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Your money is either in charge of you or you're in charge of it, there's no middle ground. Using some type of personal finance software can help alleviate some of that money stress and better allow you to manage your money effectively. Without it, you may just be setting yourself up for constant financial worry. Life is already tough enough and there's no need to make it more difficult by simply hoping your money issues will all work out in your favor. Hint: they won't.
Open an Etsy store. If you have a creative talent or skill – whether it's creating art, sewing clothes, or making keepsakes – you can open an online store on Etsy.com and sell your wares for some quick cash. With your own Etsy store, you're left in charge of pricing and, ultimately, how much you make. See our detailed primer, "How to Make Money on Etsy."
We have a saying that "good data" is better than "big data." Bid data is a term being thrown around a lot these days because brands and agencies alike now have the technology to collect more data and intelligence than ever before. But what does that mean for growing a business. Data is worthless without the data scientists analyzing it and creating actionable insights. We help our client partners sift through the data to gleam what matters most and what will aid them in attaining their goals.
If your YouTube channel has a million subscribers, you can earn from $100,000 to $300,000 per month. Mostly it depends on how interesting your videos are. Secondly, it depends on the how many people have subscribed to your YouTube channel. The stories of Olga Kay and Mimi Ikonn will tell you that they definitely earn more than $100,000 every year from their YouTube channels. This is the minimum. No one really knows the exact amount but many sources say that they could actually be touching one million dollars every year.
Now, I know what you're thinking. Most of the software and apps you use on a regular basis are made by massive companies or established development studios. Well, yes. But many successful apps, particularly those in the Apple and Google stores, are created and marketed by individuals and small businesses. In fact, independent developers made $20 billion in the App Store in 2016 alone.
The ad exchange puts the offer out for bid to demand-side platforms. Demand side platforms act on behalf of ad agencies, who sell ads which advertise brands. Demand side platforms thus have ads ready to display, and are searching for users to view them. Bidders get the information about the user ready to view the ad, and decide, based on that information, how much to offer to buy the ad space. According to the Internet Advertising Bureau, a demand side platform has 10 milliseconds to respond to an offer. The ad exchange picks the winning bid and informs both parties.
While mobileXpression doesn't dole out a set rate of cash, installing the app and answering an array of questions does put you in the running for all kinds of rewards and free merchandise. Once you install the app on one of your devices and keep it installed, you can earn everything from Amazon gift cards to electronics like iPads or flat-screen televisions, just for searching the web as you normally would.
Have you got a deep baritone voice? or a bubbly, chirpy one? or may be the one that has the chime and clarity of a tolling bell! If yes, you can online lend your voice as voice over artist. You can narrate stories, news and other text items. A friend of mine uses Umano and he is pretty thrilled with the experience. You can also use VoiceBunny or Voice123 for the same purpose.
GoodFinancialCents.com has an advertising relationship with some of the offers included on this page. However, the rankings and listings of our reviews, tools and all other content are based on objective analysis. For more information, please check out our full disclaimer. GoodFinancialCents.com strives to keep its information accurate and up to date. The information in our reviews could be different from what you find when visiting a financial institution, service provider or a specific product's website. All products are presented without warranty.
Furthermore, advertisers may encounter legal problems if legally required information doesn't actually display to users, even if that failure is due to technological heterogeneity.[86]:i In the United States, the FTC has released a set of guidelines indicating that it's the advertisers' responsibility to ensure the ads display any required disclosures or disclaimers, irrespective of the users' technology.[86]:4–8
Market your course: The beauty of using a course to make money online is that you can continue to sell it for as long as you'd like. Look for niche communities on Facebook, LinkedIn, or Reddit that might benefit from your content. Guest post on relevant blogs and sites. Look for anywhere you might be able to get in front of the right people. With just a few hours a month you can continue to generate sales.
Obviously, this was going to be the first on my list (I am also a professional blogger after all!). Professional blogging is one of the best known method of getting income from computer based jobs. We all arrive at blogs while surfing and we see advertisements on many blogs. So, we can easily know that the owner of that blog is earning some amount of money from writing the blog content.
Understanding just how big the internet is may be the first step to understanding why internet marketing is becoming so important to businesses. As previously mentioned, the internet connects millions of people from all over the world. Look at social media websites such as Facebook which has over 800 million active users every day. Imagine the exposure your business could get from having a proper advertising campaign on social media website. With that in mind Social Media Marketing through popular websites such as Twitter and Facebook is only a small part of the internet marketing process.
The Nielsen Global Connected Commerce Survey conducted interviews in 26 countries to observe how consumers are using the Internet to make shopping decisions in stores and online. Online shoppers are increasingly looking to purchase internationally, with over 50% in the study who purchased online in the last six months stating they bought from an overseas retailer.[23]
Working from home takes a lot of self-discipline. Before you become a remote worker, make sure you have a good system for keeping yourself organized and on task. Use whatever works for you – make a bullet journal, organize your duties on Trello, keep a detailed Google Calendar with Calendly for setting meetings. Just make sure you have a self-management scheme that keeps you going. I really like the new Focused Collection from Erin Condren.
Many work-from-home jobs that you find will require you to sit at your computer for long periods each day. So it's super important that you have a comfortable space to work! Even if it's just a comfortable chair pulled up to an old table that's the right height, or a spot on your couch, put some thoughtfulness for your future self into your home office. Your body will thank you. (Oh, and make sure it's in a quiet spot!)
Going into network marketing? Understand that if you're not close to the top of the food chain there, your ability to generate any serious amount of income will be limited. Be wary of the hype and the sales pitches that get you thinking that it's going to work the other way. Simply understand that you're going to have to work hard no matter what you pick to do. Email marketing? Sure. You can do that. But you'll need a massive and very targeted list to make any dent.
Working from home takes a lot of self-discipline. Before you become a remote worker, make sure you have a good system for keeping yourself organized and on task. Use whatever works for you – make a bullet journal, organize your duties on Trello, keep a detailed Google Calendar with Calendly for setting meetings. Just make sure you have a self-management scheme that keeps you going. I really like the new Focused Collection from Erin Condren.
Usually you can earn to the tunes of $2 per survey. Filling online surveys does not pay handsome money —it is just good for earning some extra bucks. Companies involved in online surveys pay very little amounts and in many cases they don't pay cash. When you will open an account and fill surveys, these companies will award you points. When you earn a certain number of reward points, you can get an equivalent gift (and sometimes cash).
Mechanical Turk is Amazon's take on micro-jobs. These are small miniscule-jobs that you can do for other people, which they call HITs, or Human Intelligence Tasks. These are super simple tasks that anyone can do. Some examples are listing off some URLs with certain kinds of images for one cent, or recording a few phrases with a microphone for 6 cents.
Many new users prefer to start off with the 100% Free Offers, which is what we usually recommend. These offers are great for beginners because nothing is required other than several minutes of your time. The payouts on these offers are lower, but you're not paying a dime to complete them and they'll get you use to the CashCrate system. After you've started making money online with our free offers, you can always move on to trial offers and increase your earnings.
PPC advertising is a method of advertising on search engines like Google and Bing. As mentioned earlier, with PPC ads, you pay each time that ad is clicked on. PPC ads also exist on social media platforms like Instagram and Facebook as well. However, if you're going to engage in PPC advertising, it's important that you determine conversion rates by using tracking pixels.
More and more companies and startups especially are embracing remote work—where you use online collaboration and communication tools to do your work from wherever you want. And you don't have to be a 20-something hotshot designer or coder to reap the benefits of working remotely. Many remote positions are for customer support positions or other customer-facing positions that don't require specialized skill sets.
Research other listings in your city on AirBnB and see what the going rate is for a place like yours. You could also just rent out a private room as well or even a bed in a shared room. In fact, that's how AirBnB got its start. However, you might find it hard in the beginning without reviews, but as long as you take really good care of your guests and provide a lot of value, the reviews will eventually come rolling in.
Focus on the surveys and offers at the top of the list. Here's a tip not many people know. CashCrate has a special algorithm that automatically selects the best surveys and offers and places them at the top of the list. There are several factors that go into determining which are the "best", including payout, approval rate, and a variety of other factors. But all you need to know is that these surveys and offers are great, so you can do them in confidence!
Consumers also face malware risks, i.e. malvertising, when interacting with online advertising. Cisco's 2013 Annual Security Report revealed that clicking on ads was 182 times more likely to install a virus on a user's computer than surfing the Internet for porn.[105][106] For example, in August 2014 Yahoo's advertising network reportedly saw cases of infection of a variant of Cryptolocker ransomware.[107]
Don't sacrifice morals for a quick buck — At the outset, you'll want to do all sorts of things to make money online, but don't sacrifice your morals for a quick buck. Not only will you put people off, but you'll lose Google's trust. You also shouldn't concern yourself with things like Adsense or other ads on a blog before you have around 100,000 visitors per day. Yes, per day.
If you're an expert in a particular field (like technology, health, cars, business development or anything else) you can write guest posts for bloggers who run profitable blogs on these subjects. Some big bloggers who earn money from blogging, can pay good amount of money if you can write an authoritative post for them. You can even earn as much as $500 by writing just one article! I have written an article about what is guest blogging and how should be done.
As well as selling your eBooks, Amazon can also create paperback copies of your book. CreateSpace, an Amazon-owned company, will help you self-publish your book. A relatively straightforward process, you must upload your work to CreateSpace and submit it for review. Your book will then be displayed on Amazon, and printed on demand and shipped by CreateSpace when purchased. And you can collect up to 70% royalties for each book sold.
The peer-to-peer concept is coming to virtually every corner of human existence. That includes the hospitality industry. Through AirBnB you can actually rent out your house for various lengths of time for a predetermined fee. That gives you an extra income on your house and gives the visitor the benefit of having an entire house, rather than being crammed into a single hotel room.
Infographics are currently an extremely popular type of media. An infographic showing information or data in an interesting and fun way can help engage an audience and generate numerous likes and shares. As infographics take more time to create than just simply uploading an image, busy website owners are prepared to pay for custom infographics. And don't worry, an infographic can be created using simple design software such a Canva. So extensive graphic design skills are not necessary, just a creative eye.
What's the authority of your website or webpage, or any other page on the internet for that matter where you're attempting to gain visibility? Authority is an important component of trust, and it relies heavily on quality links coming from websites that Google already trusts. Authority largely relates to the off-page optimization discipline of SEO that occurs away from the webpage as opposed to the on-page optimization that occurs directly on the webpage.
The larger the company, the more requirements and prerequisites they likely have in place. That's not necessarily a bad thing. Even though you may need a newer computer, they may be offer health insurance and a full-time schedule. There's always a trade-off. Know that more scheduling freedom and flexibility and less management oversight may mean lesser pay or no benefits.
Write and publish an eBook. You don't need a traditional publisher and financial backing to publish your own book anymore. In fact, Amazon.com makes it possible for you to publish your own eBook and sell it independently – with no financial investment on your part. And with Kindle eBook publishing, your book will appear on Kindle stores worldwide within 24-48 hours. Just be prepared to market it yourself on social media, your blog, or elsewhere if you expect to generate sales.
What these opportunities typically turn up to be is a pyramid scheme. You pay a fee to signup. You then receive a flyer saying how great stuffing envelopes is and list of names and addresses to mail said flyer. IF those people pay that signup fee so they can mail that flyer too, you get paid a small referral fee. ONLY if they take the bait do you get paid.
Merch by Amazon is another service offered by Amazon that will enable you to make money online without any initial outlay. To get started you need to create an account with Merch by Amazon. Then simply design some T-shirt logos or slogans, and upload them to your Merch account. You will need to choose a T-shirt type, color, and price, and Amazon will create product pages for each T-shirt. When someone makes a purchase, Amazon takes care of production and shipping. And you are paid a royalty for your design.
Not only will this multiply the money you're bringing in in a serious way, but it protects you against any sudden changes in the market or in your business. Remember that old saying about putting all your eggs in one basket? A few hours a week committed to just one or two of the following opportunities will put you in a much stronger position to be financially safe and independent.
The truth? Today, rising above the noise and achieving any semblance of visibility has become a monumental undertaking. While we might prevail at searching, we fail at being found. How are we supposed to get notice while swimming in a sea of misinformation and disinformation? We've become immersed in this guru gauntlet where one expert after another is attempting to teach us how we can get the proverbial word out about our businesses and achieve visibility to drive more leads and sales, but we all still seem to be lost.
LinkedIn is a social media site for professionals. This isn't a direct way to make money, but it's a great way to connect to your current and potential peers, customers, clients, vendors, and more here. You'll build a reputation and get in the loop on important developments in your chosen career path and/or industry. One day an old college buddy may hit you up for a dream job you never considered at the exact moment you are looking for a new vocation. Whether you like it or not, keeping your LinkedIn profile current is a great way to get surprised with new work opportunities out the blue.


