Sports for a causeNepal’s flag could fly high at Paralympics in a few years if it invests in disability sports
Recently, new the executive members were elected to the National Olympic Committee, ending the long-standing controversy of having parallel Olympic committees in the country. This, along with a new and strong leadership at the National Sport Council, could rejuvenate Nepal’s sports sector. Though the timeframe is too short to expect any major achievement in the upcoming Olympic Games in Rio de Janeiro next year, the country does have hopes for Tokyo 2020.
Unnoticed and invisible
Sports can be a unifying element in the national reconciliation project as citizens regardless of their caste and creed rally behind the national teams. Unfortunately, we tend to forget that sports can unite the nation in another different but equally powerful way: by cheering up and rallying behind the athletes living with disabilities who often remain neglected and invisible.
Yet you can find them out there on the basketball courts, on the cricket and football fields, training and sweating hard day by day. Athletes living with disabilities are able to defy the unequal structures of society. Deprived of the connections and often lacking the skills to land a job, and highly unequal and inaccessible urban and rural spaces, an increasing number of youths and young adults living with disabilities are taking up sports as way to empower themselves. Inclusive sports can help to create a new narrative for an inclusive and cohesive nation that does not discriminate and prevent anyone from succeeding in life. But even as these athletes are left to their own devices, they strive to improve their performances and achieve new goals.
If only more resources were dedicated to the development of disabled sports and more importance was given to building disabled friendly sports infrastructure. If only there was more awareness among the people to mainstream disabled sports in the country and to bridge the existing divide between sportsmen and sportswomen with and without disabilities. By giving sportspersons with disabilities the same level of recognition and importance bestowed on their colleagues without disabilities, the country could set an example for other nations on ways to include disability in the national agenda.
In the past two years, the Nepali national team has played some memorable football and cricket games in Kathmandu. And it was not only the people in Kathmandu but those from across the country who stood in the line for hours to get the tickets to those matches. The same could happen with disabled sports. There is much potential. For example, last year, the best among the wheelchair basketball athletes had the opportunity to play in a major sport competition in Bangladesh. But Nepal does not have a national wheelchair basketball team. If it did, the team could excel in wheelchair basketball not only in South Asia but the entire Asia-Pacific region. Oftentimes, we think that the only problem keeping Nepal from having a strong paralympics team is the lack of resources to do so. And when we talk about infrastructures, we end up discussing only about physical playgrounds that are certainly indispensible for trainings and practice. But it is not a sufficient condition to develop the sector.
We need to go beyond the ‘hardware’ component and think about how the entire ‘governance’ of disability sports movement is organised and how we can strengthen it or maybe even radically change it. We also need to talk about coaching, training standards and about challenges the differently-abled face while commuting to take part in trainings.
Nepal often prides itself on having a strong disability movement but within it, disabled sports remains unnoticed. It is a pity because in terms of visibility, inclusive sports could boost the entire disability movement. Athletes who chase a ball while on a wheelchair or bat on a cricket pitch despite being visually impaired could become the real ambassadors of a new and more inclusive nation. If we want a change and really develop disability sports in Nepal, the actors and other stakeholders involved in it, must forge a new consensus and work together with a renewed sense of unity.
For encouragement, Nepal could turn to China: it took at least 20 years for the country to emerge as a global sports player. It would certainly be unfair to compare Nepal to China. Even India has only just begun to invest in sports and it could take at least a decade to reap some results. Regardless, Nepal with all its limitations and challenges should play smart and set ambitious and realistic goals. And disability sports could do even better than so called ‘mainstream’ sports. Imagine the flag of Nepal flying high during the Tokyo Paralympic Games in 2020. Imagine if a strong paralympics team from Nepal would do well in Tokyo. Now fast-forward to 2024 Games with the Paralympic venue still undecided. Just imagine the happiness a paralympics games medal could bring to Nepal. A 10-year long framework could be the basis on which Nepal could launch a new disability sports movement. The corporate sector should also be actively involved in it.
Sometime back, Pete Hughes, one of the most successful wheelchair basketball coaches in the US told me, “Remember the most important thing about wheelchair sports is the effort in the chair. It does not matter if you can score a basket, but maneuvering that chair is the primary objective.” At the end of each day it brings a smile on the faces of the players and a sense of accomplishment. “This,” according to Hughes “is called empowerment”.
Therefore, the bid to promote disability sports in the country could be listed as one of the national pride projects. This should encourage us all to build a more inclusive country by leveraging the power of sports.
Galimberti is co-Founder of ENGAGE and Editor of Sharing4Good


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Product Review of The Elements in Action
The home screen is an attention-grabber that may actually seem a bit overwhelming to some users, but it’s simply a periodic table with tappable video clips for each element. Students tap an element and a video pops up, along with the element’s name, symbol, atomic number, and a brief description of the video. Kids can tap a speaker icon to hear the name of the element pronounced. It’s important to note that several direct links to social media and the app store are included, so be sure to monitor kids’ use of these features as needed.
This could be a great resource for kids’ research. Arrange kids into small groups and assign them three or four elements to explore. Have them use the app for initial research, and provide additional resources so they can learn more about the assigned elements. Have each group present its research to the class.
Not a conventional teaching tool, but an innovative way to get kids interested in science. Kids learn the names and locations of elements on the periodic table, their chemical symbols and atomic numbers, and fun, interesting facts about each element. Before using this reference tool, kids should have some prior experience with the periodic table and should understand the terms element and atomic number.
Overall User Consensus About the App
Watching videos that demonstrate unique properties of elements is fascinating.
Curriculum and Instruction
Primary appeal of this app is that it gets kids interested in science. Videos are both educational and fun, but additional facts about the elements could boost learning.
Navigation is easy — just tap an element and watch the video clip. Text below each video is small and may be hard for some kids to read.

domain: chemistry


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03.11.2019 | Case | Eastern Europe | Energy Efficiency | Street Lighting | Ukraine
The project envisaged implemenatation of energy saving measures in the public buildings of Pavlograd city, Ukraine.
Energy saving projects in Pavlograd
Pavlograd is an industrial city in Eastern Ukraine with a population of around 110 000 people located some 550 kilometres southeast of Kyiv. NEFCO has financed three large energy-efficiency projects in the city since 2012.
Within the first project energy saving measures were implemented in 9 day-care centres between 2012 to 2013. New energy-efficient windows were installed and heating systems were modernised.
The project has resulted in higher indoor temperature and better conditions for the benefit of children and teachers
The total project costs amounted to EUR 348,000. NEFCO contributed with EUR 260,000 loan from its Energy Saving Credits Facility and the city with own financing of EUR 88,000.
Cooperation continued in 2015, when a comprehensive thermal modernisation of school No. 5 was carried out. The refurbishment included insulation of the façade, replacement of windows, installation of automatic heat regulation as well as modernisation of the lighting system. All these measures have contributed to the large savings of the school’s yearly consumption.
The project was financed by a grant provided by the Nordic Energy Efficiency and Humanitarian Support Initiative for Ukraine (NIU). NIU was established in 2014 by all Nordic Governments and is administered by NEFCO.
Energy saving measures were implemented in six more schools and a health care centre in 2016 and the street lighting was modernised. A total of 1254 energy efficient windows have been installed in 7 buildings.
The new street lighting improved the safety conditions for the city’s inhabitants
Considerable energy savings were achieved with the modernisation of the street lighting system in Dniprovska street. The 12 km long street is the main road in Pavlograd. Within the project a part of the street, about 800-900 metres, was renovated and 142 street lighting fixtures were replaced with LED lamps. As a result, savings of electricity consumption comprised 80%.
The quality of the street lighting has also improved. As LED-lamps provide bright and comfortable light, the city has continued the modernisation using its own funds and replaced all the lights in the street. More than 7000 people have benefitted from the implemented measures.
Upon implementation of these three energy saving projects, the annual savings of the city amount to 2,600 Gcal of heat energy and about 200 MW of electricity consumption which corresponds to about EUR 230,000 annually.
Altogether, 18 projects have already been implemented with a total amount of EUR 16 million of NIU grant financing.

