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The Eastern Cape region of South Africa is home to traditional villages on rolling hills, giant hardwood forests, rocky coves, sandy beaches and the some of the best game viewing opportunities available in Africa. Stay in luxury Eastern Cape accommodation wherever you choose to base yourself and explore the myriad attractions of the region.
Start your Eastern Cape journey in the Tsitsikamma, a treasure trove of attractions; this indigenous forest is part of a huge conservation area boasting an incredibly diverse ecosystem. Walk the forest, climb the mountain or follow the water – in spectacular gorges and crystal clear rock pools as it makes its way through kloofs and waterfalls to the river mouth and the sea.
Enjoy a malaria-free safari in the Eastern Cape, home to some of the country's leading luxury game reserves. The Big Five are on show here in game reserves such as Shamwari, Addo and Kwandwe; you will see elephant, lion, leopard, rhino and buffalo as well as a host of other mammals and predators. Luxury accommodation is available on the game reserves and safaris include game drives, unique bush experiences and fabulous cuisines and wines.
Head to the country's fifth largest city and capital of the Eastern Cape, Port Elizabeth if you need some city life after time spent in the bush on safari. Geared up for visitors, Port Elizabeth's attractions include safe swimming beaches and warm water! The Snake Park, Aquarium and delightful performing wildlife at the Oceanarium are guaranteed to be a hit with both adults and children. Stroll along the beachfront at the end of the day before heading out to dinner at one of the city's fine dining establishments.
Get into the warm Indian Ocean waters to swim, surf, yacht or ski and the beautiful white sand beaches are the perfect place to relax and rejuvenate. The coastal waters are also teeming with life from the abundance of fish to dolphins and even the occasional whale is spotted.
If you travel a few hours up the coast from Port Elizabeth you will reach the laidback city of East London, the only river port in South Africa. Long, white sandy beaches that are a surfer's paradise appeal to all who love being on the water. It also boasts a natural history museum that is home to the only dodo egg in existence!
Stretching from the lush Tsitsikamma Forests, inland to the Great Karoo, the Bavianskloof Wilderness Area and the southern slopes of the Drakensberg, the Eastern Cape offers you seaside escapes, Big Five encounters on malaria-free game reserves, the chance to discover the history of the frontier and sophisticated city entertainment. Contact Southern Africa Travel today.
Malaria-free Game Reserves
Choose a luxury safari experience when visiting the game reserves of the Eastern Cape. Home to the Big Five - elephant, lion, rhino, buffalo and leopard – these luxury game reserves offer game drives and unique bush experiences.
Addo Elephant National Park
Visit one of our national treasures, the Addo Elephant National Park to view one of the densest African elephant populations on earth. The highlight of any trip to Addo is watching these large matriarchal herds interacting with their babies, but there is plenty of other big game too including buffalo, giraffe, zebra and lion.
Warm Ocean and Sandy Beaches
The long, sandy beaches and safe swimming around Port Elizabeth make this stretch of coastline a popular choice with families, or for something a little wilder continue north up the coast to Oceana Beach & Wildlife Reserve.
Be entertained at the Grahamstown Arts Festival
For 10 days every June or July, thousands of people gather in the small town of Grahamstown to celebrate the phenomenal artistic talent of South Africa. This National Arts Festival has been running since 1974 and offers simply superb entertainment – look forward to theatre, music, art, dance and goodie-packed craft markets.
Journey to the Valley of Desolation
Surrounding the historic town of Graaff-Reinet is the Camdeboo National Park and Valley of Desolation: incredible rock formations with vertical columns teetering 120m above the valley floor and views opening up to the vast open spaces of the Great Karoo. Visit in the late afternoon for warm, rich colours and photos to 'wow' your friends.
Experience the Owl House
Another attraction close to Graaff-Reinet is the hamlet of Nieu-Bethesda, where you'll find the famous Owl House. Wander around the house and its fantastical gardens where the artist Helen Martins, inspired by the bible, the poetry of Omar Khayyam and the works of William Blake, created hundreds of concrete and ground glass sculptures depicting scenes from these books as well as her own fantastical imaginary beings.
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The Cederberg Mountains are a great place to rejuvenate mind, body and soul. This pristine wilderness area with its dramatic…
For a vibrant fun-filled vacation the region around Durban is a great choice of destination with its warm waters, amusement parks…
South Africa's Garden Route is holiday perfect: white sand beaches, forests, lakes and world-class accommodation. Start…
You can choose from the many luxury accommodation options in the leafy suburbs of Johannesburg city, close to world-class malls…
Plan a visit the Kalahari Desert with Southern Africa Travel and take in the famous red sands. The only permanent surface water…
Good For: Bird Watching, Eastgate Airport, Hoedspruit
The Kruger National Park is South Africa's largest reserve offering visitors the chance to experience Africa's Big Five in…
The towns and surrounding areas in the KwaZulu Natal battlefields region all have an intriguing story to tell you of battles for…
Divide your time between the coastal reserves and the inland game viewing opportunities that abound in the mountain foothills,…
Choose a classic African luxury safari adventure for memorable experiences and a touch of the African magic in the Limpopo and…
Whether you are coming for the game lodge experience or a gaming experience at Sun City, the North West province offers you…
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stay in luxury accommodation in Swaziland and take a leisurely journey through Swaziland, the jewel in the African crown, by…
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Happiness, creativity, passion, equality. Personal and professional growth. Having fun while making it happen. Sharing the fun. Are you feeling it? We do too.
In 2018 we share inspiring Growth Stories by people such as artist Roisin Murphy, designer Satu Maaranen and fashion influencer Cipriana Quann to name a few. They have challenged themselves on different fields of creativity, design, ambition and personal growth to become who they are today.
Our story as Marimekko was born from a vision to create something unforeseen. Since the 1950s we've followed that passion and empowered people with our bold prints and colours. This year we'll be sharing encouraging Growth Stories from people who have inspired us.
Stay tuned to hear what Satu Maaranen, Head Designer of Marimekko, and Roisin Murphy, internationally acclaimed artist, have to tell about their paths to become who they are today. We will release new interviews monthly.

subset: commoncrawl


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- This event has passed.
Non-Profit Accounting Updates
September 21, 2017 @ 8:00 am - 10:30 pmFree
Event Navigation
Non-Profit Accounting Updates presented by Brian Carter, Mauldin & Jenkins.
Registration & breakfast begins at 8:00 a.m. Earn 2 hours of FREE A&A CPE. For more information or to RSVP email Cara Krusch at email@example.com or by calling 941-747-4483 by September 15th.


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In celebration of The Empire Strikes Back's 30th anniversary, LucasArts is releasing a slew of costume items for your Xbox Live Avatar.
These are the following Star Wars costumes to be available on the Xbox Live Marketplace Thursday, May 20 – the eve of the movie's actual 30th birthday:
- Imperial snowtrooper (male and female)
- Boba Fett (male and female)
- Lando costume with cape (male)
- Chewbacca (male and female)
- Rebel snowspeeder pilot (male and female)
- Han Solo Hoth outfit (male)
- Leia Hoth outfit (female)
- Bossk (male and female)
- C-3PO (male and female)
- AT-AT pilot (male and female)
In addition to the lightsaber which is already available, LucasArts will also release a Tauntaun pet and ballcap with Yoda ears. Check StarWars.com for the complete set of images.
Read more at Kotaku
Meghan's Opinion
This is great and all, but where are the costumes people actually want? Why Bossk and not the good stuff like Vader, Luke, Han Solo (not Hoth), Obi Wan, Greedo, and obviously Leia's slave costume? Oh right. We'll buy these and then they'll release those. Also, I would kill for an Ewok pet. Or maybe a mini AT-ST. Or maybe an actual AT-ST for myself.