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With a view of the medieval fortress town of Monemvasia,the Byzantine-era Kinsterna mansion has been restored with the utmost respect for its long history and now is an entire settlement unto itself, with a central manor house, adjacent out-buildings and nearby dwellings. At the same time, the owners have also set out to restore the estate's rich natural environment, emphasizing various traditional agricultural activities such as: harvesting grapes, producing olive oil, distilling tsipouro and making soap –with the aim of making the estate as autonomous and self-sustaining as possible.
Today, the Kinsterna produces its own wine, extra-virgin olive oil and table olives, tsipouro (traditional Greek spirits), bread, marmalade and other fruit preserves, soap and much more. The fruit-bearing trees and fertile vegetable gardens, the beds of aromatic herbs and flowers and the springs with their fresh, flowing waters all contribute to this ethos and model of sustainability.
In addition to its exciting history as a centuries-old fortified manor, today the Kinsterna is a year-round pole of attraction as a comprehensive vacation experience. A Mediterranean adventure would not be complete without a spa experience, especially since Kinsterna's Spa treatments use pure natural local ingredients, such as olive oil, honey and grape extracts. Fresh aromatic plants and herbs from the property's gardens, such as rosemary, peppermint, lemongrass and chamomile, play a major role in a series of specially formulated treatments.
Four unique dining experiences await guests including fine dining at the renowned
"Sterna" restaurant, which serves traditional Greek recipes prepared with the latest culinary craft and techniques, as well as the more casual taverna "Linos" located outdoors next to the old stone olive press. All day dining is available at the "Mouries" restaurant while cocktails are served throughout the day at the hotel bar and pool bar.

subset: commoncrawl


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ROWAN COUNTY, N.C. — A youth football coach at Power Cross Ministries in Salisbury is facing child abuse charges after being accused of letting two boys fight in September.
"The child physically assaulted my son and again, this is my son's first fight ever. Like, he's never been in a fight before in his life," the boy's mother, Kelly Hosch, said.
The assault happened at the church on North Ellis Street.
"When he came home that afternoon and I opened the door, I automatically just saw him being distraught, like, 'What's wrong?'" she said.
Her 10-year-old son Allahzion had returned home from football practice.
"Through his tears, what he was able to say, he explained to me in his own words, 'Coach Anthony got mad.'"
Salisbury police and Hosch said it started as a fight among other kids at Power Cross ministries, and coach Anthony Shegog got upset that no one intervened to stop it.
"The coach called the child in, confronted him, wanted to know why he didn't do anything, and the child didn't have any kind of response at that point, and the coach spoke to several other kids there and pretty much inferred to them that they needed to do something to the other child to get him to talk," Lt. Greg Beam said.
That's when Beam said another child assaulted Allahzion by punching him and internal video appeared to capture the coach watching it.
"He's never been in a fight before in his life," Hosch said. "He's never been written up."
Shegog was charged with misdemeanor child abuse and Hosch said the coach apologized to her and her son.
The attorney for Shegog told Channel 9 the claims are unfounded. He said Shegog never instructed any child to assault another, never witnessed any assault and never intentionally put any child in harm's way. He said the coach is still employed here.
Statement from Power Cross founder Jeff Storment:
"For 13 years, Power Cross Ministry has strived to provide hope to the young men of the communities we serve. We provide tutoring, character development, Bible study, meals and athletic training, among many more activities and experiences for children.
"We provide transportation for each kid to and from their home when needed. All of this for free with an open-door policy to all of the community's youth. The well-being of each and every child we work with has been and will continue to be our #1 priority.
"In this current situation, we have been accommodating and transparent with anyone involved and all law enforcement agencies. We believe in our justice system, and we believe that when this is over, the truth will overcome. We have worked hard to build a working staff and we trust them, their training and their testimonies.
"We have worked with thousands of children over the past 13 years without any legal issues, and we expect that record to continue. Children, their safety and their future are the number #1 priority at Power Cross Ministry."
>> Reading this story in our app? The new "Follow the Lead" feature allows you to tap the blue tag indicated with a '+' to subscribe to alerts on the very latest breaking news updates.
© 2019 Cox Media Group


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Client: Mrs N.M.D. Sheffield
Work undertaken: Survey, drawings, liaising with engineers and surveyors, planning application re construction of detatched bungalow with double garage (Pavillion).
Contains: Correspondence between the office of Francis Johnson and Mrs Sheffield, North Yorkshire County Council, Hambleton District Council planning department, National Rivers Authority, Prospect Forestry Ltd, Department of the Environment, E.C. Harris & Partners Engineers, Joseph Hardgrave Ltd, James Martin & Co Surveyors. Also notes, location plans, schedules, meeting notes, query list, plan drawing, report on Sutton Park.


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Desde la Sociedad Andaluza de Patología Digestiva @sapdes os deseamos a tod@s una Feliz Navidad y un Próspero Año N… https://t.co/pX4R5qTSuL
Pylephlebitis is septic thrombosis of the portal venous system due to thrombophlebitis at some of the draining veins related to infectious diseases.
Its diagnosis has increased possibly because of the improvement in imaging methods and the finding and early treatment has contributed to a decline in the rate of associated mortality.
This study presents the case of a woman who developed a pylephlebitis secondary to recurrent cholangitis due to stenosis of the bile duct.
Key words: Pylephlebitis, portal thrombosis, hepatic abscess.


