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Colorado / Re: Introduce Yourself« on: April 17, 2009, 11:09:08 am »
I am Steve, and my wife Lin & in I live in Boulder. I have managed to spend about 3% of my life on tour, starting with a Athens to London ride 30 years ago. We have a set of local rides we like to do every spring to train for our summer tour. You can see some of these at http://tsteven4.qwestoffice.net/BoulderRides/index.html. If you would like to join us for some of these warm ups login and click the email link by my user name at the left margin.
Happy Cycling,


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Wednesday, 15 January 2014 19:06
Ozobot - Augmented Virtuality in Gaming?
The small (1" diameter) Ozobot 'robot' gaming pieces bridge the physical and virtual online world with robots that respond to what they see beneath them on the tablet screen or tabletop or both. Basically, they move themselves! An interesting new innovation that could bring tabletop social gaming back to life. Devices and Apps not available until later this year, but I will be looking forward to trying this one.
The fun will come when lots of games and Apps appear.
see video of basics here: http://www.youtube.com/watch?v=-xFjTXQ8uho
Published in Other amazing stuff

subset: commoncrawl


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Osho,Many things have happened in your world during the past fourteen months that were very controversial and looked strange for a spiritual group.Just reading the facts and figures set forth in the 156-page, two-part article in recent issues of The New Yorker could make most people angry.Though some sannyasins left, I notice that many of us, including myself, were undisturbed in our inner beings. With no regrets, many of us jumped into the new way, the new adventure.Family, friends, strangers, are either curious about this or suspect we're brainwashed dummies, or irresponsible. Is there any way to explain this inner trust that happens to a disciple with a master? Is it possible for any of us to explain anything beyond these facts and figures - the love, the fun, the mystery of spiritual transformation that we are so fortunate to participate in with you? Should I even bother, or just concentrate on you and the inner journey and ignore the curious and the skeptics?
It would have been very easy if life were only facts and figures - easy but boring; easy but flat; easy but not worth living. And the mind is concerned only with facts and figures. It is a great blessing of existence that the mind is not all, that there is much more to life, which cannot be confined to facts, explanations, theories. Something mysterious remains always unexplained, and that is the most valuable, the most significant part of life. It is impossible to explain love, trust, beauty, grace, gratitude, silence. All that is meaningful seems to be beyond the mind, and all that is meaningless seems to be the boundary of the mind.
I am reminded of a great Sufi master, Junnaid. One day one of his disciples who had managed somehow to trust in Junnaid, who had with effort remained non-skeptical, had gone hunting in the forest. And he saw Junnaid sitting by the side of a beautiful lake with a beautiful woman. He saw from far away, and from far away everything is beautiful, and particularly a Mohammedan woman. No Mohammedan woman is ugly; her face is veiled. It is a great strategy of ugly women against the beautiful; in this way the beautiful are lost.
All his repressed suspicions and doubts arose, surfaced - and it was not only that Junnaid was sitting with the woman; the woman was pouring wine from a flask into a cup for Junnaid. All his trust was shattered, all his love was finished: "There is a limit to everything. This is going too far. This man is a fraud!"
And if he had returned without going to Junnaid to say something, he would have remained with the idea that the man was a fraud. He had all the facts, he had seen with his own eyes, he was a witness. He needed no other evidence, no other proof. No argument would have convinced him that he could have been wrong.
But Junnaid shouted loudly, "Don't go back! Come close, because when you come close many facts prove to be fictions. The closer you come, the more fictitious they are. Just come close."
A little bit afraid, but he came.
Junnaid lifted the veil from the woman's face. She was Junnaid's mother, an old woman. And he said, "What about the beautiful woman you had seen? - and you had seen her with your own eyes. Could you have imagined an old woman, my own mother? It was beyond your imagination. And take this flask and look closely, taste it; it is pure water, not wine. Just the flask is of the wine. But you were thinking with absolute certainty that this man is a fraud: women and wine in privacy, in the forest; and in public he has another face, of a great master."
The disciple fell at his feet and said, "Please forgive me."


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COURSE: L5-306 Concepts of Professional Nursing (4)
The professional identity of the individual nurse and the position of the nursing profession in the health care system and society are explored in this course. Emphasis is placed on critical inquiry to evaluate practice and accountability for leadership in the pursuit of safety, quality, and improved outcomes for patients and populations. Concepts relevant to the practice of nursing in a global society, such as professionalism, professional values, the image of nursing, inter-professional communication and collaboration, resource allocation, and practice in a diverse environment are addressed. Prerequisite: RN/BSN track student, LN 111.


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Us home redecorators know that there's nothing more frustrating than pinning paint colors on the wall and having no one to help you choose the right color, or worse yet, being vetoed by your significant other who doesn't share your zest for punchy colors.
Well, be frustrated no more — My Colortopia has assembled a team of relatable, Do-It-Yourself bloggers with painting and home-decorating experience who can help you with your questions. From finding the right coordinating colors to creating a mood for your living room, learning paint techniques like wall stripes, adapting design trends, and more, the Colortopia Team has you covered (big score). Submit a question to the advice forum here. Happy decorating!


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The 1999 Camaro SS
SLP Engineering is at it again.
The new Camaro SS is on its way, and is bringing some new optional styling queues with it. We were fortunate enough to be the first enthusiasts outside of SLP to attend a promotional photo shoot, and here's what we observed….
First of all, the look that everyone loves about the Camaro SS is still standard on the SLP package. The aggressive hood with functional ram-air still remains, as well as the uplevel spoiler with LED third-eye brake lights. For 1999 SLP will offer two exciting new options that will be shipped from SLP direct to the ordering dealership. Those options are then installed by the dealer and are fully warranted by SLP. Here we got a chance to see these two exiting new options.
[imagebrowser id=5]
Coming at you, the Camaro SS with SLP's new high-gloss grill looks radically different. Instead of the standard black plastic insert, SLP's version offers aggressive open ports on the right and left sides of the grill. It also has the SS logo boldly positioned in the center, letting the world know you are driving a mighty '99 Camaro SS.
Another new optional feature of the SS is the rear of the car, which is more than likely the part of the car you will see the most. A new version of SLP's exhaust has four exhaust tips, all positioned in the center directly under the car. This gives the SS a completely different look from all other Camaros currently on the road. On both sides of the exhaust tips are rear fascia pieces that allow the exhaust tips to seamlessly integrate with the rest of the car. The fascia pieces wrap around the rear pan and head toward the rear wheels. We think that the new body pieces finish out the rear of the car a little more. There is still the distinctive and familiar rumble to the exhaust, which adds 10 horsepower to the car. Total everything up, and the new Camaro SS with SLP's dealer-direct exhaust has a total of 330 horsepower. Quite impressive for a car that you can buy right off the showroom floor with both warranty protection from GM and SLP.
There is also good news for registered 1998 Camaro SS owners. If you are one of the lucky ones to own a '98 SS, SLP's grill and rear exhaust treatment can be ordered and installed on your car at any Chevrolet dealership. Don't forget, the options descibed above are only available direct from SLP to the dealer. Production of the grill is slated to begin in September, with the exhaust following shortly. SLP's optional dual-dual exhaust is still available for those who still want a traditional look to their '98 or '99 SS.
In short the Camaro SS continues to remain awe-inspiring year after year. SLP has always done a fantastic job of making the SS powerful and attractive and continues to do so with the new 1999 Camaro SS.