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Review: La Maison Du Chocolat – Un Ete en Bretagne (Summer in Brittany)
Posted on June 14 2012 by Jenny Linford
France's acclaimed Maison du Chocolat is noted both for the quality of its chocolate creations and their chic appearance. This new collection Un Èté en Bretagne ( Summer in Brittany) lives up to expectations, smartly presented in a dapper, vibrant blue box, discreetly decorated with a few curving lines, elegantly evoking waves and the sea. Inside the box contains an assortment of five chocolates, with flavours inspired by Brittany.
Four of the chocolates are restrained, classic rectangles, while the fifth is a textured 'twiglet', suggesting the idea of finding a branch of driftwood on a beach. Made from milk chocolate, studded with slivers of nut and slivers of crepe, this (Pays de Saint-Brieuc) was the sweetest chocolate in the box, with a short finish.
The smooth, dark rectangle (Pays de Dol) had a silky ganache filling which melted cleanly in the mouth and packed a powerful chocolate flavour, with almond and coffee notes lingering on in the mouth and a slight saltiness right at the finish.
The rippled dark rectangle (Cornouaille), in contrast, contained a nicely crunchy, fine-textured praline filling with a pleasant grittiness to it and a salty-sweet flavour; shades here of sand and sea spray.
The milk chocolate rectangle (Pays de Saint-Malo), yielded nicely on biting to reveal a succulent, salted caramel filling which filled the mouth with a rich toffee flavour with a long finish, coating the tongue with a distinct butteriness. The saltiness of the caramel filling worked well with the sweet milk chocolate, countering it to create a very nicely balanced creation.
Finally, elegantly striped with one pale green, textured line came the surprising apple chocolate (Trégor), filled with both a layer of ganache and a textured apple jelly. Biting into this filled the mouth at once with a full-bodied apple flavour, which made me think of cider and Calvados.
Maison du Chocolat's Artistic Director Gilles Marchal has taken classic ingredients from Brittany – crepes, apples, butter caramel – and cleverly created a truly elegant collection of appealingly contrasting textures and evocative flavours which were a pleasure to eat. A very enjoyable collection indeed; eating these is a treat which would put anyone in a holiday mood.
(Available in two sizes: 12 chocolates (£12.50) or 32 chocolates (£33.50)) More info from http://www.lamaisonduchocolat.co.uk/uk/en/
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Words containing bartlesvill
Found 1 words containing bartlesvill. Browse our Scrabble Word Finder, Words With Friends cheat dictionary, and WordHub word solver to find words that contain bartlesvill. Or use our Unscramble word solver to find your best possible play! Related: Words that start with bartlesvill
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- Trending Now: More Trending:
- hogwash probity sequacious inadmissible oligarch, pejorative
foolscap was our Word of the Day on 08/08/2014. Hear the podcast!
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Did You Know?
These days, we are most likely to encounter foolscap as a reference to a sheet of paper or, more specifically, to a sheet of paper that is similar in size to a sheet of legal paper. In the early 17th century, when the use of foolscap was first attested to in English, we would have encountered it as a reference to an actual fool's cap-the cap, often with bells on, worn as part of a jester's motley. How did we get from this colorful cap to a sheet of paper? The connection is attributable to the former use of a watermark depicting a fool's cap that was used on long sheets of writing or printing paper. There are various explanations for the introduction of this watermark-including the claim that a 1648 British parliamentary group substituted it for the royal arms during exceptionally turbulent times-but such explanations remain unsupported by historical facts.
First Known Use of foolscap
in the meaning defined at sense 1See Words from the same year
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ESCP Europe offers a broad range of general and specialised programmes in various subjects with a cross-cultural approach to management.
ESCP Europe's Executive Education combines top-notch knowledge with a hands-on approach across our 6 campuses and beyond.
Corporate Relations provide companies with a unique gateway into the School and its highly dynamic and very culturally diverse student body.
ESCP Europe's strong network of 49,000 alumni in over 150 countries worldwide represents more than 200 nationalities.
ESCP Europe > Executive Education > Custom programmes > Our clients > Our clients
L'Oreal (Spain) HR Director's testimonial
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Estate planning with your lawyer is serious business in Tampa. In order to avoid unnecessary estate planning complications, the best advice we could give is to talk to a Tampa lawyer, but going into the conversation prepared is always your best bet. Here's some information on what to take into consideration when thinking about the future your loved ones will have when you're no longer around.
The first step in making sure your estate is properly planned is to streamline your assets and take an inventory. In matters this complicated, you want to be thorough, and you can start by making a list of all your bank and credit card accounts, but you should also take time to talk to insurance professionals about your policies, and making sure your contact information is up-to-date with all of the firms you deal with. Consolidating your assets and working with a single Tampa financial adviser is also not a bad idea, but first consult with your lawyer in Tampa.
The biggest hurdle to making sure you avoid complications is probably going to be gathering the necessary documents. It can be hard to know what you need, but your lawyer should be able to guide you through the process. A good estate plan should take into consideration not only the possibility of death, but also the possibility of incapacitation. Having a power of attorney named in writing should be one of the first things you do, so you can make sure that the process moves swiftly should the unthinkable happen.
In addition to power of attorney documentation, having a living will or a healthcare directive in place will help your loved ones remove the guesswork from their lives when you are unable to make the decisions on your own. Think about it, the situation is already a stressful one, adding more onto their plate when they're trying to deal with an emotional situation is the last thing you want to do.
Finally, having a last will and testament in place is important to spell out, in writing, all the instructions that you want followed in the event of your death. Having a good executor and lawyer named in these documents can be key, so make sure you don't overlook those. Check out our blog for more tips and info. Contact Fresh Legal Perspective today by calling (813) 448-1042 and ask about meeting one of our lawyers!


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The Blue Canyon Boys are proud to announce the addition of the award-winning Chris Elliott on banjo and vocals. As a founding member of the Spring Creek Bluegrass Band, Chris has spent the past seven years travelling and performing at many of the country's top bluegrass festivals and solidifying his place in the banjo world. "I am honored to be joining the Blue Canyon Boys. It will be a great vehicle for my banjo playing and they're a great bunch of guys. I'm looking forward to playing some very exciting bluegrass!"
Chris Elliott joins band members: Jason Hicks (guitar, lead/harmony vocals); Gary Dark(mandolin lead/harmony vocals); Drew Garrett (upright bass/harmony vocals). For more information on the Blue Canyon Boys, including upcoming shows, please visit their website at www.bluecanyonboys.com
The Blue Canyon Boys, based in Denver, Colorado, have been performing together since 2002 performing across the United States as well as Canada and Malaysia. First place winners of the 2008 Telluride band contest, the Boys have released four well received CDs that have garnered heavy airplay across the country and around the world. In 2011 the Blue Canyon Boys were selected to represent the United States and bluegrass music at the 14th annual Rainforest World Music Festival in Sarawak, Malaysia on the island of Borneo, which has been rated in the top 25 International Festivals by Songlines Magazine. National Geographic had this to say about their performance:
"The Blue Canyon Boys from Colorado were one of the festival highlights with their pitch-perfect harmonies and sweet sounding blue-grass music performed with real panache"
The band also hinted about new CD very soon!


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This electronic archive for preprints in the philosophy of science is offered as a free service to the philosophy-of-science community. The goal of the archive is to promote communication in the field by the rapid dissemination of new work. The archive is sponsored by the Philosophy of Science Association, the Center for Philosophy of Science, and the University of Pittsburgh Library System.
The Center for Philosophy of Science, which celebrated its 50th anniversary in 2011, promotes scholarship and research, encourages scholarly exchanges, and fosters publications in the philosophy of science as well as in philosophically informed history-of-science and related fields. Among its many activities are an extensive Visiting Fellows Program, frequent conferences and workshops, biweekly lunchtime talks and the Annual Lecture Series.
In recent decades the University of Pittsburgh has established itself as a leader in scientific philosophy, one of the 20th century's most important intellectual currents. Furthermore, the University has committed itself to assembling archival resources for investigating the history of scientific philosophy. Known as the Archives of Scientific Philosophy (ASP), these holdings include the scholarly papers of Rudolf Carnap, Hans Reichenbach, Frank Plumpton Ramsey, Paul Hertz, and Rose Rand. In addition, the archives hold microfilm copies of the papers of Herbert Feigl (the originals being housed at the University of Minnesota). The archives also include the private working libraries of Carnap and Reichenbach and microfilm copies of the manuscripts of Ludwig Wittgenstein and the Archives for the History of Quantum Physics. Most materials are housed in the Special Collections Department of the University's Hillman Library.
The Department of History and Philosophy of Science owns a lab. We use it to run experiments that bear on philosophical debates and to reconstruct important experiments in the history of science. PDF e-books and videos of experiments that document our projects are posted on the lab Web page.
The departments of Classics, Philosophy, and History and Philosophy of Science at the University of Pittsburgh cooperate in offering a program leading to the PhD degree in classics, philosophy, or history and philosophy of science, with a special concentration in ancient philosophy and/or science. The program also sponsors a lecture series and hosts colloquia and conferences.
The CNBC is a joint venture of the University of Pittsburgh and Carnegie Mellon University. The center leverages the strengths of the University of Pittsburgh in basic and clinical neuroscience and those of Carnegie Mellon in cognitive and computational science to support a coordinated cross-university research and educational program of international stature. In addition to a Ph.D. program in Neural Computation, it sponsors a graduate certificate program in cooperation with a wide variety of affiliated Ph.D. programs.
The Center for Bioethics and Health Law (CBHL) brings together clinicians, scholars, and researchers from many schools and disciplines across the University to investigate issues in bioethics and health law.
This collaborative project is funded in part by a Faculty Collaborative Research in the Humanities grant from the Humanities Center. Collaborators include James Lennox, Dennis Looney of the department of French and Italian, and Domenico Bertoloni Meliof the HPS Department at IU Bloomington. They also include advanced and recent graduate students from Pitt and IU Bloomington: Peter Distelzweig, Benjamin Goldberg, and Evan Ragland. The project aims to cultivate collaborative work on the interactions between medicine and natural philosophy during the 'Scientific Revolution' across several departments and institutions. It involves an ongoing working group and occasional small, one-day and afternoon workshops, and it will culminate in a large international conference on the topic hosted by the Center for Philosophy of Science, and co-sponsored by the Humanities Center, the World History Center, our department, the Medieval and Renaissance Studies Program, and the HPS Department at IU Bloomington. The conference took place in Fall of 2012.
The HPS graduate students organize a series of "WIP" (Work in Progress) talks each term.
The Department of HPS works closely with Pittsburgh's world-class philosophy program at both graduate and undergraduate levels. The strengths of the two departments are assessed together in The Philosophical Gourmet Report.
The Department of HPS has close links with the Department of Philosophy at Carnegie Mellon University.
Duquesne University, located in downtown Pittsburgh, has a philosophy department and is also home to the Simon Silverman Phenomenology Center.
The Pittsburgh Area Philosophy Colloquium was first held in 2010 in an effort to bring together philosophers in the region in a spirit of collegiality. Its program has been a mix of traditional hour-long paper presentations followed by discussion, and small working groups of philosophers with similar interests to share works-in-progress.


