text:
The Aboriginal Medical Services Alliance NT (AMSANT) recently called for a minimum floor price on alcohol.
In the UK, the conservative Prime Minister David Cameron is supporting such a policy, and some of the background to this breakthrough has been revealed today by a leading British authority on alcohol policy, Professor Sir Ian Gilmore.
At an alcohol policy forum hosted by the Australian Medical Association in Canberra, Sir Ian, the Royal College of Physicians’ Special Advisor on Alcohol and Chair of the United Kingdom’s Alcohol Health Alliance, described how a mix of persistent advocacy, serendipity and evidence created a cocktail for change in alcohol policy.
Thanks to Adrian Rollins, Editor of the AMA’s publication, Australian Medicine, for filing the report below on the forum.
To reduce alcohol-related harm, focus on regulation
Adrian Rollins writes:
Governments have to tighten rules governing the price, marketing and availability of alcohol if they are to curb the harm caused by dangerous drinking, a leading international expert on alcohol reform has warned.
Renowned UK physician Professor Sir Ian Gilmore told an AMA forum on alcohol policy at Parliament House that the consumption of alcohol was driven by price, promotion and availability, and efforts to reduce harmful drinking needed to focus on the product, rather than blaming individuals and ‘culture’.His comments came against the backdrop of ongoing debate about alcohol regulation in the NT.
Sir Ian backed warnings from the AMA that the alcohol industry was actively promoting drinking among teenagers and adolescents, and needed to be subject to tough external regulation.
Studies in the UK and the Netherlands had shown that children were more likely to see alcohol ads than their parents, and many were specifically pitched to appeal to young people, he said.
AMA President, Dr Steve Hambleton, said the proliferation of such advertising, including through sports sponsorships, social media games and product promotions, was very worrying because such exposure could lead to damaging drinking habits.
“Too many adolescents and teenagers are starting to drink alcohol,” Dr Hambleton said. “The earlier they start drinking, the more likely they will become problematic drinkers throughout their lives.”
Sir Ian, a former President of the Royal College of Physicians and current Chair of the UK Alcohol Health Alliance and the European Alcohol Health Forum Science Group, linked Britain’s position atop Europe’s binge drinking table to marketing and to the ready availability of cheap booze.
He said Britons, on average, drank 25 standard drinks a week – twice the amount they did in the 1950s – with serious affects on health, both in the short and long term.
Sir Ian said alcohol was the “number one risk factor” among those in the UK who died before 60 years of age – contributing to almost a third of all deaths of men aged between 16 and 25 years – though the peak period for alcohol-related deaths was between 45 and 65 years.
Sir Ian, who is a leading UK specialist on liver disease, said alcohol was not only linked to car fatalities, physical assaults and violence, but was also a major cause of chronic disease and fatality, noting that drinking caused 80 per cent of all deaths in the UK from liver disease.
He said it was these complaints, rather than injuries and accidents, that accounted for most of the one million admissions made to UK hospitals each year stemming from alcohol.
Sir Ian said the approach taken by the drinks industry to alcohol policy was to emphasise that its consumption was a normal part of life, and to try to confine any policy action to the relatively small section of the population deemed to be heavy drinkers.
But he said this merely protected the industry’s own interests, and governments needed to take a much broader and more thoroughgoing approach.
The product is the problem
“Alcohol is not ordinary. It is a drug of dependence,” he said. “And the problem lies not with individuals, but the product.”
He said that, as in Australia, industry self-regulation in alcohol advertising in the UK had been a failure, and he endorsed the AMA’s call for a parliamentary inquiry into the marketing of alcohol to young people.
Sir Ian said the promotion of alcohol and drinking in all its forms, from traditional advertising to sports sponsorship, product placement in television shows and films, through social media games and even mobile phone apps, was an important target of policy reform.
But he said equally significant in the UK was action on the cost of alcohol.
Sir Ian said the rise of heavy drinking in Britain had been driven by the availability of cheap alcohol sold by supermarkets and off-licenses (bottle shops).
While the volume of alcohol sold through pubs, clubs and restaurants had remained relatively stagnant, it had surged through other outlets, with supermarkets using heavy discounts on wine, cider, spirits and other drinks to lure shoppers.
For example, he said, one retailer was selling three litres of alcoholic cider for the equivalent of $4.35.
Alcohol reform campaigners in the UK have been heartened by political moves to introduce a minimum unit price for alcohol.
Sir Ian said such a measure could be very effective, because it would raise the price of the very cheap drinks that were favoured by heavy drinkers, while not affecting the cost of beer and wine sold at pubs and restaurants.
The Scottish parliament has approved an alcohol floor price of 50 pence, though it has yet to enact the legislation pending the outcome of legal challenges being mounted by the alcohol industry.
Just as promisingly, Sir Ian said, British Prime Minister David Cameron had thrown his support behind a minimum floor price in England, though at a lower rate of 45 per cent per unit of alcohol.
Asked what had brought the British Government to this position after years of resistance to the idea, Sir Ian ascribed it to a slow build-up of pressure over time from reform advocates, combined with a serendipitous and unexpected circumstance.
He said the breakthrough came when an MP, who was a doctor, asked Mr Cameron during parliamentary Question Time about a minimum floor price on alcohol.
The Prime Minister then sought a briefing on the issue and threw his weight behind the proposal, which is yet to be submitted as a Bill.
Developments in the UK have leant fresh impetus to efforts in Australia to tackle the curb the visibility and availability of alcohol, particularly products targeted at the young.
Dr Hambleton said governments in Australia should take note of what is happening overseas, including in the UK.
“Young people continue to be exposed to alcohol marketing at an unprecedented level, and from multiple sources,” the AMA President said. “It is vital that we reduce this exposure.”
• Declaration: Professor Gilmore was guest speaker at the AMA’s Alcohol policy reform forum, at Parliament House, Canberra, on 18 February.


text:
VAGABUNDO BAY CRUISE
Free Discount Card with your reservation! SEE DISCOUNTS
TO ADD THIS TOUR TO YOUR CART, CLICK "BOOK NOW"
OUR ADVICE: Don't forget your camera! In addition to your regular camera, get an underwater camera to take photos while snorkeling!
Exclusive Online Discount!
LOS ARCOS & LAS ANIMAS
If you don't want to take up your whole day, this might be the cruise for you. Aboard the Vagabundo, a smaller and faster boat than most of the other bay cruises, you'll visit Los Arcos Underwater Marine Preserve for fantastic snorkeling, and visit beautiful Las Animas beach.
THIS TOUR IS NO LONGER AVAILABLE
Runs: Monday through Saturday at 7:55AM from April through Dec 15th Only. In the winter the Vagabundo turns into a Whale Watching boat.
Duration: 5 hours
Includes: Snorkeling Equipment, On-board activities, Breakfast, Unlimited Beer & Soda, Snacks
Departs From: Marina Vallarta. Directions will be provided with your confirmation.
NOTE: THIS TOUR NO LONGER HAS A DEPARTURE FROM NUEVO VALLARTA. IT NOW DEPARTS FROM MARINA VALLARTA, ABOUT 10-15 MINUTES BY TAXI DOWN THE ROAD. See Puerto Vallarta Tours.

domain: travel


text:
If you want to gain some Adobe Photoshop skills, Adobe Photoshop CS5-Classroom in a Book is the perfect go-to guide. It explains what each tool is and breaks down the step by step process of how to use it.
I am really green when it comes to new technology and software and Photoshop is no exception. Before this book I managed to figure out a few basics in Photoshop but it took me hours to do what could have been accomplished in a few minutes had I picked up this book first.
I’m just getting my feet wet, experimenting with all of the tools, but so far the water feels fine.

domain: design


Physical oceanographic real-time system
From Glossary of Meteorology
Revision as of 17:36, 25 April 2012 by
Physical Oceanographic Real-Time System
(Abbreviated PORTS.) A system of current, water level, and meteorological stations telemetering their data to a central location for real-time dissemination, processing, and archival.
Available to pilots, mariners, the U.S. Coast Guard, and all marine interests in voice, graphical, and digital form and via the World Wide Web. The PORTS concept was first introduced in the United States by NOAA's National Ocean Service in Tampa Bay, Florida, and subsequent systems have been installed in the Port of New York and New Jersey, in Galveston Bay, and in San Francisco Bay.


text:
Difference between revisions of "Category:About Inkscape"
|Line 15:||Line 15:|
Revision as of 20:08, 20 May 2009
Inkscape is an open source drawing tool that uses the World Wide Web Consortium's (W3C) scalable vector graphics format (SVG). Some supported SVG features include basic shapes, paths, text, markers, clones, alpha blending, transforms, gradients, and grouping. In addition, Inkscape supports Creative Commons' metadata, node-editing, layers, complex path operations, text-on-path, text-in-shape, and SVG XML editing. It also imports several formats like EPS, PostScript, JPEG, PNG, BMP, and TIFF and exports PNG as well as multiple vector-based formats.
About Inkscape in other languages
Pages in category "About Inkscape"
The following 38 pages are in this category, out of 38 total.


text:
Techtextil Focus | November 2019
Mobiltech & Protech hold high potential: Dr. N Saraf of Sarex
For over 60 years, Sarex Chemicals has been one of the leading textile chemical manufacturing companies. Sarex exports its products to continents like North America, South America, Europe, Asia, and Africa. Sarex is India’s largest exporter of textile chemicals, and the Government has recognised Sarex with a two star export house status.
Dr. Naresh M Saraf, Executive Director of Sarex Chemicals, speaks on the recent products offered by the company, and their salient features.
What are the recent products and technologies that your company offers to the industry? What are their salient features?
We offer wide range of products from pre-treatment, dyeing printing, finishing and value-added finishes. Sarex focuses more on providing solutions rather products to the industry. Our products are based on latest technologies and are free of any restricted substances, and many of our products are also compliant to the latest certifications like bluesign, ZDHC, Oeko-Tex, GOTS, etc. We offer products like oil water repellents, fluorine-free water repellents, flame retardants, chlorine fastness improvers, moisture management chemicals, water-based pu coating agents, wet rubbing fastness improvers, tear strength improvers, products for polyamide, and many energy-saving products for pretreatment of textiles.
In India, technical textiles, know how or end-products, have been experiencing slow growth. How has been the market response to your company?
Overall the technical textile market in India is on the rise as lot of enquiries and requirement are being generated and received by our company on a daily basis. Fortunately, we are doing well in this. Slowly but surely, we are growing in the technical textile market. The only reason for this is our ability to understand the requirements of our customers and provide timely solutions to them.
If your company has been in export, give us a glimpse of the export achievements?
We are a government recognised two star export house and are exporting to more than 40 countries. We have also received “Certificate of Merit” by the Council under the category of Panel-II basic inorganic and organic chemicals including agro chemicals (SSM) for the outstanding performance of the year 2017-2018.
What are the challenges faced in the industry you are in? What are your strategies to overcome them?
The biggest challenge faced by any textile chemical manufacturing company is to the follow the environmental regulations, and to provide products at a lower cost without using any restricted substances. This is followed by the stiff competition faced by the manufacturers locally and from foreign suppliers.
How do you think is R&D important for technical textiles sector in the current scenario? What is your company’s contribution in this?
As technical textile is more about performance than aesthetic, R&D plays a very important role in this industry. Being a doctorate in textiles myself, I encourage in constant R&D in my organisation so that we can focus on providing solutions to the industries where they can increase their output, reduce the wastage, save energy and time. We have a dedicated team of technical team, which is well qualified to cope up with the exiting need of industry.
Sarex has developed many products, where we can save time and energy in pretreatment of textiles. We have developed a fluorine-free water repellent based on sustainable raw materials and many more.
What investments have you planned for in the next five years? How do you look at the future of your products/company in the next few years?
We regularly invest some of our profits for expansion of our manufacturing facilities and also in the R&D so that we are well versed with the latest developments.
Tell us which segments in technical textiles hold higher potential, both in the domestic as well as export markets? What are your reasons?
According to me, mobiltech and protech are two major segments, which will hold very high potential in both domestic as well as export markets. If you take an example of a car or an aeroplane, you will find that 70 per cent of the material used is textile, which requires various finishes. In protech, if you see today, average personnel with uniform wear’s multilayered clothing, which is finished with different chemicals.