domain: economics


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The location of attractions is intended only as a guide. Distances are 'as the crow flies'.
With two bars, this welcoming pub offers food daily and real ales. There is a garden for summer months and children are very welcome. Parking is available.
The Wolsey Down
Not quite my idea of a suitable family dining pub, and certainly not welcoming
The oddly-named Strangles Beach is made up of two sand and shingle beaches that join together at low tide. Framed by dramatic cliffs reaching up to 200 meters tall, the beach has a fairly difficult access route with a 20-30 minute walk from the car park and a steep cliff path. That said, the beach below is well worth the descent and provides a striking spot to sit and escape the everyday. The beach is popular with naturists.
A gentle three and a half mile stroll from the church through valleys, woodland and fields. The walk is fully described on the iwalkcorwall.co.uk website and here you'll find lots of other places to go.
Located on the edge of Bodmin Moor, Altarnun lies in one of Cornwall’s prettiest valleys and is perfectly located for exploration of the north Cornish coast and Dartmoor. The wild beauty of Bodmin Moor is right on the doorstep, with plenty of excellent walking and panoramic views from the many rocky tors that fill the skyline. Classic granite architecture dominates the village itself, including the rectory which is featured in Daphne du Maurier’s novel ‘Jamaica Inn’, and a 15th century packhorse bridge spans the river that runs through the village.
A 16th century inn a couple of miles from Altarnun, The Rising Sun is a great single-room pub with slate floors and an old oak bar worn smooth by years of resting elbows. Known locally for excellent food served in the bar or restaurant, there is a great selection of real ales, mouth-watering food and a welcoming atmosphere.
Wonderful pub on the edge of Bodmin moor
We visited the a few times during our holiday and each time made me want to move the Rising Sun to my local village! Great welcoming people and atmosphere, fabulous food and decent ales. We will be back.
......a pub should be. Lovely owners, wonderful staff and very, very good food cooked to order and well worth the wait. Can’t wait to go back
Lovely old pub, open fire, slate floors and warm welcome. Lots of good food, locally sourced and cooked with style. Eat filling snacks in the bar or splash out on the full three courses in the restaurant. Also does an excellent Sunday lunch.
This is a really nice, tucked away pub. As well as the main atmospheric pub area, there is a newly refurbished restaurant area, which is more modern in design and nice and spacious. The food is lovely, reasonably priced and the service is friendly and good. Lots of nice fish dishes on the specials board!
The food is fantastic very good value for money.
The sanctuary is home to orphaned otters and also runs an otter breeding programme, don't miss feeding time at 12pm and 3pm. There are other animals to see including fallow deer, wallabies, muntjac, and a variety of bird of prey.
Well worth a visit
A lovely place to visit. The staff are so friendly and helpful, from the ladies on the reception desk and in the cafe to the gentleman who gave an interesting talk on his wild birds and the enthusiastic 'otter man' who could not hide his fascination of these wonderful delightful creatures. he obviously hold a great affection for them. There is so much more as well, deer, wallabies, meerkats, serval etc etc.
The Otter Sanctuary is a lovely day out for adults and children alike. We enjoyed watching the otters being fed and listening to the knowledgeable staff talk about them. We strolled throught the woodland, fed deer, patted the rabbit and held a kestrel. There are plenty of places to enjoy a picnic and also a lovely cafe.
Tamar Otter Park
A great place to visit with children. An enclosed wooded walk area where the deer and wallabies are free to roam. Watch the otters swimming about and being fed. A lovely place for a picnic or alternatively there is The Countryman Inn at the end of the road that serves great food.
One of the few sheltered harbours on the north coast, Boscastle became famous in 2004 due to the devastating floods that swept through the village. The community has recovered remarkably well, and Boscastle is once more a thriving resort and a lovely village to visit.
A very dog friendly village, a wide variety of shops. Good range of eating places.
Staying in Boscastle
Boscastle was a wonderful place to stay for our holiday. Good local shops, like the post office and Spar for provisions. We tried several pubs. The Napoleon, the Wellington (for Sunday lunch = yum) and the Old Manor house. I would recommend the Boscastle made ice-cream from a shop half way down the harbour walk on the left hand side as you walk towards the sea.
We thoroughly enjoyed sitting for ages, watching the blowhole in the harbour. It 'blow's about 1-1.5 hrs either side of low tide, and is amazing to watch, and listen to aswell.
There is a really enjoyable walk from the main car-park along the Valency river, which is flat, unless you decide to go up to Minster church, and that path is very steep.
We didn't rate the Tourist shops very highly, because everything was so expensive.
Delightful place; harbour and old village up the hill. Fine coast walks to either Tintagel or Crackington Haven with options of bus connections. Also local valley/wood walks. Recommend Cobwebs pub at the harbour and The Napoleon up in the village. If self catering, the farm shop about half mile out of village towards Crackington Haven on B3263, is worth a visit.
Wonderful old harbour village - beautiful scenery can be seen from the headland which can be reached by two different paths, one on the right by the Harbour Light and one on the left. A tiny cove is also accessible where the blowhole can also be seen. Some lovely places to eat in Boscastle. We ate a lovely meal at the Cobweb Inn and also at the Riverside. We had cream teas and breakfast at the Harbour Light and Bridge Guest House, but the Riverside provided the best breakfast EVER. Some lovely shops such as the Mill and Things as well as an art gallery, fudge and Rock shop and 2 other fab gift shops. We love Boscastle and have been there many times. The Museum of Witchcraft also interesting. Visitor centre provides maps and info and also information about the flood and general history. Tintagel is 5 mins away and Crackington Haven about 10-15 mins drive as is Port Isaac. Padstow about 40 mins.
This 15th Century village pub was once home to the masons and craftsmen who constructed the nearby church of St James the Great. Today, you will find a welcoming bar and restaurant, with blazing fire in the winter, and a large beer garden for those balmy Cornish summer evenings. Look out for the resident ghost.
St Kew Inn
Excellent food and friendly service to our party of six. Would recommend fish meal of the day.
excellent food very friendly staff
Very good food
Lovely food. Service was a little slow at lunchtime but they were aware of this, apologised and tried to resolve.
So good we visited twice
Superb food, varied menu, good service. We were a group with 4 vegans and 2 carnivores and we all thought this Inn was top notch.
A must visit
Fantastic pub, great menu for veggies, beautiful garden and loads of space
The St Kew Inn does excellent food. It is not cheap but very good value for money. My wife said that the hake she had one evening was the best fish she had tasted for many years - cooked to perfection. You need to book early to get a table in the restaurant, but can also take pot luck in the bar where the tables are not reserved. The staff are very friendly and helpful.
Best pub in the arae
Excellent pub food. Best in the area. Good real ale and great pub food consistent performance and very popular so need to book in season.
idyllic country pub
A warm welcome and helpful staff. The food was great - the haddock kedgeree was worth the trip alone.
Great country pub
Great atmosphere and food. Nice seating inside and outdoor summer barbeque.
Beautiful and Atmospheric
Picturesque 15th Century village pub serving beer from wooden barrels and excellent homecooked food prepared to order by hosts Paul Ripley and Sarah Allen. One of the most beautiful and atmospheric pubs in Cornwall.
- Mr and Mrs Whitten
Traditional farmhouse food, homecooked on the Aga. Good vegetarian and gluten-free options are usually available here.
A great little find
We stopped here for afternoon tea and cake. The have a lovely selection of fresh food, cake and ice cream. There is a lovely terrace and a little garden with views over the valley. A great place for a quick bite to eat.
Perfect for lunch
My favourite place for lunch. All local and organic, with their own home grown vegetables and salads. Beautiful patchwork on display with a shop where you can buy materials and gifts. Book for one of Jo's patchwork courses if you're here when one is on.
Articles | From around the area
Places to Go
Places to Go
Places to Go

domain: travel


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Moc Chau Travel Guide
Little more than a road with a bunch of houses and shops on each side, it's all about the tea, here in Moc Chau, which thrives on the mountainous hillsides, and has put the town well and truly on the map — for tea fans at least.
The other reason it's on the map is that it's a possible place to break up a trip from Hanoi to Son La or Dien Bien Phu, and there are certainly worse places to be stuck for the night. Not that there's anything of interest here, but there are a couple of decent guesthouses, a restaurant or two and some very friendly locals.
If you have an honest reason to head here specifically, take a through bus to Dien Bien or Son La from My Dinh or Luong Yen bus stations in Hanoi and jump off in town.
Cyclists in particular should take comfort in the knowledge there is accommodation available.
There's a BIDV and an Agribank in town, along the main road, both of which have ATMs, and Western Union is available at BIDV.
Agribank: Tieu Khu 7 (Highway 6), Moc Chau. T: (022) 386 6236, F: (022) 386 6133. Hours: 07:30 to 11:30 and 13:30 to 15:30.
BIDV Bank: Tieu Khu 4 (Highway 6), Moc Chau. T: (022) 386 6591, 117, F:
Post Office: Tieu Khu 4 (Highway 6), Moc Chau. T: (022) 386 7667, F: (022) 386 7000. Hours: 07:00 to 20:00.
How to Get there :
There's a small bus station in Moc Chau but the best bet if you are heading towards Hanoi or Dien Bien Phu is to wait on the main road outside town and pick up a through bus.
Moc Chau is on the main through-route so transport will be passing through on a pretty regular basis during daylight hours.
If all else fails, stand around looking lost for a while and someone hanging around the bus station will point you in the right direction.

domain: geography


text:
Scientists will be probing the waters of Loch Ness in coming weeks in a bid to solve the mystery of what lies beneath the surface.
Experts from all over the world will form the Super Natural History team, carrying out DNA sampling of the waters.
Members will be drawing up a list of all life in the loch and making comparisons with other sites.
The study is being led by Professor Neil Gemmell, who said: "Large fish like catfish and sturgeons have been suggested as possible explanations for the monster myth and we can very much test that idea and others.
"While the prospect of looking for evidence of the Loch Ness monster is the hook to this project, there is an extraordinary amount of new knowledge that we will gain from the work about organisms that inhabit Loch Ness - the UK's largest freshwater body."
Prof Gemmell, of the University of Otago, New Zealand, said he would be surprised if there's any evidence of DNA from a large extinct reptile - the so called "Jurassic hypothesis".
Professor Gemmell has assembled a team of global science leaders from the UK, Denmark, US, Australia and France, as well as Adrian Shine of the Loch Ness Project.
The team is carrying out its investigation at the loch this month.
VisitScotland said the Nessie phenomenon is worth millions to the Scottish economy, with hundreds of thousands of visitors travelling to Loch Ness and Drumnadrochit every year to catch a glimpse of the mythical monster.
Chris Taylor, VisitScotland regional leaderships director, said: "The mystery and the intrigue of the Loch Ness monster attracts visitors from all over the world to the stunning area every year.
"It is exciting to welcome Professor Gemmell and his team to the loch and we will be eagerly anticipating their findings.
"While the research could provide an insight into one of the world's most famous enigmas, it will certainly offer a closer look at all of the different creatures living in the loch."
Unusual items previously found in the loch include a 30ft long Loch Ness monster model discovered on the loch bed in 2016 during a sonar search by Kongsberg Maritime and supported by The Loch Ness Project and VisitScotland.
The model was a prop from the 1970 film The Private Life of Sherlock Holmes, directed by Billy Wilder and starring Robert Stephens and Christopher Lee. It is believed the model sank after its buoyant humps were removed.

domain: news


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The Police Force has formed a Rugby team as it seeks to find ways to provide recreation for its ranks. Force training officer Senior Superintendent Paul Williams said he has many talented athletes in the force who will make the team competitive.
Superintendent Williams said his goal is to have a team in each division
The force will be getting assistance from the Guyana Rugby football union
East Ruimveldt will battle Tutorial High in a female matchup at 11this Saturday morning and Tutorial High tackles Campbellville Sec. in male action at 9:30 Sunday in the GSDF scotia/pepsi Schools football tournament.
Both games will be played at the Ministry of Education Complex on Carifesta Avenue. At the launching chairman of the GSDF Victor wright said that the round robin format will benefit the players who will be in the 10-15 yr age range.
The Coaches association will also be involved and according to Sampson Gilbert the players and coaches will benefit from their technical expertise
DDL and Scotia bank are again fully behind the 4 month long tournament and they both expressed satisfaction with how the tournament has been evolving
And in cricket Merissa Aguillera will once again lead the WI women in the ICC World 20 20 cup due for Bangladesh starting this weekend. The Squad includeds last year’s player of the Tournament Stefanie Taylor as well as Anisa Mohammed who was one of the highest wickettakers. Guyanese pair Shemain Campbelle and tremayne Smartt are part of the squad. On Tuesday the wi take on SA in a warm up match before opening their Campaign on March 24 against England.
And this Year’s Caribbean Premier League 20/20 tournament will have its semi-finals and Finals in St Kitts and Nevis. Over 120 overseas players have signed up for this year’s tournament a number the organisers say is five times more than those signed for last year.Australians lead the way with 30 while Englishmen will make their debuts if selected. The full list is to be released on Monday. Jamaica’s Tallawahs defeated Guyana’s Amazon warriors to lift the inaugural cup.

