text:
HRV's House: Henderson Community Stadium celebrates its 20th anniversary
Over the past twenty years, it's been the place where parents wiped tears as they watched their children walk across the stage to collect their diplomas.
All you need is ... to come on the Magical History Tour
Join the Cascade Locks Historical Museum on The Magical History Tour on the Columbia Gorge Sternwheeler on Friday, Oct. 9, from 5:30-9:30 p.m.
Yesteryears: Lower valley apple harvest in full swing in 1945
Hood River Valley's apple harvest was in full swing in the lower valley Monday of this week, and with fine weather prevailing and an increasing number of both white and Mexican pickers available, fruit was coming off the trees in considerable volume. Contrary to the usual rule, Newtowns were the first of the late apples to be harvested, and the picking of both Delicious and Spitzenbergs was delayed to add to the already good color attained by these red varieties.
Fruit and Craft Fair adds ranch sorting and other events
One of the Columbia Gorge's most popular autumn events — the Gorge Fruit and Craft Fair — arrives Saturday and Sunday, Oct. 17-18, from 10 a.m. to 5 p.m., at the park-like Hood River County Fairgrounds, 3020 Wy'east Road, near the community of Odell. Admission and parking are free.
Harvest Quilt fundraiser on now
The Harvest Quilt is a yearly community fundraiser for a local non-profit. This year, Hood River Adopt A Dog is honored to be the recipient of the quilt and is raising money by selling raffle tickets. The quilt is being shown around town and tickets are being sold at ETC — Every Thread Counts — and at Gorge Dog and Hood River Adopt A Dog, 3910 Heron Drive, in Odell. Tickets are 1 for $2, 3 for $5, or 7 for $10.
'Romeo and Juliet' staged with masks and music
William Shakespeare's best-known tragedy comes to the Hood River stage this weekend for the first time.
Entertainment Update for Oct. 7
Power, Einhorn play Hood River; Visionseekerz at Moth lounge; more
A wish and a zip: Hawks Ridge residents enjoy adventure aloft
Three residents of Hawks Ridge Assisted Living in Hood River wanted to go on a zip line, so after some research by staff, they headed to Skamania Zip Tours, located at the Skamania Resort Lodge, on Sept. 8 .
'Paws for a Cause' event returns Oct. 15
On Oct. 15, the fourth annual Paws for a Cause silent auction and social event will be held by Hood River Adopt A Dog (HRAAD) at Springhouse Cellar Winery, 13 Railroad Ave., Hood River, from 5:30-8 p.m.
Harmony of the Gorge invites women to sing for the holidays
Oct. 6 is the kickoff night for Christmas Chorus with Harmony of the Gorge, a local chapter of Sweet Adeline's International.
CHURCH NEWS: Revival in Parkdale, Odell, Hood River
Updated for Oct. 3
Dr. Jim Schuppe will again lead this year's Fall Revival, which will rotate between Parkdale Baptist, Odell Baptist and Tucker Road Baptist Oct. 9-12.
Entertainment Update for Oct. 3
Wasco Bros. in The Dalles Oct. 2; Tim Snider, more at CEBU; more
Travelogue: Hood River backpacker follows trail mantra: 'Hike Your Own Hike'
When I found myself jobless at the end of May, I decided to follow a long-held dream to solo-hike sections of the PCT (Pacific Crest Trail).
Roots and Branches: Reviving 'Nisei picnic': What's old can be new again
Saturday evening kindled fond memories of a Japanese tradition we let slip away decades ago.
Parkhurst Place supports fire fighters
Residents and staff of Parkhurst Place Assisted Living have put together care packages for firefighters "battling the front line fires near John Day, Ore.," reports Community Relations Manager Terri Hansen.
Previous Next
- Dar Williams is concerned with bees, sings about myth and power and likes towns with 'positive proximity'
- Expect familiar and new songs from Quarterflash
- Sounds of Summer series kicks off with the Pat Travers Band July 21
- Lavender Daze features new music from MMB
- All she wants to do is dance
- 'Here' is there and everywhere
- These guys will probably grow on you
- Quiet on the set: An interview with Aaron Keim
- Lost radios and happy memories
- Welcome to our new site!


text: Featured comments + Add comment Comments (30) Featured SoulSercher620 ""Titanfall breathed new life in FPS games" How by adding mechs? Something that has been done in shooters already and done better? " 484d ago Utalkin2me "@gamefan0909 Funny you say respawn is multiplayer focused, but then in turn name tons of things they need to do to improve the multiplayer. " 484d ago Lukas_Japonicus "@ mrsonystalker Ughhh, typical Xbox fanboy attitude, all you care about is MP. So one dimensional. " 484d ago

subset: commoncrawl


text:
Here we go - the cover reveal and some exciting information about my newest book!
Gratitude Renewed is a personal journal of faith and healing. By journal, I mean that you, the reader, are going to be doing some of the writing. This book is meant as a tool to start your own journey of renewal. The idea for this book came from the Lazarus Filmworks motion picture God, Where Are You?, staring Wade Wilson and Kibwe Dorsey. My co-author on this book and father, De Miller, also wrote and directed the movie. In this movie, the main character feels that God has abandoned him. He then receives Gratitude Renewed as a gift with instructions to fill the pages with his life.
Gratitude Renewed will be available in paperback for you to do the same. If you know someone having troubles in their life, this book could be the blessing on which they are waiting.
Synopsis: This is not a traditional self-help book. It is a life plan designed to renew gratitude and strengthen faith. The authors of this book are not trained psychologists or ordained clergy (although they got lots of input from the pros). They are a father and son with a combined one hundred years (plus) experience of Life. They have faced challenges, experienced failure and explored their faith.
There are several journal pages included at the end of each chapter. They are intentionally left blank for you, the reader, to fill.
Gratitude Renewed is already garnering praise through reviews and endorsements:
"It is well written and easy to understand and is prospectively a great self-help tool."
"Mark and De have a great gift in writing given to them by God. What a blessing...from God."
"Putting the principles from the book into action will renew the gratitude and help you discover the blessings in your life. I recommend this book to any individual that wants to live on the other side of why in a land called peace."
"...an interactive challenge to their readers that will have them reexamining their lives and turning those lives around." 5 Stars
"...a very motivational and positive read." 5 Stars
"...you really feel like you're learning something without a lot of extra noise...definitely a book I will hold onto for a long time." 4 Stars


text:
Cooler Master NotePal U Stand Notebook Cooler Review
Cooler Master has really kept me busy this last month. We already saw a new pair of CPU coolers with the Hyper 612 PWM and the GeminII S524. This showed us one thing that carries over to what we are about to discuss today and that is the sheer amount of ideas and concepts that are developed to fit everyone's particular needs. We have seen a few notebook cooling solutions from Cooler Master in the past, but I have just had a pair of them sent to me to look at what is new and interesting in the notebook cooling lineup.
Most of the laptop coolers aren't very efficient at cooling my particular Lenovo that I use to test with, but they do in fact keep the top of your lap cool by removing the whole notebook from direct contact. When I think notebook cooling, I feel the notebook is what needs to be cooled primarily and not necessarily do I need it to cool my legs. For my usage of many of the coolers I have tested, they sit on my desk. While lights and flashy finishes are all nice and have their market, I find myself gravitating towards the aluminum solutions that come with either one large fan, or a pair of smaller fans attached in some manner.
The cooler we are about to look at today, the NotePal U Stand from Cooler Master takes on what I expect from a cooler of this nature, and takes it a couple steps further. What we are about to see offers plenty of additional USB connectivity, offers an adjustable support system and has positional cooling with the use of a pair of 100mm fans included in the kit. I don't want to give away everything or you won't have a reason to stick around. For those looking for a notebook cooling solution with all the right tricks, the U Stand is definitely worth following me deeper into this rabbit hole to see Cooler Master's new approach.
