text:
Camilla Pagliaroli tells her story about moving from New York to Livorno
About two years ago it all started with a possibility. A Bologna connection, one that was a long shot. It put Italy on the map for us. So we started talking about moving here, about what it would be like to jump off the rat race into the slow lane. In the middle of life, and not wait till we retire. But we knew it was a very long shot so we didn't want to get too excited. What if nothing would come of it?
And that's when it hit me. All of a sudden I saw us a decade down the road, sitting back there in New York, wondering what our life would have been like had we taken the plunge and moved to Italy? And that's when you know, you just KNOW, you have to do it. Come what may.
The Bologna connection did not work out, and in hindsight - we were glad it didn't. But the arrows started pointing towards a place called Livorno. I knew nothing about it. Literally. Wow, it's in Tuscany? I found out it was right on the west coast, which is great; we prefer sunsets to sunrises, and we love being by the sea. We both grew up by the sea. Different ones, on different continents but still, there's nothing like the calming effect of waves hitting the shore and the endless horizon stretching out before you.
A year and a half later I feel like I am just beginning to scratch the surface of Livorno. It goes slowly, as language fluency is still a way off and I have that Scandinavian look about me, which just screams: "she's a tourist off today's cruise ship". But I'm learning, mixing with the locals, and I look forward to continuing to do so.
It isn't pretty here, like Florence or Pisa with their world-famous attractions and pretty classical facades reflecting in the waters under the bridges of the river Arno. Oh well; these places are close enough for me to visit when I want, and when I come home at night I don't have to wade through camera-ladened tour groups, or look for that hidden (tourist) service charge on the multilingual food menu.
Livorno is not an easy place to love. I see lots of graffiti, overgrown bombed-out ruins left from the second world war, but there is a simple straightforwardness in its people I admire. I do miss that certain intellectual feeling & all its cultural offerings that comes with living a university town. I miss being able to pick food of any ethnicity and find a restaurant that serves it, and the possibility to have a good conversation in English.
But the 'Livornese' have their own way of doing things, and it's pretty fascinating when you take time to get to know them. So I hop on the local bus to the beach, get my fruit and vegetables fresher and cheaper at the Livorno market, and I'm making a point to talk to some of the shop-keepers on our street.
Livorno has potential, and it's easy to see glimpses of its by-gone charm, and I wonder: will we be here long enough to see some of it restored? It's a blue-collar town flying a red flag. Which doesn't make it easy for progress to take root. But we didn't come here to change things. We wanted to be changed by Italy. Slow down.
So I am learning to live here and enjoy life the Italian way: the food, the culture, the wine, the country-side, and life itself. As I'm slowing down enough to enjoy these things, I find that I am more willing to contend with the bureaucratic hurdles and the equally slow pace with which things get accomplished here.
You can follow Camilla's experiences on her blog
- Add new comment
- 3144 views


text:
724 Felix Street
Saint Joseph, MO 64501 St Joseph Rate and review
Saint Joseph, MO 64501 St Joseph Rate and review
We are a traditional Japanese Ramen shop serving up fresh authentic artisan ramen here in St. Joseph.
Upcoming Events
Places You Should Consider
About us
Japanese Restaurant
Business Listed in Categories
News & Articles
People Connected
Is Komatsu Ramen St. Joseph one of your Favorite Places?
Add to My Connections
Add to My Connections
Plug Updates
Say Something or Ask a Question about Komatsu Ramen St. Joseph
Recommended Ratings & Reviews
Rate & Review
Restaurant Atmosphere
Bang for your Buck
The Crowd
Parking & Accessibility
Is This Your Listing?
Are you the owner of Komatsu Ramen St. Joseph in Kansas City Missouri? Send us more details! Claim Your Business Or Contact Us
Related places
Sweet Al's Donut Shoppe808 N Walnut St , Cameron MO, 64429
Biscuits and Gravy is a mix of the gravy, so not top notch, but it tastes good and the biscuits are great. Donuts, Pastries, and Cookies.
Easton's Pub & Grub106 Elijah St , Easton MO, 64443
Known for it's great food, lively and entertaining atmosphere. Filled with good food and good people it is a place any person can enjoy. Come in and...
The Big Biscuit139 N. Belt Hmy , Saint Joseph MO, 64506
They were light and fluffy. We had a basic breakfast with eggs, biscuits and hash browns. All was cooked to our requests. Fantastic southern chicken sandwich...
Just Desserts by Mom505 Francis St , Saint Joseph MO, 64501
It's all about desserts at Just Desserts by Mom. We start and finish each order with only the best of ingredients available. Freshness is our staple...

subset: commoncrawl


text:
Thank you for your interest.
Medical coverage goes right to the heart of why we buy insurance in the first place. We buy insurance as a way to protect ourselves against loss. If you've ever had a major injury or surgery, you understand the importance of having good medical insurance.
After completing the form, please click on the "Submit" button. Your information will be emailed to our offices and we will process your request. All information will be kept confidential.
» Required Fields
410 12th Street, Suite 325,
Oakland, CA 94607
|Phone: 510-869-9990
|Fax: 510-869-9994


text:
Buy Adrianna Papell Off Shoulder Beaded Gown, Rose Gold | John Lewis Women ZMCUJDPQW
- Product Code: ZMCUJDPQW
- Availability: In Stock
- £144.81 £60.30
Make a dazzling entrance in this Off Shoulder Gown by Adrianna Papell.
In a gorgeous pink hue, this off the shoulder evening dress boasts a tulle overlay applied with glittering rose gold sequins in various sizes. With a sensational column shape, it skims your figure flatteringly before falling to a short train.
The dress leaves your décolletage exposed with its feminine bardot neckline. It comes fully lined in soft fabric for comfort and a seamless fit, and is finished with concealed strap securers.
Let it speak for itself by teaming it with understated strapped heels and a slick of lipstick.
Shoulder to hem length: 179cm (on a size 12 cut).
- BrandAdrianna Papell
Buy Adrianna Papell Off Shoulder Beaded Gown, Rose Gold | John Lewis Women ZMCUJDPQW


text:
November 20, 2017 - 10:03am
1 Trainer available for Northern Tale 4, see below
In Northern Tale 4, Ragnar's kingdom is in danger once again. After defeating the evil witch Gesta and the Dragon of Ether, the Viking lands are on the verge of a new invasion. Collect, repair and build to defend against a vast army of Giants, Witches and Black Knights.


text:
The perfect Las Vegas spring weather set the tone for BOHO HOSPITALITY'S Tiki Party Open House benefiting Wagging Tails Pet Rescue on April 25th.
"Brynn" is a Pomeranian rescue who had a great time at the party (you see her here sporting a pink lei on the lap of a Wagging Tails volunteer) and is available for adoption to the right home.
Julie decided to have her first charitable event for BOHO HOSPITALITY be a pet rescue. Her beloved border collie mix, Bennett, was adopted from the Dumb Friends League in 2006 when she lived in Denver, Colorado. "Benny" is still a terrific companion and host dog twelve years later. He loves having friends (human and canine) at the house.
Wagging Tails Pet Rescue is currently looking for volunteer foster families to assist with rescued animals who need forever homes. Please message Jodi on their website if you can help in the Las Vegas Valley.


text:
Interest rates have been at their lowest levels in more than a year and a half for quite some time now, but with the U.S. Federal Reserve expected to raise short-term borrowing rates as early as June, this could push mortgage rates back up in the coming months. For the meantime, interest rates are still well below 4 percent for 30-year fixed-rate mortgages in most surveys, with the national average currently at 3.75 percent. Last year, that average rate was at 4.28 percent.
Keeping that number and a possible Fed rate hike in mind, it stands to reason that there couldn't be a better time for consumers to stop sitting the fence and start shopping for a new home and the lowest rates possible.
According to Bankrate chief financial analyst Greg McBride, rate hikes can definitely affect affordability, but it's not the only thing one should consider. "You don't buy a house because of low interest rates any more than you get married because of a sale at the bridal shop," he said.
So what can you, as a consumer, do if you're looking for the lowest interest rates possible? For one, it would be a good idea to take a long, hard look at your credit score. Ideally, a FICO score of 740 or higher would qualify you for the lowest possible rates, but we have to admit that that's not exactly the easiest score to achieve. Chances are you'll be paying a higher mortgage rate if your credit score is lower than 740, but if it is below that threshold, it's a good idea to review your credit report for errors, as you get a free credit report every 12 months from major "credit bureaus" Equifax, Experian, and TransUnion.
It is also advisable to do a lot of comparison shopping, and to get all your quotes on the same day, so as to allow you to accurately compare rates side-by-side. Remember that mortgage rates tend to fluctuate on a daily basis.
Also, you should also keep in mind the fees you may have to pay, and also, if possible, try to leverage a reduction in fees and never forget the Good Faith Estimate; when comparing offers, you want more than one of those estimates for a better idea of each lender's terms and conditions.
In order to search for live mortgage rate quotes from some of the top U.S. lenders, please click on the link below. To calculate your monthly mortgage payment, feel free to use our featured mortgage calculator.