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Britten's conscientious objectors certificate, dated 1943.
Teens in Colorado are struggling. Rates of mental health issues, substance abuse and suicide are rising. We investigate the causes — and possible fixes.
This year marks the 10th anniversary of Colorado Gives Day, a statewide movement to celebrate and increase philanthropy in our state. Colorado Public Radio is proud to be a part of this nonprofit community. We're in good company so we're highlighting nonprofits doing great work.Learn more.


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Hello all, For the last couple of weeks my Runecast VM widgets DID NOT look like this: They showed an error message about not being able to connect with the Runecast appliances. Today, I noticed that the Runecast appliance console DID NOT look like this: It had a pink banner on it that said please … Continue reading Runecast VM widgets not connected!
Category: How To
Making vRealize Automation v8 work
BTW, I found the VMware doc's on this a little less than useful. They seem to have the info I needed for a basic install scattered about in different places. But below is what I used to get things to work so it should work good for you too. This is likely the install that … Continue reading Making vRealize Automation v8 work
vRealize Automation v8 install fails hard!
I thought my vRA 8 install was going good. But then..... It says to check the vRSLCM UI. So I access the UI, and select Lifecycle Operations and see an issue. It suggests I need to look at the deploy.log file. And yes, I tried the retry but guess what happened. Same issue. So I … Continue reading vRealize Automation v8 install fails hard!
Using vVols on NetApp HCI
I have a new work lab, and I am turning on vVols so I am documenting what I do. I have a few questions that I am not finding good answers on. So working through it and taking notes will help! I am doing thing in a lab with vSphere 6.7 U3 and Elements 220.127.116.11. … Continue reading Using vVols on NetApp HCI
My Catalina Upgrade
Hello there, This is about my Catalina upgrade. I was on the road when it dropped and I do not control the upgrades on my work Mac. So at home now and time for the upgrade. I like very much Catalina as I have been using it for a while in beta. Learning / Links … Continue reading My Catalina Upgrade
Moving to my new iPhone 11
Hi all, I moved from my old iPhone to a new iPhone 11. I use an Apple Watch, and I use - a lot - Apple Pay so wanted to make sure I had no trouble with either. So that is why I am sharing this info - in case this is different for you … Continue reading Moving to my new iPhone 11
Patching vRealize Lifecycle Manager v2.1
So we have our vRLCM installed and it is time to patch. Hre is our series on the subject: Installing vRealize Lifecycle Manager - find it here Patching vRealize Lifecycle Manager - this article Configuring vRealize Lifecycle Manger - find it here - cancelled Deploying with vRealize Lifecycle Manager - find it here - cancelled … Continue reading Patching vRealize Lifecycle Manager v2.1


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Brighton Faculty of Arts sculptor and ceramicist Alma Boyes has headed up a campaign entitled 'Firing Up', to relight school kilns and introduce children to clay and ceramics.
The faculty has led the Crafts Council countrywide initiative in South East England which has provided an ambassador, a technical demonstrator and five students who have been working with professional ceramists, teachers and children in five secondary schools in the Sussex area to teach children in Years 9 and above. In preparation for the project, the secondary school teachers learnt about ceramics at faculty workshops held at the faculty during autumn 2012.
Boyes says that by providing school children with access to the specialist facilities and hands-on tuition the faculty has built partnerships that are both educational and rewarding. She said: "Ceramics is a subject that is rarely taught in secondary schools despite huge benefits that children receive from learning with their hands. 'Firing Up' has raised awareness of the importance of clay in our everyday lives, supported schools to use clay creatively and confidently, and encouraged children to view ceramics as a viable career option.
"This project offered a fantastic opportunity for teachers and pupils to engage with ceramics as a means of expression and to experience ceramics professionally. It will also enabled students on the faculty's 3D Design & Craft courses an opportunity to work with established artists within an educational context and to share their experience of pursuing ceramics professionally through higher education. We are proud to have taken an active role in facilitating the success of this initiative."
Funding for 'Firing Up' was provided by the Esmée Fairbairn Foundation which aims to improve the quality of life for people and communities in the UK, and the Paul Hamlyn Foundation whose mission is to maximise opportunities for individuals and communities to realise their potential and to experience a better quality of life.
An exhibition of work produced by the pupils from Varndean School, Hove Park School, Longhill School, Portslade Aldridge Community Academy and Holy Trinity School, Crawley is on show in the Cafe Foyer Gallery at The University of Brighton Faculty of Arts at Grand Parade until 26 July, 2013.
'Firing Up' is a national programme of networked learning in ceramics and has been developed by the Crafts Council, supported by the Arts Council of England. For more information visit: www.craftscouncil.org.uk/learn/firing-up.