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HANOVER COUNTY, Va. -- From Florida to Maryland, first responders across the country are showing their support for a young Hanover boy by posting videos of their fire trucks and police cars with their lights on.
Caleb Lambert, 3, was diagnosed with stage 3 Neuroblastoma last week when doctors found a large tumor in his abdomen after he kept complaining of stomach pains.
Richmond firefighter Betty Migliaccio started a campaign to help Caleb feel better as he starts his cancer treatments.
She did not realize it would spread beyond central Virginia.
"All of a sudden people from all over the U.S. were sending in pictures of their apparatus and saying Hi to Caleb and they hope he gets better soon," Migliaccio said.
Caleb's mom and dad have spent years serving others as a nurse and firefighter, now they are leaning on the community to help them in their son's fight.
As of Monday night, a GoFundMe page set-up to raise money for Caleb's family, has raised over $16,000 toward a $50,000 goal.
"Caleb will have to endure a blood transfusion, biopsy, and a bone marrow test followed by five or six rounds of chemotherapy before any surgery can be performed," the GoFundMe page's organizer stated. "Caleb will have to travel back and forth to the hospital every 28 days for five days of treatment."
President of the local firefighters union, Keith Andes, said from picking up shifts to getting food to the lamberts at the hospital everyone is chipping in to help.
"I don't know this young three-year-old that well," Andes added. "But he's mine and we are going to make sure he has everything that we need to get him."
"It's a lot easier to help someone than to receive help," Lt. Taylor Flowers. "I think that's especially true with police officers, firefighters, first responders, and nurses because that's what everyone is used to doing."


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{ The J. Crew Fringe Slingback Sandals; image from jcrew.com }
1. History repeats itself: This article shows all the top dress styles from previous decades and similar ones you can buy now.
2. Cool idea for a DIY manicure!
3. Thought this round up of Etsy shops from style.com was awesome.
4. This article cracked me up, and, of course, made me love Grace Coddington even more (I wrote a review on her memoir last year).
5. Right now, J.Crew is offering 30% off your purchase! Just in time to snatch up cute spring sandals like the ones above.


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Go-Go Sport
The Go-Go® Sport delivers high-performance operation and easy disassembly for convenience on the go. With a 325 lbs. weight capacity, charger port in the tiller, and standard front LED lighting, the Go-Go Sport is feature rich and travel ready.
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|"Benjamin Young (Commented) (JIRA)" <j...@apache.org>
|[jira] [Commented] (COUCHDB-642) Support rev in PUT URL
|Thu, 06 Oct 2011 20:45:29 GMT
[ https://issues.apache.org/jira/browse/COUCHDB-642?page=com.atlassian.jira.plugin.system.issuetabpanels:comment-tabpanel&focusedCommentId=13122275#comment-13122275 ] Benjamin Young commented on COUCHDB-642: ---------------------------------------- >From a REST perspective nothing in the query string or header should take precedence over the content body. Meaning, the "rev" query parameter value really shouldn't override the "_rev" keys value in the document. Updating the _rev in the JSON object seems like a very small "cost" in exchange for the cumbersome "order of preference" given to various query parameters or headers. The content body is the canonical source for the document--especially in regards to a PUT. The DELETE case doesn't require a content body, so issuing it with a "rev" is mandatory as it semantically specifies the resource your deleting. You can delete documents via POST, but in that case the _rev is included in the content body (as it should be)--which matches the PUT use case. -1 as it's just not worth the "confusion cost" for a nice-to-have. > Support rev in PUT URL > ---------------------- > > Key: COUCHDB-642 > URL: https://issues.apache.org/jira/browse/COUCHDB-642 > Project: CouchDB > Issue Type: New Feature > Components: HTTP Interface > Environment: trunk 08 Feb 2010 > Reporter: Brian Candler > Priority: Minor > Attachments: 0001-Allow-for-the-current-revision-number-to-be.patch > > > A DELETE request lets you append ?rev=xxxx to the URL. But this doesn't work with a PUT request; you have to put the _rev in the body instead (even though the _id is taken from the URL path) > $ curl -X PUT -d "{}" http://brianadmin:firstname.lastname@example.com:5984/briantest/foo > {"ok":true,"id":"foo","rev":"1-967a00dff5e02add41819138abb3284d"} > $ curl -X PUT -d "{}" http://brianadmin:firstname.lastname@example.com:5984/briantest/foo?rev=1-967a00dff5e02add41819138abb3284d > {"error":"conflict","reason":"Document update conflict."} > $ curl -X PUT -d '{"_rev":"1-967a00dff5e02add41819138abb3284d"}' http://brianadmin:email@example.com:5984/briantest/foo > {"ok":true,"id":"foo","rev":"2-7051cbe5c8faecd085a3fa619e6e6337"} > Allowing ?rev in the URL would make PUT and DELETE more consistent, and would allow you to replace an existing JSON doc with another one without having to merge the _rev into it first. -- This message is automatically generated by JIRA. If you think it was sent incorrectly, please contact your JIRA administrators: https://issues.apache.org/jira/secure/ContactAdministrators!default.jspa For more information on JIRA, see: http://www.atlassian.com/software/jira
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1996 -
Below is the text of the written answers relating to Prime Minister's Question Time from 16th July 1996.
National Debt
10. Mr. Wilson: To ask the Prime Minister if he will make a statement on trends in the level of national debt since 1990.
The Prime Minister: There are a number of different measures of public debt. On the measure used for the European Union excessive deficits procedure, the general Government gross debt-
Since 1991, the GGGD/GDP ratio has averaged 44 per cent. Under the last Labour Government, the GGGD/GDP ratio averaged 62 per cent.
European Court of Justice
Mr. Austin Mitchell: To ask the Prime Minister what assessments he has made of the (a) advantages and (b) disadvantages to the United Kingdom of the European Court of Justice.
The Prime Minister: The advantages and disadvantages to the United Kingdom of the European Court of Justice were set out in the White Paper, "A Partnership of Nations".
Sir Peter Tapsell: To ask the Prime Minister if he will list his official engagements for Tuesday 16 July.
Mr. Harry Greenway: To ask the Prime Minister if he will list his official engagements for Tuesday 16 July.
The Prime Minister: This morning I had meetings with ministerial colleagues and others. In addition to my duties in the House I shall be having further meetings later today.
Berlin Bombing 1986
Mr. Dalyell: To ask the Prime Minister, pursuant to his answer to the hon. Member for Southend, East (Sir T. Taylor) of 24 June, Official Report, column 14, what discussions Her Majesty's Government have had with (a) the German, (b) the Lebanese and (c) the United States authorities about the activities of Yassar al-
The Prime Minister: We have had occasional contact with the German and United States' authorities about the activities of Yassar al-
Bilateral discussions are conducted on the basis of confidence and trust between interlocutors. The substance of such discussions cannot be made public.


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Birth and Newborn Sessions: Baby Corban
This is one of the most unique stories I've ever had the honor of telling via photography.
It's a story about struggle, adoption, acceptance, and love.
Stu and Lauren of Atlanta came all the way to Oregon to adopt baby Corban. Immediately after Corban was due, he had some complications and instantly had to be swept off to the NICU. Luckily, I was able to visit them again a few days later and he was feeling much better. It was the first birth I had ever witnessed in person and it was the first time filming an adoptive situation. It was really moving and I hope I have conveyed a tiny piece of the magic.
Keywords: Kylie Keppler,, Kylie Marie Photography, Oregon Photographer, Springfield Oregon, adoption photography, baby corban,
No comments posted.
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Domestic violence matters are filed in the Family Law courts under the Domestic Violence Protection Act (DVPA). Courts take DVPA actions very seriously. Courts do not look kindly on individuals who use a domestic violence action as a strategic move in a divorce.
Courts have different views of what constitutes domestic violence and thus it is critical to avoid any conduct that could potentially be construed as domestic violence. There may be harsh consequences if a judge determines domestic violence has been committed.
Documented domestic violence may impact the court's orders relative to spousal support, child support, custody and attorney's fees.


