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This paper falls into three sections.
The first section provides a brief overview of organised violence and torture in Zimbabwe, mainly drawing on statistics from the 2001-2009 period, but in some cases going back further, Figures showing the kinds of abuse inflicted and its frequency provide evidence of the extent to which Zimbabweans have suffered during this period of complex emergency, when the infrastructure of the country has collapsed around them. Further surveys are presented to support the argument that damage to the psychological well-being of the population is likely to be one of the most serious and long-lasting effects of the ongoing crisis. Information gathered from war veterans underlines the persistant impact of unresolved trauma after the liberation war and figures relating to the gukurahundi, to elections and to Operation Murambatsvina show how specific events have increased the level of mental suffering. Furthermore, the effects of organized violence and torture in Zimbabwe must be contextualized within the global HIV epidemic (of which Zimbabwe is an epicentre) and the social effects of economic decline and mass impoverishment.
It is also argued that attention must be paid to the psychological well-being of those who have suffered indirectly , particularly women and children, as research suggests that the impact of violence and torture extends beyond those directly victimised. A second category of indirect' sufferers includes the impact of these forms of suffering on mental health professionals and human rights workers.
The effect of organised violence and torture on the social fabric of communities and the disempowerment which results both from individual suffering and from the fragmentation of relationships and systems which might have provided support or offered resistance is acknowledged as another important area for attention.


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As the province marks important acknowledgement of traditional Indigenous lands, Metrolinx is highlighting a map of our routes to show customers the hidden tapestry of historic territory and treaties right outside the windows of GO buses and trains. The view from the passenger seat may have seemed largely unchanged over the years. As passengers look … Continue reading Ontario Treaties Recognition Week – Find the lines of history on your GO Transit route
A sea of support for Orange Shirt Day
Traditional knowledge keeper shared history - passed along important message. Metrolinx employees gathered today (Sept 30) to commemorate Orange Shirt Day and learn about the impact of Indian Residential Schools, then and now Orange Shirt Day began in 2013 to educate and build awareness about the history and continued impact of the Indian Residential School … Continue reading A sea of support for Orange Shirt Day
Indigenous history explored through unique exercise
Program is an interactive and participatory lesson. This blanket is made to uncover. As part of Indigenous History Month, Metrolinx's Employee Resource Group, Anishinaabe Circle, in partnership with Oxford Properties, held a culturally immersive event on June 19, to help staff travel back 500 years into Canada's past. The colourful 'KAIROS Blanket Exercise' is an interactive … Continue reading Indigenous history explored through unique exercise

subset: commoncrawl


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The OnePlus 8 series is just around the corner, and the leaks are ramping up ahead of its April 14 launch date. The latest leaks reveal the stylish and colorful official cases you'll be able use to accentuate both the OnePlus 8 and OnePlus 8 Pro.
OnePlus 8 Pro Stories April 1
Last week, official renders of the OnePlus 8 leaked showing off all three color options the phone would release in. Now, another set of images from the same source — WinFuture — is giving us a look at official OnePlus 8 Pro renders, including a never-before-seen "Ultramarine Blue" color.
OnePlus 8 Pro Stories March 31
We're just 15 days away from the debut of the OnePlus 8 series, and there have already been many leaks of the lineup to give us an idea of what to expect. Now, in traditional OnePlus fashion, the company itself is officially confirming a few OnePlus 8 specs in a forum post.
OnePlus 8 Pro Stories March 30
Well, it's official. The launch date for the next OnePlus series of smartphones — the OnePlus 8 — is set for April 14th.
OnePlus 8 Pro Stories March 29
The OnePlus 8 series is set to debut in just a couple of weeks and in these past several days a ton of big details have trickled out on the web. This morning, a pair of leaks reveal some official renders of the OnePlus 8 series along with a unique new "Interstellar Glow" color variant.
OnePlus 8 Pro Stories March 28
The OnePlus 8 Pro isn't far from its official debut, and this week we've learned a lot about the device. To add a cherry on top, Ishan Agarwal has posted more tidbits about the OnePlus 8 Pro, including its display and more.


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11th Edition of MakeUp in Paris: hybrid makeup in the spotlight!
Skin care and hybrid makeup care: a flagship theme of the show A trend straight from Asia that is not to be missed! Hybrid makeup has become THE most demanded product by consumers, who want to go for multi-tasking products and no longer wish to choose between beauty and skincare. This new improved makeup with benefits and textures similar to skin care products, perfectly answers the new consumer demands. Indeed, with Millennials and Gen-Z who are ultra-aware of their impact on the planet but who also want to save time by lightening their beauty routine, brands have to think benefits more than products! MakeUp...READ MORE


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The British car industry suffered twin disappointments in 2019, with sales falling to a six-year low and average carbon dioxide emissions increasing further above new regulatory limits.
UK car sales fell by 2.4% year on year to about 2.3m, according to figures from the Society of Motor Manufacturers and Traders, with the industry body blaming Brexit uncertainty and the slump in diesel sales as the main factors.
This indicates the worst year for the UK market since 2013, when sales were 2.26m. They reached a peak of 2.7m in 2016 but have declined steadily since.
However, the increase in CO2 emissions may point to greater longer-term problems for carmakers selling in Britain. All manufacturers selling in the EU are rushing to meet emissions regulations that came into force at the start of the year. Average CO2 emissions rose for the third year in a row, up 2.7% year on year to 127.9g of CO2 per kilometre, the figures suggested – far above the target of 95g per kilometre they need to achieve over this year and next for all cars sold.
Mike Hawes, the SMMT chief executive, acknowledged the figures showed the challenge facing the industry. He said: "The step change that is required is significant."
About half of the headline increase was caused by a change to testing standards. Another quarter of the change was caused by the 21.8% drop in diesel sales over the year. Newer diesels on average have lower CO2 emissions than petrol cars, despite a backlash prompted by air quality concerns. The final quarter was caused by increased sales of SUVs, which are often heavier and have much worse aerodynamic profiles than smaller cars. Increased fuel use by SUVs was the second largest contributor to the increase in global CO2 emissions from 2010 to 2018, according to the International Energy Agency.
Under the EU regulations carmakers face fines potentially running into billions of euros across the UK and the rest of Europe if they surpass individual limits designed to hit that target. Continued increases in emissions from cars would make it much more difficult for the UK to reduce overall CO2 reduction targets.
One bright spot in an otherwise difficult market was the rapid increase in sales of battery electric and hybrid vehicles, which combine an internal combustion engine with a battery-powered motor. Annual sales of alternatively fuelled vehicles rose by 20.6% to a record market share of 7.4%. That was driven by the surge in battery electric sales, which were up by 144%.
However, sales of battery electric vehicles would need to rise from the 1.6% market share for 2019 to 27% to hit the 95g target alone, according to the SMMT's calculations.
Hawes repeated his industry's plea for a post-Brexit trade deal that preserves frictionless trade between the EU and the UK, and that prevents the imposition of tariffs. He added that Brexit uncertainty remained his number one fear for the industry.
"Undoubtedly consumer confidence around our big-ticket items is weak," said Hawes, speaking at a briefing in London ahead of the publication of final figures, due to be confirmed on Monday morning.Source : The GuardianLink : https://www.theguardian.com/business/2020/jan/06/uk-car-sales-brexit-diesel-electric-vehicles-emissions
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text: Hot water bottles have been in use for centuries. Today, research has revealed, just how dangerous hot water bottles can be. In response to these concerns, new safer products have been introduced- for both children and adults. Warm Buddy warm up animals are the perfect aid for your child`s aches and pains. They make a great child-friendly alternative to the traditional hot water bottle. Warm Buddy...[Read More]


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The perfect pair to the trousers or bib and brace.
Tough, roomy, classic and durable. What's not to love about our range of cover up outer wear. Waterproof and machine washable. Designed with lots of clever features to keep your little ones warm and dry. Don't let bad weather get in the way of outdoor play and exploration!
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To ensure the health and well-being of our staff and customers, we have decided to temporarily close EYR office and warehouse from March 25th 2020 until further notice.
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The Adventurer Classroom serves children ages 12 to 28 months and maintains a ratio of 2 teachers for 6 children. Children of this age need to investigate everything and find out, "what happens if…" and this sets the tone for the room. The main focus of the room is cause and effect and every day is an adventure. The teachers support the children in their investigations by setting up the space so that children can explore independently while remaining safe. Teachers sing and read to children all through the day. They also create all types of messy projects to engage the children focusing on the process not the product. Children in this age group love sensory explorations and they are given many opportunities to play with water, sand, mud, paper and oatmeal to name just a few. Children are bundled up and taken outside to play every day where they continue to use all their senses to observe and investigate all the things found in the natural world. The Adventurers are also learning to separate from mom and dad and build trusting, caring relationships with other adults. Teachers are sensitive to separation issues and help with these transitions.
Some of the goals for the Adventurer classroom include:
- Appreciating and guiding the expression of emotions.
- Encouraging the sense of self and body awareness.
- Foster positive learning experiences where the process is more important than the product.
- Support the emerging development of expressive and receptive language.