text:
Did you know that by next year, more time will be spent in the digital world than on all other media combined? That's just one of the key ideas shared by Michelle Anderson, The Warehouse Group's Chief Digital Officer, at her address during last week's NZ Retail Summit.
From steam in the 1700s, to Electricity and Scientific approach to mass production in the 1800s, followed by computing in the 1900s, the world as we know it has been shaped by several industrial revolutions, and as Anderson and others see it, we're at the cusp of the fourth. "The fourth industrial revolution is the digital age and the era of the connected device, and it's bringing companies and customers together like never before," said Anderson.
The convergence of advances in Artificial Intelligence (AI), robotics, Internet of Things and other technologies are part of the collective force behind many products and services that are increasingly becoming indispensable to modern life, she added.
"From GPS systems that suggest the fastest route to a destination, voice-activated virtual assistants such as Apple's Siri, personalized Netflix recommendations and Facebook's ability to recognize your face and tag you in a friend's photo, these technologies paving the way for transformative changes in the way we live and radically disrupting almost every business sector," added Anderson.
To stay competitive, Anderson says that retailers need to take notice and understand the implications of all of this to their brands. One way to accomplish delivering experiences that are both world class to the consumer and deliver value to the business is through personalization.
"Developing tailored recommendations, content, offers, and experiences, across all channels and devices, along the entire customer journey is how retailers can stay ahead of the curve," said Anderson. "Personalization at scale has the potential to create $0.45 trillion to $0.8 trillion in new value to the retail industry according to a recent report by McKinsey. 81% of consumers want brands to get to know them and understand when to approach them, but implementing and integrating the right technologies create significant complexity and call for a lot of coordination."
At the core of personalization is one important thing: data. 2.5 quintillion bytes of data are created every day, and 90% of the world's data has been made in the last two years alone. There's estimated to be 50 billion connected devices by 2020, which opens up vast opportunities for marketers, but also raises the question of privacy, shared Anderson.
"We at The Warehouse Group believe that data can be a tool to build trust," said Anderson. "When you listen, respect, and earn consent appropriately, you can power incredible end-to-end customer experiences - cross-channel, cross-device and across lifecycle – but with all of this opportunity for personalisation."
For The Warehouse Group, the vision for this data-saturated future is a fully-scaled and integrated marketing capability that delivers for customers and for the businesses. As Anderson put it, "We don't just want to turn up, we want to be there for our customers when it matters across their various shopping missions. We want to leverage our rich data set to understand our customers to deliver personalised and relevant experiences to them. It all boils down to right message, right time, right channel."
Anderson believes that part of finding the right mix involves developing customer journeys for each brand, where they can map out interaction points and touch points across all channels to see the moments that matter to customers. It's something that The Warehouse Group brands are exploring by offering personalised recommendations across desktop sites and mobile apps that customers interact with, and Anderson believes that bridging the physical and digital experiences will be even more important moving forward.
She wrapped up her presentation by sharing her key takeaways on how to fine-tune an organisations approach to digital. "Focus on optimising the customer experience, but be transparent with how you're using the data that comes as a result of that," added Anderson. "While unlocking siloed data can be difficult, it's key to build a solid foundation to assist in that area. Investing in decisioning and analytics capability will open your eyes as marketers to what's missing and what's possible with your digital efforts."


text:
Kylie Jenner's former BFF Jordyn Woods has been spotted on-stage at Coachella, helping out Jaden Smith with his performance.
Wearing a white face mask covering her nose and mouth, the 21-year-old danced her away on to the stage alongside her long-term friend Jaden.
Jordyn's friendships with musicians seems to be blossoming at the moment, as Coachella isn't her only recent foray into the limelight.
YouTuber Justin Roberts has featured Jordyn in his latest music video for Way Too Much, which sees Jordyn passed over by the rapper for fellow model Sofia Jamora.
The women play life-size dolls stuck inside a toy factory and the YouTube description explains the song is 'a homage to young stars in the spotlight of social media and the consequences when fame becomes Way Too Much.'
After the year she's had so far, perhaps the song struck a chord with Jordyn.
Jordyn is trying to move on from the Tristan Thompson cheating scandal, in which she was caught out kissing her best friend's sister – otherwise known as Khloe Kardashian's – boyfriend.
As a result of the news, Jordyn was dumped by best mate Kylie and asked to move out of the billionaire's home. She has since been ostracised by the Kardashian family.
Jordyn admitted to and subsequently apologised for kissing Tristan on an episode of Red Table Talk, which is of course hosted by Jaden's mum, Jada Pinkett Smith.
Jada herself had beef with the Kardashian clan over the way they treated Jordyn during the scandal and eventually unfollowed Khloe, Kim and Kris on Instagram.
The ultimate celeb burn.
During the emotional interview, the former Good American model denied that she and Tristan had been in a relationship, adding,: 'I see what has been done to my life in only a week.
'The media put their focus on a young black woman that made a mistake and not one that's worth public crucifixion.'
Got a showbiz story?
If you've got a celebrity story, video or pictures get in touch with the Metro.co.uk entertainment team by emailing us email@example.com, calling 020 3615 2145 or by visiting our Submit Stuff page - we'd love to hear from you.
MORE: Jordyn Woods' split from Kylie Jenner 'best thing to ever happen' as she steps out of ex-BFF's shadow
MORE: Kylie Jenner denies putting Jordyn Woods lip kit on sale after Tristan Thompson cheating scandal


text:
We all know Fine Lines, Wrinkles, and Age Spots become a nuisance as we age, but that doesn't mean you have to let it take over your life. Fight back, and look 10 years younger! Don't let your appearance hold you back from doing amazing things, or life!
In this presentation, Judith is performing a step by step Makeup demonstration using Special Care Cover and the Everything Pencil in Ultra Light, Pure Beige and Sun-Kissed Beige.
This combination will easily make fine lines, wrinkles, age spots, and any other skin imperfections disappear. All of this can be done within a few minutes, and is designed to be portable. So, if you ever need an emergency touch up, no problem, it only takes a minute or 2 to freshen up and look great!


text:
Volunteer Stories
All of our volunteers make a huge impact...
Meet Kelly - Assistant Vice President and Community Engagement Officer at Independent Bank. Kelly and a fellow volunteer, Mark facilitated a CHAMPS class together, focused on Financial Empowerment. The goal at Frisco Family Services is to provide tools that enable our clients to achieve or maintain self-sufficiency. Our CHAMPS (Challenging Adult Minds for Personal Success) classes, facilitated by working professionals, are a big part of helping them reach that goal.
Five women from all different backgrounds attended this class. Kelly and Mark came into Frisco Family Services to teach a class about budgeting and credit, and along the way ended up learning the impact that serving others can have.
"It was an eye-opening experience to meet these ladies and hear a bit about their life stories," said Kelly. "I know the importance of empowerment when it comes to finances, but meeting those beautiful women yesterday drove home the reality of educating myself, my daughters and all women on basic financial matters. I think Mark would agree, our goal was to share some practical financial tools; but we quickly realized, just being present with these ladies and hearing their individual concerns was as important as having them learn new skills. Each of these individuals had a unique and powerful story; and as always happens when serving others, I gained much more than I gave."
Close to 100 local Paycor employees took part in their 'Paycor it Forward' event which provides their team with the opportunity to give back to a cause they believe in. An employee at their Frisco office, Nate led his team to Frisco Family Services.
He was introduced to Frisco Family Services at a local networking event, and immediately felt a strong connection to our mission. Frisco is home to Nate and his family, and he understands the importance of being part of a community that takes care of one another, having been in a situation where he was close to reaching out for assistance himself. "Our whole lives are in Frisco, and I really like that a place like this exists, as well as the holistic type of approach it provides," said Nate.
Strengthening their bond as a team was something they wanted to accomplish while they were volunteering, and Frisco Resale was a great place for their entire group to come and invest in their community together. They even joked that talking about work was prohibited.
"Our group had a great experience at Frisco Resale, and we felt good that the work we were doing would help fund such a good cause."
Meet Caryn
"Volunteering with Frisco Family Services made me feel like I was part of something bigger, like we were all working towards having a huge impact that can't happen if you attempt it on your own," said Caryn. "Knowing you're part of a group that is so passionate about their community and is doing such amazing things, makes you feel connected in a meaningful way in the community."
Caryn is a volunteer who wanted to make a unique impact on her community... and she did! Along with her serve team, Impact Frisco, a special event was organized for those assisted by Frisco Family Services that are part of our community's growing senior population.
Caryn and her team wanted to help others in a way that focused on interacting with people and building relationships with them. The event took place this past February, where more than 30 of our senior clients joined together for an exciting day of food, bingo, giveaways and conversation. The event went so well that Frisco Family Services is already planning the next one, and is in the process of adding it to the list of the programs regularly provided to our seniors.
"It was really special to be part of the first senior luncheon and build a foundation to grow on in the future. I hope to see familiar faces become people with stories we know personally and are able to be an encouragement during their struggles."


text:
Britain to close Yemen embassy at weekend over security
Britain's embassy in the Yemeni capital Sanaa will be shut this weekend, the Foreign Office said, after the United States announced the temporary closure of two dozen embassies over fears of an Al-Qaeda attack.world Updated: Aug 03, 2013 11:59 IST
Britain's embassy in the Yemeni capital Sanaa will be shut this weekend, the Foreign Office said, after the United States announced the temporary closure of two dozen embassies over fears of an al Qaeda attack.
Several British embassy staff have been withdrawn from Sanaa over the security fears, a spokesman for the Foreign Office in London said.
"The embassy will be closed on the 4th and 5th of August," the spokesman said.
"We have withdrawn a number of staff from Sanaa, due to increased security concerns." He added: "We are particularly concerned about the security situation in the final days of Ramadan and into Eid." The Muslim holy month of Ramadan ends next week with the feast of Eid al-Fitr.
The United States had issued a worldwide alert on Friday warning of plans by al Qaeda to launch an attack in the Middle East or North Africa in August.
On Thursday, the US State Department had announced that at least 22 US embassies would close on Sunday as a precaution.
The State Department said attacks were possible "particularly in the Middle East and North Africa, and possibly occurring in or emanating from the Arabian Peninsula."
The Foreign Office advises British nationals against all travel to Yemen, and strongly advises any remaining Britons to leave the impoverished Arabian Peninsula country.
"Our travel advice advises particular vigilance during Ramadan, when tensions could be heightened," a ministry spokesman said.
On its website, the Foreign Office warns of a "high threat from terrorism throughout Yemen", and says British nationals face "a very high threat of kidnap from armed tribes, criminals and terrorists".
There are no current plans to close other British embassies this weekend, a Foreign Office spokesman told AFP.


