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HTML5 removes the 'frameBorder' and 'scrolling' attributes for the iframe element. So in order to have a validating HTML5 document, you're not allowed to use them.
In theory, CSS can pick up the task instead, so the HTML5 changes makes sense on the surface. HTML5 also introduces a new 'seamless' attribute to achieve what most people are trying to achieve anyway.
However, it all goes down hill from here…
IE does not support changing them via CSS, either via setting styles on the iframe element or by setting styles on the html/body elements of the frame contents.
And, no browsers support the 'seamless' attribute yet (so next time you hear someone say Chrome or Firefox supports HTML5, remember they're misinformed – there are actually quite a few HTML5 features not supported by any browser yet).
Unfortunately IE is so buggy you're not allowed to set 'frameBorder' or 'scrolling' after the iframe has rendered (you can do it for a new progmatically created iframe though).
One could progmatically re-generate the iframes, but that would result in the iframes double-loading, with pretty horrible consequences.
So the solution is missing: there is no choice but to produce invalid HTML5 if you want to support all browsers and use iframes.
Some might say iframes are a bad idea, but I think the opposite – if you need a small part of the page to work independently, you shouldn't (for accessibility reasons) rely on Javascript to manipulate it individually. I am reliably informed many (or all?) braille-readers do not run Javascript. Refreshing the whole page is not good either (it's also an accessibility issue, and just plain annoying).
So that kind of sucks, but in the end, validation is not the most important thing in the world. There's one simple trick we can use – only feed the invalid HTML to IE, via server-side detection. Then the compliant browsers and tools won't be tainted.
Another HTML5 problem…
IE (prior to version 9) will not understand CSS applied to tags it does not recognise, so you can't use the new HTML5 block-level tags because IE will default to them being inline and you can't style them with CSS. Fortunately there's a workaround:
HTML5 enabling script
Moving forward with Composr
ocPortal has been relaunched as Composr CMS. ocPortal 9 is superseded by Composr 10.
Head over to compo.sr for our new site, and to our migration roadmap. Existing ocPortal member accounts have been mirrored.
HTML5 and iframes -- a mess
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Seiwert, Franz W. (1894-1933). Medium: oil on card. Date: 1926. In the foreground are four men; the art dealer Aloys Faust; Josef Haubrich, the art collector from Cologne; art and labour unite in one painting; Provenance: Hamburger Kunsthalle, Hamburg, Germany. Photographic rights held by The Bridgeman Art Library.
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February 25, 2014
"To live in the world without becoming aware of the meaning of the world is like wandering about in a great library without touching the books."
– Dan Brown
I used to think of the time when people realize that they were the ones who made life today possible. In years of yellow fever, of political dynasties, of KKKs, of Media interpolation, of 20++ years of celebrating EDSA People Power I, would there be a book emphasizing the role of every Filipino in the movement? None, of course. Some got published but died the natural propagandistic death. The freedom we know is not the freedom we earned. We hail the great vultures that eat the meat of our labor. We keep on electing their kinds because of the belief that they are sent by God to free us, where in fact they are the ones pulling us to hell.
EDSA Revolution is about the people, the Filipino people. It was not just about Ninoy and Cory by the way. They are the powerful and the power-trigger that's why they got noticed. But the focal point shakes from the gizzards of every mighty patriot of this country, who expressed their sincere love for the motherland.
They're not just the Aquinos who have been walking in EDSA then, but the asphalted roads and pavements were kissed by the feet of every single Filipino who started the move toward a better Philippines.
Remember how it all started. The glory does not only bound the powerful and the popular, it is a piece that requires fair share to every Filipino who stood.
EDSA People Power is not just for the Aquinos. It is the day, written in history, acknowledging that once in civilization of the universe, Filipino made their race a remarkable one. And that single act of valor for democracy will be forever remembered by the world.
Remember that. Filipino made it.


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MOVIE SYNOPSIS [ General Audiences ] Being a creation directed by Jeff Leroy, this feature film in English (subtitles) was presented in 2015. This film written by Leslie Morris is an original production of USA. You can watch Dracula's Sorority Sisters online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2015.
Rating: 5 by 10 users./
Runtime: 100 minutes
Alejandra Morin, Antoinette Mia Pettis, Nicole Laino, Kelly Erin Decker, Robert Rhine, Missy Martinez, Jacqui Holland, Al Smithie, Michael Dougherty, Ryan Izay, Michael Beardsley, Shawn C. Phillips and Tom Newth.
100-minute film (full movie) directed by Jeff Leroy.
The original audio of Dracula's Sorority Sisters has dialogues in Dolby Digital, it was produced in USA.
It premiered in cinemas or television in 2015.
Screenplay written by Leslie Morris.
Tom Newth.
David S. Sterling and Laura Azevedo.
This film was produced and / or financed by Sterling Entertainment.
Composed by Andrew Crandall.
Without commercial breaks, the full movie Dracula's Sorority Sisters has a duration of 100 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, Dracula's Sorority Sisters in FULLTV Movies.


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Other Services
Pulmonary Function Test
Hearing Test
Joint Injection
Home Sleep Study
Select Dermatology Services – A dermatology visit may be focused on a specific skin condition or involve a full screening exam for skin cancer. Patients with newly diagnosed skin cancer will be referred to a dermatologist and/or a surgeon for follow up treatment or monitoring. If there is a history of skin cancer it is recommended that you be followed by a dermatologist.
Shared Medical Appointment – This effective approach blends the power of group dynamics with personalized medicine. Patients receive individualized care in a confidential group setting.
Home Visits – We offer this as an alternative to an office visit for our patients that are homebound.


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Witty and thought provoking, two Vatican astronomers shed provocative light on some of the strange places where religion and science meet.
"Imagine if a Martian showed up, all big ears and big nose like a child's drawing, and he asked to be baptized. How would you react?" - Pope Francis, May 2014.
Pope Francis posed that question - without insisting on an answer! - to provoke deeper reflection about inclusiveness and diversity in the Church. But it's not the first time that question has been asked.
Brother Guy Consolmagno and Father Paul Mueller hear questions like that all the time. They're scientists at the Vatican Observatory, the official astronomical research institute of the Catholic Church. In Would You Baptize an Extraterrestrial? they explore a variety of questions at the crossroads of faith and reason: How do you reconcile the The Big Bang with Genesis? Was the Star of Bethlehem just a pious religious story or an actual description of astronomical events? What really went down between Galileo and the Catholic Church - and why do the effects of that confrontation still reverberate to this day? Will the Universe come to an end? And - could you really baptize an extraterrestrial?
With disarming humor, Brother Guy and Father Paul explore these questions and more over the course of six days of dialogue. Would You Baptize an Extraterrestrial will make you laugh, make you think, and make you reflect more deeply on science, faith, and the nature of the universe.
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A primer on Catholic doctrine
- zandperl
Faith and Reason Kiss
Yes, because it shows religion and science in their true light as complimentary disciplines and not mutually exclusive.
It was casual, witty, and yet deeply insightful
Aside from one passing reference to Teilhard de Chardin , which they ascribed no doctrinal weight to, I was pleased with the whole performance. I love when authors narrate, it is so much more real and you get that emotion that you sometimes miss in print.
- Cory


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Perry backs wife's 'rough month' campaign comment
Mark B Eureka, CAOct. 15, 2011 2:44 p.m.
Perry must have thought all the competitors would melt away when he flashed his oil money stash. It's a tough old business, pal. Just ask Bob Bennett.
Little Child like me... Highland, UTOct. 15, 2011 11:55 a.m.
Considering that "The Church of Jesus Christ of Latter-day Saints" (commonly called Mormon) is about the 4th largest church in the United States, I would assume that Perry may have offended a large portion of the voters in the U.S.A.
I was favorable toward Perry for his day of Prayer, until his attempt to 'put down Romney', then, that made my decision for me. Now I am Romney all the way! He is a good man, and has raised a good family and this is something I admire very much!
atl134 Salt Lake City, UTOct. 15, 2011 2:12 a.m.
I don't remember stories about Perry's religion... I do remember a lot (especially on Maddow's show) about some of the pastors he invited to his prayer meeting thing and of course there's this recent flareup over Jeffress but I have a hard time thinking of anything about Perry's own reigion.
MoJules Florissant, MOOct. 14, 2011 11:48 p.m.
I so agree with you Hellooo :) Kind of the pot calling the kettle black. But honesty, I would not condone others going after his religion any more then his going after the Mormons. If Obama were 50% Muslum, and people found fault with that, I would not condone that.
Hellooo Salt Lake City, UTOct. 14, 2011 9:51 p.m.
Of course, this is from the candidate whose campaign supporter voice such tolerance and kindness to Mormons. Nice try, but I have a lot of trouble finding the abuse of which Mr. Perry's wife is referring. Most came because he took front runner position and his record and comments exposed positions on issues that voters do not like. But, a victim of religious intolerance really?