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HAHAHA! Sometimes schadenfreude is a wonderful thing. A group of four people vandalize a Trump sign at a store (apparently) and then get shocked when the store owner, Taren Meacham, refuses to put up with it.
This story doesn't paint a pretty picture of Meacham (since he's pro-Trump that's no surprise), but in all honesty it sounds like the vandals are trying to justify themselves and their criminal behavior by acting like THEY are the victims in all of this. Listening to their tone you'll hear it's quite whiny. Which is typical of liberals who think there should be no consequences for their actions.
The end of the piece reveals Meacham has had to deal with vandalism before, as if that's irrelevant. For all he knew these were the same a-holes that messed with him before.
Was this an overreaction? Yeah, probably. I wouldn't react the way Meacham did, but it should be lost on nobody that these vandals (who could easily be a metaphor for the government) were a lot less proud of what they did when they saw an armed citizen lay down the hammer on their shenanigans.
That Meacham wasn't arrested tells you all you really need to know here.


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Marketers Hold Keys to Shifting Consumer Awareness to Adoption of Digital Wallets
Digital wallets and mobile payment apps are on the minds of consumers—whether it be Apple's Passbook, Google Wallet, PayPal or Starbuck's mobile app. While the ubiquitous use of mobile devices has given rise to an especially high awareness of digital wallets and fewer people are carrying cash, consumer usage of digital wallets actually remains low. These are among the key findings of our recent Digital Wallet Usage Study, which was conducted via an online survey of more than 2,000 U.S. smartphone, tablet and desktop users in June.
Results showed that nearly 80 percent of consumers are aware of digital wallets—an extraordinarily high stat—but usage remains fairly low at less than one-third. Security proves to be the main barrier to widespread usage, followed by lack of usability in comparison to cash and credit cards, and not being top of mind as a payment option at the time of a purchase. This situation offers senior marketers a significant opportunity, both on the digital wallet provider side and for retailers, to proactively educate their audiences on the benefits of the digital wallet shopping experience and to support digital wallets as a form of payment to encourage adoption.
Additional study results showed that demographics play a key role in digital wallet usage, especially when combined with the fact that fewer consumers are carrying cash overall. On-the-go females represent a strong target demographic for digital wallets as 71 percent of females carry less than $30 in cash, compared with 54 percent of males. Additionally, three out of four consumers who don't carry any cash are under the age of 43. What's surprising is that nearly 60 percent of digital wallet users are male, despite carrying less cash than females. As far as the types of digital wallets used, females aged 18-29 tend to use merchant apps such as Target, Macy's and Home Depot, while males gravitate more toward discounts/coupons and price shopping. Males who do use merchant apps are mostly engaged in browsing and service related activities like paying bills and looking up information. With these stats in mind, marketers are poised to operationalize consumer demographic usage profiles and purchase patterns and thus extend their monetization opportunities through digital wallet payments.
Another finding of the study showed that the majority of digital wallet transactions are mobile (60 percent) and that three out of four transactions amounting to less than $10 are conducted weekly via a mobile phone. [pullquote align="left|center|right" textalign="left|center|right" width="45%"]Digital wallet purchases are most commonly coffee, drinks, retail items, games, books and groceries.[/pullquote] Given that the heaviest digital wallet users are on mobile and that the mobile industry continues to see consumers relying more and more on their devices for everyday activities, the potential for increased mobile-driven sales via digital wallets is enormous. As such, marketers and retailers that accept digital wallet payments will likely reap the benefits of demonstrating their investment in more mobile-friendly payment options.
As far as the leading providers, the top digital wallets used are PayPal (79 percent) Google Wallet (40 percent) and Apple Passbook (17 percent) with Google Wallet leading the large transaction pack with purchases of $30 or more (35 percent). However, Apple Passbook leads the way as far as usage on a weekly basis. The study also showed that 70 percent of digital wallet users take advantage of one or more digital wallets at least monthly, and daily digital wallet users tend to use more types of digital wallets (3) than infrequent users (1).
Ultimately, the digital wallet space is currently very fragmented with many players focusing on different areas without a clear market leader piecing everything together. As the market evolves, we expect to see more companies working to educate their customers on the value of using a digital wallet, optimizing their mobile merchant apps and/or partnering with businesses to secure the necessary equipment. However, proper demographic targeting is also needed to increase consumer adoption and wallet share. To be successful in the digital wallet market, marketers will need to develop new innovative ways to demonstrate value and transform current consumer behaviors, such as seamless loyalty integration, redemption options and helpful shopping tools.


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|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|"Daniel Farinha" <dan...@ktorn.com>
|RE: coding conventions
|Fri, 15 Aug 2003 13:58:12 GMT
I doubt that the 80 cols limit will be a coding convention. Personally I keep Eclipse with the print margin on at 80, just to keep the code readable on screen and, if needed, paper. But sometimes it's better to go over the margin than break the line in weird places. I consider the 80 margin more of a good practice than a strict convention. Daniel farinha -----Original Message----- From: email@example.com [mailto:email@example.com] On Behalf Of Fabrice M. Sent: 15 August 2003 13:42 To: firstname.lastname@example.com Subject: Re: coding conventions Aditya Gore <email@example.com> writes: > I am something of a newbie to open source... > Was browsing through the code and came across many files > that have lines that extend way beyond 80 characters. Cutting these > into multiple lines could be a task in itself. Do such things qualify > for a patch? Frankly said, and with all due respect, no. Nobody codes a J2EE application server with 80x25 passive terminals these days. Fabrice
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Future Kingdom Workers Ready for Self-Sufficiency
Uttar Pradesh, India — Christian Fellowship is embarking on a new, exciting journey to train their future pastor students to become self-sufficient. Ministering to such poor communities often means they are unable to provide for their own families from the church's tithe.
They are now offering classes in candle making, vegetable gardens, and sewing. In the future we plan to expand this program to include a microloan component. This training will provide great Hope for these future kingdom workers.


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Audi e-tron Concept | All electric. All Audi.
The first fully-electric Audi will be available to order late in 2018, with the first deliveries following in early 2019Register your interest | Find out more
Motability | Audi Q2
Available from £399 Advance PaymentFind out more
Browse the New Audi Range
Featured Audi Offers
Welcome to Yeovil Audi in Somerset.
Yeovil Audi strives to deliver an experience as prestigious as the vehicles we sell. As the only Audi approved Centre in the Somerset and Dorchester area, we are proud to represent the Audi brand.
Yeovil Audi has a dedicated sales team, combined with the skills of our specialist Audi trained technicians, ensure that a seamless service is provided to all our customers in every aspect of their Audi experience.
To achieve this, we invest for the future with our customers as the highest priority. Our customer-oriented view on decisions is driven by customer feedback and we always strive to ensure our customers' convenience comes ahead of our own. This is reflected in our customer satisfaction scores, as measured by Audi UK.


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When reporting a problem or requesting a service from IT, we make calls, send emails—or in most modern enterprises—go directly to a web portal to create a service ticket. Service tickets can also be auto-generated when something goes down such as an ATM machine, toll scanner, or some other Internet of Things (IoT) device.
Property fields and categorizations are part of the ticket as incident data. If not auto-generated, information must be manually entered along with choices from drop-down lists – for anything ranging from priority, department, category, or region to specifying a time and date for the ticket to be resolved.
The information that is contained in these incident service tickets within complex digital environments is a great source of actionable insight for C-level executives, data scientists, and IT operators.
"The digital transformation required to remain or become competitive means innovating and doing more for less. Enterprises need to get the right digital experience to the right users at the right time to achieve a business outcome regardless of costs." -IT Analyst
Service tickets and IT operations
The infrastructure and apps that run the business are the responsibility of IT operations while the IT service desk handles anything from employees wanting help with their digital office equipment to customers submitting tickets for a service that is provided directly to them. Some enterprises have multiple ticketing systems and do record keeping for just about every process including the delivery of software and documentation. Getting the most out of service ticket data requires not only responding to them in a timely manner but also integrating them into a comprehensive IT operations strategy.
Improved MTTR with efficient event management
Depending on the enterprise, service tickets can be associated with a configuration management database (CMDB) that is made up of the elements or configuration items (CIs) that are related to a service provided by IT. In some cases, there might not be a CMDB but there are IT infrastructure elements that can be related to the IT service—the network elements, the servers running the apps, the apps themselves, or even the vast storage needed by most enterprises.
It is a best practice to discover the CMDB and associate elements with monitored IT services. When there's a performance issue with any one of the elements related to the service, other related elements can be impacted and troubleshooting begins based on priority. This event in operations management can be associated with a service ticket depending on the level of integration that is configured:
Level 1—Incident created in ticketing system and linked to IT operations event
Level 2—Incident created in ticketing system for CI and linked to IT operations event
Level 3—Two Incidents created in ticketing system, one for the selected CI that is causing the outage and a second the for service that is impacted by the causal CI and both linked to the IT operations event
These levels of integration help with the more efficient resolution of IT issues. Moreover, once patterns of incidents are identified, automation can be configured to remediate issues without human intervention.
Insights from machine learning and analytics
Another way to elevate IT operations and incident management is to apply machine learning and analytics to the volume and velocity of service tickets to help achieve the digital transformation of decision making. For example, a service ticket analysis of 1,000's of tickets per day – or even per week – could reveal that password resets have tripled in the last month and the amount of time to address them with IT staff could be greatly reduced if the resets were handled automatically instead of with human intervention.
What if the volume of incident data unexpectedly spikes? Using a machine learning and analytics approach, the condition would first be flagged as abnormal based on what has already been learned from the past. Correlating this spike with trends based on properties like geography, problem, application, and so on could help quickly identify a source. Furthermore, if needed, you could apply natural language processing to the description field and quickly understand problem details.
Many enterprises are realizing that applying these ways of integrating service ticket incident data with the core of IT operations can help advance the business by being more efficient and putting valuable insight in the hands of decision makers across the enterprise.
Artificial Intelligence for IT Ops (AIOps) is the term that Gartner analysts have introduced to describe this approach that leverages machine learning, analytics, and a big data platform, as well as remediation through automation.
BMC's TrueSight helps with analytics and remediation on time-series and event performance data, incident tickets, business metrics, and social sentiment by leveraging the BMC portfolio of market-leading solutions.
For more information, see:
What Is AIOps? AIOps Explained
BMC AIOps Solutions
Enable proactive incident management
These postings are my own and do not necessarily represent BMC's position, strategies, or opinion.