text:
In the property development market we have seen a growing sense of excitement from the initial slow start to 2017. Competition for land tenders is increasingly aggressive as both local and international clients look to increase their portfolios. Another trend we have been seeing is that developers have tended to be more active in acquiring existing properties for full-scale development or major refurbishment, restacking and/or rebranding such as TripleOne Somerset, AXA Tower and Golden Shoe.
Further investment into the regional markets has continued with cash flow into the likes of Malaysia, Vietnam, Thailand and Indonesia remaining strong – all of which points to an exciting 2018 with the market returning back to form. In recruitment terms, we have experienced a sharp increase in the need for quality Project and Development Managers. In addition, on the investment side, the hiring of good Business Development and Investment Managers has upturned.
The construction sector, not surprisingly, has echoed the development market with many main contractors cutting back on project staff and teams early in 2017. But projections suggest that between $20 billion and $24 billion has been awarded/spent in infrastructure construction alone, up from last year's $15.9 billion. Hence, we saw a noticeable change in the recruitment market in Q3 most likely down to some major packages being awarded across both civil infrastructure and building sectors such as DTSS Phase II, Circle Line 6 and Project Glory.
As we come to the end of the year the Singapore construction market is looking poised to have a busy 2018 with many contractors hiring ahead of plan and before Christmas and the CNY. The desire to find good Project and Construction Managers has been the flavour of recent months as well as Architectural Managers with large scale project experience.
Whilst the hospitality and retail sectors have been struggling in Singapore and will remain a key focus for change in 2018, the outlook for other major sectors remains strong. We anticipate strong levels of hiring to remain in commercial fit-out, data-centres and publicly funded projects.
All in all 2018 is a year the Cobalt team are looking forward to.
If you're looking for a new Construction role in 2018 contact our team today.
Article written by Leong Siu Meng, Principal Consultant - Property, Construction & Engineering
You may also be interested in:
Rising demand for engineers across Asia
Find out more about the Cobalt Asia team


text:
Social Security Surviving Divorced Spouse Benefits
Program Description
The Social Security Surviving Divorced Spouse Benefits are a federally funded program administered by the U.S. Social Security Administration (SSA). These are benefits paid to the divorced spouse of a deceased worker who had earned enough work credits.
General Program Requirements
To qualify for this benefit program, you must meet all of the following requirements:
- Be at least age 60
- Be divorced from the deceased worker
- Have been married to the deceased worker for at least 10 years
- Not be entitled to an equal or higher Social Security benefit on your own work record
- Be unmarried (unless an exception applies).
Based on the information you gave you currently receive benefits based on a former spouse's work record. You also indicated that you were married for at least 10 years to more than one former spouse. You might be able to get a higher benefit on the other spouse's record.
PLEASE NOTE: If you get (or will get) a pension based on work for an employer who did not withhold Social Security taxes, such as a government agency or an employer in another country, your Social Security benefits may be reduced. Visit SSA's Publications Home Page and review A Pension from Work Not Covered by Social Security, Social Security Publication Number 05-10045 and Government Pension Offset, Social Security Publication Number 05-10007.
Your Next StepsThe following information will lead you to the next steps to apply for this benefit.
Application Process
Widows, widowers, and surviving divorced spouses cannot apply online for survivor's benefits. For application information, please visit the How To Apply page.
Program Contact Information
Would you like more information about surviving divorced spouse's benefits?
Visit SSA's Publications Home Page and review the publication "Survivors," Social Security Publication Number 05-10084 or review Chapter 4 of the Social Security Handbook.
Would you like more information about social security credits?
Visit SSA's Publications Home page and review "Social Security: How You Earn Credits," Social Security Publication Number 05-10072.
Or for additional information on this and any SSA benefit, call:


text:
In October 2013, Barcelona City Council restricted access to the Monumental Area of Park Güell, with the primary objective of preserving this emblematic city area, which is a UNESCO heritage site, and keep it in the best possible state of conservation.
On one hand, the idea is to protect, study and provide information about Gaudí's work for humanity, as established in UNESCO's requirements, and provide a high-quality visiting experience.
On the other hand, the park has to remain an urban park, a place for the community and even a place for individual and collective memories.
A ticket is required to visit 1.7 hectares of the Park's total surface area of 19 hectares. This area is considered to be the most vulnerable part, and in order to protect it, regulations were established to control the number of people entering. Enjoying the rest of the park is totally free of charge.
The money raised by this restricted access is reinvested in the park, through various projects to improve and renovate green areas, viewpoints, paths and play areas. Etc.
Local residents in the neighbourhoods around Park Güell (El Coll, Vallcarca-Penitents, La Salut, El Carmel and Can Baró) enjoy free access every day of the year, by using a personal, non-transferable card. Registered residents of these neighbourhoods can apply for a card on the City Council website (online procedures) or by calling 010. They can also go to the Citizen Help and Information Offices in Gràcia, Horta-Guinardó or Plaça de Sant Miquel.
The Gaudir Més cultural register also offers free access to the restricted area to all its members. For further information: www.barcelona.cat/gaudirmes/en
Income reinvestment/Improvements in the Park
The money obtained from ticket sales in the restricted monumental area is used to carry out improvement work, both inside Park Güell and in the surrounding area.
The size of the park and its various green areas, the fragility of its architectural heritage and the millions of people who visit it every year all mean that a large amount of economic resources have to be invested in keeping this Heritage of Humanity site in good condition.
For example, Plaça de la Natura has recently been completely renovated, as has the Sant Salvador Fountain and the area around it, the biodiversity path has been recuperated and in the coming months, improvement work will also begin on the Hypostyle Room and other historic features.
Monumental axis has its own rate an schedule.


text:
Exciting news! We awarded $120,000 to 12 organizations through our Community Innovation Grant program. The one-year, project-based grants ranged from $500 to $10,000 and are made possible by an ongoing partnership with the Bush Foundation.
The investments will help Minnesota communities find more effective, equitable, and sustainable solutions to challenges and opportunities. "Headwaters is here to support the best of community-centered problem solving, and it's clear these organizations are leading that work," said David Nicholson, Headwaters Foundation Executive Director.
The 12 Community Innovation Grant recipients (and the cities they are headquartered in) are:
|4-Directions Development, Inc.
|(Red Lake)
|APIA MN Film Collective
|(Saint Paul)
|Black Visions Collective
|Central Minnesota Sexual Assault Center
|(Saint Cloud)
|Centro de Trabajadores Unidos en la Lucha
|Family Tree, Inc.
|(Saint Paul)
|Native America Community Development Institute
|New Arab American Theater Works
|(Saint Paul)
|Solutions Not Suspensions Coalition
|Trans United
|(Fond Du Lac)
Applicants were asked to define their community, identify a challenge or opportunity, and outline their proposed solution. Throughout July, a committee of community members reviewed applications against program criteria and made funding recommendations to the Headwaters Board of Directors.
Headwaters accepts Community Innovation Grant applications online three times per year. The third and final round of 2018 will open on Thursday, September 13 at 9 a.m. Program staff will host three hour-long information sessions next month. The first is scheduled for Thursday, September 6 at 5:30 p.m.; the second is set for Minneapolis on Monday, September 17 at 6 p.m.; and the third will be on Thursday, September 20 at Noon. Visit our events page for registration details.
Applications are due Thursday, September 27 at 5 p.m. . Grantseekers will find a new, user-friendly application system. Instructions will be included in the application. Visit our program page for more information. Organizations that are interested in grants between $10,000 and $200,000 should apply through the Bush Foundation.


text:
[SongFest Home]
Below is some information on people involved with IOCA music collections over the years. If you know any of these folks, or have any information, please let us know.
Dick and Beth Best
The later editions of the IOCA Song Fest collection were edited by Dick and Beth Best. Their full names, as indicated by the Library of Congress, were Richard Lundelius Best and Beth Arlene Best. According to the prefaces in the Song Fest books, Dick was a member of the Cornell class of 1944, and Beth was a member of the Radcliffe (now Harvard) class of 1947. Dick passed away some years ago, but as of summer 2005, Beth was still living in New Hampshire.
Will Brown
Will Brown, along with Gerry Richmond, wrote the "Preface to the Original Collection". According to the signature on the preface, Will was in the Dartmouth class of 1937. Will was involved with music and singing his entire life, and remained friends with Beth Best upto his death in 2005. His name has come up often in relation to the Song Fest collection, and any more information on his involvement would be greatly appreciated.
Gerry Richmond
Gerry Richmond, along with Will Brown, wrote the "Preface to the Original Collection". According to the signature on the preface, Gerry was in the Brown class of 1936. Any information on his involvement with the collection would be greatly appreciated.
Lou Heermance and John Ehrenfeld
Louise Heermance and John Ehrenfeld are the two signatures on the "Forward" of the IOCA Barn Dance. I have not been able to determine their level of involvement in the Song Fest collection. Lou's signature includes "M.S.C.O.C '42", and John's includes "A.O.C. '41". These apear to be acronyms for college outing clubs. Any information on these two would be very helpful.
David Hunt
David Hunt created the illustrations used in "The New Song Fest" and "The New Song Fest Deluxe".
Eileen Chandler
Eileen Chandler illustrated the 1948 edition of "Song Fest".


text:
One Week In...
A little bit of reflective rambling about the first week of school:
Admittedly, I like schedules. I like routine. I like to know what to expect. However, I also like to think I'm pretty good at being flexible and going with the flow when necessary (that's what makes trips like this work!) But, overall--I think schedules and routines are good and they generally suit me well.
So, imagine my surprise, when this first week of school came around and actually got going...I've found that I'm a little bit out-of-sorts and...bored(!). Getting up at 6am everyday and completing about 15 tasks by the end of breakfast had been WAY forgotten over this wake-up-late, get dressed when you feel like it, watch-a-lot-of-television summer.
I've noticed that I've spent a good part of each day this week making sure I'm ready for the next section of the day--which means I'm wandering a little aimlessly inbetween because my mind won't focus on what I could be doing NOW.
It's funny, because I dread the summer arrival when both kids will be home ALL DAY EVERY DAY, all the fighting, the noise, the messes, and overload of kid-themed activities. I anticipate with excitement when they'll head back to school to see their friends and go to their classes. I'm thrilled that I'll have more quiet work time by myself, and that then I can focus, create, and produce more easily.
So, how come now that I have all this quiet, my brain feels like mush and I can't focus on anything? Waking up too early? Too many days of exercise? Too much schedule to keep straight? By 3:00 when Ayla comes home and Paxton wakes up from rest time, all I want to do is stretch out on the couch and veg out till bedtime.
I'm sure this feeling will pass quickly - it always does, and then I'll be back to my get-er-done self, but for this week, I think I spent a lot of time with a blank stare on my face!
So, I'm wondering if anyone else goes through a week or two of bewilderment at the beginning of each school year?
5 comments so far:
Yep, I go through it for a full two weeks at the beginning of summer break and when school starts up again. That's a MONTH lost of productive time!
Thats where I am right now as well...and our school breaks are shorter than yours! Feeling lazy & unproductive has been my MO lately, I'm going to really try to snap out of it next week because I'm slowly making myself crazy!!!
and by the way, the thought of having a high schooler and a toddler is making my head spin a little...
I have a love/hate things with summer & back-to-school. By May, I'm always excited for Summer and some time to be unscheduled and free to come and go whenever. Then, August hits and I am just itching to get some sort of structure to my days. It'll be different this year with a 2 1/2 hour stretch where the older 3 are in school and Levi will be napping. I am almost giddy at the thought of it!
I absolutely feel the same way! I remember the feeling from last year too so I have tried to give myself permission this week to feel lost. But I still feel unproductiver. A friend on FB, who has gone through the experience more than we have, said it well, "I am always thrown off by the feeling I get when the kids first go back to school...that feeling of 'hey, I actually have a chance to pause...and think about life and work and what domestic priority might emerge...hmm, laundry or dog poop? I'm going to think a while longer..."