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Aries: your daily horoscope on the 07 of December 2019
Your astral forecast
You will make exciting contacts who will open doors for you. Very positive burgeoning friendships are in the offing. As compensation, your effervescent brain is demanding moments of relaxation. Give yourself the means to do so and all will be for the best!
If you were trying to make a life-changing decision, the doors are going to open positively for you today.
There will be some connection, be it close or distant, between friendship and love. Don't hesitate to ask advice from someone you trust, and who has the distance you don't from the situation. You'll figure out the rest.
Your foresight and good sense will allow you to resolve a big problem. Don't count on anybody but yourself.
You're going to free yourself of a tiresome piece of work, and it will be to your own credit. You'll be efficient at everything you do today, so make the most of it.
|Horoscope for after tomorrow
What about your Decan today ?
The moon is in Aries, in position 19 degres, 41 minutes: Fits of fancy, fits of impulse, fits of gloom, everything is spontaneous and no half measures.
First decan(21st March to 1st April)
Avoid occasions where your heart may mislead you today, as you will be particularly vulnerable and your spontaneous enthusiasm could lead you to make mistakes. Wait for a day or two, when the climate will be more realistic and measured, so that your reactions will be better thought-out.
Second decan(2nd April to 11th April)
Your reactions will be close to the surface. Watch out for falling in love at first sight or crushes on another, shouting matches or a fit of depression! Everything will be heightened today and your reactions could flare up over very little. Think things over and avoid damaging outbursts.
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As long as you manage to keep yourself under control, you will have everything to gain from this influence. But watch out for outbursts, excessive behaviour and impulses. The door will always be open for misadventure, if you behave too unwisely or carelessly.
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Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering
Edited by Arnold L. Demain, Massachusetts Institute of Technology, Cambridge, MA, and approved April 27, 1998 (received for review February 26, 1998)
A previously unknown chemical structure, 6-desmethyl-6-ethylerythromycin A (6-ethylErA), was produced through directed genetic manipulation of the erythromycin (Er)-producing organism Saccharopolyspora erythraea. In an attempt to replace the methyl side chain at the C-6 position of the Er polyketide backbone with an ethyl moiety, the methylmalonate-specific acyltransferase (AT) domain of the Er polyketide synthase was replaced with an ethylmalonate-specific AT domain from the polyketide synthase involved in the synthesis of the 16-member macrolide niddamycin. The genetically altered strain was found to produce ErA, however, and not the ethyl-substituted derivative. When the strain was provided with precursors of ethylmalonate, a small quantity of a macrolide with the mass of 6-ethylErA was produced in addition to ErA. Because substrate for the heterologous AT seemed to be limiting, crotonyl-CoA reductase, a primary metabolic enzyme involved in butyryl-CoA production in streptomycetes, was expressed in the strain. The primary macrolide produced by the reengineered strain was 6-ethylErA.
Erythromycin (Er) is a broad-spectrum macrolide antibiotic produced by Saccharopolyspora erythraea. The backbone of the molecule is a 14-member macrocyclic ring (Fig. 1) that is produced through the sequential condensation of one molecule of propionyl-CoA and six molecules of methylmalonyl-CoA by a modular polyketide synthase (PKS). This enzyme complex comprises three large multifunctional polypeptides, DEBS1, DEBS2, and DEBS3, each of which contains two modules, and, in the case of DEBS1, a loading domain to initiate synthesis of the polyketide chain (1, 2). Each module contains the enzymatic activities necessary for one condensation and subsequent reduction of the extender to the growing chain. Selection of the appropriate extender unit is accomplished by the acyltransferase (AT) domain present in each module (3).
Recently, genetic engineering has joined synthetic chemistry in the production of novel macrolide structures. Hybrid PKSs have been constructed through the replacement of AT domains with those that specify different starter or extender units (3–6). These manipulations have resulted in the production of Er derivatives lacking methyl groups at C-10 and C-12 (5) and those incorporating the branched chain starter units of avermectin biosynthesis (6). To date, however, there have been no manipulations that allow the methyl side chains of Er to be replaced by ethyl groups. This type of modification would be useful in expanding the structural diversity of hybrid polyketides produced by combinatorial biosynthesis.
Niddamycin (Nd) is a 16-member macrolide with an ethyl side chain at C-6 (Fig. 1). The module 5 AT, which is believed to be responsible for incorporation of the ethyl side chain into the polyketide backbone, was identified by sequence analysis of the Nd PKS genes (7). In this paper we describe the construction of a hybrid PKS through the replacement of a methylmalonyl-specific AT of the Er PKS with that of the ethylmalonyl-specific AT of the Nd PKS. This substitution alone, however, was not sufficient to produce an Er derivative with an ethyl side chain; additional manipulations of the carboxylic acid precursor pools were necessary to generate the desired compound.
Strains, Plasmids, and Media.
The wild-type Er producer is S. erythraea ER720 (8). Plasmid pWHM3 (9) is an Escherichia coli–Streptomyces shuttle vector that is maintained in S. erythraea only when it contains heterologous DNA for chromosomal integration. Plasmid pDPE81 is a derivative of pKAS37 (10) in which a 1.7-kb BglII fragment containing the hygromycin resistance marker was inserted into the BglII site of pKAS37. E. coli DH5α (Life Technologies, Gaithersburg, MD) was the host used for plasmid construction and isolation. Liquid cultures of S. erythraea strains were grown in SGGP (11) for production of protoplasts and SCM (5) for metabolite or enzyme analysis. Plate cultures of S. erythraea strains were grown on R3M medium (5). Thiostrepton (Ts)-resistant strains of S. erythraea were grown in 25 μg/ml and 10 μg/ml Ts for plate and liquid cultures, respectively.
Plasmid pEAT4 (Fig. 2A) was constructed as follows. Cosmid pAIBX85, a pWHM3 derivative containing DNA from modules 3 and 4 of the Er PKS (corresponding to nucleotides 979-9349; GenBank accession no. M63677), was used to clone DNA flanking eryAT4. The 5′ flanking region was isolated by digesting pAIBX85 with MscI and BstEII (nucleotides 4247–6033), treating with the Klenow fragment of DNA polymerase, and ligating the fragment into the SmaI site of pUC19 to generate pUC/5′ flank. An AvrII site was engineered 13 bp downstream of the BstEII site by PCR amplification of a 306-bp region of DNA from the PmlI site (nucleotide 5739) to 12 bp 3′ of the BstEII site (nucleotide 6045). The engineered AvrII site does not change the Pro-Arg residues encoded by this region (Fig. 2B). A BamHI site was also included on the PCR primer just downstream of the AvrII site. The resulting fragment was digested with PmlI and BamHI and cloned into the PmlI/BamHI site of pUC/5′ flank, replacing the native sequence and resulting in vector pUC/5′ flank/AvrII. The 3′ flanking DNA was isolated by digesting pAIXB85 with PmlI and MscI (nucleotides 6999 and 8977), treating with the Klenow enzyme, and ligating the fragment into the SmaI site of pUC19, generating vector pUC/3′ flank. nidAT5 was isolated by PCR amplification of cosmid 13f5 (7), which contains DNA spanning module 5 of the Nd PKS cluster. The 5′ oligonucleotide was designed to create an AvrII site 12 nucleotides upstream of the beginning of the nidAT5 domain (nucleotide 25290; GenBank accession no. AF016585) and to place the amino acid sequence Pro-Arg-Lys-Pro in front of nidAT5 to correspond to the sequence that is found upstream of the eryAT4 domain. The 3′ oligonucleotide generated an FseI site at the end of the nidAT5 domain (nucleotide 26284, GenBank accession no. AF016585), resulting in a conservative Val to Ala change. A BamHI site was also incorporated after the FseI site. The fragment was digested with AvrII and BamHI and ligated into AvrII/BamHI-digested pUC/5′ flank/AvrII, creating vector pUC/5′ flank/nidAT5. The 3′ flanking DNA was then cloned onto the 3′ end of nidAT5 by first digesting pUC/3′ flank with FseI and BamHI, gel purifying the 1920-bp fragment, and ligating it into FseI/BamHI-digested pUC/5′ flank/nidAT5, creating vector pUC/nidAT5/C6-flank. To generate pEAT4, the nidAT5/flanking DNA cassette was isolated from pUC/nidAT5/C6-flank by digestion with EcoRI and HindIII and then ligated to EcoRI/HindIII-digested pWHM3.
Plasmid pDPE-ccr (Fig. 3) was constructed as follows. The Streptomyces collinus crotonyl-CoA reductase (Ccr) gene (ccr) was subcloned from plasmid pZYB3 (12) by digestion with XbaI and BamHI, which releases ccr and the upstream T7 ribosomal binding site. This fragment was treated with the Klenow enzyme and ligated into the polylinker of pDPE81 that had been digested with EcoRI and treated with the Klenow enzyme. The polylinker of pDPE81 is in the center of a 10-kb fragment of S. erythraea chromosomal DNA, which directs integration into the chromosome at a site that does not seem to affect Er production. Plasmid pDPE-ccr was designed so that the ccr gene expressed from the ermE* promoter (13) and the Ts-resistance marker are left behind in the chromosome following a double-crossover event.
Genetic Manipulations.
Standard molecular biology techniques were performed as described (14). Enzymes and reagents were purchased from Life Technologies. Protoplast transformation and marker replacement in S. erythraea were performed as described (5). One microgram of plasmid DNA was routinely used for protoplast transformations. Putative transformants were grown in SGGP medium containing 10 μg/ml Ts to confirm resistance. For chromosomal eviction of pEAT4, transformants were passaged twice in SGGP medium without Ts, cells were plated for spores, and individual colonies arising from spores were screened for Ts sensitivity. Electroporation was performed as described (15) by using 1 μg of pDPE-ccr DNA. Because the plasmid is unstable when integrated into the S. erythraea chromosome, two consecutive platings on Ts-containing R3M plates results in resolution and eviction of plasmid sequences. PCR and Southern hybridization were as described (7).
Ccr Assay.
Seed cultures of S. erythraea grown in SCM and S. collinus grown in medium A (16) were diluted 5-fold into SCM and medium A, respectively, and grown for 48 h at 30°C. Cells were harvested, opened with a French pressure cell, centrifuged to obtain cell-free extracts, and assayed spectrophotometrically for Ccr activity as described (16). One unit of Ccr activity is defined as 1 μmol of NADPH oxidized in 1 min.
Isolation and Identification of Metabolites.
Small-scale (milliliters) isolation of metabolites from S. erythraea, TLC, and MS analysis were performed as described (5). Large-scale isolation for compound identification was as follows. Fermentations were conducted in 42-liter LH fermentation series 2000 stainless steel vessels (LH Fermentation, Maidenhead, UK). Each fermentor was charged with 30 liters of a medium consisting of 1.5% soluble starch, 2.2% soybean flour, 0.2% CaCO3, 0.15% brewer's yeast (Wind Gap Farms, Baconton, GA), 0.102% MgSO4⋅7H2O, 0.0027% FeSO4⋅7H2O, and 1% soybean oil. Sterilization was at 121°C and 15 psi (1 psi = 6.89 kPa). A two-step seed protocol was used. Vegetative growth from a frozen stock of S. erythraea strain EAT4-ccr was inoculated at 1.5% into a 500-ml Erlenmeyer flask containing 100 ml of the following medium: 1.5% glucose monohydrate/1% soluble starch/1% Soytone (Difco)/0.9% yeast extract (Difco)/0.21% Mops/0.001% Ts. For the second step, a 2-liter Erlenmeyer flask containing 600 ml of the same medium was inoculated at 5% with the first-step growth. Both steps were incubated at 28°C on a rotary shaker at 225 rpm for 48 h. The fermentor was inoculated at 5% with the second-step seed growth. Fermentor temperature was controlled at 32°C, agitation was 250 rpm, aeration was 1 vol⋅vol−1⋅min−1, and head pressure was 6 psi. Silicone antifoam was added at 0.01% initially and was available on demand. Harvest was at 108 h.
The fermentation broth (28 liters) was filtered, the pH was adjusted to 9 with NH4OH, and the broth was extracted with CH2Cl2 (twice with 14 liters each). The combined extracts were concentrated, and the residue was partitioned between the two phases of the heptane/methanol/0.02 M K2HPO4 system (pH 6; 1:1:1). The aqueous phase was evaporated, and the residue was dissolved in 0.05 M aqueous potassium phosphate buffer (100 ml), adjusted to pH 9 with NH4OH, and extracted twice with equal volumes of CH2Cl2. The CH2Cl2-soluble fraction (1.65 g) was separated by droplet countercurrent chromatography (100 vertical columns; 0.4 × 24 cm) by using the upper layer from the hexane/ethyl acetate/0.02 M K2HPO4 (pH 8; 1:1:1) system as the mobile phase. The fractions were analyzed by bioassay against Staphylococcus aureus and by 1H NMR. The bioactive fractions that were shown to contain macrolides by NMR were pooled, dried (0.4 g), and chromatographed on a Sanki Engineering (Kyoto) HPLC centrifugal partition chromatograph by using the hexane/ethyl acetate/0.02 M K2HPO4 (pH 6; 1:1:1) system. The active fraction was further purified by HPLC on C18-derivatized silica by using an acetonitrile/methanol/0.01 M (CH3)4NOH/0.05 M KH2PO4 (pH 6; 73:10:59:59) system. Two-dimensional NMR experiments, including double-quantum correlation, heteronuclear multiple quantum correlation, and heteronuclear multiple-bond correlation, performed in C2HCl3, were used for structure elucidation and the complete assignment of the 1H and 13C NMR signals.
In Vitro Antibacterial Activity.
Antibacterial activity was determined by the broth microdilution method (National Committee for Clinical Laboratory Standards (Villanova, PA) M7-A4, 1997), except that Brain Heart Infusion (Difco) broth was used as the test medium. Assays were incubated overnight at 35°C. Minimal inhibitory concentrations were defined as the lowest drug concentration (μg/ml) inhibiting bacterial growth.
Construction of S. erythraea EAT4.
Plasmid pEAT4 was constructed to replace DNA encoding the AT domain of module 4 in the Er PKS (eryAT4) with DNA encoding the AT domain from module 5 of the Nd PKS (nidAT5) (7). S. erythraea ER720 protoplasts were transformed with pEAT4 DNA, and 10 transformants were obtained. Genomic DNA was extracted from one of the transformants for Southern analysis, in which probing with pWHM3 DNA confirmed the integration of pEAT4 at the appropriate location in the chromosome (data not shown). Nonselective growth to allow plasmid eviction through a double-crossover event yielded 96 colonies, of which 9 were found to be Ts sensitive. Southern analysis of the 9 clones showed that 3 had nidAT5 DNA sequences in place of eryAT4 chromosomal sequences, and the remaining 6 had segregated to wild type (data not shown).
Characterization of S. erythraea EAT4.