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It is a completely ready-to-install kit of the Smart Home system. The kit includes the main MF-10 unit, 3 motion sensors, 2 FW-WL leakage sensors, 4 FW-TS temperature sensors, 4 reed switches, and a 12V 1,25A PSU.
Metaforsa can be controlled via cloud with a securely encrypted signal. The system history from all other devices and sensors is stored on the device for one year. Its plug and play feature means that it automatically detects and configures new Larnitech devices that are added to the system. A large and constantly updated database of ready-made scripts ensures that your home is able to satisfy your most complex requests. This universal unit is able to control lights, various types of valves, blinds, gates, locks and fan coil units (see full list below). Get things going by telling your home what to do with the help of Siri, Alexa or Google Home. Get push notifications on your phone whenever the system has things to tell you and enjoy the ultimate control at your fingertips. The Metaforsa kit is ready to be installed and serve your needs as soon as you take it out of the box!


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I know this post is a few months old but I stumbled onto an app called Quickthoughts, a survey site. Unfortunately they only give out Amazon gift cards. You redeem them every time you hit $10 which is shockingly easy because every survey if you qualify pays $2 and then id you don't qualify they credit you .10. I was skeptical because I hit 10 bucks in less than 2 hours and have cashed out 4 $10 Amazon gift cards .i took a screen shot as proof but not too sure how to post or if I am able my current valance is 5.70 doing a few short surveys just today.
And yes, if you do commit to something like blogging, or writing and freelancing, you can and will make a living (you could even make way more than you could ever at your day job. That said, no matter how much time and effort you put into things like surveys, paid to click sites and things like that, they are not gonna replace your day job. They are just an attitudinal income generation options that you can use in your free time.
You'll also need ecommerce software, fulfillment software, worry about warehousing, customer service and refunds. But that's not all. You'll also need traffic. Think search engine optimization, Facebook ads, and other social media campaigns. It is hard work, especially on your own. You could opt for Amazon's platform, which might be the easier route. But, then again, at the end of the day, this is a serious business, which could produce significant profits. So you're either all in or you're not.
Another champion that I have a lot of respect for in this arena is Ramit Sethi. He has an amazing continuity program (Ramit's Brain Trust) and each month provides useful and specific information for his passionate community. The community thrives off of his wise direction and its members pay him monthly to continue getting access to the community and resources. The beauty of this model is you sell something once and get paid each month for it.
I suppose I should have said She instead of He, and I should not be surprised. Women are notorious for making sure someone is corrected. It is just so frustrating. This person wrote so much helpful information and after reading all that they had provided, this asshat thought to say…….Swagbucks isn't instant? Really? Why even comment at all? You look so freaking inconsiderate. The age of entitlement……….ho hum. The planet is doomed.
And yes, if you do commit to something like blogging, or writing and freelancing, you can and will make a living (you could even make way more than you could ever at your day job. That said, no matter how much time and effort you put into things like surveys, paid to click sites and things like that, they are not gonna replace your day job. They are just an attitudinal income generation options that you can use in your free time.
If you're ready to enter the ecommerce fray, you could sell your own stuff. Of course, along with selling your own stuff on your own website comes a whole slew of both responsibilities and technical configuration and requirements. For starters, you'll need a website and a hosting account. You'll also need a merchant account like ones offered by Stripe or PayPal. Then you'll need to design that site, build a sales funnel, create a lead magnet and do some email marketing.
Clearly, there's a lot of demand on Amazon, and if any product is going to sell, it's going to sell well on Amazon. But the goal here is to source the right products that will easily sell at the world's largest online retailer. Generally, products between $10 and $50 sell very well here. Just be sure to do the right market research before jumping on this bandwagon.
I think Drop shipping is the best way that can help you make money online. It is the only way that allows you to start an online business without investing capital in inventory, warehouse and to buy products individually from a wholesaler and ship them directly to your customer. Moreover, with my drop shipping experience, you will learn key strategies that you won't learn anywhere else to help your online business grow and thrive.
Fiverr is one of the biggest providers in the gig economy on the web and you can sell a wide variety of services and products through this medium. Do the research and find out what you can offer. However, keep in mind that like any other money-making task, it takes time to succeed here. And stellar reviews will help you generate more and more income over time.
There are loads of resources for making money online as an affiliate. You could source products from ClickBank, Commission Junction, Rakuten Marketing, Share-a-Sale, Impact Radius and many others. Plus, many of the larger companies have their own affiliate programs as well. Do your due diligence and find the right company with a relevant product or service to your audience that you can sell as an affiliate.
Now, making money online should seem like a pleasurable activity. Why wouldn't we do just about anything to see things through, since it would be a major source of pleasure, right? Wrong. In the beginning, like anything else, we might get really excited about it. We might also set our hopes very high. But that all comes crashing down when we begin to fail.
Are you good with research and data? Are you an expert in any particular facet of business? If so, this could be a windfall for you. Trust me. I do a lot of this myself. I use my experience and expertise as an entrepreneur to offer clients advice on how to launch their own businesses. I do it all virtually, and I usually charge around $200 an hour for the service.
Awesome to hear :) i'm new at all this don't even own a computer just an iPhone, I am very interested in getting started in this world of making money on Internet. I've been a hairdresser for 16 years and developed health issues from my job and will not be able to do it like I used to so I am trying to find other sources to make income so I can still continue to do whaf i love part-time and supplement my income in different ways where I can still grow myself financially . Thank you everybody for all input
Amazon locked me out of my account because of something so dumb. Their reasoning was because I asked for too many refunds, plus I had 258$ on my balance from gift cards that I purchased, and now I can't even use my balance. I was shocked because I never heard of anything like this. However, it's true that I had asked for a refund several times but it was only for one purchase because they messed up and only gave me 4 out of the 6 gift cards that I had ordered, but it's not like I ever got the refund because when they sent me the 2 gift cards that I was owed, their codes were locked by amazon, so of course I am going to Hassel them for my refund and there you have it. Locked out of my account didn't get my refund and now there's a balance of $258 and I cannot use.
19. eBay – Of course you can't read an article about making money online that doesn't mention eBay. You can start an eBay store and get serious about it or you can just sell some stuff to declutter your home. Either way, I've made my fair share from selling on eBay and it's still a popular way to earn money. If you decide to start an actual eBay store, you'll want to find a drop-ship business like Doba that will store and ship items straight to your customers so you don't have to deal with an inventory.