domain: chemistry


text:
Blue Bell's New Holiday Ice Cream is Here
Nabisco Chips Ahoy! Soft Chunky Peanut Butter Cookies 10.5 oz. Pack
These cookies are the best cookies for when someone is visiting you! They are delicious! Nice big cookies maybe to big, its with choclate love that!! Its very original , and coups wat these every day too
Yum yum who doesn't love cookies?! I love these ones. Purchased at our local foodland for the first time a few weeks ago and they taste absolutely delicious. Not as good as homemade but pretty darn slose
These are amazing. Although I hate to think how much sugar is in them. They are really tasty if your craving that peanut butter and chocolate combo. Would buy again, but not on a regular basis.
Chewy chocolate chip cookie meets peanut butter bits... Match made in heaven! Its chewy, its chocolatey, chunks of peanut butter, melting in your mouth.. I want a box right now!
Love the product and I love peanut butter so that was an added bonus that a good price for my daughter loves it also and it's a very simple product you get a lot
I love for this chips ahoy cookies! i love them, they are the best cookies and they have a lot of chocolate and taste so good
No me canso de comer esto..lo como con leche y se deshace en la boca...simplemente fantástico hay varios pero me encanta este
Love the taste, i eat these and my kids love these! packaging is good, id rather these bceuase of the nice smooth tatse
Me encanta Me encanta Me encanta Me encanta fabuloso increíble sabroso. malo para la dieta jaja pero me encanta
Deliciosas , a un buen precio me encantan, excelente sabor, ademas me parece saludable y de muy buen gusto!
Q & A
Be the First to Ask a Question

domain: food


Kerala Chief Minister Pinarayi Vijayan has written to Prime Minister Narendra Modi against the Centre’s decision to hand over Thiruvananthapuram airport to the Adani Group for 50 years, NDTV reported on Thursday. Vijayan said the move was against “the assurance given during a personal meeting with prime minister in New Delhi”.
On Wednesday, the Union Cabinet approved a proposal to lease out the Jaipur, Guwahati and Thiruvananthapuram airports through a public-private partnership model. Currently, these airports are managed by the Airports Authority of India.
“The assurance was that as and when the indication of a private player is considered, Government of India would factor in contributions made by the state government for the development of the Trivandrum Airport,” Vijayan said in his letter to the prime minister. He said Kerala had repeatedly requested that the airport’s operations and management be transferred to a Special Purpose Vehicle in which the state government is a major stakeholder.
Vijayan said the Kerala government had in 2005 transferred 23.57 acres of land free of cost to the Airports Authority of India, to build an international terminal, on the condition that the land value should be reflected as the state’s share capital in a Special Purpose Vehicle. Vijayan told Modi in his letter that the state government will “not be able to fully cooperate” due to the unilateral decision taken by the Centre to give the airport to private players.
All-party meeting called to discuss matter
The Kerala government has called an all-party meeting at 4 pm to discuss the Centre’s decision to transfer the airport to the Adani Group, PTI reported. The meeting, called by Vijayan, will be held via videoconferencing.
Adani Enterprises had won the rights to manage six airports – Lucknow, Ahmedabad, Jaipur, Mangaluru, Thiruvananthapuram, and Guwahati – through the public-private partnership in February 2019.
Congress opposes privatisation, Shashi Tharoor backs it
The Opposition Congress has also opposed the Centre’s move. “The Union government’s decision to award operations of Trivandrum Airport to Adani Group on a 50-year lease amidst the COVID crisis is a deplorable move,” Leader of the Opposition Ramesh Chennithala said in a tweet. “The airport is the state’s property, and must not be privatised.” He claimed that the Centre is trying to use the coronavirus pandemic to hand over all public sector units to private companies.
Kerala Pradesh Congress Committee President Mullapally Ramachandran said “none of us need to be on the payroll of these corporate giants”. He called for protests to force the Narendra Modi-led government to withdraw its decision.
But Congress leader and Thiruvananthapuram MP Shashi Tharoor welcomed the Centre’s move. “The people of Thiruvananthapuram want a first-class airport worthy of the city’s history, status, and potential,” he tweeted. “The reality is that a private entity running the operations competitively is the only way this airport could flourish.” Tharoor also said he would have explained his views to his fellow Congress members had they consulted him.
State minister Kadakampally Surendran attacked Tharoor, calling his stand “treacherous”. He claimed that Tharoor had been “batting for this businessman [Adani]”. “Tharoor is taking a shameless stand for Adani,” Surendran said. “He should withdraw the statement and apologise to the people.”
Senior Congress leader Jairam Ramesh said that six airports had now been sold to the Adani Group. “At this rate, AAI (Airports Authority of India) could well mean ‘Adani Airports of India’,” he tweeted.


Two-time Grammy winning Cajun band BeauSoleil, with master fiddler Michael Doucet, is back in town, riding the wave of its latest album, “From Bamako to Carencro,” an allusion to the band’s musical journey from West African roots to the suburbs of Lafayette, La. BeauSoleil has been sailing through scintillating two-steps and waltzes about life, love and loss on the bayous — sung in fetchingly nasal French and pumped along with button accordion — for 37 years now. Credited with sparking the revival of all things Cajun (including food, thank you very much), Beausoleil thrives by sending down deep roots but also being open to influences, from jazz to country. They let the good times roll at 7 p.m. Wednesday at the Showbox SoDo, 1700 First Ave. S., Seattle; $21.50 (800-745-3000 or www.showboxonline.com).
Paul de Barros, Seattle Times music critic


text:
Lucknow super soft wool block print scarf KS126
Score a wardrobe win with this fabulous block print scarf. The navy motif print is bordered with a vibrant crimson stripe to give it that extra edge.
Made from super soft wool with eye lash fringes for a relaxed finish
Hover over the photos so you can zoom in on the photo to see more detail.
FREE POSTAGE WORLDWIDE!
- 70cm x 190cm
- 100% wool
- Dry Clean Only


text:
The slot machine is one of the most popular gambling games today. It has five reels and up to one hundred paylines. The Ancient Fortunes Zeus slot machine is based on Yunani and has an RTP of 96.02%. You can use four deposit methods to fund your account. There is a wide variety of online slot machines available today, so you can find one that suits you perfectly. This article will provide you with some information about the different types of slot machines available.
Pragmatic Play creates striking graphics for its slots. The graphics resemble those of traditional 2D tools but they give a three-dimensional feel to the game. The slot also contains many of the traditional features of the classic slot game such as the fruit symbols, three reels, and a few paylines. Players are sure to enjoy the variety of Pragmatic Play’s games, so you should check them out!
The payouts on this slot machine are quite high, so you can expect to make a lot of money. This is because the slot machine uses random number generators to determine the winning numbers. This process can be very complicated, but it will ensure that you get the best payout. In addition, the slot machine has a high RTP, so it will give you more chances of winning.
PONDOK777 is an online gambling website that provides players with a great selection of slot machines. PONDOK777 also offers bonus jackpots, so there’s no need to worry about not finding the best site for you. The site also offers a variety of arcade games and poker games. All of these games come with a bonus jackpot.
Pragmatic Play also introduces Special Tournaments each time a new game is released. These Tournaments typically consist of three E xtra Cash tournaments, each with a EUR200 prize pool. Players can also earn experience by completing daily missions with their Social Pass. This rewards the player with Coins to spend in the Store and levels them up.
Pragmatic Play is one of the most popular online slot machines. They offer a huge range of slots and offer great customer service. The site accepts several payment methods, including e-money, bank lokal, and pulsa. In addition to these, they offer a mobile version of the games.
PONDOK777 Slot Machine is one of the highest-rated slots available. It has become the premier online slot in the world. PONDOK777 is an online gambling site backed by a team of experienced casino operators. With this reputation, you can rest assured that playing in the PONDOK777 slot machine is as safe and fun as playing at an offline casino.
Besides a high RTP, another important feature of slot games is volatility. This is a measure of how often you’ll receive a payout in the casino. Some slot machines offer large payouts regularly, while others offer small wins more often. This difference in volatility can affect the overall gameplay of a slot machine. If volatility is high, it means that you’ll get large payouts in a short period of time. If volatility is low, it means that you’ll win smaller amounts more often.

domain: game


Welding, Brazing, and Soldering
This article presents a glossary of terms related to welding. The terms are arranged in the conventional dictionary letter-by-letter alphabetical sequence.
Supplementary Information, Welding, Brazing, and Soldering, Vol 6, ASM Handbook, Edited By David LeRoy Olson, Thomas A. Siewert, Stephen Liu, Glen R. Edwards, ASM International, 1993
Download citation file:


text:
Medium Ellipse Chimnea & Lid real glaze black and hand painted glazed dragon inside S1 table stand and small chim burner kit
Medium Ellipse Chimnea & Lid real glaze black and hand painted glazed dragon inside S1 table stand and small chim burner kit. Make a style statement with this very unusual glazed fireplace, with a glazed dragon on the inside of the chimenea. Supplied complete with table stand and small Chim Burner. Ideal for indoor- outdoor setting.
Gardeco bio-ethanol is a man-made plant based alcohol (ethanol) produced by industrial distillation process, but treated so it is not fit for human consumption (denatured). It is used as fuel for indoor fireplaces. Being made from plants it is a renewable resource and not a fossil fuel, and consequently is environementally-friendly. Gardeco bio-ethanol is completley clean burn. Only water and carbon dioxide are released by burning it. There are no noxious smokes or gases released, and there is no soot. This means that fireplaces using bio-ethanol do not require any special installation, flues or chimneys and can therefore be used immediately after purchase.It also give more flexibility as to where to locate the firepalce, and enables people in houses without chimneys to enjoy a real fire. Bio-ethanol burns very hot.
It is important you observe the instructions and maintenance recommendations. Not all fuels and lighting fuels can be used in Mexican chimeneas and they will need protection from the weather.
Dimensions 32 x 25 x 65 cm

domain: artistic


text:
- 1 kilo Chicken
- 1tsp ginger paste
- 1tsp garlic paste
- fresh dhuniya leaves (1 bunch) (cilantro / coriander leaves)
- 3-4 green chilies
- 1 medium Onion
- 1 tsp garam masala
- 4-5 whole dried red chilies
- salt according to taste
- 6 tbs yogurt
- 3 tbs lemon juice
- Mix all the spices together.
- Put in a blender and make a paste.
- Make 4-5 deep cuts on each side of the Chicken pieces.
- This will help it cook better on the inside as well as giving it a better flavor.
- Coat the pieces with the paste and leave to marinate for a few hours preferably overnight in fridge.
- Then BBQ or bake in oven.
- If you're going to bake it in the oven:
- Bake it in an aluminum tray covered with foil for the first 20 minutes at 375 F. Then uncover, turn the Chicken over, and cover it up again. Bake for an additional 10–15 minutes. By this time the Chicken should be cooked. Now increase the temp to 450 F and uncover and bake until the top is dry and the cuts that you made have 'opened up'. Turn over and when it looks done - its done!
Community content is available under CC-BY-SA unless otherwise noted.