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Digital Download Stephanie Fowers 2014
Andrea Emmes's narration is perfect for this fluffy story aimed at Jane Austen fans. Emmes's high tones and fast pace capture the voice of Jane, an event coordinator who is launched into a comedy of romantic chaos and familial absurdities when she agrees to plan her friend's wedding. Emmes captures the haughtiness of the English in-laws-to-be and creates a wide range of caricatured voices for other characters, reflecting the lightheartedness of this romance. Emmes's enthusiasm keeps the listening dynamic, even as the author makes the somewhat laborious connections between the characters in Jane's life and those in Austen's novels. M.F. © AudioFile 2015, Portland, Maine [Published: APRIL 2015]
Digital Download Stephanie Fowers 2014
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Two different heights for counters.
industrial bar, industrial bar design, rustic bar design, rustic and industrial bar design in Australia, The Nelson- recycled timber bar counter - - italianbark 3
Wood Ceiling Polished Concrete Floor Exposed Brick Wall Light Fixtures Inspirational Design Loft Living Bar Warehouse Home Design Magazine
Create your own cozy cafe using string lights and a basic bistro set!
A very artsy and strong design perhaps could use some color but is overall very strong. Important areas are highlighted or are bolder while various typefaces add flare and variety. The overall layout is thought out and well organized.
These fashionable dining spots in New York City offer chic minimalist design…


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pizza in Ada, OK
Domino's Pizza908 Arlington Ctr, Ada, OK map
Visit your Ada Domino's Pizza today for a signature pizza or oven baked sandwich. We have coupons and spe... more
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Nathan Hansen
Primary tabs
Nathan Hansen has been a reporter and editor with the Farmington Independent and the Rosemount Town Pages since 1997. He is very tall.
Contact Email
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- 4 years 8 months
Author Content
Education has been on a quest to create schools where every student is successful, meeting and exceeding the standards, while achieving their highest aspiration. While this is a noble goal it is complicated by today's student who comes to school with widely different sets of knowledge, skills, talents, strengths, motivations and support.
What exactly do the Minnesota Wild have to do to put more tallies in the win column? This past off season they acquired star forwards Dany Heatley and Devin Setoguchi from the San Jose Sharks, which was quite a bold move for the Wild -- usually, new names on the ice are those "developing players" from the Houston Aeros. Why has this year been another horrible season for scoring goals? We fans need to send a message to the Wild's head honchos who have the ability to make a change.
Consider this: When the Great Depression began in 1930, Walter Klaus was 18 years old. It was 55 years ago that voters in rural Dakota County first elected Walter Klaus as their representative. On April 19, that man turned 100 years old. Klaus, a longtime Farmington resident who has lived in Hastings for the past four years, had a birthday party in his honor Saturday at Augustana Health Care Center.
I have something of a poster problem. I'll admit, as problems go, this falls somewhere on the low end of the scale. It's not a serious illness. It's not a family tragedy. It's not even as bad as being strapped into one of those Clockwork Orange machines and forced to watch reruns of Mike and Molly. Well, not quite. Still, it's a lot of posters. And I'm not quite sure what to do with them. This is not a new problem. It started maybe eight years ago, when I went through a bit of a concert-poster phase. I bought them at concerts.
The Farmington School District has a pretty good idea where it would like to be in five years. Now superintendent Jay Haugen is looking for some help figuring out exactly how to get there. Haugen presented a draft of the district's new strategic plan at an April 9 school board meeting, but he's turning to the public to provide some of the concrete steps to make the plan's seven overarching strategies reality. He's looking for a group of 150 to 200 volunteers from school staff and the community to work on the project over the next several months.
The media tells us the world is going to end, we're not good enough and everyone is crazy. What ever happened to optimism? The whole, "The world is your oyster," philosophy? In high school, being a pessimist isn't even an option. It's basically setting yourself up for failure. Whether it's about schoolwork, athletics or relationships, having an optimistic view on life can make the world of difference in any situation. High school can be a horrid breeding ground for pessimistic thoughts. Girls beat you down, guys beat you down and everyone seems to always have it out for somebody.
It starts, as these things often do, with a mad dash. Children scramble across four adjoining yards in search of the colorful plastic eggs that dot the grass and sit nestled in tree branches. Younger kids scour the nearby yards, while older hunters sprint to the far end of the field. It's a little bit noisy and a little bit chaotic, but it also looks like a lot of fun. Gina Meihofer is definitely enjoying herself, and she's not even involved in the hunt. She's the one who organized the event, which took place Thursday in a yard filled with neighbors, friends and relatives.
I've spent a lot of time in recent years riding my bike. Maybe you know that already. I tend to mention it a lot in this space because, well, I don't have a whole lot of interesting stuff going on. I've taken my bike on long rides and short rides, hilly rides and flat rides. I've gone on rides that ended with people being hauled away in an ambulance. I don't recommend that. It turns into a big hassle. Hills are kind of a pain too, now that I think about it. Through all of the years and all of the rides, though, I've never tried to commute by bike. There are reasons for that.
There are stories hidden away in Farmington homes. There are colorful ceramic plates handed down from grandmother to mother to daughter. There are meat cleavers that belonged to a long-ago ancestor who was a butcher. There are bits of the past tucked away in cabinets and chests and closets, and the organizers of an event this weekend at Faith United Methodist Church would like to hear all about them. The church's Women of Faith group will hold a brunch and antique appraisal event starting at 11 a.m. Saturday.
The latest plan for the Farmington School District's German program could shift the language entirely to the high school, a move that has at least one board member concerned about the long-term future for the class. Superintendent Jay Haugen presented a plan Monday that would move the first offering of German I to ninth grade. He hopes the move will make it easier to offer the class to what has been a shrinking population of interested students. About 40 students signed up for German I for the 2012-13 school year, but they are spread among two middle schools and Farmington High School.


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Error Fixes for Titanfall 2 PC- Black Screen, Crash, Lag, Dll Errors Fix
Titanfall, the successful FPS game from Respawn entertainment is now back into the market with the sequel Titanfall 2. The Titanfall was a huge success in the market with around 60+ awards to it's name. Now, the sequel is also getting positive reviews in the market after it's release. However, there are quite good number of negative reviews as well for the Titanfall 2. They are mostly because of the errors/ crashes and bugs that are encountered while installing/ running Titanfall 2 on PCs. To make your task easier, we have come up with the error fix guide for Titanfall 2.
First of all, we would like you to have a look at the system requirements of Titanfall 2. Just make sure that your PC stands above all the requirements of the game. Also, if you are running on a low end PC, just upgrade it as quickly as possible. Here's a guide for you recommending the best Graphics cards to buy in 2016.
System Requirements for Titanfall 2:
OS – Win 7/8/8.1/10 64bit
CPU – Intel Core i3-3600t or equivalent
HDD Free Space – 45GB
GPU – NVIDIA Geforce GTX 660 2GB, AMD Radeon HD 7850 2GB
DirectX – 11
Internet Connection – 512Kbps or faster
OS – Win 7/8/8.1/10 64bit
CPU – Intel Core i5-6600 or equivalent
HDD Free Space – 45GB
GPU – NVIDIA Geforce GTX 1060 6GB, AMD Radeon RX 480 8GB
DirectX – 11
Internet Connection – 512Kbps or faster
The requirements for recommended settings are pretty high. You would need atleast 16 gigs of RAM to maxout TitanFall 2. Okay, now let's get into the error fixes list for Titanfall 2.
# How to Fix Errors/ crashes and Bugs in Titanfall 2:
# Unlock 30 FPS cap in Titanfall 2: Workaround
There's a 30 FPS cap for Titanfall 2 for some people who are running on Nvidia Graphics cards. The probable workaround for this issue would be
- Turning on the adaptive Vsync in nvidia Control panel.
# Black Screen on Startup: Titanfall 2 fix
Well, the black screen issues are most certainly because of the conflicts between the drivers of harware components. Mainly, when the Display drivers are involved in this, this black screen issue often occurs. So, to solve this, we recommend you to make a clean install of the latest drivers on your PC. This is time consuming and hence we recommend Driver scanner that can update all the drivers in one click. Just follow the instructions given below to solve the black screen issue in Titanfall 2.
- Go to this page
- Now, click on "scan for drivers"
- It'll prompt you to download the Driver scanner.
- After downloading it, install it.
- After finishing the installation, run a PC scan using it.
- From the results, you can update all the drivers by one click.
# Cannot Find Multiplayer Servers : Titanfall 2:
There are few servers issues going on with the game at present. Just stay calm and patient for few moments and it'll be fine.
# Lag issues on PC: Titanfall 2 Fix:
As you can see, the requirements for this game are huge and it demands a high performing PC Gaming Rig from your side. So, if you think you have one, you can try these workarounds to tweak up your FPS a little.
- In the Nvidia control panel, go to Manage 3D settings.
- In the drop down settings menu, find "Power Management Mode" option.
- Set it to "prefer maximum performance"
- Also, go to task manager and in the "details tab", search for the exe file of the game and set high priority to the game.
# Error 0xc000007b in Titanfall 2 fix:
This issue is related to the Microsoft Visual C++ software. You might be running on an older version of it. So, check for the latest version on the official Microsoft website, install it and restart your computer.
# Dll Error in Titanfall 2 Fix:
Dll files are the lifesavers for the hardware as they make the task of computer simpler and enhance better performance of the hardware components. So firstly, whenever you install any software/ game, it installs few dll files into your PC library and uses them while running. However, there can be multiple software which use the same dll file and when you uninstall one of the software, the dll file gets wiped away and it causes the error while using other software which uses the same dll file. Generally, the best solution for this error is the Dll-Files Client, the tool which is specifially designed to solve the dll errors.
- Just download it from here
- In the search tab, enter the name of the Dll file that you are having error with
- From the search results, click on the first result and click on install.
That's it. The problem is solved. You can sit back and enjoy the game.
Having any more issues in Titanfall 2 ? Do comment below and allow us to help.


