text:
Sections of this page Sections of this page Skip to Top navigation Skip to NYUAD Home Skip to News Navigation Skip to News Skip to Contacts Skip to Footer
World and Societies
NYU Abu Dhabi and National Health Insurance Company – Daman Collaborate on a Healthier Future for the UAE
National Health Insurance Company – Daman, the leading health insurance provider in the UAE, has joined hands with NYU Abu Dhabi (NYUAD)'s Public Health Research Center (PHRC) to collaborate on broader promotion and support of the UAE Healthy Future Study.
The UAE Healthy Future Study is a first of its kind study aimed at building a healthier future for the UAE. Investigators from NYUAD and the NYU Langone Medical Center have collaborated with local partners in Abu Dhabi to conduct this long-term study in order to understand why obesity, diabetes, and heart disease are common in the UAE. Toward this, the study is looking to recruit 20,000 UAE nationals between the ages of 18-40. To date, over 4,000 participants have signed up to take part in the study.
With the signing of this Memorandum of Understanding (MoU), a formal framework of cooperation has been agreed upon by both parties. Daman will support the study's recruitment drive by displaying information about the Study at its Thiqa branches and service points. Additionally, personnel from the study will be present at Daman's premises and ActiveLife events in order to promote the study and recruit willing volunteers.
Thiqa, managed by Daman on behalf of the Government of Abu Dhabi, delivers healthcare services to UAE nationals in the emirate of Abu Dhabi. This partnership ensures that the message of the study reaches its target audience.
Daman Chief Executive Officer Dr. Michael Bitzer, said: "Our collaboration with NYU Abu Dhabi marks a step in the right direction as we look to identify effective ways of dealing with common UAE health epidemics, such as obesity. Through our extensive Thiqa network, service points and branches, we are working to recruit and provide access to UAE nationals to partake in the study. Those who agree to participate will provide valuable data that supports this significant research – crucially, they will also be part of a positive movement for the UAE, as collectively, we look to get to the root-cause and combat the UAE's health concerns. Our work with NYU Abu Dhabi enables us to continue working towards creating healthier communities and achieve a better health outlook for the country."
NYU Abu Dhabi Provost Fabio Piano, said: "Research is a central component of NYU Abu Dhabi's mission, and this collaboration between Daman and our Public Health Research Center demonstrates NYUAD's continued commitment to develop strong, meaningful connections within the local community and to help improve the overall health of the UAE."
Research, Partnerships
About NYU Abu Dhabi
NYU Abu Dhabi is the first comprehensive liberal arts and research campus in the Middle East to be operated abroad by a major American research university. NYU Abu Dhabi has integrated a highly selective undergraduate curriculum across the disciplines with a world center for advanced research and scholarship. The university enables its students in the sciences, engineering, social sciences, humanities, and arts to succeed in an increasingly interdependent world and advance cooperation and progress on humanity's shared challenges. NYU Abu Dhabi's high-achieving students have come from over 115 countries and speak over 115 languages. Together, NYU's campuses in New York, Abu Dhabi, and Shanghai form the backbone of a unique global university, giving faculty and students opportunities to experience varied learning environments and immersion in other cultures at one or more of the numerous study-abroad sites NYU maintains on six continents.
More Science and Technology News
Turning Waste into Sustainable Fish Feed Engineering Jobs student life Global Education
NYU Abu Dhabi's Water Research Center Partners with Emirates Water and Electricity Company to Advance Water Management Research and Sustainability
Talking Stars
NYUAD Researchers Discover Occasional Low-Oxygen on Abu Dhabi Coral Reefs, a New Potential Contributor to Coral Bleaching
NYU Abu Dhabi Represents UAE in Joining the Atlas Experiment at CERN
Email: nyuad.public.affairs@nyu.edu
Reem Fekri
Associate Director, Public Affairs and Communications
Email: reem.fekri@nyu.edu
Maisoon Mubarak
Public Affairs and Communications Manager
Email: maisoon.mubarak@nyu.edu
Farah Shamma
Email: farah.shamma@nyu.edu


text:
Q: DocumentDB vs Cosmos DB - Are both same? I was using DocumentDB for the product and now I hear that there's no DocumentDB! There's this new Cosmos DB which Microsoft is building which is superset of DocumentDB. I'm really confused now.
Is it only the name they changed or there are many more changes?
Can someone shed a light on this?

redpajama_set_name: RedPajamaStackExchange


This module outlines the underlying causality of major accident and incidents. We cover the development and use of the DIM-ICE model and provide a template to the delegates for their review. We outline the principles and applications of crowd science and crowd risk analysis. We cover different perspectives of the model for crowd safety and risk analysis and we outline the ways in which design, information and management influences crowd behaviour in the built and complex environment.


text:
Epic CIC
Play 6-14
Children's Forum
Sport 7-19
School sport services
School Games
SEND Activities
After School Programme
Sparks Holiday programme
KaBoom! Fun Science
Epic Learning school
Holiday Programme
Flashpoint Centre Play
Wiltshire Close Adventure Playground
LWS Adventure Playground
Golborne Centre
Chelsea Centre
Lancaster Centre
Canalside Centre
Cremorne Centre
Volunteering and mentoring
Epic October Half Term
Thank you for an EPIC October half term! Watch this space for our February half term activities- COMING SOON!
Hello October!
This autumn we are BACK with even more EPIC activities for children and young people.
Looking to explore the great outdoors? Make unforgettable memories while meeting new people? Then pick and choose from our fantastic activities list below.
Thrilling Watersports 9-19 year olds
Join us on adrenaline filled kayaking and canoeing sessions where you will be able to explore the great outdoors in and outside of London, participate in team challenges and even learn to build a raft!
When? Click here for the timetable
Where? Canalside Centre
Energising Sports
If you're up for the challenge, get active with our active sports sessions in basketball, weightlifting, squash and more!
When? Football: Tuesday 5 - 7pm @Westway Sports & Fitness Centre 14-16 year olds
Weightlifting 4.30 - 6pm @Canalside Centre 14-19 year olds
Squash 4.40 - 6pm @Portobello Fitness Club 14-19 year olds
Adventurous Play 6-14 year olds
The fun for children continues! Our Community & Adventure Play invites all children to climb, swing, jump, play and learn in an informal setting.
When? Little Wormwood Scrubs Adventure Playground: 10 - 4pm
Flashpoint Centre Play: 10 - 5pm
Wiltshire Close Adventure Playground: 12 - 4pm
Where? Little Wormwood Scrubs Adventure Playground, Wiltshire Close Adventure Playground, Flashpoint Centre Play
Evening Chill Time 13-25 year olds
Try new things, meet new people. Our Youth Centres are offering a safe space to relax, socialise and take part in exciting evening activities.
When? Monday - Thursday 6.45 - 10pm
Where? Golborne Youth Centre
Interested in our regular sessions?
Click HERE for our Term- Time Activities Schedule.
Let the Autumn fun begin!
Author: Jone Kociunaite
Epic February
It's 2020. Time to have fun! Our team has put together an exciting schedule filled with fun and engaging activities for children and young people. Join us this February from Monday 17th to Friday 21st for unforgettable fun in one of our centres. We can't wait for February half term!
Lancaster Youth Centre
North Hub
We are partnering with The Science Museum, Natural History Museum, L'Oreal, Imperial College London, The Design Museum, Scientists, Artists, and Technologists to design STEM (science, technology, engineering, and mathematics) activities that integrate community & adventure play and are accessible for all children. The idea of this project is to develop the natural curiosity children have in a play environment. In the past 12 months we have empowered 163 children to see science as an everyday, re
Engaging and fun activities for children and young people aged 13-19 (up to 25 for those with Lifelong Learning Disability and/or Disability) in Kensington & Chelsea.
Little Wormwood Scrubs Adventure Playground
Slides, swings, games and more. Based in North Kensington, Little Wormwood Scrubs is a supervised play area for children to explore, play games and have an adventure!
Our mission is to inspire children and young people to achieve their potential and to make a positive difference to the communities in which we work. A pathfinder in the Cabinet Office Employee Mutual's programme, the creation of Epic was a response to the changing landscape of public sector funding and is leading the way in new approaches to financing and delivering youth services.
Epic Learning, based in West London, is a small independent school providing an alternative provision for KS4 and KS5 children sitting GCSE's and/or Functional Skills.
Chelsea Youth Centre
Young people learn while having fun and engaging in positive activities, which help them make positive choices to meet their needs, in a safe place.
Competitive sports leagues and festivals for local Primary and Secondary schools as part of the National School Games competition framework.
Epic operates under the terms of its Articles of Association and is governed by a Board of Directors. We have assembled a high-quality Board of Directors to govern the activities of the enterprise. The Board consist of an independent chair person, our Managing Director, four non-executive directors and five elected staff representatives.
Welcome to Flashpoint Centre Play situated on the World's End Estate in Chelsea open to children aged 6 to 14 years. Play allows children the opportunity to grow in an informal setting by encouraging a whole host of developmental experiences through the programme of activities we offer.
The Kensington and Chelsea Youth Grants is a pot of money for young people aged between 13 and 19 or up to 25 with a lifelong learning difficulty and/or disability (LLDD).
Epic has shown we are a thriving social business creating value for young people and communities. We know from listening to young people and community groups, that Epic's programmes helps young people to achieve outcomes that are meaningful to them.
For more information on how your data is stored and used, please see our privacy policy.
Company registered no. 8488968 © 2017 | Epic ELM C.I.C, First Floor, 36C Oxford Gardens, London, W10 5UQ

redpajama_set_name: RedPajamaCommonCrawl


text:
Trasierra-Tierras de Granadilla County Tourist Office
Come and discover all that the Trasierra-Tierras de Granadilla county has to offer.
Oficina de Turismo de la Mancomunidad de Trasierra-Tierras de Granadilla
Trasierra-Tierras de Granadilla County Tourist Information Centre
A-66 motorway. Exit 455
10667 Oliva de Plasencia , Caceres (Extremadura)
Contact person: Roman City of Caparra Interpretation Centre
Tel.:+34 927 19 94 85
Email: turismo@trasierratierrasdegranadilla.es
Website address: www.trasierratierrasdegranadilla.es
Tuesdays-sundays, 10am-2pm and 6pm-8pm
Closed on Mondays, unless it is a public holiday
Granadilla, History and Art
It was evacuated after being declared a flood risk area, currently the village is being rehabilitated to return it to its former glory.
Gabriel and Galán House-Museum
Roman City of Cáparra Interpretation Centre
Discover this old city on the Silver Route.
The Roman town of Cáparra
Caparrá, which has uncertain origins, became a Roman municipium under Emperor Vespasian. The town's growth was propelled by its privileged position along the Silver Way
Roman arch of Cáparra
Cáparra was an important town in Roman times. Nowadays some vestiges of that glory remains, most outstandingly its peculiar four-sided arch.
Granadilla Castle
Probably built by order of the Great Duke of Alba, Granadilla Castle is, without a doubt, a singular piece of architecture.
Cáparra Roman bridge
Cáparra Roman bridge is in perfect condition because it has been remodelled many times over the centuries.
Guijo de Granadilla Roman Bridge
In the 2nd century AD this bridge connected the two banks of the river Alagón.
Zarza de Granadilla Bullring
Renowned bullfighters have performed in this small, modern bullring.


