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The National Association of Real Estate Investment Managers (NAREIM) provides members the opportunity to refine strategy, improve operations, and provoke new thinking through meetings, surveys and thought leadership activities.
The NAREIM Innovation Summit is the real estate investment management meeting where industry leaders sharpen their understanding, knowledge and skills about innovation across technology, buildings and people. NAREIM brings together senior executives in commercial real estate to discuss new ideas and strategies emerging within organizations, within the asset class and within the investment management industry - and deliver practical solutions that can be implemented today.
If you'll be attending and want to hear how we can make real estate run better for you, connect with Brent McFerren and John Seaton who will be representing RealFoundations.
Learn more about the event here.
About RealFoundations RealFoundations is a professional services firm focused on the real estate industry. With offices on four continents, 400+ client-serving professionals and off-shore delivery capabilities in China and India, RealFoundations provides Management Consulting, Managed Services and Energy Solutions to developers, owners/operators, service providers, institutional investors and corporate occupiers. From the building itself to the way it is used, operated and financed, no firm understands the inner workings of the entire real estate ecosystem as well as RealFoundations. We Make Real Estate Run Better.


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UK digital station CBS Drama has acquired the rights to repeat Footballers' Wives and Bad Girls.
UK digital station CBS Drama has acquired the rights to repeat Footballers' Wives and Bad Girls. The two dramas' were produced for ITV by Shed Media and ran respectively for five seasons and eight seasons. Although the two dramas were initially separate, with actors having roles in both shows, the two later crossed over during the sixth season of Bad Girls. The crossover involved the lead character of Footballers' Wives Tanya Turner [Zoe Lucker] sent to Larkhall prison on drug charges. Zoe Lucker appeared in three episodes of Bad Girls prompting rumours of further crossovers between the two dramas – which never happened – and even cross others with other ITV dramas such as The Bill.
CBS Drama has picked up all five seasons of Footballers Wives and will begin repeating it from the end of the month. The series was a "glossy" and "camp" take on the world of 'WAGS' and was described as a British version of Dynasty. Appearing in the drama alongside Zoe Lucker across its five year run were Gary Lucy, Gillian Taylforth, Laila Rouass, Alison Newman, Jessie Birdshall and Joan Collins. Bad Girls ran for eight seasons and was initially considered to be a gritty drama before losing the plot somewhat taking inspiration from Australian series Prisoner Cell Block H. Amongst the cast members of Bad Girls over the years were Linda Henry, Helen Fraser, Debra Stephenson, Victoria Alcock, Claire King, Kika Mirylees and Stephanie Beacham.

subset: commoncrawl


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EDM/Dub-step DJ, Skrillex, released the album Recess earlier in March that features the song Dirty Vibe, which recently received a music video, above. The trippy clip also gets some flavor from the east with appearences from K-Pop superstars CL and G-Dragon, with DJ/Producer Diplo also lending a hand on the sonic front.
Although Skrillex nor Diplo are in the video, it's hyper-sonic beat and entrancing vocals – and visuals – from CL and G-Dragon pull you in the whole time you're wondering what the hell you're watching.


text: Larj Bash ft Innocent , "YES SIR" , Money Making Machine. by Philip Walter Wasrock January 29, 2018, 4:24 pm Share this:Click to share on Twitter (Opens in new window)Click to share on Facebook (Opens in new window)Click to share on Google+ (Opens in new window) Related See more Previous article Ideas & Tips To Start Making Money From Your Online Home Business Next article HOW TO START ONLINE STORE BUSINESS | HOME BASED BUSINESS IDEAS Leave a Reply Cancel reply Your email address will not be published. Required fields are marked *Comment Name * Email * Website Notify me of follow-up comments by email. Notify me of new posts by email.


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Dissertation on youth homelessness. Outed and outside : the lives of LGBTQ youth experiencing homelessness
Furthermore, a strengths based approach among persons with mental illness shows a positive association with number of hospitalizations, quality of life, social functioning and social support [ 33 ].
Many have lost trust in professionals and services [ 15 ]. Randomization of homeless youths was not feasible since entire teams were trained prior to the baseline measurement in the strengths based method. Both internal factors e.
Social Support from Family and Friends Procidano and Heller's measures, designed to assess adolescents' perceived social support from family and from friends, were adapted, deleting items that might be confounded with psychological health. Keywords: Psychological distress, Runaway, Adolescence, Youth, Sexual minority, Homosexual Homeless LGB youth are at far greater risk for psychological symptoms than homeless heterosexual youth see Gattis for review.
The minimum age of developing a same-sex or other-sex sexual orientation composed the measure of sexual orientation development used here. Second, homelessness may lead to experiencing additional stressors by the mechanism of stress dissertation on youth homelessness Pearlin et al.
The success of provision was widely agreed to depend to a significant degree upon the quality of individual project staff and their relationship with young people. There is a strong association between homelessness and withdrawing from education, employment or training, with a discord evident between the welfare benefit system and employability initiatives.
This gender and sexuality paradox kept the youth in this study cycling between the streets and the shelter, but not achieving and maintaining housing stability. The current study examines the role of homelessness versus no history of homelessness on the psychological symptoms of LGB youth.
Interviewers were college educated and of the same sex as the youth. Most studies found heightened rates of substance abuse [ 9 ].
Youth homelessness in the UK | JRF
Agencies were more effective and coordinated in their approach to meeting the needs of young people aged 16 and 17, and those looked after by the local authority, than a decade ago. Other impacts are more mixed. Statutorily homeless young people are very unlikely to have an ethnic minority background in Scotland, Wales or Northern Ireland but are significantly over-represented in England, most particularly London.
The research questions of the present study are: 1.
Secondary Menu
Research has motivation letter for apply university shown that homeless youth, a highly vulnerable and heterogeneous population, suffer from a wide range of problems. Another shortcoming of the existing research on homeless LGB youth is its exclusive reliance on the cross-sectional design Gattis New targets on bed and breakfast use in Wales and Northern Ireland with England to follow were welcomed but those who took part in the study felt that more emergency accommodation was required.
Services could usefully develop policies to support better youth transitions across housing, employment and family formation. Although an average of 4 years had elapsed between the first episode of homelessness and the Time-1 assessment of symptoms i.
The youth also were asked the ages when they first experienced any of several sexual activities with the same sex, with the earliest age selected as the age of first same-sex sexual behavior or activity. Abstract Background Custom writing socks homelessness among youth is a serious problem, there is little information about evidence-based interventions for homeless youth.
Outed and outside : the lives of LGBTQ youth experiencing homelessness
Many youths have become dependent on services for homeless youth, have lived on the streets, or have found temporarily shelter with friends. Furthermore, the study examines factors i. On average, they have little money to spend because of lack of income or high debts, a low level of education, are struggling to maintain a daily routine, and frequently, experience limited support of their social network which mostly consists of other homeless people [ 6 - 8 ].
Psychological symptoms are often reported both by currently homeless youth and those with a history of homelessness Clatts et al.
It also incorporated the findings from two national consultations with young where can i buy essays led by Centrepoint, and a policy roundtable event with national experts. Given the relationships suggested relatively independent outcomes, we examined the outcomes separately.
For most European countries this information is lacking as well. Instead, an adult at each CBO served in loco parentis to safeguard the rights of every minor in the study. Youth were asked the ages when they were first erotically attracted to, fantasized about, and were aroused by erotica focusing on the same sex. Model fidelity will be tested around T1.
The two resulting item measures were administered at Time 1, using a yes 1 or no 0 response format e. Likewise, the developing sexual orientation of LGB youth may lead to conflicts with parents and result in youth being evicted from the home Gangamma et al.
Because such studies would focus exclusively on LGB youth, they would hold constant sexual orientation and, therefore, any potential health disparities associated with sexual orientation. Comparable items assessed the age at first other-sex sexual behavior or activity.
Yet, even though there is little information available on evidence-based interventions, some studies mention a supportive working relationship between professionals and youths as a crucial element in an effective intervention, [ 425 - 28 ].
Indeed, the differences found between homeless LGB and homeless heterosexual youth may be a function of sexual orientation and related stressful factors rather than homelessness, given the disparities found between LGB and heterosexual youth in the general, youth population Bontempo and D'Augelli ; Fergusson et al. The introduction of homelessness strategies has placed a new emphasis on prevention, paying explicit attention to young people.
A strengths based method for homeless youth: Effectiveness and fidelity of Houvast
Linear regression was used to examine the role of homelessness and potential mediators in understanding the internalizing symptoms i. Third, homelessness may lead to lower availability or erosion of resiliency resources that serve to protect youth from the negative effects of homelessness.
Mar 27, Journal Article Journal Article A brief group-based motivational interview intervention was effective in helping homeless young adults make positive changes in their alcohol and condom use.
Only recently has research included young homeless lesbian and bisexual women Walls et al.
Site-wide navigation
Effective prevention also requires the creation of affordable housing pathways for young people. This included 43, young people aged 16—24 who were accepted as statutorily homeless in the UK and at least 31, non-statutorily homeless young people using Supporting People services during — Operational joint working between service providers was seen to have made some significant steps forward in the last five years, assisted by factors such as policy and legislative change; youth homelessness forums; and joint protocols.
Shortages of social motivation letter for apply university were acute in many areas, necessitating often very lengthy stays in temporary literary magazines for creative writing.
Yet, even though there is little information available on evidence-based interventions, some studies mention a supportive working relationship between professionals and youths as a crucial element in an effective intervention, [ 425 - 28 ].
I show how the family and other institutions i. Limited data also suggests that considerably more young people may experience rough sleeping over the course of a year than on any given night.