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2 interesting reads on Lowry.
Line of the articles:
Williams, now the Raptors director of player development, first met Lowry when he was in high school. Williams alma mater, Villanova, was trying to recruit Lowry. Villanova wanted Williams to help with the pursuit, which was ultimately successful.
Point blank: Kyle, he was an a when I first met him, Williams says. I didnt have a great relationship with him.
He was dismissive. He was rude. But you know what? He was focused. At that age, at that time, he thought he had to be a certain way. Being from Philadelphia, not only do they have to be tough, but they have to have that persona that they are tough.
While the Houston situation proves Lowry can still get defensive, he has matured as his career has progressed. Heimerdinger says Lowry seemed far happier when he last saw him, when he was injured with the Grizzlies. Lowry is now a father to Karter, his son with Ayahna Cornish, who excelled on the court at both Cardinal Dougherty and St. Josephs University. He eschews a lot of the glamour that comes with the life of a professional basketball player. He frequently makes unpublicized trips to childrens hospitals.
Williams has been monitoring Lowrys growth for a decade now.
Now, its a beautiful relationship, Williams says. He has an older brother, but I feel like Im an older brother, too.
I like the comment about the unpublicized trips to children's hospitals. It says something about his character that it isn't a part of the NBA marketing crap.
I'm guessing they're will be a press conference tomorrow introducing Fields and LowryKyle Lowry @Klow7
Headed up north
Really when I think about it, heading into the draft I wanted the team to find a way to get TRoss, and either DLillard or KMarshall. For me, TRoss and KLowry is a huge upgrade to that 'plan' of mine so I am very happy with those two pieces of this off-season.
Lowry posted a pic of the training facility at 6 am saying he was ready to start working. Awesome.
this is going to be a good year
I don't know if this was posted anywhere.. but here it is http://www.hoopsworld.com/nba2-lowry...a-playoff-team
Twitter: @ReubenJRD NBA, Raptors writer for Daily Hive Vancouver, Toronto.
Lowry/Fields are introduced to Toronto at noon today.
Here is the stream:
Dont be surprised if the Toronto Raptors are one of the NBAs most improved teams in 2012-13, and dont be surprised if that success has a lot to do with their new starting point guard. The Raptors brain trust of Bryan Colangelo and Ed Stefanski have been looking to put their team in the Eastern Conference playoff picture with their offseason moves, and while they have made a number of nice moves, the best may be the acquisition of point guard Kyle Lowry from the Houston Rockets.
Lowry came into his own over the last two seasons in Houston after beginning his career in a platoon role with Mike Conley in Memphis. Houston starter Aaron Brooks suffered a severe ankle injury two seasons ago, opening the door for Lowry to prove himself as a starter for the Rockets. Lowry played so well that Brooks lost his starting job, and was eventually traded to Phoenix. The situation in Houston soured for Lowry when head coach Rick Adelman left for Minnesota and Kevin McHale took his place, and the rift that developed led to Lowry being traded. Things may not have ended on a great note, but Lowry is grateful for the opportunity the Rockets gave him.
Houston was a great situation while it lasted, Lowry tells HOOPSWORLD. I personally think things have changed in that organization and it is what it is. I definitely had an opportunity and was happy to have the opportunity to grow as a player, with them giving me the ball and letting me go and prove that Im an NBA starter.
In 2011-12 Lowry averaged career-highs of 14.3 points, 4.5 rebounds and 1.6 steals during the 2011-12 season. He also contributed team-highs of 6.6 assists and 32.1 minutes in 47 games (38 starts). As a starter, he upped his averages to 15.9 points, 5.3 rebounds, 7.2 assists, 1.8 steals and 35.5 minutes.
I believe Im a pretty capable point guard in this league, Lowry says of his best season as a pro. Having a career year last year definitely attracted some new teams that wanted me to be there after the year I had. But you know, Im only 26 years old and Im still getting better as a player. I see every year as a chance for me to get better and prove myself every chance I can.
Now Lowry is looking forward to proving himself as the starter on a team that expects to be among the best in the Eastern Conference next season.
Im excited to be a part of a young team, to have an opportunity to be part of the rise of a young team. I think Mr. Colangelo has done a great job with what hes implemented, players hes signed, players hes traded for. I know Coach Casey is a great coach; hes all about defense, which is good for me, and I know (assistant coach) Johnny Davis so Im comfortable with that staff.
While Lowry is just 26 years old, he will be one of the grizzled veterans on what is a very young team in Toronto. Still, he sees himself and Andrea Bargnani as the kind of veterans who can help a young team come together quickly.
I believe, with me and some of the other moves the team has made, that we are a playoff team. We have great young guys with potential, me and Andrea Bargnani are kind of the elder statesmen on that team right now. Its definitely one of those things where we can be the leaders and instill a playoff mentality with the younger guys on that team.
Of course, making the playoffs in the Eastern Conference is one thing, advancing is something else. The HEAT have improved already, Brooklyn is gearing up and the Celtics are expecting to be back in contention with Jeff Green and Jason Terry in the mix. Lowry isnt quite ready to say that his new Raptors team is ready to hang with the Easts best.
I dont know. I think we have to come together as a team, really become one cohesive unit, we can go far, but its too early to tell, admits Lowry. We definitely have the talent and the ability to match up with the best teams in the East, but we have to put it together, and that will happen during the two weeks of training camp. So I cant say for certain yet, but I do believe that with the talent and ability we have we have a chance to be very, very good.
As for Lowry himself, hes not satisfied with just having a career year. After recovering from sports hernia surgery hes back in the gym getting ready to push his game to new heights in Toronto.
Im working on everything, says Lowry, who wants to be an All-Star one day. Im sure Raptors fans arent too familiar with me, but Im working on everything. Im working on three-point shooting, mid-range, getting better at every part of the game. I want Raptors fans to see that Im one of the hardest-working players in the game.
If his work ethic and his desire to win catch on, the Toronto Raptors could be one of the most improved teams in the NBA next season, and back in the playoffs, as well.
Article by Bill Ingram on Lowry
that pass between dragics legs is breathtaking
A few other Toronto notes:
Casey, a defense-first coach, was ecstatic when he learned the team had a chance to nab Lowry in exchange for a first-round pick. Casey coached Randy Foye during his time as an assistant in Minnesota, and he remembered Foye, Lowrys college teammate at Villanova, raving about Lowrys crazy toughness.
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text: Hey Croatian - glad to see you back and in the coolest bistro around. Fee is the social one - I just pour the ale. LOL. I'm going nuts waiting for the season, but I hope you make this a regular stop on your travels. Just got up for a bit to give my cat his shot, so back to bed I go. Take care all!!


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Try This Trend: Miley Cyrus, Rashida Jones + More Tackle Bright, Bold Blues
Even celebs get the blues. And right now, bright, deep and attention-seeking blue shades — like cornflower, cobalt and electric — are everywhere. Examples? Miley Cyrus donned a bright but airy blue Pucci ensemble in NYC, while actress Rashida Jones rocked a leather skirt that appeared purplish-blue.
You can't be a shrinking violet (which, we might add, is also in the blue family) when wearing bold blue hues, but there's a variety of ways to try the blue trend.
Miley Cyrus
Cyrus is a fashion magnet — that is, we're drawn to her, even when she's wearing strange, weird or hideous ensembles. This Pucci two piece was as ladylike as we've seen from her in a while, and the color played up her baby blues. If you've got the same eye color, this hue is for you.
Rashida Jones
Leather is the fabric of the summer, and actress Jones paired a purple-blue leather mini with black and white stripes — proving the color really does go with anything.
Sandra Bullock
'The Heat' star chose a rich cobaltish hue for her pantsuit. If you are a devotee of basic black, this shade allows you remain in the dark color family and stay in your fashion comfort zone, all the while branching out a little.
Christina Milian
'The Voice' social media correspondent — yes, that's a job in 2013, millennials — went bright and tight. Her clingy, cornflower dress popped against her dark skin tone, so if you're lucky enough to have skin with that same richness and depth, this is the shade for you.
Mischa Barton
Once upon a time, 'The O.C.' alum was a fashion darling — but she fell on hard times. Still, this deep blue frock was modern and youthful on her, and proves the shade works with all cuts and personal styles.
Miranda Cosgrove
The 'iCarly' star's two-piece dress was all kinds of flirty, thanks to the color and peplum'ish waist. This gradation is prime for those of you with porcelain complexions.
Amy Heidemann
The Karmin singer's patterned body-con dress played up and accentuated her curves. You can also wear cobalt shades with black, like so, and look smashing — like so.


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Totowa New Jersey Family History
For your convenience, here are Totowa, New Jersey, family history resources currently being sold on eBay. These are live auctions. Click on any of the links for more information.
If there are no listings above, it could be a bug in the program. I recommend clicking over and searching for "Totowa New Jersey family history" or "Totowa NJ genealogy" or "Totowa Irish family history" or whatever suits your fancy. You might be surprised at what you find. The variety of local family history tools being auctioned every day on eBay is amazing.
If there's still nothing, your best bet is to contact your local Totowa public library, or Totowa used book stores.
Happy Hunting!
Chris Whitten
© 2001-2010 Interesting.com
Search for Totowa books at Amazon.
Search for New Jersey posters at AllPosters.
Search for Totowa, NJ family history resources at eBay.


