text:
Clean eating is a lifestyle, and with any lifestyle you choose, you will have curves and bumps in the road. I have never driven down any road that was a straight shot, nor do all things in life go as planned. This isn't always a bad thing; some things in my life have gone way better than I could ever have imagined or planned. This 'things don't always go as planned' doesn't just go for major life events, this goes for every day things too. For example; say you plan a delicious crockpot meal for your family and you walk in the door from work only to find that you forgot to turn your crockpot on. (Ruh-roh) What matters then, is what you choose to do in that unplanned moment. Maybe you choose a salad from a fast-food restaurant or an Amy's frozen meal from the freezer–is this what you planned to have? No, but it is a lot better than some alternative fast food choices or eating junk food. My point is that you have to give yourself grace with clean eating because there will never be such a thing as perfection.
Clean eating can mean different things to different people. I think it is important to experiment with what clean eating means to you. Once you know what your own definition of clean eating is, then you can take small steps to eat that way. To give you a frame of reference to start, I want to share with you my own clean eating rules. I don't expect myself to be 100% perfect all of the time (insert Yesterday's Reese's Cheesecake – how do you say no?) but I do expect myself to adhere to these rules 90% of the time. I hope my rules inspire you to create your own. Heck, I don't even care if you steal all of mine 🙂
Eat real food. This rule is as simple as it sounds. When you are making your food choices, choose things that grow on a plant, not things that are made in a plant (a factory). Choose foods the way nature intended them to be–in their whole, real form, instead of processed, puffed, and in a box on a shelf. When you fill your plate with real foods, you simply have less room for junk.
If it grows on a plant, eat it. If it is made in a plant, don't.
Eliminate artificial ingredients. Your body knows how to recognize, break down, and use the nutrients found in real foods. When you eat chemicals, artificial ingredients, and preservatives, your body sees them as foreign invaders. In response to them, your immune system is stimulated. Constant stimulation in response to these additives in food can result in widespread silent inflammation, which can eventually present itself as a chronic condition. The less inflammation you have, the less likely you are to develop disease. My rule of thumb: If you can't pronounce it, don't eat it.
If you can't pronounce it, don't eat it.
Eliminate added sugar. Sugar stimulates the same receptors in the brain that drugs like heroin and cocaine do, so you can literally become addicted to it. I would argue that it is the most readily available drug in America. Most Americans have a constant drip of sugar–think Starbuck's foo-foo drinks, granola bars, dressings, ketchup, BBQ sauce, cereals, yogurts, coffee creamers, pop, candy, and more. Eating sugar not only gives you a doughnut around your middle but it also puts you at greater risk for developing heart disease, diabetes, cancer, and more.
Sugar is the most readily available drug in America. Stay clean.
Choose organic, grass-fed, and non-GMO when possible. Look guys, I know you are going to tell me that this type of food is more expensive. You know what? You're right. Personally, I would rather pay more money up front for quality food then more money down the road for healthcare.
I am never going to tell you not to eat a vegetable, organic or not, but I do think it is important you pay attention to the quality of the food you buy. The organic label on produce ensures that it hasn't been sprayed with harmful chemicals, is non-GMO, and has been grown using sustainable farming methods. Conventional produce is sprayed with harmful pesticides to kill off bugs and herbicides to kill weeds around the crops and make up for unfit soil conditions. Eating residual pesticides and herbicides stimulate your immune system in the same way that artificial ingredients do, contributing to inflammation. Would you allow a farmer to spray a chemical directly into your mouth? I didn't think so. Then why be ok with eating it on piece of produce?
In an effort to maximize your dollar, I recommend using the Environmental Working Group's Dirty Dozen and Clean Fifteen list when shopping for fruits and vegetables. The Dirty Dozen is a list of produce that has been shown to have the highest amount of residual pesticides after washing and the Clean Fifteen is a similar list of the cleanest fruits and vegetables. I always purchase produce from the Dirty Dozen organic and think it would be a good practice for you to adopt too, especially if it is something your family eats a lot of.
I recommend sourcing quality meat as well. I typically look for the labels 'organic' and 'grass-fed'. The organic label ensures that no hormones or antibiotics have been administered. Conventional farmers give antibiotics to cattle because the living conditions are so unfit, that without them, disease would run rampant. Actually, 80% of antibiotics sold in the U.S. are administered to animals, not humans. (NRDC) Growth hormones may also be administered to fatten the animal up. The fatter the animal, the more $$$ for the farmer.
Grass-fed means that the animal has been raised in its natural environment and was fed from grass, as nature intended. This results in much leaner and richer tasting meat. Conventional cattle farmers typically feed their cattle corn and grain because it's cheap and it fattens them up. Knowing that you are what you eat, why would you want to eat something that has been fattened up? Once you make the switch from conventional to grass-fed, you won't be able to go back. There are many more labels to get to know and understand but organic and grass-fed will get you started.
I also think it is important to familiarize yourself with what GMO's (genetically modified organisms) are. GMO's are crops that have been altered at their genetic level to be able to withstand pesticides like Round-Up (yeah, that Round-Up) and to be able to grow in unfit conditions to maximize profits. Approximately 90% of soy and corn in America is genetically modified. Considering that corn and soy are in almost every packaged food item available, it is important to seek out non-GMO packaged grocery items. A great place to start is with corn chips because they are made of corn (thank you Mrs. Obvious) and crackers because they typically contain both corn and soy under names like soybean oil, corn oil, corn syrup, and more. Defenders of GMO's say that there is no difference between GMO crops and non-GMO crops, but considering that this new way of growing food hasn't been around all that long, I am not willing to be part of the test group. You can learn more about GMO's and what products are non-GMO certified here.
Focus on the quality of your food. Source organic, grass-fed, and non-GMO when possible.
Do the best you can with what you have. I just spewed a lot of information to you. Sometimes, it is just entirely not possible to choose organic, grass-fed, or non-GMO and that is ok. You just have to make the best choice possible in any given situation. Altering from the cleaning rules above from time to time is not going to kill you. Give yourself grace for the moments when the 'perfect' choice is not possible. What you do most of the time is what matters, not what you do once in awhile.
Make the best choice possible in any given situation.
Enjoy food. Food is love and it is meant to be enjoyed. Creating a positive mindset around food is an important aspect to clean eating. A lot of Americans, specifically women, view food as an enemy when it should really be viewed as a friend. It is what gives you the energy to power through your day, muscles to hold your children, and and the building blocks for your body to function optimally. Make friends with food, not enemies.
Make friends with food, not enemies.
All of these rules may seem overwhelming at first. Personally, I didn't change everything at once. I did it over a long period of time by taking small steps. What can you do today to take one step in the direction of cleaner eating? Please leave your answer in the comments below.


text:
Team Nargis Dutt Foundation visited and distributed start-up kits to the families affected by floods, landslides and cyclones in the Konkan belt of Mahad, Poladpur and Chiplun on 4th and 5th October 2021.
The visit was to give hope to the people of Raigad and Ratnagiri and let them know that they are not alone in their sufferings. The visit gave us an opportunity to interact with the families and understand the ground realities of life. It made us realise the fragility of life, the fury of nature and the importance of humanity coming together to help those in need. The villagers felt emotional that someone somewhere thought about them. We believe it will provide some strength to pass through this difficult phase in their lives.
We are grateful to our donors who supported this cause wholeheartedly. You are the reason behind their smiles.
Special thanks goes out to Mr. Prasad More, Team Samatawadi Pratishthan and Team Lalbaug Yuva Group for shortlisting the families in need.

subset: commoncrawl


text:
When I originally heard of Madame Alexander Washable Dolls I knew that they would be adorable and more "playable" then the collectable dolls I always think of when I hear of the brand. I knew they would be adorable because well… It's Madame Alexander and they have been creating gorgeous dolls for 90 years! Then while on vacation this summer I took my daughter Hayley to a doll shop and the first thing we saw when we walked in was Glinda the Good Witch
Hayley was immediately taken by Glinda's beauty. She has a beautiful pink skirt with a shiny tutu type overlay and her own wand.
And then there she was, I felt like a little girl again, I saw Dorothy! It reminded me of Friday nights as a child curled up on the couch with popcorn watching one of my favorite movies, "The Wizard of Oz"
These dolls are not only adorable for children but the nostalgia behind them makes me, as a mother, extra excited to see my little girl loving her. Hayley has Dorothy and loves her. Dorothy is a 12 inch machine washable doll. She is super soft and the perfect size for little ones to snuggle with. Hayley sleeps with hers every night
12-inch soft, cloth Dorothy™, has an embroidered face and brown fabric hair that's decorated with a blue bow that's tacked on in back. She wears a blue and white gingham pinafore dress with a white blouse that has short, puffed, ruffled sleeves. Blue and white gingham trim on the collar, blue socks and red cloth shoes, with bows, completes our new, yet still iconic, Dorothy™. Washer and Dryer Safe on Gentle Cycle.
Madame Alexander has offered to give one of my readers an amazing and special prize!! One lucky reader will win their very own Scarecrow, Cowardly Lion and Tin Man 12 inch Washable Dolls (US Only)
All three dolls just for entering via the rafflecopter below and be sure to comment and share daily for extra entries!
Disclosure: We were sent Dorothy for review and Hayley just loves her!! All the opinions in this post are my own