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Infinita SYSTEM4 Open-Back Steel Bookcase in Multiple Finishes
$1269 - $1519
No Sales Tax, Except CT
Product Reviews
Pulp fiction, creative classics, and more come together on this modern SYSTEM4 Steel Bookcase designed by the folks over at Infinita. Designed with modernity and functionality in mind, this product delivers on both, resulting in an item that enhances any room in which it is placed. The higher than average shelves and open back remove the relegation of book-only storage from his product; in addition to any literary works you may want to store, this bookcase can store much more. Add a row of vinyl to the shelf, or maybe a row of knick-knacks or collectibles; whatever you've got to display, go ahead and add it. This bookshelf comes in two (2) different size options to suit your storage needs, and is crafted out of durable steel to ensure enhanced quality and lasting viability. Read it all then store it away on this chic minimalist Infinita SYSTEM4 Steel Bookcase.
(W = side to side) (D = front to back) (H = top to bottom)
Four (4) Shelf Option Overall Dimensions: 30" W x 16" D x 61-3/10" H
Six (6) Shelf Option Overall Dimensions: 59-1/2" W x 16" D x 46-1/2" H
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7 Tips for Easy Photo Management
with ASUSTOR's Photo Gallery
ASUSTOR NAS offers users a cloud storage solution that allows them to centrally store all of their photos in one place while also providing the Photo Gallery App that allows users to effortlessly manage and share their photos.
Asustor NAS AS3204T: Hardware Upgraded From AS-304T
Check out the sharing from the youtuber, AskUncleDave.
" I have owned an Asustor AS-304T NAS for couple years now. I Decided to Buy a Second unit, a more powerful unit to dedicate for Store and Stream all of my media. AS3204T is the Perfect upgrade. "


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Maximize your French gîte bookings with fast broadband
One of the silent killers of gîte bookings today is the aggregate of all those potential guests that simply click away and skip a listing because the gîte doesnt offer fast broadband. With most homes having six or more internet connected devices, and many of them being brought along on holiday with their owners, holiday makers have quickly raised this service towards the top of their list of requirements.
The difficulty lies in the location of these gîtes. The vast majority are set in beautiful and picturesque -but rather remote- rural areas, away from the large metropolis. Consequently, the local telecom services may not be up to the same standards as those in urban areas.


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The only brand I endorse is Saluda Cymbals. They make 100% custom cymbals in both look and sound.
I've used Saluda since 2010, and endorsed them exclusively since 2015. Over the years I tried many of the big brand cymbal companies and their different lines. What I found though was that finding that perfect cymbal with the right amount of wash, cut, and darkness was always a struggle. A cymbal would cut well but sound too gongy when crashed. Or a cymbal would have a beautiful wash but no definition on stage.
Saluda have developed a system that bypasses this problem completely. You describe the cymbal sound you want, and then they make it. If you know the cymbal sound you are after, it makes finding that perfect cymbal a lot easier. It saves a lot of time rather than hitting every cymbal in a different cymbal shop each weekend.
You can check out everything Saluda HERE
You can look at pricing and current models HERE
If you need more customisation, Jamie and his team at Saluda are always capable. In the past, I've really gone in depth with weights, sounds and descriptions to get exactly what I'm after.
I hate heavy, thick & bright cymbals (though for some styles they're needed) but as I'm found with playing more and more gigs, really dark washy cymbals get lost on the stage. To have a beautiful cymbal have enough cut to be able to be heard is a delicate balance. This is always the sound that I strive for. I'm after musicality and complexity in a cymbal but I also need usability when you're up against PAs and amps.
Current cymbal Setup
Here's a look at my current setup. I often switch out my crashes and rides depending which sound I want for the performance.
14″ Symbolic Med-thin Hi Hats
These Hi Hats have the perfect combination of cut and darkness. These hats are almost darkly pronounced. You can lay into them without them sounding like hubcaps. They have a high profile from the bell to the edge which gives them a nice dryness. The cymbals have a really interesting range of sounds when opening up the hi hat pedal. I always tend to prefer slurps and half open hi hat sounds over the boisterous and callous sounds of wide open hats. This is where these hats shine. You ride them half open and run 8th/16th note patterns while still hearing each note. You can also lay full, heavy quarter notes which still sounds lush.
18″ Diamond Thin Crash
This crash I use for every gig. It opens nicely you can still ride it just under the bell at small acoustic gigs. It's very thin at the edge so it opens up easily. The bell is chimey and cutting.
20″ Diamond Extra Thin Crash
This cymbal is the thinnest cymbal I have ever owned. It opens up with the last touch. You can't ride this cymbal, or play the bell without the whole cymbal opening up. This is exactly what i wanted in a 20″ crash. As soon as you touch it, it opens up to a wall of darkness and wash, this is the sort of cymbal that you can't throw at the guitarist due to the danger of beheading This is the sort of cymbal you can bend in half with your hands.
20″ Symbolic Thin Ride
The thin ride is almost a crash ride. I don't think of it as a ride or a crash because it really fits both categories. It opens up as a complex and dark crash. This cymbal tapers off hard which is why it such a great multipurpose cymbal.
21″ Symbolic Med-Thin Ride
This is my main ride. It has a nicely pronounced bell, a great wash/crash sound and rides without losing definition. It has a nice amount of weight, not too heavy and it tapers off from the bell to the edge. This means the edges still open up when it's crashed.
Saluda know their cymbals and they operate differently to most other companies. Check them out for a different experience!


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The need for Automation in a Post-Truth World
2016 has seen the rise of Extreme Confirmation Bias. It's also been the year where new technology has become the norm in our everyday lives. As we enter 2017, one of the biggest changes we'll see is the idea of the Internet of Things, specifically the industrial IoT. …Read more →


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I see it all the time. Parents bringing so much with them to the hospital or birth center it looks like they are moving in. The non-laboring parent is running back and forth to the car getting everything they could possibly need.Read More
Chiropractic spinal and pelvic adjustments can reduce or resolve pregnancy discomforts such as back pain, hip pain, sciatica, and heartburn. Chiropractic helps with optimal fetal positioning by helping the pelvis to be symmetrical. This in turn helps the uterus to be more symmetrical. Adjustments can help the baby to fit the brim better to engage in time to help stimulate cervical ripening. Adjustments also make the pelvis more flexible so that pelvic joints move more easily in laborRead More
Articles pop up all the time on Facebook and other social media sites about birth and the latest studies. Almost every time I scroll through the comments, there is at least one "I am a labor and delivery nurse, and any mom who comes in with a birth plan is guaranteed a c-section/epidural/episiotomy." As if the care providers will abuse the mother for daring to have goals for her birth. Even if this is true, care providers like this would make up a very small portion of hospital staff. After 7 years of being a doula, I have never encountered one.Read More


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Editorial published in the 20th March Print edition of Dreamingless Magazine! Styled by me. 'Modern Retro' shoot. Credits in the photos.
Tag: Bev K MUA
Styling 'Modern-Retro' Shoot for Dreamingless Magazine: 26/02/2016
I have wanted to work with Dreamingless magazine for a while as a stylist, and so I e-mailed the magazine with my portfolio etc. to which I received a response from the editor – in – chief of the magazine; Zoé Ashiotis (whose role on the shoot was Creative Director and Photographer). The theme was…


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Western Cowboy shoeing his horse,
giving a pedicure while the dog eats lunch on the trimmings!
Art Pencil Art
Giclee Prints
Christmas Cards
Note Cards
Commissioned Art
Contact Us About The Artist News Links FAQS Shows Workshops
Copyright © 1982-2018, Virgil C. Stephens, All Rights reserved. No part of this site, including but not limited to
the original artwork may be reproduced in any format without written permission.
Website design Emily Stephens firstname.lastname@example.com