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Lionel Rose Biography
Nationality: Australian
Famous: Boxers Australian Men
Died At Age: 62
Sun Sign: Gemini
Also Known As: Rose, Lionel
Born in: Warragul
Famous as: Boxer
Spouse/Ex-: Jenny
place of death: Warragul
Recommended For You
Lionel Rose was a professional boxer from Australia who won the world bantamweight boxing championship in 1968. He became the first Aboriginal or Indigenous Australian to win a world boxing title. As a child, he grew up amidst hardships, staying in a single-roomed hut with his large family. To avoid school truancy charges, the family had to change their locality and the children's school but to no avail; Lionel disliked the strict rules at school and eventually dropped out. In the meantime, he picked up boxing skills from his father who was a skilled fighter. At the age of 15, he fought his first amateur fight. At the age of 19, he won the world bantamweight title with a 15-round decision over Masahiko 'Fighting' Harada of Japan. He successfully retained his title thrice before being defeated by Mexican Ruben Olivares in 1969. After a while, he put on a significant amount of weight and moved up several classes to the lightweight division. During this time, he also experimented in a moderately successful singing career. He retired from professional boxing in 1976 with a career record of 42 wins of which 12 were a knockout, and 11 losses. He was the first Aboriginal to be named 'Australian of the Year' for his achievement in boxing.
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- Lionel Rose was born on 21 June 1948, in Victoria, Australia. His father, Roy, was a skilful fighter and his mother's name was Regina. His was a big family and he had several siblings. The family used to reside in a single room tin cabin in Jackson Track.At the age of eight, he was registered at the Labertouche State School. The school was three miles away from his home, leading to irregular attendance.When he turned 10, the Aboriginal Welfare Board helped the family shift into a double roomed house in Drouin, hoping that the children would attend school more regularly. Nevertheless, Rose was a rebellious child and disliked the stringent rules and the crowded bus. He left school at the age of 14.He learnt boxing from his father at an early age. Soon, he was gifted a pair of gloves by his teacher, Ian Hawkins, who noticed his skills.He travelled to Melbourne for the first time in 1958. The trip was sponsored by Save the Children. Later in Melbourne, he met Graham Walsh and watched his first professional fight.During his teen years, he would hang around with his friends at Warragul, an energetic town not very far from Drouin. Eventually, he met Dave Proctor there who arranged his first amateur fight at Sale.He was just 15 years old at the time of his first fight. He fought against a well-built opponent and got badly injured, leading to a two year sabbatical. He received coaching from Frank Oakes.Two years later, he travelled to Sale again with his coach Frank. However, they couldn't find anybody in his weight division to fight with.Continue Reading BelowYou May LikeRecommended Lists:
Recommended Lists:Career
- Lionel Rose began his career by working at a saw mill near Drouin. The job lasted for nine months only as he did not like the role. Following his father's death, he had to consider employment more seriously. However, he failed to keep any job for long because of his poor work ethics.After failing to qualify for the 1964 Olympic selections, he embarked on his professional boxing career in Warragul. He was 16 years old and defeated Mario Magriss over eight rounds.After Warragul, most of his fights were held in Melbourne. There he stayed with his first professional trainer, Jack and his wife Shirley Rennie. He would train regularly in their backyard gym.He won five fights in a row, before being re-matched with Singtong Por Tor in July 1965 whom he had earlier defeated in a 12-round decision. Por Tor defeated Rose in six rounds; it was Rose's first defeat. Later that year, he fought his first international fight at Christchurch, New Zealand. He defeated Laurie Ny by a decision in 10 rounds.In the following nine matches, he won eight and lost one. The single loss was to Ray Perez. In October 1966, he defeated Noel Kunde in a 15-round decision for the Australian bantamweight title at Melbourne.In 1967, he won eight belts including a thirteenth round knockout win against Rocky Gattellari to shield his Australian championship.On 26 February 1968, he won the World Bantamweight title in Tokyo by defeating Masahiko 'Fighting' Harada in a 15-round decision. He became the first Indigenous Australian world champion boxer. This win made him a national hero and an icon.In July 1968, he defended his title in Tokyo with a 15 round decision win over Takao Sakurai. Subsequently, in December same year, he defeated Chucho Castillo in Inglewood, California. This enraged Castillo's supporters leading to a riot that injured 14 fans and the fight referee Dick Young.In March 1969, he defended the title with a 15-round decision over Alan Rudkin. However, in August, he lost the world bantamweight title to Rubén Olivares in a fifth-round knockout at Inglewood.Continue Reading BelowNot to be disheartened easily, he continued boxing. However, when he started losing against unheard of fighters, he lost his popularity as well. Nevertheless in October 1970, he regained his fame again by defeating Itshimatsu Suzuki in a 10-round decision.After losing to WBC world junior lightweight champion Yoshiaki Numata, in a fifteen round decision in May 1971, he declared his retirement.During this time off, he began his singing career and delivered hits like 'Please Remember Me' and 'I Thank You', which went on to become a top five hit in Australia.In 1975, he tried his hands in boxing again but lost four of his six fights, including one against Rafael Limón. This led him to retire from boxing permanently. Post retirement, he became a successful businessman.Awards & Achievements
- As a professional boxer, he recorded 42 wins and 11 losses, with 12 wins by knockout.In 1968, he became the first Indigenous Australian to win the world bantamweight boxing championship.In 2003, he was inducted into the Australian National Boxing Hall of Fame. Two years later, he was featured on a stamp (2005 edition) and was awarded the title of 'King of the Ring'.In 2010, he was featured in Wendy Lewis's book 'Australia's Greatest People'.Personal Life & Legacy
- Lionel Rose married his childhood darling Jennifer, daughter of his first coach Frank Oakes, at Mernda in December 1970. The couple's son, Michael, was born in 1974.In 2007, he suffered a stroke that impaired his speech and movement. He died on 8 May 2011 after suffering from prolonged illness.Trivia
- He was the God-father to model and actress Ruby Rose.In 1991, a television mini-series and period drama on his life called 'Rose against the Odds', was produced. It was later released as a feature film in 1995.Translate Wiki to Spanish, French, Hindi, Portuguese
How To CiteArticle Title- Lionel Rose BiographyAuthor- Editors, TheFamousPeople.comWebsite- TheFamousPeople.comURL- https://www.thefamouspeople.com/profiles/lionel-rose-7145.phpLast Updated- November 12, 2017
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This randomized phase II trial is studying how well T4N5 liposomal lotion works in
preventing the recurrence of nonmelanoma skin cancer in patients who have undergone a kidney
transplant. Chemoprevention therapy is the use of certain drugs to try to prevent the
development of or recurrence of cancer. T4N5 liposomal lotion may be effective preventing
the recurrence of nonmelanoma skin cancer in patients who have undergone a kidney
I. Compare the incidence of nonmelanoma skin cancer (NMSC) (average per patient) on the
sun-exposed skin of renal transplant recipients with a history of NMSC treated with T4N5
liposomal lotion vs placebo.
I. Compare the proportion of these patients who develop NMSC on sun-exposed skin during
treatment and after cessation of treatment with these regimens.
II. Compare the incidence of NMSC on the sun-exposed skin of these patients after cessation
of treatment with these regimens.
III. Compare the incidence of recurrent and de novo actinic keratoses (AKs) in patients
treated with these regimens.
IV. Determine whether either of these regimens induces regression of AKs left untreated on
the sun-exposed skin of these patients.
V. Compare the proportion of these patients who develop melanoma, in both treated and
untreated sites, during and after cessation of treatment with these regimens.
OUTLINE: This is a randomized, double-blind, placebo-controlled, multicenter study. Patients
are stratified according to study center. Patients are randomized to 1 of 2 arms.
Six months before randomization, lesions suspicious for nonmelanoma skin cancer (NMSC) are
surgically removed and histologically analyzed. All but 10 randomly selected non-suspicious
lesions are removed. Of these 10 lesions, 5 are shave biopsied immediately pre-treatment for
histologic and surrogate endpoint biomarker (SEB) analysis and to determine a baseline
actinic keratosis: wart ratio. Patients also undergo a pre-treatment biopsy of normal
appearing sun-exposed and non-sun-exposed skin (buttocks).
Arm I: Patients apply T4N5 liposomal lotion topically to non-occluded, sun-exposed areas of
the head, neck, face, and upper extremities once daily for 12 months.
Arm II: Patients apply placebo topically to non-occluded, sun-exposed areas of the head,
neck, face, and upper extremities once daily for 12 months.
Treatment in both arms continues in the absence of the development of metastatic cutaneous
squamous cell cancer or melanoma. Patients are followed every 3 months.
PROJECTED ACCRUAL: A total of 100 patients (50 per treatment arm) will be accrued for this
study within 6 months.
Inclusion Criteria:
- History of histologically confirmed nonmelanoma skin cancer
- Renal transplant recipient ≥ 4 years ago
- Currently receiving standard multi-agent pharmacologic immunosuppression
- Fitzpatrick skin type I, II, or III
- Sun-damaged skin with ≥ 10 lesions consistent with actinic keratoses OR wart on the
upper extremities (arms, forearms, hands), neck, face, and exposed scalp combined
- No history of keloid formation
- No known photosensitivity disorder
- No history of malignant melanoma
- Not pregnant or nursing
- Negative pregnancy test
- Fertile patients must use effective contraception
- No diagnosis of acute allograft rejection within the past 30 days requiring an
increase in immunosuppression
- No invasive malignancy within the past 4 years except curatively excised NMSC, cured
polyclonal posttransplantation lymphoproliferative disease, carcinoma in situ of the
cervix, stage 0 chronic lymphocytic leukemia, unless all of the following criteria
are met:
- No current evidence of disease
- No treatment for the invasive malignancy within the past 6 months
- No concurrent or planned therapy for the invasive malignancy
- Has an expected disease-free survival of at least 5 years
- No diagnosis of melanoma or melanoma in situ
- No other medical or psychosocial condition that would preclude study participation
- No likelihood, in the opinion of the transplant surgeon/nephrologist, to experience
graft loss and/or discontinue standard immunosuppressive therapy during study
- More than 30 days since prior and no concurrent topical chemotherapy (including
topical fluorouracil) to areas being studied
- No concurrent topical preparations containing corticosteroids
- More than 30 days since prior and no concurrent local radiotherapy to a study area
- More than 30 days since prior and no concurrent cryotherapy to target lesions
- No prior or concurrent experimental immunosuppressive agents
- More than 30 days since prior investigational medication
- More than 30 days since prior and no concurrent systemic psoralens or retinoids
- More than 60 days since prior and no concurrent laser resurfacing, dermabrasion, or
chemical peels to a study area
- No other concurrent investigational agents
- No other concurrent topical medications, including prescription and over the counter
preparations, to the areas being studied (e.g., upper arms, forearms, neck, face, and
- Concurrent moisturizer, emollient, and sunscreen allowed
- No concurrent topical preparations containing vitamin A derivatives
- No concurrent nonsteroidal anti-inflammatory drugs
- Concurrent cardioprotective doses of aspirin (< 100 mg/day) allowed
Principal InvestigatorCraig ElmetsUniversity of Alabama at Birmingham
Birmingham, Alabama 35294United StatesThere is no listed contact information for this specific location.Site Status: N/A
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text:
Sienna is a strapless ballgown with a modified sweetheart neckline. Scatters of sequins stream through the lace with dimension added through floral lace appliques along the surface.High above the chapel length train sit a set of crystal buttons. A thin velvet ribbon around the waist create a luxurious finish to the dress.
Location: Armadale
Sizes: 2-32
Colours: Ivory/Ivory/Ivory, White/White/White, Champagne/Ivory/Champagne