text:
SOOTHING THE SICK AND HURTING OF JERUSALEM An unlikely harpist, this Israeli believer is living a ministry dream that seemed impossible...Story & photo by Rebekah Kolber, Staff Journalist; The Messianic Times - July/August 2005 |
Twelve-year-old Rena Glaser was lying in bed at Jerusalem's Hadassah Hospital when she first met Elisheva Margolis, a Jewish believer who ministers to the sick and injured - particularly those who have been hurt in terror attacks - using music and prayer. Glaser, who was being treated for a secondary infection after chemotherapy treatment after cancer, remembers that Margolis popped into her room with a big smile and offered to play music for her. "I had never heard a harp before or seen one up close so I was like, sure, play it," she remembers. After playing a few songs Margolis let Glaser try out the harp, then prayed for her to have peace and healing, before moving on to the next room. "It made me really happy that Elisheva came to see us because when you're sick all the time kids are scared to hang out with you. They don't want to visit the hospital because they think you'll give them cancer. And being in the hospital all the time is boring. It's fun to get a visit and I really like the harp music now," Glaser said. For two years, Margolis has been visiting hospitals throughout the Jerusalem metropolitan area, sharing music and the love of Yeshua. Margolis, who made aliyah from California in 1997, felt the Lord prompting her to minister to others through harp music for years before her ministry became a reality. "Music from the harp helps people to have a calm spirit and it helps change the spirit, the way David did with Saul," says Margolis. "For years I had a burden to minister in this way to those in the hospital and to people with difficulties such as autism." In 2002 Margolis found an inexpensive child-sized toy harp and began to practice on it, trusting that one day the Lord would provide her with a real harp. "I asked the Lord to bring a harp to me and I had a dream that one day in fact I would receive a harp and be playing a harp to minister to those in pain. I didn't know how that would be possible but I was sure the Lord was speaking to me." That same year, Messianic Connie Buck purchased a Davidic harp from Rick and Mary Woods of Jubilee Harps, Inc., in Evansville, IN. Although Buck did not need the harp she felt called to purchase one. Together, the three prayed for an appointed person to receive the harp when Buck visited Israel several months later for an international Christian gathering. Shortly after arriving in Israel, Buck said the Lord spoke to her again about the harp. "I took the harp to a room where we had prayer and I was reading in the Psalms concerning the importance of playing the harp, especially on Shabbat. I had such a joy playing the harp for the Lord, even though I am not a musician, that I wanted to leave it in our prayer room so that others could play it for the Lord as well," she said. The next day Buck was standing in the hallway of her hotel when friends returned the harp to her. At that moment, Margolis happened to walk by looking for another hotel guest. She noticed the harp and struck up a conversation with Buck, who began to wonder if this was the person the Lord wanted her to leave her harp with. When Buck heard that Margolis was on her way to minister to terrorism survivors she decided then and there to give her the harp. The next day she too was on her way to Hadassah hospital to minister in the children's ward. "Bringing the harp to the hospital was a way of bringing comfort just as Isaiah's words said 'Comfort ye my people.' It was a great honor, a great privilege to be the bearer of comfort one beautiful day in Jerusalem." In the three years since, Margolis has performed for thousands of patients. She plays for paralyzed children, cancer patients, injured soldiers, and terror victims. Margolis also uses the harp at healing prayer services for believers. Those she ministers to are just as moved by the music as by the story of how she came to play the harp, says Margolis, "Sometimes I take the harp onto the streets of Jerusalem and I just play and praise the Lord and worship Him. When people ask I tell them about how I got this harp from a Gentile as a gift from her and an answer to prayer from the Lord. I tell them that as a Levite I feel so honored and special to have this blessing because it came from God." People are amazed that a Christian tourist would just give away such an expensive gift, says Margolis; it touches them because it makes them realize how much Gentile believers love the Jewish people. "When I tell the story they can't believe it at first and then they do believe it and they wonder what it is that makes Christians love them so much. I tell them it is Yeshua the Messiah because He is both their Messiah and our Messiah." Without the Lord's provision in Connie
Buck's gift, Margolis says she never would have had the resources to start
her musical ministry. Harps can easily retail at several thousand dollars,
well beyond the reach of the average music ministry. When Rick and Mary
Woods of Jubilee Harps finally met Margolis at the Messiah 2003 conference
they immediately recognized who she was from Buck's story and decided
to give her a harp case, amplifier, tuner and music books for her ministry.
"The Lord's working through Elisheva and we wanted to support that,"
said Mary Woods. "The Lord is touching people through her."
Home | Request a Catalog | Our Business Ministry | Our Family | F.A.Q. | Kinnor History | King David Harp | Watchman Harp
Paul Wilbur | Greg Buchanan | Testimonies | Media Exposure | Photo Gallery | Audio Gallery | Shop Online
Site Map


text:
#1 Funny Airline Photo
This picture is very funny. The man in the photo is flying on Czech Airlines. His hairline has almost completely receded and he has just a little hair on the sides of his head. Under his head is a seat protector, which is normal on flights. What makes this photo so funny is that the seat protector reads, czechairlines.com. If you split it up properly, it reads Czech Airlines. If you look at it the wrong way, it reads czec hairlines. This is quite the funny coincidence.
#2 Wedding Day Warning
This is one of the craziest coincidences you won't believe. A young woman got married and she asked her brother to buy a newspaper on her wedding day. She wanted it to be something that she could look back on years later and see what was going on that day. Unfortunately, the newspaper didn't have the best headline that day. The major headline on the paper read, Tragic Mistake. Hopefully the woman's marriage wasn't defined by the headline.
#3 He Fits Right In
There is nothing worse than road construction. Traffic gets backed up and there are orange and white barrels and barriers everywhere. This picture makes road construction look funny. The man in the photo looks just like one of the orange and white barrels in the road. If he planned it, it is pretty funny. If he didn't, this photo is hilarious.
#4 Crazy Coincidence License Plates
The license plates on these cars aren't really funny alone. One reads, WE WRITE and the other reads, WEREAD. If you put the two together in a parking lot side by side the way they are in this picture, it is quite the coincidence. At least you know that one driver can read and the other can write.
#5 The Coffee and the Tee
Many people own stripped shirts. It is a very common pattern for clothing. The woman in this photo loves her black and white stripped t-shirt so much that she go coffee cups to match. When her friends discovered the coincidence and decided to take a picture, she seemed to be a bit embarrassed about the whole thing.
#6 A Vampire at a Blood Bank
Everyone knows that vampires love to suck blood, which makes this photo so funny. The license plate on the car at the blood bank reads VAMPYR. The blood bank is the perfect place for a vampire to get the nourishment that he needs.
#7 The Road To Success?
The tractor trailer truck in this photo just smashed into an overpass. On the truck it reads, On the road to success, there are no shortcuts." Sadly, the person who was driving this truck isn't on the road to success. They must have taken this route thinking it was a shortcut only to find that the area is not a thru area for trucks.
#8 Cereal
This photo can ruin breakfast for cereal lovers everywhere. On the left is a rabbit and a bowl of rabbit food. On the right is a box of Nesquik cereal and a bowl of cereal below. Both bowls of food look the same and suddenly the Nesquik doesn't look very appetizing.
#9 Converging Into Reality
These two photos are pretty amazing. In the photo on the left, a dog is walking behind a Chew Moo sign in just the right place to make it look as though the dog has a cow head. The photo on the right is of two billboards. The one on the top simply reads, Jesus. The one on the bottom reads, "beefy cheesy glory." Both billboards refer to religion, even though one is for McDonald's it is still a coincidence.
#10 Ironic Photos
These two photos are pretty ironic. The photo on the left has a sign that reads, "Thank you fro driving carefully." Right beside it is a car on its side that was clearly in an accident. The second photo was taken at an amusement park near a ride called the Wind Seeker. The sign beside the ride reads, "This ride is currently CLOSED due to high winds. Maybe the Wind Seeker found the wind and it shut the ride down.
#11 No-Chew?
The man in this photo bought a spray that is designed to keep dogs from biting, chewing, and licking places where it is sprayed. It is supposed to have a bitter taste. Unfortunately, the taste wasn't too bitter because his dog had no problem chewing up the bottom of the bottle.
#12 Twins?
Some people say that after a while, a person starts to look just like their pet. In this case it is actually true. The man in the photo and his pet hedgehog have the exact same haircut.
#13 The Perfect Chair the Perfect Shirt
The boy in this photo is wearing a Kiss t-shirt, one of the greatest bands of all time. The lead singer is known for sticking his very long tongue out like he is doing on this t-shirt. The boy just happened to sit in a chair where someone drew a picture of an ice cream cone and he sat in exactly the right position to make it look like he is eating the ice cream cone.
#14 He Was Only Following Directions
The person driving this truck was going to park when he saw the Crush soda machine. Rather than parking in a regular space, the driver chose to follow the directions on the soda machine that read Crush.
#15 Turn To Your Right!
These women are sitting side by side. One is reading the newspaper and the other is biting her nails. The irony is that the headline on the newspaper reads, "Killed By Nail Biting."
#16 We Don't Fit
Here is another example of a truck driver who didn't know the limits of his truck. The ironic part is that on the back of the truck it reads, we fit. Unfortunately, it didn't fit.
#17 The Devil?
The man on the news is trying to talk is way out of a scandal. The man sitting behind him is wearing a button-up shirt. The collar makes it look like the speaker has devil horns. It makes you wonder what he thought when he saw himself on television.
#18 What a Coincidence
The photo on the left is of a person about to give his cat a treat who looks exactly like the cat on the package. The photo on the right is of the back of a man's head. The person taking the photo happened to be eating a piece of chocolate that looked just like the man's head.
#19 Taylor Swift
Taylor Swift is one of the most popular musicians today. The person in this car happened to be driving behind two trucks, one was a Taylor truck and the other was a Swift. This is quite the coincidence.
#20 The Batman Song
The mother of these children got her kids dressed and couldn't figure out why her husband thought that it was so funny. If you read the girl's shirt and then her brother's it is the Batman song.
#21 The Dog Can Read
The dog in this photo looks like he loves the car and he can also read. The license plate reads, 1EARUP. He just so happens to have one ear up.
#22 Baby Needs Beers & Wines
Most stores have signs at the beginning of each aisle to let you know what is in the aisle. Whoever designed this store made a mistake. The sign looks like it is telling you that the baby needs some wine and beer.
#23 Perfect Placement
The two businesses in the corner of this plaza are Girls Night Out and Planned Parenthood. They go together quite well.


text:
Despite a reasonable number of wine aficionados out there, most wine and spirit buyers wouldn't know a claret from a clarinet. And those who do frequent the upscale shelves have one thing in common with their less fussy comrades: packaging is often what makes the sale.
This month, FiberMark unveiled a premium portfolio of folding carton products that can help designers give their wine and spirit packaging an elegant touch.
These dyed-through board materials:
- Ensure color consistency from lot to lot
- Are available in three calipers (12 pt, 18 pt and 26 pt)
- Provide custom color duplex solutions
- Come in a wide variety of embossed patterns
- Are all FSC certified
- Provide deep color saturation that does away with those maddening white edges in the finished product.
This new portfolio includes:
- Grafton – Premium board with an uncoated vellum finish
- Dorset – Premium board with a matte pigment-coated finish
- Metal-X and Shimmer (K-Series) – Luxury board products with distinctive, decorative coatings.
For more on these, don't miss the Sept. 25th's PaperSpecs Newsletter, where you will find an exclusive look at these premium solutions. (Click here to begin your free subscription today if you're not already a subscriber.)


