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Published at Monday 30th, July 2018, in Desks. By Agda Chauvet.
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The romantic dreamer enters your life and this card often predicts love and romantic adventures. He has a lot of feelings and knows how to express them. You may be falling in love with a man you believe is perfect. This is the coming or going of matters concerning your love life, often a love that is entering your live. If you're in a relationship, this card represents a romantic phase; arguments and harsh words are forgiven and forgotten.
I've always found fall to be a good time to experiment with what I want in my relationships. Maybe it's falling victim to "cuffing season" — or maybe it's just because I've come to associate autumn with life changes. But whatever the reason, these chillier months always have me feeling like I need to start trying something new. I studied abroad in London from September through December two years ago. While I was there, I had a psychic reading done in Greenwich Market. The man delved right in, and told me that my next adventure in ~love~ would be with a Virgo who has a quirky personality. I was also supposed to have already met this person, but they weren't a constant in my life.
The "three-card spread" is one of the most simple and effective tarot spreads. You can adjust the categories to accommodate any situation (past, present, future; yourself, the other person, the relationship; opportunities, challenges, outcomes; mind, body, spirit). The cards and their corresponding positions will effortlessly expose bonds and dynamics. But before reading the straightforward explanation of each card, take a moment to create your own story based on observation. How do the cards you drew make you feel? What are the colors and symbols? If there are characters, are they facing towards or away from each other? Do the illustrations seem cohesive or disjointed?
In recent years, we've seen the emergence of "celebrity mediums," who are people that have become famous simply for being mediums. This, in turn, has led to some fairly intense scrutiny of those who claim to have mediumship ability. People like the "Long Island Medium," Theresa Caputo and Allison DuBois, who inspired the hit television show Medium, have often been criticized for taking advantage of their clients' grief. Still worse, many are accused of being frauds.
Mediums are souls in the world who still have their physical bodies, yet they can attune to souls who don't (and communicate with them). In other words, we tune-in to those who have moved over to the Other-Side. I am the type of Medium who uses her mind to connect mentally with energies on the Other-Side. There are other Mediums who use their bodies to channel spirit.
You can find the psychics on various online websites who can offer Tarot Card readings, Palm readings, numerology readings, astrological (birth chart) readings etc. and also help you with issues related to relationships, career or personal life. However, they are generally expensive. I find that local psychics and sensitives ask a more reasonable hourly rate. Because they are in the room with you, you get a more personal relationship as well.
AuKeeRa has aided the public for over 25 years using her abilities. She aids you to 'see around corners' providing insight (which she believes you already have within you). She is able to aid within personal, past, finances, health, relationships, career, pets as well as communicate with those who have passed. Speaking with her is like talking to someone who has known you your whole life. You will find your time with her refreshing and empowe
As the thirst for all things mystical, spiritual, occult and witchy expands into the mainstream, tarot cards are experiencing a huge, almost cultish following. Tarot cards are a form of divination, which literally means working with the divine, or your higher self, which is the ultimate purpose of tarot cards, just like yoga. We've broken down everything you need to know about them, from types of decks to what each card represents.
Keen is a Leader in Psychic Advice Keen also has many psychic reading options outside of spiritual healing readers. If you have questions about your love life or relationships, you can get a love reading from a Keen psychic. If you are interested in knowing your future through cards, Keen has online tarot readers, cartomancers, and angel card readers who are proven experts in that medium. If you wish for an astrology reading, Keen has astrologers who specialize in subjects such as Vedic astrology, Mayan astrology, and Chinese astrology. No matter what you're looking for, Keen.com can get you in touch with specialists in all of these very diverse and enlightening subjects.
Our free love tarot is a relationship tarot spread casting light upon the present and the future of you sentimental relationship. It is an oracle of love rather than a love horoscope. One of the most important differences between horoscope and tarot lies in the fact, that the tarot does not attempt to predict the future, but to stimulate reflection and to enable us to grasp subtle developments and aspects of our major questions in life.
Between 8 November and 31 December 1920 Gustav Geley of the Institute Metapsychique International attended fourteen séances with the medium Franek Kluski in Paris. A bowl of hot paraffin was placed in the room and according to Kluski spirits dipped their limbs into the paraffin and then into a bath of water to materialize. Three other series of séances were held in Warsaw in Kluski's own apartment, these took place over a period of three years. Kluski was not searched in any of the séances. Photographs of the molds were obtained during the four series of experiments and were published by Geley in 1924.[123][124] Harry Houdini replicated the Kluski materialization moulds by using his hands and a bowl of hot paraffin.[125]
The psychologist and psychical researcher Stanley LeFevre Krebs had exposed the Bangs Sisters as frauds. During a séance he employed a hidden mirror and caught them tampering with a letter in an envelope and writing a reply in it under the table which they would pretend a spirit had written.[68] The British materialization medium Rosina Mary Showers was caught in many fraudulent séances throughout her career.[69] In 1874 during a séance with Edward William Cox a sitter looked into the cabinet and seized the spirit, the headdress fell off and was revealed to be Showers.[70]
Keen is a Leader in Psychic Advice Keen also has many psychic reading options outside of spiritual healing readers. If you have questions about your love life or relationships, you can get a love reading from a Keen psychic. If you are interested in knowing your future through cards, Keen has online tarot readers, cartomancers, and angel card readers who are proven experts in that medium. If you wish for an astrology reading, Keen has astrologers who specialize in subjects such as Vedic astrology, Mayan astrology, and Chinese astrology. No matter what you're looking for, Keen.com can get you in touch with specialists in all of these very diverse and enlightening subjects.
For example, if a spirit wants to give the psychic medium the name Sandy, they might flash (in the psychic medium's mind) the face of a person whom the psychic medium knows named Sandy. If they want to get the psychic medium to say the word "coffee," they might show the psychic medium someone's cat that had the name Coffee. This can be confusing and requires that the psychic medium interpret what they are seeing in their mind. For instance, the psychic medium might say cat rather than coffee; and to the person being read, the psychic medium might appear wrong when the psychic medium is actually just "misinterpreting" the message.
Because the earliest tarot cards were hand-painted, the number of the decks produced is thought to have been small. It was only after the invention of the printing press that mass production of cards became possible. The expansion of tarot outside of Italy, first to France and Switzerland, occurred during the Italian Wars. The most important tarot pattern used in these two countries was the Tarot of Marseilles of Milanese origin.[1]
In accordance with law, all psychic services are for entertainment purposes only. Please read our terms, conditions, and copyright policy before using this site or our services. If you enjoy the cards used on our website please click on our Tarot Deck Resource page where you can purchase these Tarot Decks from Amazon. Tarot Deck Resources Thank you.