text:
No Products in the Cart
There are few foods that are more Spanish than ham. Alongside gazpacho and paella, it might just be the country’s most iconic food.
Spanish ham (jamón) is the one food that all Spaniards, by default of just being Spanish, have to like. Whenever I’ve heard a foreigner say they don’t like jamón, the response of every single Spaniard has been, “well then you haven’t tried the good stuff!”.
Because for Spaniards, there’s no way that anyone couldn’t like ham!
The “good stuff” is jamón ibérico de bellota, a melt-in-your-mouth, otherworldly ham experience. This is one of the world’s most sought-after foods, and holds records for its headache-inducing prices. The world’s most expensive legs of ham are these Spanish beauties, going for $4,500 each.
But before you reach this ridiculously delicious (and very expensive) level of Spanish ham, there is a plethora of styles, breeds, cures, and colors to choose from. Let’s break them down.
Spanish ham is the best thing that can happen to the back leg of a pig. Rather than cooking the ham, you make jamón by dry-curing the leg. After slaughter, the legs are trimmed and cleaned, and then stacked in a barrel full of salt. They’re left for roughly two weeks to drain as much moisture as possible!
After that, the salt is wiped off and the legs are left to hang and cure. It’s carved off of the bone and served alongside other cured meats, sausages, and cheeses as an appetizer.
The Spanish tradition of ham making dates back more than 2,000 years to when the Roman Empire controlled most of the country. Even to the exotic palates of the Romans, Spanish ham was a delicacy.
But for hundreds of years, cured ham legs were a specialty reserved only for royalty and clergy. It wasn’t until the 13th century that ham made its way to the everyman’s table. Today, the average Spaniard eats more than 7 pounds of cured ham per year!


text:
The Mists of Pandaria release date was revealed last month. The panda invasion will kick off on September 25. To help World of Warcraft fans ready themselves for the furry apocalypse, Blizzard have released the Mists of Pandaria intro cinematic. As we've seen from Diablo 3 and Cataclysm, Blizzard are really, really good at this sort of thing. If you've ever wanted to watch a panda school an Orc in the art of combat, you'll want to give this one a watch.
(opens in new tab)

domain: entertainment


Good Thursday morning. Here’s a glance at opinion content published today in The Journal News:
Tax cap and mandate relief: Editorial
As public-school teachers and education advocates rally against a property-tax cap that would limit annual levy increases at 2 percent, we encourage the Legislature to act quickly on the compromise brokered last week to create such a stop-gap. We also beseech our leaders in the state capital to take up real and meaningful mandate relief with haste. We write:
From New City to Albany, teachers and others opposed to a proposed property tax cap picketed outside state legislators’ offices this week, waving signs that read “Scrap the cap!” and “What happened?”
Here’s what happened: While the Great Recession ravaged the economy, New York homeowners watched their property values plummet as they dug deeper to pay ever-growing property taxes.
New Yorkers with underwater mortgages demand immediate relief. Their ranks are swelling. According to the Standard & Poor’s/Case-Schiller 20-city housing index, home values have fallen to their lowest levels since 2002 in the New York metro area.
Opponents of the cap, including teachers and education advocates, argue that a cap would be detrimental to sustaining high-quality public schools — a hallmark of suburban life — and therefore detrimental to property values. That position doesn’t hold water for house-poor residents already drowning in debt. …
Sam Zherka: Reisman
Phil Reisman checks in with Sam Zherka, the controversial publisher of the weekly Westchester Guardian. Zherka, a proponent of streamlined government, was recently named to a commission to examine Westchester’s charter.


text:
Laser hair removal impacts the root of the hair, resulting in smooth and hair-free skin over the course of several treatments
All reviews are from people who have redeemed deals with this merchant.
What You'll Get
- Valid only for select treatment areas.
- Click here for answers to frequently asked questions about laser hair-removal.
“View Refine Medical Spa’s other offers:
The Fine Print
Promotional value expires 120 days after purchase. Amount paid never expires. Valid only for select treatment areas. Not valid for red or gray hair. Not valid for darker skin tones. Appointment required. Consultation required; non-candidates and other refund requests will be honored before service provided. Limit 1 per person, may buy 1 additional as gift(s). Valid only for option purchased. All goods or services must be used by the same person. Not valid for clients active within the past 12 month(s). Merchant's standard cancellation policy applies (any fees not to exceed voucher price). Merchant is solely responsible to purchasers for the care and quality of the advertised goods and services.
About Refine Medical Spa
As guests walk in the doors at Refine Medical Spa, they're greeted by a tranquil waiting room area decorated with soft sea blues and vases lush with lively plants. Whether they've arrived for an injectable appointment—designed to tighten facial features and reduce fine lines—or they've scheduled a revitalizing chemical peel, they'll be ushered to their own individual treatment room for a hands-on consultation with a licensed medical practitioner. There, guests will receive professional care as their specialist performs treatments ranging from B-12 and Dysport injections to microneedling and laser hair removal, all with the goal of helping guests to maintain a healthy and youthful appearance as they age.

domain: medical


text: I may have went a little bit crazy with the camera the other day! She is changing so quickly and i want to remember her early puppy days.\n\n\n\n\n I love this one because she is running to me! She wanted that camera!\n\n\n\n\nSee total overload of pictures! I'm obsessed. With my puppy and my camera!

domain: pet


Americans have always been a people on the move—on rails, roads, and waterways (for travel through the air, visit the National Air and Space Museum). In the transportation collections, railroad objects range from tools, tracks, and many train models to the massive 1401, a 280-ton locomotive built in 1926. Road vehicles include coaches, buggies, wagons, trucks, motorcycles, bicycles, and automobiles—from the days before the Model T to modern race cars. The accessories of travel are part of the collections, too, from streetlights, gas pumps, and traffic signals to goggles and overcoats.
In the maritime collections, more than 7,000 design plans and scores of ship models show the evolution of sailing ships and other vessels. Other items range from scrimshaw, photographs, and marine paintings to life jackets from the Titanic.
"Transportation - Overview" showing 1 items.
- In the later 19th century, guns with explosive charges shooting the harpoons took the place of hand tools for catching and killing whales. They were much safer, for they could be shot at a whale from greater distances than a hand lance could be applied. They also penetrated the whale’s skin deeper and were harder for the animal to dislodge.
- Gun harpoons were also far more efficient, for the steam whalers could approach the prey directly and did not need labor-intensive whaleboats and their highly trained crews any longer.
- Designed to be fired from a shoulder gun, this nonexplosive style of harpoon was invented by Oliver Allen of Norwich, Conn. to fasten to whales prior to killing.
- date made
- harpoons replaced hand tools
- late 19th century
- Allen, Oliver
- Allen, Oliver
- ID Number
- catalog number
- accession number
- patent number
- Data Source
- National Museum of American History, Kenneth E. Behring Center


text:
1900S Fashion. By 1910 women were growing tired of bulky evening dress, and in particular, french women began to remove the trains from their dresses and embrace the new empire lines, especially poiret’s ballet russes inspired creations. It was the era of the titanic, the first world war.
“morning coats” happened to be the regular morning clothes worn by the 1900s men. As mentioned, the early 20th century was defined by a conservative fashion in combination with nature. The silhouette was slim at the hip, accomplished by pleating and smocking the excess fabric.
Any Fullness In The Skirt Began Below The Knee.
Social rituals, especially the custom of formal visiting, dictated the use of each of these dresses, or gowns, as the fancier garments were called. But, of course, the 1900s men had many other important fashion essentials! The 1900s were known as the edwardian era in england because it was during the reign of edward vii.
Jun 3, 2012 | By Admin | Fashion Timeline | 1900S Read More.
In 1900s the key trends were la belle époque and the gibson girl. Some men, including younger men, donned sack suits (similar to modern business suits) all day. London for men and pais for women dominated the fashion world.
These Selections Mainly Depended On The Wearer’s Age.
The fashion of the edwardian era is popular again thanks to the titanic movie, downton abbey, suffragette, and mary poppins. The prosperous 1900s was a time of great elegance. From the middle of the 19th century, world fashion was dictated by designers of the fashion capital of the world, paris.
Men’s Fashion In This Time Was Dependent On The Time Of Day.
Women's elegant dresses weretrimmed with fur, feathers and jewelry. It was designed to create an “s” shape by pushing the chest forward and the hips back. Edwardian fashion for men and women had some unique patterns.
Clothes For Women In The 1900S Were Frilly And Layered, Worn Over A Stiff Corset.
By the end of the decade, most fashionable skirts still brushed the floor, but approached the ankle. Not only was fashion important for wealthy ladies of the 1900s, it was practically a pastime. This was a popular fashion trend for women to wear while they rode their bikes.

domain: history


text:
|A quan họ singer (right) introduces artefacts related to folk music in Bắc Ninh Province. — VNA/VNS Photo Diệp Trương|
BẮC NINH — An exhibition to mark the 10th year anniversary of quan họ (love duet singing) receiving the title of intangible cultural heritage of humanity from UNESCO is opening in the northern province of Bắc Ninh.
With the theme Dân ca Quan họ Bắc Ninh - Truyền thống và Đương đại (Quan họ Bắc Ninh-Traditional and Contemporary), the exhibition is being held by the provincial Department of Culture, Sports and Tourism. It is displaying 500 photographs, documents and artefacts related to folk music at the Bắc Ninh Province Museum.
Photos of various historical and cultural relics including Diềm Temple, Vua Bà Temple, Ngọc Well and Quan Họ Communal House are also exhibited.
As part of the exhibition, a programme called Không Gian Văn Hóa Quan Họ (Quan họ Culture Space) will introduce various forms of quan họ folk songs, which have long been popular among local people in the province.
Organisers said the event was designed not only to promote public awareness of the preserving quan họ as an important cultural practice, but also to promote Bắc Ninh Province and show its achievements in culture and tourism.
It is also a chance to honour folk singers specialising in quan họ Bắc Ninh. — VNS
|Students learn about items used during quan họ performances — VNA/VNS Photo Diệp Trương|

domain: news


text:
The Youth Employment & Skills Strategy for Gloucestershire
The YES Strategy is Gloucestershire County Councils commitment to help young people from 14 to 24 years old, to get the information, gain the skills, work experience and abilities they need to make a successful transition into employment.
It’s also aimed at supporting economic growth by helping businesses to access young people who can develop the skills that they need.
The Youth Employment & Skills Strategy is part of Grow Gloucestershire – our commitment to investing in better infrastructure, more jobs and new homes.
What can this site offer to you?
This site contains searchable employment & development opportunities along with resources to assist employers, young people and education providers along the way.