text: Written by Mimi Tasheva on September 30, 2020. Posted in Programme Closures Untitled Document body {font-family: Tahoma, Verdana, Segoe, sans-serif; background-color:#efefef} a, a:link, a:visited {color:#ff6600;} p {line-height:18px;} img {display: block;} /* Shrink Wrap Layout Pattern CSS */ @media only screen and (max-width: 600px) { td[class="responsive"] { width: 100%; } } @media only screen and (max-width: 480px){ td[class="button"] { max-width:600px !important; width:100% !important; } td[class="button"] a { display:block !important; font-size:18px !important; } } Affiliate Program CLOSED: Found Hea Dear Affiliates We regret to inform you that Found Hea, ID 20072, is now closed. Please remove all existing links. All remaining commissions will be submitted to the advertiser for validation. Thank you for your efforts in promoting this advertiser. We suggest the following list of advertisers in this category that you can swap your links to: List of similar advertisers on the network: MID: 4244 - Tessuti MID: 4318 - Coggles UK MID: 1669 - Boden UK Alternatively you can click here to view more advertisers in the same category. Kind regards Awin Support email@example.com


text:
Do you consider time to work a waste of time?
I do. I don't have to drive or use bicycle, so I don't need to pay my attention to other things on the road. I walk or take public transport listening to a mp3 player in my ears. Another opportunity is to read, but this one is suitable only for long distance public transport. What to do if you don't have any?
Train your creativity with the objects in the street! Here're 5 simple tips to try.
1. Color scheme
Even a simple blue summer sky consists of few shades of blue and maybe a bit of grey. Life around us is full of colors.
Try to imagine what you see presented in just 3-5 colors. It can be some specific object or a whole scenery in general.
It doesn't only helps to be more creative but also helps to take a more careful look at a world around and like it more.
2. Going minimal
Going minimal is dangerous, there's a big chance to go too minimal when the point and functionality are being lost.
To practise doing it just the right amount take some object – for example a big advertising banner next to a bus stop – and try simplifying it as much as possible still keeping the idea of the message in.
3. Find geometry
Geometry is all around us. Square boxes of houses, round stop signs etc.
Try looking at the street you're in in terms of geometrical objects they consist of. Lines, shapes, forms.. how they make all together the thing you're looking at.
Personally I also find entertaining also looking at one side of the building imagining it whole as a stereometric drawing.
4. Going cartoon style
This is probably my favourite because it's a real fun.
Look at the person close to you and image him as a cartoon character (person or animal). Think of the impression that disney cartoon characters give to you – it's easy to imagine a life person behaving like that. Try to do the reverse. It's especially fun with people you know for a longer time.
5. Shoot a video
This one is very useful for people who wear headphones walking everywhere like me.
Try not just to listen just to the lyrics and sing along, but also imagine what kind of video you'd make for this song if you were a director.
Would you try these tips? )
image on top from here


text:
As part of Frankfurt Fashion Week, 23-year-old designer Samuel Gartner presented his show at the Senckenberg Natural History Museum.
Dressed in out-of-the-box looks, models walked past dinosaur skeletons, as they unveiled the collection.
Only 100 guests were invited to be a part of this fashion extravaganza due to COVID-19 restrictions.
Please click on the images for a better look at what the models are wearing.
IMAGE: Designer Samuel Gartner, right, and a model during the fittings.
In the background you'll spot the skeleton of a dinosaur.
All photographs: Thomas Lohnes/Getty Images
IMAGE: Models get clicked as they wait backstage for the rehearsals to begin.
IMAGE: A model poses in front of a dinosaur during the fittings.
The collection featured big collars and outfits with contrasting details.
IMAGE: Dressed in a tiger print dress and a matching hat, this model awaits her turn to walk the ramp.
IMAGE: The show will definitely go down in history as a fashion memory.
IMAGE: A better view of the settings at the museum.
Concept: Hitesh Harisinghani/Rediff.com
Feature presentation: Ashish Narsale/Rediff.com
Source: Read Full Article


text:
Prices reduced to clear an older batch. Part B is more viscous.
A revolutionary translucent silicone rubber that self-degases without the need for a vacuum machine. Now you can create bubble-free silicone moulds easily within a short duration of 2 to 3 hours. Non-toxic and safe to use indoors. Cures at room temperature.
An excellent choice for beginners due to its ease of use and self-degassing properties.
Mix Ratio: 1:1 by weightDemould: Approximately 2-3 hours (depending on volume mixed and temperature)
Hardness: Shore 15A
Cure inhibition:
Please take note that all silicone rubbers may be subject to cure inhibition by contaminants on the surface of the master, or the material of the master being used for mould making.
If your silicone rubber does not cure, there could be a chance that your master is reacting with the silicone rubber.
Materials that may inhibit the curing of platinum silicones are: Sulphur-based clay, tin-cured silicone rubber, latex rubber, some paint products, some 3D-printed plastic materials, etc.
Uncured rubber can be difficult to remove from your masters and may stain them. We recommend performing a small test on your master before making your actual mould.
Get the latest updates on new supplies, workshops and promotions!


