Welcome back, friends of the OCC, to another week of fun! We're so happy you dropped by this week to see what our design team has been up to. We have a wonderful sponsor this week and a special guest. But first, let's take time to recognize an outstanding challenge entry from this past week. Our featured designer this week is Debbie and below you can see her beautiful entry from last week's Try a New Technique challenge. We hope you will take time to drop by her blog to congratulate her and see more of her wonderful designs.
Our sponsor this week is a brand new digital stamp company Sugar Creek Hollow. So new, in fact, that their Grand Opening is March 1st. They are a Christian company that is giving all of their proceeds to a food pantry. They are offering $10 in free digital images to the lucky winner of this week's random selection. But really everybody wins because as part of their Grand Opening there is a fabulous Friday Freebie (from yesterday), a blog hop, a drawing for $50 in free digital stamps and the opportunity to receive a free digital stamp for every $10 you spend on the Grand Opening day of March 1st. So be sure to drop by their site on Monday and check out all the goodies!
This week our February host, Linda Coughlin, has challenged the OCC to a sketch challenge. We can't wait to see your take on the sketch, feel free to use your favorite stamps and supplies!!
This week we have Joseline Capre from the Sugar Creek Hollow DT with us as a special guest designer.
Hello OCC friends! We're so happy you have stopped by. Thanks to everyone who came by last week and special thanks to those who stayed to play in our challenge! We're glad you sorted through your stash and showed some love to previously neglected supplies and tools. We have another fun challenge for you this week, but before we tell you about it, we want to share a wonderful entry from last week's challenge. Our featured designer this week is Birgit and here is her beautiful sample from last week's challenge. Please take a moment to stop by her gallery to see more of her awesome work.
This week, our February host, Linda Coughlin, is challenging us to try a new technique. We all stay in our comfort zone way too much, but it really is fun to try something new. Our challenge to you this week is to try out something that you have never done before. Surely there is a technique that has caught your eye and you've been wanting to try for yourself ~ Now is the time! We can't wait to see your new creations, if you're stumped for a new technique, check out the Resources on SplitcoastStampers or the Tutorials on Papercraft Planet.
Remember, there is no time limit on our challenges. Please feel free to participate whenever you are able. To play along, upload your creation to your blog or gallery, then leave us a comment linking to your project so we can check it out. If you upload to SCS, remember to use keyword: OCCC.
Here are the samples our designers made this week with their new techniques. Be sure to stop by their blogs to read about what they learned and how they created their samples.
Hello OCC friends & thanks for stopping by!! We would like to announce that we have a NEW spot for our weekly sketch challenge - OCC Sketch Blog, that way our sketch is always at the top. Everyone is invited to play along, feel free to use your favorite stamps and supplies.
Thanks to all those that stopped by and played along with our music challenge – our hearts were singing with inspiration!! Before we show you today's samples, we would like to announce our featured designer Gardenia from A Little Less Useless. Below you will see her musical layout. Be sure to stop by her Blog for more inspiration.
NEW month means we have a NEW hostess, please help us welcome our February hostess, Linda Coughlin. This week Linda has challenged the OCC to a masculine Valentine card/project. The challenge is to create a card/project for the men in our lives, a significant other, Dad, Brother or friend. We can't wait to see where our inspiration will take you this week!!
Hello OCC Friends!! Our weekly sketch challenge got a little crowded so we moved it to a NEW spot - OCC Sketch Challenge Blog. We warmly invite you to play along, feel free to use your favorite stamps and supplies.


The Professional Women's Alliance (PWA) is a new committee of the Rockwall Chamber of Commerce that will empower and unite women through business and community events. Membership is open to women business owners and women in an executive level within their organization.
Leadership Rockwall is to identify and motivate emerging leaders, and to develop their potential for community leadership by introducing them to the realities, opportunities, and challenges of our civic and business communities.


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One of the world's most impressive feats of engineering, the Panama Canal joins the Atlantic and Pacific Oceans. Panama Canal cruises usually call at several Caribbean cruise ports, as well as Panama City or Colon.
Visit Panama and see the beaches, islands, rainforests and architecture as they reveal the history and secrets of this intoxicating country. Dubbed the 'Miami of the South', Panama City boasts charming colonial architecture mixed with modern high-rise buildings, making it a destination delight for international visitors. Try the worldly cuisines and iconic attractions at the city centre, or walk the breath-taking beaches and Panama Canal on the Pacific coast.
The Panama Canal is a 48-mile stretch from the Caribbean Sea to the Pacific, a gateway for vessels crossing the Atlantic and Pacific Oceans. Explore the region on a Panama Canal holiday, with the help of our expert Cruise Concierge team who can help plan the perfect cruise today. Please don't hesitate to pick up the phone and give us a call.
Cruising the panama canal
Even if you are familiar with the concept of a canal lock, nothing can prepare you for the awesome scale and power of the locks you'll encounter on your Panama Canal cruise. Closest to Panama City are the two-step Miraflores Locks, making them a possible excursion if you're visiting the city as there's an informative Visitor Centre which explains how they function. The locks are named after the lake they give way to and, once you've crossed it, you'll arrive at the Pedro Miguel Locks, which serve the other end of the lake. Lying close to the Atlantic coast are the famous Gatun Locks. Gatun Lake is another of the most popular areas of the Panama Canal and one of the world's largest artificial lakes. It is crucial to forming a passage between both oceans in both directions and is bordered by dense rainforests which make for a truly spectacular natural backdrop. The lake's water is what makes operation of the canal's locks possible and it also provides the people of Panama City and Colon with drinking water.
Located at the Pacific end of the canal, Panama City is both a starting and finishing point for many full transits of the waterway. Even if you are visiting the city as a cruise port on your itinerary and not travelling on the canal itself, excursions to the canal are readily available from Panama's capital and, indeed, it remains the city's most popular attraction. If your canal transit begins with a stay in the city, there are also a number of other popular attractions, such as the Old City with its wealth of historic buildings and the altogether more contemporary bayside Cinta Costera precinct.
Overlooking Limon Bay, Colon lies at the Atlantic Ocean end of the Panama Canal and, much in the same way as Panama City's biggest attraction is the canal, so too is it in Colon, specifically the Gatun Locks and the watery expanse they give way to, Gatun Lake. Aside from the attractions the canal offers, Colon is a great port to visit if you want to explore the wider region's breath-taking rainforest, and there are a number of historic landmarks to discover, such as the commanding Fort San Lorenzo, built on the orders of King Phillip II of Spain in 1595.
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Simple Symphony & Other Works – The Season Heats Up With A Burst Of New & Original Works
Because I said so… | Music by Claude Bolling
Sylvia pas de deux | Music by Leo Delibes
Simple Symphony | Music by Benjamin Britten
And a new work with music by Sergio Assad performed live on stage by the Fog City Guitar Duo
Friday, February 27, 2015 – 7:30pm
Saturday, February 28, 2015 – 4:00pm
Eissey Campus Theatre
At Palm Beach State College
11051 Campus Drive, Palm Beach Gardens
Tickets at balletpalmbeach.org or 561.207.5900
Palm Beach Gardens, FL – Simple Symphony & Other Works is a mixed repertory program with four ballets featuring new and original works with humor, romance, and live classical guitar music. Performances are at the Eissey Campus Theatre at Palm Beach State College, Friday, February 27 at 7:30 pm and Saturday, February 28 at 4pm. Enjoy a special post performance surprise after Saturday's performance and meet the dancers of Ballet Palm Beach including the company's newest members Mauris Martinez and Luise Miguel. Tickets start at $15 and can be purchased at balletpalmbeach.org or by calling the Eissey Campus Theatre at 561.207.5900.
The program opens with Because I said so… a whimsical original work with music written by present day French jazz musician Claude Bolling. Inspired by childhood memories of things your mother said, this fun-filled relatable piece brings a dose of humor to the stage.
Next, romance heats up the stage in a mythical setting with the pas de deux from the ballet Sylvia. The ballet is choreographed by Clara Cravey, associate professor of ballet at the University of Oklahoma, to music written in the late nineteenth century by French composer Leo Delibes. This exquisitely challenging pas de deux showcases the ballet talent and artistry of principal dancers Rogelio Corrales and Lily Ojea.