domain: economics


text:
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline. Among the widespread neuronal and synaptic deficits in AD, degeneration of basal forebrain cholinergic neurons (BFCNs) and loss of cholinergic innervation to the cortex (CTX) and hippocampal formation (HF) substantially contribute to cognitive decline in AD (1). During the prodromal and earliest stages of AD, cholinergic neurons undergo trophic changes, including alterations in nerve growth factor (NGF) processing or transport, expression of NGF receptors, and activation of downstream NGF signaling components (2). The loss of neurotrophic support to BFCNs, beginning in early-stage AD, contributes to impaired cholinergic neurotransmission, followed by neuronal degeneration that is observed in late-stage AD (2).
In the brain, NGF promotes neuronal survival, growth, and synaptic plasticity by binding to tropomyosin receptor kinase A (TrkA) and p75 neurotrophin receptor (p75NTR) (3). NGF is produced in the medial septum (MS), diagonal band of Broca (DBB), and nucleus basalis of Meynert (NBM), which together compose the basal forebrain, HF, and CTX. In response to NGF binding, TrkA receptors expressed by BFCNs trigger activation of the tyrosine kinase domain, receptor phosphorylation, and intracellular signaling via the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt cascades (3). NGF also binds p75NTR and can, in the absence of TrkA, induce neuronal apoptosis and glial inflammation through Jun N-terminal kinase (JNK) signaling (3, 4). In addition, BFCNs respond to NGF-induced activation of TrkA by enhancing their cholinergic phenotype. TrkA activation engages the cholinergic gene locus to increase choline acetyltransferase (ChAT) activity, the enzyme that synthesizes the neurotransmitter acetylcholine (ACh), which, in turn, promotes ACh release at cholinergic nerve terminals (3).
The importance of NGF in neuronal health and disease has been long recognized, yet translating a treatment strategy for AD remains a major challenge. The therapeutic efficacy of NGF is limited by its short half-life, detrimental effects related to p75NTR activation, and its inability to cross the blood-brain barrier (BBB) (5). Furthermore, assessment of the preclinical efficacy of NGF-based therapeutics has been limited to injury models, namely, the fimbria-fornix lesion-induced degeneration of BFCNs or aging models (5), in the absence of toxic amyloid-β (Aβ) peptides and tau pathology. Native NGF can bind to both TrkA and p75NTR, and in the presence of defective TrkA signaling (as observed in human AD), p75NTR activation facilitates Aβ- and tau-induced signaling and neurodegeneration (6–8). Recent clinical trials for NGF therapy in AD showed some growth of cholinergic afferents but ultimately failed to slow cognitive decline (9, 10). Further elucidation of the molecular interplay between NGF and pathological features of AD is necessary to facilitate the development of successful NGF-based therapies.
In comparison to native NGF, ligands with desirable pharmacological properties, designed to specifically activate TrkA, and not p75NTR, may be a preferable therapeutic approach to up regulate the cholinergic phenotype of BFCNs (11–14). Saragovi and colleagues (11–13) have previously demonstrated that D3, a peptidomimetic agonist of TrkA, promotes cell survival, outgrowth, cholinergic activity, and rescued learning and memory deficits in aged rats and a transgenic (Tg) mouse model of AD. However, like NGF, D3 does not cross the BBB.
In this study, we achieved noninvasive delivery of D3 to the basal forebrain in a mouse model of AD using magnetic resonance imaging–guided focused ultrasound (MRIgFUS). MRIgFUS, combined with intravenous microbubbles, allows a localized and transient increase in BBB permeability (15). Therapeutic agents injected intravenously enter MRIgFUS-targeted areas, thereby facilitating site-specific delivery of drugs, genes, and cells to the brain (16–18). The safety and feasibility of MRIgFUS BBB opening in humans with early to moderate AD was recently reported in a phase 1 clinical trial, validating its use translationally (19).
Our study was performed in the TgCRND8 murine model of AD. TgCRND8 mice harbor two human amyloid precursor protein mutations linked to familial forms of AD (i.e., Swedish, KM670/671NL; Indiana, V717F), leading to robust production of Aβ, with progressive Aβ plaque deposition and memory deficits starting at 3 months of age (20).
Deficits in several neurotransmitter systems occur in TgCRND8 mice (21, 22). Cholinergic atrophy and cell loss have been reported, coinciding with a reduction in the levels of ACh release in the prefrontal cortex of 7-month-old TgCRND8 compared to non-Tg mice (22). Consistent with TrkA deficits observed in AD postmortem tissue (23, 24), single-cell RNA sequencing also revealed lower mRNA levels of TrkA in TgCRND8 mice compared to non-Tg littermates (25).
The status of NGF signaling in relation to cholinergic function remains poorly understood in animal models of AD. Here, we report deficits in NGF levels, TrkA receptor levels, and TrkA-mediated signaling cascades in parallel with cholinergic dysfunction in the TgCRND8 model of AD, thus providing a disease-relevant phenotype to test NGF-based therapeutics. To this end, we delivered D3 to BFCNs using MRIgFUS in TgCRND8 mice. D3 was physiologically active in vivo after MRIgFUS delivery, significantly increasing TrkA phosphorylation and downstream signaling cascades in both TgCRND8 mice and non-Tg littermates. Moreover, we report that D3—and not NGF—successfully activated TrkA signaling pathways in TgCRND8 mice, representing an additional advantage of our therapeutic approach favoring TrkA activation without engaging p75NTR. MRIgFUS-mediated D3 delivery in the basal forebrain increased ChAT activity locally and enhanced ACh release from hippocampal and cortical slices. These results suggest that noninvasive, selective TrkA activation in BFCNs is sufficient to increase cholinergic function in the presence of Aβ pathology and that it can be achieved via MRIgFUS delivery of D3. TrkA receptor ligands, combined with MRIgFUS targeted delivery, merit consideration as a treatment strategy for AD.
NGF/TrkA signaling declines with age in TgCRND8 mice
We comprehensively evaluated age-dependent changes in NGF signaling in an AD animal model to identify a relevant, early-stage, AD phenotype for testing NGF-related therapies. We measured regional differences in the levels of NGF, TrkA, and p75NTR mRNA [quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization] and protein [enzyme-linked immunosorbent assay (ELISA) and Western blot] in TgCRND8 and non-Tg mice at 4, 6, and 8 months of age (Fig. 1 and fig. S1).
Relative to age-matched non-Tg mice, NGF mRNA levels in the MS/DBB and NBM were unaltered in TgCRND8 mice (Fig. 1A). In contrast, NGF protein levels were reduced in 6- and 8-month-old Tg mice (Fig. 1B). Similar analysis in the HF and CTX revealed no change in NGF mRNA and protein levels between groups at all time points examined (fig. S1, A and B).
TrkA mRNA and protein levels in 6- and 8-month-old Tg mice were decreased in the basal forebrain, whereas only TrkA protein levels were reduced in the HF and CTX at these ages, consistent with anterograde transport of the receptor to projection sites of BFCNs once it is translated (Fig. 1, C and D, and fig. S1, C and D). TrkA gene expression is under positive feedback from NGF signaling and may be disrupted by reduced availability of NGF to BFCNs (2, 3). In addition, reduced levels of phosphorylated TrkA (pTrkA) were found in 6- and 8-month-old Tg mice (Fig. 1E and fig. S1E). Levels of p75NTR mRNA and protein were stable between genotypes and across all age groups and brain regions (Fig. 1, F and G, and fig. S1, F and G). These data are in line with the relative imbalance of TrkA/p75NTR at the protein level (Fig. 1H and fig. S1H) and mRNA level (Fig. 1, I and J) in 6- and 8-month-old Tg mice, as a result of a shift in the balance of NGF and TrkA expression. Our results demonstrate that deficits in the NGF/TrkA system observed in TgCRND8 mice resemble those observed in human AD and provide a relevant model to evaluate NGF-based therapeutic strategies for AD.
D3, not NGF, stimulates TrkA-dependent signaling in TgCRND8 mice
Next, we directly compared the in vivo efficacy of native NGF and compound D3, a selective TrkA agonist, to induce TrkA-dependent signaling cascades in 6-month-old TgCRND8 mice. Following direct intraparenchymal injection to the MS/DBB and NBM in TgCRND8 mice, D3, unlike native NGF, stimulated neurotrophin signaling (Fig. 1, K to T). Specifically, we found elevated levels of pTrkA, pAkt (Thr308), pMAPK (Thr202/Tyr204), and pCREB (phosphorylated cyclic adenosine monophosphate response element-binding protein; Ser133) within 90 min after D3 treatment relative to NGF-treated and phosphate-buffered saline (PBS)–treated TgCRND8 mice (Fig. 1, K to O). Downstream of p75NTR, we found reduced JNK activity (Thr183/Tyr185) in D3- and NGF-treated mice relative to PBS controls (Fig. 1, K and P). No differences were detected in total protein level normalized to β-tubulin-III (βtubIII) among all conditions (Fig. 1, Q to T). As a positive control, we performed intraparenchymal injection of D3 to the basal forebrain in age-matched, non-Tg mice to confirm signal transduction pathways activated by D3 (fig. S2, A to F) and NGF (fig. S3, A to F). Total protein levels normalized to βtubIII remained unaltered across all treatment groups (figs. S2, G to J, and S3, G to J). These findings provide experimental evidence that Aβ pathology interferes with NGF bioactivity in vivo and demonstrate that selective TrkA stimulation, in the absence of p75NTR interaction, is required to promote TrkA-dependent survival and plasticity in the presence of Aβ pathology.
Localized MRIgFUS-mediated BBB disruption in the basal forebrain
To rescue NGF/TrkA signaling deficits in the TgCRND8 model, we delivered the selective TrkA ligand, D3, to BFCNs using MRIgFUS. The MRIgFUS experimental setup is illustrated in Fig. 2A. The MS/DBB region of the basal forebrain was targeted bilaterally with one spot (Fig. 2B, green dot) and the NBM with two foci (Fig. 2B, purple dots) on T2-weighted (T2w) MR images (Fig. 2C). BBB opening was confirmed on T1w contrast-enhanced MR images, visualized by the influx of Gadovist (MRI contrast agent) from the bloodstream into sonicated areas (Fig. 2D). We found no difference in the level of contrast enhancement between TgCRND8 and non-Tg mice (Fig. 2E). The mean increase in signal intensity in the sonicated areas was 20 ± 8% and 25 ± 9% in non-Tg and TgCRND8 mice, respectively. Concomitantly with relative enhancement, no difference in the average peak pressure required to induce subharmonic emissions was observed between genotypes. The mean peak pressure reached during sonication was 1.29 ± 0.15 MPa in the TgCRND8 mice and 1.21 ± 0.11 MPa in the non-Tg controls (Fig. 2F). Relative contrast enhancement and mean acoustic pressure were comparable between treatment groups as well as subregionally in the basal forebrain (fig. S4). These data suggest that the increase in BBB permeability was consistent between genotypes, treatment groups, and distinct nuclei within the basal forebrain.
Evans blue dye, which binds to serum albumin, and endogenous antibodies, immunoglobulin G (IgG) and IgM, proteins that normally do not cross the BBB, were used to histologically confirm the sites targeted by MRIgFUS. Immunohistochemical analyses revealed a 1-mm-wide focal spot of Evans blue dye at the level of the MS/DBB and two 1-mm-wide focal spots in the NBM (Fig. 2G). In contrast, Evans blue was not detected in brain regions nontargeted by FUS (Fig. 2G) and in control, non-FUS–treated, mice (Fig. 2H). Extravasation of blood-borne IgG and IgM into the basal forebrain was observed 90 min after MRIgFUS (fig. S5). Immunohistochemical (fig. S5A) and Western blot (fig. S5, B to E) analyses revealed greater levels of IgG and IgM in MRIgFUS-treated regions relative to unsonicated areas, in both TgCRND8 and non-Tg mice. Furthermore, there was a positive correlation between IgM levels and Gadovist contrast enhancement (fig. S5F), as previously reported in the same experimental model (26).
We found no evidence of erythrocyte extravasation or neuronal cell death in sonicated areas. Ter119-positive erythrocytes were confined to vascular structures identified by tomato lectin in MRIgFUS-targeted regions, without red blood cell infiltration into the brain parenchyma (fig. S6). We did not detect either cleaved caspase-3 or terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) immunoreactivity in neuronal nuclei (NeuN)–positive cells (fig. S7). The olfactory bulb was used as a positive control for caspase-3 and TUNEL due to the relatively high rate of granule cell turnover in this region (18). Together, these findings suggest that MRIgFUS allows BBB opening in the basal forebrain without overt tissue damage.
MRIgFUS effectively delivers D3 to the basal forebrain