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Kentucky Wildcats Area Rug - Tailgater, Mascot Design|
Show your team pride and add style to your tailgating party with FANMATS area rugs. They are made of 100% nylon carpet and has non-skid Duragon® latex backing. This officially licensed rug is chromojet printed in true team colors. 60"x72" in size. Made in U.S.A. This is a special order item. Please allow 2-3 for delivery.
Share this Product
Shipping is a flat rate $4.99 for the continental US. Orders are mailed via first-class or priority mail. Most products arrive 3-5 business days after processing time. Shirts and Pre-Sale items are Made to Order and may take up to 7-14 Business days to arrive after ordering.
Return Policy
If you're not 100% satisfied, let us know and we'll make it right.


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If you are looking for a permanent finance analyst position, we recruit for organisations across a variety of sectors.
Our highly experienced team has helped companies throughout England and Wales to find the right staff for the right job. This means we are often aware of vacancies before other recruiters, giving you an advantage over other candidates.
Begin your search for a permanent financial analyst role below, and ensure you do not miss out on any new vacancies by signing up for our daily job alerts. Alternatively, you can submit your CV and a member of our team will get in touch.


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Had a bit of fun tonight, helping my daughter with her science project. We made an Orrery, and it was surprisingly easy (well, especially when you have a CNC router at your disposal!)
It is a basic design, very similar to one we found online. Although the Earth spins as it moves around the sun, and the moon spins around Earth, the two are directly linked, so the timing of each orbit is the same. I roughly played with the dimensions of the two pulleys, so the moon orbit is approximately 12 times the orbit around the sun.
Now Jess just has the hard bit to go – getting it all painted up!
Filed under: Uncategorized |


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Cyneburga: A Plague of Lies
Glerothain of Theinburgh
Player: Brendan
Level 5 Dwarf Monk
Glerothain is known for his accent, for not taking any guff, and for the beating he can dish out with his mighty fists. A monk from the coastal mountain city of Theinburgh, he is currently searching for the remnants of his clan. Theinburgh was one of the first places to be hit by the plague, and Glerothain's kin were scattered in the exodus.
Glerothain has a kind heart that sometimes brings him into conflict with the more unscrupulous elements of the party. However, he seems to get along well with T'sabo, whose sense of morality more closely matches his own.


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Stiles Cove Path: Flatrock Cove to Ferry Point is a 15.8 kilometer lightly trafficked out and back trail located near Flatrock, Newfoundland and Labrador, Canada that offers scenic views. The trail is rated as moderate and primarily used for hiking, walking, and trail running.
trail running
2 months ago
Beautiful, highly recommend it as a day hike.
2 months ago
My favourite on the east coast trail.
8 months ago
À river of fantastic viewpoints
hiking11 months ago
absolutely breathtaking! i started in flatrock and went to the halfway point and turned back. was doing it alone so couldnt go the full way as only 1 vehicle. if you want to do thw full trail suggest having a vehicle at either end.


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Here is Mariah Carey captured in the process of your actual acting. We've seen her do it before in Precious. We've seen her not do it before in Glitter. We imagine this will be more along Precious lines.
It is from her new film The Butler directed by Lee Daniels of – what do you know? – Precious fame, who is a gay. An African American one. And she is playing what is known in the slave business as a 'field slave', by which we understand a slave who works in a field as against a 'house slave', who… oh, you can work the rest out, surely!
The film is about a White House butler who served at *checks notes* the White House from the 50s to the 80s, so we're not sure where slaves come in, unless she is his mum or something. It's a starry cast with everyone from John Cusack, Robin Williams and Forest Whitaker to Vanessa Redgrave and, our favourite, Jane Fonda as First Cunt, Nancy Reagan.
Anyway, anyway… we bet that 'Riah's hair under that do-rag is a riot of colour and movement. You can't supress the glamour so don't try.
Don't you love it when Mariah goes frumpy and does proper acting?,


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Here is a collection of articles from A Guy and His Monkey that look at the funny side of society.
By Nero Calrissian 4/25/17
We rank the greatest long form music videos of all time.Beastie Boys
By Chip White 4/17/17
Some chant about the death of chauvinism as if it was a positive thing. Not too long ago, a man knew his place and a woman knew hers. The question boiling in a real man's mind is, what happened?Death of the Chauvinist
By R.W. Phields 3/20/17
With all of the various wieners in the world, picking out the perfect one is not an easy task so we asked an adult film star, a florist and a Catholic priest to help us out.The Perfect Wiener
By Horse Dancer 2/27/17
Football and Salvation Ruminations. Who really is more Godlike, the handsome quarterback or the hippie carpenter?Brady or Jesus
By Chip White 2/13/17
We sent ace writer and proud womanizer Chip White to the Women's March in Washington to pick up chicks. This is his report.Pussy Grabbing
What's NewThe Greatest Long Form Music Video Ever