To analyze the Er derivatives produced by S. erythraea EAT4, the three isolates and four of the wild-type segregants were grown in SCM medium for 4 d. The four wild-type segregants produced spots indistinguishable in color and Rf from the ErA standard. The three strains in which nidAT5 replaced the AT of module 4 of the Er PKS produced spots similar in Rf and color to the wild-type segregants, but the spots were much less intense (data not shown).
To determine the mass of the compound produced by S. erythraea EAT4, the supernatant of a 50-ml SCM culture of one of the isolates was extracted with ethyl acetate. The extract was subjected to TLC, but only the edges of the plate were sprayed with anisaldehyde to locate the region of interest. A 1-cm band of resin was scraped from the unsprayed portion of the plate at the Rf of ErA. The resin was extracted twice with 500 μl of an ethyl acetate/methanol (2:1) solution, and the organic phase was dried and then analyzed by electrospray ionization MS. Surprisingly, the mass of the protonated molecular ion of the compound was observed at m/z 734, which corresponds to the mass of ErA, not an ethyl-substituted ErA derivative.
Feeding of Ethylmalonyl-CoA Precursors to S. erythraea EAT4.
One hypothesis for the failure of S. erythraea EAT4 to produce an ethyl-substituted derivative is that ethylmalonyl-CoA, the substrate for the nidAT5, is lacking. To test this hypothesis, cells were grown for 4 d in SCM medium containing precursor compounds, and ethyl acetate extracts of the cultures were analyzed. When either 50 mM butanol or 50 mM butyrate was added to the medium, a second spot running slightly faster than the Rf of ErA was seen. Butyrate-fed cultures (Fig. 4A) produced about a 1:1 ratio of the two spots, whereas the butanol-fed culture produced more of the ErA-like spot (data not shown). Addition of 10 mM ethylmalonate failed to produce significant amounts of either of the two spots described above. However, addition of 10 mM diethylethylmalonate was found to yield much more of the faster migrating compound than that seen with the other precursor compounds tested and very little compound migrating at the Rf of ErA (Fig. 4A). Cells grown in unsupplemented SCM medium produced only material migrating like ErA.
To characterize the newly synthesized compound, extracts of the butyrate- and the diethylethylmalonate-fed cultures were subjected to TLC, the region of interest was scraped from the plate and reextracted for electrospray ionization MS. The results showed that the butyrate-fed culture of S. erythraea EAT4 produced approximately equal amounts of compounds with protonated molecular ions at m/z 734 and 748. The 748 species is consistent with an additional methylene group on ErA, e.g., with an ethyl group replacing a methyl group on the macrolide ring. The diethylethylmalonate sample contained primarily a compound with a mass of 748, with only a trace of ErA (734) present.
Genetic Manipulation of Ethylmalonyl-CoA Levels in S. erythraea EAT4.
Successful production of an Er derivative with a mass consistent with the addition of an ethyl side chain by butyrate- and diethylmalonate-fed S. erythraea EAT4 suggested that the levels of ethylmalonyl-CoA, the likely substrate of the nidAT5, must be limiting. In streptomycetes, Ccr catalyzes the last step in the reductive biosynthesis of butyryl-CoA from two molecules of acetyl-CoA (12). Because butyryl-CoA can then be carboxylated to form ethylmalonyl-CoA, the possibility that Ccr could be used to increase the levels of the ethyl-substituted Er derivative was investigated. The ccr gene of S. collinus was expressed from the strong ermE* promoter at a site unlinked to the Er biosynthetic cluster in the S. erythraea EAT4 chromosome. Plasmid pDPE-ccr was electroporated into S. erythraea EAT4. About 40 Ts-resistant colonies were obtained, of which 7 were confirmed to be stable transformants. Genomic DNA was isolated from 2 of the transformants for Southern analysis. Probing with the S. collinus ccr gene confirmed that both isolates carried the gene at the expected location in the chromosome (data not shown). The strain was named S. erythraea EAT4-ccr.
To assess expression levels of the ccr gene, Ccr activity of S. erythraea EAT4-ccr was compared with that of S. erythraea EAT4 (parental strain) and wild-type S. collinus. We found (Table 1) that, although there was no detectable activity in the S. erythraea strain without the ccr gene, S. erythraea EAT4-ccr had about 20 times the relative activity of the wild-type S. collinus strain from which the gene originated.
To determine the effect of the ccr gene on production of an ethyl-substituted Er derivative, ethyl acetate extracts of supernatants of 4-d SCM-grown cultures of the EAT4 strains with and without the ccr gene were examined (Fig. 4B). TLC analysis of 10 ml of extracted cells demonstrated that EAT4 without the ccr gene produced a spot that migrated with ErA, whereas EAT4-ccr produced a spot that migrated faster than the ErA spot, with no ErA-like material visible.
Extracts were also analyzed by electrospray ionization MS. The EAT4 strain produced a compound with a protonated molecular ion at m/z 734, indicating ErA production. The EAT4-ccr strain produced a compound with a mass of 748, which is consistent with the production of the 6-ethylErA. No compound with a mass of 734 (ErA) was detected in extracts of this strain.
Structural Analysis and Biological Activity of Compound Produced by S. erythraea EAT4-ccr.
The structure of 6-ethylErA was confirmed by spectroscopic methods. The high resolution fast atom bombardment MS analysis of the sample gave an M + H+ ion at m/z 748.4846 [calculated for C38H70NO13 m/z = 748.4842 (Δ 0.5 ppm)], indicating that this molecule has an additional methylene function compared with ErA. The presence of an ethyl moiety in the molecule at the C-6 position and its relative stereochemistry were confirmed by two-dimensional NMR. The stereospecificity of the ethyl moiety was found to be the same as that of the methyl group present at C-6 of ErA (Fig. 1). The 1H and 13C NMR assignments are shown in Table 2.
Antibacterial activity of 6-ethylErA was compared with that of ErA against a panel of S. aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and Enterococcus faecium strains from the Abbott culture collection. Although 6-ethylErA does retain biological activity, it was found to be 15 to 60 times less potent than ErA against these strains. Representative minimal inhibitory concentrations (μg/ml) for 6-ethylErA and ErA, respectively, were as follows: S. aureus (4 and 0.06), S. epidermidis (4 and 0.12), E. faecium (4 and 0.12), and S. pyogenes (1 and 0.06).
In this work we have shown that an ethylmalonate AT can be substituted for a methylmalonate AT to produce a hybrid PKS that synthesizes the predicted macrolide product, 6-ethylErA. This suggests that Er analogs containing ethyl substitutions at positions other than C-6 or that ethyl analogs of other macrolides, such as rifamycin or rapamycin, can be prepared in a similar fashion by using the ethylmalonate-specific AT from the Nd PKS.
One key feature for the successful generation of 6-ethylErA was availability of ethylmalonyl-CoA as substrate for the ethylmalonate-specific AT of the Nd PKS in S. erythraea. This AT clearly demonstrates a relaxed specificity by reacting with both ethylmalonyl-CoA and methylmalonyl-CoA. In S. erythraea EAT4, the desired product could be obtained only by boosting the levels of ethylmalonyl-CoA by either precursor feeding or expression of Ccr, an enzyme involved in butyryl-CoA biosynthesis. These results suggest that ethylmalonyl-CoA derived from butyryl-CoA is not available at sufficient levels to compete with methylmalonyl-CoA for the ethylmalonate-specific AT. Ccr activity is not detectable in S. erythraea and attempts to detect a ccr gene in this organism by hybridization with the S. collinus ccr gene have failed (results not shown). In contrast, the ccr gene appears to be present in most streptomycetes (16), and in certain cases it appears to be clustered with antibiotic biosynthetic gene clusters. For example, a ccr homolog has been located in the biosynthetic cluster for tylosin (18), another 16-member macrolide with an ethyl side chain. Southern analysis of cosmid clones containing Nd biosynthetic genes also indicates that a ccr homolog may reside close to the Nd PKS (unpublished results).
It was demonstrated previously (5) that production of desmethyl Er derivatives by replacement of methylmalonate-specific ATs of the Er PKS with malonate-specific ATs from Streptomyces hygroscopicus and Streptomyces venezuelae was successful only in Er modules 1 and 2 (corresponding to C-12 and C-10 of Er, respectively) and did not give rise to a detectable polyketide when placed in module 4. In contrast, we successfully produced ethyl-substituted Er by replacement of the methylmalonate-specific AT of Er module 4 with the ethylmalonate-specific AT from the Nd cluster. In fact, in S. erythraea expressing the S. collinus ccr gene, replacements in modules 1 through 4 with the ethylmalonate-specific AT produced compounds with electrospray ionization mass spectra consistent with production of ethyl substitutions for methyl groups at C-12, C-10, C-8, and C-6, respectively. However, those in modules 5 and 6 did not produce compounds of mass 748 (data not shown). It is not known whether failure to produce the predicted compounds was caused by physical distortion of the PKS or its mRNA, inability of the PKS to process certain altered growing chains, or some structural instability of the macrolide itself because of the introduced change. Thus, even though genetic information may be present for the production of novel compounds, it is still not possible to predict which substitutions will yield detectable levels of product.
It has also been demonstrated that the malonyl-specific ATs can be distinguished from methylmalonyl-specific ATs through sequence alignments (3). By using this strategy, Ruan et al. (5) predicted that an AT from an unidentified PKS of S. hygroscopicus was a malonyl-specific AT, and they used it successfully to produce desmethyl Er derivatives. The Nd ethyl-specific AT was found to cluster with methylmalonyl-specific ATs (7) in similar AT alignments. This similarity may explain why methylmalonate was used as a substrate by nidAT5 in S. erythraea EAT4 to produce ErA when ethylmalonyl-CoA was not available.
Finally, structural determination by NMR not only confirmed the presence of the ethyl side chain on the Er derivative produced by S. erythraea EAT4 but also showed that the absolute configuration at this chiral center is the same as that in ErA. In Er biosynthesis, epimerization is required at C-6 because the PKS uses (2S)-methylmalonyl-CoA for chain extension (19). It is unclear whether the cognate epimerization occurs when ethylmalonate is used in the synthesis of 6-ethylErA because the stereochemistry of the ethylmalonate incorporated at C-6 is not known.
We thank Angela Nilius for performing minimal inhibitory concentration assays and Jim McAlpine, Rich Summers, and Thomas Vanden Boom for helpful discussions. This work was supported in part by Grant GM50542 (to K.A.R.) from the National Institutes of Health.
↵‡ To whom reprint requests should be addressed at: Abbott Laboratories, CAPD, D-4FR F3-3, 1401 Sheridan Road, North Chicago, IL 60064. e-mail: diane.stassi{at}abbott.com.
This paper was submitted directly (Track II) to the Proceedings Office.
Abbreviations: 6-ethylErA, 6-desmethyl-6-ethylerythromycin A; AT, acyltransferase; Ccr, crotonyl-CoA reductase; Er, erythromycin; Nd, niddamycin; PKS, polyketide synthase; Ts, thiostrepton.
- Received February 26, 1998.
- Copyright © 1998, The National Academy of Sciences
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You need affordable tires, whether you're trucking wheat from farm to market, hauling wind turbines through the Midwest, or delivering local construction materials. That's where GCR Tires & Service near Lapeer comes in, with the right commercial tires! Our tire options include forklift, off-the-road, mining, tractor, construction, logger, RV, skidder, and even dune buggy tires— all in a variety of sizes and from commercial tire brands you trust most! We also operate ISO-certified Bandag Retread shops across the United States for fleet owners and operators in need of uncompromised performance at a great price.
Have you heard about your options for GCR semi-truck tires near Lapeer? We have the stock and knowledge to equip every wheel position on your truck and trailer. Steer tires, drive tires, trailer tires —whatever the need, our crew will help you select your fleet's best semi-truck tires. Your local GCR Tires near Lapeer is here to keep your truck rolling with the best commercial truck tires for the condition, distance, and load.
Then there's our affordable off-the-road tires, which are ready for anything— construction, mining, forestry, waste transportation, and more! All types of equipment operators seek out commercial Firestone and Bridgestone OTR tires. These top tire brands can handle heavy loads and tough conditions.
But our experience doesn't stop when the pavement stops. Our farming tires are hard at work in North America's countryside, too. Our extensive selection of farming tires near Lapeer means we can almost any piece of equipment on the move, including skid steer loads, combines, sprayers, and tractors. We also offer tractor tire maintenance and tractor tire repair, so harvest time can go off without a hitch! When you need farm tire repair, just call one of our service trucks. We'll come to you.
GCR Tires & Service goes where you go. Even below ground with tires for mining. Your job site is our destination. We'll be there with some of the best mining tire solutions to help you maximize output. Turn to GCR for your around the wheel needs, like earthmover and mining tires, but also Bel-Ray lubricants, conveyor belts, hose solutions, flat-proofing, and inflation pressure tracking near Lapeer.
We know life is about more than just work. It's about spending time with friends and family, too! Your GCR Tires & Service store near Lapeer has affordable tires for your RV, motorhome, or camper, plus RV tire service and repair along your route. Our experienced staff can help you select the best RV tires for your next expedition, then check the balance, rotation, and alignment. If your kids' go-to phrase is, "R-V there yet?" then make sure the nearest GCR Tires & Service store is always right around the corner! We'll help you log mile after mile of memory with quality motorhome tires and maintenance you trust.
When you're shopping for some of the best commercial tires, not many tire companies can compare to GCR's variety of services and tires. Got a passenger car or minivan that needs new tires? A forklift that needs non-marking forklift tires? The best tires for a tractor? Compare tire options online or at your local GCR Tires & Service near Lapeer.
Get Repairs & 24-Hour Truck Road Service Near Lapeer
It takes more than great tires to get work done. You also need regular vehicle maintenance and timely tire repairs. GCR has one of the largest networks of commercial tire stores across the country, and that means we're there when you need us most. Contact a nearby GCR Tires & Service near Lapeer for roadside truck service, rotation, trailer repair, commercial tire mounting/dismounting, alignment, flat repair, pressure checks, wheel refurbishing, and more. You'll enjoy quick service at fair prices. Your hardworking equipment deserves a hardworking support team that does what they say they're going to do when they say they're going to do it. That's who we are at GCR Tires & Service near Lapeer.