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Maya Bistro
Maya Bistro is a family-owned restaurant in Arlington, VA. in Maya Bistro our delicious meals can be anything from a simple snack to an elaborate multi-course meal celebrates good food, hospitality and the heart of Turkey.
It is an upscale-casual dining experience that celebrates the rich diversity of Turkish & Mediterranean food and culture through a blend of classic and contemporary menu offerings and superb hospitality.
With the 35 years of experience in a variety of cuisines, our family draws on a deep knowledge of classic and contemporary Turkish & Mediterranean cooking as well as the many regional influences on Turkish & Mediterranean cuisine.
Wonderful food and very attentive service. Atmosphere is cheerful and very clean.
Exquisite dining experience, from the service and most abundantly the quality of the good. Without question, the best Middle-Eastern fare in the NOVA region.
This is my third visit to Maya Bistro this summer. So, you can see I like it. The last time I went with a friend who is from Turkey and another from Greece. They declared it excellent. So, that is a ringing endorsement. One note: we ordered 3 cold mezzes and the mixed grill platter for two. The platter is quite generous and it fed us well.
Birthday of my friend. We both enjoyed our experience.
Manti and Chicken shish entrees were very delicious. The Greek wine which was the special of the night was wonderful. Glad we listened to the server and we took the bottle home. Appetizer of the zucchini cakes were like potato pancakes and the mint leaves in it made it pop. Would definitely order that again. Everything was fresh. Baklava was the best that I have eaten in a very long time. Will be back.
4 of us shared the Maya Mix Grill which includes both beef and chicken shish deliciously seasoned and perfectly grilled along with tender lamb chops and Adana (ground beef & lamb) served with grilled veggies and rice. The most delicious meal! Ended with baklava which was sweet and crispy.
The food is delicious and there is a large selection. I'm looking forward to going back and trying other dishes! Service is great.
A wonderful local spot with great cooking and caring waitstaff.
Family run, with care and skill!
This is now our go-to neighborhood restaurant. We were happy to introduce good friends to it. The food was excellent as always. The service is usually spectacular, but on this visit it was merely good. If that's your floor, you're doing pretty well.
Love, love, love that we have this yummy restaurant in our neighborhood. We receive personal service every time we go....love the wide variety on the menu
An excellent new family-run neighborhood restaurant. We had Hummus and Maya cigars for starters and Moussaka and Adana Yogurt for mains. All were fresh and spiced to our requests. The style is Turkish with Greek and Italian influences. Service was prompt and efficient. A real gem.
Great food and service in a neighborhood environment.
Lovely place, great food and service. Wine and beer list a bit limited but charming due to the size of the place. Nice ambiance and sorely needed for the neighborhood!
Hummus and baba ganoush delicious; wish the bread was a bit thinner. Excellent Greek salad and Spinach Pie, and yogurt dishes. Desserts pretty good.
Overall, very satisfied and we'll be back!
A wonderful neighborhood restaurant with an especially interesting wine list.
Excellent food, as always. The waiter and server tried to take plates away a bit too soon, and it was a little hard to get the waiter's attention when we were ready for the check. Minor problems, not major. I'll be back. Maya Bistro is my current favorite!
Fills the need for a quality neighborhood restaurant perfectly! Have been several times, trying different meals and all were delicious! The service is wonderful. Everyone very welcoming and efficient. We will go often!
Yummy as always. Made a point of trying some new dishes, which we enjoyed. Prompt and attentive service.
This was our first visit to Maya Bistro. My friend is high maintenance and requested a number of minor changes to her order. All were graciously accommodated. The restaurant is airy, light, quiet, and a wonderful addition to our neighborhood. Our food was delicious, and the apricot dessert was both yummy and beautifully presented. I'm already going back with friends next week. I do wish they had handicapped parking for my friend and more parking in general.
Loved it. Great menu!!!!! Great prices and value!!!! Nice staff too! You should go or better yet call for delivery so I can have a table.
Lovely staff; great food; friendly service. We'll be back!
Maya Bistro was a wonderful find in North Arlington. The service was great. Our wait person was attentive and friendly. The staff who brought the meals were on the mark.
Each of us ordered something different and noone was disappointed from lamb dishes to chicken to veggie choices.
I am sure that this will be on our friends list as they still live nearby! We may also frequent the bistro from time to time even though Arlington is not our home.
Very nice and attentive staff. A little too expensive relative to other Mediterranean restaurants and portion sizes. Overall, it's a nice neighborhood restaurant.
Our first time and all three of us very much enjoyed dinner at Maya Bistro on Lee Highway in North Arlington. (Entrees of grilled salmon, lamb chops and a beef and lamb special entree.) Warm and prompt service, delicious food and a nice comfortable setting. Highly recommend.
Service and food were excellent. Atmosphere is lacking.
The food is so fresh and delicious. My daughter and I enjoyed it all!
Great food, ambiance and service. An excellent new gem!
Please add this restaurant to your regular rotation or date night. Uncomplicated, high quality food at reasonable prices. You can have kabobs and traditional Turkish dishes. Next time I'm getting the whole branzino because it looked great.
Great neighborhood restaurant! We especially love the fresh bread and spreads!
We've dined there several times always a great experience. Excellent food, friendly staff, and fair prices!
Falafel salad was delicious. Had a fun time and got my fortune read from my Turkish coffee!
Just a great restaurant with ambiance and flair. Delicious Food. Excellent Service. Neat, clean... a real keeper. Bravo.
Very nice evening with delicious food, excellent service and a lovely setting
A very welcome to Arlington's restaurant scene. Well prepared dishes and tasty.
A great new addition to North Arlington--featuring Mediterranean dishes and delicious Turkish wines. The tapas for 4 was gigantic and we could barely finish it...then among the 4 of us, we shared 3 dishes-a grilled fish, a shrimp and a chicken dish.
Staff were friendly and helpful-when our server had difficulty guiding us for a wine choice, she brought someone over to the table who offered to let us try three wines so we could decide.
After hearing good things about a neighborhood restaurant, we went to Maya Bistro for lunch Sunday. Brunch (mostly egg dishes) was available as well as main menu items. I ordered the baked zucchini while my friend chose the dolmas and an omlette. All were delicious. The service was attentive and prompt. All in all, a great dining experience.
Wonderful Turkish menu; meats were perfectly cooked to our medium rare request. Will definitely return!
Great new neighborhood treasure, good quality home made Turkish food, no wait time and don't feel rush to leave.
The mezze platter was a nice introduction to the menu and filled us up enough to share one entree be the two of us. Easy parking and lovely staff. We'll be back.
Our dishes were filled with fresh vegetables and crisp greens, and were tasty. The staff are very friendly, and remember you from past visits. I plan to be a regular here.
Extensive delicious menu, labels for gluten free and dairy free, nice spacious airy light setting, attractive looking bar, good soundproofing for a reasonable noise level allowing good conversation. And they make mojitos! Definitely will be going back.


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Good dental hygiene starts as a young child but remains just as important throughout your adult life. While brushing and flossing twice a day is extremely important, so is getting your 6-month professional cleaning.
Brushing your teeth and cleaning between teeth (flossing) is most effective in removing plaque and preventing tooth decay. An antimicrobial toothpaste also helps prevent tooth decay.
Your toothbrush should have soft bristles and fit the shape and size of your mouth to be able to reach your back teeth easily for better cleaning. Replace your toothbrush every 6 months. If you are getting your teeth cleaned every 6 months, your dentist office will provide you with your replacement toothbrush.
Flossing is also very important as it gets in between the teeth where a brush can't reach. Food gets trapped between your teeth and allows for uninhibited bacterial growth, causing cavities to form. Flossing is also essential for preventing gum disease.
A lot of people skip flossing, some because they say it takes too much time and some because they think it is too difficult to get done. It doesn't have to be a chore. There are floss picks now that make it easier, because they have a handle for better manipulation. There are also water flossers that aim water between your teeth to remove debris.


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|Punjab University Research Assistant, Laboratory Attendant Vacancy
|No. of Vacancies
|Age Limit
|Below 30 years
|Selection Process
|Last Date to Apply
|Job Mode
|Contractual Basis
|Job Location
Recruitment at Punjab University for the Vacancy of RA, Lab Attendant Job
Punjab University has invited applications for recruitment on the vacancy of Research Assistant and Laboratory Attendant Interested and suitable qualification holder person may send their application up to 09 th of August, 2012. Details regarding this job vacancy about no. of vacant seat, educational qualification, how to apply, age limit, Salary etc are given below in next few passages like following —
Punjab University RA and Laboratory Attendant Vacancy Details
1. Research Assistant
No. of vacancies – 01
Educational qualification – Post Graduation in science with Microbiology / Life Sciences with minimum obtained 55 % marks.
Preferential qualification – should be qualified in UGC NET Exam (for Ph.D)/ PU Ph.D Entrance Exam qualified will be preferred or Experienced person will get preferred.
Stipend – Fixed amount of Rs. 16,000/- Per Month and 20% HRA.
2. Laboratory Attendant
No. of Vacancies – 01 seat
Educational Qualification – should be passed Tenth Class with Science Subject and having at least 02 year relevant working experience or
should be qualified in Intermediate with Science from any recognized University.
Desirable Qualification – Experienced person will be preferred.
Salary – Rs. 9896/- Per Month (Fixed)
Selection Process – The mode of selection for this post is Interview.
How to Apply – Applicant who have above said qualification for any particular job and want to apply may do this. They should send their recent bio data with attested copies of all supporting documents to address of Chairperson, Department of Microbiology, Punjab University latest by 09 th of August, 2012.
Important Facts – 1. Date of Interview will be intimated on Email Id or Contact no of Applicant
2. No TA/ DA will be paid to any one for attending interview
For knowing full details regarding notification of recruitment at Punjab University for the job of RA and Lab Technical Assistant go through the following link –
Punjab University Recruitment notification Details
More recent updates can be read at the official website of Punjab University at this Web URL —