Using quantitative high-performance liquid chromatography (HPLC), we measured D3 concentrations in MRIgFUS-targeted and nonsonicated brain regions of TgCRND8 mice and non-Tg controls after intravenous D3 injection. D3 was detected in D3/FUS-treated tissue 30 min after treatment (Fig. 3, A and B). The mean D3 levels in the MS/DBB were 15 ± 2 and 13 ± 3 ng/mg tissue in TgCRND8 and non-Tg mice, respectively. Similarly, D3 levels in the NBM were 16 ± 2 and 12 ± 2 ng/mg tissue in TgCRND8 and non-Tg mice, respectively. The drug concentration achieved after MRIgFUS was not significantly different between genotypes in both neuroanatomical locations. By contrast, D3 levels were below the limit of detection by HPLC in D3-treated, non-FUS controls (Fig. 3B). The mean D3 concentration in the serum and plasma was comparable between D3- and D3/FUS-treated mice for both genotypes (Fig. 3C).
The mean D3 concentration and relative contrast enhancement were positively correlated, with no apparent differences in correlation between non-Tg and TgCRND8 mice (Fig. 3D). In addition, there was a positive correlation between TrkA activation and relative contrast enhancement, consistent with enhanced D3 bioavailability in the MRIgFUS-targeted regions (Fig. 3E). Overall, these data support the feasibility of using MRIgFUS to deliver D3 locally to the basal forebrain in a noninvasive manner.
Given that the sonication scheme used to achieve MRIgFUS BBB opening in this study would potentially allow the entry of serum NGF (26 kDa), or factors stabilizing NGF and/or its release, we evaluated NGF protein levels by ELISA. Elevated levels of NGF were found in PBS/FUS relative to PBS-treated mice and D3/FUS relative to D3-treated animals in the MS/DBB and NBM for both TgCRND8 and non-Tg mice (Fig. 3F). There was a positive correlation between NGF levels and contrast-enhanced T1w voxel intensity in MRIgFUS-treated non-Tg and TgCRND8 mice, indicating that Gadovist enhancement may provide a means of estimating NGF alterations in the targeted tissue volume (Fig. 3G). The elevated local NGF levels resulting from MRIgFUS lend support to the hypothesis that our therapeutic paradigm may be used to promote endogenous brain plasticity and repair for the treatment of AD.
MRIgFUS-mediated delivery of D3 activates TrkA-dependent signaling pathways
To further elucidate TrkA signaling deficits in the TgCRND8 mouse model of AD, we compared signal transduction targets modulated downstream of TrkA phosphorylation in the basal forebrain (Fig. 4). In an independent cohort of 6-month-old mice, we confirmed the reduction in pTrkA levels in TgCRND8 relative to non-Tg mice in the MS/DBB and NBM (Fig. 4B). Parallel to reduced levels of pTrkA, pAkt, pMAPK, and pCREB were also decreased in TgCRND8 mice (Fig. 4, C to E). Downstream of p75NTR, we found increased JNK activity in TgCRND8 compared to non-Tg mice (Fig. 4F).
Thus, we assessed the functional effects of MRIgFUS delivery of D3 on TrkA-dependent signaling pathways in 6-month-old TgCRND8 and non-Tg littermates (Fig. 4). We found elevated pTrkA levels in the MS/DBB and NBM within 90 min after D3/FUS treatment relative to PBS/FUS-, PBS-, and D3-treated mice in both genotypes (Fig. 4, A and B). TrkA activation was achieved in 6-month-old TgCRND8 mice in the presence of decreased TrkA expression, and the level of pTrkA was not significantly different from D3/FUS-treated non-Tg mice (Fig. 4B).
We observed elevated pAkt in PBS/FUS- and D3/FUS-treated mice compared to PBS- and D3-treated animals, respectively (Fig. 4C). These data are in line with recent studies, which demonstrate that MRIgFUS BBB opening leads to elevated pAkt levels, 90 min after sonication (27, 28). Similarly, we found an increase in MAPK activation in D3/FUS-treated compared to PBS/FUS-, D3-, and PBS-treated mice (Fig. 4D). CREB function was acutely increased after D3/FUS treatment (Fig. 4E), likely to be due to phosphorylation at Ser133 by multiple kinases, including MAPK and Akt (3). JNK activity was decreased after D3/FUS relative to control conditions in TgCRND8 mice (Fig. 4F). For each protein in the NGF/TrkA signaling pathway, no difference was detected in total protein level normalized to βtubIII among all treatment groups (fig. S8).
MRIgFUS-mediated delivery of D3 enhances cholinergic function
We established whether deficits in TrkA signaling correspond to diminished cholinergic function in TgCRND8 mice. ChAT activity in the basal forebrain (enriched in cholinergic cell bodies) and ACh release in the HF and CTX (enriched in cholinergic axon terminals) were measured (Fig. 5). In 6-month-old TgCRND8 mice, ChAT activity was reduced by 30 to 40% relative to non-Tg littermates (Fig. 5, A and B). Furthermore, basal and potassium-induced ACh release was lower in TgCRND8 compared to non-Tg slices (Fig. 5, C and D), consistent with the loss of enzymatic activity related to ACh synthesis in TgCRND8 mice. These results indicate that 6-month-old TgCRND8 and non-Tg mice represent valid experimental models of impaired and unimpaired cholinergic function, respectively.
Thus, we evaluated the potential of MRIgFUS delivery of D3 to increase cholinergic function in TgCRND8 and non-Tg mice. Three days after sonication, elevated ChAT activity was found in the D3/FUS-treated MS/DBB (Fig. 5A) and NBM (Fig. 5B) relative to animals that received intravenous D3 administration only, for both genotypes. In addition, we examined the impact of D3/FUS treatment on basal and depolarization-evoked ACh release in hippocampal and cortical slices, also at 3 days after FUS. Potassium-evoked ACh release was elevated in D3/FUS compared to intravenous D3 treated animals of both genotypes in hippocampal (Fig. 5C) and cortical preparations (Fig. 5D). Together, our data demonstrate that a single D3/FUS treatment can stimulate cholinergic function in TgCRND8 mice up to 3 days after sonication.
Overall, these findings highlight the in vivo efficacy of our treatment paradigm—that is, to selectively engage TrkA receptors and regulate critical intracellular signaling cascades that are sufficient to promote cholinergic function in the presence of Aβ pathology (Fig. 6). Thus, enhancing cholinergic neurotransmission by MRIgFUS-mediated delivery of D3 represents a possible therapeutic approach to limit cognitive decline in AD.
There is an urgent need for therapeutics and delivery methods that slow or reverse the degeneration of neurons in AD, including cholinergic neurons. Acetylcholinesterase inhibitors, which represent the most common pharmacological compounds used for the treatment of AD, do not restore dying or degenerating neurons. In contrast, NGF-related therapeutics bode well for the maintenance of cholinergic morphology, survival, and function. In particular, strategies in which NGF-mediated activation of BFCN function can be safely and effectively accomplished hold therapeutic potential for AD.
In this study, we show that MRIgFUS delivery of D3 is a novel therapeutic approach to enhance cholinergic function, addressing fundamental limitations of current NGF-based treatment modalities for AD. MRIgFUS-mediated delivery of D3 led to selective TrkA signaling in BFCNs while reducing p75NTR activation, and elevated ChAT activity and ACh release in a mouse model of amyloidosis with deficits in NGF, TrkA and cholinergic function—pathologies that are present in human AD. Our findings are consistent with the suggestion that NGF administration directly to cholinergic cell bodies, rather than in axon terminals, bypasses transport defects and allows NGF/TrkA signaling at the soma (29–31). Even in the presence of reduced TrkA levels, D3/FUS treatment increased TrkA signaling in TgCNRD8 mice, as assessed by activation of TrkA and downstream effectors including Akt, MAPK, and CREB (Fig. 4). Although p75NTR expression levels remain stable, downstream p75NTR activity decreased in response to D3/FUS treatment as measured by JNK activation. TrkA signaling has been shown to silence p75NTR-mediated pathways such as JNK via PI3K/Akt (3). Because D3 does not interact with p75NTR directly, Akt activation may represent a potential mechanism by which D3/FUS treatment decreases JNK phosphorylation. Moreover, MRIgFUS delivery of D3 rescued cholinergic function, i.e., depolarization-induced ACh release in amyloid-burdened hippocampal and cortical regions, and ChAT activity in the basal forebrain. The levels of evoked ACh release and ChAT activity for up to 3 days after D3/FUS treatment were similar to those observed in untreated, non-Tg controls, which have levels of cholinergic signaling sufficient to support normal cognitive function (Fig. 5). In this way, localized D3 delivery to the basal forebrain by MRIgFUS led to widespread restoration of cholinergic transmission in the associated projection regions of the HF and CTX.
To date, NGF-based therapies have been validated in preclinical models of aging and injury. Although these studies lend further support to the rationale of NGF as a therapeutic agent for AD, the predictive validity of these models was limited by the absence of Aβ and tau pathology. Multiple lines of evidence suggest that Aβ and tau are inextricably linked to NGF-related signaling cascades in vitro and in vivo (6–8). Here, we discovered alterations in the status of NGF bioactivity related to NGF mRNA and protein levels, NGF receptor levels, and downstream signaling components in a preclinical model of amyloidosis, which, taken together, appear to recapitulate the signaling alterations reported in early-stage AD patients (2). Evidence from preclinical and human studies indicate that the shift in TrkA/p75NTR levels may represent a switch favoring the binding of NGF to p75NTR, possibly activating apoptosis over survival pathways in BFCNs and contributing to cholinergic dysfunction before cell loss in AD (2, 3). Our signaling data also suggest a TrkA/p75NTR signaling imbalance, which can contribute to cholinergic dysfunction in AD. Previous studies suggest that increased p75NTR activity coupled with increased Aβ load exacerbates Aβ-induced neurodegeneration (6–8). Moreover, our data in the TgCRND8 model indicate that the amount of ACh synthesized (Fig. 5, A and B) and released (Fig. 5, C and D) is impaired with disease progression and may, in part, underlie the cognitive deficits observed in this model of AD (20). Likewise, Bellucci et al. (22) reported decreased ChAT immunoreactivity in the NBM and release of cortical ACh in 7-month-old TgCRND8 mice relative to non-Tg controls. To this end, the TgCRND8 model is suitable for testing NGF-related therapeutic agents as a means to improve cholinergic function in the presence of Aβ pathology.
Within this framework, selective activation of TrkA, avoiding p75NTR, was necessary to induce TrkA-related signaling pathways and sufficient to up-regulate cholinergic function in the context of amyloid pathology. Our results demonstrate that D3, in contrast to native NGF, stimulates TrkA signaling in the TgCRND8 mice (Fig. 1, K to T). Our findings draw attention to a previously unknown role of amyloid on NGF bioactivity in vivo, including the relative imbalance in TrkA/p75NTR signaling, amyloid pathology, and potentially other mechanisms affecting NGF stability, and could partly explain the limited efficacy of NGF-directed treatments for AD to date (9, 10, 32).
A major concern for NGF-based therapy is that undesirable pleiotropic effects can reduce the therapeutic benefit of NGF. For example, p75NTR activation can negate the benefit of engaging TrkA (14). Delivery of D3, a highly selective and potent partial TrkA agonist, is a promising approach to promote TrkA signaling (11), and studies in animal models of aging and AD have shown improvements in memory performance (12, 13). Clinically, D3 has been shown to be a well-tolerable and effective treatment for dry eye, without inducing potential treatment-related side effects such as aberrant growth of sensory fibers (33).
In addition, the delivery of NGF-related therapeutic agents to the brain, to date, required invasive surgery due to their limited ability to cross the BBB (5). We demonstrate that MRIgFUS is an effective method for targeted drug delivery to the basal forebrain in a noninvasive manner with real-time feedback monitoring. In human AD, clinical trials for NGF and vector-based NGF delivery all require complex intracranial surgical procedures that produce BBB breakage and tissue damage and carry substantial risk for severe hemorrhages and infection. Moreover, these surgeries pose a higher risk of adverse events among elderly individuals. These detrimental effects could rapidly outweigh the potential benefit of NGF therapy (9, 10, 32). The MRIgFUS parameters used here did not induce erythrocyte extravasation (fig. S6) or neuronal apoptosis (fig. S7). These data are in line with previous studies, which demonstrated that these MRIgFUS settings are below the threshold of damage (34, 35). Focused ultrasound combined with MRI as an imaging method for targeting the brain allows ultrasound to be applied throughout the entire basal forebrain, without significant diffusion of the drug to distal brain regions, especially in the ventricles and meninges containing cerebrospinal fluid. Here, an intravenous dose of 500 μg of D3 was sufficient to activate TrkA in the basal forebrain targeted by MRIgFUS, without drug diffusion to other brain regions (D3 levels below the detection threshold by HPLC).