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By Lex Adelman, IBA Marketing/Communications Intern
In elementary school the best days were field trip days because nothing was better than getting out of class to go somewhere new and exciting. Titonka State Bank (TSB) had a "Bank Day" and invited fifth-grade students to visit their Forest City branch for a field trip after they had completed the EverFi financial literacy course online.
"Bank Day" was a hit with not only the students but also those working for TSB, because they were able to see how much the students had learned about finance. Jan Anderson, who works for Titonka State Bank said, "The students enjoyed seeing how the bank worked and asked a lot of questions based on what they learned." The program was able to give the students enough background information so they could ask detailed questions and continue to learn throughout the visit. The field trip was so much fun for everyone involved that TSB has already invited a summer school group for another visit.
Everfi, the online learning program that TSB has sponsored, has financial literacy programming geared toward both elementary and high school students. Between TSB's three branches they are able to provide the programming to six different elementary and high schools between Algona, Forest City and other cities in northern Iowa.
While fifth-graders were invited to tour the bank, high schoolers also benefited from EverFi. By completing the program, they are able to meet their math and ELA Common Core standards, which are required for graduation. They are taught about things such as how to apply for loans, good credit vs. bad credit and what a credit score means. Anderson hopes the skills the students learn through the program will be useful in the students' lives after graduation.
The bank has received very positive feedback from parents, teachers and students since the start of the program. "It's nice for teachers because they don't have to come up with curriculum," said Jan, as EverFi has done a good job of keeping the information interesting and fun.
TSB is committed to supporting their community and plans to continue funding the Everfi program for the next three years. The results are worth the financial investment in the program, says Anderson. Anderson is also looking forward to more excited faces as students visit for the next "Bank Day" field trip.