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The electrical is adopted PLC programmable controller. This machine can be applied in components processing in large quantity. The drilling and threading processing can be finishing once. The guide rail(guideway) quenches by super audio frequency. Slide moving use with buffer, low vibrate, and adopts pneumatic clamp, loading and unloading fastly.
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Emergence of antibiotic resistance from multinucleated bacterial filaments
Contributed by Robert H. Austin, November 3, 2014 (sent for review August 26, 2014)
Understanding how bacteria rapidly evolve under antibiotic selective pressure is crucial to controlling the development of resistant organisms. We show that initial resistance emerges from successful segregation of mutant chromosomes at the tips of filaments followed by budding of resistant progeny. We propose that the first stages of emergence of resistance occur via the generation of multiple chromosomes within the filament and are achieved by mutation and possibly recombination between the chromosomes.
Bacteria can rapidly evolve resistance to antibiotics via the SOS response, a state of high-activity DNA repair and mutagenesis. We explore here the first steps of this evolution in the bacterium Escherichia coli. Induction of the SOS response by the genotoxic antibiotic ciprofloxacin changes the E. coli rod shape into multichromosome-containing filaments. We show that at subminimal inhibitory concentrations of ciprofloxacin the bacterial filament divides asymmetrically repeatedly at the tip. Chromosome-containing buds are made that, if resistant, propagate nonfilamenting progeny with enhanced resistance to ciprofloxacin as the parent filament dies. We propose that the multinucleated filament creates an environmental niche where evolution can proceed via generation of improved mutant chromosomes due to the mutagenic SOS response and possible recombination of the new alleles between chromosomes. Our data provide a better understanding of the processes underlying the origin of resistance at the single-cell level and suggest an analogous role to the eukaryotic aneuploidy condition in cancer.
Bacteria can evolve remarkably quickly to be resistant to antibiotics under certain conditions, as we showed in an earlier paper (1) using microfabrication techniques to create a structured environment of bacteria in a gradient of the genotoxic antibiotic ciprofloxacin (cipro), which perturbs chromosome replication (2). The need for the structured environment in which steep gradients facilitate the rapid evolution of resistance by multiple mutations confirmed a prediction from recent modeling (3). Our earlier work on the rapid emergence of cipro resistance in a microfabricated complex ecology did not delineate the stages in the emergence of resistance but only presented the ultimate, resistant cells (1). However, high-resolution imaging of the cells within the device before the full emergence of resistance showed that the cells went through a transient filamentation phase. Fig. 1 shows images from our microecology that Escherichia coli bacteria have changed their shape into long filamentous structures shortly after exposure to the cipro antibiotic. The filamentation response is not permanent, because after 4 h some cells have reverted to a normal phenotype (Fig. 1B) and have become resistant to high levels of the antibiotic by de novo mutation in genes encoding gyrase A topoisomerase and efflux pumps (1). We wished to explore the role filamentation played in the initial stages of antibiotic resistance.
Many microorganisms adopt a filamentous shape caused by cell-division arrest yet continue cell-volume growth in response to a variety of stressful environments, including nutrient deficiency (4), extensive DNA damage through the SOS response pathway (5, 6), host innate immune responses (7), desiccation (8), high pressure (9), and antibiotic treatment (10⇓–12). Although filamentation has long been considered an overstressed and/or sick phenotype or solely for completion of DNA replication before division (13), it has only recently been described as a more general survival strategy (7, 8, 14). For example, if the stress is relieved quickly enough, division of the filamentous cell resumes synchronously and rapidly at regular intervals along the entire length of the filament, resulting in multiple viable normal-sized offspring cells (8). However, the mechanisms underlying the evolution of filamentous bacteria when the selective pressure is maintained have been poorly described, in part because most studies have used stress conditions that were evolutionarily unfavorable and instead led to irreversible bacterial growth arrest and/or rapid death.
Bacterial cell-division arrest resulting from inhibition of DNA replication after cipro exposure (2) is caused by the induction of a division inhibitor (e.g., SulA in E. coli) (13, 15). sulA gene induction is part of a set of over 30 induced genes involved in DNA repair pathways and error-prone mechanisms, collectively known as the SOS response (5, 16, 17), a sequential transcriptional process that is tightly controlled by two master regulators, LexA and RecA proteins (18). When the replication forks are stalled, LexA autoproteolytic cleavage facilitated by RecA protein initiates the SOS stress pathway until the chromosome damage has been repaired and replication of the chromosome resumes. However, when the SOS response is fully engaged, the induction of low-fidelity replication polymerases known as error-prone polymerases increases the rate of mutation μ during DNA replication from the normally low value of 10−9 mutations per base pair per generation up to a high rate of 10−5 mutations per base pair per generation (19), so that a single bacterial chromosome such as the 4.6-Mb E. coli chromosome can be expected to have multiple mutations with each round of replication. Current research aims at targeting the error-prone polymerase pathway to slow down the evolution of antibiotic resistance and thus improve the long-term viability of some antibiotic drugs (20).
Here we address how filamentation is probably the first step in the evolution of resistance to mutagenic antibiotic exposure, internally generating mutant chromosomes via the SOS response until a solution is obtained: a mutant chromosome with resistance to the stress. We examine how individual bacteria exit from the filamentous state caused by subminimal inhibitory concentrations of the genotoxic antibiotic ciprofloxacin. We show that when cipro is maintained at low levels, E. coli filaments undergo asymmetric divisions at the filament tips, giving rise to budded resistant offspring cells. The generation of multiple mutant chromosomes that are successfully segregated toward the filaments tips favors the birth of antibiotic-resistant cells. We discuss how this filamentation step is a precursor to the final highly resistant bacteria in a complex ecology.
We focused on the role that the SOS mutational network, triggered by the presence of stalled replication forks, plays in the filamentation process and bacterial evolution of drug resistance. The transient filamentation events were studied on agar plates containing cipro at varying levels. The primary experiment involves spreading a low-density population of less than 103 per mm2 of E. coli bacteria in the presence of 0.125 of the minimal inhibitory antibiotic concentration (0.125× MIC) of cipro. Time-lapse imaging revealed that 99.5% of the bacteria formed long filaments (up to 200 μm) and expanded their length exponentially (Fig. 2A; see Movies S1 and S2 for videos of the growth of filaments). The remaining 0.5% of the bacteria that did not elongate or divide in the presence of the antibiotic may be persister cells (21) or dead cells. A lexA3 mutant strain that cannot activate the SOS response and a recB mutant strain, which reduces SOS response induction, general recombination, and DNA repair, were not able to filament or grow at these low cipro concentrations, verifying that both filamentation and growth rely on the induction of the LexA-dependent SOS response and possibly on recombination and repair pathways (SI Appendix, Fig. S1A shows the growth of filaments over a 3-h time period and the failure of the lexA3 and recB mutant strains to grow under the same conditions). We observed that the growth of a filament is transiently interrupted by asymmetric division events (Fig. 2B and Movies S1 and S2) that occur repeatably and predominantly at the tips of the filament (Fig. 2C) and give rise to short offspring cells that often resumed normal division (Fig. 2B and Movies S1 and S2). This budding process was inherently distinct from the synchronous, symmetrical division recovery profile of the filament obtained shortly after drug removal (SI Appendix, Fig. S1).
Interestingly, some degree of heterogeneity was observed in the bud fate at this low concentration (0.125× MIC) (Fig. 3A): 35% of budded cells divide symmetrically without further filamentation, indicating that they are now resistant to 0.125× MIC cipro; 35% cease division and growth; 10% of the buds show abnormal asymmetrical division (minicell formation) with little propagation; and 20% resume filamentation, indicating that they do not show evidence of cipro resistance. Buds that fail to divide and propagate may either contain a damaged chromosome or may not contain a chromosome at all [we found that 11% of the buds do not contain a chromosome, by using 4′,6-diamidino-2-phenylindole (DAPI) staining]. Conversely, the fate of the filaments after prolonged growth on low cipro showed less heterogeneity than that of the buds, since the bulk of parental filaments (70%) undergo death as evidenced by their inability to exclude the propidium iodide (PI) dye, reflecting loss of possibly both membrane integrity and efflux mechanisms (Fig. 3B). These findings suggest that filamentation at low cipro is a complex phenomenon that gives rise to short progeny and eliminates the unfit filamentous parent cell.
We were most interested by the buds that divide symmetrically without further filamentation and examined whether these buds show a lower level of SOS response. We used a sulA-gfp transcriptional reporter in a nongenic chromosomal location in cells with an intact native sulA gene (22) to quantitatively measure induction of the SOS response within the bacterial filament. We observed a decrease in the intensity of the gfp expression within buds, indicating that buds had decreased expression of the SOS regulon, as would be expected from the observed increase in cipro resistance in the buds and subsequent suppression of the SOS response [Fig. 3 shows that the buds have on average about 25% less expression of PsulA-gfp than the filaments, whereas the intensity of constitutively expressed RFP protein (lac-mrfp1) shows identical levels both in the buds and in the filaments, indicating that protein expression levels are similar in the buds and within the filaments].
Because most buds do contain a chromosome, although cipro directly affects DNA replication by targeting DNA gyrases (23) and thus perturbs chromosome dynamics (2), we wished to examine both the content and the spatial arrangement of the chromosomes in filamentous cells to understand how low doses of cipro allow the generation of short viable nucleated offspring. We used standard DAPI staining and live imaging of a specific chromosomal locus, R2 (17), as a reporter of an individual chromosomal copy (SI Appendix, Fig. S2) to enhance chromosome counting. R2 loci were visualized by the binding of fluorescent TetR-CFP fusions to the tetO operator array located 21 kb upstream of the lacZ gene (24) (SI Appendix, Fig. S2). Individual chromosomes localize as discrete CFP foci. We found that the filaments contain multiple chromosomes (Fig. 4 and SI Appendix, Fig. S2) that are partially or completely segregated, with a linear relation between filament length and chromosome number (Fig. 4A), indicating that as the filament grows it replicates multiple chromosomes. The presence of multinucleated filaments occurred only at a low level of cipro (0.125× MIC) (SI Appendix, Fig. S2C). At levels of cipro above 0.125× MIC, the chromosome morphology was severely impaired (SI Appendix, Fig. S2C) and events of budding at the filament tips were rare or nonexistent. At low levels of cipro, we found that some chromosomes localize near the tips (three-quarter positions of cell length) (Fig. 4B), coinciding with the position of the division sites at the tips (Fig. 1C). Time-lapse experiments showed heterogeneous dynamics of the chromosomes within the filaments where some chromosomes move tipward as the filaments grow whereas other remain essentially stationary in the midcell regions (Fig. 4 C and D; SI Appendix, Fig. S3; and Movie S3). In addition, two individual chromosomes were eventually observed to alternate between overlapping in a single cluster and then separating into two distinct foci (Fig. 4 C and D and SI Appendix, Fig. S3). We also noticed that the chromosomes within the same filament undergo replication or separation of foci in an asynchronous way (Fig. 4 C and D). All together, the chromosome dynamics in cipro-stressed filaments are complex and do not indicate simple replication, but nevertheless the filamentous cell succeeds in coordinating chromosome placement and asymmetric division at the tips, leading to the birth of resistant daughter cells.
We verified that the offspring evolved resistance in that normal-sized and dividing bacterial buds arose at the tips of the filaments under continued low cipro treatment, and SOS expression levels, as determined by the sulA-gfp reporter, were low. Wild-type bacteria were cultured in low cipro and filtered using a sterile 5-μm syringe filter to isolate normal-sized progeny from filamentous parent cells (SI Appendix, Fig. S4). About 70 ± 11% of the isolated progeny survived when reexposed to low cipro (0.125× MIC). The frequency of progeny resistant to high levels of cipro (1× MIC) was determined and defined as the number of cipro-resistant colonies that arose after 24 h on solid media containing 1× MIC cipro per viable cell. Progeny showed a 250-fold increase in resistance frequency [2.8 (±0.3) × 10−6] at 1× MIC cipro compared with that of the parent cells [7.2 (±3) × 10−8] that had not been preexposed to low cipro. This result indicates that a subpopulation of budded cells has indeed evolved resistance during the 24-h exposure to low cipro. We assumed that the frequency of persisters was kept low in the progeny population, even after filtration, because progeny either formed filaments or resumed normal division when exposed over again to low cipro. Instead, persisters would have resumed division and growth only once the antibiotic is removed (21).
Resistant clones were isolated by selection using subinhibitory (0.5× MIC) or inhibitory (1× MIC) doses of cipro, and a subset of genomic sequences of known resistant genes was analyzed by using targeted genome sequencing (Table 1 and SI Appendix, Table S2). The results showed that the level of resistance was a function of the cipro concentration at selection. Resistance at the bottom of the selection window was associated with overexpression of AcrAB efflux pumps, in that mutations in marR were found and none of the other genes sequenced had mutations. Deletion(s) of 1 or 11 nt in the marR gene encoding the MarR efflux pump regulator were identified in 37% of the sequenced clones. Conversely, at the top of the selection window, the resistant clones isolated were mainly mutated in the GyrA gyrase subunit, and 87% of the sequenced clones had a single mutation in gyrA (GyrA S83A), which is located in the quinolone resistance-determining region, a hot spot region for mutations (25). Mutations in gyrA or marR are likely to be sufficient to promote intermediate-level resistance of the tested clones (Fig. 5). The rest of the cipro-resistant clones presumably carry a resistance-conferring mutation(s) at a place not sequenced.
