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Titan Blaze 20" Boys' Bmx Bike
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Titan Blaze 20\" Boys' Bmx Bike
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|20" (Age 8-12)
|20\" (Age 8-12)
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|Adjustable Seat,Adjustable Handlebar
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- Walmart will assemble the bike free of charge.by healthyhousewife4/28/2014Was this answer helpful? (0) (0)Was this answer helpful?
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- Yes, the bike will arrive 85% built. You will need to do minor assembly. Install handlebar, pedals, seat, and front wheel. Installation should take you 20-30 minutes if you take your time.by bikeusainc12/5/2013Was this answer helpful? (2) (0)Was this answer helpful?
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Customer Reviews | 5 reviews | 4 out of 5
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Customer review by shelilybee
The whole process with Walmat. com was smooth. Using the site to store deliverywas fast, the bike arrived a day before the time frame began. I was quoted 48 hours to have the bike assembled; it was done the next morning. It was a Christmas present for my 7 year old son. He loves it and has had no trouble handling the bike either. No complaints here.
Customer review by healthyhousewife
My 9 year old son is still in a 20" size bike but had to pedal so much harder to keep up with the family for bike rides. We chose this bike because of the size and 6 gears. It is perfect for him, he loves to be able to switch gears to make it a little easier to go up hills. I would highly recommend this bike to everybody that wants their child to keep up on family rides. I had this bike sent to store and assembled by Walmart. We did raise the handle bars slightly for comfort.
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Learn About Precisely How To Rent Out A House Without As Much Work
A property manager is a professional who comprehends the process of letting a property. They are aware of exactly how to conduct background checks and also work to locate the best renters. They understand exactly how to help make sure the rent will be paid promptly as well as precisely how to manage any kind of scenarios that could occur. In the event there is a concern, they understand the eviction process and also just what can or cannot be done when a renter needs to be evicted. This may be extremely beneficial for an individual who wants to rent their residence but does not comprehend how it all works or who doesn't wish to do the work by themselves.
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I found this timeline about breast changes during pregnancy and knew my fellow first time moms would find it as interesting as I did! It describes everything week by week. Kind of scary, kind of cool, such an adventure we're in for...
Breast changes during pregnancy
EDD for baby #2 October 6, 2015
You mean these things are going to get bigger??
"Some women gain well over 680g (1.5 lbs) in each breast"... what?
I'm already up a cub size and about a qtr I guesstimate
Baby NJ made his debut... I'm in love!
UGH! I am not looking forward to this part I was a 42H before I got pregnant and now I just keep putting off ordering new bras even though these are super uncomfortable and way too small and I can't wait to get home to take them off. I know I need to order a few in a larger size so I am more comfortable but honestly I have no clue what to do when it comes time to order nursing bras they have next to nothing in my size as it is.
Tara (27) & Dan (30) together since 10/01 married 09/11.
2 Angels - Rainbow - Carter Camden surprised us on 5/31/13 6 weeks early
I'm more freaked out about the discharge. I had no idea. Do we need to put some kind of lining in our bras?
EDD for baby #2 October 6, 2015
I'm not looking at this. I'm going to keep my head in the sand until something happens and then ask y'all if it's normal.
I love my As!
I started off a small A, and have only progressed to a "full" A. My boobs haven't changed much at all except my nipples have gotten huge and brown-er. But I honestly didn't think something could be wrong. Well, to the doctor I go.
Great read though! But I an curious why they showed an image of expressing colostrum at only 16 weeks !?
Jelly. 21. "Supermom."
my breast have also only gotten fuller and the areola bigger and more brown. The veins are also becoming transparent in my breasts. Is it really that early, 16 wks, that you can express colostrum!?
There was actually a post on the July board about waking up with colostrum crusties. From what I have read, and personally sense/observe, I've been gradually "leaking" tiny amounts that dry up over night and result in the crusties. Some of the women have more extensive leaking, where they get wet spots on their shirts or sheets or bras. Fortunately, I don't have that much leakage and it's only really enough to cover the nipple (bugged me our since I have nipple rings and thought I had an infection). Some lucky women NEVER leak. If you prod/pole/squeeze your nips, you may be able to express some colostrum on your own. Otherwise it'll happen naturally.
Hope that helps!
Jelly. 21. "Supermom."


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The first Chia planter, "Chia Guy," was created on Sept. 8, 1977.
The first real Chia "pet," designated as a "ram," came out in 1982, and launched the product into the national consciousness.
The product's second wave included a kitten version.
Shockingly, it took until 2000 for licensed "character" Chias to be introduced: Elmer Fudd, the Tasmanian Devil, and Tweety Bird were the first wave.
Naturally, Chia Mr. T wasn't far behind.
Chia Homer Simpson followed in 2002.
Chia Obama was introduced in 2008; in 2009, the company introduced a smiling "happy" Chia Obama. (The initial model was deemed "determined" Chia Obama.)
Chia SpongeBob debuted in 2009, a full-figure model (as opposed to just a head).
Chia Gnome came out in 2012.
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Palo y a la bolsa is a music and image live project from Argentina. The band plays at the same time that the animations are created and projected. All real time. No computers involved.
The trailer includes images from 4 songs of the show:
Hawai-Bombay: a composition for three guitars, a singer, and a indoor washing line with some towels and cheerleader poms.
Fumito Ueda: a composition for two guitars, keyboards violin and a ps2 -using the game Shadow of the Colossus-
Pecera: composition for two guitars, keyboards, viola and a fishbowl with several liquids and christmas ligths.
Yonqui poor: composition for three guitars, a glass, water, oil, poms and a variety of led ligths.
All the trailer images were recorded on the rehearsals for the show Palo y a la bolsa is preparing for march. The show will be recorded and edited as a dvd of the live performance.
The trailer´s song is "Juguete", also by Palo y a la bolsa.


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Westside Film, Food and Friends Message Board › Meetup details changed: WHAT MAISIE KNEW @ THE LANDMARK, SAT. MAY 25, 5:05 P.M.
Santa Monica, CA
I've updated this Meetup. For more details, see the full listing:
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| Guest chapter 19 . 6/25/2015
This was a nice discourse from the usual buffy fics too bad it seems a long time coming
| Guest chapter 9 . 4/3/2015
Well, most people can't separate Ranma from his curse. But I doubt he would find himself in China if he ended up being taken in by the Hino Shrine, and that direction seems the most interesting.
| erik chapter 19 . 3/10/2015
No nothing about either of these genres (Ranma / Sailor Moon) but I love what you have done with them.
| cko2 chapter 19 . 1/13/2015
Some very amusing story takes in this section and others down right laughing my butt off at.
| Grizzmon chapter 19 . 1/12/2015
This should be its own fic.
| Grizzmon chapter 18 . 1/12/2015
Well, looks like Ranma will get another unwanted fiancee.
| Grizzmon chapter 17 . 1/12/2015
I love this!
| uo-chou chapter 19 . 12/3/2014
Good work, kimosabe!
| Chief Minion chapter 19 . 9/9/2014
I love this story series.
| KrisB-71854 chapter 19 . 9/2/2014
Hmm, so is Ranma setting Tofu up with Kasumi or is he safe guarding Kasumi for his own purposes? I love how she adores his house hold remodeling.
The cult thing at the end is rather funny and actually makes sense.
| KrisB-71854 chapter 18 . 9/2/2014
I had to look up the characters involved. You know funnily enough, I think Ranma will still come across as the Hero to the public while Triana seems to be The Villain.
They'll have the entire anti stereotype thing going for them. Let's face it Ranma will almost always act like a hero except that in his day to day life and his various rivals coming after him he just so happens to cause or be the root of tons of Chaos.
You know this thread deserves tons more development. I can see "everyone" in the scene thinking that Triana and Ranma make a cute arching couple. Instead of various heroes breaking into their arch relationship though, they'd have his normal rivals and hmm, (ex) fiancees trying to jump into things.
I can see Ranma and Triana playing by the rules and having fun until his normal psycho stalkers start trying to kill her off. ;) Oddly, due to the rules of his relationship, he would than be authorized extreme force on the various folks that tried to harm his arch. I'd love to see what magic that she'd unleash on the various folks trying to harm him though. I want a scene where P-chan tries to peek at her and unlike Akane, she gets Ranmas hint and tosses P-chan into her closet.
