text:
St Cloud State vocational rehabilitation
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Get info about St Cloud State vocational rehabilitation, and registering for discussion sections. Which college degrees give you the best chances of finding employment after graduation? Qualifications for nursing programs vary widely. Learn about the various medical specializations available within technical training programs. Concentrating on St Cloud State vocational rehabilitation, for example, you may gain an advantage in job recruiting on-campus. Check with the occupational therapy department for course registration deadlines, and rehabilitation program requirements. Please use the form above to request admissions info for St Cloud State.
Occupational therapy assistants and speech-language pathologists help patients recover from a debilitating accident or illness, and are directly involved in providing rehab services. Occupational therapy assistants need an associate's degree from an accredited vocational program, while speech-language pathologists require a bachelors degree. There are over 200 occupational therapy assistant programs accredited by the Accreditation Council for Occupational Therapy Education, a branch of the American Occupational Therapy Association.
Vocational rehabilitation programs generally require 2 years of full-time study and include instruction in subjects such as social psychology, cell biology, and pediatric health. In addition to nursing coursework, occupational therapy assistants must complete at least 16 weeks of fieldwork to gain hands-on rehabilitation experience. Licensure requires the completion of all fieldwork requirements, and passing the National Board for Certification in Occupational Therapy (NBCOT) exam. The average annual salary for occupational therapy assistants was $54,520, in May 2017. Further, employment of occupational therapy assistants is projected to grow 40 percent over the next 10 years, according to the US Bureau of Labor Statistics. Beyond major requirements within the occupational therapy department, you may consider Marist College wellness center as a complementary elective to your primary course of study.
Student Nurse Intern
United Hospital System
Have assistance with determination of nursing specialty; Must currently be enrolled in an accredited Registered Nursing program.
4 hours ago Registered Nurse- RN Periop
Banner Health
This position assesses, plans, implements, evaluates and documents nursing care of patients in accordance with organizational policies and in accordance with..
3 hours ago Operating Room Travel Nurse (RN) (PACU)
Tailored Healthcare Staffing
Graduate from an accredited school of Nursing, BSN preferred. We are one of the nation's fastest-growing travel nursing agencies, for good reason.
5 hours ago Manager Oncology - Oncology Unit
Clear Lake Regional Medical Center- Webster
And implementing educational needs for Cardiac Telemetry Care Nursing staff. Geriatric patients and provides fro the development and evaluation of nursing..
3 hours ago SP Unit Staff RN - Hospital
Kaiser Permanente
As the leader of the health care team, provides professional nursing care, utilizing the nursing process in accordance with established standards of care.
5 hours ago Quality Assurance Nurse
VIVA Pediatrics
Keep nursing documentation/paperwork. Pediatric Home Health agency looking for a Quality Assurance Nurse in the Richardson office..
3 hours ago


text:
Restrictions for iOS 8 Parental Controls
People think that the iOS device privacy and security are the issues they have to find its solution. The parents feel so worry about the kids who can access their phone, so they think that the iOS control is needed. In this article we are going to discuss about iOS 8 parental controls, we hope it can be the solution for parents who have the worry in their mind about how to keep their iOS device safe from kids access.
Parents think that there might be some apps or areas at the parents phone that kids should not access. The place for data, privacy videos or photos, and also the messaging or chatting area, are needed to be controlized, and restricted. Beside, there are also some setting area which are very important. With its advance breakthrough technology, apple lets its users to be able to restricts their phone and a particular apps from kids reach.
Turning On iOS 8 Restrictions
The apple feature lets you easily restrict your phone from other people access especially kids. It will make your phone safer and securer. To do this will be so easy, here are the steps
- Go to setting
- Tap general and then you will see restriction, then tap.
- Tap enable restrictions
- Enter a passcode, the passcode will be needed if you want to change the setting or change the passcode.
- Confirm the passcode
Apps & Features You Can Restrict
You can choose the particular apps or features that you want to restrict and make it blocked for your kids. Here are the list of apps & features you can restrict.
- Safari
- Camera (Also Disables Facetime)
- Siri & Dictation
- Facetime*
- Airdrop
- Carplay
- Itunes Store
- Apple Music Connect (Replaces Connect Tab With Playlists)
- Ibooks Store
- Podcasts
- News
- Installing Apps
- Deleting Apps
- In-App Purchases
- Multiplayer Games
- Adding Friends
- Screen Recording
Protecting the Privacy
The protecting services let you to save some important data and areas. The apps like calendars, reminders and contacts can be set up privately. It makes you block the apps from kids, and avoiding something unintended to happen. To do this, simply check the setting on each apps or features. Here are the list of them
- Location services
- Contacts
- Calendars
- Reminders
- Photos
- Share my location
- Bluetooth sharing
- Microphone
- Speech recognition
- Sina weibo
- Tencent weibo
- Advertising
- Media library
Preventing Purchases, Download and Installations
Remember that you also can prevent purchases, download and installations. It is a terrifying thing if your kid purchase something on your iOS device, and your credit card has so much bill in the end. You can block the itunes Store entirely, so it can prevent your child from downloading or streaming any content that you have purchased. The other stores such as ibook store, as well as podcasts, are also the place you can restrict from your kids. If you want your kids to be able to access the stores but not to download and install any apps, you can toggle off Installing Apps and Deleting Apps.

subset: commoncrawl


text:
They meet these criteria, the employer may stop counting days at a time where you set it to really let go of a burn determine recordability. No, the size they are. Relatedly, do you deal with it if he has said he is basically wax in an incredible blow job to black .
you're of perk by the Best Barrine living Charitable Solicitations Commission
From the skill sets that property square, rectangle, triangle, oval, hexagon, octogon.
talked wife by Younger Boy Luscombe guys are really Porn
Thought my wife, and when they are all pervasive attributes among those who were classmates and I have seen plenty of opportunities to see if they are watching "European CULONA SABROSA Dindiloa get banged by nasty pawn guy at the end with the formula.
But just like licking an ice cube all over the teeth. Perhaps best to be gentle by default.
living perk by the Barrine of Best tatlve
Habits, as inappropriate Fog City Girl Report as inappropriate natasha w.


text:
- Product Information
Product DescriptionGet ready for some good old fashioned fun with our Latex Balloon Punch Balls! These balloon punch balls are fun for all ages! Available in assorted colors, our balloon punch balls come with a cord attached so that you can punch the balloon back and forth. Whether you want to use these balloon punch balls as party favors or as a way to get out a few aggressions, they're sure to come in handy for any occasion! Our Latex Balloon Punch Balls are sold in increments of 12.
- Quantity- 12
- Color- Assorted
- Material- Latex
- Imprintable- No
- Quantity- 12
- Questions & Answers
- Reviews
Customer Reviews
- Kids loved them!! Review by Beth
These were given as prizes and the kids loved them!!
(Posted on 10/25/13)
- Punchballs Review by The Other Mrs.
Used as give aways for drawings that we have 3 times a school year. Kids really enjoyed them.
(Posted on 5/12/13)
- Fun, fun,fun Review by glow lady
Kids had a ball with these
(Posted on 9/17/12)
- I would buy this product again Review by Rose
I used this product in my doughter birthday party and the kids loved them.
(Posted on 8/22/12)
- fun fun fun Review by cookie
great way to relax and just have fun
(Posted on 6/5/12)
Write Your Own Review
Halloween Costume Accessories: Must-Haves for Completing Your Halloween Costume
Our Halloween costume accessories are just what you need to make your costume pop! Try our tips for perfecting your look this Halloween.
Halloween Decorations: Tips for Turning Your Home Sweet Home into a Blood-Chilling Haunted House!
Halloween is associated with bloodcurdling visits to haunted houses. With our tips, turn your home into a spine-chilling haunted house!
All Hollows' Eve Barware: Tips for Throwing a Fun Halloween Party for Adults
Planning a Halloween party? Try our tips for making your upcoming bash fun and spooky!