domain: sports


text:
A Mother's Love
Modern Marble Relief Sculpture of a Mom...
Hand Crafted Marble Chinese Checker Game...
Mexican Marble Chess Set
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Hand Carved Marble Chess Set
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Handcrafted Mexican Marble Chess Set Game
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Circle of Life
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Marble jewelry box
Inca Water Jar
Andean Inca Style Copper on Bronze Paperweight Sculpture
Buddha's Calm Blessing
Buddha Handcrafted White Marble...
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Andean Llama Sculpture Golden Bronze Paperweight
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Rose on Black
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Marble mosaic cross
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Hand Painted Makrana Marble Coasters...
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Indian Buddhist White Marble Sculpture
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Indian Jewelry Box Inlaid with Marble
The Colors of Holi
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White Agra Lily
Six White Indian Marble Carved Coasters...
Handcrafted Marble Inlay Jewelry Box

domain: design


text:
While China played its part in the coup against President Trump, they succeeded in something far greater. They took down a legitimate government, and replaced that government with a puppet.
Thus, it comes as no surprise to anybody that Biden screwed the pooch in Afghanistan. Even Democrats have come to realize that only a brain-damaged moron would implement an exit strategy like that of Biden’s. And the media isn’t buying the, “It’s Trump’s fault!” trope.
Biden is being hung out to dry.
Jake Tapper and Jim Acosta torched the Biden “administration” for its handling of the Afghanistan pullout. Leave it to Trump to showcase Leftist buffoonery, even if only from the outside looking in.
Trump proved to NOT be a warmonger, as he said he wanted our troops out. He would have gotten the Taliban and Afghan government to sit down together and formulate a deal, had he not been taken out. Instead, as one pundit assessed the situation in Kabul, Biden did a “handover” of Afghanistan to the Taliban.
Fox News’ Chris Wallace affirmed that the Taliban will take over Afghanistan. All they needed to do was wait for a feckless Leftist to take the helm, then strike. And that’s what they did.
The Taliban takeover of Afghanistan confirmed what most Americans know intimately. Leftism sucks, and it selected one of its weakest members as its leader.
Now the Taliban is a legitimate government. And American’s new overseers confirmed that.
Zero Hedge wrote:
Late last month the world beheld the unusual footage of Taliban commanders being warmly received by China’s foreign minister Wang Yi in the Chinese city of Tianjin. That trip included the Islamic terror group’s co-founder Mullah Abdul Ghani Baradar, in a rare visit widely seen as an attempt of the jihadists to gain “legitimacy” abroad.
It appears to have paid off, given that now at a moment the Taliban is scoring victory and victory on the ground they are eyeing the sought after price of Kabul. And now US News and World Report writes that “China is prepared to recognize the Taliban as the legitimate ruler of Afghanistan if it succeeds in toppling the Western-backed government in Kabul, U.S. News has learned, a prospect that undercuts the Biden administration’s remaining source of leverage over the insurgent network as it continues its startling campaign to regain control.”
You can’t get any more legit on the world stage than China these days. And China’s top tier legitimacy happened due to the ousting of Trump.
Understand that the Chinese would never have made such a move with Trump in office. However, with Trump gone the Chinese now set the course for how the rest of the world behaves. Biden can ride out what remains of his term in his basement.


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Fort Mill EMT charged with sexually assaulting a minor in ambulance
The girl was being transported from South Carolina when the alleged assault happened.
FORT MILL, S.C. (WBTV) - An EMT is facing charges after he was accused of sexually assaulting a minor in an ambulance.
Charlotte-Mecklenburg police said the alleged assault happened while the ambulance was in Mecklenburg County in January.
Mecklenburg County Sheriff’s Office records show that the man, identified as Akingbiwaju Opadele, was arrested March 7. He was charged with felony sexual contact under pretext of medical treatment and given a $15,000 bond.
Jail records show he has since bonded out about 37 minutes later.
“What message are we sending to the most vulnerable in our society?” CMPD Chief Johnny Jennings tweeted out following a press conference with the department.
Watch: CMPD press conference regarding the alleged assault
The girl was being transported from South Carolina when the alleged assault happened and told hospital staff about the assault.
Fort Mill EMS provided a statement on the incident on Thursday afternoon.
The statement reads:
“Fort Mill EMS principally wants to emphasize that we prioritize patient safety. We prioritized patient safety before this incident, we are handling the incident in a way that ensures we will continue to prioritize patient safety, and we will always prioritize patient safety.
Fort Mill EMS is a non-profit provider of emergency services and transport to local medical facilities. It is subject to strict confidentiality obligations regarding privacy of the patients and its employees. As such, we are limited in what we can disclose at this time.
Throughout the investigation, Fort Mill EMS has respected the rights and obligations of the accusing patient and the authorities, all patients and the community, and its employee. Most importantly, we have continued to prioritize the safety of the patients we care for.
With respect to the ongoing safety and wellbeing of all patients and the community: At the time of the incident, Fort Mill EMS immediately placed the employee on administrative leave pending the outcome of the criminal investigation and prosecution. We have always maintained strict procedures for screening and monitoring employees. We provide a vital and scarce service to the community. We handled 4,526 emergency dispatches in 2022 and have provided a high level of service for decades. In all that time, this is the first instance of any criminal accusation against an employee related to their service.
With respect to the patient and the investigation: Fort Mill EMS has cooperated with the authorities, including CMPD and the South Carolina DHEC Bureau of EMS, and will continue to do so. This has included voluntarily and promptly submitting to requests for interviews and information. In addition, we are in the process of conducting our own thorough internal investigation.
With respect to the employee: Fort Mill EMS lacks information to take a position on his guilt or innocence. Under our justice system, the accused are innocent until proven guilty in a court of law. While that process is running its course, the employee is not at work. Once his guilt or innocence is determined and our own internal investigation has concluded, we will act accordingly. At all times, however, we will continue to put our patients’ wellbeing first, in keeping with our seven decades of excellent and unblemished service to the community.”
This is a developing story and more information will be provided as it becomes available.
(NOTE: Bold added for emphasis)
Anyone with information is asked to call Charlotte-Mecklenburg CrimeStoppers at 704-334-1600.
Download the free WBTV News app for the latest information as it comes in.
Copyright 2023 WBTV. All rights reserved.

domain: law


By Jun Yang and Mariko Yasu, Bloomberg |
August 30, 2012
After beating household names in televisions, memory chips and mobile phones in the past decade, South Korea’s biggest company is targeting rivals including Canon Inc. in the $32-billion-a-year market for copiers.
Weather continues to drive grains
Chinese demand low supply boost crude
S&P 500 trading plan for tomorrow
Protection from market spikes
How low can corn go?
Natural gas at an inflection point?


