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Deep V Neck Bikini with Tortoise Shell Chain Details
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Tortoise Shell or Gold Chain Accessory
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High Waist and Low Waist Bottom Option
Removable Padding
Lycra XtraLife® Certificate
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Red: 72% Polyamide 28% Elastane
Jeans: 94% Polyamide 6% Elastane
Valentina Bikini is the bikini option of Lina One-Piece, because everybody loves havingoptions. The deep V neck top is styled with the same chains all over the inner bust in tortoise shell or gold. The top has removable paddings and gold clasps closure. The bottom comes with 2 different style: high waist with good coverage and low waist with chain details on the sides and low coverage.
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Located in Broward County, Florida, the town of Davie has a population of over 90,000 residents and extends across 35 lush square miles. Davie is home to the Miami Dolphins, an NFL team. The town also boasts many attractions for residents and visitors to enjoy, including Flamingo Gardens, Bar-B-Ranch, and the Butterfly Pavilion. Although it is a pleasant place to live or visit, a number of people there are harmed in accidents every year. If you have been hurt in Davie by someone else's carelessness, the experienced injury lawyers at Frankl & Kominsky can help. Our firm handles virtually all types of personal injury cases, ranging from ordinary car accidents to claims arising from an individual's wrongful death.File a Florida Negligence Lawsuit to Seek Compensation
Typically, an accident victim can file a negligence claim against whoever was responsible for causing his or her injuries. Negligence is defined as the lack of reasonable care. Someone held liable for negligence has failed to use the level of care that an ordinary person would use in a similar situation. In this type of lawsuit, a victim must prove that the defendant had a duty to use reasonable care, breached this duty, and caused foreseeable injuries to the victim because of the breach. The injured person also must have incurred damages that can be compensated, such as medical expenses, lost wages, or pain and suffering endured in the accident.
In the context of car crashes, for example, a motorist who fails to observe traffic laws or drives while distracted may have breached the duty of care by needlessly endangering others on the road. Alternately, a medical malpractice claim based on negligence can aim to hold a surgeon accountable for operating on the wrong body part. Either the driver or the doctor may be held liable for damages or injuries caused because of their failure to use reasonable care.
In some cases, such as truck accidents caused by careless commercial drivers, a victim may be able to sue the driver's employer as well as the driver under the principle of vicarious liability. Florida law allows you to seek damages from a company after you are hurt by one of its employees, assuming that the worker was acting within the scope of his or her employment when the accident happened.
To seek the compensation that you deserve, you must file a personal injury claim within the applicable statute of limitations. Under Florida law, a negligence lawsuit must be filed within four years from the date of the accident. When a plaintiff is pursuing a wrongful death claim, the time frame decreases to two years from the date of the death.Discuss Your Davie Injury Claim With an Experienced Lawyer
Since negligence claims are highly fact-intensive, getting the right legal team on your side can make all the difference. At Frankl & Kominsky, our accident attorneys spare no effort in maximizing the chances of recovery for victims in Davie and other South Florida areas. We investigate the facts, collect evidence, consult experts, and build your case to the best of our ability. For a free case evaluation, you can contact us online or call us toll-free at (855) 800-8000. We are fluent in Spanish and Creole.

subset: commoncrawl


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Jessica van Loben Sels had a fascination with microbiology when she first started college at the University of Kansas. Like many students, however, she was unsure what role research could play in her aspirations. The Goldwater application was Jessica's first significant exposure to a personal research project, and she credits the process for where she is today.
When applying for the Goldwater, Jessica's project focused on the intersection between immunology and virology which helped her develop her course of study in her PhD where she's working to improve vaccine design and antiviral therapies to treat persistent norovirus infections. Jessica currently holds a position in the National Institutes of Health Oxford-Cambridge scholarship program, through which she also attends graduate school at the University of Cambridge. Additionally, Jessica travels to different parts of the world, including Vietnam and Uganda, where the pathogen she studies is particularly endemic to the human population.
"I feel the experience of coming up with an independent research project for Goldwater in undergrad has allowed me to accept the challenge of this accelerated graduate study," says Jessica. "[The Goldwater application] requires thinking beyond the facts you memorize for class and beyond the small, discontinuous experiments you carry out in laboratory classes. It challenges you to use your knowledge in an innovative context to address a novel question that interests you and would inevitably have an impact on others, both within and outside of the science realm."


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By/ August 15, 2014
"The key issue is trying to equivocate the liberal democratic Israel with what's happening in Gaza. Gaza is currently controlled by Hamas. Hamas is an internationally recognised terror organisation, they are the equivalent of ISIS in Iraq, they have the most extremist, fanatical, ideas you can have. What they do on a daily basis is think about how they can kill Israelis.
"The people of Israel want to be able to live their lives in peace without the threat of rocket fire, without the physiological and ongoing impact on people's lives.


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A parliamentary candidate has been suspended from his position as a parent governor of a Catholic school because of his party's critical position on faith schools policy.
A report in the Herts Advertiser newspaper says that Loreto College decided to suspend Jack Easton, Green parliamentary candidate for St Albans, because it believed that the Green Party policy criticising the idea of religious organisations running state schools could be incompatible with his role at the school.
Mr Easton explained that while he is not religious, his wife is a Catholic and his children have been brought up as believers.
Defending the policy on which he is running for parliament Mr Easton said: "The Green Party's underlying vision is that every child and young person should be entitled to attend their local community school and for their needs to be met on an inclusive basis."
He continued: "The Greens believe that education should include a celebration and recognition of religious and cultural diversity and spirituality and Green Party policies include requiring schools to provide prayer space for those who wish to practice their religion."
"Schools could choose to hold acts of worship but would be expected to provide alternative activity for pupils that do not wish to take part," he added.
The Accord Coalition (which includes Ekklesia, and a wide range of organisations and individuals from both religious and non-religious backgrounds) also campaigns for major reform to laws governing school admissions, religious discrimination against teachers, and compulsory worship.
However, unlike the Greens at present, the Coalition does not take a position against or for faith schools in principle - instead bringing together all those who favour reform.
Polls suggest that the Green Party may win its first MP in the General Election on 6 May 2010.


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"Good dressing isn't necessarily rocket science. Remove a collar from a shirt and what do you have? A less formal shirt, of course. On their own or underneath a jacket, our collarless shirts form the basis of a modern understated off-duty look. Leave the complicated stuff for your morning crossword."
Slim fit collarless check blue shirt
Three month quality guarantee


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Imperial Ride is one of the finest Chauffeur Company providing impressive and up to the mark Chauffeur services in the UK as well as around the world. Imperial Ride has been standing head and shoulders above the rest Chauffeur services in London.
Our hand-picked Chauffeurs, adorned with professional attire and attitude know how to deal professionally with high-valued customers. They know the London streets more than others and provide secure and discreet Chauffeur services for Leisure, business travel, road shows, and other events.
Imperial Ride services has extensive years of experience of serving global and local business executives, CEO's, Hollywood and Bollywood celebrities and Government officials. We have a fleet of Chauffeur driven Mercedes Benz S-Class, E-Class and V-Class, Range Rover, Rolls Royce Phantom and Bentley Flying Spur Chauffeur executive cars.
We are open to provide you services for Financial Roadshows, Airport Chauffeur, Wedding and other important events.Imperial Ride offers tailored executives highly-professional Chauffeur services which are highly reliable, wholly dedicated and entirely skilled to provide the best to our clients around the planet.
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There is no doubt to the fact that travelling can be stressful and a crucial thing. There is a lot of planning involved including hiring a chauffeur company and chauffeur pickup from the airport. You have to take care of a lot of things, and in such a scenario a few can be overlooked. For your safe travel from the airport to the hotel, let our professional London chauffeur service ensure that it goes as you please. You will be tired at the time of arrival and we don't want you to stress out more about how to get to the hotel. Your tiring journey will end the minute our London chauffeur receives you. From thereon, all your stress and worries are taken care of by our professional chauffeur pickup service providers.
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It's so easy to get caught up in this idea of reading as a writer that it becomes this rule, a dogma. Some writers, like Stephen King in his famous book on writing drives home this point where it's easy to internalize in this way. I have to admit it's easy to feel guilty if the reading falls by the wayside for whatever reason.
When this happens, reading becomes a labor. This takes whatever joy there is in reading a work someone else created out of their own dreams and desires that it destroys the point of reading altogether.
Reading is about listening, hearing the story a writer is telling you. Or, it is to take in the view someone is presenting you. This is what it is to be an active reader.
Sorry, that is the English teacher part of me talking, but it is true. We do live in an age where context is missing and public figures are constantly shouting that that's not what they said. Often, the context is deliberately hid, but a bigger issue is that many of us aren't fully equipped to truly see it, especially when we are caught up in what outrages us.
I got it in my head as a young man that somehow reading a book is like eating it and writing creatively is like shitting out your literary meals. I've never seen it explicitly said like that. However, when you are advised to read to shape your own language and learn the form of wiring, it's easy to come to this view. And I'm starting to realize how warped it is.
I suppose it's better than plagiarism, which is like gobbled up prose that's vomited up too soon and the sick is still very recognizable as the food. Often, it's voluntary like the inaccurate image of Romans going into the vomitorium to disgorge the contents of their stomachs so they can eat more.
This year, I do intend to make reading a more regular practice. I also resolve to get past the baggage so that reading is more the act of listening than it is looking for something to steal. In cultivating the act of listening as a reader, I myself can present my story in a way that the audience can listen themselves.


