LETTERS OF NOTE: CATSTo clarify: there soon will be a book filled with nothing but approximately 30 letters about cats and it will be as amazing as you are imagining (i.e. These will be published by Canongate. When we're certain of the publication date, I'll let you know. And I'll show you some covers soon.In other news, this blog will relaunch when those books arrive on the scene.In other news, I'm finally, after years of never having the time, starting work on the Letters of Note podcast.In other news, Letters Live continues to grow and delight. For upcoming events.In other news, the Letters of Note newsletter launches tomorrow. It'll come out every week and will contain news about all of the above and general letter chat, delivered to your inbox each Sunday.In other news, I've missed you.All the best,Shaun. On April 18th of 1961, it was announced that iconic Hollywood star was dying of cancer after a glittering 36 year career that saw him amass countless fans, plaudits, and awards across the globe.
Weeks after that news broke, and just days before he died, Cooper received the following fan letter from, who at the time was producing and starring in (titled The Last Hero during production), an adaptation of 's Western novel,.(Huge thanks to. Photos: Gary Cooper in The Winning of Barbara Worth, 1926, via; Kirk Douglas in 1955, via.). May 4, 1961Mr. Gary CooperBeverly HillsDear Coop:When for years you’ve had affection for a guy and you find it suddenly turning to resentment you begin to think it deserves some kind of comment. When the guy you find yourself disliking is loved by the entire world you know damn well you better explain.What I'm talking about is me not liking you.Put yourself in my spot.
I'm doing a picture that should have been done by only one guy. I know it-my entire company knows it.Start with the title-”The Last Hero.' Now whom does that fit-me? Hell no!Next the author.
Edward Abbey-a ranger working in the Petrified Forest. They tell me before I meet him that he's written about himself.
So now he comes to Albuquerque where we're shooting and I go to meet him at the airport. Fifty guys step off the plane but I spot him immediately. He looks like Gary Cooper.
To make matters worse when I meet him he talks like Cooper!So now we start shooting and I learn first that I have an insensitive director who doesn't give a damn about anything except making the picture real. I give you verbatim my first-and only-direction-'Just try and play this the way Gary Cooper would.' When I say “only' I don't mean I get this 'hint' once-I mean it's the only thing I hear before each shot-and by the fourth day I have now decided I must get close to being Coop just so I can stop being hounded.Ah-but there's the rub.
It sounded easy to me-because I say to myself Coop is a simple man- natural. So I'll just be natural. Then I learned the big-big lesson.
It ain't easy. My temptation is to ask how the hell have you done it? What is the secret to this peace with yourself and your world? But then I know you couldn't possibly tell me-I'd have to live your entire life-grow-adjust-mature-as you have done.And I know now that at best I will come remotely close. But more important-I do know also, that just trying to be you-will make a better me.So, Coop-even though I may be sore as hell at you now-thanks.
6.15 p.m.Dearest Mother,I will call the place from which I’m now writing 'The Smoky Cellar of the Forester’s House'. I write on the first sheet of the writing pad which came in the parcel yesterday. Luckily the parcel was small, as it reached me just before we moved off to the line.
Thus only the paraffin was unwelcome in my pack. My servant & I ate the chocolate in the cold middle of last night, crouched under a draughty Tamboo, roofed with planks. I husband the Malted Milk for tonight, & tomorrow night. The handkerchief & socks are most opportune, as the ground is marshy, & I have a slight cold!So thick is the smoke in this cellar that I can hardly see by a candle 12 ins. Away, and so thick are the inmates that I can hardly write for pokes, nudges & jolts. On my left the Coy.
Commander snores on a bench: other officers repose on wire beds behind me. At my right hand, Kellett, a delightful servant of A Coy in The Old Days radiates joy & contentment from pink cheeks and baby eyes. He laughs with a signaller, to whose left ear is glued the Receiver; but whose eyes rolling with gaiety show that he is listening with his right ear to a merry corporal, who appears at this distance away (some three feet) nothing but a gleam of white teeth & a wheeze of jokes.Splashing my hand, an old soldier with a walrus moustache peels & drops potatoes into the pot. By him, Keyes, my cook, chops wood; another feeds the smoke with the damp wood.It is a great life. I am more oblivious than alas! Yourself, dear Mother, of the ghastly glimmering of the guns outside, & the hollow crashing of the shells.There is no danger down here, or if any, it will be well over before you read these lines.I hope you are as warm as I am; as serene in your room as I am here; and that you think of me never in bed as resignedly as I think of you always in bed. Of this I am certain you could not be visited by a band of friends half so fine as surround me here.