text:
WIN55,212-2-Induced Expression of Mir-29b1 Favours the Suppression of Osteosarcoma Cell Migration in a SPARC-Independent Manner
Department of Biomedicine, Neurosciences and Advanced Diagnostics (BIND), University of Palermo, 90127 Palermo, Italy
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, 90128 Palermo, Italy
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2019, 20(20), 5235; https://doi.org/10.3390/ijms20205235
Received: 4 September 2019 / Revised: 9 October 2019 / Accepted: 18 October 2019 / Published: 22 October 2019
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
WIN55,212-2 (WIN) is a synthetic agonist of cannabinoid receptors that displays promising antitumour properties. The aim of this study is to demonstrate that WIN is able to block the migratory ability of osteosarcoma cells and characterize the mechanisms involved. Using wound healing assay and zymography, we showed that WIN affects cell migration and reduces the activity of the metalloproteases MMP2 and MMP9. This effect seemed to be independent of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein involved in tissue remodeling and extracellular matrix deposition. SPARC release was indeed prevented by WIN, and SPARC silencing by RNA interference did not influence the effect of the cannabinoid on cell migration. WIN also increased the release of extracellular vesicles and dramatically upregulated miR-29b1, a key miRNA that modulates cell proliferation and migration. Interestingly, reduced cell migration was observed in stably miR-29b1-transfected cells, similarly to WIN-treated cells. Finally, we show the absence of SPARC in the extracellular vesicles released by osteosarcoma cells and no changes in SPARC level in miR-29b1 overexpressing cells. Overall, these findings suggest that WIN markedly affects cell migration, dependently on miR-29b1 and independently of SPARC, and can thus be considered as a potential innovative therapeutic agent in the treatment of osteosarcoma.
Osteosarcoma (OS) is a primary malignant bone tumour that most often affects males with two incidence peaks, one of which is between 10 and 40 years of age and the other in the elderly [1,2]. In particular, the marked osteoblastic and osteogenic activity of adolescent age seems to be a predisposing factor for the onset of the neoplasia . The first indication of the use of chemotherapy in patients with OS dates back to 30 years ago, and there is currently a full consensus in considering the combination of surgery and chemotherapy as a standard procedure in treating high-grade OS. However, although the tumour can respond to chemotherapy treatment, in patients with metastatic disease, the prognosis remains adverse . Thus, a better understanding of osteosarcoma biology represents an important challenge for researchers to optimize treatment strategies and develop new therapeutic agents, thus improving the prognosis.
Cannabinoids (CBs), the active constituents of Cannabis sativa, are known to exert a wide range of neuronal central and peripheral effects. Recently, a role of cannabinoids in the regulation of cell death and survival has largely emerged [5,6,7]. In particular, numerous studies have explored the anti-proliferative effects of these compounds in various tumours . Following the interaction with their specific receptors, cannabinoids can trigger several different signalling pathways , including the accumulation of ceramide, the activation of c-Jun N-terminal kinase (JNK) and p38 MAPK, as well as the increase in calcium concentration, reactive oxygen species (ROS) production and the modulation of pro- and anti-apoptotic members of the Bcl-2 family [10,11,12,13]. More recently, a relationship between the cannabinoid system and miRNA expression has been evidenced. MiR-Let-7d has been demonstrated to be a target of cannabinoid receptors , and the anticancer activity of WIN was related to the miR-27a-mediated repression of specificity protein (Sp) transcription factor in colon cancer cells .
In recent years, oncology research has investigated specific aspects of the cellular processes involved in cell death caused by synthetic cannabinoid derivatives with anticancer activity. Our previous studies have shown the in vitro effects of the synthetic cannabinoid WIN55,212-2 on different cancer cell lines [16,17,18]. In particular, we showed that treatment with increasing doses of WIN induces a significant reduction of the proliferative ability of MG63 osteosarcoma cells and sensitizes them to apoptosis induced by TRAIL, a cytokine with selective anticancer activity . The analysis of biochemical pathways evidenced an important role played by the WIN-dependent increase in the control of the intracellular level of SPARC (secreted protein acidic and rich in cysteine), a multi-faceted glycoprotein which is involved in a number of cellular processes [20,21].
In addition to pro-apoptotic and anti-proliferative roles, other studies reported that synthetic cannabinoids can also reduce the migration, angiogenesis, invasion and metastasis of cancer cells by modulating the levels of proteins involved in these processes . On the other hand, it is well known that SPARC participates in the regulation of cell adhesion, migration, and tissue remodelling . In particular, it has been shown that low expression levels of matricellular SPARC can modulate cell migration in different types of cancer cells, and these observations led researchers to hypothesize a specific role of SPARC in the inhibition of tumour progression and invasiveness. In contrast, many other studies demonstrated an oncogenic function of SPARC, thereby highlighting the divergent roles of SPARC in human carcinogenesis .
Other interesting players in the regulation of cell migration in different types of cancer cells are the members of the miR-29 family, including products from two gene loci: miR-29a/b1, located on chromosome 7 (7q 32.2); and miR-29b2/c, located on chromosome 1 (1q 32.2) . It has been demonstrated that the forced overexpression of miR-29 is able to inhibit cell migration and proliferation and promote the apoptosis of tumour cells, while its reduced levels are a frequent occurrence in osteosarcoma tissues [26,27]. However, although it is known that miR-29 is a regulator of SPARC expression , the exact correlation between these two players in cannabinoid action is a subject of investigation.
The aim of the present study was to evaluate the effects of the cannabinoid WIN in osteosarcoma MG63 cell migration and the possible involvement of SPARC and miR-29b1 in this event. Collectively, our results show for the first time that WIN is able to inhibit osteosarcoma cell migration in a SPARC-independent manner. Moreover, a crucial role seems to be played by the WIN-mediated induction of miR-29b1. Therefore, the cannabinoid has the potential to be an efficient anti-cancer drug in new therapeutic strategies for osteosarcoma.
2.1. WIN Treatment Reduces the Migratory Ability of MG63 Cells and Affects MMP Activity
In a previous paper, we demonstrated the ability of WIN—a synthetic agonist of cannabinoid receptors—to induce a significant reduction of osteosarcoma cell proliferation, and we characterized the biochemical mechanisms involved . Here, we aim to evaluate whether WIN can also induce a modification in the migratory ability of osteosarcoma MG63 cells. To this end, a monolayer of cells was scraped longitudinally, and the rate of the re-population of the area between the wound edges after the lesion was evaluated. Untreated MG63 cells showed a rapid healing process of the lesion, reaching almost full monolayer repair at 48 h (T48) after scratching (not shown). As shown in Figure 1A, 5 µM WIN markedly prevented cell migration, as indicated by the entity of the wound that was almost identical to T0 at all time points observed. In contrast, untreated MG63 cells migrated in the gap, reaching about 75% of migratory activity after 36 h (T36).
In other experiments, before WIN treatment, we added a conditioned medium from a highly confluent untreated cell culture to the cultured cells. As shown in Figure 1B, the presence of conditioned medium accelerated the wound closure of untreated cell cultures, almost achieving total confluence at T36, and partially prevented the anti-migratory effect induced by WIN. These data suggest that the cells produce factors to sustain cell migration and that WIN treatment blocks their extracellular release or reduces their action.
Next, we analysed the levels and the activity of metalloproteinases (MMPs), a family of enzymes which are responsible for normal tissue remodelling and angiogenesis. Gelatin zymography assay showed that 5 µM WIN significantly reduced extracellular MMP9 and MMP2 gelatinolytic activity after 36 h of treatment, thus indicating that WIN inhibits metalloproteinase activity (Figure 2A). In addition, Western blot analysis revealed that WIN was capable of inducing a dramatic decrease in the level of intracellular MMP9 and a slight decrease in the level of MMP2 (Figure 2B).
2.2. WIN Treatment Prevents SPARC Release in the Extracellular Environment and Increases the Release of Extracellular Vesicles
Among the numerous players implicated in regulating tumour cell migration and invasiveness, we specifically focused on SPARC protein and miR-29b1. The matricellular factor SPARC plays different roles in extracellular processes, and its function is strictly related to the cancer model and/or the metastatic grade of the tumour . Since we previously demonstrated that, in osteosarcoma cells, the cytotoxic effect of WIN was accompanied by an increase in the level of SPARC , here, we aim to analyse a possible role of this factor in the anti-migratory effect of WIN in MG63 cells. To this end, we first analysed the level of SPARC in conditioned media collected from untreated or WIN-treated cells after dialysis and lyophilization. Although we have previously demonstrated that intracellular levels of SPARC increase under treatment with WIN , Western blotting analysis evidenced that the level of released SPARC was much more abundant in the medium from untreated cells compared to that obtained from WIN-treated cells (Figure 3A).
In light of this observation, we wondered whether SPARC release was associated with extracellular vesicles (EVs). Therefore, we isolated EVs from the culture supernatants of untreated or WIN-treated MG63 cells by ultracentrifugation and quantified EVs obtained by both cytofluorimetry and acetylcholine esterase activity measurement (AChEase). As shown in Figure 3B, WIN induced a considerable increase in the amount of released EVs, which resulted to be about seven-fold higher than EVs isolated from untreated cell media. This result was confirmed by the consistent increase in AChEase activity (Figure 3C).
Subsequently, we evaluated SPARC levels in EVs by Western blotting analysis. The results showed a not significant level of SPARC in the vesicles, although the protein was present in the relative supernatants from untreated cell cultures (Figure 3D,E).
Therefore, considering that SPARC is poorly represented in EVs, we evaluated whether this protein can be secreted by MG63 cells via the canonic secretory pathway. For this purpose, we used thapsigargin and BAPTA-AM, two specific inhibitors of this pathway. As shown in Figure 4, the level of SPARC was significantly reduced in the medium from thapsigargin or BAPTA-AM-treated cells. Accordingly, in the presence of secretory pathway inhibitors, the intracellular levels of SPARC were higher than in control cells (Figure 4). Thus, we concluded that WIN could restrain SPARC inside the cell by inhibiting the secretory pathway and that the effect of WIN on cell migration is independent of the presence of SPARC in the extracellular compartment.