The MIC of resistant progeny was 2.5× increased (10 clones analyzed) but remained five times lower than that of the high-level resistant strain QZ030, which evolved four mutations both in gyrA and the efflux pumps when cultured in a complex environment (1). Although the budded progeny show enhanced resistance to cipro, they do not show the full resistance observed in the cells that grow in the microfabricated devices, indicating that the budded progeny are part-way along the path to a fully enhanced resistance (Fig. 5 compares the resistance of filtered bud progeny to cipro with that of the strain of bacteria QZ030 retrieved from our previous experiment, where evolution to resistance to cipro emerged in a complex gradient in a metapopulation, as opposed to strains extracted from this experiment, where evolution occurs in a fixed concentration of cipro). In all, the data reveal that a low level of adaptive evolution to low cipro can occur in a few hours within a single filament.
Our data show that there are five fundamental features that characterize the growth of the bacterial filaments during exposure to subminimal inhibitory concentrations of cipro (Fig. 6): (i) Exposure to 0.125× MIC cipro initiates the SOS response (Fig. 3C and Fig. S1); (ii) the filament contour length L(t) increases exponentially with time (Fig. 2A); (iii) the filaments contain multiple copies of the bacterial chromosome (Fig. 4); (iv) the filamenting process is transiently interrupted by a budding process that generates resistant progeny at the tips (Fig. 1 and Movies S1 and S2); and (v) most filaments die, but not some of the budded progeny (Fig. 3B).
Such an evolutionary path to antibiotic resistance encompasses two remarkable events that occur during the filamentation process: the presumably local decision to transiently exit from SOS and bud a resistant daughter cell from the end of the filament, and the generation of multiple chromosomes that evolve resistance via mutagenesis and possibly recombination within a single filament.
The budding events are presumably triggered by the local cessation of the SOS response in the vicinity of an evolved resistant chromosome with the mutations found in Table 1, because the SOS response is responsible for the filamentation phenotype caused by a lack of septation. It could be either that better-adapted chromosomes are generated near the tips or that better-adapted chromosomes generated internally migrate toward the tips, where they are then budded off by formation of a single septum. We can guess whether the cessation of the SOS response is a global or local event in the cell by estimating how far the SOS division-inhibitor SulA can diffuse during the replication time of a chromosome, about 30 min in our case. SulA has a molecular mass of about 18 kDa and measurements of the diffusion coefficient of similar-size GFP proteins in E. coli have yielded an average value for the diffusion constant of about 7 μm2/s (26), and hence we would expect that in 30 min the SulA protein should diffuse about 150 μm, easily the length of a filament. However, the budding process is local and not evenly distributed along the length of the filament, indicating that either SulA does not diffuse freely in the cytoplasm or other players such as the Min division control proteins are determining the local decision to bud. Identifying the molecular actors that govern the decision of local budding will be of valuable interest to answer the question of why asymmetric division is important to generating resistant progeny.
This view differs from current paradigms for cell-cycle checkpoint control in bacteria and other organisms. Cell filamentation during SOS is thought to reflect the need to complete DNA replication before cell division to protect chromosomes after DNA damage (13). Similarly, filamentation allows repair of DNA damage by recombination between chromosomes by keeping more than one chromosome per cell until repair is completed (27). Similar DNA-protection roles underlie eukaryotic cell-cycle checkpoint control (28). We suggest that in addition to DNA preservation, cell filamentation may accelerate evolution by adding recombination, which is possible only with more than one chromosome per cell, to the increased mutagenesis documented during SOS. Recombination between chromosomes can potentially separate beneficial mutations from linked deleterious mutations or unrepaired DNA damage. The dramatic acceleration of evolution by recombination accompanying mutagenesis, relative to mutagenesis without recombination, is supported by modeling (29, 30).
Coupling of mutagenesis with recombination is observed throughout life, including being predicted in cancer genomes (31, 32), and observed in the immune system (33), yeast (34, 35) and bacteria (17, 36⇓–38). Because increased mutagenesis creates both deleterious and beneficial mutations, recombination that unlinks these can be beneficial.
Further, the chromosomes display a distinct dynamics pattern in the same filament. Events of merging/splitting between two chromosomes were frequently observed during the experiment (Fig. 4C; SI Appendix, Fig. S3; and Movie S2), possibly reflecting the intimate interplay between DNA replication and genetic information exchange during cipro exposure. In addition, replication of sister loci did not occur synchronously, as shown in Fig. 4C, where one chromosome out of six was captured while replicating, and suggests potential differential DNA damage load between the chromosomes (39). Although we found these observations captivating, it remains unclear to us why there is such heterogeneity in the chromosome dynamics. One can speculate that the nonstationary chromosomes are the chromosomes with increased fitness (probably containing mutations conferring cipro resistance) that would not be eliminated but chosen for replication. Instead, stationary and/or nonreplicating chromosomes that are likely to contain neutral or deleterious mutations would eventually be eliminated in the dying filament or would recombine to separate beneficial mutations from linked deleterious damage and hence be chosen for survival (Fig. 3B). Therefore, the multiple chromosomes that are generated in the filament could enhance the chances of producing resistant chromosomes as well as allow repair. The ultimate decision to release the progeny would be done by pinching off the successful mutant chromosome that is resistant to the stress by local loss of the SOS response. Further in-depth mechanistic studies of the interplay between processes of stress-induced mutagenesis, recombination, chromosome partitioning, and local budding are needed to deepen our understanding of how antibiotic resistance stems from a single multinucleated bacterium. The chromosome dynamics in an elongating filament might possibly resemble a form of Turing machine (40), where the "computation" of fitness is like the writing of mutations on a chromosome and erasure of marks would correspond to the elimination of chromosomes that were less fit and to recombination between chromosomes reverting deleterious new mutations, and the halting of the Turing machine upon solution of the problems would be represented by the pinching off of the mutant, more fit chromosome. However, this is highly speculative at this point in our work.
Perhaps a phenomenon similar to bacterial multichromosome resistance occurs in aneuploid cells. For instance, cancer cells typically contain 60–90 chromosomes (41). Aneuploidy is a ubiquitous feature of cancer that may contribute to a more rapid accumulation of mutations, increasing survival and proliferation (42⇓⇓–45). Therefore, the strategy of generating multiple mutant chromosomes within a single cell may represent a widespread and conserved mechanism for the rapid evolution of genome change in response to unfavorable environments (i.e., chemotherapy drugs and antibiotics).
Materials and Methods
Long time-lapse movies (images recorded every 2 min for 10 h) of low-density bacteria [JEK1036 (gfp+)] growing on LB agar pads containing 5 ng/mL cipro and isopropyl β-d-1-thiogalactopyranoside (IPTG) were made using a Nikon 90i microscope equipped with a Nikon Plan Apo 60× objective and a Rolera-XR cooled CCD camera and processed with Micro-Manager (NIH, www.micro-manager.org) and ImageJ (NIH) software. All other images were collected on a Nikon Eclipse 90 microscope equipped with a Nikon Plan Apo 100× objective and an Andor Neo camera and processed with Nikon NIS-Elements software. In all experiments, cells were transferred from liquid culture to 1.4% agarose-padded slides containing LB medium (or 0.5× LB for strain AB1157), with low ciprofloxacin and IPTG when required. A coverslip was placed on the pad and then sealed with valap (1 vol vaseline/1 vol lanolin/1 vol paraffin). Filament death was visualized by fluorescence microscopy using propidium iodide, a membrane-impermeable nucleic acid stain commonly used as a cell-death marker. Nucleoids in the filaments were visualized by adding DAPI (live-cell DNA staining) directly to the agarose pads. R2 loci were visualized in E. coli AB1157 cells placed on 1.4% agarose-padded slides containing 0.5× diluted LB medium (to reduce fluorescence background). Images were recorded every 2 min right after the cells were placed on the agarose pad in the no-cipro conditions, or every 5 min after the cells were allowed 2 h of growth on the agarose pad in the low-cipro conditions. See SI Appendix, Materials and Methods for details of experimental protocols.
Bacterial Strains and Growth.
E. coli strains were cultured at 37 °C in liquid LB medium and on an LB agar (1.7%) plate (solid), unless noted. In all experiments, cipro antibiotic was added at a subminimal inhibitory concentration of 5 ng/mL (0.125× MIC) referred to as "low cipro," unless indicated (see SI Appendix, Materials and Methods for details of strain growth). Strains and primers used in this study are listed in SI Appendix, Tables S1 and S2, respectively.
Minimum Inhibitory Concentration Determination.
The MIC defined as the lowest concentration of ciprofloxacin that prevents any growth of the bacteria is known to be 40 ng/mL. For each strain, a volume of 3 mL of overnight culture was used to inoculate, in triplicate, 150 μL of LB medium containing increasing concentrations of ciprofloxacin (0, 5, 10, 15, 20, 25, 30, and 40 ng/mL) using 96-well flat-bottom plates. Growth was monitored by reading the absorbance at 630 nm every 20 min for 10 h of incubation at 37 °C with shaking in a ChroMate 4300 Microplate Reader (Awareness Technology). MICs for ciprofloxacin-resistant offspring clones (10 clones analyzed) were determined using the same conditions described above; in addition, wells contained LB plus ciprofloxacin at 50, 100, 200, and 500 ng/mL.
Determination of Cipro-Resistant Mutant Frequency.
The frequency of the cipro-resistant (ciproR) mutant was determined and defined as the number of ciproR colonies that arose after 24 h of growth on solid media containing 1× MIC cipro, per viable cell. Three independent cultures of wild-type strains were subcultured at 1:100 dilution from an overnight starter culture and grown to midexponential phase. A volume of 0.1 mL of cell culture was plated on LB agar containing low cipro antibiotic and incubated at 37 °C for 24 h. The bacterial lawn was resuspended in 5 mL LB using a sterile scraper. The resulting cell culture contained a mixed population of filaments and short bud offspring. When needed, the cell culture was recovered by filtration through a 5-μm sterile syringe filter to harvest the progeny and remove the filamentous cells. All cultures (mixed-population or filtered progeny) were serially diluted, and 0.1-mL samples of appropriate dilutions were plated onto nonselective plates to determine total viable cell counts, as well as on plates containing cipro 1× MIC (40 ng/mL) to score high-level ciproR mutants. CiproR frequencies were calculated by dividing the number of high-level cipro-resistant mutants by the total number of viable bacteria. A total of three experiments was conducted independently. Means of mutant frequency are given in the text and the "±" values represent SD.
Cipro-resistant colonies were selected on LB plates containing either cipro 0.5× MIC (20 ng/mL) or 1× MIC (40 ng/mL), after 24 h incubation at 37 °C. See SI Appendix, Materials and Methods for details of the experimental protocol.
We thank Thomas Silhavy for helpful comments on the manuscript and Douglas Austin for helping in writing the custom MATLAB code. This project was supported by National Cancer Institute Grant U54CA143803 (to R.H.A.) and National Institutes of Health Grant R01-GM53158 (to S.M.R.).
- ↵1To whom correspondence should be addressed. Email: austin{at}princeton.edu.
Author contributions: J.B., Q.Z., S.V., E.R., S.M.R., and R.H.A. designed research; J.B., Q.Z., and R.H.A. performed research; J.B., E.R., and S.M.R. contributed new reagents/analytic tools; J.B., Q.Z., S.M.R., and R.H.A. analyzed data; and J.B., S.M.R., and R.H.A. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1420702111/-/DCSupplemental.
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$8,400 for the week, includes taxes, daily house keeping and 24 hour security!
Luxurious boutique hotel can be all yours! The property offers 8 spacious and well appointed rooms, each complete with private baths, AC, hot water, refrigerator, and cable TV. Each room has a queen bed and a twin bed, for sleeping up to 3 people. Maximum 24 people.
The management offer a full concierge service to help you arrange for anything your heart desires during your vacation. We can arrange airport transfers, tours and activities, surf lessons, massages, private chef and more! The price includes daily housekeeping and 24 hour security, and laundry service is available for an additional cost.
There's an onsite restaurant which is open to the public, but the rest of the hotel and the pool is completely yours to enjoy! The restaurant can cater to your needs if so desired.
It's located just a 2 min walk to the beach and the main surf break in Playa Grande.
Peninsula 49
Welcome to Peninsula #49 - "Casa Dolce Vita", located in beautiful Playa Langosta of Tamarindo, this 2 bedrooms + 2 bathrooms condo offers 1450 sf / 135 m2 of indoor / outdoor living areas. The condo is fully furnished and equipped and it is only a few steps away from a white sandy beach on the Pacific Ocean and a bicycle ride from down-town Tamarindo night life.
The space
The master suite features a King size bed, walk-in closet, shower, dual sinks, 42" plasma TV and Jacuzzi tub.
The second guest bedroom provides a Queen size bed plus easy access to the second full bathroom.
The living room features a Queen Size sofa bed and 50" LCD TV.
Fully equipped kitchen including a dishwasher.
Extensive terraces in living room and master bedroom.
Washing machine and dryer.
Two private covered parking spaces available.
Guest access
Peninsula count with professionally maintained tropical gardens, massive pool for adults, clubhouse with BBQ grill area, game room, private fitness center, pool and playground for kids.
Peninsula is a private and gated resort with 24-hour guarded access. It's just a short 5 minutes walk to the Langosta grocery store and beautiful Playa Langosta (certified "Blue Flag" for water and beach quality). Tamarindo Down Town is just 15 minutes walking away.