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After having presented the "Brazil" and "England" models earlier this week, we present the nicest in the lineup – the "France" version. France is just very lucky, because their flag colors look great on clothing and have gained massive popularity, with Moncler and Thom Browne using the tricolor heavily these last years. That said, the Nike Roshe Run NM SP "France" comes with a navy upper, red heel tab, blue Swoosh and white accents all over. You can buy the sneaker now from SlamJam Socialism.
Nike Roshe Run NM SP "France"
Highsnobiety Sneakers


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I was only a mere daytripper into Pongwe... as I was staying for a month at villa on the Island for a month over Christmas. My friend and I did weekly trips to the Pongwe Beach Hotel to indulge in some luxury and and good food. We were made to feel so welcome by Robin and the rest of the staff... wow what a wonderful place you all run. The beach was beautiful and clean, the service was so friendly and genuine the food was lovely and fresh and the cocktails a real treat.. the welcome my friend and got was so warm... Thank you so much Robin and staff... if I manage to get out to Zanzibar again... I will most definately be booking at the Pongwe Beach hotel.. :-)
Best wishes
Sarah and Brenda...
- Official Description (provided by the hotel):
- Situated on what could perhaps be described as the best beach in Zanzibar, Pongwe Beach Hotel is a tranquil place where one can escape the hustle and bustle of modern day life. It is modestly sized, with 4 Sea View Suites, 13 traditional Beach Villas as well as 3 Garden Rooms and has an intimate, personal atmosphere. Set within a secluded bay on the east coast of Zanzibar, Pongwe Beach is a true paradise retreat. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com, Priceline, Expedia, Travelocity, Orbitz, Hotwire and Hotels.com so you can book your Pongwe Beach Hotel reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Pongwe Beach Hotel Zanzibar Island