text:
FRC calls for better governance and reporting, with a heightened emphasis on corporate sustainability and trust
The Financial Reporting Council (FRC) has published its annual review of the UK Corporate Governance Code. In doing so, it urged firms to enhance their governance practices and reporting in order to demonstrate their positive impact on the economy and wider society.
What have firms been told to do?
The independent regulator said that while the bar had been raised 'considerably' by alterations to the 2018 UK Corporate Governance Code leading to some high-quality reporting, there was a need for more emphasis to be placed on longer-term sustainability.
Such a sustainability focus, the body said, needed to encompass such matters as stakeholder engagement, diversity and the importance of corporate culture, and that it expected these changes to 'take time to bed in'.
The UK Corporate Governance Code was updated to aid in building trust in business by forging strong relationships with key stakeholders. It urged firms to align purpose, strategy and culture in addition to promoting integrity and valuing diversity as part of a focus on long-term sustainability.
The FRC's annual review looked into reporting against the 2016 Corporate Governance Code and assessed FTSE 100 'early adopters' of the modified 2018 Code. This revised Code came into force in 2019, with all premium listed companies set to report against it in 2020.
Some eye-catching highlights in the FRC's analysis
The regulator said it had found some good examples of reporting by firms that were increasingly using incentives relating to non-financial matters and embracing a longer-term strategic perspective.
Many companies, however, were evidently grappling with defining purpose, and what an effective culture means, the body criticising some firms for 'substituting slogans or marketing lines for a clear purpose'.
The FRC added that the companies it examined were still insufficiently considering the importance of culture and strategy and the views of stakeholders. It said that in the wake of the regulator's 2016 report on culture, organisations should be commenting on culture, in addition to outlining how they are monitoring and assessing it.
A lack of reporting on diversity also disappointed the regulator, which nonetheless said that "those companies that did report well had clear plans to meet targets – beyond just gender – and understood the long-term value of diversity".
It was noted, too, that many firms described the use of engagement surveys as an effective means of gaining insight into employee engagement and culture. However, while the FRC said such surveys can be useful, it warned against the use of them in isolation, explaining that "companies must be able to demonstrate that the engagement methods used are effective in identifying issues that can be elevated to the board, and how this affects company decisions."
Companies urged not to pay mere 'lip service' to the Code
Chief executive of the FRC, Sir Jon Thompson, said of the review's findings: "While there are examples of high quality governance reporting from 'early adopters', looking ahead, we expect to see much greater insight into governance practices and outcomes reporting on a range of key issues from diversity to climate change."
"Concentrating on achieving box-ticking compliance, at the expense of effective governance and reporting, is paying lip service to the spirit of the Code and does a disservice to the interests of shareholders and wider stakeholders, including the public."
Are all aspects of your own organisation's governance and reporting well aligned with the principles of the latest UK Corporate Governance Code, or would you possibly benefit considerably from the know-how in this area of our own experienced professionals here at London Registrars?
Either way, we would be delighted to talk to you about our wealth of company compliance services, to help to ensure that the most specific and demanding aspects of corporate governance are not a cause of headache to you and other stakeholders in your organisation in 2020. Simply email [email protected] or call 020 7608011 for further information.


Many travellers are searching for Shopping Experience Bangkok,Bangkok can well be described as a city of humble beginnings. In fact around 200 years ago, it was simply a small trading center and port community. Despite the fact that the city had grown rapidly and become renowned for being a modern and vibrant place, it has impressively retained its splendid and enchanting glory. That's why a visit to Bangkok should be on everyone's bucket list. You can find Shopping Experience Bangkok travel information.
Otherwise known as Wat Chetuphon, this is a temple that was designed for King Rama in Bangkok a huge selection of yrs ago. It really is thought to be a spot where people can receive feelings, and was considered the first pharmacies in most of Bangkok. Additionally it is the positioning of the first university which had been started by way of a descendent of King Rama, a place where you may now get not simply traditional medicine but a foot massage at highly affordable prices. It really is known mostly for that Temple of your Reclining Buddha and that is a Buddha which is resting perched on his arm. It really is enormous, roughly 15 m high by 45 m long, and might basically be viewed in sections due to the size. There are lots of beautiful gemstones that have been in lead within the soles of the feet, an overall total number of 108 stones total. This can be a number relevant to procession, the wobble from the earth, something which is made into lots of the temples throughout Thailand.
It is really an absolutely beautiful structure containing a substantial amount of history, and is the home of what is called Wat Phra Kaeo. It is here that this emerald or jade Buddha can be obtained, with this palace built-in 1782, the residence of your royalty that were in command of the land during the time. This palace includes two hours of not only the palace itself but the Wat Phra Kaew Tour. It is recommended that you begin early every morning to take full advantage of everything that you are able to see, or simply just conduct a half day sightseeing tour to discover either one.
You will see a beautiful representation of your ancient battles that used to occur between Burma and Siam. This is really a variety of Royal Palace and Chapel, and if you are able to climb to the peak right before sunset, you will see direct sunlight setting in to the Chao Praya River, a beautiful sight to behold.
Before precisely what is called Wat Suthat is the thing that the locals call the giant swing. It is a 27 m high structure, use for religious ceremonies, and its particular purpose is to endure a ritual that requires teams of three people coming to the top and walking across on the narrow board. There have been a tremendous quantity of fatal accidents during the 1930s which make them not utilize this being a ritual again. However, it is a beautiful sight to see, especially in front of Wat Suthat.
This is literally something out of your movies, a genuine floating market that may be about an hour or so away from Bangkok. It can be considered by some to become the Venice from the East, comparable to the way the people that element of Italy do it a lot of things on the water. Also, it is a tourist trap as a consequence of how people are seeking to see this market that may be completely floating on boats manned by people selling their goods. However, if you do go, you will end up treated to among the most delicious food that is certainly absolutely fresh, served inside an authentic way.
In case you have time, you may head over to Lumpini Park that is a literal oasis in the midst of the architecture and buildings of Bangkok. There are numerous lawns you could spend time at, Chinese pagodas, and you will discover a small lake that one could take a boat out on. Is good for relaxing for the reason that park is indeed tranquil, despite every one of the skyscrapers which can be surrounding it in all directions. The park has been a place where antigovernment protests have occurred, so just be sure that when you choose to look there, it's just people hanging out to unwind.


ProSportsDaily.com > Basketball > NBA > Dwyane Wade: Lakers are the team to beat.
View Full Version : Dwyane Wade: Lakers are the team to beat.
"The Lakers are the champions and we know the Lakers are very good. That's the team that everyone's shooting for and they should be. Not the Miami Heat. The Los Angeles Lakers."
Thoughts on Dwyane Wade's comments!!??
its true but hes only saying that to take the pressure off the heat. inside the entire heat organization believes they are the team to the beat.
He's stating the obvious. The champions are always the team to beat. Why is this a thread again?


text:
(function() {

    var website = angular.module("modules.website", []);

    website.config(['cfpLoadingBarProvider', function(cfpLoadingBarProvider) {
        cfpLoadingBarProvider.includeSpinner = false;
        cfpLoadingBarProvider.includeBar     = true;
    }]);

    website.config(['ngToastProvider', function(ngToastProvider) {
        ngToastProvider.configure({
            animation: 'slide',
            verticalPosition: 'bottom'
        });
    }]);

})();

redpajama_set_name: RedPajamaGithub


How many people have poured into your life? How many people have come alongside you so that you can be and do all that God created you to be and do? Are there specific individuals who stand out among those who have walked with you? What would your life be like today if these special people were not able to come alongside you?
That sentence is the briefest way I know how to communicate our calling. They are words that I have heard myself saying again and again to others and to the mirror. I have said them at home and abroad, to leaders and emerging leaders, to believers and not yet believers. They are very true words and I believe that they are heaven sent.
This is another sentence that has been profoundly influential in our life and our ministry. God has indeed given us much and continues to do so. We are called to give it all away for His glory and the furthering of His Kingdom.
Who are you called to come alongside?
We are the Fletcher Family and we are going to Cuenca, Ecuador to come alongside emerging and existing pastors, missionaries, and leaders. When someone is called into ministry, God often sends people to come alongside with encouragement and help in discovering their inheritance and identity in Christ. The Fletchers come alongside others. We are helpers, encouragers, mentors, teachers, equippers, and most of all friends! We are called to give what we have and to learn and grow as we do so.
Will you come alongside us so that we can come alongside others?
If the answer is yes, we want to meet you!
You can follow the Fletchers on their website at http://fletchersteps.org and pray for them.
You can help them get to Ecuador by becoming a monthly Faith Partner: Go to https://onemissionsociety.org/give/TheFletchers to partner with them in mission.
3.) Contact Them with Words of Encouragement. We would want to get to know you!


This submission provides functions (and examples scripts) for estimation, simulation and forecasting of a general Markov Regime Switching Regression.
I developed this interface some time ago, so I finally decided to refine and make it available here.
C++ source is now available.