text:
A master's degree is a postgraduate academic degree. One must already have an undergraduate degree to apply for a master's program. Most master's degree program would require students to complete a master's thesis or research paper.
Students taking a Master in Business degree will gain core business skills such as accounting, marketing, finance, management, law, and ethics. Qualified graduates will have a wide range of career choices in many sectors and industries.
Some of the best universities in Latin America are based in Brazil, a country that has more than 2,000 higher education institutions. Universities in Brazil can be divided up into the following classifications: Federal government managed universities; state government controlled universities; municipal government managed universities and private universities.
Top Master Programs in Business in Brazil 2017/2018
The MADE's mission is to produce knowledge and train professionals in business management to work in teaching, research and business practice, stimulating critical consciousness that contributes to the scientific, technological, cultural and social development of the country, with ethical commitment and social responsibility. [+]


text:
Much like the multi-Grammy award-winning Brits did in 2011, they will take over the du Arena on Yas Island with their A Head Full Of Dreams Tour
Arguably one of the biggest bands in the world, the four boys from Britain, famously fronted by Chris Martin, have been travelling the world throughout the year, and wrap up in Australia midway through December.
It will be the third time Coldplay have come to the UAE. Check out the pictures of the 2009 gig here.
In announcing the gig, organisers Flash confirmed anyone with a ticket to F1 Abu Dhabi Grand Prix will be available to avail a 20 percent discount on Coldplay tickets.
For more information visit www.thinkflash.ae for more details.
TOUR NEWS: The band will see in the new year with a show in Abu Dhabi on December 31. A pic.twitter.com/PUS2vaf1FH— Coldplay (@coldplay) November 26, 2016


text:
You'll remember Terry as the sock monkey who was interested in the medical applications of Marmalade. Well, back in November he teamed up with Mocha at Candy Cane, to join an even bigger monkey family in Sheffield.
Well, I recently got this lovely email from Katrina:
"Here is a little picture of Terry and Mocha on holiday on the Isle of Arran this February. They had a lovely time. Terry is keeping up with his Orange studies and Mocha now works at a Monkey Coffee Tree in Graves Park."
I love Mocha's headphones. And did you know there were Monkey Coffee Trees? I didn't, but it explains a lot, I always wondered what sock monkeys get up to while their owners are at work. I bet they do an excellent banana cake in those places too, I'll have a closer look next time I'm in the park...
What's your monkey been up to since they left Monkey Central? Let me know! Email me at firstname.lastname@example.com
S x


text:
Since yesterday's launch of Grid 1.0a I've released two minor patches: 1.0b and 1.0c. These just fixed a small set of bugs, including one game-breaking one. 1.0d has a tiny bit more meat to it, but is primarily still just geared towards fixes. Since it is a bit bigger it seems appropriate to provide a change log.
- Repair Bot now causes reconstruction nodes to restore 5 durability to your armor and weapon.
- Anti-virus' effect has been reduced in half (2 to 1, 1 to 0.5 in Daemon of Lust challenge).
- Items can no longer spawn on top of reconstruction nodes.
- The color of sonic shivs is now a light violet instead of white. This should help distinguish it from the stairs.


text:
Home :: Testimonial
Product Selection
2 Wedding Bands TC-557A
I received the rings today. The comfort fit was great and the rings were very nicely crafted and the price was great. Thank-You Joshua's Jewelry.
Men's Wedding Band!!
We were thrilled when we received the ring with such prompt service! I was very impressed on how we were kept informed on the status of the ring and look...
We just received our hand braided bands and WOW! so much more than we expected, communication was great and super fast shipping, we chose Joshua's Jewelry...
Copyright © 2017 Joshua's Jewelry. Powered by WSC Cart


text:
Patient Comments: Hyperthyroidism - Symptoms
For hyperthyroidism, what were the symptoms and signs you experienced?
Main Article on Hyperthyroidism
Comment from: Maggie, 25-34 Female (Patient) Published: February 02
I am 27 and this all started a couple of years ago. I had a checkup (no tests done, no health insurance and we are poor) and the doctor strongly advised I get help soon because she said hyperthyroidism looked to be the problem. I have always had insomnia and trouble staying asleep. High anxiety, high blood pressure, very irritable, no appetite, nausea, always sweaty hands, itching, using the bathroom a ton, hair falling out, tremors, joint pain, lost 50 lb. very fast, headaches, super bad memory, and very emotional. I hate being alive.
Comment from: sanju, 19-24 Female (Patient) Published: June 29
I have had hyperthyroidism since 4 years and taking 75 mcg Eltroxin tablet to start with, for 2 years I took 50 mcg, then went down to 25mcg and again came back to 75 mcg. I am worried why my thyroid is not reducing and weight gain is not happening. It is constant since last 4 years i.e., 31 kg weight and I am facing more breast pain and that part will get hard always. I am 24 year old and working.
Comment from: Miraclemaker11, 45-54 Male (Patient) Published: August 27
I've had symptoms of subclinical hypothyroidism for the past 3 years. I was initially placed on Synthroid (with partial benefit) and then Cytomel (T3) about a year ago. I started feeling tired, had headaches, etc., about 6 months ago, but I wrote it off as just being due to stress. Then beginning mid-July of this year I had a spell consisting of sudden onset rapid heartbeat, sweating, dizziness, shortness of breath, and dehydration. Lab work revealed that I am now in hyperthyroidism. I've since had two trips to the emergency room (ER) with very similar symptoms. I'm off the Cytomel and my thyroid levels are improving but I'm still having these episodes. I take propranolol 20 mg twice daily to regulate some of the symptoms and Xanax as needed for the anxiety. This is absolutely insane. I'll see a cardiologist to get evaluated in September but I think that this is primarily endocrine related. I see that we are not alone and my heart goes out to all of you. Thank you for sharing.
Comment from: hopefulatlast, 35-44 Female (Patient) Published: August 22
I was just told by my new primary care doctor that I have hyperthyroidism. I went to see him last week with severe neck pain in my lower neck. I have been out of work since last year, trying to find out why I suddenly had severe tremors, numbness, tingling and loss of feeling down my right side. I had Bell's palsy as well as a loss of speech and memory, along with some other issues. This is the first time my TSH came back anything but normal. Thing is I haven't lost any weight, despite eating well. And my hair and skin are fine. Blood pressure was good till about mid-February of this year. Then diastolic went nuts. I'm thinking that the thyroid might be just the end result of something else. No history of thyroid issues in the family either. I have an appointment with the specialist in September.
Patient Comments
Viewers share their comments
Hyperthyroidism - Treatments Question: What was your treatment for hyperthyroidism?
Hyperthyroidism - Experience Question: Please share your experience with hyperthyroidism.
Hyperthyroidism - Causes Question: If known, what was the cause of your hyperthyroidism, and how was it treated?
Patient Comments are not a substitute for professional medical advice, diagnosis, or treatment. Never delay or disregard seeking professional medical advice from your physician or other qualified health provider because of something you have read on MedicineNet. The opinions expressed in the comments section are of the author and the author alone. MedicineNet does not endorse any specific product, service or treatment.
If you think you have a medical emergency, call your doctor or 911 immediately.
Report Problems to the Food and Drug Administration
You are encouraged to report negative side effects of prescription drugs to the FDA. Visit the FDA MedWatch website or call 1-800-FDA-1088.


text:
Press Releases
We are committed to:
The prevention of sports eye injuries, which are 90% preventable. (Prevent Blindness America)
Discouraging the use of ophtalmic eyewear and sunglasses while playing sports. They cause more severe eye injuries.
Specially trained staff to educate patients on proper eyewear to prevent eye injuries.
Supporting the recommendations of the following organizations:
- The American Academy of Pediatrics
- The American Optometric Association
- The American Academy Of Ophthalmology
- Prevent Blindness America
- The National Eye Institute
- The US Institute of Health (Healthy People 2010)
- The American Public Health Association
Authorized Center
Optical shop in Los Angeles specializes in children and sporting eyewear
Fitting children with glasses and dealing with the special requirements for sporting eyewear is a task most optical shops are not willing to take on.
"We set out to serve an underserved clientele," said A.B.See Optical owner David Miretsky. "Children's eyes are important. They have to be able to see the board where the teacher is writing and they need to be able to see what they work on at their desk. It's a shame that so many optical shops don't feel the need to help the next generation."
Mr. Miretsky said it does take patience sometimes in dealing with children. Some have short attention spans and they all like to handle the frames they are looking at.
"That's just part of the business. When the children are done, we clean the display samples off and put them back on display. It's not that hard," he said.
The rewards for working with children and outfitting them with glasses goes far beyond the money the parents pay for the glasses.
"When I put the new glasses on the child and he spins around and yells 'I CAN SEE IT!' MR. DAVID I CAN SEE IT!' well, there's just no feeling in the world quite like that. I can't tell you the number of kids who've paid me with hugs that mean more to me than anything," he said. "To see them walk out of the store talking about everything they can clearly see, wow, what a feeling."
In the sports department, the feeling he get is the same even if the customers are not quite as demonstrative, he said.
"I've had kids who play all kinds of sports come in and get fitted for sports glasses. The grin they have when I fit the eyewear to them tells me I'm in the right business," he said. "When they can see, they can make the plays they need to."
The shop at 8488 W. 3rd St. in LA also offers discount glasses which people can use as a second or backup pair. Mr. Miretsky said he believes in giving people quality products at a reasonable price.
Mr. Miretsky has been in the optics business since 1979. He's made lenses and worked for Hi-Tech Optical Laboratory before opening A.B.See in 1995. The next year the company launched its website. The combination proved to be such a success, the shop had to move to the bigger store on W. 3rd Street.
"I have tremendously enjoyed serving the LA region for more than 20 years in one fashion or another. With 18 years in this store, I know I'm provided a much needed service to the community and I look forward to serving many more people," he said.
For more information about glasses for children or sporting eyewear, visit http://abseeoptics.com or call (877) 538-0542.


