At Dumpster Blog, we strive to bring you the best service at the best price available. We have a wide selection of dumpster sizes in stock in Marion, CT, with flexible & timley pickup and delivery. Give us a call today for all your dumpster rental and roll off needs.
Total capacity & Weight restriction: For both front loading and roll-off containers the better the dumpster the more high-priced it is. Nevertheless, renting a dumpster that's too small will also be costly. You could possibly accrue extra charges should the weight with the materials placed in the dumpster meets the maximum weight limit with the size and expense. For example, The Florida service provider rented some sort of 20-cubic-yard roll-off dumpster for about $400, packed it with heave construction trash and ended up being charged another $400 because the weight of the trash exceeded the particular 3-ton limit. In the same manner, a dumpster that's too small would certainly require extra pick-ups to take away the garbage. Although some firms automatically contain dump/disposal fee of their quoted charges, depending on community garbage costs and the sort of waste placed there may a further charge of 25-250 dollars or more for each and every pickup.
The frequency of which have you acquired something or perhaps put something to the side because you might use that one day? This stuff can be modest, like coupon codes, or huge like safe-keeping boxes or perhaps machinery or perhaps furniture. End keeping this stuff that you don't want! You are just collecting junk! For high-priced items, offer them on-line or over the classified part of your local document. When selling things, restriction how long you are going to wait prior to dropping your current price and how long you are going to keep the piece. Less expensive goods can either visit charity or perhaps go right into the trash, according to whether another individual could use the item.
Did you know that you won't need to be a skilled contractor (and have an "in" while using dumpster fairy) to be able to receive an industrial type of dumpster for your home advancement projects? That's right. There are firms out there that realize Do-It-Yourselfers make just as much development waste as a professional crew, and they've committed themselves to make sure householders like you have access to a large type of dumpster whenever they want it.
You can clean the yard, dispose of that trash immediately after your home's renovation and can do a lot more for keeping the home clean with these services. The actual disposal trash can and Cost of a Roll Off Dumpster in Marion, CT providers help you say bye to individuals heaps of junk of your house in a very convenient technique only.
If you want a dumpster maybe you have a lot of junk or reuse material to acquire hauled away. The totally best method to get these often bulky bins is to get in touch with a preferred and local company. Simply talk to these and they'll help you to get the best possible dumpster to your requirements. Some types of materials are more nearly impossible to find rid of or perhaps recycle as opposed to runners, and local polices may establish where your current container could dumped. Almost any price quoted is at the mercy of how much you've, and what it truly is, and the articles taken as a whole.
You will end up purchasing the free area if you employ a roll off dumpster when your junk disposal is extremely less. Consult the Cost of a Roll Off Dumpster in Marion, CT firms if they rent junk dumpster that are modest in size when compared with roll off dumpsters. These junk dumpsters usually range between 2 yard to 8 yard.
You may be doing extensive work towards your bathroom and plan on tearing out the ceramic tile in your restroom or knowing a person else take action. The person performing it will be able to effortlessly throw the outdated tiles and old damaged tiles available safely in to the dumpster.
Another moment when you might choose to go this route can be when you're shifting. Sometimes, you have large trash that you are not able to fit into a new black trash bag, similar to old bookcases, tables, or cardboard boxes. Guaranteed, you might not populate the jar to the top, but it'll sure make your life quicker to toss the trash in and have someone else haul it out when you're completed.
Some organizations offer Cost of a Roll Off Dumpster in Marion and garbage bin rental services that collect garbage from you and recycle the idea. Hence they aid you in getting rid of the trash along with doing it in an eco-friendly approach. The company you actually assign for the position will provide you with a new bin or maybe dumpster of a big or maybe small dimensions depending on the variety of trash. A standard example of this sort of dumpsters and packing containers can be seen with the construction web-sites. They may be available with the company providing you services in a variety of shapes and sizes. Place the order of a giant bin or maybe dumpster for more trash and smaller ones for no more garbage.
Just about every project is made simpler and much easier with swift trash removing. Cleaning out your shed and removing unwanted damaged equipment like old rakes and shovels can be carried out quickly and never having to pile the trash out by the street and hope that the regional trash collection takes the full pile. It is possible to take the anxiety out of cleansing your storage area, basement, or maybe shed by reduction of the need to use local trash pickup. Building projects may also be made easier as one dumpster can handle outdated appliances, drywall, bathroom fixtures, twisted precious metal and damaged glass. Simplify your home initiatives by calling or ordering online a new Maryland Cost of a Roll Off Dumpster in Marion.
Once you have a major cleanout job to do, you may need help receiving all of that trash and trash out of the constructing and over property. Make a professional trash removal team to speed the process.
Your current den may be the place you head to, to get away from our planet's every day worries. Maybe you sit in your living room to read a magazine or the magazine. You may even bring clients in your den when you work from home. If this describes the case, you will need a den that's comfortable but professional looking.
Understand that you are ordering a trash dumpster for the metropolis with no dump so be sure of the size of the particular dumpster required if you're not sure of the size and style, get the assistance of the Cost of a Roll Off Dumpster in Marion service company. Be sure of the particular Cost of a Roll Off Dumpster in Marion period prior to going to order just one. A company that's transparent to use pricing may help you at the best in giving you the first sort notice in regards to the fee that you are going to be charged. These companies will probably best assist you in getting the right sized dumpster at best value.
According to their demands, the different types connected with containers include varying prices, depending on the dimension, the period of time it would be required and the buyers need to find more information about most of these. Having a common idea about the price of dumpster that they need, will help them within coming up with a budget and also in choosing a company that provides affordable prices without having compromising around the quality of service. Although researching around the price, they need to also make an effort to find out the form of lingo employed in this field in order to find an alternative way to communicate with their staff.
To learn more about Cost of a Roll Off Dumpster in Marion, CT services investigate Budget Dumpster company. They are the national company with a good track record of affordable, upfront, all inclusive costs and great customer service. 1-866-284-6164 If you are planning to choose a home renovation task or wish to construct a home for yourself; you can't simply do from the or a home rehabilitation activity without hiring a professional dumpster rentals company. By simply dumping all of the waste materials inside their dumpster, you are completely finished with your hard work.Following a few major reasons you simply must seek the services of Cost of a Roll Off Dumpster in Marion, CTs organizations for your home building or restoration project.
Using a roll-off dumpster is essential for virtually every construction task, major home rehabilitation venture along with other numerous industrial works by using. However, if you've never hired before, this process could seem too much to handle and nerve-racking.
With every task, there is gonna be waste to clear out. When you have a practical partnership that has a Cost of a Roll Off Dumpster in Marion, CT company, both you and your team will know what is appropriate dumping material. Dumpster rental companies will show you what components are appropriate because of their dumpsters, but they could also let you know how to properly discard those undesirable materials far too, like untouched paint or any other hazardous material. Improperly discarded material may result in heavy fees and actions against that could amount to more than just a couple of bucks. It could amount to your business. That's why it is important to build those robust working interactions.
There are many belongings in your garage and surface buildings on your property that you may no longer use. Maybe you have been in your house for over two decades. You know how considerably junk you may accumulate within twenty years. If you have had a family you should have even more what to get rid of.
Examine the dumpster companies plan on what cannot be added to their dumpster(s). For example, most dumpster organizations will not let concrete inside their dumpster. Concrete can easily stick along with cause the dumpster to be able to weigh additional, take up area and essentially make the dumpster less useful because it should be.
Some junk treatment companies present labor, which means they'll not solely provide the dumpster and take the trash, however they'll likewise help you bring items out of your home to the18 wheeler or dumpster. Consider whether you will need this service in accordance with the scale from the cleanout and your family along with friends' ability to accomplish this heavy lifting yourselves. It will likely cost more, nevertheless it can get the task done rapidly. Note that if you're paying for toil, especially if you might be paying per hour, you may not be capable of take the time to dig through every pack and case in search of expressive items.


