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About me
For a solid four years, food controlled my entire life. I was obsessed with dieting in order to look a certain way and I never truly accepted my body. I thought if I stuck to "clean eating", I would achieve the "perfect" body and a happier life. Because of this, my life revolved around food and exercise.
During these years, every decision revolved around food. If I was going to a social event, I made sure to drink a protein shake beforehand. If I was going on a road trip, I stocked up on anything I thought was clean so that I would not have to cheat on whatever diet I was following at the time. Not only was food in total control but my mind was also full of body-hate. At times, I judged my legs for being too muscular. My stomach was never flat enough. The cellulite on my legs constantly bothered me.
I felt alone in these struggles and ended up (ironically) turning to food to avoid my emotions. Eventually, this turned into binge eating and I used to hide what I ate from everyone. I had never felt more horrible and my body was not thriving. My mind was being overtaken by so much food anxiety and body-hate.
This anxiety I was having around food and my body was stopping me from living my life.
I finally started seeking help via therapy and started to educate myself at the Institute for Integrative Nutrition. Through therapy and self-discovery, I began the healing process. Once I finally stopped thinking of my food anxiety as a will power issue, my mentality began to change. I learned how to ditch the diet mentality and began listening to my own body. I started making food choices that my body was telling me to make. I started to love my body for everything that it was instead of wanting it to be something else.
This self-love and trust in my body ultimately helped me reach a place of total food freedom and body-love.
Flash forward to now and I have never felt better about life.
It took me a while to get out of that place of obsessive dieting and body-hate but I'm forever grateful for this journey. This journey taught me a lot about myself and what I'm meant to do in the world. I wholeheartedly believe that my mission in life is to help educate and empower women who are going through similar struggles.
I hope my story inspires you. I hope my social media brings you hope and positive energy that makes you believe in yourself. That is why I am here. My blog is there to empower you and help you take back your life. Stay up to date with current happenings via my bi-weekly newsletter.
My Education
In September 2015, I decided to go back to school to receive my health coaching certificate from the Institute for Integrative Nutrition. My education provided me with a solid background in how food affects our bodies and also the importance of holistic living.


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In This Section
Supported Playgroups are run by a qualified facilitator in a fun safe play environment and incorporates the 10-week parenting program Small Talk and in-home support. Small Talk is an evidence-based parenting program and has five key elements.
It aims to increase:
For eligible families with children 0-5 years the program includes:
Cost: Free
Eligibility information:
If one of the following applies to a family member in the household:
2019 - Term 4 timetable (please contact our Customer Focus Team on 03 5950 1099 for further information):
Crib Point
Briars Bush Playgroup
Vern Wright Reserve
Wallaroo - Hastings
Mornington Park
Seawinds -Capel Sound
In home support:
Join a playgroup:
To join a playgroup or receive in home support just complete the online form.
Join a playgroup
Service Providers:
Service providers can refer a client to the program through our simple online form.
Make a Referral
For further information contact:
Please get in touch with the Shire's Playgroup Facilitator, phone: 5950 1099 or email: firstname.lastname@example.com
Click here to view a promotional video from the state government on the importance of playgroups.
If you are deaf, or have a hearing or speech impairment, you can contact us through the National Relay Service.
Visit: National Relay Service
TTY/voice calls: 133 677 Speak and listen: 1300 555 727 SMS relay: 0423 677 767
Please be aware that CCTV cameras are in operation at a number of Shire buildings, including municipal offices, recreation centres and youth centres.
Mornington Peninsula Shire acknowledges and pays respect to the Boon Wurrung / Bunurong people, the traditional custodians of these lands and waters.

subset: commoncrawl


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Jenny Ye
Realtor & Broker Owner
With over 10 years of experience in the Boston area real estate market, Jenny brings local insights to our clients, with highest level of professionalism and personal attention.
Direct: 781-285-5898
Email: email@example.com
Long Li
Long is a a successful and experienced real estate agent coming with Biotech industry background.
Direct: 978-394-2668
Email: firstname.lastname@example.com
Xinyu Wu
Xinyu is fluent in English and Chinese (both Mandarin and Cantonese). She is ready to serve your real estate needs!
Direct: 781-698-9361
Email: firstname.lastname@example.com
Suzz Zhang
Direct: 781-201-0665
Email: firstname.lastname@example.com
Jianhua Zhou
Direct: 617-953-4160
Email: firstname.lastname@example.com
Leo Liu
Direct: 617-309-7549
Email: firstname.lastname@example.com


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Leica Camera has just unveiled it's latest camera in their prosumer TL-System line - the new Leica TL2. For those of you who have read my Leica T review, you'll remember that it was launched at a time where mirrorless cameras were showing signs of becoming the mainstream 'go-to' camera for many amateurs due to the performance to size ratio. Following the T came the world's first professional mirrorless camera in the SL, which set new standards for the mirrorless system by offering all the benefits of a mirrorless camera with the performance of a full-frame professional SLR. Leica then made some major firmware improvements to the T, and also released the upgrade in the Leica TL. Fast forward to the present, and we now have the Leica TL2.
Taking Street Photography to new limits using the Huawei P9 by Muhammad Imam Hassan
Social Media has taken online communications to the next level, giving photography enthusiasts a way to connect and share their work and inspire like-minded enthusiasts, be it of amatuer or professional status. Personally, I set very high standards for my own work, and the same set of standards for those photographers I admire. Every now and then you come across a new photographer you haven't heard of and I had that moment recently while viewing my Facebook timeline.
While doing my usual run down my news feed filled with the usual selfies, complaints about daily life, and crazy posts about Trump, I came across a picture by a picture posted by a guy named Muhammad Imam Hassan......
Leica T (typ 701) Mirrorless Camera Review
Quite possibly the most anticipated Leica camera since the M9, the Leica T (Typ 701) is finally here, well almost (in stores around 4 weeks from now). Launching with two lenses – a SUMMICRON-T 1:2/23 ASPH and a VARIO-ELMAR-T 1:3.5-5.6/18-56 ASPH, the camera starts off with a very sound lens combination that works very well together, with more lenses to come later this year. With the sensor being an APS-C Size with a magnification ratio of 1.5, those lenses become 35mm f/2 and a 27-84mm f/3.5-5.6 in full-frame 35mm terms. The camera will be launched in silver with a black option to come from July.
Zeiss Otus 55mm f/1.4 APO-Distagon on the Leica M (type 240) Review
If you saw my last blog post on using the Zeiss Otus on the Nikon Df, you'll know just how excited I am about this beast of a lens. Since writing my first impressions, there have been many questions as to whether this lens is as good as I say it is.......
Book Review | The Suffering of Light by ALEX WEBB
There are endless amounts of photography books available these days, and an endless line of photographers behind them. Amongst those are some of the elite photographers, and once in a while they create a body of work that leaves you absolutely speechless. One of those photographers (at least for me) is Alex Webb.
Leica M (typ 240) Field Test and Review
The latest camera in Leica's M line of digital rangefinders was a big surprise to many upon it's announcement. Looking at it's feature-set it's clear that Leica has taken a few new steps to compete against cameras in the mainstream market, including M4/3, NEX, Nikon 1 and even SLR's.


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CC writes:All the points in the KISS post are dead-on, but what if you are 40 or 45 and you haven't saved 25 times your income? This is where alternative investments MAY become viable. Are there any strategies (not get rich quick schemes) that MAY allow someone to (1) accelerate their LBYM and (2) take some risk to reach the point where their anticipated retirement needs can reasonably be met?Good point, and I think you know at least one of the answers to your question. One of the major drawbacks of the safe withdrawal rate study is that it speaks not at all to the real problem; assembling that starting nut. Because of its volatility, the stock market is an absolutely horrid place to be during the accumulation stage. For someone desiring very early retirement, you either get lucky or you fail. I prefer to eliminate luck as a consideration to the greatest extent possible. Since the volatility of the equities markets can create extended period of low returns, an equity heavy approach in the short accumulation period allowed to early retirees, is a pure crapshoot. LBYM, diversification and the use of investment approaches that involve real estate are advised.Regards,FMO
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Jesus Eats the Passover with His Disciples
12 On the first day of the Festival of Unleavened Bread, when it was customary to sacrifice the Passover lamb, Jesus' disciples asked him, "Where do you want us to go and make preparations for you to eat the Passover?" 13 So he sent two of his disciples, telling them, "Go into the city, and a man carrying a jar of water will meet you. Follow him. 14 Say to the owner of the house he enters, 'The Teacher asks: Where is my guest room, where I may eat the Passover with my disciples?' 15 He will show you a large room upstairs, furnished and ready. Make preparations for us there." 16 The disciples left, went into the city and found things just as Jesus had told them. So they prepared the Passover. 17 When evening came, Jesus arrived with the Twelve.


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"I've been in the online content world for over 25 years. Jamie is one of the best writers that I've worked with. She brought fantastic ideas to the project and brought her A+ game. Looking forward to working with Jamie again." "Easy to work with, delivered ahead of schedule, took feedback on with changes made promptly, would happily work with again and I recommend to anyone." Hi, my name is Jamie Johnson. I'm a personal finance freelance writer and blogger. I specialize in writing blog posts and articles about a variety of topics
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CKT Projects BV
Schiedamsedijk 128
3134 KK Vlaardingen
The Netherlands
+31 (0)10 2993555
CKT is a dedicated and reliable EPC contractor specialised in Design, Fabrication and Maintenance of complete (modular) Accommodations and Technical Buildings as well as Architectural Outfitting and Insulation Works for the Offshore and Maritime Industry.
With decades of experience CKT supplies:
- Turnkey Accommodation- & Technical Modules
- Outfitting Works on Naval, Merchant and Offshore Vessels
- Blast Resistant Modular Buildings
- Containerised Solutions
- Defence Logistic Support Equipment
- HVAC systems
- Architectural Outfitting and Insulation Works
CKT is also specialized in performing of multidisciplinary Maintenance Services, Upgrades and Refurbishments.
Our international footprint enables us to serve our global clients locally in the most efficient manner possible. Our services support our clients throughout the entire lifecycle: from FEED & Detailed Design to Corrective & Preventive Maintenance of their assets and Decommissioning/Dismantling.
CKT's strength lies in the extensive experience in our fields of business, combined with excellence in continuously improving safety, reliability and operational performance.
Ton Dorresteijn, Managing Director
Jean Paul van Hecke, Manager M&R
Peter van den Berge, Tender Manager


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Modelling with Molymods is absolutely essential to understanding condensation reactions between monomers and it is something that many students really enjoy.
I recommend that you do this as a whole class activity.
Careful management and guidance are needed so all students succeed in making glucose (and then proceed to carry out condensation reaction and form maltose with a glycosidic bond).
First discuss the nature of a hexose sugar and the (CH2O)n formula.
Then, count into the lid 6 Oxygen - Red, 12 Hydrogen - White, 6 Carbons - Black (make sure they all have 4 holes in them.
then join the carbons together in a chain
get the students to number the carbons from the bottom, then make a double bonded oxygen with the fifth carbon C5
Then put an Oxygen on each of the other carbons.
Then add the Hydrogens (white)
now fold the double bonded oxygen until it gets close to the C1.
this is an opportunity to discuss isomerism ....
And then, open one of the double bonds to the oxygen, remove a hydrogen from c1 and then bond the oxygen into the hole left by the removal of the hydrogen.
then move the spare H to the C5.
and you have now made glucose.
Next you should do a condensation reaction between the 1 and 4 carbons (of two glucose monomers) to form maltose, producing water and a glycosidic bond. Then join the maltose to form the polysaccharide amylose.
Finally, hydrolyse the amylose back to maltose (amylase does this in digestion).