In the early hours of July 21st, 1969, shortly after the landed on its surface, and became the first humans to step foot on the Moon. 28 years later, Buzz wrote a letter to Barry Goldman, a professor at the University of Maryland.Transcript follows.( Many thanks to Benjamin Cole.)TranscriptSeptember 25, 1997Dear Mr. Goldman,I am writing to you to share some of my personal ideas and thoughts about my experiences related to the moon landing.I have often described the moon as a “magnificent desolation.” Its rocky horizon curved against the deep black of space, making it perfectly obvious that we were standing on a ball spinning through the universe.When I planted the American flag on the dusty surface of the moon, I had an unusual thought: A billion people were watching me on television. Human beings had never been farther away than we were nor had more people thinking about them!I think the spirit and the sense of involvement exhibited by the numbers of people who remember where they were when that event happened make it even more apparent to me over the years that the moonwalk added value to the lives of all the people who participated in it.
Every person felt good about the nation achieving it-that the world, that humanity could have done this.I have snapshots of myself on the moon that will always remind me of that strange and fascinating place. Someday in the future as people are mulling over their vacation plans, I hope they’ll choose to fly into space. It’s the trip of a lifetime.Regarding your questions of space exploration in 50 years: all of the rationales reduce to one simple truth: we will walk on Mars in the spirit and wonder that sets our species apart.Sincerely,Buzz AldrinAPOLLO XI.
In 1983, at the end of an amazing career during which she was nominated for a then-record breaking ten Academy Awards for acting, two of which she won, Hollywood actress was diagnosed with breast cancer. Surgery followed, as did a number of strokes which left her partially paralysed. Then, in 1985, her daughter, released a controversial book, titled, that exposed their supposedly troubled relationship and generally painted Davis in a terrible light. Two years later, Bette Davis published —at the very end was this letter to her daughter.(This letter, along with 124 other fascinating pieces of correspondence, can be found in the bestselling book,; Photo of BD Hyman and Bette Davis: Getty Images.). The LetterDear Hyman,You ended your book with a letter to me. I have decided to do the same.There is no doubt you have a great potential as a writer of fiction.
You have always been a great storyteller. I have often, lo these many years, said to you, 'B.D., that is not the way it was. You are imagining things.'
Many of the scenes in your book I have played on the screen. It could be you have confused the 'me' on the screen with 'me' who is your mother.I have violent objections to your quotes of mine regarding actors I have worked with. For the most part, you have cruelly misquoted me. Ustinov I was thrilled to work with and I have great admiration of him as a person and as an actor. You have stated correctly my reactions to working with Faye Dunaway. She was a most exasperating co-star.
But to quote me as having said Sir Laurence Olivier was not a good actor is most certainly one of the figments of your imagination. Few actors have ever reached the towering heights of his performances.You constantly inform people that you wrote this book to help me understand you and your way of life better. Your goal was not reached. I am now utterly confused as to who you are or what your way of life is.The sum total of your having written this book is a glaring lack of loyalty and thanks for the very privileged life I feel you have been given.In one of your many interviews while publicizing your book, you said if you sell your book to TV you feel Glenda Jackson should play me. I would hope you would be courteous enough to ask me to play myself.I have much to quarrel about in your book.
I choose to ignore most of it. But not the pathetic creature you claim I have been because of the fact that I did not play Scarlett in 'Gone With the Wind.'
I could have, but turned it down. Selznick attempted to get permission from my boss, Jack Warner, to borrow Errol Flynn and Bette Davis to play Rhett Butler and Scarlett. I refused because I felt Errol was not good casting for Rhett.
At that time only Clark Gable was right. Therefore, dear Hyman, send me not back to Tara, rather send me back to Witch Way, our home on the beautiful coast of Maine where once lived a beautiful human being by the name of B.D., not Hyman.As you ended your letter in 'My Mother's Keeper' — it's up to you now, Ruth Elizabeth — I am ending my letter to you the same way: It's up to you now, Hyman. For the past few years I've been working on a new book-a collection of speeches titled, unsurprisingly, Speeches of Note-and I'm very excited to say that you can now pledge for this beautiful object and then visit the which I shall be updating regularly.The Speeches of Note book will celebrate oratory old and new, taking care not just to highlight the speeches that we know and admire but also to shine a light on those speeches which, despite their brilliance, have until now been largely ignored in these collections through no fault of their own. Some, for compelling reasons, were never actually read aloud. Speeches of all flavours will feature-enlightening, gripping, comforting, disturbing, cheering, emboldening-and the majority accompanied either by a photograph of the speaker or the speech being made, a gorgeous illustration, or even, where possible, a facsimile of the original speech itself. Each entry will feature an introduction that will offer the context necessary for the speech to be fully appreciated. Anthologies of speeches have been published before; however, none have contained a selection quite like Speeches of Note.To watch me speaking very badly about the book, and to get your name in the back of the special edition,.
As always, suggestions are very much welcome-the easiest way to do so is via the.Much love and thanks,Shaun.
NameAddressPhoneEddie A.