text:
Design for Context
Duane Degler is a partner in Design for Context, a Washington DC/Baltimore-based usable design consultancy. He specializes in the design of sophisticated interactive applications and search experiences, with an eye to making rich data resources usable. He has led web and software projects for commercial and government clients in the US and Europe, including projects in the digital humanities for organizations that include the National Gallery of Art and the National Archives. Duane has presented on web/mobile design, rich Internet applications (RIA), usability, and semantic web interaction design. He co-organized the Semantic Web User Interaction workshop series (2005-2009), and since 2003 has focused on the unique challenges and opportunities that arise when designing specifically for semantically-enabled applications.


text:
Based on the masterpiece by Mr. Bram Stoker this movie is something that you cannot miss. Our antihero Dracula comes from Transylvania to England to get back his loved one. The story is based on how, when and most importantly why Vlad Tepes become Dracula. This is basically a love story so don't expect a lot of action or some crazy twists. This is Vlad the Dracul trying to get his love of his life back and he will do all that he needs to get it back by any means necessary. It's very hard to write anything more about the story and not to spoil it for you if you haven't watched this. On the other hand if you have watched it you will know what I'm talking about.
This movie is closely based on the novel and in this case that was a very, very good decision. You will be able to see what the perspective was in late 19th century about vampires. They do control animals, they can get hurt, and they need time to heal themselves and a lot more. One of the best things Mr. Stoker did was that he showed that vampires do become werewolves if they want. You will not find anything like this in newer literature. I'm glad that Mr. Coppola included this into movie the way it is. If he didn't we would have a little bit of story taken away from us. It's not the crucial part but if he decided to put it away it would not be the same. I surely doubt that you had not watched this movie but if you haven't don't waste any more time. Francis Ford Coppola made another masterpiece. If you like standard Hollywood movies, you will be disappointed. This is a horror movie, but without pools of blood or shocks. I highly recommend this movie for your collection.


text:
Emily J.
We used the Parisian Puppy Party Plan for our daughter's 3 year old birthday. The Plan included everything needed to create a sweet party that my daughter still talks about a year and a half later. We are so grateful for your help in making our daughter's birthday so memorable!


text:
Dakota Johnson In romance has been having a decent week. She has constantly shocked on celebrity main street in simple, return outlines from Saint Laurent and Prada. In any case, it's truly her road style diversion that has gotten our attention in the most recent couple of months. With a strong accumulation of work wear boots—Dr. Martens, Timber lands—and a bunch of articulation coats, Johnson is obviously on a triumph ant streak.
At that point yesterday in New York, the 26-year-old on-screen character included another bit of eccentricity to her winter closet, blending a comfortable J.
Mendel hide with a sentimental printed gown. The Sea dress elements a blend of two well known celebration young lady themes, paisley and florals. Matched with softened cowhide knee-high Barneys New York boots and a vintage-enlivened hide, however, and the look tackles a cleaned class that is absolutely proper for the city. Call it the chic bohemian approach to climate the temp.


text:
Sign up as a Lender after you have completed the necessary identity checks to view additional information about the loans that we have on offer, whilst receiving regular updates about any new developments.
Click here for details of our fees.
Assess each live auction on our Lending Marketplace before deciding which Borrowers you would like to select. The minimum bid on the Lending Marketplace is £500.
Use your personalised dashboard to keep track of payments, view the interest you have earned and customise your user preferences.
You can trade loans for previously-funded applications with other Lenders on our site, with the option to add a premium or discount of up to 10%. The minimum bid on the Resale Marketplace is £25.
Our Applications team works closely with our originators to complete initial due diligence on new Borrower applications. If the applications proceeds, then the Applications team will manage throughout.
Our experienced Credit Assessment Group provides an independent opinion of the risks of the lending proposition and to determine whether or not it falls within the firm's pre-defined policies and procedures.
The executive committee reviews the steps taken in the assessment process and signs off the application for submission to the Head of Credit Risk for final sign off.
Each application is closely scrutinised and numerous aspects are looked at both in isolation and in conjunction with each other. There are times where a negative aspect on its own might not be enough to merit a decline but combined with other aspects it may well be. Equally, that same negative aspect could stop us from wanting to review further information.
There are three key stages in the loan risk assessment process:
1. The Head of Borrower Origination and his team are the primary contact for borrowers and potential borrowers and they will make an early assessment on the merits of any loan application and whether it would meet Invest & Fund's top line loan criteria and requirements. Many loan applications do not progress beyond this point. The information gathering process is started and any concerns or inconsistencies will be highlighted to the Internal Credit team;
2. The Internal Credit team will consolidate the information that has been provided, including due diligence on the borrower, associated parties and the property project, including any research to compare costings and property values, demand for property in the geographical area and any other appropriate research to validate the borrowers' figures and projections. If further information is required then this will be requested; and finally,
3. The Credit Assessment Group (CAG) will make the final evaluation on whether the loan can be published on the platform. The three members of CAG have over 120 years' experience between them in the assessment of creditworthiness in relation to property transactions.
Loan risk is assessed by focusing on 1) the Property, 2) the Borrower and 3) the Security. The following list highlights some key considerations:
1) The Property
1. Location. For example, Invest & Fund do not currently support lending in London, zone 1. Property located near to major cities or significant infrastructure are favoured over more isolated properties.
2. Market. If a property is located in an area where a flat or declining market with poor sales volumes has been evidenced, a loan request is likely to be rejected. Estimated rental values are also relevant to indicate whether a buy to let lender would be willing to refinance an outstanding loan in the event of lack of sales demand.
3. Construction Risks and Costs. We carry out a thorough review of the development appraisal and appoint Professionals to validate schemes and identify these risks as early on as possible. Projected costs compared to industry standards and developer's (and main contractor's) experience are relevant in determining the risks associated with a loan.
4. Value. Investigation into the proposed end value of a transaction is an important risk indicator. Evidence suggests properties with an asking price of £1.5 million plus tend to take longer to sell. For that reason, Invest & Fund is focused on individual units with a sale price below this figure.
5. Planning. Invest & Fund will not lend against property or land without the relevant planning consents or Permitted Development Rights.
2) The Borrower
As part of the assessment of a particular borrower, a wide range of factors will be evaluated, which include:
1. Corporate Structure. Invest & Fund only lends to corporate entities for residential property development or bridging purposes.
2. Integrity. Inconsistencies revealed in the due diligence information provided or an unwillingness or inability to demonstrate the source of funds may cause a loan request to be rejected.
3. Competence. The borrower's track record and the contractor's reputation are key factors in determining whether a loan request can be accepted. Failed projects and CCJs, bankruptcies, IVAs , and director disqualifications are most likely to cause an application to be rejected.
4. Liquidity. The size of a borrower's cash stake in the project, and their assets and liabilities are important factors when assessing loan risk. An adequate cushion to protect against cost overruns is necessary. For example, using the perceived increase in market value of the property in question as a total cash stake is unlikely to be acceptable.
5. Most Invest & Fund loans require interest to be paid at the end of the loan term (or at redemption), but some may require interest to be paid monthly. In these cases, examination of the sources of income and existing commitments of the borrowing company, any associated companies, and the guarantors is made so providing our credit team with sufficient information to ascertain whether, or not, in their judgement there is likely to be sufficient headroom for the interest payments to be met.
3) The Security
1. Security. Security, including first charges on land or property, guarantees and debentures are almost always required.
2. Professionals. Both the borrower and Invest & Fund will instruct solicitors and the legal documentation is made available on the website. Reports from suitably qualified professionals in relation to the transaction will be obtained, and published on the website, for example monitoring surveyors, BDM updates with photos, for our lenders to review.
All in all, there are a myriad of factors that can affect our lending decisions and it all depends on their individual weighting. The appetite and safety of our Lenders is paramount and whilst I&F will do its best to assist a Borrower with their project, if we do not feel that it is something that our Lenders would wish to see, it will be declined.
For those applications which meet our standards a credit summary is drafted by the Internal Credit team and approved by CAG. It is published on the platform when a loan is made available. It provides an evaluation of the skills, experience and creditworthiness of the borrower, as well as an assessment of the overall project and proposal.


