), it can drive you crazy, Cruce added. Ive actually told people not to call me or send me emails. I am an adult and can take criticism. But when you criticize my players thats malicious. Theyre teenagers.
Cruce has drawn on his experiences at Hanahan and Cane Bay, programs he turned into winners.
If I were a younger coach, I might have been overwhelmed, Cruce said. But I drew on my experience. We went 0-10 in my first year at Hanahan, 6-6 the second year and then had that great run (where we won 12 straight region championships). It was the same thing at Cane Bay. Its a process you go through.
Cruce dismissed four starters from the team this season and that showed his players he was committed to discipline.
You cant sacrifice the integrity of the program when a kid makes a mistake and doesnt have the right attitude, Cruce said. When you say something, you have to stick to your word. You give your players boundaries and they have to stay within it.
The Stags first victory came in Week 5 when they topped defending Division II-AA state champ Timberland 21-20 thanks to 10 Wolves turnovers.
Obviously, a win, a win of that caliber, helps us mentally, Cruce said after the game. Its hard for us because were so young.
The team has final mature as is the program. The B- and junior varsity teams have winning records and next years team will include 40 seniors if there is no attrition.
Running back Ezekiel Jenkins and quarterback Matt Harkness have shown improvement every week.
Jenkins scored three touchdowns and amassed 321 all-purpose yards to lead Berkeley to a 51-32 victory over Hilton Head that gave the Stags a berth in the playoffs.
The defense is led by outstanding defensive ends Cyrus Pinckney and Jewuan Jefferson.
Last week, Pinckney recorded a fumble recovery, 2.5 sacks to lead a Stag defense that held Midland Valley to 175 yards of total offense in their first-round 24-2 win.
We ready for the challenge of playing the No. 1 or 2 team in the state, Cruce said. We believe in ourselves.


text:
Nothing is more personal than how we feel about how we look. View Dr. Yearsley's Before & After photos to see the difference.
Begin a conversation with Steven Yearsley, MD, board-certified plastic surgeon, even before your free consultation. It's private, secure, and easy.
Copyright 2016 Mid Dakota Clinic Site by Odney

subset: commoncrawl


text:
Riverbend Academy - School of Performing and Visual Arts
Rating: 4.7 stars 10 10 reviews
PO Box 482 145 North Main Street - 2nd and Main Historic Soaper Hotel 1st & 2nd Floors Henderson KY 42419 USA
RiverBend Academy advances life in our region by infusing superior arts enrichment and culture primarily through premium instruction in various areas of the arts as an inclusive mentoring school for the arts.
Presently RiverBend Academy (RBA) serves primarily the Henderson County, Kentucky market. However, approximately 10% of students travel as much as 45 minutes away to access our expert instruction downtown at the academy in Henderson from adjacent Kentucky Counties.. We are in 5 Henderson County Schools, after-school,but growing. Presently we serve both public middle schools, 3 public elementary schools, one private elementary school & middle school, and an alternative school.
Target demographics:
Students from any financial background -- from the wealthy to those in poverty, who don't fit into academics and sports rarely find their niche and can have more of a propensity for undesirable outcomes. RBA was founded to help catch those falling in the cracks and instruct all in the arts within the region with desire and interest at any age, ability or disability regardless of financial means. This will raise the overall cultural and education bar for our region as statistics prove that art and the brain are a powerful combination in building self esteem, reducing social ills and promoting higher grades and education.. RBA's arts education begins as young as lap babies and spans to ladies in their 70's tap dancing.
Direct beneficiaries per year:
Well over 300 plus
Geographic areas served:
Henderson County with outreach to surrounding counties in KY
Tiny Notes for Pre-school to 5 years, Piano, Guitar (all types), Double Bass, Keyboard, Social Ballroom Dance, Voice, Theater/Acting, Culinary Cooking Classes, Tap Dance, Ballet, Jazz Dance and More!
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Promote This Nonprofit
GreatNonprofits badges allow you to raise awareness of your favorite nonprofits on your own web sites!
Reviews for Riverbend Academy - School of Performing and Visual Arts
Truely dedicated group focused on bringing the arts to all. This group is especially involved in providing alternatives to children who may not be involved in organized sports. A wide variety of classes are offered as alternatives to the traditional afterschool activities. Those alternative include: dance, voice, acting, film making, guitar, piano and cooking classes. Scholarship are provided to those who qualify. Additionally, River Bend provides afterschool programs onsite at several schools.
More Feedback
I've personally experienced the results of this organization in...
I've seen the pride in students faces when they realize their potential.
If I had to make changes to this organization, I would...
Somehow increase public awareness of the opportunites that are provided. Work more closely with the local schools and churches.
As a founder for RBA, I am remarkably proud of where we have come in such a short time in one of the worst economies of our time. RBA is approaching its beginning its 5th year of operation and have overcome lots to still be here with a dedicated mission to educate and instruct everyone from lap babies to those in their 70's in the arts.
More Feedback
I've personally experienced the results of this organization in...
I have personally seen the benefits to children and youth that experience our instruction. Their self-esteem is higher, their behavior improves, communication is better, there are many, many benefits when we make excellent art instruction available to all regardless of ability to pay.
If I had to make changes to this organization, I would...
We need more paid staff and funding so we are able to reach more children. RBA has been fully managed by an all-volunteer working board until recently with part-time 15 hour a week staff -- who I pray we can pay more in time, give more hours and retain. Ann is excellent! Ann and RBA needs more staff support and we need more funding in order to achieve more operationally. RBA is very good stewards of its resources and therefore we have put the emphasis on funding after school programs and those in need.
Will you volunteer or donate to this organization beyond what is required of board members?
How much of an impact do you think this organization has?
Will you tell others about this organization?
How did you learn about this organization?
What is this organization's top short-term priority?
Educate children in the arts starting more with pre-school and elementary age before stigmas are established on participating in the arts.
What is its top priority in the long run?
To bring our arts education outreach to the full western Kentucky area and serve all regardless of ability to pay, disabilities, etc.
When was your last experience with this nonprofit?


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Firm Profile: Lobbyists, 2012
Total Lobbying Income: $80,000
Total number of revolvers: 0 (0.0%)
Total number of former members: 0 (0.0%)
Lobbyists working for Bagley Group:
|Bagley, John F
|Battelle Memorial Institute
Case Western Reserve University
Commonwealth of Kentucky (Commonwealth of KY Energy & Enviro)*
Stanford University
University of Chicago
University of Delaware
* Subsidiary (Lobbied For)
NOTE: All lobbying expenditures on this page come from the Senate Office of Public Records. Data for the most recent year was downloaded on April 20, 2015.
Feel free to distribute or cite this material, but please credit the Center for Responsive Politics. For permission to reprint for commercial uses, such as textbooks, contact the Center: firstname.lastname@example.com


text:
SSL and TLS VPN Security
SSL and TLS VPN Security News
August 16, 2016
New Vawtrak Trojan variant leverages SSL pinning, HTTPS
Fidelis Cybersecurity reports notorious Vawtrak banking Trojan gets upgrades to increase security and evade detection, including SSL pinning and domain generation algorithm.
December 22, 2015
PCI DSS 3.1 deadline for TLS migration pushed back
The Payment Card Industry Security Standards Council unexpectedly pushed back the deadline for enterprises to migrate off of early versions of TLS.
December 03, 2015
Experts question customized TLS implementation after Amazon s2n flaw
Amazon's s2n passed its first test by patching a flaw quickly, but experts said enterprises still need to be wary of the complexities surrounding TLS implementation.
May 20, 2015
Logjam flaw crimps TLS encryption; NSA may have used it
A newly discovered TLS vulnerability that affects thousands of websites, servers and browser users could allow attackers to bypass encryption.
SSL and TLS VPN Security Get Started
Bring yourself up to speed with our introductory content
How to address key SSL security issues and vulnerabilities
As SSL technology evolves and changes, new vulnerabilities begin to cause problems. Expert Rob Shapland explains how security professionals can overcome these SSL security issues. Continue Reading
Transport Layer Security (TLS)
The Transport Layer Security (TLS) protocol defines a mechanism to encrypt and maintain data integrity for communications over a network. Continue Reading
Secure Shell (SSH)
Secure Shell (SSH) is a network protocol that secures data communications between computers on an insecure network using strong authentication and encryption. SSH also refers to the utility suite that implements the protocol, and which can be used ... Continue Reading
IPSec vs. SSL VPNs: Which cures your remote access ills?
August 2003 Information Security magazine: IPSec vs. SSL VPNs -- how to make the choice; 802.1X authentication and Web services security issues.
By submitting your email address, you agree to receive emails regarding relevant topic offers from TechTarget and its partners. You can withdraw your consent at any time. Contact TechTarget at 275 Grove Street, Newton, MA.
You also agree that your personal information may be transferred and processed in the United States, and that you have read and agree to the Terms of Use and the Privacy Policy.
Evaluate SSL and TLS VPN Security Vendors & Products
Weigh the pros and cons of technologies, products and projects you are considering.
Will Google's Certificate Transparency prevent certificate abuse?
Google's Certificate Transparency tool publicly logs certificates issued by CAs. Expert Michael Cobb explains how the log viewer works to improve certificate security. Continue Reading
Digital Identity Management
In this excerpt of Digital Identity Management, authors Maryline Laurent and Samia Bousefrane discuss principles of biometrics and the TLS protocol. Continue Reading
Choosing secure file transfer products for your enterprise
Are you concerned about file transfer security? Expert Karen Scarfone discusses the best products for transporting files while maintaining their confidentiality. Continue Reading
Manage SSL and TLS VPN Security
Learn to apply best practices and optimize your operations.
Can a subscription ease SSL certificate management?
SSL subscription services are emerging to help enterprises handle the daunting task of SSL certificate management. Expert Michael Cobb discusses the benefits of such a service. Continue Reading
From SSL and early TLS to TLS 1.2: Creating a PCI DSS 3.1 migration plan
PCI DSS 3.1 requires enterprises to deplete SSL and early TLS use by June 30, 2016. Expert Michael Cobb offers advice for putting a migration plan to TLS 1.2 in place. Continue Reading
What's the best way to secure VPN access for teleworkers?
The U.S. Postal Service suspended teleworking following a recent breach. Expert Kevin Beaver explains why teleworkers aren't always to blame in the event of a breach and explores methods for secure VPN access. Continue Reading
Problem Solve SSL and TLS VPN Security Issues
We've gathered up expert advice and tips from professionals like you so that the answers you need are always available.
How does the Logjam vulnerability affect TLS encryption?
Expert Michael Cobb discusses the TLS protocol flaw dubbed Logjam and explains how to detect if enterprise systems are at risk, as well as how to remediate them. Continue Reading
Can open source cryptography libraries be trusted?
After the Heartbleed fiasco, the future of OpenSSL and open source cryptography libraries is up in the air. Application Security Expert Michael Cobb discusses whether they can -- and should -- be trusted. Continue Reading
Defense best practices for a man-in-the-middle attack
Man-in-the-middle attack defense requires careful, layered security. Michael Cobb reviews the tactics enterprises should employ to stay secure. Continue Reading