Ballet Palm Beach then presents its newest original work (not yet named) to live accompaniment by the Fog City Guitar Duo. This piece is dedicated to street performers, also known as buskers, and how they inspire people when they least expect it. The music is by present day Brazilian guitarist and composer Sergio Assad.
The program finale is original work Simple Symphony with music written in the early 1930's by English composer Benjamin Britten. "Simple Symphony was inspired by Benjamin Britten's magnificent score and is dedicated to our ballet mistress Claudia Cravey. Claudia has shared her wealth of experience touching the lives of so many dancers throughout the world. She has been instrumental in the growth of our dancers and has clearly impacted how the company looks as a whole." says Artistic Director Colleen Smith.
Ballet Dance entertainment family Theatre


On July 11, 2016, the seventh tanker of Project RST54, Balt-Flot 6, built by Okskaya shipyard (Hull No 5407), was put into operation, says the shipyard. It is the sixth ship of Project RST54 built by Okskaya Shipyard for Balt Flot Tanker (BFT, Saint-Petersburg), says the Project designer Marine Engineering Bureau.
Krasnoye Sormovo Okskaya shipyard and BFT signed a contract for construction of 5 vessels including two RST-27 and three RST-54 tankers in September 2015. The contract involving State Transport Leasing Company (STLC PJSC) was signed in the framework of NEVA-2015, the International Maritime Exhibition and Conferences of Russia.
The vessel has been built under the contract signed by BF Tanker on September 23, 2015 for construction of 7 sea/river type tankers at Krasnoye Sormovo (United Shipbuilding Corporation) and Okskaya Shipyard (UCLH). Under the contract involving State Transport Leasing Company (STLC PJSC), the company is to get three RST-54 tankers (combination bulk oil / deck cargo carrier) and four RST-27 tankers. Okskaya Shipyard will build 5 vessels (two RST-27 tankers and three RST-54 tankers), Krasnoye Sormovo - two RST-27 tankers. All the vessels are to be delivered in 2016.
Okskaya Shipyard has built three tankers of Project RST54 and two tankers of Project RST27 (the first of them was launched on June 8, 2016). Two tankers of Project RST27 has been built by Krasnoye Sormovo shipyard. Okskaya Shipyard is going to deliver the forth ship of Project RST54 on August 25, 2016.
RST54 project vessels of Volga-Don Max class meet the dimensions of the Volga-Don Ship Canal and Volga-Baltic Waterway. LOA - 140.85 m, beam - 16.6 m, depth – 6.0 m. Class of the Russian Maritime Register of Shipping - M-ПР2,5 (ice30) А. Crew - 12 persons (14 places + pilot). Sea endurance - 20 days.
Okskaya Sudoverf (Shipyard "Oka") based in Navashino of Nizhny Novgorod region was founded in 1907. The modern Okskaya Shipyard JSC was established through privatization of the state enterprise Navashino Shipyard "Oka". The firm specializes in building multipurpose river and sea going vessels, container carriers, special ships, barges. The company's major shareholder is Universal Cargo Logistics Holding B.V. (UCL Holding).
UCL Holding is an international transportation group consolidating a number of Russian shipping, stevedoring, shipbuilding, railway and logistic companies. The division also includes Volga Shipping Company, V.F.Tanker, several cruise and shipbuilding companies.


"You know, sometimes I feel like I was born with a leak, and any goodness I started with just slowly spilled out of me, and now it's all gone. And I'll never get it back in me. It's too late. Life is a series of closing doors, isn't it?"


Dr. Joann MacMillan, MD is an otolaryngology (ear, nose & throat) specialist in Warwick, RI. She graduated from Universita Degli Studi Di Bologna, Facolta De Medicina E Chirurgia and specializes in ear, nose, and throat and allergy & immunology.
The dr herself is awfully rude, but her secretary is very pleasant.
×Post a ResponseAre you Dr. Joann MacMillan, MD?
Dr. MacMillan took the time to listen to what I was going through with my right ear, she was very thorough and took the time to explain my symptoms and what she would do to help me.


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Cardi B's Strip-Club Brawl Leads To Charges And A 'No Contact' Order From NY Judge
The 26-year-old avoided arrest by finally showing up to a court hearing.
John Lamparski / Getty Images
Holly Chavez
Cardi B has been accused of starting a brawl with two bartenders inside the Angels NYC strip club on August 29.
The rapper didn't appear for her first court hearing, and was facing arrest as a result, per TMZ. This time, though, Cardi B made her way to the New York courtroom at the appointed time, where she received a stern warning from the judge about her behavior.
The Inquisitr previously reported that Cardi B's attorney told the judge that his client had an "entertainment-related commitment" that had kept her from appearing at her first scheduled arraignment. The judge wasn't impressed, stating that the rapper's failure to show up was "not acceptable." Despite this, an extension was granted from Monday, December 3, to Friday, December 7.
TMZ indicated that Cardi B, whose real name is Belcalis Almanzar, asked the crowd outside the courtroom, "Am I this famous?" Once inside, the judge declared that the performer wasn't a flight risk and, therefore, could be released without the $2,500 bail that had been requested by prosecutors.
The incident from August was allegedly started by Cardi B as a way to get revenge. Although it hasn't been publicly announced whether or not the rumors are actually true, Cardi B apparently believed that one of the strip club's bartenders, Jade, had slept with the rapper's husband, Offset. An earlier TMZ report quoted Jade as saying that Cardi B had been "threatening her for months."
According to Reuters, the hip-hop star is facing three charges: assault, reckless endangerment, and harassment. During this morning's court appearance, the judge issued a "no contact" order, meaning that Cardi B could face additional legal woes if she has any form of contact with the bartenders. This edict extends toward social media — and includes not only written threats, but any type of comments that are about, or aimed toward, the two alleged victims.
In an interesting twist, CNN posted earlier this week that Cardi B and Offset are no longer together. This was initially announced via Cardi B's Instagram account, in a video that has since been deleted. It's unknown at this time if the two have reconciled.
On the other hand, things haven't been all bad news for the popular performer. As the Guardian indicated, Cardi B was nominated for four Grammy Awards, including Album of the Year. This comes just a week after being named one of the Entertainers of the Year by Entertainment Weekly.
A post shared by Cardi B (@iamcardib)
The hip-hop performer will be back in court on January 31, 2019. If Cardi B is convicted, she could end up giving her next concert from behind bars — as she faces up to one year behind bars for her role in the strip-club brawl.


The adjustable lumbar section of the Cresta offers increased support for your back, wherever it's most needed.
The Cresta's separated head section is designed to find the best way to support your back and relieve pain.
Sleek and stylish, the deck-on-deck design adds sophistication to any bedroom and creates smooth movement – even in lounge position!
The smooth deck-on-deck design and wide range of features make the Avante the top-of-the line in adjustable beds.
Sleep better – use the anti-snore position to reduce snoring, or improve blood flow in zero gravity.
A wireless remote, stylish upholstery, and an affordable price make the Edge an excellent choice in entry-level adjustable beds.
Heavy-Duty Motors Two durable motors provide a lift capacity of up to 850 lb. per base.
Love drawers under your bed? The Summit's thin profile fits smoothly on platform beds and still gives a full range of movement.
Go ahead, get the big mattress. The durable steel frame and support brackets hold up even the heaviest bedding.
Raise and lower the head and foot to get just the right setting for you and whoever is with you.
The technologically advanced wireless remote allows you to effortlessly raise the head and foot with the touch of a button and lights up for easy use in the dark.
The low-profile design is specially engineered for platform beds with footboard drawer units.
Our exclusive mattress retention system eliminates the need for a mattress retainer bar, providing a sleeker, more modern look. MicroHook may not be compatible with all mattresses or bed accessories, such as dust ruffles.


That's pretty cool for fans of the show.
My father in law would love this! He is always quoting that show!
That sounds really cool. Thanks for the info.
i just got into Duck Dynasty before Christmas, and I can't get enough they are too hilarious! I am going to become a Duck Scout thanks!!


Are You Really Taking Care of Your Teeth?
We all have been through the painfully long wait at the dentist's office and the further painful session seated in the dentist chair. A visit to the dentist is never fun. Starting from having your teeth cleaned to a root canal, teeth and gums are the more sensitive parts of the human anatomy. And not to mention every trip burns a large hole in your wallet.