text:
Stunning time-lapse shows Perseids meteors streak across night sky
By Catherine Park
Timelapse captures Perseids meteors over Oregon
A timelapse taken from the summit of Oregon's Black Crater volcano captured the Perseids meteor shower on August 7. (Credit: Mike Cohea via Storyful)
BLACK CRATER, Ore. - Stunning time-lapse footage captured the Perseids meteor shower over a volcano in Oregon on Aug. 7 and it literally looks like a scene from a fantasy movie.
The beautiful footage, shot by Mike Cohea, shows the summit of Oregon's Black Crater volcano as meteors can be seen streaking across the starry sky.
NASA said the 2021 Perseids meteor shower was set to peak this week.
"This year, even relatively faint meteors should be visible through clear skies from a dark location as the bright Moon will be mostly absent," the space agency said.
Freeze frame of time-lapse video showing the Perseids meteor shower over Oregon volcano.
RELATED: Perseids meteor shower to dazzle in July, August — here's how to watch
The Perseids are active every year from July 14 to August 24, but the meteor shower will peak between midnight and dawn on August 11, 12 and 13.
Up to 100 meteors will pierce Earth's atmosphere every hour, traveling at speeds of up to 37 miles per second. The speed at which the meteors travel creates resistance with Earth's atmospheric particles causing the meteors to burn in bright balls of fire in the night sky.
The Perseids appear to come from the region of the Perseus constellation, but they don't actually come from the constellation.
As the 109P/Swift-Tuttle comet orbits the sun, it leaves behind a trail of debris that Earth passes once a year.
RELATED: Virgin Galactic launches Richard Branson, 5 others into space
When Earth passes through 109P/Swift-Tuttle's debris, particles from the comet come into contact with Earth's atmosphere, creating a meteor shower.
109P/Swift-Tuttle was discovered in 1862 by Lewis Swift and Horace Tuttle and measures around 16 miles across.
Storyful and Skyler Rivera contributed to this report.
Weather Forecast for Tuesday, Jan. 18
Rams fans pumped after Wild Card win
Kirsten Crowley nominated to be first female LAFD Fire Chief

redpajama_set_name: RedPajamaCommonCrawl


In this lesson, your tutor will help you go over this topic: Earthquakes. First, read the following dialogue out loud with your tutor, then switch roles and try again.
Kevin and Dorothy are talking about the recent earthquake in Chile.
Did you hear about the earthquake in Chile?
Yes, I heard it was 6.8 in magnitude.
I saw there was also a tsunami warning in the news.
I'm glad it was just a warning. I was told there were a lot of aftershocks.
Were there any collapsed buildings?
I don't think so. Most of the buildings in Chile are made to withstand a powerful earthquake.
That's good. I think it's a good idea to have a disaster preparedness kit.
I agree. I'm sure the people in Chile were shaken up.
No, but I have experienced tremors before.
There was an earthquake in Ecuador last month.
The earthquake was 6.8 in magnitude.
The government gave a tsunami warning after the earthquake.
The aftershocks were very strong.
The building collapsed after the earthquake.
The buildings are made to withstand an earthquake.
The powerful earthquake caused a lot of damage.
We have a disaster preparedness kit in our house for emergencies.
My parents were shaken up after the car accident.
Did you feel the two tremors today?
No, I've never felt an earthquake.
I experienced a 7.0 earthquake.
Do you have a disaster preparedness kit?
I have never heard of such a kit.
I think I will consider buying or making one.
Can the buildings in your hometown withstand an earthquake?
I don't think the buildings in my hometown can withstand an earthquake.
Have you ever been shaken up by something?
I was shaken up in a car accident one time.
I was shaken up when my parents died.
Can you give an example of something powerful?
The rainstorm last night was powerful.
What types of disasters do you have in your country?
Is it a good idea to have a disaster preparedness kit? Why or why not?
Do you think it's possible to survive in a building that has collapsed?
What would you put in a disaster preparedness kit?
What would you do if you were in an earthquake?


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text:
Elie Bou Assi, PhD
Dr. Elie Bou Assi
Elie Bou Assi is an IVADO assistant professor in the Department of Neuroscience at the Université de Montréal and a principal scientist at the Centre Hospitalier de l'Université de Montréal (CHUM).
His principal research interests cover the application of artificial intelligence techniques and the internet of things to improve the management of neurological and psychiatric conditions.
The excellence of his work has earned him one of the three CRCHUM awards of excellence in research (year 2019) and the award of excellence in publication of the Canadian League against Epilepsy (year 2019).
Large-Scale Desynchronization during Interictal Epileptic Discharges recorded with Intracranial EEG
Predicting urinary bladder voiding by means of a linear discriminant analysis: Validation in rats
Effective connectivity analysis of iEEG and accurate localization of the epileptogenic focus at the
Elie Bou Assi, Ph.D.
© 2021 by Elie Bou Assi


Skin diseases must have been a challenge for the Hebrews living out in the desert. I can't help but feel pity for those who were forced to stay outside the camp, ostracized and forced to declare themselves "unclean" any time anyone approached them.
One of my favorites stories of Jesus is how he went up to the lepers, touched them in their uncleanness, and then healed them.
The bleeding after pregnancy and during menstrual cycles also made women ceremonially unclean. That's not such a bad idea! I get so cranky and tired, and lately I've been in so much pain, that having to spend time alone in bed for a whole week sounds almost like a vacation! Perhaps in a way this regulation was God's way to ensure that a new mother and a woman having her period had a chance to recover before jumping into life so quickly.
God provided his circle of protection, again with the regulations about what meat could and could not be eaten. I'm fairly sure they did not have access to cooking thermometers, nor did they have the knowledge that we do about the safe temperature required for certain foods.
Tonight's blog thus far is more of a summary of what I've read rather than a musing on application in my own life. I do long for Jesus to reach down and heal me of my female ailments I'm currently suffering. I know what that lady who bled for twelve straight years must have felt like (not really). I have a sonogram scheduled for tomorrow — wouldn't it be awesome if they are unable to find anything wrong? Actually, I pray that they will find out what is causing me so much pain. Endometriosis? Fibroids? Cysts? Onset of menopause? What did women do thousands of years ago, traipsing around in the desert, when they were accosted by severe cramping and bleeding? Did they have herbs or medicines to ease pain? Or were those women made of stronger stuff than me? I suspect that may be the case!
I can relax and rest, though, in the knowledge that Jesus isn't afraid to touch me even if I get myself into an unclean state — physically, emotionally, or mentally. In reaching out and healing the lepers, he took the Lord's provision for them one giant step forward and demonstrated what it looks like to serve in love, without fear.
Who are the metaphorical lepers in our current society? My niece is involved in a mission with her church where she reaches out to prostitutes. She goes in groups and gets to know them personally and tries to help them see that there is a better way. Like Jesus, she isn't afraid to touch them with her friendship and her love.
My husband and his roommates befriended a sometimes odiferous homeless, mentally ill man when they were students in college. They let him use their address to apply for food stamps, and they did all they could to try to convince him to move in with them. He had an illness, though, that kept him on the streets. Still, they sought him out, brought him food, and checked up on him at his usual haunts. They showed him love when many in today's society would not.
Tomorrow I will keep my eyes open for those the world has labeled unlovely, and I pray the Lord will give me eyes to see and hands and feet that are willing and the courage to step over my comfort zone so I can follow Jesus in freely touching the "lepers" I encounter with love and grace.


KB - Dashboard Warning: "Unregistered User"
The Buttonwood Exchange username 'user@domain.com' has not been registered with the Buttonwood Cloud Exchange. Please contact your system administrator. If your account has been recently registered please allow up to 30 minutes for activation to complete.
Other users who are logged in are not seeing this error and in addition, receive valid Cost Centre and Cost details.
The Buttonwood Broker retrieves Cost Centre and Cost information from the Buttonwood Cloud Exchange. User objects are linked between the Cloud Broker and Cloud Exchange via their usernames.
The error indicates that a user with the same username (user and domain) as the logged in user is not configured in Buttonwood Cloud Exchange.
In some circumstances, where the username is configured differently within the Cloud Broker and Cloud Exchange (eg. security reasons), the username may be mismatched.
Confirm with the organisation's Buttonwood Cloud Exchange administrator that the user exists, and note down the configured username. Refer to the Exchange article Managing Users for detailed steps.


text:
package com.hazelcast.partition;

import com.hazelcast.collection.impl.list.ListService;
import com.hazelcast.collection.impl.queue.QueueService;
import com.hazelcast.collection.impl.set.SetService;
import com.hazelcast.concurrent.atomiclong.AtomicLongService;
import com.hazelcast.concurrent.countdownlatch.CountDownLatchService;
import com.hazelcast.concurrent.lock.InternalLockNamespace;
import com.hazelcast.concurrent.lock.LockServiceImpl;
import com.hazelcast.concurrent.lock.LockStore;
import com.hazelcast.concurrent.semaphore.SemaphoreService;
import com.hazelcast.config.Config;
import com.hazelcast.config.PartitioningStrategyConfig;
import com.hazelcast.core.HazelcastInstance;
import com.hazelcast.core.HazelcastInstanceAware;
import com.hazelcast.core.IAtomicLong;
import com.hazelcast.core.ICountDownLatch;
import com.hazelcast.core.IExecutorService;
import com.hazelcast.core.IList;
import com.hazelcast.core.ILock;
import com.hazelcast.core.IMap;
import com.hazelcast.core.IQueue;
import com.hazelcast.core.ISemaphore;
import com.hazelcast.core.ISet;
import com.hazelcast.core.IdGenerator;
import com.hazelcast.core.Member;
import com.hazelcast.core.Partition;
import com.hazelcast.core.PartitioningStrategy;
import com.hazelcast.instance.HazelcastInstanceFactory;
import com.hazelcast.instance.Node;
import com.hazelcast.instance.TestUtil;
import com.hazelcast.nio.serialization.Data;
import com.hazelcast.partition.strategy.StringAndPartitionAwarePartitioningStrategy;
import com.hazelcast.partition.strategy.StringPartitioningStrategy;
import com.hazelcast.ringbuffer.Ringbuffer;
import com.hazelcast.ringbuffer.impl.RingbufferService;
import com.hazelcast.spi.impl.NodeEngineImpl;
import com.hazelcast.test.HazelcastParallelClassRunner;
import com.hazelcast.test.HazelcastTestSupport;
import com.hazelcast.test.TestHazelcastInstanceFactory;
import com.hazelcast.test.annotation.ParallelTest;
import com.hazelcast.test.annotation.QuickTest;
import org.junit.AfterClass;
import org.junit.BeforeClass;
import org.junit.Test;
import org.junit.experimental.categories.Category;
import org.junit.runner.RunWith;

import java.io.Serializable;
import java.util.Map;
import java.util.concurrent.Callable;
import java.util.concurrent.Future;

import static org.junit.Assert.assertEquals;
import static org.junit.Assert.assertNotNull;
import static org.junit.Assert.assertTrue;

