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Today, 10:37 AMspoula replied to a thread Babies dresses ? in MainHello elproct, I am sorry my reply with the chart was meant for Luna411 it was her question. I appreciate your nice reply to me, Looks like you...4 replies | 207 view(s)
Today, 10:23 AMHi Jean Marie, I like your colors the dress is so cute. I would never have thought about so many colors for that Sailor dress pattern. I still...12 replies | 201 view(s)
Today, 09:38 AMHello jean Marie, OH I couldn't tell it was purple you know every computer has different colors. It looked pink. I remember when making that puff...12 replies | 201 view(s)
Yesterday, 03:27 PMspoula replied to a thread Babies dresses ? in MainHello elproct, I make baby things all the time and I like to make them just a little bit bigger so the baby can get more wear out of it. If you have...4 replies | 207 view(s)
Yesterday, 03:14 PMHello Jean Marie, Don't you just love the look of this pattern. I like the little puff stitches and the shell stitches are so pretty. We both chose...12 replies | 201 view(s)
Yesterday, 03:05 PMHi Jean Marie. Wow! you have been busy. They all turned out really cute and colorful.. Is this the pattern you memorized? I can see how that would...12 replies | 201 view(s)
Yesterday, 08:37 AMMeToo! replied to a thread More dresses in PicturesA sailor dress - I've never seen such a thing, but it is a great idea and this highly colourful one will be seen (on a clear day) by jet passengers...12 replies | 201 view(s)
04-20-2018, 02:54 PMspoula replied to a thread Hey! in Introduce YourselfHello BreMick, Welcome to the forum. You can create a new post or you can reply to Jean Marie where she asked you what problem are you having. ...4 replies | 256 view(s)
04-20-2018, 02:18 PMspoula replied to a thread Two blue dresses in PicturesHi Jean Marie, The placket is about 2 more inches in crochet that makes the button band. It goes under the other side of the bodice back where you...5 replies | 279 view(s)
04-19-2018, 04:31 PMspoula replied to a thread Two blue dresses in PicturesHello Jean Marie, Thank you for the compliment. Are you going to make this dress too? I made the larger one different then the pattern. I did not...5 replies | 279 view(s)
04-19-2018, 01:12 PMspoula started a thread Two blue dresses in PicturesHello, I made the second dress this week for my Grand- daughter and decided to give the smaller one I had made previously to my great niece she can...5 replies | 279 view(s)
04-17-2018, 01:19 PMspoula replied to a thread HI. I'm new to crocheting only started about a week Ago in Introduce YourselfHello Seancorb, Welcome to the forum, I just saw this post and I will see if I can help you understand what to do on this pattern. You may have...14 replies | 434 view(s)
04-15-2018, 01:31 PMHello Heidi, Thank you so much for the compliment. I like to see what others are making also. I am currently making a dress for my grand daughter as...9 replies | 732 view(s)
04-14-2018, 10:23 AMspoula replied to a thread Looking for pattern for a child's poncho. in Pattern RequestsThis one ties at the waist. I looked at all the magazine covers from 1967 through all of 1970 Red Heart and Coats and Clark and found regular...15 replies | 653 view(s)
04-14-2018, 07:26 AMspoula replied to a thread Crochet bobble stitch in Links and ResourcesHello Wheels, Thank you for the complement. I can't take pictures very well but the dress is really pretty. It is still to big for my little grand...4 replies | 645 view(s)
04-12-2018, 04:14 PMspoula replied to a thread Looking for pattern for a child's poncho. in Pattern RequestsHello Linda, I found this one on the Internet it has a tie at the neck and pom poms. It came from archive patterns. It doesn't look like a poncho...15 replies | 653 view(s)
04-11-2018, 12:14 PMspoula replied to a thread Crochet bobble stitch in Links and ResourcesHello Maia, It has been awhile since I have seen any of your posts. Awhile back you posted a picture of a dress you wanted to make. I just loved it...4 replies | 645 view(s)
04-09-2018, 07:41 AMspoula replied to a thread Two color easy double crochet edging pattern in Pattern RequestsHi Jean Marie, That is a pretty way to do an edge but it would take forever on a large project maybe really cute on a baby blanket. Spoula3 replies | 440 view(s)
04-08-2018, 04:54 PMspoula replied to a thread Double pointed needles 10 inch/8 inch questions in MainHello crochetknitfan, You are welcome. I taught myself how to knit watching you tube videos so I didn't know if I would like it as much as crochet. ...8 replies | 493 view(s)
04-08-2018, 02:20 PMspoula replied to a thread Double pointed needles 10 inch/8 inch questions in MainHello crochetknitfan, Double pointed needles are used mostly in knitting to do sleeves and smaller parts of the item that you cannot do with long...8 replies | 493 view(s)
04-08-2018, 01:33 PMHello Jean Marie, Yes I did put flowers on the dress they are white with a diamond gem in the middle but they don't show up in the picture. I...9 replies | 732 view(s)
04-07-2018, 02:48 PMHi Choiyuk, I will have to search for the post myself. I will let you know if I can find it. It might have been a pattern that I had but you give me...9 replies | 732 view(s)
04-07-2018, 10:45 AMHi Jean Marie, Thanks for your reply. It is a really pretty pattern you should make this one it is easy to do. Spoula9 replies | 732 view(s)
04-07-2018, 06:50 AMspoula started a thread Oned down one to go baby dress in PicturesI finished the baby dress for my granddaughter now I will start on one for my great niece. This pattern is the Purple Dress pattern that choiyuk...9 replies | 732 view(s)
04-05-2018, 09:58 AMspoula replied to a thread Grandmother's baby booties in MainHi Suewin, Good job on figuring out an old crochet pattern. It is even more special because of all the generations that made that pattern and you are...2 replies | 292 view(s)
04-05-2018, 09:54 AMspoula replied to a thread I've Been So Bad... in The Crocheted ButterflyHi Nova55, These Bunnies are so cute and I like the write up even cuter. Sppula4 replies | 264 view(s)
04-04-2018, 10:52 AMspoula replied to a thread Basket finished in MainHello Nova55, I have only done one stiffening project and that was the baby hat I made and posted on this forum. I used corn starch on that one as I...5 replies | 429 view(s)
04-04-2018, 07:55 AMspoula replied to a thread Would I be able to use a DK?... in MainHello Hekel, Here is a tip I hope will help you. If the number of stitches is less than the number of stitches given in the gauge ( you used a...4 replies | 220 view(s)
04-04-2018, 07:53 AMMeToo! replied to a thread baby cardigan in PicturesSuch a sweet little set.12 replies | 1025 view(s)
04-03-2018, 05:56 AMspoula replied to a thread Crocheter's Guide in Amy's Creative CreationsHello Amray767, I noticed that your hook size for H through J is not the standard number. I know that different sizes could be according to the...2 replies | 238 view(s)
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About MeToo!
Basic Information
About MeToo!
- Biography:
- Re-learning crochet after many years.
- Location:
- Canada
- Interests:
- Gardening.
- Occupation:
- Housewife.
Total Posts
- Total Posts
- 162
- Posts Per Day
- 0.47
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- Last Activity
- Yesterday 10:54 PM
- Join Date
- 05-10-2017
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Showing Friends 1 to 1 of 1
- 07:38 AM - MeToo! clicked Likes for this post: baby cardigan by TeriThank you....me...


text:
Phone Numbers
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Echo Limousine Phone Number
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Customer service
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6 mins
(4m avg)
Hours: 24 hours, 7 days; best time to call: 10:15am
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Rank: #1 of 1 ways to contact Echo Limousine
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Get talking points and pro tips before you call Echo Limousine.
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Customer Number 773-774-1074 - by the Numbers
Phone number to dial
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24 hours, 7 days
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Echo Limousine's Best Toll-Free/800 Customer Phone Number
This is Echo Limousine's best phone number, the real-time current wait on hold and tools for skipping right through those phone lines to get right to a Echo Limousine agent. This phone number is Echo Limousine's Best Phone Number because 54 customers like you used this contact information over the last 18 months and gave us feedback. Common problems addressed by the customer care unit that answers calls to 773-774-1074 include Make a booking, Change booking, Cancel booking, Overcharge/Strange charge, Loyalty program and other customer service issues. Rather than trying to call Echo Limousine we recommend you tell us what issue you are having and then possibly contact them via phone. In total, Echo Limousine has 1 phone number. It's not always clear what is the best way to talk to Echo Limousine representatives, so we started compiling this information built from suggestions from the customer community. Please keep sharing your experiences so we can continue to improve this free resource.
Contacting Echo Limousine - by phone or otherwise
While 773-774-1074 is Echo Limousine's best toll-free number, it is also the only way to get in touch with them. The next best way to talk to their customer support team, according to other Echo Limousine customers, is by telling GetHuman about your issue above and letting us find somebody to help you. Besides trying to call, best to start by telling us what the issue is above. If you think this information is inaccurate or know of other ways to contact Echo Limousine please let us know so we can share with other customers.
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text:
Copyright © 2018 Guaranteed Dental Solutions PLLC
4530 E. Ray Rd. Suite 170 Phoenix · AZ 85044 · The United States (480) 477-3090
4530 E. Ray Rd. Suite 170 Phoenix · AZ 85044 · The United States (480) 477-3090
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Romance profile dating friends site
Two thirds of online daters—66%—tell us that they have gone on a date with someone they met through a dating site or dating app.That is a substantial increase from the 43% of online daters who had actually progressed to the date stage when we first asked this question in 2005.Many online daters enlist their friends in an effort to put their best digital foot forward.