I'm an Angel Therapy Practitioner® and Certified Angel Card Reader(TM) (certified & trained by Doreen Virtue), Certified Meridian Technologist, Certified Law of Attraction Practitioner & have earned bachelor's & master's degrees in metaphysics & a Ph.D. in metaphysical counselling through the University of Metaphysics/Sedona. As an ATP®, I communicate with Angels & Archangels to answer questions & provide guidance. Services include: Ange
On 4 February 1922, Harry Price with James Seymour, Eric Dingwall and William S. Marriott had proven the spirit photographer William Hope was a fraud during tests at the British College of Psychic Science. Price wrote in his SPR report "William Hope has been found guilty of deliberately substituting his own plates for those of a sitter... It implies that the medium brings to the sitting a duplicate slide and faked plates for fraudulent purposes."[128] The medium Kathleen Goligher was investigated by the physicist Edmund Edward Fournier d'Albe. On July 22, 1921 in a séance he observed Goligher holding the table up with her foot. He also discovered that her ectoplasm was made of muslin. During a séance d'Albe observed white muslin between Goligher's feet.[129]
He is the King and head honcho. He symbolizes masculine creativity. He represents authority, power, responsibility, leadership, passion and action, and is seen as a symbol of sex, the warrior and defender. He symbolizes new beginnings, competition and aggression. He can represent the father, husband, man in your life, boss, or any authority figure.
Likewise, the entire audience can affect the clarity of the spirit communication at a psychic medium demonstration. If the audience as a whole is very open, accepting and light-hearted, this helps the psychic medium, as it would help any speaker on stage. If the audience as a whole is skeptical, apathetic or angry, this can negatively affect the psychic medium, as it might affect any speaker in front of such a group. Spirit communication is about energy. If the energy in the room is low, it makes it more difficult for the psychic medium to raise his or her energy to connect with spirit. If the energy is high, it can assist the psychic medium to raise his or her energy.
One day I travelled to a witch village in Sedona, Arizona. Once I entered a psychic boutique, the woman told me I was a gifted light bearer and that I would help people that would require my gifts. So I started from there, getting seriously into it and learning to understand what I could do with my gifts. My biggest rewards were knowing that I could help people understand their situation.
When someone crosses over, they see the world through a new perspective. Each lifetime in the physical plane, if lived correctly, leaves the soul enlightened. When we go to the other side, we become enlightened, especially through the process of having a life review. So don't be surprised if the deceased have some really important messages to pass on to you.
I'm 3rd generation Psychic . I have GOD gifted abilities with remarkable psychic powers which I got by my Forefathers I do energy and meditation reading, Tarot, love astrology cards, crystal ball and pendulum readings.I also do Healing to save ur life .My main area of expertise is love relationship, career, business, health, finance, luck, dream interpretation and any issues related to your life.
In the later half of the 20th century, Western mediumship developed in two different ways. One type involved clairaudience or sensitives who hear spirit, and then relay what they hear to their clients.[29] The other incarnation of non-physical mediumship is a form of channeling in which the channeler goes into a trance, or "leaves their body", allowing a spirit entity to borrow their body, who then speaks through them.[30] When in a trance the medium appears to come under the control of the spirit of a departed soul, sometimes entering into a cataleptic state,[31] although modern channelers may not.[citation needed] Some channelers open the eyes when channeling, and remain able to walk and behave normally. The rhythm and the intonation of the voice may also change completely.
A love & relationship psychic reading can help restore your peace of mind and understanding regarding your relationship. The information and relationship advice that your psychic guide provides you with during your conversation will help you see your emotional attachment from an entirely different perspective. Seeing things from this new point of view can provide you with the reassurance and tranquility of knowing that you are doing what is best for you and for making your relationship continue to work.
According to the magician John Booth the stage mentalist David Devant managed to fool a number of people into believing he had genuine psychic ability who did not realize that his feats were magic tricks. At St. George's Hall, London he performed a fake "clairvoyant" act where he would read a message sealed inside an envelope. The spiritualist Oliver Lodge who was present in the audience was duped by the trick and claimed that Devant had used psychic powers. In 1936 Devant in his book Secrets of My Magic revealed the trick method he had used.[159]
Some scientists of the period who investigated spiritualism also became converts. They included chemist Robert Hare, physicist William Crookes (1832–1919) and evolutionary biologist Alfred Russel Wallace (1823–1913).[13][14] Nobel laureate Pierre Curie took a very serious scientific interest in the work of medium Eusapia Palladino.[15] Other prominent adherents included journalist and pacifist William T. Stead (1849–1912)[16] and physician and author Arthur Conan Doyle (1859–1930).[17]
Hi, My name is Tara Arnold. I am a Conscious/ Trance Channel Medium, which means I can be in deep trance and conscious at the same time. When I am doing this the right brain and left brain work together to translate light language into English. The spirit guide blends their energies with mine so they can use my voice box, hand movements, body etc to translate their messages. The deeper I go into trance the more of their personality is present. If interested in viewing my Conscious / Trance Channelling videos Visit my you tube page -Channelling St. Germain-Tara Arnold
I thought i would mention that all Psychic related abilities are GIFTS that some of us receive, not everyone who claims to be gifted is genuine and 100%. This could be because they're a fake or are not advance to a higher level there are different reasons for this. Genuine Psychics, Mediums, Clairvoyants etc are given the gift for a reason decided by The Spiritual Realms, these gifts and more are not of your own choice and most often you will notice such abilities at a very young age, for me it was when i was 13 years old some people notice earlier. We all know the golden rule which applies to those who have been fortunate enough to have been blessed which such an amazing gift or gifts the rule states "When you have a gift it is your duty to share it with the rest of the world." It is never about you it is never about making money or fame it is for the good of humanity.
She invited me to her apartment in Brooklyn. When I got to the brownstone-lined street, I found my final destination: a beautiful old converted hospital building that was positively witchy. After I was buzzed in and made my way to the correct floor, I was greeted by a small dog in the hallway that led me exactly to where I needed to be. Mama Donna was waiting by the door, and invited me to sit on her pink floral couch in front of a wicker table. Her pup, Poppy, hopped onto my lap and we began the reading.
Cold reading also explains why psychics have consistently failed scientific tests of their powers. By isolating them from their clients, psychics are unable to pick up information from the way those clients dress or behave. By presenting all of the volunteers involved in the test with all of the readings, they are prevented from attributing meaning to their own reading, and therefore can't identify it from readings made for others. As a result, the type of highly successful hit rate that psychics enjoy on a daily basis comes crashing down and the truth emerges – their success depends on a fascinating application of psychology and not the existence of paranormal abilities.[50]
This is an organized event that can take place in an auditorium, church or even a TV studio. Participants are picked out from the audience randomly, and the spiritualist or medium passes messages, either directly from the spiritual realm or via his or her personal spirit guide. It's a good way to see how it all works, but you are playing a numbers game as the chances of you receiving a message are remote.
Drop all fear, negativity and resistance to live the life you deserve today! Part of being able to release is being able to know and trust that something better will replace the released emotions, beliefs and states. Know that there is purpose behind "all this"! Mediumship is a powerful tool to be able to help you do this, showing you a larger perspective and view on life, showing you that there is more going on than we see, and that you can tr
Mina Crandon claimed to materialize a "spirit hand", but when examined by biologists the hand was discovered to be made from a piece of carved animal liver.[143] The German apport medium Heinrich Melzer was discovered to be a fraud in 1926. In a séance psychical researchers found that Melzer had small stones attached to the back of his ears by flesh coloured tape.[144] Psychical researchers who investigated the mediumship of Maria Silbert revealed that she used her feet and toes to move objects in the séance room.[145]
If everything is currently going well and you have a healthy relationship, your psychic reading can provide the reassurance you are looking for that your instincts are right – everything is fine. On the other hand, the reading may also alert you to a lurking issue that you will soon have to overcome in order for the intimate relationship to survive.
