text:
MARK AUSTIN THOMAS: A budget standoff in the state of New Jersey continues today. All but essential state services are closed. That could leave thousands of government workers without a paycheck. It also spells trouble for one of the state's biggest industries. Marketplace's Amy Scott explains.
AMY SCOTT: The state of New Jersey has ordered casinos in Atlantic City to shut down by Wednesday morning. That's because the agency that regulates them will close as part of the government shutdown.
Gambling and the lottery bring the state a $3 million jackpot every day. Milton Leontiades is former dean of the Rutgers business school. He says a shutdown would ripple throughout the New Jersey economy.
MILTON LEONTIADES: It not only affects the casino industry per se in terms of revenues, but the employees who work at the casinos and the suppliers and vendors that rely on the casinos for their businesses.
Gambling operators have gone to court. They argue the Casino Control Commission counts as an essential service. New Jersey governor Jon Corzine is battling fellow Democrats over his proposal to raise the state sales tax to help close a $4.5 billion budget gap.
In New York, I'm Amy Scott for Marketplace.


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Please note images are for illustration purposes and may differ from the product(s) you receive
Valentina by Valentino is a sensual, irresistible scent. The notes of bergamot, white truffle, tuberose and blossom, exude sophistication and elegance making this Valentina gift set a fresh, floral and very feminine fragrance. With Italian inspitration this Eau De Parfum is a classic Valentino scent perfcet for evening wear.

subset: commoncrawl


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Duncan v. Nevada
In June 2015, the Nevada governor signed into law S.B. 302, which created the Education Savings Account Program. Through the program, parents may receive money from the state's public-school fund, which is deposited into an Education Savings Account, to pay for their child's education at a religious private school. Once the private school receives this funding, there are no prohibitions on how the public funds may be used, meaning that private schools are free to use these funds for religious instruction.
In August 2015, we joined with the national ACLU and the ACLU of Nevada to file suit on behalf of several Nevada residents. Our lawsuit argues that the Program violates the Nevada Constitution, which bars any public funding from being "used for sectarian purpose[s]."
Parents who wish to use the Program to send their children to private school intervened in the lawsuit, and both the parents and Nevada moved to dismiss the case. We filed a brief in opposition to their motion and also moved for a preliminary injunction, to bar the state from releasing the funds. The trial court held a hearing in December 2015.
In May 2016, the trial court ruled in favor of the state, finding that the voucher program did not violate the Nevada Constitution because the Legislature passed S.B. 302 into order to promote education, rather than to promote religion or religious observance.
We have appealed the trial court's decision to the Nevada Supreme Court. In July 2016, we filed our opening brief with the court. Oral argument is scheduled for July 29, 2016.


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LiverSupport Health News
Living with Liver Disease: Why Am I So Tired?
Your fatigue is real. Learn more about a handful of biological phenomena that explain why liver disease frequently causes severe exhaustion.
UltraNourish Will Change Your Life
Is your protein shake packed with herbs, vitamins and nutrients that support your liver and positively impact nearly every nook and cranny of your body? If not, start researching UltraNourish now, a liver-loving protein shake with head to toe benefits.
The Advantages and Disadvantages of Liver Fibrosis Evaluation Methods
Liver fibrosis tests are necessary for those with chronic liver disease. Find out why your physician may order a combination of liver biopsy and/or non-invasive liver fibrosis testing, as well as the pros and cons of these methods.
The Worst Type of Sugar for Your Liver
This type of sugar can cause a host of health issues, including damage to your liver.
Liver Support Essentials: 5 Ways to Reduce Stress
Stress is not good for your liver! Consider these five stress reduction approaches for your liver's health.
Is Green Tea Good or Bad for Your Liver?
As long as you are respecting the adage that too much of a good thing can be harmful, green tea is a powerful protector of liver health.
Spring Is the Ideal Time to Clean Your Liver
Find out why your liver often becomes congested during wintertime, and learn five ways to prepare your liver for the energetic spike in spring.
New Study Links Monsanto's Weedkiller With a Fatty Liver
New evidence connects glyphosphate, an active ingredient in RoundUp, to liver damage. This demonstrates that modern agricultural practices are now a major factor in our society's increase in chronic liver disease.
Damage to Your Liver With Just ONE Cheeseburger
Learn more about a new study that finds eating even one high-fat meal can injure your liver.
How Your Medicine Cabinet Is Affecting Your Liver Health
There are more than the usual suspects, like over-the-counter (OTC) painkillers, that you should know the facts about when it comes to liver health.
5 Ways to Reduce Liver-Related Gas
People with liver disease are likely to have some digestion issues, often resulting in excessive gas. Nobody welcomes gas in their stomach and intestines, especially when it is expelled as flatulence. Easy for anyone to incorporate, these five suggestions can help a person reduce their quantity of gastrointestinal gas.
What Is Liver Cirrhosis and How Is it Prevented?
Liver disease, regardless of the cause, is considered to take place in four basic stages. Find out how you can prevent liver cirrhosis.
Announcing a Financial Support Program for Patients with PBC
"The Assistance Fund is eager to help patients facing financial barriers to treatment afford the medications that will preserve their quality of life."
4 Foods That Cleanse Your Liver
Do you want to improve liver health and function? You need to add these four items to your next grocery list.
10 Liver Damage Symptoms That Could Be Trouble
Are you experiencing any of these warning signs of liver damage?
Did You Know? Garlic Lowers Liver Inflammation
Garlic is wonderful for reducing inflammation in your body, and is especially beneficial in reducing inflammation in your liver. It has antibacterial, antiviral, anti-fungal, and antioxidant properties. Garlic can also improve our immune system.
How Will I Know If I Need a Liver Transplant?
Find out the signs of liver failure, as well as the factors that go into determining if someone is a good candidate for a liver transplant.
Foods to Avoid for Liver Health
While we all indulge once in a while, choosing a steady diet of these types of foods may lead to liver damage over time.
Did You Know? Walnuts Can Help Detoxify Your Liver
Walnuts have high amounts of arginine, an amino acid that helps your liver detoxify ammonia – a toxic waste product of protein metabolism. Moreover, these nuts are high in glutathione and omega-3 fatty acids, both of which support detoxification.


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We promise to do whatever it takes to ensure you're satisfied, or you don't pay. You can count on us. GUARANTEED. The Hilton Garden Inn Promise reflects our focus on hospitality and integrity. We are committed to providing an excellent hotel experience for every guest, every time.
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Located within five miles of the Chevron Refinery, the Singing River Hospital and many shipbuilding, manufacturing and chemical companies, Hilton Garden Inn Pascagoula offers easy access to Interstate 10 on US Hwy 90. Vegas-style casinos, tempting restaurants and stunning Gulf Coast beaches - all easily reached from our beautiful hotel in Pascagoula, MS. Explore downtown Pascagoula, home to the Gulf Coast's newest specialty retail and dining destination, the Anchor Square Shopping Complex, and visit Biloxi and D'Ibervill – just 20 minutes' drive from our Pascagoula hotel.
Feel at home in a comfortable and modern guest room or suite at this hotel in Pascagoula, MS, equipped with complimentary WiFi, a refrigerator, a microwave, a Keurig® coffee maker and an HDTV.
Cool down in the inviting outdoor swimming pool or whirlpool, or work out with the latest exercise equipment in the complimentary 24-hour fitness center. Begin your morning with a cooked-to-order breakfast and buy snacks, travel essentials, alcoholic and non-alcoholic drinks in the 24-hour Pavilion Pantry®. Unwind with a beer or a cocktail, before enjoying delicious American cuisine in the Garden Grille & Bar or dine in the privacy of your guest room with our evening Room Service.
Stay connected at this hotel in Pascagoula, MS, with the complimentary 24-hour business center, and hold a conference or celebration for up to 400 guests in our 6,000 sq.ft. ballroom. Whether it's for a meeting, training seminar or wedding reception, our onsite catering service and dedicated staff will ensure your event is a success.
Our hotel has received the 2015 TripAdvisor Certificate of Excellence Award. This honor is awarded to establishments that achieve outstanding traveler reviews on TripAdvisor, the world's largest travel site. Our staff takes great pride in consistently offering an exceptional experience to our guests.


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(8)Yellow Mello Of The Marsh Variety!(8)
By the time they reached Wammy's House once more, Seul, Abby and Megan were asleep in the back and somehow, Ciel still sat purring on Abby's lap. The front door was opened, and two children ran out, followed by a third, shuffling slowly. L got out, going inside without another word. The three were woken.
Ryuk, bored of trying to make Seul jump, or annoy her, slugged along, moaning for apples. Abby, Seul, Megan and Ciel got out. Seul was almost knocked down by Mello, who bowled into her. She laughed. Matt hugged her too, but Seul slapped him across the cheek, giving him a warning glare, after he had grabbed her bottom.
"Abby, This is Matt, Mello, and that's Near. Boys, this is the newest Wammy's girl, and this is Megan." She said. After that, they all went inside. Abby chose her name after a while; Neko, meaning cat in Japanese. After Abby gave L her new name, she wandered around with Seul and the boys. In the end, they all ended up in Seul's room. She was tapping away at the computer, sending the pictures and report to L, before turning it off again, putting it away quickly. The rest of the day was uneventful. There was no point in going to the rest of the lessons that day; the boys had skipped, waiting for Seul, and Abby and Seul had no clue what to do, so they spent the day in B's old room. They all went down for dinner, but after that, everyone went back to Seul's room.
Megan stayed in her clothes, while Neko changed into some grey pyjamas, with cats on, Seul in a large, long-sleeved black top and some shorts, Mello and Matt discarded their tops, and Near stayed as he was.
Megan, who had already fallen asleep, was curled up on the floor with her rabbit, a blanket over her, and Ciel purring beside her. The others all piled into the double bed, Mello inbetween the two girls, Near on Seul's side, and Matt on Abby's side, who had promised to keep his hands to himself.
"Oi, Seul, Neko, is this what a family's like?" Mello asked. Seul certainly didn't expect that coming from him.
"A little, I guess, Mello." Neko said thoughtfully. "First thing I'm gonna do tomorrow is eat some chocolate ice-cream. I don't know why." She said. They laughed.
"Chocolate. Mm." Mello smiled.
"Neko, you should have seen him this morning, his whole face was covered in whipped cream!" Seul giggled. Mello turned red.
"Yummy." She answered.
"Indeed." Seul said seriously. Mello, red as anything, shrunk under the covers.
"Shut uppp, your so embarassing." he moaned.
"Can we just go to sleep, guys? Those two are probably on the case again tomorrow, too, so shut up, Mello."
"Yeah Mello!" Both Neko and Seul said together. They looked over Mello's head at each other, before singing out loudly; "Yellow Mello, of the marsh variety, whipped cream and chocolate flavour!" they burst about laughing. They finally settled down, and they all said goodnight, even with the two girls still winking at each other.