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Print Green tomato chutney
This is a great use for tomatoes that stubbornly won't ripen. If you're not using new jars, to help prevent re-used jars smelling of their previous contents, add a little bicarbonate of soda and hot water, leave for a couple of minutes, then rinse well before using. To sterilise jars: wash and leave jars to air dry. Place cold jars on a baking tray in a cold oven. Heat to 150C/Gas 2 for 15 minutes. Sterilise lids by boiling for 10 minutes then leaving to dry thoroughly before using, or once you've potted your chutney, pop on clean, dry lids and invert the jars for 10 mins to let the hot chutney sterilise them, before turning right-way up. Always pot hot chutney into hot jars. This recipe should make about 1kg chutney.
Key facts
- Preparation time
- 30 mins
- Cooking time
- 1 hr 30 mins
- Total time
- 2 hrs
- Serves
- 4
- 750g green tomatoes, washed & chopped
- 350g shallots or onions, chopped
- 200g cooking apples, peeled, cored & chopped
- 300ml malt vinegar
- 2 red chillies
- Small piece fresh root ginger, peeled
- A few yellow mustard seeds (optional)
- 100g raisins
- 1 tsp salt
- 200g brown sugar
- Put the tomatoes, shallots or onions and apples in a large, heavy-based, non-reactive pan, with half the vinegar.
- Bring to the boil then gently cook for about 30 minutes, until tender. Tie the chillies, ginger and mustard seeds (if using) in a muslin bag. Bruise with a heavy rolling pin or hammer and add to the pan with the raisins.
- Cook on a low heat, stirring from time to time, until the mixture thickens (about 1 hour).
- Add the salt, sugar and the rest of the vinegar, stirring well until the sugar dissolves. Continue cooking until the mixture has thickened.
- Remove the muslin spice bag. Pot into sterilised jars while the chutney is still warm. Seal. Leave for 6 weeks to mature.
Nutritional Info
|per tbsp
|of which Saturates (g)
|of which Sugars(g)


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Emily Estefan's debut album, "Take Whatever You Want," has landed on several Billboard Charts.
The 22-year-old daughter of music legends, Gloria and Emilio Estefan, debuted at No. 27 on R&B Current and No. 28 on Heatseekers charts, under her own label.
The eclectic body of work grabs inspirations from jazz, neo-soul and funk genres and tops it off with Estefan's smooth vocals and musicianship.
"It's the first expression where I didn't confine myself to any barriers and you can take whatever you want from it and interpret it however you want," Estefan said of her self-written, produced and recorded album.
Estefan recently played her first-ever show to a sold out crowd at the University of Miami's Frost School of Music's 33rd Annual Festival Miami.
Estefan's live show is making its way across the United States and is expected to make an appearance at the SXSW festival next month.


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Pest Prevention in Chicago
It's sad to say it, but winter is around the corner. Anyone that had the pleasure of being in the Chicagoland area last winter is probably not looking forward to another long, cold, snowy winter. Summer seemed to take forever to arrive and now it's gone already. I guess that is just how the story goes when you live in a four season state like we do. The good news is that there are many things you can do as a homeowner that will help make living in your home this winter a little more tolerable no matter how cold it gets outside. It's one thing to wake up in the middle of the night cold, but it's another to wake up cold and see a mouse scurry across your floor. Below you will find tips that will help keep your home pest free and also might even save you money in the long run.
1. Seal Up Entry Points. This will not only make it tougher for pests to invade your home, but it will also prevent drafts and save you money on your gas and electric bills. Repair any damaged screens or windows and make sure they are all properly caulked on the outside and the inside. Apply weather stripping and/or door sweeps around all doors. Use caulk to seal up any other holes or cracks you may have on the outside of your home. Also, be sure to keep the garage door closed as much as possible.
2. Clean Out Gutters. Doing this will eliminate insects from nesting in your gutters and eventually invading your home. It will also prevent a potential roof leak if water gets backed up on your roof from the gutters being clogged.
3. Clean Up Yard & Relocate Firewood. Leaves and brush should be picked up and properly disposed of (check with your local municipality for a brush/leaf pickup schedule). Don't forget to clean out your window wells also. Ideally you should have window well covers installed. Firewood and brush piles should be stored at least 2 ft away from your home. In addition, try to avoid bringing firewood indoors until you are ready to burn it since many pests like to hang out in the wood piles.
4. Eliminate Moisture. Make sure you inspect your basement, crawlspace, bathrooms, kitchen, attic, and roof to be sure you do not have any water leaks or moisture content. Not only will accumulation of water or moisture cause damage to your property it can also attract pests into your home. To help eliminate dampness in a basement or crawlspace you can set up dehumidifiers to alleviate some of the moisture. For any major leaks that you find, we recommend using a licensed contractor to make the necessary repairs.
5. Remove Clutter. Pests love cluttered conditions since there are so many places for them to hide. Since wintertime can leave you stranded inside with nothing to do, why not go through your storage in your basement and garage and get rid of what you no longer need. Doing this will help you be more organized and also eliminate potential hiding places for pests.
Hopefully you will find these tips useful and will enjoy a pest free winter. If you do end up with any unwanted visitors inside your home be sure to give us a call and we will get a technician out to take care of the problem. Stay warm and enjoy your winter!