@RunWith(HazelcastParallelClassRunner.class)
@Category({QuickTest.class, ParallelTest.class})
public class PartitionControlledIdTest extends HazelcastTestSupport {

    private static HazelcastInstance[] instances;

    @BeforeClass
    public static void startHazelcastInstances() throws Exception {
        Config config = new Config();
        PartitioningStrategy partitioningStrategy = StringAndPartitionAwarePartitioningStrategy.INSTANCE;
        config.getMapConfig("default")
                .setPartitioningStrategyConfig(new PartitioningStrategyConfig(partitioningStrategy));

        TestHazelcastInstanceFactory instanceFactory = new TestHazelcastInstanceFactory(4);
        instances = instanceFactory.newInstances(config);

        warmUpPartitions(instances);
    }

    @AfterClass
    public static void killHazelcastInstances() {
        HazelcastInstanceFactory.terminateAll();
    }

    private HazelcastInstance getHazelcastInstance(String partitionKey) {
        Partition partition = instances[0].getPartitionService().getPartition(partitionKey);
        Member owner = partition.getOwner();
        assertNotNull(owner);

        HazelcastInstance hz = null;
        for (HazelcastInstance instance : instances) {
            if (instance.getCluster().getLocalMember().equals(owner)) {
                hz = instance;
                break;
            }
        }
        assertNotNull(hz);
        return hz;
    }

    public NodeEngineImpl getNodeEngine(HazelcastInstance hz) {
        Node node = getNode(hz);
        return node.nodeEngine;
    }

    @Test
    public void testLock() throws Exception {
        String partitionKey = "hazelcast";
        HazelcastInstance hz = getHazelcastInstance(partitionKey);

        ILock lock = hz.getLock("lock@" + partitionKey);
        lock.lock();
        assertEquals("lock@" + partitionKey, lock.getName());
        assertEquals(partitionKey, lock.getPartitionKey());

        Node node = getNode(hz);
        LockServiceImpl lockService = node.nodeEngine.getService(LockServiceImpl.SERVICE_NAME);

        Partition partition = instances[0].getPartitionService().getPartition(partitionKey);
        LockStore lockStore = lockService.getLockStore(partition.getPartitionId(), new InternalLockNamespace(lock.getName()));
        Data key = node.getSerializationService().toData(lock.getName(), StringPartitioningStrategy.INSTANCE);
        assertTrue(lockStore.isLocked(key));
    }

    @Test
    public void testSemaphore() throws Exception {
        String partitionKey = "hazelcast";
        HazelcastInstance hz = getHazelcastInstance(partitionKey);

        ISemaphore semaphore = hz.getSemaphore("semaphore@" + partitionKey);
        semaphore.release();
        assertEquals("semaphore@" + partitionKey, semaphore.getName());
        assertEquals(partitionKey, semaphore.getPartitionKey());

        SemaphoreService service = getNodeEngine(hz).getService(SemaphoreService.SERVICE_NAME);
        assertTrue(service.containsSemaphore(semaphore.getName()));
    }

    @Test
    public void testRingbuffer() throws Exception {
        String partitionKey = "hazelcast";
        HazelcastInstance hz = getHazelcastInstance(partitionKey);

        Ringbuffer ringbuffer = hz.getRingbuffer("ringbuffer@" + partitionKey);
        ringbuffer.add("foo");
        assertEquals("ringbuffer@" + partitionKey, ringbuffer.getName());
        assertEquals(partitionKey, ringbuffer.getPartitionKey());

        RingbufferService service = getNodeEngine(hz).getService(RingbufferService.SERVICE_NAME);
        assertTrue(service.getContainers().containsKey(ringbuffer.getName()));
    }

    @Test
    public void testIdGenerator() throws Exception {
        String partitionKey = "hazelcast";
        HazelcastInstance hz = getHazelcastInstance(partitionKey);

        IdGenerator idGenerator = hz.getIdGenerator("idgenerator@" + partitionKey);
        idGenerator.newId();
        assertEquals("idgenerator@" + partitionKey, idGenerator.getName());
        assertEquals(partitionKey, idGenerator.getPartitionKey());

        AtomicLongService service = getNodeEngine(hz).getService(AtomicLongService.SERVICE_NAME);
        assertTrue(service.containsAtomicLong("hz:atomic:idGenerator:" + idGenerator.getName()));
    }

    @Test
    public void testAtomicLong() throws Exception {
        String partitionKey = "hazelcast";
        HazelcastInstance hz = getHazelcastInstance(partitionKey);

        IAtomicLong atomicLong = hz.getAtomicLong("atomiclong@" + partitionKey);
        atomicLong.incrementAndGet();
        assertEquals("atomiclong@" + partitionKey, atomicLong.getName());
        assertEquals(partitionKey, atomicLong.getPartitionKey());

        AtomicLongService service = getNodeEngine(hz).getService(AtomicLongService.SERVICE_NAME);
        assertTrue(service.containsAtomicLong(atomicLong.getName()));
    }

    @Test
    public void testQueue() throws Exception {
        String partitionKey = "hazelcast";
        HazelcastInstance hz = getHazelcastInstance(partitionKey);

        IQueue queue = hz.getQueue("queue@" + partitionKey);
        queue.add("");
        assertEquals("queue@" + partitionKey, queue.getName());
        assertEquals(partitionKey, queue.getPartitionKey());

        QueueService service = getNodeEngine(hz).getService(QueueService.SERVICE_NAME);
        assertTrue(service.containsQueue(queue.getName()));
    }

    @Test
    public void testList() throws Exception {
        String partitionKey = "hazelcast";
        HazelcastInstance hz = getHazelcastInstance(partitionKey);

        IList list = hz.getList("list@" + partitionKey);
        list.add("");
        assertEquals("list@" + partitionKey, list.getName());
        assertEquals(partitionKey, list.getPartitionKey());

        ListService service = getNodeEngine(hz).getService(ListService.SERVICE_NAME);
        assertTrue(service.getContainerMap().containsKey(list.getName()));
    }

    @Test
    public void testSet() throws Exception {
        String partitionKey = "hazelcast";
        HazelcastInstance hz = getHazelcastInstance(partitionKey);

        ISet set = hz.getSet("set@" + partitionKey);
        set.add("");
        assertEquals("set@" + partitionKey, set.getName());
        assertEquals(partitionKey, set.getPartitionKey());

        SetService service = getNodeEngine(hz).getService(SetService.SERVICE_NAME);
        assertTrue(service.getContainerMap().containsKey(set.getName()));
    }

    @Test
    public void testCountDownLatch() throws Exception {
        String partitionKey = "hazelcast";
        HazelcastInstance hz = getHazelcastInstance(partitionKey);

        ICountDownLatch countDownLatch = hz.getCountDownLatch("countdownlatch@" + partitionKey);
        countDownLatch.trySetCount(1);
        assertEquals("countdownlatch@" + partitionKey, countDownLatch.getName());
        assertEquals(partitionKey, countDownLatch.getPartitionKey());

        CountDownLatchService service = getNodeEngine(hz).getService(CountDownLatchService.SERVICE_NAME);
        assertTrue(service.containsLatch(countDownLatch.getName()));
    }