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The ESP E-II FRX sports an alder body with a flamed maple top, neck-thru maple neck, 25.5" scale and 24 extra-jumbo frets on an ebony fingerboard on a thin-U neck. And a pair of Seymour Duncan SH-14 (B) & SH2 (N) pickups pushes all your shreddy tone down the signal path. A Floyd Rose Original tremolo bridge allows for all the dive bombs and squeals your brain can conjure, while Gotoh tuners keep everything cinched down and in tune through heavy crunches and string-stretching leads.Neck-thru construction 25.5" scale Alder body Flamed maple top Maple neck Ebony fingerboard 42mm locking nut Thin U neck contour 24 XJ frets Black hardware Gotoh tuners Floyd Rose Original bridge Seymour Duncan SH-14 (B) & SH2 (N) Finish: STBLK
Order today!
Custom Guitar's Friend Gold Coverage
Gold Coverage goes above and beyond the manufacturer's warranty to protect your gear from unexpected breakdowns, accidental damage from handling and failures. This plan covers your product for one, two, three or up to five years from your date of purchase, costs just pennies per day and gives you a complete "no-worry" solution for protecting your investment.
Gold Coverage Includes:Unexpected and unintentional spills, drops and cracks Normal wear and tear Power surges Dust, internal heat and humidity
Other plan features include:For products over $200, No Lemon Policy applies. If it fails for the same defect 3 times, it is replaced on the 4th breakdown. (Does not apply to failures due to drops, spills, and cracks) For products under $200 experiencing the above failures, a Custom Guitar's Friend gift card will be issued for the full price of the product + tax. Shipping fees covered if required for repair or replacement Plans are transferable in the event merchandise is sold Plans are renewable on new gear
*Limitations and exclusions apply. See terms and conditions for program details
Term for New Gear begins on date of purchase but does not replace the store return policy or manufacturer coverage.
Displaying reviews 1-2
Back to topCame with a hi e string fret buzz that a pro guitar tech could not fix, so it went back today. It may have overheated shipping to FL in Aug. That said, the pickup sound was a bit higher tone than my other Seymour Duncan guitar. It sounded great for leads but not so much for chugging. It had a beautiful clean sound. The looks of this small bodied guitar were gorgeous. Neck has good feel, Trem was ok but a bit hard to get perfect tuning first try as it's a floating bridge. Case it came in is great. I'm sad it wasn't right since I would have liked it as my only hot pickup guitar with a trem.
ESP E-II Horizon-3 Electric Guitar With Floyd Rose See-Thru Black
.Excellent craftsmanship, great rock/metal tone. Very smooth neck.
ESP E-II Horizon-3 Electric Guitar With Floyd Rose See-Thru Black