I want to see Ranma "accidentally" recruit Nabiki as his personal spy mistress and accountant. I could only imagine the fun things that you could do with Nabiki not realizing that she has joined the Guild and also having various minions protect her or have her own arch boyfriend. ;) Nah, Nabiki is the type that would be a national level boss within 3-6 months without trying.
| ranma hibiki chapter 19 . 9/1/2014
*falls over laughing* really,this is gold mine of comedy!
| ranma hibiki chapter 17 . 9/1/2014
i'm not sure which is more hilarious..setsuna trying to match up ranma, or a "Evil Guild" taking in said martial artist who thinks it's sort of like a college for those who don't do well with books
keep up the good work!
| dialNforNinja chapter 19 . 8/27/2014
Kasumi considering Indiana Jones type traps a fun work-out routine and Tofu being set up to drop himself into her bed gift-wrapped is what really makes this series of snips sing, I have to say. The Guild of Calamitous Intent really does seem like a good match for Ranma, if he's been stirred upenough to be a more proactive about pursuing goals of his own - most of the time he comes off as rather passive and reactive, not really interested in anything beyond his next meal and practicing The Art without even looking for new challenges, just waiting for them to appear. Wonder of wonders, Genma might actually be onto something with calling him a lazy student lacking in dedication...
| Guest chapter 18 . 8/23/2014
Type your review for this chapter here...


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It could take a month, but Anna Gladstone of Hollywood Hills put a deposit down on a Prius at Toyota of Hollywood. The gas-electric car averages 46 miles per gallon. Gladstone is canceling the lease on her Volvo Cross Country.Allen J. Schaben, Los Angeles Times
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What Does The End Of The Recession Look Like?
I thought maybe I found a bit of good news on the economic front in Foreign Policy. They asked a series of experts what the end of the recession might look like:
Their consensus? The end of the recession looks much like the recession itself. We are at the end of the beginning of bad times.
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Alabama-Notre Dame: Oh My!
Here's how I feel about Alabama playing Notre Dame for the national college football championship:
You're dining at a restaurant—a pretty good restaurant—and you've had the bread and the fancy olives and the beet salad (again!), and the lamb chops were tasty and substantial, and there were also a few handfuls of sweet-potato fries off of somebody else's plate, and you know you should call it a night, when the waiter appears at the table and asks, calmly but firmly, if anyone would care for dessert. And before you have time to say no, the waiter is ticking off the establishment's desserts, the last of which is an astonishing confection called The Sinful Devil's Deep-Fried Doughnut Straight-to-Rehab Sugar Bender. And The Sinful Devil's Deep-Fried Doughnut Straight-to-Rehab Sugar Bender is something in which handmade doughnuts are deep-fried by sugar elves in a sugar barrel, then wrapped in a chocolate mattress and baked into the floorboards of a gingerbread house, where they rest for a thousand years until they are excavated and bathed anew in honey and butter and sugar. Somewhere along the way, a chocolate truffle, a peanut-butter cup and a wheelbarrow of whipped cream join the party. And this dessert sounds at once like the best thing you've ever heard of and also totally insane, but before you can resist and offer a meek, No, that's OK, we'll take the check, someone else at the table interrupts and announces, "Sounds amazing. We'll take five."
That's how I feel about the Crimson Tide facing the Fighting Irish for the national championship. It's delicious and too much. Power versus power, legends atop legends, history upon history, with big ladles of obnoxiousness and hysteria. I fear it and can't wait at the same time. Alabama vs. Notre Dame is the The Sinful Devil's Deep-Fried Doughnut Straight-to-Rehab Sugar Bender. À la mode.
Notre Dame was already in this game. Alabama got in Saturday by beating Georgia in the Southeastern Conference championship game in Atlanta. Georgia almost won, but in the closing minutes of the contest, the Bulldogs fell under the impression they were playing a leisurely game of Ultimate Frisbee, and time ran out before they had a chance to score and ruin everything. I know everybody feels bad for Georgia, and Georgia played great, but those Bulldogs were messing around with the desired narrative. The Crimson Tide versus the Fighting Irish was the dreamy matchup (non-Georgia football-loving) people wanted to see. Sorry. That fact is both annoying and true.
And so now here it comes, on Jan. 7, in a concrete pit in Miami Gardens, Fla., where the NFL Dolphins play something that swings wildly between football and running around the backyard with plastic mop buckets on their heads. I am going to spare you the rant in which I howl that this Notre Dame-Alabama game, like every big college-football bowl, should really be happening on New Year's Day, and not a week later, on a Monday, like a PTA meeting. College football long ago sold the remaining threads of its soul to television and they have quite nearly ruined the whole thing under a tacky pile of sponsorship and a BCS system which makes sense to about 2.7 humans. The coming playoff system will only drive everyone battier. Just wait.
This year, however, there's not a ton of confusion. Notre Dame is ranked No. 1. Alabama is No. 2. I know Ohio State fans are cranky because the on-probation Buckeyes finished undefeated but postseason ineligible, and I will agree to be as sad as I can possibly be for an institution under the aegis of Urban Meyer. Ohio State advocates are now sentenced to a long life of cocktail parties in which they will overhear people talking about the hallowed national-championship game between Alabama and Notre Dame, and feel obligated to jump in to remind everyone of the underappreciated 12-0 Buckeyes, who could have beaten either, except for some lame-o NCAA sanction involving a tattoo parlor. And then the Notre Dame-Alabama fans will say, "Yeah but Ohio State gave up 49 points to an Indiana team that finished 4-8" and then the Buckeye fan will claim Notre Dame got lucky versus Pittsburgh and note that Bama lost to Texas A&M and the whole thing will go around and around and sound as tedious 40 years from now as it does today.
Alabama and Notre Dame is what we get, and it's not a bad get. Two schools with 25 claimed national championships between them. The Irish and Tide have met only six times in their histories, with Notre Dame winning five. And the legends! There are enough football ghosts between these institutions to produce an epic paranormal event, and be assured that in the coming weeks you will be rendered numb by all of the misty, water-colored memories getting pumped out of South Bend and Tuscaloosa.
I give Alabama a slight edge on the Insufferable Index, not because of its excellent team, but because it plays in the SEC. Half of the pleasure of watching SEC football, it seems, is reminding everyone else how good SEC football is, and, by extension, how bad everyone who doesn't play in the SEC is. There's real truth to the strength of the conference (the SEC has produced college football's last six national champions) but after a while, all that talk about SEC superiority and "big-man football" becomes repetitive and a little unbearable, like the guy who stops by your desk every Monday morning, does unsolicited calf stretches in his khakis and talks about the awesome half-marathon he ran over the weekend. I get it. You guys rule. Please stop.
It's inevitable that this title game will be presented as a referendum on How Good Notre Dame Actually Is. Alabama has won two of the last three national championships, and Notre Dame is the recovery act with something to prove, and you could fuel a rocket to Mars with all of the underestimation of the Fighting Irish. That's fine. That's OK. We will find out soon enough. Alabama and Notre Dame are playing for everything. The Sinful Devil's Deep-Fried Doughnut Straight-to-Rehab Sugar Bender is in the oven. It's ridiculous but irresistible. You probably should take a couple of brisk laps around the block.
Write to Jason Gay at firstname.lastname@example.com
The WSJ projects Team USA to win the overall medal race in Rio—thanks largely to its dominant contingent of female stars.
Perfect, Freaky Olympic Bodies
We focused on four types of athletes—cyclists, weightlifters, swimmers and high jumpers—to imagine just how different 'faster, stronger, higher,' could look.
The Olympic Fantasy Confronts Reality
The scrutiny facing Rio epitomizes the changing perception of the Games.
Brazil Calls in the Military to Win Olympic Gold
Brazil has enlisted athletes in its military to boost the country's chances for gold.
How to Make Olympic Basketball Interesting
Team USA's dominance means the most intriguing thing to watch in Rio is how the subtle differences of the international game
Rio Olympics 2016


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how wonderful, like it due to these beautiful colours and image formating.
→ vote for you
kind regards from Dresden
‣ erzhof
Beautiful colors! Great shot! Greetings from Slovakia
stunning, VOTED Please visit my gallery!
Meine Stimme dafür!
Amazing! :) to drift along in an air balloon, It would be nice to do that one day. A beautiful photo. Regards, L Anita
Charming ...! I like very much and YS! Best wishes, Simona
Photo taken in 2620 Hemiksem, Belgium


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February 2016 - Volume 43 - Supplement 1 2S
June 2014 Supplement, Volume 41, S1
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More STD Prevention for the Money: Maximizing Impact,
Efficiency & Return on Program Investments
Noda, Angel A.; Blanco, Orestes; Correa, Consuelo; Pérez, Lissette; Kourí, Vivian; Rodríguez, Islay
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Pine wilt disease is caused by the pine wood nematode, Bursaphelenchus xylophilus, which threatens pine forests and forest ecosystems worldwide and causes serious economic losses. In the 40 years since the pathogen was identified, the physiological changes occurring as the disease progresses have been characterized using anatomical and biochemical methods, and resistant trees have been selected via breeding programs. However, no studies have assessed the molecular genetics, e.g. transcriptional changes, associated with infection-induced physiological changes in resistant or susceptible trees.