text:
This post is part of a series called Logo Reveals.
It's amazing how much pseudo-3D you can do in After Effects today. This tutorial will focus on using a new plugin that brings 3D bicubic deformations to layers that allows you to truly bend and twist layers in 3D space. If you are familiar with 3D packages, this is true displacement as opposed to just bump mapping.
Republished Tutorial
Every few weeks, we revisit some of our reader's favorite posts from throughout the history of the site. This tutorial was first published in March of 2009.
Music and Sound Effects
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Nathan Ross
Nathan Ross, b. 26 May 1814, Somerset Co, PA, son of Adam Ross and Phoebe Miller. Adam had at least two other marriages.
Nathan married Barbara Braun/Brown in Columbiana Co, OH. They raised their family in Brush Valley, Indiana Co, PA.
Click on the City or Town name right under the name of the photograph, to view more photographs from that city or town. If there's a county listed, you can view more photographs from that county. If you see a surname listed under the title of the photograph, you can click on that surname or surnames to view more photos that are associated with that surname. Same thing works with any of the other "tags" listed right under the title of the photograph. You may be able to make the photo larger, if the original photograph was larger than the photo shown here. Click on the word "original" above and a window will open with a the photo in a larger size. Want to add some information or comment on this photo? Click on "Add new comment" above.


text:
There are high-quality 2012 NFL free agents not named Peyton Manning. I know; I was surprised too.
Stalled in the logjam of the Manning Sweepstakes, quite a few free agents were left pacing the floor and driving their wives crazy last weekend as they wait for the call that will determine the next phase of their lives.
One week after the official start of the NFL's annual "do-si-do," it certainly did feel at first as if "all the good ones are married." And some of them are.
Peyton Manning, Mario Williams and Vincent/DeSean Jackson have tied the knot. Even Calvin Johnson is off the market—and he wasn't on the market to begin with.
But when you really start searching, it's amazing how many good players are still out there.
So, take a deep breath, pull up your team's depth chart and let's take a look. Even if you didn't land Peyton Manning, there's a lot of free-agency joy to spread around.


text:
SonRock Kids Camp: VBS Starter Kit 2009
More in SonRock Kid's Camp VBS Series
Gospel Light VBS / 2009 / Other
$9.99 (CBD Price)|
Save 75% ($30.00)
Availability: In Stock
CBD Stock No: WW307358
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Product Media
Product Description
At Son Rock Kids Camp, campers will build bonds offriendship with each other and with caring counselors as they discoverthat their true identities are not formed by where they go, what they can do or what others think. They will explore who they are in Jesus Christ - the most important relationship of all.
Grades Pre-K to 6. Uses the NIV and KJV.
Starter Kit includes:
- Big Rock Bible Games
- Look-out Point Bible Stories Grades 1 - 2, 3-4, and 5-6
- Camp Director's Guide
- Son Rock Music CD
- Son Rock Overview DVD
- Hungry Bear Snacks and Games
- Camp Creations Crafts for Kids
- My Camp Journal- all 5 age levels.
- Critter Corner Teacher's Guide (Pre-K)
- Wilderness Trail Theme Adventures
Director's Sample Resources include:
- Attendance Chart
- Single Balloon
- Single Bookmark
- Single Bulletin insert
- Growing as God's Child Booklet - 1 copy
- God Loves you Booklet - 1 copy
- Single Doorknob Hanger
- Jesus Helps me Coloring book
- Counselor Trail Guide
- Single Promo Postcard
- Single Iron on Transfer
- Single Name TagSingle Camp Stuff Sticker Sheet
- Single Daily Truth Sticker Sheet
- Single Animal Sticker Sheet
- Single Photo Frame
- Single Skin Decal
- Single Promo Poster
- Single Student Certificate
- Single Volunteer Certificate
- Single SonRock Magnet
Product Information
|Format: Other
Vendor: Gospel Light VBS
Publication Date: 2009
Availability: In Stock
Series: SonRock Kid's Camp VBS
Other Customers Also Purchased
SonForce Starter KitGospel Light VBS / 2007 / Other, N/A$14.99 Retail:
$33.50Save 55% ($18.51)Availability: In StockCBD Stock No: WW738703Video
My Camp Journal, Kindergarten (Ages 5-6)Gospel Light VBS / Trade Paperback$0.25 Retail:
$1.00Save 75% ($0.75)Availability: In StockCBD Stock No: WW747337
SonRock Clip Art and Publicity CDROMGospel Light VBS / Compact disc$0.25 Retail:
$10.00Save 98% ($9.75)Availability: In StockCBD Stock No: WW747970
SonQuest Rainforest VBS Super Starter KitGospel Light VBS / 2010 / Other$9.99 Retail:
$119.99Save 92% ($110.00)Availability: In StockCBD Stock No: WW752508Video
Related Products
SonRock Director's Guide with CDROMGospel Light VBS / Trade Paperback$0.25 Retail:
$9.00Save 97% ($8.75)Availability: In StockCBD Stock No: WW747420
Camp Decor and MoreGospel Light VBS / Other$0.25 Retail:
$3.00Save 92% ($2.75)Availability: In StockCBD Stock No: WW747436Video
Big Rock Bible GamesGospel Light VBS / Trade Paperback$0.25 Retail:
$3.00Save 92% ($2.75)Availability: In StockCBD Stock No: WW747440
Hungry Bear Snacks and GamesGospel Light VBS / Trade Paperback$0.25 Retail:
$3.00Save 92% ($2.75)Availability: In StockCBD Stock No: WW747451
Product Reviews
of customers would recommend this product to a friend.
Page 1 of 1
RondaGender: female5 Stars Out Of 5August 9, 2012RondaGender: femaleQuality: 5Value: 5Meets Expectations: 5This was excellent for camp ministries. I was able to stretch it out to 10 days. You do need to order the extra items that would be needed for the amount of children you have. The children loved it.
Page 1 of 1
Ask Christianbook
Q: In the SonRock VBS Starter Kit do I get piano music? How about with the Super Starter Kit?
No, the SonRock VBS Starter Kit does not include piano music. The SonRock VBS Super Starter Kit, CBD WW830735, includes Son Rock Songbook in PDF format on the DVD/CD-ROM.
Have a question about this product? Ask us here.
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text:
Keepsake Barbie® Doll & Fashions Gift Pack - Weekend Getaway
Item #: T03453Bá
Was: $29.99Now: $14.99
Get free shipping on orders of $39+!More Promotion Details
When anything is possible, a girl needs a wardrobe for any occasion!
More Details
- Details
This set features fabulous pieces that can be mixed and matched to really make a style statement. Perfect for a fashionable doll's weekend away!
Barbie® doll comes with six trendy wardrobe pieces: A hot pink ruffled top, two-toned black and silver pants, comfy hoodie, denim shorts, trendy travel-graphic mini dress and a basic black tee.
Accessories include a purple wristlet purse, black strappy heels and a silver statement necklace.
- Exclusive keepsake box contains your doll and fashion giftset!
- For a unique personalized experience, young fashionistas can write their name on the front of the box!
- Pack the box with multiple Barbie fashions and accessories and take it on your next Barbie dress-up play date!
- It's a complete fashion closet!
- Collect all of Barbie® doll's fashion packs for the ultimate mix-and-match fun to put your own spin on fashion!
Battery Required: No
Warranty: Yes
Age Grade: 3+
Country of Origin: CN


text:
inspiration for celebration
Reception Style: Modern (Yellow & Black)
- posted in:
- Engagement
- Weddings
There's a great feature on Brides.com titled "What's Your Reception Style?" that highlights 3 different styles of receptions: Romantic, Modern, and Natural. There are really so many different ways that you can interpret each of these 3 styles – and many of us might lean towards a combo of the two – but for this […]
REAL PARTIES: Lollipop, Lollipop….
- posted in:
- In the Spotlight
Today's Real Party feature is pretty much the cuteness. Wait… make that the sweetness! This "Yummy Lollipop Birthday" theme was created by the ultra-creative Kim from TomKat Studio for her daughter Kate's second birthday celebration! Everything from the invitations & decorations to the food & favors played up Kim's colorful lollipop theme. (P.S. You can […]
Guyot Designs: Smart + Cool + Eco-Chic
- posted in:
- In the Spotlight
I came across an image of these "Squishy Bowls" in this month's issue of Real Simple (June 2009) and was instantly intrigued by both the name and mod look. You can use these bowls to serve dishes like pasta & salad on when your on a picnic/at an outdoor BBQ, then squash them into your […]
Outdoor Entertaining: Twist, Axis, Hammered
- posted in:
- In the Spotlight
These fabulous new dishes from West Elm are the perfect excuse for a backyard party! Made from sturdy, outdoor-friendly materials like melamine and acrylic, there's no need to worry about slippery hands or broken glass 'round the pool. More bonuses: cool, contemporary designs + affordable prices! Pictured above: Twist Melamine Dinnerware ($14 – $18 per […]
Cute Enough to Eat (Fun Favor Ideas)
- posted in:
- In the Spotlight
Looking for a unique party favor idea? With designs ranging from "Enlightened Desserts" to "Sushi" to "Savory Foods", these tiny, japanese-inspired erasers are sure to stand out! More favorites pictured below, like this pretty Tea Ceremony Set featuring Mocci, Matcha, Manju, Delicacy, and Pancakes:
REAL PARTIES: Li'l Tweet Baby Shower
- posted in:
- Baby Boy
- Baby Showers
This adorable "Li'l Tweet" Baby Shower was created by Kathleen Gibson of the wonderfully creative Twig & Thistle blog. Read on for the details, as told by the hostess herself – plus info on where you can get these charming owl-themed invites & party supplies for your own celebrations! ***** "Last year I had the […]