Few Americans had online dating experience when Pew Research Center first polled on the activity in 2005, but today 15% of U. adults report they have used online dating sites or mobile dating apps.
Striking up a friendly rapport with a newcomer becomes the exception, not the norm. These days, there are apps for pretty much anything, from getting a cleaner in after a party to chartering a private jet and booking a massage. 'Wiith', a new San Francisco-based app, is designed to buddy you up with people looking for buddies. No romance, no sex, just pure, unadulterated friendship. The premise is simple: if you're new to a city, or just want to meet someone new, the app connects you with like-minded others inside a set radius.
You sign up via Facebook, build a basic profile, then approach people for friendship or arrange social events and invite others along.
Either way, you wouldn't be in the minority of adults if you sometimes wished for a new friend or two to enter your life.
After school and university – both moveable feasts of friend-making opportunities – men in particular often forget how to make close buddies.


text:
Can I Get a Pet if I Live in a Small Apartment? »
Living in small space doesn't mean you can't have a furry friend -- it just means you have to do some planning.
YP - The Real Yellow PagesSM - helps you find the right local businesses to meet your specific needs. Search results are sorted by a combination of factors to give you a set of choices in response to your search criteria. These factors are similar to those you might use to determine which business to select from a local Yellow Pages directory, including proximity to where you are searching, expertise in the specific services or products you need, and comprehensive business information to help evaluate a business's suitability for you. "Preferred" listings, or those with featured website buttons, indicate YP advertisers who directly provide information about their businesses to help consumers make more informed buying decisions. YP advertisers receive higher placement in the default ordering of search results and may appear in sponsored listings on the top, side, or bottom of the search results page.
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We found13results
Living in small space doesn't mean you can't have a furry friend -- it just means you have to do some planning.
We've put together a small list to get you started on your journey -- and the first step starts with a single box.


text:
Journal Articles
Affordable Care Act: Medicaid Expansion Key to Increasing Access to Care
- Author
- Chesney Mary L, Duderstadt Karen G
- Journal
- Journal Of Pediatric Health Care SCI(E) SCOPUS SSCI
- Publisher
- Elsevier
- Cited Count
- 1
- Partners
- Elsevier
- 10.1016/j.pedhc.2013.04.001
- Paid
- Elsevier
- Category
- Medicine > Pediatrics
- Keywords
- Medicaid expansion, ACA, CHIP, children, access to care
- References open button
- Cited Articles open button
References 12
1.Updated projections for the insurance coverage provisions of the Affordable Care Act.(March 12, 2012)
2.Medicaid expansion opt-outs and uncompensated care. New England Journal of Medicine. Vol. 367. 23652367(2012)
3.Kaiser Family Foundation, Kaiser Commission on Medicaid and the Uninsured. (2012). Medicaid financing: An overview of the Federal Medicaid Matching Rate (FMAP). Retrieved from http://www.kff.org/medicaid/upload/8352.pdf.
4.Kaiser Family Foundation, Kaiser Commission on Medicaid and the Uninsured. (2013a). Getting into gear for 2014: Findings from a 50-State survey of eligibility, enrollment, renewal, and cost-sharing policies in Medicaid and CHIP, 2012–2013. Retrieved from http://www.kff.org/medicaid/upload/8401.pdf.
5.Kaiser Family Foundation, Kaiser Commission on Medicaid and the Uninsured. (2013b). Medicaid: A primer. Retrieved from http://www.kff.org/medicaid/upload/7334-05.pdf.
6.Kaiser Family Foundation, Kaiser Commission on Medicaid and the Uninsured. (2013c). Medicaid: Three key issues to watch in 2013. Retrieved from http://www.kff.org/medicaid/quicktake_medicaid_in_2013.cfm.
7.Kaiser Family Foundation, State Health Facts. (March 5, 2013). Health reform: State activity around expanding Medicaid under the Affordable Care Act. Retrieved from http://www.statehealthfacts.org/comparereport.jsp?rep=158&cat=17.
8.Lucile Packard Foundation for Children's Health. (2013). Issue brief: Children with special health care needs and California's Healthy Families transition to Medi-Cal. Retrieved from http://lpfch-cshcn.org/wp-content/uploads/2013/01/Healthy-Families-Issue-Brief-1-16-132.pdf.
9.Patient Protection and Affordable Care Act, Pub. L. No. 111–148. (2010)..
10.The Supreme Court's surprising decision on the Medicaid expansion: How will the federal government and states proceed?. Health Affairs. Vol. 31. 16631672(2012)
Articles referencing this article
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I thought when we moved to Los Angeles, I'd never endure spring allergies again. My dreams of swirling in fragrant orange blossoms and jasmine would blossom into a (sneeze-free) reality. I didn't... read more >>
Polly BlitzerApril 19, 2018 - 10:04pm
At the risk of sounding old school, do you guys remember when red eye was a thing to be avoided? That demonic glare beaming from people's eyes in photos. Back when we used cameras with flashes and... read more >>
Polly BlitzerJanuary 9, 2018 - 1:40pm
Twelve tubes of lipstick? Poetry scribbled on Sephora receipts? Keys to a Harley? You can tell a lot about a person by the contents of her bag. In our What's in My Bag series, we ask beauty and... read more >>
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Forget surreptitious selfies to check your makeup. You'll never need a powder room again with the reflective minaudieres spotted on the SAG Awards red carpet. Miu Miu Mirrored Box Clutch, ... read more >>
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Shay Mitchell can do no wrong in our book. Whether she's rocking a bright coral lip on vacation in Thailand, or giving us mega sultry vibes on the red carpet, the Pretty Little Liar's star just gets... read more >>
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En route to Neutral Instinct are these gorgeous concrete lamps and we are excited. Order now for dispatch next week. Beautiful styling by Sofie Brunner and photo by Enok Holsegaard.
Black and White Scandinavian interior
my scandinavian home: A light and airy Danish home inspiration with natural touches
Black & white living room design - DIY Home Decor
Kiva synttärilahja, Fokus tarjotin, Marimekko. String. Fokus trade, Marimekko.
new homewares collection from ferm living / sfgirlbybay / sfgirlbybay.


text:
Secluded and private Territorial House 10 Min.-Red River 20 Min. to Taos
About the property
- Cabin
- Bedrooms1
- Sleeps8
- Bathrooms1
- Half Baths0
- Minimum Stay2 nights
2400 sq ft home, a bar area, 3 queen beds, 1 Bath, (Sleeps 6)
If you are looking to relax and soak up fresh mountain air, Paradise Creek is the best of Northern New Mexico in the mountains of Questa. Questa is a small, quaint, heritage rich town that is just north of Taos about 20 enchanting miles. It is also the place where you turn east, at the only light in town, and drive 10 miles east to Red River - a mountain resort.
Questa is located on the Enchanted Circle scenic highway, near the confluence of the Rio Grande and the Red River and is at the western base of the Taos Mountains. To the southeast lies Flag Mountain, a northwestern spur of the group of mountains that includes Wheeler Peak, the highest peak in New Mexico. To the north and west, you will be struck by the Rio Grande Gorge and how it cuts a volcanic plateau. A great private place to relax.
Hikers enjoy the beauty of the mountains with a variety of different hikes available only minutes away. Fishermen relax and enjoy the quiet serenity of Eagle Rock lake only l/4 mile away. Northern New Mexico has an incredible number of fishing spots. ATV trails are numerous. Goose Creek and Goose Lake is just 15 minutes away. To the South, 10 miles away, relax and fish at the local's favorite fishing spot - The John Dunn Bridge in Arroyo Hondo. Take a drive 10-15 miles north of the property and you will be in awe of the Valle Vidal or the Wild Rivers. Visit Taos 26 miles south - take time to see the Taos Pueblo, a national historic landmark along with many other historical sites.
Great for summer vacation get aways for large family outings, retreats, gatherings or family reunions. There is plenty of space to have adventures, play softball, horseshoes or football. The property is gated by a tall New Mexico traditional 'Latilla' fence which provides for wonderful privacy and security. Tall cotton wood trees surround the property with a creek cutting through.
Enjoy and explore the mysteries this 3 acre land has to offer. Mystical stories abound about treasures buried in this land 200 years ago. Guests have found small Indian artifacts.
A traditional New Mexican Territorial style home, it is a large house with an upstairs open loft with 2 queen beds separated by a tall divider. A futon sleeper and double bed sofa sleeper downstairs. Can sleep up to 6 people. Price includes up to 6 people - addition guest are $25.00 per person. There is plenty of room to dance, play and enjoy the bar area. Ceiling has accoustical treatment which makes the Bose Surround Sound system and the plasma T.V. and washer and dryer. Fully equipped kitchen with pots and pans, dishware, toaster, coffee maker, etc. There is also a 2 bedroom GUEST HOUSE (Casita) on the same 3 acre property that is also available for rent. See VRBO #372713. Please inquire if you would like the extra room!
Keywords: House on 3 acres with running creek. Plenty of room for large families.
Patricia Rael
- Response rate:
- 100%
- Response time:
- within a few hours
- Speaks:
- english, spanish
- Calendar last updated:
- Apr 26, 2018
About Patricia Rael
Patricia Rael is a member of the Rael Family. Her father was the historian of Questa, a school teacher and writer. Her mother and father raised 6 children. She remembers her father inviting the school children to enjoy the land.. At an Easter Egg Hunt, holding her dad's hand, she decided one day she would have a an affordable beautiful place in the country for people to get away from the city and have enough room to enjoy nature and each other.