We constructed seven subtractive suppression hybridization (SSH) cDNA libraries using time-course sampling of trees inoculated with pine wood nematode at 1, 3, or 7 days post-inoculation (dpi) in susceptible trees and at 1, 3, 7, or 14 dpi in resistant trees. A total of 3,299 sequences was obtained from these cDNA libraries, including from 138 to 315 non-redundant sequences in susceptible SSH libraries and from 351 to 435 in resistant SSH libraries. Using Gene Ontology hierarchy, those non-redundant sequences were classified into 15 subcategories of the biological process Gene Ontology category and 17 subcategories of the molecular function category. The transcriptional components revealed by the Gene Ontology classification clearly differed between resistant and susceptible libraries. Some transcripts were discriminative: expression of antimicrobial peptide and putative pathogenesis-related genes (e.g., PR-1b, 2, 3, 4, 5, 6) was much higher in susceptible trees than in resistant trees at every time point, whereas expression of PR-9, PR-10, and cell wall-related genes (e.g., for hydroxyproline-rich glycoprotein precursor and extensin) was higher in resistant trees than in susceptible trees at 7 and 14 dpi.
Following inoculation with pine wood nematode, there were marked differences between resistant and susceptible trees in transcript diversity and the timing and level of transcripts expressed in common; in particular, expression of stress response and defense genes differed. This study provided new insight into the differences in the physiological changes between resistant and susceptible trees that have been observed in anatomical and biochemical studies.
Pine wilt disease is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, and was first reported by Tokushige and Kiyohara [1]; this disease threatens pine forests and forest ecosystems around the world and causes significant economic losses [2]. Pine wilt disease is a chronic problem in pine forests (Pinus thunbergii and Pinus densiflora) in Japan, where approximately 40,000,000 m3 of pine forests have been blighted by the PWN [3]. Over the past 40 years, public administration and central, prefectural, and city governments have attempted to stem the spread of PWN and pine wilt disease by controlling the pine sawyer beetle (Monochamus sp.), the vector of PWN, with insecticides and cutting down infected trees. Additionally, national and prefectural forestry institutes have established breeding programs to develop resistant pine varieties. A breeding project to develop pine varieties resistant to pine wilt disease was started in 1978 in western Japan, and related projects were promoted throughout Japan, excluding Hokkaido Island, as the damage spread. In this breeding project, trees are screened for resistance using an artificial inoculation test that follows a strict protocol; during the first breeding program, which ran from 1978 to 1984, 92 resistant P. densiflora individuals were selected from 11,000 candidate trees, and only 16 resistant P. thunbergii individuals were selected from 14,000 candidate trees. The breeding projects continued, and 204 resistant P. densiflora and 90 resistant P. thunbergii individuals were generated. Resistant P. densiflora (n = 92) and resistant P. thunbergii (n = 16) were ranked with regard to resistance (levels 1-5) based on the survival rate of openly pollinated progeny following inoculation; higher survival rates are thought to indicate greater resistance. Average rates of survival of openly pollinated progeny from the selected pines (i.e., resistant trees) were 64% for P. densiflora and 53% for P. thunbergii, which was respectively 16% and 40% higher than for unselected populations [4].
Since the causative pathogen was identified [1], many researchers have characterized the physiological changes associated with progression of pine wilt disease, and by the mid-1990s, more than two thousand papers on the disease had been published [5]. Many symptoms associated with PWN infection, including decreased photosynthesis, denaturation of xylem and cortex parenchyma cells, traumatic resin canal formation, cambium destruction, production of phytotoxic substances and ethylene, reduced water potential and transpiration in leaves and heat pulse velocity have been studied (for review, see reference [6]). Based on the anatomical and biochemical evidence gathered during these 40 years, development of symptoms is thought to occur in two stages, early and advanced stages [6]. In the early stage, a small number of nematodes migrate and symptoms such as necrosis and destruction of cortex and phloem tissue and cambium, destruction of cortex resin canals, formation of wound periderm in cortex parenchyma around resin canals, and ethylene release all occur near the inoculation site. In the advanced stage, ethylene production is enhanced and coincides with the broad destruction of cortex parenchyma, cambial destruction, and cavitation-induced embolism of the tracheids in the xylem. The cavitation-induced embolism causes a decrease in leaf water potential and cessation of photosynthesis. After cessation of photosynthesis, severe symptoms develop rapidly with a burst in the nematode population; this population increase results in wilting and eventual tree death. Resistance against PWN infection depends on stopping the symptoms at the early stage or preventing the progression of infection to the advanced stage. While the physiological changes that occur as pine wilt disease progresses have been characterized anatomically and biochemically, molecular genetic events such as changes in transcript profiles that may be associated with the physiological changes in either resistant or susceptible trees remain poorly understood.
Recently, the gene expression profiles of resistant (resistant variety of P. thunbergii 'Sendai-290'; resistant rank 1) and non-resistant Japanese black pine (P. thunbergii) trees were assessed using the LongSAGE technique on stems collected 3 days after PWN inoculation [7]. The researchers found that catalase and proteins in the dienelactone hydrolase family were highly expressed in resistant trees, but not non-resistant trees, whereas pathogenesis-related (PR)-1, 2, 3, leucoanthocyanidin dioxygenase and cell wall-related genes were expressed at higher levels in the non-resistant trees. Although the study assessed a difference of one time point in the defense responses of resistant and non-resistant P. thunbergii following PWN inoculation, the defense response is continuous, and the differences in resistance and susceptibility do not depend only upon qualitative differences in the activated defense genes, but also on differences in the timing and magnitude of their expression [8]. To characterize the differences in transcript profiles of resistant and susceptible trees as pine wilt symptoms develop, it is necessary to sample resistant and susceptible individuals over a defined time course.
Subtractive suppression hybridization (SSH) is a powerful tool for gene expression profiling that effectively identifies genes differentially expressed under different conditions or in different tissues [9]. This method is relatively simple and easy, it can be used with species for which there is little or no genomic information, and the cDNAs isolated are typically longer than 100 bp and can be effectively annotated using comparative genomics (e.g., BLAST analysis). This method is often used to isolate plant genes specifically expressed in response to pathogen infection and to identify differences in the transcript profiles of infected resistant and susceptible plants [10-14]. SSH selection reduces the cloning of abundantly expressed housekeeping genes or genes commonly expressed in both "tester" and "driver" samples, and therefore normalizes the expressed cDNA profiles during library construction. As a result, SSH selection significantly enhances the chances of cloning differentially expressed genes.
The goal of this study was to identify differences in the transcript profiles of PWN-inoculated P. thunbergii to understand the difference in the defense responses of resistant and susceptible individuals. Three important experimental design elements enhanced the clarity and relevance of our findings: 1) We used the most resistant variety of P. thunbergii, 'Namikata-73'; resistant rank 5. 2) We sampled inoculated trees (both resistant and susceptible) 1, 3, and 7 dpi before any macroscopic changes usually occur; resistant trees were also sampled 14 dpi, when macroscopic changes are usually evident. 3) We used SSH, a powerful approach for identifying differentially expressed genes regardless of their abundance.
Six subtractive libraries constructed from samples taken at three time points, 1, 3, and 7 days dpi, were used to identify genes that are differentially expressed in resistant and susceptible trees as disease symptoms develop following PWN inoculation. In addition, a seventh library contained genes expressed predominantly in resistant trees at 14 dpi compared to susceptible trees at 7 dpi (see Methods). We sequenced nucleotides from the 5'-end of 3,299 cDNA inserts that were recovered in the seven libraries (Table (Table1).1). Insert length varied from 100 to 780 bp; the median length ranged from 366 to 429 bp, depending on the library. The redundancy within each library varied from 3.9% to 27.2% in libraries from resistant trees and from 28.4% to 72.8% from susceptible trees. The obtained non-redundant expressed sequence tags (ESTs) ranged from 351 to 435 in libraries from resistant trees and from 138 to 315 in libraries from susceptible trees. Importantly, the overlap between the two libraries at each time point was extremely low; for example, 0.4% of the cDNA inserts were shared between libraries from resistant and susceptible trees sampled at 1 dpi. Similarly, only 3.7% of the cDNA inserts were shared by the library from resistant trees sampled at 14 dpi and from susceptible trees sampled at 7 dpi. This high level of specificity is expected from efficient SSH procedures.