Inaugurated in 2016, the Borgeson Artist in Residence Program is a twelve-week summer artist residency, hosted by the Department of Art & Art History at Hope College, that runs from mid-May through mid-August of each year.
The residency, created through the generosity and enthusiasm of Hope alumni Clarke '72 and Nancy Rayner '72 Borgeson, supports the creation of new work through provision of a stipend as well as studio and living space on Hope's campus. In addition to the time and space the residency provides, the Borgesons envisioned the artist in residence as invigorating the experience of the department's top students through a series of encounters, which might include workshops, studio conversations, critiques and assistantships. The artist in residence concludes their residency with a month-long solo exhibition and public lecture at the college's De Pree Art Gallery.
The artist in residence is provided with 24 hours a day, 7 days a week access to a private studio, housed in the De Pree Art Center on Hope's campus. The 1,308 square foot, air-conditioned south-facing studio offers outstanding natural and track lighting, hardwood floors, tall ceilings, and a Mac-adaptable projector and screen. Proposals that utilize other department studios and facilities, including an intaglio print shop, darkroom, Mac lab, fully-equipped ceramics and sculpture studio are welcome. Access to these additional department facilities is granted based on prior experience and the artist in residence's proposal.
The residency further supports the selected artist through a financial stipend.
The residency further supports the selected artist through provided on-campus housing in a residential cottage minutes away (walking distance) from the studio.
The Strand Cottage is a two-story, three-bedroom, air-conditioned cottage located at Lincoln and 15th Street. There is a stackable washer and dryer located in the main floor bathroom. The main-floor bedroom has two twin beds, two desks and two dressers. The second-floor bedrooms each have one twin bed, a dresser and a desk.
All furniture (i.e., living, dining and bedroom) is provided. A linen packet is provided upon arrival, which includes pillows, pillow cases, sheets, towels and blankets. Additional comforters and lamps are provided.
Basic kitchen furnishings, including a microwave, toaster and all necessary utensils, pots and pans, dining ware, kitchen linens, etc… are provided. A detailed inventory is available.
De Pree Art Gallery, to coincide with the start of the academic year.
As part of the residency's goal to create opportunities for our students and the larger campus community, the department hosts an artist's lecture, closing reception and related programming in September.
Caleb Paul Kortokrax (b. 1987) is an American painter living and working in Baltimore, MD. His current work is about forming new connections between different image traditions and human perception/ context investigation. His studio practice is rooted in the interdisciplinary spirit, and his recent body of work builds bridges between disparate painting traditions and time periods for the present. In the process of making a painting, he resamples quality material traditions of the past into the current omni-dimensional state of imagery. Through combining past and present visual signals in his studio process, he reanimates the painted image—similar to a DJ giving a renewed vigor to an old sound sample by placing it into a fresh context. He uses the image and the physical surface of the paintings to navigate a middle way full of more possibilities and cross-pollination, which doesn't particularly favor any one style or dogma.
constructed identity and the performance of the self. She is best known for her interactive performance projects which often involve printmaking techniques and engaging viewers in the creative process, connecting their actions to an aesthetic object, and as a result, revealing something about themselves.
Nancy grew up in Southeastern Massachusetts and pursued a career teaching and producing exhibitions throughout the United States, Europe and Turkey. She received her BFA from Rhode Island School of Design and her MFA from The City College of New York. Over the years, Nancy also worked extensively as a bookbinder and in rare book and paper conservation.
In her current body of work, Nancy is interested in addressing the aesthetics of cultural identity and how it relates to the natural world, specifically, how and why we construct and portray ourselves the way we do, what that says about contemporary culture and what it looks like from a global perspective.
Summer 2015 – Chris interned at Small Editions, in Brooklyn; A small artist book publishing house and book bindery. He published two artist books since graduating from Hope: Spiritual Lake, 2012, edition of 50; and Beholder, 2014, edition of 150. Beholder was exhibited at New York Art Book Fair, 2014, MoMA PS1, New York and is for purchase through the International Center of Photography, New York.
2016 – Borgeson Artist in Residence, Hope College.
All of this publishing, lots of exhibitions and collaborative projects were done through Gaspard, an artist group and gallery that he ran from 2012–2014. Gaspard operated out of storefront on Division Ave. in downtown Grand Rapids, Michigan.
Chris did a fair amount of commercial work, working with Herman Miller, doing photography assisting, digital asset archiving and styling for photoshoots and showrooms. Chris participated in variety of group shows, primarily in Grand Rapids, Chicago, and New York.


text:
Cumberland Democratic Chairman fined $200 for ethics violation
On December 13, 2017, the Local Finance Board, the primary enforcer of the Local Government Ethics Law (LGEL), fined a former Deputy Director of the Cumberland County Board of Chosen Freeholders $200 for voting to appoint the former mayor of Willingboro Township (Burlington County) to head the County's Office of Purchasing while the former Deputy Director served as Willingboro's Labor Counsel.
The Board, which has the statutory authority to issue fines between $100 and $500 for LGEL violations, found that Douglas Long, who formerly served as Freeholder Deputy Director and presently chairs the Cumberland County Democratic Committee, violated the LGEL by voting in favor of appointing former Willingboro Mayor Jacqueline Jennings to head the purchasing office while he simultaneously served as Willingboro's Labor Counsel. According to its Notice of Violation, the Board determined that Long's vote in favor of Jennings' appointment constituted an official act where Long had an interest or involvement "that might reasonably be expected to impair his objectivity or independence of judgment."
The Board also dismissed a related ethics complaint brought against Jennings, finding that her participation in the appointment did not constitute an LGEL violation. In deciding to dismiss, the Board noted that the Cumberland Freeholders rescinded Jennings' appointment two weeks after it was made and that Jennings never collected any salary or benefits for the two week period. Jennings presently serves on Willingboro Township Council.
According to the Notice of Violation, the $200 fine will not be finally imposed until thirty days have elapsed. During that thirty-day period, Long can request an administrative hearing to contest the violation and the fine.
The matter was initiated by a September 30, 2014 complaint filed by the author and the New Jersey Libertarian Party.
In February 2015, Long announced that he would not seek a second term on the Freeholder Board. On November 4, 2016 the New Jersey Supreme Court reprimanded Long, who is an attorney, for an unrelated ethics matter. On November 9, 2017, the Election Law Enforcement Commission charged Long with violating the New Jersey Campaign Contributions and Expenditures Reporting Act.


text:
Republicans Scramble to Contain Trump's Damage, but Path Is Unclear
Near unanimous bipartisan majorities in both the House and the Senate passed tough new sanctions authorities in part to punish Russia for its election interference last...
Donald Trump, Japan, Brexit: Your Wednesday Briefing
(Want to get this briefing by email? Here's the sign-up.) Good morning. President Trump makes a U-turn, the E.U. signs a major trade deal and Brexit...
Hong Kong May Ban Political Party That Seeks Independence From China
HONG KONG — The Hong Kong authorities said Tuesday that they were considering banning a political party that advocates the city's independence from China, an aggressive...
Would It or Wouldn't It Be Russia: Trump Goes Double Negative
WASHINGTON — It was really just a mix-up, he insisted. Like confusing "effect" and "effect," but for unprecedented global cyberaggression. "I thought I made myself very...
Guilty, Again: Dean Skelos, Former Senate Leader, Is Convicted of Corruption in Retrial
By 2015, the companies had sent Adam Skelos more than $300,000, ostensibly for consulting or employment contracts, even though he did not bring in any clients,...
In Russia, Summit Is Seen as a Triumph for Putin
Breaking Political Isolation Russian analysts, including Kremlin critics, agreed that the meeting had allowed Mr. Putin to show that Russia is indispensable in world affairs —...
After Putin Meeting, Trump Voters Mostly Dig In. But Cracks Are Showing.
So it went in interviews with Republican voters across the country on Monday and Tuesday, most of them devoted to the president's policies generally. They quickly...
Vladimir Putin, India, Myanmar: Your Wednesday Briefing
(Want to get this briefing by email? Here's the sign-up.) Good morning. A Trump U-turn, troubling climate trends in South Asia and the danger of Myanmar's...
I.R.S. Will No Longer Force Kochs and Other Groups to Disclose Donors
The Trump administration will end a longstanding requirement that certain nonprofit organizations disclose the names of large donors to the Internal Revenue Service, a move that...
Trump Now Says He Accepts U.S. Intelligence Reports on Russian Election Meddling
WASHINGTON — President Trump said on Tuesday that he had misspoken a day earlier in Helsinki, Finland, when he appeared to take the word of President...
The content writing has become popular as the world of Internet has evolved and more people have adapted to modern...
More Title


A while ago I was asked to consult with a group of very driven people who wanted to create something. They were a SaaS startup and ready to go for their next round of funding (basically a second, larger angel raise) and they were trying to plan out a timeline to show investors where and when they expected to deploy the capital. The team came in and laid out a fairly exhaustive plan for the next three years of their business. They had financial projections, hiring projections, feature projections, you name it and they probably had it.
The only problem was, they were projecting that it would take two full years until a working beta would be available.
Now this startup was in the consumer SaaS space. Two years there is like 5 in other industries. So we worked together to cut as much fat as possible out of their plan and eventually got them down to a beta in about 3-5 months. My point in writing this is this: If you can't create and test something with users within a few months you need to reevaluate your featureset. Ideally you should be able to get something to the users much faster than this because their feedback should be the driver of what you build next. The tighter you can make that feedback loop the better.
So please, if you ever need help with an idea I'm happy to take a look, just don't walk in with an expected beta launch that's 24 months away.


Defenceman Dale Dunbar spent parts of two years with the Vancouver Canucks and Boston Bruins in the 80s. His strength was taking the body and clearing the puck out of his own zone.
Born in Winthrop, Massachusetts, Dunbar spent four years anchoring the blueline at Boston University. In May, 1985 he was signed as a free agent by Vancouver and made it into one game the following season. The bulk of his first two pro seasons came with the AHL's Fredericton Express and the Peoria Rivermen of the IHL.
Dunbar came to terms with the Boston Bruins in August, 1987 and was a farmhand on the AHL's Maine Mariners before retiring in 1989. He made it into one NHL game for Boston in 1988-89 and helped Maine reach the Calder Cup semifinals the previous year.
Signed as a free agent by Vancouver, May 10, 1985. Signed as a free agent by Boston, August 25, 1987.
Suffered career-ending knee injury in Norwich Union Cup game vs. Cardiff, October, 1990.


This how-to guide gives you the basics on starting your vegetable garden in a small spot ready for containers.
Select a good potting soil for optimum plant growth.
For best results, don't use soil from your garden. Purchase potting soils from recognizable brands or those recommended by your local garden center. Look for a potting mix, rather than a bagged product labeled "garden soil." Avoid those that are dense and heavy when wet. If in doubt, try small bags of different kinds. You'll find your favorite pretty quickly, based on plant growth.
Do not overfill your pots.
Leave about an inch of the rim above the soil so that water is forced to drain down through the potting mix, rather than spilling over the edge. The soil warms quickly in a container, so gardeners may find containers helpful in getting an early start for a few plants.
Use pots with drainage holes or drill sizable ones.
Containers are available in a variety of materials to suit your space and aesthetic. Pot feet (or any piece of brick or stone) raise a container off the surface below so as not to block the drainage hole. This also preserves surfaces like wooden decks that may rot under constant moisture.
Terra-cotta pots are traditional and look great, but they dry quickly due to evaporation from the sides of the pot. Also, if you have freezing temperatures in your area during winter, the pots need to be stored in a dry place. The moisture that is absorbed into the terra-cotta will freeze and crack the pot.
Glazed ceramic pots come in a variety of colors, sizes, and shapes for design-conscious gardeners. They survive mild freezes, but where the soil freezes, it may crack the pot as it expands.
Resin or plastic pots have the advantage of being lightweight and durable. Nice ones can be costly, but they'll last for years.
Wooden pots or whiskey barrels are favorites of gardeners everywhere because of their generous size and rustic appearance. You can grow mixed plantings in a container this size.
Concrete pots are an excellent choice, as long as you don't like to rearrange your container garden very often. They can be extremely heavy, but they're durable. It's a good idea to place the empty pot where you want it permanently, fill it with potting mix, and then plant.
Fiberglass pots resemble stone, wood, terra-cotta, or any material the maker wants to replicate. They're lightweight and durable, albeit costly.


press to up and down arrow buttons and press control button to fire.
Description Find five differences on two pictures. Interesting game, the beautiful images, great hanging in your free time. Instructions Find differences between two images; click on 5 things and get points after that you advance to the next level.


