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Nancy Pelosi Net Worth: Is Nancy Pelosi Gavin Newsom's Aunt?
eliza grace
Nancy Pelosi Net Worth
Nancy Pelosi Net Worth: Nancy Pelosi is an American politician. Nancy and her husband Paul Pelosi estimated their personal net worth to fall somewhere in the range of $43 million and $202 million depending on the value of real estate assets, stock investments, and other private assets.
Nancy Pelosi Early Life: Where Was She Born?
Nancy Patricia D'Alesandro was born in Baltimore in 1940. She was the only girl of seven kids. Her parents are Italian. Her family was political. Her father was a Democratic Congressman from Maryland and later Baltimore's mayor.
Thomas D'Alesandro III was Baltimore's mayor from 1967 to 1971. Her mother also organized Democratic women. Pelosi got involved in politics when she helped her father with campaign events.
She graduated from Notre Dame Institute in 1958 and Trinity College in 1962 with a BA in political science. Nancy met Paul Pelosi, a Georgetown University student, at Trinity College.
Nancy Pelosi Career: How Did She Start Her Career?
Pelosi interned for Maryland Democrat Daniel Brewster in the 1960s. She moved to San Francisco, where she befriended Phillip Burton. She was a California DNC member from 1976 to 1996. She served as finance chair of the Democratic Senatorial Campaign Committee in the late 1970s and 1980s.
Sala succeeded Burton in 1983. Sala Burton chose Pelosi as her successor when she decided not to run for reelection, ensuring that her supporters would back Pelosi. Pelosi became 5th District congresswoman in June 1987 after Sala's untimely death a month into her second term.
She represented the 5th, 8th, and 12th districts between 1987 and 2013. (as of April 2020). Pelosi has also served as House Minority Whip from 2002 to 2003, Leader of the House Democratic Caucus since 2003,
House Minority Leader from 2003 to 2007 and 2011 to 2019, and Speaker of the House of Representatives from 2007 to 2011 and again in January 2019. She's the first Speaker re-elected for non-consecutive terms since Sam Rayburn in 1955. To regain the speakership, she agreed to a four-year limit.
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Kelly Mi Li Net Worth 2022: How Much Does She Make In A Year?
Nancy Pelosi Personal Life
Nancy Pelosi is active in the Italian-American community, serving on the National Organization of Italian American Women and National Italian American Foundation boards (NIAF). In 2007, the NIAF gave her a Special Achievement Award for Public Advocacy.
The two met in college. In 1963, they married. Paul Pelosi owns Financial Leasing Services, INC. Paul owns large stakes in Facebook, Apple, Comcast, Shutterfly, and Walt Disney Co. Paul Pelosi owns San Francisco office buildings and California commercial properties.
Paul bought the Oakland Invaders for $10 million in 2009. The team renamed the Sacramento Mountain Lions, eventually folded. They have five kids and eight grandchildren. Pelosi lives in a $10-20 million mansion in San Francisco's Pacific Heights neighborhood. They own a $5-$25 million vineyard in St. Helena, California.
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Is Nancy Pelosi Gavin Newsom's Aunt?
Gavin Newsom's aunt isn't Nancy Pelosi, but there's more to the story. Nancy married Paul Pelosi in 1963. Ron is Paul's brother. Pelosi married Barbara Newsom in 1956. Barbara's brother William Newsom had Gavin in 1967. The couple divorced in 1977.
So, Nancy Pelosi was Gavin's aunt's sister-in-law for his first 10 years. Gavin was Nancy's brother-in-spouse law's during those years. Do you have an aunt? Imagine your aunt's husband's brother. He's married.
But! Imagine your blood aunt hasn't been married for decades. That's Gavin and Nancy's connection. Imagine you have an aunt named Katie to illustrate. Imagine Aunt Katie married Joe Example. Katie and Joe are your aunt and uncle. Consider Joe's brother Bill.
Bill Example married Sarah. Bill Example isn't an uncle. Sarah Example, Bill's wife, is not your aunt. To continue the Pelosi analogy, imagine Katie and Joe Example divorced 50 years ago.
Nancy Pelosi Net Worth: How Rich Is She?
Pelosi has a $120 million net worth. Nancy and Paul Pelosi estimated their personal net worth to be between $43 million and $202 million in their most recent wealth disclosure.
Mark this page as a favorite and check back often to see any changes. Please send the link to this article to anyone you think would get something out of reading it. You can find the most up-to-date version of this article at thetecheducation.com.
Categories: Net Worth


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Un tunnel de dérivation est un type de tunnel servant à détourner tout ou partie d'un cours d'eau ou les eaux de pluie, en les faisant passer sous un obstacle naturel comme une montagne mais sans nécessairement les faire changer de bassin versant. Il n'a pas vocation à être utilisé pour la navigation, l'irrigation, le transport d'eau potable ou encore la production hydroélectrique ; il permet une alternative à l'écoulement naturel de l'eau d'un talweg.

Utilisation
Une telle infrastructure peut être construite dans un but préventif afin de constituer une voie de secours au cours d'eau si son lit venait à être subitement obstrué à grande échelle, par exemple par un glissement de terrain. C'est le cas en France du tunnel de dérivation de la Tinée à la hauteur du glissement de terrain de la Clapière ; un autre exemple est celui devant être aménagé dans une ancienne galerie le long de la Romanche au niveau des Ruines de Séchilienne. Dans cette configuration, le tunnel vise typiquement à empêcher le stockage de l'eau en amont du barrage naturel par la formation d'un lac. Outre l'inondation provoquée par le lac en lui-même, une crue subite et dévastatrice menace les populations et les infrastructures en aval si le barrage naturel venait à se rompre soudainement, entraînant la vidange rapide du lac.

Il peut aussi avoir une utilité pour écrêter une crue en absorbant un excédent d'eau comme c'est le cas pour la Kanda-gawa, une rivière du Japon dont le cours de situe en intégralité dans la partie urbaine de Tokyo et dont le régime hydrologique a provoqué de nombreuses inondations par le passé.

Un tunnel de dérivation peut être une solution de détournement d'un cours d'eau dans le cadre de la construction d'un barrage comme ce fut le cas pour le barrage de Vouglans dans le Jura français.

Article connexe
 Barrage naturel

Derivation
Ouvrage hydraulique
Sécurité civile

redpajama_set_name: RedPajamaWikipedia


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Heqet (Egyptian , also  "Heqtit"), sometimes spelled Heket, is an Egyptian goddess of fertility, identified with Hathor, represented in the form of a frog.

To the Egyptians, the frog was an ancient symbol of fertility, related to the annual flooding of the Nile.
Heqet was originally the female counterpart of Khnum, or the wife of Khnum by whom she became the mother of Her-ur.
It has been proposed that her name is the origin of the name of Hecate, the Greek goddess of witchcraft.

Name

The name is written as  with the determinative "frog" (I7).  The phonetic spelling may use the biliteral  hieroglyph (S38) in place of uniliteral  (V28). The alternative form   adds an explicit feminine ending, used alongside the "egg"  determinative (H8) to emphasize the deity's femininity.
The  Middle Egyptian pronunciation of the name may have been close to ,  which has been proposed (among other possibilities) as the origin of the name of Greek Hecate ().

Worship

The beginning of her cult dates to the Early Dynastic Period at least. Her name was part of the names of some high-born Second Dynasty individuals buried at Helwan, such as prince Nisuheqet, and was mentioned on a stela of Wepemnofret and in the Pyramid Texts. Early frog statuettes are often thought to be depictions of her.

Heqet was considered the wife of Khnum, who formed the bodies of new children on his potter's wheel.

In the Osiris myth, it was Heqet who breathed life into the new body of Horus at birth, as she was a goddess of the last moments of birth. As the birth of Horus became more intimately associated with the resurrection of Osiris, so Heqet's role became one more closely associated with resurrection. Eventually, this association led to her amulets gaining the phrase I am the resurrection in the Christian era along with cross and lamb symbolism.

A temple dedicated to Horus and Heqet dating to the Ptolemaic Period was found at Qus.

As a fertility goddess, associated explicitly with the last stages of the flooding of the Nile, and so with the germination of corn, she became associated with the final stages of childbirth.
This association, which appears to have arisen during the Middle Kingdom, gained her the title She who hastens the birth (cf. the role of Heqet in the story of The Birth of the Royal Children from the Westcar Papyrus). Frog amulets representing Heqet were probably worn by women during childbirth to ensure an easy delivery.

Gallery

References

External links

Agricultural goddesses
Childhood goddesses
Egyptian goddesses
Fertility goddesses
Legendary amphibians
Piscine and amphibian humanoids
Hecate
Hathor


Today saw the end of our visit to Moravian congregations. One late request saw our stay in Herrnhut extended by an hour in order to play at the local high school. The journey to Meerbeck lasted 8 hours, and on our arrival in Meerbeck we found our hosts waiting for us at the church. We were pleasantly surprised to find Sherwyn Roussouw, from Port Elizabeth, amongst those waiting for us. We were then taken to our hosts families for supper and to refresh before meeting again for a night out with members of the Meerbeck Brass Band.