In order to annotate the putative gene and functional Gene Ontology (GO) categories for the transcripts of each SSH library, we compared non-redundant ESTs of each SSH library with the GenBank non-redundant and EST databases using the search programs blastx, tblastx, and blastn using the Blast2GO program [15] (Additional file 1). The transitional hit rate of the BLAST analysis and the annotation rate per library are indicated in Table Table1.1. For each library, 98% or more of the clones were matched in a BLAST search using the nr and EST databases at the National Center for Biotechnology Information (NCBI). Similarly, 97% or more of the clones in each library were matched in a BLAST search (blastn) using the Dana-Farber Cancer Institute Pinus taeda Gene Index (DFCI PGI, release 8.0). The annotation rates for GO terms resulting from automated and manual annotation varied from 75.45% to 91.00% per library.
Non-redundant ESTs recovered in the seven libraries were classified by function into the three principal Level 1 GO categories: biological process, molecular function, and cellular component. Furthermore, the biological process category was split into 24 subcategories at Level 2, molecular function was split into 61 subcategories at Level 3, and cellular component was split into 39 subcategories at Level 7. The level presented herein corresponds to the depth of hierarchical categories within each principal GO category, and higher levels represent more general classifications. The classification for biological process, divided into 15 major subcategories and 24 lower-level subcategories, is shown in Figure Figure1a.1a. Of the 15 subcategories under biological process, the subcategory of response to stimulus was present at a higher percentage in the susceptible libraries at each time point, and three categories-cell wall organization or biogenesis, immune system process, multi-organism process-were present at a higher percentage in the susceptible libraries than in the resistant libraries at each time point. In contrast, transcripts related to cellular process and metabolic process were present at a higher percentage in the resistant libraries. In addition, the category of response to stimulus was present at a higher percentage even in the resistant libraries at 7 and 14 dpi. The classification for molecular function, divided into 17 major subcategories and 61 lower-level subcategories, is shown in Figure Figure1b.1b. At each time point, susceptible trees expressed more transcripts in the four subcategories of carbohydrate binding, enzyme inhibitor activity, hydrolase activity, and pattern binding than did resistant trees, whereas transcripts related to several molecular binding categories such as ion binding and protein binding and to several molecular activity categories such as oxidoreductase activity and transferase activity were present at a higher percentage in the resistant libraries. The classification for cellular component, divided into 18 major subcategories and 39 lower-level subcategories, is shown in Figure Figure1c.1c. At each time point, susceptible trees expressed more transcripts in the cytoplasmic membrane-bounded vesicle category than did resistant trees. In contrast, the transcripts related to organelle categories such as chloroplast, plastid and mitochondrion were present at a higher percentage in the resistant libraries at each time point.
In order to evaluate the specificity and diversity of the transcripts that were specific in a time course from infected resistant and susceptible trees, ESTs were selected from each SSH library (Table (Table2,2, Additional file 2). The libraries from susceptible infected trees showed very limited transcript diversity and were chiefly composed of PR proteins, such as basic PR-1, PR-2 (beta-1,3-glucanase), PR-3 (class I, IV chitinase), PR-4 (chitinase type I & II), PR-5 (thaumatin-like protein), PR-6 (type II proteinase inhibitor family protein), and antimicrobial peptide. In contrast, the ESTs from resistant trees were more diverse. Transcripts encoding metallothionein-like protein, heat shock protein 70 (HSP70), xyloglucan endotransglycosylase (XET), and cytochrome P450 (CYP450) were discernible in the resistant library at 1 dpi and 3 dpi. The libraries from resistant trees sampled at 7 dpi and 14 dpi had transcripts encoding PR-5, PR-9 (peroxidase), PR-10 (ribonuclease-like), hydroxyproline-rich glycoprotein (HRGP) precursor, and extensin.
To validate the results of the SSH procedures, expression of 16 ESTs recovered in one or more of the seven libraries was assayed using qRT-PCR; samples for the qRT-PCR analysis were collected from resistant and susceptible trees at 0, 1, 3, 7, and 14 dpi. (Figure (Figure2).2). Furthermore, expression of 16 ESTs of mock samples at each time points was also assayed using qRT-PCR to monitor expression changes induced either by PWN infection or by cutting (Additional file 3). The results of the qRT-PCR analysis and SSH were consistent.
Expression of 12 of 16 ESTs was clearly upregulated following inoculation with PWN. Among the upregulated ESTs, expression of putative PR-1b, PR-2, PR-3 (class I & IV chitinase), PR-4, PR-5, PR-6, and antimicrobial peptide was much higher in the susceptible trees than in the resistant trees at each time point. Expression of these ESTs was much higher in susceptible trees at 1 dpi and expression had increased further by 7 dpi. In the resistant trees, expression of these ESTs was at a relatively low level at 1 dpi and 3 dpi, but was abundant at 7 dpi and 14 dpi. ESTs encoding PR-9, PR-10, HRSP, and extensin were discernible in resistant SSH libraries. Expression of PR-9 and PR-10 was much higher in the susceptible trees at 1 dpi and 3 dpi; however, levels of these two transcripts were much higher in resistant trees at 7 dpi, though it was lower at 14 dpi than 7 dpi. HRGP and extensin transcript accumulation in resistant trees was high at 14 dpi, although expression of both ESTs was higher in susceptible trees than in resistant trees at 1 dpi, 3 dpi, and 7 dpi.
Based on qRT-PCR analysis, three ESTs--CYP450, metallothionein-like protein, and XET--were downregulated in both resistant and susceptible trees after PWN inoculation, with greater downregulation in susceptible trees. Transcript levels of these three ESTs decreased in susceptible trees within 1 dpi, and were even lower at 3 and 7 dpi. The levels of these ESTs in resistant trees at 7 and 14 dpi were similar to those in susceptible trees at 7 dpi. The transcript level of HSP70 was lower than the other downregulated genes, and its decrease was more moderate in resistant trees, with an expression pattern similar to that of mock samples.
We sequenced cDNAs in seven SSH libraries to characterize transcriptional differences between resistant and susceptible P. thunbergii trees in response to inoculation with PWN. In susceptible trees, transcript diversity was statistically lower than in resistant trees at the three time points jointly tested, 1, 3, and 7 dpi. In susceptible trees, several transcripts encoding pathogenesis related proteins were present at a higher constitutive level than in resistant trees. In resistant trees at 14 dpi, several transcripts encoding cell wall proteins were identified. The results of the SSH approach were validated by qRT-PCR. We clearly demonstrated that transcript composition, temporal changes, and levels of gene expression involved in the stress/defense response to PWN inoculation in resistant trees differs from susceptible trees.
There was a significant difference in transcript diversity between resistant and susceptible trees after infection with PWN. The percentage of unique sequences in susceptible libraries ranged from 71.59 to 27.17%, whereas the percentage of unique sequences in resistant libraries ranged from 72.82 to 96.11%. Although transcript diversity of the susceptible library at 3 dpi was higher than the susceptible libraries at 1 and 7 dpi, transcript diversity in resistant libraries was higher than susceptible libraries at all time points after PWN infection. According to the GO classification of the differentially expressed transcripts, a large percentage in susceptible trees was involved in stress/defense response categories such as the response to stimulus, multi-organism process, and immune system process within the biological process category and the enzyme inhibitor activity, carbohydrate binding, pattern binding, and hydrolase activity within the molecular function category. Furthermore, a large percentage of transcripts in the cellular component category were in the cytoplasmic membrane-bounded vesicle subcategory. On the other hand, a large percentage of transcripts in resistant SSH libraries was categorized into the cellular process, metabolic process and response to stimulus subcategories of the biological process category and in the ligand binding and transferase activity subcategories of the molecular function category. Many transcripts recovered in libraries from resistant trees were assigned to the cellular component subcategory and further classified into organelle subcategories of plastid, chloroplast, and mitochondrion. The difference in transcript diversity between libraries from resistant and susceptible trees revealed by the GO classification indicated a qualitative difference in the stress/defense response of resistant and susceptible trees to PWN infection.