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Start chatting to our centre now by clicking the button above
Our commitment to you is to maintain the highest possible standards at all times.
No news found with your current search criteria.
Our centre star rating is based on customer satisfaction scores from real customers. It is based on information collated by BMW UK over the past 90 days of new car sales and aftersales service transactions, where the customers have subsequently elected to complete a satisfaction survey online or by phone. You will also see below customer comments, which are unedited.
The service took 2.5hrs and I had sufficient facilities to keep me amused with coffee and newspapers and staff looking after my welfare
Expert diagnosis.. all necessary work carried out and completed as advised.
On arrival was seen promptly and service carried out within expected timescales and car returned fully valeted
Great experience as always!
Derek and Darran sorted the oddities of dead batteries, use of auxiliary heating at point of low battery. Battery accepted charge when RAC failed and car is back to normal.
Derek dealt with booking in and returning the car. He's a thoroughly nice chap to deal with, very competent, knowledgeable and friendly
You liaised on our behalf to ask BMW to fund the air conditioning part without any prompting from us. Can't ask for a better service than that. It's was also lovely getting a clean car back.
Very good service - clean, helpful, efficient
No problems
After ringing about my X3 recall notice, BMW Hailsham were absolutely brilliant, booked my car in within days of calling,they also checked over a few other things I was worried about,even cleaned the car for me before returning it, brilliant service
Abby was extremely professional
I know how much cost it will be for my service and mot will cost. If I keep my car.
See above. Car was not serviced/repaired properly.
Excellent service, Chandlers collected my X1 for service and MOT, and repaired a grazed bumper at a competitive price. Well done.
Very good and easy.
First class service from the friendly team.
If it was a bit cheaper, you would get 5 stars
long list of items I wanted checked, along with recall work - completed within 5 hours of dropping it off with them on a very busy Friday. Staff were friendly and communicative, answering all of my questions.
quite promp on arrival but expensive and unfinished
Car booked in am and waiting for MOT. Icon on dashboard then showed SERVICE. Was advised I needed full engine oil change so arranged whole day. Service dept rang to say service unnecessary until next year. Car passed MOT.
Always ready to meet you on arrival and deal with your every need.
Was left waiting for 20minutes after the receptionist told me to take a seat, but then didn't tell anyone I had arrived for a service. I then had to find another staff member to serve me after waiting.
As above - key is to be clear in your comms and meet my expectations - you did!
Because the staff at the Hailsham branch are always very polite and helpful !
I took my car in for 1st service, drove less than 20 miles, warning on the car screen. 'Fill up with oil'! Oil leak, told it was an oil seal on the filler cap, I do not believe that.
Fantastic customer service
The guys in the service department were friendly, courteous and knowledgable throughout and made the experience hassle free by booking a courtesy car at very short notice to enable the warranty work to be completed
The process broke down. If it had been done as advised I would not have been delayed. I was given a courtesy car though.
I did have a slight issue when booking car in with the department however this was quickly sorted out and the work was done and explained well
Water leak mostly fixed, we hope. Not sure yet. What was odd was to report that brake pads were very low, but not give me enough time to authorise that work before delivering the car back to me.
The final cost was as quoted. Car returned cleaned. Car available on time.
Check in was efficient . Update efficient and collection very efficient
Time given for service and waiting was accurate. Reception and staff always exceeds service
good service - times & price as quoted
Car is running too hot after aircon condenser and idler pulley replacement.
See reasons to previous question
The work was completed on time as arranged.
Explained fully the procedure, time it would take and cost. An added bonus was cost cheaper than quoted
Good, friendly service. Time to suit me. Easy drop off and collect. Car washed at same time foc.
Welcomed and kept informed.
The pick up of my car was very well communicated via phone message to introduce himself and accurately predict what time he would arrive ( Bill ) But customer service were not really so on the ball at times and gave contradictory information
I was misinformed about the servicing requirement at the time of booking etc but this was sorted out in a friendly, helpful and considerate manner by Derek Ashford who redeemed the situation well
See above. The car was serviced correctly. The MOT was done correctly. It was all the other things around the edge that failed badly.
Communication and updates so I knew the whole situation.
Car was serviced on time with minimal fuss or delay
They keep you updated how the works are going
I liked the fact that service and repairs communicated together
Met my expectations.
Staff usually got their finger on pulse
I booked the service online and chose a specific service based on what I could budget for that month. When at the centre the service that was listed on the work sheet was a full, and much more expensive, service.
Because you did what you said and on time
Would have given maximum score but service indicator was not updated on the dash board and I have to return for that to be completed.
Service team and work were very good. Complimentary vehicle wash was mediocre.
Very happy Chandlers was able to fit me in at short notice to undertake my end of warranty check. However I was promised a call back after it was discovered my alloy wheels have corrosion and whether these would be covered by the warranty. No call !
Friendly efficient staff, loyal customer recognition.
the time taken was rather more than promised
So we'll looked after.
No problem with the service /MOT - Car Valet more dissatisfied as I paid for the additional valet and the car windows were not clean and the bodywork had streaks .
My car was collected and returned at the times stated and was very satisfied
They carried out an additional service requirement to save me another appointment in a month or and were generally extremely helpful.
When I rang to book the first service I was told I did not have a service package despite buying this from the company. However this was quickly sorted out and everything went fine from then on.
Very helpful and attentive staff
Every endeavour to be efficient.
Nothing not to like other than the cost
The recall was done , curtesy car supplied
The entire process was slow and methodical. All options were explained and fair negotiations took place.
Staff very helpful.
MOT passed and other items carried out well.
Service carried out within time and vehicle presented back to the customer in a clean and most acceptable standard.
Actual service very good but see comments above which detracts severely from overall experience.
Work carried out promptly. Good feedback via vehicle health check.
Car had to go in three times in total to fix the issue for a total of 2 weeks. The whole process from initial booking to completion took nearly 2 months. I also still have a rattle from a speaker which I was told had been replaced
Disappointed that having had the car for two days, it was handed back without being washed & vacuumed. Why was it necessary to change the seat position to something resembling the layout of a deck chair?
Just not hitting the mark!
I decided to purchase the model as it fitted most of my criteria. I informed the sales person that I would need to use chains on the car as I ski. After purchase the owner manual tells me that I cannot use chains on the model I purchased.
All good
Quick response, efficiency in dealing with our enquiry and professional from start to finish.
I felt like I well looked after and that my continued custom was valued. The team were committed to working out a deal. This would make me want to continue using Chandlers time again.
I turned up on a Saturday without booking an appointment and it was busy but I was met straight away and I was there for a few hours and didn't feel rushed or in the way
Quick professional service
The staff were very pleasant and approachable in making us welcome.
Tom was very helpful and gave us time to take things in . Very informative and did exactly what he said he would do . Service that day was excellent
From the start to the finish our experience has been excellent
I'll give a high score because of the individual performances some of the processes were not as good
The BMW Centre is very well organised and looks good. The staff were very professional, respectful and friendly.
Extremely good service