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Who were the non-Western women delegates who took part in the drafting of the United Nations Charter and the Universal Declaration of Human Rights (UDHR) from 1945-1948? Which member states did these women represent, and in what ways did they push for a more inclusive language than "the rights of Man" in the texts? This book provides a gendered historical narrative of human rights from the San Francisco Conference in 1945 to the final vote of the UDHR in the United Nations General Assembly in December 1948. It highlights the contributions by Latin American feminist delegates, and the prominent non-Western female representatives from new member states of the UN.
I denna uppsats kommer jag att behandla Karl XII och hur denne har framställts i läroböcker från 1884-2003. I min uppsats har jag valt att behandla 14 läroböcker, som i sin tur jag delat upp i tre stycken epoker: Epok I, Epok II och Epok III. Analysen i sin tur har utgått i från tre teman. Vilket jag med hjälp av ett diskursanalytiskt förhållningssätt försökt redogöra kring hur författarna framställt Karl XII, hans personlighet, centrala skeenden samt värdeladdade ord kring Karl. Analysen kommer fokusera på uttalanden från författarna som kan anses vara värderande, samt koppla författarens ställningstagande och sedan se om det stämmer överens med den rådande historieforskning som gällde när läroboken skrevs. Genom att behandla mina epoker i en kronologisk ordning är tanken att ett förändringsmönster i diskursen ska uppstå. Samt att se huruvida läroboksförfattarna faller ifrån opartiskhet och visar sina personliga värderingar i texterna. Tanken med denna uppsats är alltså att se ett utvecklingsmönster, tillika skåda hur pass mycket en författare låter sina egna tankar belysa en lärobok.
The aim of this thesis is to investigate how politics was done at the level of the parliamentary constituencies at the time of the treaty of Amiens 1802-1803. This is achieved through two case studies of the elections in Middlesex and Nottingham, which are investigated as social practices. This thesis argues that understandings of masculinity and national identity, as well as questions about the nature of the constitution and citizen rights were central to participants in the extraparliamentary political process. Collective emotions were also highly important in the process of mobilising political support, and this thesis emphasises that participation in these elections was a collective effort; men and women from all levels of society were significant political actors. Moreover, this thesis demonstrates the importance of competences such as knowledge about the organisation of crowds and political violence in the performance of the election.
The collection covers topics of interest to both the historical and linguistic study of the contacts between speakers of African and Iberian languages in the constitution of Latin American societies. Supported by historical and demographic data, the twelve chapters cover topics of interest to the discussion on the formation of Latin American varieties of Portuguese and Spanish. Moreover, the book draws attention to the need to articulate the fields of Linguistics and History and contributes to the discussion on the formation of varieties of Latin American Portuguese and Spanish.
This article presents the "Smolensk Archives", which consist mainly of documents from the Chancellery of Smolensk from the time of the Polish king Sigismund III's siege of the city (1609–1611). Most of the documents are now in the Swedish National Archives in Stockholm, but a significant number are also to be found at the Institute of History of the Russian Academy of Sciences in St Petersburg. The archives have a complex history. Following the fall of Smolensk in 1611, they were taken as spoils of war, ending up on the Sapieha family estate in present-day Belarus. In the mid‑17th century they were seized as war booty once again — now by Swedish troops — and taken to Skokloster Castle in Sweden. There they were discovered in the 1830s by Helsinki professor Sergej Solov'ev, who in turn removed a relatively large part of the collection to St Petersburg. Portions of Solov'ev's collection of documents from Skokloster were published in 1841 in Akty istoričeskie. Around 1900, Russian historian Jurij Got'e worked on the documents from the Swedish National Archives, and in 1912, he published a text edition comprising much of the collection, Pamjatniki oborony Smolenska 1609–1611. The article includes a brief overview of the contents of the material in Stockholm, with a few examples of individual documents. A digital catalogue of the Smolensk Archives is currently being prepared, including brief descriptions of the contents and information on external characteristics such as condition, inscriptions and watermarks.
Is it possible to survive on a deserted farm: Manors, tenants and farming systems during the Middle Ages in the Lägerbo area, Östergötland.
This study approaches the late medieval farm desertion from a landscape perspective. It focuses on the area of a former medieval estate in southern Östergötland, Sweden. Based on a retrogressive analysis of cadastral maps and historical records the medieval settlement is reconstructed. In this process three formerly unknown deserted farms were identified, with abandoned field systems and building remains. The volume provides the archaeological documentation of field systems and settlements at these sites. These data provide the background for investigating the shifting social and ecological circumstances that once made it possible for tenant families to survive on these farms. During the height of the manorial system the small farms were specialised units in a redistributive system. In the late 14th century the estate and all tenant farms were donated to the convents of Vadstena and Vreta. Rents were no longer paid in labour but in butter. In the fifteenth century several farms were abandoned and turned into meadows under the surviving farms. The new tenurial relations prevented the recolonization of the farms. The study is the result of an interdisciplinary project involving medieval archaeology, historical geography, palynology and medieval history.
Könspolitiska nyckeltexter är en mångfacetterad introduktion till svensk genushistoria. Genom ett pärlband av originaltexter, från C.J.L. Almqvists roman Det går an 1839 till #metoouppropen 2017, ges en fördjupad förståelse av hur kön har diskuterats, politiserats och iscensatts under nästan 200 år. Varje nyckeltext är kommenterad och analyserad av en forskare.
Arbete, sexuella rättigheter, familjeliv, diskriminering, våld, försörjning, värnplikt, rösträtt, preventivmedel, skönhet och barnomsorg är några exempel på de många frågor som behandlas i boken, nu i omarbetad och utvidgad upplaga.
In the 1780s the medieval weapon of war; the English longbow, enjoyed a renaissance, as historical archery became a fashionable recreation among the English aristocracy. Later, during 1819-1845, longbow archery developed into a mass movement, as it spread downwards in the English class system, into the bourgeoning middle class. During the entire time period of 1780-1845, the "English warbow" was instrumental in producing a specific English (i.e. not British) nationalistic memory culture regarding the medievalmilitary triumphs of the "English bowmen" in battles of old, against French and Scottish forces, as well as reproducing a nationalistic narrative surrounding the English national hero and master-archer Robin Hood. The English longbow, as an object, became a mani- festation of English nationalism. An important fact was that both men and women were included as archers, despite the masculine context of the memory culture surroundingmilitary archery, the celebration of medieval English battlefield victories and the man- liness of the English "bowmen". How did England come to view the female archer as an ideal for English women, while at the same time publicly upholding a patriarchal doctrine of a feminine "private sphere" womanhood, whereby women should be constrained to the domestic space as housewives, mothers and daughters? How was the English inclusion of females in the nationalistic public sphere of longbow archery made possible, communica- ted and reproduced? In summary, this study is about how longbow archery was manife- sted in the context of the rise of English modern nationalism and how women were inclu- ded – or rather included themselves – as English longbow archers. As the study shows, the answers exists in an inter-relating web of English memory culture regarding warfare and historical archery; gender constructions and female agency; constructions of English national identity and English nationalism within a British context; and class developments in English society. This accounts for how the Amazon Archers of England came to exist from 1780-1845.
This thesis addresses the issue of how the Swedish governmental authorities – the secular (the State) and the ecclesiastical (the Swedish church) – aimed at treating the population during the early modern era. The question asked is whether the authorities always strived to foster the people, or whether there were areas where care for individuals was more important. This is explored by a study of the authorities' general normative principles for the handling of death.
The study focuses two normative problems: the first concerns how the authorities decided that the deceased should be taken care of; the second concerns the norms formulated regarding the actions of the survivors. These problems are explored through normative source material – laws, royal ordinances, church regulations, service books and hymnbooks – and to some extent illustrated by funeral sermons and grief correspondence. A framework for interpreting the handling of death is used where care is placed on a level equal to social support, and fostering to that of demands.
The handling of death was characterised by great continuity. The norms concerning the handling of the deceased foremost aimed at getting the deceased interred in a respectable fashion, and they were motivated by care. The prescriptions concerning the survivors are more complex and the amount of care and fostering respectively varied, depending on whether the regulations had ecclesiastic or secular origin. The study shows that the authorities and especially the Church were mainly acting out of care for individuals. There were also obvious fostering elements, but it is impossible to say that the authorities always displayed a single fostering ambition. Hence care and fostering must be considered parts of a whole, since all fostering in some sense could be understood as motivated by care.


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Hack cartoons – Political cock-up of the day
Isn't it amazing what donations made during an election for the deputy leadership of the Labour party will lead to? This was originally made in June at the height of the excitement which got Harriet Harman to the heady heights of Gordon's deputy.
27th November 2007
The best British political cartoons