text:
Abstract. Binding of a T cell to an appropriate antigen-presenting cell (APC) induces the rapid reorientation of the T cell cytoskeleton and secretory apparatus towards the cell–cell contact site in a T cell antigen receptor (TCR) and peptide/major histocompatibility complex–dependent process. Such T cell polarization directs the delivery of cytokines and cytotoxic mediators towards the APC and contributes to the highly selective and specific action of effector T cells. To study the signaling pathways that regulate cytoskeletal rearrangements in T lymphocytes, we set up a conjugate formation assay using Jurkat T cells as effectors and cell-sized latex beads coated with various antibodies as artificial APCs. Here, we report that beads coated with antibodies specific for the TCR-CD3 complex were sufficient to induce T cell polarization towards the bead attachment site, as judged by reorientation of the microtubule-organizing center (MTOC) and localized actin polymerization. Thus, these cytoskeletal changes did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-ζ and CD3ε, were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-ζ and once in CD3ε, revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling requirements.
Polarization of a T cell response towards a triggering antigen-presenting cell (APC)1 is thought to contribute to the specificity of the immune response. Upon encountering an APC, T cells rapidly undergo cytoskeletal polarization, which includes the formation of a tight collar of polymerized actin at the T cell–APC interface and the reorientation of the microtubule-organizing center (MTOC) towards the bound APC (Geiger et al., 1982; Ryser et al., 1982). Whereas F-actin accumulation at the cell–cell interface was suggested to stabilize and favor continuous T cell antigen receptor (TCR)–antigen interactions (Valitutti et al., 1995b), MTOC reorientation is thought to position the T cell secretory apparatus into close proximity with the APC. Indeed, several studies showed that repositioning of the MTOC was accompanied by the polarized concentration of cytokines and cytotoxic mediators at the T cell–APC/target interface (Kupfer and Dennert, 1984; Kupfer et al., 1991, 1994). Moreover, Poo et al. (1988) have demonstrated that limited TCR cross-linking in a cloned helper T cell line triggered the release of cytokines preferentially over the area of receptor cross-linking. Thus, the engagement of only a fraction of TCRs on a helper T cell results in the polarized delivery of cytokines towards the site of the stimulus.
Cytoskeletal polarization of a T cell is dependent on the presence of specific antigen (Kupfer and Singer, 1989), suggesting the involvement of a TCR-mediated signaling pathway. A variety of cellular responses, including gene activation, proliferation, and secretion of cytokines and cytotoxic mediators, have been reported to be initiated by TCR engagement. Interestingly, activation of Jurkat T cells with immobilized TCR-specific antibodies could induce actin polymerization and formation of F-actin-rich pseudopods, suggesting that TCR-mediated signaling pathways may indeed regulate cytoskeletal organization (Melamed et al., 1991). Furthermore, a recent report implies a role for the small GTPase CDC42 in APC-mediated T cell polarization (Stowers et al., 1995). Members of the Rho subfamily of small GTPases (Rho, Rac, CDC42) are evolutionary conserved regulators of the actin cytoskeleton in a variety of different cell systems (Hall, 1994). Consequently, TCR-mediated signals might activate small GTPases by an as yet unidentified mechanism. However, T cell–APC conjugate formation involves the clustering and engagement of various other T cell surface receptors besides TCR–antigen interactions, including the CD8/CD4 coreceptors and integrins (Kupfer and Singer, 1989; Kupfer et al., 1987). It is conceivable that triggering of these additional receptors is required for or at least contributes to APC-induced T cell polarization.
The TCR is coupled to intracellular signaling pathways by two noncovalently associated signal-transducing complexes, namely the CD3 (γ, δ, and ε) and the TCR-ζ chains. Signaling through these complexes requires a conserved sequence motif (YxxLx(6–8)YxxL), termed the immunoreceptor tyrosine-based activation motif (ITAM) (Irving and Weiss, 1991; Romeo and Seed, 1991). The CD3 and TCR-ζ chains each contain one and three ITAMs, respectively. Upon TCR engagement, the tyrosine residues in the ITAMs are rapidly phosphorylated by the Src-family protein tyrosine kinase (PTK) Lck (van Oers et al., 1996) and serve as docking sites for src-homology-domain 2 (SH2) containing signaling molecules such as the PTKs of the Syk/zeta-associated protein 70 (ZAP-70) family. After recruitment, Syk/ZAP-70 is activated by phosphorylation and contributes to the initiation of downstream signaling events such as calcium mobilization and activation of the Ras pathway. Single CD3 and TCR-ζ chains can independently activate a variety of qualitatively similar proximal and distal signaling events (Irving and Weiss, 1991; Letourneur and Klausner, 1991; Romeo and Seed, 1991; Shinkai et al., 1995), suggesting that the presence of multiple ITAMs in the TCR mainly serves the purpose of signal amplification. However, amino acids surrounding the ITAMs differ in the CD3/TCR chains and have been shown to bind to distinct SH2-containing signaling molecules (Osman et al., 1995, 1996; Songyang et al., 1993). This suggests that, in addition to signal amplification, distinct ITAMs might couple the receptor to qualitatively different signaling pathways. For example, chimeric proteins containing the cytoplasmic tail of CD3ε or TCR-ζ differ in their ability to induce apoptosis in a murine T cell line (Combadiere et al., 1996). Furthermore, the third TCR-ζ ITAM and the CD3ε ITAM have been implicated in coupling the TCR to the actin cytoskeleton in an activation-dependent manner (Rozdzial et al., 1995). Thus, individual subunits might couple the TCR to effector molecules involved in the regulation of cytoskeletal changes.
To investigate the role of TCR-mediated signaling pathways in APC-induced T cell polarization, we set up a conjugation assay using Jurkat T cells or mutants derived from it as effector cells, with antibody-coated, cell-sized latex beads as APCs. We show here that TCR cross-linking alone is sufficient to induce MTOC polarization and actin polymerization at the T cell–bead interface. Cytoskeletal reorientation required the presence of at least one intact ITAM. In support of this result, no cytoskeletal changes could be observed in the Lck-deficient Jurkat mutant JCaM1.6. Furthermore, our studies demonstrate that MTOC reorientation occurs in the absence of Ras activation. Our results imply that APC-induced T cell polarization is a TCR-mediated event that is coupled to the receptor by the same signaling motif as TCR-mediated gene activation, but diverges in its distal signaling requirements, since Ras is not required. The assay described in this study might be a useful tool to characterize signaling molecules involved in T cell polarization.
Materials And Methods
Cells and Reagents
The Jurkat T cell subclone E6, JCaM1.6 (Goldsmith et al., 1988) and large T antigen (TAg) Jurkat cells (kindly provided by Dr. G. Crabtree, Stanford University, Palo Alto, CA; Clipstone and Crabtree, 1992) were maintained in RPMI 1640 medium supplemented with 5% fetal calf serum (Intergen Co., Purchase, NY), 2 mM glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin. Jurkat-derived stable clones were cultured in complete medium containing 10% FCS and 2 mg/ml G418 (GIBCO BRL, Gaithersburg, MD). JCaM1.6/Lck cells (Straus and Weiss, 1992) were maintained in complete medium containing 10% FCS and 250 μg/ml hygromycin (GIBCO BRL). Poly-l-lysine was obtained from Sigma Chemical Co. (St. Louis, MO). The MAPKK inhibitor PD 098059 and Wortmannin were purchased from Calbiochem-Novabiochem Corp. (San Diego, CA). Polystyrene latex microspheres (diameter 6 μm) were purchased from Polysciences Inc. (Warrington, PA). Antibodies were absorbed to the beads as previously described (Mescher, 1992). Briefly, 5–10 μg of purified antibody were mixed with 107 polystyrene beads in a final volume of 1 ml PBS, and incubated for 90 min at room temperature with constant tumbling. Beads were then blocked in 1.5 ml of PBS/1% BSA for 30 min. After three washes in PBS, latex beads were resuspended in PBS and stored at 4°C. Efficient antibody absorption was verified by flow cytometry.
Antibodies used for stimulation and immunofluorescence microscopy are as follows: the mAb C305 (IgM) specifically recognizes the Jurkat Ti β chain (Weiss and Stobo, 1984). Leu 4 (IgG1) is directed against the human CD3ε chain. RBC4 (IgM) recognizes the transferrin receptor. The mAb 9.1 (IgG3) is specific for human CD2 (Yang et al., 1986). Mouse mAb OKT8 recognizes an extracellular epitope of human CD8 and was acquired from American Type Culture Collection (Rockville, MD). The mAb 7G7B6 is directed against murine CD25 (Tac) and was obtained from American Type Culture Collection. A mouse mAb to human CD11a (IgG1, SPV-L7) was purchased from Zymed Laboratories, Inc. (S. San Francisco, CA). A rat mAb to α-tubulin (YOL1/34) was obtained from Harlan Sera-Laboratories (Crawley, UK) and was detected with an FITC-conjugated, affinity-purified donkey anti–rat (Fab′)2 antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). F-actin was detected with rhodamin-phalloidin (Molecular Probes, Inc., Menlo Park, CA). Antiphosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). An mAb against human Ha-Ras was purchased from Transduction Laboratories (Lexington, KY).
The dominant-negative ZAP-70 constructs SH2 (N+C) and SH2 (N+*C) are described elsewhere (Qian et al., 1996). The N17Ras plasmid was a kind gift from D. Cantrell (ICRF, London, UK).
Chimera Construction and Clones
Stable clones expressing the TT-ε chimeras were kindly provided by Dr. Nicolai S.C. van Oers. Plasmids encoding for chimeric proteins containing the external and transmembrane portions of CD25 fused to the cytoplasmic tails of either CD3ε (TT-ε) or TCR-ζ (TT-ζ) were a gift from Dr. R.D. Klausner (National Cancer Institute, Bethesda, MD; Letourneur and Klausner, 1991). A truncated version of TT-ε was generated by PCR mutagenesis of the original TT-εTo construct (kindly provided by Dr. R.D. Klausner) using the following primers: 5′ primer (within Tac): 5′ ACAGATCTCCAGGTAGCAGTG 3′; 3′ primer (within CD3ε): 5′ TCGATATCACCTATTCTTGAGCCCACT 3′. Wild-type TT-ε in pCDL SRα was replaced by the BglII/EcoRV-digested PCR fragment. The new truncated TT-ε encompasses the three first amino acids of the CD3ε cytoplasmic tail. All constructs were subcloned into pcDNA3 (Invitrogen Corp., San Diego, CA) and transfected into Jurkat T cells by electroporation. Briefly, 107 cells were transfected with 20 μg of plasmid in a Gene Pulser (Bio-Rad Laboratories, Hercules, CA) at 250 V and a capacitance of 960 μF. After transfection, cells were grown for 2 d in complete RPMI before plating out in medium containing 2 mg/ml of G418 (GIBCO BRL). Clones were obtained by limiting dilution. Generation of the CD8-ζ clone has been described elsewhere (Irving and Weiss, 1991). Stable cell lines expressing the wild-type CD8-ζT76 truncation or the CD8-ζ4F mutant were generated as described elsewhere (Irving and Weiss, 1991). Construction of CD8-ζT76 and CD8-ζ4F vectors was based on modifications of the original CD8-ζ plasmid. The CD8-ζT76 truncation mutant was created by PCR mutagenesis from the CD8-ζ wild type using the following oligonucleotides: 5′ primer (within CD8): 5′ CACCATCGCGTCGCAGC3′; T76 truncation: 5′ GATCTCGAGAGGATCCTATTACATCCCAATCTCACTG 3′.
After PCR amplification, the CD8-ζT76 truncated fragment was digested with EcoRV and BamHI, and substituted for the wild-type CD8-ζ in the ptf neo CD8-ζ expression vector (Irving and Weiss, 1991). The mutation was confirmed by sequencing. The CD8-ζT76 mutant truncated the ζ chain two amino acids after the second ITAM. The CD8-ζ4F mutant was created from the CD8-ζT76 truncation by sequential PCR mutagenesis. The outside primers and fragment substitution were the same as for the creation of the CD8-ζT76 truncation. After PCR mutagenesis, the construct was sequenced to verify that only the intended mutations existed.
To mutate the first tyrosine to phenylalanine of the first ITAM of CD8-ζ, the following primers were used: 5′ CAGCTCTTTAACGAGCTCAA 3′, and 3′ TCTTGGTCGAGAAATTGCTC 5′.
To mutate the second tyrosine to phenylalanine of the first ITAM of CD8-ζ, the following primers were used: 5′ GAGGAGTTCGATGTTTTGGA 3′, and 3′ CTTCTCTCCTCAAGCTACAA 5′.
To mutate the first tyrosine to phenylalanine of the second ITAM of CD8-ζ, the following primers were used: 5′ GGCCTGTTCAATGAACTGCA 3′, and 3′ TCCTTCCGGACAAGTTACTT 5′.
To mutate the second tyrosine to phenylalanine of the second ITAM of CD8-ζ, the following primers were used: 5′ AGGCCTTCAGTGAGATTGG 3′, and 3′ ACCGCCTCCGGAAGTCAC 5′.
Conjugate Formation and Immunofluorescence Microscopy
Jurkat T cells or derived clones and mutants were mixed with antibody-coated latex beads at a 2:1 ratio. After centrifugation for 5 min at 100 g, the cell–bead mixture was incubated for an additional 5–25 min at 37°C. Conjugates were then resuspended, plated onto poly-l-lysine-treated slides, and fixed for 30 min in 3.4% paraformaldehyde at room temperature. Fixed cells were permeabilized for 4 min in PBS/0.1% Triton X-100 and blocked for 10 min in PBS/0.2% BSA. Cells were labeled for 30 min with antibody diluted in PBS/0.2% BSA, followed by two washes of 10 min each. After secondary antibody labeling, cells were incubated for 10 min in 33 nM rhodamin-phalloidin. After three final washes, slides were mounted. Samples were either viewed under a laser scanning microscope (LSM 410; Carl Zeiss, Inc., Thornwood, NY) (1st experiment) or a conventional Microphot-FXA microscope (all other experiments). The MTOC was scored as reoriented if it was located in close proximity to the T cell plasma membrane between the T cell nucleus and the bead contact area. At least 100 conjugates were scored in each experiment.
Transient Transfection Conjugate Formation Assay
Cell (107) were transiently cotransfected by electroporation, as described above, with 30 μg of the indicated TT chimera and 40 μg of a vector containing no insert or expressing dominant-negative mutants of ZAP-70 and Ras. After 36 h, dead cells were eliminated by centrifugation over a Ficoll gradient (Sigma Chemical Co.). After two washes, conjugates were formed and analyzed as described above.
Anti–TCR-coated Latex Beads Induce MTOC Reorientation and Polarized Actin Polymerization in Jurkat T Cells
Previous T cell polarization studies performed in a B cell–T cell system demonstrated the dependence of cytoskeletal rearrangements during conjugate formation on the presence of antigen, implying the participation of the TCR in these events (Kupfer and Dennert, 1984). The involvement of accessory molecules including integrins and coreceptors such as CD4/CD8 made it difficult to evaluate to what extent TCR-linked pathways were involved and sufficient for T cell cytoskeletal polarization. To address this question, we set up an in vitro assay using Jurkat T cells as effectors, and antibody-coated, cell-sized latex microspheres as APCs. Previous studies have demonstrated that such artificial APCs, either coated with TCR-specific antibodies or antigen–major histocompatibility complexes (MHC), could induce early and late signaling events, such as tyrosine phosphorylation and exocytosis, in T cells (Anel et al., 1994; Mescher, 1992). In contrast to soluble or plate-bound TCR-specific antibodies, the use of latex beads allowed us to study signaling events in response to a focal stimulus. In a first set of experiments, we investigated whether triggering of the TCR alone was sufficient to induce reorientation of the microtubule cytoskeleton and polarized actin polymerization. Latex beads coated with the CD3ε-specific antibody Leu4 were mixed with Jurkat T cells at a 1:2 ratio and incubated for 30 min at 37°C. Formed conjugates were fixed and stained for tubulin and F-actin. The position of the MTOC within the T cells was analyzed microscopically. Only MTOCs directly underlying the bound latex bead were scored positive. In the majority of all conjugates (73 ± 2%), the T cell MTOC was facing the latex bead contact area (Fig. 1,a). Polymerized actin was concentrated in a dense collar at the bead attachment site. F-actin-rich pseudopods protruded from the T cell surface, and in many cases surrounded the bead (Fig. 1,b). Similar results were obtained at shorter incubation times (10–20 min) and with latex beads coated with the Jurkat Tiβ-specific antibody C305 (data not shown). In contrast, the position of the MTOC was random when conjugates between Jurkat T cells and poly-l-lysine-treated beads were analyzed (Fig. 1,a). Furthermore, no F-actin accumulation at the bead contact site could be observed (Fig. 1 b).
To determine whether cytoskeletal polarization was specifically associated with TCR cross-linking, antibodies directed against a variety of T cell surface molecules (CD11a/ LFA-1, transferrin receptor [TfR] and CD2) and poly-l-lysine were bound to latex beads and used in the conjugate formation assay. All surface molecules analyzed were expressed at comparable levels in Jurkat T cells and similar numbers of bead–cell conjugates were formed (data not shown). As summarized in Fig. 2, TCR/CD3-specific beads displayed MTOC polarization in ∼80% of all scored conjugates, whereas beads coated with either anti–CD11a, anti–TfR, anti–CD2, or poly-l-lysine were randomly positioned (31 ± 5, 26 ± 2, 39 ± 4, and 26 ± 6% of scored conjugates, respectively), demonstrating that cytoskeletal polarization is specifically associated with TCR/CD3 cross-linking.
Clustering of Chimeras Containing the Cytoplasmic Domain of CD3ε or TCR-ζ Can Induce MTOC Reorientation and Actin Polymerization
Several studies support the view that the presence of multiple ITAMs in the TCR might serve the purpose of signal amplification (Irving et al., 1993; Shinkai et al., 1995). However, single TCR subunits can associate with distinct SH2-containing signaling molecules and show qualitative differences in the induction of certain cellular responses (Combadiere et al., 1996; Osman et al., 1996). This suggests that distinct TCR subunits could couple the receptor to qualitatively different signaling pathways. To evaluate the ability of single TCR chains to induce cytoskeletal changes, we analyzed Jurkat T cells stably expressing a chimera between the extracellular and transmembrane domain of CD25 (Tac) with either the cytoplasmic tail of TCR-ζ (TT-ζ), CD3ε (TT-ε), or a truncated form of CD3ε (TT-εT) containing only the first three amino acids of the cytoplasmic tail. An ITAM motif is present once in the CD3ε chain and three times in the TCR-ζ chain. The ITAMs in both chains differ in their nonconsensus sequences and were shown to bind signaling molecules with different affinities (Osman et al., 1996). As demonstrated previously (Letourneur and Klausner, 1991; Shinkai et al., 1995), TT chimeras are expressed as monomeric molecules and do not associate with endogenous CD3 subunits. Clones with similar surface expression of the chimeric proteins (Fig. 3) were chosen and mixed with anti–Tac-coated latex beads for 10 min. The percentage of conjugate formation was comparable (data not shown). As shown in Fig. 4,A, clustering of the cytoplasmic tail of either TCR-ζ or CD3ε was sufficient to induce MTOC reorientation. The extent of MTOC reorientation triggered by either chimera was comparable and was similar to levels induced by cross-linking of the complete TCR-CD3 complex. Furthermore, both chimeras could induce actin polymerization (Fig. 4, B and C). In contrast, no cytoskeletal polarization was detected towards beads bound to the truncated TT-εT chimera. These results suggest that both the ζ and ε chains can trigger qualitatively similar signals, which are normally generated by the TCR and induce T cell polarization. Furthermore, the presence of one ITAM motif was sufficient to induce cytoskeletal rearrangements, and the number of ITAMS had no obvious quantitative effect on the induction of cytoskeletal polarization. However, in transient expression experiments where chimera surface expression was lower, the TT-ε chimera induced MTOC reorientation less efficiently than TT-ζ (see below and data not shown). This result supports the view that multiple ITAMs can act as signal amplifiers under suboptimal activation conditions.
MTOC Reorientation and Actin Polymerization Require the Presence of an Intact ITAM