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Gullipuds are small non-sapient quadrupeds native to the swamps of planet Naboo. They are able to inflate their body when threatened, becoming extremely hard to swallow. When inflated, Gullipuds can bounce like a rubber ball. Gulliball, played with inflated Gullipuds, is a popular game among Gungans.


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RPSC School Lecturer Admit Card 2015
Rajasthan Public Service Commission is going to release the RPSC School Lecturer Admit Card 2015 soon through the main portal i.e. rpsc.rajasthan.gov.in. Get latest updates regarding RPSC School Lecturer Exam Date Call Letter 2015 from here.
Examination Date: Updated Soon
A Few weeks back, the Rajasthan Public Service Commission has released an employment notification for the appointment of well educated. This notification is about to hire 13098 vacant posts of the School Lecturer posts under School Education.
The enormous digits of applicants have submitted their applications for the above mention posts and now waiting to see the exam date. As per the details, once the application process has been finished the department will issued the admit card for each appeared candidate.
Organization Name
Posts Name
Exam Name
Rajasthan Public Service Commission
School Lecturer
Written Exam
Rajasthan PSC School Lecturer Exam Date Call Letter 2015
The Rajasthan Public Service Commission will conduct the written test in the upcoming months. The exact date of examination will be announced afterward with the hall ticket document. After releasing the exam date, the status of hall ticket and its availability will probably be cleared by the department which we update here on this page.
Call Letter Availability: Soon Declared
Candidates keep in touch with us or more updates, by the help of this page applicant will also get the direct link to download call letter. Dear candidates, if you were here since for the Rajasthan PSC School Lecturer Exam Date Call Letter 2015 then let me notify you it can be updated here shortly. After declaration of hall ticket, aspirants must download it and bring t the day of examination.
Steps to download RPSC School Lecturer Admit Card 2015
- Firstly, the candidates need to open the official site which is www.rpsc.rajasthan.gov.in
- Here check the current news and notification section which is available on the homepage.
- Click on the suitable link under this section.
- Now enter your details like registration id and date of birth.
- After entering the details, hit on submit button and wait for a while till your entry ticket won't be generated.
- After few second, the hall ticket will open on the PC.
- Take a hard copy printout of it for examination use.
Download the call letter through visiting http://rpsc.rajasthan.gov.in/


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This charming hotel has been lovingly restored from two buildings on a registered historical site. Named after a large rubber tree which sits in the garden, its owners brought in special craftsmen to restore the wood and iron work in 2009. There is a paved garden with small pool and a beautiful courtyard. The interior is furnished with antiques and local craftwork. The historical hotel offers 11 guest rooms and facilities include a lobby with currency exchange facilities, Internet access and a laundry service (fees apply).
The charming historical hotel is located in the historical part of Antalya city known as Kaleii or 'Inner Bailey'. This area has lots of narrow cobbled streets and is in an elevated position above Antalya's pretty harbour and close to Hadrian's Gate, which dates back to Roman times. Antalya's modern facilities with excellent shopping, bars, restaurants and nightlife are just a short stroll away. Guests will find links to the public transport in the immediate vicinity, the beach a 3-minute stroll away and Antalya Museum approximately 3 km from the hotel. The ancient city of Perge is a 25-minute drive from the hotel, while Termessos is around 45 minutes by car. Antalya Airport is approximately 10 km from the beach hotel.
Each room has its own individual style and is comfortably furnished with wooden carved ceilings and special artefacts, all reflecting the history of the two houses that they once were. Each room features an en suite bathroom with shower and hairdryer, a double bed, direct dial telephone, Internet access, a safe and minibar and individually regulated air conditioning and heating.
Guests may take a dip in the heated outdoor swimming pool or relax by the poolside snack bar using the sun loungers and parasols laid out for use. There is also a sauna. Sun loungers and parasols can be hired for a fee on the rocky beach.
A traditional Turkish breakfast is served.
American Express, MasterCard and VISA are accepted as methods of payment.
Pets are not allowed and rooms are non-smoking.
Room Facilities:
Direct dial telephone
Internet access
Double bed
Individual airconditioned
Individual heating


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Lyrics Depot is your source of lyrics to Betty Lou's Gettin' Out Tonight by Bob Seger. Please check back for more Bob Seger lyrics.
Betty Lou's Gettin' Out Tonight Lyrics
Artist: Bob Seger
Album: Against The Wind
Have you heard the news
It's all over town
If you ain't heard it boys
You better sit down
I got the story here
It's hot off the press
Brace yourself now
And take a deep breath
Grob a hold of something
Hold on tight
Betty Lou's gettin' out tonight
First heard the rumor down on 12th and Main
The poor druggist he was going insane
His stuff is selling out like never before
He finally had to up and close the store
All the boys were getting ready to fight
Betty Lou's gettin' out tonight
Betty Lou's gettin' out tonight
Betty Lou's gettin' out tonight
She was bad
Her momma got mad
But now her momma said it's all right
All the boys are getting ready and right
Betty Lou's gettin'out tonight
Betty Lou
Betty Lou
It's all true
It's really true
What do you think about that boys
Bob Seger Lyrics
Bob Seger Against The Wind Lyrics
More Bob Seger Music Lyrics:
Bob Seger - Cat Lyrics
Bob Seger - Come To Poppa Lyrics
Bob Seger - Comin' Home Lyrics
Bob Seger - Get Out Of Denver Lyrics
Bob Seger - Mainstreet Lyrics
Bob Seger - Momma Lyrics
Bob Seger - The Aftermath Lyrics
Bob Seger - Understanding Lyrics


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This is a 26oz stainless steel water bottle with a screw-top lid, pop-up spout, and karabiner clip. It is decorated with a high quality metal team logo. Eco-Friendly and BPA free.
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Improved Track-Etched Filter Technology
Award Information
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Adelphi Technology Inc.
2181 Park Boulevard, Palo Alto, CA, 94306
Principal Investigator
Name: Louis Lombardo
Phone: () -
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Business Contact
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Research Institution
AbstractWe will improve microfilter technology with a superior track-etched filter. Microfilters are used ivariety of analytical procedures including the concentration of macromolecules, the sterilization ofpharmaceuticals, and to analyze microbiological impurities. These track-etched filters will be madebeams, polyimide membranes and a novel 'varying-incidence' irradiation technique. Recent advances intechnology allow accelerator-ions to be economically competitive and technically superior to the trafission-fragment sources. The resulting filters will be chemically cleaner and have no residual radibe used instead of traditional materials (such as polycarbonate) due to its superior mechanical, cheproperties. We will also attempt to increase the porosity of the track-etched filter by varying theduring irradiation. This work will consist of several aims: i) irradiating the membranes, ii) etchincharacterizing the resulting filter, and iv) repeating the procedure using varying-incidence irradiaresult in a robust track-etch filter that combines uniform pore size and high porosity with superiorproperties.
* information listed above is at the time of submission.
An Official Website of the United States Government