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Nucleic Acid Arrays
Development and Applications of High Density RNA Arrays
Overview. We have recently developed a powerful new approach for the fabrication of high-density RNA arrays. In this approach, a high-density DNA array is fabricated by standard photolithographic methods using photoprotected nucleoside phosphoramidites, the surface-bound DNA molecules are enzymatically copied into their RNA complements (also tethered to the surface), and the DNA templates are enzymatically destroyed, leaving behind the desired RNA array. These arrays are RNA analogs to the high-density DNA arrays that have proven over the past two decades to be tremendously powerful tools for biomolecular analysis, particularly in the areas of global transcriptomics and genome-wide genetic variation analysis. We seek to further develop and optimize this novel technology for RNA array fabrication, and to develop and demonstrate its power and utility in important application areas.
Development and optimization of RNA array fabrication. The utility of RNA arrays depends strongly upon the array quality. Two key interrelated quality parameters are the RNA strand sequence fidelity (what fraction of RNA molecules in the array have the correct sequence?) and the RNA lengths that can be obtained (how long are the RNA molecules that can be made without excessive loss of sequence fidelity?). Other parameters of interest are the surface density of the RNA strands, the nature of the substrates employed for array fabrication, and the ability to employ modified nucleosides of various types. In this first aim, we will develop and apply the tools needed to measure these parameters, and use them to guide optimization (maximize sequence fidelity and length) of the chemistry and enzymology employed for array fabrication. We will also develop the ability to control strand density (which is important as inter-strand interactions on the surface can interfere with RNA strand folding and secondary structure), vary substrate types (important to be able to access different array synthesis chemistries and technologies for monitoring array binding), and employ various modified nucleosides (important for RNA and DNA aptamer projects as well as for therapeutic applications of nucleic acids).
Applications. The importance of RNA array technology will depend upon the extent to which RNA arrays open new possibilities in biomolecular analysis. Here are two interesting applications:
Identification and characterization of fluorescent RNA mimics of GFP. We will fabricate RNA arrays comprised of hundreds of thousands of variants of the recently reported fluorescent RNA mimics of GFP, and seek to identify variants with stronger chromophore binding affinities and improved fluorescent properties.
Determining the binding specificity of RNA-binding proteins. We will fabricate RNA arrays containing hundreds of thousands of permutations of the known binding sequences for the model RNA-binding protein PUM2, and use them to determine the rules governing its target binding specificity.
Significance. In 1991 Steve Fodor and his colleagues at Affymax ushered in the new field of biomolecular array analysis with the publication of their Science paper entitled "Light-Directed, Spatially Addressable Parallel Chemical Synthesis" [1]. In this work the authors showed how the photolithographic methods developed for integrated circuit fabrication could be adapted for the light-directed synthesis of addressed arrays of biomolecules on planar substrates. Although their initial proof-of-principle work was limited in scope compared with present day standards (they fabricated a peptide array of 1024 elements and demonstrated the light-directed synthesis of a dinucleotide), it has been transformed over the ensuing years into a powerful analytical platform with widely available DNA arrays containing millions of oligonucleotide features and employed for myriad applications such as SBH (Sequencing by Hybridization) [2], Genome-wide Gene Expression Analysis [3], ChIP-chip (Chromatin ImmunoPrecipitation and analysis by DNA "chip") [4], and CSI (cognate site identification) [5].
Remarkably, while DNA arrays became mainstream and ubiquitous tools of the modern molecular biologist, the seemingly straightforward extension of the concept to RNA arrays never became practical. The fabrication of DNA arrays relies upon the phosphoramidite chemistry developed in Marv Caruthers lab at CU Boulder [6, 7]. This chemistry is remarkable because of its extraordinary efficiency, which manifests itself in stepwise yields for monomer addition over 99% [8]. This high efficiency is what allows DNA molecules as long as 150 nt in length [9] to be synthesized, and is the fundamental reason for the widespread availability of both individual high quality oligonucleotides and high-density DNA arrays. Although the same chemistry can be used for RNA synthesis, it does not give results of comparable quality. For RNA synthesis it is necessary to protect the 2' hydroxyl to keep it from coupling during synthesis; in spite of much effort by many groups, it has not been possible to find a protecting group for this position that does not interfere with coupling at the adjacent 3' hydroxyl, and/or give rise to undesired side reactions during deprotection [10]. Because of these issues, there has not existed to date any viable technology for the fabrication of RNA arrays. (We note that it is possible to obtain fairly short individual RNAs, since they can be purified from even quite inefficient syntheses.)
Figure 1. Enzymatic fabrication of high-density RNA arrays.
We have recently made a breakthrough in this area, with a simple yet powerful new strategy for the enzymatic synthesis of a high-density RNA array [11]). The key idea is to use RNA polymerase to copy surface-attached DNA molecules on a high-density DNA array into their RNA complements (Figure 1). The surface is first partially deprotected (e.g. light is used to effect removal of 50% of the NPPOC photolabile protecting groups covering the surface), an array of the DNA complements to the eventual desired RNA sequences is synthesized by standard light-directed synthesis on the exposed sites, and the remaining surface sites are then deprotected, followed by synthesis of an RNA primer sequence. These primer sequences on the second group of sites then hybridize to their complements on the first group, whereupon they may be extended with T7 RNA polymerase to yield an RNA:DNA duplex. The DNA is removed with DNase I, leaving behind the desired single stranded RNAs.
A powerful aspect of this enzymatic approach to RNA array fabrication is the ease with which chemically modified nucleosides may be incorporated. For example, natural (unmodified) RNA molecules are resistant to DNase I but susceptible to RNase A, whereas 2'-fluoro RNA is resistant to both DNase I and RNase A. This resistance to nuclease digestion is very important for therapeutic applications of RNAs, and also extends the chemical diversity which can be obtained in combinatorial chemistry applications. Figure 3 shows the effects of RNase or DNase treatment upon either a natural RNA array or a 2'-fluoro RNA array. The natural RNA array is totally resistant to DNase but destroyed by RNase within 30 min, whereas neither DNase I nor RNase A has any effect upon the 2'-fluoro-RNA array. We note that the use of a very long, flexible, hydrophilic spacer (we employed three tandem PEG 2000 moieties) between the substrate and the oligonucleotides was critical to success of the approach, which is not surprising as it is necessary for the DNA complement and RNA primer sequences to anneal while both are still attached to the surface.
To illustrate some of the many potential applications of such arrays, we briefly outline several below.
Deciphering the binding specificities of RNA-binding proteins. A powerful strategy for determining the binding specificities of DNA-binding proteins such as transcription factors has been pioneered by our Wisconsin colleague Aseem Ansari [5]. His group synthesized high-density arrays of double-stranded DNAs in which the binding sequence was systematically varied, and measured the relative binding intensities of the transcription factors to the different elements. This experiment reveals the binding specificity of the transcription factor, which is essential knowledge for unraveling its effects in vivo on gene regulation. High-density RNA arrays will allow similar experiments to be performed to look at the specificity of RNA-binding proteins, by synthesizing a combinatorial array of RNA targets with systematically varying sequences, and measuring the relative binding intensities for the RNA-binding proteins of interest. This information is essential to understanding the roles they play in critical biological processes such as alternative splicing and translational regulation .
Fabrication of RNA aptamer arrays to discover those with the best binding affinity and specificity. It is widely recognized that there is a pressing need for high affinity and high specificity ligands directed against all of the proteins in the human and other proteomes (http://commonfund.nih.gov/proteincapture/highlights.aspx). Aptamers are molecules selected from DNA or RNA libraries that fold into shapes whereby they behave as high affinity and high specificity ligands [15]. The ability to fabricate high-density arrays of RNA aptamer candidates will allow them to be screened in parallel against protein targets to ascertain their binding affinity and specificity, simply by incubating the array with the target protein and measuring binding by either fluorescence [16, 17] or using a label-free binding assay such as surface plasmon resonance [18, 19].
Identifying RNA mimics of Green Fluorescent Protein (GFP). It is known that the fluorescent chromophore in GFP and related proteins is not fluorescent when separated from the protein; binding of the chromophore to the protein reduces its conformational flexibility and thereby shuts down other (non-fluorescence) pathways for relaxation from the excited state. This knowledge was leveraged in recent work by Jaffrey et al. [20, 21], who showed that it is possible to identify RNA molecules that similarly interact with the chromophores to render them fluorescent. This is exciting because, as shown by the authors, it is possible to engineer RNA molecules to include such fluorescence domains, thus allowing them to be visualized and tracked in vivo in a similar manner to how GFP fusion proteins are now ubiquitously employed to track proteins of interest. High-density RNA arrays will permit a dramatic simplification of the process for determining the correct RNA sequences, as one can simply fabricate an array of candidate RNA molecules, add the pre-fluorophore (a small organic compound similar in structure to the fluorophore in GFP), and observe which elements of the array produce a fluorescence signal.
Engineering sequence-specific RNA-binding proteins. Filipovska et al. showed recently that it is possible to engineer sequence-specific RNA-binding proteins based upon the sequence-recognition properties of the PUF proteins [22]. RNA arrays provide a beautiful and efficient means of monitoring and optimizing the binding affinities and specificities of such engineered proteins, by measuring binding of the candidate proteins to RNA arrays. This is similar to the measurement of binding specificities of naturally occurring binding proteins as described above, but employed instead as a tool to assist in the engineering of specific binders.
RNA-based therapeutics. There continues to be much interest and activity in the area of RNA-based therapeutics [23]. As with all drugs, binding activity is a sine qua non for biological activity. RNA arrays offer an elegant means to characterize the binding behaviour of large numbers of drug candidates, by simply monitoring binding of the target protein to the array.
Tiling arrays of RNA viral genomes. A powerful tool for studying the function of parts of RNA genomes will be to fabricate a tiling array that steps through it systematically (e.g. for a 100kb RNA genome, make an array of 100,000 hundredmers each starting one base further through the sequence), and use the array to look for binding interactions with viral or host proteins or other RNA components.
Arrays of miRNA. There are now over 1500 miRNAs known in human alone (www.miRBase.org), and this number will likely continue to grow as more are discovered. It is straightforward to synthesize arrays with all of these molecules and thereby create a tool for evaluating their binding interactions with other biomolecules. We note that the "miRNA arrays" sold by Affymetrix, for example, are actually DNA arrays, not RNA arrays, employed for measuring the binding of the miRNAs in biological samples by nucleic acid hybridization, and thus have a very different purpose or capability than actual arrays of miRNAs will have. The short length of miRNAs makes it very straightforward to fabricate them.
To save space, we will just mention some additional applications without explaining them fully. Engineering ribozyme arrays, discovering new ribozymes, studying ribozyme function, engineering artificial siRNAs and miRNAs, fabricating mRNA tiling arrays, and searching for miRNA "sponges" (molecules that bind to and inactivate miRNAs) [24] are other exciting applications of RNA arrays that may be considered. Clearly, although this vast compendium of possible applications is far too great to be developed fully in any single grant proposal, the point we seek to make for the reviewers is that the RNA array technology we seek here to develop has vast and far-reaching potential to accelerate research in many important emerging areas of biology. In the Approach section below we describe how we envision the further development, optimization, and implementation of the technology for selected critical applications.
RNA array fabrication. Our process for the photolithographic synthesis of DNA and RNA arrays employs a maskless array synthesizer (MAS) that was developed at UW-Madison by Professors Franco Cerrina, Michael Sussman, and Fred Blattner [25, 26]. This instrument uses photolithography and light-protected nucleotide building blocks to create surfaces covalently modified with hundreds of thousands of different oligonucleotide sequences/cm2. The key concept of the MAS is that the chromium masks used by Affymetrix for light-directed array fabrication[1] are replaced with a digital micro-mirror device (DMD). The DMD is a 1.4cm x 1.1cm electronic "chip" containing an array of 1024 x 768 independently controlled mirrors. The MAS is a bench top machine that uses the DMD to produce 786,000 different 20-100nt long oligonucleotides on a single substrate. These sequences can be changed every 4 hours, as easily as one changes the text being fed to a laser printer [25, 26]. The instrument is a key enabling technology for the present work.