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- Research
- Open Access
Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures
© da Silva et al.; licensee Springer. 2013
- Received: 28 September 2012
- Accepted: 23 June 2013
- Published: 17 July 2013
In spite of the large number of reports on fed-batch cultivation of E. coli, alternative cultivation/induction strategies remain to be more deeply exploited. Among these strategies, it could be mentioned the use of complex media with combination of different carbon sources, novel induction procedures and feed flow rate control matching the actual cell growth rate. Here, four different carbon source combinations (glucose, glycerol, glucose + glycerol and auto-induction) in batch media formulation were compared. A balanced combination of glucose and glycerol in a complex medium formulation led to: fast growth in the batch-phase; reduced plasmid instability by preventing early expression leakage; and protein volumetric productivity of 0.40 g.L-1.h-1. Alternative induction strategies were also investigated. A mixture of lactose and glycerol as supplementary medium fully induced a high biomass population, reaching a good balance between specific protein production (0.148 gprot.gDCW -1) and volumetric productivity (0.32 g.L-1.h-1). The auto-induction protocol showed excellent results on specific protein production (0.158 gprot.gDCW -1) in simple batch cultivations. An automated feed control based on the on-line estimated growth rate was implemented, which allowed cells to grow at higher rates than those generally used to avoid metabolic overflow, without leading to acetate accumulation. Some of the protocols described here may provide a useful alternative to standard cultivation and recombinant protein production processes, depending on the performance index that is expected to be optimized. The protocols using glycerol as carbon source and induction by lactose feeding, or glycerol plus glucose in batch medium and induction by lactose pulse led to rSpaA production in the range of 6 g.L-1, in short fed-batch processes (16 to 20 h) with low accumulation of undesired side metabolites.
- SpaA antigen
- Erysipelothrix rhusiopathiae
- Bioreactor cultivation
- Auto-induction
- Lactose feeding
Swine erysipelas causes great economic losses in swine culture worldwide, and has been controlled by the use of live attenuated or inactivated vaccines (Wood 1992). These formulations offer good levels of protection but can aggravate arthritic problems (Freeman 1964; Wood 1992). The surface protein A (SpaA) from Erysipelothrix rhusiopathiae is the most studied antigen from this pathogenic bacteria and has shown promising results as a candidate to be used in a subunit vaccine against swine erysipelas (Imada et al. 1999; Kitajima et al. 2000), replacing the cellular vaccines in use.
The production of recombinant antigenic proteins for subunit formulations is among the technologies proposed for the next generation of vaccines (Ashtekar et al. 2012). In case of bacterial antigens, prokaryotic expression systems, such as the E. coli bacterium, are the most popular choice due the low costs of production, high productivities of heterologous protein and availability of many genetic tools (Demain and Vaishnav 2009).
Recombinant protein production in E. coli has been extensively investigated, particularly as high cell density cultivations (HCDC) employing bioreactors operating in fed-batch mode and using defined medium (Shiloach and Fass 2005; Choi et al. 2006; Shojaosadati et al. 2008). HCDC studies usually focus on strategies to achieve high biomass concentrations, whereas the induction is triggered by applying simple techniques, such as addition of IPTG pulse (Babaeipour et al. 2007; Carvalho et al. 2011; Khalilzadeh et al. 2004). Thus, induction strategy, feeding control and media formulation are among the main process conditions eligible to be further improved. Besides that, many reports point out for the need to study case by case all these process variables in order to identify the best operation conditions to produce a specific protein.
Induction conditions for proteins under the lac promoter control have been largely studied but, in spite of that, just a few new alternative induction strategies have been proposed, such as the so called "auto-induction" (Studier 2005) and the "continuous lactose induction strategy" (Horta et al. 2012; Lim et al. 2004). The auto-induction medium contains glucose, which encourages fast cellular growth in the early stages of the culture, while also inhibiting expression leakage by catabolic repression. Lactose is also present, and is consumed after glucose depletion, activating the lac promoter to induce recombinant protein expression. Glycerol is included to (together with lactose) provide sustained growth during the induction phase (Studier 2005). In spite of the widespread use of auto-induction in shaker flask experiments, only few bioreactor cultures carried out with auto-induction media in batch (Giomarelli et al. 2006; Blommel and Fox 2007a, b) or fed-batch mode (Jia et al. 2011; Xu et al. 2012) have been reported so far. In these studies, bioreactor media contain basically the same carbon and nitrogen sources, but the total carbon source concentration was increased by factors of approximately two (Blommel et al. 2007a, b), three (Giomarelli et al. 2006) or four (Xu et al. 2012) in comparison to the sum of glucose, glycerol and lactose concentrations present in the original auto-induction ZYM-5052 complex formulation (Studier 2005). Consequently, maximum optical densities (600 nm) in the range of ~ 20 to 40 (Giomarelli et al. 2006; Blommel et al. 2007a, b; Jia et al. 2011) were reported for batch experiments and of 140 for fed-batch cultures (Xu et al. 2012). Nevertheless, none of these studies aimed to compare induction strategies. They were focused on biomass production for further purification studies (Giomarelli et al. 2006; Blommel et al. 2007b), on the role of yeast extract or peptone components as inducers or on the influence of aeration on the auto-induction process (Blommel et al. 2007a).
Concerning media formulation, the majority of reports rely on defined media using glucose or glycerol as carbon sources (Shiloach and Fass 2005).The formulation proposed by Neidhardt et al. (1974) containing only simple inorganic salts and a defined carbon source (generally glucose), and improved by the addition of citric acid, EDTA and thiamine-HCl (Riesenberg et al. 1990) is commonly used for E. coli fed-batch cultures. Korz et al. (1995) used this medium and showed that higher cell densities could be achieved replacing glucose by glycerol. The present work shows that balanced mixtures of both carbon sources can also be used to speed up growth with negligible acetate accumulation. Complex media formulations with glycerol as the main carbon source can also be employed with excellent results.
Regarding the control of supplementary feed flow rate in the fed-batch phase, the conventional strategy manipulates the input of nutrients so that the specific growth rate would not exceed a pre-selected, heuristic, critical value (μcritic), to prevent toxic by-products formation. Since glycerol did not trigger acetate accumulation under aerobic conditions (Pinsach et al. 2008), an innovative approach was developed by Horta et al. (2012) to control the carbon source supply in HCDC carried out in defined medium with glycerol. In this work, the exponential feeding flow was tuned using actual rates estimated on-line during the cultivation by a capacitance probe, assuming that the feed of supplementary medium should be limited by the actual growth rates, which reflect the dynamics of the cell metabolic state.
Protein synthesis is also influenced by the chosen cultivation conditions. Usually, defined media and limited growth rates are employed during induction phase (Korz et al. 1995; Lee 1996), which may hamper protein production. However, when cells are in a rich nutritional condition they increase their ribosome content in order to grow faster. Indeed, a linear correlation between cellular ribosome content and growth rate was reported elsewhere (Keener and Nomura 1999). Complex medium and a high growth rate (not limited by a pre-set value during fed-batch operation) probably favor the increase of ribosomal content, which may also improve the productivity of the desired recombinant biomolecule.
In summary, this work presents studies of different cultivation and induction strategies for the production of a recombinant fragment (42 kDa) of the N-portion of SpaA, whose antigenic potential has already been demonstrated in swine vaccination tests (Imada et al. 1999). Gene fragment coding for rSpaA was cloned under the lacUV5 promoter (Silva et al. 2012). The performance of the conventional bioreactor operation protocol, comprising high biomass accumulation prior to induction of expression by a pulse of lactose or IPTG, was compared with alternative protocols using real-time updated exponential feeding profiles of two supplementary media: a glycerol-based complex medium containing lactose, and the auto-induction medium.
Materials and methods
Experiments were carried out with E. coli BL21(DE3) cells producing a recombinant fragment of the SpaA protein from Erysipelothrix rhusiopathiae. Cloning procedures were done according to standard protocols (Sambrook and Russell 2001), as previously described (Silva et al. 2012). Briefly, the 1026 pb fragment codifying for the antigenic region of the SpaA protein (Imada et al. 1999) was amplified by PCR from the E. rhusiopathiae chromosomal DNA and cloned into pGEM-T vector (Promega). After sequencing, the spa A fragment was cloned into the expression vector pET28a (Novagen) and used to transform E. coli BL21(DE3) cells.
Media composition used for bioreactor cultivations
Experiment #1
Experiment #2
Experiment #3
Experiment #4
Nutrient (g.L-1)
Yeast extract
kanamycin (μg.mL-1)
Antifoam PPG 30% (mL.L-1)
Metal solution
Metal solution composition
Batch and induction medium (mg.L-1)
Fed-batch medium (mg.L-1)
Ferric citrate
Zn (CH3COO)2.H2O
Experiment #1: the conventional process strategy was used in this cultivation. A modified LB medium containing glucose as carbon source was used in both batch and fed-batch phases. An exponential feeding profile was set by Eqs. 1 and 2, assuming a constant μSET = μcrit. Induction was carried out by adding a pulse of IPTG, for a final concentration of 0.25 mM in the medium. Two hours after induction, the feeding profile was changed to a constant flow rate, in order to avoid substrate accumulation.
Experiment #2: the auto-induction strategy was employed in this cultivation. Here, induction occurs naturally after glucose exhaustion, as the cells start consuming the lactose present in the medium. The supplementary medium, containing glycerol and lactose as carbon sources was supplied at an exponential feeding flow rate (Eqs. 1 and 2), assuming a constant μSET = μcrit.