text:
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Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from 15 to 100 mM Na+. From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A•T- and G•C-containing DNA polymers. We find that temperature and salt dependences of the stacking term fully determine the temperature and the salt dependence of DNA stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.
Stability of the DNA double helix with respect to separation of complementary strands is known to depend on the base composition of the duplex (1–4). A classical study of Marmur and Doty (1) on DNA polymer stability gives a linear relationship between the G•C content of the polymer and its melting temperature. The simplest explanation of the linear dependence is that A•T- and G•C-pairs differ in stability independently of their neighbors. Accordingly, base-stacking interactions have been thought to constitute only a small correction to the major effect of the differences in G•C and A•T pair stabilities (5). Nearest-neighbor stability parameters have been introduced to account for sequence effects in DNA stability. These parameters are obtained from the analysis of the melting data for DNA polymers (5), DNA oligomers (6–8) and DNA dumbbells (9,10), and present the cumulative (base pairing and stacking) contribution of each dinucleotide stack to the overall stability of the molecule. In fact, DNA melting experiments do not allow separation of the two contributions.
Partitioning of base pairing and stacking contributions to DNA stability not only delivers a new aspect in the fundamental understanding of DNA structure and energetics, but also it has significant implications in a number of biological processes. Fluctuations in local helical conformation of DNA, the phenomenon known as DNA breathing, lead to infrequent events of base pair opening thus making normally buried groups available for modification and interaction with proteins (11,12). Fluctuational base pair opening implies disruption of hydrogen bonds between the complementary bases and flipping the base out of the helical stack disrupting two contacts. Heterogeneous stacking at these contacts determine sequence dependence of the base pair fluctuational motility. Moreover, single-stranded break (a nick) in the DNA double helix is stabilized by stacking interactions between base pairs flanking the lesion; these interactions are sequence-dependent (13). In the cell, DNA nicks are substrates for DNA damage-detecting and DNA-repair proteins (14–17). In vitro, DNA nick-based approaches are often used in DNA detection and amplification protocols (18,19). Also, stabilization achieved through coaxial stacking interactions has been put into effect to improve the efficiency of short primer hybridization for standard sequencing protocols (20) and within the format of sequencing by hybridization approach (21). By using partially double-stranded probe with a 5 nt overhang, the latter method takes advantage of the enhancement of the stability of the short duplex formed between the probe overhang and single-stranded target by coaxial stacking with the duplex interface of the probe. Another biotechnology application involving nicked DNA is the formation of padlock probes via ligation of a nick formed by two termini of artificial oligonucleotide hybridized to single-stranded or locally opened duplex DNA (22–24).
A number of theoretical studies addressed characterization of stacking and base pairing contributions to the duplex stability (25–30). Experimentally, energetics of base-stacking interactions in nucleic acids has been evaluated by studying the effect of dangling (unpaired) terminal bases on the overall stability of duplexes (31–35) and in the coaxial stacking hybridization experiments where binding of a short oligonucleotide to the single-stranded DNA template is assisted by stacking interaction with the nearby duplex interface (36–41). Both of these approaches rely on thermal denaturation measurements of short duplex molecules.
We have recently introduced an entirely new approach for characterization of stacking interactions in the DNA double helix (13). For this purpose we study DNA molecules with solitary nicks positioned in a strictly defined sequence context. We subject these molecules to PAGE. Structurally, introduction of a break to one of the strands of DNA perturbs only slightly the double helix conformation with stacking and base pairing at the nick site remaining intact (42–49). Nicked molecules have been shown to move somewhat slower during PAGE than intact molecules of the same size; this retardation is enhanced at higher temperatures (14,50,51).
The model of the DNA nick underlying our approach involves an equilibrium between the two conformations as shown in Figure 1a (13). One conformation is very close to that of the intact double helix where stacking between the base pairs flanking the nick is conserved. We assume that no optimization of stacking interactions—like in case of duplexes with dangling nucleotides (52)—occurs at the site of the nick in DNA. The other conformation corresponds to complete loss of stacking at the nick site thus inducing a kink in DNA. The fast equilibration between stacked/straight and unstacked/bent conformations of the nick directly affects the mobility of DNA molecule during PAGE leading to a differential retardation characteristic to a particular dinucleotide carrying the nick, i.e. KL. For each contact, stacking free energy parameter, , is ascribed governing the distribution of the molecules between the two conformations. Using PAGE mobility data of DNA molecules with solitary nicks flanked by all possible combinations of the base pairs we extract stacking parameters of the DNA double helix (13). Using heterogeneous stacking parameters and a well-known Marmur–Doty (1) dependence of DNA stability on its content, we have previously arrived at a revised set of the nearest-neighbor stability parameters which is in full agreement with the DNA melting data (13).
Here we measure, for the first time, temperature dependence and salt dependence of base-stacking contribution to the DNA duplex stability. Contributions of A•T and G•C pairing are estimated from the comparison of A•T- and G•C-containing polymer stability parameters with the stacking terms. We find that throughout the temperature range employed, base-stacking interactions stabilize DNA double helix. Temperature dependence of the base-stacking term fully determines the temperature dependence of the DNA stability parameter. Base pairing term is destabilizing in case of A•T and somewhat stabilizing in case of G•C pairs. Differential contribution of base-stacking in A•T- and G•C-containing contacts is responsible for 50% of the dependence of DNA stability on its G•C content. Salt conditions affect stacking parameters leading to stabilization upon an increase in sodium concentration. In this case, we also find that salt dependence of stacking term determines the salt dependence of the DNA stability term.
The 300 bp-long DNA molecules used in this study are PvuII/PvuII (all enzymes used in this study were purchased from New England Biolabs, Ipswich MA) restriction fragments of pUC19 derivatives described before (13). Briefly, pKL (were K and L are DNA bases) carries recognition sites for nicking endonucleases each of which introduces a single nick to the forward (N.BstNB I) or reverse (N.Alw I) strand of KL/K′L′ dinucleotide stack (or simply KL; K′ and L′ are complementary to K and L) yielding KL/F and KL/R fragments, respectively (Figure 1b). The nick is located 104 bp apart from the terminus of PvuII/PvuII fragment. Molecules with a single 2 nt gap in place of a nick are obtained by digesting pG2F and pG2R by N.BbvC IA followed by N.BstNBI digestion. As shown in Figure 1b, nicking sites of these nicking endonucleases are 2 nt apart and located on the forward strand of G2/F and reverse strand of G2/R. To remove 5′ phosphoryl groups, nicked and gapped molecules were incubated with Alkaline Phosphatase (CIP). Following treatment with enzymes, all DNA samples were purified by phenol extraction, precipitated with ethanol and resuspended in TE buffer [10 mM Tris (pH 7.4) and 1 mM EDTA].
Nicked, gapped and intact DNA fragments 300 bp in length were concurrently subjected to electrophoresis through 6.7% (w/v) polyacrylamide gels run at 17 V/cm with 1× TBE [90 mM Tris (pH 8.0), 90 mM boric acid and 1 mM EDTA] as a running buffer. We have shown before that under this set of PAGE conditions stacked-unstacked equilibrium at the nick site of DNA is not affected by specific experimental design (13). Specifically, Figure 5 of (13) demonstrates that the presence of the gel matrix and force applied to the molecule during PAGE do not affect apparent free energy parameters of stacked-unstacked equilibrium at the DNA nick. We also conducted PAGE in the presence of increasing concentrations of sodium by adding NaCl up to 85 mM to the gel and to the running buffer; the strength of electric field for these experiments was reduced to 12 V/cm. The gel electrophoresis was performed using Protean II xi Cell (BioRad, Hercules, CA) where the gel-plate sandwich is mounted against the temperature-controlling core connected to a circulating water bath set to a desired temperature. The temperature during PAGE was measured by a thermocouple (Oakton, Vernon Hills, IL) with a type T wire probe inserted directly into the gel. DNA band patterns were visualized by ethidium bromide staining and detected by the CCD camera.
Relative mobilities of DNA fragments with a nick, μ, were calculated as a ratio of the distance migrated by the nicked fragments to the distance migrated by the intact fragment during PAGE. All experiments were conducted at least in triplicates. To enhance the separation of the molecules with solitary nicks from the intact molecules we added various concentrations of urea (0.4 to 8 M) to the gel thus allowing subsequent extrapolation of measured parameters to zero urea (13). We also conducted PAGE in the presence of dimethylformamide (DMF) in place of urea. In this case, a smaller range of concentrations was used since the gels became fragile at concentrations of DMF above 1.5 M.
Two synthetic 65 nt-long complementary DNA strands (MWG Biotech Inc., High Point, NC) were annealed in TE buffer appended with a desired concentration of NaCl (from 20 to 200 mM) at 0.5 µM of each strand. The same duplex was also prepared in the 1×TBE buffer. The purity of the duplex was checked by PAGE. Melting curves of DNA duplexes were collected using a CARY Bio100 spectrophotometer equipped with the peltier thermocontroller (Varian Instruments, Walnut Creek, CA). Both denaturation and renaturation curves were collected at slow heating/cooling rate of at 0.08 °/min; no hysteresis between heating and cooling curves has been observed. Melting temperature, TM, was estimated as a temperature at the midpoint of the duplex-to-coil transition.
In melting studies we determined stabilities of four DNA duplexes, which differed in G•C content, χ (DNA sequences are listed in Supplementary Table 1). Salt dependence of A•T-containing duplex stability (χ = 0%) was measured directly for 22 mM < [Na+] < 202 mM (i.e. 20–200 mM NaCl plus 1 mM EDTA). Salt dependence of more stable G•C-containing duplex was obtained by extrapolating TM's measured for four duplexes with χ = 0%, χ = 14%, 32% and 41.5% to χ = 100%. Corresponding plots are shown in Figure 4a.
During gel electrophoresis, a 300 bp-long DNA molecule with a solitary nick migrates slower with respect to the DNA fragment without the lesion (Figure 1c). Retardation of nicked fragments depends on the identity of the base pairs flanking the nick like in case of the molecules with nicks flanked by As and Ts shown in Figure 1c. Differential retardation of nicked DNA is due to specific interactions characteristic to each nicked dinucleotide stack. Quantitatively, equilibrium between stacked/closed and unstacked/open conformations at the nick site is governed by stacking free energy, ΔGST, so that:
where Nclosed and Nopen are occupancies of stacked and unstacked conformations at the DNA nick, respectively, R is the universal gas constant and T is the absolute temperature.
At a given temperature, due to fast equilibration between the two states, mobility of nicked DNA is a weighted average of the mobility of molecules in closed state and mobility of molecules in open state (13). Since stacked conformation of the DNA nick is very close to the DNA molecule without the lesion μclosed is given by the mobility of the intact DNA fragment. The presence of a single-stranded stretch in a DNA molecule with a short gap prevents stacking interactions between the base pairs flanking the gap thus forcing the molecule into the open state. We use the mobility of molecules with a single 2 nt-long gap to estimate μopen. Thus stacking parameter ΔGST describing stacked-unstacked equilibrium at a nick site can be calculated directly from the mobility data using Equations 1 and 2 (13):
Since the perturbations brought about by the nick are often minor we introduce urea to the gel in order to enhance the separation of nicked molecules from intact molecules (Figure 2c) (13,51). Data obtained in urea-enhanced PAGE is then extrapolated to zero urea concentration resulting in stacking parameters for each nicked dinucleotide stack in the absence of urea, (Figure 2d). The use of denaturant during PAGE is an imperative part of our experimental approach since it allows characterization of even subtle differences in nicked molecule mobilities. To ensure that the nature of the denaturant does not affect the resulting stacking parameters we also conducted PAGE in the presence of varying concentrations of DMF (Figure 2d). Additionally, for the least stable stacks, we can measure stacking parameters directly at zero urea without extrapolating the mobility data collected in the presence of denaturant; this parameter is also plotted in Figure 2d. DMF-enhanced and urea-enhanced PAGE yield quantitatively similar dependencies of apparent stacking parameters on the concentration of the denaturant. This finding is quite unexpected and is most likely due to a narrow range of DMF concentrations used in PAGE; an increase in DMF concentration could yield more significant differences in apparent values measured in the presence of the two denaturants. We are unable to conduct these experiments since the gels containing more than 1.5 M DMF are too fragile to handle. Extrapolation of the data obtained in DMF-enhanced and urea-enhanced PAGE experiments to zero denaturant concentration is consistent with values measured directly in the absence of denaturant.
Our further experiments involve conducting urea-enhanced PAGE at different temperatures to obtain temperature dependence of DNA stacking parameters. Molecules with solitary nicks flanked by A•T pairs shown in Figure 1c, display a defined dependence of the electrophoretic shift towards the molecules with a 2 nt gap when temperature increases from 22 to 52°C. Relative electrophoretic mobilities of nicked AA/F fragment and gapped G2/F fragment are plotted as a function of temperature in Figure 2a. A 30 degrees increase in temperature leads to gradually decreasing PAGE mobility of the nicked fragment while mobility of the gapped fragment remains largely unaffected. In terms of occupancies of the two states of the nicked site, the shift in mobility is equivalent to the shift in the equilibrium towards the open conformation of the nicked stack. Similar results were obtained for the G•C-containing contacts. More narrow temperature range has been employed in this case since molecules with nicks located in more stable G•C-containing stacks do not resolve well enough from intact molecules at temperatures under 30°C.
Stacking free energies were calculated from the mobility data for all four A•T- and four G•C-containing nicked stacks using Equations 1 and 2. For each stack, molecules with solitary nicks located on the forward strand, KL/F and on the reverse strand, KL/R, were examined. Our further analysis is grounded on the assumption that the stacking free energies of the stacked-unstacked equilibrium at the site of the DNA nick are close to the stacking parameters of the intact DNA double helix (13). In this case we calculate the by averaging (i) the parameters obtained for KL/F and KL/R and (ii) the parameters obtained for the stacks equivalent due to dyad symmetry of the double helix (in our case there are two pairs of stacks: AA/TT and TT/AA pair and GG/CC and CC/GG pair). Individual stacking parameters are listed in Supplementary Table 2 and are plotted as a function of temperature in Figure 3a. Error bars on this plot correspond to the scatter of individual stacking parameters around the mean. As expected, throughout the temperature range employed, values for both A•T- and G•C-containing contacts increase (become less stable) gradually with increasing temperature.
To address the effect of ionic strength on stacked-unstacked equilibrium at the site of DNA nick we conducted urea-enhanced PAGE at different salt concentrations by adding NaCl to the gel and to the running buffer. Salt dependence of relative PAGE mobilities of AA/F nicked fragment and corresponding G2/F gapped fragment is shown in Figure 2b. Unlike in case of increasing temperature (Figure 2a), opposite tendencies are displayed by the two molecules with increasing salt concentration. Mobility of the fragment with a gap decreases. The decrease is likely due to more efficient screening of phosphate charges reducing electrostatic repulsion between the two double-stranded segments on either side of the gap thus making the gap more flexible (53). Similar effect of the salt concentration is expected at the nick site in open conformation. Mobility of the nicked fragment increases corresponding to the salt-assisted shift towards stacked state of the nick.
To facilitate the comparison of DNA stacking parameters we determine experimentally, with the DNA stability parameters we have to account for the difference in salt conditions. Our PAGE-based experiments were conducted in a standard gel electrophoresis buffer 1× TBE. In order to find concentration of NaCl equivalent to PAGE conditions with respect to DNA stability, we conducted melting experiments, i.e. we determined TM of duplex DNA in 1× TBE and in the presence of various concentrations of NaCl (Figure 4). For A•T-containing DNA, melting temperatures under different salt conditions were determined directly. A DNA molecule consisting only of guanines and cytosines is much more stable and melts around or above 100°C depending on the concentration of NaCl. Melting temperatures of G•C-containing DNA were obtained by extrapolating TM's measured for four DNA duplexes with different G•C-contents (χ = 0, 14, 32 and 41.5%) to χ = 100% (Figure 4a). Thermal stabilities of A•T- and G•C-containing DNA duplexes in the presence of NaCl and in 1× TBE are compared in Figure 4b. Melting temperatures of both molecules in 1× TBE correspond to the TM of these molecules in the presence of 15 mM [Na+].
We studied eight A•T-containing and eight G•C-containing nicked dinucleotide stacks. Like in the experiments on the temperature dependence of stacking parameters described above, for each KL contact we examined KL/F and KL/R molecules and the stacking parameter was obtained as their average in case of unique contacts (AT, TA, GC and CG). In case of stacks with dyad symmetry, we also average values of equivalent stacks. Individual stacking parameters are plotted in Figure 3b and listed in Supplementary Table 3. Note that all values are plotted as a function of the 'effective' concentration of sodium, i.e. the fist point at [Na+] = 15 mM corresponds to the experiment conducted in 1× TBE, the last point at [Na+] = 100 mM corresponds to 1× TBE appended with 85 mM of NaCl. All values become more negative gradually as concentration of sodium increases.
Site-specific nicks in KL/F and KL/R fragments were introduced enzymatically. Nicking enzymes, like any restriction enzymes, cleave DNA strand producing a 5′ phosphate and a 3′ hydroxyl termini, which translate into two negative charges at the nick site of the double helix. To estimate the effect of the phosphate negative charges on the stacked-unstacked equilibrium at the DNA nick we considered DNA molecules where the phosphate has been removed from the nick site. Dephosphorylated nicks carry no charge.
The molecules with dephosphorylated nicks and gaps were studied by urea-enhanced PAGE in the same manner as the molecules with phosphates. As expected, treatment of DNA fragments with Alkaline Phosphatase, which results in the removal of terminal phosphates as well as dephosphorylation of the gap, had no effect on PAGE mobility of intact and gapped fragments (data not shown). ΔGST values obtained for molecules with nicked stacks before and after dephosphorylation are compared in Figure 5a. A defined trend is observed for both A•T- and G•C-containing contacts—there is a significant ~0.4–0.6 kcal/mol shift towards more negative energies upon removal of a phosphate from the nick site only if phosphorylated 5′ nt is a purine nucleotide, like in case of AA/F and TT/R, but not in case of AA/R or TT/F. When the phosphate is located on a pyrimidine nucleotide, its removal has no effect on ΔGST values. This trend holds for 15 out of 16 nicked fragments shown in Figure 5a; the only exception is GC/F. The origin of such diverse behavior cannot be addressed by our approach and it deserves a separate detailed study. We can only suppose that removal of the phosphate from purine nucleotides allows rearrangement—in a sequence-independent manner—of the local structure benefiting stacked conformation. Alternatively, differential effect of phosphate positioning on purines or pyrimidines might be entropic in origin due to larger conformational flexibility of purine nucleotides without 5′ phosphates (54).