Today, because of the kind of lifestyles we lead with our vegan phases, liquid diets and fluctuation nutrition intake and green tea binge drinking, our teeth are bearing the brunt of our experiments. So it is possibly more important than ever to make dental hygiene a habit.
After a long weekend of binge drinking with friends, a friend's wedding or after a weekend getaway, here are the two words going through your mind: liquid diet. This trend of detoxing your body caught on a few years back. Having proved effective in providing sufficient hydration to the body, it strengthens your muscles and is much easier to execute than a regular diet with solid food.
A recent study by the JAMA showed that more than two thirds of Americans around the age of twenty five are obese. UK reports that one in ten children face childhood obesity in kindergarten and the number is one in five by the time they hit school. The numbers in India may not be as alarming, but medical research institutes predict that they will be dangerously close to their Western counterparts in the next decade. A latest report by The Times of India reiterates this fact. India is already the third most obese country in the world. "Latest estimates show prevalence of obesity among adolescents (13-18 years) has grown from 16% to 29% over the last five years," the report says.
Are You Working Out at Work?
The 21st century office-goer in large metros or smaller cities, spends more than half his day commuting to work and back and sitting before his desk. This behavior is making us all unhealthy. There are no immediate repercussions but every minute you spend staring at your screen, bustling through meetings and skipping lunch to read the brief one last time, you're either unwittingly gaining weight, adding to your stress levels and fueling hypertension and anxiety. It is not exactly the healthiest thing for your heart either.
If the last time you let your anger get better of you and it took you by surprise, you need to take a more serious look at the problem. If you've raised your voice more than once at a dinner party, punched a wall after a fight, stormed out of a room in anger or more importantly, felt the increasing urge to do any of the aforementioned things, you're probably angrier than you think you are.
Anger and anxiety are problems that can easily go undiagnosed and untreated. The issues may be boiling under layers of denial and ignorance years before they finally burst out. The more serious worry though is that people may not even be aware that they may be suffering from serious anger issues.
Even if you think you have your anger in control, here are some things you probably didn't know your anger could lead to. No matter in what stage you think you are, inadvertently your anger is taking control of you with every temper tantrum. There is a list of scary and serious effects of anger including, mild to severe headaches, digestion problems, such as abdominal pain, stomach uneasiness, insomnia, depression, high blood pressure and skin problems. If you've been battling with anger for a while, you must have also been warned against the high level of stress it causes you, the risk to your heart .
Notice your behavior carefully for a week. If you think your problem is not chronic, or not as serious, be vigilant with yourself. The next time you get angry, make a mental note. If your anger episodes are occurring more frequently than you thought, Houston, we have a problem.
Here's the tricky part. Even if you've not yelled at the cashier, packed a punch at the punks who ticked you off or raised your voice in anger, you may still want to take a closer look at yourself. You may be repressing anger by keeping it bottled up. It is just harmful as aggressive anger, possibly even unhealthier.
Usually when a person gets angry, the body reacts to that anger. If a colleague is inefficient, your employees have walked in late or your spouse is driving you up the wall, your body will react first. That pain in your neck will start acting up, your joints will probably call for attention or the throbbing pain in your temple will return. This is your body telling you that a storm is coming.
A visit to a doctor is probably the smartest thing you will do all day. Not only will you know how serious your problem is, a therapist might help subdue the problem before it gets worse.
If you see the familiar symptoms of the imminent anger outburst, excuse yourself and walk away. Find an activity that will help you cool off. Take a walk or go for a run. Go talk to a more friendly voice or put on your favourite music playlist. Change the mood and return when you feel calmer.
You can be better than that. If you've already realised you have no patience for incompetent drivers on the road, or your child's messy bedroom, don't return to the same issue. Accept some inevitable life truths, and try moving on from the small stuff.
Distraction is the key here. People who have anger issues usually hate the Hulk monster they become once they turn green. So add an aspect to your personality that you really enjoy. Take up cooking, music or sports. Become a cinephile for a month. Change your activity monthly.The important positive to remember here is the amount of advancement medicine has made in terms of mental health. So today you have therapists, pain relievers and medication to help you pull through. The equipment is ready, so it's now time to let go and breathe easy.
To put it more simply, almost half the deaths caused by cancer were linked to smoking. Though these figures were limited to the United States of America, it is not too difficult to extrapolate these numbers globally, or to India.
These figures that scream to you across news headlines are not new. With every research paper published, the case against smoking is getting stronger. But to quit smoking is never easy. If you're reading this, you've probably tried to quit, more than once perhaps, and undergone a relapse.
But there is no shame in admitting defeat and giving it another try. So if you're trying to quit, helping a friend/spouse or child cut down or have never tried to cut down because you don't know how to, here are some pointers to nudge you to stub it out.
Identify the problem you're dealing with. Make a note of the number of cigarettes you smoke daily. Be completely honest, even if you smoke more than you thought you do. The aim is to cut that number down. Once you give a face to the problem, it gets easier to deal with it. So if you smoke ten cigarettes a day, the number needs to go down to eight, then five. Set yourself a target for each week. After two or three weeks, cut the number of cigarettes by one. Go slow, set a reasonable number for yourself.
Even though quitting to stay healthy is reason enough, give yourself a separate reason. Are you bringing up kids? Did you quit swimming after you started smoking? Does your husband hate the fact that you smoke? Is it making your skin look darker and making you look older? All you need is one reason. You'll find that it is easier to latch on to your resolve, once you have a strong reminder of why you're quitting.
Find a friend who wants to quit too. But it is important to keep in mind that this friend must be equally motivated to quit. Once you have your 'person', make a pact of how many you're allowing each other to smoke. If you cross the line, tell him, he'll understand. If you do well, tell him, he'll want to do better. Two is always better than one, whether it's to complain about craving a smoke, or discuss withdrawal symptoms. Drag this buddy to parties so that you can keep a tab on each other.
Promise yourself you won't smoke after dinner, in the car or in the bedroom. Stay away from your usual smoking zones such as the smoke room at work, your terrace and your balcony. Don't buy a packet of cigarettes on your way home. Throw away your ashtray and lighter. You'll find that refusing a smoke after a couple of beers is hard, but physically trying to clean out your life is much easier.
Try Nicotine Replacement Therapy, which means supplying your body with nicotine but not through tobacco. Try options such as nicotine patches, gum, lozenges, inhalers, and nasal sprays. You can also ask your doctor for prescription pills to help with the withdrawal.
Discover a new activity that will excite you. Take up cycling, running, swimming (exercise will also help you feel better about quitting). Pick up a musical instrument or a fresh hobby such as cooking. gardening or painting. You don't need to be very good at this, just good enough to be distracted.
Don't be too hard on yourself. If you decided to smoke four cigarettes a day, and have not stuck to your word, try again tomorrow. Don't abandon your plan midway. It's a long road, and a few bumps along the way never hurt anyone.
Once you have your cigarettes per day in control, take the plunge for the next step. If you've cut down successfully and feel comfortable enough, concentrate on quitting. It's only the next step.
Anyone who has quit smoking successfully will tell you that it was not the easiest thing in the world for them to do. But the rush of taking your life in control is going to make the long and hard journey worth it.


The Lumber Mill produces timber for your turf, one of the four main resource generating building in Lords Mobile. Upgrading your lumber mills increases your timber production per hour and total timber capacity.
Building ten lumber mills will complete a turf quest rewarding you with 120,000 resources and a good boost to your might.
As you progress through Lords Mobile, you will require significantly more resources to upgrade buildings, research and train troops. If you have a balanced setup you will not be able to generate enough timber from your lumber mills alone.
One strategy is to form a resource sharing group with three other members of your guild. Each of you will choose one of the four main resources: food, timber, ore or stone and concentrate solely on this resource alone.
Taking timber as an example; you would build solely lumber mills in your turf and maximize your lumber mill production research and hero gear. You can then share resources with the other four guild members. This provides you with a much greater production rate and capacity than you could achieve alone.
Once maxed your lumber mill will produce 30,560 lumber per hour and provide 2.3 million storage.


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Made in Flanders: 400 hp in a shoebox-sized e-motor
The next Elon Musk could very well come from Flanders. Based in the town of Kortrijk where it employs around 30 people, tech start-up Magnax – short for 'magnetic axial' – has raised EUR 16 million for the further development of its 'electric motor of the future'. Lighter, cheaper, stronger and more energy-efficient: the prototype weighs in at 25 kilograms and is no bigger than a turkey, but can produce up to 400 horsepower (hp).