    @Test
    public void testObjectWithPartitionKeyAndTask() throws Exception {
        HazelcastInstance instance = instances[0];
        IExecutorService executorServices = instance.getExecutorService("executor");
        String partitionKey = "hazelcast";
        ISemaphore semaphore = instance.getSemaphore("foobar@" + partitionKey);
        semaphore.release();
        ContainsSemaphoreTask task = new ContainsSemaphoreTask(semaphore.getName());
        Future<Boolean> f = executorServices.submitToKeyOwner(task, semaphore.getPartitionKey());
        assertTrue(f.get());
    }

    private static class ContainsSemaphoreTask implements Callable<Boolean>, HazelcastInstanceAware, Serializable {
        private transient HazelcastInstance hz;
        private final String semaphoreName;

        private ContainsSemaphoreTask(String semaphoreName) {
            this.semaphoreName = semaphoreName;
        }

        @Override
        public void setHazelcastInstance(HazelcastInstance hazelcastInstance) {
            this.hz = hazelcastInstance;
        }

        @Override
        public Boolean call() {
            NodeEngineImpl nodeEngine = TestUtil.getNode(hz).nodeEngine;
            SemaphoreService service = nodeEngine.getService(SemaphoreService.SERVICE_NAME);
            return service.containsSemaphore(semaphoreName);
        }
    }

    @Test
    public void testObjectWithPartitionKeyAndMap() throws Exception {
        HazelcastInstance instance = instances[0];
        IExecutorService executorServices = instance.getExecutorService("executor");
        String partitionKey = "hazelcast";
        String mapKey = "key@" + partitionKey;
        IMap map = instance.getMap("map");

        map.put(mapKey, "foobar");

        ISemaphore semaphore = instance.getSemaphore("s@" + partitionKey);
        semaphore.release();

        ContainsSemaphoreAndMapEntryTask task = new ContainsSemaphoreAndMapEntryTask(semaphore.getName(), mapKey);
        Map<Member, Future<Boolean>> futures = executorServices.submitToAllMembers(task);

        int count = 0;
        for (Future<Boolean> f : futures.values()) {
            count += f.get() ? 1 : 0;
        }

        assertEquals(1, count);
    }

    private static class ContainsSemaphoreAndMapEntryTask implements Callable<Boolean>, HazelcastInstanceAware, Serializable {
        private final String mapKey;
        private transient HazelcastInstance hz;
        private final String semaphoreName;

        private ContainsSemaphoreAndMapEntryTask(String semaphoreName, String mapKey) {
            this.semaphoreName = semaphoreName;
            this.mapKey = mapKey;
        }

        @Override
        public void setHazelcastInstance(HazelcastInstance hazelcastInstance) {
            this.hz = hazelcastInstance;
        }

        @Override
        public Boolean call() {
            NodeEngineImpl nodeEngine = TestUtil.getNode(hz).nodeEngine;
            SemaphoreService service = nodeEngine.getService(SemaphoreService.SERVICE_NAME);

            IMap map = hz.getMap("map");
            if (map.localKeySet().contains(mapKey)) {
                return service.containsSemaphore(semaphoreName);
            } else {
                return false;
            }
        }
    }
}

redpajama_set_name: RedPajamaGithub


The upcoming Android device headed to T-Mobile HTC Amaze 4G (Ruby) design press image leaks out showing the smooth slightly rounded edges.
According to rumors the back cover of the phone is similar to HTC Flyer cover white plastic separated with silverish aluminium. HTC Amaze 4G (Ruby) features a 1.5GHz dual-core CPU, 4.3-inch qHD display (540x960p), a 8 mega-pixel rear-facing camera and a 2 mega-pixel camera on the front for video chat, 1GB of RAM and the latest Bluetooth 3.0 technology.