text:
Jason and Todd Talk Through Lousy Films (Episode 81)
about 8 days ago.
Los Angeles critic Jason Rohrer and actor Todd Robert Anderson talk incessantly through cult films. It would be annoying if you were trying to watch the movie, but this is a podcast...so it's just delightful!RSS
Updated 10 months ago.
The Seventh Son is a movie about the seventh son of a seventh son, we guess, but it's really hard to understand a word Jeff Bridges is saying. Words you can understand are supplied by Jason and Todd, who talk about other bad movies that'd rather be watching and what an actual alien invasion would do to the political divide in the United States. Also, they talk about their weight.


text:
Engineering & Packaging Jobs
"We completely reshaped the infrastructure of a wet factory's process area, coming up with a new way to make the product more efficient and of even higher quality. The company invests back into the business, so there is always an opportunity to foster continuous improvement and new idea."
"Working at Purina feels like working for your family as we try to solve business problems. The fact that we are independent thinkers, our ideas count, and we collaborate across departments with little or no overhead is the best thing about this group."
Management Development Associate Positions Now Posted!
As a Management Development Associate within our manufacturing facilities, you'll begin a fast-track career to management through a tailored rotation to learn end-to-end operations of our factories.
Available Engineering & Packaging Jobs
There are not any jobs available at this time.
Who From Nestlé Purina Are You Connected To?
To see your connections, make sure you're logged in to your LinkedIn account.


text:
See Koalas and Kangaroos IN THE WILD! Walk with wild animals in their natural habitats on this full day, fully inclusive small group (maximum 10 people) eco-tour, led by an experienced Wildlife Guide.
Walk through natural bush-land to meet wild koalas, then learn about each one's history and social life from your Wildlife Guide and Koala Researcher. Help create new koala habitat for future generations of baby koalas by participating in the 'Make a Home for Clancy' Koala project. Join wild mobs of Kangaroos on the plains and in open forests. Their natural instinct is to flee humans, but with care, you often get close enough to take photographs and observe their complex social life. The natural environments visited are also home to parrots, fairy-wrens, emus, honeyeaters, possums, wallabies and lizards. Your Wildlife Guide will help you find and understand them, and show you how to look for wildlife wherever you travel in Australia.
The tour is vehicle-based, with short walks during the day; includes a substantial bush picnic lunch and pick up/drop off to Melbourne city between 4 and 5pm. NOTE: this itinerary is an indication only, due to conditions on the day it can change slightly to maximize wildlife viewing opportunities.


text:
Dating and Autism, Myth or Reality?
Dating is something most autistic parents cannot even begin to fathom for their children on the autistic spectrum. Eventually though, every parent faces the reality that their autistic son or daughter will express some interest in learning how to date. Can autistic romantic relationships exist? Can they succeed?
When I was first diagnosed with autism in 1995, the condition had only just been recognized and even the psychiatrist who diagnosed me was relatively new to it as a clinical condition. What he did do was inform my parents that I would never have any normal relationships and that they should prepare themselves for the fact that I would need lifetime care. With them, as with most autistic parents, they were so caught up in the day to day care and drama of raising an autistic child that such thoughts were relegated to the army of never ending worries and fears that keep us all tossing and turning at night.
Fortunately we have come a long way since then, not only diagnostically, but therapeutically and there are significantly more advanced therapies and programs available than the bare bones experimental Frankenstein programs used back in the late 90's.
Autistic people by and large have the same needs and desires as neurotypicals. Despite touch aversions and sensory restrictions, we still want the comfort of a relationship, the companionship that comes with a committed partner and we still have the same sexual needs. What makes relationships difficult to handle is the emotional and social components of a relationship. I leave behind me a string of failed relationships and a failed marriage. I could coldly chalk them up to experience and growth in the absence of resources and knowledge, but I have learned that neurotypicals generally don't find that sort of objectivity comforting or acceptable.
I still need partnership. I don't like to be touched, but I crave validation and acceptance that in my
experience can only come from a romantic partner. Emotional entanglement makes me very uncomfortable and despite my 36 years, I still find it nearly impossible to react appropriately to externally emotional situations. I don't feel guilt, I don't understand many shades of emotion beyond my direct experience and while I've grown to emulate certain reactions I have little emotional depth beyond happy, angry or sad.
I've grown to understand that my own neuroses and my own compulsive need for control and routine have to develop some measure of compromise and I've learned to develop my own ways of bending those needs in such a way that they are still relatively satisfied without unduly pressuring or stressing a partner. I've learned that there are situations in which it is necessary to touch and be touched, to even welcome and draw comfort from that touch from a partner or a child.
I have loved and I do love, though not in the way that neurotypicals love. Much as I've tried to capture the passion and seemingly magical sensation we see on television and read about in magazines and books, I've not developed the capacity to replicate that sensation, I don't understand it and I doubt I ever will. What I have done is, to me at least, far more intimate than any of those fantastical love stories. I have allowed these people into my routine, into the structure of my life, the very delicate and tender scaffolding that defines my existence and I have made them a permanent part of it. I am used to them and they are a part of my world. That is to me a far more intimate expression of feeling than roses and chocolate and dinner dates. I have at times tried to express this experience to my wife, but she becomes upset because she doesn't understand the depth behind the statement "I am used to you". To me it is far more intimate than 'I love you' and far more passionate than any physical touch, although I provide that as often as needed because I understand that she needs it, even when I don't.
We build around ourselves such complex structures to define us and order a world which is beyond chaos and which all too easily overloads and overwhelms us.
I am married to a woman who has supported, understood and put up with my very neurotic and often unintentionally hurtful self for eight years. We have three beautiful, difficult, painfully complex children and another on the way. Yes, it's possible for autistics to find and keep relationships, but it requires work from both people and a support system that can explain the insanity of both each to the other.
To my children and my wife, I am used to you.


text:
RECORD breaking climber Vanessa O'Brien talked to students about challenge and resilience, in a visit to Ilkley Grammar School.
The British-American mountain climber, explorer, public speaker and former business executive - who holds the 2015 Guinness World Record holder for climbing the Seven Summits in 295 days and The Explorers Grand Slam in 11 months - visited the school on Friday (September 18).
She talked to the students about challenge and resilience, key qualities for both record breaking mountain climbing and doing their best at school. The school were also thrilled to receive a signed copy of the Guinness Book of records for the Library.
Assistant Headteacher, Chris Washington, said: "It's fantastic that Ilkley Grammar school has been lucky enough to host Vanessa, when the opportunity came up it was simply too good to miss, particularly for Year 7 who are just starting their KS3 'mountain challenge' in English.'
Vanessa, who was clearly impressed with the students later tweeted: "It was a top day at Ilkley Grammar School; such inspirational students."
Vanessa's next challenge is to climb Chimborazo in Ecuador to bring awareness to Climate Change for 25zero.com.


text:
Dear Church Family,
Last Sunday
Last week Pastor Chuck preached from Matthew 18:15-20. In these verses, Jesus calls the church to take an active role in the discipleship and discipline of its members. We are to be lighthouses for one another, putting a spotlight on the jagged rocks of sin that would dash our souls. The culture tells us that it is loving to ignore someone's sin, when in fact it is hateful and dangerous. We love each other best when we help each other grow up into Christ-likeness. When is the last time you lovingly pointed out a sinful blind spot to a brother or sister? How do you respond when someone tries to help you see a sinful blind spot in your own life? Let's be a church loving enough to do the hard work of watching out for each others' souls!
Financial Update
Thank you for your generosity in giving. Our finances are not ultimately ours but God's—and yet he is pleased when we are faithful stewards of what he has entrusted to us, and he uses our resources to further his gospel purposes on the earth. Currently, we are about $20,000 behind in our annual budget. Let's continue to give sacrificially as God leads, always remembering that our true treasure is in heaven. To give online, click here.
Your brother in Christ,
Phil Hoshiwara


text:
Full disclosure – this upcycling idea and tutorial was first published in July of 2014. But in the years since, the product I used to make these glass fishing floats went off the market. So, I started from scratch with new products, MUCH better photos, and the cutest Japanese glass floats that are simple and inexpensive nautical decor. Who says beach home decor can't come from the thrift store anyway? Read more ›
Glass Fishing Floats / Japanese Glass Floats for Nautical Decor
Upcycling a Beverage Cooler into a Wine Bucket or Small Wine Cooler
How about a crazy easy summer time project that requires no tools or effort whatsoever? This is one of those ideas of mine that *almost* doesn't warrant a blog post because it's so dang simple. BUT- I've learned through your comments that even when I have a simple project idea, I should always publish it here on the blog. So, here we are…with a thrifted beverage cooler that I upcycled into a wine bucket or small wine cooler!Read more ›
Cheese Shakers as Milk Glass Vases with a Flower Frog
There are certain things that I always find on my thrifting adventures and yet, always pass by- and one of those used to be cheese shakers. I never had a fun or fresh idea for them…until recently. And once I had a vision in mind, I just couldn't shake it (pun intended)! I wondered if a shaker top would act as a flower frog or floral frog to create flower centerpieces. Read more ›
From Outdated to Outstanding: My One-Stop Snack Station
The time has come for a new post for The Good Life Blog. And this time around, I took a couple of frequently-spotted thrift store items…fused them together…and gave them a fun makeover. In particular, I'm working with an outdated wooden coaster set- you've probably seen them yourself! Coaster sets (usually a dark stained wood) that look like small pieces of furniture…Read more ›
How to Upcycle Vintage Planters into Mosquito Candles / Citronella Candles
Let's face it – summer is NOT my favorite season in the South. Between a constant case of "humidity hair" to a melted make-up sweaty glow, it just leaves much to be desired. But, I still love sitting out back and listening to the summer yard sounds as the sun goes down. So, I desperately needed to make some citronella candles or mosquito candles that would keep the bugs away.
Avoid Plastic / Reduce Plastic with Reusable Produce Bags
Like so many other eco/cost-conscious shoppers, I love to bring my own tote bag(s) to carry my groceries home. Any time I can avoid plastic or reduce plastic, I take it! So, I decided to make some reusable produce bags (i.e., veggie bags or fruit bags) by upcycling some kids t-shirts. They are plentiful at thrift stores and on a smaller scale that is perfect for produce!Read more ›
EmbROADery: A Summer Road Trip Keepsake
It probably comes as no surprise that Jon and I are road trip junkies. Living in Upstate South Carolina gives us access to much of the southeast and mid-Atlantic… and vacations often have a junkin'/Sadie-blogging aspect. Plus, driving our car allows us to fill it up with vintage and thrifted treasures. So marrying our love of road trips with my love of vintage road maps inspired this fun and easy project. Read more ›