text:
The Chronicle Gets Its Feelings Hurt By Treme; Possibly Offers A Solution
In case you hadn't heard -- and there's no way you haven't heard, since seemingly everyone who writes for a living in America worships David Simon's TV shows -- Treme premiered Sunday.
It's a story about New Orleans in the aftermath of Katrina. It contained two passing references to Houston: One, a mention of a guy getting killed "in one of those Telephone Road bars"; two, a character saying "all the crime moved to Houston."
Pretty innocuous stuff; Telephone Road isn't exactly murder central, but the name is irresistible.
Still, the Houston Chronicle, in the guise of an assistant features editor, has had its feelings hurt. Ronnie Crocker wrote in their pop-culture blog:
That's the only recognition Treme could muster for a city that took in perhaps 240,000 storm victims, many of whom arrived in buses with nothing but the clothes they were wearing?
Our area, according to my recollection, played a pivotal and overwhelmingly positive role in the Katrina story. Who couldn't have been impressed by the masses of volunteers who showed up at the Astrodome to feed and shelter the evacuees? Houstonians swamped clothing and furniture drives with so much that donations had to be halted....All I know for sure is that Houstonians can be proud of the role they played in the Katrina disaster and secure in the knowledge that, should the need ever arise, we would do it all over again.
Upcoming Events
Rice University Owls Football vs. Prairie View A&M University Football
TicketsSat., Oct. 22, 2:30pm
University of Houston Cougars Football vs. UCF Knights Football
TicketsSat., Oct. 29, 11:00am
Rice University Owls Football vs. Florida Atlantic University Owls Football
TicketsSat., Nov. 5, 2:30pm
University of Houston Cougars Football vs. Tulane University Football
TicketsSat., Nov. 12, 11:00am
Houston Proud!!
To: David Simon
From: Houston Chronicle
Re: Proposed Treme episode
Please read and use the attached script for the second episode of Treme. We realize shooting and editing the episode in time for Sunday might require some extra hours, but we feel it is absolutely necessary. Not to mention the script is, if we say so ourself, damn good. Here's an outline.
Title: There Are Angels in Texas
Act One: The episode opens with a montage of Houstonians enjoying their city -- going to the museums, enjoying the white tigers at the Aquarium restaurant (or any other Tilman Fertitta establishment), cheering the Astros, listening to our world-class symphony, strolling in Discovery Green. (Length of Act One: 35 minutes.)
Act Two: As word hits of Katrina, Houstonians of all races, creeds and colors leap into action, rummaging through their closers for clothes that no longer fit. Steely-eyed government officials say things like "Damn the red tape, just do it!!! This is Houston, where we get things done!!" (Length of Act Two: 10 minutes.)
Act Three: Pan over the Astrodome like the after-battle scene in Gone With the Wind. Mariah Carey singing Leonard Cohen's "Hallelujah" plays, with the title word in the chorus replaced by "Thank You, Houston." Show plenty of shots of refugees gratefully accepting Members Only jackets from soccer moms who tell them "it's nothing, really." Close with testimonials from actual Katrina evacuees saying how great it is. Last shot: As you pan back from the Dome floor, fade to black. The the screen fills with white lettering:
"What I'm hearing, which is sort of scary, is that they all want to stay in Texas. Everyone is so overwhelmed by the hospitality.
And so many of the people in the arena here were underprivileged anyway, so this is working very well for them."
-- Barbara Bush
Fade to black. Credits roll over a montage of Apollo 11's first words on the moon, Jean Michel Jarre's 1986 downtown concert and....and...we'll get back to you with other highlights.
Thank you for your consideration,
Houston Chronicle
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TicketsThu., Sep. 29, 11:00am
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text:
This iReport is part of an assignment:
Tell us the Good Stuff!
About this iReport
- Posted June 17, 2014 by
- Ethicist1
Louisville, Kentucky
Louisville, Kentucky
More from Ethicist1
- Annual 2015 St. James Art Festival in Louisville, Ky as colorful as ever under a clear blue sky on a sunny day
- VindaLou, Indian festival and Irish fest make Louisville, KY final September weekend lively
- Worldfest 2015 is the best ongoing labor day weekend event celebrating diversity of cultures and cuisines in Louisville, KY, USA
- Dr. David Baltimore, a brilliant American mind, Nobel laureate, President Emeritus of Caltech, a father and a recent grand father
- City of Louisville, KY mayor Gregory Fischer and his father George Fischer are truly the city fathers
Kale is the new Yale of Vegetables and good for you stuff
Last week I was driving to the scenic river road in Louisville to get to town for the Greek Festival and on the way outside a church I saw a sign for fresh blueberries and inside was a farmer's market. While looking at the fresh produce I found Kale, a vegetable about which I had heard for a while now but never bought it. I have compared it to Yale because Yale is one of the top Universities in the world. Three of the 4 presidents in the past 25 years have been Yalees. A first lady of one of the most admired president (Bill Clinton) of the past 25 years is also a Yalee and with a good chance (40%) for being the first woman president of the USA. Hopefully having convinced you that Yale is a top school even though most of you already knew that let me go on to why has Kale taken the top position amongst vegetables. Why do we eat vegetables? We eat to get balanced nutrition and part of good nutrition is getting essential minerals like Iron (more than in beef?), calcium, Magnesium and vitamins (C and K), other antioxidants and fiber and Kale meets the requirements in good measure. Read more on the mind body green site. But we would not eat Kale, which has been a key vegetable as a part of Soul food and Southern style greens. if it did not taste good. So how best to make food tasty. Apply a thin coating of oil with a spray along with some seasoning of your choice and bake it until crispy. Crispy Kale tastes good. Another way to cook it is to stir fry it in olive oil and spices and herbs along with lentils and cabbage your other vegetables of your choice. I am not a Yalee and will never be the president of the USA but in less than a week have converted to being a Kalee. Delicious and nutritious what more of good stuff can you ask for. By the way if you have a child that does not want to eat anything green just slip the veggies into something they like. Maybe a pistachio ice cream or even a chocolate ice cream.
More from this assignment - Tell us the Good Stuff!