text:
"A PEPTIDE WHICH IS CAPABLE OF BINDING TO AN MHC CLASS I OR II MOLECULE"
A peptide which is capable of binding to an MHC class I or II molecule without further processing, and which is selected from the following myelin basic protein peptides: YKSAHKGFKGVDAQG (134-148), KSAHKGFKGVDAQGT (135-149), SAHKGFKGVDAQGTL (136-150), AHKGFKGVDAQGTLS (137-151), HKGFKGVDAQGTLSK (138-152), GFKGVDAQGTLSKIF (140-154).
TOLEROGENIC PEPTIDES FROM MYELIN BASIC PROTEIN
The present invention relates to peptides from myelin basic protein. In particular, the invention relates to peptides which comprise a portion of the region 131-158 of myelin basic protein and their use in the treatment and/or prevention of a disease.
In an adaptive immune response, T lymphocytes are capable of recognising internal epitopes of a protein antigen. Antigen presenting cells (APC) take up protein antigens and degrade them into short peptide fragments. A peptide may bind to a major histocompatability complex (MHC) class I or II molecule inside the cell and be carried to the cell surface. When presented at the cell surface in conjunction with an MHC molecule, the peptide may be recognised by a T cell (via the T cell receptor (TCR)), in which case the peptide is a T cell epitope.
T cell epitopes play a central role in the adaptive immune response to any antigen, whether self or foreign. The central role played by T cell epitopes in hypersensitivity diseases (which include allergy, autoimmune diseases and transplant rejection) has been demonstrated through the use of experimental models. It is possible to induce inflammatory or allergic diseases by injection of synthetic peptides (based on the structure of T cell epitopes) in combination with adjuvant.
By contrast, it has been shown to be possible to induce immunological tolerance towards particular peptide epitopes by administration of peptide epitopes in soluble form. Administration of soluble peptide antigens has been demonstrated as an effective means of inhibiting disease in experimental autoimmune encephalomyelitis (EAE - a model for multiple sclerosis (MS)) (Metzler and Wraith (1993) Int. Immunol. 5:1159-1165; Liu and Wraith (1995) Int. Immunol. 7:1255-1263; Anderton and Wraith (1998) Eur. J. Immunol. 28:1251-1261); and experimental models of arthritis, diabetes, and uveoretinitis (reviewed in Anderton and Wraith (1998) as above). This has also been demonstrated as a means of treating an ongoing disease in EAE (Anderton and Wraith (1998) as above).
The use of tolerogenic peptides to treat or prevent disease has attracted considerable attention. One reason for this is that it has been shown that certain tolerogenic epitopes can down-regulate responses of T cells for distinct antigens within the same tissue. This phenomenon, known as "bystander suppression" means that it should be possible to induce tolerance to more than one epitope (preferably all epitopes) within a given antigen, and to more than one antigen for a given disease, using a particular tolerogenic peptide (Anderton and Wraith (1998) as above). This would obviate the need to identify all of the pathogenic antigens within a particular disease.
Peptides are also a favourable option for therapy because of their relatively low cost and the fact that peptide analogues can be produced with altered immunological properties. Peptides may thus be modified to alter their interactions with either MHC or TCR.
One possible problem with this approach is that it has been shown that not all peptides which act as T cell epitopes are capable of inducing tolerance. The myelin basic protein (MBP) peptide 89-101 is an immunodominant antigen after immunisation and is also a very effective immunogen both in terms of priming for T cell reactivity and induction of EAE. However, this peptide has been shown to be ineffective at inducing tolerance when administered in solution (Anderton and Wraith (1998), as above).
A number of explanations for the observed hierarchy in the ability of T cell epitopes to induce tolerance have been proposed (reviewed in Anderton and Wraith (1998) as above). In particular, it has been proposed that there is a correlation between the affinity of the peptide for the MHC and tolerogenicity (Liu and Wraith (1995) as above), but this does not tally with some of the observations. For example, MBP[89-101], which is not tolerogenic, binds to I-A with relatively high affinity. It is thus not straightforward to predict which peptides will induce tolerance.
The present inventors have shown that if a peptide epitope is of an appropriate size to be presented by immature APC without antigen processing, it can induce immunological tolerance (International patent application number PCT/GBOl/03702). The observation
that some T cell epitopes are tolerogenic and others are incapable of inducing tolerance can therefore be explained by the fact that some epitopes require further processing before they are capable of being presented by an MHC molecule. These epitopes which require further processing do not induce tolerance when administered in a soluble form, despite their capacity to induce disease when injected in combination with adjuvant.
The epitopes which do not require further processing are capable of inducing tolerance, and have been termed "apitopes" (Antigen Processing Independent epiTOPES) by the inventors.
The present inventors have examined the region 131-158 of MBP, and found a number of peptides which can be presented by fixed antigen presenting cells to T-cells
These peptides are defined as apitopes, because they are capable of binding to MHC class I or II without further processing.
In a first aspect, therefore, the present invention provides an apitope which comprises a portion of the region 131-158 of myelin basic protein.
The present inventors have also identified two minimal epitopes in this region which are recognised by particular T-cell clones. The peptide may comprise one or both of these epitopes which are MBP 142-152 and 140-148.
In a preferred embodiment the peptide is selected from the following myelin basic protein peptides: 134-148,135-149,136-150,137-151,138-152,139-153,140-154.
In a second aspect, the present invention provides a pharmaceutical composition comprising one or more peptide according to the first aspect of the application.
In a third aspect, the present invention provides a method for treating and/or preventing a disease in a subject in need of same which comprises the step of administering a peptide according to the first aspect of the invention to the subject.
The present invention also provides the use of a peptide according to the first aspect of the invention in the manufacture of a medicament for use in the treatment and/or prevention of a disease.
The peptides of the present invention are useful in the prevention and/or treatment of multiple sclerosis.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the response of T cell clone MS60:D2 to presentation of nested MBP peptides in the region 131 -158 by APC.
Figure 2 shows the response of T cell clone N5 to presentation of MBP peptide 140-154 by APC.
Figure 3 shows the response of T cell clone N5 to presentation of nested MBP peptides in the region 136-157 by APC.
DETAILED DESCRIPTION OF INVENTION
In a first aspect, the present invention relates to a peptide.
The term "peptide" is used in the normal sense to mean a series of residues, typically L-amino acids, connected one to the other typically by peptide bonds between the a-amino
and carboxyl groups of adjacent amino acids The term includes modified peptides and synthetic peptide analogues.
A peptide of the present invention may be any length that is capable of binding to an MHC class I or II molecule without further processing.
Peptides that bind to MHC class I molecules are typically 7 to 13, more usually 8 to 10 amino acids in length. The binding of the peptide is stabilised at its two ends by contacts between atoms in the main chain of the peptide and invariant sites in the peptide-binding groove of all MHC class I molecules. There are invariant sites at both ends of the groove which bind the amino and carboxy tennini of the peptide. Variations is peptide length are accommodated by a kinking in the peptide backbone, often at proline or glycine residues that allow the required flexibility.
Peptides which bind to MHC class II molecules are typically between 8 and 20 amino acids in length, more usually between 10 and 17 amino acids in length, and can be much longer. These peptides lie in an extended conformation along the MHC II peptide-binding groove which (unlike the MHC class I peptide-binding groove) is open at both ends. The peptide is held in place mainly by main-chain atom contacts with conserved residues that line the peptide-binding groove.
The peptide of the present invention may be made using chemical methods (Peptide Chemistry, A practical Textbook. Mikos Bodansky, Springer-Verlag, Berlin.). For example, peptides can be synthesized by solid phase techniques (Roberge JY et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). Automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
The peptide may alternatively be made by recombinant means, or by cleavage from a longer polypeptide. For example, the peptide may be obtained by cleavage from myelin
basic protein. The composition of a peptide may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure).
The peptide of the invention is derivable from region 131-158 of MBP. Preferably the peptide is derivable from a fragment of the antigen which arises by natural processing of the antigen by an APC.
For practical purposes, there are various other characteristics which the peptide should show. For example, the peptide should be soluble at a concentration which permits its use in vivo. Preferably the peptide should be soluble at concentrations of up to 0.5 mg/ml, more preferably the peptide should be soluble at concentrations of up to 1 mg/ml, most preferably the peptide should be soluble at concentrations of up to 5 mg/ml.
For intranasal administration the maximum volume of dose which can be taken up using current procedures is approximately 200µ1 per nostril. If the peptide is soluble at 1 mg/ml, a double dose to each nostril enables 800ug to be given to the patient. It is unusual to give more that 5mg in any individual dose.
It is also important that the peptide is sufficiently stable in vivo to be therapeutically useful. The present inventors have found that in vivo, 30 minutes after administration the total amount of a test peptide drops to about 50%, 4 hours after administration the amount drops to about 30%, but that after 5 days the peptide is still detectable (at about 5%). The half-life of the peptide in vivo should be at least 10 minutes, preferably at least 30 minutes, more preferably at least 4 hours, most preferably at least 24 hours.
The present inventors have found that following intranasal administration, the amount of peptide in the draining lymph node peaks at about 4 hrs following administration, however peptide is still detectable (at levels of about 5% maximum) after 5 days. Preferably the peptide is sufficiently stable to be present at a therapeutically active concentration in the draining lymph node for long enough to exert a therapeutic effect.
The peptide should also demonstrate good bioavailability in vivo. The peptide should maintain a conformation in vivo which enables it to bind to an MHC molecule at the cell surface without due hindrance.
Myelin Basic Protein (MBP)
MBP is an antigen from myelin, which has been previously characterised and sequenced (Roth et al (1987) J. Neurosci. Res. 17(4) 321-328; Accession number M30516).
The peptides of the present invention are derivable from MBP region 131-158.