text:
|N·mero de publicaci¾n
|Tipo de publicaci¾n
|N·mero de solicitud
|Fecha de publicaci¾n
|27 Dic 2011
|Fecha de presentaci¾n
|1 Oct 2010
|Fecha de prioridad
|4 Jun 2002
|Tambiķn publicado como
|N·mero de publicaci¾n
|Anand Jagota, Steven Raymond Lustig, Siqun Wang, Hong Wang
|Cesionario original
|E.I. Du Pont De Nemours And Company
|Exportar cita
|BiBTeX, EndNote, RefMan
|Citas de patentes (34), Otras citas (23), Clasificaciones (21), Eventos legales (1)
|Enlaces externos: USPTO, Cesi¾n de USPTO, Espacenet
This application is a Divisional of granted U.S. patent application Ser. No. 11/352,582 filed Feb. 13, 2006, now granted U.S. Pat. No. 7,829,504, which under 35 U.S.C. 120 claims the benefit of U.S. patent application Ser. No. 10/453,415—filed on Jun. 3, 2003—now U.S. Pat. No. 7,304,128 granted Dec. 4, 2007.
The invention relates to methods, and compositions useful for manipulation, purification and characterization of carbon nanotubes. More specifically, the invention relates to peptides that bind carbon based nanostructures, their synthesis and methods of use.
Carbon nanotubes (CNT) have been the subject of intense research since their discovery in 1991. CNT's possess unique properties such as small size and electrical conductivity, which makes them suitable in a wide range of applications, including use as structural materials in molecular electronics, nanoelectronic components, and field emission displays. Carbon nanotubes may be either multi-walled (MWNTs) or single-walled (SWNTs), and have diameters in the nanometer range. Depending on their atomic structure CNT's may have either metallic or semiconductor properties, and these properties, in combination with their small dimensions makes them particularly attractive for use in fabrication of nano-devices.
One of the drawbacks to the implementation of CNT's in nano-device fabrication processes is the difficulty in obtaining samples of CNT's that have uniform lengths, or chirality. Additionally, no facile method is available for the immobilization and manipulation of CNT's for nano-device fabrication.
Most methods of CNT synthesis produce a product that is a mixture of entangled tubes of "ropes", giving CNT's differing in diameter, chirality, and in the number of walls. Various methods such as acid washing, ultra-sonification, polymer wrapping and use of surfactants have been employed for nanotube separation (J. Liu et al. Science 280, 1253 (1998); A. G. Rinzler, Appl. Phys. 67, 29 (1998); A. C. Dillion et al. Adv. Mater. 11, 1354 (1999); (Schlittler et al. Science 292:1136 (2001)).). However, there has been no report of a method for the specific disentangling of nanotube ropes or their separation into populations having discrete sizes, chirality or conducting properties.
Because of their ability to specifically recognize substrates, various proteins represent one possible route to solving the CNT separation/purification problem as well as providing a possible means for CNT immobilization. Some attempts have been made to raise antibodies to various carbon based structures. For example, Chen et al. (WO 01/16155 A1) used conjugated fullerenes to raise monoclonal antibodies to C60 fullerene as a hapten. However, the population of antibodies raised by immunization of mice with this C60 fullerene derivative which was conjugated to bovine thyroglobulin included a sub-population that cross reacted with a C70 fullerene. No attempts have been made to date to raise antibodies to carbon based nanotubes.
Since its introduction in 1985 phage display has been widely used to discover a variety of ligands including peptides, proteins and small molecules for drug targets. (Dixit, S., J. of Sci. & Ind. Research, 57, 173-183, 1998). The applications have expanded to other areas such as studying protein folding, novel catalytic activities and DNA-binding proteins with novel specificities. Whaley et al (Nature, 405:665 (2000)) has used phage display technique to identify peptide sequences that can bind specifically to different crystallographic forms of inorganic semiconductor substrates. Although the method of generating large, diverse peptide libraries with phage display has been known for some time, it has not been applied to the problem of finding peptides that may be useful in the binding and manipulation of CNT's.
The problem to be solved, therefore, is to provide materials that have binding specificity to CNT's and other carbon based nanostructures so that they may be used in separation and immobilization of these structures for the fabrication of nano-devices. Applicants have solved the stated problem by providing a series of carbon nanotube binding peptides with high affinity and specificity for CNT's.
In one aspect the invention provides a process for generating a carbon nanostructure binding peptide comprising a composition comprising a solid substrate coated with a peptide having an amino acid sequence selected from the group consisting of SEQ ID No: 147-156.
Provided herein are also compositions comprising a solid substrate coated with a carbon nanotube binding peptide, as well as compositions comprising a solid substrate coated with a carbon nanotube binding peptide having at least one carbon nanotube bound thereto.
The following sequences conform with 37 C.F.R. 1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules") and consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. ¦1.822.
SEQ ID Nos:1-24 and 35-39 are carbon nanotube binding peptides of the invention.
SEQ ID NOs:25, and 32 are derivatized carbon nanotube binding peptides having a polyglycine tail.
SEQ ID Nos:26 and 27 are mutant carbon nanotube binding peptides having a serine substituted in place of a tryptophan at position 6.
SEQ ID NO:28 is a control peptide that have little or no binding affinity for carbon nanostructures.
SEQ ID NO:29 is a charged portion of a carbon nanotube binding peptide.
SEQ ID NO:30 is a polar portion of a carbon nanotube binding peptide.
SEQ ID NO:31 is a hydrophobic portion of a carbon nanotube binding peptide.
SEQ ID NO:33 is a primer used for sequencing M13 phage.
SEQ ID NO:34 is a non-CNT binding peptide.
SEQ ID NOs:40-85 are peptides raised against and binding to single walled nanotubes.
SEQ ID NOs:86-147 and 177 are peptides raised against and binding to multiwalled carbon nanotubes.
SEQ ID NOs:148-176 are peptides raised against and binding to granphite cleaned carbon nanotubes.
The present invention provides various carbon nanotube binding peptides generated by the process of peptide phage display. The peptides are useful for the manipulation of carbon based nanostructures in the fabrication of nano-devices as well as in the separation and purification of nanotubes from mixed CNT populations.
The peptides of the invention are particularly useful as ligands for the assembly of carbon nanotubes and related molecules into conducting nano devices for use in electronic applications such as field-emission transistors, artificial actuators, molecular-filtration membranes, energy-absorbing materials, molecular transistors, and other optoelectronic devices as well as in gas storage, single-electron devices, and chemical and biological sensors.
In this disclosure, a number of terms and abbreviations are used. The following definitions are provided.
"CNBP" means Carbon nanotube binding peptide
"HRTEM" means high-resolution transmission electron microscopy
"MWNT" means Multi-walled nanotube
"SWNT" means Single walled nanotube
"PEG" means polyethylene glycol
"pfu" means plaque forming units
"TEM" means transmission electron microscopy
"CNT" means carbon nanotube
The term "peptide" refers to two or more amino acids joined to each other by peptide bonds or modified peptide bonds. Peptides include those modified either by natural processes, such as processing and other post-translational modifications, but also chemical modification techniques. The modifications can occur anywhere in a peptide, including the peptide backbone, the amino acid side chain, and the amino or carboxyl terminal. Examples of modifications include but are not limited to amidation, acylation, acetylation, cross linking, cyclization, glycosylation, hydroxylation, phosphorylation, racemization, and covalent attachment of various moieties such as nucleotide or nucleotide derivative, lipid or lipid derivatives (see, for instance, Proteins-Structure and Molecular Properties, 2nd Ed Creighton, W. H. Freeman and Company, New York (1993) and Post-translation covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York (1983)).
As used herein, the term "peptide" and "polypeptide" will be used interchangeably.
The term "nanotube" refers to a hollow article having a narrow dimension (diameter) of about 1-200 nm and a long dimension (length), where the ratio of the long dimension to the narrow dimension, i.e., the aspect ratio, is at least 5. In general, the aspect ratio is between 10 and 2000.
By "carbon-based nanotubes" or "carbon nanotube" herein is meant hollow structures composed primarily of carbon atoms. The carbon nanotube can be doped with other elements, e.g., metals.
The term "carbon nanotube product" refers to cylindrical structures made of rolled-up graphene sheet, either single-wall carbon nanotubes or multi-wall carbon nanotubes.
The term "carbon nanotube rope" means a population of non-aligned nanotubes.
The term "carbon nanostructure binding peptide" refers to peptides that were selected to bind with a carbon nanostructures. Where peptides are generated with specific affinity to carbon nanotubes, these peptides will be referred to as carbon nanotube binding peptides or CNBP's.
The term "stringency" as it is applied to the selection of CNBP's means the concentration of eluting agent (usually detergent) used to elute peptides from CNT's.
The term "peptide-nanotube complex" means structure comprising a peptide bound to a nanotube via a binding site on the peptide.
The term "nano-structure" means tubes, rods, cylinders, bundles, wafers, disks, sheets, plates, planes, cones, slivers, granules, ellipsoids, wedges, polymeric fibers, natural fibers, and other such objects which have at least one characteristic dimension less than about 100 nm.
The term "solid substrate" means a material to which a carbon nanotube or binding peptide may be affixed either by direct chemical means or via an intermediate material such as a coating.
The term "identity" refers to a relationship between two or more polynucleotide sequences or two or more polypeptide sequences, as determined by comparing the sequences. "Identity" and "similarity" can be readily calculated by known methods including but not limited to those described in (Sequence Analysis in Molecular Biology, Von Heinje, G., Academic Press, 1987, Sequence analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, and Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds, Humana Press, New Jersey, 1994.
The term "amino acid" will refer to the basic chemical structural unit of a protein or polypeptide. The following abbreviations will be used herein to identify specific amino acids:
Amino Acid
Aspartic acid
Asparagine or aspartic acid
Glutamine acid
Glutamine or glutamic acid
The term "variant(s)" refers to a polynucleotide, or polypeptide, that differs from a reference polynucleotide, or polypeptide, respectively, but retains essential properties. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, deletions, additions, fusions, and truncations in the polypeptide encoded by the reference sequence. A typical variant of a polypeptide may differ in amino acid sequence from another reference polypeptide by one or more substitutions, additions, deletions in any combinations. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as allelic variant, or may not be known as naturally occurring variant. Non-naturally occurring variants of polynucleotides and polypeptides may be made by direct synthesis, mutagenesis techniques, or by other recombinant methods known in the art.
The term "chemically equivalent amino acid" will refer to an amino acid that may be substituted for another in a given protein without altering the chemical or functional nature of that protein. For example, it is well known in the art that alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded protein are common. For the purposes of the present invention substitutions are defined as exchanges within one of the following five groups:
"Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A "foreign" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.
"Synthetic genes" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments which are then enzymatically assembled to construct the entire gene. "Chemically synthesized", as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.
"Coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "Suitable regulatory sequences" refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.
"Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.
The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.
The term "host cell" refers to cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.
The terms "plasmid", "vector" and "cassette" refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. "Transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell. "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
The term "phage" or "bacteriophage" refers to a virus that infects bacteria. Altered forms may be used for the purpose of the present invention. The preferred bacteriophages are derived from two "wild" phages, called M13 and lambda. Lambda phages are used to clone segments of DNA in the range of around 10-20 kb. They are lytic phages, i.e., they replicate by lysing their host cell and releasing more phages. The M13 system can grow inside a bacterium, so that it does not destroy the cell it infects but causes it to make new phages continuously. It is a single-stranded DNA phage.
The term "phage display" refers to the display of functional foreign peptides or small proteins on the surface of bacteriophage or phagemid particles. Genetically engineered phage could be used to present peptides as segments of their native surface proteins. Peptide libraries may be produced by populations of phage with different gene sequences.
Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter "Maniatis"); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).
The present invention provides peptides that bind carbon nanostructures as well as methods for generating the same and uses thereof.
Carbon Nanostructures
The invention relates to the generation of peptides with binding affinities for carbon nanostructures, and particularly nanotubes. Carbon nano-structures of the present invention are those structures comprised at primarily of carbon which take the form of tubes, rods, cylinders, bundles, wafers, disks, sheets, plates, planes, cones, slivers, granules, ellipsoids, wedges, polymeric fibers, natural fibers, and other such objects which have at least one characteristic dimension less than about 100 nm. Preferred carbon nanostructures of the invention are nanotubes.
Nanotubes of the invention are generally about 1-200 nm in diameter where the ratio of the length dimension to the narrow dimension, i.e., the aspect ratio, is at least 5. In general, the aspect ratio is between 10 and 2000. Carbon nanotubes are comprised primarily of carbon atoms, however may be doped with other elements, e.g., metals. The carbon-based nanotubes of the invention can be either multi-walled nanotubes (MWNTs) or single-walled nanotubes (SWNTs). A MWNT, for example, includes several concentric nanotubes each having a different diameter. Thus, the smallest diameter tube is encapsulated by a larger diameter tube, which in turn, is encapsulated by another larger diameter nanotube. A SWNT, on the other hand, includes only one nanotube.