text:
THE Harare City Council could find itself forking out $460 800 as compensation to former business ventures manager, Gadzamoyo Dewa after the latter sought to register an arbitral award granted in his favour by the Labour Court.
According to the court papers, Dewa was fired from Harare City Council over a decade ago following his unearthing of massive corruption in the EasiPark joint venture deal, which he alleged benefited council's top brass at the expense of ratepayers.
In March last year, Dewa filed a $1,5 million litigation against his former paymasters seeking compensation for his unlawful dismissal and ill-treatment by the top council officials, but the matter was never finalised.
Dewa said he worked for the City of Harare as an administrative assistant responsible for health services prior to being promoted to business ventures manager, a position he held until he resigned at the end of 2008.
The former manager further said he was re-engaged by the council to represent its interest in its joint venture with EasiPark in February 2011, but after some time the City of Harare's top brass allegedly subjected him to painful working conditions, which he alleged was meant to make him a sacrificial lamb, while plundering the council's resources.
Dewa said he was eventually dismissed from employment in December 2011 after which he then approached the Labour Court and was granted an order which he has now taken to the High Court for registration.
"Applicant (Dewa) contested against the unfair dismissal in the Labour Court and an order was issued affirming the arbitrator's arbitral award remains in force. Applicant, therefore, now approaches this honourable court seeking to have order granted by the Labour Court registered with this court," Dewa said in his declaration.
"The arbitral award sounds money and provides for a monthly salary of $7 200. That money should be paid from the date of suspension and deprivation to date. I have 64 months of deprivation and a total sum of $460 800 which should be paid within seven days.
"I should also receive my benefits that include, use of personal issue twin cab vehicle or equivalent, I must be restored any outstanding fuel of 20 480 litres being 80 litres per week for the 256 weeks. In addition I should get my laptop, phone, medical aid, funeral benefit and this should be paid with effect from December 12, 2011."
In his application Dewa also said sometime in December last year EasiPark, now called City Parking, sought confirmation of a draft ruling order, but the application was dismissed by the High Court, which then ruled that the arbitral award granted by the Labour Court remains untampered with and in force.


text:
Saturday, August 27, 2011
My Magazine Modeling Superstars (the evidence)
Adoptive Families Magazine arrived in today's mail along with our runner-up prize (a pretty cool Putamayo CD) and I just thought you'd like to see the evidence (or, perhaps, the genesis) of our profound celebrity. We're on page 6 - pick it up... um... wherever magazines for adoptive families are sold.
Labels: adoption
3 comments:
- Shelly said...
YAY!!! Love it!
- August 27, 2011 at 11:00 PM
- Lou Ann said...
Cool!! Can I get your autographs when I get my copy?
Thanks for your kind words about Kit.
Lou Ann & Lexie too
- August 28, 2011 at 1:05 AM
- Rayna said...
love it!! so cool!
- August 28, 2011 at 1:21 PM
Subscribe to: Post Comments (Atom)


text:
|Rasa Sayang
|Season 5, Episode 4
|Air date
|August 6, 2011
|Episode guide
| Previous
Bila Besar Nanti
| Next
Kisah Raja Pemburu
At home, Upin and Ipin are exciting to go picnic tomorrow and they must prepare everything that Ms Jasmin adviced. The next day, everyone started to line up when Upin and Ipin come. Then the entire class with Ms Jasmin walk into the forest for the picnic area while singing the "Rasa Sayang" song. After that, they have a great picnic together with treats, foods and many things.
Community content is available under CC-BY-SA unless otherwise noted.


text:
Group Tasting for 13-25 People
Groups of 13-25 may book a sit down tasting conducted on our pavilion. Theses group tastings are only scheduled at 11am. Tasting fees include a logo glass to keep. Every person in the group must choose one of the following tasting options: 10 tastes for $20, 5 tastes for $15, non-tasters $6. Non-tasters also get the logo glass to keep and may choose from a selection of our Hosmer sodas or bottled water. The group must stay together during the tasting. Please be aware that groups larger than 12 are not permitted on our covered pavilion and so options for remaining on site after the tasting are limited to the picnic or patio area.
Reservations of this size require a deposit of $50.
Please fill out the inquiry and you will be contacted to confirm your reservation.


text:
The American Hospital Association recently held its Rural Hospital Policy Forum in Washington, D.C.
The American Hospital Association gave leaders of hospitals in rural areas updates on the organization's legislative and policy priorities, and leaders heard from several speakers, according to a press release.
Sen. Chuck Grassley (R-Iowa), the primary supporter of both the Rural Emergency Acute Care Hospital Act and Rural Hospital Access Act, discussed the legislation and how crucial such hospitals are in the economy of their areas, the release said.
Grassley also discussed the 340B Drug Pricing Program, which gives hospitals that serve those who are poor, disadvantaged or don't have insurance the ability to reduce the cost of outpatient medications and offer more health services.
George Sigounas, Health Resources and Services Administration administrator, also addressed the 340B Drug Pricing Plan at the opening meeting and he stressed that hospitals in rural areas need the plan, according to the release.