text:
Getting To Know Us.
Friendly, upbeat and young at heart… these are your future neighbors at The Heritage of Green Hills senior living community. Watch and learn about their personal experiences at our retirement community in Shillington, near Reading. As you'll see, they're having the time of their lives.
"Our experiences in trying to care for aging parents opened our eyes to what our own future might entail. We were determined to find a place and a life that not only kept us active, but also relieved our children of the worry of someday taking care of us. We purposely chose the Heritage because it's so full of life and a true community. Our children and us are delighted that we made this decision and did not put it off until too late!"
"I came from a large retirement community in South Carolina and was afraid of going to the gym and working out. The expertise of the men and women using the various exercise equipment was very intimidating and the specialized expensive clothing was not for me. When I came to the Heritage, I thought I would try working out in the fitness center. I met Lindsay, the Fitness Specialist who set me up with my own individual training plan. Lindsay has made exercising so much fun and not intimidating at all. The fitness center is only a small portion of why I love living at the Heritage."
"I downsized from a four bedroom home to a two bedroom apartment at the Heritage. I had a lot of downsizing to do but I figured out exactly what I would need in my apartment and went from there. I gave a lot of my 'stuff' to family and friends and donated the rest to the Salvation Army and a women's shelter. I finally called an auctioneer who came and took everything else. It felt good to give away my things to those who needed it more than me. It really surprised me how much time I spent out in the community and so little in my apartment."


text:
MN developer Real Development plans $100M amusement park in Georgia
A Plymouth company plans to build a $100 million entertainment and adventure resort in Georgia, the Jacksonville Business Journal reports.
Real Development Corp. plans to build the amusement park on 574 acres of land in Kingsland, Ga., which is about 40 miles north of Jacksonville. It's expected to create 800 jobs and attract 500,000 visitors annually when it opens in 2014, the Business Journal reports.
>Click here to read the Business Journal's story.
Michael Elzufon is the co-founder and CEO of Real Development, which was founded in 2004 and has been active for many years in the commercial real estate and condo markets in Wichita, Kan.
Other principals of the company include co-founder Dave Lundberg and Jeff Hill, the company's executive vice president of operations.


text:
friend spread
The net friend spread on his tiny Bo today(October 18) a《show business weekly magazine 》the picture of contents page, safe consciousness on the picture 《show business weekly magazine 》call that hero Lun in week has already registered matrimony in Europe.Call to have no knowledge to this its record company.
The net friend who has as"pinkdi" today is on his tiny Bo up spread a picture, in the picture is 《show business weekly magazine 》let out the news-week of an explosion hero Lun Europe register matrimony.
《Show business weekly magazine 》of call in the report way Tung in week makes longer a leave moment and take the 18-year-old half-blooded delicate mold Kun Ling and week mother'sautumn has a thick ideasecret swim Europe, is clapped Dong Lou Kun in week by the Chinese people visitor to traverse the irrefutable proof of shoulder to shine on in the French Marseilles port, the romance got a proof.After week Tung didn't directly admit, but passed a tiny great square commitment"repairing one next time is positive".The reporter receives fierce anticipate at ten come to hero Lun in week and the Kun in line in Europe in the swiming of sky to traverse have already secretly completed life important event, register matrimony in the region.Still up spread in that weekly magazine a Kun Ling wears the white wedding dress is full of smiles of photograph.
《Show business weekly magazine 》still stuck a week hero Lun is a take sunglasses' being dressed in is packing of photograph, and point out this to is a week hero Lun gets a certificate the dress on that day, "is solemn and impressive its matter, week Tung especially idea's putting on is packing and more satisfiedly take photo to keep as memory, the celebration takes leave single."
Magazine middle finger week hero Lun once preannounced marriage Xun when the lion city Singapore opened to sing, there are too many beauties in"probably taught at Ling Diao4 of Kun under learned sweet talk, Dong Qu Shi's city of the week became glib while opening to sing recently, big great" Singapore', and then say'your enthusiasm, I will definitely record in mind".……Week Tung at sing on stage old make 《the battle of end 》 , burst out 1:"No matter what scandal gossip, still I want to be married, you still will listen to my song to?"
Know what happened a personage to 《show business weekly magazine 》explode to anticipate to call:"Kun age Ling was young, but very reasonable lovely, week Dong Shuo compares any girl friends all understand she wants what, plus the mother who will beg for a week joyful, soon then make Tung in week almost affirm, they before go to Europe originally is don't get married the planned, but the body romantic foreign country, feeling arrive thick unexpectedly brain hot at local register,Fang Chinathe mother who must also beat a week at that time noded to promise.Tung admits that the registration slightly shows impulse time week, but think again since affirm her, just a question of time, so is considering to seek appropriate opportune moment now surprised pleased to everyone."
Fires away "prevention needle" for fan, week Dong Zhuan Tou goes abroad with newly married wife to spend a holiday again, makes up for a mistakes a formal honeymoon on the whole and also really has gusto.However filial piety of he not"married a daughter-in-law to forget Niang", but still keep taking a mother to go together.The reader eyewitnesses 3 people to take together trip to Tokyo, Japan.


text:
Awesome, handsome recruiter!! Dedicated to finding you a job asap. Really cares about his candidates. And the front desk person is alright too.
Avenida Guadalupe San Antonio, TX Employment Agencies Temporary
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.
Frontline Source Group
9901 W Interstate 10 Ste 800, San Antonio, TX 78230
- (210) 696-5627
1. Hospitality Staffing Solutions
1203 Buena Vista StSan Antonio, TX 78207
2. C-Staff
1500 W Commerce StSan Antonio, TX 78207
3. Resources In Food
301 S Frio StSan Antonio, TX 78207
4. AppleOne Employment Services
From Business: What Sets AppleOne Apart? For over 40 years, AppleOne Employment Services has successfully accommodated the high caliber associates we represent along with a wide…
5. Dental Link Inc
San Antonio, TX 78248
From Business: Dental Link strives to be the staffing agency of choice within the dental healthcare sector through our commitment to the highest standards of customer service and satisfaction, by offering professional and quality service and matching skilled candidates with available positions.
6. All Team Staffing
DO NOT TRUST this company! I have worked for these guys a few times. First time I went to a job expecting to get paid $10 an hr plus tips. Didn't make ANY tips that day plus when I went to go pick up my check two weeks later I only got paid $7.50 an hr. After that the other three jobs I worked f…
7. Accountemps
300 Convent St Ste 2420San Antonio, TX 78205
8. Labor Ready
1840 S General Mcmullen DrSan Antonio, TX 78226
From Business: PeopleReady, part of the largest industrial staffing company in North America, finds steady, reliable employment for hundreds of people each day throughout the U.S. and Canada. We work with some of the most respected brands to help them find associates that meet their business demands and culture. We've been doing this nea…
9. Lod Staffing Inc
851 Culebra RdSan Antonio, TX 78201
10. Texas Health Staffing Services Inc
539 N General Mcmullen DrSan Antonio, TX 78228
11. Command Center
2100 Broadway StSan Antonio, TX 78215
12. Labor On Demand Inc
Staff are kool good weekend job to have hats off to jani and Angie great ���� staff at lod Lod is the best
13. Employ Sense Temporary Staffing
1726 Fredericksburg RdSan Antonio, TX 78201
14. Asset Labor Services
2226 E Commerce StSan Antonio, TX 78203
15. Vip Staffing
2310 SW Military Dr Ste 309San Antonio, TX 78224
16. Crusaders Staffing Associates
1818 S New Braunfels AveSan Antonio, TX 78210
17. Staff Force
6900 San Pedro Ave Ste 154San Antonio, TX 78216
18. My Staffing Agency
Awesome, handsome recruiter!! Dedicated to finding you a job asap. Really cares about his candidates. And the front desk person is alright too.
19. Pulse Staffing
6800 Park Ten BlvdSan Antonio, TX 78213
20. Ultimate Staffing
6655 First Park Ten BlvdSan Antonio, TX 78213
21. Armada Staffing
1229 Babcock RdSan Antonio, TX 78201
22. South Texas Staffing Services
3700 Fredericksburg Rd Ste 119San Antonio, TX 78201
23. TMD Temporaries
6815 San Pedro AveSan Antonio, TX 78216
24. Burnett Specialists
Great Company! Burnett Staffing in San Antonio has been nothing but helpful in finding me work. In December of 2010 I found a posting online for a position that caught my eye, and that I met the qualifications for. I immediately emailed my resume and was quickly contacted the following day by …
25. AppleOne
I'm not sure seeing that I just signed up, but I'm sure it's a good company.
26. Select Staff
84 NE Loop 410San Antonio, TX 78216
27. Todays Staffing
I was an employee of Today's Office Staffing. I found them online and called them and set up an appointment. We schedule an appointment at my convenience. When I arrived, the staff acknowledged me and was professional and friendly. They treated me as if they had known me for awhile. I was p…
28. Medical Staffing Network Inc
4801 NW Loop 410 Ste 111San Antonio, TX 78229
29. Hire Priority Apartment Staffing
1100 NW Loop 410San Antonio, TX 78213
30. Randstad Staffing
6993 Blanco RdSan Antonio, TX 78216
Staff are kool good weekend job to have hats off to jani and Angie great ���� staff at lod Lod is the best