Magnax
The axial flux motor sounds like a device from a sci-fi movie, but Magnax co-founders Peter Leijnen, Daan Moreels and Kester Goh seek to conquer the world with this cutting-edge technology. Supported by the University of Ghent (UGent), the engineers built an electric motor that delivers three to four times more power than best-in-class motors found in electric cars today.
Most electric cars on today's market work according to the principle of radial flux, where magnetic flux moves perpendicular to the car's axis. In an axial motor, however, flux occurs in parallel with the axis of the motor. The concept has existed for at least a hundred years but was considered too complex for mass production. However, in the last ten years, the technology has resurfaced and is gaining momentum.
Greater power, smaller size
"We have already produced dozens of prototypes," says Daan Moreels. "Tests in June 2020 showed that, with our new prototype, we can generate 2.5 times more power than a Tesla engine of the same weight. For production versions, the generated power will easily be 3 times higher. Due to its efficiency, our engine can also increase a car's driving range by up to 20%. For a BMW i3, our calculations showed an increase of 25%, which exceeded our expectations."
Our technology is revolutionary. It has much more power density, is much more efficient, is made with much less material and is being developed for mass production. We're just in time for the next generation of e-cars.
Daan Moreels
co-founder of Magnax
"As far as radial flux is concerned, Tesla has taken it to the highest level," adds Peter Leijnen. "But we are still developing our axial flux prototype. We achieve the same yield from an engine that weighs a third of Tesla's, and it's cheaper to produce. Even more, our engines are recyclable, which is also better for the environment."
The biggest stumbling block for electric motors is the price, and we're getting rid of that obstacle.
Peter Leijnen
CTO and co-founder of Magnax
From garage to international headlines
Like many other start-ups, the story of Magnax began in a garage. "I was working as an independent engineer and had to design a wind turbine generator for a customer," says Leijnen. "There, I met Daan, also an engineer, and we started talking. We also saw opportunities in this technology for electric engines. Daan brought Kester Goh on board, and that's how Magnax was born."
The choice to start a company in this highly specialized niche proved to be a high-potential one. "In recent years, we have been contacted by over 4,000 companies interested in the high power density and compactness of our prototypes," says Daan Moreels. "These are all companies active in e-mobility and the development of electric vehicles, boats and airplanes."
Magnax will now refine its prototypes and aims to start producing them in 2022. Daan Moreels: "Our technology is mature enough to be used in vehicles that will be on the market for many years to come. We are initially targeting manufacturers of sports cars, off-highway vehicles, vans, trucks, tracked vehicles and drones. These customers need compact, light, high-powered engines. Next on our list are electric passenger cars."
It's our ambition to capture a large part of the electric motor market: a billion-dollar market in full growth.
Three to four years
According to the engineers, their engine will be used in passenger cars within three or four years. "To produce the engines, we have entered into a partnership with a manufacturing subcontractor," explains Daan Moreels. "We cooperate closely with this subcontractor, but we stay in charge of commercial matters."
What's more, various investors and business angels have channeled funds into the Flanders-based start-up through Magnax's investment fund – including Vlerick Group, Torus Capital and Hirschvogel Automotive.
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It's a balmy evening on Jaffa's sea-front and we're in a busy restaurant overlooking a jumble of small fishing boats. Our table is spread with a feast of salads presented Arab-style in 20 tiny metal dishes. There's sweet red cabbage, chilli and avocado, roasted cauliflower, and six incarnations of tahini which we scoop on to pitta bread, washing it all down with a crisp, tangy white from Mount Hermon. On the promenade in front, cool kids whizz by on electric scooters. At the bar next door dancing has started.


text:
DPC fines WhatsApp €225m over data sharing policies
Finalised penalty almost five times larger than draft fine issued in December 2020
Image: WhatsApp
Facebook-owned messaging service WhatsApp has been hit with a €225 million GDPR fine for a lack of transparency in the way the service shares user data.
The penalty, which has been issued by the Irish Data Protection Commission (DPC) and approved by the European Data Protection Board (EDPB), is several times higher than the €50 million draft fine issued in December last year.
Following a two-year investigation, WhatsApp was found to have been unclear about the way it had processed and shared data with Facebook, as well as between WhatsApp and other Facebook-owned companies.
Specifically, the investigation found that WhatsApp violated Article 14 of GDPR, which states that data controllers must provide data subjects with sufficient information about the way data is collected and processed.
The provisional €50 million fine, issued under the one-stop-shop principle, was submitted to the regulator's European counterparts for approval once it was issued. The data watchdog was chosen as the lead supervisory authority because WhatsApp is headquartered in the country.
After eight of its counterparts raised a dispute, the EDPB issued a binding decision in July with a "clear instruction" for the DPC to increase its provisional fine.
The regulator subsequently raised the level of its draft fine several times higher, alongside issuing requirements for WhatsApp to take steps to improve its GDPR compliance.
A summary published by the EDPB found the GDPR Article 14 infringements were "very serious in nature" and "severe in gravity", with these violations amounting "to a high degree of negligence".
WhatsApp has branded the fine "entirely disproportionate", and claims it will appeal the penalty.
"We have worked to ensure the information we provide is transparent and comprehensive and will continue to do so," a WhatsApp spokesperson said. "We disagree with the decision today regarding the transparency we provided to people in 2018 and the penalties are entirely disproportionate. We will appeal this decision."
This fine is likely to be the first of many the regulator will issue against Facebook and its subsidiaries, with the regulator currently working through a backlog of cases against big tech firms. The regulator is also investigating more than 10 complaints against Facebook-owned companies alone.
This is the biggest GDPR fine issued to date, although it might soon be dwarfed if a provisional €746 million (approximately £637 million) fine issued by Luxembourg's regulator against Amazon is finalised.
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text:
Therapeutic Potential of Tricyclo-DNA antisense oligonucleotides
Article type: Review Article
Authors: Goyenvalle, Aureliea | Leumann, Christianb | Garcia, Luisa; *
Affiliations: [a] Université de Versailles St- Quentin, U1179 INSERM, UFR des Sciences de la Santé – LIA BAHN CSM, France | [b] Department of Chemistry & Biochemistry, University of Bern, Switzerland
Correspondence: [*] Correspondence to: Luis Garcia, U1179 UVSQ-INSERM Biothérapies des Maladies du Système Neuromusculaire UFR des sciences de la santé Simone Veil, Université de Versailles saint Quentin, 2 Avenue de la source de la bièvre, 78180 Montigny le bretonneux, France. Tel.: +33 1 70 42 94 16; E-mail: luis.garcia@uvsq.fr.
Keywords: Antisense oligonucleotides, splicing correction, exon-skipping, exon-reinclusion, nanoparticles, delivery
DOI: 10.3233/JND-160146
Journal: Journal of Neuromuscular Diseases, vol. 3, no. 2, pp. 157-167, 2016
Oligonucleotide therapeutics hold great promise for the treatment of various diseases and the antisense field is constantly gaining interest due to the development of more potent and nuclease resistant chemistries. Despite a rather low success rate with only three antisense drugs being clinically approved, the frontiers of AON therapeutic applications have increased over the past three decades and continue to expand thanks to a steady increase in understanding the mechanisms of action of these molecules, progress in chemical modification and delivery.
In this review, we will examine the recent advances obtained with the tricyclo-DNA chemistry which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration. We will review their specific properties and their therapeutic applications mainly for neuromuscular disorders, including exon-skipping for Duchenne muscular dystrophy and exon-inclusion for spinal muscular atrophy, but also aberrant splicing correction for Pompe disease. Finally, we will discuss their advantages and potential limitations, with a focus on the need for careful toxicological screen early in the process of AON drug development.