text:
Pseudomonas aeruginosa is an opportunistic pathogen that causes a number of infections in humans, but is best known for its association with cystic fibrosis. It is able to use a wide range of sulfur compounds as sources of sulfur for growth. Gene expression in response to changes in sulfur supply was studied in P. aeruginosa E601, a cystic fibrosis isolate that displays mucin sulfatase activity, and in P. aeruginosa PAO1. A large family of genes was found to be upregulated by sulfate limitation in both isolates, encoding sulfatases and sulfonatases, transport systems, oxidative stress proteins, and a sulfate-regulated TonB/ExbBD complex. These genes were localized in five distinct islands on the genome and encoded proteins with a significantly reduced content of cysteine and methionine. Growth of P. aeruginosa E601 with mucin as the sulfur source led not only to a sulfate starvation response but also to induction of genes involved with type III secretion systems.
Pseudomonas aeruginosa is a well-studied, opportunistic pathogen that causes a wide range of infections in humans, posing particular risk to patients with burn wounds, immune system deficiencies (e.g., AIDS), and cystic fibrosis (31, 32). It displays a high level of metabolic versatility, manifested in the presence in its genome of an unusually high number of genes encoding transcriptional regulators and transport systems for uptake of organic compounds into the cell (45). This allows the cell to make a flexible and tightly regulated response to conditions where particular nutrients become limiting. Under conditions where the supply of inorganic sulfate becomes limiting, for example, P. aeruginosa PAO1 can grow at similar rates by desulfurizing a range of organosulfur compounds such as sulfate esters and sulfonates, since it responds to inorganic sulfate limitation by synthesizing a range of proteins for uptake and utilization of these alternative sulfur sources (34). However, synthesis of these enzymes is controlled by several different nutritional constraints, and whereas expression of the arylsulfatase AtsA is controlled by sulfur supply and provides sulfur for cell growth (3, 19), the SdsA1 alkylsulfatase catalyzes the first step in mineralization of long-chain sulfate esters (14) as a carbon source, and its expression is independent of sulfate concentration. Proteomic studies have shown that sulfur supply also controls the synthesis of some of the enzymes of sulfate assimilation to cysteine in P. aeruginosa (34), and at least two corepressors are important for this control (18). Similar responses have been observed in Escherichia coli (35) and Bacillus subtilis (2), where they appear to be controlled in part by the availability of adenosine-5′-phosphosulfate and S-adenosylmethionine, respectively (2, 4). Further studies of the E. coli sulfate starvation response by microarray expression analysis (13) showed that it is dominated by increased expression of the cysteine biosynthetic pathway but is also controlled by nitrogen supply as part of an overall starvation response dependent on the RpoS protein (12).
Studies of the sulfate starvation response in P. aeruginosa have concentrated on the well-characterized isolate PAO1 (18, 34). However, P. aeruginosa strains isolated from the cystic fibrosis lung are often genetically significantly different from strain PAO1 (55). They are also exposed to a very different set of sulfur-containing molecules, since they colonize the bronchiolar lumen as microcolonies attached to mucus components such as the sulfated respiratory mucin (28, 44). A number of reports have shown that the level of mucin sulfation is enhanced in cystic fibrosis patients (7, 29, 57), and this may well be a defense response in itself, since mucin sulfation is known to prevent degradation of the mucin by bacterial enzymes (48). Mucin sulfatase activities have been found in anaerobic bacteria such as Bacteroides and Prevotella spp. (36, 48), but mucin desulfation has also been reported for several clinical isolates of P. aeruginosa (20). In the P. aeruginosa strains, mucin sulfation seems to be independent of the characterized arylsulfatase activity, suggesting that cells may react differently to exposure to mucin as a sulfur source than to sulfate limitation alone.
This study was initiated to address the questions of which sets of genes are regulated by sulfur supply in P. aeruginosa and whether these are altered in a cystic fibrosis patient isolate on exposure to mucin. Here, we report that under sulfate-limited conditions, a cystic fibrosis P. aeruginosa isolate was able to utilize mucin as the sole sulfur source, although P. aeruginosa PAO1 did not do so. Under these conditions, P. aeruginosa cells showed three groups of transcriptional responses, including a complex sulfate starvation response, a specific response to mucin as the sulfur source, and a more general response to mucin exposure.
Bacteria and growth conditions.Pseudomonas aeruginosa PAO1 (45) and the nonmucoid cystic fibrosis isolate P. aeruginosa E601 (20) were grown at 30°C in minimal medium with 25 mM succinate as the carbon source (24), supplemented with all proteinogenic amino acids with the exception of cysteine and methionine (40 μg/ml of each amino acid). Sulfur sources were added as described in the text, at concentrations of 500 μM for sodium sulfate or cyclamate and of 25 μg/ml for human colon cell line mucin LS174T or pig mucin. Glassware used for sulfur-limited work was washed with 3 M HCl before use. Cultures used for RNA preparation were grown as aerobic 5-ml batch cultures in 50-ml Erlenmeyer flasks on an orbital shaker (225 rpm). Growth experiments were performed with 200-μl cultures in microtiter plates with continuous shaking, using a Synergy HTi plate reader (BioTek Instruments), and growth rates were determined in the exponential growth phase by measurements of A600 taken every 10 min over 18 h.
P. aeruginosa K1040M was constructed from P. aeruginosa 1040 (met-9011 pvd-9 tonB1::Hg) (58) by replacement of the methionine auxotrophy by transduction using phage E79-tv2 grown on strain PAO1. This strain was grown in iron-supplemented medium as previously described (58). Other cystic fibrosis P. aeruginosa isolates used included three epidemic strains (C3373, LES400, and LESB58 [42]) and a selection of other isolates from the University of Liverpool laboratory collection (strains 49211, 49244, 59032, 59039, 59070, and 59079).
Preparation of mucin.The human colon adenocarcinoma cell line LS174T, a cell type which forms well-differentiated goblet cells, was obtained from the European Collection of Animal Cell Cultures (Porton Down, United Kingdom). The presence of O-sulfate esters on LS174T secretory mucins has been demonstrated previously in [35S]sulfate incorporation studies (5). For production of human mucin LS174T, cells were grown as monolayers in Dulbecco's modified Eagle's medium supplemented with 4 mM glutamine, 10% (vol/vol) fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin in 5% CO2-95% air at 37°C. The medium was harvested 2 to 7 days after confluence was achieved, and the mucin was purified by gel filtration over Sepharose CL-2B minicolumns (5 by 1.5 cm), eluting with phosphate-buffered saline (pH 7.4) containing 0.01% (wt/vol) thimerosal. Mucin-containing fractions were desalted over PD10-Sephadex GM25 columns, lyophilized, and stored at 4°C. Pig gastric mucin (type III) was obtained from Sigma.
RNA preparation and reverse transcriptase-PCR.Cells used for the preparation of RNA were grown to exponential phase (optical density at 600 nm, 0.2), harvested at 4°C, and immediately frozen in liquid nitrogen. Total RNA was prepared with an RNeasy mini kit and an RNase-free DNase set from QIAGEN (Hilden, Germany), by following the protocol of the manufacturer. For each growth condition, RNA was prepared from three or four independent batch cultures harvested at the same optical density at 600 nm (within 0.05 optical units).
The quality of RNA preparations was routinely checked on 1% agarose gels (39), and by reverse transcriptase-PCR of the sbp gene PA0283 (reverse primer, CGACGATCTCGAAGTTTTCC; forward primer, ATCGACGAACTGCACAAGC). Reverse transcriptase-PCR was performed by a two-step protocol, using 1 μg of RNA and the First Strand cDNA synthesis kit (MBI-Fermentas). The same PCR setup was used to check RNA preparations for the absence of contaminating DNA prior to reverse transcription.
DNA microarray hybridization.Frozen batches of RNA were thawed and used only once. Reverse transcription of RNA, cDNA fragmentation, and hybridization followed the protocol recommended and provided by Affymetrix (Santa Clara, CA). Briefly, reverse transcription of 5 to 10 μg of P. aeruginosa RNA was performed using 750 ng of random hexamer primer mix and Superscript III transcriptase (Invitrogen). The cDNA product was purified with the QIAquick PCR purification kit (QIAGEN). Purified cDNA (3 μg) was then fragmented for 6 min at 37°C using DNase I, and the fragmented cDNA was biotin labeled and hybridized for 16 h at 50°C to an Affymetrix P. aeruginosa chip (P. aeruginosa genome array; Affymetrix). Microarrays were further processed in accordance with the recommended protocol of Affymetrix. Experiments were performed with either threefold or fourfold biological replication. Hybridization of genomic DNA was carried out similarly, after fragmentation of genomic DNA samples using the protocol provided by Affymetrix.
Analysis of microarray data.Image analysis of the scanned arrays for the presence and absence of RNA transcripts was done using the MAS 5 software package with the following parameter settings: α1 of 0.04, α2 of 0.06, τ of 0.015, TGT value of 400, and norm factor of 1.0. Array images were checked visually for any irregularities in hybridization using dChip 1.3 (30, 59). Array data were normalized using the GC-RMA algorithm (56), and the data were then further analyzed using the microarray software package Genespring GX 7.3 (Agilent). Genes whose levels of expression were significantly influenced by sulfur source (P < 0.05) were identified with Genespring GX, using a Benjamini-Hochberg multiple-testing correction and a false detection rate of 5%. Significance analysis of the microarray results was performed using the SAM algorithm (49), with false detection rates of <5%.
Validation of microarray results with quantitative reverse transcriptase-PCR.The accuracy of the microarray data was verified by quantitative reverse transcriptase-PCR analysis of 10 selected genes displaying high and low levels of change in the microarray analysis (see Table S1 in the supplemental material). PCR amplification was performed using an ABI Prism 7000 cycler (Applied Biosystems) with two-step TaqMan reverse transcription and a SYBR green PCR master mix kit (Applied Biosystems) and 100 ng of template RNA, in accordance with the protocol of the manufacturer. The calibration curves for each primer pair were constructed with purified PCR product as the template. Experiments were repeated three times for each of the biological triplicates of the corresponding microarrays.
Microarray data accession number.Lists of induced and repressed genes can be found in the supplemental material. The gene expression data are available from the Gene Expression Omnibus (GEO) under accession number GSE8408.
Growth of P. aeruginosa with mucin as the sulfur source.Although many Pseudomonas species express arylsulfatases (22), the ability to desulfate mucin is less common. Pseudomonas mucin sulfatase activity was first identified in the cystic fibrosis isolate P. aeruginosa E601 and is not present in the sequenced strain P. aeruginosa PAO1 (20). Growth of these two P. aeruginosa strains was tested in a succinate-based minimal medium with sulfur supplied as sulfate, cyclamate, model human mucin LS174T, pig mucin, or cyclamate mixed with human mucin LS174T (Table 1). For comparison, growth with mucin LS174T or with sulfate was also tested for a range of nine cystic fibrosis isolates selected from a previous study (42), including two different isolates of the Liverpool epidemic strain (strains LES400 and LESB58) and an isolate of the Manchester epidemic strain (strain C3373). Minimal medium containing no sulfur source was used as a negative control. Strain E601 grew with sulfate, cyclamate, human mucin, and the combination of cyclamate with mucin but not with pig mucin. A small pulse of growth (up to an A600 of 0.1) was observed in the initial part of the experiment, due to exhaustion of residual sulfate in the growth medium, and exponential growth rates (Table 1) were determined after exhaustion of this contaminating sulfate. Growth was fastest with sulfate, as a preferred sulfur source (23). Although growth with mucin as the sulfur source was 35-fold slower than with sulfate, it was consistently above the slow growth observed in sulfur-free medium or with pig mucin, demonstrating that the human mucin acts as a specific growth substrate. The other cystic fibrosis P. aeruginosa isolates tested grew at rates similar to that of strain E601 with human mucin as a sulfur source but were unable to grow with pig mucin. The ability to grow slowly with LS174T mucin as a sulfur source therefore seems to be typical for these clinical isolates. By contrast, strain PAO1 was unable to desulfurize either human mucin or pig mucin, though it grew well with sulfate, cyclamate, or the combination of cyclamate and human mucin (Table 1).
Genome comparison of P. aeruginosa strains PA01 and E601.Pseudomonas aeruginosa E601 is a recent clinical isolate and was therefore expected to be genetically different from strain PAO1, which has been in laboratory culture for several decades. Differences between the genomes of different isolates of P. aeruginosa and strain PAO1 have been reported to vary, with 10 to 17% of genes being present in other isolates but not in P. aeruginosa PAO1 (43). The genomic differences between strains PAO1 and E601 were investigated by hybridization of the total genomic DNA from each strain to the Affymetrix P. aeruginosa PAO1 GeneChip. The P. aeruginosa E601 DNA hybridized to 5,198 of 5,570 annotated open reading frames (ORFs) of the P. aeruginosa PAO1 GeneChip and to six intergenic regions and one non-PAO1 gene that did not give a signal with genomic DNA from P. aeruginosa PAO1. Overall, the genome cross-hybridization showed 97% coverage for the detection of transcripts between the two species, thus allowing the reasonable usage of the P. aeruginosa PAO1 chip with P. aeruginosa E601. No further attempt was made at this stage to identify additional genes that were present in the genome of strain E601 but not in strain PAO1.
P. aeruginosa sulfate starvation response—upregulation of gene families.Expression levels of specific P. aeruginosa genes are expected to change when mucin rather than sulfate is supplied as the sole sulfur source, primarily because many genes are upregulated in the absence of sulfate as part of the sulfate starvation response (34). However, changes were also expected to include specific mucin-induced genes and changes in gene expression due to growth rate effects, since growth with mucin is considerably slower than with sulfate (Table 1). To avoid these growth rate effects, we compared gene expression in sulfate-grown cells with that in cells grown with cyclamate (N-cyclohexylsulfamate) as an alternative sulfur source. Cyclamate allows a growth rate comparable to that observed with sulfate (Table 1) and acts as a synthetic, chemically defined surrogate for N-linked mucin. In addition, the use of cyclamate-grown cells facilitates a comparison with previous proteomic studies of P. aeruginosa PAO1 (34). The sulfate starvation responses of P. aeruginosa strains PAO1 and E601 were examined by isolating the total RNA of each strain from exponential-phase batch cultures grown with sulfur supplied as cyclamate, sulfate, or cyclamate and sulfate together, followed by GeneChip analysis. Cells were harvested at the same optical density in order to minimize growth-phase-dependent effects.
Growth with cyclamate in the absence of added sulfate led to the differential upregulation (greater than twofold; P < 0.05) of 132 genes in both of the two strains studied. It is important to note that under these conditions, cells are not starved for sulfur, but the absence of inorganic sulfate leads to the change in gene expression (34). Transcriptomic responses can vary dramatically between different strains of the same species (38), and comparison of these gene lists showed that 155 and 134 genes were upregulated in strain PAO1 and strain E601, respectively, while 67 and 273 genes were downregulated more than twofold in the two strains, respectively (Fig. 1; see Table S3 in the supplemental material). Of the 132 upregulated genes, 43 were strongly upregulated (>10-fold) (Table 2). When differentially regulated genes were classified by metabolic category (Fig. 1), broadly similar patterns were observed for the two strains. The most strongly upregulated groups of genes correspond to hypothetical and conserved hypothetical proteins, but a large group of genes related to transport functions was also upregulated. Interestingly, we detected significant downregulation of genes related to energy metabolism (Fig. 1). Comparison of sulfate-grown cells with those provided with mixed cyclamate and sulfate revealed that only two cyclamate-upregulated genes were not repressed by cyclamate-sulfate (PA2298 and PA2299), suggesting that these may be specifically cyclamate induced.
