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Wholesale Story Shipping and Return
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What's better than finding a piece of cobalt blue sea glass on the beach? Your five-year old finding a piece of cobalt blue sea glass on the beach.
The shrieks of delight and enthusiasm from my daughter still echo in that happy place in my brain where good memories are filed away for keeps.
She found it a couple of weeks ago under a driftwood log.
"Look what I found mom!!!" she announced, holding up the piece as if it were the Devil's Eye Diamond from The Rescuers.
Maddie knows that big chunks of cobalt blue sea glass are hard to come by – especially ones with words on them.
Today I did some research on the piece. I simply plunked in "A. J + cobalt blue bottle" into Google, and presto-mundo (the internet really is magic!) – there it was: a Laxol bottle.
Yes, it's a laxative. And likely more than 100 years old. My daughter will get a serious kick of these two facts.
The bottle was from A. J. White (Andrew Judson White), New York. A. J. was a proprietary medicine maker in New York City who teamed up with the Shakers in New York to produce medicines using botanical ingredients supplied by the Shakers. In 1957, A. J. White was purchased by Smith, Kline and French Laboratories.
You know you've got it bad when a laxative bottle (or piece of it) turns your crank.
I have a special fondness for sea glass with words. It's like I've found a clue. Or sometimes it's a message from the Great Beyond.
Chase. What am I chasing?
I'm chasing something. With fierce determination.
Freedom. That's why there are flames coming out of my heels and my reflection is a blur. I don't relax. I chase.
But the biggest irony of all is that in my quest for freedom, I have given up so much freedom. The freedom to read novels in the evening and paint on Saturdays. The freedom to write poetry late into the night. The freedom to do nothing.
No time for that.
But there is still time for sea glass.
Photo: Cobalt blue sea glass found on north shore of Lake Erie. By Christina Friedrichsen.

subset: commoncrawl


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2nd June, 2010 - Today's post is a continuation of the survey of playground in Sapporo. OK, it's not like we're trying to go to every single playground in Sapporo (though sometimes it feels that way), it's just that if we go somewhere, we tend to look around to see if there's a good playground around the area. How useful is this to you, the 0.75 readers of this blog? Perhaps only marginally more useful than knowing the stock levels of the local drink vending machine.. However, it's perhaps not a side of Sapporo (or Japan) you might have seen that often. And if you have children, and you're thinking of moving to Sapporo (the odds of this being my 0.75th reader are astronomical), then perhaps it's sort of interesting.
Enough of the verbage. Let's get on with it.
Today, I'm writing about a small playground that T-chan took our son L-kun to. Not an overly remarkable playground (but not bad either). The playground is just across the road from the Ishiya Chocolate factory (hmmm - did I mention having a good reason to be somewhere?) and right behind the Consadole Soccer Club rooms (and pitch).
View Sapporo
Now the Consadole Soccer Team is Sapporo's black sheep (or is that prodigal son?) that is still at centre of Sapporo's other sports dreams (the Sapporo Nippon-Ham Fighter Baseball team is the number one attraction). Now whether you call it Soccer or Football, it's a universal game. And Sapporo's Consadole's team is an almost universal constant in terms or relegation to the 2nd division from the 1st division. You gotta give it to them, even though they've been relegated three times since it's inception in 1996, they still keep plugging away (and of course, being relegated that many times also means they've been promoted that many times as well... taking the glass half full view).
|Consadole club rooms
Consadole, as a name, comes from a very bizarre (alchhemist-like) formula where they took the name of the iconic Hokkaido draft horse (known as the dosanko), and reversed the Japanese syllables (do-sa-n-ko = ko-n-sa-do) and added a bit of Spanish exclamation (ole!) to the end. And all of that turned into Consadole. A word of advice... keep it simple, stupid. Sapporo's baseball team, the Nippon-Ham Fighters might be an odd name, but it at least well reflects their team sponsors, Nippon-Ham.
Enough about soccer. And baseball.
The thing I like about Japanese playgrounds (a clue, it isn't the ground cover) is the variety of play equipment. Back here in Adelaide, Australia, there's a certain... hmmm... predictability of the equipment you'll find. In Sapporo, there seems to be (outside the very small local suburban playgrounds) something equating to pride at the uniqueness or variety of their playgrounds.
There also seems an infinite variety of contraptions that make up the play equipment... and whilst some of it sometimes seems to have come from an SAS training course, I always feel impressed at the imagination. And the proof is in the pudding... or more precisely the eating of the pudding (what happens after that is perhaps best not discussed on this blog). L-kun loves playgrounds in Japan. Ok, he loves playgrounds everywhere, but he seems to love them particularly in Sapporo.
There's always a slightly different challenge, or skill to master. After all, there's only so many slippery slides you can go down... hold on... they have them too (but the in thing in Japan is the roller slide). L-kun's quite partial to those as well.