Experiment #3: Glycerol was used as sole carbon source during the batch phase. Induction was done along with feed supply, using a feeding medium containing glycerol plus lactose. By this protocol, heterologous protein expression was induced gradually as the supplementary medium was added to the bioreactor. An exponential feeding flow rate was employed based on the actual growth rates of the bacterium to reset both feed control parameters (C1 and C2 in Equation 1).
Experiment #4: the batch medium was prepared using a combination of glycerol and glucose. Fed batch was carried out exclusively with glycerol as carbon source, using an exponential feeding, again based on the actual growth rates as implemented at Experiment #3 (C1 and C2 automatically update in Eqs. 1 and 2). Induction was done by a single addition of 400 mL of induction medium containing lactose, after a high biomass concentration was reached.
Inocula were prepared picking up cells from a glycerol stock suspension. The cells were aseptically transferred to the culture medium. Overnight incubation in a rotary shaker was done at 37°C, 250 rpm using the medium of the batch phase of each experiment, with a volume corresponding to 10% of the bioreactor cultivation volume. For the auto-induction strategy (Experiment #2) the inoculum medium was prepared with 0.5 g.L-1 glucose and 5.0 g.L-1 glycerol, but without lactose.
Experiments were carried out in a 6.0 L bioreactor (in-house made), connected to a gas analyzer (Sick/Maihak S710). Two mass flow controllers (GFC Aalborg, USA) were employed to supply air and/or oxygen, keeping dissolved O2 concentration at 30% of saturation (O2 sensor Mettler Toledo, model Inpro 6800) by changing the agitation speed (between 100 and 900 rpm) and enriching air with oxygen when the maximum agitation was reached. The total gas flow rate was restricted between 1 and 2 VVM. The pH was controlled at 7.0 ± 0.2 by adding NH4OH 25% (v/v) and H3PO4 10% (v/v). All experiments were conducted at 37 ± 0.5°C (growth and induction phases) by controlling the temperature of the water re-circulating in bioreactor jacket. A capacitance probe (Biomass System - Fogale Nanotech) was used for on-line monitoring of biomass formation. The permittivity data collected from the capacitance probe was smoothed by the classical moving average filter (Eilers 2003). The filter used had a size vector of 80 rough data. Online data were used to estimate cell growth rate. The supervisory system SuperSys_HCDCR (Horta et al. 2011Horta et al. 2012), based on LabView (National Instruments), was used to monitor and control the experimental apparatus.
Cx 0 and V 0 are cellular concentration and volume, respectively, which were updated at every 10 min, Y X/S is the biomass yield, m the maintenance coefficient (both assumed equal to 0.6 gDCW.gsubstrate -1 and 0.025 h-1, respectively), while Cs 0 is the carbon source concentration in the supplementary medium (Table 1), and Cs R the expected residual concentration of the carbon source in the reactor (assumed as 5 g.L-1). When more than one sugar was present in the medium, lactose and/or glucose and/or glycerol were pooled together as one limiting substrate. In Experiments #1-2, the tuning parameters C1 and C2 were kept constant during all the growth phase, with C2 = μSET = 0.13 h-1 (the "critical" specific growth rate, μcrit) in Equation 1 and μ= μSET = 0.13 h-1 in Equation 2 (the "classical" approach).
In Experiments #3 and #4, both C1 and C2 were continuously retuned at each 10 min using the values of μ obtained online from the permittivity measurements provided by the biomass sensor (Horta et al. 2011). Thus, for Experiment #3 and #4, Equations 1 and 2 are no more based on a phenomenological reasoning that imposes a fixed value for μ. Instead, Equations 1 and 2 must be faced as empirical, open loop control laws.
Analytical methods
Cellular growth: culture broth optical density reading (OD, λ = 600 nm), dry cell weight measurements (gDCW.L-1), counting CFU.mL-1 and on-line estimation by measuring broth permittivity (pF.cm-1).
Metabolites and carbon source concentrations were analyzed by HPLC as previously described (Silva et al. 2008). Glucose and glycerol were also monitored throughout the fermentation by a glucose oxidase reaction kit (Laborlab) and a triglycerides determination kit (Laborlab), respectively. Protein expression was checked by SDS-PAGE (Laemmli 1970) and the gels were photographed to estimate the protein production using the software ImageJ (Abramoff et al. 2004). BSA samples with known concentration (0.1, 0.2, 0.4, 0.8 and 1.0 mg.mL-1) were used as standards to estimate rSpaA concentration on gels.
To assess plasmid loss during the cultivations, diluted samples of culture broth were initially spread on LB agar plates and incubated for 16 h at 37°C. Seventy five colonies from each sample were simultaneously transferred using sterile toothpicks to new plates with and without kanamycin (30 μg.mL-1). After incubation, colonies grown at both conditions for each sample were counted for comparison.
Results and discussion
Substrate uptake, biomass formation and metabolite accumulation
The capacitance probe measures the dielectric permittivity of cell suspensions, which is related not only to biomass concentration, but also to the cell structure as well as to its morphological and physiological states (Matanguihan et al. 1994). A relationship between capacitance and cell viability has also been suggested (Matanguihan et al. 1994; Asami 2002).
Comparison of cell growth results, including standard deviations of estimated kinetic parameters
Experiment #1
Experiment #2
Experiment #3
Experiment #4
μmax (h-1)
1.12 ± 0.05
0.80 ± 0.27
0.62 ± 0.05
1.08 ± 0.02
Yx/s (batch phase)
0.60 ± 0.01
0.57 ± 0.04
0.59 ± 0.05
0.64 ± 0.04
Final Cx (g.L-1)
[acetate] maximum (g.L-1)
[formate] maximum (g.L-1)
Maximum specific growth rate was 0.80 h-1, and biomass yield coefficient was 0.57 gDCW.gsubstrate -1, both a little lower than with glucose (Experiment #1), as expected (Table 3).
Permittivity data detected the drop in cellular viability from the mid fed-batch phase on, as also shown by CFU counting. Such a reduction cannot be observed in the dry cell weight data as this method does not distinguish between viable and non-viable cells.
From the growth profile depicted at Figure 2, it seems that the anticipated expression overloaded cellular metabolism and affected cell division, despite the abundance of nutrients. It caused glycerol accumulation and low biomass formation. However, lactose remained intensely consumed until the end of cultivation.
Glycerol was used as sole carbon source in the batch medium for Experiment #3. An alternative induction strategy was also studied here, where the feeding medium was prepared with a combination of glycerol and lactose to promote rSpaA expression and sustain growth at the same time.
As it has been reported in the literature, E. coli cells grow slower on glycerol than on glucose due to differences in the substrate assimilation mechanisms (Korz et al. 1995; Lee 1996). But, on the other hand, growth on glycerol leads to lower toxic byproducts formation as this lower assimilation rate does not cause metabolic overflows (Lee 1996; Tripathi et al. 2008).
As also observed in Experiment #2, glycerol accumulation occurred after the mid fed-batch phase but lactose was almost completely consumed, showing that glycerol concentration can be reduced in the formulation of the supplementary medium. In this experiment, the growth deceleration during heterologous protein expression was not as intense as in Experiment #2, neither was the accumulation of lactose. Therefore, lower lactose concentration– for instance, in the range of 80 g.L-1 (half of that used in Experiment #2) – might be enough to promote an efficient induction in a gradual manner. The absence of lactose in the batch medium and the control of μ matching the actual growth rate were other successful changes implemented in this experiment. Together, they allowed intense growth towards 40 gDCW.L-1 within 20 h of cultivation. As discussed by Sandén et al. (2002), higher specific growth rates favor protein production by influencing both the protein synthesis apparatus and the energy production.
Biomass formation was improved by a combination of glucose and glycerol in the batch phase and after only 7 h a cellular concentration around 30 g. L-1 was reached. Despite such an intense cellular growth, there was no accumulation of organic acids.
Permittivity data matched the biomass formation profile up to 8 h from the onset of the experiment, when induction, by a lactose pulse, took place. As also observed in Experiment #3, as protein expression triggers an increase of metabolic activity, the capacitance probe signal is affected (see Figure 4, between 8–10 h).
A maximum of 66 gDCW.L-1 was achieved after only 16 h of cultivation. Such a high biomass productivity can be associated mainly to two aspects of this experiment: i) the improved batch medium, containing glycerol plus glucose and; ii) the feeding rate controlled in such a way to sustain the real growth rate of the cells, which was inferred on-line from data collected by the capacitance probe. In fact, at the beginning of the exponential feeding phase (6.8 h of cultivation), the value of μSET was around 0.35 h-1, which is significantly higher than the pre-defined μSET used in Experiments #1 and 2 (0.13 h-1).
The free software SuperSys_HCDCR (Horta et al. 2011 2012) was used to control the feeding flow rate. Based on the broth permittivity signal from the capacitance probe, the supervisory system retuned the parameters of the feed flow control each 10 min. Thus, biomass growth was limited by the cells own growing capacity, and not by the parsimonious feed of a limiting substrate. The feeding flow decreased with time after induction, accompanying the growth deceleration caused by the recombinant protein expression.
Glycerol as carbon source in the supplementary medium did trigger the aerobic acetic fermentation as reported by Korz et al. (1995), but the production of acetate was negligible. This is a remarkable difference with respect to growing cells on glucose at high rates. The saturation of the cellular catabolic fluxes, typical for cultivations using glucose, is not observed for glycerol, due to its lower uptake rate by the cells. This fact allows one to feed the bioreactor in such a way that cells are able to grow at high specific rates without accumulation of toxic by-products. On the other hand, during the batch phase, a balanced mixture of both carbon sources leads to fast biomass formation but also without acetic acid accumulation.
During the fed-batch phase, lactose consumption led to a slight accumulation of glycerol, as lactose, besides its intrinsic induction activity, acts as a carbon source. However, after lactose depletion, the accumulated glycerol was readily consumed.
A comparison of results from the four cultivations is presented in Table 3.