PRICING: You can find products similar to this one for sale below.
United States: Find other tech and computer products like this over at Amazon's website.
United Kingdom: Find other tech and computer products like this over at Amazon UK's website.
Canada: Find other tech and computer products like this over at Amazon Canada's website.
- Page 1 [Introduction]
- Page 2 [Specifications, Availability and Pricing]
- Page 3 [The Packaging]
- Page 4 [The Cooler Master NotePal U Stand]
- Page 5 [Accessories and Documentation]
- Page 6 [The Final Look & Testing Results]
- Page 7 [Final Thoughts]
- We at TweakTown openly invite the companies who provide us with review samples / who are mentioned or discussed to express their opinion of our content. If any company representative wishes to respond, we will publish the response here.
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text:
After the audi commercial, there was no doubt in our minds, the voice of Theo James is mesmerizing. We are in love with everything about him, but his voice…. amazing! So it is no surprise he is featured, along with Ansel Elgort, in Teen's article, '10 Hot Actors You Forgot (or Never Knew) Are Hot […]
This website (TrisandFour.com) is a non-profit "FANSITE" dedicated to the Divergent Series and it's actors. We do not affiliate with Veronica Roth, or Lionsgate Entertainment or any other related entity. All photos and media are being used under the Fair copyright law 107. I, the admin, or any of the bloggers do not claim any of the books/movies information as our own, nor are we receiving any profit from them. Anything that should be removed, please contact first, and we will cooperate completely to resolve any problem. © 2010-2014 TrisandFour.com FULL Disclaimer can be found under the "About Us" section located on the Menu Bar
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text:
Game Credits
|Puyo Puyo Sun (1996)
|(Sales Promote)
Public Relations
|Neutopia II (1991)
|(Public Relations)
|Puyo Puyo 2 (1994)
|(Special Thanks)
|Seiryū Densetsu Monbit (1991)
|(Special Thanks (スペシャルサンクス))
Developer Biography
There is no biography on file for this developer.
[add bio]
Cca. 2013
(source: Kazuhiko Kitade facebook)
Related Sites
There are no related websites on file for this developer. If you know of any related websites for the person, please consider contributing them.


text:
What is Address Search?
Spokeo reverse address search helps people connect with their neighbors and find people by address. Search your neighborhood today to find out current residents, past residents, and family information. Find out who lives on W 45th Ave today.
Other Spellings
W 45th Ave is also spelled 45th Avenue, 45th Avnue, 45th Avenu, 45th Aven, 45th Avn, 45th Av.
Gary, IN
Time Zone & Current Time
Gary, IN lies within the Central Standard Time (or CST). The current time in Central Standard Time is 02:44 AM on 10/07/2015.
Gary, IN
Top Streets
10th Ave, 10th Pl, 11th Ave, 11th Ct, 11th Pl, 12th Ave, 12th Pl, 13th Ave, 13th Pl, and 14th Ave
Gary, IN
Population Demographics
Household Income
Home Value
33 years old
53% female
Local Ethnicity
African American
Ethnicity Ratio
About Gary, IN
Gary is a city in Lake County, Indiana, United States. The city is in the southeastern portion of the Chicago metropolitan area and is 25 miles from downtown Chicago. The population is 80,294 at the 2010 census, making it the seventh-largest city in the state. It borders Lake Michigan and is best known for its large steel mills. Gary is adjacent to the Indiana Dunes National Lakeshore. (more on Wikipedia)


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Fanatical Support for Azure adds Around-the-Clock Expertise to a Powerful Cloud Platform
The launch this week of Fanatical Support for Azure is an exciting addition to the Rackspace portfolio of expertise and service for a wide array of products and platforms.
Filed in
New Products
on Jul. 14, 2015 by Abe Selig
How Do You Improve Collaboration In An Increasingly Mobile World?
When a team knows how to collaborate well, it's no accident. The chemistry needed for effective teamwork can be months, or even years, in the making. And that's for people who work in the same office every day.
Filed in
on Jan. 20, 2015 by Brooke Jackson
Top New Year's Resolutions For IT [Infographic]
Happy 2015 to everyone in the IT industry! May all of your technology dreams and predictions come true! As many post their predictions for the cloud industry (including our very own CTO John Engates, with his
Cloud Predictions for 2015
) we'd like to offer up a few New Year's Resolutions for IT decision makers to consider as they transition into the Managed Cloud era.
Filed in
Cloud Industry Insights
on Jan. 7, 2015 by Scott Whitright
MongoDB DIY vs. MongoDB On ObjectRocket [Infographic]
NoSQL technologies like MongoDB are often very simple to configure and deploy. They're typically free, which makes it easy for users to boot-strap in development and early production. While configuration, development and deployment are relatively straightforward, scaling NoSQL technologies can be anything but. In fact, many users report significant pain at the critical stages where they need to scale. Compounding matters, many users may not have the expertise or access to best practices they need to do it in a smart and economic way. In addition, the technology is so new that building a team of NoSQL database professionals is often prohibitive.
Filed in
Cloud Industry Insights
on Nov. 13, 2014 by Sean Anderson
A DevOps Snapshot: Rackspace Survey Shows Businesses Benefits Of DevOps [Infographic]
You've heard the usual arguments for why practicing
is good for your IT department – faster time to market and increased innovation are just two. A recent Rackspace-sponsored survey shows that
DevOps can deliver business benefits
, too, such as an increase in sales and customer conversions. The
infographic below
highlights just some of the survey's data, including these wider – and measurable – business benefits tied to DevOps tactics.
Filed in
Cloud Industry Insights
on Oct. 31, 2014 by Andrew Hickey
Infographic: The State Of SMB Cloud Adoption In 2014
It's 2014 and the cloud has been adopted to some degree by nearly 90 percent of all businesses. That is in part because cloud offerings have evolved. Several years ago cloud meant "public cloud." Today, new hybrid offerings have emerged—combinations of cloud hosting and dedicated hardware—letting businesses tailor their cloud infrastructure to their business demands.
Filed in
Cloud Industry Insights
on Oct. 22, 2014 by Franklin Morris
How Downtime Impacts The Bottom Line 2014 [Infographic]
This year's research confirms that downtime is still causing headaches for end-users and IT professionals. However, in addition to the frustration caused by outages, it's also costing businesses more than ever. Take a look below in the
How Downtime Impacts the Bottom Line 2014 infographic
Filed in
Cloud Industry Insights
on Sep. 8, 2014 by Lizetta Staplefoote
Tapping Into Mobile Commerce 2014 [Infographic]
What a difference a year makes! We published the first mobile ecommerce infographic last year. Since then, all the stats suggest that mobile has continued its meteoric rise as an ecommerce powerhouse.
Filed in
on Jul. 24, 2014 by Lizetta Staplefoote
Evaluating Enterprise Content Solutions
Content has always been king on the web, but there has been a dramatic shift in how it rules. Enterprises have moved beyond the simple content management systems (CMS) of yesteryear in favor of adopting robust services to manage the end-to-end visitor experience. These businesses have deeper needs than just scheduling and publishing content. Enterprises now require sophisticated systems with the ability to deliver information to a unique user persona, tailored by geography and presented with consistency across an array of devices.
Filed in
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on Jul. 10, 2014 by Garrett Heath
Six Types Of B2C Customers And Their Purchase Journeys [Infographic]
For years, marketers have been writing obituaries for the "purchase funnel," that cone-shaped path that customers traverse before clicking "Buy." But reports of the funnel's death may have been greatly exaggerated.