Custom Reserved Parking Signs Rapid Ship Custom Parking Signs Reserved Parking Tow Away Seton Uploaded by masrbook on Monday, September 10th, 2018 in category Design.
See also Custom Reserved Parking Signs Custom Parking Signs Custom Parking Lot Signs from Design Topic.
Here we have another image Reserved Parking Signs Custom Reserved Parking Signs Rjm Systems Custom Reserved Parking Signs featured under Custom Reserved Parking Signs Rapid Ship Custom Parking Signs Reserved Parking Tow Away Seton. We hope you enjoyed it and if you want to download the pictures in high quality, simply right click the image and choose "Save As". Thanks for reading Custom Reserved Parking Signs Rapid Ship Custom Parking Signs Reserved Parking Tow Away Seton.


text:
Journal Clubs
Cellular/Molecular
Neurosteroid Access to the GABAA Receptor
Gustav Akk, Hong-Jin Shu, Cunde Wang, Joe Henry Steinbach, Charles F. Zorumski, Douglas F. Covey and Steven Mennerick
Journal of Neuroscience 14 December 2005, 25 (50) 11605-11613; DOI: https://doi.org/10.1523/JNEUROSCI.4173-05.2005
Gustav Akk
Hong-Jin Shu
Cunde Wang
Joe Henry Steinbach
Charles F. Zorumski
Douglas F. Covey
Steven Mennerick
GABAA receptors are a pivotal inhibitory influence in the nervous system, and modulators of the GABAA receptor are important anesthetics, sedatives, anticonvulsants, and anxiolytics. Current views of receptor modulation suggest that many exogenous drugs access and bind to an extracellular receptor domain. Using novel synthetic steroid analogs, we examined the access route for neuroactive steroids, potent GABAA receptor modulators also produced endogenously. Tight-seal recordings, in which direct aqueous drug access to receptor was prevented, demonstrated that steroids can reach the receptor either through plasma membrane lateral diffusion or through intracellular routes. A fluorescent neuroactive steroid accumulated intracellularly, but recordings from excised patches indicated that the intracellular reservoir is not necessary for receptor modulation, although it can apparently equilibrate with the plasma membrane within seconds. A membrane impermeant neuroactive steroid modulated receptor activity only when applied to the inner membrane leaflet, demonstrating that the steroid does not access an extracellular modulatory site. Thus, neuroactive steroids do not require direct aqueous access to the receptor, and membrane accumulation is required for receptor modulation.
GABAA receptor
neurosteroid
Certain neuroactive steroids are among the most potent known potentiators of GABAA receptor activity. Neuroactive steroid modulation of GABAA receptors has enjoyed increasing recent attention because endogenous steroids may regulate GABAergic tone in the CNS, neurosteroid dysregulation may underlie neuropsychiatric disorders, and GABA-active steroids may be clinically useful anesthetics, anticonvulsants, and anxiolytics (Zorumski et al., 2000; Belelli and Lambert, 2005). Thus, understanding the mechanisms by which neuroactive steroids access and modulate GABAA receptors is important for understanding endogenous modulation of GABAA receptors and for developing new drugs that target GABAergic transmission.
Current models of drug-receptor interactions tend to view a molecule dissolved in extracellular aqueous medium encountering and interacting with an extracellular binding site on the ligand-gated ion channel. This model accommodates GABA and benzodiazepines, the binding sites of which are well characterized (Amin and Weiss, 1993; Buhr et al., 1996; Wingrove et al., 1997; Wagner and Czajkowski, 2001; Newell and Czajkowski, 2003). In contrast, some anesthetics bind to a hydrophobic pocket formed by membrane-spanning domains (Mihic et al., 1997; Wick et al., 1998). A clear neurosteroid binding site on GABAA receptors has been elusive, and neuroactive steroids are remarkable for their lipophilicity, with an oil-water partition coefficient of >50,000 (logP of 4.89, calculated with Advanced Chemistry Development, version 4.67; Advanced Chemistry Development, Toronto, Ontario, Canada) for the endogenous neurosteroid allopregnanolone (3α5αP). Furthermore, we have recently shown that steroid effects on GABA receptors can be much slower than predicted by simple aqueous diffusion (Shu et al., 2004). Therefore, neuroactive steroids might access receptor sites by typical (but inefficient) aqueous access, through lateral membrane diffusion, or perhaps even through an intracellular site if the steroid is capable of completely permeating the cell membrane. Investigating steroid routes of access to the receptor may be a useful indirect approach to identifying potential receptor binding sites but also needs to be generally considered in drug design. If the plasma membrane interactions are important for access to the receptor site, then steroid membrane mobility and perhaps orientation will be important considerations in the potency and effective lifetime of steroid actions. Likewise, if neuroactive steroids accumulate intracellularly, this may be an important limit on the clearance time of steroids.
We investigated the route of access of neurosteroids to GABAA receptors. Combining experiments on excised and cell-attached membrane patches with whole-cell electrophysiology and imaging of novel, GABA-active fluorescent steroid analogs, we draw several conclusions about steroid access to the GABAA receptor. First, we show that direct, extracellular aqueous access to the receptor is not necessary for potentiation. Second, we show that intracellular accumulation of GABA-active steroid is prominent, but this accumulation is not necessary for actions of the steroid on receptor function. Rather, steroid effects persist in excised patches after access to intracellular steroid is removed. Third, we show that under some conditions, intracellular steroid can resupply the membrane reservoir responsible for GABAA receptor modulation. Intracellular steroid appears to be in relatively rapid equilibrium with membrane steroid, because loss of intracellular steroid temporally tracks a slow component of decay of steroid-mediated current, and wash of cells with membrane-impermeant cyclodextrin speeds the loss of intracellular steroid. Finally, we show that a membrane-impermeant steroid analog effectively potentiates GABAA channel activity when applied to the inner but not outer membrane leaflet, excluding the possibility that aqueous steroid accumulation in the local extracellular environment near the membrane accounts for GABAA receptor effects. In summary, our results show that the plasma membrane is apparently the most direct and relevant access route governing neurosteroid interaction with the GABAA receptor.
Cells, transfection, and drugs. Rat GABAA receptor α1, β2, and γ2L subunit cDNAs were sub-cloned into pcDNAIII vector (Invitrogen, San Diego, CA) and transiently expressed in human embryonic kidney 293 (HEK293) cells using a calcium phosphate precipitation-based transfection technique (Akk et al., 2001). Most drugs and chemicals were purchased from Sigma (St. Louis, MO). (3α,5α,17β)-3-Hydroxyandrostane-17-carbonitrile (ACN) was synthesized as reported previously (Hu et al., 1993). 7-Nitrobenz-2-oxa-1,3-diazole (NBD)-3α5αP was prepared by reacting 4-chloro-7-nitrobenzo[1,2,5]oxadiazole with (3α,5α,11β)-11-amino-3-hydroxypregnan-20-one. Alexa-3α5αP was prepared by reacting (3α,5α,17β)-17-aminomethylandrostan-3-ol hydrochloride with Alexa Fluor 546 carboxylic acid, succinimidyl ester. Synthetic and spectroscopic details for NBD-3α5αP and Alexa-3α5αP will be published elsewhere. Steroid stock solutions were prepared at 10 mm in DMSO and diluted in pipette or bath solution on the day of the experiment with final DMSO concentrations typically ≤0.1% (0.5% for Fig. 4 B). For Figure 5, primary cultures of postnatal hippocampal neurons were grown as described previously (Shu et al., 2004). Cells were used 5– 8 d after plating.
Electrophysiology. The single-channel currents were recorded using the patch-clamp technique in the cell-attached and inside-out configurations (Hamill et al., 1981). The bath solution contained the following (in mm): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.4. The pipette solution in single-channel recordings contained the following (in mm): 120 NaCl, 5 KCl, 10 MgCl2, 0.1 CaCl2, 20 tetraethylammonium chloride, 5 4-aminopyridine, 10 glucose, and 10 HEPES, pH 7.4. The pipette potential was held at +60 to +80 mV, for a total potential across the patch membrane of approximately –100 mV (Vrest, approximately –40 to –20 mV). The channel activity was recorded with an Axopatch 200B amplifier, low-pass filtered at 10 kHz, acquired with a Digidata 1200 series interface at 50 kHz using pClamp software (Molecular Devices, Union City, CA), and stored on a personal computer hard drive. For whole-cell recordings, we used a pipette solution containing the following (in mm): 140 KCl, 4 NaCl, 5 EGTA, 0.5 CaCl2, and 10 HEPES, pH 7.25. Drugs were delivered with a multibarrel, gravity-driven local perfusion system (whole cells), or by bath exchange (patch experiments).
Steroid access from extracellular solution is not necessary for robust GABAA receptor modulation. Cell-attached recordings from transfected HEK cells were performed under the conditions schematized to the right of the panels. Channel openings are shown as downward deflections. The graphs represent log-binned open and closed time histograms from the patch as indicated. A, Channel activity in a patch exposed to 50μm GABA in the recording pipette. The open times were 0.16 ms (49%), 3.1 ms (37%), and 7.8 ms (14%). The closed times were 0.19 ms (61%), 1.7 ms (20%), and 21.7 ms (18%). B, Channel activity in a patch exposed to the combination of GABA and 1 μm ACN in the patch pipette. The open times were 0.27 ms (29%), 1.4 ms (24%), and 19.6 ms (47%). The closed times were 0.19 ms (68%), 1.5 ms (28%), and 25.6 ms (5%). Note the increased duration and amplitude of the third component of the open time distribution and the decreased relative weight of the third component of the closed time distribution, as described previously (Akk et al., 2004). C, Potentiation of receptor function was qualitatively similar when the steroid was applied not via the patch pipette but rather to the bath solution after seal formation. The open times were 0.34 ms (33%), 9.2 ms (34%), and 21.3 ms (33%). The closed times were 0.11 ms (57%), 1.0 ms (35%), and 25.0 ms (8%). D, Channel activity from a cell pretreated with ACN. After removal of bath ACN and exposure of the channels to GABA in the pipette, potentiation of channel activity remained. The open times were 0.42 ms (19%), 1.7 ms (38%), and 34.5 ms (43%). The closed times were 0.17 ms (73%), 1.2 ms (23%), and 22.7 ms (4%).
Kinetic analysis. The kinetic analysis has been described previously (Akk et al., 2001, 2004; Steinbach and Akk, 2001). In short, analysis was performed on clusters of openings occurring within a critical closed-time interval of 500 ms. Typical cluster duration is 2–3 s, with many seconds usually separating one cluster from the next (Akk et al., 2001). Clusters isolated from the recording were low-pass filtered at 2–3 kHz and idealized using the segmented-k-means algorithm (QuB Suite, www.qub.buffalo.edu). The intracluster open and closed times were estimated using maximum likelihood methods, which incorporate a correction for missed events (Qin et al., 1996, 1997).
Imaging. Images were obtained with a Nikon C1 confocal laser-scanning attachment to a TE300 inverted microscope (Fig. 2) or with conventional epifluorescence illumination and a SPOT RT digital camera (Diagnostic Instruments, Sterling Heights, MI) (Figs. 5, 6). For confocal data, images represent z-series projections excited from the 488 and 543 nm laser lines.
We used cell-attached patch recording from HEK cells transfected with rat α1β2γ2 GABAA receptor subunits to test whether steroid requires direct aqueous access to receptors to modulate channel activity. Figure 1A shows channel activity in a typical patch elicited by 50 μm GABA in the cell-attached pipette. When 1 μm ACN, the neuroactive steroid (Hu et al., 1993), was included with 50 μm GABA (Fig. 1B), channel activity was potentiated in a characteristic manner, as reported previously (Akk et al., 2004). At least three effects can be distinguished. Mean open time and the fractional contribution of the longest duration openings increased, and the contribution of the longest closed time decreased. Previous studies (Akk et al., 2004) have indicated that at least two sites are likely involved in mediating these effects; the "A" site underlies the changes in the relative contributions of the long open and closed time components, whereas the "B" site underlies the increase in duration of the longest open time component. An abbreviated summary of effects on channel kinetics for all single-channel analyses performed in our studies can be found in Table 1. Complete analysis, including channel parameters unaffected by steroids, can be found in supplemental Table 1 (available at www.jneurosci.org as supplemental material).
Potentiating actions of steroids on GABAA receptors
The potentiation of GABAA channel activity was equally robust when we exposed the cell-attached patch directly only to GABA and applied ACN to the bath solution after seal formation (Fig. 1C). As a control, we compared channel activity in patches directly exposed to 1 μm GABA (which corresponds to <EC2) in the cell-attached patch pipette with activity in the same patches after adding 1 mm GABA (saturating concentration) to the surrounding bath solution. We found no effect of the increased GABA in the bath on channel open or closed times (n = 3 patches), indicating our glass/membrane seal effectively excluded direct access of small molecules to the patch. Overall, the result from Figure 1C indicates that steroid does not require direct aqueous access to the receptor to effectively potentiate channel activity.
If steroid in aqueous solution is not directly important for modulating receptor activity, then removal of free steroid from the extracellular solution may not terminate drug action. To test this idea, we pretreated cells with ACN for 5 min before exchanging total bath solution three to four times with drug-free saline. We found that even after extensive washing, potentiation of GABAA channel activity continued (Fig. 1D) for as long as 30 min after wash. These results suggest that steroid can modulate the receptor without being present in the extracellular bath, most likely because of accumulation in the plasma membrane, or possibly even inside the cell.
We also tested the effect of preincubation (followed by wash) ofa5β-reduced steroid (B285) (Akk et al., 2004) and an inactive steroid (3β5αP). As expected from previous single-channel (Akk et al., 2004) and whole-cell (Wang et al., 2002) experiments, preincubation with a 5β-reduced steroid resulted in prolonged potentiation, whereas preincubation with 3β5αP was ineffective at potentiating the receptor function (see supplemental material, available at www.jneurosci.org). We then examined whether steroid applied by preincubation exhibits concentration dependence. We preincubated cells expressing GABAA receptors in 10 nm ACN for 8 –10 min, followed by five full-bath exchanges. We found no evidence of potentiation when cells were subsequently patched with GABA in the pipette solution (supplemental material, available at www.jneurosci.org). These results are consistent with the lack of effect of 10 nm ACN on single-channel currents when this low concentration of ACN is coapplied with GABA in the pipette (Akk and Steinbach, 2003).
A fluorescent GABA-active stero id suggests plasma membrane and intracellular steroid accumulation. A, Structure of NBD-3α5αP. B1, Potentiation of whole-cell GABA responses in HEK cells. The superimposed traces represent the response of a transfected HEK cell to 2μm GABA alone (middle trace), GABA plus 3 μm NBD-3α5αP (largest trace), and GABA, NBD-3α5αP, and 50 μm bicuculline(flat trace). B2, Direct response of another transfected HEK cell to 3μm NBD-3α5αPalone and coapplied with 50μm bicuculline (flat trace). C, NBD-3α5αP (green fluorescence, C1, C3) colocalization with DiI (red fluorescence, C2, C3). Note some colocalization with plasma membrane (arrow) but the prominent intracellular staining (arrowhead) that is excluded from the nucleus(n). C3 is a merged image of C1 and C2. D, NBD-3α5αP (green, D1,D3) colocalization with Nile Red (red, D2, D3), a cell-permeant lipophilic marker. D3 is a merged image of D1 and D2.