Neighbourhood playgrounds tend to (strangely enough) have neighbourhood children... and that means there's a certain degree of calm, or community and of camaraderie amongst the children. As a case in point, T-chan told me a story about this particular playground where L-kun was playing. L-kun's always a bit nervous in a new environment, but two older children decided to take him under their wing and look after him. It was nice to see children being so supportive of each other (and not just hitting one another).
But at the end of the day - it's all about having fun. After all... shouldn't that be what life's about (when you strip away all of the social convention, mortgage stress, peer pressure, and all sorts of other largely self-induced angst and anxiety). As we get older, we just seem to forget it, wrapped up in our own social balance statement.
Now if only Consadole can show a little bit of wonderful as well!
(note - we'd actually been to Ishiya and the Consadole club rooms on our first trip to Sapporo in 2003 - but then it was covered in snow, and snowmen... and where we went into the Guinness Book of Records).


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While original TV movies continue to struggle on broadcast TV as evident by the underwhelming ratings for the Hallmark Hall Of Fame film on ABC last night, the genre's resurgence on basic cable continues. TNT has decided to extend its Mystery Movie Night franchise into the spring, greenlighting today the original movie Hornet's Nest, an adaptation of the Patricia Cornwall novel that will star Sherry Stringfield and Virginia Madsen. The green light for Hornet's Nest comes ahead of the debut of the TNT Mystery Movie Night, which features original movies based on popular crime novels. The franchise is set to premiere tomorrow with Scott Turow's Innocent and continues Wednesday with the Sandra Brown novel Ricochet, starring John Corbett, Julie Benz and Gary Cole. The series will feature six movies this month and in December before returning in the spring with Hornet's Nest and three other movies to be announced. Hornet's Nest, based on Cornwell's New York Times bestseller, centers on a serial killer in Charlotte, N.C., who targets out-of-town businessmen. Police Chief Judy Hammer (Madsen) pairs her top deputy (Stringfield) with a cub reporter (Robbie Amell) to address the crisis. Michael Boatman and Michael Silver co-star. Dee Johnson (TNT's Rizzoli & Isles) is writing the script. ER alumna Stringfield is with APA and John Carrabino.
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- 2 organic pears, halved and pitted
- 1/4 cup dried cherries, finely chopped
- 1/4 cup walnuts, finely chopped
- 1 tbsp. honey
- 1/2 tsp. ground cinnamon
- 2 tbsp. grass-fed butter
Preheat the oven to 350 degrees. Slice a small piece off the bottom of each pear so it will sit upright in the baking dish. Sprinkle cinnamon over each pear in a baking dish. Mix cherries and walnuts together, spoon 2 tbsp. of the mixture into each pear center. Drizzle honey over each pear and top with 1/2 tbsp. of butter and bake for 20 minutes or until easily poked with a fork.
Nutritional Information: Total Calories 198, Total Fat 11g, Carbohydrates 26g, Sugars 19g, Dietary Fiber 4g, Protein 2g, Sodium 3mg


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Previsions of the future
In Deus Ex: Mankind Divided, man has fused with adolescent cybernetic technology and the results are…erratic.
Media propaganda is spoon-fed to the public by corporate controlled intelligence, while revolutionary journalists coordinate their insurgency beneath the streets of Prague. Cultural uncertainty, in regards to a marginalized augmented population devolves into virulent paranoia, as law enforcement deploys brutal tactics to handle the "opposition". Political leaders seek to destroy autonomy via the guise of benevolent technological innovation for all. In the shadows, a clandestine group seeks to establish governance without opposition by obliterating civilized institutions with carefully calculated maneuvering. This is the world of Mankind Divided, a future gone astray…a future not far off from our own.
After 60 hours of exploring every nook and cranny of an exhaustively detailed Prague 2029, I am fixated on a disturbingly plausible future, in which the technological singularity is knocking on the door of artificial general intelligence.
In Mankind Divided, the primary narrative handles these themes overtly and tends to take a backseat to the well-crafted subtlety of its side missions. While the main story ends abruptly, the side missions are well fleshed out and provide consequential and meaningful resolutions for each action and decision you make. Side missions offer compelling twists and turns while exploring the more existential themes the game has to offer.
Cybernetic Security
Mankind Divided retains the same stealth/action and RPG elements of its predecessors, while vastly improving the fluidity of its mechanics by allowing the player to efficiently implement a strategic chain of augmented maneuvering at a faster pace than prior iterations. The developers have incorporated more limiting energy usage mechanics this time around, which require calculated movements throughout the environment if the player wants to remain the hunter and not the hunted. A plethora of interesting augmentation options open up several creative avenues for the player to navigate the environment and snuff out the enemy. The new "experimental augmentations" enhance the protagonist with prototype technology, such as remote hacking, and my personal favorite, the Tesla, which functions as an offensive stealth option, projectile stun with multi-targeting functionality. These new augmentations are not only a blast to use, they're viable and just as accessible as their more "standard" counterparts.