Filed in
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on Jun. 10, 2014 by Franklin Morris
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text:
ScriptDC is the premier conference for Mid-Atlantic writers, producers and talent to achieve their creative dreams by connecting them with accomplished teachers, consultants and industry professionals. The 2015 edition will be November 13-15, 2015 at The George Washington University. Early Bird Registration available to September 18, 2015. Confirmed presenters include Joan Darling, Agnieszka Holland, Dawn Porter, Ligiah Villalobos, Tom Schulman, Aviva Kempner, Laurie Scheer, Kate Pearson, Sheree Guitar, Monica Lee Bellais, Jonathan Zurer, Arla Bowers, Brigid Maher, Ron Newcomb, and more! Saturday Schedule of Workshops here. Script and Pitch Critiques on Sunday, November 15. Download Registration Form here or register online through the WIFV calendar here.
Like us on Facebook to stay up to date on ScriptDC news!
What is Included in Your Registration?
We'll open with a special program on Friday with Story District and DC Shorts. PLUS a full day of workshops on Saturday with a screening that evening. PLUS pitch and script critiques on Sunday.
Workshop tracks include a directing master class (Friday night and all-day Saturday); four workshops for emerging writers; four workshops for advanced writers including a pitching intensive; four workshops for producers; and four workshops that concentrate on documentaries.
Bill Svanoe's one-on-one sessions with writers are SOLD OUT.
"Here's what I know about ScriptDC: I won't miss it ever again! ScriptDC is on my list of development opportunities permanently." Jen Whiting
"I figured out how to sell my screenplay. A great learning experience!!!" Brian Streaty
"I am always amazed at how you gather such high-quality presenters and create such a warm, welcoming atmosphere for everyone from beginners to the very experienced." Shoshana Rosenbaum
"I had such a fun time this weekend being a panelist. Thank you for organizing a great event and including me on the panels. It was like being on American Idol for screenplay writers." Jonathan Stein
"Thank you for putting on such a wonderfully helpful conference…Hearing Joan Darling comment on my screenplay is one of the highlights of my life. Thank you for making that possible." Renee Tynan
"You make a big difference in many people's lives, I just wanted you to know that we thank you and appreciate you. Again, I can't thank you enough." Maureen Sasscer
2015 Sponsors include Office of Motion Picture and Television Development, DC Commission on the Arts and Humanities, Interface Media Group, The Actors' Center, School of Communications American University, The Documentary Center at The George Washington University, and GemStar Foundation.


text: More from SI Report: Dodgers release pitcher Freddy Garcia Report: Dodgers release pitcher Freddy Garcia Marshawn Lynch sports gold cleats for Super Bowl Marshawn Lynch sports gold cleats for Super Bowl Louisville vs. FIU: Game time, live stream, TV coverage Louisville vs. FIU: Game time, live stream, TV coverage Pittsburgh-area high school football player dies after... Pittsburgh-area high school football player dies after... Close Tweet0 Share0 Comment0 Share0 Share0 Stumble It0 Print0 More SI Wire LSU DB Jalen Mills suspended indefinitely after battery arrest by Scooby Axson Email Posted: Wed Jun. 11, 2014 Updated: Thu Jun. 19, 2014 LSU's Jalen Mills had 67 tackles and three interceptions last season. (AP Photo/Margaret Bowles)LSU indefinitely suspended junior defensive back Jalen Mills after he was arrested and charged with second-degree battery.Mills turned himself to authorities and is awaiting bond.According to WAFB.com and a probable cause report, Mills allegedly punched a woman in the mouth at his residence on May 4. The report states that the woman "fell to the pavement and was briefly unconscious." She suffered a cut lip which required four sutures to close.Authorities made contact with Mills by phone May 29 but he said was not in Baton Rouge, so an appointment was made for him to meet with police on Tuesday. When Mills did not show and did not respond to calls, an arrest warrant was issued.More from WAFB:The report said Mills is listed at 6'0" and 195 pounds, according to his identification. The victim is listed at 5'3" and 113 pounds. The report also stated a witness gave the same account as the victim.According to the probable cause report, the victim said she was at the apartment complex and knocked on Mills' door because she was looking for her friend. It stated Mills confronted the woman and closed the door in her face. It added she knocked again, but when no one answered, she walked away.Mills was third on LSU with 67 total tackles and had three interceptions in 13 games last season. He has started all 26 games in the past two seasons for the team and was named of 42 players chosen to the 2014 Lott Trophy watch list. SI.COM STAFF: Ed O'Bannon v. the NCAA trial: The key figures to know in the landmark sports case Promoted Stories Comments


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Page 1
Improvement of downstream processing of recombinantproteins by means of genetic engineering methodsFlaschel, Erwin; Friehs, KarlFlaschel, Erwin; Friehs, KarlSuggested CitationFlaschel, Erwin ; Friehs, Karl (1993) Improvement of downstream processing of recombinantproteins by means of genetic engineering methods. Biotechnology Advances, 11(1), pp. 31-77Posted at BiPrints Repository, Bielefeld University.http://repositories.ub.uni-bielefeld.de/biprints/volltexte/2009/3201
Page 2
Improvement of downstream processing of recombinantproteins by means of genetic engineering methodsAbstractThe rapid advancement of genetic engineering has allowed to produce an impressive numberof proteins on a scale which would not have been achieved by classical biotechnology. At thebeginning of this development research was focussed on elucidating the mechanisms ofprotein overexpression. The appearance of inclusion bodies may illustrate the success. In themeantime, genetic engineering is not only expected to achieve overexpression, but to improvethe whole process of protein production. For downstream processing of recombinant proteins,the synthesis of fusion proteins is of primary importance. Fusion with certain proteins orpeptides may protect the target protein from proteolytic degradation and may alter its solubility.Intracellular proteins may be translocated by means of fusions with signal peptides. Affinitytags as fusion complements may render protein separation and purification highly selective.These methods as well as similar ones for improving the downstream processing of proteinswill be discussed on the basis of recent literature.
