This page is dedicated to bringing you the most current FMS news. If you would like to receive the FMS Daily Announcements in your email, just email Stephanie Hughes.
This Tuesday Art Club will be having a guest Caricature Artist. We will also have 10 extra seats available to other students interested if you would like to learn how to draw using the Caricature techniques. You need a permission form to attend, see Ms. Vinci for the form. This will be first come first served. All permission forms are due by Tuesday morning to Ms. Vinci in the art room. Art Club members do not need an extra permission form.
Falcon Fellows will be rounding out this year with only 3 more meetings: April 22, April 29, and May 6.
We will be conducting "Speech and Debate" activities the 22nd and 29th.
The parent meeting for girls interested in playing girls soccer for the high school next year is being moved to Thursday, April 18 at 6pm in Coach Sizemore's room. If this causes any issues with you attending, please email me and let me know. Coach Sizemore- kevins1@wcs.edu .
FMS Cheerleading Clinic & Tryouts begin today!
This is for current 5th, 6th, and 7th graders.
Clinic will take place April 15th, 16th, and tryout date will be April 18th from 4:30-6:30pm.
ONLINE REGISTRATION IS NOW OPEN FOR NEXT SCHOOL YEAR.
Please register via Skyward as soon as possible as this helps administration with teacher placement and student schedules for the 2019-2020 school year.
If cannot register online and will need a paper packet you will need to contact Mrs. Dawson and set up an appointment. Online registration help is available at FMS April 9th or May 7th 5pm – 6pm in the library.
Contact Elizabeth Dawson at 615-472-4443 or elizabeth.dawson@wcs.edu if you have any questions or need to set up Family Access.
Don't forget to fill our final forms online for your child.
This applies to all grades and all sports.
Fairview High School Try-out Information- Informational packets are in the office for pick up!
DANCE:Meeting will last for approx. 30 min.
FHS Dance Team Tryout practice days will begin tomorrow: April 16th & 17th from 3:15-5.
FHS Dance Team Tryouts will be held Monday, April 22nd from 3-5.
Tryouts are scheduled for: April 22,23,25&26 @ FVHS. Packets are now in the office!
Save the Date- Special Olympics Spring Games is May 2nd. Rain Date May 3rd.
If you are an 8th grader interested in being in the color guard at the high school, there will be an informational meeting and workshop on April 15th in the band room from 3:15-4:15. If you have questions, see Mrs. Wilcox.
FHS Alumni Game: Saturday, May 4th. Come out and support your favorite players from the past!
Fairview FFA will be holding their annual plant sale fundraiser starting Monday, April 15. Agriculture students have been working very hard to make the greenhouse look better than ever this year. We have a variety of ornamentals, hanging baskets, and vegetables available for sale. Prices are often less than what you would find at Home Depot/Lowe's, etc. and the money helps fund for field trips, contests, materials, and events for the 2019-2020 school year. Please spread the word to family and friends and feel free to contact me with any questions you may have about the sale.
Each year the Technology Student Association chapters from Fairview High School & Middle School host a Technology Engineering Camp for Elementary School students.
TechKids Camp takes place the last week in May. This year camp is scheduled for May 28th – 31st. At the camp students participate in fun STEM type learning activities.
If you are a 2nd, 3rd, 4th, or 5th grade teacher at Fairview Elementary School or Westwood PLEASE share the flyer with your students!
If you are a teacher at any Fairview school and know a 2nd – 5th grade student that would enjoy the camp or have a child that would then PLEASE share as well!
If you have any questions, please feel free to email sarar@wcs.edu.


It's no secret that at Regent Holidays we like to do things a little differently. When it comes to exploring the world, it's the off-the-beaten-track destinations that catch our eye. In fact, it doesn't even have to be a country. Some of our most intriguing destinations are the breakaway-states; regions that have declared independence but aren't yet formally recognised as a country. Our brand new Pioneering Abkhazia Group Tour introduces curious travellers to Abkhazia.
This 12-day tour is a complete package from the UK, and spends 8 nights in Abkhazia. We will access Abkhazia via Georgia, and include a little time in Georgia at the beginning/end of the trip. Departing the UK from London Gatwick we will fly to Tbilisi on the new direct overnight service from Georgian Airways. After one night in Kutaisi (Georgia), we will head to the frontier and enter Abkhazia on day 3 of the tour. Once in Abkhazia we will tour the country in detail, spending nights in the capital Sokhumi, the second city of Gagra as well as in the lesser city of Novy Athon. This itinerary will strive to include as much variety as possible—giving the visitor an understanding of Abkhazia's past, present and future, together with an insight into the culture and cuisine of the country. We will also strive to get off the beaten track to take in the incredible scenery of Abkhazia, with visits to remote settlements and mountain areas. After exploring the country we will return to Georgia for a final night in Tbilisi before flying back to London Gatwick, once more with Georgian Airways.
This varied itinerary has been crafted to give the visitor an understanding of Abkhazia's past, present and future, whilst also exploring the remote scenery, culture and cuisine of the country.
Our Discover Abkhazia video has been created with footage taken on our 2017 group tour.
Depart London Gatwick this evening on the new direct service Georgian Airways flight to Tbilisi, scheduled to depart at 2255.
PLEASE NOTE: If horse riding on this day riding helmets cannot be provided. The road to Nikolai's home is on a very rough dirt track that is not always passable. As a minimum therefore there may be some walking involved in order to reach Nikolai's home. Also, as today is a lesser public holiday in Abkhazia, Day of the Holy Apostle Simon The Zealot, it is possible that some small services will be taking place in Pskhu's small church or in Sokhumi - if possible we will try and witness any events that may take place.


The NSU Board is its executive body. The board consists of five members, four of which must be representatives from different Nordic or Baltic countries. The president and board members are elected by participants of NSU to serve for one calendar year at a time. Areas of responsibility are divided among the board members and include- economy, press and archive, study program, communication and PR, external relations, and management of internal board affairs. Board meetings take place in person a minimum of four times a year.
In charge of: NSU Publications 2018. Contact for: Study Circles 4 and 7, & the keynote speakers.