According to the magician John Booth the stage mentalist David Devant managed to fool a number of people into believing he had genuine psychic ability who did not realize that his feats were magic tricks. At St. George's Hall, London he performed a fake "clairvoyant" act where he would read a message sealed inside an envelope. The spiritualist Oliver Lodge who was present in the audience was duped by the trick and claimed that Devant had used psychic powers. In 1936 Devant in his book Secrets of My Magic revealed the trick method he had used.[159]
To up the difficulty for me and to make it easier for the psychics (it's one thing to lie over an email or call, but another to lie to someone's face), I decided to see my next psychics in person—who knows, maybe getting readings via email didn't provide a strong enough spiritual connection or clues to see I was lying. I also looked for people who charged for readings (maybe you get what you pay for?) and settled on one for $20 and another for $40. Both told me to bring a photo so I pulled one off a (very much alive) friend's Facebook and, armed with Emily's backstory and a few years of high school acting/improv experience, headed out for my third reading of the day. At the point, I was almost hoping to be called out soon—it was too easy.
Welcome, cosmic warriors. I'm Aliza Kelly Faragher, Allure's resident astrologer, and this is Aliza's Allure Astrology, a column dedicated to astrology, esotericism, and all things magick. (That's not to be confused with magic, which has more to do with entertainment — think pulling rabbits out of hats — than inner growth.) In each installation, I'm taking a closer look at an otherworldly subject, exploring both important celestial events and time-honored traditions of the occult. Today, we're getting acquainted with the ancient art of taromancy, or divination through the use of tarot cards.
In 1992, Richard Wiseman analyzed the Feilding report of Eusapia Palladino and argued that she employed a secret accomplice that could enter the room by a fake door panel positioned near the séance cabinet. Wiseman discovered this trick was already mentioned in a book from 1851, he also visited a carpenter and skilled magician who constructed a door within an hour with a false panel. The accomplice was suspected to be her second husband, who insisted on bringing Palladino to the hotel where the séances took place.[181] Massimo Polidoro and Gian Marco Rinaldi also analyzed the Feilding report but came to the conclusion no secret accomplice was needed as Palladino during the 1908 Naples séances could have produced the phenomena by using her foot.[182]
German-suited decks for Bauerntarock, Württemberg Tarock and Bavarian Tarock are different. They are not true tarot/tarock packs, but a Bavarian or Württemberg pattern of the standard German-suited decks with only 36 cards; the pip cards ranging from 6 to 10, Under Knave (Unter), Over Knave (Ober), King, and Ace. These use Ace-Ten ranking, like Klaverjas, where Ace is the highest followed by 10, King, Ober, Unter, then 9 to 6. The heart suit is the default trump suit.[1] The Bavarian deck is also used to play Schafkopf by excluding the Sixes.
Known for her accuracy, integrity, and flowing reading style, Psychic Sophia Elise is a relationship specialist, but can advise you on any aspect of your life. Sophia Elise is an internationally known, multi-dimensional psychic, Transformational Life Coach and expert Tarot reader, with over 25-years experience, specializing in love, romance, relationships, soulmates and twinflames. Sophia is Clairvoyant (seeing), Clairsentient (feeling), Claircognizant (knowing) and Empathic and will use all of these gifts to help you find the right path.
Whether you're looking for love advice or are trying to work through a problem in your current relationship, love & relationship psychic readings can provide you with valuable insight that you seek regarding matters of the heart. The information gained from these readings can help you confidently continue forward with your romantic relationship in the best possible way. Connect with a psychic guide on PsychicCenter for your live love & relationship reading.
Professional Psychic Medium since 2009. Elizabeth's readings are a combination of Mediumship, Psychic Visions and Spiritual Messages. Valuable insight into your past, present and future to help you gain clarity and confidence in your decision making during times of crossroads. Detailed messages and information that provide you with comfort and peace. Elizabeth uses a special process of meditation and automatic writing to connect to her client'
This card predicts that you will have a lot to do, many projects running at the same time and you manage it well. Change is on the way and you're able to adapt and balance at the same time. A change in your financial situation and you need to find stability and balance again. You feel that you're alive and kicking, having some fun. In a relationship this card predicts a fun and exciting time. If you're single this is a time when you're not very serious but play around for a while. In matters of work you seem to have your hands full but you manage to balance it well.
The British direct-voice medium Frederick Tansley Munnings was exposed as a fraud when one of his séance sitters turned the lights on which revealed him to be holding a trumpet by means of a telescopic extension piece and using an angle piece to change the auditory effect of his voice.[126] Richard Hodgson held six sittings with the medium Rosina Thompson and came to the conclusion she was a fraud as he discovered Thompson had access to documents and information about her séance sitters.[127]
The original purpose of tarot cards was to play games. A very cursory explanation of rules for a tarot-like deck is given in a manuscript by Martiano da Tortona before 1425. Vague descriptions of game play or game terminology follow for the next two centuries until the earliest known complete description of rules for a French variant in 1637.[11] The game of tarot has many regional variations. Tarocchini has survived in Bologna and there are still others played in Piedmont and Sicily, but in Italy the game is generally less popular than elsewhere.
Physical mediumship is defined as manipulation of energies and energy systems by spirits. This type of mediumship is claimed to involve perceptible manifestations, such as loud raps and noises, voices, materialized objects, apports, materialized spirit bodies, or body parts such as hands, legs and feet. The medium is used as a source of power for such spirit manifestations. By some accounts, this was achieved by using the energy or ectoplasm released by a medium, see spirit photography.[26][27] The last physical medium to be tested by a committee from Scientific American was Mina Crandon in 1924.
The poet Robert Browning and his wife Elizabeth attended a séance on 23, July 1855 in Ealing with the Rymers.[64] During the séance a spirit face materialized which Home claimed was the son of Browning who had died in infancy. Browning seized the "materialization" and discovered it to be the bare foot of Home. To make the deception worse, Browning had never lost a son in infancy. Browning's son Robert in a letter to The Times, December 5, 1902 referred to the incident "Home was detected in a vulgar fraud."[65][66] The researchers Joseph McCabe and Trevor H. Hall exposed the "levitation" of Home as nothing more than his moving across a connecting ledge between two iron balconies.[67]
Professional Psychic Medium since 2009. Elizabeth's readings are a combination of Mediumship, Psychic Visions and Spiritual Messages. Valuable insight into your past, present and future to help you gain clarity and confidence in your decision making during times of crossroads. Detailed messages and information that provide you with comfort and peace. Elizabeth uses a special process of meditation and automatic writing to connect to her client'
"The picture reading shows me what goes on in the past life and in this life ... So if it shows me she was your guardian angel at one point, she was a guardian angel," she said. She added that the reading wasn't accurate because I had come in with the intention to trick, that the energy I came in with was one about my sister which is what she picked up on, that the whole thing wasn't very "civilized" of me and that she'd be calling other psychics and warning them about me. She told me she's been tested by people who declared their intentions and passed their tests, and offered to give me a reading that would reveal secrets about me that not even my closest friends know. I declined.