Note that ΔGST values of nicked stacks with and without phosphate display similar dependences on salt concentration (Figure 5b). For nicks flanked by A•T pairs, this is the case for AA/R, TT/F, AT/F and AT/R fragments which display no change in ΔGST values upon removal of the phosphate from 5′-T nt and for AA/F, TT/R, TA/F and TA/R fragments which shift towards more negative ΔGST values upon dephosphorylation of 5′-A nt. This result indicates that the phosphate charge at the nick does not contribute to the salt dependence of the measured stacking component; differences seen in Figure 5a between the nicked stacks with or without phosphate on 5′-purine nucleotides are not due to electrostatic interactions. In our further analysis we consider only molecules carrying phosphates at the nicks.
We obtain stacking free energy parameters by studying the equilibrium between stacked and unstacked form of DNA nick schematically presented in Figure 1a. The DNA molecule in the stacked state is very close the molecule without the lesion (42–49). Strictly speaking, the conformation of the unstacked state is unknown and our analysis is based on the assumption that unstacked state is close to the molecule with a short gap in place of a nick. Indeed, presence of a single-stranded gap precludes stacking interactions between two helical interfaces. Note, that this is not the case for 1 nt gaps—it appears that stacking between the base pairs flanking the gap is restored to some degree leading to anisotropic, directional bending of the molecule reducing the size of the gapped cavity (48,55). Molecules with longer gaps however, have been shown to possess isotropic bending flexibility which manifests itself in the absence of helical periodicity in electrophoretic mobility and cyclization kinetics measurements (50,53). Molecules with gaps 2, 3 and 4 nt in length migrate very closely during PAGE revealing similarity of their effective conformations (13,50). Additional factors [i.e. sequence of single-stranded linker (56)] come into effect once the gap size is longer than persistent length of single chains—in this case, the gap is likely to act as a hinge.
Thus, we use molecules with 2 nt-long gaps positioned in forward or reverse strand to approximate the open state of the nick; the site of the gap is thought of as a point of increased isotropic bending flexibility, which leads to retardation of the molecule during PAGE (57). Perturbations of DNA fragment at the gap sites are largely unaffected by ambient conditions—temperature, salt and denaturant concentration (Figure 2a–c).
Nicked DNA undergoes transition between the stacked (closed or intact-like) and unstacked (open or gapped-like) states (13). Note, that like in case of short gaps, perturbation at the nick site has been shown to be isotropic with no preferential directionality (50,58). The partitioning of molecules between these conformations determines the electrophoretic mobility of the nicked fragment and is the basis for measuring stacking parameters using Equations 1 and 2.
For further analysis we assume that the parameters describing the equilibrium at the nick site of DNA that we measure are in fact the same stacking parameters that stabilize intact double helix and determine the first term on the right-hand side of Equation 3:
where ΔGKL is the total melting free energy parameter (per 1 base pair) of a long DNA molecule (neglecting the end effects) and ΔGBP terms are the free energy contributions due to pairing between complementary bases (5). There are two ΔGBP parameters and for A•T and G•C pairs, respectively.
How accurate is the above assumption? Experimentally, the assumption proves to stand a critical test when we compare the values obtained for KL/F and KL/R series: these values indeed are very close [see Figure 4b in ref. (13)]. The values for dinucleotide stacks equivalent due to dyad axis of symmetry of the double helix (e.g. AA/TT and TT/AA) are close as well.
Let us examine the stacked-unstacked equilibrium at the nick of the dinucleotide stack and compare it to the helix-to-coil transition of a DNA doublet. The schematic in Figure 6 depicts these two transitions. Hydrogen bonding between complementary bases and stacking of pairs along the helical axis are disrupted upon melting; duplex melting to single strands is accompanied by the gain in conformational entropy, release of counterions and change in interactions with the solvent. Strictly speaking, partitioning of these effects between ΔGST and ΔGBP terms is not known. For example, base pairing terms in Equation 3 include breakage of hydrogen bonds between bases and formation of hydrogen bonds with water molecules rearranging the water structure around DNA chains. Likewise, base-stacking interactions have electrostatic and hydrophobic components.
The stacked-unstacked transition of a nicked DNA doublet involves loss of stacking interactions while hydrogen bonding between the complementary bases is preserved (Figure 6). It also involves removal of structural constraints imposed in the double helix making most of the conformational space of the intact sugar-phosphate backbone accessible. This part of backbone conformational entropy gain is close to the gain per one strand upon duplex melting. The conformation about the glycosidic bond in an open stack, however, is preserved as in duplex form. Nicked strand is lacking O3′-P linkage; the absence of this constraint allows for additional conformations when second-order interactions (interactions depending on two torsional angles) are repealed (59). Note that the backbone conformational entropy change is independent of the particular sequence and does not affect heterogeneity of values. We assume that conformation of base pairs flanking the nick is preserved as in stacked duplex. Like in case of helix-to-coil transition, other factors contributing to transition parameters come from interactions with solvent and counterions.
We assign free energy difference to stacked-unstacked equilibrium at the nick site of DNA—this parameter describes interaction of two neighboring base pairs and is sequence-dependent. As noted above, it also includes a part of conformational entropy change and other relevant interactions. These factors are universal for all neighboring pairs and do not contribute to heterogeneity of measured . We assume further that this parameter represents the stacking term of intact DNA duplex in Equation 3. Note that this assumption explicitly implies that stacked/closed state of the nicked stack is equivalent to the stack in duplex without the lesion and no optimization of the base pair stacking occurs upon introduction of the nick like in case of dangling unpaired nucleotides (52). If this assumption is valid, then parameters of stacked-unstacked equilibrium at the nicks positioned on one or the other strand of the each dinucleotide stack will be close. Indeed, we have shown previously that stacking parameters for KL/F and KL/R molecules are similar (13). Parameters obtained for stacks equivalent due to dyad axis of symmetry, i.e. AA and TT, GG and CC, are also similar. Note that marked difference is observed in case of coaxial stacking parameters determined in melting experiments (39).
Let us consider a DNA polymer comprised of adenines and thymines. The set of nearest-neighbor stability parameters of all A•T-containing dinucleotides, i.e. ΔGAT, ΔGAA = ΔGTT, ΔGTA, fully describes the contacts found in this polymer. According to Equation 3, stability parameter per 1 bp, ΔGA•T, of the A•T-polymer with a random sequence is given by [see ref. (13)]:
Stacking contribution to the polymer stability is the first term of the right-hand side of Equation 4; the second term presents the base pairing contribution. Experimentally determined stacking term is the mean of stacking parameters we obtained for all A•T-containing contacts. Stability parameter for G•C-containing polymer, ΔGG•C, is calculated analogously from stacking, and base pairing contributions:
DNA stability parameters in terms of melting temperatures, and , are salt-dependent and are given by the following empirical equations for A•T- and G•C-containing polymers, respectively (60):
where and are in degrees Kelvin. Equation 6 translates into free energy difference stability parameters using ΔS° = −24.85 cal/mol K (61) and Equation 7.
Stability parameters of A•T-containing and G•C-containing polymers calculated for [Na+] = 15 mM using Equations 6 and 7 are plotted in Figure 7a. Salt dependences of these parameters calculated at 37°C are presented in Figure 8a.
Stability of a DNA polymer comprised only of A's and T's arranged in a random order is given by two terms—the stacking term and the base pairing term—that appear on the right-hand side of Equation 4. Stacking term is calculated as an average of stacking parameters of A•T-containing contacts, which we determine in PAGE experiments conducted under certain set of ambient conditions (temperature and salt concentration). Stacking term of the G•C-containing polymer is obtained analogously.
Stacking contributions to the stability of A•T- and G•C-containing polymers for different temperatures are plotted in Figure 7a. Comparison of the temperature dependence of stacking terms with the temperature dependence of stability terms for both polymers reveals remarkable similarity between the two. Linear regression analysis of the temperature dependences of ΔGST values gives mean slope of kcal/molK, which is very close to the slope of temperature dependence of stability parameters described by Equations 6 and 7. It is evident that temperature dependence of stacking component fully determines the temperature dependence of DNA stability parameters throughout the temperature range employed in our experiments.
According to Equations 4 and 5, the difference between the melting stability of the polymer and the stacking term gives the contribution of A•T or G•C base pairing. The plots shown in Figure 7b present the base pairing term calculated for different temperatures by subtracting the experimentally determined stacking term from the polymer stability parameter. With good accuracy, the base pairing term is independent of temperature within the temperature range used in our experiments. The horizontal lines give the mean values of base pairing parameters at 15 mM Na+ of and . We conclude that stability of the DNA polymer with respect to strand separation is mainly determined by stacking interactions with base pairing being destabilizing (A•T pairs) or contributing almost nothing (G•C-pairs) (Table 1).
Furthermore, throughout the temperature range used in our experiments, heterogeneity of base-stacking interactions of A•T- and G•C-containing contacts, , is responsible for a substantial part of the difference in the stabilities of A•T- and G•C-polymers, (Figure 7a). In the presence of 15 mM Na+, heterogeneous stacking accounts for δΔGST = 0.6 kcal/mol out of δΔG = 1.2 kcal/mol stability difference of A•T- and G•C-containing polymers. Thus, the dependence of DNA stability on the G•C content is not only due to the fact that G•C pairs are stronger than A•T pairs (Figure 7b) but also is due to the stronger stacking component of G•C-containing contacts (Figure 7a). This understanding of stacking domination constitutes a paradigm shift in the view on DNA stability.
Stacking terms of A•T- and G•C-containing polymers are calculated from stacking parameters obtained in PAGE experiments that have been carried out under different salt conditions (Figure 8a). Salt dependences of the stability parameters of both polymers at 37°C calculated using Equation 6 and 7, are also shown in Figure 8a. The two dependencies are very similar indicating that salt dependence of stacking term determines salt dependence of DNA stability parameters throughout the range of salt concentration used in our experiments. In case of A•T-containing contacts, salt dependence of stacking component reproduces salt dependence of DNA stability parameters with a great degree of accuracy. Salt dependences of stacking terms of A•T- and G•C-containing contacts are similar to . The dependence of stacking parameters on salt concentration was unexpected. In fact, in our original publication we assumed that base pairing term is fully responsible for ionic strength dependence of DNA melting stability (13). Figure 8a shows that this assumption is incorrect and base pairing term plotted in Figure 8b is virtually independent of salt concentration. Horizontal lines correspond to the values of base pairing parameters averaged over the whole range of salt concentrations: and (Table 1).
What is the origin of the ionic strength dependence of stacking parameters measured in PAGE experiments? It is clear that stacking is a net phenomenon involving hydrophobic, electrostatic and dispersion components (28,29,62–65). There is no agreement between researchers, however, what is the dominant force in this interaction. Luo et al. (29) emphasize a dominant role of nonelectrostatic interactions. Gellman and co-workers (65,66) report that neither solvophobic effects nor dispersion forces are important in aromatic stacking; stabilization is achieved through attractive interactions between positive and negative partial charges on bases. Guckian et al. found that hydrophobic effects dominate stacking but dispersion forces and electrostatic interaction between partial charges contribute substantially to the total base-stacking as well. This is particularly true for natural bases, which have significant charge localization (28,67). In either case, electrostatic component of base-stacking interaction (substantial or not) will depend on the ambient ionic strength through local screening of partial charges at the solvent accessible positions. In the context of the DNA double helix, however, this effect is likely to be overwhelmed by the salt-dependent interactions between highly negatively charged DNA backbones as described by the polyelectrolyte theory of DNA stability (68,69). Similarity between salt dependences of DNA stacking parameters measured in PAGE experiments and DNA stability parameters indicates that electrostatic component contributes exclusively to of Equation 3. Note, that charge of the phosphate at the 5′ nt of the nicked stack does not affect salt dependence of measured parameters.
Separation of the two contributions to thermal stability of the DNA double helix is achieved by studying PAGE of DNA molecules with solitary nicks and gaps. For the first time, the dependence of DNA stacking parameters on ambient conditions (temperature and salt concentration) is determined. Throughout the temperature and salt concentration range of our experiments, base-stacking interactions are always stabilizing for both A•T- and G•C-containing contacts in the DNA double helix. In fact, DNA stability is mainly determined by base-stacking interactions. G•C pairing does not contribute to stabilization of DNA duplex, while A•T pairing is always destabilizing. This finding presents a paradigm shift in the understanding of the interplay of the forces stabilizing DNA double helix. For all temperatures heterogeneity of stacking interactions in A•T- and G•C-containing contacts accounts for at least half of heterogeneity in the stability of A•T- and G•C-polymers; the other half is due to the difference in the energetics of A•T and G•C base pairing. The data on separation of stacking and base pairing contributions have made it possible to describe sequence-dependent fluctuational opening of the DNA double helix (70).
Supplementary Data are available at NAR Online.
The authors thank Dr R.M. Georgiadis for providing access to spectrophotometer and Ms J. Ruemmele for technical assistance with melting experiments. The authors thank Dr Y. Mamasakhlisov and Dr P. E. Nielsen for fruitful discussions. Funding to pay the Open Access publication charges for this article was provided by NIH.
Conflict of interest statement. None declared.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Q: Rear End on 2008 Chrysler Pacifica
We had new tires put on our Pacifica and now it is making a noise that sounds like loud tires. My husband says the "rear end " is going out. What does that mean and what does it take to fix it. Thanks
sounds like a tire issue or a hub bearing..most of the tires i see are the 3rd tier tires that all these major tire stores sell...good price but not worth a poo as far service life comfort or noise is concerned.........you may just need to invest in a set of decent tires and keep them rotated..rotation is the key to tire life and not replacing them before you should have too....get a decent set of tires buy the road hazzard and keep up on it...,most road hazzard warrantys cover you rotations so take advantage of it
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Old post office restaurant project delayed again
The Hudson City Council has extended another deadline on a project to convert the former post office building on Locust Street into an Italian American restaurant.
The council on Tuesday, May 15, set June 25 as the new deadline for Russell Evenson to close on his purchase of the building from Don Maysack.
If the transaction is completed by then, the council will consider renewing the restaurant's liquor license for a year and extending the deadline for owner Evenson to put the license to use.
The council previously had set May 15 as the deadline for Evenson to finalize the purchase of the building and keep his license.
Council members were sympathetic about the difficulty Evenson has had obtaining financing for the purchase and renovation of the building.
He said he had bank and Small Business Administration approval for loans, but then an appraisal of the old post office came in too low to satisfy the bank.
Evenson said the bank wanted him to put up either more collateral or cash. He said he wasn't willing to invest more cash in the project, but was having his Amery restaurant appraised as collateral.
He explained the situation to both the Finance Committee that met immediately prior to the City Council and to the full council.
Evenson said he would soon know what his Amery restaurant was appraised at, and that he hoped to close on the Hudson building by June 1.
"I can guarantee that I'm 100 percent behind the project. I want to get this done as fast as I can," he said.
He said he has hired four new employees who are training at the Amery restaurant and will come to the Hudson restaurant when it opens.
"I do guarantee that I will get this project done if you let me," Evenson told the council. He said the restaurant would be good for Hudson -- and for Locust Street in particular.
Evenson threw himself at the mercy of the council and said, "If you want to approve it fine. If you don't, I can get some sleep."
He added that he had never failed at anything but public speaking.
Evenson has owned and operated Village Pizzeria in Amery since the mid-1990s. The restaurant is a converted grocery store that offers more than the name implies. The menu features a variety of pasta entrees, steak dinners and salads in addition to pizza.
Evenson started The Village Pizzeria in Dresser in 1979 and sold it about five years ago. He renovated a former truck garage to be the Dresser restaurant.
Last January, he told the Star-Observer that the old Hudson post office would make the nicest restaurant he has opened. He plans to name it Postmark Grille.
The City Council approved the liquor license for the restaurant last December and set a deadline of April 30 for the opening.
In April, Evenson reported to the council that he had been unable to close on the purchase of the building. He asked for and received a 120-day extension on deadline to begin using the liquor license.
The city requires businesses to put liquor licenses to use within 90 days of receiving them, because a limited number of licenses are available. In recent years, the City Council has loosened the restriction on the number of licenses to the maximum allowed by the state -- one Class B license for every 500 city residents.
The council in February also amended the ordinance on the time limit for putting a liquor license to use. The council now has leeway to grant an extension "as it deems reasonable and appropriate" to the 90-day limit. Previously, only a 30-day extension was allowed.
The council recognized that more time is often needed to start up a restaurant, and that construction can't begin until the owner receives a liquor license.
Evenson told the City Council that was the situation in his case. His bank financing is contingent upon him having a liquor license. And he can't begin renovations to the old post office until the financing is in place.
He previously told the council that the renovations to the old post office, and construction of a kitchen addition, would take time.
Last week, he said would likely be back before the council asking for an extension to the Aug. 31 deadline for opening the restaurant. He said Oct. 1 was a more realistic opening date.
City Attorney Catherine Munkittrick said the building purchase would have to take place before the council decides whether to renew Evenson's license for another year. All alcoholic beverage licenses are up for renewal for each summer. The license period runs from July 1 through June 30.
Munkittrick said the council could consider giving Evenson more time to open the restaurant when it considers renewal of his liquor license.
The council members were convinced of Evenson's effort to move the project forward.
In response to a question from Alderperson Randy Morrissette, City Clerk Nancy Korson said two reserve Class B liquor licenses are still available.
That also appeared to be a factor in allowing Evenson more time to close on the building purchase. Alderperson Mary Yacoub said the availability of the other licenses made it an easy decision for her.
Evenson's lawyer, Brian Byrnes of Amery, said the building purchase is close to being completed.
He also vouched for the quality of Evenson's Amery restaurant.
Byrnes said his office window faces the restaurant and he can see from the number of cars in the parking lot that it is a popular place.
"Sunday mornings, you can't find a spot to park," he said. "Russ has done an incredibly good job with what he has done in Amery."