The process of photolithographic synthesis on the MAS begins with preparation of a hydroxyl-terminated surface. Most DNA array manufacturers employ glass substrates for this purpose, modified with a silane reagent [1, 25, 35, 36] to provide a surface hydroxyl group as the start point for DNA synthesis. Our group has pioneered the use of carbon substrates for DNA array fabrication, which have a number of advantages over glass, the most important of which is increased stability [32]. The carbon substrate is overlaid with neat 9-decene-1-ol, and then illuminated with a low pressure Hg arc lamp to initiate a radical-mediated coupling of the alkene to the surface [32, 37]. This coupling reaction produces carbon-carbon bonds linking the hydroxyl functionality to the surface, a critical improvement compared to the hydrolytically unstable silyl ethers conventionally employed for glass functionalization. After rinsing, nucleotides with a photolabile protecting group are chemically coupled to the surface hydroxyl groups. Light is directed onto the surface by the DMD according to a programmed spatial pattern in order to photolithographically cleave the protecting group (NPPOC, 3'-nitrophenylpropyloxycarbonyl). For the RNA arrays, the surface hydroxyls are then reacted successively three times with a commercially available DMT-protected PEG-2000 phosphoramidite (Glen Research), with standard acid deprotection steps in between to remove the DMT group. (Note: DMT is an abbreviation for dimethoxytrityl, the standard acid-labile protecting group employed for conventional solid-phase DNA synthesis). This produces a hydrophilic spacer of approximately 45 x 3 = 135 ethylene oxide groups, with an approximate end-to-end length of around 300 angstroms. An NPPOC-protected nucleoside phosphoramidite is then coupled to the surface to give the first nucleotide (3'-terminal) of the sequences to be synthesized. The subsequent steps are depicted in Figure 1 and briefly described as follows. Partial deprotection (ca. 50%) with UV light is followed by coupling of DMT-rU to those exposed hydroxyls; then, the other 50% of the NPPOC protecting groups are removed to allow photolithographic synthesis of various DNA sequences on different spots of the substrate. These DNA strands are capped at their 5' termini with acetyl groups. Then, RNA primers (12-base-long, same everywhere on the surface) are synthesized using acid-labile DMT protection chemistry onto the initial DMT-rU that was incorporated prior to the DNA strand synthesis. After base deprotection, these RNA primers are hybridized to the DNA strands, providing a start point from which T7 RNA polymerase can then copy the DNA strands into their RNA complements. The result is an array of RNA-DNA duplexes. Treatment with DNaseI to destroy the DNA molecules yields the desired array of single-stranded RNA molecules.
Strand density measurement and optimization. We and others have shown the importance of strand density (number of strands per unit area) on DNA arrays [38, 39]. If DNA strands are packed too closely together on the surface, they become less available for hybridization, but if too far apart, then hybridization rates and signal intensities will decrease. Such considerations are also important for RNA arrays. For example, protein binding to RNA arrays is likely to require sufficient distance between the RNA strands that the binding protein molecules do not impede one another. In order to control and optimize this variable, it is necessary to a) be able to vary the strand density on the surface, and b) be able to measure the effect of that strand density upon the measurement of interest (e.g. hybridization of complementary strands, binding of RNA-binding proteins, or aptamer folding). We have previously published methods [39] to control strand density by partial deprotection of the surface, followed by capping to permanently inactivate the exposed sites; strand synthesis after this procedure occurs on sites of reduced density on the surface, and thus the strands are synthesized at lower density. A necessary aspect of optimization is careful measurement of the resulting strand density. We have extensive experience with this sort of measurement, which we perform by hybridizing fluorophore-tagged oligonucleotides to the surface, followed by elution from the surface and measurement with a spectrofluorimeter [40]. This measurement yields the density of hybridizable oligonucleotides on the surface. Measuring the density in this way of both the single-stranded DNA (prior to extending the RNA strand) and the single-stranded RNA (after DNAseI digestion to remove the DNA template) will allow us to evaluate the efficiency of the RNA fabrication process (primer duplex formation and polymerase extension). These measurements will enable optimization of numerous aspects of the RNA arrays including the substrate preparation, incorporation of the flexible PEG linker, and most importantly, the RNA strand surface density optimal for each of the three key applications discussed below.
Sequence fidelity and length. Recent work on gene assembly in our laboratory revealed a high degree of sequence fidelity (1.63 errors per kb) for the DNA molecules fabricated on our MAS arrays [34]. It is thus possible, or even likely, that the RNA arrays already have sufficient sequence accuracy to accomplish the various applications mentioned in this proposal. Accordingly, we do not anticipate spending much time further optimizing the fidelity. However, a good measurement of sequence accuracy is required to inform our decisions and to aid in optimization experiments, if they become necessary. The sequence fidelity of DNA arrays on surfaces is usually estimated based upon the fluorescence signal obtained in hybridization experiments. Although expedient, hybridization is not the best means of evaluating sequence fidelity, as it is relatively tolerant of sequence mismatches. Therefore, we will employ the much more accurate method of Sanger sequencing, as follows. In order to cleave the synthetic RNAs from the surface we will use the commercial reagent Thiol-Modifier C6 S-S from Glen Research to incorporate an internal disulfide bond in the synthesis of the RNA primer, which may then be cleaved from the surface using dithiothreitol. If a problem is encountered with this cleavage strategy, there are several other approaches that can be employed for release of the RNA molecules. These include using a free RNA primer for transcription, rather than a surface-bound primer; using RNase H for directed cleavage; or reverse-transcribing the RNAs on the surface into DNA. The released RNAs will be reverse transcribed into DNAs using specific DNA primers flanking the regions to be analyzed, and the DNA copies will be cloned into plasmids for Sanger sequencing. We employed a similar strategy to evaluate sequence fidelity for work described in our recent Angewandte Chemie paper on RNA-mediated gene assembly [34]. We will analyze the sequence fidelity of both the DNA grown on the MAS array and the RNA strands created from it. If fidelity or length analyses reveal inadequate RNA quality, then we will work to improve the RNA synthesis using different extension conditions (e.g. salt and manganese concentration) and by evaluating different commercially available RNA polymerases (e.g. T7, T3, and Durascribe (a T7 mutant available from Epicentre Technologies that has been developed for use with modified ribonucleotides)).
Key Applications. The utility of RNA array technology will depend on the information that it is able to provide. Some may feel that the era of high density array technology has passed, given the advent of next generation sequencing and its revolutionary impact on experimental design (e.g. the replacement of ChIP-chip by ChIP-Seq). The importance of RNA array technology thus lies in its ability to provide important information that is not more readily obtained in other ways. We describe below two important applications of RNA array technology that explore, respectively, the identification and characterization of fluorescent RNA mimics of GFP, and determination of the binding specificity of RNA-binding proteins.
Identification and Characterization of fluorescent RNA mimics of GFP. Fluorescent reagents continue to have a profound impact on cell and molecular biology with well over 2000 new articles a month documented in PUBMED. Scientist's ability to track, localize, image and quantify molecules of interest in complex environments such as the cell cytoplasm or nucleus is steadily improving as the diversity of fluorescent reagents grows, concomitant with the increased availability of low-cost laser sources providing a wide variety of wavelengths, efficient optical filters and light-collection optics, and high-sensitivity detectors. One reagent in particular (Green Fluorescent Protein, GFP) has had such an amazing impact that its discoverers and developers, Osamu Shimomura, Martin Chalfie and Roger Y. Tsien, were awarded the 2008 Nobel Prize in Chemistry for its development. GFP along with its variously colored fluorescent cousins is remarkable because it is a naturally fluorescent protein molecule that can be expressed in a living system (e.g. a cell) either by itself or as a fusion protein with little or no cytotoxicity and brilliant fluorescence. The family of GFP-like proteins can be employed in concert to visualize many different molecules simultaneously.
A fascinating new set of RNA reagents with properties quite similar to GFP was recently described by the Jaffrey lab [20, 21]. These reagents, referred to as "RNA mimics of GFP," are RNA molecules rather than proteins. The fluorophore in GFP is formed from three adjacent residues Ser65, Tyr66, and Gly67, which undergo an autocatalytic intramolecular cyclization to yield 4-hydroxybenzlidene imidazolinone (HBI). This molecule, normally nonfluorescent, becomes fluorescent when it interacts with folded GFP, which holds it in a manner that disrupts its molecular motions, and thereby closes down excited state relaxation pathways other than fluorescence. The "RNA mimics" are RNA aptamers that were selected using the SELEX process [15, 49] for binding to this or other similar molecules, and by binding it, also convert it from a non-fluorescent species to a fluorescent species. The aptamer sequence can be inserted into a gene of interest to yield a "fusion" RNA comprising the natural RNA and the aptamer tag; addition of the normally non-fluorescent and cell-permeable chromophore to the cells causes the aptamer tag to fluoresce upon binding, allowing direct fluorescence visualization of the associated RNA species within the cell! As with GFP, these RNA mimics can be expressed within cells and variants have been developed to fluoresce in different colors.
Table 1. Photophysical and binding properties of RNA-fluorophore complexes
(adapted from Table S1 of Ref. 20).
Although a fascinating and important new concept, these "RNA mimics of GFP" are not yet perfect. In particular, they are not yet adequate for monitoring low abundance RNAs in vivo. The best RNA mimic reported in the Jaffrey paper is referred to there as 24-2, or "Spinach." This aptamer complexes with the fluorophore DFHBI (3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), commercially available from Lucerna Technologies), whose photophysical and binding properties are given in Table 1. Comparing the free and complexed form, the molar extinction coefficients differ by a factor of two, and the fluorescence quantum yield by a factor of 1000. Thus there will be a background fluorescence level of 1 part in 2000 from the uncomplexed fluorophore, relative to the complexed fluorophore. Accordingly, if the abundance of the RNA to be monitored is more than ~2000-fold lower than the concentration of free fluorophore employed, the RNA will be difficult to discern over background. The reported KD for "Spinach" is 537 nM, and accordingly relatively high concentrations of 10 uM free fluorophore are utilized for visualization experiments [20]. Thus RNAs present at concentrations below ~5 nM will be near or below the fluorescence background level. In a hypothetical spherical cell 10 microns in diameter, 5 nM corresponds to 1500 copies per cell, a relatively high abundance RNA species. Hence the difficulty in monitoring less abundant species. As the photophysical properties of the DFHBI-Spinach complex are already quite excellent (reasonably high extinction coefficient and fluorescence quantum yield), the most obvious area for improvement is in the area of binding constant – if a thousand-fold increase in binding affinity could be obtained, this would allow a corresponding reduction in the concentration of fluorophore, and hence background level, needed for the assay.
Figure 4. Secondary structure predictions for aptamers 24-2 (Spinach) and 24-2-min by mFold, adapted from Figure S4C of Ref. 20.
Figure 4A shows the secondary structure predicted by mFold (http://mfold.rna.albany.edu) [50] for "Spinach". Jaffrey and colleagues explored alterations to this sequence in their paper and used this information to come up with a minimal size variant called "24-2-min" which exhibits 95% of the fluorescence of the full-length sequence but is 18 nt shorter than 24-2 (Figure 4B). Interestingly, all of the changes that gave rise to increased fluorescence were located in one region of the structure, as indicated by the green positions in Figure 4A. We propose to extend the study of Jaffrey et al, exploring the effects of changes in the region identified in that study as being the one area of the structure in which changes led to increased fluorescence. The 786,000 features that can be synthesized in parallel on the MAS will allow us to make all combinatorial variants at 9 base positions (49 = 260,000). We will make RNA arrays containing all possible variants of 9 bases on one side of the hairpin, or on the opposite side of the hairpin, and either varying the opposing strand to maintain base complementarity, or not (4 designs in total). Thus in four experiments we will sample over a million sequence variants in the critical region of the structure for their effect upon fluorescence yield. Each of these will also be evaluated for the effect upon fluorophore association constant. To do this the RNA arrays will be exposed to the fluorophore at various concentrations and imaged by a fluorescence scanner. This approach will permit measurement of both the equilibrium binding strength [16, 17] as well as a comparative measure of the fluorescence produced by each RNA (relative to the other RNAs on the surface). Arrays will be imaged using our GENETAC UC4X4 4-color laser scanner, which we have used for high-resolution fluorescence imaging of biomolecule arrays for many years.
We will perform similar experiments to explore the possibility of finding tighter-binding variants of 24-2 min; again we will follow the pioneering experiments of Jaffrey et al, who showed that the sequences within the short hairpins are critical to binding (red bases in Figure 4A) – happily, the short lengths of these hairpins are within the range that can be explored with the achievable MAS combinatorial complexity. We will make all possible variants of 9 positions for each hairpin, and measure fluorescence intensity and binding coefficients by fluorescence imaging as described above. In this way we will find out, at a minimum, if the 24-2 min sequence is the optimum sequence within that combinatorial space; and if we are fortunate, we may find better sequences that confer advantages in sensitivity for in vivo imaging experiments, and thus provide important advantages for the in vivo imaging of specific RNA sequences. More generally, this important and interesting system will serve as a testbed for the development of RNA arrays as a platform for the discovery, characterization, and optimization of RNA ligands for small molecules.