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Cornish housekeepers are very professional, reliable and trustworthy. The standard of their service is exceptionally high which is imperative for my holiday let property. Many of our guests comment on how clean and well maintained our property is. Cornish Housekeepers provide this quality of service week after week. The team are very approachable, friendly and professional. I really couldn't ask for more.....
Sue Watt: 11 Fish Street St Ives
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Morna Luscombe Samphire
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"Working in the hotel industry and staying in top hotels world wide leaves me with high expectations generally and I have to say that this properties cleanliness leaves some hotels to shame……..look forward to using this property again....
Visitor at Dolphinhome


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WWE RAW Results: September 10, 2018
How did Roman Reigns lure Braun Strowman in for a fight before Hell in a Cell? What big announcement did Mick Foley have regarding the WWE Hell in a Cell pay-per-view this Sunday? How did Dean Ambrose & Seth Rollins outsmart Baron Corbin to get a title match this Sunday? How was Alexa Bliss able to weaken Ronda Rousey ahead of their rematch at WWE Hell in a Cell? Could The B-Team reclaim the RAW Tag Team Titles? What did Triple H have to say about last week's confrontation between Shawn Michaels and The Undertaker? What perk did Kevin Owens reveal that he received for agreeing to stay on RAW?


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MorganBennett Coaching
At MBC we provide professional coaching at grassroots level. Our aim is to improve your game, give you an insight into how to make it to the very top and most importantly, enjoy playing the beautiful game. We look to create a fun environment and cater for all abilities, with the aim to send each and every player home with improvements as an individual, some inspiration and a huge smile.
Combined with our mentoring program this is where we are very unique. football fun numeracy, literacy and short courses are intgrated working alongside mentors and teachers. not only will you improve as a footballer so will your education and you as a person. We are always looking to pass on our expertise and knowledge. Whether it's inviting a professional player down to one of our courses or a question and answer session, we are always looking to inspire our kids at MBC.
MBC believes football is more than just a game. So whether you're looking to learn the very basics, make friends, improve your game or education and pursue a career within football with links to many pro/semi professional clubs we are suited to you. With great coaches, mentors, teachers, fantastic facilities and equipment, MBC will always provide a professional service.
Will you be the next Wes Morgan or Julian Bennett?
Why choose Morgan Bennett Coaching?
Copyright 2015 Morgan Bennett Coaching Tel: 07725809769 Email: email@example.com - email@example.com Follow us on Twitter: @mbcoachinguk Instagram: mbcoachinguk Facebook.com/mbcoaching2015


text: In Kustoms Illustrated issue #28 we have started a new series of articles on the Larry Watson Photo Collection. A collection of absolutely amazing photos and snapshots. I'm busy working on the second installment of this article for Kustoms Illustrated #29, and I know the fans will love the photos I have selected for this. Since there are so many photos in this collection there is no way these can all be printed in the magazine, or even in books. So I have started to share some of them on my daily post on my Facebook. And also on my Custom Car Photo Archive site. If you are a Larry Watson fan.... check them out... you will not be disappointed. Here is a selection of what has been posted. There are photo captions on both the Facebook an my site. And don't forget to pick up a copy of Kustoms Illustrator to see the best photos of the collection in print. (top) This is a really cool photo of the Shop from where Bill DeCarr worked. In the photo we can see Larry's 1959 Caddy parked in front of the shop. We can also see Terry Holloway's 1957 Plymouth, and that car is most likely in for a redo at Bill's shop. The car was first painted in this wonderful olive green with beige, and was later redone with new molded in with bumperete front- and rear-end. It is parked on the left side in this photo. There is another 59 Caddy, and what looks like a 1955? Buick and a 1959 Chevy, all crammed in in front of the Ed's Auto Service Shop. This is the Shop that Bill DeCarr took over from Ed Schelhaas, and Larry would rent a spray booth from Bill. Bill and Larry where very good friends... like brothers. Larry's Firestone Blvd. Shop. (top) This very clean 1953 Ford Convertible was painted by Larry in one of his trademark outline paneled paint jobs. This time in candy red over gold with - hard to see in this photo - white striping. The car had been mildly customized with de-chromed and shaved body. Custom hubcaps and an aftermarket tube grille. Larry's shop was is the perfect backdrop... (Top) Larry Watson painted Ron Aguirre's Corvette the "X-Sonic" several times. This Platinum Pearl and Royal candy blue fogging version was done in 1963. Larry took this photo of the car and must have really liked it. Most likely it was hanging on his shop or office wall for a years hence the faded colors. (Top) Here is another photo of the Bill DeCarr Shop (former Ed Schelhaas) where Larry rented a space to paint his Custom Cars. I now have been able to identify this as the shop located at 10116 Artesia Boulevard, Bellflower, CA. and this was around 1960. The car is a wonderful mild customized 1958 Chevy owned by Dave Roberson. Painted Cherry burgundy over platinum pearl. Front of the side trim is swapped with a rear fender piece. Nice sculptured front fender tops. The interior is done in wonderful white tuck&roll. And we can see the Johnny Zupan/Bettancourt Mercury sitting against the wall behind the car. The building which is actually next door to Bill and Larry's shop is still standing today, and so is the shop. (Top) Here is a photo of bike that Larry painting. The paint almost looks like a semi gloss black, but in fact it is a pearl or platinum black. In the background we can see the 1955 Studebaker of Dick Gonzales.... (Top) This photo, taken at another Watson Shop shows the Watson painted Jaguar for H.O. MacLean. The paint job in Candy Gold cost a staggering $165.00... In the back ground we can see Jim Streets (Skonzakis) Golden Sahara truck. This was the truck Jim got to drive around the Golden Sahara. Bill DeCarr customized it with among other things a 1960 Mercury grille, and Larry Watson painted it. This is the first version of the truck, later on it was redone with more custom work, and a flake gold paint-job. (Top) Another very interesting photo of the Artesia Blvd. shop is this one where Ed Roth is pushing his Rotar around. Perhaps pushing it into Larry's Paint booth with is at the end of this drive way... We can see the Jeep again on the right side, and the hay trucks across the street.