Further work to optimize experimental design parameters should be conducted with the goal of achieving consistent BBB modulation of permeability and simultaneously minimizing potential damage. Factors such as sonication scheme, microbubble composition, and brain subregion can influence the extent of BBB permeability and associated biological effects following FUS treatment. Microbubble dose, size, shell properties, and half-life circulation can affect the degree of BBB permeability (36). In this study, we used the recommended clinical imaging dose of Definity microbubbles (20 μl/kg) to approximate an equivalent microbubble dose between animals. Recently, McMahon and Hynynen (34) reported that Definity at 20 μl/kg compared to 100 μl/kg (10 times higher than the clinical imaging dose) can be used to increase BBB permeability without activation of inflammatory markers (i.e., nuclear factor κB signaling pathway genes), tissue damage, or MRI evidence of edema and hemorrhage. The probability of FUS-induced BBB permeability using a clinical imaging dose of Definity and Optison is similar across a range of pressure amplitudes, as the ones achieved in our study and established previously by McDannold et al. (37).
We monitored and controlled FUS-mediated BBB modulation in real time using an acoustic controller. The applied acoustic pressure was incrementally increased until subharmonic emissions (characteristic of stable cavitation) were detected, thereby increasing BBB permeability below the threshold for tissue damage (35). This method has been shown to improve sonication consistency and safety. A similar approach was used in the first clinical trial for BBB opening in AD using MRIgFUS with no adverse events after sonication (19). Immunohistochemical analysis in our study revealed no neuronal loss or erythrocyte extravasation in the basal forebrain 90 min after sonication (figs. S6 and S7). These findings are in accordance with other preclinical studies that demonstrate increased BBB permeability in the absence of edema, neuronal damage, and minimal to no erythrocyte extravasation using real-time feedback monitoring (34, 35, 38, 39).
Previous evidence suggests that local differences in vasculature and brain region can lead to variable effects of FUS-induced BBB permeability (40). Here, we show that similar pressure amplitudes were required to induce subharmonic emissions in the subregions of the basal forebrain (fig. S4), and acoustic pressures were comparable to those achieved following FUS-mediated BBB opening in the hippocampus of TgCRND8 mice and non-Tg littermates using an identical transducer/hydrophone configuration and feedback controller (38). We observed no differences in BBB opening between TgCRND8 and non-Tg mice (Fig. 2F) despite vascular dysfunction in TgCRND8 mice by 6 months of age (41). Future work is necessary to evaluate whether BBB recovery varies temporally in different basal forebrain regions and due to the presence of AD pathology.
In recent years, the potential use of MRIgFUS for AD has evolved beyond a method for drug delivery. In several mouse models of AD, FUS-mediated BBB opening reduced amyloid and tau pathology, even in the absence of drug delivery (26, 38, 39, 42, 43). Here, we combined FUS, as a delivery tool with disease-modulatory effects, with a pharmacological agent that is designed to enhance plasticity and resilience of BFCNs vulnerable to degeneration in AD. In this way, our therapeutic approach targets multiple pathologies implicated in AD and thus may prove more efficacious than a single target paradigm to slow or reverse disease progression. We found elevated levels of NGF in the brain parenchyma following MRIgFUS (Fig. 3, F and G). The source of increased NGF levels at sites of FUS-BBB modulation remains to be identified. Candidates include circulating NGF present in the bloodstream, cells capable of rapid NGF release residing on the parenchymal side of the BBB and known to be first responders [e.g., mast cells; reviewed in (44)], and microglia and astrocytes known to be activated by FUS (26, 28) that secrete NGF as early as 3 hours after stimulation in vitro (45). However, it remains to be tested whether glial cells can release sufficient amounts of NGF within 90 min in vivo, to account for the levels detected here.
In the adult mouse, basal NGF levels in the serum equivalent to 50 to 100 pg/ml may be physiologically meaningful in view of NGF binding affinity for the TrkA receptor [Kd (dissociation constant) of ~10−11 M] (3, 11, 46). Our results demonstrate that elevated levels of NGF are not sufficient to induce TrkA phosphorylation in non-Tg and TgCRND8 mice at 90 min following MRIgFUS alone. This result may be explained by NGF instability in solution and sensitivity to proteolysis and supported by the fact that NGF bioactivity is optimal at nanomolar concentrations (11). Nevertheless, this finding has important implications for the therapeutic action of D3 in MRIgFUS-treated areas. Because NGF and D3 can bind TrkA concomitantly, D3 can potentiate TrkA signals activated by low picomolar levels of NGF (11, 47). Therefore, MRIgFUS-induced increase in NGF and D3 may together further potentiate TrkA-dependent signaling cascades in the context of Aβ pathology.
An important part of preclinical development toward clinical translation will be dosing optimization for D3/FUS treatment. Previous work has shown that a single intraparenchymal injection of NGF in young adult and aged rats induces a rapid TrkA tyrosine phosphorylation response that persists for up to 5 days after injection (48, 49). Notably, our results demonstrate that BFCNs remain responsive to TrkA stimulation in the presence of amyloid pathology and impaired NGF-related signaling mechanisms. They further suggest that the biological response of a single D3/FUS treatment, at the level of TrkA receptor phosphorylation, is similar in healthy, adult cholinergic neurons and those burdened by amyloid pathology. For successful clinical application of D3, it will be essential to define the dosing interval needed to elicit TrkA phosphorylation leading to sustained cholinergic function and cognitive benefit. Prolonged TrkA signaling may be required to rescue degenerating BFCNs (i.e., for maintenance and remodeling of cell bodies, axons, and dendrites of BFCNs) and affect cognition. Preclinical studies demonstrated that administration of NGF and specific agonists of TrkA for at least 2 weeks in aged or lesioned animal models reversed BFCN atrophy and ameliorated behavioral impairments; however, the deficits reappeared after cessation of treatment (5, 12, 50, 51).
The first AD BBB modulation clinical trial recently revealed that single and repeated FUS treatments, monitored in real time using passive cavitation detection, are well tolerated in AD patients (19), supporting the feasibility of FUS delivery of D3 in AD patients. Subsequent larger clinical trials will help determine the potential therapeutic benefit of FUS-induced BBB permeability alone. An important question for future work will be to assess whether repeated administration of a TrkA agonist using FUS can act as a combination therapy to restore cholinergic function and reduce Aβ deposition and tau pathology in ways that promote cognitive function.
Evidence suggests that administration of NGF or TrkA ligands can, on one hand, improve performance in cognitively impaired rodents and, on the other hand, compromise intact function in healthy animals (12, 13, 51–53). This differential effect in diseased compared to healthy brains has to be taken into consideration when treatment strategies are planned. NGF modulation of cholinergic function is tightly regulated such that either excess or lack of the neurotrophin can be detrimental. Excessive and adverse neuronal plasticity as a result of TrkA activation is unlikely to occur in the presence of toxic Aβ species that are negatively influencing TrkA signaling continuously; however, this remains to be tested.
Pharmacological treatment of AD with D3 will require personalized strategies to address the disease process of each individual. Promising advances in neuroimaging may be eventually used to identify patients who might be most responsive to a therapeutic regimen incorporating agonists to TrkA. For instance, Trk-targeted radiotracers for positron emission tomography imaging may reveal spatiotemporal changes in TrkA receptor expression level—an early molecular alteration in mild cognitive impairment (MCI) and AD correlated with cognitive impairment (54). Alternatively, radioligands designed to quantify and map in vivo degeneration of cholinergic terminals in AD patients could be used for clinical identification, as well as monitoring the efficacy of TrkA-based interventions (55).
In conclusion, our study demonstrates that, in contrast to native NGF, only a selective TrkA agonist, without engaging p75NTR, can stimulate prosurvival and regenerative signaling in the context of Aβ pathology. The compound D3, and not NGF, activated TrkA-dependent signaling cascades in the TgCRND8 model of AD. Our results reveal a relative imbalance of TrkA/p75NTR activity in TgCRND8 mice, as observed in human AD, which may partly explain the failure of NGF reported here and, more broadly, why NGF therapy in AD clinical trials failed to display the efficacy observed in preclinical animal models without Aβ and tau pathology. As an alternative therapeutic strategy, we demonstrated that noninvasive MRIgFUS-mediated delivery of D3 localized to BFCNs stimulated TrkA signaling, and it was sufficient to rescue cholinergic function, even in the presence of Aβ pathology. Further investigation of the long-term effects of D3 delivery to the basal forebrain using MRIgFUS and after repeated treatments may reveal a promising therapy to promote cognition regulated by cholinergic neurotransmission in AD. Overall, our study highlights an alternative and complementary approach to amyloid- and tau-targeted therapies to counter neurodegeneration in AD.
MATERIALS AND METHODS
Our study aimed to (i) investigate how the NGF/TrkA signaling system is altered with disease progression in a mouse model of AD, (ii) compare the efficacy of native NGF and selective TrkA agonist D3 in a disease-relevant phenotype, and (iii) establish the feasibility of noninvasive and localized D3 delivery to the basal forebrain with MRIgFUS. Accordingly, we treated and analyzed four cohorts of TgCRND8 and non-Tg littermates. A first cohort of sex-balanced TgCRND8 and non-Tg mice at 4, 6, and 8 months of age (n = 8 per group) was used to characterize the genotype- and age-associated alterations in the NGF/TrkA signaling system using qRT-PCR, in situ hybridization, ELISA, Western blotting, and confocal microscopy.
In a second cohort, sex-balanced, 6-month-old TgCRND8 and non-Tg mice were randomly assigned to receive PBS, 2 μg of NGF, or 2 μg of D3 injected to the basal forebrain via intraparenchymal injection. Mice were sacrificed 90 min after injection (n = 4 per group). Using immunoprecipitation and Western blotting, we measured TrkA-dependent signaling pathways activated in the basal forebrain following PBS, NGF, and D3 administration.
A third cohort of sex-balanced, 6-month-old TgCRND8 and non-Tg littermates was tested. Mice were randomly assigned to treatment groups as follows: (i) D3/FUS-treated (500 μg, intravenous dose of D3 with MRIgFUS), (ii) PBS/FUS-treated (intravenous PBS with MRIgFUS), (iii) D3-treated (500 μg, intravenous dose of D3, no MRIgFUS), or (iv) PBS-treated (intravenous PBS, no MRIgFUS). Using histological methods, Western blotting, ELISA, and confocal microscopy, we evaluated the extent of BBB permeability in the basal forebrain 90 min following MRIgFUS (n = 6 PBS/FUS- and PBS-treated non-Tg and Tg mice). HPLC was used to measure the concentration of D3 delivered to the basal forebrain targeted with MRIgFUS, and mice were sacrificed 30 min after treatment to maximize therapeutic detection (n = 4 per group). Using immunoprecipitation and Western blotting, we evaluated the effect of D3/FUS treatment on TrkA signaling cascades 90 min after sonication (n = 6 per group). Treatment allocation was kept blinded until analysis. All animals were included in the analysis.
In the fourth cohort, sex-balanced, 6-month-old TgCRND8 and non-Tg mice were randomly assigned to either a D3/FUS-treated (500 μg, intravenous dose of D3 with MRIgFUS) or D3-treated (500 μg, intravenous dose of D3, no MRIgFUS) experimental group. Mice were sacrificed 3 days following treatment (n = 5 per group) for the assessment of ChAT activity in the basal forebrain, in addition to basal and depolarization-evoked ACh release in cholinergic terminal regions following D3 or D3/FUS treatment.
TgCRND8 mice and their age-matched non-Tg littermates were used in this study (20). All procedures were conducted in accordance with guidelines established by the Canadian Council on Animal Care and protocols approved by the Sunnybrook Research Institute Animal Care Committee.
Anesthesia was induced and maintained with isoflurane in oxygen. Mice were placed in a stereotaxic apparatus (David Kopf Instruments). The skull was exposed, and burr holes were made with a Stoelting drill to the following coordinates: MS/DBB: anteroposterior, +0.8 mm; mediolateral, +0.3 mm; dorsoventral, −4.4 mm (measured from bregma with a 4° vertical angle alignment); NBM: anteroposterior, −0.3 mm; mediolateral, +1.5 mm; dorsoventral, −5.6 mm (measured from bregma). A 33-gauge needle attached to a 5-μl Hamilton syringe containing the solution to be injected was lowered to the injection site, and 1-μl injection was made over 10 min. Following each injection, the needle was held in place at the injection site for 5 min and then removed from the brain.