Patricia Rael purchased this Cabin in 1990
Why Patricia Rael chose Questa
I was fortunate to have inherited this history rich property from my parents. One of the acres which is now the main house was an artist's studio converted to an open loft and easily sleeps 6 people
What makes this Cabin unique
I enjoy the beauty of the mountains closeby, the large cottonwood trees, the small creek that weaves through the property and the serenity and privacy - a place of peace and space to relax and not be burdened by the city noise.
- Internet
- Pets Welcome
- Fireplace
- Satellite or Cable
- Kids Welcome
- Parking
- No Smoking
- 1 Bedrooms, Sleeps 8
- Queen size beds in the loft (2), Full sofa sleeper (1), Futon queen sleeper (1) **Beyond 6 people, contact Elizabeth (property manager) at 505-697-9293.
- 1 Bathroom
- 1
Location Type
- Lake ViewEagle Rock is a mile away, Cabresto Lake - 5 miles, Goose Lake 12 miles away.
- Mountain ViewSangre de Cristo mountains are the backyard. Hear the running creek while relaxing outside. Creek approximately 30 feet away.
- Ski-In10 minutes from Red River Ski Area - 26 minutes from Taos World Class ski Area
- Water ViewWild Rivers 12-15 miles away. John Dunn Bridge 15 miles away
- Guests provide their own meals
- Linens Provided
- Parking Off Street
- Parking for RV/Boat/Trailer
- Wireless Internet
- Wood Fireplace(2)
- Cooking Utensils
- KitchenFully Stocked pots and pans, dishes, and glassware
- Microwave
- Refrigerator
- Dining Area
- CD Player
- DVD Player
- Satellite / CableBoise surround sound speakers
- Stereo
- TelevisionDVD for movies and has Boise surround sound speakers
- Outdoor Grill
- churches
- cinemas
- library
- museums
- restaurants
- ruins
Leisure Activities
- bird watching
- gambling casinos
- horseback riding
- horseshoes
- photography
- scenic drives
- sight seeing
- sledding
- walking
- wildlife viewing
Local Services & Businesses
- ATM/bank
- groceries
Sports & Adventure Activities
- mountain biking
- cross country skiing
- fishing
- fly fishing
- golf
- hiking
- hunting
- cycling
- rafting
- rock climbing
- skiing
- snowboarding
- snowmobiling
- water tubing
- whitewater rafting
- Cleaning Fee $100.00 Pet's allowed for additional fee. Contact property manager Elizabeth at 505-697-9293. Occupancy greater than 6 people for additional fee. Contact property manager Elizabeth at 505-697-9293.
House Rules
Check-in: 3:00 PM
Check-out: 11:00 AM
Cancellation Policy
Canceled bookings will not receive a refund
40 Reviews
Latest reviews
We loved staying at this charming place. Pat went over and beyond to make sure our stay was comfortable. We will try and stay here again next year.
Patricia is the most welcoming and accommodating host I think I have ever encountered. Not only was her place absolutely stunning, she drove all the way to Questa in order to make the place ready for us to check in at 8am instead of 4pm because we were making the drive from Texas to NM overnight and wanted to be able to get there and get situated. The only problem that we had was with the internet, but we let Patricia know right away and she worked super hard to get it fixed, but the company couldn't get somebody out there until after we had left so that is no fault of hers. I would absolutely recommend this residence, and Patricia, to anybody looking for an easy, relaxing and beautiful stay in New Mexico. Thank you to Patricia for everything she did for us, you are the best!!!
Ahhh thank you. You all were great! Internet did get fixed btw. Thanks for letting me know.
Great getaway with quick access to the surrounding mountains
Awesome place to get out of the elements after a long day in the woods. Rustic character is awesome and everything worked basically as would be expected for a mountain adventure base camp. Kind and accomodating host. Thanks for a great week!
This party was a wonderful group to rent to!! I wish I could have personally met them!
Very clean, spacious, and peaceful!
The rooms were cozy and warm and the property owner was VERY hospitable. Definitely makes me want to stay there again sometime. I felt right at home, especially with all the accommodations.
Great Property!
Good property and amazing location. The owner is very nice and very helpful, would recommend this property to anyone!
Beautiful property!!
This is a beautiful piece of property. The stream running through it is calming. It is on a main road coming into/out of the housing area. Although surrounded by coyote fencing, there is still road traffic so if you are looking to be out and away from all that, this may not work for you. The home layout is different that it seems by the pictures. The kitchen is very small and confined by the bar. Not a big deal as it was only my husband and I, but for a larger group may be difficult. The owner Patricia is amazing. She is very helpful and accommodating. Super great to work with. The home is equal distance between Taos and Red River.
Thank you for your review! We look forward to your
next stay
Quick and easy access to the Rio Grande Gorge hiking, Wild Rivers, Goose Creek, Valle Vidal and Cabresto Lakes, Rafting 'The Box'. Skiing, snowboarding, hiking, fishing, hunters. Family vacation, gatherings, family reunions, weddings, or a getaway .This property has a creek running through the property and a perfect place to relax. This main house can be rented along with another guest house in the property which accomodates 4-5 and perfect for large groups.
Rates & Availability
Additional information about rental rates
Includes a washer and dryer


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We Service your area with the Best in commercial and residential – handicap access construction alterations and service estimates.
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Track or Rail – The stair lift track sustains the chair, supplies a device (typically a geared strip) to thrust the chair and also overviews it up as well as down the stairs just as a train track guides a train.
Track Overruns – Some stair lifts offer the choice of an overrun at either the top of the track, bottom of the track or both. The purpose of a stair lift track overrun is to relocate the customer away from the stairway to securely accessibility or dismount the stair lift in a location where, if they drop, they won't landed on the stairways.
Power supply – While many stair lifts are battery operated today all design will call for a power supply to either bill the battery or power the system directly when it comes to non-battery powered units. Non-battery powered stair lifts attract a great deal of power as well as generally require a devoted circuit.
Call/Send Controls – Call/Send controls are normally placed on the wall surface near each end of the stair lift track to give a means for a user to call the chair to them or send it away when not required. They can be either wireless or wired to the lift depending on the model.
Seat – The stair lift seat is merely a seat for the individual to rest on while utilizing the lift. Depending upon the design it could rotate at the top and/or base of the track and will normally fold off the beaten track when not being used.
Seat Belt – As in an auto the stair lift seat belt is meant to hold the customer securely seated on the seat while the lift remains in usage.
Foot Rest – The foot remainder of the stair lift is a security feature to avoid the the users feet from striking the actions while making use of the lift and a convenience function to sustain the user's feet when resting on the seat. The foot rest will certainly have buttons constructed into them to quit the lift ought to it strike anything on the stairs and also will turn up off the beaten track when the lift is not in usage.
Motor – The electric motor that owns a stairlift chair along the track is located in the base of the seat.
Transmission – Because the electric motor turns at too expensive a speed to securely move the stair raise the track at kind of transmission is utilized to minimize the speed while enhancing the power readily available.
Battery Charger – Battery powered stair lifts have to have a charger of some sort to maintain the battery billed. The battery chargers are generally found under the track or near one end of the track. The battery charger works automatically without any activity needed by the customer.
Battery – The batteries made use of in stair lifts are fairly pricey as well as located within the base of the seat near the electric motor. They will certainly have to be changed at a long time yet their life expectancy is determined in years and also their replacement need to not be a huge worry.
If a ramp run has a rise greater than 6 inches or is longer than 72 inches, after that it's recommended to have hand rails on both sides of the ramp. For ramps over 30 inches from ground degree, pins should be used. And also a curb or prop stop need to be put along both sides of the ramp to stop wheels from leaving the ramp.
A home elevator could not be set up in any kind of kind of public building, including in locations of prayer. For any type of buildings that are not personal residences, you will need an industrial elevator or lift. The exception is a private condo with exclusive accessibility.
Do not build a hoist-way without recognizing the requirements required for your elevator. An elevator likewise needs overhead area (room above the top of the elevator at the leading touchdown), and also typically a pit at the base.
A hoist-way has a number of demands for construction consisting of specifics for the wall surface where the elevator rails will certainly be secured (the assistance wall). Each producer offers these specifics for each and every design of elevator.
There are 2 standard kinds of stair lifts that are sold today: straight and also bent. The type you require will certainly rely on the style of your stairs.
A straight stair lift is one that travels in a straight line up a flight of staircases continuous by landings, bends or contours, and prices in between $3,000 and also $5,000 installed. Rounded lifts, however, are much more intricate as well as will certainly go around edges, bends as well as reversals. Rounded lifts are also far more expensive generally running in between $10,000 and $15,000 or more relying on the complexity of the installment.
Additionally readily available with particular companies are weatherproof lifts for outdoor steps, and standing stair lifts (likewise called perch lifts) for those that have problem flexing their knees.
You also have to understand that all stair raises place to the stair treads, not to the wall, so they are extremely durable and can be installed in practically any house.
We could set up grab bars as well as safety rails to guarantee you won t lose your balance as well as provide a safe, available surrounding. Safety bars offer security for every person, consisting of the elderly and those with physical constraints. It s one of the most economical residence modification we can make.