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Alessia Bruchi
Talk about my biography & it seemed a little presumptuous to me, because often, reading the bio of the others, I remember a certain sense of inadequacy, because unlike many adults, I have never attended famous and expensive schools of photography, but I have learned through the years, trying and experimenting with everything, while I was attending university and working to win my longed-for journalist's card.
Born in 1977, graduated in communication, I began to become familiar with the camera from an early age, using as a first tool a fantastic polaroid, a gift from the first communion. And from that moment I never stopped, studying the technique in the manuals that I passed on hand and practicing in the field, photographing anything that passed under my nose. In the following years then, I switched to the various analog reflexes and finally to the digital ones after 2005.
Today I am a professional photojournalist, mainly dealing with Palio photography, reportage, wedding, and photography, collaborating with various local agencies and newspapers.


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BaoBao Tea
10 Rue Dauphine 75006 Paris France Map
- Ile-de-France
- ...
Average price €20.00
32 reviews
305 reviews
Pictures from our users
Special offers and Menus
Special offers are only available by booking online with thefork.
You'll be able to select the menus in the last step of booking, if available for your criteria.
You'll be able to select the menus in the last step of booking, if available for your criteria.
Special offers
-20% off the "à la carte" menu!
Preset menus and drinks not included.
Available on the booked timeslot.
Restaurant prices
Average à la carte price
(average à la carte price based on a two-course meal excluding drinks)
Drink prices
- €3.50Half bottle of mineral water
- €5.50Bottle of mineral water
- €3.50Glass of wine
- €14.90Bottle of wine
- €2.00Coffee
Prices and menu can change. Those prices don't take into account any promotion and are VAT included.
How to get to the restaurant BaoBao Tea
How to get there?
- Public transportsMetro Pont Neuf (ligne 7) ou Odéon (ligne 4, 10)
- Credit Card , Mastercard , Visa , Voucher
- Air-conditioning , English spoken , Open in August , Open sunday , Wifi
Only clients who have dined at the restaurant, after booking on TheFork, can leave their opinion.
Value for price
Noise level
Waiting time
11/305 reviews
Abdes L.
Conqueror(9 reviews)
- Meal date : 21 Apr 2019
Muy sabroso
Nicolas B.
Explorer(3 reviews)
- Meal date : 11 Mar 2019
Toujours un excellent service, un bo bun et un Bubble Tea au top!
Mylène B.
Foodie(26 reviews)
- Meal date : 19 Jan 2019
Très sympa !
Peter S.
Foodie(13 reviews)
- Meal date : 20 Nov 2018
Petit restaurant à recommander !
Virginie H.
Foodie(13 reviews)
- Meal date : 11 Sep 2018
Super service, la serveuse était au top ????
Karen M.
Adventurer(1 review)
- Meal date : 5 Sep 2018
Je recommende vivement
Louisa W.
Conqueror(5 reviews)
- Meal date : 29 Jul 2018
Simple restaurant with good, simple Asian food and great boba tea. Great spot for lunch.
T-Jay K.
Explorer(4 reviews)
- Meal date : 24 Jul 2018
Quick friendly service. Food was lovely, would have liked a little more pork with my Ramen, but all in all, very nice.
Francis M.
Conqueror(8 reviews)
- Meal date : 14 Apr 2018
super dessert
Sibille D.
Explorer(4 reviews)
- Meal date : 27 Mar 2018
Tres joli restaurant simple et charmant.
Mona K.
Conqueror(5 reviews)
- Meal date : 25 Feb 2018
Had a Bao Burger and it was much sweeter than I was expecting otherwise excellent food and service.


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In this timely and innovative book scholars from Europe, the UK, North America and Australia, explore their own sense of identity, reflecting both on their research and scholarly interests, and their work experiences. Taking the form of a debate, Changing Identities in Higher Education helps to…
Paperback – 2009-09-18
Key Issues in Higher Education
Improving Learning by Widening Participation in Higher Education presents a strong and coherent rationale for improving learning for diverse students from a range of socio-economic, ethnic/racial and gender backgrounds within higher education, and for adults across the life course. Edited by…
Paperback – 2009-09-17
Improving Learning
The 'Key Issues in Higher Education' series aims to raise both awareness and the standards of debate on the fundamental issues that lie at the very heart of higher education and intends to assist national and international debate. Higher Education and Sustainable Development examines whether it is…
Paperback – 2009-06-29
Key Issues in Higher Education
There are two key questions at the heart of the ongoing debate about education and training for all young people, irrespective of background, ability or attainment: What counts as an educated 19 year old today? Are the models of education we have inherited from the past sufficient to meet the…
Paperback – 2009-05-26
In today's complex and ever-changing world it has become obvious that even highly developed knowledge and skills are no longer sufficient to meet new challenges, situations and problems facing individuals, organisations and nations. This raises an enormous and potentially confusing issue for…
Paperback – 2009-05-18
Interest in learning at work has captured the attention of many people around the world, often taking centre stage in policy debates about improving economic performance, prosperity and well-being. This book is about the learning that goes on in workplaces – ranging from offices, factories and…
Paperback – 2009-04-23
Improving Learning
What's the problem with literacy at college? How might everyday literacy be harnessed for educational ends? Based on the first major study of literacy practices in colleges in the UK, this book explores the reading and writing associated with learning subjects across the college curriculum. It…
Paperback – 2009-02-16
Improving Learning
How should you prepare for the first day of class? How can you encourage all students to participate in discussions? How do you ensure disabled students can take part in field work? Increasingly, universities are drawing from a less traditional group of students – international students, disabled…
Paperback – 2008-10-28
Key Guides for Effective Teaching in Higher Education
Student engagement with digital learning resources and online social networking are strong forces in education today. How can these resources best be utilized by educators and course designers in higher education? This book aims to provide the reader with enough background information to appreciate…
Paperback – 2008-04-09
How can opportunities for teaching and learning be improved to ensure that many more people participate, gain qualifications and obtain decent jobs? Will government policies enable us to achieve these goals? What new ideas do we need to ensure a more inclusive, equitable and efficient learning…
Paperback – 2008-03-19
Improving Learning
This book is the first to show how to integrate Personal Development Planning (PDP) activities into teaching in higher education. It is packed with activities, exercises, lesson plans, resources, reflective questionnaires, skills audits and case studies, and with suggestions for how these may be…
Paperback – 2008-01-31
This book examines an important aspect of the relationship between higher education and the public - especially secondary - system of schooling in Britain. Higher education has influenced secondary schools in a number of ways, and not least in the development of school examinations. The…
Paperback – 2007-12-24


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Some East End residents are being asked to voluntarily evacuate their homes as others are bracing for the arrival of a nor'easter that is expected to hit the area starting on Wednesday afternoon, making it the second major storm to slam the Eastern Seaboard in less than two weeks.
On Wednesday morning, representatives from the National Weather Service said the winds were already picking up and gusts have already been recorded at 30 mph. Meteorologists also said the storm is a slow moving system and conditions will gradually deteriorate throughout the day.
According to the NWS website, www.weather.gov/nyc, there are high wind and flood warnings for our area through Thursday.
Lauren Nash, a meteorologist with the NWS in Upton, said on Tuesday that the nor'easter, based on its current path, was expected to drop between a half inch and an inch of rain across the area, and also bring with it sustained winds of 20 to 30 mph and gusts possibly topping 50 mph.
As of Tuesday afternoon, meteorologists said they did not expect the center of the storm to make landfall on Long Island, as its trajectory has it heading out to the ocean, meaning that the strongest winds, located on the eastern side, should remain out at sea. But even if the storm's strongest winds stay to the east, local residents can still expect the storm's surge to raise water levels between 2.5 and 4 feet.
That could spell bad news for an area still digging out from last week's devastating Superstorm Sandy, which caused severe flooding in several areas of East Hampton Town, including Gerard Drive in East Hampton, certain points in Napeague, and along South Emerson Avenue and in front of the Royal Atlantic Beach Resort in Montauk—and in Southampton Town, including East Quogue, Flanders, Hampton Bays, North Sea and Sag Harbor.
On Tuesday afternoon, Southampton Town officials issued a voluntary evacuation order for all low-lying areas in the town, most notably zone A homes. In Westhampton Beach, the village has also issued a voluntary evacuation for all homes and businesses south of Main Street.
In Montauk, the water level is forecasted to be 5 feet over the mean low water mark, with slightly higher levels to the west due to the strong easterly winds. In comparison, the water was 7.2 feet over the mean low water mark in Montauk when Superstorm Sandy slammed into the region last week.
While Ms. Nash said the expected northeast winds should be good news for oceanfront properties along the south shore, they could cause problems for the north-facing shorelines in the area, like the Culloden areas of Montauk, Gerard Drive in East Hampton and Pine Neck in Flanders. All three areas sustained massive flooding last week, and are expected to see higher water levels again on Wednesday evening and Thursday.
East End officials, however, are stressing that the nor'easter is not expected to be as powerful as Superstorm Sandy, explaining that they are different types of events. While they are closely monitoring the storm, East Hampton Village Administrator Larry Cantwell said officials there are not taking any extra precautions while preparing for its arrival.
Southampton Town Supervisor Anna Throne-Holst said Tuesday that officials in her town have been working to shore up the town's beaches, most of which were heavily damaged by Sandy. On Monday and Tuesday, the town's Department of Public Works employees created temporary berms, using sand displaced by last week's storm, to protect areas where washovers occurred. Ms. Throne-Holst also said the town has opted to post voluntary evacuation notices in low-lying areas and is encouraging residents to heed warnings about flooding.
In order to accommodate the voluntary evacuees, the town will keep two emergency shelters open during the storm. Those who opt to leave their homes can seek shelter at either the Hampton Bays Senior Center on Ponquogue Avenue or the David W. Crohan Community Center on Flanders Road in Flanders. The town's Emergency Operation Center (EOC) also will remain open for information. "All of the low-lying areas on the north and south shores ... should consider the voluntary evacuations," Ms. Throne-Holst said.
Aram Terchunian, a coastal geologist for the consulting firm First Coastal Corporation in Westhampton Beach, said Southampton Town is doing an excellent job in shoring up Dune Road, considering it has had little time to prepare for the nor'easter. He said town workers have been using bulldozers to build temporary berms along the shore that will be between 4 and 6 feet tall and 10 and 20 feet wide.
"The objective is to limit the amount of water that can come across the island because of the storm surge," he said on Tuesday. "The town is working very closely with Suffolk County, and they have been out there since early this morning doing a great job. I am very impressed."


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Photo By Baird & Warner, a member of Luxury Portfolio International
This stylish sitting room is perfect for formal gatherings or entertaining friends. The light neutral palette makes the room inviting while pops of color from the abstract canvas and decorative throw pillows add life to the sophisticated look.
16 Photos