As it can be seen in Table 3, the highest μmax value was achieved when cells were cultivated using glucose (Experiment #1); cultivation #3, where glycerol was the carbon source, showed the lower maximum specific growth rate, as expected. Intermediate values of μmax were observed where a mixture of substrates was used (#2 and #4). The balanced composition used in Experiment #4 led to a high μmax value, of 1.08 h-1. Table 3 also shows that biomass yield coefficients were not significantly influenced by the carbon sources present in the rich media. As expected, growing on glucose (#1) led to significant amounts of formic and acetic acids production, while in the other experiments no formic acid nor other toxic metabolic by-products were detected at all. Lactose feeding in Experiments #2 and #3 induced acetate production, which is related to the degradation of this sugar into glucose and galactose. The latter is not consumed by E. coli BL21(DE3) cells, but glucose metabolism could favor acetate formation under the stressing conditions of heterologous protein over expression.
Induction strategy and protein production
The rSpaA protein was expressed in the form of inclusion bodies and different levels of protein production were reached according to the induction strategy adopted. For the classical process, where cells were grown in modified LB medium and induced by an IPTG pulse (Experiment #1), the maximum rSpaA specific production reached 0.14 gprot.gDCW -1 after four hours of induction. This value corresponds to approximately 25% of total cellular protein content – considering that total cellular protein content is around 55% of its dry weight (Neidhardt and Umbarger 1999).
A great efficiency for recombinant protein production was registered for the auto-induction protocol (Experiment #2). Lactose from batch and feeding media gradually activated rSpaA expression and the highest specific protein production from all experiments performed (0.16 gprot.gDCW -1) was achieved after 8 h of culture. On the other hand, the high metabolic load, caused by prolonged and intense induction, impaired cellular growth.
The auto-induction protocol showed to be very efficient to achieve high levels of protein expression within a short period. However, it does not seem to be appropriated for fed-batch cultures because the high cellular concentrations desired in this mode of operation cannot be reached due to the intense metabolic burden associated to the prolonged and early induction process.
Feeding lactose as inducer in the supplementary medium (Experiment #3) resulted in high rSpaA specific and volumetric productivity at the end of the cultivation (0.15 gprot.gDCW -1 and 0.323 gprot.L-1.h-1, respectively), which confirm the good performance of the alternative induction protocol. Recombinant SpaA accumulated up to almost 6.0 gprot.L-1, corresponding to around 27% of total cellular protein content after 8.5 h of induction.
Addition of a lactose pulse to induce recombinant protein expression was tested in Cultivation #4. Although the rSpaA specific production was the lowest registered in all the four experiments, the volumetric productivity reached was the highest among all the strategies, due to the great biomass productivity.
The lactose concentration of 20 g.L-1 in the moment of induction was superior to values commonly reported in the literature (Li et al. 2006; Neubauer et al. 1992). It led to an amount of 76.5 g of lactose totally consumed within 3.3 h after its addition. Compared to the total amount of lactose fed in the other experiments in the same 3.3 h (10.5 g in #2 and 36.1 g in #3), the value of 76.5 g used in #4 was not too low to sustain the same pattern of induction in the continuously growing cell population.
Once biomass concentration during induction phase of Experiment #4 was significantly higher than in Experiments #2 and #3, a better comparison is provided by estimating the overall specific lactose uptake rates (OSLUR) and overall specific glycerol uptake rates (OSGUR) for each of them during the first 3.3 h of induction. Rather similar values for OSLUR (0.09 for Experiments #2 and #3 and 0.11 glactose.gDCW -1 h-1 for Experiment #4) and for OSGUR (0.47 and 0.41 gglycerol.gDCW -1 h-1 for Experiments #3 and # 4, respectively) were observed. The OSGUR of Experiment #2 was rather lower (~ 0.23 gglycerol.gDCW -1 h-1) due to the reduced glycerol uptake associated to the low biomass build-up caused by the early protein expression triggered by auto-induction protocol. Thereby, these results suggest that the inferior specific protein production observed at Experiment #4 was not caused by lack of lactose. Instead, it seems to be related mainly to the way lactose was supplied. It could be associated to the modulation of the transcription rate provided by the continuous inducer feeding (Exps #2 and #3), which led to extended production phase and better yields (Striedner et al. 2003). The same differences between pulse induction and continuous inducer dosage using IPTG were reported elsewhere (Pinsach et al. 2008), and better results were also achieved using the inducer feeding protocol.
For the auto-induction experiment (#2), the reasonable values of specific protein production and volumetric productivity reached after only 8 h of cultivation (that corresponds to a few hours after the end of the batch phase) shows that such protocol is better suited for short experiments, namely batch cultures, where high protein production can be achieved within a few hours by the early induction of expression.
The protein production profile observed in the auto-induction, fed-batch culture carried out in the present study (Figure 5) is different from the continuously increasing protein production profile attained for the auto-induction, fed-batch cultures of Xu et al. (2012). These authors used higher glucose concentration (30 g/L) and lower inducer concentration (~0.2 g/L) in the batch medium. In addition, a lower temperature (28°C) and a constant rate of feeding supply were adopted. So, protein production during batch phase was not expressive, and during the fed-batch phase the induction of protein synthesis took place at slower rate, relieving the metabolic burden and resulting in the increasing profile, even after 18 h of culture. On the other hand, such a strategy impairs productivity, which is an important performance index to be optimized.
In the other experiments, with shorter periods of induction, specific protein production and volumetric productivity did not decrease when the growth rate decelerated. Experiment #1, induced by IPTG, had the shortest induction phase and specific protein production and volumetric productivity were evaluated only at the end of the process (0.136 gprot.gDCW -1 and 0.25 g.L-1.h-1, respectively).
In fact, Figure 5 shows that time plays a less important role than expected during the induction phase. First of all, data depicted at Figure 5a, b show that significant protein accumulation in the cell was already attained after 2 – 3 h of continuous induction with lactose, what is rather shorter than the usual, minimum 8 h of induction. Generally speaking, the specific production (protein/cell mass) increased slightly with time (with one exception at the beginning of induction of Experiment #2) while the effect on volumetric productivity of the protein was not monotonic, due to the different growth rates after induction. Experiment #4, in particular, showed a trend towards increasing productivities as time goes by.
Comparative performance for production of some biopharmaceutical proteins by E . coli BL21(DE3) fed-batch cultures in complex medium
Product & molecular weight
Carbon source/inducer
Culture time (h)
Biomass productivity (gDCW.L-1.h-1)
Protein volumetric productivity (gprot.L-1.h-1)
Specific protein production (gprot.gDCW -1)
Product content (g.L-1)
Human soluble B lymphocyte stimulator (hsBLyS) 17-18 kDa
Li et al. 2006
Tilapia insulin-like growth factor 2 - 7 kDa
Hu et al. 2004
Streptokinase 47 kDa
Goyal et al. 2009
Anti-cancer drug TATm-survivin (T34A) 17 kDa
Zhang et al. 2009 (t = 14 h)
Anti-HIV protein Griffthsin (GRFT) 14.5 kDa
Auto induction (Glucose + glycerol + lactose)/lactose
Giomarelli et al. 2006
this work:
SpaA from E. rhusiopathiae 42 kDa
Exp #1 (t = 11.5 h)
Auto induction (Glucose + glycerol + lactose)/lactose
Exp #2 (t = 8 h)
Exp #3 (t = 20 h)
Glycerol + glucose/lactose
Exp #4 (t = 16 h)
Both specific protein production and volumetric productivity impact on process performance. In addition, for bioprocess optimization, the economics of each solution must be assessed, including the cost for disruption of cells and separation and purification of the protein.
Cellular productivity reflected the induction strategy and media formulation that were adopted. Conditions with prolonged periods of induction (Experiments #2 and #3) led to lower biomass productivity caused by the intense stress imposed to the cells. Formulations with glucose (#1) or glucose plus glycerol (#4) promoted fast growth, reducing the total time of the process.
Plasmid maintenance is also strongly influenced by the induction strategy employed. It remained between 96 and 100% before induction for all experiments, and decreased in different intensities according to the induction regimen adopted, ranging from 70% (Exps #2 and #3) to 95% (Exp #4) at end of the processes. The metabolic burden imposed to plasmid-carrying cells after induction causes a growth deceleration, which favors the growth of plasmid-free cells, generated by unequal partition of plasmids between daughter cells (Summers 1991). Shorter periods of induction may prevent the accumulation of plasmid-free cells, avoiding a reduction in the protein productivity with time. Glucose in the batch medium is also supposed to have a beneficial effect on plasmid maintenance, as it represses expression leakage before induction by catabolite repression (Zhang et al. 2003).
The production of heterologous proteins under the control of lac promoter in rE.coli fed-batch cultures has been widely studied in the last 15 years. But, the majority of the reported studies focus on IPTG as inducer and defined media containing glucose as major carbon source. Here we showed that lactose supplied continuously as part of feeding medium formulation is an effective approach to fully induce protein expression.
Concerning carbon sources, the balanced combination of glucose and glycerol in the batch medium is a simple way to boost growth even with glycerol as main substrate. Furthermore, during the fed-batch phase, growth on glycerol can be kept as high as 0.35 h-1 in complex medium without metabolite formation. This enables faster biomass production leading to shorter cultivations. Even when glycerol accumulated to 30 g.L-1, neither metabolite formation nor inhibition of protein synthesis occurred, as it would actually happen if glucose was used, instead.
The auto-induction protocol also studied here showed to be best suited for simple batch cultivations, where great amounts of recombinant protein can be reached in a short period of time by running an easy-to-do experiment.
We thank Amadeus Azevedo for HPLC analysis and technical assistance. Financial support for this work was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP Process 2007/04743-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Process 475.128/2007-9).
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