2.3. Silencing SPARC did not Affect the Anti-Migratory Effect of WIN
To validate the hypothesis that SPARC is not involved in the WIN-mediated anti-migratory effect, we silenced SPARC expression by RNA interference. Figure 5A shows that SPARC was downregulated in silenced cells at both the intracellular and extracellular level in the presence or absence of WIN. The analysis of cell migration in SPARC-silenced cells was then evaluated. The results reported in Figure 5B show that—also in this case—WIN almost completely prevented the closure of the wound after 36 h in both control (siScr-transfected) and siSPARC-transfected cells. In the absence of the cannabinoid, transfected cells closed the gap with a migratory activity of about 80% regardless of the level of SPARC.
Considering the substantial increase in the amount of released EVs after WIN treatment, we evaluated whether EVs play a role in the control of cell migration. For this purpose, MG63 cells were incubated in the presence of the EVs isolated from conditioned medium of WIN-treated cell cultures. As shown in Figure 6, EVs isolated from WIN-treated cells halted the wound closure similarly to that observed after WIN treatment, whereas EVs isolated from untreated cultures did not induce any modification in cell migration (not shown).
2.4. WIN Induces the Upregulation of miR-29b1 which Plays a Role in Cell Migration
To explore the mechanism by which WIN prevents cell migration, we focused on miR-29b1, which is considered a mediator of the migratory ability of cancer cells [28,30]. Real-time PCR experiments showed that WIN induces an upregulation of this miRNA of about 700-fold compared with untreated MG63 cells (Figure 7A). To investigate the role of this miRNA on cell migration, we transfected MG63 cells with a vector carrying miR-29b1 and selected a stable line overexpressing this miRNA (Figure 7B). Then, we analysed the motility of these cells by means of wound healing assay. Similar to WIN-treated cells, after 36 h, only a small percentage of transfected cells migrated into the wound, whereas mock transfected cells showed a migratory activity of about 80% with respect to that observed in untreated cells (Figure 7C). Moreover, miR-29b1 transfected cells showed a reduced extracellular activity of matrix metalloprotease 9 and 2 (MMP9 and MMP2) (Figure 7D), mimicking the WIN effect. In addition, also the intracellular levels of both MMPs were reduced in miRNA transfected cells (not shown).
Since miR-29b1 is considered a putative post-transcriptional regulator of SPARC expression , we evaluated the level of SPARC in miR-29b1 overexpressing cells. As shown in Figure 7E, the overexpression of miR-29b1 did not cause any change in the level of SPARC, so we concluded that, in our experimental model, SPARC is not a direct target of miR-29b1, as predicted by in silico analysis.
Cell migration represents an essential step during tumour progression, and therefore inhibitors of this process can be considered as potential antimetastatic drugs. On the basis of our previous results on apoptosis induction by WIN55,212-2 in osteosarcoma cells [19,20], in the current study, we show the ability of this compound to block osteosarcoma cell migration and we characterize the mechanism involved. Specifically, we demonstrate here that WIN markedly reduced the migratory ability of osteosarcoma MG63 cells, and this effect was accompanied by a dramatic reduction in the extracellular activity and intracellular levels of MMP2 and MMP9 metalloproteases. Although conflicting data are present in the literature regarding the expression levels of metalloproteases in osteosarcoma cells [31,32], we found that, in our serum-free conditions, both the intracellular levels and extracellular activity of MMP9 were higher than those of MMP2. Fragmentary data in the literature demonstrate that WIN can inhibit the epithelial mesenchymal transition and migration in different cancer cell models [33,34] In an attempt to clarify the mechanism of the anti-migratory effect of WIN in osteosarcoma cells, our study specifically focuses on a possible involvement of the matricellular protein SPARC and a hypothetical role exerted by miR-29b1. SPARC is often considered as an ambiguous gene product because it plays opposite roles in extracellular matrix remodelling and carcinogenesis . Despite the fact that we have previously demonstrated a crucial role exerted by intracellular SPARC in WIN-induced apoptosis in osteosarcoma cells, in this paper, we demonstrate that SPARC is not involved in the anti-migratory effect of WIN. Indeed, although WIN upregulated intracellular SPARC, it markedly prevented its release and restrained the protein inside the cells by inhibiting the canonical secretory pathway. This conclusion is based on the observation that, in our model, WIN mimicked the effects of BAPTA-AM and thapsigargin, two selective inhibitors of the secretory pathway. We also demonstrate that WIN increased the production of extracellular vesicles, in which SPARC was not present. Moreover, as a confirmation that the anti-migratory effect of WIN was independent of SPARC, the knockdown of this factor by RNA interference did not influence the migratory ability of osteosarcoma cells either in the presence or absence of WIN.
In our opinion, an interesting point in this study is the evidence that WIN induced a marked increase in the amount of secreted EVs and that EVs isolated from WIN-treated cells exerted a significant anti-migratory effect in untreated cultures. Ongoing studies in our laboratory attempt to characterize by proteomic analysis the content of WIN-induced EVs and this will shed light on the factors involved in WIN-mediated intercellular communication.
Osteosarcoma cells are characterized by the downregulation of miR-29 family members which exert an anti-proliferative and pro-apoptotic role in this cancer and sensitize the cells to the effects of chemotherapeutic agents . Interestingly, we demonstrate that WIN induced a dramatic increase in the level of miR-29b1, and that the stable overexpression of this miRNA produced a decrease in cell migration and a reduction of MMP2 and MMP9 activity similar to those observed in WIN-treated cells. This represents the first evidence that miR-29b1 is able to induce the downregulation of both MMP2 and MMP9. Figure 8 shows a schematic representation of the WIN-induced mechanism responsible for its anti-migratory effect in osteosarcoma MG63 cells. The preventive role of miR-29b1 on cell migration is in accordance with data recently reported by Zhu et al.  which demonstrate a role of miR-29 in the regulation of osteosarcoma cell proliferation and migration in connection with CDK6. Our current study aims to verify the presence of miR-29b1 in WIN-induced EVs and discriminate the vesicle types. Moreover, another intriguing aspect concerns the relationship between cannabinoid effects and the specific receptors (CB1-R and/or CB2-R) expressed in the different cancer models . In this regard, we consider it relevant to characterize the cannabinoid receptor in osteosarcoma cells that is responsible for the anti-migratory effect of WIN.
In conclusion, considering that the five-year survival rate in metastatic osteosarcoma is about 15–30% and that no specific drug has been found to date, the understanding of molecular mechanisms that regulate osteosarcoma migration and the potential role exerted by the cannabinoid WIN may have important implications for more specific osteosarcoma treatment.
4. Materials and Methods
R-[2,3-Dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenyl methanone mesylate (WIN55,212-2) was purchased from Sigma, (Sigma Aldrich, Milan, Italy). Stock solutions were prepared in DMSO and opportunely diluted in culture medium. The final concentration of DMSO never exceeded 0.04%, which is a concentration that was experimentally determined to have no discernible effect. All the experiments were carried out using vehicle alone as a control. Antibody against SPARC was purchased from Takara (Takara Bio Clontech, Mountain View, CA, USA), anti β-actin from Sigma (Sigma Aldrich, Italy) and anti-MMP2 and MMP9 from Santa Cruz Biotechnology (Santa. Cruz, CA, USA).
4.2. Cell Cultures
Human osteosarcoma MG63 cells were acquired from Interlab Cell Line Collection (ICLC; Genoa, Italy). Cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS), 2.0 mM L-glutamine, and antibiotic antimycotic solution (100 U/mL penicillin, 100 µg/mL streptomycin and 250 ng/mL amphotericin B; Sigma, Milan, Italy) in a humidified atmosphere containing 5% CO2. For the experiments, cells were seeded in 96 or six-well plates, and after 24 h, culture medium was replaced with fresh serum-free DMEM before cannabinoid treatment.
4.3. Wound Healing Assay
Changes of migration and motility of cancer cells were examined using a wound-healing assay . Cells (106) were seeded in six-well dishes to achieve approximately 90% confluence. Using a sterile 200 µL pipette tip, a straight scratch simulating a wound in a monolayer was made. After scratching, wells were gently washed with medium to remove the detached cells, and fresh serum-free DMEM medium was added. MG63 cells were treated with the vehicle (untreated cells), 5 µM WIN or 50 µg/mL EVs. In other experiments, before WIN treatment, the medium was replaced with serum-free medium from highly confluent untreated cultures. The plates were then incubated at 37 °C, and the speed of cell movement across the gap was observed. Digital documentation at the same position was made after scratching at time zero (T0) and after 8, 24 and 36 h and captured by a computer-imaging system (Leica DC300F camera and Adobe Photoshop for image analysis). The effects on cell migration and motility were estimated by using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The area of the remaining wound was determined as the ratio between the residual area at a given time point and the original wound area (T0) × 100.
4.4. Gelatin Zymography
The enzymatic activity of MMP2 and MMP9 was measured by gelatin zymography in 10% SDS-polyacrylamide separating gels in the presence of gelatin. Cells were treated with 5 µM WIN for 24 h, and the conditioned media from untreated or WIN-treated cells were collected and centrifuged (2000× g for 5 min) to remove cells and cell debris. The amount of total protein in the supernatants was assessed by the Bradford assay (Bio-Rad, Segrate, Milan, Italy) according to the manufacturer’s protocol. Supernatants were incubated with an adequate volume of SDS sample loading buffer without β-mercaptoethanol and separated by electrophoresis. Following electrophoresis, gels were rinsed with enzyme renaturing buffer containing 2.5% Triton X-100 in 50 mM Tris-HCl (pH 7.5) for 60 min at room temperature. Subsequently, gels were incubated in developing buffer (0.15 M NaCl, 10 mM CaCl2, in 50 mM Tris-HCl, pH 7.5) for 24 h at 37 °C, stained with Coomassie brilliant blue R-250 solution for 2 h and destained in methanol, acetic acid and water (50:10:40) solution until clear bands of MMPs activity were visible on the dark blue background. Digestive activity of MMPs was confirmed by the presence of two bands on zymograms, slower migrating MMP9 and faster migrating MMP2 in comparison with a standard of molecular weight.
4.5. Western Blot Analysis