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Sur restaurant in la
Storms close Highway 1 at Big Sur's slide areas, possibly
Catch RestaurantsDiscover restaurants in La Roche-sur-Yon: great places to eat and dining recommendations from other travelers.
Château De La Bourdaisière - Hotel in Montlouis-sur-Loire
Big Sur Bakery & Restaurant - Big Sur, CA
We will be open with regualr service New Years Eve and Bakery Service on New Years Day.
Astrolabe Restaurant – ASTROLABE – Restaurant L'AstrolabeLa Peniche is on the barge MV Riasc (peniche is the French for barge) moored on the Grand Canal alongside Mespil Road between Baggot and Leeson Street bridges.Drawing on his experiences in kitchens across the country, Corsentino crafts coastal cuisine—using Pacific-sourced seafood delivered by local fishermen, free-range meats from local farmers, produce from nearby.
La Terrasse proposes seasonal and original recipes, blending mediterranean savors with an exotic touch.
Explore Big Sur, CA | Monterey County Regions
Le CirqueSUR Restaurant, featuring eclectic cuisine, brings a taste of the exotic to the trendy corner of Melrose and Robertson.Our guide to Las Vegas restaurants will help you make reservations at the best restaurants with reviews, pictures and more.
Le Soir RestaurantSUR offers guests friendly, attentive service and sumptuous international cuisine for a truly unique.
La Coupole / Restaurant / PARISThough there are no active slides, Highway 1 has been closed at two spots south of Big Sur where falling debris has been a problem in the past as a storm passes through the area.Under responsibility of Didier REVILLIOD, restaurateur Master, the restaurant will welcome you in the field of golf of St Donat, with traditional and Provencal cuisine.
Big Sur Lodging & Dining | Big Sur River Inn & Restaurant
Tango Sur – Steakhouse · Latin American Restaurant
The room is decked out in chalet decor, and is a little getaway to the country.in the middle of Old Montreal.There will be no dinner service from December 3rd through the 19th.SUR offers one of the most exciting dining destination in the city that is now recognized internationally with a South-European Flair, luxurious atmosphere, and friendly service.Worldwide food experts have ranked it among the best restaurants in the world.See up-to-date pricelists and view recent announcements for this location.Sur La Table is a specialty retailer known for its cookware, kitchen tools, appliances and other kitchen products.Partake in the pleasures and delights of renowned Belgian executive chef Dany Lamote.
La Terrasse Restaurant | Le Méridien Nice | Rooftop
West Hollywood, CA Restaurants Guide - menupages.com
20% Off Sur La Table Coupons & Promo Codes - January 2019
Chef Andre' Returns in a New Stylish Way - lecontinental.ca
Deetjen's Big Sur Inn | RestaurantExplore menus for restaurants in West Hollywood, CA on MenuPages, your online source for restaurant menus in West Hollywood.
Explore SUR SUR offers guests friendly, attentive service and sumptuous international cuisine for a truly unique dining experience.A high point of the menu is the prix fixe wine tasters meal with wine pairings for each course.
Lovers of French cuisine cannot fully describe the taste of the food presented in the form of cold and hot appetizers, salads, soups, classics and side dishes and sauces.