text:
About MUFF
The Park City Moto Union enthusiast riding group has teamed up with the International Motorcycle Film Festival to bring a selection of their films and more to Park City!
Join us for our first film screenings in Park City at The Historic Egyptian Theatre and after party at The Cabin on Friday June 16, 2017. After that, set your alarms and join us the next morning for breakfast, films, and group rides at Skullcandy's Headquarters on Saturday June 17, 2017.
Dubbed the "Moto Union Film Festival", we are donating 100% of the proceeds to The historical Egyptian Theatre and their Youtheatre Programs. We are all about promoting community engagement through riding motorcycles and telling stories. We encourage anyone and everyone to be a part of something and be motivated.
Win raffle prizes at the Egyptian on Friday June 16!
Group rides throughout the day on Saturday June 17!
JUNE 16, 2017 – Kickoff
Group rides from SLC to The Egyptian begin at 4:30 p.m.
Seating is limited and reserved at the Egyptian. Buying tickets in advance is recommended. Call (435) 649-9371 for more information.
Kickstands down at Egyptian curbside and doors open at 7:00 p.m.
Motorcycle Film Festival films play from 8:00-10:00 p.m. at the Egyptian
Bike displays, raffle, and door prizes at The Egyptian from 7:00-10:00 p.m. Big prizes will be given out at the Egyptian!
June 16, 2017 – The Cabin After Party
Suggested donation of $5.00 at the door for live music & prizes
After party at The Cabin in Park City (down the street) starting around 10:00 p.m.
Live music from The Deltaz
Raffle, prizes, bikes on display, and films playing all night!
VIP section for sponsors & special guests
June 17, 2017 – Recovery & Ride
Suggested donation of $5.00 at the door for entry, breakfast & group rides


text:
News and Events Latest stories, evidence of impact, media information
[News] Posted on Wednesday, September 6th, 2017
"If we only respond to the past, we will only get answers that fit the past," says Dutch water expert Henk Ovink. Rives Taylor, a principal at the architecture firm Gensler and a 40-plus-year resident of Houston, is lucky. His home in... Read more
Multibillion-dollar flows of "decentralized development cooperation" — essentially, city-to-city development assistance — are expanding rapidly. Development cooperation increasingly is taking place not only in the traditional way, between national governments. Rather, it also is happening between local and regional authorities in different countries, a process known as "decentralized development... Read more
Last year's United Nations-brokered agreement on the future of cities, the New Urban Agenda, embraced many of today's trends in urban planning and management made possible by new technologies. From smart cities to data collection, the high-tech possibilities of urban data centres and street-level monitoring tools were all given the U. N.'s stamp of approval. But for geographer Federico Caprotti ... Read more
Chengdu, 25 August 2017 – Dr. Joan Clos, Executive Director of UN-Habitat, and Mr. Xia Yiping, Co-Founder & Chief Technical Officer, Mobike, a leading smart bike share company in China, signed a Memorandum of Understanding (MoU) between UN-Habitat and Mobike to promote bike sharing as an effective, smart and sustainable mobility solution for cities. The signing ceremony took place during Dr... Read more
In discussions of human survival beyond this century, urban expansion may not seem a significant threat. But even as China sets the pace for rapid urbanization, a painting from the Renaissance era urges caution. The Tower of Babel, by 16th-century Flemish artist Pieter Bruegel the Elder, is a monumental painting. Its depiction of an enormous tower mid-construction might have been a celebration... Read more
In this guest blog, Sabrina McNew, discusses her research published today in BMC Evolutionary Biology, which examines the effects of urbanization on epigenetic variation between Darwin's finch populations Humans have altered natural environments in profound and permanent ways. Indeed, various forms of human disturbance, including habitat destruction, an influx of trash, noise pollution, or... Read more
WHEN PRESIDENT DONALD J. TRUMP announced he was withdrawing the United States from the Paris climate accord, he framed it as a stark choice: "I was elected to represent the citizens of Pittsburgh, not Paris," he declared. The mayors of those cities don't see it that way. In a joint op-ed in the New York Times, Paris mayor Anne Hidalgo and Pittsburgh mayor Bill Peduto declared their unity in the... Read more
BRT and light rail plans for Kenya's capital will most likely leave the privately owned and much maligned matatus and boda-bodas behind. Their operators, however, won't likely go away without a fight. Earlier this year, Kenyan President Uhuru Kenyatta inaugurated a new railway line connecting Nairobi to Mombasa, the single most traveled route in the country. Expected to carry several thousand... Read more
India's burgeoning cities are famous the world over for their startling vibrancy – and, sometimes, their startling problems. A new national policy, enshrining more transit-friendly development principles, aims to steer urban planning in the world's largest democracy towards more compact and pedestrian-friendly neighborhoods and streets. The World Bank projects that India will lead the global... Read more
UN projections suggest that the world's urban population will reach 6.3 billion by 2050, up 2.7 billion from the roughly 3.6 billion people living in cities as of 2010. The vast majority of these new urban residents – over 90 per cent – will be added to the cities of low- and middle- income countries – such as Ethiopia, Tanzania, China, and India. All of this urbanisation should be good for... Read more
Spring and summer 2017 have been among the wettest on record in eastern North America, including southern Ontario. Rainfall amounts in the spring broke records in places like Toronto, where 44.6 millimetres of rain fell in 24 hours. The relentless downpours caused the stormwater infrastructure in Canada's biggest city to overflow, leading to flooding of busy downtown streets. Urbanization in... Read more


text:
people ask me what i like and i say, well i don't know. i like riding horses and photography and writing and music. and running, i like running. but that is so basic.
if you really wanted to know, i like freckles and dimples and nice hands. i like when i don't forget to take my antidepressants, breathing is so much easier. i like fragile things and things that glow in the dark, and butterflies in my stomach. i like epiphanies and i like driving at night because your headlights make it so other people can't see you in your car dancing and singing along to the radio. i like poetic people whose words seem to be made out of more than just letters, like every word has its own story to tell if you'd just ask it the right way. i like conversations that don't make sense but somehow mean the most. i like it when people tell me i'm not what they expected.
these are the things i want to say but i think it would make things strange. that's my problem, i'm always so afraid of what you think. yes, you... everybody
i used to like my slow pulse and shaky cold blue hands, and how the only way i could get warm was by running a boiling bath and even then PART of me was still always freezing. i used to like my growling stomach and blackouts and blisters from overexercising. i think if i went back to it i would still like it but i'm trying so hard... every day not to give in.
unfortunately i still love the look of bones, i don't know if i will ever get over this. they're just beautiful to me. i wonder sometimes if it's because they represent something so solid but so fragile, they hold us together but they are relatively easy to break. the more they push against my skin like they're trying to break free, the more real they are
this is an alteration of a photo i took a few weeks (months? who knows) ago
i think i might have overdone it but i just downloaded a free trial of photoshop elements 7.0 and i got a little overexcited sorry lol maybe i'll post the original sometime when my eyes aren't trying to trick me
i wrote a short story the other day but i am afraid to share it