MRI-guided focused ultrasound
Mice were anesthetized with 5% isoflurane and maintained at 2% isoflurane in medical air. Head fur was removed with depilatory cream, and a 26-gauge angiocatheter was inserted into the tail vein. The MRIgFUS experimental setup is illustrated in Fig. 2A. MRIgFUS was performed using the in-house build prototype system (similar to LP100, FUS Instruments Inc.), and animals were imaged using a 7.0-T MRI (Bruker). The acoustic beam was generated using a spherically focused transducer (1.68-MHz frequency, 75 mm diameter, 60 mm radius of curvature), with standard BBB disruption parameters (10 ms bursts, 1 Hz burst repetition frequency, 120-s duration). Axial T2w MR images were used to target three focal spots, covering the basal forebrain bilaterally.
At the time of sonication, mice received an intravenous injection of D3 (500 μg) diluted in PBS, or PBS only, Definity microbubbles (0.02 ml/kg; Lantheus Medical Imaging), and Gadovist (0.2 ml/kg, Schering AG) via the tail vein. For animals that received Evans blue, the dye (2% solution in PBS; 4 ml/kg; Sigma-Aldrich) was injected immediately after the sonication. Each injectable was administered individually, separated by a 150 μl saline flush to ensure transfer into the bloodstream.
Acoustic emissions were recorded using a polyvinylidene fluoride (PVDF) hydrophone coupled to an acoustic controller (34). The applied acoustic pressure was increased incrementally after each pulse until subharmonic emissions were detected, then reduced to 25%, and maintained for the remainder of the sonication. BBB permeability was visualized by an increase in Gadovist signal intensity on T1w MR images acquired 5 min after sonication. Contrast enhancement was assessed by measuring pixel intensity in a 2 mm × 2 mm target region normalized to a reference region of the brain. A custom image analysis program was used to calculate relative contrast enhancement (MATLAB, MathWorks).
Biodistribution and pharmacokinetic analysis of D3
D3 was quantified using HPLC versus standard curves as previously described (11). Mice were intracardially perfused with saline 30 min after treatment, and brains were removed and flash-frozen in liquid nitrogen. Tissue was homogenized in methanol using a mini-pestle fitted in a pellet pestle motor (Kontes) for 1 min at 4°C and centrifuged at 12,000 rpm for 15 min at 4°C for phase separation. The organic phase was collected and evaporated using nitrogen gas and resuspended in 40 μl of methanol for further analysis.
RNA isolation, reverse transcription, and qRT-PCR
Mice were intracardially perfused with ice-cold saline, and brains were removed, flash-frozen in liquid nitrogen, and stored at −80°C until analysis. Samples were homogenized (Minilys homogenizer, Bertin Technologies) in 1 ml of TRIzol (Invitrogen), and RNA was isolated with the PureLink RNA Mini Kit (Invitrogen). RNA concentration and purity were confirmed by spectrophotometry (NanoDrop Technologies). Synthesis of complementary DNA (cDNA) was performed using the SuperScript III First-Strand Synthesis SuperMix Kit (Invitrogen), followed by qRT-PCR using the ViiA 7 Real-Time PCR System (Applied Biosystems) with SYBR Green qPCR Master Mix (Applied Biosystems). Relative gene expression of NGF, TrkA, and p75NTR was normalized to the mean Ct value of three housekeeping genes (b-actin, GAPDH, and HPRT1), using the ΔΔCt method. All samples were run in triplicate. Primers used in this study are listed in table S1.
In situ hybridization
Mice were intracardially perfused with ice-cold saline, followed by 4% paraformaldehyde (PFA). Brains were isolated, postfixed in 4% PFA for 24 hours, and equilibrated in 30% sucrose (0.1 M PO4 buffer). Brains were sectioned axially at 40 μm and stored in cryoprotectant [50 mM phosphate buffer, 30% ethylene glycol, and 25% glycerin (pH 7.4)] at −20°C. Fluorescence in situ hybridization was performed using RNAscope (ACD Newark, CA, USA) according to the manufacturer’s instructions with slight modifications. Briefly, brain sections were washed in PBS, and endogenous peroxidase activity was quenched with 3% (v/v) H2O2 in PBS for 45 min at room temperature (RT). Antigen retrieval was performed at 95°C for 20 min using ACD antigen retrieval buffer. Sections were washed with PBS, mounted on Superfrost Plus slides (Fisher Scientific), and dried overnight at 37°C. Sections were then permeabilized with 0.3% Triton X-100 (Sigma-Aldrich) in PBS for 30 min at RT and then washed in double-distilled H2O. Probes for TrkA (ACD ref.: 435791-C1), ChAT (ACD ref.: 408731-C2), and p75NTR (ACD ref.: 494261-C3) were added in a 50:1:1 ratio. Sections were covered with parafilm and incubated overnight at 40°C in a humidified chamber (HybEZ oven, ACD). Subsequent washes were performed with 0.025% Tween 20 in 0.1× SSC (SSCT). Incubation times for additional steps were as follows, each succeeded by three washes in SSCT for 2 min at RT: Amp1-3 for 60 min at 40°C, horseradish peroxidase (HRP)–C1-C3 for 25 min at 40°C, TSA Plus fluorophore (TSA dyes, PerkinElmer) for 30 min at 40°C, and HRP blocker (ACD) for 20 min at 40°C; nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI; ACD).
Mice were intracardially perfused with ice-cold saline, and brains were removed, flash-frozen in liquid nitrogen, and stored at −80°C until analysis. Tissue lysates were prepared by homogenizing tissue in ice-cold lysis buffer containing 20 mM tris-HCl (pH 7.5), 137 mM NaCl, 2 mM EDTA, 4% NP-40 detergent, and protease and phosphatase inhibitors (Sigma-Aldrich), using the Minilys homogenizer (Bertin Technologies), and then centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was collected and used to measure total protein concentration with the BCA assay (Pierce Biotechnology).
Brain homogenates were resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to activated PVDF membranes. The membranes were blocked with 5% bovine serum albumin in tris-buffered saline/Tween 20 (TBST). The following primary antibodies were incubated overnight at 4°C: TrkA (1:1000; Cell Signaling, 2505), p75NTR (1:1000; Santa Cruz Biotechnology, sc-271708), pAkt (1:1000; Cell Signaling, 4060), Akt (1:1000; Cell Signaling, 9272), pCREB (1:1500; Cell Signaling, 9198), CREB (1:1500; Cell Signaling, 9104), pMAPK (1:2000; Cell Signaling, 9101), MAPK (1:2000; Cell Signaling, 9102), pJNK (1:1000; Cell Signaling, 9251), JNK (1:1000; Cell Signaling, 9252), βtubIII (1:5000; Covance, MMS-435P), and β-actin (1:5000; Sigma-Aldrich, A5316). Membranes were washed in TBST, incubated with corresponding IRDye secondary antibodies at 1:15,000 for 1 hour at RT (LI-COR Biosciences), and imaged with an Odyssey infrared imaging system (LI-COR Biosciences). Densitometry analysis was performed using ImageStudio software.
NGF levels were quantified using a commercially available ELISA kit from RayBiotech (ELM-bNGF-1) according to the manufacturer’s protocol. Samples (1:5 dilution in tissue lysis buffer) and controls were analyzed in triplicates.
Tissue lysates containing 500 μg of protein were left shaking overnight at 4°C with 5 μg of TrkA antibody (Santa Cruz Biotechnology, sc-118). Protein A agarose beads (50 μl; Santa Cruz Biotechnology, sc-2001) were added and left shaking at 4°C for 4 hours. The beads were washed with lysis buffer. SDS-PAGE and Western blotting were performed as described above. TrkA phosphorylation was measured by blotting for phosphotyrosine (1:1000; Upstate, 16-103) and total TrkA (1:1000; Cell Signaling, 2505).
Mice were intracardially perfused with saline, followed by 4% PFA. Brains were isolated, postfixed in 4% PFA for 24 hours, and equilibrated in 30% sucrose. Brains were sectioned axially at 40 μm and stored in cryoprotectant at −20°C. Sections were rinsed with 0.1 M PBS and then incubated for 1 hour at RT in 10% donkey serum and 0.3% Triton X-100 in PBS. Sections were incubated with mouse anti-IgM (1:400; Jackson ImmunoResearch, C839P24), mouse anti-IgG (1:400; Chemicon, AP192C), goat anti-ChAT (1:500; EMD Millipore, AB144P), tomato lectin (1:400; Vector Laboratories, DL1174), or Ter119 (1:400; BioLegend, 116207) for 72 hours at 4°C. Sections were incubated with fluorescently labeled secondary antibodies appropriate for the primary antibodies used (1:200; Jackson ImmunoResearch) for 2 hours at RT and then counterstained with DAPI. For TUNEL, we used the DeadEnd Fluorometric TUNEL System (Promega) as per the manufacturer’s instructions. For double immunostaining with cleaved caspase-3 and NeuN, TUNEL-stained sections were incubated in blocking solution (10% donkey serum, 0.3% Triton X-100 in PBS) for 2 hours at RT and then with anti-cleaved caspase-3 (1:300; Cell Signaling, 9661) and anti-NeuN (1:500; EMD Millipore, ABN90) for 24 hours at 4°C. Sections were rinsed with PBS and then incubated with fluorescent secondary antibodies (1:200; Jackson ImmunoResearch) for 24 hours at 4°C.
Immunofluorescence was detected by laser scanning confocal microscopy (Nikon A1) using 20× and 60× magnification objectives and visualized using NIS-Elements (Nikon). Z-stack images were acquired with an optical thickness of 1 or 0.2 μm using the 20× and 60× objectives, respectively. Maximum intensity projections were generated from Z-stacks.
[14C]ACh release and ChAT activity
Three days following D3/FUS or D3 (i.e., intravenous D3, no MRIgFUS) treatment, mice were deeply anesthetized, decapitated, and selected brain regions (i.e., MS/DBB, NBM, HF, and CTX) were dissected rapidly on ice. The MS/DBB and NBM were flash-frozen in liquid nitrogen and kept at −80°C until ChAT activity quantification. Tissue was homogenized in 5% (w/v) buffer containing 50 mM phosphate buffer, 10 mM EDTA, 1× protease inhibitor, and 5% Triton X-100 (pH 7.4). ChAT activity was measured by incorporation of [14C]acetyl–coenzyme A into [14C]ACh as described previously (56). ChAT activity is expressed as nanomoles of radioactive ACh formed per milligram of protein per hour. To measure [14C]ACh release, the freshly dissected CTX and HF were cut into 500-μm-thick traverse slices. The release of labeled ACh was determined following a preincubation with labeled choline as described previously (56). [14C]ACh release is expressed as a percentage of total radioactivity. The total radioactivity was measured using liquid scintillation counting (Beckman Coulter). The BCA assay was used to determine the protein content of the samples (Pierce Biotechnology).
Prism 7 software (GraphPad) was used for statistical analysis and graphical presentation. All values are presented as the means ± SEM. Differences between two groups were assessed using unpaired Student’s t tests. Three groups were compared using a one-way ANOVA. Correlation analyses were performed using linear regression analysis with 95% confidence intervals. All other analyses were performed using a two-way ANOVA (with or without repeated measures, as indicated). Multiple comparisons between groups were performed with Bonferroni’s post hoc analysis for significant ANOVAs. In all cases, P < 0.05 was considered statistically significant.
Acknowledgments: We thank P. Fraser, D. Westaway, and P. St George-Hyslop for supplying breeding pairs of TgCRND8 mice. We acknowledge K. Mikloska and S. Seerala for assistance with MRIgFUS; S. Rideout-Gros, V. Chan, and M. Theodore for animal care; and H.Y. Lin for graphics. S. Josephy offered insights on signaling pathways. P. Nagy provided helpful advice on cholinergic assays. Funding: This work was supported by the Canadian Institutes of Health Research (grants FRN 93603 to I.A. and FDRN 154272119312 to K.H.). Additional funding was received from the FDC Foundation, the WB Family Foundation, Gerald and Carla Connor, the Weston Brain Institute (TR130117 to I.A.), The National Institute of Biomedical Imaging and Bioengineering of the NIH (R01 EB003268 to K.H.), the Canadian Consortium on Neurodegeneration and Ageing (to H.U.S.), and the Canada Research Chair Program (to K.H.). The LI-COR Odyssey CLx was purchased with funds given by the James H. Cummings Foundation. K.X. was awarded a Frederick Banting and Charles Best Canada Graduate Scholarship (GSD 152271). Author contributions: K.X., H.U.S., K.H., and I.A. conceived and planned the experiments. K.X., K.M.-C., H.N., and S.H. performed the experiments. K.X. drafted the manuscript. All authors discussed the results and edited the manuscript. Competing interests: K.H. is a founder and a board member of FUS Instruments, a startup company manufacturing preclinical focused ultrasound devices. He receives research support from the company. K.H. and H.U.S. are inventors on patents related to this work (nos. EP1265605B1 and US6514221B2). The authors declare no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors. The drug-agents used, and related analogs or monoclonal antibodies, can be provided by H.U.S. pending scientific review and a completed material transfer agreement with the Lady Davis Institute. Requests for any agents should be submitted to. In accordance with the material transfer agreement between the University of Toronto and Sunnybrook Health Sciences Centre, TgCRND8 mice can be provided by P. Fraser pending scientific review and a completed material transfer agreement. Requests for TgCRND8 mice should be submitted to .