Determining the binding specificity of RNA-binding proteins. Understanding gene expression has been a goal for researchers since it was discovered that DNA is the genetic material responsible for the production and heritability of discrete biological traits. It has become evident that a major stage in the regulation of gene expression is the control of protein translation through protein-RNA interactions. Post-transcriptional regulation involves many intertwined layers of mRNA processing such as polyadenylation, splicing, cellular translocation and ultimately degradation. The timing and location of each of these processes is controlled by interactions with specific proteins and/or protein complexes called RNA binding proteins (RBPs). The elucidation of these interactions, in particular the binding specificities of RNA binding proteins, is critical to understanding the mechanisms by which gene expression is regulated.
High-density RNA arrays provide a flexible and comprehensive tool to identify and study protein binding sites. We will evaluate the effectiveness of using an RNA array to determine all possible binding sequences of a commercially available prototype RNA-binding protein, PUM2. PUM2 is an extensively studied RNA binding protein in the PUF family of proteins. The significance of the PUF protein family has been established by their evolutionarily conserved binding domain (Pumilio-homology domain) [73] and their known involvement in stem cell maintenance and self renewal [74] and neuronal function [75]. PUF proteins generally bind the 3'-UTR of their target mRNA transcripts, at positions where the mRNA is single stranded. Furthermore, their binding does not appear to depend upon the recognition of RNA secondary structure, which will be conducive to the use of the RNA array that presents relatively short 40nt sequences capable of forming limited secondary structure [76]
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26. Nuwaysir, E.F.; Huang, W.; Albert, T.J.; Singh, J.; Nuwaysir, K.; Pitas, A.; Richmond, T.; Gorski, T.; Berg, J.P.; Ballin, J.; McCormick, M.; Norton, J.; Pollock, T.; Sumwalt, T.; Butcher, L.; Porter, D.; Molla, M.; Hall, C.; Blattner, F.; Sussman, M.R.; Wallace, R.L.; Cerrina, F.; and Green, R.D., "Gene expression analysis using oligonucleotide arrays produced by maskless photolithography." Genome Res, 2002, 12(11), 1749-1755.
27. Lockett, M.R.; Carlisle, J.C.; Le, D.V.; and Smith, L.M., "Acyl Chloride-Modified Amorphous Carbon Substrates for the Attachment of Alcohol-, Thiol-, and Amine-Containing Molecules." Langmuir, 2009, 25(9), 5120-5126. PMC2824164.
28. Lockett, M.R.; Shortreed, M.R.; and Smith, L.M., "Aldehyde-Terminated Amorphous Carbon Substrates for the Fabrication of Biomolecule Arrays." Langmuir, 2008, 24(17), 9198-9203. PMC2575013.
29. Lockett, M.R. and Smith, L.M., "Attaching Molecules to Chlorinated and Brominated Amorphous Carbon Substrates via Grignard Reactions." Langmuir, 2009, 25(6), 3340-3343. PMC2765564.
30. Lockett, M.R. and Smith, L.M., "Formation and Stability of Alkylthiol Monolayers on Carbon Substrates." The Journal of Physical Chemistry C, 2010, 114(29), 12635-12641. PMC2917760.
31. Lockett, M.R. and Smith, L.M., "Halogenation of Carbon Substrates for Increased Reactivity with Alkenes." Langmuir, 2010, 26(22), 16642-16646. PMCID Policy Exempt - not NIH funding.
32. Phillips, M.F.; Lockett, M.R.; Rodesch, M.J.; Shortreed, M.R.; Cerrina, F.; and Smith, L.M., "In situ oligonucleotide synthesis on carbon materials: stable substrates for microarray fabrication." Nucleic Acids Research, 2008, 36(1), e7. PMC2248760.
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34. Wu, C.-H.; Lockett, M.R.; and Smith, L.M., "RNA-Mediated Gene Assembly from DNA Arrays." Angewandte Chemie International Edition, 2012, 51(19), 4628-4632. PMC3422211.
35. Pease, A.C.; Solas, D.; Sullivan, E.J.; Cronin, M.T.; Holmes, C.P.; and Fodor, S.P.A., "Light-Generated Oligonucleotide Arrays for Rapid DNA-Sequence Analysis." Proceedings of the National Academy of Sciences of the United States of America, 1994, 91(11), 5022-5026.
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37. Sun, B.; Colavita, P.E.; Kim, H.; Lockett, M.; Marcus, M.S.; Smith, L.M.; and Hamers, R.J., "Covalent photochemical functionalization of amorphous carbon thin films for integrated real-time biosensing." Langmuir, 2006, 22(23), 9598-9605.
38. Guo, Z.; Guilfoyle, R.A.; Thiel, A.J.; Wang, R.; and Smith, L.M., "Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports." Nucleic Acids Research, 1994, 22(24), 5456-5465.
39. Chen, S.; Phillips, M.F.; Cerrina, F.; and Smith, L.M., "Controlling Oligonucleotide Surface Density in Light-Directed DNA Array Fabrication." Langmuir, 2009, 25(11), 6570-6575. PMC2693259.
40. Peelen, D. and Smith, L.M., "Immobilization of Amine-Modified Oligonucleotides on Aldehyde-Terminated Alkanethiol Monolayers on Gold." Langmuir, 2004, 21(1), 266-271.
41. Lockett, M.R. and Smith, L.M., "Fabrication and Characterization of DNA Arrays Prepared on Carbon-on-Metal Substrates." Analytical Chemistry, 2009, 81(15), 6429-6437. PMC2797128.
42. Burnett, J.C. and Rossi, J.J., "RNA-based therapeutics: current progress and future prospects." Chem Biol, 2012, 19(1), 60-71. 3269031.
43. Hafner, M.; Landthaler, M.; Burger, L.; Khorshid, M.; Hausser, J.; Berninger, P.; Rothballer, A.; Ascano, M., Jr.; Jungkamp, A.-C.; Munschauer, M.; Ulrich, A.; Wardle, G.S.; Dewell, S.; Zavolan, M.; and Tuschl, T., "Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP." Cell, 2010, 141(1), 129-141.
44. Hafner, M.; Landthaler, M.; Burger, L.; Khorshid, M.; Hausser, J.; Berninger, P.; Rothballer, A.; Ascano, M.; Jungkamp, A.-C.; Munschauer, M.; Ulrich, A.; Wardle, G.S.; Dewell, S.; Zavolan, M.; and Tuschl, T., "PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins." J Vis Exp, 2010, (41), e2034.
45. Castello, A.; Fischer, B.; Eichelbaum, K.; Horos, R.; Beckmann, B.M.; Strein, C.; Davey, N.E.; Humphreys, D.T.; Preiss, T.; Steinmetz, L.M.; Krijgsveld, J.; and Hentze, M.W., "Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins." Cell, 2012, 149(6), 1393-1406.
46. Capodici, J.; Karikó, K.; and Weissman, D., "Inhibition of HIV-1 Infection by Small Interfering RNA-Mediated RNA Interference." The Journal of Immunology, 2002, 169(9), 5196-5201.
47. Aurup, H.; Williams, D.M.; and Eckstein, F., "2'-Fluoro-2'-Deoxynucleoside and 2'-Amino-2'-Deoxynucleoside 5'-Triphosphates as Substrates for T7 Rna-Polymerase." Biochemistry-Us, 1992, 31(40), 9636-9641.
48. Campbell, Zachary T.; Bhimsaria, D.; Valley, Cary T.; Rodriguez-Martinez, Jose A.; Menichelli, E.; Williamson, James R.; Ansari, A.Z.; and Wickens, M., "Cooperativity in RNA-Protein Interactions: Global Analysis of RNA Binding Specificity." Cell Reports, 2012, 1(5), 570-581.
49. Tuerk, C. and Gold, L., "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase." Science, 1990, 249(4968), 505-10.
50. Zuker, M., "Mfold web server for nucleic acid folding and hybridization prediction." Nucleic Acids Res, 2003, 31(13), 3406-15. 169194.
51. Strack, R.L.; Disney, M.D.; and Jaffrey, S.R., "A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA." Nat Methods, 2013, doi: 10.1038/nmeth.2701.
52. Jiang, Q.H.; Wang, Y.D.; Hao, Y.Y.; Juan, L.R.; Teng, M.X.; Zhang, X.J.; Li, M.M.; Wang, G.H.; and Liu, Y.L., "miR2Disease: a manually curated database for microRNA deregulation in human disease." Nucleic Acids Research, 2009, 37, D98-D104.
53. Garzon, R.; Calin, G.A.; and Croce, C.M., "MicroRNAs in Cancer." Annu Rev Med, 2009, 60, 167-179.
54. Bartel, D.P., "MicroRNAs: Target Recognition and Regulatory Functions." Cell, 2009, 136(2), 215-233.
55. Guo, H.; Ingolia, N.T.; Weissman, J.S.; and Bartel, D.P., "Mammalian microRNAs predominantly act to decrease target mRNA levels." Nature, 2010, 466(7308), 835-U66.
56. Lewis, B.P.; Shih, I.H.; Jones-Rhoades, M.W.; Bartel, D.P.; and Burge, C.B., "Prediction of mammalian microRNA targets." Cell, 2003, 115(7), 787-798.
57. Lal, A.; Navarro, F.; Maher, C.A.; Maliszewski, L.E.; Yan, N.; O'Day, E.; Chowdhury, D.; Dykxhoorn, D.M.; Tsai, P.; Hofmann, O.; Becker, K.G.; Gorospe, M.; Hide, W.; and Lieberman, J., "miR-24 Inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to "seedless" 3'UTR microRNA recognition elements." Mol Cell, 2009, 35(5), 610-25. 2757794.
58. Brodersen, P. and Voinnet, O., "Revisiting the principles of microRNA target recognition and mode of action." Nat Rev Mol Cell Bio, 2009, 10(2), 141-148.
59. Shin, C.; Nam, J.W.; Farh, K.K.H.; Chiang, H.R.; Shkumatava, A.; and Bartel, D.P., "Expanding the MicroRNA Targeting Code: Functional Sites with Centered Pairing." Molecular Cell, 2010, 38(6), 789-802.
60. Elkayam, E.; Kuhn, C.D.; Tocilj, A.; Haase, A.D.; Greene, E.M.; Hannon, G.J.; and Joshua-Tor, L., "The Structure of Human Argonaute-2 in Complex with miR-20a." Cell, 2012, 150(1), 100-110.
61. Tan, G.S.; Garchow, B.G.; Liu, X.; Metzler, D.; and Kiriakidou, M., "Clarifying mammalian RISC assembly in vitro." Bmc Molecular Biology, 2011, 12.
62. Kawamata, T. and Tomari, Y., "Making RISC." Trends in biochemical sciences, 2010, 35(7), 368-376.
63. Wang, B.; Li, S.; Qi, H.H.; Chowdhury, D.; Shi, Y.; and Novina, C.D., "Distinct passenger strand and mRNA cleavage activities of human Argonaute proteins." Nature structural & molecular biology, 2009, 16(12), 1259-66.
64. MacRae, I.J.; Ma, E.; Zhou, M.; Robinson, C.V.; and Doudna, J.A., "In vitro reconstitution of the human RISC-loading complex." Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(2), 512-517.
65. Rivas, F.V.; Tolia, N.H.; Song, J.J.; Aragon, J.P.; Liu, J.D.; Hannon, G.J.; and Joshua-Tor, L., "Purified Argonaute2 and an siRNA form recombinant human RISC." Nature structural & molecular biology, 2005, 12(4), 340-349.
66. Garzon, R.; Marcucci, G.; and Croce, C.M., "Targeting microRNAs in cancer: rationale, strategies and challenges." Nature Reviews Drug Discovery, 2010, 9(10), 775-789.
67. Vergoulis, T.; Vlachos, I.S.; Alexiou, P.; Georgakilas, G.; Maragkakis, M.; Reczko, M.; Gerangelos, S.; Koziris, N.; Dalamagas, T.; and Hatzigeorgiou, A.G., "TarBase 6.0: capturing the exponential growth of miRNA targets with experimental support." Nucleic Acids Research, 2012, 40(D1), D222-D229.
68. Maragkakis, M.; Reczko, M.; Simossis, V.A.; Alexiou, P.; Papadopoulos, G.L.; Dalamagas, T.; Giannopoulos, G.; Goumas, G.; Koukis, E.; Kourtis, K.; Vergoulis, T.; Koziris, N.; Sellis, T.; Tsanakas, P.; and Hatzigeorgiou, A.G., "DIANA-microT web server: elucidating microRNA functions through target prediction." Nucleic Acids Research, 2009, 37, W273-W276.
69. Ule, J.; Jensen, K.B.; Ruggiu, M.; Mele, A.; Ule, A.; and Darnell, R.B., "CLIP identifies Nova-regulated RNA networks in the brain." Science, 2003, 302(5648), 1212-1215.
70. Ule, J.; Jensen, K.; Mele, A.; and Darnell, R.B., "CLIP: A method for identifying protein-RNA interaction sites in living cells." Methods, 2005, 37(4), 376-386.
71. Wang, Z.; Tollervey, J.; Briese, M.; Turner, D.; and Ule, J., "CLIP: Construction of cDNA libraries for high-throughput sequencing from RNAs cross-linked to proteins in vivo." Methods, 2009, 48(3), 287-293.
72. Konig, J.; Zarnack, K.; Rot, G.; Curk, T.; Kayikci, M.; Zupan, B.; Turner, D.J.; Luscombe, N.M.; and Ule, J., "iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution." Nature Structural & Molecular Biology, 2010, 17(7), 909-U166.
73. Galgano, A.; Forrer, M.; Jaskiewicz, L.; Kanitz, A.; Zavolan, M.; and Gerber, A.P., "Comparative Analysis of mRNA Targets for Human PUF Family Proteins Suggests Extensive Interaction with the miRNA Regulatory System." PLoS One, 2008, 3(9).
74. Lee, M.H.; Hook, B.; Pan, G.; Kershner, A.M.; Merritt, C.; Seydoux, G.; Thomson, J.A.; Wickens, M.; and Kimble, J., "Conserved regulation of MAP kinase expression by PUF RNA-binding proteins." Plos Genet, 2007, 3(12), 2540-2550.
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I've been pretty quiet as I plug through marathon training, I'm now entering week 8 which means that I'm almost half way through this crazy thing.
Overall training has been going well! It's been a sweaty and hot and muggy summer in Massachusetts and I've been drinking everything in sight after each run but we all groove on and muster through it. Some runs feel great and others feel like an absolute slogfest, it happens, especially when you're training in the summer.
The toughest part I've had with my training so far was during week 4. The weekday runs went well but when it got to Saturday, my body simply wouldn't push through a 6 mile run. It was hot, it was very windy, and I hadn't been getting enough sleep. I ran/walked the 6 miles and felt so frustrated and defeated, my legs felt completely empty. Blech! Going into Sunday's long run in week 4 with that horrible run in my mind set me up for failure, especially after another night where I wasn't sleeping well. My 11 mile long run turned into 6 awful miles and they were run/walk again. 100% failed training week. I'm calling it out though because I think it's important to mention that not every training run is going to be perfect, there WILL be bad days. I think it's good to acknowledge, learn whatever you can from it, and make sure it doesn't affect the rest of your training. Because how you react is when you and your training are tested. Every week is a new opportunity and I came back in week 5 and completed all of the mileage and felt great – always need to get back on that metaphorical horse, people!
As for the rest of the training weeks, I've been running the miles that are pre-determined for each day using Hal Higdon's training guide. Even though I've been completing my mileage I still get nervous, especially before my weekly long run. Part of me knows the routine, part of me feels like I'm teaching myself all over. Where I'm struggling the most is with my speed. Ideally I'd like to be running my longer runs around an 8:10 pace. I've been logging my shorter runs around an 8:00 pace which is pretty standard for me, but as soon as I start to up the miles my long training runs come in at around 8:40 which is pretty much where I was last year. I know the heat and humidity have been slowing me down, and I work some pretty serious hills into my long runs which Chicago doesn't have, so I'm hoping with cooler temps and a flatter course I'll be able to compensate for the speed issue – but I also don't think that's enough so I've been trying to work in some speed work on my short weekday runs.
My cross training day is for yoga which I'm pretty sure isn't the typical recommended cross training but that's alright. I'm loving the Monday stretch sessions! In my previous post I mentioned that Pure Barre was integrated into my training, that's no longer the case since some of the exercises began to aggravate my IT band and I'm not taking any extra risk. Instead, I have strength training exercises that I typically try to work in at least 3-4 times a week. In addition to the strengthening exercises, I've been getting grastin treatments on my left and right IT bands every 1-2 weeks which has helped immensely to relieve a lot of tension that had built up.
What an update! I need to post more frequently because while writing this I went off in about 6 other tangents about training and had to keep deleting things. So much to say, who knew. More to come!
What are you training for? Any plans for the fall race season?