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Under the VolcanoBy: Anthony Bourdain, CNNAmericans love Mexican food. We consume nachos, tacos, burritos, tortas, enchiladas, tamales, and anything resembling Mexican in enormous quantities.We love Mexican beverages, happily knocking back huge amounts of tequila, mezcal and Mexican beer every year.We love Mexican people - as we sure employ a lot of them. Despite our ridiculously hypocritical attitudes towards immigration, we demand that Mexicans cook a large percentage of the food we eat, grow the ingredients we need to make that food, clean our houses, mow our lawns, wash our dishes, and look after our children. As any chef will tell you, our entire service economy - the restaurant business as we know it - in most American cities, would collapse overnight without Mexican workers. Some, of course, like to claim that Mexicans are 'stealing American jobs.'
But in two decades as a chef and employer, I never had ONE American kid walk in my door and apply for a dishwashing job, a porter's position - or even a job as prep cook. Mexicans do much of the work in this country that Americans, provably, simply won't do.
We love Mexican drugs. Maybe not you personally, but 'we,' as a nation, certainly consume titanic amounts of them - and go to extraordinary lengths and expense to acquire them.We love Mexican music, Mexican beaches, Mexican architecture, interior design, Mexican films.So, why don't we love Mexico?We throw up our hands and shrug at what happens and what is happening just across the border. Maybe we are embarrassed.Mexico, after all, has always been there for us, to service our darkest needs and desires.
Whether it's dress up like fools and get pass-out drunk and sun burned on Spring break in Cancun, throw pesos at strippers in Tijuana, or get toasted on Mexican drugs, we are seldom on our best behavior in Mexico. They have seen many of us at our worst. They know our darkest desires.In the service of our appetites, we spend billions and billions of dollars each year on Mexican drugs - while at the same time spending billions and billions more trying to prevent those drugs from reaching us.
The effect on our society is everywhere to be seen. Whether its kids nodding off and overdosing in small town Vermont, gang violence in LA, burned out neighborhoods in Detroit - it's there to see.What we don't see, however, haven't really noticed, and don't seem to much care about, is the 80,000 dead - mostly innocent victims in Mexico, just in the past few years. 80,000 families who've been touched directly by the so-called war on drugs.Mexico. Our brother from another mother. A country, with whom, like it or not, we are inexorably, deeply involved, in a close but often uncomfortable embrace.Look at it. It's beautiful. It has some of the most ravishingly beautiful beaches on earth.
Eddie C Parts Unknown Zip Download
Mountains, desert, jungle. Beautiful colonial architecture, a tragic, elegant, violent, ludicrous, heroic, lamentable, heartbreaking history. Mexican wine country rivals Tuscany for gorgeousness. Its archeological sites鈥攖he remnants of great empires, unrivaled anywhere.And as much as we think we know and love it, we have barely scratched the surface of what Mexican food really is.
It is NOT melted cheese over a tortilla chip. It is not simple, or easy. It is not simply 'bro food' halftime. It is in fact, old - older even than the great cuisines of Europe and often deeply complex, refined, subtle, and sophisticated. A true mole sauce, for instance, can take DAYS to make, a balance of freshly (always fresh) ingredients, painstakingly prepared by hand. It could be, should be, one of the most exciting cuisines on the planet.
If we paid attention.The old school cooks of Oaxaca make some of the more difficult to make and nuanced sauces in gastronomy. And some of the new generation, many of whom have trained in the kitchens of America and Europe have returned home to take Mexican food to new and thrilling new heights.It's a country I feel particularly attached to and grateful for.PHOTO GALLERY:In nearly 30 years of cooking professionally, just about every time I walked into a new kitchen, it was a Mexican guy who looked after me, had my back, showed me what was what, was there - and on the case - when the cooks more like me, with backgrounds like mine - ran away to go skiing or surfing - or simply 'flaked.' I have been fortunate to track where some of those cooks come from, to go back home with them. To small towns populated mostly by women - where in the evening, families gather at the town's phone kiosk, waiting for calls from their husbands, sons and brothers who have left to work in our kitchens in the cities of the North. I have been fortunate enough to see where that affinity for cooking comes from, to experience moms and grandmothers preparing many delicious things, with pride and real love, passing that food made by hand, passed from their hands to mine.In years of making television in Mexico, it's one of the places we, as a crew, are happiest when the day's work is over. We'll gather round a street stall and order soft tacos with fresh, bright, delicious tasting salsas - drink cold Mexican beer, sip smoky mezcals, listen with moist eyes to sentimental songs from street musicians. We will look around and remark, for the hundredth time, what an extraordinary place this is.The received wisdom is that Mexico will never change.
That is hopelessly corrupt, from top to bottom. That it is useless to resist - to care, to hope for a happier future.But there are heroes out there who refuse to go along. On this episode of 'Anthony Bourdain Parts Unknown,' we meet a few of them. People who are standing up against overwhelming odds, demanding accountability, demanding change - at great, even horrifying personal cost.This show is for them. ❚Share Share This Facebook Twitter Google+ Linked In.