Stealth is relatively the same as it was in its predecessor, Human Revolution. Unlike Human Revolution, a more aggressive style of play is enhanced by complimentary augmentations such as Titan armor (temporary invulnerability) and a more expansive set of weapon handling augments. I decided to go with a non-lethal ghost playthrough my first time around, after briefly experimenting with a lethal approach which felt a bit clunky in terms of target acquisition. Environmental exploitation, coupled with hastily adaptive stealth, made for some truly memorable moments that left me feeling like a cybernetic predator.
Dystopian Beauty
Exquisite, gothic architecture overshadows the cobbled streets of Prague. Claustrophobic and dizzying corridors harbor the occult and criminal alike below. Graffiti and ivy contest one another over surface domain, while alleyways shelter capitulated prey within the shadows. Ebony towers loom ominously over denizens with a scornful facade. Streets are illuminated by the glow of screens perpetuating 24 hour propaganda, while flyers rallying the resistance occupy every corner. Colossal, prison like structures harbor the demonized and afraid within a maze of wire and metal.
The environment is alluring and immersive, as it pulls the player through a nebulous glimpse of a future slowly overriding antiquity. The experience is enhanced by the games wonderful lighting and nuanced textures, which meld the noire and dystopian themes into a plausible glimpse of the future. The graphical fidelity of this game does come with a hefty cost for graphic enthusiasts looking to run ultra-settings on PC. My GTX 1070 goes from 65 fps avg. with MXAA off, to 40 fps avg. with MXAA 2x enabled. Fortunately, the graphics options are expansive and allow the user to manipulate multiple, lighting, shadow and anti-aliasing settings. The Dawn Engine is quite capable of rendering large, intricate maps without additional loading; however, I feel there was room for further optimization.
Sweeping orchestral piano, layered over top trip hop and ambient electronic elements provide an ethereal, resonant backdrop for the environment. The soundtrack, composed by the brilliant Michael McCann, ebbs and flows with the pulse of the games action. Activated augmentations provide a satisfyingly unique sound cue just before the bone crushing impact of a takedown. Shots from a tranquilized sniper rifle utter a muzzled wisp just before they strike their target. Mechanized enemies vibrate the floor with a threatening stride of hydraulic impact. The atmosphere is a richly innovative blend of visual and sonic ambition.
Swallow the Red Pill
Deus Ex: Mankind Divided offers the player a chance to explore its expansive universe with freedom and creativity. Each encounter provides the opportunity to navigate challenges in uniquely satisfying ways. Viable stealth, action, lethal, or nonlethal playstyle hybrids provide enough replay value, coupled with a multi-layered narrative that rewards environmental exploration with context. Fully realized, gorgeous environments within the alluring, dystopian world of Deus Ex, challenge the player to embrace the future of humanity and all of its consequences.
|+Vast arsenal of augmentations allow for a truly unique playstyle
|-Poorly optimized for PC
|+Compelling side stories provide rich context for the world and its inhabitants
|-Guns don't handle well enough to effectively utilize with aggressive playstyles
|+Meticulous attention to detail throughout each environment. Each environment tells a story and serves a purpose to the underlying themes


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Writing in the Winona Daily News, Maggie Modjeski doesn't identify the city and parents who allegedly made life miserable for her kid's soccer team last weekend. But, the ongoing anecdotes tell us, it could be just about anywhere.
The travel team's visit to the home field of Team Unnamed started well enough. When they arrived the host parents welcomed the players and parents and thanked them for traveling so far to play their team in a rescheduled game.
Then the game started, she writes.
The physical aspect of the game was overshadowed by the commentary. From the opposing parents any time a call went against them, to the players whose language was fouler than the smell of their cleats. The coach joined in the fun as well. The official who was a young lady took a pretty good beating, as our parents and team remained quiet as hard as it was.
Officiating a game is not easy. The calls aren't always perfect. But having adults yelling at you from the sidelines and a player argue and shout obscenities at you make the job that much more difficult. This official kept her cool, and kept making the calls.
Sitting there hearing the nonsense and not replying was very difficult. My first instinct was to yell back or comment. Fortunately, I was surrounded by great parents who help everyone keep their cool. At halftime I ran into the welcoming parent, and she wasn't so much anymore. She made a few comments, I smiled, nodded and bit off my tongue and walked away.
This is all learned behavior, as is sportsmanship, she concludes. Today's soccer parents are raising tomorrow's soccer parents.
Her kid's team lost.
"My son shared that one particular player was embarrassed at how much his dad was yelling. Our boys didn't feel bad about the loss; we knew we played hard, we didn't make a scene, and no one was hurt," she says.


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In the World War II drama Phoenix, Nelly (Nina Hoss) is liberated from the concentration camp Auschwitz, but she has been wounded to the point of facial disfigurement. Following reconstructive surgery that leaves her appearance slightly altered, Nelly spends her nights searching for her husband Johnny (Ronald Zehrfeld) at the night club Phoenix.