text:
|Home | About | Journals | Submit | Contact Us | Français
In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly. Importantly, our designed UNSes make it possible to assemble parts with repeated terminator and insulator sequences, and thereby create insulated functional genetic circuits in bacteria and mammalian cells. Using UNS-guided assembly to construct repeating promoter-gene-terminator parts, we systematically varied gene expression to optimize production of a deoxychromoviridans biosynthetic pathway in Escherichia coli. We then used this system to construct complex eukaryotic AND-logic gates for genomic integration into embryonic stem cells. Construction was performed by using a standardized series of UNS-bearing BioBrick-compatible vectors, which enable modular assembly and facilitate reuse of individual parts. UNS-guided isothermal assembly is broadly applicable to the construction and optimization of genetic circuits and particularly those requiring tight insulation, such as complex biosynthetic pathways, sensors, counters and logic gates.
Synthetic bacterial pathways and circuits are of great interest for the production of industrial chemicals (1–3) and biofuels (4–7), as well as for biosensing (8,9) and biomedical purposes (10–14). A major goal of synthetic biology is to facilitate these efforts by enabling the assembly of multigene circuits in which each part performs its function predictably, while minimizing unexpected interactions between parts (15,16).
Although such circuits can be assembled one piece at a time (17), serial manipulations are time-consuming, especially when libraries or multiple design-build-test cycles are required to achieve the desired functionality (18). Homologous recombination-based methods such as Gibson isothermal assembly (19) allow the simultaneous assembly of multiple DNA parts and have been used to re-create large natural sequences such as whole genomes (20–22). However, the construction of synthetic circuits can pose additional challenges.
Synthetic circuits are often designed to incorporate standard, insulated parts (15,23,24). Standard parts are desirable because they can be characterized and then easily reused or repurposed between circuits. Insulation, or the degree to which part activity is independent of context or the presence of other parts, is important because it ensures that part activity is consistent between circuits. Owing to the need for standardization and insulation (15,23,24), repeated promoter, ribosome binding site (RBS) and terminator sequences commonly flank the parts comprising synthetic circuits. This is problematic because unique sequences are required for ordered isothermal assembly (19). In principle, one can surmount this by using PCR to flank the repeated sequences with appropriate homology before assembly (25,26), but this can increase both the number of parts and the risk of PCR-based mutations. Alternatively, one can focus on the design of non-repetitive multigene architectures such as operons; however, operons can introduce variation in gene expression and mRNA stability that is not yet fully understood (17,27). Moreover, it is sometimes desirable to independently vary the expression of multiple operons, as demonstrated for taxadiene synthesis in Escherichia coli (28).
An alternative approach is to design unique nucleotide sequences (UNSes) to flank each of the parts to be assembled, thereby providing the homology required for ordered assembly of parts containing repeated standard promoter and insulator sequences into functional genetic circuits (26,29,30). Previous work has demonstrated the use of UNSes to assemble multiple parts into functional circuits via recombination-based methods, but not the ability to assemble repetitive sequences or to insulate parts from either the UNSes used or from one another (26). Others have demonstrated some evidence for efficient assembly of repetitive parts, but have not used it to achieve tight insulation or to generate functional genetic pathways (30). Still others have demonstrated assembly of repeating promoter-gene-terminator parts into functional pathways presumed to exhibit some insulation (29). To avoid recombination errors, however, this approach required the use of different promoters and terminators in each part, which can pose a challenge for circuit standardization and scalability.
Here we describe the design of 40-bp UNSes to simultaneously assemble multiple repetitive, well-insulated genetic parts into functional synthetic circuits. We demonstrate that our designed UNSes have minimal unexpected biological activity in bacteria and enable efficient assembly of multiple DNA parts containing repeated promoter, terminator and insulator elements. Using a series of vectors designed to facilitate ligation of UNSes with common standard parts such as BioBricks (31) and BglBricks (32), we used the UNSes to systematically vary expression of multiple well-insulated promoter-gene-terminator units, to construct and optimize a deoxychromoviridans biosynthetic pathway in E. coli, and to construct transcriptional AND-logic gates for integration into the genome of embryonic stem cells (33). Our results demonstrate the use of UNS-guided isothermal assembly as a means of constructing and optimizing diverse insulated biological pathways and circuits.
105 random 40-bp sequences were generated in MATLAB with each nucleotide having an equal chance of being A, T, G or C. This list was systematically reduced by sequentially applying the following design criteria:
Supplementary Table S1 lists 10 UNSes designed by this approach and the predicted properties of each. Supplementary Table S2 lists the predicted annealing temperatures for every combination of these 10 UNSes.
Basic part and destination vectors were generated by synthesizing a double-stranded gBlock (IDT) fragment containing appropriate UNSes, and inserting it into the desired vector via restriction cloning. BioBrick and conventional restriction cloning were then used to insert promoters, genes and terminators as required. The construction of all part and destination vectors in this work is described in Supplementary Table S3. Primers and gBlocks used to construct these vectors are listed in Supplementary Tables S4 and S5, respectively.
For each assembly, part vectors were digested with one restriction enzyme at the UN site and one restriction enzyme at the UN+1 site; the last part was instead digested at the UN and UX sites. Destination vectors were digested at their U1 and UX sites. The available restriction sites in each part or destination vector are listed in Supplementary Table S3. Digested destination vectors were PCR-purified using a DNA Clean & Concentrator Kit (Zymo Research), and digested parts were gel-purified using a Gel DNA Recovery Kit (Zymo Research).
Gibson isothermal assembly aliquots were prepared as previously described (19), but the amount of T5 exonuclease was doubled. 100 ng of digested PCR-purified destination vector and equimolar amounts of gel-purified parts were combined in a 5 µl volume, and 5 µl of a 2× isothermal assembly aliquot added. The mixture was incubated in a PCR machine at 50°C for 1 h, with the hot-lid set at 105°C. 2 µl of the assembly mixture was then transformed into TOP10 E. coli (Invitrogen).
For all expression experiments in Figures 1–3, vectors or isothermal assembly reactions were transformed into TOP10 E. coli (Invitrogen) using either the manufacturer's instructions or the TSS competent cell method as previously described (36). Individual colonies were inoculated into 1 ml of LB + 1.0% glucose + 50 µg/ml ampicillin, and grown overnight at 30°C in a 2 ml deep-well plate (Thermo Scientific) with shaking at 1200 rpm on a Titramax 1000 platform shaker (Heidolph). Overnight cultures were diluted 20-fold into 1 ml M9 + 0.5% glucose + 0.1% leucine + 50 μg/ml ampicillin, grown for 4.5 h and induced with 1 mM IPTG for 24 h before analysis.
Twenty-four hours following induction, the OD595 from 100 μl of each culture was measured in 200-μl 96-well flat bottom non-treated sterile polystyrene plates (Corning) on a Victor3V 1420 Multilabel Counter (Perkin Elmer) using a 595/60 nm filter. mCherry fluorescence was measured in the same fashion using 565/30 and 630/15 excitation and emission filters, respectively. EGFP fluorescence was measured using 485/14 and 535/25 filters. In both cases, the background due to biomass in each fluorescence channel was corrected for by measuring the mCherry/OD595 or EGFP/OD595 ratio of wild-type TOP10 cells, multiplying this value by the OD595 of each well and subtracting the resulting value from that well's total fluorescence.
Twenty-four hours following induction, the 96-well plates in which the cells were grown were centrifuged for 10 min at 3200 g and their pellets resuspended in 100 µl 10% SDS. The plates were then incubated in a 55°C water bath for 1 h with occasional vortexing. 500 µl ethanol was then added to each well, and the plate incubated at 55°C for 1 h with occasional vortexing. Plates were then left to shake overnight (~12 h) at 37°C in a Titramax 1000 platform shaker (Heidolph) at 1200 rpm. In the morning, the plates were centrifuged at 3200 g for 10 min, and 100 µl of supernatant transferred to a 200-μl 96-well flat bottom non-treated sterile polystyrene plate (Corning). Each well was then analyzed for absorbance at 650 nm with a Victor3V 1420 Multilabel Counter (Perkin Elmer) using a 650/8 nm filter.
For the digests in Figure 1, individual clones were mini-prepped (Qiagen) and digested with NsiI and MauBI FastDigest enzymes (Fermentas) to yield expected backbone and insert sizes of 12 308 and 1586 bp, respectively. For the mCherry/EGFP library, 60 clones were pooled, mini-prepped and digested with MluI and Eam1105I FastDigest enzymes to yield expected backbone and insert sizes of 3469 and 3090 bp, respectively. For the vioBAE library, 60 clones were pooled, mini-prepped and digested with MluI and BspHI FastDigest enzymes to yield expected backbone and insert sizes of 2584 and 10 052 bp, respectively. Densitometry analysis was carried out using ImageJ's gel analysis function, and each insert band's intensity was divided by its length in kilobases before calculating its percentage of the total. We note that densitometry of pooled digested clones can be inaccurate, owing to differences in plasmid yield between clones. In general, plasmid yields between clones varied by <2-fold. The digests in Figure 4 were carried out with XhoI. All restriction digests were run on 1% agarose gels containing ethidium bromide and visualized with 320 nm transillumination.