Resistance and susceptibility do not depend only upon qualitative differences in the activated defense genes, but also on differences in the timing and magnitude of their expression [8]. The gene regulation patterns of pathogenesis related defense proteins such as PR-1b, PR-2, PR-3, PR-4, PR-5, PR-6 and antimicrobial peptide indicated temporal and quantitative differences between resistant and susceptible trees in response to PWN infection. In regulating the plant defense response, most pathogenesis related proteins are induced through the action of the signaling compounds salicylic acid (SA), jasmonic acid (JA), or ethylene [16]. PR-1b, PR-2 and PR-5 genes are SA-responsive genes and also indicators of systemic acquired resistance; additionally, the PR-6 gene is a JA and ethylene responsive gene [17]. Although the relationship between phytohormones and the defense response in the PWN-nematode interaction is not clear from this study, it is interesting that expression of pathogenesis related genes associated with phytohormones such as SA and JA/ethylene and with antimicrobial activities were induced more quickly and to a higher level in susceptible than in resistant trees.
The same three phytohormones, SA, JA, and ethylene, are important for both basal and R-gene mediated defense responses to foliar pathogens and insects. The JA and ethylene signaling pathways seem to work synergistically, whereas the SA and JA/ethylene signaling pathways inhibit each other, and negative cross-talk exists between SA and JA/ethylene signaling pathways [18]. In interactions between Hero A-resistant tomatoes and cyst nematode, expression of SA-responsive genes PR-1 and PR-5 is a hallmark of the resistant cultivar, and expression of JA-dependent PR-6 is higher in the susceptible cultivar, indicating that SA plays some role in the resistance to the nematode and that JA and ethylene in susceptible tomatoes are likely to interfere with the SA-inducible resistance pathway [19,20]. The involvement of SA in resistance and expression of SA-responsive genes in resistant plants has been observed in interactions between other plant species and nematodes (e.g., an Arabidopsis thaliana mutant [21] and a root knot nematode resistant peanut [13]). In our study, the higher induction of both SA-responsive genes such as PR-1b, PR-2, PR-5 and JA/ethylene-responsive genes such as PR-6 in susceptible trees indicates that the defense response mediated by these phytohormones was not very effective in controlling PWN infestations.
We detected notable levels of putative HRGPs, extensin and peroxidase (PR-9) in resistant trees at 7 dpi and 14 dpi, though expression in susceptible trees was higher than in resistant trees at 1 and 3 dpi. Cell wall-mediated resistance is the first line of plant defense against pathogens, and the components of plant cell wall are modified by production of reactive oxygen species (ROS) such as H2O2 during attack by pathogens [22]. The structural cell wall proteins extensin and HRGPs play an essential role in biotic and abiotic stress responses due to their oxidative cross-linking, which contributes to the strength of cell walls and is catalyzed by an oxidizing system based on peroxidase and H2O2 [23-25]. The cross-linking of HRGPs and extensin is involved in cell wall-mediated resistance and has a major role in arresting pathogens at the site of entry [23]; these proteins accumulate in the walls of a number of plant species during interactions with microbes [26]. A number of reports describe the response of plant cell wall HRGPs and extensin to pathogens such as viruses, bacteria, and fungi (for review, see reference [27]). In plant-nematode interactions, high extensin gene expression was observed in the cortical region of tobacco at 7 and 14 days after inoculation with root knot nematodes [28]. In rhg1 resistant soybean, extensin was identified as one of the genes characteristically expressed in syncytia after inoculation with soybean cyst nematode, indicating that altered cell wall composition is important in the defense response [29]. Also, in Mi resistant tomato, extensin and glycosyltransferase may play a role in cell wall synthesis, which is an essential defense against root knot nematode [30,31]. In anatomical studies of PWN infection, Ishida et al. [32] inoculated virulent nematode (B. xylophilus) isolate S6-1 and avirulent nematode (B. mucronatus) isolate B. m to Japanese black pine, and observed accumulation of lignin- and suberin-like substances around the resin canals in the cortex with both nematode isolates at 7 dpi. Kusumoto et al. [33] also inoculated a virulent nematode (B. xylophilus) isolate, Ka-4, to Japanese black pine and found more frequent accumulation of phenolic compounds around the cortex resin canals in resistant trees at 7 dpi and 14 dpi after inoculation with PWN (B. xylophilus), and suggested that this accumulation was a very effective defense against infection due to restricting PWN migration. Although the relationship between HRGPs or extensin and other substances in the cell wall was not examined in this study, it is possible that upregulation of expression of cell wall-related genes such as those for HRGPs or extensin and oxidative cross-linking of these proteins by peroxidases plays a role in the effective defense response of trees resistant to PWN infection at 7 dpi and 14 dpi.
PR-10 was also one of the characteristically significantly upregulated genes in libraries in the 7 dpi subtraction library from resistant trees. Although the biological function of PR-10 remains unclear, many PR-10 genes are upregulated when plants are exposed to abiotic stresses, such as SA, CuCl2, H2O2, cold, darkness and wounding [34], and biotic stresses, such as viruses [35], bacteria [34,36], fungi [37-40] and insects [41,42]. We observed synchronized expression of PR10 with peroxidase in resistant trees, which indicates this gene may be induced by ROS such as H2O2. However, PR10 (CpPRI) acts against a digestive proteinase from the root knot nematode Meloidogyne incognita [43]. Therefore, PR10 may act as a proteinase against cellulases, beta-1,3-glucanase, and pectate lyases secreted from PWN [44-46].
Heat shock protein (HSP) ESTs were characteristically recovered in libraries from resistant trees. In particular, stable HSP70 expression in infected resistant trees was validated by qRT-PCR. HSP family members, which consist of HSP70, HSP60 and HSP90, are required for folding of nascent proteins and intracellular transportation in addition to stress responses, and are collectively called molecular chaperones [47]. In the interaction between soybean and soybean cyst nematode, Klink et al. [48] observed the induction of HSP70 and ROS responsive genes such as lipoxygenase and superoxidase dismutase isolated from 3 dpi syncytial cells showing an incompatible response to soybean cyst nematode infection, and suggested that HSP70 may be involved in maintaining a properly functioning environment for other defense responses. In the present study, it is unclear how HSP70 is involved in the defense response to PWN infection.
Three ESTs--putatively encoding CYP450, metallothionein-like protein, and XET--were detected in resistant SSH libraries at 1 dpi and 3 dpi, which depend on the genes significantly downregulated in susceptible trees at 1 and 3 dpi. In plants, CYP450 monooxygenases play paramount roles in the synthesis of lignin intermediates, sterols, terpenes, flavonoids, isoflavonoids, furanocoumarins, and a variety of other secondary plant products [49]. In conifers, diterpene resin acids are prominent defense compounds against insect pests and pathogens in conifers [50-52], and CYP450s are involved in the formation of a suite of diterpene resin acids of conifer oleoresin; they oxidize abietadienol and abietadienal to abietic acid in vitro and in vivo [53,54]. Keeling and Bohlmann [50,51] indicated that oleoresin may contain specific components that are toxic or deterrent to insect herbivores or may affect adults or broods physiologically and thus prevent successful colonization or reproduction. The downregulation of CYP450 observed in the present study may cause a reduction in diterpene resin acids in pine trees infested with PWN. Consequently, PWN may expand its invasion and habitat, and the rapid reduction in CYP450 expression in susceptible trees may trigger PWN expansion.
Metallothioneins are involved in ROS scavenging, and in rice, downregulation of metallothionein expression is observed during the oxidative burst phase in elicitor-treated cells, and metallothionein expression is important for defense signaling [55,56]. The metallothionein expression we observed indicated that ROS accumulation and defense signaling may have been induced by 1 dpi in susceptible trees infected with PWN, whereas it may not have been induced much in resistant trees 3 dpi later; this provides evidence for rapid induction of defense response genes such as those encoding pathogenesis related proteins in susceptible trees.
XET action is thought to regulate wall loosening during turgor-driven expansion by rearranging load-bearing xyloglucan cross-links between cellulose microfibrils, and its activity and expression have been detected in growing tissues [57-64]. We found that XET was downregulated in both resistant and susceptible trees following PWN infection, and its regulation was induced relatively early, by 1 dpi. These findings suggest that the expansion of cell walls in xylem, phloem or both is inhibited by the downregulated XET during PWN infection; alternatively, the cell wall may be immobilized by the cross-linking of HRGPs or extensin.