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BackgroundBrassica napus is the third leading source of vegetable oil in the world after soybean and oil palm. The accumulation of gene sequences, especially expressed sequence tags (ESTs) from plant cDNA libraries, has provided a rich resource for genes discovery including potential antimicrobial peptides (AMPs). In this study, we used ESTs including those generated from B. Napus cDNA libraries of seeds, pathogen-challenged leaves and deposited in the public databases, as a model, to perform in silico identification and consequently in vitro confirmation of putative AMP activities through a highly efficient system of recombinant AMP prokaryotic expression. ResultsIn total, 35,788 were generated from cDNA libraries of pathogen-challenged leaves and 187,272 ESTs from seeds of B. Napus, and the 644,998 ESTs of B.
Napus were downloaded from the EST database of PlantGDB. They formed 201,200 unigenes. First, all the known AMPs from the AMP databank (APD2 database) were individually queried against all the unigenes using the BLASTX program.
A total of 972 unigenes that matched the 27 known AMP sequences in APD2 database were extracted and annotated using Blast2GO program. Among these unigenes, 237 unigenes from B. Napus pathogen-challenged leaves had the highest ratio (1.15%) in this unigene dataset, which is 13 times that of the unigene datasets of B. Napus seeds (0.09%) and 2.3 times that of the public EST dataset. About 87% of each EST library was lipid-transfer protein (LTP) (32% of total unigenes), defensin, histone, endochitinase, and gibberellin-regulated proteins.
The most abundant unigenes in the leaf library were endochitinase and defensin, and LTP and histone in the pub EST library. After masking of the repeat sequence, 606 peptides that were orthologous matched to different AMP families were found. The phylogeny and conserved structural motifs of seven AMPs families were also analysed. To investigate the antimicrobial activities of the predicted peptides, 31 potential AMP genes belonging to different AMP families were selected to test their antimicrobial activities after bioinformatics identification.
The AMP genes were all optimized according to Escherichia coli codon usage and synthetized through one-step polymerase chain reaction method. The results showed that 28 recombinant AMPs displayed expected antimicrobial activities against E. Coli and Micrococcus luteus and Sclerotinia sclerotiorum strains. BackgroundGene-encoded anti-microbial peptides (AMPs) are widespread in nature, and are essential lines of host defence against pathogens. These peptides are evidently less susceptible to bacterial resistance than traditional antibiotics, and could form the basis for a new class of therapeutic agents. As eukaryotes, plants have innate AMP defense that usually consists of small Cys- or glycine-rich peptides that are effective against a variety of pathogens. The main classes of AMPs are represented by the alpha/beta-defensins, lipid-transfer proteins (LTP), thionins, cyclotides, snakins, and hevein-like proteins according to their amino acid sequence homologies.
Interestingly, a series of novel plant AMPs has been discovered as processed forms of large proteins. Plant AMPs provide novel strategies not only in therapeutic use, but can also potentially increase agricultural yields through phytopathogen or pest control.To date, in spite of the increasing number of reported AMPs from plants, developments in gene expression methodologies and computational algorithms lead to new prospective strategies of biomining AMPs in plant systems. Computational and bioinformatics approaches has allowed the application of silico-associated molecular tools aiming to screen and identify novel potential candidates that code for these peptides, starting from substantial amounts of genomics, proteomics, transcriptomics, metabolomics, and other ‘-omics’ data from various cultivated or wild plants.
Expressed sequence tag (EST) databases are increasing in number and size, especially regarding cultivated plants. Currently, more than 63 million ESTs are available through the dbEST entry of GenBank. As expected, crop species have been more frequently targeted for AMP research and application due to the highly available molecular data. Recent bioinformatics analyses of sequenced plant genomes have revealed a previously unrecognized abundance of genes encoding AMPs.B. Napus is one of the most important oil crops worldwide, providing a challenge for understanding innate immune systems and resistances to phytopathogens in Brassicaceae plants.
The large number of EST sequences of B. Napus provides a timely opportunity to discover a complete repertoire of AMP sequences. Candidate AMP-coding genes identified from in silico approaches require further biological validation. In this study, after bioinformatics analysis of potential AMP genes from three different B. Napus EST databases, an effective method has been performed for large-scale validation of the biological activities of these candidate AMP genes. EST-based search for discovery of novel B. Napus AMPsIn this study, a general search aiming to identify AMPs was performed on three different B.
Napus EST databases including: 136,202 ESTs generated from immature seeds of two rapeseed lines ; 35,788 ESTs generated from an cDNA library which was constructed from mixed mRNAs of B. Napus leaves inoculated with S. Sclerotiorum or treated with chemicals benzothiadiazole (BTH), methyl jasmonate (MeJA), or oxalic acid (OA, a toxin and pathogenicity factor produced by S. Sclerotiorum); 643,944 ESTs downloaded from the EST database of PlantGDB (refreshed until Oct-1-2009). After screening for low-quality DNA and trimming the vector sequences, the three EST datasets were clustered and produced unigenes using CAP3 program respectively (Table ).