The street outside of Phoenix is a wash of red light that gives the bar a foreboding aura. Nelly, her face masked in a black veil that further conceals her identity, looks on in almost a surprised horror as she discovers her husband. She calls out to him, but he only hears enough of it to hesitate for a moment before walking away. She returns to Phoenix the next day to find that Johnny has also altered his identity, calling himself Johannes and working as a server at the club.
When Johnny and Nelly meet again, Johnny does not recognize his wife. Thinking that Nelly had died in the camps, he has been trying to get her weighty inheritance. Johnny decides to take this new woman in and have her assume the identity of Nelly in order to obtain the inheritance. All the while, he is unaware that the woman he is asking to impersonate his wife is in fact his wife.
Phoenix is an experiment in dramatic irony and an exploration of a reformed identity given a traumatic, identity destroying experience. There is a fascination in watching Nelly accept being continually unrecognized by her own husband. There may be a drabness to the pacing and a stripped down simplicity to its setting, but there is something undeniably engrossing about the back and forth between Nelly and Johnny as he attempts to transform her into his image of Nelly. His Nelly is an old Nelly, one that lives in the past and likely died the moment that she was arrested by the Gestapo.
Hoss's almost silent performance as Nelly is hauntingly majestic, her face a range of complex emotions that reflect bitter dimensions of thought laying underneath. Zehrfeld's Johnny plays off of this incredible depth well by being completely–almost willfully–ignorant to the undercurrent of trauma that exists inside the woman in front of him. His one track mind forces the real Nelly behind him, leaving in its stead some specter of the past that he can now only recreate for his own selfish benefit.
The film is quiet, scored mainly by lazily tweeting birds or the occasional ticking of a clock. The silence creates a mood of tense isolation that remains throughout, helping guide us toward the isolation that must exist in Nelly, an isolation that allows her to consent to the exploitation at the hands of Johnny.
Accompanied by a brilliant performance by its lead and a passionate, tense climax, Phoenix overcomes early pacing issues to become a welcome addition to the expansive WWII film canon.
The Post-Script
When viewed as a case study, Phoenix is chilling. The protagonist Nelly has just recently escaped from a place whose goal is to strip away the very identity of its prisoners, and she is quickly thrown into a similar environment at the hands of her husband. Only this second time, her identity is being stripped away for the sake of returning her to some dream version of her past self. This leaves Nelly deeply conflicted, a conflict that is evident on Hoss's face throughout the film. Hoss's performance truly makes this movie, and it is a performance that is not to be missed.
As always, thanks for reading!
Have you seen Phoenix? If so, what did you think? Let me know in the comments!
–Alex Brannan (@TheAlexBrannan)


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Talk to an advisor to get tailored advice.


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Game of Thrones Season 3 Episode 2 Quotes
Game of Thrones Season 3 Episode 2 has some awesome quotes from Jaime Lannister, among others including lesser known characters bringing in some good quotes. Here are the best quotes Game of Thrones Season 3 Episode 2 provided.
1. "I try to know as many people as I can. You never know which one you'll need" – Tyrion Lannister
Defending himself for knowing who Ros the whore is to Shae, Tyrion ends up explaining why he makes a point to know as many people as he can. He says that having an arsenal of contacts is beneficial as you never know who you might need if you're in a tight spot.
2. "Yes, she is. Objectively. Very. Her face is… quite pleasing… to other men and to women, people, in general. But not to me of course" – Tyrion Lannister
Tyrion immediately faces Shae's wrath after referring to Sansa as a "great beauty" when he is telling Shae that she will not lack for suitors. Trying to extricate himself from the situation, he begins an entertaining line where he tries to backtrack away from his comment to appease his fuming lover.
3. "You think Lady Stark's going to want a..uh.. giant, toe-headed plank following her around for the rest of her life?" – Jaime Lannister
No one can conjure an inventive insult quite like Jaime Lannister, and here he has another awesome one for his captor Brienne on their trek to King's Landing. In this, he says that part of being in service to someone is being good company, and that Brienne won't be that to Lady Stark. He then questions if Catelyn will want a "giant, toe-headed plank" always trailing her if she isn't good company. Funny lines from him are prevalent in Game of Thrones Season 3 Episode 2 and its quotes.
4. "Ah… Renly. Really? He wasn't fit to rule over anything more important than a 12-course meal" – Jaime Lannister
Renly takes quite the bashing in Season 3 Episode 2, with several quotes defaming the dead man. After Brienne recoils from the suggestion she was in Stannis' army, Jaime connects that this means she was with Renly. He then belittles Renly's ability to manage things, saying a meal was the height of the responsibility he could handle.
5. "It's all true about Renly; his proclivities were the worst kept secret at court. It's a shame the throne isn't made out of cocks, they'd have never got him off it" – Jaime Lannister
Ah, one of the most iconic Game of Thrones quotes from Jaime Lannister comes in Season 3 Episode 2. Brienne's denial of Renly's homosexuality brings a joke forth from Jaime, who says if the throne was made of cocks he'd never have been off it.