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30 LISTS Super-Franchise MeLists that rank the best and worst film series, sequels, prequels, threequels, and other ways movie studios chew up and regurgitate good ideas to squeeze out every last dignity-free penny.
The Worst Remakes Ever Made Greatest Action Film Franchises The Best Horror Movie Franchises The Best Sequels Ever Made The Funniest Comedy Franchises The Worst Sequels of All Time Movies That Totally Need Sequels Franchises with Only One Great Movie Best Third Films in a Series The Best Remakes Ever Remade Sequels That Missed Their Window Best Sequels to Comedy Movies Sorta Sly Actor Replacements When Horror Franchises Lost Their Grooves When Sequels Changed Major Characters Beloved Nerd Franchises You Actually Hate Best Animated Film Franchises Concessions Made for the Stars The Greatest Franchises That Were Books First Sequels That Were Worth the Wait
The Very Best Movie Franchises
Originally by Ranker Community
299.8k votes 23.6k voters 418.7k views 118 items
List Rules Vote up the favorite franchise with the best story arc.
The best movie franchises of all time have brought us some of the best movies ever as well as some billion dollar films and many of them are still cranking out classics (Toy Story 4?? Yes, folks, Pixar has confirmed that it's happening!) This is your chance to vote for what you think is the greatest movie franchise ever and see how your favorite movie franchises rank against other movie enthusiasts' choices.
Some people will judge a franchise based on its best parts (*cough*Star Wars*cough*) while others will take all the parts of a franchise into account when ranking the top movie series, so don't get offended if you see The Lord of the Rings further down this list than you think it should be. Also, try to keep in mind that movie franchises that will have three or more movies but don't yet do not yet qualify for this list of the best movie franchises of all time.
Vote on these top movie franchises based on the series as a whole, or just because one or two of the movies mean something special to you. If you don't see your favorite movie franchise on the list, make sure to add it so you can see where it shakes out among the greatest movie franchises ever.
list ordered by
all voters
rerankers: 59
rank your version
Star Wars Franchise
Episode IV - A New Hope (1977)
Episode V - The Empire Strikes Back (1980)
Episode VI - Return of the Jedi (1983)
Episode I - The Phantom Menace (1999)
Episode II - Attack of the Clones (2002)
Episode III - Revenge of the Sith (2005)
Episode VII - The Force Awakens (2015)
Episode VIII - The Last Jedi (2017)
The Fellowship of the Ring (2001)
The Two Towers (2002)
The Return of the King (2003)
Iron Man series (2008-2013)
The Incredible Hulk (2008)
Thor series (2011-2017)
Captain America series (2011-2016)
Avengers Series (2012-2019)
Guardians of the Galaxy series (2014-2020)
Ant-Man series (2015-2018)
Spider-Man series (2017-2019)
Dark Knight Trilogy
Batman Begins (2005)
DylanMcCracken added Harry Potter Franchise
Harry Potter and the Philosopher's Stone (2001)
Harry Potter and the Deathly Hallows – Part 1 (2010)
Toy Story Franchise
Avengers Franchise
Back to the Future Franchise
Back to the Future (1985)
Back to the Future Part II (1989)
Back to the Future Part III (1990)
The Godfather Trilogy
The Godfather Part II (1974)
The Godfather Part III (1990)
Jurassic Park Franchise
Jurassic Park (1993)
The Lost World: Jurassic Park (1997)
James Bond Franchise
Indiana Jones Franchise
Temple of Doom (1984)
The Last Crusade (1989)
Kingdom of the Crystal Skull (2008)
Kali Mcdaniel added X-Men Franchise
X-Men (2000)
X-Men: First Class (2011)
X-Men: Apocalypse (2016)
X-Men: Dark Phoenix (2019)
The Curse of the Black Pearl (2003)
Dead Man's Chest (2006)
At World's End (2007)
On Stranger Tides (2011)
Dead Men Tell No Tales (2017)
Spider-Man Trilogy (Sam Raimi)
Spider-Man 2 (2004)
Rocky Franchise
Rocky (1976)
Rocky III (1982)
Rocky IV (1985)
Rocky V (1990)
Rocky Balboa (2006)
Creed (2015)
Creed II (2018)
BaruchLalaev added The Hobbit franchise
An Unexpected Journey (2012)
The Desolation of Smaug (2013)
The Battle of the Five Armies (2014)
Iron Man Franchise
Iron Man 2 (2010)
Shrek Franchise
Shrek (2001)
Shrek the Third (2007)
Shrek Forever After (2010)
The Bourne Identity (2002)
The Bourne Supremacy (2004)
The Bourne Ultimatum (2007)
The Bourne Legacy (2012)
Jason Bourne (2016)
bojanthebest added Die Hard Franchise
Die Hard (1988)
Die Hard 2 (1990)
Die Hard with a Vengeance (1995)
Live Free or Die Hard (2007)
A Good Day to Die Hard (2013)
Mission: Impossible Franchise
Mission: Impossible (1996)
Mission: Impossible 2 (2000)
Mission: Impossible – Ghost Protocol (2011)
Mission: Impossible – Rogue Nation (2015)
Alien Franchise
Aliens (1986)
Alien 3 (1992)
Alien Resurrection (1997)
Prometheus (2012)
Alien: Covenant (2017)
Men in Black Franchise
Men in Black II (2002)
Men in Black 3 (2012)
LanaYoshii added The Terminator Franchise
Terminator 2: Judgment Day (1991)
Rise of the Machines (2003)
Genisys (2015)
Filed Under: Polls FilmEntertainmentSequelspollBest Moviestop 10Entertainment Franchises
Entertainment 249.7k readers Kelly Slater's Loves & Hookups READ 135.6k people have read Famous People Who Died of Brain Tumors Teen TV 131 people have voted on The Best Shows That Speak to Generation Z America 32.4k people have read The Most Historic Restaurant In Every State
Movie Quotes 171 voters The Greatest Zapp Brannigan Quotes From Futurama Baseball 2k people have voted on The Best Minor League Baseball Team Logos Netflix 207 people have voted on Ranking the Best Seasons of 'Orange Is the New Black' READ 25.4k people have read Celebrity Warriors Fans
best teriyaki sauce chevy chase young carolands chateau castle in hillsborough best looking male singers 2018 actresses named teri best loot crate ever bobby crawford actor jacket brands list who lives in malibu famous leo youtubers

redpajama_set_name: RedPajamaCommonCrawl


Since 1994 Nevis has emerged as a premier international trust jurisdiction offering the user simplicity and flexibility within a sophisticated and business-friendly regulatory environment.
More than 25 years of financial services experience backed up by centuries of Common Law.
Ordinance allows for many different formats including charitable and non-charitable trusts, spendthrift, protective, asset protection and family office trusts.
Trusts may be used as investment vehicles.
No need for the trust deed to be registered. Statutory confidentiality.
No requirement for filing of accounts or annual returns.
Absolutely no tax on profits, income, dividends or capital gains provided trust property does not include Nevis real property.
At least one trustee must be a Nevis incorporated entity or individual. This may be a professional trustee such as NTL or a privately-incorporated special purpose vehicle.
A protector may be privately appointed and given overriding power.
A settlor may also be beneficiary with no loss of protection.
The Statute of Elizabeth does not apply to a Nevis Trust; therefore, transfer of property may not be voided with respect future unknown creditors.
A creditor seeking to void a trust must file action in Nevis and must post a substantial bond before commencing action. An elevated standard of proof is required.
Foreign judgements are not applicable against Nevis Trusts.
Two year statute of limitations on fraudulent transfer claims.
Overall, Nevis compares very favorably to comparable trust jurisdictions. The client has substantial leeway in designing the trust and professional assistance is readily available. Most importantly, the law provides a cast-iron guarantee that the settlor's wishes will be followed.
NTL offers professional trustee services and can assist with all aspects of trust administration and asset management. If you are interested in finding out more, please contact our offices for an initial discussion.


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PhDr. Dušan Machovec (* 12. ledna 1929, Praha – 20. dubna 2005) byl český filosof, specialista na dějiny antické filosofie, pedagog a hudebník. Jeho starší bratr byl filosof Milan Machovec.

Dílo
 Co je filosofie, 1957
 Dějiny antické filosofie (skripta), 2. vyd. 1962, 2. dotisk 7. vyd. 1980, knižně 1993
 Technika psaní ročníkových prací z dějin antické filosofie (učební pomůcka), 1963, dotisk 3. vyd. 1980
 Dějiny antického atheismu (skripta), 1963.

Sborníky
 Atomistická filozofie starověkého Řecka, její zrod, ohlasy a kritika, Čtení o antice 1978–79, 1979
 Několik poznámek k nábožensko-teologickému pojetí boha a filozofického pojetí absolutní, Problémy křesťanství, 1986
 Geneze myšlenky stvořenosti světa "z ničeho" v počátcích křesťanství, Příspěvky k dějinám křesťanství, 1991.

Reference

Externí odkazy
 Za Dušanem Machovcem

Muži
Čeští filozofové
Úmrtí v roce 2005
Úmrtí 20. dubna
Narození v roce 1929
Narození 12. ledna
Narození v Praze