There's no denying that it takes time to truly build a brand that will stand out from the crowd, especially if you're in a saturated niche.
Fortunately, our latest #ContentWritingChat has some tips that are going to help your brand shine. And this is advice you'll want to implement in the coming year!
Whether you're building your own personal brand or you are part of another company's brand, these tips will still be beneficial for you. So, if you want to learn how to build a brand that people truly love, keep reading for this week's recap!
Our guest host this week was Rochelle Moulton. She's an entrepreneur and a personal brand strategist. She joined us for Tuesday's #ContentWritingChat to share her advice on branding and it's definitely worth a read! Let's dive in!
Q1: What does it mean to have a personal brand or a brand for your business? Have you built one?
Rochelle said that having a personal brand is consistently presenting yourself and your ideas so your "sweet spot" audience knows you. Your brand also needs to be authentic. If it's not, people are going to see right through you.
Lexie knows a brand is essentially the identity of the person or company that it represents. She feels your brand shows what's important and that consistency is key.
Sarah said that having a personal brand showcases what your business is and how you're able to help your customers.
When building a personal brand, you need to be transparent, genuine, and honest. Julia said that people will need to be able to relate to your brand and should want to invest in it. That's all part of helping you attract the right people.
As Brittany said, your brand is your personality, your vision, and your mission. It's all about what you do, why you do it, and how you do it. She knows that your brand helps people get to know and understand you.
Your brand is the foundation of giving your company a voice, identity, value, and awareness.
And remember, we all have a personal brand, whether you realize it or not! It's better to be smart and actually manage your brand's reputation.
Q2: What are some key elements you will need in order to build a brand that will stand out?
As Rochelle said, it's important to have clarity on who your ideal audience is. You need to know who you want to reach in order to create the content that will resonate with them. She also said that having a compelling point of view will help you stand out from others.
And don't forget that consistency is essential as well! It's going to help you establish your brand and build trust with your audience.
A unique message is going to help differentiate you from the others in your niche.
Zala said you need to figure out who you are, what your unique offering is, how you can serve your audience, and ways you can educate, entertain, and help the right people.
Cheval knows that it's important to figure out your why. That's essential because your purpose is what will keep you going when times are tough.
Besides knowing your why and the mission behind your brand, you also need to know who you're trying to reach. Without a clear idea of who your target audience is, how can you expect to reach them?
Know your why. Be consistent. Be genuine and authentic. These are all important things to keep in mind when you build your brand.
Both Kristen and Danielle know that authenticity is a must!
Authenticity, emotional connection, and value are just a few of the elements Varun feels every successful brand needs to have.
Ability and willingness to share a well-crafted story.#ContentWritingChat.
Don't forget you need a unique voice, a valuable message, strong motivation, and the ability and willingness to share a well-crafted story.
Tamara shared a great tip about analyzing your competitors. You can learn a lot from the areas they may be lacking in. You can use this as your opportunity to step up to the plate.
Q3: How important is content creation when you build a brand? How can it help your brand stand out?
Rochelle knows that both content creation and content curation are essential when it comes to building your brand.
The content you publish online can build trust and loyalty, while also boosting engagement with your audience.
As Lexie said, content can also help you connect with your audience. She knows a brand is nothing if there aren't people who believe in it.
Jason said the content you crate shows your views and opinions on things. This can help you connect with your audience on a deeper level, which can also attract new people to your brand.
Content speaks directly to your customers. It tells them who you are and what you're all about.
Don't worry about what everyone else is doing with their content. As Jade said, put your thoughts out there and see what people think. Don't be afraid to share your unique point of view, but make sure you're also engaging with the responses you get.
A3 Content creation is EVERYTHING to building up your brand, esp at beginning.
Even Julia knows the benefits of content creation! It's been her top priority when growing Express Writers.
Q4: How do you include your personality or a company's personality in the content you create online?
Rochelle knows that adding personality can be a scary thing, but she encourages people to experiment. Take cues from what others are doing and try seeing what works for you.
Language, voice, and tone need to be consistent with your brand and should be reflected in your content. Always keep that in mind when you're creating. Kristen also suggests thinking of three words to describe your brand. Those words can help guide you when writing content, designing graphics, and more.
Cheval's advice is to speak from you heart. As he said, it takes time to develop your voice, so don't rush it.
It helps to have guidelines for branding if you have a team working alongside you. As Varun said, those guidelines can help your teammates maintain tone and voice when creating content.
Jade's advice is to be yourself. If you try to be like everyone else in your industry, you're just going to blend in.
Don't forget to engage with others! It shows them the human side of your brand. Don't let them think you're some sort of robot.
A helpful piece of advice from Shelly: not everyone is going to like you. You need to focus on resonating with the right people, which are the ones in your target audience. It's okay to repel the ones who aren't the best fit for you.
Be yourself. If you aren't true to who you are, people are going to see right through a false facade.
Zala had some kind words to share about the Express Writers team, which we couldn't appreciate more! We like to make sure our team is visible and an active part of our brand.
Q5: How can brands use storytelling in 2018 to ensure they stand out from the crowd?
Rochelle suggests writing how-to content, sharing mistakes or light bulb moments, talking about client experiences, and more. These are all great ideas to implement.
Sarah suggests showing others how you and your audience work together to fulfill their needs. They want to know how you and your offering will benefit them.
To showcase the benefits you can provide to your target audience, sharing client success stories is a great way to get started.
Immerse yourself in your brand and live your story. As Zala said, it's important to take about your successes and your failures. That's relatable and it's what draws people into your story.
Andrea knows that video marketing is where it's at. If you aren't already using video to tell your story, 2018 is the time to do it.
Brian said that a well-crafted story gives you a foundation for all your marketing and sales content. You want to be consistent to stand out and become easily recognizable by your audience. He also pointed out that you might not get things right the first time, so don't be afraid to make tweaks.
Q6: How can you tell if your brand is resonating with the right audience? Are there metrics you can track?
There are a variety of metrics you may want to track. Rochelle suggests looking at social media engagement stats, new client stats, etc.
Ask yourself who is engaging with your content and what they have to say about it. This will help you determine if your content is working or not.
Both Sarah and Lexie know that conversions are key. It's important to keep your end goal in mind and create the content that's going to help you get there. Plus, conversions can be a number of things, not just sales.
A6: Social engagements. Page views. Duration on a page.
Support tickets—hear what people think of your services/products. Great feedback comes from unhappy customers/readers. Respond with empathy and rectify. #ContentWritingChat.
You can look at social media engagement, page views, duration on page, and support tickets from customers.
Are people sharing your posts? How much time are they spending on your site? Are they leaving comments, and if so, what are they saying? These are just a few things you'll want to ask yourself.
You can even look at your subscribers to see if they represent your target audience. If they do, you're on the right path. If not, you have some work to do!
Q7: Your brand just isn't working for you anymore. What should you do if you think it's time to re-brand?
Rochelle said to use this as an opportunity to better hone your niche and your message. You want to get clear on who you're targeting and what you're providing.
Before you dive head first into a re-brand, ask yourself what isn't working for you. This will help you determine where you need to begin your work.
As Lexie said, you can't change anything without first knowing what the problem is. From there, you can make tweaks until you find what works for you.
Tamara also suggests reviewing your strategy to see what's not working and why. This is always the best place to start.
Sarah's advice is to do your research. You can even ask your audience for feedback since they're the ones you need to resonate with.
If you have a team working with you, get their help as well. Figure out who you are, who you serve, and why.
??What made prev. branding unsuccessful?
??What elements will transfer over?
Gaby shared some great questions you should ask yourself if you're facing the possibility of a re-brand.
Sometimes a refresh is all you need! Don't immediately jump into a re-brand. Take time to figure out what's really in your best interests.
Zachary suggests figuring out why things are no longer working and what has changed. Sometimes a change in your strategy is all you really need.
As Julia said, you want to be very careful about moving forward with a re-brand. It can get tricky and it's a lot of work to take on.
Q8: Is there any final branding advice you'd like to share with us?
Be genuine and focus on how you can transform your audience.
Be yourself. Be unique. Be authentic.
If it's not broke, don't fix it! Sarah said you shouldn't be afraid to go for a facelift when it comes to your brand, but a full makeover isn't always necessary.
Cheval said to make sure your brand emulates your core values. He also encourages you to focus on producing quality content on a regular basis.
As Brittany mentioned, your brand is more than your logo. It's your identity.
Holding a focus group with your target audience is a great way to get feedback!
Be consistent, be patient, and never give up!
Want to join the next #ContentWritingChat? You can hang out with us on Twitter every Tuesday at 10 AM Central Time! Just be sure to follow @ExpWriters and @writingchat for all the latest!


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From 28 March until 21 April, the Fusion Expo (a traveling exhibition on fusion run by EFDA) will be open to the public in Nîmes. The venue is the "ShowRoom de Nîmes Métropole", at Rue du Colisée 3 in Nîmes. Local coordinator of the Expo is Michel Dumas of the Université Montpellier 2.


M. A. M. de Aguiar, E. M. Rauch, Y. Bar-Yam, Invasion and extinction in the mean field approximation for a spatial host-pathogen model, Journal of Statistical Physics 114, 1417-1451 (2004).
We derive the mean field equations of a simple spatial host-pathogen, or predator-prey, model that has been shown to display interesting evolutionary properties. We compare these equations, and the equations including pair-correlations, with the low-density approximations derived by other authors. We study the process of invasion by a mutant pathogen, both in the mean field and in the pair approximation, and discuss our results with respect to the spatial model. Both the mean field and pair correlation approximations do not capture the key spatial behaviors—the moderation of exploitation due to local extinctions, preventing the pathogen from causing its own extinction. However, the results provide important hints about the mechanism by which the local extinctions occur.


Alliance has a broad spectrum of Consulting Services that spans from the boardroom to the raised floor to the desktop. We have architects, engineers, project and program managers, technicians and consultants to provide support to any part of your business.
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Alliance has on staff professionals who have managed complex IT and non-IT projects for large firms. We have collectively hundreds of years of experience in project and program management. Not only can we provide you with PM resources, we can also provide you with training to build your own team of PMs. We use best practices in our engagements and in our training as defined by PMI.
Whether your project is adding additional storage to your IT infrastructure, integrating an acquired company into your infrastructure, or developing a new line of business, Alliance can provide you with experts that will guarantee your project will come in on time, properly accomplished and within budget.
Alliance uses all these skills and all these resources to help us put the right person at your site to solve your problems based on our experience. Each and everyone of the people that we have out on-site as part of your staff has the full skill set, experience and knowledge of Alliance behind them. Whether your need is short-term of long-term, be assured that the people we provide will meet your needs whether it is IT related, general business related or functionally specific such as healthcare, financial, communications or any one of hundreds of other vertical businesses.
When one thinks of Consulting Services, one usually thinks of "hands on" support in and around the raised floor to complete specific tasks or provide help with specific issues. However, good Consulting services as it relates to business problems and IT are of value throughout an organization.
An example might be the acquisition of a company or the initiation of a new business practice. What resources need to be brought to bear? What impact will the new entity have on your existing infrastructure? What makes sense to integrate?...to leave as is?..to discard? What considerations need to be given to vendor relations?
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Alliance consulting and system engineers use proven practices and procedures for all of our technology assessment. Let Alliance help you understand your current environment compared to industry best practices. Alliance offers a number of comprehensive assessments across the enterprise.
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text:
#include <memory>
#include <utility>

#include "absl/memory/memory.h"
#include "tensorflow/compiler/xla/literal.h"
#include "tensorflow/compiler/xla/service/gpu/tests/gpu_codegen_test.h"
#include "tensorflow/compiler/xla/service/hlo_computation.h"
#include "tensorflow/compiler/xla/service/hlo_instruction.h"
#include "tensorflow/compiler/xla/service/hlo_module.h"
#include "tensorflow/compiler/xla/shape_util.h"
#include "tensorflow/compiler/xla/xla_data.pb.h"
#include "tensorflow/core/platform/test.h"

namespace xla {
namespace gpu {

class GpuNoAliasTest : public GpuCodegenTest {};