Page 3
Biotech. Adv. Vol. 11, pp. 31-78,1993 Printed in Great Britain. All Rights Reserved. 0734-9750193 $24.00 @ 1993 Pergamon Press Ltd IMPROVEMENT OF DOWNSTREAM PROCESSING OF RECOMBINANT PROTEINS BY MEANS OF GENETIC ENGINEERING METHODS ERWIN FLASCHEL and KARL FRIEHS Universittlt Bielefeld, Technische Fakulttlt, Arbeitsgruppe Fermentationstechnik, Postfach 10 01 31, 4800 Biel~Celd 1, Germany ABSTRACT The rapid advancement of genetic engineering has allowed to produce an impressive number of proteins on a scale which would not have been achieved by classical biotechnology. At the beginning of this development research was focussed on elucidating the mechanisms of protein overexpression. The appearance of inclusion bodies may illustrate the success. In the meantime, genetic engineering is not only expected to achieve overexpression, but to improve the whole process of protein production. For downstream processing of recombinant proteins, the synthesis of fusion proteins is of primary importance. Fusion with certain proteins or peptides may protect the target protein from proteolytic degradation and may alter its solubility. Intracellular proteins may be translocated by means of fusions with signal peptides. Affinity tags as fusion complements may render protein separation and purification highly selective. These methods as well as similar ones for improving the downstream processing of proteins will be discussed on the basis of recent literature. KEY WORDS Downstream processing, recombinant protein, genetic engineering, fusion protein, fusion complement, purification tag, affinity handle, protein secretion, protein export. 31
Page 4
E. FLASCHEL and K. FRIEHS INTRODUCTION Proteins are the primary products of the translation of the genetic code. They are therefore of central interest for life science research as well as applications in a multitude of areas. Thus, they are a very important class of biotechnology-derived products. Enzymes have found a large area of applications due to their catalytic properties. Spectacular advancements in molecular genetics (regulation of transcription and translation, gene transfer, polymerase chain reaction etc.) and in the cultivation of animal and human cells (cell fusion, hybridoma-technique, transfection) have given the opportunity that intensive efforts are undertaken in order to develop processes for the production of species-specific peptide hormones (insulin, somatostatin, growth hormones, etc.), immunologically active proteins (interferons, interleukins, etc.), coagulation factors (urokinase, t-PA, factor VIII, etc.), as well as of vaccines and monoclonal antibodies. Such proteins may only be obtained economically by means of biotechnological processes, since they represent polymers composed out of the 21 different proteinogeneous L-s- amino acids (including selenocysteine). Chemical synthesis of proteins [1] is only competitive for the production of short chain peptides. However, chemical synthesis is important when non-proteinogeneous amino acids have to be incorporated into peptides. The chemical route suffers from a laborious and expensive protection group chemistry and an error rate which is still too high. In addition, it has to be taken into account that proteins are only active in their natural folding state and that proteins of eukaryotes often need posttranslational modifications (glycosylation, proteolysis). In consequence, protein synthesis will need the use of microorganisms and of cells of animal or human origin. This also means that proteins will need to be separated from complex natural media. Cell-free translation systems are another possible method to produce recombinant proteins. The usefullness of such systems in bioprocessing is under investigation [274]. The importance of downstream processing very likely will at least remain or even increase [2, 3] because the separation and purification of proteins will remain the main source of costs for the manufacturing of proteins. A whole spectrum of downstream processes has been developed for the recovery of proteins [4-7] and these have been documented in recent books and monographs [8-15]. Uncertainty with the use of novel processes, the partiality for problem-specific solutions and new possibilities for the production of a multitude of pharmacologically active proteins have considerably promoted the interest in downstream processing. Since the tool box of genetic engineering is more and more used for improving the production of proteins, it has been straightforward not only to use these techniques in order to achieve overexpression but to aim at the improvement of downstream processing as well. Quite some novel approaches for improving the downstream processing of proteins have already been worked out [16- 19]. These novel methods may lead to the opportunity that very different proteins might be
Page 5
IMPROVING DOWNSTREAM PROCESSING 33 purified by means of one unique process strategy. On the one hand, the physico-chemical properties of the proteins may be changed or amplified by means of protein design or site- directed mutagenesis. Recombination of different genes for the production of fusion proteins, on the other hand, is of primary importance because unique fusion partners may be chosen for the complementation of the target protein. Such fusion partners are adequate which are carriers of a specific affinity useful for the downstream process applied. The class of fusion proteins englobe the use of signal sequences in order to change the localization of the target proteins. Such approaches and similar ones will be reviewed - taken from recent literature. The following chapter will deal with questions referring to the choice of host organisms and the requirement for producing biologically active natural proteins. Only a few issues can be emphasized with respect to host organisms and expression in general for not overloading the article. A further chapter discusses possibilities of manipulating the localization of proteins and the properties of cells which have direct impact on downstream processing. The last chapter is dedicated to strategies of protein modification for the improvement of protein separation and purification. PRODUCT CONCENTRATION AND BIOLOGICAL ACTIVITY The economy of downstream processing of proteins highly depends on the achievable product concentration. Therefore, recombinant protein production aims at the improvement of the overall protein concentration. However, aiming at a high product concentration may fail, unless a high proportion of biologically active proteins is obtained. Overexoresslon The first step for improving the concentration of the target protein is the choice of an adequate biological system by means of which overexpression may be achieved. Many such expression systems are available. For the optimisation of these systems a series of factors have to be taken into account referring to the replication, transcription and translation of the genes. The replication of genes coding for recombinant proteins strongly influences the stable transmission as well as the copy number of these genes. Most expression systems for bacteria use plasmids as vehicles of recombinant DNA. Plasmid stability depends on both the structural and the segregative stability. The structural stability refers to the error rate during plasmid replication, whereas the segregative stability depends on the transmission of the plasmid on to daughter cells. Besides the correct replication of plasmids the structural stability is influenced by the rate of mutation and the gene repair facilities of the host organism. The segregative stability, commonly called plasmid stability, depends on a
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34 E. FLASCHEL and K. FRIEHS series of factors which cannot be discussed here in detail [20]. Means for stabilizing or enhancing plasmid stability have been discussed by Kumar et al. [21]. The rate of gene expression may be accelerated by increasing the number of plasmids per cell. This may be achieved by using so-called multi-copy plasmids, the copy number of which may reach 100 owing to their particular mode of replication. Since the plasmid copy number has to be high only at the time when the target protein shall be produced, a controlled induction of plasmid replication represents an advantageous strategy. This may be achieved by using so-called runaway replication plasmids the copy number of which may easily reach 1,000 [22]. An obvious additional strategy consists of integrating several copies of the coding gene into the plasmid. However, it has to be kept in mind that a high plasmid copy number may affect the physiological state of the host organism leading to a limited rate of gene expression. Both a promoter-operator system and an appropriate termination sequence are required in order to achieve efficient transcription. In addition, transcription should be inducible. During the development of expression systems different sequences have been recognized as strong promoters. A strong promoter is the prerequisite of a high transcription frequency. The best known inducible bacterial promoter-operator systems are those using operators derived from the lac- [23], trp- [24] and the ~,- [25] systems. The lac-operator is often used for the induction of different promoters like lac and tac. Induction is achieved by the addition of IPTG (isopropyl-J3-D-l-thiogalactopyranoside). Promoters coupled with the trp- operator may be induced by iAA (3-(3-indolyi)-acrylic acid). An induction by means of a temperature jump is applied in the case of X-promoters using a temperature sensitive mutant strain with respect to the ~.-repressor. Chemical inducers may be quite expensive and may lead to contamination of the product. Temperature induction may cause undesirable secondary products to appear due to an altered metabolism. Therefore, there is still a need for improved novel promoters [281] - like ones inducible by oxygen depletion [26] or by means of a pH-shift [279]. When strong promoters are applied, a strong terminator is required as well. Without an appropriate termination sequence transcription would proceed beyond the target gene and transcription would lead to useless products. Appropriate sequences like the trpA-transcription terminator are commercially available [e.g. 27]. Transcription is the first step during gene expression leading to mRNA. The second step involves the ribosomes which are responsable for the translation of the mRNA into the amino acid sequence of the recombinant protein. Translation efficiency depends on the stability of the mRNA, the ribosomal binding site (RBS), the correct termination of translation as well as the use of special codons. The stability of mRNA is a function of its susceptibility for hydrolysis by ribonucleases. It is commonly assumed that the lifetime of mRNA is influenced mainly by the presence of 3'-ribonucleases, because the secondary