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What to expect
On your first visit you will have a consultation with Dr. Durrer, a chiropractic exam, a thermograph and an EMG scan, and a foot scan. At your follow-up appointment you will have a report of findings consultation with Dr. Durrer at which time he will explain what your scans revealed and will share the care plan he recommends for you. At that time you will also receive your first chiropractic adjustment.
"Why do you offer these plans to people?"
It is my responsibility to have a plan for your care. That is a requirement that my license mandates I provide for the patient. There must be a course of action. "Without vision, the people perish." My own interpretation of this is, "Without a plan, the body perishes." I create a plan based on my experience, the patient's expectations, and the health status of the individual.
"What is the purpose of the care plan?"
The purpose is to give your body the best opportunity to heal and function with as little subluxation interference to your nervous system as possible. Have you ever learned something new? How hard is it the first few times you do it?
I have a patient who's an avid golfer. He decided to change his swing. For the next few months, his game was ruined. He played the worst golf of his life. But with time, he learned a new way, a better way. Now he is playing the best golf of his life and doing it pain-free. His old way was neurologically ingrained in his body. It was a habit.
Your old way of walking, thinking, carrying yourself, eating, moving, living, and working all change when you are under corrective care. It takes time to learn a new way of doing all these things! There is no magic pill to health or healing.
"Why don't you accept insurance?"
Quite simply, it is my responsibility as a care giver to give you the best care possible. When there is a third party involved it can often hinder that process. While we do not directly deal with insurance companies, what we are willing to do for you is provide you with a printed statement that you can submit to your insurance company for reimbursement as your personal policy allows.


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FSD Dual Stage schokdemper VB airsuspension voor de vooras Ducato type 250 nr. prijs per stuk
€ 577,65€ 477,40 excluding VAT
Item code
Op voorraad (4)
Give your opinion and be the first to write a review.
This form is meant for reviews only. Questions will not be published, please ask them by phone or e-mail instead, also see our contact details.


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SOME of the newest and most fascinating developments in applied science lie in the field of high polymers—and yet the story of high polymers, since they form the principal physico-chemical basis of life, is one of the oldest and most fundamental in the world. Most natural chain-molecules still cannot be synthesized by man; but he can build many others that are not found in Nature. Among the latter were thought to be the polyesters, first synthesized by Carothers and Arvin in 1929. It is reported now by A. R. Kemp and H. Peters (India Rubber World, 110, 639; 1944) that what seems to be very likely a polyester constitutes the highly elastic skin that fits tightly round the seeds of Smilax rotundifolia Linn. The ripe berries usually contain three seeds about ½ cm. in diameter, each enclosed in a membrane about 0-003 cm. thick. On removal, the membrane is found to be stretchable by 300–400 per cent and to give then a typical X-ray fibre photograph with a probable fibre period of about 22½ A. This finding, taken in conjunction with chemical analyses carried out on the skins both before and after hydrolysis with alcoholic caustic potash, indicates that the main component is a polyester formed by the repeated condensation of a unit having 17 or 18 carbon atoms in the chain with two hydroxyl side groups, the suggested empirical formula of the monomer being C18H36O5. The conclusions are for the present tentative, but they are by no means unconvincing, and the results of further investigation—very much worth while—will be awaited with interest.
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Further reading
Naturally Occurring Polyesters
Nature (1945)
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Woodland Meadow Farms Snohomish Wedding Photography by Entwined Weddings Seattle Snohomish Photographers PNW Wedding and Elopement PhotographyRead More
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Intimate Seattle Wedding at Parsons Gardens downtown Seattle elopement photography by Entwined WeddingsRead More
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Seattle couples photo session at Bothell Landing Park by Entwined Weddings Seattle elopement and wedding photographer.Read More
Discovery Park Engagement Photos by Seattle wedding photographers | Entwined Weddings Seattle
Intimate sunset engagement photo session at Discovery Park West Seattle | Entwined Weddings Seattle elopement proposal couples and wedding photography in Seattle and beyond!Read More
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Seattle engagement photos at the beach by Entwined Weddings Seattle Wedding Photographer. Elopement wedding and engagement photography in the PNWRead More
Romantic Winter Engagement Photos Snoqualmie Pass WA | Entwined Weddings PNW Photographers
Snowy Winter Engagement Photos in Snoqualmie Pass by Entwined Weddings PNW Adventure engagement photographers. Wedding Elopement and Engagement Photography in the PNW based in SeattleRead More


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Nathan Sutherland, the former Arizona nurse who is accused of raping and impregnating an incapacitated patient, has pleaded not guilty.
According to the New York Daily News, Sutherland entered the plea in Maricopa County Superior Court on Tuesday, and this comes two weeks after he was arrested on sexual assault and abuse of a vulnerable adult.
Recall that the 36-year-old was still working as a practical nurse at Arizona's Hacienda HealthCare, where an unidentified patient—who was originally reported to be in a vegetative state for over a decade—gave birth in late December.
Although the lawyer for the woman's family, John Micheaels, has clarified that the woman was not comatose or in a vegetative state, they insist, "The important thing here is that contrary to what's been reported, she is a person, albeit with significant intellectual disabilities. She has feelings and is capable of responding to people she is familiar with, especially family."
After the scandal surfaced, a DNA test of all every man who worked at the Hacienda, and Sutherland's DNA reportedly matched the woman's newborn.
Now, he pleads not guilty and we can't wait to see how this pans out.


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Joe Pichler has been missing from #Washington state since 2006!
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Today, I am thanking God for my infertility. I am not always good at doing this. Well, to be honest, frequently I can let the pain of infertility weigh heavily on my heart. It is easy to do this because the reminders of my barrenness are everywhere. However, I know that I can and I... Continue Reading →
Always Expecting
It wasn't supposed to be like this. In fact, it was supposed to be completely different. I glance a few feet from me to the Christmas tree, decked with ornaments my husband and I each brought from "another life," and those we share as witnesses to a union that took us by surprise. Our first... Continue Reading →


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All posts tagged "Charlotte Hornets"
Lopez, Mirotic, DiVincenzo make big contributions in Bucks victory over Hornets
Brook Lopez, Nikola Mirotic and Donte DiVincenzo carried enough of the load Saturday night, so when Giannis Antetokounmpo did his usual...
Big quarter for Lopez gives Bucks lead entering fourth
Brook Lopez scored 18 third-quarter points, all of them inside the arc, as he helped the Milwaukee Bucks turn a 59-55...
Bucks off to poor start against Hornets
Cold shooting has put the Milwaukee Bucks into a deficit after one quarter. While the Bucks have made just 4 of...
Bucks host Hornets looking for 50th victory
The schedule favors the Milwaukee Bucks tonight as they take on the Charlotte Hornets at Fiserv Forum. The Hornets are playing...
Live Updates/ 8 months ago
Connaughton brings needed energy as Bucks rally past Hornets
Pat Connaughton didn't score. He collected two rebounds and one assist in 12 minutes. And yet he may have been the...
Live Updates/ 8 months ago
Bucks struggle, trail Hornets by two at half
Poor shooting, even on opportunities in the paint, have plagued the Milwaukee Bucks tonight, and they take a 54-52 deficit into...
Page 1 of 212


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As the polls open for the 2014 European Parliament elections, the PDU gives ten reasons why should bother voting.
- The European Parliament will only get stronger, compared to theTroika, if it has a democratic mandate. If we want a more democratic Europe, we need to ask for it at theballot box.
- We need a pan-European response to the events in Ukraine. If the crisis on our eastern border has shown anything, it is that member-states cannot afford to follow their completely separate agendas with regards to security.
- After so much effort to promote the idea of democratic change in Europe, a failure to support theParliament would send a very bad signal about the tolerance of bureaucratic rule.
- Voting is a symbol of continental solidarity 100 years after the start of the First World War.
- An unused vote is effectively a vote for the Eurosceptics – and if you're thinking of voting Eurosceptic, see if our appeal changes your mind.
- Under the Lisbon Treaty, this could be the first time that Parliament nominates the president of the European Commission. As we have seen from the TV debates, we have a strong range of candidates to represent the people of Europe in the higher bureaucratic echelons.
- The elections are run on proportional representation, so you can actually vote for who you want to rather than for who might win. This will be of particular novelty to voters in those countries, like Britain, who are used to the first-past-the-post system.
- We need a pan-European approach to environmental issues, particularly climate change. After the sluggish outcome of the Warsaw Conference, this is even more important.
- We need to keep democracy and the rule of law as attractive ideals for future member-states. The possibility of accession to the EU has provided a major incentive for countries to improve their governing structures – we should not sacrifice this now.
- It is the first chance to express opinion on a European level since the start of the eurozone crisis. Whether you are for austerity, or against it, or somewhere in between, then you can have your say on Europe's economic future.
Image: 'The hemicycle of the European Parliament in Brussels.′ courtesy of Ash Crow via Wikimedia Commons, released under Creative Commons.