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Peptide synthesis
This article has multiple issues. Please help improve it or discuss these issues on the talk page. (Learn how and when to remove these template messages)(Learn how and when to remove this template message)
In organic chemistry, peptide synthesis is the production of peptides, which are organic compounds in which multiple amino acids are linked via amide bonds, also known as peptide bonds. The biological process of producing long peptides (proteins) is known as protein biosynthesis.
- 1 Chemistry
- 2 Liquid-phase synthesis
- 3 Solid-phase synthesis
- 4 Solid supports
- 5 Protecting groups
- 5.1 C-terminal protecting groups
- 5.2 N-terminal protecting groups
- 5.2.1 t-Boc and Fmoc protecting groups
- 5.2.2 Other protecting groups
- 5.3 Side chain protecting groups
- 6 Activating groups
- 7 Regioselective disulfide bond formation
- 8 Synthesizing long peptides
- 9 Microwave-assisted peptide synthesis
- 10 Cyclic peptides
- 11 See also
- 12 References
- 13 Further reading
- 14 External links
Peptides are synthesized by coupling the carboxyl group of one amino acid to the amino group of another amino acid molecule. Due to the possibility of unintended reactions, protecting groups are usually necessary.[citation needed] Chemical peptide synthesis most commonly starts at the carboxyl end of the peptide, and proceeds toward the amino-terminus.[citation needed] This is the opposite direction of protein biosynthesis.
Liquid-phase synthesis[edit]
Liquid-phase peptide synthesis is a classical approach to peptide synthesis. It has been replaced in most labs[citation needed] by solid-phase synthesis (see below). However, it retains usefulness in large-scale production of peptides for industrial purposes.[citation needed]
Solid-phase synthesis[edit]
Solid-phase peptide synthesis (SPPS), pioneered by Robert Bruce Merrifield,[1] caused a paradigm shift within the peptide synthesis community, and it is now the standard method for synthesizing peptides and proteins in the lab. SPPS allows for the synthesis of natural peptides which are difficult to express in bacteria, the incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins, which consist of D-amino acids.
Small porous beads are treated with functional units ('linkers') on which peptide chains can be built. The peptide will remain covalently attached to the bead until cleaved from it by a reagent such as anhydrous hydrogen fluoride or trifluoroacetic acid. The peptide is thus 'immobilized' on the solid-phase and can be retained during a filtration process while liquid-phase reagents and by-products of synthesis are flushed away.
The general principle of SPPS is one of repeated cycles of deprotection-wash-coupling-wash. The free N-terminal amine of a solid-phase attached peptide is coupled (see below) to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached. The superiority of this technique partially lies in the ability to perform wash cycles after each reaction, removing excess reagent with all of the growing peptide of interest remaining covalently attached to the insoluble resin.
The overwhelmingly important consideration is to generate extremely high yield in each step. For example, if each coupling step were to have 99% yield, a 26-amino acid peptide would be synthesized in 77% final yield (assuming 100% yield in each deprotection); if each step were 95%, it would be synthesized in 25% yield. Thus each amino acid is added in major excess (2~10x) and coupling amino acids together is highly optimized by a series of well-characterized agents.
There are two majorly used forms of SPPS – Fmoc and Boc. Unlike ribosome protein synthesis, solid-phase peptide synthesis proceeds in a C-terminal to N-terminal fashion. The N-termini of amino acid monomers is protected by either of these two groups and added onto a deprotected amino acid chain. Automated synthesizers are available for both techniques, though many research groups continue to perform SPPS manually.
SPPS is limited by yields, and typically peptides and proteins in the range of 70 amino acids are pushing the limits of synthetic accessibility. Synthetic difficulty also is sequence dependent; typically amyloid peptides and proteins are difficult to make. Longer lengths can be accessed by using native chemical ligation to couple two peptides together with quantitative yields.
Since its introduction over 40 years ago, SPPS has been significantly optimized. First, the resins themselves have been optimized.[2][non-primary source needed] Furthermore, the 'linkers' between the C-terminal amino acid and polystyrene resin have improved attachment and cleavage to the point of mostly quantitative yields.[3][4][5][non-primary source needed] The evolution of side chain protecting groups has limited the frequency of unwanted side reactions. In addition, the evolution of new activating groups on the carboxyl group of the incoming amino acid have improved coupling and decreased epimerization. Finally, the process itself has been optimized. In Merrifield's initial report, the deprotection of the α-amino group resulted in the formation of a peptide-resin salt, which required neutralization with base prior to coupling. The time between neutralization of the amino group and coupling of the next amino acid allowed for aggregation of peptides, primarily through the formation of secondary structures, and adversely affected coupling. The Kent group reported that concomitant neutralization of the α-amino group and coupling of the next amino acid led to improved coupling.[6] Each of these improvements has helped SPPS become the robust technique that it is today.[according to whom?]
Solid supports[edit]
The name solid support implies that reactions are carried out on the surface of the support, but this is not the case. Reactions also occur within these particles, and thus the term "solid support" better describes the insolubility of the polymer. The physical properties of the solid support, and the applications to which it can be utilized, vary with the material from which the support is constructed, the amount of cross-linking, as well as the linker and handle being used. Most scientists in the field believe that supports should have the minimum amount of cross-linking to confer stability. This should result in a well-solvated system where solid-phase peptide synthesis can be carried out. Nonetheless, the characteristics of an efficient solid support include:[7] it must be physically stable and permit the rapid filtration of liquids, such as excess reagents; it must be inert to all reagents and solvents used during SPPS; it must swell extensively in the solvents used to allow for penetration of the reagents; and it must allow for the attachment of the first amino acid.
There are three primary types of solid supports: gel-type supports, surface-type supports, and composites.[7] Gel-type supports are highly solvated polymers with an equal distribution of functional groups. This type of support is the most common, and includes polystyrene (styrene cross-linked with 1–2% divinylbenzene), polyacrylamide (hydrophilic alternative to polystyrene), polyethylene glycol (PEG) (PEG-polystyrene (PEG-PS) is more stable than polystyrene and spaces the site of synthesis from the polymer backbone), and PEG-based supports composed of a PEG-polypropylene glycol network or PEG with polyamide or polystyrene. Surface-type supports: Many materials have been developed for surface functionalization, including controlled pore glass, cellulose fibers, and highly cross-linked polystyrene. Composites are gel-type polymers supported by rigid matrices.
Polystyrene resin[edit]
Polystyrene resin is a versatile resin and it is quite useful in multi-well, automated peptide synthesis, due to its minimal swelling in dichloromethane. The initial support used by R. Bruce Merrifield was polysytrene cross-linked with 2% divinylbenzene. This support is sometimes referred to as the 'Merrifield resin.' This resin produces a hydrophobic bead that is solvated by a nonpolar solvent such as dichloromethane or dimethylformamide. Since then, new resins have been developed that have the advantages of chemical inertness, and enhanced swelling or rigidity (a property of mechanical strength). For instance, highly cross-linked (50%) polystyrene has been developed that possesses the features of increased mechanical stability, better filtration of reagents and solvents, and rapid reaction kinetics.
Polystyrene resins are also available as PEG hybrids. An example of this type of resin is the Tentagel resin. The base resin is polystyrene onto which is attached long chains (MW ca. 3000 Da) of polyethylene glycol (PEG; also known as polyethylene oxide). Synthesis is carried out on the distal end of the PEG spacer making it suited for long and difficult peptides. In addition, it is also attractive for the synthesis of combinatorial Peptide libraries and on resin screening experiments. It does not expand much during synthesis, making it a preferred resin for robotic peptide synthesis.
Polyamide resin[edit]
Polyamide resin is also a useful and versatile resin. It seems to swell much more than polystyrene, in which case it may not be suitable for some automated synthesizers, if the wells are too small.-->
PEG-based resin[edit]
Improvements to solid supports used for peptide synthesis enhance their ability to withstand the repeated use of TFA during the deprotection step of SPPS.[8][non-primary source needed] Furthermore, different resins allow for different functional groups at the C-terminus. The oxymethylphenylacetamidomethyl (PAM) resin results in the conventional C-terminal carboxylic acid. On the other hand, the paramethylbenzhydrylamine (pMBHA) resin yields a C-terminal amide, which is useful in mimicking the interior of a protein.
Along with the development of Fmoc SPPS, different resins have also been created to be removed by TFA. Similar to the Boc strategy, two primary resins are used, based on whether a C-terminal carboxylic acid or amide is desired. The Wang resin was, as of 1996,[needs update] the most commonly used resin for peptides with C-terminal carboxylic acids.[9] If a C-terminal amide is desired, the Rink amide resin is used.
Protecting groups[edit]
Amino acids have reactive α-carboxylic acid and α-amine groups that allow for their linking into polymers, but that also complicate the aim coupling specific pairs of amino acids, in precise order.[citation needed] In addition, many amino acids have reactive side chain functional groups, which can also react in a variety of ways, including with free α-carboxylic and α-amino groups during peptide synthesis (given the very reactive reagents present), "side-reactions" that would negatively influence yield and purity. Chemical groups have been developed to facilitate the synthesis of peptides with precise amino acid sequences, with minimal side reactions, groups that block or "protect" all functional groups present in amino acids except that pair whose coupling is desired.[citation needed] These protecting groups, while very many in practice, can be described in three groups: α-carboxylic acid (C-terminal) protecting groups, α-amino (N-terminal) protecting groups, and side chain protecting groups.[citation needed]
C-terminal protecting groups[edit]
Protecting groups of the carboxylic acid are the least used given the direction and methods applied in solid-supported peptide synthesis,[citation needed] but are covered first because to the brevity of the topic. These protecting groups are mostly used in liquid-phase synthesis,[citation needed] and there is some redundancy in the chemistry with respect to groups used to protect carboxylates in side chains (see below).
N-terminal protecting groups[edit]
Protected amino acids being added in a given step of peptide synthesis are added in excess to ensure maximal yields during each synthesis step.[citation needed] Without N-terminal protection of the added amino acid, its self-coupling (polymerization) would compete with the desired peptide synthesis, resulting in low yield of even failure of the desired peptide synthesis.[citation needed] N-terminal protection requires an additional step of removing the protecting group in each peptide synthesis cycle, a deprotection step, prior to the next coupling step.[citation needed]
As of this date,[when?] two protecting groups, tert-butyloxycarbonyl (t-Boc) and 9H-fluoren-9-ylmethoxycarbonyl (Fmoc) are most commonly used to protect the α-amine group of the "incoming" (newly added) amino acid in a solid-phase peptide synthesis cycle.[citation needed]
t-Boc and Fmoc protecting groups[edit]
Tert-butyloxycarbonyl (t-Boc) protection[edit]
The original method for the synthesis of proteins relied on tert-butyloxycarbonyl (or more simply "Boc") to temporarily protect the α-amino group. In this method, the Boc group is covalently bound to the amino group to suppress its nucleophilicity. The C-terminal amino acid is covalently linked to the resin through a linker. Next, the Boc group is removed with acid, such as trifluoroacetic acid (TFA). This forms a positively charged amino group (in the presence of excess TFA; note image on the right illustrates neutral amino group), which is neutralized (via in-situ or non-in-situ methods) and coupled to the incoming activated amino acid.[6] Reactions are driven to completion by the use of excess (2- to 4-fold) activated amino acid. After each deprotection and coupling step, a wash with dimethylformamide (DMF) is performed to remove excess reagents, allowing (by year 2000)[needs update] for high yields (~99%) during each cycle.[7]
t-Boc protecting strategies retain usefulness in reducing peptide aggregation during synthesis. t-Boc groups can be added to amino acids with t-Boc anhydride and a suitable base. Some researchers prefer Boc SPPS for complex syntheses . In addition, when synthesizing nonnatural peptide analogs, which are base-sensitive (such as depsipeptides), the t-Boc protecting group is necessary, because Fmoc SPPS uses a base to deprotect the α-amino group.
Permanent side-chain protecting groups are typically benzyl or benzyl-based groups. Final removal of the peptide from the linkage occurs simultaneously with side-chain deprotection with anhydrous hydrogen fluoride via hydrolytic cleavage. The final product is a fluoride salt which is relatively easy to solubilize. Importantly, scavengers such as cresol are added to the HF in order to prevent reactive t-butyl cations from generating undesired products. In fact, the use of harsh hydrogen fluoride may degrade some peptides, which was the premise for the development of a milder, base-labile method of SPPS—namely, the Fmoc method.
9H-fluoren-9-ylmethoxycarbonyl (Fmoc) protection[edit]
The capacity for anhydrous hydrogen fluoride to degrade proteins during the final cleavage conditions led to a new α-amino protecting group based on 9-fluorenylmethyloxycarbonyl (Fmoc). The Fmoc method allows for a milder deprotection scheme. This method utilizes a base, usually piperidine (20–50%) in DMF in order to remove the Fmoc group to expose the α-amino group for reaction with an incoming activated amino acid.[7] Unlike the acid used to deprotect the α-amino group in Boc methods, Fmoc SPPS uses a base, and thus the exposed amine is neutral. Therefore, no neutralization of the peptide-resin is required, but the lack of electrostatic repulsions between the peptides can lead to increased aggregation. Because the liberated fluorenyl group is a chromophore, deprotection by Fmoc can be monitored by UV absorbance of the runoff, a strategy which is employed in automated synthesizers.
The advantage of Fmoc is that it is cleaved under very mild basic conditions (e.g. piperidine), but stable under acidic conditions, although this has not always held true in certain synthetic sequences. This allows mild acid-labile protecting groups that are stable under basic conditions, such as Boc and benzyl groups, to be used on the side-chains of amino acid residues of the target peptide. This orthogonal protecting group strategy is common in organic synthesis. Fmoc is preferred over BOC due to ease of cleavage; however it is less atom-economical, as the fluorenyl group is much larger than the tert-butyl group. Accordingly, prices for Fmoc amino acids were high until the large-scale piloting of one of the first synthesized peptide drugs, enfuvirtide, began in the 1990s, when market demand adjusted the relative prices of the two sets of amino acids.
Semipermanent side chain protecting groups are t-butyl-based, and final cleavage of the protein from the resin and removal of permanent protecting groups is performed with TFA in the presence of scavengers. Water and triisopropylsilane (TIPS) present in a 1:1 ratio are often used as scavengers. Thus, the Fmoc method is orthogonal in two directions: deprotection of any α-amino group, deprotection of side groups and final cleavage from the resin occur by independent mechanisms. The resulting final product is a TFA salt, which is more difficult to solubilize than the fluoride salts generated in Boc SPPS. This method is thus milder than the Boc method because the deprotection/cleavage-from-resin steps occur with different conditions rather than with different reaction rates.
Comparison of t-Boc and Fmoc solid-phase peptide synthesis[edit]
Both the Fmoc and Boc methods offer advantages and disadvantages. The selection of one technique over another is thus made on a case-by-case basis.[11]
|Requires special equipment
|Cost of reagents
|Solubility of peptides
|Purity of hydrophobic peptides
|May be lower
|Problems with aggregation
|Less frequently
|More frequently
|Synthesis time
|~20 min/amino acid
|~20–60 min/amino acid
|Cleavage from resin
|Potentially dangerous
|Relatively safe
Boc SPPS uses special equipment to handle the final cleavage and deprotection step, which requires anhydrous hydrogen fluoride.[citation needed] Because the final cleavage of the peptide with Fmoc SPPS uses TFA, this special equipment is not necessary.[citation needed] The solubility of peptides generated by Boc SPPS is generally higher than those generated with the Fmoc method, because fluoride salts are higher in solubility than TFA salts.[citation needed] Next, problems with aggregation are generally more of an issue with Fmoc SPPS, primarily because the removal of a Boc group with TFA yields a positively charged α-amino group, whereas the removal of an Fmoc group yields a neutral α-amino group.[citation needed] The electrostatic repulsion of the positively charged α-amino group limits the formation of secondary structure on the resin.[citation needed] Finally, the Fmoc method is considered orthogonal, since α-amino group deprotection is with base, while final cleavage from the resin is with acid.[citation needed] The Boc method utilizes acid for both deprotection and cleavage from the resin.[citation needed] Hence, both methods possess advantages and disadvantages for their application in specific situations, and several factors must be considered to decide between the methods.[citation needed]
Other protecting groups[edit]
The (Z) group is another carbamate-type amine protecting group, first used by Max Bergmann in the synthesis of oligopeptides.[12] It is removed under harsh conditions using HBr in acetic acid,[citation needed] or milder conditions of catalytic hydrogenation.[citation needed] While it has been used periodically for α-amine protection in peptide synthesis,[citation needed] of this date,[when?] it is almost exclusively used for side chain protection.[citation needed]
Alloc and miscellaneous groups[edit]
The allyloxycarbonyl (alloc) protecting group is sometimes used to protect an amino group (or carboxylic acid or alcohol group) when an orthogonal deprotection scheme is required.[citation needed] It is also sometimes used when conducting on-resin cyclic peptide formation, where the peptide is linked to the resin by a side-chain functional group.[citation needed] The Alloc group can be removed using tetrakis(triphenylphosphine)palladium(0).[citation needed]
For special applications like synthetic steps involving protein microarrays, protecting groups sometimes termed "lithographic" are used, which are amenable to photochemistry at a particular wavelength of light, and so which can be removed during lithographic types of operations.[citation needed]
Side chain protecting groups[edit]
Amino acid side chains represent a broad range of functional groups and are sites of nonspecific reactivity during peptide synthesis. Because of this, many different protecting groups are required that are usually based on the benzyl (Bzl) or tert-butyl (tBu) group.[7] The specific protecting groups used during the synthesis of a given peptide vary depending on the peptide sequence and the type of N-terminal protection used (see next paragraph). Side chain protecting groups are known as permanent or semipermanent protecting groups, because they can withstand the multiple cycles of chemical treatment during synthesis and are only removed during treatment with strong acids after peptide synthesis is completed.[citation needed]
Because N-terminal deprotection occurs repeatedly during peptide synthesis, protecting schemes are established such that different types of side chain protecting groups (e.g., Bzl, tBu, etc.) are matched to either Boc or Fmoc, respectively, for ultimate deprotection that is optimized.[citation needed] Because multiple protecting groups are normally used during peptide synthesis of peptides greater in length than oligomers, care must be taken such that all side chain protecting groups are compatible, so that when deprotection of individual protecting groups is required (e.g., to selectively modify the side chain of one amino acid of a synthetic peptide), the one deprotection step does not affect other side chains.[citation needed] Protecting schemes are therefore developed in each particular case of a peptide synthesis to assign protecting groups to each amino acid residue.[citation needed]
Activating groups[edit]
For coupling the peptides the carboxyl group is usually activated. This is important for speeding up the reaction. There are two main types of activating groups: carbodiimides and triazolols. However the use of pentafluorophenyl esters (FDPP,[13] PFPOH[14]) and BOP-Cl[15] are useful for cyclising peptides.
These activating agents were first developed. Most common are dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC). Reaction with a carboxylic acid yields a highly reactive O-acylisourea. During artificial protein synthesis (such as Fmoc solid-state synthesizers), the C-terminus is often used as the attachment site on which the amino acid monomers are added. To enhance the electrophilicity of carboxylate group, the negatively charged oxygen must first be "activated" into a better leaving group. DCC is used for this purpose. The negatively charged oxygen will act as a nucleophile, attacking the central carbon in DCC. DCC is temporarily attached to the former carboxylate group (which is now an ester group), making nucleophilic attack by an amino group (on the attaching amino acid) to the former C-terminus (carbonyl group) more efficient. The problem with carbodiimides is that they are too reactive and that they can therefore cause racemization of the amino acid.
To solve the problem of racemization, triazoles were introduced.[citation needed] The most important ones are 1-hydroxy-benzotriazole (HOBt) and 1-hydroxy-7-aza-benzotriazole (HOAt), though others have also been developed.[citation needed] These substances can react with the O-acylurea to form an active ester which is less reactive and less in danger of racemization. HOAt is especially favourable because of a neighbouring group effect.[16] As of this date,[when?] HOBt has been removed from many chemical vendor catalogues; although almost always found as a hydrate, HOBt may be explosive when allowed to fully dehydrate and shipment by air or sea is heavily restricted.[citation needed] Alternatives to HOBt and HOAt have been introduced. One of the most promising and inexpensive[citation needed] is ethyl 2-cyano-2-(hydroxyimino)acetate (trade name Oxyma Pure), which is not explosive and has a reactivity of that in between HOBt and HOAt.
Newer developments omit the carbodiimides totally: the active ester is introduced as a uronium or phosphonium salt of a non-nucleophilic anion (tetrafluoroborate or hexafluorophosphate): HBTU, HATU, HCTU, TBTU, PyBOP.[citation needed] Two uronium types of the coupling additive of Oxyma Pure is also available as COMU or TOTU reagent.[citation needed]
Regioselective disulfide bond formation[edit]
The formation of multiple native disulfides remains one of the primary challenges of native peptide synthesis by solid-phase methods. Random chain combination typically results in several products with nonnative disulfide bonds.[17] Stepwise formation of disulfide bonds is typically the preferred method, and performed with thiol protecting groups.[18] Different thiol protecting groups provide multiple dimensions of orthogonal protection. These orthogonally protected cysteines are incorporated during the solid-phase synthesis of the peptide. Successive removal of these groups, to allow for selective exposure of free thiol groups, leads to disulfide formation in a stepwise manner. The order of removal of the groups must be considered so that only one group is removed at a time. Using this method, Kiso and coworkers reported the first total synthesis of insulin in 1993.[19]
Thiol protecting groups used in peptide synthesis requiring later regioselective disulfide bond formation must possess multiple characteristics.[citation needed][verification needed] First, they must be reversible with conditions that do not affect the unprotected side chains. Second, the protecting group must be able to withstand the conditions of solid-phase synthesis. Third, the removal of the thiol protecting group must be such that it leaves intact other thiol protecting groups, if orthogonal protection is desired. That is, the removal of PG A should not affect PG B. Some of the thiol protecting groups commonly used include the acetamidomethyl (Acm), tert-butyl (But), 3-nitro-2-pyridine sulfenyl (NPYS), 2-pyridine-sulfenyl (Pyr), and triphenylmethyl (Trt) groups.[citation needed] Importantly, the NPYS group can replace the Acm PG to yield an activated thiol.[20]
Important to the discussion of disulfide bond formation is the order in which disulfides are formed.[citation needed] The synthesis insulin by Kiso and coworkers is illustrative of the logic and methods for regioselective disulfide bond formation.[citation needed] In this work, the A-chain of insulin was prepared with following protecting groups in place on its cysteines: CysA6(But), CysA7(Acm), and CysA11(But), leaving CysA20 unprotected.[19] Synthesis of the B-chain was performed with the following protecting groups in place CysB7(Acm) and CysB19(Pyr). The first disulfide bond, CysA20–CysB19, was formed by mixing the two chains in 8 M urea, pH 8 (RT) for 50 min, while the second disulfide bond, CysA7–CysB7, was formed by treatment with iodine in aqueous acetic acid to remove the Acm groups.[19] The third disulfide, the intramolecular CysA6–CysA11, was formed after the removal of the But groups by methyltrichlorosilane with diphenyl sulfoxide in TFA.[19] Importantly, formation of the first disulfide in 8 M urea, pH 8 does not affect the other protecting groups, namely Acm and But groups. Likewise, formation of the second disulfide bond with iodine in aqueous acetic acid does not affect the But groups.[citation needed][non-primary source needed] From a logical standpoint, the order in which the thiol groups are exposed to form disulfides should be of little consequence, since the other cysteines are protected; however, it is observed, practically, that the order in which disulfides are formed can have a significant effect on yields.[citation needed][21]
Synthesizing long peptides[edit]
Stepwise elongation, in which the amino acids are connected step-by-step in turn, is ideal for small peptides containing between 2 and 100 amino acid residues. Another method is fragment condensation, in which peptide fragments are coupled. Although the former can elongate the peptide chain without racemization, the yield drops if only it is used in the creation of long or highly polar peptides. Fragment condensation is better than stepwise elongation for synthesizing sophisticated long peptides, but its use must be restricted in order to protect against racemization. Fragment condensation is also undesirable since the coupled fragment must be in gross excess, which may be a limitation depending on the length of the fragment.
A new development for producing longer peptide chains is chemical ligation: unprotected peptide chains react chemoselectively in aqueous solution. A first kinetically controlled product rearranges to form the amide bond. The most common form of native chemical ligation uses a peptide thioester that reacts with a terminal cysteine residue. Other methods applicable for covalently linking polypeptides in aqueous solution include the use of split inteins,[22] spontaneous isopeptide bond formation[23] and sortase ligation.[24]
In order to optimize synthesis of long peptides, a method was developed in Medicon Valley for converting peptide sequences.[citation needed] The simple pre-sequence (e.g. Lysine (Lysn); Glutamic Acid (Glun); (LysGlu)n) that is incorporated at the C-terminus of the peptide to induce an alpha-helix-like structure. This can potentially increase biological half-life, improve peptide stability and inhibit enzymatic degradation without altering pharmacological activity or profile of action.[25][26]
Microwave-assisted peptide synthesis[edit]
Although microwave irradiation has been around since the late 1940s, it was not until 1986 that microwave energy was used in organic chemistry. During the end of the 1980s and 1990s, microwave energy was an obvious source for completing chemical reactions in minutes that would otherwise take several hours to days. Through several technical improvements at the end of the 1990s and beginning of the 2000s, microwave synthesizers have been designed to provide both low and high energy pockets of microwave energy so that the temperature of the reaction mixture could be controlled. The microwave energy used in peptide synthesis is of a single frequency providing maximum penetration depth of the sample which is in contrast to conventional kitchen microwaves.
In peptide synthesis, microwave irradiation has been used to complete long peptide sequences with high degrees of yield and low degrees of racemization. Microwave irradiation during the coupling of amino acids to a growing polypeptide chain is catalyzed not only by the increase in temperature but also by the alternating electric field of the microwave.[27] This is because the polar N-terminal amine group and peptide backbone continuously try to align with the alternating electric field, thus helping prevent aggregation and increasing access to the solid phase reaction matrix. This increases yields of the final peptide products. There is however no clear evidence that microwave is better than simple heating and some peptide laboratories regard microwave just as a convenient method for rapid heating of the peptidyl resin. Heating to above 50–55 degrees Celsius also prevents aggregation and accelerates the coupling.
Despite the main advantages of microwave irradiation of peptide synthesis, the main disadvantage is the racemization which may occur with the coupling of cysteine and histidine. A typical coupling reaction with these amino acids are performed at lower temperatures than the other 18 natural amino acids. A number of peptides do not survive microwave synthesis or heating in general. One of the more serious side effects is dehydration (loss of water) which for certain peptides can be almost quantitative like pancreatic polypeptide (PP). This side effect is also seen by simple heating without the use of microwave.
Cyclic peptides[edit]
On resin cyclization[edit]
Peptides can be cyclized on a solid support. A variety of cylization reagents can be used such as HBTU/HOBt/DIEA, PyBop/DIEA, PyClock/DIEA. Head-to-tail peptides can be made on the solid support. The deprotection of the C-terminus at some suitable point allows on-resin cyclization by amide bond formation with the deprotected N-terminus. Once cyclization has taken place, the peptide is cleaved from resin by acidolysis and purified. The strategy for the solid-phase synthesis of cyclic peptides in not limited to attachment through Asp, Glu or Lys side chains. Cysteine has a very reactive sulfhydryl group on its side chain. A disulfide bridge is created when a sulfur atom from one Cysteine forms a single covalent bond with another sulfur atom from a second cysteine in a different part of the protein. These bridges help to stabilize proteins, especially those secreted from cells. Some researchers use modified cysteines using S-acetomidomethyl (Acm) to block the formation of the disulfide bond but preserve the cysteine and the protein's original primary structure.
Off-resin cyclization[edit]
Off-resin cyclization is a solid-phase synthesis of key intermediates, followed by the key cyclization in solution phase, the final deprotection of any masked side chains is also carried out in solution phase. This has the disadvantages that the efficiencies of solid-phase synthesis are lost in the solution phase steps, that purification from by-products, reagents and unconverted material is required, and that undesired oligomers can be formed if macrocycle formation is involved.[28]
See also[edit]
- ^ R. B. Merrifield (1963). "Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide". J. Am. Chem. Soc. 85 (14): 2149–2154. doi:10.1021/ja00897a025.
- ^ Mitchell, A. R. K., S.B.H.; Engelhard, M.; Merrifield, R.B. (1978). "A new synthetic route to tert-butyloxycarbonylaminoacyl-4-(oxymethyl)phenylacetamidomethyl-resin, an improved support for solid-phase peptide synthesis". J. Org. Chem. 43 (13): 2845–2852. doi:10.1021/jo00408a022.
- ^ Wang, S.-S. (1973). "p-alkoxybenzyl alcohol resin and p-alkoxybenzyloxycarbonylhydrazide resin for solid phase synthesis of protected peptide fragments.". J. Am. Chem. Soc. 95 (4): 1328–33. doi:10.1021/ja00785a052. PMID 4687686.
- ^ Matsueda, G. R. a. S., J.M. (1981). "A p-methylbenzylhydrlamine resin for improved solid-phase synthesis of peptide amides". Peptides. 2 (1): 45–50. doi:10.1016/S0196-9781(81)80010-1. PMID 7243625.
- ^ Sieber, P. (1987). "A new acid-labile anchor group for the solid-phase synthesis of C-terminal peptide amides by the Fmoc method". Tetrahedron Lett. 28: 2107–10. doi:10.1016/S0040-4039(00)96055-6.
- ^ a b Schnolzer, M. A., P.; Jones, A.; Alewood, D.; Kent, S.B.H. (2007). "In Situ Neutralization in Boc-chemistry Solid Phase Peptide Synthesis". Int. J. Peptide Res. Therap. 13 (1–2): 31–44. doi:10.1007/s10989-006-9059-7.
- ^ a b c d e Albericio, F. (2000). Solid-Phase Synthesis: A Practical Guide (1 ed.). Boca Raton: CRC Press. p. 848. ISBN 0824703596.
- ^ Feinberg, R. S.; Merrifield, R. B. (1974). "Zinc chloride-catalyzed chloromethylation of resins for solid phase peptide synthesis". Tetrahedron. 30 (17): 3209–3212. doi:10.1016/S0040-4020(01)97575-1.
- ^ Hermkens, P. H. H.; Ottenheijm, H. C. J.; Rees, D. C. (1997). "Solid-phase organic reactions II: A review of the literature Nov 95 – Nov 96". Tetrahedron. 53 (16): 5643–5678. doi:10.1016/S0040-4020(97)00279-2.
- ^ Jones, J. (1992). Amino Acid and Peptide Synthesis. Oxford, UK: Oxford University Press.[full citation needed]
- ^ Nilsson BL, Soellner MB, Raines RT (2005). "Chemical Synthesis of Proteins". Annu. Rev. Biophys. Biomol. Struct. 34: 91–118. doi:10.1146/annurev.biophys.34.040204.144700. PMC 2845543 . PMID 15869385.
- ^ Bergmann, Max; Zervas, Leonidas (1932). "Über ein allgemeines Verfahren der Peptid-Synthese". Berichte der deutschen chemischen Gesellschaft. 65 (7): 1192–1201. doi:10.1002/cber.19320650722.
- ^ K. C. Nicolaou; Natarajan, Swaminathan; Li, Hui; Jain, Nareshkumar F.; Hughes, Robert; Solomon, Michael E.; Ramanjulu, Joshi M.; Boddy, Christopher N. C.; Takayanagi, Masaru (1998). "Total Synthesis of Vancomycin Aglycon – Part 1: Synthesis of Amino Acids 4–7 and Construction of the AB-COD Ring Skeleton". Angew. Chem. Int. Ed. 37 (19): 2708–2714. doi:10.1002/(SICI)1521-3773(19981016)37:19<2708::AID-ANIE2708>3.0.CO;2-E.
- ^ Schmidt; Joullié, Madeleine M. (1998). "Synthetic studies of 14-membered cyclopeptide alkaloids". Tetrahedron Lett. 39 (40): 7211–7214. doi:10.1016/S0040-4039(98)01589-5.
- ^ R. Baker; J. L. Castro (1989). "The total synthesis of (+)-macbecin I". Chem. Commun. (6): 378–381. doi:10.1039/C39890000378.
- ^ L. A. Carpino (1993). "1-Hydroxy-7-azabenzotriazole. An efficient peptide coupling additive". J. Am. Chem. Soc. 115 (10): 4397–4398. doi:10.1021/ja00063a082.
- ^ Zhang, J.-W.; Wu, Cui Rong; Liu, Wen; Zhang, Jing Wen (1991). "Disulfide bond formation in peptides by dimethyl sulfoxide. Scope and applications". J. Am. Chem. Soc. 113 (17): 6657–6662. doi:10.1021/ja00017a044.
- ^ Sieber, P.; Kamber, B.; Hartmann, A.; Jöhl, A.; Riniker, B.; Rittel, W. (1977). "Total synthesis of human insulin. IV. Description of the final steps (author's transl)". Helvetica Chimica Acta. 60 (1): 27–37. doi:10.1002/hlca.19770600105. PMID 838597.
- ^ a b c d Akaji, K.; Fujino, K.; Tatsumi, T.; Kiso, Y. (1993). "Total synthesis of human insulin by regioselective disulfide formation using the silyl chloride-sulfoxide method". Journal of the American Chemical Society. 115 (24): 11384–11392. doi:10.1021/ja00077a043.
- ^ Ottl, J.; Battistuta, R.; Pieper, M.; Tschesche, H.; Bode, W.; Kuhn, K.; Moroder, L. (1996). "Design and synthesis of heterotrimeric collagen peptides with a built-in cystine-knot. Models for collagen catabolism by matrix-metalloproteases". FEBS Lett. 398 (1): 31–36. doi:10.1016/S0014-5793(96)01212-4. PMID 8946948.
- ^ This may be because the formation of the CysA20–CysB19 disulfide may place the thiol group of CysB7 in close proximity with both CysA6 and CysA7, leading to multiple disulfide products.[original research?][speculation?][citation needed]
- ^ Aranko AS, Wlodawer A, Iwaï H (2014). "Nature's recipe for splitting inteins". Prot Eng Des Sel. 27: 263–71. doi:10.1093/protein/gzu028. PMC 4133565 . PMID 25096198.
- ^ Reddington SC, Howarth M (2015). "Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher". Curr Op Chem Biol. 29: 94–9. doi:10.1016/j.cbpa.2015.10.002. PMID 26517567.
- ^ Haridas V, Sadanandan S, Dheepthi NU (2014). "Sortase-based bio-organic strategies for macromolecular synthesis". Chembiochem. 15: 1857–67. doi:10.1002/cbic.201402013. PMID 25111709.
- ^ Kapusta, D. R.; Thorkildsen, C; Kenigs, VA; Meier, E; Vinge, MM; Quist, C; Petersen, JS (2005). "Pharmacodynamic Characterization of ZP120 (Ac-RYYRWKKKKKKK-NH2), a Novel, Functionally Selective Nociceptin/Orphanin FQ Peptide Receptor Partial Agonist with Sodium-Potassium-Sparing Aquaretic Activity". Journal of Pharmacology and Experimental Therapeutics. 314 (2): 652–60. doi:10.1124/jpet.105.083436. PMID 15855355.
- ^ Rizzi A, Rizzi D, Marzola G, et al. (October 2002). "Pharmacological characterization of the novel nociceptin/orphanin FQ receptor ligand, ZP120: in vitro and in vivo studies in mice". Br. J. Pharmacol. 137 (3): 369–74. doi:10.1038/sj.bjp.0704894. PMC 1573505 . PMID 12237257.
- ^ Palasek, Stacey A.; Cox, Zachary J.; and Collins, Jonathan M. (2007). "Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis". Journal of Peptide Science. 13 (3): 143–148. doi:10.1002/psc.804. PMID 17121420.
- ^ Peter Scott (13 October 2009). Linker Strategies in Solid-Phase Organic Synthesis. John Wiley & Sons. pp. 135–137. ISBN 978-0-470-74905-0.
Further reading[edit]
- Stewart, J.M.; Young, J.D. (1984). Solid phase peptide synthesis (2nd ed.). Rockford, IL: Pierce Chemical Company. p. 91. ISBN 0935940030.
- Kent, Stephen B. H. (1984). "Chemical Synthesis of Peptides and Proteins". Annual Review of Biochemistry. 57. Palo Alto, CA: Annual Reviews. pp. 957–989. doi:10.1146/annurev.bi.57.070188.004521. Retrieved 12 November 2016.
- Atherton, E.; Sheppard, R.C. (1989). Solid Phase peptide synthesis: a practical approach. Oxford, England: IRL Press. ISBN 0199630674.
- Chan, W. & White, Peter, eds. (2000). Fmoc Solid Phase Peptide Synthesis: A Practical Approach. Practical Approach Series, Issue 222. Oxford, UK: Oxford University Press. ISBN 0199637245. Retrieved 12 November 2016.
- Stawikowski, Maciej & Fields, Gregg B. (2002). "Unit–18.1. Introduction to Peptide Synthesis". Curr Protoc Protein Sci. Chapter 18: Unit 18.1. doi:10.1002/0471140864.ps1801s26. PMC 3564544 . PMID 18429226.
- Bodanszky, M. (2012). Principles of Peptide Synthesis. Reactivity and Structure: Concepts in Organic Chemistry, Volume 16. New York, NY: Springer Science & Business Media. ISBN 3642967639. Retrieved 12 November 2016.
- Bodanszky, M.; Bodanszky, A. (2013). The Practice of Peptide Synthesis. Reactivity and Structure: Concepts in Organic Chemistry, Volume 21. New York, NY: Springer Science & Business Media. ISBN 364296835X. Retrieved 12 November 2016.
- Benoiton, N. Leo (2016). Chemistry of Peptide Synthesis. Boca Raton, FL: CRC Press / Taylor & Frances. ISBN 1420027697. Retrieved 12 November 2016.
- Laconde, Guillaume; Desroses, Matthieu (2016). "Synthetic Protocols Coupling Reagents in Amide Synthesis" (commercial blog). Montpellier, France: Helixem. ISBN 1420027697. Retrieved 12 November 2016.
External links[edit]
|Wikimedia Commons has media related to Peptide synthesis.