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The accumulation of thick stagnant mucus provides a suitable environment for the growth of Pseudomonas aeruginosa and Staphylococcus aureus within the lung alveoli of cystic fibrosis (CF) patients. These infections cause significant lung damage, leading to respiratory failure and death. In an artificial mucin containing medium ASM+, P. aeruginosa forms structures that resemble typical biofilms but are not attached to any surface. We refer to these structures as biofilm like structures (BLS). Using ASM+ in a static microtiter plate culture system, we examined the roles of mucin, extracellular DNA, environmental oxygen (EO2), and quorum sensing (QS) in the development of biofilm-like structures (BLS) by P. aeruginosa; and the effect of EO2 and P. aeruginosa on S. aureus BLS.
Under 20% EO2, P. aeruginosa strain PAO1 produced BLS that resemble typical biofilms but are confined to the ASM+ and not attached to the surface. Levels of mucin and extracellular DNA within the ASM+ were optimized to produce robust well developed BLS. At 10% EO2, PAO1 produced thicker, more developed BLS, while under 0% EO2, BLS production was diminished. In contrast, the S. aureus strain AH133 produced well-developed BLS only under 20% EO2. In PAO1, loss of the QS system genes rhlI and rhlR affected the formation of BLS in ASM+ in terms of both structure and architecture. Whether co-inoculated into ASM+ with AH133, or added to established AH133 BLS, PAO1 eliminated AH133 within 48–56 h.
The thick, viscous ASM+, which contains mucin and extracellular DNA levels similar to those found in the CF lung, supports the formation of biofilm-like structures similar to the aggregates described within CF airways. Alterations in environmental conditions or in the QS genes of P. aeruginosa, as occurs naturally during the progression of CF lung infection, affect the architecture and quantitative structural features of these BLS. Thus, ASM+ provides an in vitro medium in which the effect of changing levels of substances produced by the host and the bacteria can be analyzed to determine the effect on such structures and on the susceptibility of the bacteria within the BLS to various treatments.
Cystic fibrosis (CF), an inherited disorder caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator, affects approximately 30,000 Americans, primarily those of Northern European origin [1,2]. These mutations cause a deficiency in chloride secretion with ensuing accumulation of thick, stagnant mucus within the lung alveoli of the patients [1-4]. Nutrients in the thick mucus facilitate the colonization of various bacterial pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae[3,5]. Colonization by these pathogens elicits a strong host inflammatory response which leads to destruction of the lung tissue and, ultimately, death from respiratory failure [1,6,7]. P. aeruginosa is one of the significant pathogens in chronic lung infections of CF patients [1,8].
Among the different factors that contribute to the virulence of P. aeruginosa is its ability to form a biofilm, a community within which bacteria are attached to a substratum or to each other [9]. Within the biofilm, the bacteria are surrounded by extracellular polymeric substance (EPS), which protects them from the effects of the host immune system and from diverse antibiotics [10-12]. Biofilm development occurs in stages that require specific bacterial factors at each stage. For example, during the initial (attachment) stage of biofilm formation, bacteria depend on both the flagellum-mediated swimming motility and the pili-mediated twitching motility [13]. A number of P. aeruginosa infections are associated with biofilm formation, including chronic otitis media, heart valve endocarditis, and chronic lung infections in CF patients [9,14].
Previously, studies have described synthetic mucin-containing artificial sputum media (ASM) that mimics the thick mucus within the lung of CF patients [15,16]. When grown in ASM, P. aeruginosa formed in tight microcolonies suspended within the medium rather than attached to the surface or free swimming as in standard broth media [15,16]. Mucin is the main component of secreted mucus, which also contains a large number of plasma and non-plasma proteins, carbohydrates, amino acids, nucleic acids, lipids, and electrolytes [17,18]. It has been shown that mucin-P. aeruginosa interactions promote biofilm formation in the continuous culture flow-through system [19].
In this study, we utilized a static microtiter plate culture system to investigate the effect of different conditions on the development of P. aeruginosa biofilms in mucus medium. Within the medium, P. aeruginosa strain PAO1 formed structures that are biofilm-like, but are not attached to the surface. The amount of mucin and extracellular DNA in the medium, as well as the level of environmental oxygen (EO2), are critical for the development of these biofilm-like structures (BLS). Additionally, one of the P. aeruginosa quorum sensing systems, rhl, affects formation of the BLS. Furthermore, as it develops its BLS, P. aeruginosa eliminates already established S. aureus BLS by a bactericidal mechanism.
Previous studies described a synthetic medium, ASM, which closely mimics the sputum of CF patients [15,16]. When grown in ASM, PAO1 formed clusters, or microcolonies, that are attached to the components of the ASM but not the abiotic surface [16]. In this study, we analyzed the influence of different conditions on the formation of these unique structures. We then examined the growth of the P. aeruginosa strain PAO1/pMRP9-1 in the static microtiter plate culture system using ASM+. First, we eliminated the possibility that the addition of antibiotics (either carbenicillin or erythromycin) to ASM+ to maintain the GFP plasmid had an adverse effect on either the growth of the tested strains or BLS development by these strains (data not shown). Inoculated plates were incubated at 37°C under 20% EO2. In situ CLSM of the gelatinous masses at 48 h revealed the formation of structures composed of numerous coalescing microcolonies that closely resemble mature well-developed PAO1 biofilms (Figure 1A, B). Quantitative analysis of the BLS using the COMSTAT program [20], supported these findings: a total biovolume of 6.52±0.43 μm3/μm2 and a mean thickness of 11.57±0.28 μm was seen at 48 h (Table (Table1).1). Unlike the development of PAO1 biofilms in other media, these unique suspended biofilm-like structures (BLS) are induced only within the gelatinous mass, as PAO1 did not form any biofilm on the surface of the microtiter well (Figure (Figure1C).1C). To further confirm these results, we grew PAO1 as described above but inserted a small plastic coverslip within the well of the microtiter plate. PAO1 again formed the unique BLS but failed to form any biofilm on the plastic surface (data not shown). In contrast, when we repeated the experiment with TSBDC, an iron-deficient medium in which P. aeruginosa grows planktonically and develops conventional biofilm, PAO1 formed a thick mature biofilm attached to the coverslip surface (data not shown).
One condition that may influence the development of the BLS in the ASM+ is length of incubation. Since the growth of PAO1 in ASM+ appears similar to the macrocolonies reported within the lungs of CF patients with chronic P. aeruginosa infection [21], we inoculated ASM+ with PAO1/pMRP9-1 as described above and incubated the cultures in 20% EO2 at 37°C for up to 16 d. From days 2 to 6, the BLS gradually developed to resemble a complete, mature and well developed biofilm (Figure (Figure2A).2A). Three-dimensional (3-D) images constructed from the CLSM scans clearly show the gradual increase in the size and the thickness of the BLS (Figure (Figure2B).2B). Structural analysis revealed that between 2–3 and 2–6 days, the BLS significantly increased in total biovolume and mean thickness (Tables (Tables11 and and2).2). In contrast, portions of the BLS that are exposed to nutrients (the surface to biovolume ratio) and roughness coefficient values were significantly reduced (Tables (Tables11 and and2).2). The total surface area was significantly (P<0.0001) decreased between 2–6 days only (Table (Table1).1). For the 16-d growth experiments, we maintained the growth of the PAO1 BLS by adding fresh ASM+ to the media remaining in the wells to maintain the original volume every 4 d to replace volume lost to evaporation. At 16 d, PAO1 BLS appears to be greater than at any time during the course of the experiment (Figure (Figure3).3). Due to enhanced growth by the replacement of the medium, new microcolonies appear to have developed atop the underlying thick growth (Figure (Figure3).3). Alternatively, these microcolonies may represent detached segments of the well developed biofilm (Figure (Figure3).3). Such detachment may occur mechanically and would not represent the well known bacterial dispersion phenomenon. In bacterial dispersion, individual planktonic cells and not biofilm segments are released from the mature biofilm [14]. No biofilm attached to the surface of the well of the microtiter plate at any time point throughout the experiment (data not shown). These results suggest that dynamic changes within occur PAO1 BLS during growth in ASM+ over an extended period of time.
Mucin, together with extracellular DNA, contributes to the viscosity of the CF sputum [17,18]. Mucin is one of the main components of ASM+. To determine if variations in the amount of mucin or DNA in ASM+ would affect the formation of the BLS, we adjusted the concentration of each component individually. With 0.5X mucin (2.5 mg/ml) or 2X mucin (10 mg/ml), PAO1 formed BLS, but the architecture was more diffuse in appearance than BLS seen with 1X mucin (5 mg/ml) (Figure (Figure4).4). In general, varying the mucin concentration altered the structural parameters of PAO1 BLS. Either reduced or elevated mucin concentrations increased the biovolume and thickness significantly while the roughness was significantly decreased in both cases (Tables (Tables11 and and2).2). Additionally, 0.5X mucin significantly increased the total surface area, while 2X mucin reduced the surface to biovolume ratio significantly (Table (Table2).2). We eliminated the possibility that variations in the amount of mucin simply affected the growth of PAO1 by determining CFU/ml for PAO1 grown in ASM+ with 1X, 0.5X or 2X mucin. After 3 d, comparable growth was observed in each condition, approximately 5 X 109 CFU/ml (Figure (Figure44D).
Variation in the amount of DNA produced more dramatic differences. When the amount of DNA was reduced to 0.5X (2 mg/ml), BLS were detected but confined to a small area of the gelatinous mass rather than spread throughout the medium as observed with 1X DNA (Figure (Figure5A,5A, B). When we increased the amount of DNA to 1.5X (6 mg/ml), individual cells were found scattered throughout the gelatinous medium, but no defined structures were detected (Figure (Figure5C).5C). The total biovolume, mean thickness, and total surface area of BLS developed in the presence of either 0.5X or 1.5X DNA were significantly less than those of BLS developed in the presence of 1X DNA (Tables (Tables11 and and2).2). In contrast, the values of the roughness coefficient and surface to biovolume ratio were significantly increased (Table (Table2).2). This resembles the initial stage of biofilm development on an abiotic surface in which P. aeruginosa colonizes the surface and forms a single monolayer. As for the variations in mucin, we enumerated the CFU/ml for PAO1 grown in ASM+ with 1X, 0.5X or 1.5X DNA, and again, comparable levels of growth were obtained in each condition (Figure (Figure55D).
Previous studies suggested that within the lung alveoli of CF patients, P. aeruginosa survives and grows under an oxygen gradient of 10% EO2 to 0% EO2[5,21,22]. The experiments described above were conducted under 20% EO2. Therefore, we compared the development of the PAO1/pMRP9-1 BLS in ASM+ under 20%, 10% and 0% EO2. Cultures were incubated for 3 d under 20% and 10% EO2. For growth under 0% EO2, ASM+ was supplemented with 10% potassium nitrate as a terminal electron acceptor [5] and incubated for 6 d. Under anaerobic conditions, P. aeruginosa grows rapidly using anaerobic respiration, which requires nitrate (NO3−), nitrite (NO2−), or nitrous oxide (N2O) as alternative terminal electron acceptors [5]. As P. aeruginosa penetrate the thick mucus within the lung alveoli of CF patients and reach the hypoxic zone, they transit from aerobic to anaerobic metabolism and begin to utilize the NO3− and or NO2− present within the CF mucus [5]. Compared with structures that formed under 20% EO2, those that formed under 10% EO2 appeared more developed by CLSM (Figure (Figure6A),6A), much more dense and reaching almost twice the maximum depth (Figure (Figure6B).6B). Quantitative structural analysis by COMSTAT confirmed that compared with 20% EO2, the growth of PAO1 under 10% EO2 significantly increased the biovolume and mean thickness of the BLS (Tables (Tables11 and and2).2). However, the values for the roughness coefficient, surface area, and surface to biovolume ratio were significantly reduced (Tables (Tables11 and and2).2). In contrast, structures developed under 0% EO2 were smaller and limited to only a small portion of the gelatinous mass within the well (Figure (Figure6).6). These structures were much less developed than BLS formed under 20% EO2 as shown by the significantly reduced mean thickness, total biovolume, and surface area (Tables (Tables11 and and2).2). However, the roughness coefficient and surface to biovolume were significantly increased (Tables (Tables11 and and2).2). These results suggest that in ASM+, maximum development of the PAO1 BLS occurs under 10% EO2, whereas the growth under 0% EO2 severely limits their development. Based on this finding, we conducted the rest of the PAO1 BLS analysis under 10% EO2.