Carbon nanotubes (CNT) may be produced by a variety of methods, and are additionally commercially available. Methods of CNT synthesis include laser vaporization of graphite (A. Thess et al. Science 273, 483 (1996)), arc discharge (C. Journet et al., Nature 388, 756 (1997)) and HiPCo (high pressure carbon monoxide) process (P. Nikolaev et al. Chem. Phys. Lett. 313, 91-97 (1999)). Chemical vapor deposition (CVD) can also be used in producing carbon nanotubes (J. Kong et al. Chem. Phys. Lett. 292, 567-574 (1998); J. Kong et al. Nature 395, 878-879 (1998); A. Cassell et al. J. Phys. Chem. 103, 6484-6492 (1999); H. Dai et al. J. Phys. Chem. 103, 11246-11255 (1999)).
Additionally CNT's may be grown via catalytic processes both in solution and on solid substrates (Yan Li, et al., Chem. Mater.; 2001; 13(3); 1008-1014); (N. Franklin and H. Dai Adv. Mater. 12, 890 (2000); A. Cassell et al. J. Am. Chem. Soc. 121, 7975-7976 (1999)).
Peptide Generation
Peptides of the invention are generated randomly and then selected against a population of carbon nanostructures for binding affinity to CNT's. The generation of random libraries of libraries of peptides is well known and may be accomplished by a variety of techniques including, bacterial display (Kemp, D. J.; Proc. Natl. Acad. Sci. USA 78(7): 4520-4524, 1981, and Helfman, D. M., et al., Proc. Natl. Acad. Sci. USA 80(1): 31-35, 1983) yeast display (Chien C T, et al., Proc Natl Acad Sci USA 1991 Nov. 1; 88(21): 9578-82) combinatorial solid phase peptide synthesis (U.S. Pat. No. 5,449,754, U.S. Pat. No. 5,480,971, U.S. Pat. No. 5,585,275, U.S. Pat. No. 5,639,603) and phage display technology (U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,571,698; U.S. Pat. No. 5,837,500). Techniques to generate such biological peptide libraries are described in Dani, M., J. of Receptor & Signal Transduction Res., 21(4), 447-468 (2001).
A preferred method to randomly generate peptides is by phage display. Phage display is an in vitro selection technique in which a peptide or protein is genetically fused to a coat protein of a bacteriophage, resulting in display of fused protein on the exterior of phage viron, while the DNA encoding the fusion residues within the virion. This physical linkage between the displayed protein and the DNA encoding it allows screening of vast numbers of variants of proteins, each linked to a corresponding DNA sequence, by a simple in vitro selection procedure called "biopanning." In its simplest form, biopanning is carried out by incubating the pool of phage-displayed variants with a target of interest that has been immobilized on a plate or bead, washing away unbound phage, and eluting specifically bound phage by disrupting the binding interactions between the phage and target. The eluted phage is then amplified in vivo and the process repeated, resulting in stepwise enrichment of the phage pool in favor of the tightest binding sequences. After 3 or more rounds of selection/amplification, individual clones are characterized by DNA sequencing.
Thus it is an object of the invention to provide a process for generating a carbon nanostructure binding peptide comprising:
After a suitable library of peptides has been generated they are then contacted with an appropriate population of carbon nanostructures or nanotubes. The nanotubes are presented to the library of peptides typically while suspended in solution, although it will be appreciated that CNT or peptides could also be immobilized on a solid substrate to facilitate binding. In such an embodiment suitable solid substrates will include but are not limited to silicon wafers, synthetic polymer substrates, such as polystyrene, polypropylene, polyglycidylmethacrylate, substituted polystyrene (e.g., aminated or carboxylated polystyrene; polyacrylamides; polyamides; polyvinylchlorides, etc.); glass, agarose, nitrocellulose, and nylon.
A preferred solution is a buffered aqueous saline solution containing a surfactant. A suitable solution is Tris-buffered saline with 0.1% Tween 20. The solution can additionally be agitated by any means in order to increase binding of the peptides to the nanotubes.
Upon contact a number of the randomly generated peptides will bind to the CNT's to form a peptide-nanotube complex. Unbound peptide and CNT may be removed by washing (if immobilized) or by any other means such as centrifugation, or filtering, etc. After all unbound material is removed, peptides, having varying degrees of binding affinities for CNT's may be fractionated by selected washings in buffers having varying strengths of surfactants. The higher the concentration of surfactant in the wash buffer, the higher the stringency of selection. Increasing the stringency used will increase the required strength of the bond between the peptide and nanotube in the peptide-nanotube complex.
A number of materials may be used to vary the stringency of the buffer solution in peptide selection including but not limited to acidic pH 1.5-3; basic pH 10-12.5; high salt concentrations such as MgCl2 3-5 M, LiCl 5-10 M; water; ethylene glycol 25-50%; dioxane 5-20%; thiocyanate 1-5 M; guanidine 2-5 M; urea 2-8 M; various concentrations of different surfactants such as SDS (sodium dodecyl sulfate), DOC (sodium deoxycholate), Nonidet P-40, Triton X-100, Tween 20« wherein Tween 20« is preferred. The materials can be prepared in buffer solutions including but not limited to Tris-HCl, Tris-borate, Tris-acidic acid, triethylamine, phosphate buffer, glycine-HCl wherein 0.25M glycine-HCl solution is preferred.
It will be appreciated that peptides having greater and greater binding affinities for the CNT substrate may be eluted by repeating the selection process using buffers with increasing stringencies.
The eluted peptides can be identified, sequenced, and produced by any means known in the art.
Carbon Nanotube Binding Peptides
Peptides of the invention selected by the above process have been identified. A large number of peptides having particularly high binding affinities to carbon nanotubes were isolated having the amino acid sequences as set forth in SEQ ID NOs:1-24 and 35-177
It will be appreciated by the skilled artisan that the invention is not limited to these specific sequences but will include amino acid sequences comprising chemically equivalent amino acid substitutions that do not interfere with the ability of the peptide to bind CNT's. So for example, the chemically equivalent substitutions for each of the amino acids in SEQ ID NO:14 are detailed in the following table:
Alignment and analysis of the selected peptides of the invention suggests that the carbon nanostructure or nanotube binding properties are related to the secondary characteristics of the peptide. For example a simple pendant model was developed for the peptides of the instant invention, which accounts for hydrophilicity or hydrophobicity. It demonstrates that all of the consensus sequences are essentially symmetric surfactants-hydrophilic on the ends and hydrophobic in the middle. The model describes the degree of hydrophilicity or hydrophobicity of an amino acid pendant group by classifying all pendant groups as either hydrophilic (h=−1) or hydrophobic (h=1). Side chains which are either basic, acidic or uncharged polar are be hydrophilic while side chains that are nonpolar are hydrophobic. Several of the peptides selected by the methods of the invention are modeled below:
These peptides were selected over a broad range of detergent concentrations (0.6%-3%) and yet show the same pattern of hydrophilicity and hydrophobicity. With a few exceptions, the h=−1 are predominantly on the ends and h=1 are concentrated in the middle.
It is thus an object of the invention to provide a carbon nanotube binding peptides having the general structure:
N is the N-terminal portion of the peptide having about 4 amino acids, 75% of which are hydrophilic;
M is the median portion of the peptide having about 4 amino acids, 75% of which are hydrophobic; and
C is the C-terminal portion of the peptide having about 4 amino acids 75% of which are hydrophilic.
Peptide Production by Recombinant Methods
Once a peptide having suitable binding properties is identified it may be produced recombinantly in large quantities. Genes encoding nanotube binding peptides may be produced in heterologous host cells, particularly in the cells of microbial hosts.
Preferred heterologous host cells for expression of nanotube binding peptides are microbial hosts that can be found broadly within the fungal or bacterial families and which grow over a wide range of temperature, pH values, and solvent tolerances. Because of transcription, translation and the protein biosynthetic apparatus is the same irrespective of the cellular feedstock, functional genes are expressed irrespective of carbon feedstock used to generate cellular biomass. Examples of host strains include but are not limited to fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterial species such as Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes, Synechocystis, Anabaena, Thiobacillus, Methanobacterium and Klebsiella.
A variety of expression systems can be used to produce the peptides of the present invention. Such vectors include but are not limited to chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from insertion elements, from yeast episoms, from viruses such as baculaviruses, retroviruses and vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain regulatory regions that regulate as well as engender expression. In general, any system or vector suitable to maintain, propagate or express polynucleotide or polypeptide in a host cell may be used for expression in this regard. Microbial expression systems and expression vectors contain regulatory sequences that direct high level expression of foreign proteins relative to the growth of the host cell. Regulatory sequences are well known to those skilled in the art and examples include but are not limited to those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory elements may also be present in the vector, for example, enhancer sequences. Any of these could be used to construct chimeric genes for production of the any of the nanotube binding peptides. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high level expression of the peptides.
Vectors or cassettes useful for the transformation of suitable host cells are well known in the art. Typically the vector or cassette contains sequences directing transcription and translation of the relevant gene, one or more selectable markers, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene which harbors transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host. Selectable marker genes provide a phenotypic trait for selection of the transformed host cells such as tetracyclin or ampicillin resistance in E. coli.
The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator or control element, so that Initiation control regions or promoters, which are useful to drive expression of the instant ORF's in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, ara, tet, trp, IPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli) as well as the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus.
Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is most preferred if included.
The vector containing the appropriate DNA sequence as here in above described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the peptide of the present invention. Cell-free translation systems can also be employed to produce such peptides using RNAs derived from the DNA constructs of the present invention.
Optionally it may be desired to produce the instant gene product as a secretion product of the transformed host. Secretion of desired proteins into the growth media has the advantages of simplified and less costly purification procedures. It is well known in the art that secretion signal sequences are often useful in facilitating the active transport of expressible proteins across cell membranes. The creation of a transformed host capable of secretion may be accomplished by the incorporation of a DNA sequence that codes for a secretion signal which is functional in the host production host. Methods for choosing appropriate signal sequences are well known in the art (see for example EP 546049; WO 9324631). The secretion signal DNA or facilitator may be located between the expression-controlling DNA and the instant gene or gene fragment, and in the same reading frame with the latter.
Nano-Device Fabrication
The carbon nanotube binding peptides (CNBP) of the instant invention could be one element in an entity with bi-, tri- (or higher) binding functionality. A CNBP can be depicted graphically as shown below,
where the "C" is suggestive of a carbon nanotube and a binding functionality depicted by "⊂". However, despite its being drawn at one end, it should be interpreted as a collective, not localized, property of the peptide sequence. The overall entity would be constructed by "fusion" of the CNBP with another body, depicted by "B", with a binding functionality depicted by ">", and the combination is represented graphically below
This is meant to represent a minimal example; the fusion could create higher order (enumerative) functionality. Examples of B include but are not limited to a DNA binding protein, a metallic electrode, for example Au bound directly to an amino-acid residue like cysteine, or a hard (e.g., Si or SiO2) substrate for immobilization of the CNBP.
Directed self-assembly of carbon nanotubes into useful structures could be achieved by combining the binding of CNBP with a pre-patterned substrate. For example, if the binding functionality "B" was a series of cysteine residues, the sequence of (a) preparation of a dilute suspension of carbon nanotubes, (b) functionalization of selected types by CNBP, and (c) washing over a substrate with patterned Au electrodes would result in the attachment of carbon nanotubes to metal electrodes via the peptide, within distances of relevance to nano-electronic devices. Because of the diversity of the bio-chemical toolkit in combining elements to obtain higher order functionality, many other such methods can be conceived, once the fundamental binding motifs have been identified.
A major obstacle to the use of carbon nanotubes in a variety of applications is the fact that all manufacturing processes produce a mixture of entangled tubes. Individual tubes in the product differ in diameter, chirality, and number of walls. Moreover, long tubes show a strong tendency to aggregate into "ropes". These ropes are formed due to the large surface areas of nanotubes and can contain tens to hundreds of nanotubes in one rope. Furthermore, the structure of individual tubes varies widely from armchair, zig-zag or other chiral forms which coexist in the material and their electrical properties also vary dramatically accordingly (metallic or semi-conductive). Therefore, a need exists for the isolation of a single form (such as armchair, zig-zag or a chiral form) of carbon nanotubes.
Existing methods for separating such product, for example acid washing, ultra-sonification, and use of surfactants, is non-specific with respect to the type of nanotube. Because peptide binding is usually highly specific, a major utility of a CNBP is to effect specific separation. One possible method would use dilute suspensions of carbon nanotubes separated by having them flow over substrates patterned with different types of binding CNBP's. One would choose to order the patterning based on strength and specificity of binding, i.e., strongly selective binding peptides would be positioned to act on the mixture in advance of less specific ones. Many other ways to achieve separation can be conceived. If "B" binds to a magnetic particle, the joint entity could be used in one stage of a continuous flow to bind to carbon nanotubes, while in another stage the bound nanotubes could be separated magnetically.
Thus it is an object of the invention to provide a method of dispersing a population of carbon nanotube ropes comprising:
N is the N-terminal portion of the peptide having about 4 amino acids, 75% of which are hydrophilic;
M is the median portion of the peptide having about 4 amino acids, 75% of which are hydrophobic; and
C is the C-terminal portion of the peptide having about 4 amino acids 75% of which are hydrophilic;
whereby the carbon nanotube ropes are dispersed.
One of skill in the art will appreciate that it will be useful to sort populations of nanotubes to select for various binding properties. It is contemplated that CNT-binding peptides or isolated phage expressing a CNT binding peptide, may be used for this purpose. For example, CNT-binding peptides or phages expressing the same, having an affinity to a specific population of CNT's may be immobilized on a solid substrate and then contacted with a mixed population of CNT's. The desired CNT's will bind to the immobilized peptides or phage and the undesired CNT's may be washed free. Alternatively, solid substrates such as beads or microshperes may be coated with CNT-binding peptides or phage and used to assemble CNT's. Materials suitable as solid supports may be made of synthetic polymers such as polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), or other materials such as glass, ceramics, metals, and the like. These materials be used as films, microtiter plates, wells, beads, slides, particles, pins, pegs, or membranes.
Accordingly the invention a method for assembling carbon nanotubes comprising contacting a solid substrate coated with at least one species of carbon nanotube binding peptide with a population of carbon nanotubes whereby the carbon nanotubes bind to the coated substrate and are assembled.
It will be appreciated by the skilled artisan that patterning of CNT-binding peptides on a particular solid support will be a useful technique in the design and fabrication of nanodevices. In some instances patterning may be achieved by partially and selectively masking portions of the support with materials that repel or have no affinity for CNT's. A variety of materials may be used for this purpose, however non-CNT binding peptides of similar physical characteristics to CNT-binding peptides will be particularly suitable. Non-CNT binding peptides are easily selected and identified in the early rounds of any selection process for CNT-binding peptides. These may be used to mask a solid support to effect the pattering of CNT binding on the support. Several examples provided herein include the peptides set forth in SEQ ID NO:28 and 34.
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
General Methods
Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).
Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified.
Preparation of Phage Library
The phage library used in the present invention was purchased from New England BioLabs (catalog number E8110S, Ph.D.-12 Phage Display Peptide Library Kit). The kit is based on a combinatorial library of random peptide 12-mers fused to a minor coat protein (pill) of M13 phage. The displayed peptide 12 mer is expressed at the N-terminus of pill, i.e. after the signal peptide is cleaved the first residue of the coat protein is the first residue of the displayed peptide. The library contains 2.7ū109 (100 μl) variants in the displayed epitope. A volume of 10 μl contains about 55 copies of each peptide sequence. To avoid introduce bias into the library, each initial round of experiments were carried out using the original library provided by the manufacture.
Sequencing of Phages
Random M13 phage plaques were picked and single plaque lysates were prepared following manufacture's instruction (New England Labs, Beverly, Mass.). The single stranded phage genome DNA was purified with Qiagene kit (QIAprep Spin M13 kit, Cat. No. 27704). The single stranded DNA were sequenced with −96 gill sequencing primer (5′-CCCTCATAGTTAGCGTAACG-3′). SEQ ID NO:33 The displayed peptide is located immediately after the signal peptide of gene III.
Multi-Sequence Analysis
Sequences from the phage display experiment were analyzed using software DNA Star (version 5.02) or ClustalW according to software instructions. The alignment provides similarities among the selected sequences and predict important functions of certain amino acid residues.
Carbon nanotubes designated CNT-7 were obtained from Yet-Ming Chiang, Department of Materials Science, MIT, Cambridge, Mass. The nanotubes were prepared by heating SiC (silicon carbide) at 1700░ C. under vacuum. Silicon "evaporated" from the sample and left behind the carbon, which formed folded structures of carbon including nanotubes.
Single-walled carbon nanotubes were purchased from CNI (Carbon Nanotechnology Incorporated, Houston, Tex.). These nanotubes were produced by a laser oven technique or a HiPCO (high-pressure carbon monoxide) process (P. Nikolaev et al. Chem. Phys. Lett. 313, 91-97 (1999)).
The CNT-7 sample contained various carbon structures including multi-wall and single-wall carbon nanotubes whereas CNI samples were mostly single wall carbon nanotubes.
CNT-7 and CNI carbon nanotubes were suspended in Tris-Buffered-Saline with 0.1% Tween 20 (TBS-T) at a concentration of 1 mg/ml. The carbon nanotube solution was then sonicated by a Branson Sonifier model 450 (Branson Sonic Power Co., Danbury, Conn.) with power output setting between 4 and 5, duty cycle 70-80% for three times. Ten microliters of M13 phage library (containing about 1011 phage) were added to 1 ml of carbon nanotubes. The mixture was incubated at room temperature with mild agitation for 60 minutes. Unbound phages were separated from the nanotube sample by high speed spin at 14,000 rpm (16,110ūg) in an Eppendorf 5415C centrifuge (Brinkmann Instruments Inc., Westbury, N.Y.) for 10 minutes. Subsequently the phage/nanotube complex was washed 10 times each with 1 ml TBS-T in which concentration of Tween-20 increases according to the cycles of selection, as shown in the data below. For example, in one experiment the Tween 20 concentration was increased from 0.2% in round one, to 0.3% in round two, to 0.4% in round 4, to 0.5% in round 5, to 0.6% in round 6, to 0.7% in round 7, to 1% in round 8, to 2% in round 9, 3% in round 10, 6% in round 11 and 10% in round 12. After the last (tenth) washing step, the bound phages were eluted off by incubating with 0.5 ml of 0.25 M glycine-HCl, pH 3.0 for 10-15 minutes at room temperature. The phages and nanotubes were separated by centrifuging at 14,000 rpm (16,110ūg) for 10 minutes, with the cleared supernatant containing the eluted phages. The presence and concentration of phages in the supernatant were determined by phage titering. Once the sample confirmed the presence of phages, they were used to inoculate E. coli for phage amplification, and the amplified phage sample was used as the "pool" for next round. In a typical experiment, the entire eluent was added to a 20 ml E. coli culture at early log phase. The culture was further incubated for 4.5 hours at 37░ C. to allow phage to propagate. At the end of the incubation, the cultures were spin at 16,000ūg for 10 minutes at 4░ C. The phages in the cleared supernatant were precipitated with PEG/NaCl at 4░ C. After centrifugation, the phages were resuspended in 200 μl PBS and the concentration was determined by titering. This sample is used subsequently as the stock for the next round experiment. To carry out the next round experiment, 1011 phages were used as input "pool" and the selection process was repeated as described above with increased stringency for washing, i.e. increased concentration of Tween-20. Useful peptides were obtained by selection at detergent concentrations of 0.5% and higher and the amino acid sequences of these peptides are shown in Table 1.
M13 peptide sequences for CNT-7
Binding of these peptides to CNT's is further confirmed by TEM micrographs as shown in
The following example illustrates the importance of conserved amino acids to the binding affinity of peptides for carbon nanotubes.
Alignment of the selected peptide sequences suggests strongly that histidine at position 1 and tryptophan at position 6 are important for binding. Further analysis of more than one hundred phage clones, shown below in Table 3, revealed that His and Trp are two dominant amino acids in the composition of peptides selected by the display.
TABLE 3 Original Change Number % by % by library relative to Amino Acids count weight frequency (%) original (%) Charged: 391 36.03 33.59 RKHYCDE (SEQ ID NO: 29) Acidic: 37 2.84 3.18 DE Basic: 89 8.51 7.65 KR Polar: 305 22.51 26.20 NCQSTY (SEQ ID NO: 30) Hydrophobic: 376 37.62 32.3 AILFW (SEQ ID NO: 31) A 57 2.69 4.90 6.0 — C 6 0.41 0.52 0.5 0.52 D 35 2.67 3.01 2.8 3.01 E 2 0.17 0.17 3.1 0.17 F 5 0.49 0.43 3.3 0.43 G 30 1.13 2.58 2.6 2.58 H 217 19.73 18.64 6.3 — I 27 2.03 2.32 3.4 2.32 K 38 3.23 3.26 2.8 3.26 L 55 4.13 4.73 9.3 4.73 M 25 2.17 2.15 2.6 2.15 N 66 4.99 5.67 4.6 5.67 P 85 5.47 7.30 12.2 7.30 Q 37 3.14 3.18 5.1 3.18 R 51 5.28 4.38 4.7 4.38 S 97 5.60 8.33 10.0 8.33 T 57 3.82 4.90 11.1 4.9 V 6 0.39 0.52 3.9 0.52 W 226 27.90 19.42 2.2 — Y 42 4.54 3.61 3.6 3.61 B 0 0 0 Z 0 0 0 X 0 0 0 Ter 0 0 0 1. Data is from analysis of 100 clones
2. Original library data is adapted from manufacturer's manual and is from analysis of 104 clones
Site-directed mutagenesis was used to introduce mutations in peptides SEQ ID NO:13 (HWKHPWGAWDTL) and SEQ ID NO:14 (HWSAWWIRSNQS) Trp→Ser at position 6, to produce peptides HWKHPSGAWDTL (SEQ ID NO:26) and HWSAWSIRSNQS (SEQ ID NO:27), respectively. The phages carrying these mutations were assayed for their binding activity against CNT-7 at detergent concentration 0.4% as described above. The mutation at Trp6 reduced binding to CNT-7 for both peptides. The data is shown in Table 4 below. The number of plaque forming units is charted for a control peptide (LPPSNASVADYS) SEQ ID NO:28 and peptides SEQ ID NO:13, 14 and mutant peptides SEQ ID NO: 26 and 27. The binding data shown in Table 4 confirms the critical role of Trp in binding to nanotubes.
4.58 ū 106
5.4 ū 106
6.7 ū 106
15.9 ū 106
59.2 ū 106
The following example illustrates the ability of carbon nanotube binding peptides to disentangle carbon nanotube "ropes".
Experiments were carried out with synthetic peptides and single-wall carbon nanotubes (CNI/Laser oven) and binding peptides sequence HWKHPWGAWDTLGGG [SEQ ID NO: 25]. This peptide was selected as described in Example 2 and represents the peptide as set forth in SEQ ID NO:13, with the addition of a poly-glycine tail.
At a concentration of 4 mg/ml peptide of SEQ ID NO:25 was seen to disperse the nanotube ropes as examined by HRTEM whereas the mutant peptide SEQ ID NO:26, containing a polyglycine tail HWKHPSGAWDTLGGG [SEQ ID NO:32] and a control peptide SEQ ID NO:28 did not disperse the nanotube at the same concentration. The results are shown in
In order to find peptides with specific binding to carbon nanotubes, phage display experiments were performed as described in Example 2 on CNT-7 carbon nanotube substrates using a "graphite-cleaned" phage library. The graphite-cleaned phage library was generated by first washing the complete phage library on a pyrolytic graphite substrate. The washed or cleaned library was thus denuded of phage that would bind to Graphite. Highly ordered pyrolytic Graphite (HOPG SPI-2, SPI Supplies, West Chester, Pa.) was attached to a petri dish and a fresh layer of graphite was exposed using a Scotch tape. About 1011 pfu M13 phage in TBS-0.1% Tween-20« was added to the graphite substrate and allowed to sit for binding for 45-60 minutes at room temperature. Unbound phages were washed away with excess amount of (TBS-T) at defined concentrations of Tween-20«. Bound phages were eluted with Glycine-HCl buffer at pH 2.3. The unbound phage (graphite-cleaned library) were then used to perform phage display experiments on CNT-7 as described in Example 2. Individual phages were isolated and DNA sequences were obtained using standard molecular biology methods described above.
After four rounds of phage display on CNT-7 with the graphite-cleaned library (round 4 with 0.5% concentration of Tween-20«), two consensus sequences emerged. These are:
The implication is that the binding of these sequences is specific to the CNT-7, as compared to a graphitic clone. The significance of the finding is in the close relationship between the graphene sheet that bounds freshly cleaved graphite, and the surface of carbon nanotubes. Carbon nanotube surfaces are essentially curved or graphene sheets. As such, objects may bind both to carbon nanotubes and to graphite. Additional significance may be attached to those whose binding discriminates between the two. This result illustrates that peptides can recognize different allotropes of carbon.
Example 6 illustrates that microspheres coated with CNT binding peptides are effective in binding single walled nanotubes and forcing assembly of the microspheres.
Preparation of Phage-Coated Microspheres.
Purified phage clones were amplified. Anti-mouse antibody IgG-coated microspheres (seven microns in diameter from Bangs Laboratories, Inc, 9025 Technology Drive, Fishers Ind. 46038-2886) were coated with an anti-M13 monoclonal antibody (Amersham pharmacia biotech Inc. 800 Centennial Avenue PO Box 1327 Piscataway N.J. 08855). Purified phage clones were coated onto these microspheres in TBS buffer. The phage-coated microspheres were incubated overnight with 10% Triton-X-165 dispersed SWNTs 7.5 μg/ml in a dialysis tube against 1 L of TBS buffer containing 10 grams of Amberlite XAD-4 (Sigma, P.O. Box 14508 ST. Louis, Mo. 63178). The microspheres were then washed three times with water. The beads were examined under SEM.
Preparation of Peptide-Coated Microspheres.
Selected sequences were synthesized as free peptides, including, NH2-HWKHPWGAWDTLGGG-COOH (SEQ ID NO:25) and NH2-HWKHPWGAWDTL-COOH (SEQ ID NO:13). Amino-modified microspheres (0.66 microns in diameter from Bangs Laboratories, Inc, 9025 Technology Drive, Fishers Ind. 46038-2886) were cross-linked to synthetic peptides at the C terminus through an EDC linker (Pierce Inc. 3747N. Meridian Road, P.O. Box 117, Rockford, Ill. 61105 Sigma, P.O. Box 14508 ST. Louis, Mo. 63178). The peptide-coated microspheres were incubated overnight with 10% Triton-X-165 dispersed SWNT 7.5 μg/ml in a dialysis tube against 1 L of TBS buffer containing 10 grams of Amberlite XAD-4. The microspheres were then washed three times with TBS buffer. The beads were examined under SEM.
Results of contacting CNT's with microspheres either coated with CNT binding phage or isolated peptide are illustrated in
This example illustrates that carbon nanotube binding peptides may be generated to a variety of carbon nanotube substrates including those made by the HiPCo process and those that have undergone various cleaning processes.
A series of experiments to select carbon nanotube binding peptides were performed as described in Example 2. The first substrate used was SWNTs from CNI prepared using the HiPCo process that was prepared only by acid cleaning, dispersion in toluene and drying to form a mat. Peptides resulting after the selection process are listed in SEQ. ID Nos:39-85.
The second substrate used in the selection process as described in Example 2 were MWNTs obtained from Yet-Ming Chiang, Department of Materials Science, MIT, Cambridge, Mass. Peptides that were selected are listed in SEQ. ID Nos:1-4,6,8,10-13, 15, 16, 18-22, 28, 36, 92, 114-147, and 177. The above example was repeated used fresh MWNTs obtained from the same source. Resulting peptides are listed in SEQ. ID Nos:94-113 and 177.
Graphite-cleaned MWNTs obtained from Yet-Ming Chiang, Department of Materials Science, MIT, Cambridge, Mass. prepared as described in Example 5 and were also used as substrates. Resulting peptides are listed in SEQ. ID Nos:148-176.
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|Clasificaci¾n de EE.UU.
|530/327, 977/705, 506/14
|Clasificaci¾n internacional
|Clasificaci¾n cooperativa
|Clasificaci¾n europea