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Engelsk ELT - A1/A2 - Elementary
Level A1-A2
A game in which players must try to be the first to buy all the food on their list. The game can also be used as Bingo.
• 36 shopping lists with bingo cards on the reverse side;
Pack your bag
Engelsk ELT - A1 - Beginner ELT - A1/A2 - Elementary
A1 (Beginner to Elementary)
The players have to be the first to pack their bag with all the clothes and accessories on their list. Useable also as a bingo-style game, Pack your Bag helps students learn and reinforce language associated with clothing, set in the fun situation of preparing for a journey.
• 36 lists with the bingo cards on the reverse side;
A close shave
Easy Readers - Elementary Engelsk ELT - A1/A2 - Elementary
When Wallace the inventor meets Wendolene in her wool shop, he falls in love with her at once. But why does her dog, Preston, hate Wallace's dog, Gromit?
Then, after Wallace's new sheep-shaving and pullover-making machine falls into the wrong hands, things start to go very wrong.
Can Gromit save Wallace from the danger of a 'close shave'?
Dominoes is a full-colour, interactive readers series that offers students a fun reading experience while building their language skills. With integrated activities, an interactive MultiROM, and exciting, fully dramatized audio for every story, the new edition of the series makes reading motivating for students while making it easy for you to develop their reading and language skills
Shirley Homes and the Cyber Thief
Bassett, Jennifer
Easy Readers - Beginner Easy Readers - Elementary ELT - A1 - Beginner ELT - A1/A2 - Elementary Engelsk
Bookworms - Starter (A1) - Audio CD Pack : Book + Complete Recorded text
Shirley Homes, private investigator. Like Sherlock Holmes a hundred years ago, she lives in London, enjoys working on difficult cases, and has some helpful friends. She understands people, is a good listener, and of course, she is clever with computers. In today's world that is important, because a lot of crime is cyber crime. In this second Shirley Homes detective story, Shirley must catch a cyber thief. But how? You can't see a cyber thief, you can't hear a cyber thief. Only the computer knows, and the computer isn't talking . . .
Shirley Homes and the Lithuanian Case
ELT - A1/A2 - Elementary ELT - A1 - Beginner Easy Readers - Elementary Easy Readers - Beginner Engelsk
Bookworms 1 - book + CD)
Shirley Homes is a private investigator. She is clever with computers, and knows London like the back of her hand. She laughs when people say, 'Was Sherlock Holmes your grandfather?' Sherlock Holmes, of course, was not a real person, but, like Sherlock, Shirley has good eyes, and good ears. And she knows the right questions to ask.
And in the Lithuanian Case, the right questions are important. Because Shirley must find a missing person - Carrie Williams, aged fifteen. Where is she? Who is she with?
The Lottery Winner
ELT - A1/A2 - Elementary Easy Readers - Elementary Easy Readers - Beginner Engelsk ELT - A1 - Beginner
Everybody wants to win the lottery. A million pounds, perhaps five million, even ten million. How wonderful! Emma Carter buys a ticket for the lottery every week, and puts the ticket carefully in her bag. She is seventy-three years old and does not have much money. She would like to visit her son in Australia, but aeroplane tickets are very expensive.
Jason Williams buys lottery tickets every week too. But he is not a very nice young man. He steals things. He hits old ladies in the street, snatches their bags and runs away . . .
Degnan-Veness, Coleen
Nelson Mandela's life is the story of a country boy and his fight for the black people of South Africa. The extraordinary story of a great man, a freedom fighter and one of the world's most important leaders.
Voodoo Island
Duckwotrh, Michael
Bookworms - stage 2 - 700 headwords. 1 Audio CD + 1 Book.
Mr James Conway wants to make money. He wants to build new houses and shops - and he wants to build them on an old graveyard, on the island of Haiti.
There is only one old man who still visits the graveyard; and Mr Conway is not afraid of one old man.
But the old man has friends - friends in the graveyard, friends who lie dead, under the ground. And when Mr Conway starts to build his houses, he makes the terrible mistake of disturbing the sleep of the dead . . .
Bookworms Club Bronze - Stories for reading Circles
In these seven short stories there are marriages and murder, mistakes and mysteries. People fall in love, and fall out of love; they argue, and talk, and laugh, and cry. They go travelling, they go dancing - they even see ghosts. All of human life is there...
- Faik, S. The horse of death
- Ural, Y. The Little Hunters at the Lake
- Bassett, J. Mr Harris and the Night Train
- Escott. Sister Love
- Bassett, J. Omega File 349: London, England
- Henry, O. Tildy's Moment
- Hardy, T. Andre, Jane, the Parson, and the Fox
Oxford Word Skills - Basic
Gairns, Ruth & Stuart Redman
Oxford Word Skills - Basic - enables students to:
* See how the words are used in spoken and written English.
* Practise using the vocabulary.
* Revise what they have learned.
* Study at home or in class.
* Learn the words as preparation for major exams.
* Use the 'test yourself' card to check which words and phrases they know, and which ones need more practice.
Book + Interactive Super-Skills CD-ROM which helps students to:
* Learn how to say the words.
* Practise listening to the words and using them.
* Consolidate what they have learnt in the Students Book with linked interactive exercises.
Business Result - Elementary - Student's Book & CD-ROM
Grant, David, John Hughes & Rebecca Turner
With a highly communicative syllabus and interactive multimedia support materials, plus expert tips and advice from one of the world's leading business schools, Business Result helps learners develop the skills they need, quickly and effectively. Key features
* Communicative syllabus provides pick-up-and-use business skills.
* Real-world case studies deliver authentic insights into key business issues.
* The Expert View from Cranfield School of Management offers authoritative business commentary on every case study.
* Interactive Workbook on CD-ROM - included with every Student's Book - gives students a complete self-study resource.
* Teacher Training DVD with every Teacher's Book shows classroom footage, includes author commentary, and covers three pedagogical areas per level.
Book + CD-ROM
In Clanton, Mississippi, not much happens. There aren't many interesting stories for Willie Traynor's newspaper. But then there's a murder. When the killer goes to jail, the story does not end. Everybody is afraid now of this man and his family. Will there be more killings? Who will be next?
'It's a good place for gold,' said people in the 1840s, and they came from all over the world. 'It's a good place for a prison,' said the US government in the 1920s, and they put Al Capone there on the island of Alcatraz. 'It's a good place for love,' said the hippies in the 1960s, and they put flowers in their hair and came to Haight Ashbury. And San Francisco is still a good place - to take a hundred photographs, or see the Chinatown parade, or just to sit in a coffee shop and be in this interesting, different city . . .
The Bird of Happiness and Other Wise Taler
Herdon, Tim
What is the secret of happiness, or the best thing for a wife to take with her when she leaves home?
How does a man pay for the smell of bread, or decide if he is lucky?
What happens when a friend steals a gift meant for you, or is careless when he tries to make his dreams of a better life come true?
How can you change dirt into gold, or get what you want?
The eight wise tales in this collection can teach us some important lessons about life. Dominoes is a full-colour, interactive readers series that offers students a fun reading experience while building their language skills. With integrated activities, an interactive MultiROM, and exciting, fully dramatized audio for every story, the new edition of the series makes reading motivating for students while making it easy for you to develop their reading and language skills.
Freddy Finds the Thief
Heward, Victoria
Freddy Finds the Thief gives children their first taste of English in the form of a play: they read and act out the adventures of Freddy, who helps the king to unveil a thief in the castle. They will meet an assortment of lively characters who add colour to the story and personality to the script. In addition, the exercises offer the opportunity to 'play' with words, listen to and act out a series of amusing sketches and above all, lose their inhibitions and develop fluency when speaking English.
- Activities in the four language skills
- A full recording of the text plus additional listening activities.
Hill, Susan
A2 / Book + Audio CD
The Children wanted Arthur to tell them a ghost story. But he could not do so. His story was too horrible to tell.
His story was of a lonely house among the dangerous marshes. There a woman in black with hate in her eyes waited and watched. And a child cried out in the mist.
A retold version of a gripping ghost story set in a lonely house on a bleak and foggy marshland.
Hobart, Ruth
Some festivals are popular all over the world. Festivals! tells children how Christmas, Easter, Halloween and other festivities are celebrated in many countries of the world. Young readers are encouraged to use fine-motor skills to colour in, cut out, glue on to create fun decorations and greeting cards. Festivals! offers an innovative approach to teaching English: teachers and children alike won't be able to do without it! In fact, the book is not only a unique language learning experience, but an vive cultural one as well.
- Clear illustrations and pictures make understanding easy
- Subject areas like basic science, mathematics and music are integrated into the activities and games
- Songs, games and cut and stick activities make learning really fun
- An audio recording of the text is downloadable free of charge from the Black Cat website.
Easy Readers - Elementary Engelsk Easy Readers - Pre-intermediate ELT - A1/A2 - Elementary ELT - A2/B1 - Pre-Intermediate
Dominos -Level 2
A young woman arrives at a large country house. Her job is to look after the two children who live there, but she soon discovers that there is something very strange about both the house and the children. The longer she stays, the more she feels that the two children are in danger - or is it that the children are the danger, and the person in danger is herself?
One-Way Ticket
Jennifer Bassett
Easy Readers - Elementary Easy Readers - Beginner Engelsk ELT - A1/A2 - Elementary ELT - A1 - Beginner
Oxford Bookworms Level 1 - 400 headwords. Book + CD
Tom Walsh had a lot to learn about life. He liked travelling, and he was in no hurry. He liked meeting people, anyone and everyone. He liked the two American girls on the train. They were nice and very friendly. They knew a lot of places.
Tom thought they were fun. Tom certainly had a lot to learn about life. This is a collection of short stories about adventures on trains.
Strange, wonderful, and frightening things can happen on trains - and all of them happen here.
Bad Love
Leather, Sue
Beginner/Elementary (A1) - Book + 1 CD
Detective Flick Laine meets handsome Dr Jack Daly at a party in Denver, USA.
When Daly calls later, inviting her to meet him to 'talk about something', she accepts. But before they can talk, the doctor is found dead in an apparent case of suicide. Flick is put in charge of the investigation.
How did the doctor die? And what has love to do with it?
The Bridge and Other Love Stories
Lindop, Christine
Bookworms Stage 1 (A1-A2). Audio CD Pack : Book + Complete Recorded text
Luke is a good-looking young man, but he's not very clever with words. Gemma is clever with words, but what does she want? Lucy and Becky are good friends, but what about Sam? He makes wonderful cakes, but does he make mistakes too? Nina and Dragan are in love, so deeply in love, but they live in the wrong place, at the wrong time ...
All love stories have moments of happiness, pain, misunderstanding, laughter, and sometimes great sadness. But love will nearly always find a way ...
Everybody took photos of Prince William when he first arrived at the University of St Andrews. Crowds of photographers came to the little Scottish town next to the sea and took pictures of this new student - the nineteen-year-old grandson of the Queen of England.
But nobody photographed Kate Middleton on her first day at the university. She moved in quietly, ready to begin her studies in art history. She was just an ordinary student with an ordinary future in front of her. Or was she?
A Little Trouble in Amsterdam
MacAndrew, Richard
Engelsk Easy Readers - Elementary Easy Readers - Pre-intermediate ELT - A1/A2 - Elementary ELT - A2/B1 - Pre-Intermediate
Andy and Mary are twins. During the school holidays, they accompany their mother on a business trip to Amsterdam.
Andy and Mary witness what they believe to be two criminals hiding a painting from an art robbery. But they can't prove anything and nobody will believe them. This paperback is in British English.
Download the complete audio recording of this title and additional classroom resources at cambridge.org/experience-readers
Level A2
Logan's Choice
Easy Readers - Elementary Engelsk ELT - A1/A2 - Elementary ELT - A2/B1 - Pre-Intermediate
Elementary/Lower-intermediate level (A2 - KET) Book + 1CD - British English.
"I'm Inspector Logan of the Edinburgh police," Jenny said. "I'm very sorry about the death of your husband."
Who killed Alex Maclennan? His friend, his wife or her brother? It isn't easy, but Logan has to choose.
Business Vocabulary in Use - Elementary
Mascull, Bill
Business Vocabulary in Use Elementary provides basic, essential vocabulary in a business context for learners of Business English.
Business Vocabulary in Use Elementary follows the same successful approach as the tried and tested In Use vocabulary and grammar books, with vocabulary presentation and explanation on the left-hand page and practice on the right-hand page.
The book comprises a number of thematic sections including work, time, money, products, services; and Skills units including numbers, telephoning, emails and faxes, meetings and presentations.
McLean, Alan C.
Anna is a new student at Oxford University. When she arrives in Oxford, she meets Selim, and they become good friends. But Selim is not English, and living in a different country is not easy for him. Anna tries to help but she knows that her father isn't going to like it.
Selim and Anna have each other. But is that enough? And can they find true happiness together?
Five Children and It
Nesbit, Edith
Bookworms - 700 Headwords
When the children dug a hole in the gravel-pit, they were very surprised at what they found. 'It' was a Psammead, a sand-fairy, thousands of years old.
It was a strange little thing - fat and furry, and with eyes on long stalks. It was often very cross and unfriendly, but it could give wishes - one wish a day. 'How wonderful!' the children said.
But wishes are difficult things. They can get you into trouble . . .