The development of the antisense oligonucleotide (AON)-based approach started with the work of Zamecnik and Stephenson in 1978 when oligonucleotides were used as tools to downregulate the expression of specific genes [1]. The earliest strategies aimed to modulate gene expression through inhibition of protein translation via steric hindrance of translation initiation sites within the 5′ untranslated region of the targeted gene. AONs were originally unmodified synthetic DNA complementary to the targeted mRNA but it rapidly became evident that chemical modifications to protect AONs from nuclease degradation were necessary. Since the natural phosphodiester internucleotide linkage is highly sensitive to nucleases, it was the obvious site for chemical modification. Various backbones have been introduced over the years but it is interesting to note that the very first one, the phosphorothiate (PS) linkage is still the most widely used backbone modification in therapeutic AONs. The other prime site for chemical modification is the 2′-position in the sugar moiety and it has been widely used in the antisense field. This includes modifications such as 2′-O-methyl (2′OMe), 2′-O-methoxyethyl (2′O-MOE), 2′-fluorinated (2′F) and 2′-O-aminopropyl analogs. Incorporation of these 2′-modified sugars considerably increases binding affinity and pharmacokinetics of AONs over their 2′-deoxy counterparts. Similar characteristics are found in oligonucleotides with backbones based on Phosphorodiamidate morpholino oligomers (PMO), peptide nucleic acid (PNA), locked nucleic acid (LNA), phosphoramidate and methyl-phophonate derivatives (Fig. 1). These advances in the development of antisense chemistries have led to numerous studies investigating the therapeutic potential of antisense technology and clinical applications (for review see [2]). AONs offer therapeutic options in several ways, including the classical targeted gene knockdown, achieved through the recruitment of RNAse H1 to degrade mRNA at sites of DNA/RNA hybridization upon AON binding. For this purpose however, most AONs containing chemical modifications need to be designed as "gapmers" with a DNA core (or "gap") to induce RNAse H activity flanked by nuclease resistant-modified ends to improve drug stability and tissue distribution. Fully modified AONs on the other hand can be used for other applications which have rapidly gained attention over the last two decades, amongst others the antisense mediated elimination of mRNA toxicity or the manipulation of alternative splicing where the antisense molecules are also called splice switching oligonucleotides (SSO). In this context, oligonucleotides can be used to modulate the ratio of splicing variants or correct splicing defects, by inducing exon-skipping or exon-inclusion, which opened far reaching implications in the treatment of a variety of diseases. The field has progressed very quickly over the last few years and promising results have been achieved, leading to several clinical evaluations.
Notably, most of these advances have been made for neuromuscular disorders (NMDs) such as Duchenne muscular dystrophy (DMD) using exon-skipping, Spinal muscular atrophy (SMA) using exon re-inclusion or aberrant splicing correction for pompe disease. In this review, we focus on the recent pre-clinical developments using the tricyclo-DNA chemistry which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration. We will examine their therapeutic potential in the context of NMDs such as DMD, SMA and the Pompe disease and discuss the promises and challenges of this chemistry.
PHYSICOCHEMICAL PROPERTIES OF TC-DNA
Tricyclo-(tc)-DNA has been designed in the late nineties as a conformationally constrained DNA analogue (Fig. 1) [3, 4]. It is thus a second generation analogue of bicyclo-(bc)-DNA which has been introduced as the first member of the class of conformationally constrained DNA analogues in 1993 [5]. The basic concept behind conformational restriction consists in reducing the torsional flexibility of the sugar phosphate backbone within the repeating nucleotide units, thereby structurally preorganizing the single strand for duplex formation. This reduces the entropy of duplex formation and leads thus to more stable duplexes.
Fully modified tc-DNA containing all four natural nucleobases reveal increases in thermal stability of duplexes with complementary DNA by ca 1.2°C/mod and with complementary RNA of ca 2.4°C/mod [6]. The fidelity of Watson-Crick base-pair formation is slightly increased as compared to natural DNA. As expected the driving force for the increased thermodynamic stability of duplexes is a more favourable entropy term as determined from Tm vs concentration variation experiments. Tc-DNA residues show the highest stabilization if used as fully modified oligonucleotides. Single tc-DNA units do not mix very well with the DNA backbone and even less with the RNA backbone, due to energetic penalties arising from structural dissimilarities at the backbone junction sites [7]. However, tc-DNA can efficiently be used as gapmers as we will see below.
Structurally, tc-DNA is rather an RNA than a DNA-analogue as it prefers an overall A-like duplex conformation with complementary DNA and RNA. This has been demonstrated by molecular modelling and by CD-spectroscopy and could very recently be confirmed by a high resolution NMR structure of a fully modified tc-DNA/RNA duplex (unpublished data).
Fully modified tc-DNA in its phosphate form is highly stable against nuclease activity in murine and human serum and does not elicit RNaseH activity [8]. The latter fact can easily be explained by the preference of tc-DNA for an RNA like structure. Even tc-DNA gapmers with 5 nucleotide tc-DNA wings and a core of 8 natural DNA nucleotides in the center of the sequence are still highly stable in human serum. As expected, such gapmers are RNaseH competent due to the natural DNA window [9].
As common for the 2′-O-alkyl-RNAs or LNAs, tc-DNA can also be prepared with a phosphorothioate backbone (tc-PS-DNA). Besides negative effects like the occurrence of diastereomeric mixtures of oligonucleotides within the same sequence scheme, there are also general advantages such as a significant increase in nuclease stability and cellular uptake and transport. In contrast to most other phosphodiester based oligonucleotide chemistries tc-DNA is already highly nuclease stable in its phosphate form [8, 9]. Hence, there is no need to use tc-PS-DNA just for increasing nuclease resistance. However, it turns out that tc-PS-DNA shows increased plasma transport, cellular uptake and tissue distribution over normal tc-DNA which makes it the candidate of choice for therapeutic applications [10].
Over the years the potential for antisense applications of tc-DNA either in the phosphate (tc-DNA) or thiophosphate (tc-PS-DNA) form has been evaluated in various systems and formats. Outside therapeutic applications of tc-PS-DNA in the field of neuromuscular disorders, which is outlined in more detail below, tc-DNA has been used for splice correction in a cellular assay with HeLa cells stably expressing the human β-globin gene containing two different mutations in intron 2. These mutations activate a cryptic splice site, leading to the inclusion of an aberrant exon. In a comparative study with a tc-DNA 17-mer, masking the cryptic splice site, it appeared that tc-DNA was able to restore correct splicing by a factor of up to 100-fold more efficiently compared to 2′-OMe-PS-RNA [8]. In another set of experiments fully modified tc-DNA has been directed to the cyclophilin A gene in order to induce exon skipping. Again, in a comparative cellular assay, it was shown that a tc-DNA 11-mer showed significant exon skipping while fully modified LNA of the same length was virtually inactive [11]. In another comparative assay, tc-DNA was tested for its capacity to act as a steric blocker AON of human immunodeficiency virus type 1 tat-dependent trans-activation as a means to inhibit HIV viral replication. Here it was found that a fully modified tc-AON showed the same inhibitory activity as an LNA/2′OMe-RNA mixmer of the same length. Corresponding tc-DNA/2′OMe-RNA mixmers were significantly less active demonstrating again that tc-DNA does not mix well with 2′O-Me-RNA (see also above) [12]. In a final benchmark experiment tc-PS-DNA gapmers of lengths between 14 and 20 nt were tested for their capability to downregulate scavenger receptor B1 mRNA in mice. It turned out that the tc-DNA gapmers outperformed corresponding 2′-MOE-RNA gapmers in activity not only in the liver but also in other non-hepatic tissues such as kidney, heart, lung, diaphragm and skeletal muscles [10].
THERAPEUTIC POTENTIAL FOR NMDS
Exon skipping therapy for DMD
As mentioned above, fully modified AONs are particularly useful to manipulate alternative splicing and splice modulation therapy, where the antisense molecules, also called SSOs can be used to correct splicing defects either by inducing exon-skipping, exon-reinclusion or masking aberrant splice splices, is a promising therapy for numerous genetic diseases. One of the most studied examples till date is for DMD where the AONs aim at skipping one or several exons to restore the reading frame (Fig. 2A).
DMD is a lethal X-linked progressive muscle-wasting disease caused by mutations, typically large deletions in the DMD gene, the largest gene in the human genome [13]. Most mutations, including deletions (approximately 65%), duplications, point mutations or other small gene rearrangements disrupt the open reading frame, leading to aberrant translation and, therefore, to the absence of the essential muscle protein dystrophin. Interestingly, the allelic disease Becker muscular dystrophy (BMD), which results in a much milder phenotype, is mainly caused by mutations maintaining the open reading frame and allowing the production of a partially deleted but functional dystrophin [14]. Antisense-mediated exon-skipping strategies for DMD aim to remove the mutated exon alone or together with additional exons to restore the reading frame and consequently induce the expression of "BMD-like" shortened forms of dystrophin retaining crucial functions. Although the exon-skipping approach appears to be applicable to a large proportion of patients (possibly up to approximately 83% of all DMD patients [15]), one should keep in mind that this will not offer a definite cure but an improvement towards a BMD-like phenotype depending on the functionality of the restored dystrophin.