To evaluate the extracellular protein content, conditioned culture media were dialyzed using 5000 cut off dialysis tubing and concentrated by lyophilization. Samples reconstituted with water were separated by electrophoresis under reducing conditions. For the evaluation of intracellular proteins, extracts were prepared as previously reported [39,40]. Ponceau red staining (for extracellular proteins), AChEase activity (for SPARC in EVs) or β-actin blots (for intracellular proteins) were reported as loading control. The blots were developed using the enhanced chemiluminescence (ECL) labeling system or alkaline phosphatase colorimetric system. Optical densities of the bands were analysed with Quantity One Imaging software from Bio-Rad Laboratories. The results shown in the figures are representative of at least three independent experiments with similar results.
4.6. Isolation and Quantification of Extracellular Vesicles from Culture Media
Extracellular vesicles (EVs) were purified from medium as previously described . Briefly, conditioned media by subconfluent cells were centrifuged at 2000× g for 10 min and at 4000× g for 15 min to remove cells and large debris. The supernatant was ultracentrifuged in a 70-Ti rotor (Beckman Coulter, Brea, CA, USA), at 105,000× g for 90 min at 4 °C, and pelleted vesicles were resuspended in filtered PBS. The protein content of isolated EVs was determined using the Bradford method (Bio-Rad, Segrate, Milan, Italy) with bovine serum albumin as standard.
The number of obtained EVs was determined by flow cytometry with a FACSCanto instrument (BD Biosciences, Erembodegem, Belgium) as previously described. Briefly, 1 μL of EVs was diluted in a fixed volume of 200 μL of filtered PBS (0.1 μM filter); all samples were analysed by FACS for 30 s at medium flow rate. The event number corresponds to the number of EVs present in a specific volume of sample .
EVs release was also quantified by measuring the activity of acetylcholinesterase (AchEase) by Ellman assay . Briefly, EVs (5 µL) were suspended in filtered PBS (95 µL) and incubated with 1.25 mM acetylthiocholine chloride (Sigma, Milan, Italy) and 0.1 mM 5,5′-dithio-bis(2-nitrobenzoic acid) (Sigma, Milan, Italy). PBS was then added to a final volume of 1 mL, and the change in absorbance at 412 nm was monitored every 5 min for 20 min.
4.7. Gene Silencing Using siRNA
Small interfering RNAs (siRNAs) against SPARC (5’-AACAAGACCUUCGACUCUUCC-3’) (siSPARC) and scrambled siRNA (siScr), as a negative non-silencing control, were purchased from Dharmacon RNA Technologies (Chicago, IL, USA). For the experiments, cells (105) were seeded in six-well plates and cultured in antibiotic-free DMEM supplemented with 2.0 mM L-glutamine, until 50% confluence. Then, cells were transfected with 30 nM siSPARC or siScr in the presence of 5 µL Metafectene Pro (Biontex Laboratories GmbH, Martinsried/Planegg, Germany) in a final volume of 1 mL serum-free medium. The reaction was stopped after 6 h replacing the culture medium with fresh 10% FBS DMEM. After 48 h from transfection, silenced cells were treated with WIN for other 24 h.
4.8. Real-Time PCR for miR-29b1 Expression
RNA was extracted by Direct Zol RNA Mini-Prep (Zymo research, Freiburg, Germany). A DNase I treatment step was included. Twenty nanograms of total RNA was reverse transcripted in a final volume of 10 μL by using miRCURY LNATM Universal RT microRNA PCR kit (Exiqon, Mi, Italy) according to the manufacturer’s instructions. The resulting cDNAs were used for quantitative analysis by real-time PCR (qPCR) using miR-29b1 LNA™ primers (204261; Exiqon) and SYBR Green Master Mix (Exiqon). Reactions were performed in 96-well plates according to manufacturer’s instructions, using Bio-Rad instrument, as previously reported . qPCR was performed in triplicate and repeated for confirmation. U6 small nuclear RNA was used as internal control for miRNA detection. Data processing and statistical analysis were performed by using IQ5 cycler software. The relative quantification in gene expression was determined using the 2−ΔΔCt method.
4.9. Stable Transfection of MG63 Cells with miR-29b1 Plasmid Vector
For the stable transfection of MG63 cells expressing miR-29b1, a specific plasmid construct, prepared as reported in , was employed. Before stable transfection, cells were seeded in six-well plates until they reached 90% confluence and then transfected with 4 µg of miR-29b1 expression vector or empty vector (mock) encoding the green fluorescent protein (GFP), by using Lipofectamine 2000 (InvitrogenTM, Monza, Italy) according to manufacturer’s instructions. The efficiency of transfection was evaluated by monitoring the ratio of transfected cells showing GFP fluorescence in relation to all cells. Two days after transfections, the cells were transferred in 100 mm dishes in selective medium containing 1 μg/mL Puromycin. The medium was replaced every 3–4 days. A plate of non-transfected cells was used as a control for the selection.
4.10. Statistical Analysis
Data, reported as means ± SD from at least three independent experiments, were analysed using the Student’s t-test. Differences were considered significant at p < 0.05.
Conceptualization, M.G., A.N., investigations A.N., S.E. and F.G.; methodology, A.N., A.D. and S.E.; data curation, A.D.; writing—original draft preparation, S.E. and A.N.; writing—review and editing, M.G. and M.L.; supervision, M.G. and G.C.; funding acquisition, M.G. and A.D. All authors agree on the results and commented on the manuscript.
This work was funded in part by the Funding for Basic Activities Related to Research (Ministero dell’Istruzione, dell’Università e della Ricerca, Italy, FFBAR Year 2017 PJ_RIC_FFABR_2017_009169) and FFR D15006475 (Università degli Studi di Palermo).
The authors thank Riccardo Di Fiore for providing miR-29b1 plasmic construct.
Conflicts of Interest
The authors declare no conflict of interest.
|SPARC||Secreted protein acidic and rich in cysteine|
- Lindsey, B.A.; Markel, J.E.; Kleinerman, E.S. Osteosarcoma Overview. Rheumatol. Ther. 2017, 4, 25–43. [Google Scholar] [CrossRef] [PubMed]
- Ottaviani, G.; Jaffe, N. The etiology of osteosarcoma. Cancer Treat Res. 2009, 152, 15–32. [Google Scholar] [PubMed]
- Taran, S.; Taran, R.; Malipatil, N. Pediatric osteosarcoma: An updated review. Indian J. Med Paediatr. Oncol. 2017, 38, 33. [Google Scholar] [CrossRef] [PubMed]
- Aljubran, A.H.; Griffin, A.; Pintilie, M.; Blackstein, M. Osteosarcoma in adolescents and adults: Survival analysis with and without lung metastases. Ann. Oncol. 2009, 20, 1136–1141. [Google Scholar] [CrossRef] [PubMed]
- Calvaruso, G.; Pellerito, O.; Notaro, A.; Giuliano, M. Cannabinoid-associated cell death mechanisms in tumor models (review). Int. J. Oncol. 2012, 41, 407–413. [Google Scholar] [CrossRef] [PubMed]
- Hinz, B.; Ramer, R. Anti-tumour actions of cannabinoids. Br. J. Pharmacol. 2019, 176, 1384–1394. [Google Scholar] [CrossRef]
- Bifulco, M.; Laezza, C.; Pisanti, S.; Gazzerro, P. Cannabinoids and cancer: Pros and cons of an antitumour strategy. Br. J. Pharmacol. 2006, 148, 123–135. [Google Scholar] [CrossRef]
- Velasco, G.; Sánchez, C.; Guzmán, M. Anticancer mechanisms of cannabinoids. Curr. Oncol. 2016, 23, S23–S32. [Google Scholar] [CrossRef]
- Lu, Y.; Anderson, H.D. Cannabinoid signaling in health and disease. Can. J. Physiol. Pharmacol. 2017, 95, 311–327. [Google Scholar] [CrossRef]
- Velasco, G.; Galve-Roperh, I.; Sánchez, C.; Blázquez, C.; Haro, A.; Guzmán, M. Cannabinoids and ceramide: Two lipids acting hand-by-hand. Life Sci. 2005, 77, 1723–1731. [Google Scholar] [CrossRef]
- Chakravarti, B.; Ravi, J.; Ganju, R.K. Cannabinoids as therapeutic agents in cancer: Current status and future implications. Oncotarget 2014, 5, 5852. [Google Scholar] [CrossRef] [PubMed]
- Kim, J.; Lee, K.J.; Kim, J.S.; Rho, J.G.; Shin, J.J.; Song, W.K.; Lee, E.K.; Egan, J.M.; Kim, W. Cannabinoids Regulate Bcl-2 and Cyclin D2 Expression in Pancreatic β Cells. PLoS ONE 2016, 11, e0150981. [Google Scholar] [CrossRef] [PubMed]
- Park, S.-W.; Hah, J.H.; Oh, S.-M.; Jeong, W.-J.; Sung, M.-W. 5-lipoxygenase mediates docosahexaenoyl ethanolamide and N-arachidonoyl-L-alanine-induced reactive oxygen species production and inhibition of proliferation of head and neck squamous cell carcinoma cells. BMC Cancer 2016, 16, 458. [Google Scholar] [CrossRef] [PubMed]
- Chiarlone, A.; Börner, C.; Martín-Gómez, L.; Jiménez-González, A.; García-Concejo, A.; García-Bermejo, M.L.; Lorente, M.; Blázquez, C.; García-Taboada, E.; de Haro, A.; et al. MicroRNA let-7d is a target of cannabinoid CB1 receptor and controls cannabinoid signaling. Neuropharmacology 2016, 108, 345–352. [Google Scholar] [CrossRef]
- Sreevalsan, S.; Safe, S. The cannabinoid WIN 55,212-2 decreases specificity protein transcription factors and the oncogenic cap protein eIF4E in colon cancer cells. Mol. Cancer Ther. 2013, 12, 2483–2493. [Google Scholar] [CrossRef]
- Giuliano, M.; Pellerito, O.; Portanova, P.; Calvaruso, G.; Santulli, A.; De Blasio, A.; Vento, R.; Tesoriere, G. Apoptosis induced in HepG2 cells by the synthetic cannabinoid WIN: Involvement of the transcription factor PPARgamma. Biochimie 2009, 91, 457–465. [Google Scholar] [CrossRef]
- Pellerito, O.; Calvaruso, G.; Portanova, P.; De Blasio, A.; Santulli, A.; Vento, R.; Tesoriere, G.; Giuliano, M. The synthetic cannabinoid WIN 55,212-2 sensitizes hepatocellular carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by activating p8/CCAAT/enhancer binding protein homologous protein (CHOP)/death receptor 5 (DR5) axis. Mol. Pharmacol. 2010, 77, 854–863. [Google Scholar]
- Pellerito, O.; Notaro, A.; Sabella, S.; De Blasio, A.; Vento, R.; Calvaruso, G.; Giuliano, M. WIN induces apoptotic cell death in human colon cancer cells through a block of autophagic flux dependent on PPARγ down-regulation. Apoptosis 2014, 19, 1029–1042. [Google Scholar] [CrossRef]
- Notaro, A.; Sabella, S.; Pellerito, O.; Di Fiore, R.; De Blasio, A.; Vento, R.; Calvaruso, G.; Giuliano, M. Involvement of PAR-4 in cannabinoid-dependent sensitization of osteosarcoma cells to TRAIL-induced apoptosis. Int. J. Biol. Sci. 2014, 10, 466–478. [Google Scholar] [CrossRef]
- Notaro, A.; Sabella, S.; Pellerito, O.; Vento, R.; Calvaruso, G.; Giuliano, M. The secreted protein acidic and rich in cysteine is a critical mediator of cell death program induced by WIN/TRAIL combined treatment in osteosarcoma cells. Int. J. Oncol. 2016, 48, 1039–1044. [Google Scholar] [CrossRef]
- Arnold, S.A.; Brekken, R.A. SPARC: A matricellular regulator of tumorigenesis. J. Cell Commun. Signal. 2009, 3, 255–273. [Google Scholar] [CrossRef] [PubMed]
- Freimuth, N.; Ramer, R.; Hinz, B. Antitumorigenic effects of cannabinoids beyond apoptosis. J. Pharmacol. Exp. Ther. 2010, 332, 336–344. [Google Scholar] [CrossRef] [PubMed]
- Brekken, R.A.; Sage, E.H. SPARC, a matricellular protein: At the crossroads of cell-matrix communication. Matrix Biol. 2001, 19, 816–827. [Google Scholar] [CrossRef]
- Nagaraju, G.P.; Dontula, R.; El-Rayes, B.F.; Lakka, S.S. Molecular mechanisms underlying the divergent roles of SPARC in human carcinogenesis. Carcinogenesis 2014, 35, 967–973. [Google Scholar] [CrossRef]
- Yan, B.; Guo, Q.; Fu, F.-J.; Wang, Z.; Yin, Z.; Wei, Y.-B.; Yang, J.-R. The role of miR-29b in cancer: Regulation, function, and signaling. Onco. Targets 2015, 8, 539–548. [Google Scholar]
- Jones, K.B.; Salah, Z.; Del Mare, S.; Galasso, M.; Gaudio, E.; Nuovo, G.J.; Lovat, F.; LeBlanc, K.; Palatini, J.; Randall, R.L.; et al. miRNA signatures associate with pathogenesis and progression of osteosarcoma. Cancer Res. 2012, 72, 1865–1877. [Google Scholar] [CrossRef]
- Schmitt, M.J.; Margue, C.; Behrmann, I.; Kreis, S. MiRNA-29: A microRNA family with tumor-suppressing and immune-modulating properties. Curr. Mol. Med. 2013, 13, 572–585. [Google Scholar] [CrossRef]