After her death in the 1980s the medium Doris Stokes was accused of fraud, by author and investigator Ian Wilson. Wilson stated that Mrs Stokes planted specific people in her audience and did prior research into her sitters.[174] Rita Goold a physical medium during the 1980s was accused of fraud, by the psychical researcher Tony Cornell. He claimed she would dress up as the spirits in her séances and would play music during them which provided cover for her to change clothes.[175]
Now that you have grounded yourself, please select the cards we will use for your Tarot card reading. Remember, I shuffle my deck and draw new cards for the Tarot spreads every day, and these cards represent each one as it was laid out in front of me. Please take a moment to pause between drawing your cards, and when you are done selecting your cards for your Tarot reading you will be whisked away to a detailed interpretation of each card and what it means in its position in the Celtic Cross Spread.


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Plastic Pyrolysis Machine - Chinaprice: 50000.00 Dollar US$
Therefore, Doing makes great efforts to develop and manufacture the Plastic pyrolysis machine, which is very popular after it comes into being. The Plastic pyrolysis machine is a great recycling machine which is can be widely applied to large-scale, large-volume recycling of post-consumer, post-commercial, and post-industrial plastic scrap. The Used of plastic waste recycling machine is mainly used to reprocess plastic and make the material reusable for another product.
Plastic pyrolysis machine
Easy operation: It has automatic constant temperature control and stable performance. After adjusting well, one or two workers are enough to operate this machine, to feed the materials and change pellet receiver.
Low cost after redesign: Now this kind machine is more economic in electric power source and also water. In the long working life of this machine, it can save many sources in energy.
High output, long operating life and lower noise: It's the ideal waste plastics regenerative pelletizer machine nowadays.
Energy and space saving: Doing waste plastic recycling equipment combines shredding, extruding and pelletizing into one machine and uses the high torque gearbox with precise grinding.
Company Contact:
- Posted By: DOING
- Phone: 8637156771821
- Address: No.138 Yingxie Road,Jinshui District,Zhengzhou, Henan Province, China, Zhengzhou, Henan, China
- Email:
- Website: http://www.china-doing.com
Published date: August 31, 2016 -
- Business Description: We DOING Company as a leading manufacturer in China has specialized in all kinds of waste recycling machinery and the supporting equipments for about 10 years, 5 years experience for domestic market, and 5 years for foreign trading. Now we have the waste tire/plastic recycling machine, waste oil recycling machine, waste cable wire recycling machine, waste aluminum plastic panel recycling machine, waste PCB recycling machine and waste home appliance recycling machine which can be provided for you.
By now we have installed hundreds of machines in about 30 countries:
Africa: Nigeria, Congo, Egypt, Uganda;
Europe: Romania, Poland, Italy, Macedonia, Albania, Turkey;
Asia: Malaysia, Pakistan, India, Thailand, Iran, Bangladesh, Lebanon, Jordan;
America: Mexico, Ecuador, Haiti, Guatamala, Aruba, Panama, Jamaica, etc.
Except the machine, we have professional engineers can be sent to your factory for installation, commissioning and training your workers after you buy the machine.
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"Mom's Brownie Recipe" joke
Remove teddy bear from oven and preheat oven to 375.
Melt 1 cup margarine in saucepan.
Remove teddy bear from oven and tell Billy "no, no."
Add margarine to 2 cups sugar.
Take shortening can away from Billy and clean cupboards.
Measure 1/3 cup cocoa.
Take shortening can away from Billy again and bathe cat.
Apply antiseptic and bandages to scratches sustained while removing shortening from cat's tail.
Assemble 4 eggs, 2 tsp. vanilla, and 1-1/2 cups sifted flour.
Take smoldering teddy bear from oven and open all doors and windows
for ventilation.
Take telephone away from Billy and assure party on the line the call was a mistake. Call operator and attempt to have direct dialed call removed from bill.
Measure 1 tsp. salt, 1/2 cup nuts and beat all ingredients well.
Let cat out of refrigerator.
Pour mixture into well-greased 9x13-inch pan.
Bake 25 minutes.
Rescue cat and take razor away from Billy. Explain to kids that you have no idea if shaved cats will sunburn. Throw cat outside while there's still time and he's still able to run away.
Mix the following in saucepan:
1 cup sugar
1 oz unsweetened chocolate
1/4 cup margarine
Take the darn teddy bear out of the @#$% broiler and throw it away -
far away.
Answer the door and meekly explain to nice policeman that you didn't
know Billy had slipped out of the house and was heading for the
street. Put Billy in playpen.
Add 1/3 cup milk, dash of salt, and boil, stirring constantly for 2
Answer door and apologize to neighbor for Billy having stuck a garden
hose in man's front door mail slot. Promise to pay for ruined carpet.
Tie Billy to clothesline.
Remove burned brownies from oven.
President Clinton and his wife are at the first baseball game of the season. At the start of the game the pitcher comes up in the stands and whispers something in Clinton's ear.
All of a sudden Clinton looks at Hillary and yells, "Okay, Hillary, GET OUT!".
She more...
When Apollo Mission Astronaut Neil Armstrong first walked on the moon, he
not only gave his famous "One small step for man, one giant leap for man
kind" statement but followed it by several remarks, usual com traffic
between him, the other astronauts and more...
Stepsby justincider
OK, You're a woman, walking home alone. You suddenly notice you are being followed by a black man. What steps would you take?
Fucking long ones!
A lady says to her doctor, "My husband has been complaining that my vagina has an odor, but I bent over and took a whiff, and I don't smell anything."
The doctor examines her vagina, and then says, "There's something terribly wrong. You need an more...
Two blondes walk into a bar. You'd think at least one of them would've seen it.


text:
chat support Can Be Fun For Anyone
$F: Normally the identical difficulty! in some cases it works then some days it just cuts out or the speaker just won't get the job done for a complete day
The calls managed by several Philippine call centers can be classified into one among two types: outbound calls and inbound calls. Outbound calls consist of advisories, product sales verification, customer products and services, surveys, collections and telemarketing.
The Philippines is justifiably popular for its beach locations, but sporty varieties need not come to feel ignored. Although surfers are only catching on on the delicious (if fickle) waves that variety on both of those coasts, divers have very long been enamoured on the region's underwater charms.
Duterte's Luster Dulls as Rice Selling prices Soar in Philippines The president ordered constraints on rice imports for being dropped in an effort to tame inflation, but his administration has become broadly blamed for mismanaging the issue.
The exact scope and mechanisms of Indian cultural influences on early Philippine polities remain the subject of some debate amongst Southeast Asian historiographers,[fifty][57] but The existing scholarly consensus is there was most likely little or no immediate trade involving India and also the Philippines,[50][57] and Indian cultural qualities, like linguistic phrases and spiritual practices,[fifty six] filtered in through the 10th with the early 14th generations, via early Philippine polities' relations Together with the Hindu Majapahit empire.
In 1965, Macapagal misplaced the presidential election to Ferdinand Marcos. Early in his presidency, Marcos initiated numerous infrastructure tasks but was Read full report accused of enormous corruption and embezzling billions of pounds in community funds.
To maximize efficiencies, calls are often created with an automatic dialer after which transferred to an offered agent through an IVR system once a connection with a person is manufactured. Outbound call centers ought to be certain compliance Along with the Countrywide Will not Call Registry, a list to which citizens can increase their cellular phone numbers to avoid acquiring undesired solicitation calls.