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Posted by Yourcareerchina on 17. Jul 2012
Location: Beijing
Company Profile:
Our client is part of the elite medical investment industry with over 6 bases throughout China. Investing in high quality medical institutions, they strive on constructing revolutionary, innovative, and sustainable establishments. Our client is involved with medical strategies, output management, personnel training, among many other things. They are currently looking for a Butler to serve their premium customers in Beijing.
1. Warmly acknowledges all customers
2. Attempt to anticipate the needs of customers and provide assistance without being asked.
3. Maintains confidentiality of guest information and activities.
4. Upon guest arrival, greet and escort guests to correct department
5. Check each room daily, refreshing water and daily amenities.
6. Serve meals, snacks, tea, hors d'oeuvres, and other food and beverage items to guests.
7. Willingness to support all departments as necessary.
8. Coordinate housekeeping services by notifying the Housekeeping and Reception of appropriate times to provide cleaning, turn down services etc.
9. Report deficiencies or safety hazards to the line manager and coordinate corrective action.
10. Maintain high standards of personal appearance and grooming including wearing the proper uniform and nametag while working.
11. Performs other job related duties as assigned.
1. All nationalities welcomed.
2. English, conversational Mandarin Chinese is essential.
3. Previous customer service experience
4. Have a pleasant personality
Starting date: ASAP 2012
Salary: 12,000-30,000 RMB per month based on interview and experiences
We Offer:
Free Accommodation
Free Meals
Visa Support
Contact: Ms. Dawn Huang
Email: email@example.com
Skype: YourCareerChina
Attention please: Photos with CV will be required.
Please note: we only contact candidates who fully meet with our requirements. Sorry for any inconvenience that may occur to you
Logg inn for å skrive en kommentar.