- Kapinas, K.; Kessler, C.B.; Delany, A.M. miR-29 suppression of osteonectin in osteoblasts: Regulation during differentiation and by canonical Wnt signaling. J. Cell. Biochem. 2009, 108, 216–224. [Google Scholar] [CrossRef]
- Bradshaw, A.D.; Sage, E.H. SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury. J. Clin. Investig. 2001, 107, 1049–1054. [Google Scholar] [CrossRef]
- Qiu, F.; Sun, R.; Deng, N.; Guo, T.; Cao, Y.; Yu, Y.; Wang, X.; Zou, B.; Zhang, S.; Jing, T.; et al. miR-29a/b enhances cell migration and invasion in nasopharyngeal carcinoma progression by regulating SPARC and COL3A1 gene expression. PLoS ONE 2015, 10, e0120969. [Google Scholar] [CrossRef]
- Perissinotto, E.; Cavalloni, G.; Leone, F.; Fonsato, V.; Mitola, S.; Grignani, G.; Surrenti, N.; Sangiolo, D.; Bussolino, F.; Piacibello, W.; et al. Involvement of chemokine receptor 4/stromal cell-derived factor 1 system during osteosarcoma tumor progression. Clin. Cancer Res. 2005, 11, 490–497. [Google Scholar] [PubMed]
- Chu, Y.; Fang, Y.; Chi, J.; Li, J.; Zhang, D.; Zou, Y.; Wang, Z. Astragalus polysaccharides decrease proliferation, migration, and invasion but increase apoptosis of human osteosarcoma cells by up-regulation of microRNA-133a. Braz. J. Med Biol. Res. 2018, 51, 12. [Google Scholar] [CrossRef] [PubMed]
- Xian, X.; Huang, L.; Zhang, B.; Wu, C.; Cui, J.; Wang, Z. WIN 55,212-2 Inhibits the Epithelial Mesenchymal Transition of Gastric Cancer Cells via COX-2 Signals. Cell Physiol. Biochem. 2016, 39, 2149–2157. [Google Scholar] [CrossRef] [PubMed]
- Xu, D.; Wang, J.; Zhou, Z.; He, Z.; Zhao, Q. Cannabinoid WIN55, 212–2 induces cell cycle arrest and inhibits the proliferation and migration of human BEL7402 hepatocellular carcinoma cells. Mol. Med. Rep. 2015, 12, 7963–7970. [Google Scholar] [CrossRef] [PubMed]
- Xu, W.; Li, Z.; Zhu, X.; Xu, R.; Xu, Y. miR-29 Family Inhibits Resistance to Methotrexate and Promotes Cell Apoptosis by Targeting COL3A1 and MCL1 in Osteosarcoma. Med. Sci. Monit. 2018, 24, 8812–8821. [Google Scholar] [CrossRef]
- Zhu, K.; Liu, L.; Zhang, J.; Wang, Y.; Liang, H.; Fan, G.; Jiang, Z.; Zhang, C.-Y.; Chen, X.; Zhou, G. MiR-29b suppresses the proliferation and migration of osteosarcoma cells by targeting CDK6. Protein Cell 2016, 7, 434–444. [Google Scholar] [CrossRef]
- Kisková, T.; Mungenast, F.; Suváková, M.; Jäger, W.; Thalhammer, T. Future Aspects for Cannabinoids in Breast Cancer Therapy. Int. J. Mol. Sci. 2019, 20, 1673. [Google Scholar] [CrossRef]
- Sherman, A.B.; Gilger, B.C.; Berglund, A.K.; Schnabel, L.V. Effect of bone marrow-derived mesenchymal stem cells and stem cell supernatant on equine corneal wound healing in vitro. Stem Cell Res. 2017, 8, 120. [Google Scholar] [CrossRef]
- Lauricella, M.; Carlisi, D.; Giuliano, M.; Calvaruso, G.; Cernigliaro, C.; Vento, R.; D’Anneo, A. The analysis of estrogen receptor-α positive breast cancer stem-like cells unveils a high expression of the serpin proteinase inhibitor PI-9: Possible regulatory mechanisms. Int. J. Oncol. 2016, 49, 352–360. [Google Scholar] [CrossRef]
- Cernigliaro, C.; D’Anneo, A.; Carlisi, D.; Giuliano, M.; Marino Gammazza, A.; Barone, R.; Longhitano, L.; Cappello, F.; Emanuele, S.; Distefano, A.; et al. Ethanol-Mediated Stress Promotes Autophagic Survival and Aggressiveness of Colon Cancer Cells via Activation of Nrf2/HO-1 Pathway. Cancers 2019, 11, 505. [Google Scholar] [CrossRef]
- Barreca, M.M.; Spinello, W.; Cavalieri, V.; Turturici, G.; Sconzo, G.; Kaur, P.; Tinnirello, R.; Asea, A.A.A.; Geraci, F. Extracellular Hsp70 Enhances Mesoangioblast Migration via an Autocrine Signaling Pathway. J. Cell. Physiol. 2017, 232, 1845–1861. [Google Scholar] [CrossRef] [PubMed]
- Geraci, F.; Ragonese, P.; Barreca, M.M.; Aliotta, E.; Mazzola, M.A.; Realmuto, S.; Vazzoler, G.; Savettieri, G.; Sconzo, G.; Salemi, G. Differences in Intercellular Communication During Clinical Relapse and Gadolinium-Enhanced MRI in Patients With Relapsing Remitting Multiple Sclerosis: A Study of the Composition of Extracellular Vesicles in Cerebrospinal Fluid. Front. Cell. Neurosci. 2018, 12, 418. [Google Scholar] [CrossRef] [PubMed]
- Ellman, G.L.; Courtney, K.D.; Andres, V.; Feather-Stone, R.M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95. [Google Scholar] [CrossRef]
- Di Fiore, R.; Drago-Ferrante, R.; Pentimalli, F.; Di Marzo, D.; Forte, I.M.; D’Anneo, A.; Carlisi, D.; De Blasio, A.; Giuliano, M.; Tesoriere, G.; et al. MicroRNA-29b-1 impairs in vitro cell proliferation, self-renewal and chemoresistance of human osteosarcoma 3AB-OS cancer stem cells. Int. J. Oncol. 2014, 45, 2013–2023. [Google Scholar] [CrossRef] [PubMed]
Figure 1. WIN55,212-2 (WIN) treatment inhibits MG63 cell migration. The effect of WIN on MG63 wound healing in the absence (A) or presence of conditioned medium (B). After mechanical wounding, confluent cells were treated with 5 µM WIN and cell migration was monitored over time. In (B), before treatment, cell culture medium was replaced with conditioned medium from high confluence cultures. Representative microphotographs were taken at the indicated time points after wounding (magnification 200×). The average distances between cells at the edges of the gaps in the wound healing assay were estimated by ImageJ software. Histograms reporting the mean percentage area healed at T8, T24 and T36 post-wounding are shown on the right panels. (*) p < 0.05 and (**) p < 0.01 versus untreated cells at the same time point.
Figure 2. WIN treatment inhibits metalloprotease activities. Gelatin zymography (A) and Western blotting analysis (B) of the metalloproteinases MMP2 and MMP9 in MG63 cells treated for 36 h with 5 µM WIN. Arrows indicate the relative bands at 72 kDa (MMP2) and 92 kDa (MMP9). Blots are representative of three independent experiments with similar results. Densitometric analysis is reported in the histograms. (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001 compared to the untreated sample. In (A), volumes containing equal amount of proteins were loaded, in (B), densitometric measurements were made after normalization with β-actin.
Figure 3. WIN treatment blocks secreted protein acidic and rich in cysteine (SPARC) release and increases the number of extracellular vesicles. (A) Western blotting analysis of SPARC in media derived from untreated or WIN-treated cells for 36 h. The media were dialyzed, concentrated by lyophilization, and reconstituted with water. Protein samples were separated under reducing conditions, and immunoblotting was performed using SPARC antibody. Red Ponceau staining was reported as loading control. The results are representative of three independent experiments and densitometric analysis is reported in the histograms. (***) p < 0.001 compared to untreated cells. (B) Flow cytometry analysis of isolated extracellular vesicles (EVs) derived from untreated or WIN-treated MG63 cells. Data are representative of three independent experiments. SSC, side scatter; FSC, forward scatter. (C) Acetylcholine esterase (AChEase) activity in EVs from untreated or WIN-treated cells assessed as reported in Section 4 and relative quantification. (D,E) Western blotting analysis of SPARC in MG63 cell-derived EVs. Media from untreated or WIN-treated cells were collected and EVs isolated as reported in Section 4. The immunoblotting of isolated EVs (D) or EV supernatants (E) was performed to detect SPARC level. AchE activity (D) and Ponceau red staining (E) were used as loading control. Representative blots of three independent experiments and densitometry analysis histograms are depicted. (**) p < 0.01 compared to the untreated sample.
Figure 4. Effects of secretory pathway inhibitors on SPARC levels. Western blotting analysis of extracellular or intracellular level of SPARC after treatment with 5 µM WIN, 1 µM thapsigargin (THG) or 5 µM BAPTA-AM (BAPTA). Ponceau red staining or β-actin blot were reported as loading control for extracellular or intracellular protein levels, respectively. Representative blots of three independent experiments and densitometry analysis histograms are depicted. (*) p < 0.05, (**) p < 0.01 compared to the untreated sample. N.S. not significant.
Figure 5. The effect of WIN on MG63 cell migration is not dependent on SPARC. (A) The effect of SPARC siRNA (siSPARC) on the protein level under 5 µM WIN treatment in both intracellular or extracellular fractions. β-actin blot or Ponceau red staining were reported as a loading control for intracellular or extracellular protein levels, respectively. Representative blots of three independent experiments and densitometry analysis histograms are depicted. (*) p < 0.05, (**) p < 0.01 compared to the untreated sample at the same time point. N.S. not significant. (B) Effects of siSPARC on cell migration in comparison with scramble control (siScr). Pictures reported were taken at T36 after wounding (magnification 200×). The average distances between cells at the edges of the gaps in the wound healing assay were estimated through ImageJ software. On the right panel, a graph of the mean percentage area healed at T36 post-wounding is shown. (**) p < 0.01 compared to untreated cells at the same time point. N.S. not significant.
Figure 6. Extracellular vesicles from WIN-treated cultures halt MG63 cell migration. Wound healing migration assay of MG63 cells in the presence of EVs (50 µg/mL) isolated from WIN-treated cells. The average distances between cells at the edges of the gaps in the wound healing assay were estimated through ImageJ software. Pictures reported were taken at different time points after wounding (magnification 200×). On the right of the picture a graph of the mean percentage area healed at T8, T24 and T36 post-wounding is shown. (**) p < 0.01 compared to the original gap at T0. N.S. not significant.
Figure 7. WIN induces a dramatic increase in miR-29b1 expression, and miR-29b1 overexpression reduces cell migration. (A) Gene expression levels of miR-29b1 in untreated or 5 µM WIN-treated cells for 24 h were analysed by RT-qPCR. (**) p < 0.01 compared to untreated cells. (B) Validation of gene expression changes in cells stably transfected with miR-29b1 versus mock transfected cells. The height of each column reflects the ratio of the expression of miR-29b1 compared to U6 (a non-coding small RNA used as a control) (**) p < 0.01 and (***) p < 0.001 compared to the untreated cells at the same time point. (C) Wound healing migration assay of miR-29b1-transfected MG63 cells in comparison with cells transfected with mock alone. Pictures were taken at the indicated time points after wounding (magnification 200×). The average distances between cells at the edges of the gaps in the wound healing assay was estimated through ImageJ software. On the right of the picture, a graph of the mean percentage area healed at T8, T24 and T36 post-wounding is shown. (**) p < 0.01 and (***) p < 0.001 compared to the untreated cells at the same time point. (D) Gelatin zymography of MMP2 and MMP9 in miR-29b1-transfected cells versus mock transfected cells. Arrows indicate the relative bands at 72 kDa (MMP2) and 92 kDa (MMP9). Volumes containing equal amount of proteins were loaded. Densitometry values are averaged from three independent experiments. (***) p < 0.001. (E) Western blotting analysis of SPARC level in miR-29b1 overexpressing cells. The results were obtained by immunoblotting employing a specific SPARC antibody as reported in Section 4. β-actin blot was included as a loading control. Densitometry values are averaged from four independent experiments normalized to β-actin. N.S. not significant.
Figure 8. Schematic representation of the WIN-induced anti-migratory effect.
© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