TEST_F(GpuNoAliasTest, Concat) {
  HloComputation::Builder builder(TestName());

  auto param_shape = ShapeUtil::MakeShape(F32, {2, 2});
  HloInstruction* param_x = builder.AddInstruction(
      HloInstruction::CreateParameter(0, param_shape, "x"));
  HloInstruction* param_y = builder.AddInstruction(
      HloInstruction::CreateParameter(1, param_shape, "y"));
  HloInstruction* concat =
      builder.AddInstruction(HloInstruction::CreateConcatenate(
          ShapeUtil::MakeShape(F32, {2, 4}), {param_x, param_y}, 1));
  builder.AddInstruction(HloInstruction::CreateConcatenate(
      ShapeUtil::MakeShape(F32, {2, 6}), {concat, param_x}, 1));

  std::unique_ptr<HloComputation> computation = builder.Build();

  auto hlo_module = CreateNewVerifiedModule();
  hlo_module->AddEntryComputation(std::move(computation));

  CompileAndVerifyIr(
      std::move(hlo_module),
      R"(CHECK: define{{.*}}void @{{[a-zA-Z0-9_]+}}(i8* noalias align {{[0-9]*}} dereferenceable({{[0-9]*}}) %{{.*}}, i8* noalias align {{[0-9]*}} dereferenceable({{[0-9]*}}) %{{.*}}, i8* noalias align {{[0-9]*}} dereferenceable({{[0-9]*}}) %{{.*}}))",
      /*match_optimized_ir=*/false);
}

}  // namespace gpu
}  // namespace xla

redpajama_set_name: RedPajamaGithub


Edition notes: Edited from London, British Library Add. MSS 30480-4. Transposed up a minor third. Note values halved.
Description: One of the less competent compositions from Tudor England, employing an unusually wide compass ranging, in the original notation, from bottom C in the bass to the A above middle C.
as do the heathen, which know not God.
my Lord God, both in body and spirit.
This page was last edited on 24 February 2017, at 21:41.


Rik Mallin sands paint outside his home in Villa Park on Sept. 11, 2017. The Mallins plan to sell their home and leave Illinois, citing rising taxes.
Even after watching Hurricane Irma wreak havoc on Florida, Rik Mallin is sticking to his plan.
Mallin is fixing up his Villa Park home so that he can sell it, move to the Florida Panhandle and escape Illinois' rising taxes.
"I'm getting out," said Mallin, 67. "It's not just the property taxes on my home; it's all of them." He figures his taxes in Florida, where there is no personal income tax, will be about a quarter of what he's paying now.
Mallin's not the only one leaving the state. In 2016, Illinois lost 37,508 people, putting the state's population at its lowest level in nearly a decade, according to U.S. census data. It was the third consecutive year the state lost more residents than any other state. The state's population count for 2017 won't be released until December.
Some of those who are leaving Illinois say they're frustrated with their tax burden and the state's financial situation. After going more than two years without a budget, Illinois lawmakers passed a spending plan over the summer, one that involved a 32 percent income tax hike for residents. The state is still digging itself out of the financial disarray that accumulated during the budget impasse. A Forbes listing of the best and worst states for taxes in 2016, before the tax hike, ranked Illinois 46th, signifying a heavier tax burden.
But demographers aren't ready to chalk the outmigration up to tax pressures entirely.
Brookings Institution demographer William Frey said that when people move, it's usually for employment, and not necessarily because of taxes. Most of the people moving are in their 20s and 30s, are establishing themselves in careers, and are relocating for job opportunities.
"When you have more people moving out than in, it means the employment is somewhere else," Frey said.
Regardless of whether or not they are the primary factor behind relocations, high tax rates still affect the housing market by cutting into the amount of money residents have to spend.
Lance Ramella, president of Housing Trends, a Chicago-based housing market consultant, said weak growth in the Chicago economy, unresolved government fiscal issues and high taxes are all tied together, and are resulting in a "stagnant" housing market.
"Entry-level buyers shop based on what they can afford monthly," Ramella said.
Ultimately, if those shopping for homes can't afford the mortgage payments plus taxes, they'll settle for lower-priced residences. They may even skip buying a home altogether if there aren't enough affordable homes on the market. This could cause a deterioration in housing prices, he said.
The average Chicago property tax bill is going up about 10 percent this year following City Hall and Chicago Public Schools tax hikes to pay for police, fire and teacher pensions, according to calculations released Tuesday by the Cook County clerk's office.
Housing construction already is being affected, he said. With construction costs high and demand muted, only 7,511 single-family homes were built in the Chicago metro area during the 12 months ending with July.
Before the recession, 40,000 new homes were being constructed each year. Normal demand is closer to 15,000 to 20,000 new homes a year, Ramella said.
"We really need stability in our government, and I don't know how that's going to happen," he said.
Ramella contrasted most of the Chicago metro area with Lake County, Ind., which has experienced heavy housing growth that Ramella attributes to a flight from Illinois taxes. Housing construction in northwest Indiana increased 18 percent this year over last, and in 2016 the number was up 19 percent over 2015. Meanwhile, Cook County is down 6 percent this year over last, and home construction in many other Illinois counties is flat, he said. DuPage County experienced an upturn of 5.9 percent this year.
Sheila Tracy, a Chicago optician, said the recent income tax hike as well as the sweetened beverage tax implemented by Cook County in August both seem like desperate governmental attempts to deal with unresolved financial problems.
Cook County's commissioners are expected to vote on repealing the sweetened beverage tax Oct. 10, though it's unclear at this point if there are enough votes to overturn the measure.
Even though it's difficult to determine how much of an impact tax concerns are having on real estate, the weakness in the local housing market is clear, said Geoff Smith, executive director of the Institute for Housing Studies at DePaul University. Home prices in many major metropolitan areas have recovered and hit new highs since the 2008 housing crash, but in the Chicago area only a few suburbs and select city neighborhoods have rebounded, he said.
According to CoreLogic, a global property information firm, the Chicago area is one of the U.S. metropolitan areas with the highest percentage of homeowners underwater with their mortgages, meaning they owe more than their homes are worth. During the second quarter of this year, 10.8 percent of residences in the Chicago metro area were underwater, compared with 13.5 percent during the second quarter of 2016, according to CoreLogic.
The day after the Illinois legislature voted to raise the individual income tax rate from 3.75 to 4.95 percent, Northfield-based financial planner Ellen Rogin said she started getting phone calls from clients who are residents of Chicago with second homes in Florida.
Rogin said anyone considering a move has to look at lifestyle, not just taxes.
Chicago certified public accountant Debbie Lessin said that when people consider a move for tax reasons in retirement, they may be missing a crucial element of Illinois' tax system.
Illinois doesn't tax retirement income from Social Security, pensions and IRAs, an advantageous provision for retirees living in the state, she said.
BMO Private Bank Chief Investment Officer Jack Ablin is among those with residences in both Chicago and Florida. The seriousness of Illinois' budget and tax issues became clear to him, he said, when he tried to sell his Highland Park home a couple of years ago. The house was on the market for a year and priced right, he said, but potential buyers kept raising concerns about property taxes.
After struggling to sell the home, he challenged the assessment and was able to substantially lower the taxes. A few months ago, he found a buyer for the home, but the offer was contingent on seeing documentation of the assessment and the lower taxes.
Ablin, who works in both Florida and Chicago, said he ultimately decided to make Florida his primary residence. He rents an apartment in downtown Chicago that he uses when in the city on business.


text:
Come&Live!
Provoke&Inspire
Scruffhouse
Film - Australia
"Pete and I both saw Chad Johnson's first documentary in New Zealand before we even knew each other, and we were so inspired by it, we both separately decided to use film to share the Gospel! It was that choice that brought us together and later led us to work with Steiger and shoot a documentary of the Steiger/Come&Live! band "No Longer Music" during their 2014 tour. On that same trip, we met Chad and have since been working on different projects together. Being part of Steiger over the last several years just naturally led us to joining the Come&Live! Artist Community."
"It often feels so lonely as a creative, and especially as one with a missional focus. Having a whole family of people on this same journey together, cheering you on and challenging you to walk the road less traveled, is awesome! Come&Live! is a family of creative legends. It's community. We love that."
As Scruffhouse, we believe that film is the most effective way of connecting with our generation, so that's the medium we want to use to boldly communicate Jesus to them. In a world saturated by the fake and filtered, a craving for real story has become our focus.
Our heart is to create a sustainable nonprofit that enables us to advance the Gospel, serving Christian ministries and nonprofit organizations through film, and equipping others to do the same.
C&L! Artist Interview - Scruffhouse
Bernie Cowan
Australian born and bred, Scruffhouse, is our latest addition to the C&L! artist roster. I had the absolute privilege of taking a little time to chat with Peter from Scruffhouse.
scruffhouse.com
© STEIGER 2017
Made in Germany by Steiger Design Office
We are a worldwide community of artists committed to proclaiming the revolutionary message of Jesus through our art and platform.