text:
COMMERCE CITY, Colo. -- The LA Galaxy take the field this evening at Dick's Sporting Goods Park when they take on the Colorado Rapids.
*Saturday's match will be an emotional one for Pablo Mastroeni who will take on his former club, the Colorado Rapids. Mastroeni, who admits that he's discovered a new "spark" with LA, will be facing one of his former teams for the first time in his 16 year MLS career.
*Carlo Cudicini is quietly entering his best form this season, but can he build on it against Colorado.
*The question of who fills in for center back Omar Gonzalez has been a major one around the Galaxy camp this week, but it appears that Tommy Meyer will get the nod.
*With Robbie Rogers likely out for Saturday, it seems that Marcelo Sarvas will step into his spot on the wide yet again.
*Gyasi Zardes scored a highlight reel goal in the win against Vancouver, but he'll be asked to put in another solid performance against Colorado
*LAGalaxy.com's Larry Morgan offers his preview to the match.
*Lastly, my Three Points to the game
DILLON POWERS vs. PABLO MASTROENI
The young gun against the old salt. Mastroeni was an important mentor for Powers prior to being dealt to LA and he'll face off against the young midfielder on Saturday. Expect Mastroeni to be physical with his former midfield apprentice as he looks to limit any chances that he has going forward.
GYASI ZARDES vs. DREW MOOR
Gyasi Zardes has displayed a penchant for getting involved in the attack as of late as he's rattled off plenty of shots in recent starts, but with a goal under his belt last week, he may be on the cusp of breaking out. Look for Moor to attempt to limit Zardes' chances as he is particularly astute with his positioning and will seek to cut out any potential shot opportunities.
MARCELO SARVAS vs. MARVELL WYNNE
A battle of technical ability and sheer athleticism will likely determine the Galaxy's ability to create down the left side. Sarvas' ball skills far and away outweigh Wynne's but the right back's sheer breakneck speed make him a tough challenge for the Galaxy attacker.
Cudicini; Franklin, DeLaGarza, Meyer, Dunivant; Jimenez, Juninho, Mastroeni, Sarvas; Villarreal, Zardes