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Please notice that the apartment is "ready to live in" and fully equipped with furniture. There is one large bed of 140 x 200cm for 2 persons. Kitchenware are provided. The rate does not include bank transfer fees (no fee inside Euro countries for regular bank transfer). There are also a complete bathroom and toilets inside the apartment and a fully equipped kitchen. The flat is very quiet and very bright, located on the 6th floor with a lift and the stairs. Please notice that the flat is not convenient for persons of 5.90 ft (1m80). A baby cot can be provided on request (10 Euros per day).
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|Příjezd po
|Check-in before
|Odjezd před
|Stornovací podmínky
Ohodnoďte tomuto hostiteli i další ubytování
Nicely decorated appartment. Lots of space for your stuff and spacious enough for us (two adults, two kids), even when friends came to visit. Unfortunately, some things were broken on arrival. They were however quickly fixed after e-mailing this. Walking distance to the metro is fine. Appartment is on the third floor, no elevator, so be sure not to bring to heavy bags.
The apartment was fine, and well located close to metro, restaurants etc., however the bathroom was quite old and small. If you are on the older side (thankfully none of us were) climbing over the bath to get into the shower would be a challenge! The wire for the upstairs heater was cut so those staying there froze the first night. We requested blankets but had to go past the office (in a cab) to pick these up ourselves. Very inconvenient. Staff were nice and easy to deal with, except for the blanket incident and the fact we had to chase up the email directions for how to get the keys, which we received one day before arrival.
This was the most horrible experience I have ever had. After booking and paying an extra price for included cleaning for this flat, we arrived to a dirty apartment with dust and hair all over the floor and the most disgusting bathroom with a wet towel lying on the floor. We called the host a several times and she promised to send someone but no one came. If you think this is the worst part, the next four days of our stay we had no WIFI, no warm water and electricity coming and going. On top of all this, when we called the emergency phone nr they would either not answer at all our hang up on us when trying to tell them to help us. The ONLY good thing was the location of the flat. It is in a lively and beautiful area with many cafés, restaurants and minimarkets just around the corner. And also the metro connections were very good. But I would never book this or any other flat that is owned by this user. Because despite expecting the worst of it, it was worse than worst.
We stayed two night at that flat. and everything in flat is good. thank you so much Wimdu.
everything you need is in the house, the heating system works well, the location is great, the area is quiet, but the cleanliness is very poor, there is mold in the shower, there is a lot of dust under the bed and on the shelves, and there are webs on the chandelier and walls.
Great apartment, clean, safe and perfect location. I enjoyed my stay and highly recommend this studio. Thank you!
Terrible experience. Apartment was dirty when we arrived; dirty sheets and towels were laying on the floor. The beds were not made and the shower looked as if it hadn't been cleaned in weeks. There was no toilet paper either. We had to go out and buy towels and toilet paper for our stay as the office was already closed. There wasn't a phone in the apartment anyway. There was a note and the keys from the previous renters on the table. The note had many of the same complaints we had. Obviously since they were still on the table, nobody had come in to wash the bedding and make the beds. The Internet did not work and the t.v. did not work. There is NOT a couch as depicted in the pictures. The "view" of the eiffel tower requires one to step outside on the very small balcony and look to your far right and in the distance you can see it. As far as we're concerned, this apartment is full of misleadings and should be considered false advertisement. Save yourself a lot of headache and stay somewhere else.
I enjoyed my week-end in Paris. This apartment is really very nice.Keys were in the office so I was not worry about keeping someone in apartment in the beginning. It is about 15 minut from airport shutle and 5 minutes from Metro. There is only 15 minutes to Champs Elysees.There are shops and supermarkets not far from apartment.There is everything what is necessary for short stay in Paris. Marzena
Apartment was as described, in a good location and had everything we needed.
It was a nice stay in Paris for the hole family - 2 adults and 3 kids ind the age from 6-18 years. The appartment was bigger than we had thought from the pictures on the website.
Be aware of the parking prices if you come by car - as we did - and the traffic in Paris on a saterday night...
- Ověřenou kvalitu ubytování
- Prověření každého hostitele
- Bezpečné platby prostřednictvím SSL šifrování