Solid Mount Grab Night clubs permits the installment of grab bars right into existing fiberglass shower and also tub wall surfaces. The Strong Mount mounts through the face of the fiberglass utilizing a patented mounting setting up that connects to the stud, filling up the hollow space, securing the opening as well as leaving an installing surface area to which a conventional grab bar could be safely connected.
Call/Send switches are made use of to move the stair lift chair without any person being on it. A DC lift is connected in as well, yet it runs utilizing the power kept in the batteries that are part of the lift. Many straight stair lifts could be set up in 3 to four hrs. A straight stair lift is one that travels in a straight line up a flight of stairs nonstop by landings, flexes or contours, as well as expenses between $3,000 as well as $5,000 set up. Most stair raises offered today likewise have seats, armrests and footplates that fold up out of the means, as well as swivel seats that make getting into and also out of the chair less complicated.
Just what makes a home handicap as well as mobility device accessible? Restrooms, bed rooms, kitchens that allow the flexibility of movement without narrow doors or actions. Decreased counters & cupboards that are there to help with easy accessibility and even the capability to obtain right into or from the home without barriers can make a house a better atmosphere with a special needs.
We recommend a shower with an aesthetic but, there s times when a roll-in shower is your only option. Many existing restroom sizing limitation the alternatives to a tub to shower conversion. A wheelchair will certainly fit right into the location however it is tight.
The angle of the ramp surface areas and also the size or run of the ramp is a vital project factor to consider. The ramp incline will certainly impact the design demands, the cost entailed as well as the ramp's ultimate usefulness. Slope is the angle partnership of vertical elevation (increase) to the horizontal length or projection (run). It's typically revealed as a proportion of these 2 measurements, with the surge number often set at an unit of one. A slope of 1:12 means that as each dimensional unit (normally inches) of elevation changes, the various other side jobs (or runs out in size) 12 systems (inches).
Other slope proportions might be utilized for constructing your ramp, the (American with Disabilities Act) ADA-recommended and the most commonly utilized incline is 1:12. This means that if your veranda height is 24 inches off the ground, you'll require a 24-foot ramp to securely suit mobility device access. If you intend on drifting from this criterion, you ought to examine your local building regulations to be specific you're in compliance.
Doors and also gates are regulated with electro-mechanical locks for safety. Strong core doors are required. A door operator could be included so that the elevator doors do not have to be opened manually. Automatic sliding doors like those from Savaria offer added safety because they get rid of the gap between the landing door and the taxicab door. Try to find doors as well as gateways that include light screens or light curtains. Light screens develop a light beam of light that, when damaged by a person strolling via it, makes sure that the elevator could not move.
Your local specialist elevator specialist must know the local code as well as evaluation needs for property elevators. You could require a permit as well as additionally require government inspection prior to you could utilize your elevator, depending upon where you live. Silver Cross can assist you find a pre-qualified installer near you that will understand just what is needed in your area.
We could likewise mount the brand-new Walk-In bathtubs and also add Bath tub or shower grab bars to offer support. Many individuals with present tubs locate them difficult to make use of and also have troubles entering and also out of tub. The new Walk-In bathtubs have several functions that have a tub/shower combination. They additionally have an alternative of air jetted or Healing Health club Massage Jets. They are often referred to as whirlpool jets or hydrotherapy jets, spa massage therapy or various other such recommendations.
Hydrotherapy entails the use of re-circulating the water in the tub to press out water for pain-relief and also dealing with several illnesses. The hydrotherapy jetted tubs can be used to minimize pain connected with arthritic conditions, blood circulation, as well as fibromyalgia, rehab of busted bones or replacements in addition to general discomfort alleviation. You ought to seek advice from your doctor before installment of this kind of tub making certain that it will not make your problem worse.
The minimum, inside clear size of the opening between the opposing handrails have to go to the very least 36 inches to accommodate a mobility device. This suggests the ramp needs to be developed at the very least 42-inches vast to permit for the 1- inch spacing in between the handrail as well as any surface area and the real 1- inch hand rails.
Standard home contractors have never truly thought about the requirements of those that are impaired or elderly, and that can make life truly hard to handle sometimes. We understand these limitations could be problems, and we want to aid you make your life a little simpler and extra productive. That s why we at In The House Acquiring, Inc., offer handicap easily accessible improvement and availability adjustments in Destin, Florida and also bordering neighborhoods. We make every effort to locate the solutions to your house ease of access needs and modifications, therefore enabling you to improve the security and accessibility in your house. Let us assist you enjoy your home by fitting your requirements today and also in the future.
A number of us will experience at the very least a momentary handicap throughout our lifetime. Crashes do happen and as a house owner you can discover yourself utilizing a wheelchair or pedestrian. As we age, getting around our home could come to be more difficult sometimes. We ask ourselves How can I obtain up those actions or through that narrow door? Getting ready for this future demand originates from much spotted home owners, builders as well as remodelers who expect restrictions and also changing demands. As you age, you could think about offering your house to find something that accommodates your altering demands. Instead of experiencing the problem of offering your residence and also moving, leaving your buddies and also neighborhood, why not let In your home Acquiring, Inc. offer you even a lot more reason to stay!
Stair Lift Neshanic Station NJ
Wheel Chair Lift Three Bridges NJ
Stair Lift Skillman NJ
Wheel Chair Lift Belle Mead NJ
Walkin Tub Blawenburg NJ Home Elevator Piscataway NJ
Residential and Commercial Improvements
Neshanic Station, NJ 08853
Somerset, New Jersey (NJ)


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LUFS is a hybrid userspace filesystem framework supporting many "exotic" filesystems (localfs, sshfs, ftpfs, gnutellafs, locasefs, gvfs, cardfs, cefs, etc. ).
This is a daemon that emulates Microsoft's Internet Connection Service (ICS). It implements the UPnP Internet Gateway Device specification (IGD) and allows UPnP aware clients, such as MSN Messenger to work properly from behind a Linux NAT firewall.
A GTK+ Gnutella client, optimized for speed and scalability
A GTK+ Gnutella client, efficient, reliable and fast, written in C. It has been optimized for speed and scalability, with low-memory consumption. It is meant to be left running 24x7, using little CPU and only the configured bandwidth. It is possible to run gtk-gnutella without a GUI or using a shell interface.
CurlFtpFS is a filesystem for acessing ftp hosts based on FUSE and libcurl. Automatically reconnects if the server times out.
The Player Project: Player is a networked interface to robots and sensors. Stage and Gazebo are Player-friendly multiple-robot simulators. The software aims for POSIX compliance and runs on most UNIX-like OS's. Some parts also work on Windows.
ODABA is an terminology-oriented database management system and develo
ODABA is an terminology-oriented database management system (TODBMS) on a high conceptual level. It provides a number of enhanced features based on natural language analysis. Terminology orientation allows designing user oriented data models. Enhanced event model and Active Data Link technology support rapid development of GUI applications. Several development, documentation, test and analyzing tools are provided in order to support developers and administrators. .NET interface and a powerful object-orjented script language (OSI) improve development efficiency.
An attempt to write a driver for Canon CAPT-printers, including, but not limited to, LBP-2900. THIS PROJECT IS OUTDATED AND INACTIVE. The new and supported version is located at https://github.com/agalakhov/captdriver
DownloadDaemon is a comfortable download-manager with many features like one-click-hoster support, etc. It can be remote-controled in several ways (web/gui/console clients), which makes it perfect for file- and root-servers, as well as for local use.
NFS-GANESHA is a NFS server running in User Space. It is available under the LGPLv3 license. See website (hosted by sourceforge): http://nfs-ganesha.sourceforge.net
A scanner button daemon for Linux (and possibly other Unix-like operating systems) which allows you to trigger certain actions (e.g. run a shell script) whenever one of the scanner's front panel buttons has been pressed.
ffmpeg-php is a PHP extension that adds functions for accessing and retrieving information from movies and audio files. ffmpeg-php provides access to the many audio and video formats supported by ffmpeg. NOTE: This project is no longer maintained and completely unloved. Check out (https://github.com/char0n/ffmpeg-php/tree/master/adapter) for a drop in replacement that's actively maintained and not hosted on this horrendous sourceforge site.
wview is a unix application for weather stations. It archives weather data from the station. Generates full html web sites with graphics. ftp/ssh export capability. MySQL/PostgreSQL archiving. International. Weather alarms. Data feed. RSS feed.
This site hosts the source code for C++ version of the Broker for SBW, NOM module, advanced simulation suite, analysis applications and model editors.
tinyhttpd is a relatively simple webserver I wrote for a school project. While exceedingly simple, tinyhttpd is threaded and handles basic CGI scripts! This is an educational tool to demonstrate the concepts behind http.
Siproxd is a proxy/masquerading daemon for the SIP protocol. It allows SIP clients (softphones & hardphones) to work behind an IP masquerading firewall or router.
MySQL is a true multi-user, multi-threaded SQL database server.
This project is a random collection of Linux kernel-related projects and source code.
C++ exception (and generic) stack trace library (now libinstrument)
Project is migrating to libinstrument (https://sourceforge.net/projects/libinstrument/) Libcsdbg is a C++ exception stack tracer, offers the tools to create and process exception traces, the path an exception has propagated up the call stack, unwinding it, up to the point where it was handled. The traces are detailed with demangled function signatures and addr2line info (source file and line of each function call). Libcsdbg will perform flawlessly with multi-threaded programs, with dynamically linked shared objects (DSO) and with any statically linked library. Libcsdbg helps the developer/tester also create stack traces of any thread, at any point during, as a snapshot of the runtime call graph. This is very useful in cases of fatal errors, reception of terminating signals or process abortion/termination (such as segmentation faults)
IP Monitor notifies you when your public IP address changes
IP Monitor is a simple application which monitors your public IP address for changes and lets you set different kinds of notifications such as email, audio, pop up or executing a command. It can also run in background as a Windows service or Linux/Mac daemon.