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An air conditioner's cooling process produces condensation, which normally flows away from the equipment, causing no problem. If there is a clog in the condensate drain lines or drip pan, or if outdoor humidity levels are high, moisture may back up into your air conditioner. Excess condensation will increase indoor humidity levels and hinder the air conditioner's performance. It could also cause damage to your air conditioner's components.
We are service professionals! As licensed contractors and specialists in the field of heating and air conditioning, we have the tools, the equipment, and the experience to keep your equipment running smoothly all year long. If it is emergency service that you need, we are available to assist you! To help reduce service emergencies, we offer service agreements to keep your comfort system running at peak performance.
The three central functions of heating, ventilation, and air conditioning are interrelated, especially with the need to provide thermal comfort and acceptable indoor air quality within reasonable installation, operation, and maintenance costs. HVAC systems can be used in both domestic and commercial environments. HVAC systems can provide ventilation, and maintain pressure relationships between spaces. The means of air delivery and removal from spaces is known as room air distribution.[3]
ABM (NYSE: ABM) is a leading provider of facility solutions with revenues of approximately $5.5 billion and more than 130,000 employees in 350+ offices throughout the United States and various international locations. ABM's comprehensive capabilities include janitorial, electrical & lighting, energy solutions, facilities engineering, HVAC & mechanical, landscape & turf, mission critical solutions and parking, provided through stand-alone or integrated solutions. ABM provides custom facility solutions in urban, suburban and rural areas to properties of all sizes - from schools and commercial buildings to hospitals, data centers, manufacturing plants and airports. ABM Industries Incorporated, which operates through its subsidiaries, was founded in 1909. For more information, visit www.abm.com.
Before we get into the different AC repairs and replacements that John Moore offers, it's important to understand the basic operating principles of your AC system. There are two types of systems: electric and natural gas. Electric AC systems use an air handler to blow air through your home while natural gas systems use a furnace. Both furnaces and air handlers are typically located in the attic.
Although HVAC is executed in individual buildings or other enclosed spaces (like NORAD's underground headquarters), the equipment involved is in some cases an extension of a larger district heating (DH) or district cooling (DC) network, or a combined DHC network. In such cases, the operating and maintenance aspects are simplified and metering becomes necessary to bill for the energy that is consumed, and in some cases energy that is returned to the larger system. For example, at a given time one building may be utilizing chilled water for air conditioning and the warm water it returns may be used in another building for heating, or for the overall heating-portion of the DHC network (likely with energy added to boost the temperature).[4][5][6]
Ground source, or geothermal, heat pumps are similar to ordinary heat pumps, but instead of transferring heat to or from outside air, they rely on the stable, even temperature of the earth to provide heating and air conditioning. Many regions experience seasonal temperature extremes, which would require large-capacity heating and cooling equipment to heat or cool buildings. For example, a conventional heat pump system used to heat a building in Montana's −70 °F (−57 °C) low temperature or cool a building in the highest temperature ever recorded in the US—134 °F (57 °C) in Death Valley, California, in 1913 would require a large amount of energy due to the extreme difference between inside and outside air temperatures. A few feet below the earth's surface, however, the ground remains at a relatively constant temperature. Utilizing this large source of relatively moderate temperature earth, a heating or cooling system's capacity can often be significantly reduced. Although ground temperatures vary according to latitude, at 6 feet (1.8 m) underground, temperatures generally only range from 45 to 75 °F (7 to 24 °C).
HVAC is an important part of residential structures such as single family homes, apartment buildings, hotels and senior living facilities, medium to large industrial and office buildings such as skyscrapers and hospitals, on ships and submarines, and in marine environments, where safe and healthy building conditions are regulated with respect to temperature and humidity, using fresh air from outdoors.
The icing problem becomes much more severe with lower outdoor temperatures, so heat pumps are commonly installed in tandem with a more conventional form of heating, such as a natural gas or oil furnace, which is used instead of the heat pump during harsher winter temperatures. In this case, the heat pump is used efficiently during the milder temperatures, and the system is switched to the conventional heat source when the outdoor temperature is lower.
RepairClinic.com® is the trusted online parts store with 3,000,000+ appliance parts, lawn mower parts, power tools and heating and cooling parts for over 200 appliance, lawn equipment, power tools and heating & cooling equipment brands. Since our launch in 1999, RepairClinic.com has helped 4,500,000+ people save more than $500 million in repair costs by fixing stuff on their own.
New heating and cooling equipment installations present a wide variety of options that can save you money on your power bills and reduce the amount of HVAC maintenance and service that will ultimately be required over the life of your new unit. Energy-efficient options like heat pumps, two-stage furnaces, air-to-heat exchangers (HRVs), and programmable thermostats can all go a long way toward increasing efficiency and extending the life expectancy of your system.
A specialized air conditioner that is used only for dehumidifying is called a dehumidifier. It also uses a refrigeration cycle, but differs from a standard air conditioner in that both the evaporator and the condenser are placed in the same air path. A standard air conditioner transfers heat energy out of the room because its condenser coil releases heat outside. However, since all components of the dehumidifier are in the same room, no heat energy is removed. Instead, the electric power consumed by the dehumidifier remains in the room as heat, so the room is actually heated, just as by an electric heater that draws the same amount of power.
Our experienced technicians know how best to repair and maintain an HVAC system, keeping your family safe and your system running efficiently. Our team can also identify potential problems, addressing them before they cause serious damage to your system. Instead of attempting a repair yourself, have one of Brubaker Inc.'s trained technicians look at your system for you.
At Edwards Heating & Air, our dedication to quality extends to every service and product we provide. Since first opening our doors in 2001, we have continued to protect home and business owners in Acworth and across Acworth, Alpharetta, Cummings, Cartersville, Kennesaw & Woodstock, GA with conscientious workmanship, honest pricing, and exceptional products. By never failing to handle every project with care and dedication, we earn your satisfaction, recommendation, and business. We have continued to expand our list of clients by answering every call promptly, responding with courteous service, and delivering rewarding solutions. Whether you are in need of new installation, replacement, upkeep or repairs for residential or commercial, heating, cooling, ventilation, and air quality, know that Edwards Heating & Air gets results.
Waiting or performing repairs yourself could cause further damage to your system, resulting in more expensive repairs. If you suspect a problem with your air conditioner, schedule an appointment with a licensed, trained HVAC technician at Service Professor right away. When it comes to safe and reliable electrical service and repair, leave it to the friendly, skilled electricians at Service Professor. Your Satisfaction is 100% Guaranteed!
The Refrigeration Loop is a closed system and refrigerant should never have to be added to the unit unless there is a leak. Most residential units use R-22/R410A refrigerant. Prices of R-22/R410A are rising every year as we get closer to the date when refrigerant manufacturers will no longer be able to make R-22/R410A. Phase-out of R-22, an HCFC, begins in 2010 and expected to be complete by 2020.). Furthermore, make sure you ask what the service company charges for R-22 when you are calling an HVAC contractor.
Regardless of the scope of your project, find out what you need to do to prepare your home for their visit. For example, the HVAC professional will need access to the areas in question and may need access to other mechanical or ventilation points. Make sure pets and children are kept out of the way. Ask if your project will involve removing dry wall or making any other structural changes – you may need to hire a professional drywall contractor to patch up HVAC work. If you are adding a unit that may contribute to carbon monoxide within the home, find out if you need to purchase a carbon monoxide detector.
You haven't had a ductwork inspection in years: According to PG&E, the average home's ductwork leaks up to 30 percent of its air before it actually reaches your living space. That means you're still paying for the energy used to cool the air—without getting the relief of a cool home. Hire a technician to come take a look at your system; they can perform what's called a duct-leakage test, which measures how much air is escaping the system. If it turns out your ducts are faulty or have significant gaps, a technician should be able to seal them up or replace your pipes—meaning you might not need a complete system overhaul. However, keep in mind that modern HVAC units are almost always more efficient thanks to technology advancements. In the end, you have to weigh your options: will you need to perform repairs each month?
In variable climates, the system may include a reversing valve that switches from heating in winter to cooling in summer. By reversing the flow of refrigerant, the heat pump refrigeration cycle is changed from cooling to heating or vice versa. This allows a facility to be heated and cooled by a single piece of equipment by the same means, and with the same hardware.
One of the easiest maintenance tasks consumers can do themselves is maintaining the filters. Air filters should be inspected and cleaned once a month. This allows for regular monitoring to eventually determine when the filter needs to be replaced. Most filters can be changed every three months if no other problems exist. Maintaining clean and regularly changed filters will not only produce better air quality, but appliances will function more efficiently, which can save on heating and cooling costs.
The installment team ARS made numerous attempts to correct the issue over the last several months. They have not found the issue on why my 2018 outside unit and inside unit (4 ton unit) is not cooling the house down. I had the unit for 1 year and in the months of July and August the Nest unit reads 90 degrees from 12 noon to 7pm. My wife and I are very upset with the upgrade unit we purchase from Home Depot.
So, whether you need a furnace repair or you're searching for a new heating and cooling system—or you need a plumbing repair now–you can count on our team in North America. We're specialists in heating system and plumbing repair and offering exceptional heating and plumbing service is what we do best. Call Service Experts 24/7/365 at 866-397-3787 or request an appointment online today, and don't forget to visit our coupons page for savings on your heating system or plumbing repair service.
In order to make your everyday life easier and more enjoyable, Sky Heating & Air Conditioning specializes in state-of-the-art heating and cooling technology, partnering with Trane, Mitsubishi, and Waterfurnace. We bring you HVAC solutions that represent the best the industry has to offer, ensuring long-term performance and satisfaction. Whether you're looking for the design/build of a new system, replacement, or hoping to cut costs and enhance convenience, we answer with proven products. Our qualified HVAC technicians help you determine the ideal solution for your exact requirements, and make sure you're happy with every step of the process. From ductless HVAC to geothermal heating and cooling systems, we offer a wide selection of options and comprehensive services to answer any type of challenge. Call on Sky Heating & Air Conditioning and let us exceed your expectations today!
The employees were on time, professional, and extremely thorough while addressing a particularly difficult situation. The entire time Saul, the supervisor of the team, kept me informed, and educated me about any process that I may have not completely understood. The team was courteous to the residents they encountered, and made sure not to leave evidence that they had even been there. Great job!
As a Carrier Factory Authorized Dealer, Edwards Heating & Air meets strict requirements for business practices, such as proper licenses, insurance and liability coverage, and customer service. Our technicians participate in continued factory training, keeping current with advancements in techniques and technology, and adhere to strict installation and maintenance procedures. Our sales team is educated in leading-edge Carrier products, ensuring accurate recommendations for cost-effective, highly efficient, and reliable solutions to your specific circumstances. We specialize in heating, cooling, and air quality systems that are widely regarded as the world's best. Leading the industry in design and engineering, Carrier products offer unprecedented convenience, economy, and luxury. These systems don't just regulate temperature. They improve your everyday life.
Occupational employment projections are developed for all states by Labor Market Information (LMI) or individual state Employment Projections offices. All state projections data are available at www.projectionscentral.com. Information on this site allows projected employment growth for an occupation to be compared among states or to be compared within one state. In addition, states may produce projections for areas; there are links to each state's websites where these data may be retrieved.
Each spring and fall before you start using your air conditioner, furnace, or other heating and cooling systems, you should get a tune-up to ensure your HVAC system is ready for the season. By spending a little twice a year, you can save a lot in energy bills by keeping your system operating at peak efficiency. You also can avoid costly repairs by identifying and fixing minor problems before they turn into big ones. Plus, many homeowners don't realize that their manufacturer's warranties may be considered invalid if they aren't getting their unit serviced twice a year.
A.J. LeBlanc Heating specializes in the installation and service of heating, air conditioning and plumbing systems for residential homes. Whether you are building a new home, converting your existing heating system to a different fuel, upgrading your old unit to a more efficient system, or just adding air conditioning, LeBlanc Heating can service your needs.
In the fall you should prepare your HVAC unit for winter. Turn off your air conditioner's circuit, then hose it off to remove debris. It must dry completely. Do this when it's warm enough that there's no risk of the water freezing. Cover the exposed pipes with foam pipe insulation ($1-$3), and protect your entire unit with a weatherproof cover ($20-$50).
They did come out Tuesday and completed the job. We called the office to ask for a breakdown of the cost and they said the tech would call us back, but that never happened. Eventually Alberto called and explained the breakdown. One reason it was more was they used an OEM circuit board, rather than an aftermarket part. I agree with this but it would have been better if this had been explained from the beginning. All in all, it still seems like their price is a bit on the high side, but they did a good job and used OEM parts so I'm giving them four stars. Read more
Low refrigerant charges take away from the life of the compressor. The compressor is cooled from the return refrigerant vapor. When there is a low charge there are fewer vapors to cool the compressor, therefore it runs hotter. Any compressor (or electric motor for that matter) which runs above the recommended temperature range will have a shorter life span and cost more to operate.
In 1902, the first modern electrical air conditioning unit was invented by Willis Carrier in Buffalo, New York.[citation needed] After graduating from Cornell University, Carrier found a job at the Buffalo Forge Company. There, he began experimenting with air conditioning as a way to solve an application problem for the Sackett-Wilhelms Lithographing and Publishing Company in Brooklyn, New York. The first air conditioner, designed and built in Buffalo by Carrier, began working on 17 July 1902.[citation needed]
If you can't make up your mind about whether you should repair or replace your current unit and it's over the age of 10, buying a new unit might very well be a better investment. AC units will no doubt need repairs every once in a while. However, if your system continuously requires repairs, these can eventually cost more than installing a new unit, making investing in a new unit the wiser choice.
Need 24-hour emergency furnace repair? We can handle that too. With our same-day service and live phone and online chat service available 24/7, we make your comfort our greatest priority. Heating and air conditioning should help you live a comfortable life, so let us help you make your home the most comfortable it has ever been. It's easy, just grab your furnace repair coupon, then simply give us a call at 208-488-4892, chat or schedule an appointment with us online.
Plus, when it comes to heating and air conditioning maintenance and repair in San Diego homes, Bill Howe offers affordable pricing to provide you with everything that you need without breaking the bank. So, whether you find yourself with an inoperable AC unit that needs a complete replacement or you just want an annual tune-up, we are here for you. After all, we have specialized in heating and cooling San Diego for quite some time, and no problem is too big or small for us.
Your heating and cooling equipment is vital to your comfort and breakdowns can be stressful, inconvenient and even dangerous. You can rely on the professionals at One Hour Heating and Air Conditioning* for expert service when you need it most. Our highly-trained technicians are available around the clock to meet your heating and cooling needs. We'll install, repair and maintain your HVAC system, keeping it in peak condition. Give us a call when you need to:
An air conditioning system, or a standalone air conditioner, provides cooling and humidity control for all or part of a building. Air conditioned buildings often have sealed windows, because open windows would work against the system intended to maintain constant indoor air conditions. Outside, fresh air is generally drawn into the system by a vent into the indoor heat exchanger section, creating positive air pressure. The percentage of return air made up of fresh air can usually be manipulated by adjusting the opening of this vent. Typical fresh air intake is about 10%.
Waited a week for technician who showed up for less than 15 minutes and told me that my air filters were dirty and I needed to experiment with thermostats to be comfortable. Thermostat shows temp at 75 when set on 71. Did what he instructed and still had problems when I realized unit was short cycling. When I called technician he told me I would have to place a new request for service. He gave me the office number and hung up on me. By the time I called office he had called in and said he refused to come back out. The operator would not give me business owners name and could not tell me why technician refused to return although they did say they would refund the fee for service call. Telephone rep yelled at me.