In pine-nematode interactions, Myers [65] and Futai [66] suggested that invasion and rapid migration of a few mobile parasites through tree tissues invokes an innate hypersensitive reaction such as death of the parenchyma, production of toxins, and leakage of oleoresins and other material into tracheids. Furthermore, the population of PWN spreads throughout the whole body, and a series of hypersensitive reactions eventually leads to tree death in susceptible pine species. In this study, defense response genes, antimicrobial peptide, SA-responsive genes such as PR-1b, PR-2, PR-5 and JA/ET-responsive genes such as PR-6 were induced more quickly and to a higher level in susceptible than in resistant trees. These defense responses in susceptible trees would not be effective in controlling PWN nematode infestations, and defense signaling induced within the tree may then induce a series of hypersensitive reactions through cellular interactions that subsequently lead to death, as Myers [65] and Futai [66] suggested. In contrast, a moderate hypersensitive reaction along with upregulation of pathogenesis related genes followed by upregulation of cell wall-related genes contributing to the strength of cell walls would be a very effective defense against PWN infection, because these events might restrict PWN migration.
This is the first study to assess the difference in EST transcript diversity of activated defense genes and differences in the timing and magnitude of expression of these genes between resistant and susceptible P. thunbergii trees following PWN inoculation. In susceptible trees after PWN inoculation, pathogenesis related genes and antimicrobial-related genes were rapidly induced to high levels within 1 dpi; this finding indicated that a hypersensitive reaction-like response was induced in susceptible trees and supported the hypothesis of Myers [63] and Futai [64]. In contrast, a moderate defense response mediated by pathogenesis related protein expression followed by significant upregulation of cell wall-related genes induced by ROS was a very effective defense against PWN infection. The ESTs generated in our study will certainly advance understanding of defense mechanisms against PWN at the transcriptional level in other varieties or other Pinus species.
A resistant tree of 'Namikata 73,' which is the most highly resistant variety of the 16 resistant varieties selected out of 15,000 individuals from 1990 to 1998 [4], was planted in the Forest Products Research Institute, Forest Tree Breeding Center (FFPRI-FTBC) in Ibaraki, Japan. A susceptible tree of the variety 'Kataura 1,' selected as a plus-tree for growth traits, was also in the FFPRI-FTBC. Both clones were grafts obtained from the original trees at the FFPRI-FTBC in 2005. The PWN used in this study was the Ka-4 isolate, which has been used in pine wilt disease resistance breeding projects since 2003.
Inoculation with PWN was conducted on July 1, 2007. In four susceptible clones and four resistant clones, 2 cm at the tip of the main stem was cut off, the cut edge was quickly crushed with pliers, and 10,000 nematodes that had been suspended in 100 μl sterile water were injected into the cut edge. As a mock sample, sterile water (without nematodes) was injected into the cut edge of the main stem of another four susceptible clones and four resistant clones. Stem tissue of inoculated samples and mock samples was collected 5 cm below the inoculated stem apex at 1, 3, 7, and 14 dpi. A 2 cm segment of stem was cut, frozen immediately in liquid nitrogen, and stored at -80°C.
Total RNA was isolated from 1.5 g of stem that included bark and wood tissue using the RNeasy plant mini kit (QIAGEN) following the protocol supplied by the manufacturer. RNA concentration was determined using a GeneQuant pro spectrophotometer (Amersham Biosciences). Integrity of the RNA was tested using the Agilent 2100 bioanalyzer (Agilent Technologies).
Six subtractive libraries were constructed from samples taken at three time points; specifically, two libraries--one from resistant trees and one from susceptible trees--were constructed from samples taken 1, 3, or 7 days dpi. Forward libraries containing genes expressed predominantly in resistant trees were constructed by subtracting driver RNA sampled from susceptible trees from tester RNA sampled from resistant trees, and reverse libraries containing genes expressed predominantly in susceptible trees were constructed by subtracting driver RNA sampled from resistant trees from tester RNA sampled from susceptible trees. Additionally, a seventh library was constructed by subtracting driver RNA sampled from susceptible trees 7 dpi from tester RNA sampled from resistant trees 14 dpi; the driver RNA samples were taken at 7 dpi because the susceptible trees had died by day 14. SSH libraries were constructed using a SuperSMART cDNA Synthesis kit (Clontech) and a Clontech PCR-Select cDNA Subtraction kit (Clontech). The SSH products were purified using a QIAquick PCR purification kit (QIAGEN) and ligated into the pT7Blue vector (Merck4Biosciences). Blue/white selection was conducted on plates containing ampicillin, isopropyl-D-thiogalactopyranoside and X-gal. Clones were randomly selected and single-pass sequenced using a primer that recognizes vector sequences from the 5' -end of the inserts. On -average, 500 clones were sequenced per library using an ABI 3130xl DNA Analyzer (Applied Biosystems). The resulting sequences were trimmed and edited manually to identify the cloning vector sequences, adaptor sequences used in the SSH procedure, and regions of low-quality sequence using Sequencher 4.10.1 software (Gene Codes Corp.). Quality sequences greater than 100 bp were selected for further analysis. A total of 3,299 sequences were at least 100 bp and these sequences were submitted to the GenBank EST database with the GenBank accession numbers FY841122 to FY844420. To determine the number of contigs and EST singletons in each library, the ESTs were assembled using Sequencher 4.10.1 by the requirement for at least 98% identity over each 20 bp continuous sequence.
A similarity search and functional annotation were performed for the EST singletons in each library using online version of the BLAST2GO program (BLAST2GO 2006; [15]). The thresholds used with the BLAST algorithms were as follows: (1) blastx comparison with the non-redundant protein database of NCBI with a threshold value of e-6; (2) tblastx comparison with the nucleotide database of NCBI with a threshold value of e-6; (3) tblastx comparison with the EST database of NCBI with a threshold value of e-6; (4) blastn comparison with the EST database of NCBI with a threshold value of e-10. Additionally, the ESTs in each library were compared with 69,968 EST sequences in the DFCI Pinus Gene index release 8.0 using an E value cut-off of e-10. The ESTs were assigned to functional categories using the Blast2GO program, and manual annotations were based on the result of BLAST analysis using the PGI database. The Blast2GO program extracts the GO terms associated with homologies identified with NCBI's QBLAST and returns a list of GO annotations represented as hierarchical categories of increasing specificity. Therefore, the "level" presented in this study corresponds to the depth of hierarchical categories in each principal GO category, with the topmost Level 1 representing the most general classification (principal GO categories) of biological process, molecular function, and cellular component.
Primer pairs were designed for each sequence using Primer Express software v3.0 (Applied Biosystems) and following the manufacturer's guidelines for primer design (Table (Table3).3). For SYBR Green real-time RT-PCR assays, the amplification efficiency of all primer pairs was optimized with genomic DNA from resistant and susceptible trees using the StepOnePlus Real-Time PCR System (Applied Biosystems).
RT-PCR was performed using the total RNA used to make the SSH libraries. For the mock samples at each time point and for the reference sample (without nematodes or water), total RNA was extracted from stem tissues and used for RT-PCR. Total RNA (500 ng in a final volume of 20 μL) was reverse-transcribed using the PrimeScript II 1st strand cDNA synthesis kit (TaKaRa) according to the manufacturer's protocol. Real-time quantitative PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) on the StepOnePlus Real-Time PCR System (Applied Biosystems). PCR mixtures were prepared according to the manufacturer's instructions and contained 300 nM of both the forward and reverse gene-specific primers and 4 μL of the 20-fold diluted reverse transcription reaction (total 5 ng) in a final volume of 20 μL. All reactions were heated to 95°C for 10 min; this denaturation step was followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The PCR products were subjected to melting curve analysis; the conditions were incubation at 60-95°C with a temperature increment of 0.3°C s-1. Elongation factor 1-alpha was used as the reference gene for normalizing the transcript profiles. The real-time PCR data were calibrated against the transcript levels in resistant tree stem samples prior to nematode exposure; the 2-ΔΔCt method was used to quantify relative transcript abundance [67]. All assays were carried out in triplicate, and the data are presented as means ± S.D. of three replicates. The specificity of each amplification was checked by melting analysis and agarose gel electrophoresis of the amplified products.
TH prepared the RNA preparation, sequenced the ESTs, analyzed the EST data, and drafted the manuscript. EF helped with data analysis of the ESTs and prepared the DDBJ GenBank submissions. AW conceived of the project, carried out the inoculation of PWN and sampling, and helped to draft the manuscript. All authors assisted with manuscript preparation and read and approved the final draft.
Summary of BLAST search results for ESTs among 7 SSH libraries.
Summary of BLAST search results for specific ESTs in the expression analysis.
Quantitative real-time PCR of transcripts differentially expressed in uninfected resistant and susceptible clones. The putative functional genes from (a) to (h) were clearly discernible ESTs in susceptible libraries. The putative functional genes from (i) to (p) were clearly discernible ESTs in resistant trees. Elongation factor 1-alpha (EF1a) was used as the reference gene, and the data were calibrated relative to the transcript levels in resistant trees prior to nematode infection (at 0 dpi). The data are presented as the mean ± S.D. of three replicates. Means designed by the same letter did not significantly differ at P < 0.05 according to Tukey' s HSD test.