text:
The Houston Dynamo showed their support for the fallen firefighters of stations 51 and 68 last week by visiting both stations. The team participated in drills, talked with the firefighters and offered them tickets to the Dynamo's upcoming home game this Saturday, June 22 vs. Toronto FC.
The game is dedicated to the firefighters who lost their lives in the May 31 fire. Fans can show their support by purchasing a special ticket package, with proceeds benefitting the 100 Club Survivors Fund.


text:
Star Lobster packing label
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'VALENTINE'S DAY' AND OTHER FLICKS ... The Avenidas Movie Club will show the 2010 romance comedy "Valentine's Day" next Thursday, Feb. 13, at 1:30 p.m. Other movie club picks this month are Woody Allen's 2012 "To Rome With Love" on Thursday, Feb. 20, and the 2012 romantic drama "The Words" on Thursday, Feb. 27. Movie club, which includes refreshments and drinks, meets at 1:30 p.m. at Avenidas, 450 Bryant St. It's free for Avenidas members, and $2 for nonmembers.
AGING AROUND THE GLOBE ... An aging population is a looming economic and social burden, particularly in Europe and Northeast Asia, and to a lesser extent in the United States, according to a new report by the Pew Research Center. In many of these societies, the public recognizes the problem. How this recognition affects the emerging politics of global aging — allocation of scarce resources to pay for pensions and health care of the elderly — could prove a defining issue in graying economies around the world, the report said. Americans are less worried than most Europeans and Asians, reflecting the demographic reality that the U.S. population is aging more slowly, the report said. For example, the number of dependents (people under 15 and over 65) for every 100 people of working age (15 to 64) is projected to rise in the United States from 49 in 2010 to 66 in 2050. In Japan, the number of dependents for every 100 workers is projected to rise from 57 in 2010 to 96 in 2050.
FAMILY CAREGIVING 101 ... Emotional health, Alzheimer's, medications and art therapy are among the topics in a series of free Family Caregiving Workshops offered monthly through June by the Avenidas Rose Kleiner Center in Mountain View. This month's presentation, on Thursday, Feb. 27, from 7 to 8:30 p.m., features San Francisco psychologist Michael Priddy and psychiatric nurse practitioner Cara Hoepner in a discussion of emotional health. RSVP to email@example.com or by calling 650-289-5498.
DESIGNING FOR INDEPENDENCE ... The Stanford Center on Longevity has announced seven finalists from 52 teams around the globe in its inaugural Design Challenge focused on maximizing independence for people with cognitive impairment. The seven include Automated Home Activity Monitoring from Stanford, which is a system for automatically detecting activities of daily living and generating a call for help when necessary; CareSolver, from Harvard University, a platform intended to give caregivers support and help in coordinating with a larger caregiving team; Confage, from San Francisco State University, a game that teachers older users how to better use touchscreen devices; Eatwell, from the Academy of Art in San Francisco, a tableware set designed for the needs of people with Alzheimer's; Memory Maps, from the Copenhagen Institute of Design, a system helping a person with early-stage cognitive issues and his or her family to record memories attached to real-world locations; Taste+, from Singapore National University, a spoon that electrically stimulates tastebuds to promote better eating for those with diminished taste sensation; ThermoRing, from San Francisco State, a visual indication of a stove burner that's left on or that's too hot to touch, a significant safety issue for people with dementia. Finalists will receive $1,000 and mentorship from the Design Challenge corporate sponsors prior to the finals, to be held at Stanford in April.
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text:
Paying it forward. Great work everyone who contributed.
Welcome to the Quilting Board!
Paying it forward. Great work everyone who contributed.
The looks on those little faces was priceless, well done to all concerned,you are angels.
God Bless you all.
Thank you to all who continue to look at this thread. For most people across the country, Sandy was just a blip on the radar. But for many of us, the recovery goes on. Even now that it is January some still haven't had any work done on their homes! Many are still without heat. Thank you for remembering them.
Feel the fear and do it anyway!
Thank you for sharing such a lovely story. The smiles on the children's faces are sooooo lovely. You are doing a great job - all of you out there, keep up the great work and giving of your hearts. Best wishes.
Patchwork Crazy!!!