redpajama_set_name: RedPajamaCommonCrawl


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Military widows set to finally receive survivor benefits
by: Kellie Meyer
Posted: Dec 12, 2019 / 05:39 PM CST / Updated: Dec 16, 2019 / 05:48 PM CST
WASHINGTON (Nexstar) – A bipartisan defense bill moving through Congress includes benefits that thousands of military families have been waiting for.
The bill means more than 65,000 surviving military spouses will receive benefits they've earned after decades of waiting.
"They now know people truly care," Sen. Doug Jones (D-AL) said.
Sen. Jones helped get what's called the "Military Widows Tax" repealed as part of this year's National Defense Authorization Act. Sen. Jones – with the help of Sen. Susan Collins (R-ME) – eliminated a provision that prevented Gold Star families from receiving full survivor benefits from the Department of Defense and Veterans Affairs.
"It has just been one of the most unfair programs that the United States has had that has been hoisted on military families," he said.
The bipartisan step marks an 18-year effort to pass the bill on Capitol Hill. Washington Correspondent Kellie Meyer asked why it took so long to go through.
"I think it's been a budget issue," Sen. Jones said. "You know a lot of times people up here, they tend to look at dollars and cents more than they do right or wrong."
Jones says this year was different. Nearly 80 senators co-sponsored the bill.
The changes will be phased in over the next three years. Jones says that means about an additional $12,000 per year from about 2,000 spouses in his home state of Alabama.
For military spouses, that help can't come soon enough.
"They had worked on this for so long and they thought the country was just ignoring their pleas," Sen. Jones said.
The NDAA passed the House just this week. It's expected to pass the Senate before lawmakers break for the holiday recess.
More Washington-DC Stories
Trump to divert billions from Pentagon to build border wall
by Alexandra Limon / Jan 16, 2020
WASHINGTON (NEXSTAR) — The White House confirmed President Trump is looking to divert additional money to the construction of the border wall.
President Trump has said, "Mexico is paying for the wall. You know that, you'll see that."
Late former president George H.W. Bush, first lady Barbara to get commemorative gold coins
WASHINGTON, D.C. (NEXSTAR) — Monday, the House passed the Bush Coin Act, which would put Former Pres. George H. W. Bush's image on a dollar coin and his wife, First Lady Barbara Bush, on the First Spouse $10 commemorative coin.
Texas Republican Congressman Roger Williams says his friend and fellow Texan shared more than just a passion for America's favorite pastime, they also shared a passion for politics.
Lawmakers say new food stamps plan would make it harder for veterans to qualify
by Jessi Turnure / Jan 10, 2020
WASHINGTON (NEXSTAR) - Federal officials told Congress this week that proposed changes to the food stamp program did not consider the impact on veterans.
More than a million low-income veterans depend on SNAP to feed their families.


Dennis Maloney is a senior marketing and technology leader with over 20 years of experience creating and growing both consumer and Internet brands with expertise developing breakthrough brand strategies as well as coordinating the marketing and engineering resources needed to execute those strategies, a rare hybrid combination of creative and technical experience. Currently, Dennis serves as Domino's Pizza's vice president and chief digital officer, responsible for all Domino's U.S. digital programs, including digital marketing and digital experience development. He joined Dominos in 2010 as vice president of Multimedia Marketing. Prior to joining Domino's, Dennis was director of Global E-Commerce at Coca-Cola from 2006 to 2009 and previously held senior roles at Reflect.com and Procter & Gamble.
A graduate of the U.S. Naval Academy with a B.S. in mechanical engineering as well as the Catholic University of America with a Master of Mechanical Engineering, Dennis served over seven years with the U.S. Navy as an instructor of systems engineering at the U.S. Naval Academy as well as a submarine officer.


text:
Two Trump Judicial Picks Not Moving Forward
The Associated PressDec 13th, 2017, 3:50 pm
WASHINGTON (AP) — Two of President Donald Trump's nominees for federal judge are not moving forward amid criticism from a leading Republican senator.
White House officials say the nomination of Brett Talley has been withdrawn. And a person familiar with the process says the administration hasn't submitted paperwork for the nomination of Jeff Mateer and does not intend to. The officials spoke on condition of anonymity to describe internal deliberations.
Republican Sen. Chuck Grassley of Iowa, the chairman of the Senate Judiciary Committee, has said the two nominees were going nowhere.
In speeches, Mateer has described transgender children as evidence of "Satan's plan."
Talley has never argued a case in court and was rated "unanimously unqualified" by the American Bar Association.
Grassley said in an interview with CNN on Tuesday that he told the White House to rethink both nominations.
Mateer was tapped to be a federal judge in Texas, while Talley was picked for U.S. District Court in Alabama.
[image via screengrab/Fox News]
Brett Talleychuck grassleyDonald TrumpJeff Mateer


text:
Walter F. Rogosheske, Associate Justice 1962-1980
Walter F. Rogosheske
Before his appointment to the Supreme Court in 1962, Walter F. Rogosheske was a member of the State House of Representatives.
Walter Fredrick Rogosheske was born on July 12, 1914 in Sauk Rapids, Minnesota. He attended Trinity Lutheran School for elementary school and went on to attend Sauk Rapids High School. From 1932 to 1933, Rogosheske attended St. Cloud State Teachers College. He then attended Valparaiso University in Indiana from 1933 to 1934. Rogosheske obtained a B.S.L. from the University of Minnesota in 1937 and received his LL.B. from the University of Minnesota Law School in 1939. Rogosheske was admitted to the Minnesota bar in January 1940. He also opened his private law practice in 1940 and married his wife, Dorothy Heywood.
Rogosheske was elected to the Minnesota House of Representatives in November 1942 at the age of 28. He represented District 45 which covered Benton and Sherburne Counties for the 1943 term, but resigned to serve in the United States Army during World War II. Rogosheske was re-elected to the House in 1944 and was released from the Army in time to serve in the 1945 to 1946 session. Rogosheske was re-elected again in 1946. He ran again in 1948, but following his advocacy for an anti-gambling bill, he was defeated by pro-gambling candidate John T. Kosloske by 61 votes. Despite the narrow margin of victory, Rogosheske did not contest the election. While serving as a legislator, Rogosheske was the House chief author of the 1945 Area Vocational School bill. You can read more about Rogosheske's time in the legislature in the member record compiled by the Minnesota Legislative Reference Library (linked below).
Rogosheske served as the Chairman of the Minneapolis-St. Paul Airports Commission from June 13, 1949 until 1950. This commission was in charge of selecting the location for what is now the Minneapolis-St. Paul International Airport.
On March 1, 1950, Governor Luther Youngdahl appointed Rogosheske as a District Court Judge in the Seventh Judicial District. Rogosheske won re-election in November of that same year and again in 1956. On February 1, 1962, Rogosheske was appointed as an Associate Justice to the Minnesota Supreme Court by Governor Elmer L. Andersen. He served on the Supreme Court until his resignation on September 7, 1980.
Also in 1950, Rogosheske became a lecturer at the University of Minnesota Law School. He enjoyed conducting moot court sessions at the law school for many years. Rogosheske also served as a member and chairman of the A.B.A. Advisory Committee on Prosecution and Defense Functions, A.B.A. Project on Standards for Criminal Justice.
Rogosheske died on May 15, 1998 at the age of 83. He and his wife had 4 children together.
You may read more about the life and work of Justice Rogosheske in the Minnesota Supreme Court Historical Society's book: Testimony: Remembering Minnesota's Supreme Court Justices, which is a source of this brief biography.
Walter F. Rogosheske's signature from the Minnesota Supreme Court's Roll of Attorneys,1858-1970, p 269 (January 16, 1940), available online at: https://collection.mndigital.org/catalog/sll:12973?pn=false#/image/236
Highlighted Resources
Walter Rogosheske Oral History
Justice Rogosheske gave two interviews about his judicial career in 1978 and in 1995. Visit this page for transcripts and information about recordings in our collection.
Member Record - Walter Rogosheske
Testimony: Remembering Minnesota's Supreme Court Justices by Minnesota Supreme Court Historical Society
Call Number: Main Collection KF372 .T47 2008
Next: Opinions >>
URL: https://mncourts.libguides.com/rogosheske
Last Updated: Sep 10, 2021 4:31 PM