Convert Webmail to POP3.Is also included a SMTP/POP3 tunneling system that allow send and receive email in a private network HTTP PROXY based. All connection are plugin based. Over 450 email server supported and tested, GMAIL POP3/SMTP over Proxy Network.
A client implementation of Secure Socket Tunneling Protocol (SSTP) for Linux / Mac OS-X that allows remote access via SSTP VPN to Microsoft Windows 2008 Server.
MATE Desktop Environment, a non-intuitive and unattractive desktop for users, using traditional computing desktop metaphor. Also known as the GNOME2 fork.
GNU GetText translation tools for Borland Delphi and Borland C++ Builder
!! PJL Compressing Filter is renamed to ziplet and hosted on github now!! See here: https://github.com/ziplet/ziplet A J2EE servlet filter which compresses data written to the response. It supports several algorithms (gzip, deflate, etc.) and emphasizes minimal memory usage and high throughput. Also provides detailed performance stats. Feel free to fork pjl-comp-filter on Github: https://github.com/ziplet/ziplet
Linux/Unix driver development for Syntek Semicon USB2.0 Video device DC-1125, like the one that is found in Asus A6K laptops. The device can be recognized by the usb id 174f:a311 and maybe also be a standalone unit (not integrated).


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Colibactin is a nonribosomal peptide-polyketide synthesized by multi-enzyme complexes encoded by the pks gene cluster. Colibactin-producing Escherichia coli have been demonstrated to induce host DNA damage and promote colorectal cancer (CRC) development. In Taiwan, the occurrence of pyogenic liver abscess (PLA) has been suggested to correlate with an increasing risk of CRC, and Klebsiella pneumoniae is the predominant PLA pathogen in Taiwan
Methodology/Principal Findings
At the asn tRNA loci of the newly sequenced K. pneumoniae 1084 genome, we identified a 208-kb genomic island, KPHPI208, of which a module identical to the E. coli pks colibactin gene cluster was recognized. KPHPI208 consists of eight modules, including the colibactin module and the modules predicted to be involved in integration, conjugation, yersiniabactin production, microcin production, and unknown functions. Transient infection of BALB/c normal liver cells with K. pneumoniae 1084 increased the phosphorylation of histone H2AX, indicating the induction of host DNA damage. Colibactin was required for the genotoxicity of K. pneumoniae 1084, as it was diminished by deletion of clbA gene and restored to the wild type level by trans-complementation with a clbA coding plasmid. Besides, BALB/c mice infected with K. pneumoniae 1084 exhibited enhanced DNA damage in the liver parenchymal cells when compared to the isogenic clbA deletion mutant. By PCR detection, the prevalence of pks-positive K. pneumoniae in Taiwan is 25.6%, which is higher than that reported in Europe (3.5%), and is significantly correlated with K1 type, which predominantly accounted for PLA in Taiwan.
Citation: Lai Y-C, Lin A-C, Chiang M-K, Dai Y-H, Hsu C-C, Lu M-C, et al. (2014) Genotoxic Klebsiella pneumoniae in Taiwan. PLoS ONE 9(5): e96292. https://doi.org/10.1371/journal.pone.0096292
Editor: Dajun Deng, Peking University Cancer Hospital and Institute, China
Received: November 14, 2013; Accepted: April 7, 2014; Published: May 22, 2014
Copyright: © 2014 Lai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Ministry of Science and Technology (formerly National Science Council) of Taiwan, NSC 101-2311-B-005-002- to Ying-Tsong Chen, 100-2320-B-040-013- to Yi-Chyi Lai, and NSC 100-2320-B-040-016- to Min-Chi Lu, and also in part by intramural grants of the National Health Research Institutes (MG-102-PP-15) to Ying-Tsong Chen and by innovative research grants of National Health Research Institutes (NHRI-EX103-10327BI) to Yi-Chyi Lai. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Klebsiella pneumoniae is a member of the family Enterobacteriaceae. The ability of K. pneumoniae to cause a wide range of human diseases, from urinary tract infections to life-threatening systemic infections, has attracted increasing attention to the pathogenesis of this bacterium [1]–[4]. In 1990s, K. pneumoniae surpassed Escherichia coli as the leading cause of community-acquired pyogenic liver abscess [5]. K. pneumoniae-caused liver abscess (KLA) was recognized by researchers in Taiwan and several other areas since 1986. KLA is now considered an emerging disease worldwide [1]–[3], [6]. Distinct from E. coli-caused liver abscess, KLA is generically cryptogenic and is frequently complicated with metastatic lesions to other organs [7]–[9]. A significant proportion of KLA isolates in Taiwan belong to capsular serotype K1, with a prevalence rate ranging from 28% to 63% [10], [11]. Several K1-specific genetic loci have been identified to contribute to K. pneumoniae virulence, including magA (for capsular polysaccharide synthesis), kfu/PTS (for iron uptake), and genes for the TonB-dependent iron acquisition system (iucABCDiutA, iroAiroNDCB, and yersiniabactin island) [12]–[15].
Colorectal cancer (CRC) is a significant cause of morbidity and mortality throughout the world. Most CRC occur in people without a family history. Age, history of inflammatory bowel disease, and lifestyle are known risk factors for CRC. In Taiwan, CRC is the second most commonly diagnosed cancer. Its annual incidence increases steadily from 34.43/100,000 in year 2000 to 54.01/100,000 in year 2009 according to the report from the Department of Health in Taiwan. Pyogenic liver abscess has been suggested as a potential manifestation of CRC in Taiwan [16]–[19]. Intriguingly, an 11-year follow-up study in Taiwan demonstrated that patients with K. pneumoniae-caused PLA (KLA) were at increased risk of subsequent CRC than did patients with non-K. pneumoniae PLA [20]. This finding raises a possibility that some bacterial virulence factors may endow K. pneumoniae with a cancer-inducing capacity.
Since the identification of an oncogenic role for Helicobacter pylori [21], our knowledge regarding how bacteria contribute to tumorigenesis has just begun. Acquisition of genomic instability is crucial for initiation and progression of tumors. Chronic exposure to DNA damaging agents may cause elevated genomic instability as a result of failure to repair damaged DNA or altered activation of the DNA damage-induced checkpoint responses [22]. Two bacterial products, cytolethal distending toxin (CDT) [23] and colibactin [24] have been identified as genotoxins that can directly damage DNA, inhibit cell cycle, and trigger genomic instability in the host [22], [24]. CDT is a tripartite exotoxin consisting of CdtA, CdtB, and CdtC encoded by the cdtABC operon. Numerous bacteria have been found to produce CDT, including E. coli, H. pylori, Haemophilus ducreyi, Shigella dysenteriae, Salmonella enterica, and Campylobacter spp. [23], [25]. Colibactin, on the other hand, is a hybrid nonribosomal peptide-polyketide [24], [26] synthesized by multi-enzyme complexes that are encoded by genes carried on a 54-kb genomic region of E. coli [24]. The 54-kb chromosomal region, the pks colibactin gene cluster, was identified and fully sequenced in newborn meningitis E. coli strain IHE3034 [24]. In E. coli, the genotoxicity conferred by the pks colibactin gene cluster were demonstrated in vitro and in vivo [22], [24], [26]. By PCR, the presence of colibactin genes was detected in 53% of extraintestinal pathogenic E. coli (ExPEC) strains and in 34% of fecal E. coli isolates from healthy individuals [24]. The colibactin-positive E. coli isolates are almost exclusively classified in the phylogenic lineage ECOR-B2 [24]. In addition to E. coli, few members of the family Enterobacteriaceae were found positive on the carriage of pks colibactin gene cluster, including Enterobacter aerogenes, Citrobacter koseri, and K. pneumoniae [27].
Horizontal gene transfer contributes to the evolution and emergence of pathogens by allowing virulence-associated genes to spread among bacteria [28]. Clusters of genes can be acquired as a unit known as genomic island. Several genomic islands have been discovered in K. pneumoniae, including the high pathogenicity genomic island HPI-ICEKp1 at the asn tRNA loci of K. pneumoniae NTUH K2044 [29], fimbrial gene cluster-containing KPGI-5 at met tRNA loci of K. pneumoniae KR116 genome [30], and several genomic islands related to carbohydrate metabolism and toxin synthesis [31], [32]. Recently, we determined the complete genome sequence of K. pneumoniae 1084 (GenBank Accession No. NC_018522.1) [33], which was a K1 strain isolated in Taiwan from a KLA patient. A comparative genomics approach was performed to identify potential determinants of virulence in the newly sequenced genome. Here we report the identification of a 208-kb chromosomal region with typical features of a genomic island at the asn tRNA loci of the K. pneumoniae 1084 genome. This 208-kb genomic island, named KPHPI208, is composed of 8 genomic modules (GMs). GM1, the first genomic module of KPHPI208, contains genes ∼100% identical to those of the pks colibactin gene cluster reported in E. coli IHE3034 [24]. The colibactin-related genotoxicity of K. pneumoniae 1084 was validated by in vitro and in vivo experiments. The prevalence of colibactin genes among clinical K. pneumoniae isolates in Taiwan was also investigated and reported herein.