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According to Bloomberg, Google advertisers have been able to track the engagement level of ads they ran online: whether it has driven sales at a physical store in the US. The data in part came from Mastercard transactions that Google paid for. Most of Mastercard holders are unaware of this data extraction because the companies never made an official announcement to the public.
"People don't expect what they buy physically in a store to be linked to what they are buying online," said Christine Bannan, counsel with the advocacy group Electronic Privacy Information Center (EPIC). "There's just far too much burden that companies place on consumers and not enough responsibility being taken by companies to inform users what they're doing and what rights they have."
According to close sources, both companies settled on a business agreement where Google was able to measure consumer spend online as part of its strategy to cut through the competition. Since the deal never went public it raises privacy concerns about consumer data. However, "Before we launched this beta product last year, we built a new, double-blind encryption technology that prevents both Google and our partners from viewing our respective users' personally identifiable information," the company said in a statement. "We do not have access to any personal information from our partners' credit and debit cards, nor do we share any personal information with our partners."
A Mastercard spokesperson Seth Eisen declined to comment on Google. However, he added that Mastercard shares transaction trends with merchants and service providers in order to reinforce "the effectiveness of their advertising campaigns."
"No individual transaction or personal data is provided," he said in a statement. "We do not provide insights that track, serve up ads to, or even measure ad effectiveness relating to, individual consumers."


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The latest entry in our guestbook reads:
"We have had a totally relaxing week in this gorgeous apartment… peaceful, spacious, well-equipped and beautifully decorated! We are going home with our batteries re-charged. Many thanks for the wine."
To view more feedback from our guests, please see our guests' comments page.


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Michael Chapman
VDSQ Solo Acoustic Vol. 11
Release Notes
- Recently appeared on BBC's Later with Jools Holland for the first time
The godfather of alternative guitar returns with an all-instrumental album, Homages, in tribute to his influences and contemporaries. Summoning the spirits of some of the greatest guitarists of all time, Chapman proves again that he stands among them.