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Upcoming Body Current® events, trainings and retreats
listed on our Events page
Shanti Medina, CYT CNSF CPT created the Body Current® modality after over 15 years empowering clients through self-realization, yoga therapy, and intuitive life coaching programs. To learn more about Shanti's coaching style, visit: Shanti Personal Coaching.
Body Current® somatics is an empowering 3-step application coupled with simple and highly effective transformational and energizing body-centered practices designed to imprint new neural pathways by attuning to one's own innate wisdom, which is often clouded by day to day confusion and illusion. Creating a clear current of present moment communication and regulation through the Central Nervous System and the Vagus Nerve of the Peripheral Nervous System gently releases trauma imprints, resolves chronic pain, mitigates daily stress, and reprograms old conditioning.
The Body Current principles and practices, rooted in ancient wisdom and backed by modern day neuroscience, stimulate the Vagus nerve, which is a master regulator of the relaxation response of the nervous system. These empirically proven practices, personalized to each clients' needs, include conscious breathing, visualization, and somatosensory body-centered practices. Attuning to and literally supercharging the innate wisdom self-directs the nervous system by engaging the higher executive functioning parts of our brain as well as the "smart vagus" of what has been termed the Social Nervous System. Through awareness, intention and repetition new pathways are formed in the brain/body neural map.
There are now 4 Live Stream Body Current Video practices available: Body Current® Heart Centering Practice, Plugging into Your Body Current® Intelligence, Body Current® Rhythmic Breathing Practice, and Body Current® Protocol for Releasing Stress and Chronic Pain. These 4 somatic practices are simple and short (16-20 minutes in length) yet pack a powerful practice. They are a great opportunity to learn to regulate and navigate your inner landscape by plugging into your Body Current intelligence again and again alongside Shanti. Find out more: Body Current Video Practice
The Body Current modality is boiled down into an easy to remember 3 step process, or the 3 A's, forming the template for the tools and practices to prime the neural pathways empowering the practitioner to create simple shifts in their neurobiology by heightening the 4 areas of awareness: proprioception, introspection, exteroception, and interoception. Using this simple application, each moment is an opportunity to engage with the current of our innate wisdom stepping into our true nature again and again through body-centered awareness and leadership, breath by breath.
Clients have amazing results in just one session as they learn to empower themselves to build new neurological pathways! Individual sessions offered in person, via Skype or phone. To read a recent review about the Body Current modality published in the Daily Camera newspaper read here: Body Current Boulder Workout of the Week. Body Current was also named BOULDER'S BEST 2015 WORKOUT in the category of yoga by Aimee Heckel of Daily Camera. Read full article here: Body Current named BOULDER'S BEST WORKOUTS of 2015
Shanti is also a Certified Neurosculpting® Facilitator offering private Neurosculpting meditations to heal trauma, rewrite limiting beliefs and find wholeness. Neurosculpting meditation modality is a whole-brain approach integrating mindfulness and neuroscience into a simple five-step method to transform limiting beliefs into unlimited potential. Neurosculpting was developed in 2007 by Lisa Wimberger to empower practitioners to live a life powered by intention through self-directed neuroplasticity. Unique to the Neurosculpting® methodology is its structure and intentional whole-brain engagement designed to support the management of stress through specific languaging syntax and proven steps to down-regulate the hyper-arousal states while simultaneously up-regulating the higher executive functioning of the brain.
Step 1: Down-regulate flight/fight/freeze response to calm the nervous system
Step 2: Enhance activity in the higher executive functioning of the brain
Step 3: Increase activity and communication between the right and left hemispheres of the brain
Step 4: Imprint the meditation for easy identification and replication in day-to-day activities
Step 5: Choose a name or symbol for the meditation
To learn more about the Body Current® modality, please feel free to listen to Shanti's interview with Sandra Guy Malhortra, Ph.D. of W Cubed on VoiceAmerica Internet Radio along with Lisa Wimberger, Founder of the Neurosculpting® Institute as they share how their modalities support people in resolving trauma and chronic pain whiling mitigating daily stress. It's a free on demand audio at this link: "Transformation and Regeneration from the Inside Out"
Shanti's Body Current work supported me in working through the multiple physical and mental issues that so many professional musicians endure. Learning how to align my posture and to self-regulate my nervous system so that I could remain calm and present while performing changed my playing dramatically. The practices she gave me supported me in reprogramming and unraveling old conditioning and embedded trauma while creating alignment and ease in my mind and body. – Lisa de Alwis, Ph.D., musicologist and pianist
Wow Shanti! Thank you so much for following your heart's call. You are a profoundly gifted healer. I am so grateful that you shared Body Current with me. I have done a great deal of healing work over the years and even with all of that it didn't seem I was able to completely heal the PTSD that has been with me for so long. In one short session with you, I had miraculous results!! I feel as if I came full circle, honoring all aspects of this precious journey and have come to once again uncover a greatness inside of me that I always knew was there, but couldn't fully access. The self-imposed chains have fallen. The veil has lifted. You helped me open the door… to so many things releasing trauma that I had suppressed for years. Thank you! Thank you! Thank you! I am deeply grateful for you and the beauty you bring to the planet. -Annie Freedom, Founder and President Samadhi Center for Yoga
After years of excruciating sciatic pain and thousands of dollars working with traditional and alternative practitioners, Shanti changed my life in only 6 private sessions. – Holly S., athlete and mother
"I continually get positive feedback from patients regarding her unique combination of technical expertise and inspiring personality. I feel so fortunate to collaborate with Shanti for the benefit of my patients!" – Danielle Custode, D.C. Full Potential Chiropractic