Preferably the peptide is or comprises one or more of the minimal T-cell epitopes in the region, for example the minimal epitope MBP142-152 or 140-148.
Preferably the peptide is selected from the following myelin basic protein peptides:
(Table removed)
Antigen Processing Independent Epitopes (APITOPES)
The peptide of the present invention is capable of binding to an MHC class I or II molecule (in the peptide-binding groove) without further processing. Such peptides are known herein as "apitopes" (Antigen Processing Independent epiTOPES).
Cell surface presentation of peptides derived from a given antigen is not random and tends to be dominated by a small number of frequently occurring epitopes. The dominance of a particular peptide will depend on many factors, such as relative affinity for binding the MHC molecule, spatio-temporal point of generation within the APC and resistance to degradation. The epitope hierarchy for an antigen can change with progression of an immune response, which has important implications for self-tolerance and autoimmunity. Immunodominant determinant regions are likely to be good tolerogens. Hence, in a preferred embodiment, the apitope of the present invention is based on a dominant epitope.
However, after a primary immune response to the immunodominant peptides, epitope "spreading" may occur to sub-dominant determinants (Lehmann et al (1992) Nature 358:155-157). Presentation of sub-dominant epitopes may be important in triggering autoimmunity. The apitope of the present invention may, therefore be based on a subdominant epitope.
For any given antigen, cryptic epitopes may also exist. Cryptic epitopes are those which can stimulate a T cell response when administered as a peptide but which fail to produce such a response when administered as a whole antigen. It may be that during processing of the antigen into peptides in the APC the cryptic epitope is destroyed.
A cryptic epitope may act as an apitope in vitro, in that it may be capable of binding to an MHC molecule without further processing, and inducing anergy in a T cell which recognises the cryptic epitope. However, such an apitope would be unlikely to be therapeutically useful because it should be incapable of tolerising T cells which recognise a naturally processed epitope of the antigen.
Epitopes for an antigen may be identified by measuring the T cell response to overlapping peptides spanning the entire antigen (see below) when presented by APC. Such studies usually result in "nested sets" of peptides, and the minimal epitope for a particular T cell line/clone can be assessed by measuring the response to truncated peptides.
It cannot be assumed that a minimal epitope of an antigen will behave as an apitope. It may well be that amino acids flanking the minimal epitope will be required for optimal binding to the MHC. The apitope should be designed to cover the possibility that there may be subtle differences between the minimal epitopes of different T cell clones.
It should be emphasised mat it may not be possible to identify an apitope for all epitopes. There is clear evidence that some epitopes bind MHC in a way that is dependent on MHC-loading in endosomes and hence require processing (Viner et al (1995) Proc. Natl. Acad. Sci. 92:2214-2218). This is another reason why one cannot assume that each minimal epitope will inevitably behave as an apitope.
Identification of peptides containing T cell epitopes
There are a number of methods known in the art to identify the T cell epitopes within a given antigen.
Naturally processed epitopes may be identified by mass spectrophotometnc analysis of peptides eluted from antigen-loaded APC. These are. APC that have either been encouraged to take up antigen, or have been forced to produce the protein intracellularly by transformation with the appropriate gene. Typically APC are incubated with protein either in solution or suitably targeted to the APC cell surface. After incubation at 37°C the cells are lysed in detergent and the class II protein purified by, for example affinity chromatography. Treatment of the purified MHC with a suitable chemical medium (for example, acid conditions) results in the elution of peptides from the MHC. This pool of peptides is separated and the profile compared with peptide from control APC treated in the same way. The peaks unique to the protein expressing/fed cells are analysed (for example by mass spectrometry) and the peptide fragments identified. This procedure usually generates information about the range of peptides (usually found in "nested sets") generated from a particular antigen by antigen processing.
Another method for identifying epitopes is to screen a synthetic library of peptides which overlap and span the length of the antigen in an in vitro assay. For example, peptides
which are 15 amino acids in length and which overlap by 5 or 10 amino acids may be used. The peptides are tested in an antigen presentation system which comprises antigen presenting cells and T cells. For example, the antigen presentation system may be a murine splenocyte preparation, a preparation of human cells from tonsil or PBMC. Alternatively, the antigen presentation system may comprise a particular T cell line/clone and/or a particular antigen presenting cell type.
T cell activation may be measured via T cell proliferation (for example using H-thymidine incorporation) or cytokine production. Activation of THl-type CD4+ T cells can, for example be detected via IFNy production which may be detected by standard techniques, such as an ELISPOT assay.
Overlapping peptide studies usually indicate the area of the antigen in which an epitope is located. The minimal epitope for a particular T cell can then be assessed by measuring the response to truncated peptides. For example if a response is obtained to the peptide comprising residues 1-15 in the overlapping library, sets which are truncated at both ends (i.e. 1-14, 1-13, 1-12 etc. and 2-15, 3-15, 4-15 etc.) can be used to identify the minimal epitope.
Antigen Processing Independent Presentation Systems (APIPS)
Once an epitope has been identified, the next step is to investigate whether it also behaves as an apitope.
An apitope must be presented to T cells without the need for antigen processing. Having identified peptides containing T cell epitopes, apitopes may be identified using a processing free system. Truncated peptides and peptide analogues may be tested for activation using an antigen processing independent presentation system (APIPS).
Examples of APIPS include:
a) fixed APC (with or without antibodies to CD28);
b) Lipid membranes containing Class I or II MHC molecules (with or without antibodies to CD28); and
c) purified natural or recombinant MHC in plate-bound form (with or without antibodies to CD28).
It is known to use fixed APC to investigate T cell responses, for example in studies to investigate the minimal epitope within a polypeptide, by measuring the response to truncated peptides (Fairchild et al (1996) Int Immunol. 8:1035-1043). APC may be fixed using, for example formaldehyde (usually paraformaldehyde) or glutaraldehyde.
Lipid membranes (which may be planar membranes or liposomes) may be prepared using artificial lipids or may be plasma membrane/microsomal fractions from APC.
In use, the APIPS may be applied to the wells of a tissue culture plate. Peptide antigens are then added and binding of the peptide to the MHC portion of the APIPS is detected by addition of selected T cell lines or clones. Activation of the T cell line or clone may be measured by any of the methods known in the art, for example via 3H-thymidine incorporation or cytokine secretion.
The peptides of the present invention should be capable of inducing tolerance to MBP as they are apitopes for this antigen.
As used herein, the term "tolerogenic" means capable of inducing tolerance.
Tolerance is the failure to respond to an antigen. Tolerance to self antigens is an essential feature of the immune system, when this is lost, autoimmune disease can result. The adaptive immune system must maintain the capacity to respond to an enormous variety of infectious agents while avoiding autoimmune attack of the self antigens contained within its own tissues. This is controlled to a large extent by the sensitivity of immature T lymphocytes to apoptotic cell death in the thymus (central tolerance). However, not all
self antigens are detected in the thymus, so death of self-reactive thymocytes remains incomplete. There are thus also mechanisms by which tolerance may be acquired by mature self-reactive T lymphocytes in the peripheral tissues (peripheral tolerance). A review of the mechanisms of central and peripheral tolerance is given in Anderton et al (1999) {Immunological Reviews 169:123-137).
Tolerance may result from or be characterised by the induction of anergy in at least a portion of CD4+ T cells. In order to activate a T cell, a peptide must associate with a "professional" APC capable of delivering two signals to T cells. The first signal (signal 1) . is delivered by the MHC-peptide complex on the cell surface of the APC and is received by the T cell via the TCR. The second signal (signal 2) is delivered by costimulatory molecules on the surface of the APC, such as CD80 and CD86, and received by CD28 on the surface of the T cell. It is thought that when a T cell receives signal 1 in the absence of signal 2, it is not activated and, in fact, becomes anergic. Anergic T cells are refractory to subsequent antigenic challenge, and may be capable of suppressing other immune responses. Anergic T cells are thought to be involved in mediating T cell tolerance.
Without wishing to be bound by theory, the present inventors predict that peptides which require processing before they can be presented in conjunction with MHC molecules do not induce tolerance because they have to be handled by mature antigen presenting cells. Mature antigen presenting cells (such as macrophages, B cells and dendritic cells) are capable of antigen processing, but also of delivering both signals 1 and 2 to a T cell, leading to T cell activation. Apitopes, on the other hand, will be able to bind class II MHC on immature APC. Thus they will be presented to T cells without costimulation, leading to T cell anergy and tolerance.
Of course, apitopes are also capable of binding to MHC molecules at the cell surface of mature APC. However, the immune system contains a greater abundance of immature than mature APC (it has been suggested that less than 10% of dendritic cells are activated, Summers et al. (2001) Am. J. Pathol. 159: 285-295). The default position to an apitope will therefore be anergy/tolerance, rather than activation.
it has been shown that, when tolerance is induced by peptide inhalation, the capacity of antigen-specific CD4+ T cells to proliferate is reduced. Also, the production of IL-2, IFN-y and IL-4 production by these cells is down-regulated, but production of IL-10 is increased. Neutralisation of IL-10 in mice in a state of peptide-induced tolerance has been shown to restore completely susceptibility to disease. It has been proposed that a population of regulatory cells persist in the tolerant state which produce IL-10 and mediate immune regulation (Burkhart et al (1999) Int. Immunol. 11:1625-1634).
The induction of tolerance can therefore be monitored by various techniques including:
(a)	reduced susceptibility to contract the disease for which the peptide is a target epitope in vivo;
(b)	the induction of anergy in CD4+ T cells (which can be detected by subsequent challenge with antigen in vitro);
(c)	changes in the CD4+ T cell population, including
(i) reduction in proliferation;
(ii) down-regulation in the production of IL-2, BFN-y and IL-4; and (iii) increase in the production of IL-10.
Target diseases