To identify novel AMP genes in the B. Napus, all the known AMPs from the AMP databank (APD2 database, including 1199 known AMPs sequence, ) were individually queried first in the three unigene datasets using the BLASTX program (Additional file -blast result). A total of 237 unigenes from B. Napus leaves that were matched to the known AMP genes had the highest ratio (1.15%) in this unigene dataset, which is 13 times that of the unigene datasets of B. Napus seeds (0.09%) and 2.3 times that of the public EST dataset (Table ).
Analysis of ESTs related to AMPA total of 972 unigenes that matched the 27 known AMP sequences were extracted and annotated with gene ontology (GO) terms and the BLAST result against the Swissprot database with an E value cut-off that was equal to or less than 10 −5 using Blast2GO program (Additional file -blast2go result). Among these unigenes, roughly 90% of each EST library was LTP (32.0%), defensin (15.7%), histone (15.7%), endochitinase (13.5%), and gibberellin-regulated protein (10.5%) (Additional file -blast2go result). The most abundant unigenes in the leaf library were the endochitinase and defensin genes, which were different from LTP and histone in the pub EST library (Table ).
Phylogeny and motif analysis606 potential AMPs were aligned via ClustalW to construct the unrooted phylogenetic tree using the maximum-likelihood algorithm with 1000 bootstrap replicates (Fig. ). Six groups of known AMP families were found, including the most abundant types (39.1%) LTP, defensin-like peptides (DLPs), snakin (extracted from gast1 genes), hipposin (histone-derived AMPs, HDAPs), hevein (extracted from endochitinase genes), and thionin.
46 peptides belong to other unknown families are also found in B. Phylogenetic relationship of all potential AMPs. A multiple sequence alignment of 606 potential AMPs was used to calculate a matrix with the genetic distances for each pair of the sequences.
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Based on this matrix, successive clustering of lineages was done to construct the unrooted phylogenetic tree of all potential AMPs gene using the maximum-likelihood algorithm with 1000 bootstrap replicates. Only branches supported by a bootstrap value of at least 50% are indicated. Tree was generated using JalView. The tree includes 606 sequences, and therefore only major nodes and global clusters are depicted.
The labeling of the subfamilies is based on the location of AMPs that have experimentally confirmed functionEach of the resulting rough set of AMPs family sequence subgroups was separately realigned via ClustalW and visualized using JalView. Defensins are small (5 kDa) cysteine-rich peptides and consists of a cystine-stabilized αβ fold, in which the buried hydrophobic core is formed by four disulfide bridges that link pairs of the eight conserved cysteines (Cys1 through Cys8).
The defensin-like peptides (DLPs) of B. Napus have four distinct clusters (Additional file: Figure S1). All clusters have eight conserved Cys residue motifs forming four disulfide bonds. The evolutionary history of one DLP cluster candidate was reconstructed with MEGA5 at the protein level (Fig. ).
Multiple alignment of this DLP cluster revealed that the consensus pattern C-X5-C-X3-C-X9(10)-C-X6-C-X-C in defensin domain were highly conserved as typically observed in 1TI5 (PDB) from Vigna radiata. Comparison of amino acid sequences of some defensin-like.
Evolutionary relationship is depicted left. Linked bars representing the disulfide bonds arrangement denoted by: C1-C8; C2-C5; C3-C6; C4-C7. Blue helix and arrow respectively represent alpha-helix and beta-strand, which are extracted from the model structure of PDB:1N4N. PDB:1GPS (γ-1-P thionin: Triticum aestivum); PDB:1GPT (γ-1-P thionin: Hordeum vulgare); PDB:1JKZ (Defensin: Pisum sativum); PDB:2GL1 (Defensin: Vigna radiata); PDB:1TI5 (Defensin: Vigna radiata); PDB:1MR4 (Defensin: Nicotiana alata); PDB:1N4N (Defensin: Petunia x hybrida).
Numbers at the base of each clade correspond to bootstrap means at 1000 replicationsLTPs are stabilized by eight conserved Cys residues forming four disulfide bonds, and have a potential glycophosphatidylinositol modification site and a defined number of residues between each two of the eight conserved Cys residues –. LTPs are categorized into five types or clades by comparison of the LTP genes within and among individual plant species. Type I LTPs and their derivatives are the basic and ubiquitous clades. The major LTPs in B.
Napus belong to LTP type I (roughly 90%), with a hydrophilic residue such as arginine, glutamate, or lysine at the × position of CXC motif (C6-X-C8 motif) (Additional file: Figure S2). Snakin amino acid sequence alignments (Additional file: Figure S3) indicate that in addition to the 12 characterized Cys motif, the arginine, valine, and proline residues are highly conserved throughout the family ,. Napus hevein-like peptide sequences are about 30 amino acids to 45 amino acids long and possess eight unique Cys in a chitin-binding peptide with several strictly conserved residues, serine, and two glycines (Additional file: Figure S4) ,. Napus thionin has six conserved Cys residues, as well as conserved proline and glycine residues (Additional file: Figure S6) ,.