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The country research report on Brazil automotive sun visor market is a customer intelligence and competitive study of the Brazil market. Moreover, the report provides deep insights into demand forecasts, market trends, and, micro and macro indicators in the Brazil market. Also, factors that are driving and restraining the automotive sun visor market are highlighted in the study. This is an in-depth business intelligence report based on qualitative and quantitative parameters of the market. Additionally, this report provides readers with market insights and detailed analysis of market segments to possible micro levels. The companies and dealers/distributors profiled in the report include manufacturers & suppliers of automotive sun visor market in Brazil.
The report on Brazil automotive sun visor market provides a detailed analysis of segments in the market based on the surface material, type, vehicle type, and sales channel.
· Vinyl
· Fabric
· LCD Sun Visor
· Conventional Type
· Commercial Vehicle
· Passenger Vehicle
· Electric Vehicle
· Aftermarket
The report provides detailed insights into:
1) Demand and supply conditions of automotive sun visor market
2) Factor affecting the automotive sun visor market in the short run and the long run
3) The dynamics including drivers, restraints, opportunities, political, socioeconomic factors, and technological factors
4) Key trends and future prospects
5) Leading companies operating in automotive sun visor market and their competitive position in Brazil
6) The dealers/distributors profiles provide basic information of top 10 dealers & distributors operating in (Brazil) automotive sun visor market
7) IGR Matrix: to position the product types
8) Market estimates up to 2024
The report answers questions such as:
1) What is the market size of the automotive sun visor market in Brazil?
2) What are the factors that affect the growth in automotive sun visor market over the forecast period?
3) What is the competitive position in Brazil automotive sun visor market?
4) What are the opportunities in Brazil automotive sun visor market?
5) What are the modes of entering Brazil automotive sun visor market?
1. Report Overview
1.1. Report Description
1.2. Research Methods
1.3. Research Approaches
2. Executive Summary
3. Market Overview
3.1. Introduction
3.2. Market Dynamics
3.2.1. Drivers
3.2.2. Restraints
3.2.3. Opportunities
3.2.4. Challenges
3.3. PEST-Analysis
3.4. Porter's Diamond Model for Brazil Automotive Sun Visor Market
3.5. IGR-Growth Matrix Analysis
3.6. Competitive Landscape in Brazil Automotive Sun Visor Market
4. Brazil Automotive Sun Visor Market by Surface Material
4.1. Vinyl
4.2. Fabric
5. Brazil Automotive Sun Visor Market by Type
5.1. LCD Sun Visor
5.2. Conventional Type
6. Brazil Automotive Sun Visor Market by Vehicle Type
6.1. Commercial Vehicle
6.2. Passenger Vehicle
6.3. Electric Vehicle
7. Brazil Automotive Sun Visor Market by Sales Channel
7.1. Aftermarket
8. Company Profiles
8.1. Manufacturer's & Suppliers
8.1.1. Company 1
8.1.2. Company 2
8.1.3. Company 3
8.1.4. Company 4
8.1.5. Company 5
8.2. Dealer's / Distributor's Profile
8.2.1. Dealer's / Distributor's 1
8.2.2. Dealer's / Distributor's 2
8.2.3. Dealer's / Distributor's 3
8.2.4. Dealer's / Distributor's 4
8.2.5. Dealer's / Distributor's 5
8.2.6. Dealer's / Distributor's 6
8.2.7. Dealer's / Distributor's 7
8.2.8. Dealer's / Distributor's 8
8.2.9. Dealer's / Distributor's 9
8.2.10. Dealer's / Distributor's 10
Get free analyst support for the queries before and post purchasing report.
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The a subunit of F1Fo (F1Fo-ATP synthase) is a highly hydrophobic protein with five putative transmembrane helices which plays a central role in H+-translocation coupled with ATP synthesis/hydrolysis. In the present paper, we show that the a subunit produced by the in vitro protease-free protein synthesis system (the PURE system) is integrated into a preformed Fo a-less F1Fo complex in Escherichia coli membrane vesicles and liposomes. The resulting F1Fo has a H+-coupled ATP synthesis/hydrolysis activity that is approximately half that of the native F1Fo. By using this procedure, we analysed five mutations of F1Fo, where the conserved residues in the a subunit (Asn90, Asp112, Arg169, Asn173 and Gln217) were individually replaced with alanine. All of the mutant Fo a subunits were successfully incorporated into F1Fo, showing the advantage over conventional expression in E. coli by which three (N90A, D112A, and Q217A) mutant a subunits were not found in F1Fo. The N173A mutant retained full activity and the mutants D112A and Q217A had weak, but detectable, activity. No activity was observed for the R169A and N90A mutants. Asn90 is located in the middle of putative second transmembrane helix and likely to play an important role in H+-translocation. The present study exemplifies that the PURE system provides an alternative approach when in vivo expression of membranous components in protein complexes turns out to be difficult.
- a subunit
- cell-free protein synthesis
- F1Fo-ATP synthase (F1Fo)
- H+-pump
- membrane protein
- proteoliposome
F1Fo (F1Fo-ATP synthase) is ubiquitously found in the membranes of bacteria, chloroplasts and mitochondria, and synthesizes most of the cellular ATP coupled with translocation of H+ (and in some cases Na+) [1–3]. F1Fo is composed of two portions: a membrane integral Fo (subunit composition, ab2cn(10–15) in most bacteria) and a water-soluble catalytic portion F1 (subunit composition, α3β3γδϵ). Using the energy of downhill H+-translocation, Fo drives rotation of the cn ring and its connecting γϵ subunits, which induces a sequence of reactions of ATP synthesis in F1. Of the three kinds of Fo subunit, the a subunit is a 26 kDa protein containing five TMHs (transmembrane helices) [4–6]. Since H+ channels are present inside the a subunit it plays a central role in H+-translocation. When the a subunit was genetically eliminated, it resulted in formation of F1Fo which had lost all of its Fo-mediated functions [7], i.e. neither ATPase-driven H+-translocation nor ATP synthesis proceeded. Mutation studies have identified the key residues responsible for H+-translocation [8–11], such as Arg169 (Arg210 in Escherichia coli numbering) [12–15], and insertion-scanning studies have suggested the membrane topology of the five TMHs [4,6,16]. Fillingame and colleagues have extensively surveyed the a subunit of E. coli Fo by cysteine scanning combined with the reactivity of Ag+ [17–19]. Further mutation analyses are still necessary, but these studies often encounter the difficulty that the mutant a subunit is found only scarcely [4,6,9,17,20] or not at all [8,15] in membranes. The same difficulty has widely limited the versatility of mutagenesis studies of other membrane proteins [21–26].
Recent progress in cell-free protein synthesis methods enables us to quickly prepare the target membrane proteins in a combination with liposomes. Several active membrane proteins, such as bacteriorhodopsin [27] and GP91phox which is the catalytic core protein of the NADH oxidase complex [28], have been prepared by cell-free systems previously [29–38]. The method (hereafter called in vitro expression) is simple when compared with the recombinant cell methods (hereafter called in vivo expression). In vitro expression normally requires only addition of template DNA to the reaction mixture and incubation for several hours to produce the target proteins. With available mutant template DNA, mutant proteins can be obtained within just one day. This advantage becomes evident when a large number of proteins have to be tested, e.g. analyses of proteomic [39] and alanine-scanning [40] studies. On the other hand, it is also true that the in vitro expression method contains a basic weakness that unrecognized elements in the cell-extract solution sometimes bring unexpected side reactions, e.g. proteolytic degradation, action of chaperones and protein modification. Such effects make the result complicated, in the worst cases leading to misinterpretation of the assay results. To eliminate such risks, we had established a novel in vitro cell-free protein synthesis system (the PURE system) in which all of the components necessary for protein synthesis are highly purified and assembled [41]. This means that the components in the solution are all defined and unwanted side reactions do not occur during protein synthesis reactions.
Our previous study showed that F1Fo from thermophilic Bacillus PS3 has an ability to form the complex without the a subunit, and furthermore that the complete F1Fo complex can be formed in vitro by supplementing the purified a subunit [7]. In the present study, the a subunit was synthesized by the PURE system in the presence of a subunit-less F1Fo in the form of PL (proteoliposome). Remarkably, F1Fo recovered from the reaction mixture gained H+-translocation activity driven by ATP hydrolysis and ATP synthesis activity driven by H+-translocation. This indicates that the synthesized a subunit was inserted into membranes and integrated into a subunit-less F1Fo to form active F1Fo. F1Fos with mutant a subunits were also obtained by the PURE system in the presence of a subunit-less F1Fo. In comparison with in vivo expression, where some mutant a subunits were lost and not integrated into F1Fo, almost the same amount of a subunit, irrespective of mutation, was produced and integrated into F1Fo, enabling analysis of these mutations.
Construction of expression vectors
The expression vector for Bacillus PS3 F1Fo, pTR19-ASDS [42], expresses an active F1Fo complex in E. coli cells, but the unc operon encoding the F1Fo subunits on the vector lacks the uncI gene. Therefore a DNA fragment containing uncIBE′ was amplified by PCR from the plasmid pUC-F1Fo [43] and introduced into the vector in order to restore uncI to its original position on the Bacillus PS3 unc operon. The resulting plasmid was named pTR-ISBS2. A mutation, genetically lacking the whole of uncB (encoding the a subunit), was transferred from pTR19-ASDSΔa [7] to pTR-ISBS2. The resultant plasmid, pTR-ISBS2Δa, was used for IMV (inverted inner-membrane vesicle) analyses. For PL analysis, a derivative of pTR19-ASDSϵΔc was used instead of pTR-ISBS2Δa because the measuring system for ATP synthesis activity in PLs was optimized for the plasmid system. There is no detectable difference between the two plasmid systems when their Fo functions were compared with each other. The plasmid pTR19-ASDSϵΔc has a mutation at the ϵ subunit by which the C-terminal domain of ϵ was truncated to eliminate the inhibitory effect (long-lag phase) of the ϵ subunit [44]. This results in rapid appearance of ATP synthesis activity. The mutation can be assumed to have no effect on Fo function itself because the mutated region, the C-terminal helices, has no direct interaction with the Fo part [45]. For a subunit synthesis, uncB was amplified by PCR solely from pTR-ISBS2 and introduced into pET-29 at the NdeI/EcoRI sites. The resulting plasmid, pET-a, was used as a template DNA. In all plasmids, the regions prepared by PCR amplification were verified by nucleotide sequencing. Mutations at the a subunit were introduced using synthetic oligonucleotides produced by PCR into pTR-ISBS2 by the Megaprimer method as described previously [42,46].
Preparation of membrane vesicles and purification of F1Fo
Expression of F1Fo in E. coli cells and preparation of membrane vesicles were performed as described previously [42]. In the present study, the vesicles were further purified by the sucrose-density gradient method to isolate IMVs as follows: membrane vesicles dissolved in PA3 buffer [10 mM Hepes/KOH (pH 7.5) containing 5 mM MgCl2 and 10% glycerol] were supplemented with 10 mM EDTA and subjected to brief sonication (10 s). The resulting vesicles were carefully put on the top of a stepwise gradient of sucrose (0.8, 1.4, 2.0 M) in 10 mM Hepes/KOH (pH 7.5) containing 10% glycerol. Bacillus PS3 F1Fo did not disassemble even after this procedure because of its high structural stability. The resulting solution was ultracentrifuged at 32000 rev./min for 2 h at 4°C using a P40ST rotor (Hitachi). After centrifugation, a brown layer, located between 0.8 M and 1.4 M sucrose layers, was carefully isolated. The fraction was supplemented with 5 mM MgCl2, diluted 4-fold with 10 mM Hepes/KOH buffer (pH 7.5) containing 5 mM MgCl2, and then ultracentrifuged at 55000 rev./min for 20 min at 4°C using a S55A rotor (Hitachi). The pellets were suspended in PA3 buffer and used as IMVs for the following analyses.
Purification of F1Fo and quantification of the F1Fo preparation was performed as described previously [46]. In the present study, the F1Fo preparation was further purified by anion-exchange column chromatography (ResourceQ, GE Healthcare) in 10 mM Hepes/NaOH buffer (pH 7.5) containing 0.2 mM EDTA. F1Fo was eluted from the column with a linear gradient of NaCl (0–0.5 M), concentrated by a centrifugal concentrator (50 kDa, Amicon), frozen by liquid N2 and then stored at −80°C until use.
Cell-free protein synthesis by the PURE system
In vitro synthesis of the a subunit was performed using a reconstructed cell-free translation system, the PURE system [41], in the presence of either 0.9 mg of membrane proteins/ml IMVs or 0.12 mg/ml PLs containing F1FoΔa. Briefly, the PURE system consists of aminoacyl-tRNA synthetases, translational factors, ATP-regenerating systems, T7 RNA polymerase, 70S ribosome and low molecular mass compounds. As the template DNA, either 20 μg/ml pET-a or 5 μg/ml uncB DNA with a fragment-attaching T7 promotor sequence at the 5′ terminus, amplified by PCR, was supplemented into the reaction mixture. The reaction mixture was incubated for 30 min at 30°C and then terminated by placing the solution on ice as described previously [41]. Preliminary experiments showed that a reaction time of 30 min was sufficient to obtain an active F1Fo complex, and that no further increase in H+-translocating and ATP synthesis activities was observed even after prolonged reaction time. To analyse the H+-translocating activity, a further purification step is not necessary and therefore the reaction mixtures were tested directly. When measurement of ATP synthesis activity was required, the following purification step was added to eliminate the components required for ATP regeneration (adenylate kinase and creatine kinase) from the reaction mixture: the reaction mixture was diluted 4-fold with 10 mM Hepes/KOH (pH 7.5) containing 5 mM MgCl2. The IMVs or PLs in the mixture were then collected by ultracentrifugation [58000 rev./min for 20 min at 4°C using a S80AT3 rotor (Hitachi)], washed twice with the buffer and suspended in PA3 buffer.
In order to assess the incorporation of the synthesized a subunit into the F1FoΔa complex, after the synthesis reaction F1Fo was purified from the reaction mixture. As F1Fo has a His tag (10 histidine residues) at N-terminus of its β subunit, the purification was performed by using Ni-charged magnetic beads (MagneHis Protein Purification system, Promega), according to the manufacturer's protocol with some modifications. To the reaction mixture (50 μl), 100 μl of 50 mM Tris/HCl (pH 8.0) buffer containing 300 mM NaCl, 5 mM MgCl2, 20 mM imidazole and 1% DDM (n-dodecyl-β-D-maltoside) was added. The resulting mixtures were incubated at room temperature (25°C) for 1 h with gentle mixing prior to the purification step. After the addition of the magnetic beads, the beads were collected and washed twice with 150 μl of the Tris/HCl buffer containing 0.05% DDM. F1Fo was then eluted with the Tris/HCl buffer containing 500 mM imidazole and 0.05% DDM. The eluted fraction was subjected to acetone precipitation to concentrate F1Fo. The purifications of the wt (wild-type) and mutants were performed under the same conditions at the same time. The F1Fo preparations obtained were analysed by SDS/PAGE (10–20% gel) and immunoblotting using anti-a antibody.
Measurements of ATP synthesis
ATP synthesis activity in IMVs was measured at 35°C in 100 mM Hepes/KOH (pH 7.5) containing 5 mM MgCl2, 0.5 mM ADP, 10 μM Ap5a [P1,P5-di(adenosine-5′)pentaphosphate], 10 mM potassium phosphate and 1:10 vol. of luciferin/luciferase mixture CLSII (ATP Bioluminescence Kit, Roche) as described previously [46]. The synthesis reaction was initiated by adding 3.5 mM sodium succinate. Production of ATP was measured in real-time by monitoring luminescence (light intensity) from the reaction mixture at 560 nm by using a spectrofluorometer (FP6500, JASCO) without excitation light. To convert the light intensity into the ATP concentration value, defined amounts of ATP were added to the assay mixture at the end of each measurement. The activity that synthesized 1 μM ATP per min was defined as 1 unit.
Measurement of the ATP synthesis activity of PLs was done by the acid/base-transition method, combined with valinomycin-induced diffusion of potassium ion. This procedure was established by modifying the method of Fischer et al. [47] and was published recently [48]. Briefly, F1Fo PLs were prepared from a solution containing 32 mg/ml soybean L-α-phosphatidylcholine (TypeII-S, Sigma), 0.6 mg/ml F1Fo preparation and 6% n-octyl-β-D-glucopyranoside (Anatrace) by eliminating the detergent using Bio-Beads (SM-2, Bio-Rad Laboratories). The resulting PLs (final F1Fo concentration was 0.3 mg/ml) were acidified by soaking in acidification buffer (20 mM succinate, 0.6 mM KOH, 2.5 mM MgCl2, 10 mM NaH2PO4, 0.7 mM ADP, 1 μM Ap5a and 280 nM valinomycin, adjusted to pH 4.5 by NaOH) for 4 min at room temperature. The acidified PLs (30 μl) were then rapidly injected into Base buffer (1 ml) containing 200 mM Tricine, 130 mM KOH, 2.5 mM MgCl2, 10 mM NaH2PO4, 0.5 mM ADP, 1 μM Ap5a and 0.1 ml ATP luminescence assay kit CLS II reagent (Roche), adjusted to pH 8.8 by NaOH. Changes in the luminescence from luciferin/luciferase were monitored at 37°C using a luminometer (Luminescencer-PSN AB-2200, ATTO). To calibrate the luminescence intensity as ATP concentration, 200 nM ATP was added into the reaction mixture at the end of each measurement.
Other analytical methods
ATPase-driven H+-translocation activity was analysed by monitoring fluorescence quenching of ACMA (9-amino-6-chloro-2-methoxyacridine) (excitation at 410 nm and emission at 480 nm) as described previously [42] at 42°C in 10 mM Hepes/KOH buffer (pH 7.5) containing 100 mM KCl, 5 mM MgCl2 and 0.3 μg/ml ACMA. Additionally, 2.5 mM creatine phosphate and 25 μg/ml creatine kinase were added to the assay mixture to regenerate ATP during measurement. The reaction was initiated by adding 1 mM ATP, and terminated with 0.25 μg/ml FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone). All experiments were performed at least three times and the averages are shown. Protein concentrations of membrane vesicles were determined using a BCA (bicinchoninic acid) protein assay kit from Pierce, with bovine serum albumin as a standard. All SDS/PAGE in the present study used a gradient polyacrylamide gel (10–20%).
Expression of an F1Fo mutant lacking the a subunit, F1FoΔa, in E. coli cells
The a subunit-less F1Fo (F1FoΔa) was expressed from the expression plasmid, pTR-ISBS2Δa, in an F1Fo-deficient E. coli strain, DK8 [42]. Western blotting analysis of IMVs, prepared from the recombinant cells, confirmed that F1FoΔa was stably expressed into the membrane fraction of the E. coli host cells (Figure 1A). The band intensity of α and β subunits of the gel (Figure 1A, lanes 2 and 3) suggests that the expression level of F1FoΔa was comparable with that of the wt-F1Fo. Immunoblot analysis using anti-a antibody showed no detectable a subunit in the IMVs of F1FoΔa (Figure 1B, lane 3), but a clear band for wt-F1Fo (Figure 1B, lane 2). F1Fo complexes were purified from respective IMVs for evaluation of their subunit stoichiometry (Figure 1C). With the exception of the absence of the a subunit, other subunits were similarly observed on SDS/PAGE gels (Figure 1C), which indicate the stable nature of F1FoΔa. This result is consistent with an earlier finding that F1Fo still remains stable even when the a subunit is absent [7].
Formation of active F1Fo on IMVs by in vitro expression of the a subunit
The a subunit was synthesized by using the PURE system in the presence of the IMVs. The reaction was performed with or without the plasmid pET-a which contains the uncB gene (encoding the a subunit). After the synthesis reaction, IMVs were isolated by ultracentrifugation, washed with buffer and then analysed. Figures 1(A) and 1(B) show SDS/PAGE and immunoblot analyses of the IMVs reconstituted with in vitro-synthesized a subunit (lanes 4–6). Because ribosome components were also simultaneously precipitated by the centrifugation step, the band intensity of some proteins increased by overlaying of these components (Figure 1A). Immunoblot analysis using anti-a antibody (Figure 1B) clearly showed integration of the a subunit into F1FoΔa IMVs (lane 6), the amount of which was comparable with that of wt-F1Fo (lane 4).
ATPase-driven H+-translocating activity in the IMVs reconstituted with in vitro-synthesized a subunit was analysed by monitoring fluorescence quenching of ACMA, which is a ΔpH indicator of membrane ΔpH. In the absence of uncB DNA, the IMVs showed no quenching of fluorescence after the addition of ATP (Figure 1D, uncB− trace). The rapid quenching observed just after the addition of ATP is due to a direct interaction between ATP and the ACMA fluorophore. In contrast, significant quenching was observed when uncB was present during the synthesis reaction (Figure 1D, uncB+ trace). As a control, the synthesis reaction was performed with wt IMVs in the presence of uncB (Figure 1D, WT-uncB+ trace). The initial quenching rate of the F1FoΔa IMVs when reacted with uncB DNA showed 32% of the quenching observed for wt IMV, when fluorescence changes were assessed 2–20 s after the addition of ATP. The ATP synthesis activity of the IMVs reconstituted with in vitro-synthesized a subunit was assessed by a real-time monitoring method using luciferin/luciferase (Figure 1E). As shown in the WT-uncB+ trace, a linear increase in light intensity was observed in authentic wt-F1Fo IMVs that were prepared through the synthesis reaction in the presence of uncB. Although no increase in luminescence was observed in F1FoΔa IMVs without uncB (uncB− trace), a moderate, but obvious, increase in luminescence was observed in F1FoΔa IMVs reacted with uncB (uncB+ trace). ATP synthesis activities were determined as 13.4 m-units/mg of membrane proteins in wt-F1Fo with uncB, and 2.6 or 0.01 m-units/mg of membrane proteins in F1FoΔa with or without uncB. The result showed that the ATP synthesis function was acquired due to synthesis of the a subunit, although the activity was relatively lower than that of wt-F1Fo.
In vitro expression of the a subunit with F1FoΔa PLs
Recently, we have established a new assay system for ATP synthesis activity using PLs [48]. The expression vector (pTR19-ASDSϵΔc) necessary for the measurement has a mutation in the C-terminal domain of the ϵ subunit to eliminate an inhibitory effect of the ϵ subunit. A mutation lacking uncB was introduced into the vector to obtain pTR19-ASDSϵΔc-Δa. The a subunit-less F1Fo mutant, named F1Fo(ϵΔc)Δa, was stably expressed in E. coli DK8 cells and purified by the same method as wt-F1Fo. The purified F1Fo was reconstituted into soybean phosphatidylcholine to obtain F1Fo(ϵΔc)Δa PLs. An in vitro expression reaction of the a subunit was then performed in the presence of the PLs. Figures 2(A) and 2(B) show SDS/PAGE analyses of the reacted PLs, which were reacted without (lane 2) or with (lane 3) the pET-a plasmid. In immunoblot analysis of the reacted PLs (Figure 2B), a clear band was detected at the position of the a subunit (lane 3), although a few faint bands were also observed above the a subunit band.
The resultant PLs were further analysed for ATPase-driven H+-translocating activity (Figure 2C). PLs in the presence of uncB DNA (uncB+ trace) exhibited significant fluorescence quenching of ACMA upon the addition of ATP, whereas PLs without uncB DNA (uncB− trace) showed no quenching activity at all. This indicates that the appearance of H+-translocating activity resulted from a subunit synthesis. Quenching velocity in F1Fo(ϵΔc)Δa PLs with de novo-synthesized a subunit was approximately 52% of the wt PLs (WT-uncB+ trace). Subsequently, ATP synthesis activity of the reacted PLs was also analysed by using an acid/base-transition method combined with a K+-induced membrane potential (ΔΨ) in combination with valinomycin. The amount of ATP synthesized by F1Fo was assessed by monitoring the luminescence of the assay mixture. F1Fo(ϵΔc)Δa PLs reacted with uncB DNA were isolated by ultracentrifugation, acidified and then added to the assay mixture. The addition of PLs (indicated with an arrow) resulted in a rapid increase in luminescence light intensity (Figure 2D, uncB+ trace). When the synthesis reaction was performed without uncB, addition of PLs induced no increase in the light intensity (Figure 2D, uncB− trace). When ΔμH+ was dissipated by supplementing with the K+/H+-exchanging ionophore nigericin, the increase in luminescence almost disappeared (Nig+uncB+ trace). These results suggest that F1Fo(ϵΔc)Δa on PLs acquired the ATP synthetic function by synthesizing the a subunit. When compared with the activity of wt-F1Fo (Figure 2D, WT-uncB+ trace), the activity of F1Fo(ϵΔc)Δa PLs that reacted with uncB was 51%.
Mutation analysis of a subunits by the in vitro protein expression system
Figure 3 shows a sequence alignment for a subunits of E. coli, Propionigenium modestum and Bacillus PS3. The sequence identities of a subunit of Bacillus PS3 are moderately similar to those of E. coli (31%) and P. modestum (25%). In the regions of the five TMHs [19], eight polar residues are conserved among the three a subunits. Of the eight, five polar residues, Asn90 (TMH2), Asp112 (TMH3), Arg169 (TMH4), Asn173 (TMH4) and Gln217 (TMH5), were selected (Figure 3, marked by boxes), and were each replaced by alanine (N90A, D112A, etc.). The five mutants were then subjected to analyses using the in vitro or in vivo expression methods. First, the in vitro expression method was performed. DNA fragments of uncB including the T7 promoter and Shine–Dalgarno sequences were prepared by a two-step PCR (Figure 4A). Next, the variants of the a subunit were synthesized from the amplified uncB fragment in the presence of F1Fo(ϵΔc)Δa PLs. Immunoblot analysis by anti-a antibody of the reaction mixture indicated that similar amounts of a subunits were synthesized from the wt and mutated uncB DNAs (Figure 4B). H+-translocating activity in the reacted F1Fo(ϵΔc)Δa PLs was directly analysed after the synthesis reaction without any purification step of PLs (Figure 4C). The result from this series of experiments was that the N173A mutant showed strong H+-translocating activity, which was comparable with the wt. Two other mutants, D112A and Q217A, demonstrated weak activity, which indicated that side chains of the two residues, though important, are not obligatory for H+-translocation. On the other hand, the N90A and R169A mutants demonstrated no ACMA quenching by ATP, which suggests that these residues are required by the enzyme to function. To clarify whether these differences in the activities are faithfully reflecting the effect of the mutation, F1Fo was purified from the reaction mixture of the a subunit synthesis by using Ni-charged magnet beads (His-tags are present at the β subunit). The yields of the isolated F1Fo were almost same as judged from band intensities of α and β on the CBB (Coomassie Brilliant Blue)-stained SDS/PAGE gel (Figure 4D). An anti-a antibody immunoblot analysis showed that F1Fo of the mutants contains a similar amount of a subunit when compared with the wt (Figure 4E). These data suggest that the mutated a subunits were incorporated into F1FoΔa complex at a similar level to the wt a subunit.
Secondly, the in vivo expression method was carried out in a conventional manner. Expression plasmids for the five mutated F1Fo and wt-F1Fo were introduced into E. coli DK8, and F1Fo was expressed by cultivating the recombinant strains. SDS/PAGE analysis of prepared IMVs showed that all of the mutants more or less decreased the expression levels of F1Fo, especially R169A, judging from the band intensities of the α and β subunits (Figure 5A). Figure 5(B) shows that of the five, only N173A showed H+-translocating activity, even though substantial amounts of the mutated F1Fo was detected in IMVs in the four mutants (Figure 5A). The apparent inconsistency can be explained by results from anti-a antibody immunoblot analysis where three mutants (N90A, D112A and Q217) had no detectable a subunit in the IMVs fraction (Figure 5C).
Figure 5(D) summarizes the results of H+-translocating activities obtained from the in vitro expression (Figure 4C) and in vivo expression (Figure 5B) methods. In order to compare the results of both methods, each initial fluorescence-quenching rate was determined from the respective traces just after ATP addition, and their values relative to the wt moiety were presented. This comparison demonstrates that there are critical differences between the results from the in vitro and in vivo expression methods with regard to the D112A and Q217A mutants. The activities of these two mutants were detected only in the in vitro expression method.
In the present study, the a subunit of Bacillus PS3 F1Fo was successfully synthesized by the PURE system. Remarkably, when an a subunit-less F1Fo was added to the reaction mixture, it acquired Fo functions after synthesizing the a subunit without any additional agent. This result indicates that the synthesized a subunit was spontaneously integrated into membranes, folded correctly and complexed with the a subunit-less F1Fo to form active F1Fo. The results from mutation analysis of a subunits produced by the in vitro expression method were compared with results from a subunits produced by the in vivo expression method. The two methods produced analogous results for some mutants; however, differences were found for mutants of D112A and Q217A. As shown in Figure 5(D), the in vitro expression method provided a small, but substantial, amount of activity for these mutants (see also Figure 4C), in contrast to the 'normal' in vivo expression results which showed no detectable H+-translocating activity (Figure 5C). The reason for this difference is explained by immunoblot analysis using anti-a antibody, which revealed that the a subunit was absent in the IMVs of the mutants. The results from the two mutants might have been affected by proteolytic digestion of a subunits in the IMVs. In fact, when the Bacillus PS3 a subunit was expressed in E. coli cells without other F1Fo subunits, a short expression time (2 h) was optimal to accumulate the a subunit on the membrane fraction [7], which implies that the a subunit is sensitive to protease. Such decreased accumulation of the a subunit has been frequently observed in E. coli a subunit studies [4,6,8,9,15,17,20].
The in vitro expression procedure clarified the necessity of the five conserved residues. Of the five, Asp112 and Gln217 were important for ATPase-driven H+-translocating activity, but were not absolutely required (Figure 4C). This result is in good agreement with that from previous E. coli studies [17,18] where E. coli residues corresponding to Asp112 and Gln217 of Bacillus PS3 were suggested to lie on H+-translocating channels [17,18]. Additionally, the obligate role of the side chain of Asn90 and Arg169 was suggested. Arg169 (Arg210 and Arg226 in E. coli and P. modestum respectively) has been widely known as one of the most important residues in the a subunit [12–15]. Asn90 is located in the middle of the second putative TMH and also appears to play an indispensable role.
The PURE system represents a different environment from that of intact cells or cell extract, since, in the PURE system, protease is absent and therefore abnormal proteins are not destroyed by proteolytic digestion. This observation suggests that the PURE system conditions allow the a subunit to form the correct conformations on a lipid bilayer without susceptibility to protease attack during expression (Figure 4E). This advantage of the PURE system enables us to analyse such 'difficult-to-produce proteins' that cannot be prepared by the 'normal' in vivo expression method using cells. Simultaneous use of the in vitro expression procedure with the normal in vivo method would greatly facilitate studies that require such difficult proteins.
Yutetsu Kuruma and Toshiharu Suzuki equally contributed as primary researchers responsible for experimental design, data acquisition, data analysis and the preparation of the paper. Yutetsu Kuruma generated experimental data using the PURE system and activity assay of F1Fo; Toshiharu Suzuki prepared samples of F1Fo and optimized the assay conditions of F1Fo. Sakurako Ono prepared cell membranes containing a subunit variants. Masasuke Yoshida supervised the research and edited the paper before submission. Takuya Ueda obtained grant support and edited the paper before submission.
This work was supported in part by the Targeted Proteins Research Program and Grant-in-Aid for Scientific Research (A) [grant number 18201040 to (T.U.)] from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and the Human Frontier Science Program Organization (HFSPO).
We thank Dr Boris Feniouk, Mr Chiaki Wakabayashi, and Mr Kazumi Tanaka (ICORP, JST) for assistance with FoF1 purification and valuable discussions.
Abbreviations: ACMA, 9-amino-6-chloro-2-methoxyacridine; Ap5a, P1,P5-di(adenosine-5′)pentaphosphate; CBB, Coomassie Brilliant Blue; DDM, n-dodecyl-β-D-maltoside; F1Fo, F1Fo-ATP synthase; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; IMV, inverted inner-membrane vesicle; PL, proteoliposome; TMH, transmembrane helix; wt, wild-type
- © The Authors Journal compilation © 2012 Biochemical Society