text:
Beaver Island Hosts 4th Annual Winter Fest on Lake Geneserath
- This event has passed.
Fun for All Ages! Enjoy the fun that winter has to offer: Ski plane flying (weather permitting); wonderful food; canoe rides and special friends. It is a great way to enjoy a winter's day on Beaver Island.
Beaver Island, The Emerald Isle, is located in Lake Michigan, northwest of Charlevoix. Get there by ferry or plane from Charlevoix.


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The Team
Allen Perry was named 'Best Realtor'
in the 2011 Nashville Scene's
'Best of Nashville' Readers Poll
Allen PerryReal Estate Consultant
(615) 738-1550
We can't count the number of times a day that AP asks one of us, "Anything else?" We certainly can't accuse him of not being thorough! He is passionate about perfecting our website, staying on top of social media trends, and most importantly about his clients.
Allen got into real estate initially as an investor. Eventually, he started helping his friends buy and sell real estate and worked to get his license. Since then, he has won too many awards to list (but if you're curious, click here) for being a top agent, closing record numbers of transactions, and having a top real estate team: Nashville on the Move of Benchmark Realty. You can see exactly how he works with clients in clips from his appearances on HGTV shows in our media section!
On sunny weekends, Allen likes to get out on the lake. He's involved with many community service projects as well, including Habitat for Humanity, Mocha Club, Ellie's Run for Africa, and Young Life. "Anything else?"
Courtney Shaddox
Real Estate Consultant
(479) 957-4817
We are happy to have Courtney on our team as the agent that is truly the home showing expert. She's the go-to gal to find those "love it, gotta have it" needle in a haystack properties for our clients. One of her favorite things is having the opportunity to show our buyers properties that could potentially be the next place they build memories and call home.
Courtney's love for real estate comes from the many years she spent watching her dad build houses and seeing the detail that is put into building a home. If we are really honest, her slight obsession with HGTV home buying shows was the final straw that prompted her to jump both feet into the real estate pool.
When she's not "on the move," Courtney is renovating her home, playing with her chocolate lab Maeble, watching SEC football, at the lake or spending time with friends. Basically if it's outside and with friends, she's there!
Leah Perry
Real Estate Consultant
(615) 512-7677
If you've ever called our office phone and heard a Mississippi accent, chances are you were greeted by this girl. She's the creative genius on the team and the one responsible for all the fresh flowers and artwork in the office. In addition to being a realtor, her responsibilities include being Allen and Andrew's emergency contact for anything and everything they need while they're "on the move." She keeps a running list of ways to answer Allen's inevitable "Anything else?" at the end of every meeting. Leah is passionate about music, dance, family and friends. She loves spending time outside, either strolling around downtown Franklin or spending time at the lake. She's our go-to girl for blogging questions, and she even has her own. Leah and Allen got married in March 2013. We are happy that her new last name is Perry!
Andy Hunt
Real Estate Consultant
(615) 943-5552
We all know the old real estate adage, 'location, location, location.' But to Andy, real estate is all about people. An Ole Miss alum with a background in marketing and finance, Andy has been a Nashville resident since 2004, and he loves watching the exciting growth happening in his Music City.
Whether a first time home buyer or an experienced emptor, Andy relishes the chance to help his clients, both new and returning, see their dreams become reality. After all, buying a house is the biggest investment most people will ever make. It's an investment in joy, shelter, laughter, memories, family and community. Making a decision that big requires trust in the person guiding the process.
When he's not working, you can find Andy breaking a sweat at CrossFit 615 or exploring the outdoors with his wife Jamie and sons Miller and Nixon.
Shannon Cowart
Real Estate Consultant
(615) 429-4157
Shannon is excited to be the newest member of the NOTM team! After years of infatuation with all things HGTV and RealTracs, she found herself helping friends discover their dream homes in and around Williamson County. Wanting to make it more than just a hobby she loved, she finally decided to make her role as their realtor official just this year!
Shannon's desire to help people and guide them through difficult situations was evident in her first career as a Physical Therapist. As her family grew (including the adoption of their two youngest children from Haiti), her second career of stay-at-home Mom allowed her the opportunity to work with her photographer husband as his producer, utilizing her gifts in hospitality, organization and creativity. She is excited as she now gets to combine her past experience with her passion for real estate to serve others doing exactly what she loves!
During her free time, you can find her with her husband and their four kids (and three furry ones). They might be playing outside, watching football, cooking, riding bikes, or creating art or mischief. In the fall, chances are good she'll have candles burning and a pumpkin spiced latte within reach. And HGTV will definitely be on in the background.
Licensed Contract-to-Close Specialist
(615) 760-5393
A southern girl to her core, Brittney set out on a city adventure four years ago and has truly fallen in love with Nashville. She loves making checklists and the expensive juices at Whole Foods that taste like grass. With the heart to help others and the drive to excel, she is committed to be on the best team in Nashville. She loves knowing clients and making sure they trust her and enjoy the entire home buying experience. She wants to produce raving fans with every transaction she closes and she makes that evident in her commitment to never ending improvement.
In the world of real estate, things can quickly become fast paced but Brittney's motto is, "There's nothing that cannot be fixed with a little prayer, a cup of coffee and a Godiva truffle."
Suzanne Fletcher
Public Relations Coordinator
(615) 760-5393
A public relations specialist, graphic designer and lover of all things communication, Suzanne enjoys working behind-the-scenes to help the big picture come together – whether that's through a killer graphic design, public relations campaign or marketing strategy. As a dominantly right-brained, whimsical, visual learner, she thrives through details and creativity. She's thrilled to get everyone excited about moving to and living in Nashville!
Born and raised in West Tennessee, she has grown up visiting Music City regularly – whether it's for family Thanksgiving, to attend a concert, work an internship or enjoy a weekend with friends. She's a cat lover who appreciates the simple things in life. Despite the Southern stereotype, she'd prefer a cup of coffee to sweet tea any day. Her one wish would be to be a teenager in the 1950s. Need a rocking playlist for any occasion? She's your girl. You'll find her at a concert almost every weekend. She believes random acts of kindness rock the world and strives to understand life from all perspectives. Ultimately, she desires to be creative in every aspect of life and share God's love with all people.
Tessa Holder
Administrative Intern
A newcomer to the Nashville scene, Tessa is an aspiring entrepreneur and artist. Her love for houses and all that is real estate began at a young age while watching her mom and dad build and rehabilitate their own homes and businesses. One day she hopes to start her own family business building custom furniture and flipping houses (all thanks to the ever contagious HGTV bug). She has a deep love for Jesus, concerts, books, hiking, animals, and her family. You'll hardly ever see her without a smile because she believes that, "Every time you smile at someone, it is an action of love, a gift to that person, a beautiful thing." –Mother Teresa