Materials and Methods
Ethics statement
Bacterial strains were isolated from patients with primary K. pneumoniae infections at Chung-Shan Medical University Hospital in central Taiwan during a 15-month period from April 2002. The isolates were cultured from clinical samples with no collection of patient identifiers or interaction with subjects. All animal experiments were performed in strict accordance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the National Laboratory Animal Center (Taiwan), and the protocol was approved by the Animal Experimental Center of Chung-Shan Medical University.
Bacterial strains
A total of 207 non-repetitive K. pneumoniae isolates were collected in our previous study [11]. Among them, 35 (16.9%) were regarded as KLA strains as they were obtained from tissue-invasive cases that presented with the formation of liver abscesses, 59 (28.5%) were from cases associated with abscesses at non-hepatic sites, including lesions that occurred as empyema, endophthalmitis, necrotizing fasciitis, septic arthritis, along with lung, epidural, parotid, paraspinal, splenic, renal, prostate, muscle, and deep neck abscesses, and the remaining 113 (54.6%) isolates were obtained from non-abscess-related cases, including pneumonia without abscess, primary peritonitis, cellulitis, biliary tract infection, primary bacteremia with no original infectious foci identifiable, and catheter-related infections. The K. pneumoniae isolates were cultured in Luria-Bertani (LB) broth and stored at −80°C until use. Capsular antigens were determined by PCR detection of the K-serotype-specific wzx locus. Specific primers designed previously [7] were used to detect K1 type (GTA GGT ATT GCA AGC CAT GC and GCC CAG GTT AAT GAA TCC GT), and K2 type (GGA GCC ATT TGA ATT CGG TG and TCC CTA GCA CTG GCT TAA GT). K. pneumoniae 1084 is a K1 strain isolated from a diabetic patient with severe bacteremic liver abscess. Complete sequencing and annotation of K. pneumoniae 1084 was performed in our previous study (GenBank Accession No. NC_018522.1) [33].
Deletion and complementation of clbA
A 768-bp region spanning the coding sequence of clbA was deleted in K. pneumoniae 1084 by using an allelic exchange technique as described [34]. In general, ∼1,200-bp DNA fragments flanking the region to be deleted were amplified with specific primer sets, p465 (AAA ATC TAG ACA TAG AGT TGG AGC AAC TGT T)/p466 (AAA AGG TAC CCT CAT TCC TGT TAG CAA TGT G) and p467 (AAA AGG TAC CTC TGA GCC GTC GAT AAT ATT GA)/p468 (AAA AGA GCT CTC CTA CCC TCG TAA TAT GGA CA) and the amplified DNA fragments were cloned into pKAS46, a suicide vector containing rpsL, which allows positive selection for vector loss using streptomycin [35]. After the occurrence of double crossover, the streptomycin-resistant but kanamycin-sensitive colonies were selected. The deletion of clbA was verified by PCR and Southern blot analysis and one of the confirmed mutants was named ΔclbA. For complementation experiments, a 735-bp DNA fragment containing full-length clbA gene was amplified using primers p478 (ATG AGG ATT GAT ATA TTA ATT GGA C) and p479 (ATT CTG CCC ATT TGA CGA ATG). The amplified DNA fragment was cloned into pCR®II (Invitrogen) to generate pYC502. Complementation of clbA was performed by introducing plasmid pYC502 into ΔclbA via electroporation.
In vitro infection assay
BALB/c normal liver (BNL) cells (ATCC TIB-73) were maintained by serial passage in Dulbecco modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum at 37°C with 5% CO2. The BNL cells were synchronized by switched to serum free medium for 16 hours. Approximately at 70% confluence, the BNL cells were washed four times with phosphate-buffered saline (PBS) and infected with K. pneumoniae 1084, ΔclbA, or ΔclbA-pYC502 at a multiplicity of infection (MOI) of 100 bacteria per cell. After a 4-h transient infection, BNL cells were washed with PBS and cultured with fresh DMEM supplemented with 5% fetal bovine serum (FBS) and 100 µg/ml gentamycin. For clonogenic assay, the control and infected cells were trypsinized to produce a single cell suspension. Equal amount of cells (1.5×102) were seeded into a 35 mm culture dish. After incubation for 14 days, cells were fixed then stained with 0.5% crystal violet. The number of colonies for each group was counted under stereomicroscope in a double-blind manner. To detect phospho-H2AX (γH2AX) foci in the nuclei, cells were fixed with 95% methanol-5% acetic acid, blocked with PBS-0.1% Tween 20–2% skim milk for 30 min, probed with rabbit anti-phospho-H2AX antibody (Cell Signaling Technology), and then hybridized with goat anti-rabbit-Alexa488 antibodies (Invitrogen). A Zeiss LSM510 CLSM (Carl Zeiss) with a 60× oil objective was used for imaging. At least 300 cells per group were counted for phospho-H2AX staining from images in a double-blind manner. For Western blot, ∼107 cells were collected and lysed with 1000 µl of lysis buffer (PBS, 1% Triton X-100, with protease and phosphatase inhibitors). After denaturation by boiling for 5 min with loading buffer, 30 µg of total proteins were separated on 12% of SDS gel, transferred to PVDF membrane (BioRad), blocked with 2% skim milk, and probed with anti-phospho-H2AX (Cell Signaling Technology), or with anti-Histone H3 (Cell Signaling Technology) antibodies, followed by horseradish peroxidase-conjugated secondary antibodies, and chemiluminescence detection.
In vivo infection assay
Male BALB/c mice were purchased from the National laboratory animal center (NLAC Taiwan) at 7 wk of age and allowed to acclimatize in the animal house of Chung Shan Medical University for 1–2 wk before experiments. Mice were starved for 16 h prior to infection. PBS or 100 µl of bacterial suspension containing 109 CFU of K. pneumoniae 1084, ΔclbA, or ΔclbA–pYC502 was respectively inoculated into groups of mice (6 mice a group) via an oral route as described in our previous study [34]. The PBS-inoculated or K. pneumoniae-infected mice were sacrificed at 1 day or 2 days post-inoculation. The liver was recovered immediately. The retrieved liver was divided into two parts, one was homogenized for determination of bacterial loads and detection of the level of phospho-H2AX by Western blot analyses, and the other was fixed in 4% paraformaldehyde at 4°C for 24 hours then embedded in paraffin. After deparaffinization and rehydration, slides were prepared and incubated with boiling sodium citrate buffer (pH 6.0; 10 mM) for 10 min to unmask antigens, and stained with rabbit anti-phospho-H2AX (Thermo Fisher Scientific) antibodies. Signals were detected by using a NovoLink Polymer Detection System (Leica Biosystem). Counterstaining was done with nuclear Hematoxylin. Images were acquired with a Nikon light microscope. Three sections were randomly selected from each of the samples retrieved from experimental mice in a double-blind manner. At least 1000 hepatocytes per section were counted for phospho-H2AX-positive staining.
PCR detection of pks colibactin genes
The presence of pks colibactin genes among clinical K. pneumoniae isolated in Taiwan was determined by PCR with primers as published in a previous study [24]. Briefly, primers for clbB and clbN were clbBF (GAT TTG GAT ACT GGC GAT AAC CG), clbBR (CCA TTT CCC GTT TGA GCA CAC), clbNF (GTT TTG CTC GCC AGA TAG TCA TTC), and clbNR (CAG TTC GGG TAT GTG TGG AAG G). Primers for two internal loci within the pks colibactin gene cluster, clbA (phosphopantetheinyl transferase) and clbQ (thioesterase), were clbAF (CTA GAT TAT CCG TGG CGA TTC), clbAR (CAG ATA CAC AGA TAC CAT TCA), clbQF (CTT GTA TAG TTA CAC AAC TAT TTC), and clbQR (TTA TCC TGT TAG CTT TCG TTC). Genomic DNA (gDNA) samples for the 207 K. pneumoniae strains were prepared as described [11]. An initial denaturation at 95°C for 10 min was followed by denaturation at 94°C for 45 s, annealing at 54°C for 45 s, and extension at 72°C for 1 min for 30 cycles. K. pneumoniae 1084 was used as the positive control and K. pneumoniae CG43 was used as the negative control for the PCR experiments.