text:
Novel Modification of Lipid A of Francisella tularensis
Nancy J. Phillips, Birgit Schilling, Molly K. McLendon, Michael A. Apicella, Bradford W. Gibson
Nancy J. Phillips
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco
Birgit Schilling
The Buck Institute for Age Research, Novato, California
Molly K. McLendon
Department of Microbiology, The University of Iowa, Iowa City, Iowa
Michael A. Apicella
Bradford W. Gibson
Department of Pharmaceutical Chemistry, University of California, San Francisco, San FranciscoThe Buck Institute for Age Research, Novato, California
For correspondence: bgibson@buckinstitute.org
DOI: 10.1128/IAI.72.9.5340-5348.2004
We have investigated the lipid A of Francisella tularensis subsp. holarctica strain 1547-57, a type B strain, by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, nanoelectrospray quadrupole ion-trap mass spectrometry, and chemical methods. In accordance with the previously published structures of the lipid A from F. tularensis live vaccine strain (LVS) (ATCC 29684) (E. Vinogradov et al., Eur. J. Biochem. 269:6112-6118, 2002), all of the major lipid A forms from strain 1547-57 were tetraacylated. As in the LVS strain, the major fatty acids detected in the F. tularensis 1547-57 lipid A sample included 3-hydroxyoctadecanoic acid, 3-hydroxyhexadecanoic acid, hexadecanoic acid, and tetradecanoic acid. However, several of the lipid A components present in strain 1547-57 were of higher molecular weight than the previously published structures. A major component with an Mr of 1,666 was found to contain three C18:0(3-OH) fatty acids, one C16:0 fatty acid, one phosphate group, and one 161-Da moiety. This 161-Da moiety could be removed from the lipid A by treatment with aqueous hydrofluoric acid and was identified as galactosamine following peracetylation and analysis by gas chromatography-mass spectrometry. Detailed investigations of the Mr-1,666 species by ion-trap mass spectrometry with multiple stages of fragmentation suggested that the galactosamine-1-phosphate was linked to the reducing terminus of the lipid A. Similar to the modification of lipid A with arabinosamine, lipopolysaccharide species from F. tularensis containing a phosphate-linked galactosamine could potentially influence its intracellular survival by conferring resistance to antimicrobial peptides.
Francisella tularensis is an encapsulated gram-negative bacterium that causes tularemia, a severe disease of humans and other mammals (12, 24). Severity of disease depends on the host immune response and the route of infection, including intradermal inoculation via zoonotic transmission, ingestion of contaminated meat or water, and inhalation (7). F. tularensis is a facultative intracellular bacterium, and several studies have shown that a cell-mediated immune response may be required for controlling the infection (22, 24). Recently, interest in F. tularensis has increased because of its suitability for use as an agent of biological warfare (6).
F. tularensis possesses a lipopolysaccharide (LPS) that, when compared to Escherichia coli LPS, is not biologically active (1, 8, 23). The only biological activity attributed to F. tularensis LPS in vitro is the ability to activate complement (8). The relatively nonendotoxic nature of the LPS is putatively attributed to the unusual structure of the lipid A molecule. A recent study showed that the lipid A molecule of F. tularensis live vaccine strain (LVS) is not only tetraacylated but also lacks phosphate substituents (27). These structural features may contribute to its low toxicity. It has been shown that mutations affecting the degree of acylation or the acylation pattern of lipid A reduce the endotoxicity of LPS (21).
In this report, we have investigated the structure of the lipid A molecule of F. tularensis subsp. holarctica strain 1547-57, a type B strain. In agreement with the published structure for the LVS strain, we show that the lipid A molecule of this virulent clinical isolate is tetraacylated in vitro. The major lipid A forms described for the LVS strain were seen as components of our F. tularensis strain 1547-57 lipid A preparation. However, in F. tularensis strain 1547-57, we also found several higher-molecular-weight species. These constituents included monophosphorylated lipid A forms and an unusual lipid A species substituted with a galactosamine-1-phosphate moiety. Here we describe the application of various chemical and mass spectrometric methods to the structural characterization of this novel F. tularensis lipid A structure.
Bacterial strains and culture conditions.F. tularensis subsp. holarctica strain 1547-57 (type B) was grown on chocolate agar supplemented with IsoVitaleX (Becton Dickinson, Cockeysville, Md.). F. tularensis LVS (ATCC 29684) was grown on cystine heart medium (Difco Laboratories, Detroit, Mich.) supplemented with 9% sheep blood (Colorado Serum Co., Denver, Colo.). Both strains were incubated at 37°C in a 5% CO2 atmosphere for 48 h.
Isolation of LPS.Bacteria from 10 agar plates were collected by flooding plates with phosphate-buffered saline and scraping colonies from the surface. Samples were centrifuged, and cell pellets were washed twice with 0.15 M NaCl and suspended in 25 ml of deionized water. Cell suspensions and 90% phenol were equilibrated to 65°C, and 25 ml of 90% phenol was added to cell suspensions. Samples were incubated at 65°C for 1 h and on ice for 1 h. To separate the aqueous (containing LPS) and organic phases, samples were centrifuged at 3,300 × g for 10 min at 4°C. The aqueous layer was collected and the organic phase was back-extracted with 25 ml of deionized water. Samples were centrifuged as described above and the aqueous layer was collected and combined with the first extraction. Samples were precipitated with 0.3 M sodium acetate and 3 volumes of cold absolute ethanol, flash cooled in a dry ice-ethanol bath, and incubated for 2 h at −20°C. Samples were centrifuged for 10 min at 12,000 × g at 4°C, pellets were suspended in 6 ml of deionized water, and samples were reprecipitated as described above. Samples were centrifuged and pellets were suspended in 4 ml of 0.06 M Tris base, 10 mM EDTA, and 2.0% sodium dodecyl sulfate (SDS), pH 6.8. Samples were boiled for 5 min and cooled to room temperature. To digest contaminating proteins, samples were incubated at 65°C for 1 h with 10 μg of proteinase K/ml and then incubated overnight at 37°C. To remove SDS, samples were precipitated six times with 0.3 M sodium acetate and 3 volumes of absolute ethanol. After the final precipitation, pellets were suspended in deionized water and centrifuged at 120,000 × g for 1 h 15 min. The glass-like pellet was suspended in high-performance liquid chromatography (HPLC)-grade water and centrifuged at 120,000 × g for 1.5 h. The pellet was suspended in HPLC-grade water and lyophilized overnight in a VirTis BenchTop 2K lyophilizer.
Isolation of lipid A.LPS from F. tularensis strain 1547-57 (4.6 mg) and F. tularensis LVS (approximately 5 mg) was hydrolyzed in 1 ml of 1% acetic acid at 100°C for 3 h. The samples were centrifuged at 4°C for 30 min, the supernatants were removed, and the lipid A pellets were washed two times with water and then dried under a stream of nitrogen. For further purification, the lipid A pellets were partitioned in CHCl3-CH3OH-H2O (10/5/6), and the bottom organic layers plus interfaces were saved and evaporated to dryness under a stream of nitrogen.
Alternatively, the LPS samples (<0.4 mg) were hydrolyzed under more mild conditions using the method of Caroff et al. (3) with some minor modifications. LPS was treated with 100 μl of 10 mM sodium acetate containing 1% SDS at pH 4.5. The samples were sonicated for approximately 30 s and then heated at 100°C for 1 h. Samples were then frozen and lyophilized in a Speed-Vac concentrator. The dry samples were treated with 100 μl of acidified ethyl alcohol (EtOH) (a 1:200, vol/vol, solution of 4 M HCl in EtOH) and centrifuged at 12,000 × g for 15 min at 4°C. The supernatants were removed, and the pellets were then washed three times with 100 μl of EtOH. The lipid A samples were then extracted with 580 μl of a two-phase Bligh/Dyer mixture consisting of CHCl3-CH3OH-H2O (2/2/1.8 by volume) (28). Bottom organic layers and interfaces were saved and evaporated to dryness under a stream of nitrogen.
GC-MS analysis of fatty acids derived from lipid A.Approximately 50 μg of the F. tularensis 1547-57 lipid A sample was treated with 0.5 ml of 10% (wt/wt) BF3-methanol (Supelco, Inc., Bellefonte, Pa.) and heated at 100°C for 6 h. After cooling to room temperature, the sample was partitioned between 0.5 ml of saturated NaCl and 0.5 ml of HPLC-grade hexanes (Aldrich, St. Louis, Mo.). The aqueous layer was extracted a second time with 0.5 ml of hexanes, and the combined organic layers were back-extracted with 0.3 ml of H2O. The organic layer was then evaporated to dryness under a stream of nitrogen. The fatty acid methyl esters (FAMEs) were redissolved in hexanes and analyzed by gas chromatography-mass spectrometry (GC-MS) in the electron impact mode using a Hewlett-Packard 5890 gas chromatograph interfaced with a VG70SE mass spectrometer. The gas chromatograph was equipped with an on-column injector (J & W Scientific, Folsom, Calif.). FAMEs were separated on a 30-m by 0.25-mm BPX70 column with a 0.25-μm film thickness (SGE, Inc., Austin, Tex.) with helium as the carrier gas (≈6 lb/in2). The initial oven temperature was 100°C for 5 min, followed by a temperature gradient from 100 to 220°C at 4°C/min. For semiquantitative analysis of the GC-MS data, relative peak areas were measured from the total ion chromatogram and normalized to the C18:0(3-OH) component set at 3.0.
Dephosphorylation of lipid A.To remove phosphate moieties from the F. tularensis 1547-57 lipid A, approximately 0.5 mg of the sample was treated twice with 100 μl of 48% aqueous hydrofluoric acid (HF) for 20 h. After each treatment, HF was evaporated from the sample under a stream of N2 in a polypropylene desiccator attached to a water aspirator, with an in-line NaOH trap. Samples were then redissolved in 40 μl of deionized H2O and evaporated to dryness in a Speed-Vac concentrator.
Preparation of alditol acetates.The HF-treated F. tularensis 1547-57 lipid A sample was partitioned in CHCl3-CH3OH-H2O (2:1:2 by volume), and the upper aqueous layer was taken and placed in a clean vessel. The organic layer was then reextracted with a second portion of fresh aqueous layer (prepared separately), and the combined aqueous layers were evaporated to dryness. The dry residue was then reduced by treatment with 150 μl of 10-mg/ml NaBD4 in 1 M NH4OH for 2.5 h at room temperature. The reaction was quenched with 20 μl of glacial acetic acid, and then the sample was evaporated to dryness under a stream of N2 from 0.5 ml of CH3OH containing 1% acetic acid (eight times), followed by pure CH3OH (six times). The reduced sample was dried in a vacuum desiccator over P2O5 for 1 h and then peracetylated by treatment with 300 μl of acetic anhydride (Supelco, Inc.) at 100°C for 3 h, with occasional sonication. After cooling to room temperature, the reaction mixture was evaporated to dryness under a stream of N2, followed by four dryings from 0.5 ml of HPLC-grade toluene (Aldrich, Milwaukee, Wis.). Finally, the product was partitioned between 0.5 ml of CHCl3 and 0.5 ml of H2O, the aqueous layer was extracted two more times with CHCl3, and the combined organic layer was back-extracted with 0.5 ml of H2O and finally evaporated to dryness. As a negative control for this experiment, a sample of Salmonella enterica serovar Typhimurium TV119 Ra mutant lipid A derived from the LPS (Sigma, St. Louis, Mo.) was taken through the same steps as the F. tularensis lipid A.