The principle of the exon-skipping therapy for DMD has first been demonstrated by Pramono et al. in 1996 in lymphoblastoid cells and by Dunckley et al. in 1998 in cultured mouse cells in vitro [16, 17]. Since then, numerous in vivo studies have provided pre-clinical evidence for the therapeutic potential of an antisense strategy for DMD in several animal models. In particular, the mdx mouse model, which harbors a nonsense mutation in exon 23, has been used extensively to test efficacy of the AON approach using various oligonucleotide chemistries such as 2′OMe [18], phosphorodiamidate morpholino oligomers (PMO) [19–21], LNA or PNA [22, 23].
Recently, we have demonstrated the therapeutic potential of tc-DNA in this mouse model following systemic treatment for 12 weeks [24]. The tc-DNA-AON was stable and detected in all tested skeletal muscles as well as the heart and brain after intravenous injections. Quantitative PCR revealed effective skipping of exon 23 to levels 5-6-fold higher than that achieved with 2′OMe and PMO AONs, which are the two chemistries currently in trial for DMD. Importantly, this translated into a greater rescue of dystrophin protein levels, particularly in the diaphragm and heart, where levels reached 50% and 40% respectively, compared to wild-type mice. Notably, exon 23 skipping and dystrophin protein expression were seen in the central nervous system (CNS) of only those animals treated with tc-DNA-AON.
Restoration of dystrophin expression significantly improved the mdx mouse phenotype. The specific force of tibialis anterior muscles was essentially normalized and maintained up to 80% following eccentric contractions (used to measure the structural integrity of muscle fibres). A significant improvement in respiratory function was also noted, to a greater extent than that seen with 2′OMe or PMO AON treatment. In addition, echocardiography revealed that the tc-DNA-AON significantly improved ventricular ejection fraction and shortening fraction. For the first time, we could also demonstrate that tc-DNA-AON also had beneficial effects in the CNS. Tonic immobility (freezing) resulting from a restraint-induced fear response - a highly reproducible behavioral phenotype of mdx mice that is controlled by central mechanisms – was the same in tc-DNA-AON-treated mdx and WT mice, whereas the responses of the 2′OMe- and PMO AON-treated mdx mice did not differ from untreated mdx mice. Intravenous tc-DNA-AON treatment for 20 weeks was similarly effective in a more severe mouse model of DMD, lacking both utrophin and dystrophin (dko mouse model). It partially rescued dystrophin protein expression in all affected tissues, leading to significant phenotypic improvements.
This study demonstrated for the first time some of the unique properties of the tc-DNA-AON such as their ability to efficiently target the cardiac muscle, but also their capacity to cross the blood brain barrier and restore certain behavioural aspects linked to the absence of dystrophin in the CNS.
Using DMD mouse models, we were able to evaluate a broad biodistribution across all tissues and calculated a half life of the tc-DNA AONs of approximately 45 days in skeletal muscles. The long lasting activity of tc-DNA lead to satisfactory levels of dystrophin upon regimens using lower doses designed to balance a possible toxicity due to chronic exposure to high levels of AONs. When such an approach was carried out in mdx mice (4 weeks at 200 mg/kg/wk followed by 8 weeks at 50 mg/kg/wk), high levels of exon skipping and dystrophin restoration were detected and were comparable to that found in mice treated at 200 mg/kg over 12 weeks [24]. Interestingly, when comparing low dose treatment regimen of AONs in mdx mice, we were able to detect significant levels of exon skipping in mice treated with as low as 20 mg/kg of tc-DNA whereas similar dosing regimen using 2′OMe or PMO AONs hardly induced any detectable exon skipping (unpublished data).
It is well accepted in the field that AON uptake is facilitated in dystrophic muscles and an enhanced absorption of AON from the blood circulation of up to 8 fold as compared to wild type muscles was previously reported for 2′OMe for example [25]. This is believed to be caused by the myogenic degeneration and regeneration processes in the mdx mice, associated with a higher permeability of the myofiber membranes and/or vascular system. Interestingly, we found that tc-DNA biodistribution in wild type mice was not so different and levels of exon skipping detected after 4 intravenous injections were only 50% of the levels detected in mdx mice (unpublished data).
Exon inclusion strategy for spinal muscular atrophy (SMA)
As well as modulating splicing to remove exons from the mature transcript, tc-DNA can be used to promote exon-inclusion and a typical example of this strategy is considered as one of the most promising therapeutic approach for SMA. SMA is caused by mutations in the SMN1 (Survival Motor Neuron 1) gene. The SMN locus presents a telomeric copy of SMN1 which encodes the SMN protein, and centromeric copies of SMN2 which produce very few SMN. SMN2 differs from SMN1 by only a C to T transition in the coding sequence of exon 7 (not affecting the protein sequence), which however results in exclusion of exon 7 in about 90% of mature transcripts [26, 27]. Therefore SMN2 gene expression leads only to approximately 10% of functional SMN protein and the copy number of SMN2 thus correlates with the severity of the disease. As an example, severe forms (type I) carry 2 copies while type III SMA patients carry three or four SMN2 copies. Exon 7 inclusion has been shown as a promising therapeutic strategy for SMA by using AONs (Fig. 2B) and in particular 2′O-MethoxyEthyl (2′MOE) and PMO AONs. However, despite advances in antisense oligonucleotides chemistry and design, the delivery of AONs to the central nervous system remains a challenge, and intracerebroventricular (ICV) injections are required in mice to induce exon 7 inclusion in brain [28, 29], although best pre-clinical results in mice were generally obtained when ICV injections were coupled with IP injection, confirming the peripheral role of SMN [30]. This is where the unique properties of tc-DNA, being able to cross the blood brain barrier become particularly interesting allowing us to evaluate the contribution of central and peripheral restoration of SMN following systemic delivery. Tc-DNA AONs annealing to the TSL (terminal stem loop) or the downstream ISS (intronic splicing silencer) improving the strength of the 5′donor splice site were shown to restore SMN expression and activity in SMA cells (Robin et al.; manuscript in preparation). Furthermore, subcutaneous treatment of SMA type III mice with a 15mer tc-DNA targeting the downstream ISS sequence induced an efficient exon 7 inclusion in all tissues including the CNS, leading to an improvement in type III SMA mice phenotype, by preventing tail, ear and toe necrosis. This type III SMA mouse model, which is considered mild compared to the severe type I mouse model, survives and breeds normally [31], allowing a later start of treatment (7 days of age for example with tc-DNA) compared to day 1 for the severe model. Detection of exon 7 inclusion in brain and spinal cord following tc-DNA subcutaneous treatment starting at 7 days of age therefore confirmed the specific properties of tc-DNA AONs and their ability to cross the blood brain barrier at least at low levels (Robin et al.; manuscript in preparation).