As there are many strains of P. aeruginosa that differ in their ability to produce conventional biofilm, we compared the development of the BLS by PAK and PA103 under 10% EO2 with that of PAO1. These strains were originally isolated from infected patients and have been extensively utilized in in vitro and in vivo virulence studies [10,23-26]. Additionally, we examined the P. aeruginosa strain CI-4, a clinical isolate obtained from a patient with a chronic lower respiratory infection (30 days with the same strain) [27]. These strains were transformed with pMRP9-1 (for GFP expression) and grown in ASM+ for 3 d and the BLS analyzed as described in Methods. None of the PAK BLS parameters were significantly different from those of PAO1 BLS (Tables 3 and 4; Figure Figure7).7). With respect to PA103 BLS, only the total biovolume and mean thickness were significantly reduced in comparison with PAO1 BLS (Table (Table33 and and4;4; Figure Figure7).7). In contrast, CI-4 produced BLS that were significantly lower than those of PAO1 BLS in total biovolume, mean thickness, and total surface area but significantly higher than PAO1 in roughness coefficient and surface to biovolume ratio, indicating dispersal of poorly formed BLS throughout the gelatinous mass (Tables (Tables33 and and4;4; Figure Figure7).7). These results indicate that P. aeruginosa strains differ in their ability to produce BLS within the ASM+.
The three quorum sensing (QS) systems las, rhl, and pqs contribute to the development of P. aeruginosa biofilms [28-30]. Mutants defective in one or more of these systems failed to form well developed biofilms compared with the PAO1 parent strain [28-30]. Using a conventional biofilm medium (LB broth), we compared the biofilm developed on a plastic cover slip in a microtiter plate well by PAO1 and its lasR, lasI, rhlR, rhlI, and pqsA mutants. With the exception of the medium, the biofilms were developed under the same conditions that we used to develop the BLS. Compared with PAO1, all QS mutants produced reduced biofilms (data not shown). We then examined the contribution of the QS systems to the formation of the PAO1 BLS in ASM+, by comparing the structures formed by PAO1 with those formed by these same QS mutants. The mutants were transformed with pMRP9-1 and the development of the BLS by the transformants was examined under 10% EO2 for 3 d at 37°C.
The las mutants PAO-R1 (ΔlasR) and PAO-JP1 (ΔlasI) produced BLS that visually and architecturally resembled each other (Figure (Figure8A,8A, B). With respect to the five tested parameters, BLS produced by PAO-JP1 were not significantly different from those BLS produced by PAO1 (Tables (Tables33 and and4).4). The mean thickness of BLS produced by PAO-R1 was significantly higher than that of PAO1 BLS while the roughness coefficient was significantly lower (Tables (Tables33 and and4).4). The pqs mutant PW728::pqsA-lacZ produced BLS that were not significantly different from PAO1 BLS (Figure (Figure8;8; Tables Tables33 and and4).4). The biovolume and mean thickness of BLS produced by either the rhlI mutant (PDO100) or rhlR (PDO111) were significantly less than those produced by PAO1 (Figure (Figure8;8; Tables Tables33 and and4).4). In contrast the values of the roughness coefficient and the surface to biovolume ratio were significantly higher than those for PAO1 BLS (Figure (Figure8;8; Table Table33 and and4).4). These results suggest that among all three QS systems, rhlI and rhlR have a major impact on the development of BLS in ASM+ by PAO1.
S. aureus is one of the first microorganisms that colonize and grow within the thick mucus in the lung alveoli of CF patients [8]. Thus, we determined whether S. aureus would develop BLS in ASM+ under 20% or 10% EO2. The S. aureus strain AH133 which carries the GFP plasmid pCM11, was grown for 3 d at 37°C. Under 20% EO2, AH133 produced a well developed BLS within the entire gelatinous mass (Figure (Figure9).9). However, under 10% EO2, the structures were far less developed with individual cells/small microcolonies scattered within the gelatinous mass (Figure (Figure9).9). Compared to BLS produced under 20% EO2, total biovolume, mean thickness, and surface area of BLS produced under 10% EO2 were significantly reduced (P<0.0001 for each value) (Table (Table5).5). In contrast, the roughness coefficient and surface to biovolume ratio values were significantly increased (P<0.0001 for each value) (Table (Table5).5). This suggests that unlike P. aeruginosa, S. aureus produces more developed BLS under 20% EO2 rather than under 10% EO2.
The lungs of CF patients are colonized with a variety of pathogens, including S. aureus, P. aeruginosa, and K. pneumoniae, over the course of time [1]. However, as the disease progresses, the predominate pathogen within the CF infected lung is P. aeruginosa[1,8]. Previous studies showed that QS-controlled extracellular factors produced by P. aeruginosa, including quinoline molecules and LasA, inhibited the planktonic growth of S. aureus and S. epidermidis[31,32]. Additionally, recent studies showed that the P. aeruginosa extracellular polysaccharide as well as the organic compound cis-2-decenoic acid disrupted established biofilms produced by Gram-positive bacteria [33]. Therefore, we first determined if PAO1 inhibits the growth of the S. aureus strain AH133 in ASM+. We co-inoculated ASM+ with approximately 1 x 107 CFU/ml each of PAO1 and AH133 and incubated the culture for 48 h at 37°C under 20% EO2. After 48 h, about 1 x 109 CFU/ml of PAO1 was recovered from the gelatinous ASM+ mass, but no CFU of AH133 (Figure (Figure10A).10A). We obtained similar results when we repeated the experiments using TSBDC, a growth medium that supports planktonic growth of both organisms (Figure (Figure10B).10B). These results suggest that similar to other previous observations, P. aeruginosa eliminates S. aureus, when the two are grown together at the same time.
To simulate the scenario in which S. aureus colonizes the CF lung first and P. aeruginosa follows, we then examined the effect of PAO1 on partially developed (8 h) AH133 BLS. As AH133 expresses GFP, we transformed PAO1 with pMP7605, a plasmid from which RFP is expressed constitutively [34], to allow visualization of each strain within the BLS. Individually, the strains produced well developed BLS in ASM+ (Figures (Figures2,2, ,10A).10A). At 8 h post inoculation, AH133 formed a defined structure (Figure (Figure11A).11A). We then added PAO1/pMP7605 (at a starting density similar to that used to initiate the AH133 culture) and continued incubation for 56 h. The cultures were analyzed at 8- and 16-h intervals to 64 h for the AH133 alone and 56 h post addition of PAO1/pMP7605 for changes in the BLS produced by AH133 and the development of any PAO1 BLS (Figure (Figure11A,11A, B). At 16 and 24 h post-initiation, AH133 produced well-developed mature BLS (Figure (Figure11A).11A). The AH133 BLS changed in appearance over the rest of the time course, but did not disappear (Figure 11A). In contrast, in the dual culture, the AH133 structure was considerably reduced at the corresponding time points (Figure (Figure11B).11B). By 32 h only remnants of the AH133 BLS remained, and by 40–56 h, the AH133 BLS appeared to be completely replaced by well-developed PAO1 BLS (Figure (Figure11B).11B). The regression of AH133 structure appears to be due to the expansion of the PAO1 structure at 8, 16, and 32 h post-initiation (Figure (Figure1111B).
This observed phenomenon could be due to the dispersion of the AH133 BLS or a bactericidal effect of PAO1 on AH133. Therefore, at each time point, the gelatinous masses containing AH133 alone or AH133 plus PAO1 were vortexed, serially diluted, and the CFU/ml determined. Aliquots of each dilution were spotted on Pseudomonas isolation agar for P. aeruginosa and mannitol salt agar for S. aureus. At all tested time points, the CFU/ml of the single AH133 biofilm was similar (about 1 x 107) (Figure (Figure11C,11C, white bars). However, the CFU/ml of AH133 within the mixed BLS was visibly reduced 8 h after addition of PAO1 and significantly reduced at 40 and 56 h, with no CFU of AH133 recovered 56 h post addition of PAO1 (Figure (Figure11C,11C, green bars). In contrast, the CFU/ml of PAO1/pMP7605 within the mixed BLS dropped between 8 and 16 h post biofilm initiation but did not change significantly after 16 h (Figure (Figure11C,11C, red bars). These results suggest that PAO1 exerts a bactericidal effect, and that the development of the P. aeruginosa BLS in the co-culture proceeded at the expense of the S. aureus BLS.
CF sputum is a highly viscous secretion in which PAO1 grows readily. PAO1 forms conventional biofilms on abiotic surfaces [13,19,35], but it develops macrocolonies, tight aggregates consisting of numerous microcolonies, in ASM and the CF lung [16,21]. While PAO1 formed a typical flat undifferentiated biofilm that completely covered the substratum with a homogenous distribution of the biovolume in a continuous flow-through system, it grew almost exclusively as discrete microcolonies that eventually formed a mature biofilm on a mucin-covered glass surface [19]. Based on these results, Landry et al. suggested that mucin interacts with specific PAO1 adhesins thereby immobilizing the bacteria onto the glass surface [19]. In our analysis, the observed BLS developed exclusively within the gelatinous mass formed by ASM+ and not on the surface of the well (Figure (Figure1).1). It is likely that through the initial interaction of these putative adhesins, individual PAO1 bacteria adhere to the mucin glycoprotein forming the nuclei of the microcolonies and leaving no bacteria to adhere to the plastic surface. Individual nuclei would then recruit more PAO1 bacteria, possibly through additional inter-bacterial and glycoprotein-bacterial interactions, growing into well-defined BLS observed over time (Figure (Figure22).
Based on the work of Ghani & Soothill [15] and Sriramulu et al. [16], we utilized 0.5% mucin (1X) in our ASM+. But more recently, Henke et al. [36], showed that the concentrations of MUC5AC and MUC5B, the principal gel-forming mucins, are decreased in airway secretions from CF patients with stable disease and greatly increased during pulmonary exacerbations (by 89% and 908%, respectively). When we reduced the mucin concentration of ASM+ by 50% (0.5X), the gelatinous mass still formed in the well, possibly through the contribution of other ASM+ components (DNA and lecithin) that add to the viscosity. However, the typical multilayered BLS was eliminated and replaced with a structure that appears to consist of small microcolonies amid individual cells and tiny cell clusters distributed throughout most of the gelatinous mass (Figure (Figure4A,4A, B). Surprisingly, the effect of increasing the concentration of mucin to 2X on the development of BLS was similar to that induced by reducing the mucin concentration. Rather than the distinct highly structured BLS architecture, PAO1 produced small microcolonies distributed throughout the ASM+ (Figure (Figure4C).4C). At this time, we do not know if the increase in the availability of mucin glycoprotein interferes with the development of microcolonies that coalesce to form the well-developed BLS.
One of the hallmarks of the CF syndrome is the overproduction of mucin within the lung alveoli [1,3,7]. Yet during P. aeruginosa infection of the CF lung alveoli, the level of mucin may vary [36]. P. aeruginosa LPS induces the production of reactive oxygen intermediates, which cause release of transforming growth factor α; TGF-α then up-regulates the expression of MUC-5 AC thereby causing excessive mucin production [37-39]. However, P. aeruginosa produces other factors that may reduce the amount of mucus within its immediate vicinity; alveolar mucin is degraded by P. aeruginosa extracellular serine proteases such as LasB [40]. Ultimately, the interaction of all these factors would produce a net mucin concentration suitable for the full development of the BLS, while any imbalance in the production of these factors that reduces or increases mucin concentration would prevent the establishment of the BLS. Alternatively, BLS may form in the initial stages of P. aeruginosa infection in the CF lung. Treatment that reduces the amount of mucin present would disperse the bacteria making them more susceptible to antibacterial treatment (stable disease). Alternatively, mucin may reduce the chances of forming new BLS.
Extracellular DNA is another contributor to the viscosity of CF sputum [15,16]. Within the CF lung, there are several sources for this extracellular DNA – dead host immune cells, lysed bacteria, QS-regulated release of P. aeruginosa DNA, and outer membrane vesicles that contain DNA [41,42]. Like mucin, DNA contributes to biofilm formation as well; a similar biofilm was detected on a glass surface coated with DNA [41]. Previous studies using other biofilm development media, such as LB or minimal medium, indicated that extracellular DNA is critical for the initial establishment of P. aeruginosa biofilms [42]. The levels of extracellular DNA also vary within CF sputum, ranging from 0.3 to 9.5 mg/ml in one study of 167 CF sputum samples [43]. Variations in the level of extracellular DNA in ASM+ affected the development of BLS much more dramatically than variations in the level of mucin. In ASM+ with 0.5X DNA (2 mg/ml), a well developed BLS was visible (Figure (Figure5B),5B), but the biovolume and total surface area occupied were considerably less (Table (Table11 and and2).2). When the amount of DNA was increased to 1.5X (6 mg/ml), PAO1 did not produce detectable structures; rather, the gelatinous mass formed by the ASM+ contained scattered individual cells (Figure (Figure4C).4C). However, at this time it is not clear how an increase in the external DNA reduces the number of BLS within the gelatinous mass of ASM+.
Within the lung of CF patients and during other chronic lung infections, P. aeruginosa survives under microaerobic (10% EO2) to anaerobic (0% EO2) conditions. A steep oxygen gradient exists within the P. aeruginosa infected alveolar mucus [5,21]. Within the mucus, P. aeruginosa secretes compounds that lower the oxygen transfer rate generating optimum conditions for microaerobic growth [22,44]. We showed previously that lower oxygen tension also influences the expression of P. aeruginosa virulence genes [45]. Compared with aerobic conditions, the expression of pyoverdine genes was reduced under microaerobic conditions; in contrast, the expression of the exotoxin A gene, toxA was increased [45]. Compared with 20% EO2 and 0% EO2, microaerobic (10% EO2) conditions are optimal for the development of P. aeruginosa BLS in ASM+. BLS developed under 10% EO2 had a greater mean thickness and a larger biovolume than those developed under either 20% or 0% EO2 (Figure (Figure6,6, Table Table11 and and2).2). In the absence of EO2, PAO1 required 6 days to develop rudimentary BLS (Figure (Figure6C)6C) indicating that a low level of oxygen is essential for the full development of these structures.