text: Pleasant View Fire Department is selling raffle tickets for a private property buffalo hunt. This a two to three year old bull buffalo and the meat processing is included. A $2,500 dollar value, tickets are $25 and only 400 will be sold. You can purchase tickets at Greenline Equipment, Diamond D processing, Yoders Auto or from any Pleasant View firefighter. You will also be able to purchase tickets at Big R in Cortez on Saturdays, Oct. 6, 13 and 20. The raffle drawing will be held on Tuesday, Nov. 13 at 7 p.m. at the Pleasant View Fire Station on Hwy. 491 and CC. For additional information, call Jeff Daniel at 562-4812.


text: I spent all of last week doing some work on Sanibel at the Ding Darling Refuge and spent my evenings fishing the wildlife drive and several other spots around the island and did really well. My weeks total was 6 trout and 2 nice snook and my friend Wes caught 4 trout, 1 redfish, and a couple snook. ALL the fish were caught using DOA shrimp of varying colors.


text:
Curly Formal Updo Hairstyle - Black Hair Color
Description(Added: 12th February 2014 )
This high and fancy 'do is perfect for those wanting a head-turning style to compliment a round face for any special occasion. These naturally thick locks are styled into curls, pulled up and pinned to the crown to achieve this shape. Strong hold product is needed to keep this updo is shape all day or night.
- Hair Category: Formal
- Hair Length: Updo Long
- Hair Elasticity: Curly
- Gender: Women
- Face Shape: Round, Oval, Heart, and Triangular
- Hair Density: Medium/Thick
- Hair Texture: Medium/Coarse
- Age: 21 - 30 and 31 - 40
- Height: Any
- Weight: Thin/Average/Large
- Glasses: Without glasses suits best
- Styling Time: 30+ minutes
- Styling Tip: Allow up to 2 hours styling time for hot roller setting, teasing, pinning and spraying. This long lasting style is great for all occasions and all weather conditions.


text:
It's official. Fall is really and truly and officially upon us. The whole leaves & back to school thing has crept up. Before you know it we'll all be shopping for holiday gifts and making negotiable New Years' Resolutions. And TV is no exception. In the last few weeks the networks have unleashed a barrage of new fall shows of all shapes and sizes. There are comedies, dramas, reality shows…even a vampire-themed show (The Vampire Diaries — but hey. the CW isn't stupid…who wouldn't want to capitalize on Twilight's rabid fanbase with a good old soapy drama. And this one's got plenty of cute brooding guys, a troubled damsel and a superhunk of a baddie. I for one was a teensy bit intrigued by it.) So this was the week where lots of them premiered — or at least got moving — did catch any of the new crop? And if you did tune in, are any of them worth catching (or at least Tivo'ing) …and should some of them be skipped? We want to keep today's poll limited to 4 new shows — and one from each network …if you have lots to say we can always ask you about others next week. But for now, take a peek and voice your choice in our featured poll: which was your favorite brand new TV show?
1) Flash Forward (ABC)
2) The Good Wife (CBS)
3) Mercy (NBC)
4) Glee (Fox)
For the latest pop culture news and voting, make sure to sign up for the People's Choice newsletter!


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Children & Youth: Building tomorrow today
As young people mature, the decisions they make have an enormous impact on their families, their communities and our world. But growing up surrounded by poverty, war or in the aftermath of disaster increases their risks and limits their choices.
That's why giving children and youth the right support at this most critical time is essential to building tomorrow's strong, productive communities.
When disaster strikes, young children are especially vulnerable to developing emotional and social problems that can jeopardize their futures. Mercy Corps worked with experts to design our signature Comfort for Kids program that helps children process their trauma and recover through play, sports and art activities. See our expert's recommendations for how parents and caregivers can help children through times of crisis ▸
For adolescents faced with violence, early marriage and interruptions to schooling, we provide opportunities for community involvement, vocational training and life skills development. Our goal is to empower youth in the toughest places to make smart life choices and invest their energy in developing solutions to their countries' biggest challenges.
All stories about Children & Youth
Jordan, Syria: Getting Syria's most vulnerable children to school
Children with disabilities can easily fall through the cracks during the upheaval of war. We're making sure kids like Hayam have their needs met to continue their education as refugees.
Jordan, Syria: Life as a refugee: Spend the day with Nour
From dawn to dusk, every hour with Nour, 13, and her family in Zaatari refugee camp shows the hardships millions of Syrians endure every day, three years since the war began — and the occasional joys they hang on to far from home.
Jordan, Syria: Syrian youth in their own words
Ten young refugees in Zaatari camp talk about what it's like to grow up without their home and what keeps them looking forward.
Syria crisis & children: Why is it vital to focus on their needs?
Hear from Nigel Pont, Middle East Program Director: "If we don't invest in them now, then we'll certainly be hearing from them later."
Lebanon, Syria: A commitment to Syria's children
The trauma doesn't end when Syrian children escape the war. We're finding new ways to ensure this generation is not lost to this crisis.
Philippines: A child's safe haven helps families cope
Merlyn and her three children can't go home again. Child Centered Spaces give the young mother peace of mind as they recover from Typhoon Haiyan.
Jordan, Syria: Winter for Ibrahim: Helping refugee kids stay warm in Zaatari
His mother thought they'd be able to return to Syria by now. How does a family survive winter without the basics of home?
Philippines: Addressing children's needs during relief efforts
New safe spaces give kids a place to play and find support while we help their parents rebuild after Typhoon Haiyan.
Jordan, Syria: A message for you from Syria's children
With your support, we're making sure refugee children don't feel forgotten. See what they created to say thank you for giving them hope for the future.
Jordan, Lebanon, Syria: In the News: NPR's All Things Considered visits Syrian refugees with Mercy Corps' child protection expert
Alexandra Chen spoke with NPR about the importance of treating symptoms of trauma in children displaced by the conflict.