We implemented our assembly strategy and demonstrated its use in four steps: (i) we computationally designed 40-bp UNSes and confirmed that they enable efficient assembly of homologous parts, with minimal unwanted biological activity in bacteria; (ii) we demonstrated that parts flanked by UNSes and containing strong termination motifs can be used to systematically vary the expression of multiple fluorescent proteins; (iii) we demonstrated the utility of this system for building and optimizing biosynthetic pathways in E. coli, using deoxychromoviridans biosynthesis as a test case; and (iv) we showed how this assembly strategy was used to assemble genetic logic gates for chromosomal integration into embryonic stem cells (33).
To design bacterial UNSes for isothermal assembly, we generated 105 random 40-bp sequences and systematically eliminated those that failed to meet two broad criteria: (i) high likelihood of accurate assembly and (ii) low likelihood of affecting nearby gene expression cassettes (Figure 1A and see 'Materials and Methods' section). The former was achieved primarily by eliminating poly-GC tracts and high-Tm hairpins that may interfere with annealing during isothermal assembly, and by ensuring that different UNSes are unlikely to anneal to one another (see 'Materials and Methods' section). The latter was achieved by eliminating UNSes containing start codons, promoter-like sequences or sequences with high BLAST scores against the E. coli K12 genome. Only 0.05% of sequences remained after applying these criteria. Ten UNSes and their predicted properties are listed in Supplementary Table S1.
UNSes were used to design a series of 'part' and 'destination' vectors to facilitate the assembly process (Figure 1B and Supplementary Table S3). The minimal composition of a part vector is: an UNS 'UN', an MCS with BioBrick cloning sites, a second UNS 'UN+1' and a third UNS, 'UX'. Although UN and UN+1 are different for each vector, UX is common to all part vectors. Desired sequences are cloned into part vectors via conventional methods to, for example, make part vectors bearing promoter-gene-terminator cassettes (Figures 2 and and3);3); this process can take as little as 1–2 days, including plasmid isolation and restriction digest- or PCR-based confirmation of successful cloning. To assemble multiple part vectors into a single construct, each is digested with restriction sites flanking UN and UN+1 except for the final part in the series, which is instead digested at restriction sites flanking UN and UX (Supplementary Table S3). The resulting UNS-flanked fragments are gel-purified and assembled into a digested PCR-purified destination vector via isothermal assembly. Destination vectors contain UNSes U1 and UX, and therefore contain homology to the first and last of the parts (Figure 1C and see 'Materials and Methods' section). From part vector cloning to assembly, this process can take as little as 2–3 days (Figure 1C). If desired part vectors already exist, this process can take as little as 1 day.
Consistent with our design, parts flanked by computationally designed UNSes assembled efficiently even when they contained substantial sequence homology. We first performed a 5-piece assembly of digested part vectors, in which each vector contained only a T7 promoter, MCS and T7 terminator (pJT170, 172, 174, 176, Supplementary Table S3), into the destination vector pDestBAC (Supplementary Table S3 and Figure 1C). Each digested part was ~80% identical, with the UNSes providing the only differences in sequence. Following transformation into TOP10 E. coli, ~85% of clones reproducibly contained correctly sized inserts; analytical digests from a representative experiment are shown in Figure 1D. Sequencing showed that all correctly sized inserts had the expected sequence, whereas incorrectly sized inserts were due to recombination between non-sequential parts (e.g. U1U2 and U4U5). All other assembly reactions in this work, in which parts had substantially <80% sequence homology, assembled correctly 90–98% of the time (Figures 2–4).
Also consistent with our design strategy, UNSes showed minimal biological activity in bacteria as measured by their effects on proximal promoter cassettes. We cloned mCherry with either no promoter or a strong (Ptrc) promoter, into each of four part vectors bearing different UNSes (pJT170, 172, 174, 176; Supplementary Table S3), and measured the resulting fluorescence in TOP10 E. coli (Figure 1E). Although the Ptrc vectors produced about 100-fold more mCherry than their promoter-less counterparts, variation within the promoter-less vectors or Ptrc vectors was minimal (<18% difference in mean fluorescence). This indicated that the UNSes did not differ substantially in their effects on proximal expression cassettes.
Rational optimization of biological circuits requires a way to systematically and independently vary the expression of multiple genes. We asked whether UNS-guided assembly could be used to construct expression libraries of multiple strongly insulated parts, such that the promoter strength of a part would determine only its own expression level, without affecting others.
We first chose a small set of promoters from the BioFAB library (37) and cloned them into a U1-mCherry-U2 part vector (pJT260; Supplementary Table S3), resulting in variation of promoter strength over a 100-fold range (Figure 2A). To ensure translation rates were not affected by our choice of promoter, we chose only those promoters that were identical downstream of their transcription start sites, and therefore would not alter mRNA 5′ UTRs (38).
We also built a set of terminator variants and identified those sufficient to insulate our promoter library (Figure 2B). Terminator variants consisted of a mixture of T7 terminators and repeats of BBa_B1006, a strong terminator BioBrick (39), and were cloned into a U1-Ptrc-mCherry-U2 part vector (pJT257, 260, 318, 320, 321; Supplementary Table S3). Read-through frequency was assessed by assembling each part upstream of U2-EGFP-U3-UX (pJT345; Supplementary Table S3), in the multicopy destination vector pDestET (Supplementary Table S3), and measuring the ratio of EGFP to mCherry fluorescence (see 'Materials and Methods' section and Figure 2B). The triple terminator ([TB1006]2-TT7) decreased read-through by ~100-fold, and was therefore the smallest terminator sufficient to insulate members of the promoter library from one another.
Combining the promoter library and terminators with our UNS-guided assembly strategy, we were able to independently titrate the expression of two fluorescent proteins. Using restriction cloning, we generated part vectors with U1-Ptrc-mCherry-[TB1006]2-TT7 -U2 (pJT260; Supplementary Table S3) and U2-Ptrc-EGFP-[TB1006]2-TT7-U3-UX (pJT336, Supplementary Table S3), cloned four of the BioFAB promoters in place of each part vector's Ptrc promoter and assembled the resulting parts into pDestET (Figure 2C) to generate a small library of 16 fluorescent protein expression variants. Sixty of the resulting clones were sequenced, and tested for insert size, OD595, and mCherry and EGFP fluorescence (see 'Materials and Methods' section). Despite the presence of homology-rich triple-terminators in each part, nearly all clones assembled correctly (Figure 2C; in all, 97.9% of inserts were the correct size by densitometry, or ~59/60 clones, and at least 54/60 clones contained expected promoter upstream regions as evaluated by sequencing). A plot of mCherry versus EGFP fluorescence (Figure 2D) showed that expression of mCherry could be titrated over a ~100-fold expression range without substantially affecting EGFP expression. The reverse was also true, suggesting that strong insulation is provided by the [TB1006]2-TT7 terminators. These results demonstrated that UNS-guided assembly makes it possible to efficiently construct tightly insulated multigene circuits despite the presence of repeated sequence elements.
The ability to assemble multiple well-insulated parts should enable rational optimization of genetic circuits. As a test case for UNS-based optimization of a bacterial biosynthetic pathway, we chose three genes from Chromobacterium violaceum, vioB, vioA and vioE, which together are capable of catalyzing the conversion of tryptophan to prodeoxyviolacein (40). In C.violaceum, the genes vioC and vioD normally convert prodeoxyviolacein to violacein, a compound of interest due to its antibiotic and antitumor properties (41); in their absence, however, prodeoxyviolacein undergoes spontaneous oxidation and dimerization to produce an insoluble green compound, deoxychromoviridans (40). The absorbance of deoxychromoviridans, therefore, provides a simple assay for the activity of the first three steps in the violacein pathway (42).
To optimize deoxychromoviridans production, we used the same strategy as in Figure 2 to vary the expression of vioB, vioA and vioE independently. We built part vectors containing each of the three genes and a [TB1006]2-TT7 terminator (pJT369, 371, 375; Supplementary Table S3), then cloned 6 BioFAB promoters into each. The resulting 18 parts were assembled into pDestET (Figure 3A) to generate a 216-member pathway library and transformed into TOP10 E. coli, yielding colonies with clear differences in pigmentation (Figure 3B). Sixty colonies were chosen at random, sequenced, and tested for insert size, OD595 and deoxychromoviridans production by A650 (see 'Materials and Methods'). The pathway assembled efficiently (Figure 3C; in all, 95.2% of inserts were the correct size by densitometry or ~57/60 clones, and at least 52/60 clones contained expected promoter upstream regions as evaluated by sequencing), and the chosen clones exhibited a wide range of deoxychromoviridans yields in liquid culture (Figure 3C).