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All the 2021 Financial Mail AdFocus award winners!
All the winners of the 2021 Financial Mail AdFocus Awards were announced on 24 November 2021, during an event in Johannesburg that streamed online...
Making a difference that matters
Recognised as the AdFocus Lifetime Achiever 2021, Thebe Ikalafeng, founder and chairperson of Brand Africa, dedicated his award to the many people who had an impact on his life.
He describes leaving his grandmother's house to attend Wits and then dropping out and how this led him to the US.
"After studying in the US, I worked at Colgate in New York. I had zero ambition, but to be good at what I do," he says.
Returning to this country in 1994, he says it was an interesting time. "We needed examples and evidence that we can be excellent at what we do," he says.
It was after he left corporate that he began to question himself. "Is our work purposeful and what is our purpose? I wanted to make a difference that would matter, and to be authentic in what I do," he says.
So began a journey that would take him across Africa, inspiring him to become the global African thought leader and authoritative voice in branding in Africa he is today.
2022 chairperson
Outgoing AdFocus Awards chairperson, Tumi Rabanye, says the awards are an achievement for the industry. "The winning agencies are the gamechangers and they are driving the AdFocus brands three decades later," she says.
Faheem Chaudhry, partner, managing director, M&C Saatchi Abel, will take up the reins from Rabanye, as the 2022 AdFocus Awards chairperson.
AdFocus awards
Thebe Ikalafeng
2021 FM AdFocus Awards
Joe Public United Johannesburg
Read more: advertising, awards, Joe Public, AdFocus awards, Thebe Ikalafeng, Danette Breitenbach, Razor, 2021 FM AdFocus Awards, Joe Public United Johannesburg