A variable number of Cys residues that helped stabilize conserved scaffolds through disulfide bond formation were found. Napus Hipposin (Histone H2A-derived AMP) sequence had a proline hinge without Cys residues (Additional file: Figure S5) –. In addition, a type of proline-and glycine-rich peptide was found, which was similar to animal AMPs and has not been previously reported (Additional file: Figure S7).
Production of fusion proteins with AMPs via a fusion partner EDDIETo investigate the antimicrobial activities of the predicted peptides, 31 potential AMP genes that belong to different AMP families were selected to test their antimicrobial activities after bioinformatics identification (Additional file -sequence). The AMP genes were optimized according to E. Coli codon usage and synthetized through one-step polymerase chain reaction (PCR) technique (Fig. ). To recover their original activities without additional amino acid residues, each PCR production of AMPs genes was cloned into a unique vector with EDDIE as a fusion partner via an in vivo recombination strategy. PCR production of AMP genes prepared for expression vector construction.
Lane M: molecular mass makers; Lane 1–31: PCR production of AMP genes from Bn1 to Bn31The pET30a/His-EDDIE-AMP plasmids were transformed into the expressing host and cultured under optimized conditions. After the induction by isopropyl β-D-1-thiogalactopyranoside (IPTG), the expression of His-EDDIE-AMP proteins was analyzed using SDS–PAGE (Fig. ). Fusion proteins of about 20 kDa represented the majority of the insoluble components in the cell lysates. The recombinant His-EDDIE-AMPs were produced as inclusion bodies because of the properties of the fusion partner. Generation of AMPs and their activity testPurified His-EDDIE-AMP inclusion bodies were diluted in optimized refolding buffer.
After in vitro refolding, the fusion partner was released from the C-terminal end of the autoprotease through self-cleavage, leaving the AMPs with an authentic N terminus. To examine the antimicrobial activities of the recombinant AMPs, the purified supernatants were analysed using radial diffusion assay. In Fig., a total of 28 recombinant AMPs were clearly bioactive and significantly effective in destroying the sensitive strains (Additional file -activities test), but no inhibition zones were seen around the negative control spots. Among these AMPs, three AMPs have only antimicrobial activities to Gram negative strains and other three AMPs have only antimicrobial activities to Gram positive strains. Bn19 have the highest activities to Gram positive and negative strains among these 28 AMPs. Detection of the antibacterial activities of candidate antimicrobial peptides against E.
R: refolding buffer; a antimicrobial activities assay against E. Coli; b antimicrobial activities assay against M. Luteus; the number in plate from 1 to 31 indicated the name of AMPs from Bn1 to Bn31. The letter “R” in the center of plate indicated the refolding buffer as the controlThe antifungal activity assay for 28 recombinant purified AMPs with antibacterial activities to Gram positive or negative strains was carried out with S.
Sclerotiorum strains, a main agronomically phytopathogen of B. As illustrated in Fig., the growth of the tested filamentous fungal strain was inhibited by 27 AMPs, but it can grow normally with Bn30 and the control refolding buffer.
Detection of the antifungal activities of candidate antimicrobial peptides against S. In the plate containing PDA medium, a mycelial plug was placed in the center. The name of AMPs are indicated upon every plate from Bn1 to PDA medium, a mycelial plug was placed in the center. The name of AMPs are indicated upon every plate from Bn1 to Bn31. The letter “ a, b” in the plates indicated wells with the refolding buffer as the control, “ c, d” indicated wells with antimicrobial peptide samplesA total of 28 AMPs, including nine heveins, five defensins, three hipposins, three thionins, three snakins, and four LTPs, showed activities against sensitive strains. A new AMP sequence Bn19 with rich proline residues similar to SP-B (pigs, animals, and AP00889) was classified as a new member of the proline-rich antimicrobial peptide family. DiscussionBy using integrated computational approaches to systemically mine the B.
Napus EST sequence, the first B. Napus AMP repertoire was established.
The members exhibit extensive sequence and structural diversity, and can be distinguished into multiple molecular types. Napus non-redundant AMPs were organized in seven subfamily types, namely, DLPs with Cys-stabilized alpha-helical and beta-sheet (CSab) fold, LTP, thionin, Hipposin, hevein peptide, snakin, and proline- or glycine-rich peptides. Results of the bioinformatics and phylogenetic analyses of the primary structures of the B. Napus candidates support their role as antimicrobials. Furthermore, the Cys motif characteristics and other conserved residues of the main AMP families, such as defensin, thionin, and LTP gene families, were summarized.AMP candidate genes are more substantially abundant in B.