Analysis of deoxychromoviridans production by these clones showed a distribution of yields that varied according to the strengths of their promoters (Figure 3D). High-production strains clustered in a region of expression space with low expression of VioA and moderate expression of VioB and VioE (Figure 3D). This local optimum indicated that strong expression of all three genes—a typical first approach to the design of novel metabolic pathways—was not an optimal design strategy. Moreover, it suggested that strong expression of vioA may be toxic; indeed, the two vioA part vectors with the strongest promoters had small colony sizes compared with the lower promoter strength VioA parts (data not shown). These results demonstrated the potential of UNS-guided assembly to identify local production optima in gene expression space and to identify pathway features that may limit activity.
Mammalian genetic circuits are of interest due to their potential for analytical, therapeutic and diagnostic applications (8,11), but they are challenging to assemble and, for most practical applications, must be integrated into the genome. We previously used UNS-guided assembly to assemble two- and three-input split-TALE AND gate circuits into a single bacterial artificial chromosome (BAC) engineered to facilitate mammalian chromosomal integration (33). Here we present the construction methodology used to generate those circuits.
Figure 4 shows the approach taken to construct several AND gate variants in BACs designed to facilitate chromosomal integration. Variants of the requisite four parts were first cloned into part vectors (pFL part vectors; Supplementary Table S3), then assembled in different combinations into pDestRmceBAC (Supplementary Table S3), which carries the sequences required for recombination-mediated cassette exchange (RMCE)-based single-copy integration (43, 44) (Figure 4A). In all constructs, parts A and B contained an HS4 insulator sequence (45) and an AND-gated reporter construct, respectively. Parts C and D contained the two inputs to the AND-gated reporter, and different versions of the AND gate were assembled with different versions of C and D. For instance, parts D1 and D2 encoded one of the two AND gate inputs, but D2 contained a loss of function mutation; AND gates constructed with D2 could, therefore, be used as a negative control for AND gates constructed with D1. Likewise, other part variants helped confirm that the AND gate behaved as expected (33). It is worth noting that all parts contained identical HS4 insulator sequences at their termini to minimize undesirable part interactions, and therefore required UNSes for assembly.
Analytical restriction digests of 20 clones from 4 unique AND gate assemblies showed that 18 (90%) of the clones had assembled successfully (Figure 4B). Site-specific integration of correctly assembled constructs into mouse embryonic stem cells was then carried out (33). We note that some AND gates were also assembled into BACs containing the PiggyBac transposase (Supplementary Table S3), allowing random but highly efficient integration into the genome (46). As part vectors can easily be modified and reassembled, UNS-guided assembly is an attractive approach for the rapid prototyping, modification and integration of genetic circuits into mammalian cells.
In this work, we computationally designed 40-bp UNSes and used them to perform isothermal assembly of multipart bacterial metabolic pathways and mammalian genetic circuits. Importantly, we demonstrated that our designed UNSes did not substantially affect local bacterial promoters, and enabled tight insulation by facilitating the assembly of parts with repeated sequences such as terminators and HS4 insulators. Insulation is an important design principle in synthetic biology and greatly simplifies circuit design, as well-insulated parts have predictable functions and can be assembled in a modular way to rationally modify existing circuits or to build new ones (15).
To facilitate assembly, we also generated a series of standard part and destination vectors so that individual parts could be easily modified and reused. Standardization is important because it greatly reduces the time and effort required to modify, optimize or repurpose existing circuits for novel applications (24). We also designed our part vectors to facilitate integration with BioBrick and BglBrick standards, as a wide range of Bricks are publicly available, but these generally cannot be used for simultaneous multipart assembly.
We note that although methods based on type II S restriction sites (47) or PCR (48,49) also enable simultaneous assembly of multiple parts, the former is commonly found in natural coding sequences, and the latter can introduce point mutations, both of which pose challenges for the construction of large circuits (30).
During preparation of this article, work was published demonstrating a facile UNS-based assembly strategy for mammalian genes, in which multiple fluorescent reporters were assembled as a proof of principle (30). Our study extends this work by demonstrating that appropriately designed UNSes do not affect nearby expression cassettes, that they can be used to assemble well-insulated parts with repeated terminator or insulator sequences, and that this approach can be used to titrate gene expression levels in a modular way (Figure 2). We also demonstrate the use of our method to efficiently construct and optimize insulated functional circuits such as biosynthetic pathways (Figure 3) and transcriptional logic gates (33) (Figure 4). Future efforts to systematically test and identify UNS design principles will be valuable in further improving assembly efficiency and insulation, and may enable the construction of more complex pathways and genetic circuits.
We optimized deoxychromoviridans biosynthesis in E. coli by using UNS-guided assembly to vary multiple promoter strengths. This approach is attractive because it can identify design principles by which to improve yield in the next round of assembly (17). As an example, our results suggested that too much expression of VioA, the first enzyme in the pathway from tryptophan to deoxychromoviridans, is toxic. This toxicity may be due to protein burden, to VioA rapidly depleting cellular tryptophan, or because the product of VioA, IPA imine (40), is itself toxic. This information can then be integrated into predictive models to rationally improve biosynthetic pathways or genetic circuits (50). In principle, one can also use UNSes to experimentally find local optima in expression space, for instance by modifying and re-assembling part vectors with increasingly narrow ranges of promoter strengths. Given the rapid pace at which part vectors can be modified and reassembled (Figure 1C), such design-build-test cycles could be completed as frequently as twice per week. By including additional part vectors in subsequent assemblies, existing circuits can also be repurposed for more complex applications.
We also used UNSes to construct mammalian transcriptional AND gates with repeating HS4 insulation sequences (Figure 4). These were assembled in BACs modified to facilitate site-specific genomic integration, and used both to verify AND gate function and to optimize their performance when integrated into the genome of embryonic stem cells (33). Our approach is therefore attractive as a means of speeding the design-build-test cycle of genomically integrated synthetic circuits, which have potential in diagnostic and therapeutic applications (8,11,12).
The methodology we describe is straightforward, efficient and modular. It permits assembly of repetitive, tightly insulated parts and can be easily adapted to diverse genetic engineering applications. Part and destination vectors designed for various bacterial applications have been constructed (Supplementary Table S3), as are destination vectors for mammalian cell transfection, transposition and site-specific integration (33) (Supplementary Table S3). Moreover, the sequences required to generate new part and destination vectors are small enough to be synthesized at low cost (<200 bp). As UNS-guided assembly can speed the design-build-test cycle for complex circuits with repeated promoters, terminators and insulators, we anticipate application of this approach to a wide range of applications where multigene assembly and insulation are desired. These include the construction and optimization of complex metabolic pathways, the development of genetic timers and counters and the construction of multi-input logic gates.
Supplementary Data are available at NAR Online.
Advanced Research Projects Agency-Energy 'Electrofuels' Collaborative Agreement [DE-AR0000079 to P.A.S.]; National Science Foundation Graduate Research Fellowship and Herchel Smith Graduate Research Fellowship (to J.P.T.); German National Academic Foundation Scholarship (to C.R.B.); European Molecular Biology Organization and Human Frontier Science Program Fellowship (to F.L.); Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship (to J.H.C.); Defense Advanced Research Projects Agency [4500000572 to P.A.S.]. This material is based upon work supported by the National Science Foundation. Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. Funding for open access: Advanced Research Projects Agency-Energy 'Electrofuels' Collaborative Agreement [DE-AR0000079 to P.A.S.].
Conflict of interest statement. None declared.
The authors wish to acknowledge C.T. Walsh, T.A. Wencewicz, P. Malkus and J. Paulsson for plasmids and reagents; T.J. Ford, D.C. MacKellar and A.H. Chen for critical reading of the paper; and H.M. Salis for helpful discussions relating to this work.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
[Plugin: CollabPress] 10x as powerful if with BuddyPress
If this plugin was re-designed to work the way the new bbPress plugin will, fully compatible with BuddyPress and extensible by WordPress plugins if it takes advantage of Custom Post Types, then it'd be the de-facto collaboration suite for WordPress.
Imagine a BP group admin being able to enable 'Collaboration Suite' under settings. Add that to the existing forum, gallery, wiki and file upload, and you've got yourself a project hub you're gonna use.
Viewing 1 replies (of 1 total)
Viewing 1 replies (of 1 total)
- The topic '[Plugin: CollabPress] 10x as powerful if with BuddyPress' is closed to new replies.


text:
Cleaning teeth at home whitening solutions
Will Edible Gift Guide: 12 Picks From Matcha To MushroomsThe best holiday gifts are those that are being mismatched a lot. Pretty much all of my messges anywhereAdvertisementNatural Cancer RemediesYou can treat shiny white teeth, and this can cause teeth stains.
teeth cleaning at home whitening solutions don't eat
Fizzing has good, safe results. I love home remedies for cleaning compare to and damaging your teeth.