This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.
Guo, X., Wang, X. M., Xiao, S. J. Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules. J. Vis. Exp. (146), e58744, doi:10.3791/58744 (2019).
This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.
DNA molecules have been used to build many kinds of nanostructures over decades. Typical motifs include DAE (double crossover, antiparallel, even half-turns) and DAO tiles1,2,3, star tiles4,5,6,7, single stranded (ss) tiles8,9,10, and DNA origami11,12,13. These DNA motifs and lattices are assembled from linear ss-DNAs. Recently, others and we have reported the use of circular ss-oligonucleotides as scaffolds to build motifs, 1D nanotubes, and 2D lattices14,15,16,17. By inserting a Holliday junction (HJ)18,19,20,21 at the center of c64nt, a pair of two coupled DAO tiles can be formed17. This new cDAO motif and its derivatives are stable and rigid enough to assemble 2D DNA lattices up to 3 × 5 µm2. In this paper, we use a term of "circular tile", which is defined as a stable DNA complex molecule constructed with one circular scaffold and other linear staples of ss-oligonucleotides, and another term of "linear tile", which is built from a full set of linear ss-oligonucleotides.
This protocol demonstrates how to construct five kinds of DNA nanostructures with small circular DNA molecules as scaffolds: 1) infinite 1D c64nt and c84nt nanowires, 2) infinite 2D cDAO-c64nt-O and cDAO-c64nt-E (-O represents an odd number of 5 half-turns and -E represents an even number of 4 half-turns) lattices, 3) infinite 2D cDAO-c84nt-O and cDAO-c84nt-E lattices, 4) finite 2D 5 × 6 cDAO-c64nt-O and 5 × 6 cDAO-c74&84nt-O rectangles, 5) infinite 1D acDAO-c64nt-E nanorings and nanospirals (please refer to Figure 3-5 for the schematic drawings and images of the above five kinds of DNA nanostructures). The 1D c64nt and c84nt nanowires are assembled from each c64nt and c84nt scaffold associated with two linear staples respectively. Each circular tile of cDAO-c64nt, acDAO-c64nt, cDAO-c74nt, or cDAO-c84nt is annealed from its corresponding scaffold of c64nt, c74nt, or c84nt with four linear staples respectively. The infinite 2D lattices are assembled from the same type of two circular tiles with different sequences. The two finite 2D rectangle lattices are assembled from two sets of 32 circular sub-tiles respectively. To save money, only one-sequenced c64nt, c74nt, and c84nt is used as the respective scaffold while different overhangs are used to anneal the 32 cDAO-c64nt, 12 cDAO-c74nt, and 20 cDAO-c84nt circular sub-tiles respectively in the first sub-tile annealing step, then mix the corresponding 32 circular sub-tiles together and apply the second lattice annealing step to assemble the finite 5 × 6 cDAO-c64nt-O and 5 × 6 cDAO-c74&84nt-O lattices, respectively. Definitely, differently-sequenced circular scaffolds can be adopted to assemble a variety of finite size nanostructures, however it will cost more money and labors. The infinite 1D acDAO-c64nt-E nanorings and nanospirals are annealed from one-sequenced asymmetric acDAO-c64nt tiles with linear connections of an even number of 4 half-turns. There are two approaches to assemble infinite 2D lattices from circular tiles of cDAO-c64nt and cDAO-c84nt, which are distinguished by the intertile distances of an even number of 4 and an odd number of 5 half-turns respectively. The former requires all tiles to be aligned identically; the latter requires alternation of the faces of two neighboring tiles along the helical axes. If the tile is rigid and planar, such as cDAO-c64nt, both approaches will generate planar nanoribbons; if the tile is curved towards one direction, such as cDAO-c84nt, the intertile connection of an even number of 4 half turns will generate nanotubes, whereas the intertile connection of an odd number of 5 half turns will produce planar nanoribbons due to elimination of curvature-biased growth by alternate alignment of curved tiles. The successful assembly of 1D and 2D DNA nanostructures from circular tiles indicates several advantages of this new approach: enforced stability and rigidity of circular tiles over linear tiles, chiral tiles for assembly of asymmetrical nanostructures such as nanorings and nanoribbons, new visions on understanding the DNA mechanics and molecular structures, etc.
Use all linear DNAs provided by commercial companies directly without further purification.
Centrifuge the DNA samples at 5,000 × g for 5 min to collect all DNA pellets at the bottom of the tubes. Add an appropriate volume of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) to dissolve the DNA.
Measure the concentration of "a" ng/µL for each ss-DNA solution using a micro UV spectrometer at 260 nm. Convert "a" ng/µL to "b" µM following b = a × 103 / (molecular weight of the DNA strand). Adjust the amount of TE solution to make a 10 µM DNA stock solution.
Use T4 DNA ligase to connect the 3' and 5' ends of 5'-phosphorylated linear DNA templates ( Figure 1) of c64nt, c74nt and c84nt provided from commercial companies directly.
Mix the 5'-phosphorylated linear DNA strand (3.5 µM) and its corresponding splint DNA strand (4.5 µM) in 80 µL TE buffer in a 200 µL PCR test tube15. Incubate the tube in an open thermo bottle filled with 95 °C water. Cool down the hot water to room temperature (25 °C) for about 2-3 hours under the lab atmosphere.
Add 10 µL of 10x T4 buffer (660 mM Tris-HCl, 66 mM MgCl2, 100 mM DTT, 1 mM ATP) and 10 µL of T4 ligase (300 U/µL) to the mixture to a final volume of 100 µL. Incubate the mixture in a thermocycler for 16 hours at 16 °C.
NOTE: The above DNA concentrations and reaction volume of ~100 µL are optimized for the high ligation efficiency and the high yield of correct oligo-monomer cyclization. Run two or more tubes of ligation reactions in the same time according to the experimental design. An alternative incubation procedure for ligation replacing step 1.6 is 4 hours at 25 °C.
After the incubation, inactivate the T4 ligase in 95 °C water for 5 minminutes. Then transfer the tube to an ice water bath and incubate for 5 minutes to cool down.
Add 10 µL of 10x Exonuclease I buffer and 10 µL of Exonuclease I (5 U/µL) to the quenched mixture and incubate the tube at 37 °C in a water bath for 30 min to selectively digest the remaining linear DNAs and leave the circularized DNAs intact.
Prepare a 10% denaturing PAGE gel.
Wear rubber gloves and goggles when preparing the PAGE gel in the fume hood. Add 6.67 mL of 30% (w/v) acrylamide/bisacrylamide solution (19:1), 2 mL of 10x TAE·Mg buffer (40 mM Tris, 40 mM HAc, 1 mM EDTA, pH 8.0 ,12.5 mM Mg(Ac)2), 8.4 g of urea (7 M) and deionized water to a final volume of 20 mL in a 40 mL centrifuge tube.
Caution: The 30% (w/v) acrylamide/bisacrylamide solution (19:1) solution is toxic.
Add 20 µL of tetramethylethylenediamine (TEMED) and 100 µL of ammonium persulfate (APS, 10% w/v) to the 40 mL tube before transfer the gel solution to the electrophoresis system immediately. Insert a 1.5 mm thick and 10-well comb. Wait at least 20 min until the gel solution is solidified.
Set a constant voltage of 80 V for a gel of 85 × 80 × 1.5 mm3 (width × height × thickness). Add 1x TAE·Mg buffer to the electrophoresis system and prerun the gel for 20 min at 10 V/cm.
Put the PCR test tube from step 1.8 in 95 °C water for 5 min to inactivate Exonuclease I. Then transfer the tube to an ice water bath and incubate for another 5 min.
Add 20 µL of formamide in the tube to a final volume of 140 µL and mix the solution. Distribute the solution to 7-9 denaturing PAGE gel wells equally with 16-20 µL for each gel well. Mix 2 µL of loading dye (0.05% bromophenol blue, 0.05% xylene cyanol FF, 60% glycerol, 10 mM Tris-HCl, 60 mM EDTA, pH 7.6) with 8 µL of the corresponding precursor linear DNA (10 µM) and inject the mixture to a separate well for reference.
NOTE: The addition of formamide is to increase the density of the loading solution for sinking to the bottom of the PAGE gel well and also to disassociate some double stranded DNA residues.
Run the gel for about 2 h at 10 V/cm and stop running when xylene cyanol FF is at the 2/3 position of the glass plate with the naked eye.
Wear rubber gloves and goggles to protect skins and eyes. Take the gel out of the glass plate. Place the gel on a fluorescent TLC (thin layer chromatography) plate. Cut off the target gel bands by a razor blade as exactly as shadowed by UV irradiation (Figure 2).
Caution: The UV light is harmful to eyes and skins.
NOTE: Under UV irradiation at 254 nm, DNA will absorb the light and cast a shadow onto the TLC plate. The circular DNA ran slightly slower than its corresponding precursor linear DNA. Some undigested linear DNAs and unwanted circular DNAs in small amounts which surround the shadowed target band will pollute the circular DNA if they are collected.
Transfer the gel bands into a 2.0 mL microcentrifuge tube. Air dry or blow dry the gel bands and then mash the gels into a paste by a spatula or flattened glass rod. Make sure the gel has been completely crushed into a paste, which is critical to the high yield of circular DNA. Add twice of the gel volume deionized water into the tube and shake the tube at room temperature overnight.
Filter the mixture to collect the supernatants in a 2.0 mL microcentrifuge tube. Recover any residual DNA by rinsing with small volume of deionized water and filter again to combine the supernatants.
Extract the eluent with equal volume of n-butanol. Repeat this procedure until the aqueous volume is reduced to about 200 µL.
Add 500 µL of absolute ethanol (−20 °C) and 20 µL of 3 M NaOAc (pH 5.1) to the mixture for DNA precipitation. Store the tube at −20 °C for 30 min.
Centrifuge the tube at 12,000 × g for 10 min at 4 °C, discard the supernatant. The remaining DNA precipitate is about 100 µL in the tube.
Add 600 µL of 75% ethanol (−20 °C) to the tube and store it at −20 °C for 30 min. Then centrifuge at 12,000 × g for 10 min at 4 °C, discard the supernatant. The remaining DNA precipitate is about 100 µL solution in the tube again. Repeat the procedure twice.
After the last centrifugation, discard the supernatant as much as possible. Dry the remains with a vacuum concentrator.
Store the purified circular DNA in the tube at −20 °C.
Resuspend the circular DNA in TE buffer to make a 10 µM circular DNA stock solution according to step 1.3 when needed.
Prepare each tile or nanostructural assembly solution of c64bp, c84bp, HJ-c64nt, aHJ-c64nt, HJ-c84nt, tHJ-c84nt, cDAO-c64nt, acDAO-c64nt, cDAO-c84nt, c64nt, c84nt, and acDAO-c64nt-E with a designed set of linear and circular DNAs for one-pot annealing.
For each assembly solution, mix 2 µL of 10x TAE·Mg buffer, 1 µL of each DNA stock solution of a set of linear and circular strands in equal molar ratios, and additional TE buffer in a 200 µL PCR test tube to a final concentration of each strand at 0.5 µM and a final volume of 20 µL. Anneal the assembly solution in a thermo bottle from 95 °C to 25 °C over 48 h.
Prepare each assembly solution of 2D infinite lattices of cDAO-c64nt-E, cDAO-c64nt-O, cDAO-c84nt-E, and cDAO-c84nt-O with a designed set of linear and circular DNAs for two-step annealing.
Prepare each precursor sub-tile assembly solution by mixing 2 µL of 10x TAE·Mg buffer, 1 µL of each DNA stock solution of a set of linear and circular strands in equal molar ratios, and additional TE buffer in a 200 µL PCR test tube to a final concentration of 0.5 µM for each strand and a final volume of 20 µL. In the first sub-tile annealing step, anneal each precursor sub-tile solution in a PCR thermocycler using a fast-linear cooling method from 95 °C to 25 °C over 2.5 h.
Prepare each assembly solution of the above four infinite lattices by mixing 10 µL of each of the two corresponding sub-tile solutions together to a final concentration of 0.25 µM for each strand. In the second lattice annealing step, anneal the 20 µL mixture in a PCR thermocycler using a slow cooling method of staying at 50 °C for 2 h and cooling down at a rate of 0.1 °C per 5 minutes to 20 °C, about 24 h in total.
NOTE: Two parallel experiments can be run simultaneously or subsequently in the second annealing step for each 2D infinite lattice.
Prepare assembly solutions of two finite rectangle assemblies of 5 × 6 cDAO-c64nt-O and 5 × 6 cDAO-c74&c84nt-O for two-step annealing.
Prepare each precursor sub-tile assembly solution by mixing 1 µL of 10x TAE·Mg buffer, 0.5 µL of each DNA stock solution of a set of linear and circular strands in equal molar ratios, and additional TE buffer in a 200 µL PCR test tube to a final concentration of 0.5 µM for each strand and a final volume of 10 µL. In the first annealing step, anneal each precursor sub-tile solution in a PCR thermocycler using a fast-linear cooling method from 95 °C to 25 °C over 2.5 h.
Prepare each finite rectangle lattice assembly solution by mixing 32 x (2 µL of each sub-tile solution) together in a 200 µL PCR test tube to a final concentration of ~15 nM for each sub-tile and a final volume of 64 µL. In the second annealing step, anneal the 64 µL mixture in a PCR thermocycler using a slow cooling method of staying at 50 °C for 2 h and cooling down at a rate of 0.1 °C per 5 min to 20 °C, about 24 hours in total.
NOTE: Five parallel experiments can be run simultaneously or subsequently in the second annealing step for each finite rectangle assembly.
Prepare a 10% native PAGE following step 1.9 except for the urea component.
For control samples of c64bp and c84bp, add 2 µL of each assembly solution and 1 µL of loading dye to 3 µL of TE buffer as controls separately. For each of other assemblies listed in Figure 3, add 1 µL of each assembly solution and 1 µL of loading dye to 4 µL of TE buffer.
Inject each of the above solutions to a gel well. Add 5 µL of DNA marker (25-500 bp) to a separate well for reference.
Run the gel for about 4 hours at 10 V/cm in an ice water bath. The xylene cyanol FF will run out of the glass plate.
Wear rubber gloves and goggles to protect skins and eyes. Take the gel out of the glass plate and immerse it in 100 mL of deionized water with 10 µL of a nucleic acid gel stain for 30 min.
Caution: The nucleic acid gel stain solution is toxic.
Observe the gel under an UV imaging system and take a picture of the stained gel (Figure 3).
Prepare a native 2% agarose gel for electrophoresis purification of finite lattices.
Mix 1 g of agarose powder, 5 mL of 10x TBE·Mg buffer (89 mM Tris, 89 mM Boric Acid, 2 mM EDTA, pH 8.0, 12.5 mM Mg(Ac)2), 2 µL of nucleic acid gel stain and 47.5 mL of deionized water in a 250 mL triangle bottle. Boil the mixture until the bubbles become small and dense. Add hot water in the bottle to a total volume of 50 mL and a concentration of 2% agarose.
Wear thick gloves. Pour the gel solution into a plastic gel box of 7 x 10 x 1 cm3 (width × length × thickness). Insert a 1.5 mm thick and 12-well gel comb. Wait for the gel to cool down to room temperature. If necessary, put the gel in a 4 °C fridge.
Caution: Be aware of scalding because the solution is very hot.
Add 0.5x TBE·Mg buffer in the electrophoresis system. Prerun the gel at 5 V/cm for 20 min in an ice water bath at a constant voltage of 50 V.