[306] The Philippines has among the highest fees of discovery on the earth with sixteen new species of mammals found out in the last ten several years. Because of this, the rate of endemism for your Philippines has All articles risen and likely will keep on to rise.[307] Indigenous mammals incorporate the palm civet cat, the start page dugong, the cloud rat and the Philippine tarsier affiliated with Bohol.
(Not like regular American workspaces, the partitions ended up painted teal as well as cubicle dividers had been fuchsia, reflecting a Filipino flavor for brilliant colours.) "My brother performed with automobiles, but I stole my sisters' dolls," Angel mentioned. "Which was Once i started to Believe I am bakla."
That 1st knowledge of currently being affirmed and treated seamlessly as a woman by American customers profoundly influenced her desire to determine as a girl in the remainder of her everyday living.
A call center is really a centralized Division to which mobile phone calls from existing and prospective customers are directed. Call centers can manage inbound and/or outbound calls, and become Situated either within a corporation or outsourced to a different business that focuses on dealing with calls.
A tote board on one particular wall tracks in-coming calls and metrics bonus much like the time spent with each customer. (Don Lee / La Times)
He insisted that the overseas boom was not eradicating Work opportunities within the U.S. Whilst his Manila Place of work is escalating, he mentioned, so will be the just one in North Dakota, which has one hundred seventy five employees but is being expanded to 300.
1. You can begin a profession in call center in numerous techniques and the commonest is by applying straight to a call center's recruitment Office environment which is now called "walk-in" application.


text:
Man Utd news: Ryan Giggs gives Jose Mourinho advice on Alexis Sanchez problem
MANCHESTER UNITED forward Alexis Sanchez is struggling to settle in at Old Trafford because he's lacking confidence.
That is the opinion of United legend Ryan Giggs.
Sanchez is yet to score this season, and only netted twice following his move from Arsenal in January last season.
The Chile international arrived at the Manchester club on the back of a fine season at Arsenal, and was often a key source of goals for Arsene Wenger's side in the three and a half seasons he spent at the Emirates.
And following a quiet, tournament free summer, many were tipping the 29-year-old to excel during the early stages of the season.
"I'm sure he'll come good as he's a top goalscorer"Ryan Giggs
But despite his poor early season form, former midfielder Giggs has revealed he is an admirer of the ex-Barcelona man, and expects him to get himself back on the scoresheet soon enough.
"I'm a big fan of Sanchez," Giggs told Sky Sports.
"I was a big fan of his at Arsenal and sometimes it just takes players a bit longer to settle in.
"Even though he's a top player he just looks like he's a bit short of confidence, but I'm sure he'll come good as he's a top goalscorer and a good player as well."
GETTY(Pic: GETTY)
Sanchez has been used in a number of positions by United boss Jose Mourinho since signing.
Most commonly deployed on the left wing, the Chilean can also play on the right and through the middle.
But Giggs believes he should be playing on the left, suggesting his attacking partners have an impact on his role.
He said: "He can play anywhere across the front, he played on the right for Barcelona and up front in his better days for Arsenal.
GETTY(Pic: GETTY)
"I've seen him just off the left the most for United so it just depends who he's playing with and who they're playing against.
"He can also play that No 10 role but I think the best performances I've seen from him at United are just coming off the left, but it's just not quite happening for him at the moment."
Sanchez was an unused substitute for United's Carabao Cup penalty shootout loss to Derby on Tuesday.
He is expected to return to the starting line-up on Saturday when United visit West Ham.


text:
No. 2 Kansas ready to face Louisiana at home
Kansas did not figure on having a senior on this year's basketball roster.
Kansas did not figure on having a senior on this year's basketball roster.
Without 6-foot-5 guard Lagerald Vick, no one else could have filled that role, or gained the adulation of fans in what has become one of the most celebrated sendoffs in college basketball, Senior Night in Allen Fieldhouse.
But no one needs to dwell just yet on the eventual departure of Vick, who initially intended to turn pro after last season but was then allowed to return by Kansas coach Bill Self. If Vick's first performance at home is any indication, there will be much to enjoy.
Especially after Self labeled the 32-point outing Vick enjoyed against Vermont as "the best offensive game that I've ever had a player have."
Included in Vick's career high was the most accurate 3-point performance in Kansas history, an 8-for-8 salvo, as the No. 2 Jayhawks rallied from an early deficit. Kansas (2-0) resumes action at home Friday against Louisiana (1-1).
One long-range swish inside the first 30 seconds of the Vermont game provided all the confidence Vick needed.
"After the first one I just heard coach say, 'Just keep shooting the ball,' " Vick said.
So, with each consecutive make, Vick convinced himself to "shoot the next one."
Before he was through, teammates encouraged him to keep shooting.
"We were all pumping him up like, 'Man, you're probably the best shooter in Kansas history, going 8-for-8,' " said freshman guard Quentin Grimes. "So just little things like that. But yeah, we did give him a hard time for sure."
Considering that Vick netted just two points in the Jayhawks' season-opening win against Michigan State in the Champions Classic, his outburst in the home opener was additional proof of the depth Self can tap.
Consistency, however, could be an issue. A teammate who bagged a double-double with 20 points and 14 rebounds in the opener, junior forward Dedric Lawson, went scoreless against Vermont.
"He played very unathletically. He got every shot blocked it seemed like," Self said. "When they had him pushed off the spot, he shot some marginal shots. ... The biggest thing is that he wasn't engaged. We have got to have him engaged. He's way too good of a player to have nights like that."
Sophomore center Udoka Azubuike is someone the Jayhawks can feed for low-post production after he led the nation with 77 percent shooting last season. Azubuike is coming off a double-double. So too is Grimes, who had 10 points and 10 assists against Vermont.
For Louisiana, the visit to Kansas will mark its second game in a row against a top 10 opponent after falling 87-65 at Tennessee. The Ragin' Cajuns led 13-10 before they were overtaken by the Volunteers, despite a team-high 18 points from senior forward Jakeenan Gant.
"We just missed too many (3-pointers) and did take better care of the ball in the second half, but we just couldn't get it in the basket," Louisiana coach Bob Marlin said.
The Ragin' Cajuns are still averaging 11 3-point makes and a 93-point average behind Gant's team-high 18.5-point average.
Copyright 2018 by STATS. Any commercial use or distribution without the express written consent of STATS is strictly prohibited.
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text:
Adenovirus (Ad) cell attachment is initiated by the attachment of the fiber protein to a primary receptor (usually CAR or CD46). This event is followed by the engagement of the penton base protein with a secondary receptor (integrin) via its loop region, which contains an Arg-Gly-Asp (RGD) motif, to trigger virus internalization. To understand the well-orchestrated adenovirus cell attachment process that involves the fiber and the penton base, we reconstructed the structure of an Ad5F35 capsid, comprising an adenovirus type 5 (Ad5) capsid pseudotyped with an Ad35 fiber, at a resolution of approximately 4.2 Å. The fiber-penton base interaction in the cryo-electron microscopic (cryo-EM) structure of Ad5F35 is similar to that in the cryo-EM structure of Ad5, indicating that the fiber-penton base interaction of adenovirus is conserved. Our structure also confirms that the C-terminal segment of the fiber tail domain constitutes the bottom trunk of the fiber shaft. Based on the conserved fiber-penton base interaction, we have proposed a model for the interaction of Ad5F35 with its primary and secondary receptors. This model could provide insight for designing adenovirus gene delivery vectors.