Napus leaves in response to the pathogene ( S. Sclerotiorum) and signaling compounds compared with B. Napus seeds and public EST dataset. The most abundant unigenes in the leaf library are endochitinase-derived hevein and defensin genes, indicating that these genes are necessary in the protection against potential pathogen, defensins and other AMPs often expressed in an organ- or tissue-specific manner.Most of the candidate peptide sequences are new AMP genes that have no proven antimicrobial activities. Flowering plants have been previously shown to possess large gene families encoding LTPs. The results in this research also show that LTP is the most abundance type in B. Next to their inducibility upon pathogen infection, LTP genes are also responsive to abiotic stresses like drought, cold and salt , and perhaps necessary for pollen adherence to the stigma during pollen elongation in some flowering plants.
A defensin from cowpea seeds was assessed on its putative alpha-amylase inhibitory action probably involved in protection against pests.In this study, an efficient method was used for cloning and expressing AMP genes. This expression system is more efficient and is very useful for constructing genome-scale clone resources that facilitate AMP functional analysis. This approach, coupled with bioinformatics analyses of the genome and EST sequence data, will be useful in screening for new AMPs. Such tools may contribute in overcoming problems associated with yield, storage, and processing, thereby improving crop resistance and providing novel strategies not only in medicine but can potentially increase agricultural yields by phytopathogen or pest control.Thirty-one potential AMPs in this study were detected and 28 of them were confirmed to be novel AMPs of B. Napus using antibacterial activity assay. This finding indicates that large AMP genes are still undiscovered. 27 AMP candidate genes were also approved with strong activities to control the main fungal pathogens of B.
These AMPs will be further proved in enhanced crop resistance to pathogen attack through genetic breeding and transgenic manipulation in the future. Coli XL-GOLD (Stratagene, USA) was used as the host for subcloning and plasmid amplification.
Coli BL21 (DE3) was used as the host for expressing the recombinant protein. Coli ATCC2592 and M. Luteus ACCC11001, S. Sclerotiorum were used as indicators in the antimicrobial assay for the antimicrobial peptides.
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PET30a (Novagen, Madison, WI, USA) was used as a vector construction and recombinant protein expression plasmid. The restriction enzymes NdeI and SalI were purchased from Takara (Dalian, China). Database searches and peptide predictionThe B.
Napus ESTs used for the prediction of AMP genes were collected from three different EST data sets, namely, data for assembled unigenes (the PUT, PlantGDB-generated Unique Transcript) downloaded from the PlantGDB (refreshed until Oct-1-2009), B. Napus seed ESTs from B.
ZY036 (high-oil contents, HO) and B. 51070 (low-oil contents, LO), by 454 sequencing (2 weeks after flowering) , and B. Napus leaf ESTs generated from mixed mRNAs of B. Zhongshuang9 leaves inoculated with S. Sclerotiorum or treated with chemicals benzothiadiazole (BTH), methyl jasmonate (MeJA), or oxalic acid (OA, a toxin and pathogenicity factor produced by S. Sclerotiorum).The strategies for gene discovery used in this study are provided in the supplementary information (see Additional file ). Database searches were conducted using methods modified from several recent publications –.
The programs BLASTP and BLASTX were used to mine ESTs encoding putative B. Napus peptide precursors via queries using known AMP sequences.
All candidate nucleotide sequences were translated to amino acids using getORF. All hits were fully translated and manually checked for homology with the target query. Evolutionary analysisThe retrieved sequences were aligned to one another with ClustalW (Version 1.82) , and gapped positions were omitted from subsequent analyses.
A multiple sequence alignment of 606 potential AMPs was used to calculate a matrix with the genetic distances for each pair of the sequences. Based on this matrix, successive clustering of lineages was done to construct the unrooted phylogenetic tree of all potential AMPs gene using the maximum-likelihood algorithm with 1000 bootstrap replicates. Only branches supported by a bootstrap value of at least 50% are indicated.
Tree was generated using JalView. Each of the resulting rough set of AMPs family sequence subgroups was separately realigned via ClustalW and visualized using JalView. Construction of the AMP expression vector with EDDIE as a fusion partnerA total of 31 new potential AMPs were selected from different AMP families to determine their activities against bacteria. The AMP sequences were optimized according to E. Coli codon usage (Additional file -sequence). Two, four, or six primers were used to synthesize each AMP gene in a one-step PCR (Additional file -primers). The PCR reaction was performed for 25 cycles, each cycle consisting of 30 s at 94 °C, 30 s at 62 °C, and 7 min at 72 °C.
Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in E. Finger pro 9 03 windows movie. White colonies were selected and then sequenced to ensure that the coding sequence was appropriate. The resulting plasmids were named pET30a/His-EDDIE-AMPs, respectively.
Expression and purification of fusion proteinThe pET30a/His-EDDIE-AMP plasmids were transformed into the expression host E. Coli BL21 (DE3) (Novagen, Madison, WI, USA). A colony was used to inoculate 50 mL of LB (1% Bacto-tryptone, 0.5% yeast extract, and 8 mM NaCl) medium supplemented with 50 μg/mL kanamycin, and grown overnight at 37 °C in a shaking incubator. The fully grown culture was mixed with 1 L LB medium with the same antibiotics the following morning. The culture was grown at 25 °C, and IPTG was added to a final concentration of 1 mM when the OD 600 reached 0.5. The culture was harvested 5 h later, and the cells were washed and resuspended in 30 mL phosphate-buffered saline (NaCl 137 mM, KCl 2.7 mM, Na 2HPO 4 4.3 mM, and KH 2PO 4 1.4 mM; pH7.2–7.4).