Patch excision after steroid preincubation and removal does not affect potentiation, suggesting that membrane-accumulated steroid is sufficient for receptor potentiation. A, Channel activity in an inside-out patch from a cell to which ACN was preapplied and then removed before seal formation. The receptors were exposed to 50 μm GABA in the patch pipette. A1, Channel record from 1 min after excision. Histograms pertain to data from 0–2 min after excision. The open times were 0.31 ms (42%), 2.1 ms (23%), and 18.5 ms (35%). The closed times were 0.20 ms (58%), 1.2 ms (32%), and 18.5 ms (9%). A2, The addition of 5 mm methyl-β-cyclodextrin to the bath to remove steroid resulted in an immediate loss of steroid-mediated potentiation. The open times were 0.46 ms (24%), 3.4 ms (57%), and 6.9 ms (19%). The closed times were 0.15 ms (60%), 1.5 ms (9%), and 10.4 ms (31%). B, Time course of the effect of patch excision on channel activity from cells pretreated with ACN(filled circles) or in the presence of GABA alone (open circles). Mean open time was averaged over 1 min blocks. Note the prolonged potentiation of channel activity after ACN pre-exposure and removal. Data show mean ± SD from three to six patches.
Although canonical steroid signaling involves activation of intracellular receptors, GABA-active steroids do not typically activate intracellular receptors (Belelli and Lambert, 2005). Therefore, whether extracellularly applied GABA-active steroids permeate the plasma membrane and accumulate intracellularly is currently unknown. We synthesized a novel steroid with an NBD fluorescent tag at carbon 11 of the endogenous steroid 3α5αP (Fig. 2A). NBD-3α5αP retained activity at GABAA receptors (Fig. 2B). NBD-3α5αP potentiated bicuculline-sensitive GABA-gated currents in transfected HEK cells (Fig. 2B1) and neurons (data not shown) and directly gated bicuculline-sensitive currents (Figs. 2B2, 5A), although the fluorescent analog was ∼30-fold less potent than the parent compound 3α5αP (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Aside from potency, the effects of NBD-3α5αP on channel kinetics were practically indistinguishable from other 5α-reduced steroid potentiators such as 3α5αP (supplemental Fig. 1, available at www.jneurosci.org as supplemental material) or ACN (Akk et al., 2004).
Intracellular loading can result in potentiation. A, Resurgent steroid effects after cyclodextrin wash. A1, Slow direct gating of GABAA receptors in response to 1 μm ACN in a transfected HEK cell. The cell was rapidly perfused with drug-free saline after ACN removal but nevertheless had a slowly decaying off response, similar to that previously described for neurons and the natural neurosteroid 3α5αP (Shu et al., 2004). A2, In the same cell on another application of ACN, the offset current was reversibly blocked by bicuculline (bic), which is a noncompetitive antagonist with respect to steroid (Ueno et al., 1997). Therefore, in the case of bicuculline, a large part of the resurgence results from bicuculline unbinding before steroid departure. A2, A3, The offset current was partly inhibited by a brief wash with 500 μm γ-cyclodextrin (CDX), which facilitates the removal of steroid (Shu et al., 2004). The resurgent current (arrows) indicates that the replenishment of receptor-accessible steroid, possibly from the intracellular pools, which are inaccessible to cyclodextrin. To facilitate comparison of offset, the dotted trace represents a replot of the control trace in A1, scaled to account for slight rundown between sweeps. Note that the resurgence is less complete than for bicuculline and less than expected from baseline decay (dotted traces). B, Effects of intracellular steroid loading. The currents presented show representative data from a total of four cells under control conditions and five cells loaded with steroid. B1, Response to 1μm GABA in a control transfected HEK cell. Note that the GABA response is insensitive to 500 μm cyclodextrin. B2, In a cell loaded with 50 μm ACN through the whole-cell pipette, 1μm GABA gated a larger response, which was partly sensitive to 500μm γ-cyclodextrin. C1, Channel activity in an inside-out membrane patch (50μm GABA in the pipette), to which 1 μm ACN was applied by bath exchange to the intracellular face. C2, Summary of channel effects. Channel openings are shown downward. The open times were 0.42 ms (48%), 4.2 ms (15%), and 23.3 ms (37%). The closed times were 0.28 ms (48%), 1.4 ms (42%), and 14.5 ms (10%).
In imaging studies, we found that 3 μm NBD-3α5αP (incubated for at least 10 min) partially colocalized with DiI, a plasma-membrane marker (Fig. 2C1–C3), but NBD-3α5αP also colocalized prominently with Nile Red, a membrane-permeant lipophilic dye (Greenspan et al., 1985) (Fig. 2D1–D3). These results indicate that GABA-active steroids likely accumulate both in the plasma membrane and intracellularly. Thus, the data from imaging studies are consistent with conclusions from cell-attached patch recordings (Fig. 1) and again suggest that either lateral membrane diffusion or intracellular access to the receptor may be important for steroid modulation of GABAA receptors.
The bulk intracellular steroid pool equilibrates with the receptor-accessible pool in <1 min. A1, Directly gated current in a hippocampal neuron by 3 μm NBD-3α5αP. After 30 s of application, the cell was washed with saline. A2, The same neuron was rechallenged, but the wash included 500 μm γ-cyclodextrin (CDX). The dotted trace is a replot of A1 for comparison. The dots below the trace indicate time points at which images were taken of cells for B. B1, Pseudocolor fluorescence images (warm colors, high-fluorescence intensity; cool colors, low-fluorescence intensity) of a cell subjected to the protocol in A1. Photographs were from the time points indicated by the labeled dots in A2 (a– e). B2, The same cell was subjected to the protocol indicated in A2. Again, the photographs are from the time points indicated by the dots in A2. Scale bar, 20 μm. C1, C2, The data points show raw normalized decays of NBD-3α5αP-gated currents (left graphs; n = 8 saline-washed and 7 CDX-washed neurons) and fluorescence (right graphs; n = 8 saline-washed and 6 CDX-washed neurons). For current traces, only every 100th data point is represented for clarity. The lines represent superimposed biexponential fits (current decays) and single-exponential fits (fluorescence). Parameters are summarized in D. D1, D2, Summary of exponential fits to the decay phase of NBD-3α5αP-generated currents and decay of intracellular fluorescence from the individual cells represented in C. Current decays were well described by a biexponential fit in all cases. The fast component accounted for 57 ± 5% of the decay in saline and 59 ± 5% in γ-cyclodextrin. Fluorescence decays were fit to fluorescence intensities taken from an intracellular region near the nucleus. To diminish bleaching, images were obtained beginning at the end of the 30 s application of NBD-3α5αP, continuing every 4 s for 60–120 s. Fluorescence decays were well described by a single exponential. Both the fast and slow time constants of current decay were significantly speeded by cyclodextrin wash, as was the time constant of fluorescence wash(p<0.01; note the change in the y-axis between D1 and D2). Data show mean ± SEM from seven to eight cells.
We examined the rate of NBD-3α5αP accumulation with confocal microscopy. Accumulation at 3 μm NBD-3α5αP plateaued by 60 s of local application, and we could detect no difference in the time constant of accumulation in perimembrane regions versus intracellular compartments (τ = 20.1 ± 1.6 s for perimembrane accumulation vs 28.6 ± 4.5 s for intracellular accumulation at 3 μm drug; n = 4), consistent with the idea that steroid passes readily through the cell membrane into intracellular compartments (Fig. 5). These rates are similar to the slow rate of development of 3α5αP-gated currents in hippocampal cells, particularly when low concentrations of steroids are applied (Shu et al., 2004) (Fig. 5).
To determine whether intracellular pools of steroid are important for effects on GABAA receptors, we excised inside-out membrane patches from HEK cells, which had been preincubated with bath ACN, followed by subsequent ACN removal. We found that potentiation of channel activity long outlived patch excision into a steroid-free bath solution (Fig. 3A1), although there was a period of rapid decrease in potentiation within the first minute of excision (Fig. 3A1). Mean open time of patches pre-exposed to ACN subsequently slowly fell, whereas patches exposed continuously to either GABA alone (Fig. 3B) or to the combination of GABA and ACN (data not shown) retained fairly stable activity up to 13 min after patch excision. Therefore, neither extracellular water-solubilized steroid nor cytoplasmic steroid appears necessary for potentiation of channel activity. Rather, steroid retained by the plasma membrane is sufficient to potentiate channel activity. In support of this, the channel open time profile of steroid pretreated patches returned to that of GABA alone after a brief treatment with cyclodextrin to facilitate steroid removal (Fig. 3A2) (Shu et al., 2004). Although second messenger effects could mediate long-lived steroid effects, we have previously excluded a role for pertussis-toxin-sensitive G-proteins in slow steroid effects (Shu et al., 2004). Furthermore, second messenger effects are unlikely to persist in excised membrane patches.
Effects of a membrane impermeant steroid. A, Structure of Alexa-3α5αP. B, Phase-contrast (left) and fluorescence (right) images of cells incubated in Alexa-3α5αP for 10 min. Incubation times >60 min failed to label healthy cells. The arrow indicates a cell with a membrane that was purposely ruptured with a sharp patch pipette before Alexa-3α5αP application. Note that only this cell and not intact cells stain with the Alexa-conjugated steroid. Several other pieces of cellular debris also fluorescently stained.C, Cell-attached patch data with GABA (50 μm) plus Alexa-3α5αP(1 μm) in the recording pipette. Channel openings are shown downward. The open times were 0.42 ms (12%), 3.9 ms (73%), and 9.3 ms (15%). The closed times were 0.13 ms (65%), 1.2 ms (10%), and 15.4 ms(24%). D, Inside-outpatch with GABA(50 μm) in the pipette and Alexa-3α5αP(1 μm) applied to the inner membrane face. The open times were 0.41 ms (31%), 4.9 ms (13%), and 20.8 ms (56%). The closed times were 0.27 ms (54%), 1.5 ms (37%), and 16.7 ms (9%). Alexa-3α5αP potentiated receptor function when applied to the intracellular but not the extracellular side of the membrane.
Although intracellularly accumulated steroid is not directly necessary for receptor potentiation by steroid, we investigated whether intracellular pools of steroid might be indirectly important as steroid reservoirs that could supply the plasma membrane and rate limit of the decay of steroid actions at GABAA receptors. Indirect evidence for this idea comes from experiments in which we used 500 μm γ-cyclodextrin as a molecular sponge to remove steroid effects from extracellularly accessible reservoirs (Shu et al., 2004). We found that when cyclodextrin was included in the wash after exposure of transfected HEK cells to 1 μm ACN alone to directly gate GABA channels, currents were rapidly reduced by 500 μm cyclodextrin (Fig. 4A2,A3), similar to previous results in native cells (Shu et al., 2004). If the wash with cyclodextrin was sufficiently brief, we observed a small resurgent current (Fig. 4A2,A3 arrows) (n = 5 cells), indicating replenishing reservoirs inaccessible to the brief cyclodextrin wash that might represent intracellular pools. As a positive control for speed and completeness of solution exchange, we examined the effect of bicuculline (Fig. 4A2). Resurgence was also observed with bicuculline application during the decay of steroid effects, but the resurgence was stronger, reflecting the rapid kinetics of bicuculline unbinding and removal before steroid departure (Ueno et al., 1997).
The idea that such resurgent current reflects intracellular steroid accessing GABAA receptors would seem to contradict previous results suggesting that steroid is ineffective when loaded through the patch pipette (Lambert et al., 1990). However, efficiency of delivery by this method could not be verified previously. In fact, we found that NBD-3α5αP only weakly loaded HEK cells when included at concentrations up to 10 μm in the whole-cell patch pipette (supplemental Fig. 2, available at www.jneurosci.org as supplemental material), compared with the bright intracellular fluorescence observed in cells when NBD-3α5αP was extracellularly applied by the same whole-cell pipette (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). To compensate for poor loading, we directly loaded a high concentration of ACN (50 μm) by whole-cell pipette. GABA application to ACN-loaded cells exhibited a cyclodextrin-sensitive current not present in control vehicle-loaded cells (Fig. 4B). In six cells loaded with ACN, γ-cyclodextrin produced a 63 ± 4% depression of current compared with 22 ± 3% (n = 4; attributable to desensitization) in control cells (p < 0.001), suggesting that intracellular steroid, when continuously supplied through a whole-cell pipette, can access GABAA receptors on the cell surface, where it becomes rapidly accessible to extracellularly applied cyclodextrin. Similar results were also obtained by loading the 5β-reduced steroid B285. In four cells loaded with 50 μm B285, cyclodextrin reduced currents by 49 ± 5% compared with 13 ± 7% in three control cells. Note that although the experiments in Figure 4 do not address whether direct gating of receptors (Fig. 4A) and potentiation (Fig. 4B) occur through the same sites, the results indicate that both effects occur through a cyclodextrin-accessible compartment and that both sites can be replenished by steroid from the intracellular (cyclodextrin-inaccessible) compartment.
To test more directly the idea that intracellularly applied steroid is capable of modulating GABAA receptors, we challenged the intracellular face of inside-out excised patches with 1 μm ACN while exposing the extracellular face to 50 μm GABA in the pipette solution. Figure 4C shows that channel activity was potentiated in a manner indistinguishable from the effects of extracellularly applied ACN (Table 1) (supplemental Table 1, available at www.jneurosci.org as supplemental material).
Our data suggest that intracellular steroid can equilibrate with the plasma membrane pool (Fig. 4). We next addressed whether the loss of intracellular fluorescence tracked the time course of current decay after steroid application, which would imply the intracellular steroid pool is in rapid equilibrium with the membrane, receptor-relevant steroid pool. For direct comparison with previously published results (Shu et al., 2004), we used primary cultures of dissociated hippocampal neurons (Fig. 5) and examined currents directly gated by fluorescent NBD-3α5αP. Kinetics of onset were slow, similar to the natural steroid 3α5αP (Shu et al., 2004). Current decays were described by a biexponential fit (Fig. 5A–D). We also measured intracellular fluorescence loss during washout of 3 μm NBD-3α5αP (Fig. 5B–D). After NBD-3α5αP application, the slow time constant of current decline tracked the loss of fluorescence closely (Fig. 5D), implying that the intracellular pool equilibrates with the plasma membrane pool sufficiently rapidly to rate limit current decay. We could detect no difference in the time constant of dye loss near the membrane versus intracellularly (data not shown), further indicating that the two pools are in relatively rapid equilibrium. Next, we used γ-cyclodextrin to speed the rate of removal of the membrane pool. In this case, we found that intracellular fluorescence was lost more quickly (Fig. 5B2,C2) than with saline wash alone (Fig. 5B1,C1). Because cyclodextrin is membrane impermeant, this is strong evidence that the intracellular and plasma membrane pools are normally in sufficiently rapid equilibrium that loss of the membrane pool to cyclodextrin speeds overall intracellular loss. However, in this case, current decay was faster than dye loss (Fig. 5D), implying that the equilibration with the membrane pool of the bulk intracellular pool is slower than τ = 9s, the slow time constant of current decay with cyclodextrin wash.