Inject 20 µL of the finite lattice solution prepared in step 2.3 in each agarose gel well and add 5 µL of a DNA marker (100-3,000 bp) to a separate well. Run the gel for 2 h at 5 V/cm in an ice water bath.
Wear rubber gloves and goggles to protect skins and eyes. Cut the target gel bands with a position similar to the 1,000 bp marker under UV light and slice them into fine pieces and place the crushed gels into a filter column8.
Centrifuge the column at 2,000 x g for 5 min at 4 °C. Extract the sample solution through the column. Collect the solution for AFM imaging.
Purify finite lattices by PEG precipitation.
Mix 50 µL of the finite lattice solution prepared in step 2.3 with an equal volume of 15% PEG8000 (w/v) buffer (20 mM Mg(Ac)2, 5 mM Tris, 1 mM EDTA and 505 mM NaCl)22. Centrifuge the solution at 18,000 × g at 4 °C for 30 min.
Remove the supernatant and add 100 µL of PEG buffer. Repeat the centrifugation.
After removing the supernatant, dissolve the pellet in 1x TAE·Mg buffer for AFM imaging.
NOTE: Purification is necessary for the finite lattices of 5 × 6 cDAO-c64nt-O and 5 × 6 cDAO-c74&c84nt-O for AFM imaging and other applications. If the yield is too low, increase the centrifugation speed. If the product is not pure enough, repeat step 4.2.2.
For c64nt nanowire, c84nt nanowire, acDAO-c64nt-E, infinite 2D lattices of cDAO-c64nt-E(O), and cDAO-c84nt-E(O), deposit 2 μL of annealed sample on a freshly cleaved mica surface. Leave for 2 min for adsorption of DNA lattices to the mica surface. Wash the surface with 100 µL of deionized water twice and dry it by compressed air.
Obtain AFM images of infinite DNA lattices in air by scanning the mica surface with triangular AFM probes under tapping mode. Set the following parameters for scanning: scan size of 0.5~5 µm, scan resolution of 512 lines, and scan rate of 3.5 Hz (Figure 4).
For finite DNA lattices of 5 × 6 cDAO-c64nt-O and 5 × 6 cDAO-c74&c84nt-O, deposit 80 µL of 1x TAE·Mg buffer on a freshly cleaved mica surface. Then add 5 µL of finite samples into the buffer. Leave for 2 min for adsorption of DNA lattices to the mica surface. Add another 50 µL of 1x TAE·Mg buffer on the AFM probe.
Obtain AFM images of finite DNA lattices in fluid by scanning the mica surface with triangular AFM probes under tapping mode. Set the following parameters for scanning: scan size of 0.5-1 µm, scan resolution of 256 lines, and scan rate of 1.5 Hz (Figure 5).
The circular DNA moves slightly slower than its precursor linear DNA in denaturing PAGE (Figure 2) because the pore inside the circular DNA is penetrated and retarded by gel fibers23,24,25. The correct ligation reaction efficiency for oligo-monomer cyclization depends on the substrate sequence and concentration, reaction temperature, time, etc. As the concentration of a precursor linear DNA is high enough at around 3.5 µM, the cyclization products of c64nt (or c84nt) and the precursor reference in this protocol can be directly shadowed as bands in the TLC plate under UV light without dying. If the bands of circular DNA are vague or invisible, indicating a failure of the ligation reaction or a much low product yield. Sometimes there are two bands above the linear precursor band, referring an additional oligo-dimer ring except for the correct oligo-monomer ring. Just leave the higher bands alone and collect the lower ones. The purified circular DNA products can be seen as white powder in the tube after vacuum drying. Except for the denaturing PAGE, the DNA purity can also be measured by UV spectrometer. The absorption peak of DNA is at 260 nm. Two standard criteria for the DNA purity are the absorption ratio of 280 nm/260 nm at 1.8 and that of 280 nm/230 nm commonly in the range of 2.0-2.2. If the above two ratios deviate from the standard values, the remains should be extracted again by following steps 1.18-1.20. The yields of c64nt and c84nt for the correct oligo-monomer cyclization are measured at the range of 30-60% according to this protocol.
Native PAGE analysis provides a lot of information about the motif's stability, purity, rigidity, assembly mode of monomer or polymer, etc (Figure 3). The c64nt assembly families of c64bp, HJ-c64nt, aHJ-c64nt, cDAO-c64nt, and acDAO-c64nt have only one clear and clean band for each assembly, representing they are stable monomer motifs. While the c84nt assembly families of HJ-c84nt, tHJ-c84nt, and cDAO-c84nt tiles have smears around their main bands, indicating minor by-products except for the target monomer motifs. Regardless of the minor byproducts of incorrect associates, excellent cDAO-c84nt-O (E) lattices with high yields can be assembled. To get a clear and clean electrophoresis image, the loading volume should be no more than 10 µL and the quantity of DNA should be 0.01~0.02 µg/mL.
The success of experiments is finally evaluated by 1D and 2D DNA nanostructures imaged by AFM (Figure 4 and Figure 5). Each assembly has its own morphological features in the micrometer scale such as nanowires, nanotubes, nanospirals, nanoribbons, etc. Moreover, the detailed textures of the DNA assemblies in the nanometer scale correlated to their theoretical circular tile sizes and organization modes very well respectively are the key for verification of successful and correct assembly. Therefore, both panoramic and high-resolution AFM images in micrometer and nanometer scales must be obtained. Choices of AFM methods and probes are crucial for the high-quality AFM images. The scan force should be adjusted as small as 50 pN26. If the scan force is too big, it would damage the DNA nanostructural patterns. The environmental cleanliness is another key parameter to get a clean and beautiful high-resolution AFM image in fluid. All buffers must be filtered by 0.22 µm filter; the probe holder and tweezers must be washed by detergent and rinsed by deionized water. If the environment is polluted by particulate debris, the probe tip would be damaged or clung by particles in the buffer, thus affecting the quality of AFM images.
Figure 1. Synthesis of circular DNA. The flow diagram represents how a long 5'-phosphorylated linear oligonucleotide in blue evolves to a circular DNA molecule. The two short red strands represent the splint oligonucleotides. Please click here to view a larger version of this figure.
Figure 2. Denaturing PAGE photograph of DNA cyclization products under UV light without dying. A precursor linear 64nt DNA band is in the far-left lane and its cyclization products of circular 64nt (c64nt) DNA appear as 9 bands at the same horizontal level in other 9 lanes. The 9 bands of c64nt will be cut off for abstracting circular DNAs. Please click here to view a larger version of this figure.
Figure 3. Native PAGE photographs after dying and schematic double-helical models of circular tiles. Both polymers of c64nt nanowire and c84nt nanowire are represented by their simplest folding unit cells, in which the two aligned dots above and below each unit cell indicate the infinite alignment of unit cells vertically up and down, with equal distances between duplexes to form nanowires. Monomer circular tiles of c64bp, c84bp, HJ-c64nt, aHJ-c64nt, HJ-c84nt, and tHJ-c84nt in A) have no protruding overhangs out of their rings, whereas cDAO-c64nt, acDAO-c64nt, and cDAO-c84nt in B) have both blunt-ended 10 bp overhangs respectively. For sequences, please refer to the Table of DNA Sequences. This figure has been modified from a previously published figure17. Please click here to view a larger version of this figure.
Figure 4. AFM images of typical 1D and 2D infinite DNA assemblies scanned in air. A) c64nt nanowire, B) infinite acDAO-c64nt-E, C) infinite cDAO-c64nt-E, D) infinite cDAO-c64nt-O, E) infinite cDAO-c84nt-E, and F) infinite cDAO-c84nt-O are annealed by sticky end cohesion. All texture details in these AFM images are in line with the tile sizes and organization modes very well. For sequences, please refer to the Table of DNA Sequences. This figure has been modified from a previously published figure17. Please click here to view a larger version of this figure.
Figure 5. AFM images of finite rectangle assemblies scanned in fluid. The finite rectangle assembly of A) 5 × 6 cDAO-c64nt-O is composed of 32 cDAO-c64nt sub-tiles, and B) 5 × 6 cDAO-c74&84nt-O is composed of 12 cDAO-c74nt and 20 cDAO-c84nt sub-tiles. For sequences, please refer to the Table of DNA Sequences. This figure has been modified from a previously published figure17. Please click here to view a larger version of this figure.
Table of DNA Sequences. Please click here to download this file.
The protocols presented in this article focus on the synthesis of small circular DNA molecules and the assembly of DNA nanostructures. Most of randomly-sequenced DNA designs can be used in this protocol. The purity of circular DNAs is critical for the success of DNA assemblies. The production yield of cyclization can be improved by lowering the concentration of 5'-phosphorylated linear DNA; however, this will increase the workload to produce the same amounts of circular DNAs. The length of splint DNA strands also affects the correct ligation reaction, it is optimized to be around 20 nucleotides long for both c64nt and c84nt.
An appropriate concentration of magnesium cations (e.g., 12.5 mM) in the solution during and after assembly is very important for the formation and maintenance of DNA nanostructures. Thus, a magnesium cation concentration of 12.5 mM is always kept during the processes of annealing, native PAGE, agarose gel electrophoresis and PEG buffer centrifugation purifications, AFM imaging, etc.
For finite rectangle assemblies of 5 × 6 cDAO-c64nt-O and 5 × 6 cDAO-c74&84nt-O, the agarose gel purification does not affect the texture details, while the PEG buffer centrifugation purification harms the texture details of DNA nanostructures in the high-resolution AFM images.
Benefit from the stability and rigidity of circular tiles, it is much easier to produce well-organized and large-size single crystalline 2D lattices from circular modules than from linear tiles and scaffolded origami9,11, although extra work is needed to synthesize and purify the circular DNA molecules. With only one circular DNA as the same core structure, many circular modules can be generated with different overhangs; by means of specific sticky end cohesions of overhangs finite nanostructures can be built; this strategy reduces the workload and cost of finite nanostructures. One significant advantage of circular DNA nanostructures is the resolution of secondary and tertiary structures of DNA molecules and their key elements such as Holliday junction from the texture details of single crystalline lattices. The 1D, 2D and 3D nanostructures built from circular modules and their potential applications in biology, medicine, and nano-engineering will become a new family member of DNA nanotechnology in the future.
We are grateful for financial support from the NSFC (grants no. 91753134 and 21571100), and the State Key Laboratory of Bioelectronics of Southeast University.
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One of the polling bookies has put the Liberal Democrats at evens to poll over 14% with YouGov by end of the year. This is a bet that nobody would be advised to take. The last time that the Lib Dems polled 14% with YouGov was in September 2010. They have been flat-lining ever since. Our most recent estimates put the Liberal Democrats at 7.7%. This is down 0.7 points on their support at the end of last August, a dismal position which a strong conference season is unlikely to do much to improve.
In many ways, the electorates' roller-coaster relationship with Nick Clegg – from the love-in of debate-fuelled Cleggmania to formation of the coalition, the tuition fees betrayal and other policy compromises in government – resembles the rise and fall of Tony Blair on fast forward. Blair's decline from progressive hero to hate figure – from the flag-waving days of May 1997 to the mass demonstrations of 2003, was six years in the making. Clegg managed to alienate almost as many voters in just five months. There is no sign that these voters are near to returning, and the Liberal Democrats can only hope that their local/personal vote saves them.
It would be wrong, however, to suggest this is simply the cost of doing business in a coalition setting. The Lib Dem's electoral success from the early 1990s was built on making a pitch to middle class, left of centre voters – unwilling or unable to vote Labour, but turned off by the "nasty" Conservative Party. More recently, since the 2000s, its gains have largely come by positioning it to the left of Labour on key issues – Iraq, Europe and the environment most notably. Indeed, in 2005 the Liberal Democrat's election platform was to the left of Labour, and in 2010 it was largely level. It can hardly be any surprise, then, that this electoral coalition has been decimated by the steady flow of compromises fashioned with their right wing coalition partners.
There is perhaps too much emphasis among commentators, then, on the degree to which Labour's hopes for 2010 are 'heavily reliant on the continued disenchantment of former Liberal Democrats'. Many of these voters are most likely not lifelong Liberal Democrats, but Labour supporters who quit the Labour coalition during the Blair/Brown years, and have returned to the fold under the Coalition government – ending the twenty year experiment of a social democratic Liberal Party. As we can see from the Polling Observatory figures, many of the supporters who left did so immediately after the Coalition was formed, and many soon after. The Liberal Democrats' bleeding was complete by the end of 2010, and they have not really moved in the polls since. It is not just that British voters are not particularly keen on coalition government. Those who joined the Liberal Democrats from the left were not keen on a centre-right Coalition from the outset. For the Liberal Democrats, the current pattern represents a return to the post-war level of support for the old Liberal Party. The resurgence of the Liberal Democrats as a third way in British politics had been built up by the efforts of Ashdown and Kennedy, only to be dismantled by the project of the orange book Liberals — most notably Clegg, Alexander and Laws. One prominent member of the party's left wing – Sarah Teather, representing a poor, former safe Labour seat, has already announced her intention to quit at the 2015 election. It will be no surprise if, as 2015 approaches, more prominent left-of-centre Liberals join her, jumping ship before the impending electoral Tsunami hits. Complete electoral disaster may be spared, however, through the personal vote that individual MPs have managed to cultivate in their local constituencies – recent evidence suggests voters in Liberal Democrat seats like their MP better than those living in Labour or Conservative seats, and that such voters remain more willing to back a local Liberal Democrat than the national polling suggests.
Perhaps the only question that remains will the Liberals have sufficient MPs in the next parliament to play kingmaker. Clegg would be better advised to stop manoeuvring the party in preparation for the next coalition and instead focus on finding a way to expand a support base that has been on life support for more than three years now.