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City leads area with new starts, home closings
Naperville ranked first in the number of new homes under construction and also in new home closings in the Chicago area during the past four quarters, according to a market research and consulting firm.
Metrostudy, which analyzes new construction in a 12-county area around Chicago that includes Northwest Indiana and southeast Wisconsin, reports Naperville had 222 new home starts in the past 12 months. Pingree Grove, west of Elgin, was second with 215, while Elgin was third with 208. The new construction includes single family and multi-family homes..
"Naperville is still a premiere address in the Chicagoland market," said Chris Huecksteadt, director of Metrostudy. "If developers are getting houses to market in a community like Naperville, then chances for success are pretty good. That's not the case in many areas around Chicago."
While those numbers are nowhere near the 35,000 new homes started in the area in 2005 and 2006, they indicate a bit of a rebound in new housing construction since the market took a hard nose dive in 2008, Huecksteadt said. His market area has seen about 4,000 new housing starts over the past year, up from a low of about 2,500 in 2008.
"We're well below the historical high, but things are improving slightly," Huecksteadt said. He views a normal market as having about 18,000 to 20,000 new homes being built in a year.
The number of new construction permits, 214, issued in Naperville in the past fiscal year ending April 30 was nearly double that of the previous year, said John Rutkowski, development services team leader for the city.
"We're moving in the right direction. It's good for the city," he said, adding with undeveloped land in Naperville dwindling, new construction numbers should level off.
About 30 to 40 of last year's permits were for tear downs, most in Naperville's downtown, while most of the single-family homes are being built on the city's southwest side or in pockets of undeveloped land in city neighborhoods. A townhome development near 75th Street and Route 59 also accounted for much of the new construction.
The median transaction value of new Naperville homes sold for most of the months in 2012 was about $350,000, Huecksteadt said, adding that shows the strength of multi-family home sales.
Near 75th Street and Route 59, the townhome Mayfair development by M/I Homes of Chicago is 90 percent sold out, said Cheryl Bonk, vice president of sales and marketing for the company. M/I Homes entered the Chicago market about five years ago.
"We stuck to the old adage of location, location, location. And price," Bonk said. "Even in a challenging market there is success to be had. The townhome market has a little more resiliency." The price of Mayfair townhomes range from about $216,000 to about $327,000.
Meanwhile, Pete Stefani of King's Court Builders said he is heartened by the uptick in business this year. The custom home builder has six homes underway from $550,000 to $1.2 million in Naperville along with other homes in nearby communities. He closed on 26 homes in the past year, down from a high of 73 in 2005, but up from a low of 15 in 2011.
"Certainly, there has been a lot of blood let over the last number of years," Stefani said of the number of builders and developers who have had to lay off employees or go out of business. Today, he feels there is more demand than inventory in towns like Naperville.
"In comparison to recent years, I'd have to call the market hot," Stefani said.