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The High Court has directed Lakes and Waterways Development Authority (LAWDA) to take immediate steps to prohibit all transfers, alienations as well as parting of the possession of rehabilitation sites by the allottes to any third person.
Issuing directions on a Public Interest Litigation on conservation of Dal lake, a division bench of Chief Justice Gita Mittal and Justice Rashid Ali Dar said such action shall be deemed to be violation of the terms of allotment and shall invite action for cancellation of the allotment in favour of the allotees.
"Execution of Power of Attorneys and handing over the possession to such person, even if attorneys shall be treated as alienation of the plot by the allottes".
The direction came after the court observed that the COE in its report has pointed out that the persons who have been allotted the rehabilitation sites are alienating the same. "Such alienations run completely contrary to the spirit intendment and purpose of the
relocation which is for the purpose of rehabilitation," the court said.
Meanwhile, the court directed Chairman J&K Pollution Control Board (J&K PCB) to remain present on May 24 after COE reported to it that the Board has failed to furnish details to the panel about the polluting units in and around the Dal lake.
Observing that the report of the COE manifests a dismal performance of the Board, it said even no data was forthcoming from the Board to the committee about the polluting units. The court said the report of the COE "manifests dismal performance of the J&K PCB.
With regard to the status report filed by SSP Vigilance Organization Kashmir setting out the details of FIRs registered in its police station regarding illegal constructions at different locations around the Dal, the court sought within a week action taken report with regard to removal of these constructions from VC LAWDA.
The Court also directed the PCB to place before it status report about declaration of the Dal Lake as an Eco sensitive Zone. On April April 12, the Court had directed the LAWDA to examine the possibility of declaring Dal lake area formally as Eco Sensitive Zone under the Environment Protection Act.
Meanwhile, the court directed VC, LAWDA to ensure that M/s Thermax shall positively submit the technical audit report regarding functioning of the STPs within two weeks. The STPs, the court said, shall be optimally utilized by the authorities so that no untreated sewage is allowed to flow directly into the channels or the lake in any manner.