domain: literature


text:
Published by Corgi Childrens on August 18th 2015
Genres: Family, Fantasy & Magic, Friendship, Love & Romance, Mystery & Thriller, Romance, Young Adult
Format: ARC, digital ARC
Buy on Amazon
It's the accident season, the same time every year. Bones break, skin tears, bruises bloom.
The accident season has been part of seventeen-year-old Cara's life for as long as she can remember. Towards the end of October, foreshadowed by the deaths of many relatives before them, Cara's family becomes inexplicably accident-prone. They banish knives to locked drawers, cover sharp table edges with padding, switch off electrical items - but injuries follow wherever they go, and the accident season becomes an ever-growing obsession and fear.
But why are they so cursed? And how can they break free?
Accidents happen. Our bones shatter, our skin splits, our hearts break. We burn, we drown, we stay alive.
The Accident Season is an imaginative and whimsical paranormal book that’ll keep you up at night. Just a little tip: you might want to keep a teddy bear close while reading this one, too.
The main thing that turned this into a successfully haunting read was the writing style. Doyle’s writing will make you feel like you’re floating, and maybe even kind of… unsteady, which is perfect to get a good feel of the story’s spooky atmosphere. Although for some (weirdos like me), it might be a bit off-putting. Personally, it made it a bit tougher for me to feel–like I said, I was sort of floating throughout the whole book.
And now, let’s talk about the characters. While I did love the idea of them, I didn’t really love them. Let me elaborate here. See, I love how Doyle seemed to go against some character stereotypes, but I just couldn’t get a good grasp of them. Maybe it was because of the floaty feeling the book gave me. Maybe it’s because I felt like some of them were too all over the place (like everyone kissed everyone without telling each other), or because I wasn’t given enough time to really figure them out.
This book also had romance in it, which may or may not be a good thing. I mean, I didn’t love any of the relationships in particular, but I didn’t mind them being there. They didn’t necessarily add anything to the story, but they didn’t take the spotlight off the mystery, either. Okay, maybe I did sort-of like the main ship, but the side-ship (I deem this a thing now!) was pretty meaningless and had no chemistry.
If there’s one thing I appreciated most about this book, thought, it’s that it was it actually made sense. If I’m being really honest here, a lot of mystery and thriller books have these twists that come out of nowhere. The twist in The Accident Season will make you nod your head along, while still getting these chills that’ll creep up your spine.
Y’all should know that I’m a huge wuss, but for some strange reason still love reading books that’ll creep me out. And did Doyle succeed in doing just that! There were a handful of scenes in this book where I just wanted to call my mom to bring me a blanket and my teddy bear. They weren’t particularly holy-shit scary, but they were definitely spooky and mind-boggling.
Aaand Doyle also included changelings in the book, which I greatly appreciated. I love how she went with the descriptive route with them–I loved picturing these odd creatures in my mind, and it was awesome that each of them were very different and easily distinguishable.
Anyone looking for a magical realism book with a good twist and lovely chills will definitely enjoy The Accident Season. Unless you don’t like getting that floaty feeling when you read.


Guns, gaming, schools, public financing, tolls, leadership qualities and socks were among the subjects addressed Sunday night at a debate by the Democratic candidates for governor in New Haven, the city that brings the most–about 100–delegates to the state party convention on May 19. Bridgeport is number two in that category. The candidates, as part of the discussion, posed questions to each other. At one point Mayor Joe Ganim hiked up his pant leg to show off his multi-patterned sock while a nonplussed Susan Bysiewicz, the only woman on stage, looked on. Who’s buying Joe’s socks? OIB friend Paul Bass from the New Haven Independent has more on this political yarn.
Most of the debate featured candidates playing to the New Haven crowd, especially Mayor Harp.
At one moment, toward the end, Lamont gave an answer that didn’t fit neatly into what New Haven has been saying these days.
Lamont was answering a question posed by Ganim. Ganim spoke of how the state legislature has agreed to pay off $550 million in bonded debt on behalf of bankruptcy-teetering Hartford. Ganim and Mayor Harp originally blasted the state for doing that while leaving Bridgeport and New Haven to struggle with difficult budgets amid cutbacks in state aid. Last week Ganim altered his position after Hartford’s mayor dropped out of the race for governor; seeking support from Hartford’s newly freed delegates, he declared himself a convert to the idea of bailing out that city. But he continued to press for similar bailouts for Bridgeport and New Haven.
On the state Sunday, Ganim asked his opponents if they join him in seeking those similar bailouts.
“We need a comprehensive urban strategy in Connecticut that would support New Haven and would support Bridgeport and other situated cities with the same type of equity and support.” He challenged anyone who doesn’t “agree with that” to “take the microphone.”
Lamont took the microphone.
“I am going to be a champion for the cities,” he said. “But I would do it a different way than what I saw in Hartford. The bailout in Hartford in Hartford ended up bailing out a bunch of Wall Street bondholders. I want to make darned sure the money goes to the people in Bridgeport and New Haven Hartford.”
Looking at Mayor Harp seated in the audience–as candidates did often during the debate–Lamont said he would instead push for fulling funding the Payment in Lieu of Taxes (PILOT) program and increasing the amount of school aid cities get under the Educational Cost-Sharing System (ECS).
Full story here.


“Fresno Soldier is Killed in French Fighting,” Fresno Bee, September 26,1944
This article is part of a series honoring the battle-dead of the city of Fresno, California. The memorial was dedicated on November 8, 2019, at the Fresno City Hall. The following article ran in the Fresno Bee on September 26, 1944. I researched the death of PFC Potter in connection with the Fresno Veterans Memorial project.
“Fresno Soldier is Killed in French Fighting”
Source: Fresno Bee, September 26, 1944
Mrs. Ida Potter, of Route 10, Box 121, today was notified by the war department her husband, Private First Class Carl E. Potter, 20, a former ranch worker, was killed in action while fighting as an infantryman in France August 31st.
Potter, who entered the military service last year, was a native of Clovis, N.M. He lived in the Fresno area for several years.
He is survived by a 6 months old daughter, Ida Potter; two sisters, Rachel and Alpha Mae Potter, both of Fresno; three brothers, Jack Potter of Fresno, Sergeant James Potter, serving in the army air forces, and Private Frank Potter, of the medical corps, stationed in England, and his parents, Mr. and Mrs. James M. Potter of Fresno.
Mrs. Potter said her husband never wrote of his military experiences and no details of his death have been given her by the war department.
May he rest in peace.


text: Toxocara canis (Werner, 1782) is a zoonotic nematode commonly parasitizing dogs worldwide with great public health importance as the aetiological agent of human toxocariasis. In this respect, the aim of this study was to evaluate the effect of six disinfectant products commonly used in kennels, veterinary clinics and as household cleaning products on the embryogenesis and viability of T. canis eggs. The composition of active ingredients in these commercial disinfectants was sodium hypochlorite (A); a mix of N-(3-aminopropyl)-N-dodecylpropan-1.3-diamine and didecyldimethylammonium chloride (B); sodium dichloroisocyanurate dehydrate (C); a mix of glutaraldehyde, quaternary ammonium compounds, benzyl-c12-18-alkyldimethyl and chlorides (D); a mix of 2-propanol, ethanol, benzalkonium chloride and glucoprotamin (E); a mix of pentapotassium bis (peroxymonosulphate) bis (sulphate), sodium C10-13-alkylbenzenesulphonate, malic acid, sulphamidic acid, sodium toluenesulphonate, dipotassium peroxodisulphate and dipentene (F). After dilution, the tested disinfectants had the maximal concentration recommended by the manufacturer in order to achieve a biocidal effect. Each product was tested on approximately 10,000 T. canis eggs, having five different contact times (5, 10, 15, 30, 60 min). Three replicates were tested for each diluted disinfectant and for each contact time. After the treatment, eggs were washed and incubated in distilled water at 27 °C for 2 weeks. None of the tested products had a significant inhibitory effect on the embryogenesis and viability of T. canis eggs, regardless of the contact time. Moreover, after 2 weeks, in all tested samples, eggs containing motile infective larvae were identified, showing that routinely used disinfectants do not eliminate risk of infection by T. canis.


text:
1. IMIZAMO YAM
Our mothers carrying the world on their shoulders will do just about anything to put food on their children’s tables and clothes on their backs. What is waste to us, is her job. Below the hardhat lies a doek; a reminder that all work is gendered, in ways that open possibilities while presenting unspeakable challenges in a world dominated by patriarchy
2. SO NEAR YET SO FAR
South African society, is built on fences (seen and unseen) that present small openings of opportunity to a world some take for granted. A world where many fight for a small opening to survive A juxtaposed urban existence where the ‘have-nots’ are so near yet so far, as many migrants negotiate space, movement and access to the City of Gold.
We have one in our family and one or more in the community. The street smart elderly man with a youthful, township edge who always spews wise gems in the form of street metaphors and anecdotes, all said unintentionally and in unassuming jest. He wears life’s experiences on his back and lives in constant nostalgia. A conversation with him always reveals unconventional views of the world…
We’ve lost so much in the fire. Our innocence, naivety and optimism about a future we may not be assured of. This is a sentiment held by many who have sacrificed their careers, freedom and sanity, for the cause of an education promised. This is an ode to all who have lost parts of themselves in a struggle for free education, your sacrifices are written in blood, in song and inscribed in future victories
5. FIRE IN THE BELLY, ICE IN THE HEART
Cutting a figure of calm, while fires burn in the background, is an art many have mastered. We build lives while the inferno that is our communities, burns on. We find, make and maintain love, in environments where all that was expected is hate. We find ways to connect with the mundane and ordinary, in a world of dysfunction and abnormality
6.KRUGER IS THE RAND(LORD)
Statues memorialize people and their character. Placed in different times and contexts they present contradictions best captured in visual form. Here we are reminded that the places we congregate and build solidarity and politics around bear hallmarks of this ‘memory’, where the Randlords of the past hover over our lives, even when our attempts are to undo the legacy they have left us with.
7. OH’ YHINI SESGODUKA
South African cities continue to be sites of spatial injustice. Those on the margins of our society are forever in movement in flux; between home and work . Public transport is an integral part of how millions of the working class travel between home and work each day. Here, the story of space and movement in fleeting time is told.
8. IMFAMA ZIYABONA
The harsh boundaries of the city where we are all trying to create the lives we have imagined for ourselves can blur our vision and shake our foundations. Sometimes we make our way through it not knowing what to expect and sometimes slip in the abyss of despondency because the dream does not seem clear enough, but hope keeps whispering: ‘where we are going, even the blind can see’.

domain: artistic


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