text:
House Reprimands, Penalizes Speaker
By John E. Yang
Washington Post Staff Writer
Wednesday, January 22 1997; Page A01
The House voted overwhelmingly yesterday to reprimand House Speaker Newt Gingrich (R-Ga.) and order him to pay an unprecedented $300,000 penalty, the first time in the House’s 208-year history it has disciplined a speaker for ethical wrongdoing.
The ethics case and its resolution leave Gingrich with little leeway for future personal controversies, House Republicans said. Exactly one month before yesterday’s vote, Gingrich admitted that he brought discredit to the House and broke its rules by failing to ensure that financing for two projects would not violate federal tax law and by giving the House ethics committee false information. [Read Full Article]

domain: politics


text:
Movie, music, TV and tech news as seen and heard from the "heart of it all."
The Columbus Dispatch - March 22, 2013 10:18 AM
Starring James Franco, Selena Gomez, Vanessa Hudgens and Ashley Benson, Spring Break is a trip through the twisted world of writer-director Harmony Korine. Korine's resume includes the not-so-mainstream hits Kids and Gummo.
The film follows college students on an adventurous spring break that includes booze, drugs, nudity and explosive violence. The college girls end up in legal trouble, and are bailed out by Franco, who plays a highly quotable gangster.
Critic Melissa Starker thinks that the only thing more entertaining than Spring Break might be watching the audience reaction to this movie.
"True to Korine’s divisive oeuvre to date, the film is bound to draw laughs, cheers, offended huffs and more than a little head scratching," Starker said in her review.
Viewers looking for more family friendly fare would probably be better served by The Croods, a 3-D animated feature about an overly protective Stone Age family.
The movie, starring Nicholas Cage, Emma Stone and Ryan Reynolds, follows prehistoric parents protecting their brood from their dangerous landscape.
The Croods is crafted well enough to make its point, but it doesn't fully develop all of the ideas and characters in the story.
"The results are erratic and light on laughs. Still, the movie has enough colorful, fast-moving imagery to keep youngsters, the targeted audience, entertained," Starker said in her review.
For many, grilling is still a new experience, and for them, I've pulled together three recipes that have run in the last year in the Dispatch Food section.
Veteran actor-director Geoffrey Nelson is back onstage, and Columbus
fortunately still has him.
Nelson, who co-founded CATCO and led its development over the years into central Ohio’s leading professional Equity theater company, has founded a new theater company with a distinctive niche.
A Portable Theatre, focused on touring small plays, will be launched in June with a limited run of a pilot production of The Duck Variations.
In this CBS News video, Oklahoma tornado survivor Barbara Garcia is explaining how she lost her
small dog in the storm when a news crew member notices the dog, still alive, trapped under rubble.
Watch their reunion:
More than 2,000 Ohioans were helped during the first three months of the year through the National Mortgage Settlement, according to Ohio Attorney General Mike DeWine.
The number brings the total helped to 9,593 since the nation’s five largest mortgage servicers reached the settlement with state attorneys general to address complaints about the way servicers processed foreclosures.


text:
The Public Eye
Release Date: October 14th, 1992
R|1 hr. 38 min.
Tireless 1940s tabloid photographer Leon "The Great Bernzini" Bernstein (Joe Pesci) is always first to the scene of the crime, thanks to his uncanny journalistic instincts and the police scanner in his car. Known to his friends as "Bernzy," Leon is famous for capturing the gory aftermath of violent crimes that snuff the lives of New York City's famous. Bernzy gets a job offer from gorgeous nightclub owner Kay (Barbara Hershey), but finds he's way in over his head when the Mob gets involved.
Director: Howard Franklin
Genres: Crime drama

domain: library