The derivatized samples were analyzed on the GC-MS system described above, operating in the chemical ionization mode using ammonia gas. The samples were dissolved in CH2Cl2 and injected onto the BPX70 column at 120°C. The initial oven temperature was 120°C for 3 min, followed by a quick ramp to 190°C at 8°C/min, a 1-min hold at 190°C, and then a temperature gradient from 190 to 260°C at 4°C/min.
Reduction of lipid A.Lipid A samples were subjected to reduction with NaBD4 following a published procedure (15), with some modifications. Portions of the F. tularensis 1547-57 and F. tularensis LVS lipid A samples (approximately 50 μg each) were dissolved in 150 μl of 6.25-mg/ml NaBD4 (Aldrich) in 0.02% triethylamine in deionized water. As controls, equal portions of the lipid A samples were treated with 150 μl of 0.02% triethylamine in deionized water. The samples were allowed to react for 2.5 or 4 h at room temperature and then quenched with 20 μl of glacial acetic acid. Samples were then taken to near dryness by evaporation under a stream of N2, followed by evaporation from 1% acetic acid in methanol (eight times), and finally evaporation from methanol (six times). Dry samples were then partitioned in CHCl3-CH3OH-H2O (10/5/6 by volume) in a total volume of 1,050 μl. The aqueous layer was removed and the organic layer plus interface was evaporated to dryness under a stream of N2. As a control experiment, a commercial sample of monophosphorylated lipid A from Salmonella enterica serovar Minnesota Re 595 (Sigma) was also subjected to the reduction conditions described above.
MALDI-TOF MS.Lipid A samples were analyzed by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS on a Voyager DESTR Plus instrument (Applied Biosystems, Framingham, Mass.) equipped with a 337-nm nitrogen laser. Spectra were obtained in the positive-ion and negative-ion reflectron modes under delayed extraction conditions. In the positive-ion reflectron mode, the delay time was 200 to 275 ns, and the grid voltage was 66.5 to 67.3% of full acceleration voltage (25 kV). In the negative-ion reflectron mode, the delay time was 175 ns, and the grid voltage was 64.5% of full acceleration voltage (20 kV). Samples were dissolved in CHCl3-CH3OH (3:1) at a concentration of approximately 0.1 to 0.5 μg/μl, mixed 1:1 with matrix solution (a saturated solution of 6-chloro-3-mercaptobenzothiazole in CHCl3-CH3OH [3:1 by volume]), spotted on a stainless steel target, and allowed to air dry. Approximately 100 laser shots were recorded for each sample. The spectra were processed with baseline correction and a noise filter. Mass calibration was done externally with a mixture consisting of angiotensin II, renin substrate tetradecapeptide, and insulin chain B (oxidized) or a mixture consisting of angiotensin I, adenocorticotropin (ACTH) 1-17, ACTH 18-39, and ACTH 7-38 (all from Sigma).
Nanoelectrospray tandem MS (MS/MS) and fragmentation MS (MSn).The F. tularensis 1547-57 lipid A sample, dissolved in CHCl3-CH3OH (1:2 by volume), at a concentration of approximately 0.1 to 0.5 μg/μl, was analyzed by static nanoelectrospray using a LCQDeca quadrupole ion-trap mass spectrometer (ThermoFinnigan, San Jose, Calif.). In the negative-ion nanoelectrospray mode, the needle voltage was typically set to 1.6 to 2.2 kV and the temperature of the heated capillary was at 140 to 180°C. Ions were isolated for collision-induced dissociation with the "isolation width" parameter set to 1 to 3.
The LPS from F. tularensis strain 1547-57 was isolated from plate-grown organisms using phenol-water extraction, followed by proteinase K treatment (13). The lipid A was released from the LPS by mild acid hydrolysis in 1% acetic acid and then further purified by partitioning in CHCl3-CH3OH-H2O (10/5/6).
To investigate the fatty acid composition of the lipid A, a small portion of the sample was treated with 10% (wt/wt) BF3-methanol at 100°C for 6 h to prepare FAMEs. The FAMEs were analyzed by GC-MS using a highly polar capillary column optimized for FAME analysis. Figure 1 shows the total ion chromatogram of the FAMEs derived from the F. tularensis lipid A fraction, where the main fatty acids present are hexadecanoic acid (C16:0) and 3-hydroxyoctadecanoic acid [C18:0(3-OH)] in the ratio of 1.2 to 3.0. Minor components seen in the sample include tetradecanoic acid (C14:0), octadecanoic acid (C18:0), octadecenoic acid (C18:1), 3-methoxyoctadecanoic acid [C18:0(3-methoxyl)], and 3-hydroxyhexadecanoic acid [C16:0(3-OH)] in the ratio of 0.3:0.1:0.2:0.2:0.3. Trace amounts of C20:0, C22:0, and C24:0 fatty acids were also detected. Compared to the FAMEs derived from the lipid A of F. tularensis LVS (27), the FAME mixture from strain 1547-57 contains higher proportions of the longer chain fatty acids, i.e., 20 times more C16:0 relative to C14:0 and 4 times more C18:0(3-OH) relative to C16:0(3-OH).
GC-MS total ion chromatogram of the FAMEs derived from the lipid A of F. tularensis strain 1547-57.
To evaluate the heterogeneity of the lipid A preparation and measure molecular weights, the lipid A sample was analyzed by MALDI-TOF MS. Both negative-ion (Fig. 2A) and positive-ion (Fig. 2B) spectra were recorded in reflectron mode to allow for the measurement of monoisotopic masses. Table 1 gives the C-12 monoisotopic masses measured from these spectra. As indicated, the major lipid A component with a (M − H)− at m/z 1,665.06 observed in the negative-ion MALDI spectrum (Fig. 2A) is proposed to contain a diglucosamine backbone bearing three C18:0(3-OH) and one C16:0 fatty acids, one phosphate group, and one additional 161-Da moiety (proposed to be a hexosamine, HexN). The species at (M − H)− at m/z 1,503.99 is lacking the additional HexN moiety. In the positive-ion MALDI spectrum (Fig. 2B), the doubly sodiated species (M − H + 2Na)+ at m/z 1,710.92 corresponds to the major component seen in the negative-ion spectrum, and the (M − H + 2Na)+ species at m/z 1,549.88 is the lipid A lacking the additional HexN moiety. Related species lacking both the phosphate and the HexN moieties are also observed in the positive-ion mode [(M + Na)+ ions at m/z 1,447.93 and 1,429.93]. However, the major component observed in the positive-ion spectrum is the (M + Na)+ species at m/z 1,391.88, which fits for a lipid A structure bearing two C18:0(3-OH), one C16:0(3-OH), and one C14:0 fatty acids. This constituent was the major species seen in the lipid A preparation from F. tularensis LVS (27). While this component is only seen in the positive-ion MALDI spectrum of the F. tularensis strain 1547-57 lipid A sample, the novel higher-molecular-weight components present in strain 1547-57 are detected in both the positive-ion and negative-ion modes.
Sections of three MALDI-TOF mass spectra of the F. tularensis strain 1547-57 lipid A sample, acquired in reflectron mode. (A) The negative-ion MALDI-TOF MS. (B) The positive-ion MALDI-TOF MS. (C) The positive-ion MALDI-TOF MS of the lipid A sample after HF treatment for 40 h.
Molecular weights (Mr) observed for lipid A structures of F. tularensis strain 1547-57 as detected by negative-ion and positive-ion reflectron MALDI-TOF MS
To check for other potentially labile moieties and evaluate the yields of the phosphorylated components, we attempted to hydrolyze the F. tularensis LPS under more mild conditions. The LPS was treated with 10 mM sodium acetate containing 1% SDS at pH 4.5 for 1 h at 100°C (3). The resulting lipid A sample was analyzed by MALDI-TOF MS in both the negative-ion and positive-ion reflectron modes (data not shown). Compared to the lipid A preparation generated by 1% acetic acid hydrolysis, the sample generated under more mild hydrolysis conditions showed slightly higher relative amounts of the phosphorylated lipid A forms by positive-ion MALDI but did not contain any previously unseen components.
As an initial assumption, the 161-Da moiety present on the F. tularensis 1547-57 lipid A was presumed to be a HexN residue linked to the diglucosamine backbone via the phosphate group, in a manner analogous to the way arabinosamine (4-amino-4-deoxy-l-arabinose) is found linked to phosphate in the LPS of other organisms (2, 18, 20, 26). To test for the presence of a hexosamine-1-phosphate linkage, the sample was treated two times with 48% aqueous HF. The MALDI spectrum was recorded after each treatment and was used to monitor the progress of the dephosphorylation reaction. The MALDI spectrum of the sample after two HF treatments (Fig. 2C) shows the complete loss of the HexN moiety (to give the monophosphorylated species at m/z 1,549.89) and the partial removal of the residual phosphate group (to give the species at m/z 1,447.95 which lacks the entire hexosamine-1-phosphate moiety). After only a single HF treatment, the m/z 1,447.95 species was less abundant than after the second HF treatment, suggesting that the hexosamine-phosphate linkage is cleaved more readily than the lipid A-phosphate linkage. Consistent with the published structure for the (M + Na)+ species at m/z 1,391.90, which does not contain any phosphate substituents (27), there was nothing lost from that component upon HF treatment.
Further confirmation of the linkage of the hexosamine-1-phosphate moiety to the lipid A backbone of the novel species was obtained by MS/MS analysis. The lipid A sample, dissolved in CHCl3-CH3OH (1:2) was analyzed by static nanoelectrospray using a quadrupole ion-trap mass spectrometer. This approach has recently proven useful for following multiple stages of MSn of lipid A ions (4, 16). The negative-ion mass spectrum of the fraction gave an abundant (M − H)− ion at m/z 1,665.2 for the component bearing the hexosamine-1-phosphate. The MS/MS spectrum of this species (Fig. 3A) contains ions representing single- and multiple-bond cleavages. The fragment ion arising from the loss of hexosamine from the parent ion (m/z 1,503.9) indicates that the novel hexosamine moiety is in a terminal position. Fragment ions at m/z 1,408.9 and 1,364.9 arise from the losses of C16:0 (−256.2 Da) and C18:0(3-OH) (−300.3 Da) fatty acids from the parent ion, respectively. The major fragment ion at m/z 1,204.1 can be assigned as a two-bond cleavage involving the loss of a C18:0(3-OH) fatty acid (−300.3 Da) and the HexN moiety (−161.1 Da). The ion at m/z 947.9 would appear to arise from three-bond cleavages [losses of C18:0(3-OH), C16:0, and HexN].
Negative-ion nanoelectrospray MSn spectra of the lipid A from F. tularensis strain 1547-57. (A) MS/MS of m/z 1,665.2. (B) MS3 of m/z 1,408.9. (C) MS3 of m/z 1,203.9. (D) MS3 of m/z 947.8.
In an effort to fix the locations of the fatty acids and the hexosamine-1-phosphate group on the diglucosamine backbone, various fragment ions in the MS/MS spectrum were selected for further fragmentation (MS3). Both the m/z 1,408.9 and 1,203.9 fragments gave the m/z 947 ion as the base peak in their MS3 spectra (Fig. 3B and C, respectively). In the case of the m/z 1,408.9 fragment (already lacking C16:0), the 947.6 ion arises from two-bond cleavages; the loss of the HexN moiety plus the O-linked C18:0(3-OH) fatty acid (Fig. 3B). In the MS3 spectrum of m/z 1,203.9 [already lacking C18:0(3-OH) and HexN], the 947.7 fragment arises from the loss of the O-linked C16:0 fatty acid (Fig. 3C).