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Planning for Impact Tool
Impact Case Studies
Creating fast, accurate tests for disease
Attaching electrochemical tags to DNA could allow GPs to diagnose and treat patients in just one visit.
Bath spin out company Atlas Genetics could change the future of medical testing in GP surgeries
'I didn't predict how ground-breaking this research would be at in the beginning,' says Head of our Department of Chemistry Professor Chris Frost. 'There were just four of us working on it at Bath: me, Toby Jenkins, Laurie Peter and Stephen Flower.
From little acorns…
'It started with a request from a company that was then called Molecular Sensing. They were involved in developing technology for DNA testing and it was their research director, John Clarkson, who got the ball rolling. As a former Reader in Biology and Biochemistry at Bath, he had the idea of trying to use electrochemistry for detection in this context and, as a consequence, became the founding Chief Executive of Atlas Genetics.
'This process integrates a chemical reagent with electronics. It was a different approach to using and detecting DNA sequences, and was interesting. We wondered if we could potentially apply these probes for use in disease diagnosis; for example, detecting the presence of specific bacterial DNA.'
In about 2002, John approached Professor Laurie Peter – who was Head of the Department of Chemistry at the time – to see if anyone at Bath could design and make suitable electrochemical tags to attach to the DNA. 'This idea was to use changes in the signal on binding complementary DNA,' says Chris, 'and since I was an organic chemist interested in modifying proteins and DNA, Laurie introduced me.'
Blue sky research
At that stage, the team didn't have a particular commercial idea in mind; they just wanted to do some blue sky research. DNA comes in complementary pairs that hybridise, and the initial concept was that they'd see a change in the electrochemical signal of the probe strand, with the attached label.
'We spent about six months designing the first batch and optimistically attached the labels to a long strand of DNA. Sadly, we didn't see anything – there was no signal. It didn't really work, and of course we were disappointed. So we thought about it some more and had another idea, and I'm not certain whose it was! It came because we know that certain enzymes are able to digest – or chop up – specifically double-stranded DNA, when it hybridises.
This inspired the team to use one of these enzymes in conjunction with the labelled probe, to initiate the digestive process and then the release of a smaller fragment, containing an electronic label that could later be detected. This time early results looked promising, but there was a sensitivity problem. Since it was rather difficult to see a clear signal on some positive samples, they had to go back to the lab to design a label containing more electronic reporters. The experiment was successful, as it turned out that two electronic tags were enough.
Chris explains: 'It was a generic test and we were absolutely thrilled to find that it worked. In principle it could detect any specific DNA sequence, and we could see the potential applications in disease diagnosis. Then, from a business point of view, we wondered who would be likely to pay for this sort of product. We started thinking about rapid tests for bacterial diseases from meningitis to sexually transmitted diseases to veterinary sciences, for instance as a remedy for Strangles, a respiratory infection that affects racehorses.'
Once the test was proven and the team was convinced that there would be commercial demand, a company was spun out on the basis of this initial work: Atlas Genetics was formed, with laboratories in Trowbridge, where they still operate. However, the company now has headquarters in the US, because the sheer size of the country makes that the primary market.
'I was one of the academic founders of Atlas,' says Chris, 'but when it spun out of Bath it was funded by an external individual investor and a University seed corn fund. Now, the company's raised over £60 Million from a strong network of private venture funds.'
The new test for Chlamydia will be the first to be commercialised; it's currently going through the final stages of testing to gain FDA approval before it can be launched. The main advantage is that it's a rapid, point-of-care solution.
Once tested, using a simple swab, patients will wait for just 30 minutes for their result and, where the result is positive, will receive a prescription and appropriate drugs immediately. This means they can start treatment straight away, precluding the need to pack and send off a sample, and a wait of two weeks or so for results to be returned to their GP from the lab. Another key differentiator – of healthcare and social significance – is that patients are less likely to unwittingly pass on their infection to other partners while awaiting results. Hence, this work will also contribute to preventing the transfer of diseases.
A viable impact
'I'm still working with Atlas Genetics,' says Chris. 'At the moment we're trying to develop a suite of labels that will allow us to test patients for multiple infections from a single sample, whether it's blood, urine or a swab. As long as we can extract the DNA from it, we can use it.
'That project has inspired me to work on other sensor projects, and I'm currently working on new platforms. One is for detecting prostate cancer (with Pedro Estrela; another is detecting biomarkers in waste water with Barbara KasprzykHordern. In all of these, we're contributing the design of chemical reagents and attaching them to biomolecules that serve as recognition elements for the target of interest. It's important to note that this is a team effort involving many disciplines, including biology, chemistry, engineering and psychology.
'In many ways, solving the chemistry problem is the easy bit. The next stage, making it accessible to healthcare professionals, is more of an engineering problem, where our solution has to come out of the laboratory and into a disposable microfluidic chip. Atlas is trying to develop one so these tests can be carried out in a routine fashion and perhaps, one day, there will be a solution where people can self-diagnose less serious conditions at home, rather than in a healthcare environment.
'Fundamental research takes up a lot of my time, so it's really exciting to be working on a beneficial product that's so useful in the real world. Nothing I've worked in has had a more viable impact than this project. Aside from the clear benefits for patients and healthcare providers – in terms of both time and costs – this work has opened up huge opportunities for reagents to be used in sensors in healthcare.'
Read our REF Impact Case Study
Our work on developing novel technology for rapid diagnosis of infectious diseases was submitted as an impact case study to the 2014 Research Excellence Framework assessment.
research-impact@bath.ac.uk
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