Aberrant splicing correction for pompe disease
Another mutation affecting normal splicing of mRNA is found in the GAA gene encoding the acid alpha-glucosidase enzyme (GAA), causing Pompe disease or glycogenosis type II (GSD-II), an inherited recessive metabolic myopathy (incidence: 1/40,000). Hundreds of GAA mutations have been identified, but few of them are more common, particularly the [c.-32 -13T>G] mutation [32, 33], which is found in >40% of alleles in adult Caucasian patients and 90% of patients included in the French database [34]. Most severe splice mutations (e.g. totally disabling the splicing reactions) are those that typically affect the consensus donor (GU) or acceptor (AG) splice sites at the 5′ and 3′ ends of introns, thus impairing transesterification reactions. Mutations which affect cis-acting regulatory elements (silencers or enhancers) and other RNA features influencing splicing are usually less severe given that resulting inhibition is often incomplete and some normal transcripts can still be synthesised. The T to G transition provoked by the [c.-32-13T>G] mutation in the GAA gene is one of those, which strongly decreases but not completely abolish the normal inclusion of the second exon into the GAA mRNA during the maturation of the nascent pre-mRNA transcript. The consequence of such mis-splicing is that ensuing Δ2-GAA mRNAs (more than 90% of the GAA transcripts resulting from the mutated gene) lack their start codon and thus cannot be translated into a protein [35]. The [c.–32-13T>G] mutation is located at the 3′ end of the intron 1, within the polypyrimidine (Py) tract, among the branch site (in humans the branch consensus is yUnAy) and the AG splice site, which allows the recruitment of the dimeric U2 auxiliary factor protein or U2AF, identified as a major site of splicing regulation. Therefore, a common explanation for the effect of the [c.-32 -13T>G] mutation is that the T to G transition weakens the strength of the Py tract, which could not any more efficiently recruit U2AF [36]. Nonetheless, the fact that the Py consensus varies somewhat in length and sequence for every intron does not support such an assumption. Instead, the T>G transition might alter the correct removal of intron 1 by modifying the secondary structure of the pre-mRNA at the level of its 3′ acceptor splice site, thus making it ineffective. This is supported by the analysis of the intron-1/exon-2 boundary with the RNAfold software from the ViennaRNA Web Services, which shows major differences for the predicted secondary structures in normal and mutated sequences. In both cases, MFE (minimum free energy) predicted structures show that this particular region of the pre-mRNA could likely form a hairpin, although such a structure is dramatically reinforced in the mutant (Fig. 3A). As a result, the high base-pairing probability of the nucleotides involved in the secondary structure of the mutant pre-mRNA makes almost inaccessible some of the crucial consensus sequences at the 3′ end of intron-1, such as the acceptor splice site (SA1), the pyrimidine tract for U2AF recruitment and the branch site. To summarize, it appears that the T>G transition might alter the correct removal of intron-1 by adding force to a cryptic hairpin self-annihilating SA1.
According to these assumptions, one can propose a method for rescuing a full length GAA mRNA in patient cells with the [c.-32 -13T>G] mutation by using antisense oligonucleotides designed to anneal the 5′ region of exon-2 involved in the putative hairpin described above, hence allowing to set free SA1 and its key upstream cis-elements (Py & branch site) without masking downstream ESE sites (Fig. 3B). Such a rationale has been established ex vivo on skin fibroblasts derived from a compound heterozygote patient harbouring the [c.–32 -13T>G] mutation, by using tc-DNA-AONs targeting the putative hairpin and therefore rescuing the expression of a functional protein (Avril et al;, manuscript in preparation). Considering the very properties of the tc-DNA oligonucleotides, which are efficiently distributed into all skeletal muscles, in particular respiratory muscles, the heart and the CNS, one could anticipate that a splice-switching strategy might likely provide a suitable treatment option for patients with late-onset Pompe disease for whom current treatments are quite ineffective. Interestingly, a recent study using the antisense technology, but targeting the muscle specific glycogen synthase, and not the GAA gene directly, demonstrated encouraging results and could also represent a viable therapeutic strategy for Pompe disease after further development [37].
Specific properties/advantages of tc-DNA
Through these various applications, it appears that the tc-DNA chemistry may represent a new avenue for the synthesis of novel splice-switching drugs for many neuromuscular disorders. The very mechanisms responsible for the enhanced activity of tc-DNA are not yet fully understood. We have previously described using nanoparticle tracking analysis (NTA), Size-Exclusion Chromatography with Multi-Angle Static Light Scattering (SEC-MALS) and dynamic light scattering (DLS) that in contrast to the corresponding 2′OMe and PMO oligomers, tc-DNA AONs spontaneously form defined nanoparticles ranging from 40–100 nm [24, 38]. This propensity to spontaneously self-assemble into nanoparticles could imitates features of transfection reagents and nanoparticle delivery systems [39] and hence could potentially improve cellular uptake compared to 2′OMe and PMO oligomers. While it is not known whether self-assembly of tc-DNA is maintained in vivo and therefore responsible for increased potency, we have demonstrated that this process is maintained in physiological conditions and in the presence of serum proteins [38]. The involvement of class A scavenger receptors (SCARAs) together with other polyI sensitive scavenger receptors (SR) has also been shown in gymnosis conditions in vitro [38]. It is feasible to speculate that the superior activity of tc-DNA is related to its propensity to self-assemble into nanoparticles mediating better SR interaction and subsequent gymnotic uptake, which would fit with the particle-wrapping model for receptor mediated uptake of nanoparticles [39, 40]. The mechanism of spontaneous tc-DNA self-assembly is not well understood. The three additional C atoms increase the hydrophobicity of the molecule and this might impart secondary amphipathic properties on the structure enabling self-assembly via mechanisms resembling the self assembly of ampipathic peptides [41]. Alternatively, the extra rings in the tc-DNA might facilitate stacking and generation of structures resembling poly-G aggregates that were seen to enhance the uptake of AONs and binding to SR [42].
We could also hypothesize that tc-DNA as nanoparticles or free AONs might bind naturally to appropriate, not yet identified, carriers in the bloodstream allowing them to cross endothelial barriers more efficiently. Structural and modeling studies are underway to unravel the mechanism of tc-DNA self assembly and their protein binding profile.
A further crucial feature of the tc-DNA chemistry is that it displays higher RNA binding properties than 2′OMe and PMO [43], thus permitting use of AONs of decreased length. Importantly, this offers the advantage of reducing the mass of synthetic nucleotides administered while keeping biological effect, which could reduce toxicity induced by accumulation of AONs. In a recent study, we compared the effect and therapeutic potential of a 15-nt and a 13-nt tc-DNA in the mdx mouse model (Relizani et al., manuscript in preparation) and demonstrated that systemic delivery of 13nt-tcDNA allows restoration of dystrophin in skeletal muscles at similar levels to a 15nt. Remarkably in the heart, the 13nt-tcDNA induces significantly higher levels of exon skipping and dystrophin restoration than its 15nt counterpart, suggesting a potential size advantage for uptake in the cardiac muscle. However, these unique uptake properties are not entirely due to the small size of tc-DNA since treatment with a 20nt tc-DNA also leads to efficient uptake in the cardiac muscle (unpublished data).
While using small size AONs offers an advantage in terms of cost of production, decreasing the sequence length makes it increasingly more challenging to identify a unique target site. Furthermore, increased binding energy has the capacity to not only increase potency against the intended target but also to "off-targets" and the so-called hybridization-mediated off-targets effects (OTEs) may become a more prevalent concern. However because splicing modulation is highly dependent on target sequence position in the intended RNA target, effects on unintended off-target RNAs containing perfect match are extremely unlikely with splice switching AONs as opposed to gapmers AONs recruiting RNAse H [44, 45]. OTEs have not been noted with great abundance in preclinical or clinical studies to date, probably because not all off-target binding actually leads to functional effects. Not all sites on mRNA are accessible to an ASO, nor are all off-target mRNAs in tissues that receive pharmacologic concentrations of an ASO as they are differentially and temporally expressed in different tissues.
One of the unique opportunities of AONs-based therapeutics is that putative off-target interactions can be predicted using sequence alignment algorithms and pragmatic genomic screening strategies are in place in most companies developing AON-based drugs to delineate potential OTEs. The Oligonucleotide safety Working group (OSWG) recommends assessment of OTEs for AONs during drug discovery and development, both computationally and experimentally [46] and such studies are currently on-going for tc-DNA.
While these recommendations and screening are necessary, OTEs are generally not the main concern in the antisense field. Instead, the toxicities noted have been overwhelmingly associated with those expected by class. Each class of AON from first to third generation has stereotypic toxicity profiles and these nuances are important for the toxicologic pathologist to be aware of [44]. We have recently evaluated the toxicological profile of tc-DNA AONs in DMD mouse model and shown that high dose tc-DNA treatment (200 mg/kg/wk for 12 weeks) was well-tolerated in all mice. Histopathological findings in tc-DNA-treated animals were generally limited to minimal glomerular changes and few cell necrosis in proximal tubules (Relizani et al., manuscript in preparation). Serum biochemistry analysis following 12 weeks of treatment did not reveal any significant increase in transaminases or bilirubin, and only a slight variation in serum creatinine and urinary kidney toxicity biomarkers levels could be detected, which is typical of the PS-AONs accumulation in kidneys. Similarly, no toxic effects were observed at any dosage using SMA mouse models during the course of our experiments (with respect to clinical observations, organ weights, macroscopic observations at necropsy). These results suggest an encouraging safety profile for tc-DNA although one should remain cautious until full toxicological studies are completed since we cannot exclude a possible acute toxicity which would be sequence specific as it has previously been reported with the LNAs, inducing unexpected, sequence specific hepatotoxicity [47].