Although cells grown with cyclamate as a sulfur source grow at a rate comparable to that for cells grown with sulfate, it has been suggested that part of the sulfate starvation response may be the synthesis of proteins with reduced levels of cysteine and methionine (24). The predicted amino acid composition of the proteins encoded by the sulfate starvation-induced (SSI) genes was calculated (Fig. 2A) and provided startling confirmation of this, with 77% of the SSI genes encoding proteins with below-average content of S-amino acids (Fig. 2A) (the S-amino acid content was calculated as the number of encoded Cys and Met residues per ORF, not including fMet, relative to the overall ORF length) and >90% of them having a below-average cysteine content. The regulated genes also fell into distinct regions on the genome (Fig. 2B), forming five islands where most of the sulfate-starvation-induced genes were located (PA183 to PA199, PA2050 to PA2094, PA2289 to PA2359, PA3441 to PA3450, and PA3931 to PA3954) and within which >50% of the genes were upregulated greater than twofold during sulfate limitation. These islands also contained enhanced levels of genes encoding low-sulfur proteins (LSPs) (defined as ORFs in which the percentage of encoded cysteine and methionine residues [not including fMet] per ORF was <0.64%, i.e., more than 1.5 standard deviations below the average value for all the ORFs on the genome of 2.87%.) (Fig. 2C), though it should be noted that there were also many LSP-encoding genes that were not strongly regulated by sulfur supply. The clustering of genes encoding proteins with reduced sulfur content has been reported before for E. coli (37) and Pseudomonas putida (41), but a correlation with changes in gene expression was not reported.
Although only putative function can be assigned to the upregulated group of hypothetical proteins, orthologue analysis (clusters of orthologous groups [COGs]) (47) yields valuable additional information. Forty-nine of the 132 genes upregulated during sulfate limitation in both strain PAO1 and E601 belong to only nine COGs. The dominant function in these clusters is transport: eight genes appear to encode proteins related to substrate-binding proteins of sulfonate-dependent ABC transporters (COG715) while the cognate permease (COG600; six genes) and ATPase (COG1116; five genes) components of these transporters are also detected as being upregulated under conditions of sulfur limitation. A small group of HisM-type amino acid permeases is also upregulated (COG765; four genes). The other dominant COG families are classes of enzymes with representatives acting in sulfonate degradation of organosulfonates, including FMNH2-dependent monooxygenases such as SsuD and MsuD (COG2141; eight genes) and α-ketoglutarate-dependent dioxygenases such as TauD and AtsK (COG2175; five genes). Upregulation of multiple uncharacterized members of these five COGs under sulfate-limited conditions suggests that the encoded proteins catalyze the uptake and desulfurization of a variety of environmentally relevant sulfonates and/or sulfate esters.
Interestingly, the cysteine biosynthetic pathway was only weakly upregulated by sulfate limitation in P. aeruginosa, with only the sulfate transporter genes sbp and cysTWA being derepressed more than twofold during growth with cyclamate. This contrasts strongly with the changes observed in E. coli during growth in the absence of sulfate (13), where all the genes of the cys regulon are upregulated under sulfate starvation conditions and the cells do not upregulate multiple members of particular COG families. Sulfate limitation was linked to an oxidative stress response, as also observed in E. coli, with upregulation of ahpC (alkylhydroperoxide reductase), katB (catalase), ohr (organic hydroperoxide resistance protein), and lsfA (thiol-specific antioxidant). Multiple members of two additional COGs potentially related to sulfur metabolism were also upregulated. The most interesting of these was COG1960 (six genes), which includes proteins with homology to acylCoA dehydrogenases, some of which are known to be involved in desulfurization of dibenzothiophene and methanesulfonate in this and other species (DszC and MsuC, respectively) (11, 25). With the exception of DszC (which acts as an FMNH2-dependent monooxygenase in Rhodococcus [11]), the function of these proteins has yet to be clarified. A second group is made up of TonB-dependent receptor proteins (COG1629; four genes). Glutathione S-transferases (COG625) represent another large protein family connected to sulfur metabolism, with 18 members of this group on the P. aeruginosa genome, mostly of unknown function (53). No glutathione S-transferase genes, and indeed no other glutathione-related genes, were significantly regulated by shifts in the sulfur source, providing evidence that changes in the sulfur source for growth do not affect glutathione utilization or synthesis.
Role of the TonB complex in the sulfate starvation response.Three of the most strongly upregulated genes in the absence of sulfate were tonB2 (PA0197) and the two genes encoding TonB accessory proteins, exbB and exbD. The TonB-ExbB-ExbD complex is essential in the uptake of siderophores into the cell and also plays a role in the transport of vitamin B12 (8). Its potential importance in sulfur metabolism is underlined by the fact that several putative TonB-dependent receptors and transducers are upregulated during growth in the absence of sulfate. Recent work has shown that these outer membrane proteins act either as transporters or as transducers of external signals, depending on their domain structure (26), and are energized by the TonB-ExbB-ExbD complex in the cytoplasmic membrane and periplasm. The strong upregulation of the tonB2 gene under sulfate-limited conditions suggested that it might be required for uptake of alternative sulfur sources and contrasts strongly with the expression levels of tonB1 (PA5531) and tonB3 (17), which were unchanged under sulfate limitation. We cultivated a tonB2 mutant strain of P. aeruginosa (strain 1407 [58]) with a variety of different sulfur sources but found that it grew normally compared to the wild-type strain (data not shown). This suggested that the tonB1 gene (PA5531) might be able to complement the deficiency in tonB2. In contrast, TonB2 cannot fully compensate for a deficiency in TonB1, since growth of a tonB1 mutant strain (strain K1440M) required iron supplementation during growth with either sulfate or cyclamate (i.e., even when tonB2 expression was induced by sulfate limitation). We conclude that the sulfur-regulated TonB2 complex is important for uptake of an unknown sulfur-containing substrate. It is located on the genome in sulfur island I (Fig. 2B), in close proximity to a putative TonB-dependent receptor (PA0192) and a number of genes that are involved in sulfate ester metabolism (22), so it may be involved in utilization of an unidentified sulfate ester.
Sulfate starvation and the influence of mucin.Cells of P. aeruginosa E601 were grown in batch cultures containing human mucin as the sole source of sulfur, and RNA extracted from these cultures was used for GeneChip analysis. Compared to growth results with sulfate, provision of mucin as the sole sulfur source led to upregulation of a slightly larger group of genes than that observed during growth with cyclamate (see Table S4 in the supplemental material). This group included almost all the sulfate starvation-induced genes that were upregulated during growth with cyclamate, confirming that the observed sulfate limitation response is not specific to cyclamate as the sulfur source and is seen during both rapid and slow growth. Several additional genes were also upregulated under these slow-growth conditions, including exoenzyme S, components of the type III secretion apparatus, and multidrug efflux systems. However, most of these were also upregulated during rapid growth with mucin and cyclamate together, relative to growth with cyclamate alone, and they therefore constitute the cells' response to mucin independent of the overall sulfate starvation response.
Pseudomonas aeruginosa is known to attach to mucin produced by the lung epithelium of patients suffering from cystic fibrosis (6, 9), and it has been suggested that P. aeruginosa uses mucin as a carbon source (1). In this report we show that P. aeruginosa E601 and other cystic fibrosis isolates are also able to utilize a model human mucin as a sole sulfur source for growth. Strain E601 responds to these growth conditions by upregulation of a general sulfate starvation response (Table 2) and a specific response to the presence of mucin that includes genes that encode a number of virulence factors.
The homeostatic response of P. aeruginosa to sulfate limitation involved the upregulation of some 132 genes (>2-fold, P < 0.05) (see Table S2 in the supplemental material), with 44 of these showing >10-fold regulation (Table 2). Regulation of these genes by sulfur supply was observed in two different strains of P. aeruginosa, the cystic fibrosis isolate E601 and the laboratory strain PAO1. Some additional genes were upregulated in each strain specifically, but these were considered less relevant for the present study. Importantly, the same set of SSI genes was observed during growth with cyclamate and with mucin for both strains investigated, and this included the genes encoding all the SSI proteins identified in a previous proteomic study (34). Compared with that of other bacteria, the P. aeruginosa SSI response is quite extensive. E. coli, for example, responds to sulfate starvation (growth with taurine or glutathione) with changed regulation of 64 genes (13), while in Bacillus subtilis some 56 genes were similarly regulated (growth with methionine) (2). The most-expected response to sulfate limitation is increased transcription of the cysteine and methionine biosynthetic pathways, and this is indeed observed in E. coli, with all the genes of the cys regulon and several methionine biosynthesis genes upregulated under sulfate starvation conditions (13). In P. aeruginosa, however, this was not observed, and although expression of sulfate transport genes was increased, the rest of the cysteine and methionine biosynthesis pathways were not significantly affected. In B. subtilis, there was upregulation of cysteine biosynthesis, but since methionine was supplied as the derepressing growth substrate, no conclusions can be drawn concerning methionine biosynthesis (2).
The P. aeruginosa genome is characterized by the presence of larger numbers of regulatory proteins and uptake systems than in most other bacteria whose genomes have been sequenced (27). This allows a flexibility of response to different environmental conditions and helps explain the ability of this species to colonize a wide variety of habitats. The P. aeruginosa SSI response observed here reflects this genomic flexibility, since its main feature is the upregulation of families of related genes. Many of these are of unknown function, and they are likely to catalyze uptake and desulfurization of unidentified sulfur-containing substrates. B. subtilis also increases transcription of a range of transport systems under sulfate limitation, but E. coli appears to be limited to the two characterized sulfonate utilization operons, ssu and tau (50, 51). All three species respond to sulfate starvation with the synthesis of proteins that help to counter oxidative stress, such as catalases and peroxidases. It has been suggested that this may be linked to peroxide production by autoxidation of enzymes containing flavin adenine dinucleotide, such as sulfite oxidase (13). However, all three species studied respond to sulfate limitation by strongly upregulating the expression of FMNH2-dependent monooxygenases (mainly sulfonatases such as SsuD or MsuD; in P. aeruginosa, no fewer than eight genes of this family were upregulated) and the corresponding flavin mononucleotide (FMN) reductases. The resulting increase in FMN reductase activity will lead to enhanced levels of reduced FMN in the cell, and if the appropriate sulfonatase substrates are not present, rapid autoxidation will release active oxygen species, potentially causing the observed oxidative stress response.
Comparison of the sulfate starvation responses observed in E. coli during growth with taurine (rapid growth) and glutathione (slow growth), and during various shifts in sulfur supply, led to the conclusion that a set of responses that occurred both for N and S limitation were in fact consequences of slow growth and were controlled to a great extent by the RpoS protein (12, 13). We were therefore surprised to observe that in P. aeruginosa, the SSI response seen with cyclamate (rapid growth) was very similar to that observed during slow growth with mucin as the sulfur source. Of all the sulfate starvation-regulated genes (see Table S3 in the supplemental material), only six corresponded with P. aeruginosa genes found to be RpoS controlled in a previous study (40), suggesting that RpoS does not play a large role in the homeostatic sulfate starvation response in this species. However, other sigma factors are also important in the SSI response of Pseudomonas species. In P. putida, for example, the RpoN sigma factor is known to play a role in sulfur metabolism, controlling expression of the sfn genes that are required for dimethylsulfone metabolism (10). Very few of the P. aeruginosa SSI genes carried a conserved RpoN-binding site (46), but these included the P. aeruginosa sfn orthologues PA2346 and PA2347 and the nearby msuD and msuE genes, which are involved in methanesulfonate metabolism (25). Apart from these, only the porin gene oprB (PA3186) appeared to be RpoN controlled. In P. putida, expression of the sfn genes also required the CysB protein, which is a LysR family regulator of sulfur metabolism in a number of gram-negative species. In P. aeruginosa, CysB has been shown to be required for the utilization of sulfate esters and to be involved in sulfonate utilization (19, 52), but the extent to which it controls the other SSI genes reported here has not yet been investigated. A consensus CysB-binding sequence has been reported in Salmonella enterica serovar Typhimurium (16), but it is not well conserved, and a search for conserved sequences upstream of the P. aeruginosa SSI genes (Table 2) was unsuccessful.
The largest group of sigma factors in P. aeruginosa is that associated with extracytoplasmic function (the ECF group), of which the best characterized are AlgU and PvdS (15, 21). The 19 ECF sigma factors found in P. aeruginosa reflect the versatility of this species in adapting to a variety of different habitats and responding to environmental signals (33). Under sulfate-limited conditions, P. aeruginosa upregulated transcription of one of these ECF sigma factors, PA2093, and this transcriptional activation was further increased when mucin was present in the growth medium. This gene was located within a larger cluster of sulfate-regulated genes (PA2087 to PA2094) within sulfur island II and was associated with a TonB-dependent transducer system and a putative sulfonatase gene, suggesting that it is involved in sensing extracellular sulfate limitation and especially in responding to the presence of mucin as a sulfur source in the environment. Cells of P. aeruginosa also responded to the presence of mucin with increased transcription of several genes involved in the synthesis of the type III secretion system and exoenzyme S. The upregulation of the type III secretion system genes under the conditions tested here suggests that the cells recognize mucin not only as a source of sulfur but also as a component of the host environment and induce the type III secretion system accordingly. Previous studies on the response of P. aeruginosa to mucus exposure have identified the migA and npt20 genes (PA0705 and PA5499, respectively) as mucin induced (54). These genes were not induced in the present study, possibly because the concentrations of mucin applied here were much lower.
We are grateful to Gail Preston for assistance with the RpoN analysis, to Daniel Jameson, Andrew Hayes, and Leo Zeef for advice on analyzing the microarray data, and to Carola Tralau for technical assistance. We thank the University of Washington for transposon mutants of P. aeruginosa.
This work was supported by a grant from the Biotechnology and Biological Sciences Research Council to M.K. and by a CNRS ATIP grant to S.V. We are grateful for support from Cystic Fibrosis Foundation Therapeutics.
- Received 7 June 2007.
- Accepted 25 July 2007.
↵▿ Published ahead of print on 3 August 2007.
↵† Supplemental material for this article may be found at http://jb.asm.org/.
- American Society for Microbiology