Results and Discussion
Genetic structure and comparative analysis of KPHPI208
At the asn tRNA loci, an integration hotspot for foreign mobile DNA elements, we identified a 208-kb region exhibiting typical features of a genomic island and named it KPHPI208. A total of 135 protein-coding genes were contained in this island. Its overall G+C% is 49.6, which is lower than that of the entire genome of K. pneumoniae 1084 (57.4). Comparative genomics analysis of this region had identified eight genomic modules (GM1∼GM8) each displaying a conserved genetic organization and were likely exchangeable among bacteria genomes. As shown in Figure 1, KPHPI208 accommodates a pks colibactin module (GM1), 5 modules (GM2, GM3, GM4, GM5, and GM7) similar to those contained within HPI-ICEKp1 [29] and HPI-ICEEh1 [36], a microcin module (GM6), and a novel module (GM8). The insertion site of KPHPI208 is located at the asn tRNA locus near the ompS gene of the K. pneumoniae 1084 genome. Homologous regions of GM2, GM3, GM4, and GM5 were identified in the genome of Enterobacter hormaechei 05-545 spanning the high pathogenicity island HPI-ICEEh1 [36], and in the genome of E. coli ECOR31 at the integrative and conjugative element ICEEc1 [37]. GM2 is an 18-kb module containing genes for integration and conjugation. GM3 is a 30-kb module carrying genes for yersiniabactin production. GM4 and GM5 harbor genes of unknown functions (Figure 1). Homologues of GM2, GM3, GM5, and GM7 also present in the 76-kb high pathogenicity island HPI-ICEKp1 of K. pneumoniae NTUH K2044 [29]. The insertion site of HPI-ICEKp1 in the genome of NTHU K2044 is also the asn tRNA locus near ompS. Nucleotide sequence of GM6 is ∼100% identical to the microcin E492 gene cluster of K. pneumoniae RYC492 [32]. The 22-kb region consisting of genes for the production of microcin and immunity protein locates next to the second asn tRNA locus in KPHPI208. The eight modules (GM1-GM8) of KPHPI208 are bounded by 4 asn tRNA genes, 8 attO sites, and 4 intB integrase genes (Figure 1). The attO is a conserved 17-bp direct repeat that served as an integration site for integrative and conjugative elements (ICEs). In compared to HPI-ICEKp1, which has only one integrase gene (intB1) in the yersiniabactin module, KPHPI208 has three additional intB genes, intB2, intB3, and intB4, carried by GM4, GM6, and GM8, respectively (Figure 1). Acquirement of the three modules flanked by attO and asn tRNA was probably the result of insertion events occurred at the three asn tRNA loci of the K. pneumoniae genomes (Figure 1).
Modules in the genomic regions were depicted as colored boxes and the important genes were labeled (GM1∼8). Red: the pks colibactin genes; orange: the genes responsible for integration and conjugation of the ICE element; yellow: the yersiniabactin genes; light green: unknown function, similar to genomic module EhGI1 from ICEEh1 (GenBank accession No: FN297818); brown: unknown function, similar to EhGM5 of ICEEh1; dark green: microcin E492 genes, similar to the microcin genomic island in K. pneumoniae RYC492 (GenBank accession No: AF063590); purple and pink: unknown. The attO sequences were marked by triangles. The positions of the tRNA genes were also marked along the region.
GM1, the largest module in KPHPI208, consists of genes responsible for colibactin production. Nucleotide sequence of GM1 is ∼100% identical to that of the pks colibactin gene cluster reported in E. coli IHE3034 [24] (Figure 2). Colibactin is a peptide-polyketide hybrid compound. For the synthesis of colibactin, the pks colibactin gene cluster encodes several proteins including non-ribosomal peptide megasynthases (NRPS), polyketide megasynthases (PKS), hybrid NPRS/PKS megasynthases, and accessory, tailoring, and editing enzymes [24]. GM1 of KPHPI208 accommodates all of the pks genes required for the synthesis of colibactin (Figure 2). In the genome of E. coli IHE3034, the pks colibactin gene cluster is flanked by attO direct repeats at both sides, and an additional P4-like integrase gene, intP4, is located next to the end of the gene cluster near clbQ [27]. Although the pks colibactin gene cluster carried in KPHPI208-GM1 is identical to the E. coli version, the intP4, together with one of the attO next to it, are not found. This is reminiscent of a previous study, in which five out of 141 K. pneumoniae isolates from Europe were PCR-detected positive on the presence of colibactin genes. In the study the intP4 associated with the colibactin gene cluster was reported to be missing in K. pneumoniae [27]. Sequencing of KPHPI208 not only confirmed this genetic structure but also uncovered the complete genetic structure of the genomic island by discovering additional genomic modules in this region. In addition, the variable-number tandem repeat (VNTR) between clbB and clbR of KPHPI208-GM1 has 16 ACAGATAC repeats. It is 13 repeats in E. coli IHE3034 (Figure 2).
The regions spanning the genes responsible for colibactin production were depicted as arrows according to the directions of transcription. The attO sites in the left were marked by solid triangles. The VNTR locus between clbR and clbB was marked by empty triangles. The 53-kb regions indicated by dotted lines are ∼100% identical. The locations of the four PCR amplicons in studying the prevalence of the colibactin genes among K. pneumoniae clinical isolates were marked. The 768-bp region spanning the clbA gene, which was deleted in ΔclbA strain, was indicated.
Genotoxicity of K. pneumoniae 1084
Exposure to colibactin-producing E. coli was demonstrated to inflict double strand breaks (DSBs) of host DNA in vitro and in vivo [22], [24], [26]. Discovery of the pks colibactin gene cluster in KPHPI208-GM1 (Figure 2) encouraged us to examine whether this gene cluster endowed K. pneumoniae 1084 with genotoxicity. DSBs of host DNA induce phosphorylation of the histone protein H2A variant (H2AX) at serine 139 that generates γ-H2AX through the activation of ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia and Rad3-related protein) kinases. Accumulation and retention of γ-H2AX can therefore serve as an indicator for the signaling cascade induced by DNA damage [38]. In this study, BALB/c normal liver (BNL) cells were used as a cell model to examine the degree of DNA damage induced by K. pneumoniae 1084 infection. Compared to the uninfected control (Figure 3A), a number of γH2AX foci were noted in the nuclei of K. pneumoniae 1084-infected BNL cells at 4 h post-infection (green fluorescent signals in Figure 3B). Because the γH2AX foci on mitotic chromosomes represent unrepaired DNA breaks or scar of repaired lesions [39], [40], this result suggests an induction of DNA damage by K. pneumoniae 1084 infection. A phosphopantetheinyl transferase is encoded by clbA that is essential for the synthesis of colibactin [24]. It was demonstrated in E. coli that the genotoxicity in host cells was significantly attenuated by the loss of clbA [22], [26]. To verify whether the K. pneumoniae 1084-induced DNA damage was attributed by colibactin, we deleted a 768-bp region spanning the coding sequence of clbA (Figure 2) in K. pneumoniae 1084 to generate an isogenic clbA mutant and named it ΔclbA. Similarly, the γH2AX foci were hardly detected in the K. pneumoniae ΔclbA-infected BNL cells (Figure 3C). By Western blot analyses, time-dependent accumulation of γH2AX was noted in the transiently infected BNL cells, which were recovered at 2, 4, and 6 h after a 4-h infection with K. pneumoniae 1084 (Figure 3F), as compared with the uninfected control (Figure 3G). The γH2AX signal was diminished in the ΔclbA-infected group (Figure 3F). By introduction of the clbA-complementing plasmid pYC502, the reduced genotoxicity of ΔclbA was restored to the wild type level (Figure 3D, 3E and 3F). The result demonstrated the genotoxicity of K. pneumoniae 1084, which was conferred by the colibactin genes accommodated in KPHPI208-GM1. Viability of the K. pneumoniae 1084-infected cells was unaffected upon a 4-h transient infection. To demonstrate the consequence of the DNA damage induced on BNL cells by infection with K. pneumoniae 1084, we performed a conventional clonogenic assay. After 14-day incubation, the number of colonies formed in the K. pneumoniae 1084- and the complement strain (ΔclbA-pYC502)-infected group was significantly greater than that in the ΔclbA-infected group (Figure 3H and 3I). This result suggested that the accumulation of K. pneumoniae 1084-induced DNA damage might contribute to tumorigenesis.
BNL cells were left uninfected (A) or were infected with K. pneumoniae 1084 (B), ΔclbA (C), or with ΔclbA complemented with clbA coding plasmid pYC502 (D) at an MOI of 100. After 4 h infection, the cells were washed, co-cultured with gentamycin (100 µg/ml), and were examined by confocal microscopy for DNA (blue, stained with Hoechst 33342), for membrane glycoproteins (red, stained with ConA), and for γH2AX (green, recognized by Alexa488-anti-γH2AX antibodies) (scale bar, 20 µm). (E) Quantification of γH2AX-positive cells. Error bars represent SEs from three experiments. (F) Western blot analyses of γH2AX or H3 in BNL cells recovered at 2, 4, and 6 h after a 4 h transient infection with K. pneumoniae 1084 (lanes 1–3), ΔclbA (lanes 4–6), or with ΔclbA complemented with clbA-coding plasmid pYC502 (lanes 7–9). (G) Western blot analyses of γH2AX and H3 in uninfected BNL cells harvested from serum recovery for 2, 4, 6, 24, 48, and 72 h. (H) Clonogenic assays. BNL cells were uninfected (Control) or transiently infected with K. pneumoniae 1084, ΔclbA, or with ΔclbA-pYC502 for 4 hours. Colonies formed after 14-day incubation stained with 0.5% of crystal violet. A representative image is presented. (I) Quantification of colony formation. Error bars represents SEMs from three experiments. An asterisk (*) represents a significant increase in the K. pneumoniae-infected group in comparison with the uninfected control by the Student's t test (two-tailed; P<0.05).