To explore the structure of the m/z 947 fragment, it was selected from the MS/MS spectrum for MS3 analysis as shown in Fig. 3D. Fragmentation of m/z 947.8 led to losses of the N-linked fatty acids as ketenes (fragment ions at m/z 683.5 and 665.5). Also present in the spectrum were fragments at m/z 504.5 and 522.4 that appeared to arise from glycosidic bond cleavages. By using the nomenclature of Costello and Vath (5), these ions could be assigned as B- and C-type fragments if the phosphate group was on the distal glucosamine, and Z- and Y-type fragments in the phosphate group were on the reducing terminal glucosamine. To resolve this ambiguity, corresponding glycosidic bond fragments in the MS3 spectra of m/z 1,408.9 (Fig. 3B) and m/z 1,203.9 (Fig. 3C) were sought. The m/z 983.5 fragment in Fig. 3B can be assigned as a Y-type fragment if the structure of m/z 1,408.9 is as shown in the inset, with the hexosamine-1-phosphate group on the reducing terminal glucosamine. This Y ion also supports the placement of the two C18:0(3-OH) fatty acids on the reducing terminal glucosamine. Correspondingly, the m/z 522.5 fragment in Fig. 3C can be assigned as a Y ion arising from m/z 1,203.9, which is consistent with the assignment of that fragment in Fig. 3D and supports the placement of the one C18:0(3-OH) and one C16:0 fatty acid on the distal glucosamine of the structure. The assignment of the C16:0 fatty acid in an acyloxy linkage to O-3 of the N-linked C18:0(3-OH) fatty acid of the distal glucosamine was suggested by the fragment ion at m/z 683.5 in Fig. 3D (loss of a C18:1 ketene). This fragment ion could only arise from the m/z 947.8 species if the N-linked fatty acid on its distal glucosamine was a C18:1 fatty acid (Fig. 3D, inset), presumably generated by the elimination of C16:0 (as a free acid) from the tetraacylated structure.
Conspicuously lacking from the MS/MS and MS3 spectra are the strong 0,2A2 and 0,4A2 cross-ring fragments generally seen in monophosphorylated lipid A's with the phosphate group on the 4′ position and a free reducing terminus (16, 19). The lack of these A-type fragments may be an indication that the hexosamine-1-phosphate group is located on the reducing terminal glucosamine. An additional curious aspect of these spectra is the ability of many species to lose a 240-Da moiety. This moiety is proposed to be a C16H32O aldehyde eliminated from the N-linked C18:0(3-OH) fatty acid on the reducing terminal glucosamine by a McLafferty rearrangement. This type of fragmentation could potentially result from a charge-driven rearrangement process involving the neighboring reducing terminal phosphate group. When located on the 4′ position of lipid A, phosphate has been proposed to initiate a charge-driven fragmentation process affecting fragmentation at the 3′ position in negative-ion electrospray ionization-MSn (16).
To further confirm the placement of the hexosamine-1-phosphate group on the reducing terminus of the lipid A backbone, the F. tularensis 1547-57 lipid A sample was subjected to reduction by treatment with NaBD4 for 2.5 to 4 h. The reaction mixtures were analyzed by MALDI-TOF MS in both the positive-ion and negative-ion reflectron modes. Figure 4 shows the positive-ion MALDI spectrum of the 4-h reaction. Under the short reaction times used to minimize unwanted cleavage of O-linked fatty acids in the slightly basic solution, the reduction reaction did not go all the way to completion. Thus, species that were susceptible to reduction were seen to split into two sets of molecular ion peaks, with one shifted to higher mass by the addition of 3 Da (Fig. 4). Species that did not undergo reduction were unchanged by the treatment. Control samples that were incubated with solvent lacking NaBD4 did not show any evidence of mass shifts. Referring to Table 1, all of the species containing either hexosamine-1-phosphate (Mr, 1,666 and 1,638) or phosphate alone (Mr, 1,505, 1,477, and 1,427) were not susceptible to reduction, whereas the unphosphorylated species (Mr, 1,425, 1,397, and 1,369) were all able to react. As a control, the monophosphorylated lipid A from S. enterica serovar Minnesota Re 595, which has a phosphate substituent on the 4′ position of the glucosamine disaccharide, was subjected to the same procedures and found to be susceptible to reduction. In all of the reduction experiments, some minor evidence of O-linked fatty acid cleavage was detected, but the resulting molecular ion species had isotope distributions consistent with the corresponding intact structures. Thus, we conclude that the phosphorylated F. tularensis lipid A structures do not have a free reducing terminus available for reaction, supporting the assignment of the hexosamine-1-phosphate on the 1 position of the lipid A.
Section of the positive-ion MALDI spectrum of the F. tularensis 1547-57 lipid A sample after reduction by treatment with NaBD4 for 4 h, acquired in reflectron mode. Insets show expansions of the indicated peaks. Asterisks mark the monoisotopic molecular ions of reduced species that have incorporated three deuterium atoms.
To identify the purported hexosamine moiety, the HF-treated lipid A sample that had released its hexosamine (described above) was partitioned in CHCl3-CH3OH-H2O (2:1:2), and the upper aqueous layer was recovered and evaporated to dryness. This material was reduced with NaBD4 and then peracetylated by treatment with acetic anhydride to generate the alditol acetate of the hexosamine. As a control, a sample of HF-treated S. enterica serovar Typhimurium lipid A was taken through the same reaction sequence. The derivatized F. tularensis and S. enterica serovar Typhimurium samples were analyzed by GC-MS using chemical ionization. Samples were compared to a mixture of standards consisting of glucosaminitol acetate, mannosaminitol acetate, and galactosaminitol acetate, which eluted at 37.34, 38.47, and 39.21 min, respectively. The sample derived from the F. tularensis lipid A contained a component with the molecular weight of a hexosaminitol acetate, (M + H)+ at m/z 435.5, that eluted at the retention time of galactosaminitol acetate. No hexosaminitol acetates of any type were detected in the S. enterica serovar Typhimurium sample. Thus, we conclude from these results that the hexosamine-1-phosphate linked to the proximal glucosamine in the novel F. tularensis lipid A species is galactosamine-1-phosphate (Fig. 5).
Proposed structure of the 1,666-Mr component of the F. tularensis strain 1547-57 lipid A sample. The diglucosamine backbone bears four fatty acids: three C18:0(3-OH) fatty acids, and one C16:0 fatty acid. The acylation pattern shown is consistent with the arrangement determined for the lipid A of F. tularensis LVS (ATCC 29684) (27). The further modification of the Mr-1,666 component of F. tularensis strain 1547-57 lipid A with a galactosamine-1-phosphate on the reducing terminus represents a novel structural feature not previously observed. The anomeric configuration of the galactosamine-1-phosphate linkage has not been determined.
This modification seen in the Mr-1,666 component of the F. tularensis 1547-57 lipid A mixture was not reported in the lipid A from F. tularensis LVS (27). However, in our laboratory, the same modification was seen, although to a lesser extent, in the lipid A fraction from Francisella philomiragia ATCC 25015 (M. McLendon and B. Schilling, unpublished data). To address this discrepancy, we cultured F. tularensis LVS and prepared the lipid A fraction from its LPS following our protocols as described above. The lipid A from this F. tularensis LVS was analyzed by MALDI-TOF MS in the positive-ion and negative-ion modes (Fig. 6) and compared to the lipid A from the F. tularensis 1547-57 strain (Fig. 2). Under our growth and isolation conditions, the major species that we obtained from the F. tularensis LVS strain was a monophosphorylated lipid A with an Mr of 1,505.1 (Fig. 6). This species was consistent with the tetraacylated, monophosphorylated lipid A seen in the F. tularensis 1547-57 strain [consisting of three C18:0(3-OH) fatty acids, one C16:0 fatty acid, and one phosphate group]. In the positive-ion mass spectrum (Fig. 6B), we observed many of the unphosphorylated species also observed in the F. tularensis 1547-57 strain (m/z 1,391.8, 1,419.9, 1,429.9, and 1,447.9). The species at m/z 1,391.84 and 1,419.87 are consistent with the previously published major structures for the lipid A from F. tularensis LVS (ATCC 29684) (27). The species at m/z 1,565.81 is 16 Da higher than the m/z 1,549.84 species and would appear to differ by the addition of an oxygen atom (possibly a fourth hydroxylated fatty acid), but this has not been determined. Thus, although we did observe monophosphorylated lipid A structures in this preparation of F. tularensis LVS lipid A, we did not see any hint of the Mr-1,666 structure modified with the galactosamine-1-phosphate moiety.
Sections of the MALDI-TOF mass spectra of the lipid A from F. tularensis LVS recorded in negative-ion (A) and positive-ion (B) reflectron modes. For comparison to the F. tularensis strain 1547-57 lipid A sample, refer to Fig. 2. For proposed compositions, see the text and Table 1.
To evaluate the relationship of the monophosphorylated lipid A structures in this F. tularensis LVS to the corresponding species in F. tularensis 1547-57, we subjected the F. tularensis LVS sample to reduction with NaBD4. As was seen with the F. tularensis 1547-57 lipid A species, the monophosphorylated structures in this F. tularensis LVS lipid A preparation were not amenable to reduction, whereas the unphosphorylated structures were. Thus, we conclude that under our growth conditions, F. tularensis LVS can add phosphate to the 1 position of the lipid A. However, F. tularensis LVS does not appear to be capable of further modifying the monophosphorylated lipid A structure with the novel galactosamine moiety, as seen in F. tularensis strain 1547-57.
The lipid A preparation from F. tularensis strain 1547-57 contained a heterogeneous mixture of lipid A structures. In order to get a complete view of the species present in the mixture, it was necessary to analyze the sample by MALDI-TOF MS in both the positive-ion and negative-ion modes. All of the major lipid A forms detected by MALDI-TOF were tetraacylated, although there was microheterogeneity in the fatty acid constituents. One lipid A form present in F. tularensis strain 1547-57 (Mr, 1,369) had a composition consistent with the major published structure for the lipid A from F. tularensis LVS (ATCC 29684) (27), consisting of two C18:0(3-OH), one C16:0(3-OH), and one C14:0 fatty acids. The other major components found in the F. tularensis 1547-57 strain were of higher molecular weight than this species. Some forms simply contained longer chain fatty acids, whereas others were monophosphorylated as well.
The most striking constituent was a monophosphorylated species with an Mr of 1,666 bearing three C18:0(3-OH) and one C16:0 fatty acids, plus an additional 161-Da moiety that was identified as galactosamine. Through a combination of MSn studies and chemical treatments, the galactosamine was shown to exist as a galactosamine-1-phosphate moiety linked to the reducing terminus of the lipid A. MSn investigations also showed that this tetraacylated structure had two fatty acids on each glucosamine unit of the lipid A, with the nonreducing terminal glucosamine apparently bearing an N-linked acyloxyacyl group (Fig. 5). This acylation pattern is consistent with the architecture of the published structure for F. tularensis LVS (27).