ITAM phosphorylation is essential for signaling through the TCR in many cellular responses. Phosphorylated ITAMs serve as docking sites for downstream signaling molecules such as ZAP-70 (Osman et al., 1996). Furthermore, phosphorylated ITAMs can mediate the activation-induced association of TCR components with the actin cytoskeleton. In particular, the third ITAM of the TCR-ζ chain has been reported to be crucial for F-actin interaction (Rozdzial et al., 1995). To investigate the role of ITAM phosphorylation and F-actin binding in coupling the TCR to T cell polarization, we analyzed stable clones expressing a chimera between the extracellular/transmembrane domain of human CD8 and either the entire cytoplasmic tail of TCR-ζ (CD8-ζ), the cytoplasmic tail of TCR-ζ truncated after the second ITAM (CD8-ζT76), or the CD8-ζT76 chimera in which all four ITAM tyrosines were mutated to phenylalanine (CD8-ζ4F) (Fig. 5,A). Clones with comparable surface expression were used (Fig. 6). As shown before for the TT-ζ expressing clone, cross-linking of the CD8-ζ chimera efficiently induced MTOC reorientation and actin polymerization at the bead attachment site (Fig. 5, B and C). No obvious qualitative difference was observed for the CD8-ζT76 chimera, which lacks the third ITAM, suggested to mediate F-actin binding. Focal actin polymerization was as frequently detected with CD8-ζT76 as in the CD8-ζ chimera (Fig. 5,C). As judged by immunofluorescence microscopy, no significant difference in the extent of F-actin accumulation was observed (Fig. 5,D). Our data suggest that the observed clustering of F-actin at the site of TCR engagement occurs in the absence of the third ζ chain ITAM. In contrast, analysis of CD8-ζ4F demonstrated the strict dependence of both MTOC reorientation and actin polymerization on the tyrosine phosphorylation of the ITAM motif. Mixing of clone CD8-ζ4F with anti–CD8-coated latex beads still yielded conjugates, but MTOC positioning was random (38 ± 7%) (Fig. 5,B). The frequency and extent of actin polymerization was also dramatically reduced (Fig. 5, C and D). These results indicate that ITAM phosphorylation is required for TCR-induced cytoskeletal polarization.
To corroborate this finding, we analyzed cytoskeletal rearrangements in the mutant Jurkat cell line JCaM1.6, which lacks functional Lck. Src family kinases and in particular Lck have been shown to be responsible for ITAM phosphorylation in T cells (Iwashima et al., 1994; van Oers et al., 1996). Consistent with the failure to phosphorylate ζ and CD3 chains, TCR-ligation of JCaM1.6 fails to induce tyrosine phosphorylation, calcium influx, or CD69 expression (Straus and Weiss, 1992). Analyzed in our conjugate formation assay, JCaM1.6 failed to polarize towards CD3-specific latex beads. MTOC positioning was random (30 ± 4% of scored conjugates) and no actin polymerization was detected (Fig. 7, A and B). Reconstitution of JCaM1.6 with wild-type murine Lck (JCaM1.6/Lck) restored TCR-induced cytoskeletal changes comparable with that observed in the parental Jurkat cell line (Fig. 7, A and B). Similar results were obtained with the mutant Jurkat cell line J45.01, which lacks functional CD45 (data not shown). CD45 is a transmembrane tyrosine phosphatase required for the activation of Src family kinases (Weiss and Littman, 1994). Taken together, these results support the view that ITAM phosphorylation and subsequent recruitment of downstream signaling molecules to the TCR are a prerequisite for cytoskeletal alterations.
Overexpression of a Dominant-Negative ZAP-70 Mutant Impairs MTOC Reorientation
A crucial step in TCR-mediated signaling is the recruitment of ZAP-70 to phosphorylated ITAMs after TCR engagement. Recruitment leads to tyrosine phosphorylation and activation of ZAP-70, which, in turn, contributes to the activation of both early and late signaling cascades. Several studies suggest that ZAP-70 plays in important role in TCR-mediated signaling. For example, lack of functional ZAP-70 in humans and mice results in severe developmental and functional defects in the T cell compartment (Arpaia et al., 1994; Chan et al., 1994; Elder et al., 1994). Furthermore, introduction of a dominant-negative mutant form of ZAP-70 into T cells suppresses TCR-mediated tyrosine phosphorylation of several signaling molecules and activation of NF-AT, a nuclear factor essential for inducible interleukin 2 gene expression (Qian et al., 1996). To assess the role of the ZAP-70–ITAM interaction in TCR-mediated MTOC reorientation, we transiently cotransfected Jurkat cells with the activating chimera TTε and the dominant-negative ZAP-70 mutant SH2 (N+C) (Qian et al., 1996). SH2 (N+C) is thought to inhibit TCR signaling by blocking phosphorylated ITAMs (Qian et al., 1996). Transfected cells were mixed with anti–Tac latex beads 36 h later, and conjugates were formed for 10 min at 37°C. Similar to our studies in stable clones, engagement of TT-ε induced MTOC reorientation, although to a lesser extent (67 ± 1% of the conjugates). As expected, engagement of transiently expressed TT-εT failed to induce MTOC reorientation (20 ± 6%) and served as a negative control. Coexpression of dominant-negative ZAP-70 markedly reduced MTOC reorientation by ∼50–60%, whereas expression of a mutant SH2 (N+C) form (SH2 (N +*C), (Qian et al., 1996), unable to bind to ITAMs, had only minimal effects (Fig. 8). Expression of both mutants did not affect TT-ε surface expression (data not shown). Moreover, comparable results were obtained in Jurkat TAg cells (data not shown). These results demonstrate that engagement of transiently expressed activating chimeras can induce MTOC reorientation. Moreover, a dominant-negative mutant of ZAP-70 encompassing the two SH2 domains impairs both TCR-mediated MTOC reorientation (shown here) and NF-AT gene activation (Qian et al., 1996). This suggests that both responses are mediated by the same proximal signaling events requiring molecules that interact with phosphorylated ITAMs.
MTOC Reorientation Occurs in the Absence of Ras Activation
Two major signaling pathways, namely the Ras and the PLC-γ1 pathway, are activated after TCR engagement. Activation of the small GTPase Ras by the TCR induces the extracellular signal-regulated kinase (ERK 1,2) cascade involving several cytoplasmic serine/threonine kinases. ERKs phosphorylate transcription factors of the AP-1 family and are necessary for TCR-mediated NF-AT gene activation and T cell development (Alberola-Ila et al., 1995; Izquierdo-Pastor et al., 1995; Swan et al., 1995). In certain cell types, ERKs are associated with microtubules (Morishima-Kawashima and Kosik, 1996; Reszka et al., 1995) and phosphorylate microtubule-associated proteins (MAPs). MAPs stimulate the polymerization of new and preexisting microtubules. Interestingly, these microtubule-promoting properties are lost after in vitro phosphorylation by ERKs (Hoshi et al., 1992). Thus, TCR-induced ERK activation could influence microtubule dynamics. To address this question, we took advantage of the recently described compound PD 098059, a selective inhibitor of the ERK-activating enzyme MAPK/ERK kinase (Dudley et al., 1995). To characterize the effect of PD 098059 on TCR-induced ERK activation, Jurkat T cells were preincubated for 30 min with increasing concentrations of PD 098059 and were stimulated for 10 min by the addition of C305-coated latex beads. Cells were then lysed and analyzed for tyrosine phosphorylation by immunoblotting. As shown in Fig. 9,A, incubation with PD 098059 resulted in a dose-dependent inhibition of ERK phosphorylation in response to TCR engagement, whereas the phosphorylation status of various other proteins remained unchanged. The identity and quantity of ERK detected was confirmed by stripping and reblotting the same membrane with a phospho-ERK–specific antibody (data not shown). This experiment demonstrates that PD 098059 indeed specifically inhibits ERK phosphorylation. However, the inhibitor had no significant effect on MTOC reorientation (Fig. 9 B), even at concentrations that substantially reduced ERK phosphorylation. This suggests that ERKs are not involved in the regulation of cytoskeletal events associated with MTOC reorientation.
Besides ERKs, the small GTPase Rac-1 and phosphatidylinositol 3 kinase (PI-3 kinase) have been recently identified as Ras effector molecules (Genot et al., 1996; Rodriguez-Viciana et al., 1994). Interestingly, both PI-3 kinase and CDC42, a close family member of Rac-1, have been shown to be involved in B cell–induced MTOC reorientation of a murine T cell hybridoma (Stowers et al., 1995). Thus, TCR-mediated Ras activation might induce MTOC reorientation via ERK-independent pathways. To investigate this possibility, we transiently coexpressed the activating TT-ε chimera with the dominant-negative Ras mutant Ha-N17Ras in TAg Jurkat cells. Earlier studies have demonstrated that overexpression of this Ras mutant efficiently inhibits TCR-mediated events such as NF-AT/IL-2 gene activation or T cell development (Alberola-Ila et al., 1995; Rayter et al., 1992). However, when analyzed in the polarization assay, the mutant did not significantly affect MTOC reorientation (Fig. 9,C), although high expression levels could be detected by Western blotting (Fig. 9,D). Furthermore, MTOC reorientation in Jurkat T cells pretreated with the specific PI-3 kinase inhibitor Wortmannin was comparable to cells pretreated with the vehicle alone (Fig. 10). These results further substantiate our inhibitor experiment and suggest that Ras-mediated effector pathways are not involved in TCR-mediated microtubule reorganization. In contrast to earlier studies (Stowers et al., 1995), inhibition of PI-3 kinase did not affect MTOC reorientation in our assay system. This discrepancy might reflect differences in the assay systems used.
Upon binding to an APC, T cells undergo rapid and dramatic cytoskeletal changes that ensure the polarized delivery of an effector response. To date, T cell polarization has been mainly studied in a B cell–T cell system, where complex receptor–ligand interactions complicate the identification of the participating signaling pathways. In this report, we describe a simple bead-based conjugate formation assay to study this response. Jurkat T cells were chosen as model effector cells. Although this leukemic cell line may differ in many aspects from primary T cells, it has been a powerful tool in dissecting signaling pathways involved in T cell activation. Jurkat T cells can be easily manipulated by transient transfection approaches, and several TCR-signaling mutants have been generated and characterized. APCs have been replaced by antibody-coated latex beads. Since antibody-induced TCR cross-linking may induce stronger signals than TCR engagement by peptide–MHC complexes, our assay system may underscore the need for accessory molecules such as adhesion receptors or coreceptors. Furthermore, T cell activation with immobilized antibodies may not exactly reflect the role of receptor membrane motility and recycling in the generation of an intracellular signal. Nevertheless, the assay system should be useful to characterize the minimal requirements for polarized cytoskeletal rearrangements and to identify key-regulators of this mechanism. Our analysis provides evidence that TCR engagement alone is sufficient to induce MTOC reorientation and focal F-actin accumulation, suggesting that TCR-dependent signaling pathways can regulate cytoskeletal dynamics.
The induction of actin polymerization through antigen receptor activation has been previously observed in a variety of cell types including T cells, B cells, and macrophages (Greenberg et al., 1991; Melamed et al., 1991; Parsey and Lewis, 1993), but so far the underlying signaling pathways have been elusive. In contrast to cell activation by soluble factors, antigen-induced T cell activation is triggered by spatially constrained ligands; thus, TCR-induced cytoskeletal changes could influence the cellular response in several ways. First, cytoskeleton-dependent changes in cell shape and motility could increase the frequency of APC contacts, eventually resulting in the formation of stable conjugates (Negulescu et al., 1996). Second, actin polymerization could be required for the rapid reorganization and concentration of accessory molecules and receptors at the T cell–APC contact site, which would further increase conjugate stability and result in the formation of signaling complexes (Monks et al., 1997; Shaw and Dustin, 1997). Focally clustered F-actin could also serve as a spatial cue, recruiting proteins involved in MTOC reorientation. Such a role for F-actin has been postulated in the mechanism of spindle rotation during the embryogenesis of Caenorhabditus elegans (Waddle et al., 1994). Third, reorientation of the MTOC and organellar reorganization could ensure the delivery of a polarized immune response by effector T cells to a specific target cell in crowded environments such as lymphoid organs.
Two complexes within the TCR, namely the TCR-ζ and CD3 chains, couple the receptor via ITAMs to the intracellular signaling pathways. Interestingly, individual phosphorylated ITAMs bind differentially to SH2-containing signaling molecules in vitro (Osman et al., 1995, 1996) and could therefore activate distinct signaling pathways (Combadiere et al., 1996; Letourneur and Klausner, 1992). In our experiments, TCR-ζ and CD3ε were equally able to trigger MTOC reorientation and actin polymerization in stable clones. However, when transiently expressed at lower levels, CD3ε induced MTOC reorientation less efficiently compared with ζ, supporting the concept that multiple ITAMs can serve as signal amplifiers. ITAM-mediated signal amplification may be crucial when MTOC reorientation is induced by a physiological stimulus; i.e., a peptide–MHC complex. Earlier studies have demonstrated that MTOC reorientation in antigen-specific T cell–B cell conjugates was highly antigen-dose dependent and occurred inefficiently at low antigen levels (Kupfer et al., 1987). Antibody-induced TCR cross-linking, as used in our study, may induce a more potent signal than TCR engagement by peptide–MHC complexes. Thus, the requirements for multiple ITAM-containing TCR/CD3-chains may differ between the in vitro system and the in vivo situation.
A signaling molecule that binds preferentially to ζ ITAMs, but not to the ε ITAM in vitro, is the p85 regulatory subunit of PI-3 kinase (Osman et al., 1996). Interestingly, inhibition of PI-3 kinase with the specific inhibitor Wortmannin completely abolished MTOC reorientation and actin polymerization in a B cell–T cell hybridoma conjugate assay (Stowers et al., 1995). This result suggests that PI-3 kinase is a regulator of T cell polarization. Tested in our system, a TT-ε chimera readily induces cytoskeletal polarization. This indicates that PI-3 kinase is either activated independently of its recruitment to the CD3ε ITAM or is not involved in the cytoskeletal responses in our assay. Since the PI-3 kinase inhibitor Wortmannin had no effect on T cell polarization in our hands, we favor the second interpretation. The discrepancy observed between the two systems could reflect either cell line–specific differences or the different modes used to induce polarization. To generate a strong enough signal, B cell–T cell conjugates may need the contribution of adhesion molecules that may signal via a PI-3 kinase-dependent mechanism. Furthermore, efficient signaling by peptide–MHC complexes requires the serial engagement of TCRs (Valitutti et al., 1995a). PI-3 kinase could be involved in the recruitment and concentration of TCR molecules at the B cell–T cell contact area.
ITAM phosphorylation is a key event in T cell activation. Our analysis of ζ-chain chimeras and Jurkat mutants demonstrates that MTOC reorientation and actin polymerization require the presence of at least one intact ITAM and a functional ITAM-phosphorylating Src-family kinase. Similarly, Cox et al. (1996) showed recently that clustering of the ITAM-containing Fc receptor γ subunit can trigger submembranous actin assembly in the chicken B cell line DT40. Actin polymerization required ITAM phosphorylation and the presence of the PTK Syk. In T cells, the Syk/ZAP-70 family member ZAP-70 can bind to phosphorylated ITAMs of all TCR signaling subunits with very high affinity (Osman et al., 1996). Recruitment of ZAP-70 to the receptor results in ZAP-70 phosphorylation and activation. Overexpression of a dominant-negative mutant of ZAP-70, which blocks phosphorylated ITAMs, markedly reduced MTOC reorientation in our studies. This result suggests that ZAP-70 or another phospho-ITAM-binding molecule may be involved in the regulation of microtubule dynamics. In fact, both Zap-70 and Syk can associate and efficiently phosphorylate α-tubulin in vitro (Huby et al., 1995; Peters et al., 1996). Furthermore, two recent reports provide evidence that T cell activation leads to the rapid tyrosine phosphorylation of α-tubulin (Cardine et al., 1995; Ley et al., 1994). Importantly, phosphorylated α-tubulin was not incorporated into microtubules, but remained in the soluble, unpolymerized tubulin pool. Syk was found to phosphorylate a site in the COOH terminus of α-tubulin, which coincides with a region involved in MAP binding (Peters et al., 1996). Since MAPs are involved in regulating the polymerization status of tubulin, it is possible that TCR-induced activation of ZAP-70 leads to the phosphorylation of α-tubulin and the subsequent dissociation of MAPs. This in turn could destabilize existing microtubules and lower the pool of tubulin monomers available for polymerization. Such changes in microtubule dynamics would occur locally at the site of TCR engagement and might result in the repositioning of the MTOC. Although the model described is very attractive, the possibility remains that dominant-negative ZAP-70 impairs MTOC reorientation by inhibiting the binding of other SH2-containing effector molecules to ITAMs. Taking advantage of membrane-targeted ZAP-70/Syk chimeras, future experiments will assess whether independent activation of this PTK family is sufficient to induce T cell polarization.
Proteins involved in the regulation of microtubule dynamics are often good substrates for ERKs and protein kinase C (PKC) (Gelfand, 1991). Members of both kinase families are known to be activated upon T cell activation. Thus, apart from the aforementioned ZAP-70-mediated mechanism, changes in the microtubule cytoskeleton could be regulated by one of these pathways. Our experiments show that MTOC reorientation can occur in the absence of Ras activation. Neither overexpression of dominant-negative Ras, nor inhibition of MAPK/ERK significantly affected MTOC reorientation. These results suggest that Ras-independent pathways regulate microtubule rearrangements in response to TCR engagement. In earlier studies, MTOC reorientation was shown to be dependent on extracellular calcium (Kupfer et al., 1987). Incubation of B cell–T cell couples in the presence of EGTA reduced MTOC reorientation from >90% to 55 ± 5%. Chelation of extracellular calcium showed similar effects in our conjugate formation assay (data not shown). MTOC reorientation was significantly reduced, but not abolished (from 78% to 50 ± 8%). This suggests that MTOC reorientation may in part be regulated by a calcium-dependent pathway. Indeed, repositioning of the MTOC could be a complex event involving the shortening of microtubules and the subsequent anchoring of the MTOC at the site of TCR engagement. Thus, impairment of one of these events could result in partial MTOC reorientation. A more simple explanation relates to the fact that intracellular calcium stores are not depleted under our assay conditions. Thus, release of intracellular calcium may be sufficient to partially activate calcium-dependent effector molecules. Interestingly, Ca2+/calmodulin-dependent kinase family members phosphorylate MAPs (Gelfand, 1991). Thus, several kinase families might participate in the regulation of MTOC reorientation.
The assay system described here should prove useful to further characterize the effector molecules involved in T cell cytoskeleton dynamics. Once identified, it will be interesting to assess the physiological role of these effectors in maintaining a polarized intercellular immune response.
The authors thank members of the Weiss laboratory for helpful discussions, and in particular Drs. D. Yablonski and N.S.C. van Oers for critical reading of the manuscript. We gratefully acknowledge the technical assistance of T. Laroche and the laser scanning microscope.
This work was supported in part by grants from the Boehringer Ingelheim Fonds (B. Lowin-Kropf) and the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation (DRG-1356 to V. Smith Shapiro).
Address all correspondence to Dr. A. Weiss, Howard Hughes Medical Institute, U 426, 3rd and Parnassus Avenues, UCSF, San Francisco, CA 94143-0724. Tel.: (415) 476-1291. Fax: (415) 502-5081.
B. Lowin-Kropf's present address is Ludwig Institute for Cancer Research, Lausanne Branch, 1066 Epalinges, Switzerland.
1. Abbreviations used in this paper: APC, antigen-presenting cell; ERK, extracellular signal-regulated kinase; ITAM, immunoreceptor tyrosine-based activation motif; MAP, microtubule-associated protein; MHC, major histocompatibility complex; MTOC, microtubule-organizing center; PI-3, phosphatidylinositol 3; PTK, protein tyrosine kinase; SH2, src-homology-domain 2; TAg, T antigen; TCR, T cell antigen receptor; ZAP-70, zeta-associated protein 70.