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I have been selling the gemstone lamps and crystal accessories like crazy! Apparently I'm not the only one who loves them. Here are the latest:Gorgeous quartz chunk, great for table top, can be mounted to an acrylic base. [Read more…]
Bejewel Your Home With Crystal Accessories
February 20, 2012 By 2 Comments


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Modern Infrastructure
- NewsExamining hybrid data center decision points
- NewsIs VMware Horizon Suite the last word on end-user management?
- FeatureCan traditional server vendors survive the cloud push?
- FeatureServer vendors must innovate to stem bleeding
- FeatureA variety of options make data center expansion difficult
- FeatureScaling up your data center when demand exceeds computing capacity
- FeatureLooking to the future of legacy applications in the cloud
- FeatureCloud computing practices represent a fundamental shift in IT
- FeatureGet to know SoMoClo: Social, mobile and cloud technologies
- OpinionCross-cutting concerns may be the next big step for IT
- ColumnColocation facility selection requires careful planning
- OpinionHow to control desktops: Swap Windows desktop admin rights for iPads
iSTOCK/GETTY IMAGES
Can traditional server vendors survive the cloud push?
In the era of virtualization and cloud computing, can the traditional server vendors survive extinction?
- Alex Barrett, Modern Infrastructure Editor-in-Chief
Ten years from now, will we look back on the traditional server vendors like IBM, HP and Dell as dinosaurs that could not withstand the giant asteroid that is public cloud? Or will we marvel at their resourceful ability to adapt -- and survive -- in the face of rapidly changing conditions?
Can HP, IBM and Dell survive the cloud?
Part 1: Can traditional server vendors survive the cloud push?
That's the $54.9 billion question -- which is the amount that organizations spent on servers in 2012, according to IDC, with IBM, Hewlett-Packard Co. and Dell Inc. garnering a combined 74.3% of that market.
Public clouds come on the heels of server virtualization, which has already been a doozy for server unit shipments and revenues. In 2002, the ratio of physical to logical servers was almost 1:1, at 4.4 million and 4.5 million units shipped worldwide, according to IDC, and revenue was just shy of $50 billion. That heyday would soon be over: A decade later, revenue had increased a modest 11.5% to $54.9 billion, while the number of physical servers shipped had increased by 84% to 8.1 million units, and logical servers had increased a whopping 497%, to 22.4 million.
Shifting workloads from on-premises virtualized servers to the public cloud stands to be even worse for server vendors, since a workload running in the public cloud requires no infrastructure purchase. As VMware CEO Pat Gelsinger said, speaking at the company's Partner Exchange in February: "If a workload goes to Amazon, you lose, and we have lost forever."
But traditional server vendors certainly haven't given up on selling infrastructure to the enterprise. "They're going to fight," said Kuba Stolarksi, research manager for enterprise servers at IDC. "But a lot can happen in five years," he added, and come 2017, server offerings from the traditional vendors might look very different than they do today.
The cloud-first imperative
One small business has shifted the brunt of its processing from on-premises to the public cloud.
Mosaik Solutions in Memphis, Tenn. collects mobile network coverage information that it delivers as geospatial, analytical, creative and Web data. The company relied on a combination of on-premises and colocation systems from its founding in 1989 until 2008, when it began experimenting with Amazon Web Services (AWS). In 2009, it increased its AWS spend by 1,000%. In 2010, it discontinued its use of colocation and increased its AWS spend another 500%, and another 250% the year after.
A rising tide lifts all boats
While public cloud is cannibalizing some workloads, you could also make the case that it is simply meeting demand that didn't used to exist.
"It's like the [former IBM CEO Thomas J. Watson] quote: 'I think there is a world market for maybe five computers,'" said Jonathan Eunice, principal IT adviser at Illuminata Inc. Back in 1943, when Watson reportedly uttered those words, our collective imagination could not conceive of the need for more compute power. It's only in retrospect that we understand how silly that statement is.
The same dynamic is in play today.
"Our compute needs and desires are always growing faster than Moore's Law. We always want more," Eunice said.
In recent years, new compute resources have gone in large part to tackling our new interest in data analysis, Eunice said.
"We've been on a multi-decade march from transactions being the most important to analytics being the most important," he said. These days, "analyzing and figuring things out has become more important than just keeping your books. There just aren't that many books."
Today, the firm runs the lion's share of its processing on Amazon's Elastic Compute Cloud (EC2) -- an average of 335 EC2 compute units and 668 GB of memory on AWS, compared with just 64 cores and 384 GB memory in-house, mainly for its "customer-facing" applications, said Daniel Bozeman, solutions architect for the firm. It has not purchased a new server since 2011.
That's great for Amazon, but it's decidedly bad news for Cisco Systems and Dell, from which Mosaik last purchased servers. Nor is the firm particularly loyal to any one server shop.
"We go wherever the price is right," Bozeman said.
The firm is, however, looking to reduce its EC2 spend, Bozeman said, which it will do by modernizing and consolidating the services running on EC2, increasing its use of Amazon EC2 Reserved Instances, and by offloading a subset of its operations -- test, development and staging -- back in-house to a Dell server running Eucalyptus, an AWS-compatible private cloud stack.
"At least, that's the idea," Bozeman added. "We'll see how it goes."
Custom-made is in demand
Meanwhile, service providers, with their razor-thin margins and copious in-house technical talent, are increasingly backing off their relationships with tier-one server vendors -- if they ever had a relationship at all. Google, for instance, builds its own servers, and is reportedly the fifth-biggest customer of Intel server chips, according to a September 2012 issue of Wired.
The value of the support that tier-one server vendors bring to enterprises doesn't always carry over to service providers, said John Considine, CTO at Verizon Terremark, the cloud and managed services provider that has a mix of tier-one, commodity and custom gear.
"For enterprises, the real value is in service and support over the lifetime of a server," Considine said. "We see a lot less value in that support model."
Service providers also cite the customization that comes from building their own servers as the main reason for the decrease in traditional server purchases. For example, SoftLayer Technologies, the hosting-cum-cloud provider, has a long-standing relationship with contract manufacturer Supermicro, which has supplied the 100,000 servers SoftLayer has under management.
Designing servers with Supermicro "allows us to fine-tune that server to meet the application stack," by manipulating elements such as CPU, network, hard drives and memory, said Marc Jones, SoftLayer vice president of product innovation.
About the author: Alex Barrett is the editor in chief of Modern Infrastructure.