The novel modification of the F. tularensis lipid A with galactosamine-1-phosphate seen in strain 1547-57 was not reported in the previous study of F. tularensis LVS (27). In our hands, a lipid A preparation from F. tularensis LVS was shown to include monophosphorylated species but no species bearing galactosamine-1-phosphate. However, the hexosamine-1-phosphate modification was detected in a lipid A fraction from F. philomiragia (M. McLendon and B. Schilling, unpublished). Thus, it would appear that the lack of this moiety in F. tularensis LVS may result from strain variation. The phosphorylation differences seen in F. tularensis LVS by our group and by Vinogradov et al. (27) may also arise from strain variation or from different lipid A isolation and purification steps. Specifically, our protocols differed in culture conditions (plate versus broth), LPS acetic acid hydrolysis conditions (1 versus 5% acetic acid), and lipid A purification methods (extraction versus extraction plus column chromatography).
To our knowledge, the modification of a lipid A structure with galactosamine-1-phosphate has not been previously observed. However, the addition of arabinosamine to the phosphate group(s) of lipid A has been well characterized (10, 11, 17, 25, 29). In an effort to explore possible connections, we searched the F. tularensis Schu S4 genome for a homolog of the arabinosamine transferase ArnT in S. enterica serovar Typhimurium and YfbI of E. coli (25). A homolog was found on contig 34, nucleotides 16024 to 16950c, and the presumed protein product shares 27% amino acid identity to YfbI and 25% amino acid identity to ArnT. The amino acid sequence has the highest identity (33%) to the arabinosamine transferases of Bordetella parapertussis and Geobacter metallireducens, and 27% identity to the arabinosamine transferases of Pseudomonas fluorescens and Yersinia spp. ArnT and YfbI are members of the protein O-mannosyltransferase family of proteins, involved in posttranslational modification. Thus, it is possible that this homolog may be another member of the protein O-mannosyltransferase protein family.
The modification of lipid A with arabinosamine alters the net negative charge on the lipid A and is required for resistance to polymyxin and cationic antimicrobial peptides (9, 10). The addition of galactosamine-1-phosphate to the lipid A of F. tularensis is likely to have a similar effect on net negative charge. The significance of this modification for F. tularensis is unknown, although it is possible that it could confer some resistance to host antimicrobial peptides in vivo. It is interesting that F. tularensis contains a possible ortholog of a lipid A 1-phosphatase, LpxE, from Rhizobium leguminosarum (14). Thus, dephosphorylation of the lipid A structure in vivo may be a possibility. Preliminary evidence also suggests that F. tularensis may be capable of synthesizing more highly acylated lipid A structures in vivo once infection is established (M. McLendon, unpublished data). Indeed, minor constituents at molecular weights higher than the Mr-1,666 constituent were occasionally detected in the F. tularensis strain 1547-57 lipid A sample by positive-ion MALDI-TOF MS (mainly observed at m/z 1,821.2, 1,842.6, 1,849.2, 1,934.3, and 1,962.3). Thus, the various lipid A structures described in this report may represent only a subset of the lipid A modifications possible in vivo. However, the tetraacylated lipid A structures, with and without the galactosamine-1-phosphate modification, seem likely to contribute to the unusually low endotoxicity of the F. tularensis LPS.
We thank Yuequan Sun for assistance with acquisition of GC-MS data, David A. Maltby for helpful input, and the UCSF Mass Spectrometry Facility for access to the GC-MS system (supported by NIH NCRR BRTP RR01614, A. L. Burlingame, Director). We are also grateful to Karen Elkins for supplying F. tularensis LVS and Jeannine Petersen for assistance in typing F. tularensis strain 1547-57. Additionally, we acknowledge William R. Kearney of the University of Iowa for helpful discussions.
Received 15 January 2004.
Returned for modification 3 March 2004.
Accepted 2 June 2004.
Editor: J. N. Weiser
American Society for Microbiology
Ancuta, P., T. Pedron, R. Girard, G. Sandstrom, and R. Chaby. 1996. Inability of the Francisella tularensis lipopolysaccharide to mimic or to antagonize the induction of cell activation by endotoxins. Infect. Immun. 64:2041-2046.
Boll, M., J. Radziejewska-Lebrecht, C. Warth, D. Krajewska-Pietrasik, and H. Mayer. 1994. 4-Amino-4-deoxy-L-arabinose in LPS of enterobacterial R-mutants and its possible role for their polymyxin reactivity. FEMS Immunol. Med. Microbiol. 8:329-341.
Caroff, M., A. Tacken, and L. Szabo. 1988. Detergent-accelerated hydrolysis of bacterial endotoxins and determination of the anomeric configuration of the glycosyl phosphate present in the "isolated lipid A" fragment of the Bordetella pertussis endotoxin. Carbohydr. Res. 175:273-282.
Corsaro, M. M., F. D. Piaz, R. Lanzetta, and M. Parrilli. 2002. Lipid A structure of Pseudoalteromonas haloplanktis TAC 125: use of electrospray ionization tandem mass spectrometry for the determination of fatty acid distribution. J. Mass Spectrom. 37:481-488.
Costello, C. E., and J. E. Vath. 1990. Tandem mass spectrometry of glycolipids. Methods Enzymol. 193:738-768.
Dennis, D. T., T. V. Inglesby, D. A. Henderson, J. G. Bartlett, M. S. Ascher, E. Eitzen, A. D. Fine, A. M. Friedlander, J. Hauer, M. Layton, S. R. Lillibridge, J. E. McDade, M. T. Osterholm, T. O'Toole, G. Parker, T. M. Perl, P. K. Russell, and K. Tonat. 2001. Tularemia as a biological weapon: medical and public health management. JAMA 285:2763-2773.
Ellis, J., P. C. Oyston, M. Green, and R. W. Titball. 2002. Tularemia. Clin. Microbiol. Rev. 15:631-646.
Fulop, M., T. Webber, and R. Manchee. 1993. Activation of the complement system by Francisella tularensis lipopolysaccharide. New Microbiol. 16:141-147.
Gunn, J. S. 2001. Bacterial modification of LPS and resistance to antimicrobial peptides. J. Endotoxin Res. 7:57-62.
Gunn, J. S., K. B. Lim, J. Krueger, K. Kim, L. Guo, M. Hackett, and S. I. Miller. 1998. PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance. Mol. Microbiol. 27:1171-1182.
Guo, L., K. B. Lim, J. S. Gunn, B. Bainbridge, R. P. Darveau, M. Hackett, and S. I. Miller. 1997. Regulation of lipid A modifications by Salmonella typhimurium virulence genes phoP-phoQ. Science 276:250-253.
Hood, A. M. 1977. Virulence factors of Francisella tularensis. J. Hyg. (London) 79:47-60.
Jones, P. A., N. M. Samuels, N. J. Phillips, R. S. Munson, Jr., J. A. Bozue, J. A. Arseneau, W. A. Nichols, A. Zaleski, B. W. Gibson, and M. A. Apicella. 2002. Haemophilus influenzae type b strain A2 has multiple sialyltransferases involved in lipooligosaccharide sialylation. J. Biol. Chem. 277:14598-14611.
Karbarz, M. J., S. R. Kalb, R. J. Cotter, and C. R. Raetz. 2003. Expression cloning and biochemical characterization of a Rhizobium leguminosarum lipid A 1-phosphatase. J. Biol. Chem. 278:39269-39279.
Kulshin, V. A., U. Zahringer, B. Lindner, C. E. Frasch, C. M. Tsai, B. A. Dmitriev, and E. T. Rietschel. 1992. Structural characterization of the lipid A component of pathogenic Neisseria meningitidis. J. Bacteriol. 174:1793-1800.
Kussak, A., and A. Weintraub. 2002. Quadrupole ion-trap mass spectrometry to locate fatty acids on lipid A from gram-negative bacteria. Anal. Biochem. 307:131-137.
Molinaro, A., B. Lindner, C. De Castro, B. Nolting, A. Silipo, R. Lanzetta, M. Parrilli, and O. Holst. 2003. The structure of lipid A of the lipopolysaccharide from Burkholderia caryophylli with a 4-amino-4-deoxy-L-arabinopyranose 1-phosphate residue exclusively in glycosidic linkage. Chemistry 9:1542-1548.
Nummila, K., I. Kilpelainen, U. Zahringer, M. Vaara, and I. M. Helander. 1995. Lipopolysaccharides of polymyxin B-resistant mutants of Escherichia coli are extensively substituted by 2-aminoethyl pyrophosphate and contain aminoarabinose in lipid A. Mol. Microbiol. 16:271-278.
Post, D. M., N. J. Phillips, J. Q. Shao, D. D. Entz, B. W. Gibson, and M. A. Apicella. 2002. Intracellular survival of Neisseria gonorrhoeae in male urethral epithelial cells: importance of a hexaacyl lipid A. Infect. Immun. 70:909-920.
Radziejewska-Lebrecht, J., U. R. Bhat, H. Brade, and H. Mayer. 1988. Structural studies on the core and lipid A region of a 4-amino-L-arabinose-lacking Rc-type mutant of Proteus mirabilis. Eur. J. Biochem. 172:535-541.
Rietschel, E. T., T. Kirikae, F. U. Schade, U. Mamat, G. Schmidt, H. Loppnow, A. J. Ulmer, U. Zahringer, U. Seydel, F. Di Padova, et al. 1994. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 8:217-225.
Sandstrom, G. 1994. The tularaemia vaccine. J. Chem. Technol. Biotechnol. 59:315-320.
Sandstrom, G., A. Sjostedt, T. Johansson, K. Kuoppa, and J. C. Williams. 1992. Immunogenicity and toxicity of lipopolysaccharide from Francisella tularensis LVS. FEMS Microbiol. Immunol. 5:201-210.
Tarnvik, A. 1989. Nature of protective immunity to Francisella tularensis. Rev. Infect. Dis. 11:440-451.
Trent, M. S., A. A. Ribeiro, S. Lin, R. J. Cotter, and C. R. Raetz. 2001. An inner membrane enzyme in Salmonella and Escherichia coli that transfers 4-amino-4-deoxy-L-arabinose to lipid A: induction on polymyxin-resistant mutants and role of a novel lipid-linked donor. J. Biol. Chem. 276:43122-43131.
Vaara, M., T. Vaara, M. Jensen, I. Helander, M. Nurminen, E. T. Rietschel, and P. H. Makela. 1981. Characterization of the lipopolysaccharide from the polymyxin-resistant pmrA mutants of Salmonella typhimurium. FEBS Lett. 129:145-149.
Vinogradov, E., M. B. Perry, and J. W. Conlan. 2002. Structural analysis of Francisella tularensis lipopolysaccharide. Eur. J. Biochem. 269:6112-6118.
Zhou, Z., S. Lin, R. J. Cotter, and C. R. Raetz. 1999. Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate. J. Biol. Chem. 274:18503-18514.
Zhou, Z., A. A. Ribeiro, and C. R. Raetz. 2000. High-resolution NMR spectroscopy of lipid A molecules containing 4-amino-4-deoxy-L-arabinose and phosphoethanolamine substituents. Different attachment sites on lipid A molecules from NH4VO3-treated Escherichia coli versus kdsA mutants of Salmonella typhimurium. J. Biol. Chem. 275:13542-13551.
Infection and Immunity Aug 2004, 72 (9) 5340-5348; DOI: 10.1128/IAI.72.9.5340-5348.2004
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High-quality clinical placements for nursing students can help to boost nurse numbers in traditionally hard-to-recruit areas of practice, according to a study.
Researchers at the University of Bedfordshire found the quality of placements experienced by students had a direct impact on the specialty they eventually chose to practice once qualified.
The authors of the study, published in the British Journal of Nursing, say investing in better placements in hard-to-recruit areas, such as primary or older people's care, could lead to increased recruitment in those specialisms.
The university has now begun a larger longitudinal study with Canterbury Christ Church University, again focusing on placements. It will track pre-registration adult, child and mental health nursing students, and midwifery students, from 2018 over the three years of their programmes.
In the published study, a total of 110 third-year nursing students from the university were surveyed in the final week of their programmes in 2017 and 2018 to determine how placements influenced their choice of first post as a newly qualified nurse.
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'When you begin studying to become a nurse, you often come with pre-conceived ideas of where you want to end up working. In my case, I had watched a lot of [television programme] Casualty and wanted to work in A&E.