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|2 Bedrooms • Sleeps 4
|2 Full Bathrooms
|Hot Tub:
|Hot Tub (Private)
|Cable/Dish TV
Two bedroom/two bath condo with a full kitchen with granite counter tops and a living/dining area. Gas fireplace. Bedding is King and 2 Doubles. West facing.
The property has an outdoor heated pool and hot tub, steam rooms and a small workout room. Free WiFi is offered throughout the property. No smoking and no pets.
Free Parking at Vail International, which is a rarity in Vail.
The Vail International condos are located just to the east of the Lionshead Parking Structure and northwest of the Vail Valley Medical Center. It's a 5 - 7 minute walk to Lionshead, which has a variety of shops and restaurants as well as the Lionshead Gondola and Chair 8. You can ride the free in town shuttle bus to Vail Village that stops at the Dobson Ice Arena, which is adjacent to Vail International. You can also walk to Vail Village, as it takes about 5 - 10 minutes along Meadow Drive.


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MicroRNAs aid the assessment of programmed death ligand 1 expression in patients with non‑small cell lung cancer
- Authors:
- Anna Grenda
- Marcin Nicoś
- Michał Szczyrek
- Paweł Krawczyk
- Tomasz Kucharczyk
- Bożena Jarosz
- Juliusz Pankowski
- Marek Sawicki
- Justyna Szumiło
- Paulina Bukała
- Janusz Milanowski
- Published online on: April 3, 2019 https://doi.org/10.3892/ol.2019.10207
- Pages: 5193-5200
Programmed death ligand 1 (PD-L1) is a protein expressed by activated antigen-presenting cells, the majority of normal body cells and certain types of cancer cells. PD-L1 is an immune checkpoint protein that protects against autoimmunity (1). Certain types of cancer, including non-small cell lung cancer (NSCLC), are able to express this molecule on the cell surface. Interactions between tumor PD-L1 and programmed cell death protein 1 (PD-1) localized to the T-cell surface, cause lymphocyte anergy and the resistance of cancer cells to immune surveillance (1,2). Inhibition of this signaling pathway using anti-PD-1 or anti-PD-L1 monoclonal antibodies results in mobilization of the immune system, and the subsequent recognition and destruction of cancer cells (3–7).
The effectiveness of anti-PD-1 (nivolumab and pembrolizumab) and anti-PD-L1 (atezolizumab and durmvalumab) antibody therapy in patients with NSCLC depends on the expression of PD-L1 on tumor and immune cells, which is detected using immunohistochemistry (IHC) (8–12). Immunotherapy, based on the inhibition of immune checkpoints improved overall survival in patients expressing PD-L1, but certain NSCLC patients without expression may also benefit from immunotherapy (13,14). There are 2 platforms to assess PD-L1 expression in tumor cells and/or in tumor-infiltrating immune cells (9). During clinical trials with pembrolizumab and nivolumab (which bind the extracellular domain of PD-L1) the antibodies 22C3 and 28-8 were used with the Autostainer Link platform to determine the PD-L1 status of participants. Inclusion criteria for pembrolizumab therapy were >50% of tumor cells with PD-L1 expression for first line treatment, and any expression of PD-L1 (PD-L1-positive tumor cells ≥1%) for second line treatment (11). However, nivolumab may be used for second line therapy in patients without PD-L1-positive tumor cells, and testing of PD-L1 expression with the 28-8 antibody is not required to qualify patients for nivolumab therapy (9). The SP142 antibody, used with the Benchmark platform, was employed in clinical trials for atezolizumab. This clone can bind to the intracellular domain of PD-L1 (15). The scoring of PD-L1 status is complicated, due to the requirement of assessing expression on stromal immune cells that have infiltrated the tumor. However, atezolizumab may also be used as a second line of treatment in patients without PD-L1-expressing tumor or immune cells. The antibody clone SP263, used with the Benchmark platform, has the ability to bind the intracellular domain of PD-L1; this was examined in clinical trials with durmvalumab (13).
Different kinds of antibodies used in the assessment of PD-L1 give incompatible scores. This may be due to the properties of particular clones and their affinity for different PD-L1 domains. There is a requirement to supplement the diagnosis of PD-L1 with additional factors, facilitating the qualification of patients for immunotherapy. Genetic factors that influence the effectiveness of this method may be identified. One such factor may be the tumor mutation burden (TMB). There are no studies using epigenetic factors as predictors for immunotherapy. However, microRNAs (miRs) present as potential candidates. miRs are small molecules (~20 nt in length) that regulate gene expression on a post-transcriptional level. These molecules act by either promoting mRNA degradation or inhibiting translation (15,16). There are few reports concerning the regulation of PD-L1 expression by miRs in lung cancer (17–19). Therefore, the purpose of the present study was to assess the expression of miRs with the ability to regulate PD-L1 expression in patients with NSCLC, and to determine the association between these factors and the expression of PD-L1 mRNA and protein in tumor cells.
Materials and methods
A total of 43 formalin-fixed paraffin-embedded (FFPE; 91.49%) tissues from patients with NSCLC, and 4 cell blocks (8.51%) were assessed for the expression of PD-L1 protein, mRNA and miR. Adenocarcinoma was diagnosed in 31 (65.9%) cases and squamous cell carcinoma was detected in 13 (27.7%). In 3 (6.4%) cases, NSCLC not otherwise specified (NOS) was diagnosed. There were 36 (76.60%) specimens from primary tumors, 5 (10.64%) from distant metastases and 6 (12.76%) from metastases to the lymph node. Samples were collected between September 2017 and April 2018 in the Departments of Pneumonology, Oncology and Allergology, Medical University of Lublin (Lublin, Poland). The study group included 33 (70.21%) patients at stage I–IIIA, 8 (17.02%) at stage IIIB, and 6 (12.77%) at stage IV. The median age was 65 years (±9.03 years), and the group consisted of 20 male (42.55%) and 27 female patients (57.45%), of which 10 were non-smokers (21.28%). Due to the lack of reimbursement for anti-PD-1 or anti-PD-L1 antibodies in Poland at the time, information regarding the efficacy of immunotherapy was not available. Therefore patients were not treated with anti-PD-1 or anti-PD-L1 monoclonal antibodies. Furthermore, surgically resected patients were enrolled, and immunotherapy in such patients was not available at the time (with the exception of those in clinical trials).
IHC analysis
During IHC analysis, the monoclonal antibody clones SP142 (Ventana Medical Systems, Inc., Tucson, AZ, USA) and 22C3 (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) were employed. Briefly, 3-µm paraffinized tissue sections were mounted using SuperFrost™ Plus slides (Thermo Fisher Scientific, Inc., Waltham, MA, USA) on a hot-plate in 60°C for at least one hour. Deparaffinization and antigen retrieval were performed prior to staining, using the Dako PT Link device (Dako; Agilent Technologies, Inc.). Slides were washed and dehydrated twice in 96% ethanol and xylene, and mounted on coverslips. The slides were pre-heated to 59°C for at least 3 h, and IHC with the SP142 antibody was conducted using the Ventana Benchmark GX platform (Roche Diagnostics GmbH, Mannheim, Germany), with the Ventana test certified for in vitro diagnostics (CE-IVD). The OptiView Amplification kit was incorporated for signal amplification, and the OptiView DAB IHC Detection kit was used (Roche Diagnostics). As the negative control, a rabbit monoclonal antibody (standard negative control by Ventana Medical Systems, Inc., Tucson, AZ, USA; cat. no. 790-4795) was applied. IHC conducted with the 22C3 antibody was performed using the Dako Autostainer Link 48 instrument (Dako; Agilent Technologies, Inc.) incorporating the CE-IVD IHC 22C3 PharmDx PD-L1 Kit and the EnVision FLEX visualization system (Agilent Technologies, Inc.). The aforementioned staining procedures were conducted according to the manufacturer's protocols. Counterstaining with hematoxylin was also conducted according to the manufacturer's protocol. The slides were later observed by the pathologist using an Olympus BX41 microscope.
RNA isolation
Total RNA isolation was conducted using 5-µm sections of FFPE tissues or cell blocks using an miRNeasy FFPE Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturers' instructions. RNA was stored at −80°C until the synthesis of cDNA.
Quantification of PD-L1 mRNA expression level
The relative level of PD-L1 mRNA expression was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in reference to the internal control, GAPDH. Reverse transcription was conducted using a High-Capacity RNA-to-cDNA™ kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instruction. qPCR was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, Inc.) and the Illumina Eco Real-Time PCR System (Illumina Inc, San Diego, CA, USA). The 20 µl PCR mixture contained the following: 10 µl TaqMan Fast Advanced Master Mix, 1 µl TaqMan Gene Expression Assay mix (assay ID Hs00204257 for PD-L1 and Hs02786624 for GAPDH; Thermo Fisher Scientific, Inc.), 5 µl RNase free water and 4 µl cDNA. The thermocycling conditions were as follows: 95°C for 20 sec, 40 cycles at 95°C for 3 sec, and 60°C for 30 sec. Analysis was performed using the 2−ΔΔCq method (20).
Quantification of miR expression