Depending on conditions under which the biofilms were developed (medium, the biofilm development system, and the biofilm substrate), previous studies indicated the involvement of the QS systems in the development of P. aeruginosa biofilm [29,30,35,46]. In those studies, the deficiency in biofilm development was associated with either a lasI or rhlI mutation. We tested mutants defective in all three known P. aeruginosa QS systems in ASM+. PAO-R1 (ΔlasR), PAO-JP1 (ΔlasI), and PW2798::pqsA-lacZ (ΔpqsA) produced BLS that were visually and architecturally similar to each (Figure (Figure8).8). In contrast, PDO111 (ΔrhlR) BLS were visually, architecturally, and structurally dissimilar to PAO1 BLS, in that they had a smaller biovolume and mean thickness (Figure (Figure8,8, Tables Tables33 and and4).4). The loss of rhlI (PDO100) had the most profound effect on the BLS produced by PAO1 in ASM+. Both PDO100 (ΔrhlI) and PDO111 (ΔrhlR) produced BLS that were significantly smaller (biovolume, mean thickness) than PAO1 BLS (Figure (Figure8,8, Tables Tables33 and and4).4). However, BLS produced by these two strains were more heterogeneous than PAO1 BLS (a significant increase in roughness coefficient) (Figure (Figure8,8, Tables Tables33 and and4).4). Additionally, more regions of the PDO100 and PDO111 BLS were exposed to nutrients than PAO1 BLS (a significantly higher surface to biovolume values) (Figure (Figure8,8, Tables Tables33 and and4).4). Our results suggest that the production and maturation of the fully-developed complex BLS requires a potential P. aeruginosa factor that is stringently controlled by the rhl and not the las or the pqs systems. Among the P. aeruginosa factors that are stringently controlled by the rhl system are the rhamnolipid biosurfactants [47,48]. The rhamnolipids encoded by the rhlAB operon contribute to biofilm development in P. aeruginosa through multiple mechanisms including maintaining open channels by affecting cell-to-cell interaction [28], promoting microcolony formation in the initial stages of biofilm development [49], and dispersing cells from the mature biofilms [50]. Analysis of PAOΔrhlA and/or PAOΔrhlB mutants in ASM+ should allow us to determine if rhamnolipid plays a role in the development of the BLS. Interestingly, PA103, which is known to have a deletion in lasR[51], produced BLS with reduced biovolume and mean thickness (compared with those produced by PAO1 or PAO-R1) (Figure (Figure7,7, Tables Tables33 and and4).4). This suggests that the observed differences between the BLS produced by PAO1 and PA103 are not due to the loss of the lasR gene in PA103. CI-4, a clinical isolate obtained from a patient who had been continuously infected with P. aeruginosa for 30 days, has deletions in both lasR and rhlR[27]. This strain produced BLS that had less biovolume, mean thickness and covered less total surface area that PAO1; visually, the BLS were also unique in appearance among all the QS mutants, numerous small microcolonies distributed throughout the medium (Figure (Figure7,7, Tables Tables33 and and4).4). This suggests that there is a complex interaction among the QS systems in controlling BLS production within ASM+.
Using ASM+, which has the same components as our ASM+, Sriramula et al. [16] examined the growth of PAO1, PAOΔlasR, and PAOΔrhlR. Both PAO1 and PAOΔrhlR formed macroscopically visible clumps or aggregates, which they termed tight microcolonies, that could not be disturbed even with vigorous pipetting [16]. In contrast, PAOΔlasR failed to develop these tight microcolonies [16]. In our study, neither PAO1, nor any other tested strain produced macroscopically visible structures. In part, this is due to the turbidity of ASM+. Similar to the tight microcolonies described by Sriramula et al. [16], the BLS we observed in our ASM+ did not attach to a surface. The BLS are adherent when fully-developed, but cells within the BLS can be dispersed by vortexing. Differences between the two studies, despite the similarity in the growth medium, are most likely due to differences in other growth conditions. While Sriramula et al. [16], grew their cultures under 20% EO2 with shaking, we grew our cultures under static conditions regardless of the EO2 concentration. Given these differences, it is not practical to directly compare the bacterial structures observed in the two studies with respect to the role of the QS systems in their formation.
Biofilms at different infection sites often consist of multiple species of bacterial pathogens [52,53]. These bacterial species may either compete with each other or support each other's growth. Qin et al. [54] previously showed that P. aeruginosa inhibited the planktonic growth of Staphylococcus epidermidis through a QS-related mechanism. Additionally, using the static chamber cultivation system (microtiter plate assay), they demonstrated that P. aeruginosa extracellular polysaccharide disrupted an already established S. epidermidis biofilm [54]. Disruption of these biofilms, however, does not occur through the bactericidal effect observed with the planktonic cells; instead the bacteria within the biofilm were dispersed alive [54]. When we co-cultured P. aeruginosa and S. aureus statically under 20% EO2 in TSBDC or ASM+, P. aeruginosa eliminated S. aureus by day 2 (Figure (Figure10).10). Furthermore, and similar to the findings by Qin et al. with S. epidermidis[54], the addition of P. aeruginosa to S. aureus BLS established in ASM+ disrupted the S. aureus BLS (11a, b). However, P. aeruginosa disrupted the S. aureus BLS through an bactericidal effect rather than dispersion. By 56-h post addition of PAO1, no CFU of AH133 were recovered (Figure (Figure11C),11C), although it is remotely possible that our failure to detect S. aureus is due to their existence in a viable but nonculturable state. This effect is similar to the clinical observations of CF lung infections where S. aureus, an early colonizer, is gradually replaced by P. aeruginosa. The nature of the PAO1 bactericidal factor that eliminates the S. aureus BLS is under investigation.
In this study, we have demonstrated that thick, viscous ASM+ containing mucin and extracellular DNA and incubated under static conditions with lowered oxygen tension (10% EO2) – constituents and conditions similar to those within the lung alveoli of CF patients – induces the formation of biofilm-like structures by P. aeruginosa and S. aureus, two of the pathogens most commonly seen in the infected lungs of these patients. The BLS are not attached to the surface, but form within the medium as has been reported for the development of macrocolonies within the mucus in CF lungs. Thus, ASM+ represents an in vitro medium in which the effect of changing levels of substances produced by the host and the bacteria can be analyzed to determine the effect on such structures and on the susceptibility of the bacteria within the BLS to various treatments.
Bacterial strains and plasmids utilized in this study are described in Table Table6.6. Strains were routinely grown in Luria Bertani (LB) broth under shaking conditions at 37°C. To analyze the development of biofilm-like structures, bacterial strains were grown in the previously described ASM+ [16]. To attain consistency from batch to batch of medium ASM+ was made in a quantity sufficient for each planned set of experiments, a stringent method of preparing the medium was used. The components were added into sterile water in exact order with vigorous vortexing for 10–30 seconds after each addition: mucin (Sigma-Aldrich, St. Louis, MO), 0.5% (w/v); unsheared salmon sperm DNA (Sigma-Aldrich), 0.4% (w/v); NaCl, 0.5% (w/v); KCl, 0.2% (w/v); casamino acids (Amresco, Solon, OH), 0.5% (w/v); egg yolk emulsion (source of lecithin; sterile; Remel, Lenexa, KS), 0.25% (v/v); diethylene triamine pentaacetic acid (1 mg/ml stock in 1 M NaOH; sterile; Sigma), 0.0005% (w/v). Finally, the pH was adjusted to 6.8. Antibiotics were then added to maintain sterility and for maintenance of plasmids: 300 μg carbenicillin/ml and/or 50 μg tetracycline/ml for P. aeruginosa; 10 μg erythromycin/ml for S. aureus. The completed medium was then vortexed for 2 minutes and again prior to pipetting. To induce biofilm formation on the substrate surface, we used trypticase soy broth dialysate (TSBDC) to which glycerol (1% v/v) and monosodium glutamate (0.5 M) were added [55].
To allow visualization of the bacteria, all P. aeruginosa strains were transformed by electroporation [63] with pMRP9-1 from which the gene for green fluorescent protein (GFP) is constitutively expressed [56]. To visualize PAO1 grown together with AH133, PAO1 was transformed with pMP7605 in which the mCherry gene that codes for red fluorescent protein (RFP) is expressed from the tac promoter [34]. The S. aureus strain AH133 carries the constitutive GFP plasmid pCM11 (AR Horswill, personal communication).
Bacteria were grown in a static microtiter plate culture system using sterile 24-well polystyrene plates (Falcon; BD, Franklin Lakes, NJ) [64,65]. Tested strains were grown overnight in LB broth. Cells were pelleted, washed, and resuspended in PBS. For analysis of the BLS formed by individual bacterial species, resuspended cells were inoculated in ASM+ to an initial OD600 of 0.02-0.03 and dispensed into the plate wells in 1 ml aliquots. For the analysis of BLS produced by two bacterial species, individual species were prepared and inoculated at an initial OD600 of 0.015. The plates were incubated at 37°C in static (nonshaking) conditions under environmental oxygen (EO2) concentration of 20% (aerobic), 10% (microaerobic), or 0% (anaerobic). Individual GasPak jars with Campy Pak Plus envelopes (BD) or GasPak EZ Anaerobic Pouches (BD) were used to generate the microaerobic and anaerobic EO2 conditions, respectively.
This was done using confocal laser scanning microscopy (CLSM) [35,64]. The BLS were visualized within the wells of the microtiter plates using an Olympus IX71 Fluoview 300 confocal laser scanning microscope (Olympus America, Melville, NY). All images were obtained through a 203/0.40 Ph1 NA objective utilizing a green helium laser (546 nm) or argon laser (510–530 nm). Three-dimensional image reconstructions were performed using NIS-Elements 2.2 (Nikon Instruments, Melville, NY) to visualize the architecture of the BLS. All instrument settings were consistent for each individual experimental parameter tested.
The number of image stacks obtained from the BLS was based on the greatest depth of the structures formed under the test conditions and was the same for all strains/conditions within an experiment (See Tables Tables1,1, ,2,2, ,3,3, ,4).4). Each experiment was done in duplicate. Two 10-image stacks were obtained from random positions within each BLS (total 40-image stacks for each strain and/or condition). The 40-image stacks were analyzed using the COMSTAT program [20] for structural features of the BLS: biovolume, estimates the biomass of the BLS; mean thickness, a measure of spatial size of the BLS; roughness coefficient, a measure of how much the thickness of the BLS varies, or the heterogeneity of the BLS; total surface area, space occupied in each image stack; and surface to biovolume ratio, estimates the portion of the BLS exposed to nutrients (biovolume divided by the surface area of the substratum). Values represent the mean±SEM.
The highly viscous ASM+ forms a gelatinous mass in which the bacteria grow. Therefore, at the indicated time points, the mass from each well was transferred to a 1.5 ml microcentrifuge tube and vigorously vortexed to suspend the bacteria. The bacterial suspension was then serially diluted tenfold in PBS and 10 μl aliquots of each dilution were spotted on LB agar. For dual species experiments, the aliquots were spotted on Pseudomonas isolation agar (BD) to select for P. aeruginosa and mannitol salt agar (BD) to select for S. aureus. The plates were incubated at 37°C for 16 h and the colonies of microorganisms (CFU) were counted. The CFU/ml was determined using the following formula: CFU counted x dilution factor x 100.
Statistical analyses of the results were done using GraphPad InStat 3.06 (GraphPad Software, San Diego, CA). One-way ANOVA with the Tukey-Kramer multiple comparisons post-test was used to determine significant differences over time and among treatments. The t-test was used to compare two strains or two treatments.
CH designed portions of the study, conducted all experiments, and wrote the manuscript. ANH coordinated the project, designed portions of the study, and helped draft and revise the manuscript. JACH analyzed and interpreted data and critically revised the manuscript. All authors have read and approved the final manuscript.
We thank Guido V. Bloemberg and Ellen L. Langendijk (pMP7605), Alexander R. Horswill (AH133/pCM11), Barbara H. Iglewski (PAO1, PAO-R1, PAO-JP1), Dennis Ohman (PDO111, PDO100), and Matthew R. Parsek (pMRP9-1) for their kind provision of strains or plasmids; Janet Dertien for assistance with the CLSM; and Joanna E. Swickard for critical reading of the manuscript. Strain PW7298::pqsA-lacZ was made available through grant NIH P30 DK089507.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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30 years of experience
Accepting new patients
Video profile
201 Old Bank Rd
Milford, OH 45150 Phone number & directions
Patient Reviews
Patient Reviews ? Write a Review
Overall Rating:
Total Ratings
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Dr. Capurro is a very kind, caring, excellent doctor. I appreciate his concern for his patients.
Dr. Cupurro is one of a kind. One of the most "done to earth" MDs' I have ever encountered.
I found Dr. John Capurro, MD randomly when my FAA Medical Examiner moved - what a blessing. Thanks Doc and Staff
Have been with him since he was on Beechmont and follow him to Milford. They can't get any better.doesn't make you feel like you have to talk fast or he will be out the door before you get it all out. Great sense of humor.
I like Dr. Capurro. He is straight forward and to the point and he listens to you and takes care of what is needed.
After many years I have found a physician I trust and will stay with. Dr. Capurro is caring, accurate and punctual in giving me test results and responding to inquiries. I have been with him for about four years now (2011). He is the primary care physician for myself and my family.He has a dry humor, which I enjoy. He spends as much time with you as you need, listens and responds with practical advice. I recommend him to anyone searching for a great family doctor.
He is an ok doc if you are well but if there is a problem or an emergency you will be left waiting for weeks for any response on test results and in the meantime you are suffering. No bedside manner. It has been more than two weeks and I still don't know what is wrong. Look elsewhere.
I love Dr.John Capurro I have been with him for years. He is special, and I trust him.