text:
ED Magedson – Founder
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Florida Healthy KidsP.O. box 591 Tallahasee fl,33631-3105 Internet United States of America
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This report was posted on Ripoff Report on 10/11/2010 05:28 PM and is a permanent record located here: http://www.ripoffreport.com/reports/florida-healthy-kids/internet/florida-healthy-kids-florida-kidcare-florida-kidcare-account-cancelled-over-a-500-surpris-649777. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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Feathers, Fur, Fins is the theme of the next GAP Museum annual exhibit. Submissions for inclusion in the exhibit must be received by April 5, 2017. Members may obtain an entry form by signing on and selecting the "Forms" option under the Members tab. Additional annual exhibit information can be found on the Exhibitions page under Events.
Copyright ©2017 Guild of American Papercutters. All Rights Reserved.
The Guild is a tax-exempt, 501(c)(3) nonprofit organization of the Commonwealth of Pennsylvania. The operation of the Guild of American Papercutters is accomplished entirely by volunteers, with no member or officer receiving any payment for their work.


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Ultra competitive Phelps taking it slow but still hates to lose
SYDNEY (Reuters) - Almost two years of retirement has meant Michael Phelps needs to listen to his body and take it slower than in the past but it has not dulled his competitive fire as he sent an ominous warning to his fellow swimmers at next week's Pan Pacific championships in Australia.
"I still can't stand to lose," the 18-time Olympic champion told reporters in Brisbane on Saturday ahead of his first international meeting since the 2012 Olympics.
The 29-year-old Phelps, who retired after the London Games before he made a comeback earlier this year, qualified for the 100 meters butterfly and 200 individual medley at the Aug 21-24 Pan Pacs after finishing second in both events at the recently completed U.S. championships.
The fact he was pipped by Tom Shields in the 100 butterfly by 0.01 seconds and by world record holder Ryan Lochte by 0.05 seconds in the medley in Irvine, grated with him though he did recognize he was getting older.
"Losing by a hundredth (of a second) and losing by five one-hundredth at the (U.S.) nationals in two out of three events was frustrating," he added.
"I am taking it a lot slower now than what I did before because I do understand that I need to give my body time to get back and build up to where I want to be," he added.
"I can't just expect to do everything I want from the get-go. Being able to have a year-and-a-half off, I've been able to learn a lot about myself and I still have that competitive side.
"It's still the same with anything I want to do. But it's going to take a little time for me to get back. We're doing it slowly."
Phelps had said prior to the U.S. Nationals he would see where he stood before committing whether to go ahead to the next Olympics in Rio and the Pan Pacs were a step in helping make that decision. Continued...