6. "I think you lost this war the day you married her" – Lord Karstark
Lord Karstark's outward negativity causes Robb to ask if he had lost faith in their ability to win the war against the Lannisters. Lord Karstark asks if he can speak freely, before saying he thinks the war was lost for them the moment Robb married the foreign girl Talisa, which damaged their alliance with the Freys as well as turning the men against him.
7. "I like you but, if you're playing us false, it won't be hard for me to kill you" – Mance Rayder
Mance Rayder asks Jon Snow if it was hard for him to kill the Halfhand, which Jon acknowledges that it was, so Mance proceeds to ask him if Jon liked him. Jon nods, bringing this quote out from Mance.
8. "So, you know how I got moon-worshippers, and cannibals, and giants to march together in the same army? I told them we were all going to die if we don't get south, because that's the truth" – Mance Rayder
Mance tells Jon of the difficulty it took to assemble all of the wildlings in one vast army. The secret, he reveals, was telling them all that death awaited them should they stay north, and that banding together and escaping their common enemy of the White Walkers was the key to survival.
9. "Some people will always need help. That doesn't mean they're not worth helping" – Meera Reed
Osha begins to give grief to Meera's brother, Jojen, who needs her protecting as he can't fight. Meera brings some good feelings into Game of Thrones with this touching quote, where she says that his need for protection doesn't make him unworthy of help, nor should it ever for anyone.
If you enjoyed the Game of Thrones quotes from Season 3 Episode 2, be sure to check out the quotes from Season 3 Episode 1, as well as our quote compilation from Season 2.
Image credits: HBO


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On Jun 28, 2012, at 8:01 PM, Alexandre Elias <email@example.com> wrote: > It may help to reduce the vertical screen resolution in the developer tools panel as it seems to be sensitive to that. But it sounds like you are visualizing it properly anyway. > > > This means that every <h1> in the document will "stick" 10 pixels from > > the viewport top when things scroll such that the <h1> would have been > > partially off-screen. When the viewport has scrolled such that the > > visible portion of the <h1>'s containing box is too small to fit the > > <h1>, it should stop at the containing box's edge and only be partially > > displayed. > > Is it correct to say that this is equivalent to specifying "position: fixed; position-contain: parent" in the alternate proposal? I.e. the <h1> is always fixed at 10px from the top of the viewport, unless that would bring it outside the bounds of its containing box, in which case its position is clamped to the containing box's edge. In that case, I agree it addresses the google.com map example. To the best of my understanding, given that both proposals are somewhat vague: (1) I believe position: sticky with the appropriate other style rules (for margins or position) will do what you suggest. (2) It's not *exactly* equivalent to "position: fixed; position-contain: parent", as I understand it, because it leaves a placeholder behind, like relative positioning, rather than taking the element completely out of flow, like fixed positioning. So with the position-contains solution, you would have to manually insert a placeholder in the containing box, while with position: sticky that is provided for you. More generally, it seems to me that position: sticky handles a lot of different use cases, including ones previously not anticipated. Meanwhile, Tab's more elaborate proposal cannot replicate the exact behavior of position: sticky as written. And we don't have evidence of a real-world use case that can only be handled with a more elaborate proposal. Therefore, I would ask: (A) Am I wrong, and can position-contains fully replicate the proposed position: sticky behavior in some way? Or is there a natural extension that would enable it to do so? (B) Are there common real-world use cases that require the more complex proposal to be doable at all? Regards, Maciej > > > > On Thu, Jun 28, 2012 at 2:25 PM, Maciej Stachowiak <email@example.com> wrote: > > On Jun 28, 2012, at 1:52 PM, Alexandre Elias <firstname.lastname@example.com> wrote: > >> Sorry about that, looks like the behavior is currently restricted to Android. >> >> You can simulate an Android browser in desktop Chrome (I tested the following using version 20 beta) by doing the following: >> - Visit http://google.com/ >> - Click "Developer tools", then the small gear at the bottom right of the panel. >> - Click "Override User Agent" and select "Android 2.3 - Nexus S" (not Galaxy Nexus as the high resolution somehow breaks the behavior I'm describing in this desktop simulation mode -- though it does happen on a real Galaxy Nexus) >> - Reload the page to see the mobile version, then click "Restaurants" as before. >> >> You should see the map is sticky, then appears to reach a limit and gradually falls out of view if you scroll down to the bottom of the page. > > With these steps, I'm not able to replicate the behavior you describe. I see the map sticking, but not disappearing close to the bottom. I do see it tracking its container until it hits the top. What I see can definitely be replicated with position: sticky as proposed. I think the more complex behavior you describe can also be replicated with position: sticky, but I'm hesitant to say definitively without seeing the behavior first. Specifically, I think that if you make the sticky element's containing block stop short of the bottom of the page, you will get the described behavior. Again, I'm not 100% sure as I can't see it for myself yet. > > Cheers, > Maciej > >Received on Sunday, 1 July 2012 19:02:10 UTC
This archive was generated by hypermail 2.4.0 : Friday, 25 March 2022 10:08:18 UTC


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- Research article
- Open Access
- Published:
New congenital anomalies of the kidney and urinary tract and outcomes in Robo2 mutant mice with the inserted piggyBac transposon
BMC Nephrology volume 17, Article number: 98 (2016)
2579 Accesses
6 Citations
1 Altmetric
Disruption of ROBO2 in humans causes vesicoureteral reflux (VUR)/congenital anomalies of the kidney and urinary tract (CAKUT). PiggyBac (PB) is a DNA transposon, and its insertion often reduces—but does not eliminate—gene expression. The Robo2 insertion mutant exhibited non-dilating VUR, ureteropelvic junction obstruction (UPJO) not found in reported models. We studied the incidence and outcomes of VUR/CAKUT in this mutant and explored the relationship between Robo2 gene expression and the occurrence and severity of VUR/CAKUT.