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IMPROVING DOWNSTREAM PROCESSING 35 structure of the 3'-terminal sequence of the mRNA is of crucial importance [28]. In consequence, mRNA stability may be enhanced by changing the 3'-terminal sequence or shifts in growth rate [264]. The lifetime of the mRNA determines how often it may be used by the ribosomes for translation [29]. Newest results suggest that RNA degradation may depend on 5'-terminal base pairing in E. coll. [221]. Recombinant genes are commonly introduced behind an inducible promoter, but they often have their own ribosomal binding site (RBS). This may be the reason for a low translation frequency, although the rate of transcription may be high. Optimized gene expression systems include an RBS especially designed for the particular host organism [30]. Thus, AT-rich sequences may be introduced up- and downstream of the RBS in order to increase the initiation frequency of translation [31]. The distance between RBS and the start codon AUG may also be changed in order to achieve higher productivities [32]. Synthetic RBS- sequences have not led to improvements of gene expression [33]. A strong terminator is required for efficient translation, too. Appropriate sequences are commercially available [27]. If a synthetic gene is used the sequence of which has been deduced from the amino acid sequence of a protein, the base sequences of the codons for the amino acids should be chosen with care. Studies with genes of highly expressed proteins have shown that certain codons out of the pool of the degenerate genetic code are preferred [34, 266]. Protein Stability After translation the stability of the protein itself determines how much product will be obtained. Protein stability mainly depends on the susceptibility of the protein for proteolytic decomposition. Especially in the case of recombinant proteins, proteases may cause a considerable loss of product [35, 222]. It seems as if heterologous proteins were recognized as belonging to a different species with the consequence of a rapid proteolytic degradation. Different proteases may be responsible for protein digestion. In the case of E. colithe protease La coded on the /on-gene is accused of mainly being responsible for the degradation of recombinant proteins [36]. By using ~on-minus mutants this problem may be partially overcome. However, the use of such mutants may cause a series of difficulties during cultivation. The product of the Ion-gene is also controlling cell division and the development of the polysaccharide capsule. Therefore, Ion-minus mutants tend to overproduce polysaccharides causing strong production of slime and interference with cell division and growth [35]. This problem may be circumvented by controlling the Ion-gene by inducing the Ion-function during the growth phase and switching it off during the production phase. However, this control is quite delicate since the concentration of the Ion-gene product has to be kept in a very narrow band. This may not be achieved with commonly used promoters because of the constitutive gene expression taking place in spite of
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36 E. FLASCHEL and K. FRIEHS repression [37]. By inactivation of structural and regulatory genes of the slime capsule synthesis it is possible to prevent slime production and the growth problems related with that [38]. Modifications of the sulA-gene the product of which regulates the Ion-coded protease may yield a significant improvement of cell division for Ion-negative strains. However, it has to be taken into account that Ion-negative strains are very sensitive with respect to the induction of stress proteins causing additional growth problems. Additional degradation of foreign proteins by phage infection, using T4-promoter [262] and to consist the influence of growth rate on proteolysis [271]. An easier to implement strategy for reducing protein degradation consists in the synthesis of fusion proteins. Thus, small heterologous peptides which commonly are very labile have been stabilized by fusion with large proteins like J~-galactosidase or Staphylococcus protein A [39-43, 272]. In the case of such fusions it may be necessary to split off the fusion complement in order to obtain the target protein in the desired form. methods to reduce protein degradation are to inhibit the host-mediated The export of proteins is another elegant possibility to avoid the intracellular environment rich of proteases [44]. However, some secretion systems may also export proteases into the extracellular environment. The problems related to the presence of proteases have to be considered especially for the operation of cell disintegration [45]. Therefore, the addition of protease inhibitors may be necessary during cell disruption. Posttranslational Modification Many proteins, in particular eukaryotic proteins of pharmaceutical interest, require further modification after protein biosynthesis. Such posttranslational modifications may consist in phosphorylation, glycosylation, the cleavage of sequences from pre-pro-proteins of especially secretory proteins as well as the cleavage of the primary protein chain. Without appropriate modification these proteins are often obtained in a biologically inactive form. In many cases it is relatively easy to produce large quantities of protein by means of bacterial systems like E. coil However, strains of E. cofiare not provided with a pathway for the glycosylation of proteins. If glycosylation is of crucial importance, other less convenient expression systems have to be used. Yeast cells, simple eukaryotes, have glycosylating activity, but often they use other glycosidic residues then those required for the particular case [46]. There is certainly still a long way to go until bacterial systems or yeasts may have learned to achieve different posttranslational modifications by means of genetic engineering. Cells of animal and human origin will remain the systems of choice for proteins needing adequate posttranslational processing [4]. Another way to circumvent this problem is to look for the biologically active part of the target protein which, by circumstance, may not need further modification [47].
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IMPROVING DOWNSTREAM PROCESSING Inclusion Bodies, Renaturation end Protein Foldino 37 Recombinant proteins are often found as insoluble aggregates in the cytoplasm [17, 48]. Extremely high protein concentrations due to overexpression may be responsible for this phenomenon [270]. These accumulations of solid insoluble proteins are called inclusion bodies [49]. The formation of these refractile particles may represent an advantage as well as a disadvantage for the production of recombinant proteins. Thus, inclusion bodies are relatively simple to be recovered from cell homogenates [17, 50, 51], and are protected from proteolytic cleavage. However, a great disadvantage must be seen in the fact that proteins bound in inclusion bodies are biologically inactive. Therefore, these highly insoluble protein aggregates have first to be denatured under extreme conditions in order to be solubilized [51, 52]. Subsequently, these solubilized proteins have to be renatured by means of adequate methods in order to be transformed into their biologically active form. This procedure may yield heavy losses which may be acceptable if a high overproduction goes in common with a high added value. Genetic engineering methods for the reduction of aggregate formation consist in forcing protein secretion as well as in changing specific properties of the target protein which may be responsable for the occurence of inclusion bodies. Wilkinson et a/. [48] describe factors influencing the solubility of recombinant proteins. The critical factors englobe the protein concentration, the distribution of surface charge, the portion of particular secondary structures, the contents of cysteine and proline as well as the overall hydrophobicity. These properties may be changed deliberately by site-directed mutagenesis as well as by fusion of the target protein with an appropriate complement [53, 277]. Some approaches along these lines have been discussed by Kotewicz [54]. The most simple possibility for reducing or even preventing inclusion body formation seems to be by decreasing the rate of expression [52, 223], e.g. by lowering the temperature after induction [224]. In the case of heterologous proteins it is often inevitable that aggregates are formed and the whole procedure of solubilization and renaturation has to be applied [55]. The achievement of the native structure is the most crucial aspect for the production of recombinant proteins [46]. Achieving correct protein folding is a fundamental problem and of enormous economic impact [51]. Protein folding depends on a variety of different factors [56-58]. Certain additives like e.g. PEG [59] may improve folding [60]. kinetics of folding reveals a rather complex pathway [225]. However, the In recent years it has become clear that in vivo protein folding is assisted by catalytic protein complexes. Such proteins obviously play a crucial role for protein folding and assembly. They belong to the heterogeneous group of heat-shock proteins (hsp), and are now divided into two more or less distinct classes [226]. The class of the chaperonins belonging to the hspl0 and hsp60 families occur both in eubacteria, mitochondria and plastids, whereas the class of the hsp70 family seems to be present everywhere where