The rate constant of dye loss (Fig. 5) and current decays (Figs. 4, 5) appeared faster than loss of steroid in preincubation experiments (Fig. 1D), in which steroid effects persisted for many minutes. At least part of this difference may be a result of differences in experimental protocol. Cells were actively washed with saline or cyclodextrin for experiments in Figures 4 and 5, whereas patch experiments were performed in static bath (Fig. 1D). To determine whether this experimental difference helps explain the difference in longevity of effects, we examined dye loss in cells incubated in static bath versus active saline perfusion (supplemental Fig. 3, available at www.jneurosci.org as supplemental material). The results show that active perfusion speeds intracellular steroid loss and is consistent with the idea of constant equilibration between membrane and cytoplasmic compartments of steroid. Together, results of Figures 3, 4, 5 and supplemental Figure 3 (available at www.jneurosci.org as supplemental material) suggest a picture in which steroids prefer to remain in the plasma membrane compartment, or shuttle between plasma membrane and intracellular hydrophobic domains, unless steroid is actively removed from the membrane by strong external perfusion with or without cyclodextrin.
The results suggest that plasma-membrane accumulation and lateral membrane diffusion of steroid are most directly important for potentiation of GABA responses but that the intracellular pool may be rate limiting in certain situations. However, none of the results directly determine which side of the plasma membrane is most closely associated with GABAA receptor modulation. Rapid cyclodextrin accessibility (Figs. 4, 5) (Shu et al., 2004) may suggest that steroid embedded in the outer membrane leaflet is most critical. However, Figure 3A2 shows that cyclodextrins can also rapidly remove steroid when applied to the inner leaflet. Steroids may rapidly flip-flop across the plasma-membrane leaflets (Hamilton, 2003). In this case, removal of steroid by cyclodextrin from one leaflet may simply alter the equilibrium between outer-leaflet steroid and inner-leaflet steroid, thus rapidly reducing the concentration in both leaflets. To better localize the relevant membrane routes of steroid access to the receptor, we synthesized an Alexa Fluor 546-conjugated steroid (Fig. 6A), which is expected to be membrane impermeant (Revankar et al., 2005). We found that Alexa-3α5αP failed to stain live cells when extracellularly applied (Fig. 6B) (n = 3). In contrast, when the plasma membrane of cells was compromised by mechanical rupture, we detected bright intracellular fluorescence (Fig. 6B). These results confirm the expectation that Alexa-3α5αP is membrane impermeant. In excised membrane patches, we found that application of Alexa-3α5αP to the inner membrane leaflet potentiated channel activity by modifying the same characteristic kinetic parameters as other 5α-reduced steroids (Fig. 6D, Table 1) (supplemental Table 1, available at www.jneurosci.org as supplemental material). In contrast, steroid exposure of the outer leaflet of the patch membrane in cell-attached recordings (Fig. 6C, Table 1) or the addition of Alexa-3α5αP to the bath solution in whole-cell recordings (data not shown) failed to potentiate GABA responses. These results suggest that the inner membrane leaflet may be particularly important for steroid access to the GABAA receptor. Because Alexa-3α5αP is membrane impermeant, these results also indicate that accumulation of steroid in an aqueous extracellular unstirred layer (Barry and Diamond, 1984) does not play an important role in GABAA receptor modulation and strongly suggest that one or more relevant potentiating sites are readily accessible from the inner leaflet.
Our main result is that steroids can reach the site(s) involved in potentiation of the GABAA receptor function by diffusion through the plasma membrane rather than by direct binding to a site on the receptor from the external medium. Second, we show that intracellular accumulation of GABA-active steroids is prominent and that some of these intracellular pools may resupply the plasma membrane with steroid capable of modulating the GABAA receptor. Finally, we show that a membrane-impermeant steroid is active when applied to the inner face of the membrane but not the outer membrane, suggesting that steroids may more easily access at least one site on the GABAA receptor from the inner plasma-membrane leaflet.
Our results have implications for the effects of natural and exogenous steroids for nervous system function. After relatively brief steroid exposure, accumulated steroid in the plasma membrane and/or intracellular compartments could continue to modulate receptors for long after the initial exposure and limit access of the steroids to enzymes eventually responsible for terminating steroid actions (Belelli and Herd, 2003). It is worth noting that endogenous conditions may be more akin to "static bath" conditions like those in our patch experiments (Figs. 1, 3). We show that active perfusion of cells speeds up steroid loss (supplemental Fig. 3, available at www.jneurosci.org as supplemental material). Presumably, effective steroid lifetime in the brain is much longer than the ∼60 s observed in Figure 5, reflecting the strong preference of steroids to remain in the lipophilic plasma membrane and intracellular compartments. Furthermore, because neurosteroids are synthesized by neurons (Mellon et al., 2001), strong membrane and intracellular retention may suggest an important autocrine action of neurosteroids.
Our results highlight slow effects of steroid modulation of GABAA receptors. Our previous results suggest that direct gating is particularly slow (Shu et al., 2004), and the present results indicate that membrane and intracellular interactions can affect the time course of steroid actions. Although fast effects of steroids on GABAA receptors have been observed in previous experiments, it seems likely that full, steady-state effects of low steroid concentrations may not be apparent for tens of seconds, much slower than a diffusion-limited reaction. It seems likely that both slow access and slow equilibration of specific receptor states contribute to the time course of the effects. In addition, our results also suggest that the different lipid and/or sterol content of membranes may influence the kinetics of steroid actions and that onset and offset of steroid effects may therefore differ by cell type. Finally, the precise experimental protocol used may dictate the kinetics of steroid effects (supplemental Fig. 3, available at www.jneurosci.org as supplemental material).
Previous studies have suggested that intracellular application of neuroactive steroids is ineffective at modulating GABAA receptors (Lambert et al., 1990). This result may be partly responsible for the common view that modulators bind to extracellular receptor domains directly from aqueous solution, although results from other ion channels have also suggested the importance of the membrane in the access route for modulators of voltage-gated and mechanosensitive channels (Hille, 1977; Lee and Mac-Kinnon, 2004; Suchyna et al., 2004).
We found that membrane-permeable steroid applied to the inner face of excised patches is just as effective as extracellularly applied steroid (Fig. 4C). Furthermore, with high concentrations of steroid in the whole-cell pipette, intracellularly applied steroid also effectively potentiates whole-cell responses (Fig. 4B). The difference in effectiveness of intracellular steroid on excised patches versus whole cells can be accounted for primarily by poor loading through the point source of a whole-cell patch pipette (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). Although we do not completely understand the basis for this poor loading, this likely explains the failure of previous experiments to detect an effect of steroid (up to 3 μm) loaded through a whole-cell patch pipette on GABA receptors (Lambert et al., 1990).
We used an Alexa Fluor-conjugated neuroactive steroid to test whether extracellular access to the receptor is sufficient to promote potentiation. The charge on the Alexa Fluor molecule prevents membrane permeability (Revankar et al., 2005). Perhaps surprisingly, we found that impermeant steroid applied to the inner face of the membrane was effective in potentiating GABAA channel activity (n = 4) (Fig. 6C, Table 1), whereas Alexa-3α5αP applied to the outer leaflet in cell-attached patches (n = 7) (Fig. 6D, Table 1) or in whole-cell recordings (data not shown) was ineffective. This could suggest that the relevant site on the GABAA receptor is near the inner membrane leaflet. However, there are several caveats to this conclusion. First, there are likely to be several sites on the GABAA receptor (Akk et al., 2004), and differential accessibility of these sites from the outer versus inner leaflet is possible. We also do not know how deeply the steroid embeds in the membrane or the orientation of the molecule when embedded. The failure of cells to fluoresce when Alexa-3α5αP was applied to the outside of intact cells suggests that the Alexa molecule may interact only superficially with the outer leaflet. It is possible that the relevant receptor site is near the extracellular side of the bilayer but that Alexa-3α5αP permeates in the correct orientation only when applied to the inner leaflet. Future biophysical studies are needed to test these alternatives.
In summary, we show that plasma membrane access is directly relevant to GABAA receptor activity. This may help lead to pinpointing sites on the receptor at which steroids act. Perhaps more importantly, our results demonstrate that independent of steroid interactions with the receptor binding site, properties of steroid-membrane interactions (orientation, depth of embedding, rate of flip-flop) and degree of intracellular retention will influence the potency and lifetime of steroid actions at the GABAA receptor.
This work was supported by National Institutes of Health Grants GM47969 (J.H.S., C.F.Z., D.F.C.), AA14707 (G.A.), and AA12952 (S.M.) and by grants from the McDonnell Foundation (S.M.) and the Bantly Foundation (C.F.Z.). J.H.S. is the Russell and Mary Shelden Professor of Anesthesiology. We thank Bei-Wen Ma for help with tissue culture and Ann Benz for help with screening of the novel steroids.
Correspondence should be addressed to Gustav Akk, Department of Anesthesiology, Washington University in St. Louis, Campus Box 8054, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: akk{at}morpheus.wustl.edu.
DOI:10.1523/JNEUROSCI.4173-05.2005
Copyright © 2005 Society for Neuroscience 0270-6474/05/2511605-09$15.00/0
↵* G.A. and H.-J.S. contributed equally to this work.
Akk G, Steinbach JH (2003) Low doses of ethanol and a neuroactive steroid positively interact to modulate rat GABAA receptor function. J Physiol (Lond) 546: 641–646.
Akk G, Bracamontes J, Steinbach JH (2001) Pregnenolone sulfate block of GABAA receptors: mechanism and involvement of a residue in the M2 region of the α subunit. J Physiol (Lond) 532: 673–684.
Akk G, Bracamontes JR, Covey DF, Evers A, Dao T, Steinbach JH (2004) Neuroactive steroids have multiple actions to potentiate GABAA receptors. J Physiol (Lond) 558: 59–74.
Amin J, Weiss DS (1993) GABAA receptor needs two homologous domains of the β-subunit for activation by GABA but not by pentobarbital. Nature 366: 565–569.
Barry PH, Diamond JM (1984) Effects of unstirred layers on membrane phenomena. Physiol Rev 64: 763–872.
Belelli D, Herd MB (2003) The contraceptive agent Provera enhances GABAA receptor-mediated inhibitory neurotransmission in the rat hippocampus: evidence for endogenous neurosteroids? J Neurosci 23: 10013–10020.
Belelli D, Lambert JJ (2005) Neurosteroids: endogenous regulators of the GABAA receptor. Nat Rev Neurosci 6: 565–575.
Buhr A, Baur R, Malherbe P, Sigel E (1996) Point mutations of the α1β2γ2 γ-aminobutyric acid(A) receptor affecting modulation of the channel by ligands of the benzodiazepine binding site. Mol Pharmacol 49: 1080–1084.
Greenspan P, Mayer EP, Fowler SD (1985) Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100: 965–973.
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85–100.
Hamilton JA (2003) Fast flip-flop of cholesterol and fatty acids in membranes: implications for membrane transport proteins. Curr Opin Lipidol 14: 263–271.
Hille B (1977) Local anesthetics: hydrophilic and hydrophobic pathways for the drug-receptor reaction. J Gen Physiol 69: 497–515.
Hu Y, Zorumski CF, Covey DF (1993) Neurosteroid analogues: structure-activity studies of benz[e]indene modulators of GABAA receptor function. 1. The effect of 6-methyl substitution on the electrophysiological activity of 7-substituted benz[e]indene-3-carbonitriles. J Med Chem 36: 3956–3967.
Lambert JJ, Peters JA, Sturgess NC, Hales TG (1990) Steroid modulation of the GABAA receptor complex: electrophysiological studies. Ciba Found Symp 153: 56–71; discussion 71–82.
Lee SY, MacKinnon R (2004) A membrane-access mechanism of ion channel inhibition by voltage sensor toxins from spider venom. Nature 430: 232–235.
Mellon SH, Griffin LD, Compagnone NA (2001) Biosynthesis and action of neurosteroids. Brain Res Brain Res Rev 37: 3–12.
Mihic SJ, Ye Q, Wick MJ, Koltchine VV, Krasowski MD, Finn SE, Mascia MP, Valenzuela CF, Hanson KK, Greenblatt EP, Harris RA, Harrison NL (1997) Sites of alcohol and volatile anaesthetic action on GABAA and glycine receptors. Nature 389: 385–389.
Newell JG, Czajkowski C (2003) The GABAA receptor α1 subunit Pro174-Asp191 segment is involved in GABA binding and channel gating. J Biol Chem 278: 13166–13172.
Qin F, Auerbach A, Sachs F (1996) Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events. Biophys J 70: 264–280.
Qin F, Auerbach A, Sachs F (1997) Maximum likelihood estimation of aggregated Markov processes. Proc Biol Sci 264: 375–383.
Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER (2005) A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307: 1625–1630.
Shu HJ, Eisenman LN, Jinadasa D, Covey DF, Zorumski CF, Mennerick S (2004) Slow actions of neuroactive steroids at GABAA receptors. J Neurosci 24: 6667–6675.
Steinbach JH, Akk G (2001) Modulation of GABAA receptor channel gating by pentobarbital. J Physiol (Lond) 537: 715–733.
Suchyna TM, Tape SE, Koeppe Jr RE, Andersen OS, Sachs F, Gottlieb PA (2004) Bilayer-dependent inhibition of mechanosensitive channels by neuroactive peptide enantiomers. Nature 430: 235–240.
Ueno S, Bracamontes J, Zorumski C, Weiss DS, Steinbach JH (1997) Bicuculline and gabazine are allosteric inhibitors of channel opening of the GABAA receptor. J Neurosci 17: 625–634.
Wagner DA, Czajkowski C (2001) Structure and dynamics of the GABA binding pocket: a narrowing cleft that constricts during activation. J Neurosci 21: 67–74.
Wang M, He Y, Eisenman LN, Fields C, Zeng CM, Mathews J, Benz A, Fu T, Zorumski E, Steinbach JH, Covey DF, Zorumski CF, Mennerick S (2002) 3β-Hydroxypregnane steroids are pregnenolone sulfate-like GABAA receptor antagonists. J Neurosci 22: 3366–3375.
Wick MJ, Mihic SJ, Ueno S, Mascia MP, Trudell JR, Brozowski SJ, Ye Q, Harrison NL, Harris RA (1998) Mutations of γ-aminobutyric acid and glycine receptors change alcohol cutoff: evidence for an alcohol receptor? Proc Natl Acad Sci USA 95: 6504–6509.
Wingrove PB, Thompson SA, Wafford KA, Whiting PJ (1997) Key amino acids in the γ subunit of the γ-aminobutyric acidA receptor that determine ligand binding and modulation at the benzodiazepine site. Mol Pharmacol 52: 874–881.
Zorumski CF, Mennerick S, Isenberg KE, Covey DF (2000) Potential clinical uses of neuroactive steroids. Curr Opin Investigational Drugs 1: 360–369.
Vol. 25, Issue 50
Thank you for sharing this Journal of Neuroscience article.
You are going to email the following Neurosteroid Access to the GABAA Receptor
Message Subject (Your Name) has forwarded a page to you from Journal of Neuroscience
Message Body (Your Name) thought you would be interested in this article in Journal of Neuroscience.
View Full Page PDF
Gustav Akk, Hong-Jin Shu, Cunde Wang, Joe Henry Steinbach, Charles F. Zorumski, Douglas F. Covey, Steven Mennerick
Journal of Neuroscience 14 December 2005, 25 (50) 11605-11613; DOI: 10.1523/JNEUROSCI.4173-05.2005
Responses to this article
Jump to comment:
No eLetters have been published for this article.
Mu opioid receptors acutely regulate adenosine signaling in striatal glutamate afferents
Reproducibility of the rod photoreceptor response depends critically on concentration of the phosphodiesterase effector enzyme
Rapid Fluorescence Lifetime Imaging Reveals That TRPV4 Channels Promote Dysregulation of Neuronal Na+ in Ischemia
Show more Cellular/Molecular
Visit Society for Neuroscience on Facebook
Follow Society for Neuroscience on Twitter
Follow Society for Neuroscience on LinkedIn
Visit Society for Neuroscience on Youtube
Follow our RSS feeds
Copyright © 2022 by the Society for Neuroscience.
JNeurosci Online ISSN: 1529-2401
The ideas and opinions expressed in JNeurosci do not necessarily reflect those of SfN or the JNeurosci Editorial Board. Publication of an advertisement or other product mention in JNeurosci should not be construed as an endorsement of the manufacturer's claims. SfN does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of any material contained in JNeurosci.