text:
1 tube of choice - I used the wonderful artwork of Joanna Bromley which can be purchased from PSP Tube Stop. You must have a license to use.
Font of choice - I used Cuivrerie here
Gem mask 71 here
Plug Ins used:
Eyecandy 5: Nature - Snow Drift
"As the Wind Blows" is a tagger size scrap kit created by me (HorsePlay's Pasture Designs) and can be purchased at any of my stores here. My list of stores can be found in the top left column.
This tutorial assumes you have a working knowledge of PSP.
~♥~ Let's get started ~♥~
Open up a 700 x 700 blank canvas.
Flood fill white.
Copy and paste El 14 as a new layer.
Resize 70%
Duplicate the frame.
Move both frames into position - see my tag for reference.
To each frame:
Click inside of it with your Magic Wand.
Selections/Modify/Expand by 4.
New Raster layer below the frame.
Copy and paste Paper 4 Into Selection.
Add the following elements below both frames and dont' worry about what overhangs the frame edges. We'll clean that up in a bit.
El 56: Resize 60%/Duplicate/Mirror/Move below the top frame
El 55: Resize 55%
El 6: Resize 30%/Duplicate as many times as you like and move them into positions you like below both frames
Highlight the paper to the top frame.
Click anywhere outside of it with your Magic Wand.
Highlight each element below the frame and tap the delete key.
Highlight the bottom frame.
Repeat the above step.
Add the rest of your elements. I used:
El 49: Resize 60%
El 47: Duplicate
El 26: Resize 70%/Mirror
El 67
El8: Resize 70%/Duplicate/Flip/Mirror
El 61: Resize 70%/Duplicate/Mirror
To each frame apply Eyecandy 5: Nature -Snow Drift
Settings Tab:
Small, First Flakes
Basic Tab:
Lighting Tab:
Default Settings
Highlight your white background.
New Raster layer.
Select All.
Copy and paste Paper 14 Into Selection.
Apply the mask.
Resize 110%
Merge Group.
Add your copyrights.
Color Pallet:
Foreground and Background: #70130e
Make your font tool active.
Stroke 2.1
Type your name.
Convert to Raster layer.
Select All/Float/Default.
Selections/Modify/Expand by 2.
New Raster layer below your name.
Flood fill with #fbf9fb
Highlight your original name (the one that's in red)
Give it a slight Inner Bevel 3x's:
Merge Visible both names.
Copy and paste El 11 as a new layer.
Resize 35% and decorate your name with it.
I hope you enjoyed this tutorial as much as I did creating it for you.
I'd really love to see your results.
Email me!