Expression of 8 miRs, complementary to the 3′ untranslated region (UTR) of PD-L1 mRNA was assessed: miR-141-3p (478501_mir,), miR-200a-3p (478490_mir), miR-200b-3p (477963_mir), miR-200c-3p (478351_mir), miR-429 (477849_mir), miR-508-3p (478961_mir), miR-1184 (478629_mir) and miR-1255a (478661_mir) were all acquired from Thermo Fisher Scientific, Inc. The targets for miRs in 3′UTR PD-L1 mRNA were predicted using the TargetScan (version 7.1; www.targetscan.org) and miRBase (release no. 22; www.mirbase.org) systems. miR-191-5p (477952_mir) was used as an internal control. Reverse transcription was conducted using the TaqMan Advanced miRNA cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. qPCR was performed using the Illumina Eco Real-Time PCR System (Illumina, Inc.). The 20-µl PCR mixture contained the following: 10 µl TaqMan Fast Advanced Master Mix, 1 µl TaqMan Fast Advanced miRNA Assay mix, 4 µl RNase free water and 5 µl cDNA. The reactions were conducted as follows: 95°C for 20 sec, 40 cycles at 95°C for 5 sec and 60°C for 30 sec. Analysis was performed using the 2−ΔΔCq method (20).
Statistical analysis
Statistical analysis was performed using Statistica software (version 13.1; TIBCO® Software Inc., Palo Alto, CA, USA). The Spearman's rank test was used to examine the correlation between the expression of miRs and PD-L1 mRNA and protein expression. The Mann-Whitney U test was used to compare PD-L1 and miR expression in different patient groups (stratified by age, material for analysis, histopathological diagnosis, and PD-L1 expression on tumor and immune cells). Data are presented as the median ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.
The median percentage of PD-L1-postive tumor cells assessed using the 22C3 antibody was greater than that gained using the SP142 clone (35.00±39.95 vs. 2.5±37.96%). The median percentage of PD-L1-positive areas of the tumor infiltrated with immune cells, as tested with SP142, was 5±22.27%. Due to the degradation of mRNA, only 33 cases were assessed for PD-L1 mRNA expression. A positive correlation was observed between the expression of PD-L1 mRNA and the percentage of tumor cells with PD-L1 expression (R=0.35; P=0.046 tested using 22C3, and R=0.419; P=0.015 using SP142), and the percentage of the tumor area infiltrated by immune cells with PD-L1 expression (R=0.44; P=0.013).
A positive correlation was revealed between PD-L1 mRNA level and the expression of two miRs: miR-141 (R=0.533; P=0.0029; Fig. 1A) and miR-1184 (R=0.463, P=0.049, Fig. 1B). Additionally, a positive correlation was observed between the percentage of PD-L1-positive tumor cells (IHC using SP142) and the expression of three miRs: miR-141 (R=0.441; P=0.0024; Fig. 2A), miR-200b (R=0.372; P=0.011; Fig. 2B), and miR-429 (R=0.430; P=0.0028; Fig. 2C). Furthermore, there was a positive correlation between the percentage of the tumor area with immune cell infiltration and the expression of two miRs: miR-141 (R= 0.333; P=0.03; Fig. 2D) and miR-200b (R=0.312; P=0.046; Fig. 2E). The percentage of PD-L1-positive tumor cells assessed using the 22C3 antibody positively correlated with miR-141 expression (R=0.407; P=0,0055; Fig. 2F).
Figure 1.
Correlation between (A) PD-L1 mRNA and miR-141 expression levels, and (B) PD-L1 mRNA and miR-1184 expression. PD-L1, programmed death ligand 1; miR, microRNA.
Figure 2.
Correlation between the percentage of PD-L1-positive tumor cells in IHC using the SP142 antibody, and miR-141, miR-200b and miR-429 expression. (A) miR-141, n=43; (B) miR-200b, n=42; (C) miR-429, n=41. Correlation between the percentages of the tumor area infiltrated with PD-L1-positive immune cells in IHC using the SP142 antibody, and miR-141 and miR-200b expression. (D) miR-141, n=40; (E) miR-200b, n=40. (F) Correlation between the percentage of PD-L1-positive tumor cells in IHC using the 22C3 antibody, and miR-141 expression; n=39. PD-L1, programmed death ligand 1; miR/miRNA, microRNA; TC, tumor cells; IC, immune cells; IHC, immunohistochemistry.
Patients were stratified according to PD-L1 expression. Tumor cell (TC)1/2/3 or immune cell (IC)1/2/3 was defined as PD-L1 expression on ≥1% of tumor cells, or 1% of the area of tumor infiltrated by immune cells; TC2/3 or IC2/3 were defined as PD-L1 expression on 5% of the tumor cells or the tumor area; TC3 or IC3 were defined as PD-L1 expression on 10% of these cells or the tumor area; and TC4 was defined as PD-L1 expression on ≥50% of all tumor cells. TC0 was used to define PD-L1-negative samples.
Groups of patients that differed by their expression levels of PD-L1 (determined by IHC using SP142) were analyzed. The expression level of PD-L1 mRNA was significantly higher in the TC4 group compared with the TC0/1/2/3 group (P=0.0055; Fig. 3). It was also observed that the expression levels of miR-200b and miR-429 were significantly higher in the TC1/2/3 group with any PD-L1 protein, compared with the PD-L1-negative group, TC0 (P=0.043; P=0.0088 respectively; Fig. 4A and E). The expression levels of miR-200b and miR-141 were significantly higher in the TC2/3 group compared with the TC0/1 group (P=0.025; P=0.012 respectively; Fig. 4B and C). Furthermore, the expression level of miR-429 was also significantly higher in the TC3 group compared with the TC0/1/2 group (P=0.015; Fig. 4F). There were no differences in the expression of miRs in the groups that differed in the expression of PD-L1 on immune cells.
Figure 3.
Differences in the expression of PD-L1 on mRNA level depending on the PD-L1 protein expression status of tumor cells in patients with non-small cell lung cancer. Higher relative expression of PD-L1 mRNA in the group with ≥50% (n=8) compared with the group with <50% (n=25) of tumor positive cells (TC, SP142). PD-L1, programmed death ligand 1; miR/miRNA, microRNA; TC, tumor cells.
Figure 4.
Differences in the expression of miRs depending on PD-L1 expression status in the tumor cells of patients with non-small cell lung cancer. (A) Higher relative expression of miR-200b in the group with ≥1% (n=24) compared with the group with <1% (n=18) tumor positive cells (TC, SP142). (B) Higher relative expression of miR-200b in the group with ≥5% (n=14) compared with the group with <5% (n=28) tumor positive cells (TC, SP142). (C) Higher relative expression of miR-141 in the group with ≥5% (n=15) compared with the group with <5% (n=28) tumor positive cells (TC, SP142). (D) Higher relative expression of miR-141 in the group with ≥50% (n=18) compared with the group with <50% (n=21) tumor positive cells (TC, 22C3). (E) Higher relative expression of miR-429 in the group with ≥1% (n=21) compared with the group with <1% (n=20) tumor positive cells (SP142). (F) Higher relative expression of miR-429 in the group with ≥10% (n=14) compared with that with <10% (n=27) tumor positive cells (SP142). PD-L1, programmed death ligand 1; miR/miRNA, microRNA; TC, tumor cells.
Only one association was revealed between the expression of miR and PD-L1 on tumor cells analyzed using the 22C3 antibody; miR-141 expression was significantly higher in the TC4 group compared with in the TC0/1/2/3 group (P=0.009; Fig. 4D). Examples of stained NSCLC tissues visualized using the SP142 or 22C3 antibodies are displayed in Fig. 5. The expression of miRs was similar in groups of patients stratified by age, sex, pathomorphological diagnosis, status of metastasis or disease stage (P<0.05).
Figure 5.
Example of immunohistochemistry for PD-L1 expression in non-small cell lung cancer formalin-fixed, paraffin-embedded tissues. 100% of tumor cells exhibit PD-L1 expression of (magnification, ×20), using (A) 22C3 and (B) SP142 antibodies. (D) 20% of tumor cells exhibit expression of PD-L1 (22C3), and (E) 15% of tumor cells exhibit expression of PD-L1 (SP142) (magnification ×90). (G) 2% of tumor cells with expression of PD-L1 (22C3). (H) 4% of tumor cells with expression of PD-L1 (SP142) (magnification ×110). (C, F and I) Hematoxylin and eosin staining (magnification, ×20, ×90 and ×110, respectively).
Immune checkpoint inhibitors are able to prolong patient survival, and their use has been associated with fewer side effects comparison to the chemotherapy (21). These side effects are also differently classified (Immune-Related Adverse Events) (22). The greatest benefit of PD-1/PD-L1 inhibitors was observed among patients whose tumor cells expressed PD-L1 (9–11). Pembrolizumab and chemotherapy as first line treatments for patients with advanced NSCLC with PD-L1 expression on >50% of tumor cells (scored using the 22C3 antibody) were compared. This revealed a significantly higher response rate and longer progression-free and overall survival rates in patients treated with pembrolizumab, compared with those who received platinum-based chemotherapy (6). However, the association between the benefits of second line immunotherapy and the expression of PD-L1 on tumor cells is not so obvious. Brahmer et al (10) demonstrated that overall survival, response rate and progression-free survival were significantly greater when using nivolumab, compared with docetaxel, in patients with squamous-cell cancer, regardless of tumor cell PD-L1 expression. Similarly, Rittmeyer et al (23) revealed that atezolizumab treatment resulted in a clinically relevant improvement in overall survival, compared with docetaxel in previously treated patients with NSCLC, regardless of PD-L1 expression. Patients without PD-L1 expression on tumor cells or immune cells infiltrating the tumor achieved a median overall survival of 12.6 months, following treatment with atezolizumab, and only 8.9 months following docetaxel treatment. The authors indicated that the lack of an association between PD-L1 expression and immunotherapy efficacy was probably due to the complex interactions between tumors and the immune system. There are a number of studies that suggest genetic factors may also be associated with the response to immunotherapeutic treatment, in spite of PD-L1-negative tumor cell status (10,24,25).