The peptide of the invention may be used in the treatment and/or prevention of a disease.
The peptides of the present invention are particularly useful in the treatment and/or prevention of multiple sclerosis (MS). Multiple sclerosis (MS) is a chronic inflammatory disease characterised by multiple demyelinating lesions disseminated throughout the CNS white matter and occurring at various sites and times (McFarlin and McFarland, 1982 New England J. Medicine 307:1183-1188 and 1246-1251). MS is thought to be mediated by autoreactive T cells.
MBP is immunogenic and MBP-specific T lymphocytes have encephalitogenic activity in animals (Segal et al., 1994 J. Neuroimmunol. 51:7-19; Voskuhl et al., 1993 J. Neuroimmunol 42:187-192; Zamvil etal, 1985 Nature 317:355-8).
Pliarmaceutical composition
In a second aspect, the present invention relates to a pharmaceutical composition comprising one or more peptide(s) of the first aspect of the invention. The composition may also comprise one or more apitopes from another region of MBP or from a different antigen.
The present inventors predict that, despite "bystander suppression" it may be necessary to target a number of different T cell clones in order to induce tolerance effectively. Hence a plurality of peptides may be administered to an individual in order to prevent or treat a disease.
The pharmaceutical composition may, for example comprise between 1 and 50 apitopes, preferably between 1 and 15 apitopes. If there is more than one apitope, preferably the apitopes are either all able to bind to MHC class I, or all able to bind MHC class II, without further processing.
Where there are two or more apitopes, the pharmaceutical composition may be in the form of a kit, in which some or each of the apitopes are provided separately for simultaneous, separate or sequential administration.
Alternatively (or in addition) if the pharmaceutical composition (or any part thereof) is to be administered in multiple doses, each dose may be packaged separately.
The pharmaceutical composition may comprise a therapeutically or prophylactically effective amount of the or each apitope and optionally a pharmaceutically acceptable carrier, diluent or excipient.
Also, in the pharmaceutical compositions of the present invention, the or each apitope may be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), or solubilising agent(s).
The peptide may be administered in soluble form in the absence of adjuvant.
Preferably the peptide is administered by a mucosal route.
Studies have shown that peptide, when given in soluble form intraperitoneally (i.p.), intravenously (i.v.) or intranasally (i.n.) or orally can induce T cell tolerance (Anderton and Wraith (1998) as above; Liu and Wraith (1995) as above; Metzler and Wraith (1999) Immunology 97:257-263).
Preferably the peptide is administered intranasally.
Studies in mice have demonstrated that the duration of peptide administration required to induce tolerance depends on the precursor frequency of T cells in the recipient (Burkhart et al (1999) as above). In many experimental studies, it has been shown that repeated doses of peptide are required to induce tolerance (Burkhart et al (1999) as above). The exact dose and number of doses of peptide will therefore depend on the individual, however, in a preferred embodiment a plurality of doses is administered.
If a plurality of peptides is administered simultaneously, they may be in the form of a "cocktail" which is suitable for administration in single or multiple doses. Alternatively it may be preferably to give multiple doses but vary the relative concentrations of the peptides between doses.
In a preferred embodiment a "dose escalation" protocol may be followed, where a plurality of doses is given to the patient in ascending concentrations. Such an approach has been used, for example, for phospholipase A2 peptides in immunotherapeutic applications against bee venom allergy (Muller et al (1998) J. Allergy Clin Immunol. 101:747-754 and Akdis et al (1998) J. Clin. Invest. 102:98-106).
The following examples serve to illustrate the present invention, but should not be construed as a limitation thereof. The invention particularly relates to the specific embodiments described in these examples
EXAMPLE 1 - Identification of apitopes within MBP region 134-154
Human MBP is prepared from brain white matter as described by Deibler et al. (Deibler et ai, 1972 Preparative Biochemistry 2:139), and its purity assessed by SDS-PAGE. MBP and Mycobacterium tuberculosis purified protein derivative (PPD) (UK Central Veterinary Laboratory, Surrey) are used in proliferative assays at previously determined optimal concentrations; the optimum concentration for each antigen is 20 ug/ml. A panel of 15-mer overlapping peptides spanning MBP region 131-158 are synthesized using standard F-moc chemistry on an Abimed AMS 422 multiple peptide synthesizer (Abimed, Langenfeld, Germany). Each peptide is displaced by 1 aa and overlapped by 14 aa.
Tissue culture medium
RPMI-1640 medium supplemented with 20mM HEPES (Sigma, Poole, UK), penicillin (l00U/ml), streptomycin sulphate (100 mg/ml), and 4 mM L-glutamine (all from Life Technologies, Paisley, Scotland), is used as the tissue culture medium. Medium without serum is used for washing lymphoid cells and TCL. For all culture conditions and assays, medium is supplemented with 10 % heat inactivated autologous plasma.
Generation of T cell clones
MBP-specific T cell lines (TCL) are generated from 8 MS patients and 2 healthy control donors. PBMC from each subject are separated as described above and plated out at lxl06 cells/ml in 6-well plates in the presence of MBP (50 ug/ml); a portion of PBMC from each subject is regularly frozen and stored for subsequent restimulations. Seven days later the
cells are ted with fresh medium containing 2% IL-2 (Lymphocult-HT; Biotest LTD., Birmingham, UK), and on day 12 of culture all cells are restirnulated with antigen, EL-2 and irradiated (2500 Rad) autologous PBMC as a source of antigen presenting cells (APC), at a cell ratio of IT cell:5 APC. Cells are expanded in IL-2 every 3-4 days, and on day 14 are restirnulated with antigen, IL-2 and PBMC, as described above. On the day of the first restimulation cells are examined for specific proliferation to MBP. Briefly, 2x10 T cells and lxl 05 irradiated autologous PBMC are cultured in triplicate, in 96-well round-bottom plates, in the presence of MBP. Cells are cultured for 2 days and pulsed with ( H)-Thymidine at 0.4 µCi/well during the last 18 hours of the culture. Cells are then harvested as described above, and a TCL is considered to be MBP-specific with a Scpm >1000 and a . SI>3.
Following 3 restimulation/expansion cycles TCL are cloned using PEA (Sigma, Poole, Dorset, UK)) in the presence of autologous irradiated PBMC as APC. T cells are plated under, limiting dilution conditions at 0.1 cell/well, 0.3 cell/well and 1 cell/well and cultured in Terasaki plates (Nunc International, Costar) with lx104 irradiated PBMC, 5µg/ml PHA, and 2% IL-2. After 10-12 days, growth-positive wells are expanded onto 96-well round-bottom plates, using lxl05 irradiated PBMC, 5µg/ml PHA and IL-2. Three days later wells are fed with fresh medium containing IL-2, and on day 7 the clones are expanded onto 48-well plates using 5xl05 irradiated PBMC, PHA and IL-2; at this point clones are tested in proliferation assays for specific responses to MBP. MBP-specific clones are expanded a week later onto 24-well plates, using lxl06 irradiated PBMC with PHA or Dynabeads (Dynal, UK) and IL-2. The clones are maintained in 24-well plates using a 7-10 day restimulation/expansion cycle.
Proliferation assay
Live or p-formaldehyde fixed Mgar (HLA-DR2 +ve) cells were incubated with peptides in serum or in serum alone, together with T cells. The T cell proliferative response was measured by 3H-thymidine uptake as follows. Triplicate aliquots of 100 u.1 of each culture were grown in a 96-well round bottom microtitre plate for 48 hours, then pulsed with 0.4 µCi [3H]-Thymidine (Amersham International, Amersham, UK). After 20 hours cells are harvested onto glass fibre mats (LKB-Wallac, Turku, Finland) using a Mach 111 harvester
96 (Tomtec, Orange, New Jersey, USA). [3H]- Thymidine incorporation is determined using a Microbeta liquid scintillation counter (LKB-Wallac). Test wells containing antigen are considered positive when the 5cpm >1000 and the Stimulation Index (SI)3, where SI = CPM antigen containing culture/CPM culture without antigen.
The response of T cell clone MS60:D2 to presentation of nested MBP peptides in the region 131-158 is shown in Figure 1. Peptides 134-148, 135-149, 136-150, 137-151, 138-152,139-153 and 140-154 are defined as apitopes as they could be presented by fixed APC to T cells without further processing.
The response of T cell clone N5 to presentation of peptide 140-154 is shown in Figure 2. This further confirms that peptide 140-154 can be presented by fixed APC without further processing.
EXAMPLE 2 - Identification of the minimum epitope recognised by T cell clones N5 and MS60:D2
Live Mgar cells were incubated with overlapping peptides from MBP region 136-157 in serum or serum alone, together with N5 T cells. T cell proliferation was measured by 3H-thymiding uptake.
The results are shows in Figure 3. The minimal epitope recognised by T cell clone N5 is MBP 142-152.
A similar experiment was performed for T cell clone MS60:D2 (Fig. 1, unfixed Mgar). The minimal epitope recognised by this T cell clone is MBP 140-148.
various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry or biology or related fields are intended to be covered by the present invention. All publications mentioned in the above specification are herein incorporated by reference.
W CLAIM;
1.	A peptide which is capable of binding to an MHC class I or II molecule without further
processing, and which is selected from the following myelin basic protein peptides:
YKSAHKGFKGVDAQG (134-148); KSAHKGFKGVDAQGT (135-149); SAHKGFKGVDAQGTL (136-150); AHKGFKGVDAQGTLS (137-151); HKGFKGVDAQGTLSK (138-152); GFKGVDAQGTLSKIF (140-154).
2.	A peptide as claimed in claim 1 which is myelin basic protein peptide GFKGVDAQGTLSKIF (140-154).
2044-DELNP-2004-Abstract-(30-09-2010).pdf
2044-delnp-2004-abstract.pdf
2044-DELNP-2004-Claims-(30-05-2011).pdf
2044-delnp-2004-claims.pdf
2044-DELNP-2004-Correspondence Others-(30-05-2011).pdf
2044-DELNP-2004-Correspondence-Others-(30-09-2010).pdf
2044-delnp-2004-correspondence-others.pdf
2044-delnp-2004-description (complete).pdf
2044-DELNP-2004-Drawings-(30-09-2010).pdf
2044-delnp-2004-drawings.pdf
2044-DELNP-2004-Form-1-(30-09-2010).pdf
2044-delnp-2004-form-1.pdf
2044-delnp-2004-form-18.pdf
2044-DELNP-2004-GPA-(30-09-2010).pdf
2044-delnp-2004-gpa.pdf
2044-delnp-2004-pct-101.pdf
2044-DELNP-2004-Petition-137-(30-09-2010).pdf
2044/DELNP/2004
APITOPE TECHNOLOGY [BRISTOL] LIMITED
UNIVERSITY OF BRISTOL, DEPARTMENT OF PATHOLOGY AND MICROBILOLGY, UNIVERSITY WALK, BRISTOL BS8 1TD, UNITED KINGDOM,
1 DAVID CAMERON WRAITH, DEPARTMENT OF PATHOLOGY AND MICROBIOLOGY, UNIVERSITY OF BRISTOL, SCHOOL OF MEDICAL SCIENCES, UNIVERSITY WALK, CLIFTON, BRISTOL BRISTOL BS8 1TD, GREAT BRITAIN,
2 HEATHER BARBARA STREETER, DEPARTMENT OF PATHOLOGY AND MICROBIOLOGY, UNIVERSITY OF BRISTOL, SCHOOL OF MEDICAL SCIENCES, UNIVERSITY WALK, CLIFTON, BRISTOL BRISTOL BS8 1TD, GREAT BRITAIN,
3 FRANCES MARY PONSFORD, DEPARTMENT OF PATHOLOGY AND MICROBIOLOGY, UNIVERSITY OF BRISTOL, SCHOOL OF MEDICAL SCIENCES, UNIVERSITY WALK, CLIFTON, BRISTOL BRISTOL BS8 1TD, GREAT BRITAIN,
4 GRAZIELLA MAZZA, DEPARTMENT OF PATHOLOGY AND MICROBIOLOGY, UNIVERSITY OF BRISTOL, SCHOOL OF MEDICAL SCIENCES, UNIVERSITY WALK, CLIFTON, BRISTOL BRISTOL BS8 1TD, GREAT BRITAIN,
C07K7/00
PCT/GB03/00399
1 0202399-2 2002-02-01 U.K.