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1–9 of 57 posts
Yeah warrior needs an update, bring more button to smash :-P I rly want to play warrior my self, but tbh it just seems very boring right now. 1-2-3 smash, kited, 1-2-3 smash, repeat
What are the weaknesses of Rogue?Arenas
11/11/2018 13:44Posted by Atahalni11/11/2018 11:17Posted by NexintusWell you missed my point. How many classes actually have preemptive walls without something else thats passive?And lol kidney to peel myself and immunities. Right all of which require me to be out of cc and reduces the goes of my team by 40seconds leaving us playing defensive for quite some time.No but you are missing the point.It doesn't matter whether you believe they are good enough or not in the current meta. It matters that they exist.If rogue has decent mobility, great dps, great (best) cc, decent utility and sustain, an…
Balance Warlocks 1v1Arenas
What a stupid post. Cant tell if its a troll or not. By that logic rogue should be balanced up against paladins 1v1, give them mass dispell. And against mages, give rogues 4 charges of shadowstep lolol. Will make 3's balanced, hope blizzard hire you, you know this stuff!
What are the weaknesses of Rogue?Arenas
09/11/2018 22:36Posted by DamegI agree on most things but I don't really think they are that good defensively. Their "defensives" so to speak aren't really as black and white as other classes. They're pretty versatile and often used both offensively and defensively so I don't see it as something you'll always have to go through, much like death knight cooldowns for example.Their mobility isn't something I'd brag about either. They have shadowstep (which is very good) and sprint (which isn't that great) since it has the same issue as divine steed, being that it does nothing when you're slowed a…
Rogue need pruning down to othersArenas
If ur talking assas rogue. yeah lets nerf what didnt even do well at blizzcon, not what was actually considered OP during the tournament. "Great post"
WCS Arena 2018 Spec representationArenas
These stats must be wrong, as even I watched WW. Please provide link.
Fun mele pvp class?Arenas
Dh is very strong right now, and have insane mobility, but still, I don't find it too fun to play, it was fun in the beginning but I got bored really fast. I find it to be too simple IMO. I find it more entertaining to play rogue, giving me a few more buttons and outplay potential. And yeah, warrior is just super simple now a days imo. I really miss how they were back in the old days, so if you want a challange and fun I don't think it's the best either. But all of this is personal preference, and you could have a totally different opinion than everyone else and me.I recommend to watch a few v…
Is gear the reason I lose?Arenas
As others stated, no it's not the gear. You and your rogue friend need to keep practicing and set up good cc chains to climb.Also watch some high rated players playing your speccs, and look at how they play. You will probly pick up a tip or two, and try to apply this to your games.
How 2v2 looks likeArenas
29/10/2018 09:41Posted by Piksna29/10/2018 00:30Posted by DoomchampI agree, 2's is fun, but the game is balanced around 3's, if you wanna complain about this, the topic should have been "balance 2's instead of 3's"./thread2s are fun only for fotm comps that are broken. I'm not asking for Blizzard to nerf rogue and assa but buff other classes/specs that are underperforming in 3s and the result of that is gonna be bigger variety of classes in 2s aswell. For example: aff/demo locks, dks, ele shamans, ww monks, mm hunters... All those classes/specs have been utter dog!@#$ through the whole season …


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Scotland's First Minister has offered to roll out the red carpet to major City firms amid efforts by European rivals to poach financial services jobs from a post-Brexit London.
Sky News has learnt that Nicola Sturgeon met around 20 big employers in London on Wednesday to declare that Scotland would remain an ideal location for financial and professional services jobs even after the UK leaves the EU.
Among the firms represented at the meeting, which was organised by TheCityUK, a lobbying group, were Deloitte and Lloyds Banking Group, which has its legal headquarters north of the border.
Ms Sturgeon has vowed to untangle Scotland from Brexit, and has warned Westminster that momentum is building towards a second Scottish independence referendum after a majority of the country voted to remain in the EU.
The financial services industry employs tens of thousands of people in Scotland, with companies such as Lloyds, Royal Bank of Scotland, Standard Life and Virgin Money accounting for a significant proportion of them.
According to a person who was present at Wednesday's meeting with the First Minister, she made no new pledges on support for financial services but said the Scottish Government would welcome further investment by the industry there.
London is already being targeted by rival EU financial centres including Amsterdam, Dublin, Frankfurt and Paris because of uncertainty about whether the UK will continue to have access to the Continent's passporting regime once it leaves the EU.
A number of investment banks which backed the campaign to remain in the EU, such as JP Morgan, have warned that they will be forced to move operations from the UK if it is no longer part of passporting.
George Osborne, the Chancellor, has met the heads of British and international banks operating in London to attempt to reassure them about the City's continued status as a leading global financial hub.
Despite concerns about an imminent exodus, most employers are likely to wait until they see the shape of a UK-EU trade deal before relocating significant numbers of staff.
Some asset managers have taken tentative steps to do so, setting up new legal entities in jurisdictions like Luxembourg.
TheCityUK, meanwhile, confirmed on Wednesday that its new chief executive is to be Miles Celic, a Prudential executive.
Last week, the lobbying group said it was setting up a Brexit steering committee, jointly chaired by the Barclays chairman John McFarlane and the Prudential chairman Paul Manduca.
In a statement, they said they would "ensure a clear industry message is communicated to policymakers - critical if we are to achieve an outcome that is in the best interests of the UK and safeguards continued economic prosperity".
A spokesman for Ms Sturgeon and TheCityUK declined to comment.