This is the Google first basic algorithm for SEO prospects, Which looks at link between page to determine their relevance and importance.
It was named after "Larry Page", Co founder of google.
Ex. I am offering service like seo tips and many of the user like our service and they also recommended other to me for the best seo tips and so after we see more of people build with us that case our popularity is increase, value increase, quailty increase and trust increase then at that time our page rank will bi higher. so here is the logic of Google algorithms.
Default pagerank for any page is 0.15 internally.
Real Page rank is calculated based on number of pages in index, Which cab be 0.15 to Trillions.
As or march 7th 2016, Google has discontinued showing pagerank visibility is any toolbar.
Moz metrics – Moz Toolbar offers metrics like DA(Domain Authority), PA(Page Authority ) to measure the ranking factors.
Majestic Metrics – MajesticSEO offers Trust to analyze quality of site.
Ahrefs Metrics – This tool provide the calculate link value of the page.


Mike's TV, Appliance and Furniture is a family owned and operated business that was established in 1974. The owner, Michael Haas, started repairing televisions at the age of 10 in his father's hardware store (Art's Hardware) in downtown Sparta. In 1974 Mike started his own business in his dad's hardware store selling televisions and repairing electronics in the basement.
Mike and his wife Julie were married in January, 1975. They purchased a building at 115 East Oak St. in Sparta and moved their television sales and repair business in the fall of 1975 and operated out of a 10 x 40 area. A tenant moved and Mike's expanded the business display area to 20 x 40 and the repair service was operated out of the basement. In 1978 Mike's business had grown and added a 20 x 70 addition onto the building making a total of 20 x 110. At that time Mike added Whirlpool appliances to his lines of products sold and rented a building at 117 E Oak for repair and warehouse.
As business continued to grow, Mike's purchased the historic "Williams Building" (circa 1896) on the corner of Oak and Water Street and moved the business into the 10,000 sq ft building on April 1, 1989. For many years, along with television and appliance sales, Mike's rented videos. In 1995 the video portion of the business was sold to accommodate furniture sales. Mike's secured the Flexsteel Home Furnishings line, Catnapper Reclining furniture, along with Best Craft Furniture for upholstered lines. As business continued to grow, Mike remodeled three of the basements to add an additional 3,500 sq ft of showroom. In the basement Mike's has dining room furniture and bar stools, Symbol, Englander & Anatomic Global mattresses, as well as Coaster Bedroom sets, bunk beds and Oak Design bedroom furniture. Mike's also carries framed prints, plush animals, occasional tables and table and floor lamps.
Mike's also features a full line of Great Northern log furniture including sofas, recliners, cedar pub tables and bar stools, cedar tables and chairs, cedar bar, EZ-Beds with a variety of wild life prints and bedroom sets.
We enjoy working with our customers to find the right product for each customer's needs, from frames to fabrics on upholstered products, from firm, soft or just right feels (three bears effect) on mattresses, to the right television for each room in the home.
For 44 years Mike's prides themselves on selling quality products with service after the sale.
Service Area: Sparta, La Crosse, Winona, Wisconsin Rapids, Reedsburg, Richland Center, Nekoosa, Mauston, West Salem, La Crescent, Neillsville and surrounding communities.
Page Summary: Mike's, TV Appliance and Furniture offers furniture store, appliance sales, home furnishings, log furniture, Whirlpool appliances, LG televisions, satellite systems, Flexsteel Furniture, Best Craft Furniture, dining room furniture in Sparta, La Crosse, Tomah, Onalaska, Black River Falls, Bangor, Cashton, Norwalk, New Lisbon, Holmen and surrounding Monroe County WI communities.