text:
Dear Subscriber,
"One of my major frustrations is that we as an industry are spend too much time focusing on the regulation, rather than the opportunities that exist within it," – Andrea Dunlop, CEO of merchant acquiring, Europe, at Paysafe Group.
In PaymentEye this week a panel at Money 2020 discuss whether Europe's second Payments Directive (PSD2)'s Strong Customer Authentication (SCA) could be impacting customer experience.
Read, share, and enjoy.
Rebekah Tunstead


text:
The assets of Scio Diamond, a Greenville, S.C.–based producer of lab-grown diamonds, have been purchased by Nevada's Adamas One Corp., according to a Dec. 11 filing with the Securities and Exchange Commission (SEC).
The purchase agreement calls for the satisfaction of Scio's $3.3 million in secured debt in the 18 months following the deal's closing; the issuance of 350,000 shares of common stock, with a guarantee minimum resale price of $2 per share, to settle Scio's $3 million in unsecured debt; and issuance of 900,000 shares of Adamas stock to be distributed to Scio's current shareholders.
The sale is subject to shareholder approval, as well as Adamas One filing a registration statement with the SEC.
The SEC filing announcing the sale was signed by Scio CEO Gerald McGuire, who did not respond to an email request for comment.
Scio Diamond was originally Apollo Diamond, one of the first companies to commercially market lab-grown diamonds. Apollo first came on the scene in the 1990s. It went public in 2011, and soon found itself enmeshed in a series of lawsuits and proxy battles with a group of disgruntled shareholders, who eventually took over the board of directors. In 2017, its former chairman Edward S. Adams was charged with tax fraud. That case is ongoing.
Scio has not filed any public notices since February, and JCK was unable to find any current information on the company earlier this year.
The only Adamas One Corp. listed by the Nevada Secretary of State was registered on Sept. 30. It lists as its sole officer John G. Grdina of Scottsdale, Ariz.
Anthony DeMint, an attorney for DeMint Law, the Nevada law firm that is acting as registered agent for Adamas, declined comment, adding "due to the nature of this transaction, all information will be filed with the SEC when available."
Top: Diamonds produced by Scio (photo courtesy of Scio Diamond)
Follow JCK on Instagram: @jckmagazineFollow JCK on Twitter: @jckmagazine
Follow JCK on Facebook: @jckmagazine