text: Home / Categories / All Products / EQUISETUM SCIRPOIDES – Miniature Horsetail EQUISETUM SCIRPOIDES – Miniature Horsetail$12.00 This is a dwarf or miniature tropical plant known as the smallest Horsetail or Equisetum in the world. This is a cold hardy bog plant that grows well in containers. This is also a good Bonsai accent plant. Ships in 4″ plastic nursery pot. Clay pot in photo not included and is not an example of the plant for sale. Only 2 left in stock EQUISETUM SCIRPOIDES - Miniature Horsetail quantity — OR — Add to cart Description Additional information Description Cute miniature plant. It is the smallest horsetail in the World originally from the Northern parts of the US. It grows anywhere from 3″ to 12″ tall depending on the environment. It grows well around small ponds and as a potted plant as well. Make sure to keep it moist. This is a cold hardy plant. Full to partial sun is best for this plant. This will also look really nice as a bonsai companion!! FAMILY : Equisetaceae ORIGIN : North America TYPE/USES : suitable for small bogs and water gardens or kept in a container MATURE SIZE : more or less 6″ tall LIGHT REQUIREMENTS : bright filtered light is ideal WATER REQUIREMENTS : keep moist MIN. TEMP. : low 30's FLOWER : Inconspicuous All photos were taken previously of our mother plants, not the plants for sale. Additional information Weight 3.5 lbs Dimensions 6 × 9 × 24 in Related products HOYA MULTIFLORA – Shooting Stars $18.00 Out of Stock BAUHINIA GRANDIDIERI – Blue Hong Kong Orchid Tree $25.00 Out of Stock EUCHARIS GRANDIFLORA – Amazon Lily $15.00 Add to cart MALPIGHIA GLABRA – Barbado's Cherry Tree $13.50 Add to cart


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10537 Ouray St, Commerce City
Motivated Sellers and quick close date!! Move in ready home. Beautifully updated 5 bedroom plus loft and office, open floor plan.Home is located in a golf-course community. Large loft area upstairs and total of 4 bedrooms on the upper-level. The Master bedroom has a 5 piece master bath and huge walk-in-closet. Main level has new grey hardwood floors, wrought iron spindles, natural light and fireplace. Kitchen has upgraded cabinets, double ovens and granite counters. The main level also has an office with French doors and a bathroom nearby. Newer paint throughout the house and newer carpet in basement and upper level of home. Backyard has a large trex deck with covered patio, new fence gates, new concrete walkway, and is prewired for speakers. Great basement with bedroom, bathroom, storage area, small kitchenette area and living room. Don't forget about the 3 Car garage.Status: Active
8z Real Estate.
Grounds Maintenance, Trash Removal
Cable Available, Eating Space / Kitchen, Five Piece Bath, Kitchen Island, Master Bath, Open Floor Plan, Smoke Free, Utility Sink, Vaulted, Walk-in Closets
Cook Top, Dishwasher, Disposal, Double Oven, Microwave Oven, Self-Cleaning Oven, Smoke Alarm, Refrigerator (Kitchen), Water Softener
Covered Patio, Fence, Front Porch, Garden Area, Gutters, Professional Landscaping, Sprinkler System, Yard, Deck, Playground Area
Terms and Conditions: The content relating to real estate for sale in this Web site comes in part from the Internet Data eXchange ("IDX") program of METROLIST, INC., DBA RECOLORADO® Real estate listings held by brokers other than Steps Real Estate are marked with the IDX Logo. This information is being provided for the consumers personal, non-commercial use and may not be used for any other purpose. All information subject to change and should be independently verified.
Copyright 2019 METROLIST, INC., DBA RECOLORADO® -- All Rights Reserved 6455 S. Yosemite St., Suite 500 Greenwood Village, CO 80111 USA
Listing information last updated on September 20th, 2019 at 4:34am MDT.


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To see complete entry details & classes/categories, download the 4-H Catalog Here.
4-H Talent Show
July 12, 2019, 7:30PM
Have we got a show for you! We're excited to announce that, on Friday night in the Exhibit Hall, we will be hosting the 4-H Talent Show!
4-H Rabbit Show
July 13, 2019, 10AM
Join us for the 4-H Rabbit Show at the Livestock Pavilion on Saturday morning!
Visit the 4-H Youth Development website for more information here. Catalogues are also available to pick up at the Hardin County Extension Office.


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tag:blogger.com,1999:blog-7211296491534054229.post4566607970860564677..comments2019-09-22T03:42:01.374-04:00Comments on Somewhat Quirky: Late Summer Gardening Stuffkaren@somewhatquirkyhttp://email@example.com:blogger.com,1999:blog-7211296491534054229.post-72430195741035030242014-09-03T23:27:22.018-04:002014-09-03T23:27:22.018-04:00OK, I'm going to go dig through our stuff and ...OK, I'm going to go dig through our stuff and get ready for planting next year. I kill almost everything, but I love what you did and want to try again.

Shannon ~ bohemianjunktion.comShannonhttps://email@example.com:blogger.com,1999:blog-7211296491534054229.post-9564754219936052312014-08-30T08:25:50.639-04:002014-08-30T08:25:50.639-04:00Stumbled upon your plate valance photo on Pinteres...Stumbled upon your plate valance photo on Pinterest, then perused your blog. Gorgeous! Check out my blog posts on pruning Hydrangea paniculata. Love the lemon bread recipe. I can't wait to try it; I plan on throwing a little dried lavender into the mix.Linda Nelsonhttps://email@example.com:blogger.com,1999:blog-7211296491534054229.post-89672051160647272212014-08-30T07:18:00.499-04:002014-08-30T07:18:00.499-04:00Karen, I love this post! I'm beginning to thi...Karen, I love this post! I'm beginning to think about all of the stuff I can plant late fall and early spring and I had forgotten that I love nine bark. You're flowers looks gorgeous!Danni@SiloHillFarmhttps://firstname.lastname@example.com:blogger.com,1999:blog-7211296491534054229.post-18810826634511967302014-08-29T20:42:29.990-04:002014-08-29T20:42:29.990-04:00Love your spectacular collection of hydrangeas. I ...Love your spectacular collection of hydrangeas. I have Limelight + is doesn't do very well for me - just limps along year after year... Send me some of your hyacinth beans!!!Sarah Krousehttps://email@example.com:blogger.com,1999:blog-7211296491534054229.post-9137481509945880382014-08-29T15:05:43.219-04:002014-08-29T15:05:43.219-04:00Laughing all the way through this post. Beautiful ...Laughing all the way through this post. Beautiful pictures. Thanks!Minnie's Milestoneshttps://email@example.com:blogger.com,1999:blog-7211296491534054229.post-25588013043257165992014-08-29T09:22:50.201-04:002014-08-29T09:22:50.201-04:00In 936 years I will have MY to-do list completed. ...In 936 years I will have MY to-do list completed. Right? What you HAVE accomplished is absolutely beautiful. And I am totally going to copy your succulents growing through the grate.Cheryl in Wisconsinhttps://email@example.com:blogger.com,1999:blog-7211296491534054229.post-79777597745554953622014-08-29T00:57:35.337-04:002014-08-29T00:57:35.337-04:00Love the way you decorating your football field. ...Love the way you decorating your football field. We have a short little driveway- big enough for 1 car length.

We are in such a drought that I don't even want to talk about my garden.Art and Sandhttps://email@example.com:blogger.com,1999:blog-7211296491534054229.post-19622544719466477352014-08-28T18:06:18.750-04:002014-08-28T18:06:18.750-04:00That reminds me I promised you a photo of my sad h...That reminds me I promised you a photo of my sad hydrangea. Maybe tomorrow. Blisshttps://firstname.lastname@example.com:blogger.com,1999:blog-7211296491534054229.post-11094068152729622682014-08-28T16:36:55.471-04:002014-08-28T16:36:55.471-04:00i have never seen a leave like that where the colo...i have never seen a leave like that where the color is seperated so distinctly. your garden plants are so pretty . We are kinda at the point where things are a bit stale/slat with color so I will be looking forward to the fall.Laura @ Top This Top Thathttps://firstname.lastname@example.com:blogger.com,1999:blog-7211296491534054229.post-78523492117513092482014-08-28T16:21:40.767-04:002014-08-28T16:21:40.767-04:00Everything thing here looks like the desert = high...Everything thing here looks like the desert = high heat and no humidity - my plants have folded their leaves in disappointment. Yours are lovely as usual my favorite is the Quick Fire - probably because I cannot get it here - the nursery said i would be very unhappy with its performance.Donna Wilkeshttps://email@example.com:blogger.com,1999:blog-7211296491534054229.post-54676208101780807012014-08-28T16:08:58.269-04:002014-08-28T16:08:58.269-04:00Well it sure looks nice to me!! Hopefully we will ...Well it sure looks nice to me!! Hopefully we will have a nice Fall and you can do Fall gardening!! And, I need some of those purple hyacinth bean seeds! I had a bunch of seeds and cannot find them! So send me some of those!miss flibbertigibbethttps://email@example.com


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In my last post, we discussed the need for organizations to have properly written policies and procedures regarding the provision of reference information on former employees to protect the employer, the former employee, and other employers from potential litigation. In this post, I will discuss the business of obtaining and providing references and how to avoid putting your organization at risk.
Why Name, Rank, and Serial Number Reference Policies are a Double-Edged Sword
Organization leaders and Human Resources professionals are often challenged by walking the tightrope of when to share information about former employees and when to stick to facts providing only dates of employment, title, and wage. As a result many organizations have policies that closely define those who are approved to release employment information and what information they are allowed to release regarding a former employee.