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38 E. FLASCHEL and K. FRIEHS protein folding occurs. Evidence is also emerging for the presence of cytosolic chaperonins in archaebacteria as well as in eukaryotes [226, 228]. However, the term chaperone is generally used for proteins which assist in protein folding. Anyway, the division of chaperones into two classes seems to be a quite simplistic approach, because it has been shown in in vivo experiments that five chaperones cooperate in a sequential ATP- dependent folding pathway [229]. Both in vitro and in vivo studies of protein folding have already revealed quite some details about their action [61, 62, 230, 231, 267] and other proteins assisting in protein folding like peptidyl-prolyl cis-trans isomerase and protein disulfide isomerase [232, 234]. Such studies may finally lead to the fine-tuning of the cellular machinery of protein folding. This would certainly lead to a considerable improvement for the manufacturing of correctly folded recombinant proteins. (~leava_oe of Fusion Proteins Fusion proteins exhibiting the desired biological activity may be used as such, if there are no special regulations requiring them to be transformed into their natural structure, as it is still common practice for proteins of therapeutical use. In the latter case the cleavage of the fusion complement has to be accomplished. For this purpose, the amino acid sequence of the fusion protein has to be constructed with a specific cleavage side. Fusion proteins may be cleaved by chemical or enzymatic means. A chemical cleavage may be relatively unexpensive, but the rather drastic reaction conditions may lead to non- specific cleavage and denaturation of the target protein. Enzymatic methods are generally preferred, because specific cleavage sites may be introduced between the target protein and the fusion complement [63, 235]. Over 200 protein-cleaving enzymes are listed in a book of B. Keil [269]. Costs for the cleavage procedure may be reduced considerably by the development of an adequate standard cleavage sequence for a specific proteolytic enzyme. Table 1 contains an overview of available chemical and enzymatic cleavage methods and their respective cleavage sequences. At2breviations used in Table 1: BNPS-skatol = 3-bromo-3-methyl-2-(2-nitrophenyl mercapto)-3H-indol, CAT = chloroamphenicol acyltransferase, HIV = human immunodeficiency virus, ompA = outer membrane protein A, Kex 2 = yeast endoprotease
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IMPROVING DOWNSTREAM PROCESSING Table 1: Cleavage of fusion proteins Chemical methods Cleavage agent Cleavage-site sequence Hydroxylamine Formic acid Acetic acid Cyanogen bromide BNPS-skatol 2-1odo-benzoic acid N-Chlorosuccinimide Ref. Asn ~ Gly Asp ~ Pro Asp g Pro Met ~ Trp ~ Trp 8 Trp ~ [64] [65] [66] [67] [68] [69] [70] Enzymatic methods Enzyme Cleavage-site sequence Ref. Carboxypeptidase A Carboxypeptidase B Chymotrypsin Collagenase Dipeptidyl aminopeptidase Endoproteinase Enterokinase Factor Xa HIV-1 protease IgA-protease Poly His t) Poly Arg U, Poly Lys ;) Trp U, Tyr 8, Phe ~ Pro-X 8 Gly-Pro X-Tyr ~ (X not Pro) Lys ~ Asp-Asp-Asp-Lys ~ Ile-Glu-Gly-Arg ~ CAT- ~ -HIV-1 protease fusion, selfsplitting Y-Pro ~t X-Pro; Y = Pro, Ala, Gly, Thr X = Thr, Ser, Ala Pro-Phe-Arg U -Lys ~ Arg ompA-signal sequence Phe-Thr-Phe-Arg, Leu-Ser-Thr-Arg, Pro-Glu-Leu-Arg Tyr-Ile-His-Pro-Phe-His-Leu ~ Leu Glu 1~ Ala-Ala-His-Tyr Arg-Gly-Pro-Ar 9 Arg ~, Lys Ubiquitin 8 Relaxin ¢¢-chain [71] [72] [73] [74] [75] [76] [77] [78] [79, 80] [275] Kallikrein Kex 2 ompA-Signal-peptidase Protein C Renin S. aureus strain V8 protease Subtilisin Thrombin Trypsin Ubiquitin peptidase [81] [278] [82] [268] [83] [236] [84] [65] [86] [671
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E. FLASCHEL and K. FRIEHS LOCALIZATION The localization of proteins is obviously of major importance for downstream processing. If the protein is to be found in the cytoplasm, the concentrated cell mass has at first to be broken. The target protein, in consequence, has to be separated from a complex mixture of proteins, nucleic acids; eventually cellular compartments and debris. A more gentle cell disruption may be applied, if the target protein is secreted into the periplasm of Gram- negative bacteria. Obviously disruption is superfluous in the case of extracellular proteins. Separation of Cells and Cell Disruotion Biomass is most often harvested by centrifugation or cross-flow filtration [6]. For E. coli, a genetic engineering approach has been developed for changing the properties of the cells in order to facilitate biomass recovery. A gene has been cloned which codes for a protein localized on the outer membrane surface and which is responsable for the flocculation properties. This modification resulted in a higher sedimentation velocity of the cells [88]. Similar interventions should also yield better properties for centrifugation and filtration. Mechanical as well as non-mechanical techniques are applied for cell disruption on a large scale [6,89]. The mechanical methods comprise high pressure homogenization and bead milling. Both processes may exhibit considerable protein losses due to uncomplete protein liberation and thermal denaturation. Non-mechanical methods englobe chemical as well as biochemical processes. Cells may be permeabilized by organic solvents or by enzymatic lysis of the cell wall [90]. The use of organic solvents implies appropriate safety precautions [91], whereas enzymic processes are expensive on a large scale [90]. By means of genetic modifications it becomes possible to control cell lysis. The product of the kil-gene of the plasmid ColE1 may yield complete lyses of the cells [92]. A recent patent describes how the kil-gene, under the control of the lac-promoter may lyse the cells after induction with IPTG [93]. This strategy yields an expression system for which cell lyses may be induced under controlled conditions after the recombinant product has accumulated. Based on the same principle, the lysis gene E of phage ~X174 may be employed - under control of the ~,-PL-promoter [94]. In the presence of the temperature-sensitive ~,-repressor cl897, cell lysis may be induced by increasing the temperature to 42 °C. Secretion Protein export may circumvent some problems inherent to the expression of recombinant proteins [95]. It has already been discussed that the formation of inclusion bodies and protein degradation due to cytoplasmic proteases may be prevented. In addition, some
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IMPROVING DOWNSTREAM PROCESSING 41 proteins may be letal for a host when overproduced. Secretion obviously may reduce this phenomenon [82]. However, secretion also may yield uncorrectly folded proteins especially in the case of eukaryotic proteins synthesized by bacteria. Nevertheless, protein secretion into the medium considerably simplifies downstream processing. In this case the cells may be separated from the medium by e.g. centrifugation and the proteins may be isolated from the supernatant. The early separation of unbroken cells leads to reduced contamination by other proteins or cellular constituents - an obvious advantage. A minor disadvantage of this strategy is the need for handling large volumes of liquids. The transport of proteins into the periplasm of Gram-negative bacteria represents a special case of protein secretion. After separation of biomass the product may be liberated from the periplasm prior to being isolated out of a relatively small volume [265]. Contamination due to cytoplasmic constituents may be avoided by gently removing the cell capsule and the outer membrane. This strategy has actually gained much attention. The periplasmic space representing about 20 to 40% of the cellular volume in the case of E. coil is large enough for the accumulation of large amounts of proteins. Besides the advantage that proteins in the periplasm are protected against the attack by cytoplasmic proteases (but not against outermembrane bound proteases [244], the environment of the periplasm favours the correct folding of proteins. The gene of the target protein has to be coupled with an appropriate signal leader sequence and the host organism has to be provided with a cellular transport system in order to allow secretion of proteins. In addition, the target protein should not show properties preventing secretion. Signal Sequences: which enable proteins to use the sorting and transport systems of a particular organism. In bacteria signal peptides with a length of 15 to 30 amino acids are found [96,97]. These structures represent positively charged leader sequences allowing proteins to cross membranes. It may be mentioned that hemolysin of E. co/i carries its signal peptide at the C-terminus. Between signal peptide and core protein a cleavage site is found at which the signal peptide is cleaved off by means of a specific signal peptidase after transport of the core protein has occured. Signal sequences commonly are short N-terminal peptides Secreting proteins may show additional domains besides the signal sequence which may be necessary for a successful transport. In this category inner sequences are found which facilitate or even stop transport, like in the case of membrane proteins, giving rise to the development of an export competent conformation. Therefore, fusion with a signal peptide