text:
Welcome EHS Media Center. Task Definition: Reveal Top 5 Freshman Fiction Favorites Look at Different Fiction Genres and Location Look at Graphic Novels and Location Location of New Books, Magazines, and Playaways Review Check out Policies How to Recommend Books for the Collection
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Task Definition:
1. Twilight Series – Stephanie Meyer
2. Bluford Series - Anne Schraff
3. Books by Nicholas Sparks
4. The Alex Rider Series –
Anthony Horowitz
5. Where the Red Fern Grows –
Wilson Rawls
The Devil and His Boy by Anthony Horowitz
Fic (Adv) Pat
Crank by Ellen Hopkins,
Airhead by Meg Cabot
Fic (Con) Rap
Dragon Avenger by E.E. Knight,
Harry Potter series
Fic (Fan) Cas
Fic (His) Low
Fic (Mys) Bro
Fic (Mul) Mye
Twilight series, The Chronicles of Vladimir Todd by Heather Brewer
Fic (Hor) Cla
Fic (Rel) You
Fic (Rom) Ada
War of the Worlds by HG Wells, Uglies by Scott Westerfield
Fic (Sci) Shu
Fic (Spo) Mye
Chibi Vampire series, Bleach series, 100 Girls by Adam Gallardo
741.5 Chm
ABP Fic Gru


text:
Last Chance to Join Naked Archaeologist Tour!
My first ever "Naked Archaeologist" tour of Israel is about to take place December 4 – 15. It's an intimate group with an incredible itinerary. I'm going to be leading it with Eitan Chamberlin, an archaeologist, tour guide and an individual I've interviewed on many of my programs. I'm really looking forward to this. It's going to be an incredible learning experience and a lot of fun.
It's 13 days from now. If you like spontaneous decisions, make one now, hop on a plane and join us: http://samsontours.com/group-tours/1134-the-naked-archaeologist-israel-tour-2016


text:
Big Girls Don't Cry Reviews
Top Critic
September 2, 2004
A refreshing reminder that learning how to navigate danger is a big part of being a teenager, and that no kind of upbringing -- or nifty home furnishings -- can or should shield a young adult from life outside her doors.
Full Review | Original Score: 3.5/5
Top Critic
September 2, 2004
Sleek, not-quite- trashy-enough melodrama.
August 8, 2004
[A] tiresome melange of teen depression, teen sex, teen crime, and teen friendship...
October 2, 2003
It felt as heavy as a German meal of dumplings and gravy.
Full Review | Original Score: C-
September 29, 2005
Read More | Original Score: 2/5


text: Hi: I've had my HTC Hero for a few months and finally get around pretty well. Lately the battery is running out very fast, so that's how I found this site. I appreciate someone named FLIPZ who maybe supplied the clue to my problem. Anyway, am trying to learn more about this phone that has WAY more capability than I probably need.


text: Saturday Jul 17, 2010 Jul 17, 2010 0 Facebook Tweet Email Its 9am - Woke up at 7am... Decided to stay awake, and drinking Coffee Mmmmmm!!! Today is a day off then tomorrow 9am Meeting at Work - blegh. SPOILERS! (Click to view) XOXO VIEW 5 of 5 COMMENTS lalou: Oh nice thong. Jul 17, 2010 captainodle: I've got that same one. weird. Jul 17, 2010


text: Not endorsing or promoting this documentary, just letting you know about it. Think I will watch and then make up my mind as to whether they took a fair look at the subject,somehow NBC doesn't instill much in the way of fairness or objectivity. SJ 40 CNBC will air a documentary called Americas Gun: The Rise of AR-15 on Thursday, April 25th at 10pm ET/PT, which I feel may be of great relevance to you and your association. With approximately 4 million in circulation, the AR-15 is one of the most popular and misunderstood rifles in America. The documentary offers a window into the larger debate and the incendiary issues that surround it. Appealing to gun lovers and gun haters, it centers on the friction between those who abhor a rifle they believe belongs only on a battlefield, and those Americans who insist owning one is their inalienable right. Below are a few clips that showcase the life around the AR-15: Americas Gun: The Rise of the AR-15 Preview How does the AR-15 work? Why is the AR-15 popular among women? 3D Printed Gun Parts Tune-in Details: Americas Gun: The Rise of the AR-15 on CNBC Thursday, April 25th 10pm ET/PT. Please feel free to share this information with your colleagues, association, blogs and social networks . Also, join the conversation on Twitter with #RiseOfAR15 and @CNBCPrimeTV. Feel free to contact me for more information about this AR-15 special or visit http://americasgun.cnbc.com. Thank you and let us know your thoughts on the documentary.


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The mixing box is a workspace utilized to mix and prepare the paint for the vehicles that have to be painted. These paint mix rooms are equipped with lighting, ventilation, one-wing personnel door with window and thrust door handle with exit from the inside to the outside (anti-panic handle)
Double skin wall panels made of galvanized Fe/Zn, pre coated steel plate, with inner insulation
Roof made of galvanized Fe/Zn, pre coated steel plate
Light fixture fitted in the roof, equipped with a safety glass and rephrased neon tubes; IP55 class.
Entry door with safety glass and anti-panic handle
Ventilation system with exaust and in take fan
Interlocked electrical controls for lighting and ventilation
Electrical supply: 230/1/50 Hz
Light fixture/s switch on retarder
Stainless steel bench with air suction
Blowtherm invests resources in the pursuit of total quality using more and more newly developed technologies. Product quality and customer satisfaction are our main goals, and the approvals obtained from relevant authorities are a testament to our success in these fields. As the premium quality spray booth manufacturer, Blowtherm have been awarded Preferred Supplier Status by many prestigious manufacturers.
Low Res Download (1.33 MB)
The mixing box is a workspace used to mix and prepare paints for vehicle painting. It is equipped with lighting and ventilation, and a single personnel door with laminated safety glass. The door is fitter with a pull handle from the exterior and anti-panic handle from the interior.
Low Res Download (1.27 MB)


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Pope condemns bombing in Philippines
Pope Benedict XVI has condemned a bomb attack near a Catholic cathedral in the Philippines that killed at least five people, and wounded more than 29 others on Sunday.
Speaking after the Angelus in St Peter's Square yesterday, Pope Benedict said the use of violence would never solve problems.
"Sadly, today as yesterday, this cry is incessant, because human blood continues to flow as a result of violence, injustice and hatred. When will people learn that life is sacred and belongs to God alone? When will people understand that we are all brothers?" he said.
"Christ Himself did not respond to evil with evil but with good, with his infinite love."
Archbishop Orlando Quevedo was giving the homily during Mass at the Immaculate Conception Cathedral in Cotobato City when the bomb exploded. He said: "This is not just a crime, this is a sacrilege...When I heard the blast and saw smoke, I told my parishioners to stay calm and not panic."
Archbishop Quevedo appealed for prayers for the bombers, saying: "Violence does not achieve anything. Violence begets violence. Let's all pray for the conversion of the bombers."
Later Archbishop Quevedo stressed that he did not believe the bombing was intended as an attack on the church. He said he thought it was more likely the work of a rebel terrorist group.
Cotabato City is on the main island of Mindanao, about 885 kilometres southeast of Manila.
Source: VIS/UCAN


text:
SMOOTD | Jackie Montarbo
Blogger Jackie Montarbo of Love From Jackie looks like an autumn dream in her GORGEOUS boots. She lists the over-the-knee boots as one of her cold weather essentials, along with a structured leather tote, and camel wool coat. This ensemble is picture perfect against the fall foliage backdrop.


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The U.S. needs skilled workers more than ever. There are big opportunities all around you for anyone willing to learn a skill and master a trade – and it's only going to grow from here.
There are hundreds of training programs and career opportunities all throughout the state. Sign up HERE for our newly launched database to learn more about how to jumpstart your career in Tennessee's construction industry and where to locate a training program near you!


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All quotes tagged Story
(2271 quotes found)
"Finding true love is like a god, you hear stories, you believe, and you fear; you even search for signs but in the end it all seems like a fairytale."
"The greatest love stories are not those in which love is only spoken, but those in which it is acted upon."
""We each have a choice, we can continue with the same chapter of our life, or begin a new one. You are the author of your own life story, the choice is yours." ~Tom Baker"
"[All were] pivotal stories in the Australian narrative ... And obviously there is something elemental about the stories, and if that is the case, they speak across time to us today."
"Everybody has a story to tell, its just dat it may be more easier or harder compared to the other."
"Miracles are a retelling in small letters of the very same story which is written across the whole world in letters too large for some of us to see."


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Why do we exist? Where did we come from? How can one person matter? Dillard searches for answers in a powerful array of images. For the Time Being evokes the terrifying grandeur of all that remains troublingly beyond our understanding.
A spare yet exquisitely wrought narrative...by turns funny, flinty, and sublime...Dillard meshes the historical, the scientific, the theological, and the personal in a valiant effort to net life's paradoxes and wonders. Booklist