text:
Why are Small Caps in a Tailspin?
While everyone was busy talking about FAANG stocks driving the market higher this year, small caps were flying even higher, outperforming large caps by over 5% throughout most of the summer. However, as investor sentiment cooled and volatility returned in a big way, "risky" assets like small caps have taken it on the chin.
Names like Facebook, Apple, Amazon, Netflix and Alphabet dominated headlines for much of 2018, as did other high-growth large-cap companies like Tesla. Many investors were concerned over the low market breadth, meaning the small concentration of stocks moving the overall market higher. Meanwhile, as illustrated by the graph above, small cap stocks (Russell 2000 Index) were on a tear and probably didn't get the attention they deserved.
There were a few factors driving the small cap rally. The superior sales and growth rates for smaller cap companies were primary drivers, with further earnings tailwinds coming from tax-cuts. The economy was also growing at a fast clip, interest rates were relatively lower, and there was strong demand for risk assets such as small cap stocks.
As concerns over trade wars and tariffs increased, the typically-domestic footprint of small caps also became attractive for many investors. Whereas large cap companies tend to be multinational, with increased exposure to geopolitical turmoil, small cap companies tend to be much more U.S.-focused, often competing in particular geographies within the country. Similarly, many investors were betting the U.S. economy would continue to grow at a faster rate than other developed countries and preferred small caps' direct U.S. exposure.
That all changed in September. Small caps began a decline that accelerated rapidly in early October, as shown by the graph. On October 3rd, Fed Chairman Jerome Powell announced that "we are a long way from neutral," meaning further rate increases would be needed. Investors, nervous over the potential for the Fed to raise rates too far and cause a recession, began unloading risk asset classes like small-cap stocks and high-yield bonds. Investors also began covering their short positions of U.S. Treasury note futures - a very popular trade this year - which amplified the risk-off mindset in the equity market.
Despite the bearish sentiment, earnings season was largely positive. This illustrates the disconnect between strong equity fundamentals and the risk-off sentiment. Much of the bearishness continues to be driven by the bond market, as lower U.S. Treasury yields have affected the shape of the yield curve. Rates from 3-10 years have fallen and the talk of yield curve inversion - a predictor of recessionary liquidity conditions - increased considerably although corporate earnings are still quite strong.
Since the beginning of December, small cap volatility has been exacerbated by media pundits' continued chatter over a potential recession. While these concerns are likely overblown, you should find comfort in the fact that over long time periods (across market cycles), small caps have generated stronger returns than large caps, and we believe that likely isn't going to change going forward. Further, if conditions do wind up deteriorating, high-quality companies such as the ones we invest in tend to take market share from competitors during tough economic times and emerge even stronger during the next cycle. Lastly, a market repricing event, like what we are experiencing now, allows asset managers to upgrade some holdings at lower prices, since prices are effectively going on sale.
Dana Large Cap Equity Fund top-ten holdings as of September 30, 2018: Apple Inc. (2.48%), Pfizer Inc. (2.36%), Amgen Inc. (2.21%), WellCare Health Plans, Inc. (2.18%), UnitedHealth Group Inc. (2.15%), Stryker Corporation (2.12%), Alphabet Inc. Class A (2.10%), Microsoft Corporation (2.10%), Bristol-Myers Squibb (2.05%), Johnson & Johnson (2.03%).
Dana Small Cap Equity Fund top-ten holdings as of September 30, 2018: Universal Insurance Holdings, Inc. (2.34%), Bottomline Technologies, Inc. (2.14%), Ligand Pharmaceuticals, Inc. (2.05%), Marcus Corporation (2.03%), Emergent BioSolutions Inc. (2.00%), Primerica, Inc. (1.99%), Boot Barn Holdings, Inc. (1.98%), BioTelemetry Inc. (1.87%), Chefs' Warehouse, Inc (1.83%), Comfort Systems USA, Inc. (1.83%).
Price/Earnings (P/E) is the price of a stock divided by its earnings per share.


text:
Defending Premiers, the Hela Wigmen, face a tough task in their bid to retain their Digicel Cup title when they come up against the in-form Port Moresby Vipers in the opening weekend of the competition's Finals Series.
After a strong start to the regular season, the Wigmen have lost momentum in the later half of the competition, winning only two of their Final six matches, including suffering recent losses to fellow finalists, the Lae Tigers, Waghi Tumbe and Port Moresby Vipers. The Vipers on the other hand come into Sunday's Elimination Final full of confidence after adding the Mendi Muruks last wekeend to their list of high profile scalps.
The Vipers and Wigmen's Final will be one of two Elimination Finals played at the National Football Stadium on Sunday. The other Elimination Final will see the Waghi Tumbe face the Rabual Gurias.
In what has been a mixed season for the Gurias, the New Britain based franchise qualified for the Finals thanks to a 26 – 4 win over the Enga Mioks in the Final round of the regular season. The Tumbe on the other hand finished outside of the top two due to points differential and have enjoyed an eight game winning streak.
The Lae Tigers and Mendi Muruks will enjoy the first week of the Finals Series off after finishing in the top two positions of the competition ladder. The Muruks, who were on track to collect the Minor Premiership, suffered a 6 – 0 loss to the Vipers in the Final round of the regular season allowing the Tigers to leap-frog them following their 22 – 18 win over the Kimbe Cutters giving them a superior for and against. The outcome means that the top three teams of the competition all finished with 9 wins and two losses.
In other matches played in the Final Round of the regular season, the Mt Hagen Eagles defeated the Central Dabaris 26 – 12, whilst the Goroka Lahanis defeated the Gulf Isou.
As per previous years, the Digicel Cup Finals Series is made up of the top six teams in the competition. Teams ranked first and second progress to Week Two of the Finals, whilst the remaining four play in two Elimination Finals. The winners of the Elimination Finals will play off in the Minor Semi Final in Week Two, whilst the top two teams played in the Major Semi Final with a chance to qualify for the Grand Final.
Goroka Lahanis 22 – 16 Gulf Isou
Lae Tigers 22 – 18 Kimbe Cutters
Waghi Tumbe 18 – 14 Hela Wigmen
Port Moresby Vipers 6 – 0 Mendi Muruks
Rabual Gurias 26 – 4 Enga Mioks
Mt Hagen Eagles 26 – 12 Central Dabaris
Week One Finals:
Elimination Final 1: Port Moresby Vipers (4) v Hela Wigmen (5)
Elimination Final 2: Waghi Tumbers (3) v Rabual Gurias (6)
Week Two Finals:
Major Semi Final: Lae Tigers (1) v Mendi Muruks (2)
Minor Semi Final: Winner of Elimination Final 1 v Winner of Elimination Final 2
Week Three Finals:
Preliminary Final: Runners Up of Major Semi Final v Winner of Minor Semi Final
Grand Final:
Winner Major Semi Final v Winner Preliminary Final