Chris Morris @ Berryhill LTD is a well established and well known MOT service. At Chris Morris @ Berryhill LTD, our aim is to provide quality MOT and servicing in Droitwich as well as Tyres , clutch repair and car repair.
We are situated in Droitwich. We can provide quality car diagnostics across Droitwich and many of it's surrounding areas.
We are experienced in servicing who can take on any task in a reasonable timescale.


Security forces arrested a Maoist in Palamau district and seized explosives in Latehar district in separate anti-naxal operations in Jharkhand, police said.
Palamau, Superintendent of Police, Inderjit Mahata Thursday said police arrested a Maoist identified as Sudeshwar Bhuyan from a place under the jurisdiction of Manatu police station area. The SP said Bhuyan was absconding for the last 15-years.
Latehar, Superintendent of Police, Prashant Anand Thursday said that during an anti-naxal operation the security forces recovered a cache of explosives from Ranidah forest under Chipadohar police station in Latehar district.
Five IEDs, three grenades, three bundles of cotex wire, two bundles of electric wire were recovered during the operation, Anand said.


text:
iCarly er en amerikansk teenage-sitcom skabt af Dan Schneider. Serien blev sendt på Nickelodeon fra 8. september 2007 til 23. november 2012. Serien fokuserer på Carly Shay. Hun er en teenager, der opretter sit eget webshow sammen med sine bedste venner Sam Puckett og Freddie Benson. Serien kalder de iCarly.

iCarly har været et af de mest sendte programmer på Nickelodeon. I 2021 udkom serien med helt nye afsnit, som kan ses på Paramount+.

Handling
Carly bor sammen med sin 28-årige storebror Spencer. Det hele starter, da Carlys lærer sætter hende og Sam sammen til en opgave om at være dommere i skolens talentshow. Carly og Sam (Samantha Puckett) gør i pausen nar af deres lærer, og Freddie kommer til at optage det og lægge det ud på internettet. Senere får de mange fans, og teenagerne starter iCarly.

Episoder

Karakterer

Hovedpersoner
 Carly Shay (Miranda Cosgrove) er vært for sit eget populære web-show iCarly, som hun producerer med sine to bedste venner Sam og Freddie.
 Sam Puckett (Jennette McCurdy) er Carlys bedste veninde og medvært på iCarly.
 Freddie Benson (Nathan Kress) er både Carlys bedste ven, men også hendes nabo. Han er den tekniske producer på iCarly.
 Spencer Shay (Jerry Trainor) er Carlys excentriske ældre bror og værge.
 Gibby (Noah Munck) er Carly, Sam og Freddies mærkelige ven.

Tilbagevendende personer
 Marissa Benson (Mary Scheer) er Freddies anmassende og overbeskyttende mor (ofte nævnt af Sam som Freddies "freakish mor").
 Lewbert (Jeremy Rowley) er dørmanden i den bygning, hvor Carly, Spencer og Freddie bor. Han har en irriterende adfærd og en stor vorte på sit ansigt.
 Nevel Papperman (Reed Alexander) er en kritiker, der driver hjemmesiden nevelocity.com. Et websted som gennemgår andre hjemmesider herunder iCarly.com. Han er Carlys nemesis og har hele tiden forsøgt at sabotere iCarly for at få et kys af Carly.
 T-Bo (Boog!e) er leder af Groovy Smoothie. Han irriterer ofte kunderne, så de køber tilfældige fødevarer (peberfrugter, bagels, tacos), der altid sættes på en pind.
 Guppy (Ethan Munck) er Gibbys yngre bror og ofte sammen med Gibby.
 Chuck Chambers (Ryan Ochoa) er et barn i ejendommen, hvor Carly og de andre hovedpersoner bor. Han plager ofte Spencer.
 Inspektør Franklin (Tim Russ) er rektor på den skole, hvor Carly, Sam, og Freddie går. Han er meget fair og er en stor fan af iCarly.
 Mrs. Francine Briggs (Mindy Sterling) er en meget streng engelsklærer på Ridgeway Secondary School, som udviser en stor modvilje for børn.
 Mr. Howard (David St. James) er en streng og uentusiastisk lærer, der hader næsten alt herunder sin kone.

Optræden i serien

Priser

Kilder

Referencer
 Officiel hjemmeside

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The 2017 International Ice Hockey Federation Women's World Championship marked the second time Paul Mara served as an assistant coach with the U.S. Women's National Team. He served in the same capacity for the U.S. Women's National Team for a two-game series vs. Canada in December 2016, including the Team USA Winter Champions Series contest in Plymouth, Mich.
The defenseman played parts of 12 seasons in the NHL and was a bronze medalist with Team USA at the 2004 International Ice Hockey Federation Men's World Championship.
Mara played 734 NHL games and accumulated 253 points (64g, 189a) with six different franchises, including the Tampa Bay Lightning (1998-2001), Phoenix Coyotes (2001-06), Boston Bruins (2006-07), New York Rangers (2007-09), Anaheim Ducks (2010-11) and Montreal Canadiens (2009-10, 2011). His best season came in 2005-06 with the Coyotes, in which Mara led all Phoenix defensemen with a career-high 47 points (15g, 32a).
On the international stage, Mara competed in three-consecutive IIHF World Junior Championships (1997-99) with Team USA, appearing in 19 games and totaling seven points (2g, 5a). He also notched a goal and dished out a pair of assists in nine games while helping the U.S. earn a bronze medal at the 2004 IIHF Men's World Championship.
The Belmont, Massachusetts, native played two seasons of high school hockey for Belmont Hill School before playing parts of two seasons each with the Sudbury Wolves (1996-98) and Plymouth Whalers (1997-99).


Strands of Sorrow, Threads of Hope–Free all Weekend.
My book of short stories, Strands of Sorrow, Threads of Hope, including some of my mom's work as well, is available FREE for the next three days – Friday August 29th through Sunday August 31st on Amazon.
If simply FREE is not enough incentive to get you downloading – please read Roy McCarthy's review of the work.
If FREE plus a thoughtfully, stellar review doesn't cut it . . . hmmm . . . maybe short stories aren't your thing. No worries. I write novels, too!
Please feel free to re-blog this post. After all, who doesn't love a freebie?
Have a safe and wonderful Labour Day long weekend, everyone.
This entry was posted in Book Promotion and Marketing, Book Review, Flash fiction, Photography, Reading and tagged free book offer, Labour Day long weekend, short stories.
I just downloaded it and look forward to a good read!
Many thanks – hope you enjoy the snippets.
Hoping to push this up the line a wee bit with a reblog – Sale ends Sunday, August 31st.
Hi Francs, thanks for sharing your book of short stories with us. I really enjoyed the collection of work and it's really special to include some of your mother's stories. My favourite quote was from the last story "She watched as early December slipped by like sodden leaves falling battered to the dark earth." Beautiful! I'll be adding a review to Amazon too.
I'm so glad you enjoyed the book and I look forward to reading your review. I'm currently working on a full-scale, unfinished manuscript for a novel that my mom wrote and reconnecting with her through her writing is something so special.
That's great news, I hope you're enjoying working on her manuscript. What a great family talent to share. I look forward to seeing the end result.


text:
Q: NSTimer retain count increases, why? I have a problem regarding NSTimer. See the following code:
NSTimeInterval timeInterval = 1.0f;
SEL selector = @selector(executeDataRefresh);

NSMethodSignature *methodSignature = [[ExecuteDataRefesh class] instanceMethodSignatureForSelector:selector];

NSInvocation *invocation = [NSInvocation invocationWithMethodSignature:methodSignature];
[invocation setTarget:executeDataRefresh];
[invocation setSelector:selector];

NSTimer *timer = [NSTimer scheduledTimerWithTimeInterval:  timeInterval invocation:invocation repeats:YES];

The object executeDataRefresh's retain count will now increase by 1 each invocation of method executeDataRefresh. So after 1 minute the retain count is 60.
I know the method retainCount shouldn't be used, but is this method really this "incorrect"?
How come?

A: The NSInvocation retains its target because it needs the target to still be around when the timer fires. That fact is sort of buried in the documentation for -[NSInvocation retainArguments]:

If the receiver hasn't already done so, retains the target [...]
NSTimers always instruct their NSInvocations to retain their arguments, [...] because there's usually a delay before an NSTimer fires.

This is what is meant when someone says "Framework classes may be retaining things without you knowing". Don't worry about absolute retain counts.
What you should perhaps be worrying about instead* is the fact that, every time you run this code (which you seem to indicate happens fairly often), you are creating a new NSInvocation and repeating NSTimer instance with exactly the same attributes as the last time, which seems like a waste of memory.

*Unless this is just test code.

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