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Congress President Rahul Gandhi on Friday began his two-day maiden visit to the United Arab Emirates (UAE) with a breakfast meeting with business leaders here.
Gandhi, who reached here on Thursday night, held talks with the business leaders from the country accompanied by Indian Overseas Congress chairman Sam Pitroda.
He also interacted with hundreds of Indian workers at a labour accommodation in Jebel Ali.
"I am here to listen to what you all have to say," said Gandhi.
A part of his global outreach programme ahead of the Lok Sabha elections, Gandhi will also hold talks with UAE Ministers besides addressing the larger Indian expat community including students.
(This story has not been edited by Business Standard staff and is auto-generated from a syndicated feed.)


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French Pasta with Bacon and Radicchio
Prep time:
10 mins
Cook time:
15 mins
Total time:
25 mins
8 ounces linguine or tagliatelle
1 tablespoon extra-virgin olive oil
4 slices bacon, chopped
1 tablespoon butter
2 large cloves garlic
1 large head radicchio, cored and sliced
½ cup dry white wine
½ cup chicken broth
2 tablespoons snipped fresh chives
¼ teaspoon smoked Spanish paprika
Salt and freshly ground black pepper to taste
½ cup Comté, Emmental or Gruyère, or a hard-aged cheese (2 ounces)
Put the water for the pasta on to boil.
Meanwhile, in a large skillet, heat the tablespoon olive oil until it shimmers. Add bacon and cook until cooked through but not brown. Remove the bacon from the skillet and drain off all but 1 tablespoon of the fat. Melt the butter in the skillet.
Add the garlic to the skillet; cook and stir until fragrance is released. Add the radicchio; cook and stir until wilted. Add the wine and the broth; bring to boiling; reduce heat and cook until radicchio is tender about 10 minutes. Add the chives and the paprika.
While the radicchio is braising, cook the pasta according the package directions; drain and keep warm.
Toss the radichio mixture with the bacon, pasta, and half of the cheese. Divide among shallow bowls. Sprinkle remaining cheese on top.
Recipe by
Wini Moranville
at http://chezbonnefemme.com/french-pasta-with-bacon-and-radicchio/


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During May we will be repairing and refreshing the south mesa center island at the entrance on Paseo De Las Flores. This project will last approximately one week. Please follow all signage and flagman instructions.
In June asphalt repairs will be made on many streets throughout the community. Please slow down and follow all construction routing signs.
Beginning the week of July 8, all streets will receive an asphalt sealcoating. Work will be done Monday through Friday and is estimated to take three weeks. This process will protect the quality condition of our streets, decrease road noise and improve the appearance of the asphalt. A detailed plan has been developed by the construction company and will involve some street closures for a period of 24 hours. Significant attention is being focused on traffic and parking requirements. More information will be available and shared with everyone before the project begins.


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As Jeff mentioned in his blog post Top Ten Secret Pro Tools Shortcuts, learning Pro Tools shortcuts is a must for new sound editors if they want to be able to compete in our industry. Similarly, knowing the shortcuts to navigate through your OS quickly and efficiently is also really important. This is especially helpful to new editors trying to land their first sound job, as most of us come in at an assistant level, where a big part of the job is organizing files and multi-tasking among several projects. Learning basic navigational and organizational functions is a simple way to speed up your workflow and impress potential employers and clients.
Viewing entries tagged
keyboard shortcuts
The first piece of advice I give any new sound editor is to get Pro Tools and learn the keyboard shortcuts. Forget proficiency in typing, that's child's play. In order to compete in the real world of post production sound, you need to be FAST. Knowing your way around the keyboard doesn't just shorten your workday, it tells the clients - who expect requests to be carried out quickly - that you are on top of your game.
Basic keyboard shortcuts - switching the tools, changing the view - need to be second nature. But with literally hundreds to learn, there's bound to be a few that have slipped through the cracks. Here are some of the best 'lesser-known' Pro Tools keyboard shortcuts to help speed up your workflow.


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Course Overview
This qualification is designed to help learners build the knowledge and skills needed when working under supervision with children and young people from birth to 19 years of age. It covers a diverse range of job roles and occupational areas working in children's settings including early years and social care. You will need to be working, volunteering or on practical placement as you need to show competence in both knowledge and skills.
Course Structure
This qualification is made up of 11 mandatory units below, and three optional units chosen by your tutor, these units are assessed on knowledge (by your tutor in college) and skills (by your assessor in work placement.
- Introduction to communication in children's and young people's settings
- Introduction to personal development in children's and young people's settings
- Introduction to equality and inclusion in children's and young people's settings
- Child and young person development
- Contribute to the support of positive environments for children and young people
- Contribute to children and young people's health and safety
- Safeguarding the welfare of children and young people
- Understand partnership working in services for children and young people
- Contribute to the support of child and young person development
- Support children and young people's positive behaviour
- Maintain and support relationships with children and young people.
Entry Requirements
GCSE English and mathematics at Grade D/3/L1
English and/or Mathematics will not be included in your timetable, but should be enrolled on additionally alongside your course.
How Will I Learn?
The course will run for one year (1 year and a day if on an apprenticeship) and will include one day in college to complete the knowledge requirements.
Completion of this Level 2 course could lead to work as a full-time Level 2 practitioner – in early years settings or progression to a Level 3 Early Years Educator course, an apprenticeship or other level 3 qualifications relating to childcare or health and social care.
Information, Advice and Support
Careers Guidance
Help with choosing the right course and with thinking about where this might take you in the future.
Tel: 01274 433043
Email: [email protected]
Student Finance
Money matters - you may be entitled to financial support whilst you study.
Tel: 01274 433246.
Email: [email protected]
Learning Support
Support for applicants with a learning difficulty, disability, illness or other learning need.
Tel: 01274 438999
Email: [email protected]
For all other queries call 01274 088088
Tuition Fees
Tel: 01274 433172
Email: [email protected].


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This day-long seminar will dive deep into how this approach works. Providing you with the information and insights you need to get a new campaign up and running or keep an existing effort on track for success. Additional materials will be provided to those who register.
H.O.P.E. is a complement to ACES-based work that opens exciting new opportunities for promoting health in children and adults. It reverses the ways we typically understand the role of resilience, neuroplasticity and the developing brain. Research shows that positive childhood experiences both support child development and mitigate the effects of ACEs. HOPE offers a more holistic foundation for programmatic efforts to assess and address child and family needs.
In this interactive workshop, we will discuss issues related to screening for high-risk alcohol use, high-risk cannabis/marijuana use, interpersonal violence, and suicide risk. We will discuss the theory, social considerations, and the steps involved in brief interventions that utilize Motivational Interviewing in their delivery. We will provide training in and practice with specific motivational interviewing strategies. Finally, we will review how brief interventions that include norms can be part of an overall strategic prevention plan to impact the health of individuals and our communities.
Dr. Jeff Linkenbach and his dynamic team will share tools, resources, and cutting-edge science to help you jump start or recharge Positive Community Norms in your community. You'll learn from visionary leaders who are applying the Science of the Positive and Positive Community Norms in successful prevention efforts around the country. And you'll discover how to use these approaches to identify protective factors, increase healthy norms, and transform your community on issues including:


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Make it: DIY marble tray
One of my favorite things about my addiction to Pinterest is it never ceases to inspire me. I love that it's a community of people sharing ideas. I am not a DIY queen, but I do like to take on projects that are relatively simple. When I came across this one, I knew it was something I could do with ease and it would be a fantastic addition to my new bar cart.
The first thing I did was head to Home Depot to pick up the supplies: A marble slab, some drawer handles, felt pads and industrial glue. (All the materials cost less than $10!) I used a measuring tape to figure out where the handle would be centered, placed the glue on the bottom of the handle and pressed it on. Once that was dry I peeled the stickers off the felt pads and applied them to the bottom four corners. Et Voila! I mean does it get easier?!
Here is the step-by-step guide to making a DIY Marble Tray:
See Gallery
Now take a tour of Glam Latte's office:
More from Glam Latte:
One Lounge Nook Two Ways
Easy French Chic Bathroom Makeover
Idea To Steal: Dining Room Style


text: Abatina by Calypso Rose published on 2016-02-11T14:00:33Z 'Abatina' is the first extract from the 'Calypso Soundsystem feat. Calypso Rose, queen of Calypso for 40 years' compilation FB : https://www.facebook.com/queenofcalypso/ Website : www.calypso-rose.com Out in digital on March 2016 (Because Music). From Trinidad & Tobago, Calypso Rose and is the undisputed queen of Calypso for more than 40 years. After having produced 30 albums and composed 800 songs, she lives today in the Queens in NYC. Her next album "Far From Home" will be released on June 3rd 2016 on Because Music. Genre Calypso Rose Comment by Orhan Bilge <3 2018-07-20T21:11:37Z Comment by Blk Dragon Abatina ooohhh!! oooh gosshhh gal!! oh wow, Calypso Rose is a legend in trinidad and tobago!! i love all her songs!! funny how you like it too, she is granny age on this track, & still kilin it! imma have to send you the music video dj mc 2018-05-12T12:40:30Z Comment by Thenisda (SOUND Detect SOUND) YES!!! 2017-09-17T18:47:27Z Comment by Pedro Canoeiro maravilha 2017-05-26T05:38:51Z Comment by Walter Gibon ZOUK RETRO 2016-07-09T08:59:12Z Comment by yukuleletheS bringing sunshine in my ears ! thks a lot ! 2016-04-26T18:40:45Z


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How do i change php version?
2018-10-14 00:38:15
- Solved
Thinking of a bio :)
As standard, we run PHP 7+. If you wish to switch version, head to Dashboard> Manage Hosting > [Select Package] > "Switch PHP Version" Our Linux platform has the ability to run 6 different versions of PHP: PHP 5.3 PHP 5.4 PHP 5.5 PHP 5.6 PHP 7.0 PHP 7.1 PHP 7.2 Version changes may take up to 60 seconds, but are usually instant. On our Managed WordPress Platform we run PHP 7.0 and this cannot be changed.
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