Human adenoviruses (Ads) are nonenveloped, double-stranded DNA viruses and are responsible for respiratory, gastrointestinal, and ocular infections. In addition, Ads have demonstrated great potential as vaccine vectors (7, 13, 22). Ad has a large, complex, icosahedral capsid with a diameter of ∼920 Å, not including the elongated homotrimeric fibers at each of its 5-fold vertices. The Ad capsid is composed of three major proteins: 240 hexon trimers that form the 20 facets of the icosahedral capsid, 12 penton bases, and 12 fiber trimers at the 12 icosahedral vertices (8, 36). A structure of Ad5F35, comprising an adenovirus type 5 (Ad5) capsid pseudotyped with an Ad35 fiber (25, 35), has been determined at 6-Å resolution (33). Combined with secondary structure prediction for minor proteins, the 6-Å structure has enabled the precise locations of minor proteins IIIa, VI, VIII, and IX to be determined (33). The locations of these minor proteins have been further confirmed by two nearly atomic resolution Ad structural analyses: the Ad5 structure analyzed by cryo-electron microscopy (cryo-EM) and the Ad5F35 structure determined using X-ray crystallography (17, 30). The minor capsid proteins, including IIIa, VIII, and IX, are thought to form protein-protein networks to enhance the stability of the Ad capsid (17).
The Ad fiber, which is formed by three copies of fiber proteins, plays a role in the initial attachment of the Ad capsid to the host cell surface through its interaction with the cellular receptor. Although Ad fibers are of various lengths depending on the serotype, the fiber proteins of all serotypes share an overall architecture: an N-terminal tail domain that interacts with the pentameric penton base, a C-terminal globular knob domain that functions as the attachment site for the host cell receptors, and a central shaft domain that connects the tail and the knob domains. The shaft domain consists of a number of repeating β-strands, and the number of β-strands determines the fiber length for the different serotypes (23, 37, 41). The tail domain of the trimeric fiber attaches to the pentameric penton base at the 5-fold axis, resulting in a symmetry mismatch interaction. This interaction is of particular interest because the fiber and penton base play very important roles in the onset of Ad infection. Ad entry into a cell is initiated by a two-step receptor-binding process. The first step of cell entry is the attachment of the fiber to a cell receptor, tethering the viral capsid to the cell surface (3, 11, 12, 21). This attachment is followed by the interaction of an Arg-Gly-Asp (RGD) loop in the penton base protein with the host cell integrins to trigger virus internalization into clathrin-coated vesicles or macropinosomes, thereby promoting membrane penetration (4, 14, 16, 39, 40). Due to the symmetry mismatch between the trimeric fiber and penton base, it is difficult to resolve the structure of the interaction by X-ray crystallography and cryo-EM. Using multiresolution filtering and computational simulation of mismatched symmetries, a model of the fiber-penton base interaction has been proposed recently based on the in situ structure of fiber in Ad5 virion (19). According to this model, the bottom of the fiber shaft interacts with the hydrophobic ring of the penton base, and the three fiber tails of each fiber act as stay-cables, residing inside three of the five grooves between the neighboring penton base monomers. This model of fiber tail-penton base interaction is consistent with earlier observations of the crystal structure of the Ad2 penton base bound to the N-terminal fiber peptide (45) and the cryo-EM structure of a dodecahedral penton particle (10). However, this model of fiber-penton base interaction is different from that proposed by Reddy et al. (30). In the crystal structure of Ad5F35, the densities that were proposed to represent a fiber structure were located in the central pore of the penton base.
Previous studies have shown that the C-terminal knob domain, together with the length and flexibility of the Ad fiber shaft domain, determine viral tropism and infectivity (34, 41). Indeed, Ad5 and Ad5F35 have fibers that differ strikingly in their lengths and utilize different receptors for cell entry. It remains unclear whether these differences between Ad5 and Ad5F35 cause a difference in the type of fiber-penton base interaction. Similarly, it is currently uncertain how Ads with short fibers (i.e., Ad35 fiber) interact with the receptors. Aiming to address these uncertainties in the field, we reconstructed the structure of an Ad5F35 capsid with Ad35 tropism at nearly atomic resolution using cryo-EM. The Ad5F35 capsid studied here consisted of an Ad5 capsid and a chimeric fiber composed of an Ad35 fiber shaft, an Ad35 C-terminal knob, and an Ad5 N-terminal fiber tail (residues 1 to 43) (24), while the Ad5F35 capsid analyzed by X-ray crystallography (30) consists of an Ad5 capsid and an entire Ad35 fiber. Our Ad5F35 structure reveals that the fiber-penton base interaction in the cryo-EM structure of Ad5F35 is similar to that in the cryo-EM structure of Ad5 (17); however, it is different from the interaction proposed based on the crystal structure of Ad5F35 (30). The fiber-penton base interaction observed in the crystal structure of Ad5F35 could possibly reflect an intermediate stage. By comparing the fiber structure from the Ad5F35 density map with a pseudo homology model of the Ad35 fiber, comprising the C-terminal knob and shaft domains, we confirmed that the C-terminal segment (residues 22 to 43) of the fiber tail domain is located at the bottom trunk of the fiber shaft and contributes to the length of the shaft. Moreover, a model of the interaction of the Ad5F35 capsid with CD46 and integrin receptors based on our Ad5F35 structure suggests that Ads with short fibers may be capable of binding integrin and CD46 at the same time.
Sample preparation, cryo-EM imaging, and three-dimensional (3-D) reconstruction.The Ad5F35 sample was prepared as previously described (24). An aliquot of 3.5 μl purified Ad5F35 sample was applied to a 200-mesh quantifoil grid and blotted for 4 s in a chamber at 100% humidity using an FEI Vitrobot Mark IV. Viruses on the grid, embedded in thin ice, were imaged with an FEI 300-kV Titan Krios electron microscope equipped with a Gatan UltraScan4000 (model 895) 16-megapixel charge-coupled-device (CCD) camera. The microscope was carefully aligned before image collection such that a maximum visible contrast transfer function ring of a dry portion of the carbon film image (20 to 25 electrons/Å2 dose) at 1/4 to 1/3 Å−1 spatial frequency was observed. The defocus values of all micrographs were set to approximately 1.5 to 2.5 μm. The contrast transfer function correction for each micrograph was determined manually using the CTFIT program from the EMAN package (20) based on incoherently averaged Fourier transforms of each image. The orientations and centers of all virus particles were determined using the IMIRS package (15) based on the common-line strategy (9). Approximately 21,000 selected particles (68% of all particles) from 4,500 micrographs were combined and reconstructed using a reconstruction program based on icosahedral symmetry-adapted functions (18).
Structure analysis and model building of the Ad35 fiber and integrin.Protein subunit densities were segmented from the Ad5F35 map and visualized using UCSF Chimera (28). The segmented densities of the hexon protein monomer were aligned and averaged using UCSF Chimera (28) and the AVG3D program from the EMAN package (20) to improve the resolution. The fiber tail density map was segmented out from the cryo-EM density map of Ad5F35. We used the fiber-tail model from the cryo-EM model of Ad5 (PDB identifier [ID] 3IZO) to fit to our density map. We then used Coot (6) to improve the geometry and the fit of the tail atomic model to our density map. By sequence alignment of the fiber shaft, we built a pseudoatomic model of the shaft with 5 β-repeats as described previously (19). This model was then merged with the atomic model of the crystal structure of the Ad35 fiber knob (26). The density model of the fiber shaft and the knob was generated from its pseudoatomic model using the pdb2mrc program in EMAN (20). The model of integrin that adopts an extended conformation was built as a composite of the αvβ3 integrin crystal structures with PDB IDs 1L5G and 1U8C (43, 44) as described previously (16).