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The Pet Food Stamps program, a registered NY State 501 (c) (3) non-profit corporation available throughout the United States, has been created to fill the void in the United States Food Stamp program which excludes the purchase of pet food. In these rough economic times, many pet owners are forced to abandon their beloved pet to the ASPCA, North Shore Animal League or other animal shelters due to the inability to pay for their basic food supply and care. There are over 50 million Americans who currently receive Food Stamps, many with dogs or cats, who simply cannot afford to feed their animals, and these cherished companions are dropped off at animal shelters where they will most likely be put to sleep. Many people are also forced to choose between feeding their pets or themselves. A recent New York Times article states that "animal shelters have reported a steep rise in the number of cats and dogs being surrendered as owners face unemployment, home foreclosures, evictions and other financial hardships." As more families struggle with difficult choices like paying the rent or buying food, some have to choose between keeping their pet and putting food on the table.. Seven million pets are surrendered to shelters each year due to the inability of pet parents to pay for food, with four million of those pets put to sleep. The Pet Food Stamps program aims to eliminate the needless death of these loving pets and keep families together.
The Pet Food Stamps program is not a government program and exclusively funds its operation from donations from individuals, business and foundation grants. We are able to eliminate the heart-wrenching decision of surrendering a pet by making sure these pet owners are given free home deliveries of Pet Food to maintain the health, vitality and companionship of their pets.
(Due to the tremendous amount of applications we receive, there will be an extended wait to receive a response after creating your account. We ask for your patience)
To become a member of Pet Food Stamps you must be receiving some type of Public Assistance such as: Food Stamps, Welfare or Social Security (if this is your only form of income and places you under the Federal poverty level for income).
To enroll in the program, simply fill out the application above by clicking on 'Sign-up' and create an account and profile for yourself and your pet.
PLEASE NOTE: We are currently underfunded to handle all charity requests received. As funding increases more applicants will be enrolled.
Pet Food Stamps also offers a 'Pet Services Classifieds' section allowing you to post advertisements and also to find: dog walkers, pet sitters, animal trainers, boarding, grooming services and Vet services across the United States. There is a $15 fee to post for 30 days and this helps cover the cost of the Pet Food Stamps program.
Donors to the program are also able to join the 'Pet Social Network' which will allow you to see photos, videos and stories of the pet your donation is going to feed. It takes a few weeks to enroll in this program and is currently in Beta.
The PFS Pet Social Network:
www.PetSocialNetwork.org is a community dedicated to pets and the people that love them. It is open to the World. Revenue from the site is used to fund the Pet Food Stamps program. Opening toward the end of Fall 2013, The Pet Social Network experience will be second to none and a treat for pet owners and their pets. Sign up @ www.PetSocialNetwork.org
Members of the Pet Food Stamps program will have a private profile on the PFS website but may still join the Pet Social Network and enjoy all the features it will have to offer.
Once you are a verified and approved member of the Pet Food Stamps program, our staff will transmit your pet food order to www.PetFlow.com, based on the amount awarded with your application approval and your pets' needs.
PetFlow.com is an independent online retailer and has been chosen as the exclusive provider of pet food for the Pet Food Stamps program. PetFlow carries approximately 200 brands of pet products and has been chosen based on their ability to offer discounted pricing and free shipping, direct to home.
PLEASE NOTE: Pet Flow only processes orders once Pet Food Stamps provides them the applicant information & payment. They have no information regarding the program or your account or status. All questions should only be directed to PFS.
If you are not a member of the Pet Food Stamps program,www.PetFlow.com offers $10 off any $40+ scheduled order with code PFS, plus free shipping on any delivery schedule over $49+ (after discount). You can choose to have your pet's food delivered from every 2 weeks to every 16 weeks and can change, cancel or edit your upcoming deliveries at any time.
If you are receiving Public Assistance and would like to apply for Pet Food Stamps, please click HERE
This Is A Private, Non-Government Program And We Rely Exclusively On Donations To Fund The Program.
If you would like to help the program by making a secure donation, please click HERE
For any questions or comments, click HERE
The Pet Food Stamp program has received a great deal of national coverage on TV, Radio,Newspaper and online. To view some of the content click HERE.


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Call: 01229 580956
Newly Qualified Commercial Property Lawyer
We require a newly qualified lawyer to join the busy commercial team in South Lakes. The role involves commercial work comprising mainly non-contentious landlord and tenant, leases of local shops and businesses, property purchases and sales and potentially some residential conveyancing.
Good IT skills are essential to enable the successful candidate to adapt, with training, to in-house systems including audio-transcription and precedent management software and working towards a paper-light office.


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Welcome, and thank you for visiting First Reformed Church of Holland, MI online. We hope that our website gives you at least a glimpse of the wide variety of worship, fellowship and service opportunities available. If you are seeking a Holland, MI church, please feel free to read more about our church on this site, or come in for a visit. We would love to greet you and share with you our love for Jesus Christ and for you, our neighbor.
Our Church
Or maybe we should call this 'Our People,' because the Church is, after all, a body of believers. In a world where relationships are often fleeting or even trivial, we commit to walk with each other. We support each other through good times and tough times. We are the 'First Family' and together we strive to make a difference in a world that is broken and hurting, right here in Holland, Michigan and around the world. We know we need each other and God's love to make that difference. None of us claim to be perfect and you are welcome to walk with us on this journey, come on along. At First Church, Holland, MI, there is…. always a welcome home.
Our Mission
EXALT the Lord, EQUIP the saints, EVANGELIZE at home and abroad. Our God came to us in love and we are called to go and love. We strive to help our neighborhoods and our city of Holland, MI be a better place to live. We go on mission trips within the United States as well as abroad. We can use your help in making a difference, so please consider helping us help. You don't have to be a church member or believe in our mission to make a difference in our world.
SERMONS and SERVICES Online
Find an archive of First Reformed services on our VIMEO channel.


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2012 marks Opel's 150th anniversary. To honor the occasion we are celebrating the rich past of legendary Opel models. In this case the awesome line-up of compact cars such as the Kadett or the Astra. For more join us on Facebook: http://www.Facebook.com/Opel
For more info on Opel, join us on: http://www.Facebook.com/Opel Never miss an Opel video: http://j.mp/SubscribeToOpel


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Mini Athletics - Little Athletes
Fun athletics based activities encouraging teamwork and competition
Busylizzy - Mums to Bee Pilates
1.1 miles
YMCA Surbiton - St Paul's Group
1.1 mi
KOI SWIM SCHOOL - Baby, Toddler and Junior Swimming Classes
2.1 miles
Safari Hotel - Chessington World of Adventures
3 months - 9 years
2.1 mi
Busylizzy - Busylizzy First Aid Workshop
2.2 miles
Kingston Quaker Centre
0 months - 2 years
2.2 mi
Saturday 10:15–11:00
Southborough School Sports Hall miles
Kent Way
Surbiton , Surrey
View directions
3 years 6 months - 5 years
All year
Advance Booking
NB: This event MUST be booked in advance. No drop-ins.
In this class, our athletics based activities emphasise teamwork, competition and social interaction. Through team participation and working together children's confidence grows session to session. If this is your child's first experience of Mini Athletics then they may be less willing to join in with other members of their team. This is normal and usually only occurs for the first few weeks while children are getting used to each other. By week 3-4 children are co-operating together.
Booking info
Please contact provider
- Block of 12
- £99.00
- Block of 6
- £54.00


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Includes Shampoo, Conditioner and 5-in-1 conditioning spray.
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HASK LEMONADE STAND is a limited-edition collection of summer-inspired, problem/solution haircare kits designed to treat and repair all hair types. Complete with shampoos, conditioners and leave-in treatment sprays to help combat the effects of summer heat and humidity on the hair. It's time to meet your main squeeze and treat those tresses to refreshing lemonade flavor combinations and fun fragrances!
Free of: sulfates, parabens, phthalates, gluten, drying alcohol and artificial colors.
HASK is also proudly cruelty free!