To further determine whether K. pneumoniae 1084 elicited DNA damage in vivo, 109 CFU of K. pneumoniae 1084 were orally inoculated into 8–10 week old BALB/c mice. At 1-day or 2-day post-inoculation, mice were sacrificed and the liver was retrieved for tissue section. Compared with the PBS-inoculated control mice (Figure 4A), extensive distribution of γH2AX foci were observed in the nuclei of liver parenchymal cells infected with K. pneumoniae 1084 (Figure 4B) or with the complement strain (ΔclbA-pYC502) (Figure 4D). The number and intensity of γH2AX foci in the K. pneumoniae ΔclbA-infected liver was significantly reduced at 1-day and 2-day post-inoculation (Figure 4C and 4E, P<0.05). Although the loss of clbA significantly attenuated the genotoxicity of K. pneumoniae 1084, it did not affect the ability to disseminate into liver from the intestines. Bacterial loads in livers retrieved at 1-day and 2-day post-inoculation were comparable among the K. pneumoniae 1084-, ΔclbA-, and ΔclbA-pYC502-infected groups (Figure 4F). The decreased level of γH2AX in the ΔclbA-infected livers was confirmed by Western blot analyses (Figure 4G and 4H). Our results suggested that the genotoxicity may not contribute to the dissemination of K. pneumoniae into extra-intestinal tissues. It is not surprising that pks island is absent in hyper-virulent K. pneumoniae isolates from KLA patients, such as CG43 or NTUH-K2044 [12]. Collectively, similar to E. coli IHE3034 [22], [24], [26], as a carrier of the pks colibactin gene cluster, K. pneumoniae 1084 displayed a genotoxic phenotype that induces DNA damage in vivo and in vitro.
For all the experimental groups, liver sections were prepared from the liver retrieved at 2-day post-inoculation, stained with anti-γH2AX antibodies, and imaged under microscopic observation with magnification of 400×. Representative liver section of the PBS-inoculated control mice (A), the K. pneumoniae 1084-infected mice (B), the ΔclbA-infected mice (C), and the ΔclbA-pYC502-infected mice (D) are shown. Scale bar: 50 µm. (E) Quantification of γH2AX-positive hepatocytes. Each value is the mean ± SEM for 3 mice. An asterisk (*) represents a significant decrease in the ΔclbA-infected group in comparison with the K. pneumoniae 1084-infected group by the Mann-Whitney U test (two-tailed; P<0.05). (F) Bacterial loads of the liver determined at 1-day and 2-day infection with K. pneumoniae 1084 (black bars), ΔclbA (white bars), or ΔclbA-pYC502 (slash bars). Ten-folded dilutes of liver homogenates were plated onto M9 agar for enumerating CFU of K. pneumoniae. Each value is presented as the average bacterial loads ± SEM (Log CFU/g) for 3 mice a time point. Statistical analysis by the Mann-Whitney U test showed no significant difference between the K. pneumoniae 1084-infected and the ΔclbA-infected groups. (G) Liver lysates were prepared from PBS-control, K. pneumoniae 1084-, ΔclbA-, and ΔclbA-pYC502-infected mice at 1-day and 2-day post-inoculation. The sample size in each experimental group for each time point is 3. Eighty micrograms of total proteins were subjected to Western blot analyses with specific antibodies against γH2AX and H3. A representative result from at least three experiments is shown. (H) Band intensity of γH2AX and H3 was determined by Densitometry calculation and the average ratio of γH2AX to H3 is presented. Each value is the mean ± SEM for 3 mice. An asterisk (*) represents a significant decrease in the ΔclbA-infected group in comparison with the K. pneumoniae 1084-infected group by the Mann-Whitney U test (two-tailed; P<0.05).
As demonstrated by Cuevas-Ramos et al [22], DNA damage elicited by a short exposure to E. coli IHE3034 could trigger genomic instabilities. Because cell division cycles are resumed rapidly after a transient DNA damage response, in the dividing cells, accumulation of DNA breaks and propagation of breakage-fusion-bridge cycles can consequently evoke persistent chromosome aberrations, such as micronuclei, aneuploidy, ring chromosome, and anaphase bridges. If cells harboring chromosome aberrations continue to proliferate in the presence of DNA damage, the gene mutation frequency and transformed phenotype would be enhanced [22]. Given the nucleotide sequence and genetic structure of the pks colibactin gene cluster accommodated in KPHPI208-GM1 of K. pneumoniae 1084 is identical to the E. coli IHE3034 version [24] (Figure 2), we speculate that by accumulating DNA damage elicited by K. pneumoniae 1084 infection, genomic instability of host cells could also be triggered. Therefore, tumorigenesis might be promoted by colonization and/or infection with K. pneumoniae that carries the pks colibactin gene cluster by inducing DNA damage and the subsequent genomic instabilities in host cells.
Prevalence of the colibactin genes among clinical K. pneumoniae isolates in Taiwan
The presence of pks colibactin gene cluster as a part of the mobilome suggests its potential to spread among local K. pneumoniae strains. To investigate the prevalence of colibactin genes (Figure 2) among K. pneumoniae isolates in Taiwan, PCR detection was carried out on a collection of 207 clinical strains obtained from patients with primary K. pneumoniae infections. The pks colibactin gene cluster markers clbB and clbN [41] were jointly detected in 53 (25.6%) of the 207 K. pneumoniae strains. PCR detection of two additional colibactin genes clbA and clbQ yielded results concordant with those for clbB and clbN. Approximately 66% (35/53) of the colibactin-positive K. pneumoniae were the K1 type (Table 1). Statistical analysis revealed a significant correlation between the carriage of colibactin genes and K1 type (odds ratio, 19.4; 95% confidence interval, 8.8–42.9; p<0.0001). However, the presence of colibactin genes appeared to be irrelevant to the type of infection, as the colibactin-positive K. pneumoniae distributed evenly among isolates collected from cases of KLA (8/35; 22%), non-hepatic abscess (17/59; 28%), and non-abscess infections (28/113; 25%). The overall colibactin-positive rate was 25.6% (53/207). This was significantly higher than that reported by Putze et al, in which only five out of 141 (3.5%) K. pneumoniae strains isolated from Europe were colibactin-positive [27]. Our result indicates a close relatedness of the colibactin gene cluster to K1 type. The relatively higher prevalence of colibactin genes demonstrated here might be related to the fact that serotype K1 was the most frequently identified type in Taiwan [42], but it ranked eighth in European survey [43].
Chronic inflammation has been recognized as a risk factor for CRC and other cancers. In a colitis-associated CRC mouse model, the proliferation of a genotoxic E. coli commensal strain, NC101, was demonstrated to be favored by an inflammatory milieu and thus promoted tumorigenesis of CRC [26]. Several clinical studies have demonstrated the relationship between CRC, PLA and K. pneumoniae in Taiwan [17], [18], [20]. The hazard ratio of CRC was 3.36 times greater for patients with PLA as reported in a nationwide population-based 5-year follow-up study comprised of 274 PLA patients and 1,370 randomly selected subjects [18]. In another nationwide cohort study in Taiwan, of which 1257 PLA patients without prior cancers were followed-up from 2008–2012, it was reported that the incidence of CRC in PLA patients was significantly raised as compared to the control group [17]. In addition, a retrospective study of PLA patients at a medical center in Taiwan has further indicated that the risk of CRC was 2.68 times greater for patients with K. pneumoniae-caused PLA than those with non-K. pneumoniae PLA [20]. Our discovery on the high prevalence of colibactin-positive K. pneumoniae in Taiwan, predominating among K1 strains, therefore poses an intriguing opportunity in studying the link between genotoxic K. pneumoniae and CRC. Whether the CRC-associated KLA strains correlate to the presence of colibactin genes remains to be elucidated. However, as we report here that 25–28% of K. pneumoniae isolates from non-KLA infections were also colibactin-positive, the possibility that other types of K. pneumoniae infections may contribute to the development of CRC and/or other types of cancers cannot be ignored. The role of genotoxic K. pneumoniae in tumorigenesis deserves further studies.
Here we report the complete genetic structure of KPHPI208 analyzed by comparative genomics approaches. The 208-kb genomic island contains eight genomic modules (GM1-GM8), including a pks colibactin gene cluster which was first identified and characterized in E. coli IHE3034 [24]. The pks colibactin gene cluster within the GM1 of KPHPI208 shares a striking ∼100% sequence identity with the E. coli version, suggesting that their functions and regulation are conserved. Accumulating evidence has pointed out the role of colibactin-producing E. coli in the development of chronic inflammation and CRC in humans [22], [24], [26]. In Taiwan, it has also been reported that K. pneumoniae-caused PLA significantly increased the risk of subsequent CRC [17], [18], [20]. The carriage of pks colibactin gene cluster may thus serve as a molecular basis underlying the epidemiological link between K. pneumoniae and CRC. Indeed, transient infection of BNL cells with K. pneumoniae 1084 induced DSBs, as revealed by the formation and accumulation of γH2AX foci. This genotoxic capacity of K. pneumoniae 1084 required the production of colibactin because it was abolished by the deletion of clbA gene and was restored to the wild type level by the complementation with a clbA coding plasmid. Moreover, in our KLA mouse model, K. pneumoniae 1084 exhibited an enhanced ability to induce DNA damage in the liver parenchymal cells when compared with the isogenic clbA deletion mutant. Furthermore, more than 25% of K. pneumoniae strains isolated from Taiwan carried the colibactin genes, and two-third of them (66%) belonged to the K1 type which is responsible for the development of invasive disease, such as PLA. The discovery of genotoxic K. pneumoniae as well as the determination of its complete genetic environment will help to elucidate the link between K. pneumoniae, PLA, and CRC.
We thank the Instrument Center of Chung Shan Medical University, which is supported by National Science Council, Ministry of Education and Chung Shan Medical University, for providing optical and confocal laser scanning microscope.
Author Contributions
Conceived and designed the experiments: YCL MKC CYL YTC. Performed the experiments: YCL ACL YHD CCH MCL YTC. Analyzed the data: YCL YTC. Contributed reagents/materials/analysis tools: YCL MCL YTC. Wrote the paper: YCL YTC.
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