text:
Worlds collide when a barbarian warrior is ripped from his fantasy realm and dropped into a modern-day metropolis. Writer Jeff Lemire and artist Mike Deodato Jr's newly collected Berserker Unbound begins with standard sword-and-sorcery fair, but ultimately reveals itself as an affecting examination on grief and recovery.
The Mongrel King returns from battle to find his family slaughtered by the savage horde of an evil sorcerer. Wounded and distressed, the warrior wanders into a cave, where mystic runes entangle him in a wormhole and transport him to an unfamiliar modern city. A sympathetic homeless man named Joe Cobb finds and rescues the stranded warrior, and even though they cannot communicate, the two find common ground when faced with the dangers and despairs of urban life. As he struggles to return to his world, the warrior must protect himself and the homeless man from the old nemeses that threaten the new world.
If you're expecting this book to be a frivolous romp through mismatched genres, similar to the 1970 Arnold Schwarzenegger camp classic, Hercules in New York, then let me put your fears to rest. Berserker Unbound embraces some of the tropes inherent to fish-out-of-water stories, like when the Mongrel King uses a gold coin to pay for wine at a liquor store, but avoids its major pitfalls. At the same time, it exploits a few of the savage fantasy genre mainstays, like his bulging muscles and costume design, but never in a way that feels gratuitous.
Although Berserker Unbound doesn't have the sprawling scope of Descender or Black Hammer, it still bears familiar hallmarks of a great Lemire story. It has deftly subtle dialogue that's more about what's not said than what's said. And it has two main characters that are relatable and well rounded. At first, Berserker Unbound leads you to believe that it's all about revenge. The warrior says he wants to return home, avenge his family, and join them in death, but then he meets his companion in the city who has also experienced a tragic loss and is similarly displaced. Tthe narrative that began epic in issue one becomes intimate in issue four, as two characters that have nothing left to fight for end up fighting for each other.
The book's strengths are its heroes, but the villain was unfortunately one dimensional. The sorcerer's motivations were unclear; he seemed merely evil for evil's sake, and he lacked a personal connection to the warrior that would have tethered him tighter to the story.
Lemire and Deodato worked together previously on Marvel's Thanos, and the art in Berserker Unbound demonstrates the strength of their collaboration. Deodato clearly enjoyed himself illustrating the barbarian violence. The book begins and ends with gleefully rendered splash pages depicting over-the-top frenzies of bodies, blades, and blood. But Deodato also imbues the characters with a great deal of heart and employs an impressive variety of layouts to control the pace of the story and heighten the emotional impact. Some wide shots are divided by white gridlines, separating a character from their surroundings and making them feel more isolated. Likewise, characters are placed close together in a small panels, highlighting the importance of their relationship. I also loved how the cross hatching and halftone dots added a thematically appropriate roughness to all of the characters and their surroundings.
Berserker Unbound: Volume 1 has an ending that I found both satisfying and definitive. But in a recent interview with Comiccon.com, Lemire and Deodato admitted to having plans for a Berserker Unbound trilogy. I'd be excited to see that happen.
Creative Team: Jeff Lemire (writer), Mike Deodato Jr. (artist), Frank Martin (colors)
Publisher: Dark Horse Comics
Click here to purchase.


text:
Call of Cthulhu: The Card Game - Initiations of the Favored Asylum Pack
✓ All Prices in Canadian Dollars 🍁
✓ Secure Shopping
✓ Safely Packaged
✓ Orders Shipped Daily
✓ Worldwide Shipping
Live near Ottawa? Select our 'Local Pickup' on checkout!
Initiations of the Favored is the third Asylum Pack in the Rituals of the Order cycle for Call of Cthulhu: The Card Game. This cycle introduces new Monsters, Servitors, Rituals, and Items, as well as cards for the Order of the Silver Twilight. The story of Lucy Jennings, Silver Twilight initiate, continues in the serial tale by Jeff Tidball.
Initiations of the Favored is a fixed 60 card Asylum Pack that contains 20 different never-before-seen cards based on the dark mythos of master horror writer H.P. Lovecraft, designed to augment existing decks and add variety to the Call of Cthulhu metagame. |


text:
What's a better response to Covid 19 other than laughter? We decided to have some fun amidst all this craziness. Therefore, we put together a video showing each of us in our daily lives. It was an absolute blast taping it and collaborating remotely. (There are so many bloopers you wouldn't believe.) Most importantly, we wanted to communicate a message across that we are "still here." We are well, and we are still open for business. Above all, we want you all to stay well, stay safe and most of all stay positive. If you fancy a chat feel free to lift up the phone to any of us as we are always delighted to hear from you.
You can check out our video below or directly on our Youtube channel.
Let us know what your response to Covid is in our comment section below.
If you're job hunting, check out our current jobs here.


text:
The WCPA is thankful to receive a $10,000 Community Grant from the Greater Worcester Community Foundation! GWCF operating support will help us focus on our mission to celebrate the rich literary history and creative energy of Central Massachusetts through poetry.
Archives for December 2021
Nominations are open for the 2022 Stanley Kunitz Medal
On behalf of the Stanley Kunitz bequest to the Worcester County Poetry Association, the Medal committee is pleased to announce the opening of nominations for the 2022 Stanley Kunitz Medal, the eighth to be awarded since 2015.
The medal is presented annually to a person with a strong Worcester County (Massachusetts) connection who best exemplifies Stanley Kunitz's life-long commitment to poetry and poets. The award recognizes an individual's total commitment to poetry as Kunitz lived it: teaching poetry, mentoring poets, speaking poetry, publishing poetry, and supporting organizations which nurture poetry.
Letters of nomination should provide support explaining how the nominee nurtured poetry as defined above. Because the yearly award is singular and cannot honor all worthy applicants, past nominations may be reactivated for 2022 once the nominators notify the committee chair to keep the nomination active.
Nominations should be mailed to The Stanley Kunitz Medal, c/o Worcester County Poetry Association, P.O. Box 804, Worcester, MA 01613 between December 1, 2021 and March 31, 2022. The award announcement will be made in the late spring of 2022, with a ceremony at The Worcester Historical Museum coinciding with Kunitz's birthday the last week of July. Please visit the Kunitz Medal page for more details.
The 2022 Stanley Kunitz Medal committee wishes to recognize and thank the following volunteers who have served to bestow this honor in the past: Rodger Martin, chair (2013-2021), Kent Ljungquist (2017-2021), Karen Sharpe (2019-2021), Robert Steele (2015-2016), E. Robert Cronin (2015-2018).
Judith Ferrara, Committee Chair – firstname.lastname@example.com -508-757-0524
Joe Fusco, Jr.
John Hodgen
Heather J. Macpherson
Susan Elizabeth Sweeney


text: URMC / Employee Assistance Program / News & Events / UR Medicine EAP Blog / September 2021 September 2021 Ostracized at Work? If you've felt ostracized at work, read tips to create an inclusive work place in UR Medicine EAP's blog post. Practice Cheerfulness to Find Job Passion Learn how optimism can change your outlook at work in UR Medicine EAP's blog post. Teens and Video Gaming Addiction Are you or your family members reaching for video games more often? Read UR Medicine EAP's blog post to learn more about when gaming becomes too much to handle. Just This Much Fentanyl Is Fatal Unfortunately, rates of opioid addiction and opioid overdose are on the rise. Read UR Medicine EAP's blog post to learn more about how to protect you and your family. Quick Tips: Facing Personal Change Stressed over the amount of change you've experienced recently? Read on for tips from this month's UR Medicine EAP blog.


text:
This list of files includes items that you might like for use in your newsletter or other bulletins. If there's anything else that would help your associates access this great discount on floral arrangements and more, just give us a call at (704) 342-4529.
This website uses cookies. By continuing to use this site, you accept our use of cookies.


text:
PSAB Decision Summary – September 17-18, 2015
Start date:
End date:
Location: Toronto
On September 25, 2015, the Public Sector Accounting Board (PSAB) released an executive summary of discussions and decisions with respect to the topics addressed at this meeting.
The following topics were discussed:
- PSAB Planning;
- PSAB discussed a draft 2016-2019 strategic plan and provided feedback for revisions
- PSAB approved a revised 2015-2016 work plan for presentation to AcSOC
- Financial Instruments; and
- Government Transfers.
Review the Decision Summary on the PSAB's Web Site.


text:
Police in Moray and Aberdeenshire have welcomed figures that show that instances of domestic abuse fell over the festive period last month.
Patrols were stepped up in the Moray and Aberdeenshire region that held a greater focus on victim safety, perpetrator re-offending and carrying out checks that ensure bail conditions were being adhered to.
Figures released this week show that a total of 77 incidents of domestic abuse were reported to police between December 24 and January 5, a fall of 9% on the numbers for the same period in 2013.
Resulting from these incidents police say that 46 people have so far been charged while officers are expecting that to rise further as their enquiries approach a conclusion in the next few weeks.
DI Fiona Topping from the domestic abuse investigations unit said that between April 1 and December 23 last year 1301 incident were reported – also a significant fall on the 1714 reported in 2013.
She said: "My message to perpetrators of domestic abuse is this – be in no doubt, your actions have consequences for you, your family, your friends and even for your employment.
"It is not acceptable to abuse your partner, to shout at them, hit them or control where they go and who they speak to – and when instances are reported to us we will do all we can with the help of partner agencies to ensure this behaviour stops."


text:
As a part of the City of Hollywood's "Backing Business" initiative, Temporary Café Zones have been created to assist local restaurants and food establishments by expanding outdoor dining. The Program Guide gives restaurants the information needed to use these zones. CLICK HERE to learn more about how to participate in the program.
Hollywood businesses interested in participating in the Temporary Café Zones Program are invited to attend an informational session hosted by the City's Economic Development Team. Join via Webex Events using the password: CMED2020 at the links below.Monday, May 18th at 10:00 a.m.Monday, May 18th at 3:00 p.m.Tuesday, May 19th at 9:00 a.m.Tuesday, May 19th at 2:00 p.m.
The City of Hollywood's Office of Communications, Marketing and Economic Development is available to talk you through any of your business-related issues. Contact us at 954.921.3620 or by email. Please continue to monitor www.hollywoodfl.org and www.choosehollywoodfl.com for the latest updates on businesses programs and resources.
Economic Development DivisionRaelin Storey, CMED DirectorHerb Conde-Parlato, Economic Development ManagerTekisha Jordan, Principal PlannerMarina Melo, Economic Development Marketing CoordinatorCommunity Redevelopment Agency - Beach & Downtown DistrictsJorge Camejo, Executive Director954.924.2980
Copyright © 2020 City of Hollywood, Florida. All Rights Reserved.2600 Hollywood Boulevard Hollywood, FL 33020


text:
Completed in 2017/18
Featured Transactions
Acting on behalf of Aprirose Real Estate Investment Management, J D Partners brokered a forward sale and leaseback to Merlin Entertainments Group of a new hotel development at the Legoland Resort, Windsor. Merlin planned to develop a themed hotel concept on a 2.7 acre site within the grounds of their existing Legoland theme park and were seeking a funding solution.
J D Partners was able to secure a forward sale agreement with Aprirose which enabled Merlin to proceed with the development. Under the agreement, Merlin constructed the hotel and committed to a 35 year lease upon completion, renewable for a further 35 years at the end of the term. The forward sale was arranged at a price of £18.5 million, with completion of the purchase and opening of the hotel taking place in March 2012.