Structure determination.We obtained a 3-D structure of the Ad5F35 virion by cryo-EM and single-particle reconstruction (Fig. 1A). The final 3-D reconstruction was generated from approximately 21,000 particle images from 4,500 micrographs collected by CCD camera with an FEI Titan Krios microscope (Fig. 2). There are 12 copies of the hexon protein monomer in each icosahedral asymmetrical unit. By averaging the segmented density maps of the 12 hexon protein monomers, the density map of the hexon protein was further improved (Fig. 1B and C). We evaluated the Ad5F35 structure by fitting the atomic model of Ad5 capsid proteins (17) to our Ad5F35 density map. Except for the fiber structure and some hypervariable regions in the hexon protein, the major proteins and minor proteins in our Ad5F35 structure are almost identical to their counterparts in the atomic model of Ad5 capsid proteins (Fig. 1B, C, and D). An N-terminal segment of the penton base protein is resolved in our structure but not in the Ad5F35 crystal structure (Fig. 1D). Based on the clarity of the resolved features, which includes the separated β-strands, α-helix turns, and certain clearly visible side chains (Fig. 1B, C, and D), we estimated that the Ad5F35 structure has a resolution of approximately 4.2 Å. In contrast to the partially resolved fiber structure of Ad5 (19), the fiber in our Ad5F35 structure is fully resolved (Fig. 1A).
Sequence analysis and homology modeling of the Ad35 fiber.Sequence alignment indicates that amino acid residues 11 to 17 in the Ad35 fiber tail domain are conserved among the six human Ad subgroups, A to F (Fig. 3A), implying that they have conserved interactions with the penton base proteins. X-ray crystallography analysis of the recombinant Ad2 fiber protein containing the four distal repeats of the shaft and knob domains shows that the shaft domain is formed by a number of β-repeats (37). Although the amino acid sequence of the Ad35 fiber shaft domain is much shorter than that of the Ad2 fiber shaft domain, we were able to align 5 β-repeats in the Ad35 fiber (Fig. 3B) in the same way that the 22 β-repeats were aligned in the Ad2 shaft (37). The Ad35 fiber contains an insert of residues (IRAT) in the third β-repeat of the shaft from the N-terminal tail end (Fig. 3B) and is similar to the amino acid sequence of the Ad2 fiber (37). The analogous periodic sequence organization of the Ad35 fiber shaft domain, namely, the hydrophobic core residues, the residues forming the peripheral hydrophobic patches, and the conserved glycines or prolines in the turns, suggests that the shaft domains of the Ad35 fiber and the Ad2 fiber are structurally conserved. We also built a pseudo model (Fig. 3C) for the Ad35 fiber knob and shaft domains in accordance with the crystal structures of the Ad2 fiber shaft (37) and the Ad35 knob (26, 38).
Fiber structure in Ad5F35 and its interaction with the penton base.The knob, shaft, and tail domains of the Ad35 fiber are well resolved in our Ad5F35 structure (Fig. 1A), but the density map for the fiber shaft is weaker at progressively greater distances from the penton base, suggesting that the Ad35 fiber is flexible. The fiber is located at the 5-fold axis on a central pore in the penton base. The size of the central pore is approximately 25 Å in diameter in our Ad5F35 structure. No density was observed inside the central pore (Fig. 4A and B). Five fiber tails that bind at the 5 grooves between neighboring penton base monomers are clearly visible in our Ad5F35 structure (Fig. 4A and B), resembling those reported for Ad5 (19) and Ad2 (45). It is reasoned that five copies of tail fibers are observed due to the imposed icosahedral 5-fold symmetry, although only three tails bind the penton base. Considering that residues 11 to 17 in the fiber tail domain are conserved among the human Ads (Fig. 3A), we fitted these residues into the tail density map (Fig. 4C). The interactions between the fiber tail and the penton base include hydrophobic interactions and probable hydrogen bonds (Fig. 4D), which are similar to the interaction reported for Ad5. These interaction sites in the penton base protein are fully conserved among the six human Ad subgroups, A to F (Fig. 4E).
On filtering the structure of the fiber and the penton base to a 7-Å resolution and setting a display contour level of 1.5σ (σ is the standard deviation of the density map) (Fig. 5A and B), a connection between the shaft bottom and the penton base is observed, which is apparently missing in the 4.2-Å-resolution density map (Fig. 4A and B). The length of this connection is approximately 14 Å, which accounts for 4 residues (residues 18 to 21) in the fiber tail (assuming a length of 3.6 Å per residue). Alignment of the β-repeats in the shaft indicates that the shaft domain starts with residue 44, leaving residues 22 to 43 unaccounted for. We segmented out the fiber shaft and knob domains (Fig. 5C) from the 7-Å-resolution structure of Ad5F35 and compared this with a simulated fiber density map containing the knob and the shaft (Fig. 5D) generated from the pseudo model (Fig. 3C) of the Ad35 fiber. Because the 5-fold symmetry is imposed on the Ad35 trimeric fiber during 3-D reconstruction, we simulated the effect of imposing a 5-fold symmetry on our pseudo model of the Ad35 trimeric fiber for comparison. When segmented from the 7-Å-resolution Ad5F35 density map and with the display contour set to 0.75σ, an intact fiber structure is visible, showing 5 layered repeats at approximately 13-Å distances (Fig. 5C). Compared with the simulated fiber density map (Fig. 5D), these repeats correspond to the β-repeats in the shaft. However, the fiber appears longer than the pseudo model, and a 15-Å-long extra region at the bottom of the fiber is observed that cannot be attributed to the β-repeats. Considering that the pseudo fiber model only contains the knob and shaft domains (residues 44 to 323) and that residues 11 to 21 in the tail domain are assigned to the peptide that binds to the penton base and to the connection between the shaft bottom and the penton base (Fig. 5B), the extra length of 15 Å in the density map can be assigned to the residues previously unaccounted for, that is, residues 22 to 43 in the tail domain. This assignment is in agreement with the ideal suggested by a previous structural analysis of Ad5F35 (32). A clearer structure of the β-repeats is observed when the display contour is set to a higher level of 1.5σ although only β-repeats 1 to 3 are visible (Fig. 5A). When the display contour is set to an even lower level to show the weakest density, the upper part of the shaft only enlarges slightly, indicating that the fiber bend is limited to a very narrow angle and that the fiber shaft is relatively rigid (Fig. 6).