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Alignment Issues After Drop
Title says it all. I've had 3 alignments done since my Eibach drop, one of which was done at a shop that says "lowered vehicles are our specialty". All 3 alignments were done on state of the art Hunter Hawkeye alignment machines.
The first two alignments my steering wheel was turned to the right. Now it's turned to the left.
I'm not well versed in this subject, so I'll try to describe it in layman's terms. Throughout all 3 alignments the steering wheel has felt disconnected from the wheels. I feel like there is some wandering in the wheels before the steering wheel moves. Let's say the wheels move 20% from a groove in the road. The first 15% of that movement the steering wheel stays stationary. The steering feels very loose. This would make sense if I had not had an alignment, but I've had 3, all aligning within factory spec. It's only the steering wheel not centered. So how can it be loose if it's aligned with only the steering wheel being off?
The best way to sum up what it seems like is this: Right when I get the car back it feels good, tight. But after a little driving it gets looser and looser. It's as if they didn't tighten bolts enough and the settings gradually fall out of spec. I could see this happening with alignment, 1 shop. But 2 shops and 3 alignments??
My last hope is to go to the dealer and instruct them not to give it back to me until it's perfect.
Any help is appreciated. This is driving me mad! I'm a bit OCD so it's really ruining my driving experience.
Re: Alignment Issues After Drop
did a reputable shop DO the install.
suspension parts need tightened under load NOT DROOP.
the idea is that the vehicle sitting on its weight or under load places most of the components in the MIDDLE of their travel. so you tighten up that way.
perhaps something was tightened with the vehicle on the lift with weight off the wheels.
perhaps this is not the case but just throwing out ideas/
It has to be install. I have the extreme drop RMs on mine and it's been flawless from day one.
Re: Alignment Issues After Drop
I'm aware of timing/clocking bushings. When I read about it here ( Timing Bushings when lowering (How to) - Camaro5 Chevy Camaro Forum / Camaro ZL1, SS and V6 Forums - Camaro5.com) though it seems like not all the bushings were loosened. He only loosed what he had to get the springs installed.
I'm going back to the alignment shop tomorrow morning with instructions. I'm going to personally oversee everything and their most knowledgeable employee is going to do it. All this company does are alignments. They have 6 Hunter machines.
I'm just hoping after ensuring all the bushings are loosened under load, we excessively cycle the bushings, align/tighten bolts, that the Jeep will be back to normal.
I'm not sure what else could have been messed up during the install....
Re: Alignment Issues After Drop
What's your camber at? I have never clocked my bushings ever, and every car I hahbe owned I have lowered. Never had any issues unless camber was still to negative then the car felt loose
Re: Alignment Issues After Drop
Does anyone have the OEM camber specs for comparison? Also, any eibach guys have the specs they aligned to?
Re: Alignment Issues After Drop
Re: Alignment Issues After Drop
Re: Alignment Issues After Drop
Re: Alignment Issues After Drop
It's fixed! FINALLY! I took it back to the dealership and they did it right. The steering is tight. The wheel is straight. It's perfect. I guess that's what I got for going the cheap route.
I decided to go with -1.6 in the front after speaking with the Viper/SRT tech. He said bone stock SRT Jeep comes in at -2 to -2.2. He said a lot of customers are asking to go more positive to prevent tire wear. We decided to do a compromise not to sacrifice handling.
A warning for those of you doing alignments after a drop. IF you don't have a trust worthy go to shop, go back to the dealership! Pay the money. It's worth it.
Lastly, someone at the dealership stole scratch off lottery tickets I had that had $80 worth of winnings. It's Helfman Jeep in Houston. Hopefully the remedy the situation quickly.
Re: Alignment Issues After Drop
glad to hear its right/
f'ed up about the tickets. hope they fix it for you/
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Chapter 6Mature
The nurse frowned at Serena. "You shouldn't come to school emotionally exhausted like this. You can stay in here for a little bit. I will give Miss Daniels a note to take back to your teacher. For now, why don't you try and sleep?" Tiffany winked at Serena behind the nurse's back. They shared a smile before Tiffany had to leave. The nurse handed Tiffany a note and told her to head back to class.
She walked over to Serena and have her a hug. Tiffany walked back to class and silently handed the note to her teacher. He scanned it over before gruffly telling her to sit. Tiffany swayed over to her seat and settled down carefully. People looked at her, trying to guess why she was late. Whispers slithered around the class, getting louder with each pass.
The teacher noticed at told everyone to quiet down. They did until he turned around. Then they started the rumors all over again. Tiffany rolled her eyes. Some people were so shallow. But she couldn't really blame them. She would probably to the same had it not been her best friend they were whispering about.
No one came out directly and asked her for an answer. That would ruin the fun. She bit the inside of her cheek and tried to ignore them. It didn't work so well. The teacher seemed to notice the noise again and told everyone to be quiet.
"If you all don't stop talking, I will start handing out detentions." The class quieted down instantly. The English teacher had a passion for handing out detentions. Everyone knew to take him seriously.
"Stick out your tongue." Serena frowned but did as she said. The nurse looked, prodded, and poked at Serena. She was trying to find something wrong with the girl.
"I can't find anything. You are probably just emotionally exhausted like I said earlier. I can send you home if you want. Or you can just take a nap here. The home environment might make things worse." Serena mentally went through the pros and cons. She could go home and deal with her grandmother, or stay here and take a nap.
"Do you have anything that would help me sleep?"
"Yes, but I would suggest trying to sleep yourself first." The nurse walked out of the door and turned off the light. Serena stared at the ceiling. She wasn't tired, but at the same time she was too exhausted to think.
Serena watched the movements in the hall. She would probably have to see a psychiatrist now. That would be fun. Not only would she be supposed to spill her guts to a stranger, she would become a laughingstock. The door opened slightly.
Serena thought about feigning sleep. That way the nurse would leave her alone. She didn't act quickly enough, the light flashed on. Serena blinked owlishly and stared at the door. A man stood behind the nurse. He had salt and pepper hair and wore a gray business suit.
"Serena? Did we wake you?"
"No. I was still awake. Who is that behind you?" She had a feeling who it was, but she decided to ask anyway.
"This is Mr. Norris. He is going to help you though this tough time."
"I don't want a psychiatrist," Serena frowned, not trying to be polite in the least.
The man gave a small smile, "then don't think of me as a psychiatrist. Think of me as a friend, or a guide.
"I don't need one of those either. I have a friend and I don't need a guide." Mr. Norris let his smile slip slightly.
"Well, we will see what we can do. The principal has requested my help. I think you need help too. Obviously you are acting out to get attention."
"Obviously," Serena mocked, "you don't deserve to be a psychiatrist. That is no way to get a patient to open up."
The man scowled, all pretenses gone. He turned to the nurse. "She is tired. Can you schedule an appointment for tomorrow? I can come in around lunch." The nurse nodded and scribbled a note onto a piece of paper.
"Give this to the secretary. She will make sure that Serena is ready for the appointment." Mr. Norris said goodbye to Serena and walked out of the room quickly. She could tell by his tense muscles that he was angry.
She sighed. Serena hadn't meant to make him angry, but she didn't want someone analyzing her secrets. They were hers for a reason. She was upset about her parent's death, but talking to someone wouldn't help in the slightest. She didn't know how people thought that would work.
The man frowned. He had been looking at Serena's diary again. He had flipped the page over from her latest entry, but there was another. And it was from tomorrow. Something was wrong. Did she guess what he had been up to? He needed to think. This was wrong. He was missing something, and it was dancing at the edge of his mind.
Wrapped up in confusion, he forgot about the two people downstairs. He didn't remember until he was in front of the kitchen. Alice was washing dishes. He tried to move before the old woman could see her. The woman heard his movements and turned around.
She gasped and dropped the pate she had been holding. It shattered around her feet. The woman didn't seem to notice. She looked at the girl in confusion. And fear.
"Serena? Is that you?"


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This is going to be about high school, seen through many different characters. Feel free to add, but it would be much appreciated if you stayed in the situation we were talking about. c:
Here we go again, I thought as I walked through the double doors that creaked when you opened them. I looked around and saw all of the familiar faces, and people trying to find their groups. You could tell who the freshman are because their schedules were pressed to their confused faces. I remember those days, being confused, weird, but innocent. High school really changes you.
"Mel!" Hailey screamed as she ran towards me and grabbed me.
"Holy shit! Hailey?! You're back?! Why didn't you tell me!" I yelled at her. Hailey had been forced to move to Alabama two years ago. I don't know why anyone would leave New York for Alabama. It was too strange.
"Give me your schedule!" she said, grabbing for it from my bag.
"Do we have anything together?" I asked insistently.
"Oh my God!! We have the same schedule! This is great!"
We both continued shrieking like little girls in the middle of the hallway. We got strange looks from the freshman, but who cared? I sure didn't. I had my Hailey back, so nothing could ruin this moment.


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Tuesday, February 03 2009
Derrinstown Stud to sponsor Thursday's race meeting
Dubai Racing Club hosts a double header racing weekend, with Derrinstown Stud sponsoring the first leg this Thursday.
Derrinstown Stud, a world-class stud based in Ireland, has been a long-standing partner with Dubai Racing Club, having sponsored at the Dubai International Racing Carnival since its inception.
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Deep Arora had een nieuw logo ontwerp nodig en lanceerde daarom een wedstrijd op 99designs.
Een winnaar werd gekozen uit44 ontwerpen ingezonden door19 freelance designers.
This site is about creating niche products with private label rights. We will create products that our customers can buy with private label rights. That means they can put their name on our products and sell them as theirs.
Home business owners.
Have a look at the current logo:
==> http://nicherat.com/images/logo.png
The text should go as is (Hello..I am NicheRat and I create AWESOME products that you WANT to SELL. Welcome to my World)
Need a cute RAT mascot (no clipart please). The logo should represent that RAT will cut short product development time by creating products ready to sell for them.


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These guys got down on one knee with the same idea
Congrats to these happy couples! He got down on one knee & she said yes. However, is it just me.. or are these methods kinda... similar. First, this guy gets off a plane & asks his lady with the help of passengers & flowers.
Oh yea, & this guy ended up doing the same thing (kinda) a Bonaroo!
That's awesome! I'm happy for these couples, but.. it seems the originality is all gone! They did basically the same thing!
Mason & Remy had some pretty cool & original ideas to pop the question. WATCH


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This content has been marked as final. Show 1 reply
1. Re: SQLDEVELOPER 4.1 and SQL file type associations in Win7Gary Graham-Oracle Mar 13, 2014 8:03 PM (in response to Gusora)
I think you mean SQL Developer 4.0.1. Anyway, there have been issues reported in this area. See the following:
sqldeveloper64W.exe has stopped working
Since I constantly switch between versions of SQL Developer, I tend to leave sql files associated with my favorite text editor. However, I did a quick test, associating the sql file extension (in 4.0.1) with either
and it seemed to work fine. By the way, rather than setting the association in one of the ways mentioned in the referenced discussion, I just right-click on the sql file, then select Open with > Choose default program.
SQL Developer Team


text:
ED Magedson – Founder
Ripoff Report
Sarkis Mambreian/Alpha Care Transport/Tony440 HINDRY AVE D INGLEWOOD, CA 90301-2031 INGLEWOOD, California United States of America
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This report was posted on Ripoff Report on 12/23/2012 12:25 PM and is a permanent record located here: http://www.ripoffreport.com/reports/sarkis-mambreianalpha-care-transporttony/inglewood-california-90301-/sarkis-mambreianalpha-care-transporttony-tony-turgeman-stealing-clients-from-competitor-986275. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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