Within the last few years the prospect of successful AON-based splicing therapy for neuromuscular disorders has moved a step closer, particularly for DMD and SMA. Many of these promising therapies have now entered clinical trials and encouraging results have been obtained in most cases, while also showing the limitations of currently used chemistries. The large number of planned and on-going trials using the antisense technology reflects the optimism of these approaches to regulate gene expression and modulate splicing. Much of this optimism can be attributed to the development of novel oligonucleotides modifications, such as the tc-DNA described in this review or the use of cell penetrating peptide which also demonstrated extremely promising results. Recent preclinical work in animal models using these new classes of AONs, which improve tissue uptake, target binding affinity as well as resistance to nuclease degradation, suggest that solutions to the current challenges of systemic and tissue targeted delivery are close at hand. While we focused this review on DMD, SMA and Pompe disease, it should be noted that many other NMDs could benefit from the progresses described here and several preclinical studies have actually demonstrated the therapeutic potential of AONs in other pathologies as reviewed extensively by Van Roon-Mom and Aartsma-Rus [48] and Disterer and colleagues [49].
The very properties of tc-DNA described in this review make them particularly attractive as AONs drugs for such genetic diseases, and clinical evaluation of tc-DNA for the treatment of DMD is currently being planned. However, the promise of tc-DNA still crucially depends on how well it will be tolerated in humans.
With continued success, we will hopefully see a gradual implementation of novel modifications which will improve potency of these drugs over the coming years and that will increasingly extend lifespan and improve the quality of life for patients with neuromuscular diseases.
COMPETING FINANCIAL INTERESTS
Christian Leumann and Luis Garcia are co-funders of Synthena, which produces tricyclo-DNA oligomers.
The authors have financial support from the Agence nationale de la recherche (ANR – Chair of Excellence HandiMedEx), the Association Monegasque contre les myopathies (AMM) and the Duchenne Parent project France (DPPF).
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Chemical structures of a selection of oligonucleotide analogues that have been tested as antisense agents (2′OMe: 2′OMethyl, 2′MOE: 2′OMethoxyethyl, LNA: Locked nucleic acid, PMO: Phosphorodiamidate morpholino oligomer, PNA: Peptide nucleic acid, bc-DNA: Bicyclo-DNA, tc-DNA: Tricyclo-DNA).
Antisense oligonucleotides mediated splice correction for various neuromuscular diseases. A - Exon skipping rationale for DMD. Patients with DMD carry mutations disrupting the open-reading frame of the dystrophin pre-mRNA. In this example, exon 50 is deleted, creating an out-of-frame mRNA and leading to the synthesis of a truncated non-functional or unstable dystrophin. The tc-DNA AON directed against exon 51 can induce effective skipping of exon 51 and restore the open reading frame, therefore generating an internally deleted but partly functional dystrophin. B – Exon inclusion rationale for SMA. Exon 7 of SMN2 gene is spliced out in 90% of the mature mRNA leading to only 10% of functional SMN protein. Tc-DNA AON can mask the ISS inducing inclusion of exon 7 and leading to a functional SMN protein.
A - ESE finder and MFE (minimal free energy) secondary structure predictions: Notice that the beginning of exon-2 is markedly deprived of ESE (exon splice enhancer) sites binding SRSF (Serine/arginine splicing factors) but shows ESS (exon splice silencer) sites for hnRNP A1 (boxed – yellow contributes to the 5′ UTR; light purple designates coding sequence). The T>G transition might alter the correct removal of intron-1 by modifying the secondary structure of the pre-mRNA at the level of its 3′ acceptor splice site (e.g. generating a locked hairpin), thus making it ineffective. B - Splice switching rationale to rescue the [c.-32-13 T>G] common mutation: MFE analysis of the region spanning the end of intron-1 and the beginning of exon-2 in the presence of a 15-mer tcDNA-AON annealing exon-2 at position [+7 to +22]. The frequency of the new MFE structure is 25% instead of 65%. (SA1: Acceptor splice site 1, U2AF: U2 auxilliary factor, U2snRNP: U2 small nuclear ribonucleoprotein).
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On the brink of homelessness, Seattle social workers fight for raises
Human service provider contractors flood city hall on June 6, 2019, calling for new contracts that adjust for inflation. (City of Seattle)
By: Aaron Granillo, MyNorthwest
Updated: June 14, 2019 - 4:53 PM
SEATTLE — Tangled up in Seattle's homelessness crisis is the massive turnover rate among caseworkers. Some say their lousy pay puts them on the brink of homelessness, and forces them to leave the job.
"We do this because we believe that everybody should have a home," said Paul Rosenthal, program manager at Plymouth Housing. "But, a lot of us are afraid of losing our own. We could easily join the ranks of people who don't have homes."
Rosenthal runs a team of six case managers who make slightly above Seattle's $15 minimum wage. Each averages a caseload of about 25 clients. Turnover is common.
"Without improved wages for staff retention, my team occasionally falls down to five people or four people," said Rosenthal. "It creates a lot of challenges for the folks we try to serve and for the staff who remain at Plymouth."
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Seattle's Human Services Department will spend about $118 million in 2020 on provider contracts. The city allocates the funding to about 170 agencies, including non-profits that work directly with the city's homeless population. The agencies also receive county, state, and federal funding along with private donations.
Councilwoman Teresa Mosqueda believes more city funding is needed to offset rising business costs and increase pay for social service workers.
"We feel we've fallen down on these obligations," said Mosqueda, as she rolled out her newly proposed legislation. The measure would require Human Services to include an annual increase to renegotiated or renewed contracts. The raises would come from the city's general fund and be based on federal inflation rates. In 2020, that could mean a 2.4 percent increase.
"Without the front-line human service providers, we cannot provide the critical services to our most vulnerable in the community," said Mosqueda. "Without adequate funding, the workers and the organizations face tremendous burdens on a daily basis."
Seattle's case managers have been flooding city hall recently, voicing frustration over low salaries and staffing shortages. Some said they live in low-income housing or qualify for some of the same services as their clients. They've seen their peers quit after a year or even less.
"We have a really hard time getting employees. Our turnover rate is at 44 percent," said Evelyn Correa, chief human resources officer at YouthCare, a non-profit working to end Youth Homelessness.
Correa said YouthCare can afford to pay their most qualified case managers – those who have master's degrees — about $40,000 a year.
"I totally get that the market determines the value of work," said Correa. "However, we can't afford to have people come in and do this work poorly."
YouthCare and other social service agencies might see the extra funding as early as the next budget cycle. The city council could vote on the new contract rules next month.
© 2020 MyNorthwest


One of the more eye-catching technologies on display at Mobile World Congress. This smartphone iris scanner works by examining 240 unique features of a human eye. (SOUNDBITE (English) JEFF CARTER, CTO, EYELOCK, SAYING: "So if you just imagine how your eye reacts to light. So your pupil opens and closes. And your eye glazes over. You have specularities, you have water on your eyes, you have blood vessels in your eyes. So there are many many signals that allow us to get that sense that we have a real person there." A photo of an eye gets rejected. And its owner must be living and breathing. Iris scanners are considered more secure than fingerprint sensors. And both Apple and Samsung have hinted their next flagship devices will include them. The firm behind this technology says it's safe enough for banking. (SOUNDBITE (English) JEFF CARTER, CTO, EYELOCK, SAYING: "In the next two years, every smartphone in the world is going to have a biometric on it. And we believe it's going to have a combination of fingerprint and iris." But to get the full phone experience, do consumers need to close there eyes to security risks? Location trackers, public wifi and bluetooth connections can come at a cost. SOUNDBITE (English) JOHN SHIER, SENIOR SECURITY EXPERT, SOPHOS, SAYING: "When you're on those access points, if they're open, that means they're open for everybody. So not only you to do your business, but everyone else who wants to join that access point and listen in." Equipment to do that can be legally bought online. It can show who is where and when. And what they're doing. SOUNDBITE (English) JOHN SHIER, SENIOR SECURITY EXPERT, SOPHOS, SAYING: "So I've got some of their device IDs. I've got their location in space, where they were at the time. And so now I can start making a bit of a picture of you know who some of these people are, what device they're carrying, and where they're moving." Targeted advertising works in the same way. But the message here is for users to beware - others could be targeting you too.