The urinary systems of newborn mutants were evaluated via Vevo 770 micro-ultrasound. Some of the normal animals—and all of the abnormal animals—were followed to adulthood and tested for VUR. Urinary obstruction experiments were performed on mice with hydronephrosis. The histology of the kidney and ureter was examined by light microscopy and transmission electron microscopy. Robo2 PB/PB mice were crossed with Hoxb7/myr-Venus mice to visualize the location of the ureters relative to the bladder.
In Robo2 PB/PB mice, PB insertion led to an approximately 50 % decrease in Robo2 gene expression. The most common (27.07 %, 62/229) abnormality was non-dilating VUR, and no statistically significant differences were found between age groups. Approximately 6.97 % displayed ultrasound-detectable CAKUT, and these mice survived to adulthood without improvement. No severe CAKUT were found in Robo2 PB/+ mice. The refluxing ureters showed disorganized smooth muscle fibers, reduced muscle cell populations, intercellular edema and intracytoplasmic vacuoles in smooth muscle cells. Both UPJ and UVJ muscle defects were noted in Robo2 PB/PB mice.
Robo2 PB/PB mice is the first Robo2-deficient mouse model to survive to adulthood while displaying non-dilating VUR, UPJO, and multiple ureters with blind endings. The genetic background of these mutants may influence the penetrance and severity of the CAKUT phenotypes. VUR and other CAKUT found in this mutant had little chance of spontaneous resolution, and this requires careful follow-up. We reported for the first time that the non-dilated refluxing ureters showed disorganized smooth muscle fibers and altered smooth muscle cell structure, more accurately mimicking the characteristics of human cases. Future studies are required to test the role of Robo2 in the ureteric smooth muscle.
Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common causes of renal failure in childhood [1] and represent approximately 30 % of all prenatally diagnosed malformations [2]. CAKUT are phenotypically variable and can affect the kidney(s) and/or the lower urinary tract. Vesicoureteral reflux (VUR) is the most common congenital urinary tract abnormality in childhood and has been associated with an increased risk of urinary tract infection (UTI) and renal scarring, known as reflux nephropathy (RN) [3]. Ureteropelvic junction obstruction (UPJO) is the leading cause of hydronephrosis in children.
CAKUT results from faulty urinary tract development, a process for which numerous genes are critical. ROBO2, a member of the immunoglobulin gene superfamily, is a receptor for the ligand SLIT2. SLIT2-ROBO2 signaling is important for axon guidance during development [4], and both human studies and animal models have provided evidence for an association between the interruption of ROBO2 and VUR/CAKUT [5–9].
Lu et al. report the disruption of ROBO2 in one patient with a balanced translocation and a phenotype that included VUR, ureterovesical junction (UVJ) defects, and megaureter [6]. In addition, heterozygous point mutations in ROBO2 have been identified in many patients with nonsyndromic VUR [6, 10]. However, some studies [11–14] have been unable to validate the association between ROBO2 and human VUR. Heterozygous mutations have recently been identified in patients with UPJO, posterior urethral valve (PUV) or multicystic dysplastic kidney (MCDK) [15].
Robo2-deficient mouse models have been used to explore the pathogenesis of Robo2 in CAKUT [5–7]. Studies have shown that SLIT2-ROBO2 signaling plays a key role both in normal ureteric budding and in the reciprocal induction of nephrogenesis by negatively regulating GDNF/RET activity [5]. Robo2 homozygotes had multiplex, dysplastic kidneys and short ureters and died within 48 h of birth [5–7]. Possibly due to variable Robo2 gene dosage, the mosaic mice had broad-spectrum CAKUT phenotypes including renal dysplasia, short ureter, megaureter, abnormally located UVJ and hydronephrosis [6, 7]. Of the long-surviving heterozygotes, 4/26 (15 %) exhibited unilateral megaureter and a wide-open UVJ [6]. Other phenotypes reported in patients with ROBO2 mutations, such as UPJO, PUV and non-dilating VUR, were not found in these models.