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Q: Android ndk-build iostream: No such file or directory I'm having problem with compiling cpp file using ndk-build tool (windows 7 with cygwin)
Error appears when I try to compile cpp file with #include:
jni/native.cpp:5:20: error: iostream: No such file or directory

Here is my cpp file:
#include <jni.h>
#include <string.h>
#include <stdio.h>
#include <android/log.h>
#include <iostream>

#define DEBUG_TAG "NDK_SampleActivity"
#define  LOG_TAG    "hellojni"
#define  LOGI(...)  __android_log_print(ANDROID_LOG_INFO,LOG_TAG,__VA_ARGS__)
#define  LOGE(...)  __android_log_print(ANDROID_LOG_ERROR,LOG_TAG,__VA_ARGS__)

#ifdef __cplusplus
extern "C" {
#endif

void Java_com_test_ndk_SampleActivity_helloLog(JNIEnv* env, jobject thisobj, jstring logThis)
{
    jboolean isCopy;

    const char * szLogThis = env->GetStringUTFChars(logThis, &isCopy);

    __android_log_print(ANDROID_LOG_DEBUG, DEBUG_TAG, "NDK:LC: [%s]", szLogThis);

    env->ReleaseStringUTFChars(logThis, szLogThis);
}

#ifdef __cplusplus
}
#endif

And here is my Android.mk file:
LOCAL_PATH := $(call my-dir)

include $(CLEAR_VARS)

APP_STL:=stlport_static

LOCAL_LDLIBS := -llog

LOCAL_MODULE    := swingbyte-android

LOCAL_SRC_FILES := native.cpp

LOCAL_C_INCLUDES := $(LOCAL_PATH)/include-all
include $(BUILD_SHARED_LIBRARY)

I have iostream file in android ndk folder (NDK_ROOT\sources\cxx-stl\gnu-libstdc++\include) but I don't have any idea how to tell compiler to look for iotream (and other standart header files) in that folder.
It seems to that I'm missing one or few environment variables, or some comiler flags.

A: I think "APP_STL:=stlport_static" must be in Application.mk file.
Create a "Application.mk" file and write "APP_STL:=stlport_static" in it.

A: This works for me.
LOCAL_STATIC_LIBRARIES +=  libstlport

LOCAL_C_INCLUDES += external/stlport/stlport
LOCAL_C_INCLUDES += bionic

A: Adding
APP_PLATFORM := android-23

Or whatever revision you use solved it for me.

A: Update your android ndk to the latest one .
I faced error in android ndk ver 5

A: Just spent days to update my NDK from r10e to r20, there're several variables are changed.
For NDK r10e
Android.mk:
include $(CLEAR_VARS)
LOCAL_MODULE := main
LOCAL_SRC_FILES := ./main.cpp
LOCAL_C_INCLUDES += $(LOCAL_PATH)/
LOCAL_CPP_EXTENSION := .cxx .cpp .cc
LOCAL_CPPFLAGS := -fexceptions -frtti
LOCAL_CPPFLAGS += -std=c++11 -D__cplusplus=201103L
include $(BUILD_EXECUTABLE)

Application.mk:
APP_ABI := all
APP_STL := gnustl_static
NDK_TOOLCHAIN_VERSION := 4.9
APP_OPTIM := debug

For NDK r20
Android.mk:
include $(CLEAR_VARS)
LOCAL_MODULE := main
LOCAL_SRC_FILES := ./main.cpp
LOCAL_C_INCLUDES += $(LOCAL_PATH)/
LOCAL_CPP_EXTENSION := .cxx .cpp .cc
LOCAL_CPPFLAGS := -fexceptions -frtti
LOCAL_CPPFLAGS += -std=c++11 -D__cplusplus=201103L -DANDROID_STL=c++_shared
include $(BUILD_EXECUTABLE)

Application.mk:
APP_ABI := all
#In general, you can only use a static variant of the C++ runtime if you have one and only one shared library in your application.
APP_STL := c++_static
NDK_TOOLCHAIN_VERSION := clang
APP_PLATFORM := android-23
APP_OPTIM := debug

and my main.cpp (including my bin_node.h):
int main(int argc,char **argv) {
printf("****************** tree node ******************\n");
amo::BinNode<int> root(0);
amo::BinNode<int>* lchild1 = root.insertLeftChild(1);
amo::BinNode<int>* rchild2 = root.insertRightChild(2);
amo::BinNode<int>* lchild3 = lchild1->insertLeftChild(3);
amo::BinNode<int>* rchild4 = lchild1->insertRightChild(4);
amo::BinNode<int>* lchild5 = rchild2->insertLeftChild(5);
amo::BinNode<int>* rchild6 = rchild2->insertRightChild(6);
amo::BinNode<int>* lchild7 = lchild3->insertLeftChild(7);
amo::BinNode<int>* rchild8 = lchild3->insertRightChild(8);
amo::BinNode<int>* lchild9 = rchild6->insertLeftChild(9);
amo::BinNode<int>* rchild10 = rchild6->insertRightChild(10);
amo::BinNode<int>* lchild11 = rchild8->insertLeftChild(11);
amo::BinNode<int>* rchild12 = rchild8->insertRightChild(12);

printf("going to root.traversePre()\n");
root.traversePre();
printf("going to root.traversePreLoop()\n");
root.traversePreLoop();
printf("going to root.traversePreLoop2()\n");
root.traversePreLoop2();
printf("\n****************** main return ******************\n");
return 0;}

Run ndk-build and build an executable file

For more source code and information for this, check my GitHub

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