We thank Dr. Dai Kusumoto and Dr. Toshihiro Yamada at Tokyo University for helpful discussions, comments, and advice on our research; we also thank our colleagues in the field management section of FTBC for the production and cultivation of plant materials. This work was funded by a grant from the Japan Society for the Promotion of Science (No. 20380085).
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Panasonic 12-32mm kit lens, missing manual focus
Started Mar 28, 2014 | Discussions thread
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LaMesa • Senior Member • Posts: 2,357
Panasonic 12-32mm kit lens, missing manual focus
Mar 28, 2014
Many members of this forum seem to consider the acquisition of a Panasonic 12-32mm kit lens. This lens has a reputation for better sharpness than comparable kit lenses. As a disadvantage, manual focusing is not provided, except for the G1 camera.
I am surprised about the many posters in this forum, who do not regard this as a knockout drawback. My camera has the AF set for center spot focusing +MF all the time. I find myself making manual corrections quite often, and many pictures would have been impossible without the chance of selectively using the manual focus.
What is your experience as owners of this lens?
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All quotes tagged Businesses
(15017 quotes found)
"Information technology and business are becoming inextricably interwoven. I don't think anybody can talk meaningfully about one without the talking about the other."
"If your business keeps you so busy that you have no time for anything else, there must be something wrong, either with you or with your business."
"Never ask a man where's he's been. If he's out on legitimate business, he doesn't need an alibi. And, girls, if he has been out on illegitimate business, it's your own fault."
""Remember what Bilbo used to say: It's a dangerous business, Frodo, going out your door. You step onto the road, and if you don't keep your feet, there's no knowing where you might be swept off to.""
"In business, the competition will bite you if you keep running, if you stand still, they will swallow you."


text:
If you have troubles since the update to WordPress 4.5 please read this post before opening a new thread.
Tagged: customization, enfold
Here i have an issue in my website http://career.guru99.com . I want to remove link from the footer. Kindly help me.
you can see what i actually want is here : http://screencast.com/t/xvkm8cuA3MYr
Thanx & Regards.
The topic 'Remove LINKS from footer' is closed to new replies.


text:
G'day, fellow RationalWikians! Don't forget to visit the Campaign page of the 2016 Board of trustees election in order to make your voice heard. Suggested activities include:
Your participation might help other users better direct their votes. More importantly, it'll show the world that we've yet to go full Citizendium in terms of election hype!
from FuzzyCatPotato (Talk), group Site wide (urgent) at 00:24, 25 July 2016
If you are really bored, you can help RationalWiki by writing stuff for us.
If you are really really bored, you can visit articles for deletion and see what needs pruning.
This is a place where:
If you find yourself creating a stub-level article just to remove a link from the list, consider whether you should be doing it at all.


text:
The National Security Agency has routinely intercepted networking equipment that's built in the United States, added backdoor access capabilities, then shipped the devices to their intended recipients abroad.
So says a report by journalist Glenn Greenwald published Tuesday by the Guardian. Greenwald cites a June 2010 document (leaked by former NSA contractor Edward Snowden) labeled as being from the head of the NSA's Access and Target Development group.
"The NSA routinely receives -- or intercepts -- routers, servers, and other computer network devices being exported from the US before they are delivered to the international customers," Greenwald writes. "The agency then implants backdoor surveillance tools, repackages the devices with a factory seal and sends them on. The NSA thus gains access to entire networks and all their users."
According to the document from the NSA -- the US intelligence agency charged with practicing signals intelligence -- this supply chain "SIGINT tradecraft... is very hands-on (literally!)."
When the hacked networking equipment is brought online, it phones home to NSA-controlled servers. "In one recent case, after several months a beacon implanted through supply-chain interdiction called back to the NSA covert infrastructure," Greenwald quotes the leaked NSA report. "This call back provided us access to further exploit the device and survey the network."
Greenwald has worked with filmmaker Laura Poitras to publish the lion's share of the files leaked to date by Snowden, and his NSA router-backdoor report was timed to coincide with Tuesday's publication of his book, No Place to Hide: Edward Snowden, the NSA, and the U.S. Surveillance State.
The report has already triggered a flood of related questions, including the degree to which technology manufacturers and shipping firms might be colluding -- or are legally forced to comply -- with the NSA's equipment-interception program.
But it's unclear how often the technique gets used. Last year, Snowden told Hong Kong's South China Morning Post that for eavesdropping, the NSA prefers to hack into infrastructure on a grander scale. "We hack network backbones -- like huge Internet routers, basically -- that give us access to the communications of hundreds of thousands of computers without having to hack every single one," he told the Post.
Last year, however, security expert Bruce Schneier -- who also has access to the trove of NSA documents leaked by Snowden -- warned that the NSA was hacking networking equipment as well. "The NSA also attacks network devices directly: routers, switches, firewalls, etc.," he said. "Most of these devices have surveillance capabilities already built in; the trick is to surreptitiously turn them on."
The revelations over the NSA's backdoor-installation program cast new light on warnings issued last year by former CIA director Michael Hayden, who accused Chinese telecom equipment maker Huawei of actively spying for the Chinese government. Expect the latest NSA leak to stoke the ongoing debate about the agency's tactics, and their impact on the privacy rights of non-US citizens.
But some information security experts have warned that the NSA's programs -- and bigger surveillance-state analyses -- must be kept in context. "This is the problem with Greenwald: his inept analysis created a whole new urban mythology about who did what to whom," says Thomas H. Ptacek, principal at Matasano Security, via Twitter.
Indeed, if there's one espionage truism, it's that whatever one side is doing, the other side is likely not far behind -- as has been proven by tit-for-tat accusations from both China and the United States that one is hacking the other.
At the same time that Hayden singled out Chinese networking manufacturers for colluding with the military, he conceded as much, confirming what the NSA leaks have made quite clear: The United States hacks foreign targets. But Hayden portrayed US efforts in different terms. "I fully admit: we steal other country's secrets. And frankly we're quite good at it," he said. "But the reason we steal these secrets is to keep our citizens free, and to keep them safe. We don't steal secrets to make our citizens rich. Yet this is exactly what the Chinese do."Mathew Schwartz served as the InformationWeek information security reporter from 2010 until mid-2014. View Full Bio
The cybersecurity profession struggles to retain women (figures range from 10 to 20 percent). It's particularly worrisome for an industry with a rapidly growing number of vacant positions.
So why does the shortage of women continue to be worse in security than in other IT sectors? How can men in infosec be better allies for women; and how can women be better allies for one another? What is the industry doing to fix the problem -- what's working, and what isn't?
Is this really a problem at all? Are the low numbers simply an indication that women do not want to be in cybersecurity, and is it possible that more women will never want to be in cybersecurity? How many women would we need to see in the industry to declare success?
Join Dark Reading senior editor Sara Peters and guests Angela Knox of Cloudmark, Barrett Sellers of Arbor Networks, Regina Wallace-Jones of Facebook, Steve Christey Coley of MITRE, and Chris Roosenraad of M3AAWG on Wednesday, July 13 at 1 p.m. Eastern Time to discuss all this and more.


text:
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Orlando National College Fair is going to be organized in Florida, the United States of America only for one day. It is a successful educational event that holds high commercial importance in the modern day academic sector. It is intended to offer a perfect international platform for the high school students who are looking for graduate and post graduate courses and admissions in various recognized regional as well as foreign institutes. Orlando National College Fair will help the students to gather information about various latest courses and professional studies. The list of qualified attendees consists of recruitment professionals, recognized universities, high schools and colleges, enrolment management professionals, education training centers, education partners and counselors and students from all over the world.
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About the Author
Julie chose a career in education after producing a video for a non-profit organization in Los Angeles starring children who were chronically ill. Her career as an educator evolved to include both the development and acquisition of preschool programs to teach preschoolers through elementary school aged children social skills through the sports of soccer and dance, respectively, until her twin sons were born. When one of her sons was diagnosed with food allergies at 5 months old she became proactive in learning how to manage his care. Julie has been very active in advocacy, education, support, creating educational materials, and promoting legislation regarding food allergy safety.


text:
This star rating is the opinion of the property owner only. A Self-Rated hotel has not been assessed by the AA or Visit Britain, whose official star ratings we use across LateRooms.com.