PiggyBac (PB) is a DNA transposon. Its insertion often reduces but does not eliminate gene expression, facilitating the identification of specific genetic functions [16], and it is a mutagen suitable for large-scale mutagenesis in mice. The Institute of Developmental Biology and Molecular Medicine at Fudan University performed a large-scale PB insertional mutagenesis and identified one Robo2 insertion mutant. This mutant was both viable and fertile, and it exhibited non-dilating VUR, UPJO, and ureterovesical junction obstruction (UVJO) that were not found in reported models or other CAKUT phenotypes. We study the incidence and outcomes of VUR/CAKUT in this mutant and explore the relationship between Robo2 gene expression and the occurrence and severity of VUR/CAKUT.
Mouse procedure
The PB transposon was inserted in the first intron of Robo2 on mouse chromosome 16, nucleotide 74379378. The insertion direction was opposite the gene location. Robo2 PB/PB mice were crossed with Hoxb7/myr-Venus mice, which have been described previously [17], to visualize the location of the ureters relative to the bladder, as performed previously [18, 19].
Mapping the PB-inserted Robo2 allele
Offspring with the transposon inserted into the Robo2 gene were genotyped by polymerase chain reaction (PCR) amplification using the primers P3 (5′-CTGAGATGTCCTAAATGCACAGCG-3′), P4 (5′-AAGATTCCCTTTCCATGCAGAAGAG-3′) and P5 (5′-GTGTACAGGAGTTTGGCACTGG-3′). Robo2 PB/PB and Robo2 PB/+ were amplified by P3 and P5, which produced a 614 bp fragment. Wild type and Robo2 PB/+ were amplified by P4 and P5, which yielded an 864 bp fragment. PCR conditions were as follows: initial denaturation at 93 °C for 90 s; 40 cycles of 93 °C for 30 s, 57 °C for 30 s, and 65 °C for 2 min; and a final extension at 65 °C for 10 min. Genomic DNA extracted from mouse toes was used as template.
Ultrasound investigation of the urinary system
The urinary system was examined by a Vevo 770 high-frequency micro-ultrasound system. Mice were anesthetized with 2.5 % isoflurane in 1 L/min oxygen via nose cone. A heat lamp was used to keep mice warm during anesthesia. A chemical depilatory was used to remove the fur above the position of the kidneys, and ultrasound gel was generously applied directly to the depilated surface. Next, the real-time micro visualization (RMV) transducer was placed on the ultrasound gel along the spine to identify the kidneys. RMV-708 was used for newborn mice, and RMV-712 was used for juvenile and adult mice. Grossly observable phenotype were described as severe CAKUT.
VUR and urinary tract obstruction experiments
Operators who were blinded to the genotypes performed the VUR test. Mice were dissected using an anterior midline incision to expose the kidneys and the urinary tract. The bladder was punctured with a 25 gauge needle to manually inject methylene blue (1 mg/ml in PBS) at a rate of 100 μl/min until the dye exited through the urethra [20]. The severity of VUR was determined by the extent of the ureter dilation. Combined with ultrasound results, VUR was divided into dilating VUR and non-dilating VUR. To test for urinary tract obstruction, the renal pelvis was microinjected with dye, and its passage along the urinary tract was monitored to determine whether there was evidence of impaired flow along the tract [20].
Histopathological analysis
Kidneys with attached ureters and bladders were removed from the euthanized mice. Tissue samples were fixed in 4 % paraformaldehyde, embedded in paraffin, sectioned at 4 μm, stained with hematoxylin and eosin (H & E) and Masson's trichrome and examined under a light microscope.
Sections of the kidney from wild type and Robo2 PB/PB mice (VUR and renal dysplasia) were immunostained for ROBO2 (the EnVision method). ROBO2 was detected using a rabbit polyclonal anti-ROBO2 antibody (1:50; Santa Cruz Biotechnology, Inc.).
For transmission electron microscopy, ureters and renal cortices were cut into small pieces (<1 mm3) and rapidly fixed in 2.5 % glutaraldehyde, then washed in phosphate buffer (pH 7.4), post-fixed in 1 % osmium tetroxide in the same phosphate buffer and dehydrated in increasing concentrations of alcohol and embedded in araldite. The ultrathin 70 nm sections were cut on an ultramicrotome, collected on copper grids, stained with uranyl acetate and lead citrate, and examined by transmission electron microscopy (Philips CM120). RFP and GFP fluorescence were monitored and photographed using an epifluorescence stereomicroscope (MZFLIII; Leica).