text:
In progress
Raised ironwork drain cover centre of road opposite 49 Byfield Road
Reported in the Loose / Raised/Sunken category anonymously at 12:22, Saturday 18 January 2020
Sent to Northamptonshire County Council 4 minutes later. ref: 1946537.
Contractors for Taylor Wimpey repaired this last year using concrete. Raised edge of drain cover now an issue as concrete is sinking. No top coat of tarmac.
Thank you for reporting a fault. We will use the information you have provided to assess and prioritise this fault in accordance with our published response times for fault reports.
State changed to: Investigating
Posted by Northamptonshire County Council at 12:25, Saturday 18 January 2020
Thank you for your enquiry - A works order has been raised to repair defects that meet NCC investigatory levels. To be undertaken within 26 weeks.
State changed to: Action scheduled
Posted by Northamptonshire County Council at 15:33, Thursday 23 January 2020
Provide an update
Your information will only be used in accordance with our privacy policy


text:
This site uses cookies to provide you with more responsive and personalized service and to collect certain information about your use of the site. You can change your cookie settings through your browser. If you continue without changing your settings, you agree to our use of cookies. See our Privacy Policy for more information.
Have you lost someone close to you? Have you grieved properly? Who helped you walk through such a significant loss? Jennifer Fornelli joins Kelli and Steve for a Biblical look at grieving. We don't need a counseling degree to support a grieving friend, just patience, flexibiltiy and sensitivity to the Holy Spirit.
Kokomo mom of three, and now homeschool teacher, Jen Fornelli still uses her Family Counseling degree to console and encourage her fellow neighbors. Jen earned her master's degree from Indiana University, with a dual focus in Marriage and Family Therapy and Mental Health Counseling. Fornelli combines evidenced based treatment and family system approaches with a Christian worldview. She is a member of the American Association of Marriage and Family Therapists and the American Association of Christian Counselors.
Mornings with Kelli and Steve helps start your day with spiritual encouragement, fresh conversation on current topics, and Christ-centered music. Join Kelli Thompson and Steve Hocker for updates, weather, community events, interviews, and features, as well as opportunities to join the Christ-centered conversation.


text:
Timeline of Truth – Review (Genesis 1 – Exodus 10)Thursday, February 20th, 2014
As we swing back into our Timeline of Truth study series, we take time to review the series as far as we have gone to date.
As we swing back into our Timeline of Truth study series, we take time to review the series as far as we have gone to date.
Upon threats of Esau overhead by his mother Rebekah, Jacob is encouraged by Rebekah and instructed by Isaac to find a wife from Laban's daughters. Along the way, God meets Jacob.
We take a closer look at what transpired when Isaac told Esau to prepare to receive his blessing. Was Isaac ignoring the communicated will of God? God had told Rebekah that the "older would serve the younger."
Isaac makes some bad choices, but eventually shines when confront with people problems and business issues. He reflects in his life what Romans 12:17-18 command us to be like:
Romans 12:17-18
17 Repay no one evil for evil. Have regard for good things in the sight of all men.
18 If it is possible, as much as depends on you, live peaceably with all men.
We do a whirlwind review of the first 2000 years of history, represented in the first 25 chapters of Genesis, using our 12-point bible overview as a backbone.
A father obtaining a bride for his 40-year-old son? That's what Abraham did for his son Isaac. This is an amazing account!
Abraham waits 25 years for God to give him a son. Now, 15 years or more after his son Isaac is born, God commands Abraham to offer Isaac as a sacrifice? How does Abraham respond?
After 25 years of promising, God keeps his promise to Abraham and Sarah — they have a son. God can and will keep any and all promises He makes!
Abraham knew. Apparently Sarah did not. How do you tell your childless 89-year-old wife that she will have a baby? Funny? Sarah thought so!
Get this. Abram is NINETY-NINE YEARS old. God has promised to make him a father of many nations, among other things. As far as we know from the Bible, Abram hasn't heard from God for 14 years (since around the time Ishmael was born). Abram is still waiting, living one day at a time, and God again appears with great news. Funny news…


text:
A brother gives it to me
Watch video brother gives sister creampie closeup on redtube home of free creampie porn videos and teens sex movies online video length 106 starring hot amateurs gone wild in this caucasian vaginal sex video.
Watch bibi miami gives her brother a handjob on pornhubcom the best hardcore porn site pornhub is home to the widest selection of free big tits sex videos full of the hottest pornstars if youre craving kink xxx movies youll find them here.
Watch video mom my brothers horny blonde big boobs wife sucked and fucked me on redtube home of free blonde porn videos and hd sex movies online video length 1147 uploaded by mom xxx starring pornstar kayla green.
Bsu political scientist doubts that dave letterman himself could win election against greg pence.
It is cool to admit that you love brother sister porn but do you watch the best brother sister xxx clips check out best sexo tube videos to see the hottest sex videos of.
brother juniper diane gibfried on amazoncom free shipping on qualifying offers brother juniper lives in the hills of assisi with father francis and seven other brothers brother juniper is the most generous of the brothers he is so generous that he will happily give the robe off his back to someone in needeven if that leaves him with.
asahel hebrew עשהאל ancient greek ασαέλ also known as asael was the youngest son of zeruiah herself either the daughter of jesse or daughter of nahash and stepdaughter of jesse the name means made by god asahel was the nephew of king david as well as the younger brother of both joab davids general and of abishaiasahel.