Shukuya et al (26) indicated that miR may influence the efficiency of immunotherapy. Next-generation sequencing was used to determine the expression of specific miRs in the plasma, and the miR content of extracellular vesicles (EVs). A total of 26 circulating miRs and four EV-associated miRs exhibited significant differences in concentration between responders and non-responders to immunotherapy with anti-PD-1 or anti-PD-L1 antibodies. Furthermore, these miRs were potential predictive biomarkers for the response to treatment.
In the present study, 8 miRs with the ability to regulate the expression of PD-L1 transcripts in tumor cells were analyzed. A positive correlation between PD-L1 mRNA and protein expression levels and miR expression (particularly miR-141 and miR-1184) was identified. This indicates that these miRs may regulate the expression of PD-L1 at a post-transcriptional level, and thus participate in tumor-cell escape from immune surveillance. However, these miRs are not involved in mRNA degradation, which can be stored in the cell and activated if necessary. Positive correlations may indicate that these two miRs (which exhibit increased expression levels) may be able to silence PD-L1 expression in cancerous cells, but that this is restricted by the carcinogenetic characteristics of these cells, allowing them to bypass epigenetic regulation. This hypothesis evidently requires experimental confirmation, potentially with a larger patient cohort and in vitro experimentation using cell lines. Furthermore, no relevant publications on the subject were identified, thus extended studies including biochemical and molecular biological experiments will increase knowledge of the regulatory functions of miR-141 and miR-1184 in association with PD-L1. Due to the small size of the patient group, the present study may be considered as a pilot study to indicate subsequent genetic and epigenetic experimentation surrounding PD-L1 immunotherapy.
miRs are able to act as oncogenes or tumor suppressors, depending on the cancer type in question (27). There is limited evidence of miR-1184 expression in different types of cancer. Knyazev et al (28) demonstrated the potential of miR-1184 to differentiate between prostate cancer and benign prostatic hyperplasia, indicating higher levels of miR-1184 expression in the peripheral blood of patients with prostate cancer. Feng et al (29) revealed that miR-1184 was able to regulate the expression of certain oncogenes and transcription factors, including SMAD family member 5, signal transducer and activator of transcription 3 and glioma-associated oncogene family zinc finger 3. Farina et al (30) identified lower expression levels of miR-1184 in patients with breast cancer, compared with healthy controls.
Mei et al (31) demonstrated that miR-141 was upregulated in NSCLC, which was associated with aggressive disease course and accelerated tumor growth. It was indicated to regulate PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) and PHLPP2, which are involved in the AKT serine/threonine kinase 1 pathway and oncogenesis. In turn, Zuo et al (32) identified miR-141 as a tumor suppressor in gastric cancer; it was concluded that decreased expression of this miR was correlated with a more aggressive phenotype, compared with the healthy controls. Further analysis demonstrated that miR-141 targeted transcriptional co-activator with PDZ-binding motif, a transcriptional coactivator that promotes cell proliferation and epithelial-mesenchymal transition. Furthermore, ectopic overexpression of miR-141 suppressed tumor growth and pulmonary metastasis in nude mice.
There is no clear evidence of the regulation of the immune response and PD-L1 expression by miRs. However, Huang et al (33) indicated that miR-141 targets C-X-C motif chemokine ligand 12, and thus participates in the control of colonic leukocyte trafficking during Crohn's disease-associated intestinal inflammation.
Another molecule identified as a potential regulator of PD-L1 is miR-200b. A correlation between the expression levels of miR-200b and PD-L1 on tumor and immune cells was indicated. miR-200b belongs to the family which also includes miR-141, miR-200a, miR-429 and miR-200c, though its role in NSCLC development is not well understood. Xiao et al (34) postulated that the overexpression of this miR may inhibit NSCLC cell migration and invasion. It was indicated that miR-200b regulates fascin actin bundling protein 1 and therefore, is involved in the regulation of cell migration, motility, adhesion and other cellular interactions. Gibbons et al (35) stated that epithelial-mesenchymal transition (ETM) is entirely dependent on the expression of the miR-200 family, which decreased during EMT. A high miR-200 expression level inhibits tumor cell migration and metastasis.
In the present study, expression of another member of the miR-200 family, miR-429, was correlated with PD-L1 protein expression. Xiao et al (36) indicated that overexpression of miR-429 resulted in increased NSCLC cell proliferation, while knockdown of miR-429 attenuated the proliferation of H1229 cells. This was partially due to the regulation of tumor suppressor deleted in liver cancer 1 (DLC-1) expression by miR-429, suggesting that miR-429 may have an oncogenic role in the regulation of cell proliferation via the direct inhibition of DLC-1 protein expression in tumor cells.
In the present study, a significant association between PD-L1 mRNA level and PD-L1 protein expression on tumor cells was also demonstrated. Erber et al scored PD-L1 mRNA and PD-L1 protein expression on tumor cells, where a notable association between the expression level of PD-L1 mRNA and protein was identified in NSCLC (37). This suggested that in patients with PD-L1 protein expression on >50% of tumor cells, PD-L1 mRNA may be reliably detected by RT-qPCR, and that quantitative PD-L1 mRNA scoring may be considered as an alternative to IHC (37). On the other hand, Sepesi et al (38) suggested that the value of PD-L1 mRNA expression analysis in the diagnosis and prognosis of lung cancer is limited. It was demonstrated that PD-L1 mRNA expression in lung cancer was higher compared with in normal tissues and other tumor types. Furthermore, mRNA expression level was significantly higher in lung squamous cell carcinoma compared with adenocarcinoma. Other than PD-L1 mRNA, the authors suggested that further studies were required to identify novel prognostic biomarkers that are associated with improved patient survival and may be useful in identifying patients suitable for immunotherapy (38).
There is a requirement for further studies to assess the association between the expression of miR and PD-L1, as such interactions may partially explain the varied responses to immunotherapy between patients. In the present study, the positive correlation between the expression level of PD-L1 mRNA and the miR-200 family indicated no transcript degradation. The mechanism for regulation of PD-L1 expression on tumor cells remains unclear. However, miRs may serve an important role in this process, and the study of their expression may result in the discovery of novel predictors for immunotherapy.
Not applicable.
No funding was received.
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Authors' contributions
AG, MN, MSz and PK contributed to conception and design of the study. JP, MSa, JS, PK and JM acquired the data, and MN, TK and BJ conducted the laboratory experiments. MN, AG, BJ, JP and PB analyzed and interpreted the data, and AG, PK and TK were involved in drafting the manuscript or revising it critically for important intellectual content. AG, MN, MSz, PK, BJ, JP, MSa JS, PB and JM gave final approval of the published manuscript.
Ethics approval and consent to participate
The present study was approved by the Ethics Committee of the Medical University of Lublin (Lublin, Poland) (no. KE-0254/169/2014). All participants provided written informed consent to participate.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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From Difficult Dog to Comfortable Companion: Innovative Techniques to Improve your Dog's Behavior and Wellbeing
Saturday, December 7, 2019: 9:30 am – 4:00 pm
Sunday, December 8, 2019: 9:30 am – 3:30 pm
Location: Mountain Humane, Hailey, ID
In this two-day workshop you will learn techniques to help your dog release tension and build confidence, thereby eliminating undesirable behaviors related to stress, fear and anxiety.
These undesirable behaviors include leash pulling, excessive barking, fear of loud noises, reactivity to people and other pets, etc. The Tellington TTouch® Training Method uses gentle body work to release fear and tension, and groundwork to improve balance and confidence. The result is a calm, confident, happy dog.
Cost: Early Bird: $249 with your dog | $219 without a dog
After November 6, 2019: $275 with your dog | $245 without a dog
*Bring two friends and receive a 15% discount! Contact me to arrange payment at the discounted rate.
This workshop has been approved for CEUs by PPAB, IAABC, NADOI, and PSI.
Questions? See our FAQs for more information.