Model of Ad5F35 interaction with receptors.The attachment of the Ad35 fiber knob to its primary receptor, CD46 (11), is followed by the interaction of an RGD motif in the penton base with integrin, a secondary receptor, to promote subsequent steps for cell entry (16, 39, 40). The two-step receptor binding process involves concerted efforts from the fiber and the penton base. Previous studies indicated that the flexibility and length of the fiber could affect the fiber-receptor interaction or prevent the penton base from interacting with the integrin due to steric collision between the host cell membrane and the virus capsid (5, 41). Based on the model proposed in reference 5, for Ads with short and rigid fibers, only the top of the knob but not the side must be available for binding to the receptor. A recent structural analysis of the CD46 extracellular domain complexed with the Ad11 knob shows that CD46 interacts with the side of the Ad11 knob (27). Thus, how does the Ad5F35, with short and rigid fibers, interact with its receptor, CD46? Given that Ad35 and Ad11 belong to the same human Ad subgroup (subgroup B) and that their fiber knobs share a high sequence identity of 50%, it is reasonable to postulate that they have similar interactions with CD46. We docked the crystal structure of the Ad11 knob complexed with CD46 into our Ad5F35 structure in an attempt to reveal the interaction between Ad5F35 and CD46 on a host cell membrane. There are two extreme conformational possibilities of CD46 on the cell surface (27). Although it is difficult to ascertain the positioning of CD46 on the cell surface, using both of the extreme conformations of CD46 in our analysis, we concluded that Ad35 fibers can correctly interact with CD46 without introducing any steric collision between the host cell membrane and the virus capsid (Fig. 7).
The penton base protein in our Ad5F35 structure is missing a loop of 78 residues (residues 297 to 374) containing the RGD motif, perhaps due to its flexible nature. The RGD motif in the loop serves to bind integrin, a secondary receptor, on the cell surface (40, 44). The RGD motif comprises residues 340 to 342 in the middle of the loop. Based on the alignment of the Ad5 and Ad35 penton base proteins, the Ad35 penton base protein has a similar RGD loop of 59 residues (residues 307 to 365) and the RGD motif comprises residues 341 to 343 in the loop. The flexible loop can therefore project the RGD motif ∼115 Å (for Ad5) or ∼84 Å (for Ad35) away from the surface of the penton base (assuming a length of 3.6 Å per residue) to bind to the secondary receptor, integrin. We chose to use a model of integrin that adopts an extended conformation described previously (16). The sufficient mobility of the RGD loop allows the RGD motif to easily bind to the integrin irrespective of whether the integrin adopts an extended or a bent conformation (Fig. 6B and 7).
Ad5 is the most commonly used vector in gene therapy applications. However, the widespread distribution of Ad5 receptors in the host allows the Ad5 vector to infect a wide range of cells in the host, resulting in unsuccessful attempts to restrict it to specific tissues for safety and efficiency. Moreover, a number of tissues which represent important targets for gene therapy are refractory to Ad5 infection due to the lack of these receptors (2). One possible approach to alter the Ad5 tropism is genetic modification of the Ad5 fiber. Understanding the fiber-penton base interaction is thus of significance to design Ad vectors with different tropisms. The Ad5F35 capsid with Ad35 tropism (24) was resolved at near-atomic resolution. We observe a different fiber-penton base interaction from that observed for the crystal structure of Ad5F35. By comparing our structure with the cryo-EM structure of Ad5 (19) and the crystal structure of an Ad2 penton base bound with an N-terminal fiber peptide (45), we demonstrated that these fiber-penton base interactions are structurally conserved and independent of fiber length. Indeed, our Ad5F35 fiber is a chimeric fiber composed of the Ad35 fiber shaft, the Ad35 C-terminal knob, and the Ad5 N-terminal fiber tail domains, while the Ad5F35 capsid analyzed by X-ray crystallography has an entire Ad35 fiber. However, the amino acids in the fiber tails of Ad5 and Ad35 are highly conserved and the residues in the Ad5 fiber tail that are involved in binding the penton base are fully conserved with those in the Ad35 fibers (Fig. 3A). We suggest that the fiber-penton base interaction observed in the crystal structure of Ad5F35 may reflect an intermediate stage during the virus's cell entry or assembly. It is worth noting that the crystallization by Reddy et al. took many weeks at room temperature, and it is also possible that this resulted in alterations to the capsid structure (29).
Based on our Ad5F35 structure, together with the model of CD46 complexed with the Ad11 knob and the model of integrin, we propose a new model of Ad-receptor interaction that reveals the interaction of Ads with short and rigid fibers with their primary and secondary receptors. The model of interaction between Ads and their receptors indicates that Ad5F35, with its short and rigid fibers, can bind the primary receptor CD46 and the secondary receptor integrin simultaneously. Our model negates a previous model in which only the top region of the knobs of Ads with short and rigid fibers is available for receptor binding, due to steric hindrance between the viral capsid and the host cell membrane (5). The previous model did not consider the importance of the conformation of the primary receptor (CD46), which could allow the relative positions of the Ad capsid and the cell membrane be situated to avoid steric hindrance between the viral capsid and the host cell membrane. This was most likely because at that time, CD46 was not known to be an Ad receptor (42); the crystal structure of CD46 complexed with Ad11 reveals such an Ad-CD46 interaction (27) that was not previously known. It is noteworthy that Ads with long fibers (i.e., human Ads of subgroups A, C, E, and F) use CAR as their primary receptors, while Ads with short and rigid fibers (i.e., human Ads of subgroup B) use CD46 or other non-CAR receptors as their primary receptors (1). The Ads with moderate fiber lengths (i.e., human Ads of subgroup D) use both CAR and CD46 as their primary receptors (1). Note that Ad41 in subgroup F has both short and long fibers, but only the long fiber binds to CAR on the host (31). These results are consistent with our model in that Ads with short and rigid fibers can attach to receptors CD46 and integrin simultaneously and with the model proposed by Wu et al. in that the attachment of Ads to the primary receptor CAR requires long and flexible fibers (41). The two models for long-fiber Ad–receptor and short-fiber Ad–receptor interactions can be taken into consideration for designing Ads with altered tropism by genetically modifying the Ad fibers.
This research was supported by the National Natural Science Foundation of China (grants 31070663, 31170706, and 31000333), the Scientific Foundation of the Ministry of Education and the Education Department of Hunan Province, China (grants 212119 and 11A124), the Program for Changjiang Scholars and Innovative Research Team at the University of China (IRT0964), and the National Basic Research Program of China (grant 2010CB912403).
- Received 25 June 2012.
- Accepted 29 August 2012.
- Accepted manuscript posted online 5 September 2012.
- Copyright © 2012, American Society for Microbiology. All Rights Reserved.


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Reference scattering curves for polarization and intensity produced by aggregates and agglomerates of ethylene and kerosene soot are obtained for scattering angles in the 10–170° range. The polarization measurements were obtained with the Propriétés Optiques des Grains Astronomiques et Atmosphériques instrument for particles that levitate in microgravity during parabolic flights and on the ground by an air draught technique. The intensity measurements were obtained also on the ground with a Laboratoire de Metéorologie Physique nephelometer. The maximum polarization is of the order of 80% at a scattering angle of 80° at λ = 632.8 nm and approximately 75% at an angle of 90° at λ = 543.5 nm. The polarization increases by approximately 10% when the size of the agglomerate increases from 10 μm to a few hundred micrometers. The intensity curve exhibits a strong increase at small scattering angles. These reference curves will be used in the near future for the detection of stratospheric soot by remote-sensing measurement techniques.
© 2005 Optical Society of AmericaFull Article | PDF Article
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