Kerdn Electric Co.,Ltd. is a leading supplier of Lightning Protection and Surge Protection.
KERDN has a strict quality inspection process which include strict inspections of raw materials, approval of batch production samples, standard inspection of manufacturing process, random quality inspection during production and quality inspection in warehouse before shipment.
KERDN guarantees competitive pricing, our experience and contacts within China also reduce the risks of non-performance or sub-standard performance, KERDN always do our best to design, manufacture and source the products that you require to continually improve your return-on-investment, help your bottom line and take the lead in your industry.
What sets up apart? Why work with us?
1. Having a reliable quality control system. including point control in raw materials/batch production samples/inspection during production/random quality inspection(flying inspection) during production/warehouse inspection before shipment.
2. We're specialists. unlike general supplers, we offer relatively few products - so we know their strengths and their weaknesses, we state both to you.
3. Having a well-developed sourcing network around China.
5. Ensuring competitive pricing. (using lower-cost factories;purchasing volume; professional buyer).
6. We support what we sell. Long after the product reaches your place, we will be here for you to ensure your result is the best it can be.
7. Drawing clear lines between customers.


Isaac Copeland Jr. is second on the Huskers in scoring, rebounding and blocked shots.
Update on Nebraska's Isaac Copeland Jr.
Lincoln – University of Nebraska men's basketball coach Tim Miles announced Saturday evening that senior forward Isaac Copeland Jr. suffered a torn left anterior cruciate ligament (ACL) during the second half of Saturday's game against Ohio State. The injury was revealed during MRI testing following Saturday's game.
Copeland, an All-Big Ten selection in 2017-18, had started Nebraska's first 20 games this season, averaging 14.0 points and 5.4 rebounds per game. The 6-foot-9 forward from Raleigh, N.C., is second on the Huskers in scoring, rebounding and blocked shots (0.9 bpg). He has started all 53 games since arriving at Nebraska two seasons ago and including his two-plus years at Georgetown, has totaled over 1,300 points and 600 rebounds.