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42 E. FLASCHEL and K. FR[EHS Table 2: Signal leader sequences for protein secretion Signal sequence Target protein I M/P Ref. Host : Escherichia coil amy Amylase, B. stearothermophi/us bla Proinsulin, human IgG of mouse 13-Lactamase Epidermal growth factor, rat Triosephosphatase, chicken M [99] M M/P M [ 100] [lOl] [102] [lO3] [104] cgt lamB malE alk. Phosphatase, c~-amylase CD4 receptor of HIV Gene 5 protein, phage M13 Klenow-polymerase Nuclease A, S. aureus M OM P P P [282] [261] [106] [106] [106] ompA Colony stimulation factor, human Superoxide dismutase, human Interferon oc2 Antiviral protein, Mirabilis oc-Sarcin Prokallikrein, human Nuclease A, S. aureus ompF J3-Endorphin [107] [82] [lO8] [109] [1 lO] [111] [82] P M P P P M [19] pelB phoA Antibody VH-domain, mouse Trypsin inhibitor, bovine Epidermal growth factor, human Fusion : #-galactosidase-alk. phosphatase o~-neo-Endorphin Fusion : MBP-~-galactosidase Ribonuklease T1 OM [276,280] [112] [113] [98] [114] [115] [116] M/P P P P phoS Growth hormone release factor, human P [117] spA Parathyroid hormone, human Insulin-like growth factor, human Ovalbumin Ovalbum in Pullulanase 13-Lactamase M M [118] [119] [120] M [121]
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IMPROVING DOWNSTREAM PROCESSING Table 2: Signal leader sequences for protein secretion (continued) Host : E$cherichia coil (cont.) Preproineulln (rat) Proinsulin, rat Enterotoxin LTA Epidermal growth factor, human Synthetic Interferon e¢2. Metalloproteese Metalloprotease, with helper protein BRP Insulin-like growth factor, human I~-Iactamase & a-amylase hly Hemolysin Pseudomonas cholesterol esterase [122] [123] P [124] M [125] M M M [19] [283] [126] M [127] M [246] Host : Bacillus aubtilis alk. Protease B. amy/oliquefaciens alkaline Phosphatase M [128] amy, B. amy/oliquefaciens Amylase, B./icheniformis M [129] amy, prepro-peptide Amylase & human growth hormone M [247] ble, E. coil Amylase, B./icheniformis M [129] Prepro-neutral-protease Growth hormone, human M [130] Host : Saccharomyces cerevisiae Yeast killer toxin a-Amylase, mouse M [248] Prepro-a-factor Viral proteins, human papillomavirus M [131] Abbreviations used in Table 2: amy = amylase, bla = ~lactamase, BRP = bacteriocin release protein, cgt = cyclodextrin glycosyltransferase of B. circulans, hly = hemolysin, M = secretion into the medium, male = maltose binding protein, MBP = maltose binding protein, OM = secrection into the outer membrane, ompA = outer membrane protein A, ompF = outer membrane protein F, P = secretion into the periplasm, phoA =alk. phosphatase, phoS = phosphate binding protein, spa = Staphylococcus aureus protein A
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That's why we never saw Cary Grant play James Bond. Same goes for this quintet of iconic characters, who were this close to being played by different performers.
This article originally appeared on Inverse. By ...
By all accounts, anticipation is high for J.J. Abrams' upcoming Star Wars movie, The Force Awakens. In fact, according to a new online survey conducted by market research firm Harris Interactive and business information provider FusionOps, it's already the best Star Wars movie ever.
A long time ago, somebody scrawled out a trilogy about an assassin named Mara Jade who tracked down Luke Skywalker but, in a twist that blew at least one 13-year-old mind, ended up marrying him. Does Disney care? You mean about books? Not a smile's chance in Dismaland.
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When I first received word about Star Wars soups and SpaghettiOs, I was fascinated. Imagine: Star Wars and SpaghettiOs, together at last, and for once, not on some fourth graders shirt. Toys are one thing, but how can the galaxy far, far away invade your dinner table?
My mother was a sci-fi enthusiast, who successfully transmitted the virus to me, via Star Trek, Doctor Who, Star Wars, and the more obscure television program, In Search of... (ellipses intended).
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Vermont Senator Bernie Sanders, with his perpetually ahead-of-the-mainstream views, finds himself treated like old Ben Kenobi in the beginning of A New Hope, a crazy old wizard with no real shot. Now, though, it seems like maybe the tide is turning.
We remember the late, great Wes Craven, discuss what we learned on "Force Friday," and I share the story of how his heartfelt interview with Andrew Garfield about The Amazing Spider-Man caught fire all over the Internet and went viral!
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The world's first Disney park and each of the four Disney World parks in Orlando will be getting at least one new project over the next few years. Here, five upcoming park debuts that we're most excited about.


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Trumpet VoluntaryMake this regal baroque composition the centerpiece of your next wedding video, and you won't be disappointed. For a touch of majesty, this trumpet and organ duet rules the land.https://www.smartsound.com/producer/images/ps13_210.jpgSingle31311-ST$ 39.9531311-EX$ 69.9531311-W$ 149.95Production Types: Wedding Keywords: Confident, Dignified, Majestic, Regal, Spirited, Festive Genre: Traditional, Themes, Classical, Baroque Instrument: Chamber Group, Organ, Trumpet, Acoustic, Instrumental
Trumpet Voluntary from the album Wedding Classics (PS13)
Trumpet Rondeau DuetThis bright fanfare emerges triumphantly to mark the arrival of your most esteemed guests. Bold and brief, it works best when used at lengths under 1 minute.https://www.smartsound.com/producer/images/ps13_210.jpgSingle31309-ST$ 39.9531309-EX$ 69.9531309-W$ 149.95Production Types: Wedding Keywords: Confident, Dignified, Regal, Spirited, Festive Genre: Traditional, Themes, Classical, Baroque Instrument: Chamber Group, Trumpet, Acoustic, Instrumental
Trumpet Rondeau Duet from the album Wedding Classics (PS13)
Trumpet Rondeau SoloSolo This bright fanfare emerges triumphantly to mark the arrival of your most esteemed guests. Bold and brief, it works best when used at lengths under 1 minute.https://www.smartsound.com/producer/images/ps13_210.jpgSingle31310-ST$ 39.9531310-EX$ 69.9531310-W$ 149.95
Trumpet Rondeau Solo from the album Wedding Classics (PS13)
DiscoveryReflective and mellow, this rich, uncluttered cut speaks of conclusions, resolutions and the peace of mind that comes with both. Warm and spirited, it creates an image of solitary walks, stargazing, and the joy of new discovery.https://www.smartsound.com/music/images/romance.jpgSingle907-ST$ 39.95907-EX$ 69.95907-W$ 149.95
Discovery from the album Romance & Memories
Majestic MelodyAn emotional and moving theme, drifting from the darkness of a minor key, to the sparkling notes of the major.https://www.smartsound.com/music/images/inspirational.jpgSingle2610-ST$ 39.952610-EX$ 69.952610-W$ 149.95
Majestic Melody from the album Inspirational Guitar
Hope Of GloryA positive and lightly syncopated theme pleasantly fills the heart with happiness.https://www.smartsound.com/music/images/inspirational.jpgSingle2606-ST$ 39.952606-EX$ 69.952606-W$ 149.95Production Types: Wedding, Tribute/Memorial Keywords: Happy, Positive, Uplifting, Carefree, Gentle, Heartwarming, Inspiring, Light, Spirited, Mellow, Hopeful Genre: Sophisticated, Themes, Classical, Lyrical, Specialty Instrument: Solo, Guitar (Acoustic), Percussion, Acoustic, Instrumental, Guitar
Hope Of Glory from the album Inspirational Guitar
No Greater LoveDramatic, stately, and cultured - a somewhat restless piece that evokes a variety of emotions.https://www.smartsound.com/music/images/inspirational.jpgSingle2613-ST$ 39.952613-EX$ 69.952613-W$ 149.95Production Types: Wedding, Tribute/Memorial Keywords: Dignified, Lively, Refined, Spirited, Stately Genre: Themes, Classical, Lyrical, Specialty Instrument: Solo, Guitar (Acoustic), Acoustic, Instrumental, Guitar
No Greater Love from the album Inspirational Guitar
New BeginningThis cheerful and easy-going piece greets the new day with a smile.https://www.smartsound.com/music/images/inspirational.jpgSingle2612-ST$ 39.952612-EX$ 69.952612-W$ 149.95Production Types: Wedding, Tribute/Memorial, Background Keywords: Happy, Positive, Uplifting, Cheerful, Light, Refined, Spirited, Mellow Genre: Elegant, Themes, Classical, Lyrical, Easy-Going, Specialty Instrument: Chamber Group, Guitar (Acoustic), Acoustic, Instrumental, Guitar
New Beginning from the album Inspirational Guitar
Back Where We BelongThis acoustic guitar feature is fresh and outdoorsy. Just right for scenes in the woods or camping by a lake.https://www.smartsound.com/music/images/bright.jpgSingle2401-ST$ 39.952401-EX$ 69.952401-W$ 149.95Production Types: Wedding, Background Keywords: Positive, Uplifting, Carefree, Fresh, Light, Refreshing, Spirited, Upbeat Genre: Outdoorsy, Themes, Easy-Going, Easy-Listening/New Age Instrument: Guitar (Acoustic), Instrumental, Guitar
Back Where We Belong from the album Bright Horizons
Eine Kleine Nacht'Eine Kleine Nachtmusik' is a string quartet staple familiar to all. A 'Mozart must' in the classical repertoire. The 1st movement is presented here.https://www.smartsound.com/music/images/classical.jpgSingle1213-ST$ 39.951213-EX$ 69.951213-W$ 149.95Production Types: Wedding Keywords: Happy, Cheerful, Intimate, Light, Lively, Refined, Spirited, Festive, Upbeat Genre: Elegant, Sophisticated, Traditional, Themes, Classical Instrument: Chamber Group, Acoustic, Instrumental
Eine Kleine Nacht from the album Classical Masters
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We got the chance to stay in Suite #3 and Suite #9 on our honeymoon. It was ideal. Lovely, friendly and helpful staff, beautiful place, unique and full of character. The bathrooms and balconies are incredible. Location is great, so much fun to explore in the medina of Marrakech.