Highway Farm4 Self rated
West Road, Bridport, DT6 6AE
Price FromView Rooms
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About the hotel
A warm welcome awaits you! Tranquil rural location just one mile from the coastal footpaths, the Market Town of Bridport and a good local pub! We have fabulous panaramic views from every room and we like to serve your hearty breakfasts on the patio whenever possible. All rooms are spacious and ensuite and we aim to make your stay special. If you usually drive through Dorset, please don't, instead think long sweeping beaches, quaint Lyme Regis, arty Bridport, sleepy rural villages and a foodie's paradise with top chefs and some great pubs! The perfect base from which to explore and if you're walking the coastal footpaths the Jurassic bus stops outside our entrance.
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- Tea/Coffee making facilities in each room , Hairdryer in each room
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- Pets are not allowed
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text:
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Book your room at Hotel Lindrum Melbourne MGallery by Sofitel
Get an overview of this hotelLuxury Melbourne hotel in Melbourne CBD, near Melbourne Cricket Ground
Located in Melbourne CBD, this luxury hotel is within a 15-minute walk of Treasury Gardens and Melbourne Cricket Ground. Bourke Street Mall and Eureka Tower are also within 1 mi (2 km).
Hotel Features
A restaurant, a bar/lounge, and self parking are available at this hotel. WiFi in public areas is free. Additionally, 24-hour room service, a business center, and a meeting room are onsite.
Room Amenities
All 59 soundproofed rooms provide free WiFi, free wired Internet, and pillowtop beds. Comforts include Egyptian cotton sheets and pillow menus, and guests will also find conveniences like sitting areas and refrigerators.Would you like to give us feedback? Share it! Opens in a new window
Hotel Lindrum Melbourne MGallery by Sofitel26 Flinders Street Melbourne VIC 3000
Hotel Amenities
Hotel Amenities
The hotel offers a restaurant. A bar/lounge is on site where guests can unwind with a drink. This 5-star property offers access to a business center, limo/town car service, and a meeting room.
A computer station is located on site and high-speed wireless Internet access is complimentary. Event facilities measure 377 square feet (35 square meters). This Victorian hotel also offers tour/ticket assistance, multilingual staff, and concierge services. For a surcharge, a shuttle from the airport to the hotel (available on request) is offered to guests. Onsite parking is available (surcharge).
Hotel Lindrum Melbourne MGallery by Sofitel has designated areas for smoking.
- 24-hour front desk
- Airport transportation - pickup (surcharge)
- Babysitting or childcare
- Babysitting or childcare (surcharge)
- Bar/lounge
- Billiards or pool table
- Breakfast available (surcharge)
- Business center
- Free newspapers in lobby
- Computer station
- Concierge services
- Conference space size (feet) - 377
- Conference space size (meters) - 35
- Designated smoking areas
- Dry cleaning/laundry service
- Elevator/lift
- Express check-in
- Express check-out
- Fireplace in lobby
- Free WiFi
- Limo or Town Car service available
- Luggage storage
- Multilingual staff
- Number of buildings/towers - 1
- Number of floors - 4
- One meeting room
- Porter/bellhop
- Restaurant
- Self parking (surcharge)
- Television in common areas
- Total number of rooms - 59
- Tours/ticket assistance
- Free use of nearby fitness center
- Year Built 1900
Family Friendly Amenities
- Babysitting or childcare (surcharge)
- Cribs/infant beds (complimentary)
- DVD player
- Free Wi-Fi
- Refrigerator
- Soundproofed rooms
Available in all rooms: Free WiFi , Free wired high-speed Internet
Available in some public areas: Free WiFi
Self parking (surcharge)
Room Amenities
- Air conditioning
- Bathrobes
- Blackout drapes/curtains
- In-room climate control (air conditioning)
- Coffee/tea maker
- Free newspaper
- Free toiletries
- Free cribs/infant beds
- Daily housekeeping
- Desk
- DVD player
- Egyptian-cotton sheets
- Free WiFi
- Free wired high-speed Internet
- Hair dryer
- Hypo-allergenic bedding available
- In-room massage available
- In-room safe (laptop compatible)
- iPod docking station
- Iron/ironing board
- Minibar
- Pay movies
- Phone
- Pillow menu
- Pillowtop mattress
- Premium bedding
- Private bathroom
- Rainfall showerhead
- Refrigerator
- Room service (24 hours)
- Satellite TV service
- Separate sitting area
- Shower/tub combination
- Slippers
- Soundproofed rooms
Where to Eat
Full breakfasts are available for a surcharge and are served each morning between 6:30 AM and 10:30 AM.
Felt Restaurant - This restaurant specializes in local cuisine and serves breakfast and dinner. Guests can enjoy drinks at the bar.
Back Bar - Onsite lobby lounge. Happy hour is offered.
Late night fare is available from 24-hour room service.
Start your morning with a full breakfast, available each day from 6:30 AM to 10:30 AM for a surcharge. You can order up room service around the clock.
Nearby Things to Do
The recreational activities listed below are available either on site or nearby; fees may apply.
- Hiking/biking trails nearby
- Bicycle rentals nearby
To make requests for specific accessibility needs, complete the "Special or Accessibility Requests" section of "Trip Preferences" when you book your hotel.
Hotel Policies
Minimum check-in age is 18
Check-in time starts at 2 PM
Special check-in instructions:
Check-out time is 11 AM
Payment types
- Pets not allowed
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
- No onsite parking is available.
Optional extras
- Breakfast fee: between AUD 10.00 and AUD 36.00 per person (approximately)
- Airport shuttle fee: AUD 70.00 per vehicle
- Self parking fee: AUD 16.00 per night
- Crib (infant bed) fee: AUD 0 per night
- Rollaway bed fee: AUD 60 per night
Hotel Name
- Hotel Lindrum
- Hotel Lindrum Melbourne MGallery Sofitel
- Hotel Lindrum MGallery Collection
- Lindrum
- Lindrum Hotel
- Lindrum Melbourne MGallery Collection
- Lindrum MGallery Collection
- Lindrum Hotel Melbourne
- Hotel Lindrum MGallery Sofitel
- Lindrum Melbourne MGallery Sofitel
- Lindrum MGallery Sofitel
We should mention
This property offers transfers from the airport (surcharges may apply). Guests must contact the property with arrival details before travel, using the contact information on the booking confirmation. One child 12 years old or younger stays free when occupying the parent or guardian's room, using existing bedding. Only registered guests are allowed in the guestrooms.


text:
Hi everyone,
here is my problem : I have a wordpress theme I bought on themeforest 'backstreet'.
The theme uses a different page to manage posts and reviews.
Everything was working fine until 2 days ago, when I noticed that settings for comments have disappeared for Reviews. Everything is still fine for standard posts. For reviews I got a "comments are closed" and no ways to change it to open.
SO, in order to gain some time and avoid some obvious "try and tell us if it's working" here is what I did :
- Checked on WordPress Support if someone had the same problem
- I DO know about screen option. The fact is that the "comments" checkbox has disappeared from that too...
- My settings in settings > Discussions are ok, everything is as it should be
- I've already deleted every plugin (I was worried about deactivating them could be not enough)
- I can't go back to Twenty Eleven because it would be pointless. Comments are fine for post, it's the special "Review posts" that comes with the theme that miss the setting.
- It WAS working fine two days ago, I've already asked the theme owner, he never had that issue. And I do trust him, I used that theme for 4 month without problems
- I went into the DB, and manually change closed to open. Didn't change anything, 10 minutes later it was back to closed.
- I've already deleted the wp-admin folder and reupload a clean one.
So my questions are :
- Is it a known issue ?
- Can you help ?
- If not, can I force the setting to appear again in screen option (through database hack or scripts in some .php wordpress files ?
So basically, this thing is going to drive me crazy!!! I can't seem to find a logical explanation to this... Any help would be highly appreciated !
Thank you very much in advance for all the time you'll spend trying to resolve this problem with me.


text:
the field wasn't that bad last year.
the field wasn't that bad last year.
1 CB Vernon Hargreaves Florida 5-10 204
2 DT Austin Johnson Penn State 6-4 314
3 SS Jeremy Cash Duke 6-0 212
4 LB Jaylon Smith Notre Dame 6-2 223
6 WR Moritz Boehringer Germany 6-4 227
7 OT Brandon Shell South Carolina 6-5 324
7 RB Tre Madden USC 6-0 223
DE David Onyemata, LB Ian Seau, FS Jordan Lomax, CB DeAndre Elliott,
QB Jeff Driskel, TE David Morgan, OG Spencer Drango, OC Mike Matthews
2015 MNF Executive Champion!
While the turf didn't look bad last year, it never hurts to look for solutions to improve it.