The cells were lysed through freeze-thawing, and DNA was fragmented through ultrasonication. The insoluble inclusion bodies were isolated by centrifugation at 14,000 x g at 4 °C for 30 min. The pellets were washed three times with washing buffer (10 mM Tris/HCl, pH7.6; 200 mM NaCl, 2 mM 2-mercaptoethanol, and 1% Triton X-100) and then solubilized in the denaturation buffer (8 M urea; 20 mM Tris–HCl, pH7.6; and 5 mM 2-mercaptoethanol). Antimicrobial assaysPurified His-EDDIE-AMPs inclusion bodies were refolded by rapid 1:50 dilution in an optimized refolding buffer (500 mM NaCl, 20 mM Tris, 2 mM EDTA, 5% glycerol, 10 mM DTT, 0.01% Tween-20, pH 7.5) and incubated at an appropriate temperature without stirring. During refolding, EDDIE restored its correct conformation and self-cleaved at the specific sites, releasing AMPs from the fusion bodies. Renatured protein solution was then clarified by centrifugation at 15,000 x g at 4 °C for 30 min.
The insoluble sample was removed by filtering through a 0.45 μm membrane, leaving the AMPs in the supernatant. The supernatants were transferred to a Ni-NTA His-bind column for purification.Standard SDS–PAGE (12% gel) was used to assay the fusion proteins. The band density was analyzed using a GEL-DOC 2000 gel documentation system (BIO-Rad, Hemel Hempstead, UK).
Quantity One software version 4.4.0 was used to determine the fraction of the target protein. EDDIE protein was quantified using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA).The antimicrobial activities of recombinant AMPs were detected using a radial diffusion assay. Coli ATCC2592 and M. Luteus ACCC11001 were grown on the mid-logarithmic phase and washed. Approximately 2 × 10 6 cfu/mL bacteria were incorporated into a thin (1.2 mm) agarose underlay gel containing 1% ( w/ v) agarose.
Holes with 3.5 mm diameter were punched into the solidified agarose and were filled with 100 μL of AMP sample. After the plates were incubated at 37 °C for 12 h, the diameter of the clear zone surrounding each well was measured to evaluate the antimicrobial activity. The refolding buffer was used as negative controls. The above assays were performed in triplicate.The antifungal activity of the purified products was assayed using an ultra-sensitive radial diffusion method on thin potato plates (200 g potato, 20 g glucose, 15–20 g agar powder, 1 L double-distilled water) seeded with filamentous fungi. Briefly, 9-cm plates were poured with an underlay potato medium, and S.
Sclerotiorum on a 1-cm diameter potato medium were seeded on the center of the plates, which were then incubated at 22 °C until hypha grew to 2 cm in diameter. Two hundred microliters of the test sample and negative control (refolding buffer) was placed beside the filamentous fungi, and the plates were incubated at 22 °C for 72 h; the size of the clear area around the filamentous fungi was then measured. Additional files(85K, xlsx)Blast result of four B. Napus ESTs dataset against to known AMPs. (XLSX 85 kb) (111K, xlsx)The annotation with Gene ontology (GO) terms results of unigenes which matches to AMPs and the blast result against to the swissprot database with an E value cut-off equal or less than 10 −5 using Blast2GO program. (XLSX 110 kb) (3.7M, zip)Figure S1-S7.
Multiple sequence alignments of different AMPs families. Each of the resulting rough set of AMPs family sequence subgroups was separately realigned via Clustal W and via Jalview. S1: defesin family; S2: LTP family; S3: snakin family; S4: hevein family; S5: hipposin family; S6: thionin family; S7: unknown family. (ZIP 3830 kb) (13K, xlsx)Thirty-one Sequences of the AMP candidate for activities conform. (XLSX 13 kb) (9.7K, xlsx)Thirty-one recombinant AMP activities test for Gram + and Gram- strain halo diameter. (XLSX 9 kb) (43K, doc)Strategy of database searches of putative B. Napus antimicrobial peptides.
(DOC 43 kb) (14K, xlsx)The PCR primers used in this work. (XLSX 13 kb).
Tao Ke and Huihui Cao contributed equally to this work.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsSL, TK conceived the study and served as principle investigator. TK and HC prepared the manuscript. TK, HM and JY analysed the data. TK, HC JinH and FanH carried out the main experiments. XM participated in the design of the study and helped to draft the manuscript.
HC, QN, FLH, XW participated in the sequence alignment and activities analysis. JH, CD and SL participated in revising the manuscript. All authors read and approved the final manuscript.
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