- Official Description (provided by the hotel):
- Riad Farnatchi has been written up as "The City's Most Luxurious Riad" - Food and Travel Magazine 2013, "The Ultimate Hideaway" - Harpers Bazaar January 2012 and "Best Luxury Riad" - Conde Nast Traveller 2011. Riad Farnatchi been created out of five small Riads in the heart of the oldest part of Marrakech, some of our walls go back over 400 years. Riad Farnatchi with just nine suites has the feel of a beautiful, highly individual, private house, but the facilities and service of a world class 5 star "boutique" hotel. ... more less
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- Riad Farnatchi Marrakech
- Riad Farnatchi Hotel Marrakech


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Seibertron.com - The Ultimate Transformers Fansite
Burn wrote:Please don't clog this thread up with garbage. Either post a real picture of yourself, or tell Va'al how pretty he is.
dinobot-fan-88 wrote:I would love to see the Dinobots in the new Transformers movie!
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41375 Southwind Drive
A location in Canton
Commute to Downtown Wayne
18 min 30 min 60+ min View Routes
Most errands require a car.
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41375 Southwind Drive has a Walk Score of 26 out of 100. This location is a Car-Dependent neighborhood so most errands require a car.
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Home Improvement
5 signs your buyer clients need an electrician's intervention
Jul 14, 2015 by Andy Rodenhiser
Markets & Economy
The rise of gig-estate
Jun 4, 2015 by Tamir Poleg
Take a test drive of the only real estate website endorsed by Barbara Corcoran
Mar 23, 2015 by Laura Monroe
'Inbound marketing' turns strangers into real estate clients in 6 steps
Mar 18, 2015 by Laura Monroe
Approach real estate as if you were running for mayor
Mar 18, 2015 by Scott Schang
Contributor Article
8 examples of authentic leadership in real estate
Mar 17, 2015 by Don Kottick
Real estate market recap, Sept. 28-Oct. 2 Top 10 posts from last week The week in industry hires and promotions: Sept. 28-Oct. 2 Daily market update: Oct. 2, 2015 How to build a powerful real estate brand Real estate Facebook group unveils lead-generation tool 5 ways to justify your commission to clients Placester launches online marketing automation product 3 savvy ways to attract (and keep) Chinese clients Understanding the TRID Rule and fee tolerances Infographic: 12 facts about millennials homebuyers How can landlords prevent early lease termination?
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An ability to distinguish what will sell is an absolute must for any broker to survive and succeed in the real estate in...
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Review: "I can not say enough about Jennifer Muskus. she and her firm has handled many legal matter for me and quite frankly I wouldn't use anyone else. If you want the job done by someone knowledgeable, Jennifer is your pick. She looks to be meek and mil..."
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Pokes hoping for NIT bid
CHEYENNE (AP) — The Wyoming men's basketball program is waiting to see whether it will be in the postseason.
UW will find out where and when either Sunday night or Monday morning.
Coach Larry Shyatt is certain his team will be invited to either the National Invitation Tournament or the College Basketball Invitational.
Even though UW (19-13) has lost 13 of its last 19 games after a 13-0 start, Shyatt says the Cowboys will play in their second straight postseason tournament because of their body of work and what computer rankings show.
Shyatt tells the Wyoming Tribune Eagle that he believes the team's RPI rating will be good enough to get UW into the 32-team NIT.
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When the President of the United States is kidnapped by aliens and replaced with a clone, the police call on Marshal "Dick" Dix of the International Security Force to help rescue him - and quickly, before they clone the rest of the world's leaders.
First there was Airplane! Then came The Naked Gun series. This t ime Leslie Nielsen is even more far out than ever in 2001: A Space Travesty, a hila rious comedy that dares to spoof where no one's spoofed before: on another planet.
When the president of the United States is kidnapped by aliens and replaced with a clone, the police call on Marshall "Dick" Dix (Nielsen) of the International Security Force to he lp rescue him--and quickly, before they clone the rest of the world's leaders. With the f ate of the world in his clumsy hands, Dix heads to the planet Vegan, a very strange place run by an evil doctor and inhabited by aliens who, having learned to speak English from wa tching television, all talk like different movie stars. After engaging in some freaky lun ar socializing, Dix joins forces with sexy officer Cassandra Menage (Ophélie Winter) and m aster of disguise Captain di Pasquale (Ezio Greggio) to rescue the saxophone-playing presi dent. They return to Washington, D.C., and accomplish their mission of replacing the clon ed president with the real president. Or do they?
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Leslie Nielsen deserved better then this.
Horribly unfunny and ridiculous spoof.
I saw just a little of this movie and that was all I could take. Read more
My husband and I really enjoyed watching this movie. It was hilarious! We would definitely recommend this movie.Published 7 months ago by Lynda Dunbar


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"Lucy in the Sky with Diamonds" is a song written by John Lennon and Paul McCartney that was recorded by The Beatles for their 1967 album Sgt. Pepper's Lonely Hearts Club Band.
Many people thought this was about drugs, since the letters "LSD" are prominent in the title, and John Lennon, who wrote it, was known to have taken acid. In 1971 Lennon told Rolling Stone that he swore that he had no idea that the song's…
Similar Tracks
The Beatles
With a Little Help from My Friends
The Beatles
John Lennon
Pink Floyd
Picture yourself in a boat on a river
With tangerine trees and marmalade skies
Somebody calls you, you answer quite slowly
A girl with kaleidoscope eyes
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text: Remisssion (disorders) Category page Create Classic editor Talk0 This page needs content. You can help by adding a sentence or a photo. Add to this page!orSearch for "Remisssion (disorders)" "Assessment| Biopsychology| Comparative| Cognitive| Developmental| Language| Individual... Spontaneous remission Pages in category "Remisssion (disorders)" This category contains only the following page. S Spontaneous remission Retrieved from "http://psychology.wikia.com/wiki/Category:Remisssion_(disorders)"


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