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DÃa de los Muertos Art Expo seeks applicants
The Día de los Muertos Street Festival is accepting applications for the festival's Hecho-A-Mano Art Expo.
Deadline to apply is Sept. 1. Application fee is $5; booth fee/$50.
The street festival covers several blocks downtown and the 500 to 700 blocks of Mesquite Street. In addition to the expo, the festival includes three stages with live entertainment, kids' Corner, art demonstrations, altar displays, a fine art exhibit and food.
Acceptance is based on originality and quality.
Information/registration: www.diadelosmuertoscc.com/vendor-information.


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Self-host Feedback
Support for Windows XP has ended
Microsoft ended support for Windows XP on April 8, 2014. This change has affected your software updates and security options. Learn what this means for you and how to stay protected.
Article ID: 915865 - Last Review: 08/23/2007 17:09:04 - Revision: 1.4


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Job Title: City:
-- All Locations --
Murray Max
Judge Memorial
Utah Youth Soccer Association
I am interested in volunteer positions
Murray Max Technical Director Job Description Posted 3-21-2016
Murray Max Soccer seeks the right coach for this time in our growth to become our Technical Director.
This individual will be a coach who has the passion and leadership abilities to continue the tradition of excellence that our programs have enjoyed over the last 20 plus years. We require a coach who is innovative and can envision, develop and implement cutting edge ideas.
The Technical Director is responsible for leading the daily operations and technical staff of all Murray Max Soccer player and coach development programs, competitions and leagues. The Technical Director will work closely and collaboratively with the Board of Directors to build out our educational programs to ensure the progress of player and coaching development.
The Technical Director is expected to play a dynamic leadership role including, but not limited to, the following areas
The Technical Director leads and manages the technical department and all programs within the department.
The Technical Director is responsible for playing a vibrant and creative leading role in the implementation of Murray Max's Technical Development Plan.
The Technical Director must ensure that the long-range strategy achieves Murray Max's mission, and that consistent and timely progress toward that goal is achieved by continuously evaluating effectiveness and currency of existing programs and suggesting potential changes.
Specific Objectives and Areas of Development for the Technical Director to initially focus on:
· Create a comprehensive Technical Development Plan and curriculum which encompasses Murray Max's mission
· Oversee and guide the philosophy and pathways for the development of Murray Max Soccer players at all levels of the game.
· Maintain the clear philosophy and pathway to guide all programs, practices and decision laid out by the Technical Development Plan.
· Develop and implement future guidelines, based on sound principles, on the balance between training and competition at the different stages of development
· Deliver the objectives of the Technical Development Plan in an effective manner
· Maintain procedures for monitoring and evaluating the progress of the Technical Development Plan
· Facilitate clubs, leagues and affiliations by strengthening their structures
· Ensure that the Technical Development Plan is a core element of Murray Max's mission for the future Increase the quality and quantity of coaches through an extensive program of education, training and resources at all levels
· Maintain and fully implement all of the education courses outlined in the Technical Development Plan
· Increase the number and distribution of educational opportunities to Murray Max coaches, players, and parents.
· Maintain a clear development structure for emerging talent
· Implement partnership with relevant affiliates
· Develop the capacities (technical, tactical, mental, physical, personal, lifestyle) of players to reach the highest level of play to maximize the possibility of further opportunities
· Constantly monitor trends within the game and within the sport
· U.S. Soccer C License (Valid) at a minimum
· Excellent people skills, including strong consultative facilitation, listening, and decision making skills
· Ability to, and comfortable with, working with multiple supervisory and/or collaborative bodies
· Excellent computer skills (Power Point, Key note etc.)
· Demonstrated leadership and management skills, including the ability to attract, motivate and develop staff and leadership volunteers
· Excellent written, and verbal communication skills
· Outstanding public speaking skills
· Proven track record of providing strategic direction in a collaborative manner, (i.e. inspiring and implementing a Shared Vision)
· Experience with budget responsibility for a minimum of $500,000
· Ability to work flexible hours, including nights and weekends
· Experience with youth soccer and/or youth-centered organizations
· Proven track record of success coaching and developing teams
To Apply for This Position Electronic submissions are required to be considered for this position and only complete submissions will be considered. Send a cover letter addressing your desire, availability and qualifications for the position, a resume and list of three professional references firstname.lastname@example.com
· Minimum of National D License
· Previous Experience of Coaching
· Strong ability to communicate with players and parents
· Willingness to follow a set curriculum
· Full Equipment Provided For All Teams
· Future Licenses (Including travel and accommodation) paid for
· Continued education within the club
· Widest range of player support programs for players and coaches in the state
· $600 per month for National D License $850 per month for C license and above
· All Indoor Winter Training Space Provided At The Forza Fieldhouse (Completion date of October 1st 2013)
Please contact:
Director Of Coaching- Competition
Forza Futbol Club
Office: 801 298 4625


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TJUSIG toilet paper tree
July 1st, 2016 | by Jules Yap
A cute idea from Sarah. The TJUSIG hanger as toilet paper holder
July 1st, 2016 | by Jules Yap
A cute idea from Sarah. The TJUSIG hanger as toilet paper holder
April 1st, 2016 | by IH guest
IKEA items used: ORDNING utensil holder (height 13.5cm) I saw this cute repurposing of ORDNING utensil holders at a cafe
March 31st, 2016 | by IH guest
Hello guys, I'm Ela and I'd like to show you my way to hack the IKEA FULLEN washbasin cabinet.
February 26th, 2016 | by IH guest
I bought a "Variera" pot lid organizer, extended it to the max and attached it to my heater wires with
November 10th, 2015 | by IH guest
After a long basement and bathroom renovation I realized that the bathroom ceiling vent cover went missing or thrown out
August 3rd, 2015 | by jello
This is pretty self-explanatory: Tiny FEJKA fake plant. KARDAMUMMA pot optional. Works even with wide toothbrushes. Cleans very easily
May 28th, 2015 | by IH guest
When we installed the FINTORP rail system we had to trim some of it down to fit, and were left
September 8th, 2014 | by VauWeh
Godmorgon and no-Ikea wash basins At first glance it seems a normal Godmorgon-cabinet. At second glance you notice the basin
September 4th, 2014 | by Jules Yap
Materials: IDEAL candle dish, wall brackets Not a hack but a nice idea from Marina. You can repurpose the
July 31st, 2014 | by ChrisJScott
We needed some storage for our bathroom and liked the spa-like look of Ikea's Molger shelves. However, we felt that
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In Brief: Death toll rises in southern Afghanistan battle
Death toll rises in Afghanistan battle
KANDAHAR, Afghanistan -- The death toll from a week of fighting between Afghan forces and hundreds of Taliban militants in a southern province rose above 100, officials said Thursday, while the U.N. called on all parties to protect civilians who are increasingly caught in the crossfire.
The battle is shaping up as a major test of the government's ability to maintain security in volatile areas after foreign combat troops leave by the end of 2014, increasing the urgency for the government to sign a security pact with the United States that would allow nearly 10,000 American forces to stay in the country for two more years.
Outgoing President Hamid Karzai has refused to sign the deal.
Israel IDs suspects in alleged kidnapping
JERUSALEM -- Israel on Thursday identified two well-known Hamas operatives in the West Bank as the central suspects in the recent disappearance of three Israeli teenagers, in the first sign of progress in a frantic two-week search for the missing youths.
Israeli and Palestinian officials said the two men have been missing since the teenagers disappeared, and that a large manhunt was underway.
In a statement, Israel's Shin Bet security service identified the men as Marwan Qawasmeh and Amer Abu Aisheh. It said both men are activists from the Hamas militant group in the West Bank city of Hebron, near where the youths disappeared on June 12.
Israel has accused Hamas of kidnapping the three teens.
Sudanese Christian woman freed again
KHARTOUM, Sudan -- A Sudanese Christian woman whose death sentence for apostasy was overturned was freed again Thursday after being detained on accusations of forging travel documents.
Wearing a traditional white and green dress, Meriam Ibrahim, 27, walked out of a Khartoum police station carrying her newborn baby hours after lawyers said she was ordered released. Ibrahim and her husband, who is disabled and was seen being carried by two men, got into a vehicle with their other child and sped away, followed by police cars and two vehicles with diplomatic plates.
Ibrahim was sentenced to death over charges of apostasy.
Prominent female activist killed in Libya
CAIRO -- A prominent Libyan activist who had become an international face of her strife-torn country's efforts to build a democracy was assassinated by gunmen who stormed her home in the restive eastern city of Benghazi shortly after casting her ballot in the country's parliamentary elections, police said Thursday.
The slaying of Salwa Bugaighis stunned residents of her home city, politicians, activists and diplomats, among whom she was well known. International rights groups called on authorities to investigate.
Bugaighis, a lawyer and rights activist, was at the forefront in the 2011 uprising against dictator Moammar Gadhafi.
-- from wire reports


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Come on down for 'Price Is Right Live' at Riverside
Not many of us may want to admit this, but at least once in your life, you've been sucked into "The Price Is Right." You've cheered for Bob Barker and screamed at the bone-headed contestants. You've sat on the edge of your seat, watching the Big Wheel spin 'round and 'round, hoping it would land on that lucky number.
You can experience the thrill of "The Price Is Right" live at 7 p.m. Tuesday at the Riverside Theater, 116 W. Wisconsin Ave. »Read Full Article
Mac Miller makes sold-out Eagles Ballroom a massive party
There probably isn't a hip-hop show in Milwaukee this year that will offer as much bang for the buck as Mac Miller's concert Saturday night, in front of a sold-out Eagles Ballroom at the Rave.
Aside from the allure of the 21-year-old headliner, the Milwaukee date of Miller's Space Migration tour boasted four supporting acts, two of which — Earl Sweatshirt and Chance the Rapper — are among the most hyped up-and-coming MCs of 2013. »Read Full Article(3)
Looking for a farmers markets? Check out these shopping lists
If it's Saturday morning, it's time to check out farmers markets.
Check out this interactive map of southeast Wisconsin farmers markets, most of which are open Saturday mornings. »Read Full Article


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The government should focus its policies on encouraging high growth businesses that can generate jobs and wealth, instead of focusing on businesses just because they are small, says Simon Clark, managing partner at investment firm Fidelity Growth Partners Europe. He points out that many small businesses will stay small, and many will indeed want to stay small.
He said: "It is the high growth, high productivity businesses that will drive this country forward. Governments should go a lot further to support high growth businesses because that is where the interesting jobs lie and where the real growth of the economy lies. High growth companies create a lot of specialised high value jobs, and those are the kind of jobs that underpin the economy."
Clark added: "There is a big difference between a small business and a small high growth business. A small business such as a local hairdresser is not going to employ that many people and they are not going to be fundamentally creative or change the economy in an interesting way. High growth businesses are much more interesting because they have the potential to evolve into a very large valuable businesses. If the government is going to spend policy effort trying to encourage certain kinds of companies, these are the ones that can make a difference."
Clark said: "It is nice to see all the political parties trying to encourage small business and for senior politicians to turn up and say warm and fuzzy things at small business conferences, but what are they trying to achieve? If they are trying to help support a vibrant economy that creates lots of good quality jobs then more precision would be helpful along with all the good intentions."
He said that the government could encourage high growth businesses by focusing on initiatives directed at specific high growth sectors, such as greater public procurement and research and development tax credits. He pointed out that government encouragement of research in the pharmaceutical sector had reaped huge dividends.
Such a targeted approach would not cost much money, Clark said: "We don't need the government to spend a lot of money here; what we need to do is identify those sectors which the potential to create really high growth companies and then spend time making sure that those sectors have the right encouragement and the right structures to enable those small companies to be formed and then to grow."
He added: "We need to move away from thinking that small business is good and big business is bad, and start supporting high growth businesses and not just small businesses in general. We need to put the idea of high growth businesses right at the heart of everything we do."


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Top Soccer
Travel Information
The traveling program is for players U8 and older. The small sided travel teams will be composed of 100% of members that reside within the Kingsway High School district. Players may be allowed to participate from other boundaries with board approval. Large sided teams can include non Kingsway district residents subject to certain limitations. They are as follows: roster must not exceed 40% of non Kingsway district residents, the board may approve certain instances where this number may be waived.
The teams compete against other teams as members of the SJSL/SJGSL. Teams are divided by age and gender. Teams practice twice a week for about 14 weeks. One of the practices will be run by a professional soccer trainer and the other practice is run by a coach. Girls play a 10 game fall and spring season schedule on Saturday afternoons. Boys play a 10 week fall and spring season schedule on Sunday afternoons. 5 games are played at Locke Ave or High Hill West and 5 games played away.
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g_god on
g_unlimitedammo on
g_god 1
g_unlimitedammo 1
i need a honest answer pls
I have the Steam version of this game, so I went into the Steam folder on my computer. I read that you have to paste the code into the user.cfg file. The problem was, I found two of those files on my PC relating to Steam. One for Metro 2033 and the other a few sub-folders down in the "userdata" folder. I put the code in the user.cfg file that was in "userdata". I read that you may have to open and close Steam for it to work and you have you paste the code in the file again if you exist and start Metro 2033 again.
Apparently when you exit the game (and Steam) then start it again, Steam checks the file you edited with a file on Steam Cloud. So your file ends up resetting to its default. That is why you have to enter the code again, or at least that's what I remember. I haven't played Metro 2033 recently.
This also applies for other changes in the user.cfg file.
• Ammo
• Money
+ 10 more options
• 1152 x 600
• 1280 x 960
• 1280 x 1024
• 1600 x 1200
• 1920 x 1200


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Sorry, something went wrong. Please contact support .
For the words in your life only impeccable, pleasing, and perfect will do. If the goal is to cultivate an audience then stimulating, compelling and thought provoking writing is a necessity. Here you will find the solution to all of your word needs. As an English teacher and editor for the TESL newspaper, I am constantly focussed on the English language. Looking at words, phrases or concepts from various perspectives is a specialty of mine and an important feature of building rapport with readers. I can help you reach your target audie