Real-time quantitative PCR (qRT-PCR)
The human metanephric kidney is functional at approximately 11 gestational weeks, which corresponds to embryonic stages E15.5-E16.5 in mice. Total RNA from E15.5 embryos was extracted using TRIzol (Invitrogen, USA), and cDNA was prepared with the RNA PCR Kit (TaKaRa). The mixture was incubated for 15 min at 37 °C and 5 s at 85 °C. The qRT-PCR was performed with an All-in-One qPCR Mix Detection Kit (GeneCopoeia) on the Mx3000P Quantitative PCR System (Stratagene) to detect Robo2 expression with the primers P1/P2 located within exons 1 and 2. GAPDH was used as an internal control. The reaction conditions were set as follows: an initial denaturation step of 10 min at 95 °C followed by 40 cycles (10 s at 95 °C, 20 s at 60 °C, and 15 s at 72 °C) with a final elongation step of 5 min at 72 °C. Quantification was performed by measuring the threshold cycle (CT value) and using a standard curve as a reference. The primer sequences were as follows: 5′-GCGGATCTTTATTCTTTTTGCG-3′ (sense) and 5′-TCCTTTTTCCAGTAGATGGTTG-3′ (antisense) for Robo2, and 5′-TGTTCCTACCCCCAATGTGTCC-3′ (sense) and 5′-GGAGTTGCTGTTGAAGTCGCAG-3′ (antisense) for GAPDH.
ROBO2 protein analysis
Kidney and brain proteins were extracted in RIPA buffer with freshly added 1 mM PMSF and 1× proteinase inhibitor (Roche). Next, they were quantified with the BCATM Protein Assay Kit (Pierce). Equal protein were separated by SDS/PAGE and transferred onto PVDF membranes. ROBO2 expression was detected using an anti-ROBO2 antibody (ab64158; 1:1,000) as the primary antibody and biotin-conjugated goat anti-rabbit IgG (CoWin Biotech Co; 1:10,000) as the secondary antibody. SuperSignal West Pico Chemiluminescent Substrate (Thermo 34080) was used for detection and GAPDH levels were used as loading controls (CoWin Biotech Co; 1:500).
Statistical analysis
All of the statistical analyses were performed using SPSS Version 19. A statistical analysis of the RNA results was performed using Student's t-test. The prevalence of categorical variables was analyzed using the Chi-square test. Statistical significance was set at P < 0.05.
Genetic characterization of Robo2 mutant mice
The PB transposon was inserted into the first intron of the Robo2 gene (Fig. 1a). In E15.5 embryos, quantitative PCR showed that transcript levels from homozygotes (Robo2 PB/PB) were reduced by approximately 50 % compared to wild-type (Robo2 +/+) littermates. There was no significant difference between heterozygotes (Robo2 PB/+) and wild-type littermates (Fig. 1b). Western blots showed that ROBO2 protein levels were decreased in the brain tissues and kidneys of adult Robo2 PB/PB mice (Fig. 1c), consistent with the quantitative PCR results. Expression of ROBO2 in the control kidneys was higher than in Robo2 PB/PB mice. Expression of ROBO2 in the refluxing kidney was higher than in the kidney of renal dysplaisa (Fig. 1d).
The incidence of urinary malformation in Robo2 mutant mice
The urinary systems of all the newborn Robo2 mutant mice were evaluated using a Vevo 770 micro-ultrasound. Some of the normal mice and all of the abnormal mice were followed to adulthood. All mice were tested for VUR. Urinary obstruction experiments were performed on the mice with hydronephrosis after the VUR test."
The Vevo 770 micro-ultrasound showed that 6.97 % (16/229) of newborn Robo2 PB/PB mice (8 males, 8 females) displayed diverse urinary malformations, including hydronephrosis, duplex kidneys and dilated ureter (Panel A from Fig. 2). They survived to adulthood without improvement (Panel B from Fig. 2). Compared to wild-type mice (Fig. 3a–d, i–k), non-dilating VUR was more requent in Robo2 PB/PB mice (Fig. 3e–h, l–p) (27.07 % versus 6.84 %, P = 0.000, Chi-squared = 19.65). There were no differences in simple VUR prevalence between age < 60 days and age > 60 days (27.07 % versus 32.50 %, P > 0.05, Chi-squared = 0.71) in Robo2 PB/PB mice, and no gender (P > 0.05, Chi-squared = 0.42) or side (P > 0.05, Chi-squared = 0.70) biases were observed. In summary, urinary malformations were found in 34.06 % (78/229) of Robo2 PB/PB mice, and non-dilating VUR (27.07 %, 62/229) was recognized as the most common abnormality. Grossly identifiable urinary malformations were not found in heterozygotes (N = 127).
Gross anatomy, histology and outcomes of the affected ureters and kidneys in Robo2 PB/PB mice with non-dilating VUR
Compared to the histopathological analysis of the intravesical ureter in a wild-type mouse without VUR (Fig. 3b–d), normal UVJ location and ureteral orifice without dilation (Fig. 3f) were observed in a Robo2 PB/PB mouse with bilateral VUR. Paraffin-embedded sections of refluxing ureters showed normal gross structure, but contained disorganized smooth muscle fibers at the level of the bladder (arrowheads in Fig. 3g). In addition, urothelium was found to partially occlude the lumen, but cellular atypia was not obvious, and cell layer was similar to that of the control, so malignant hyperplasia was not considered (Fig. 3g). Examination by transmission electron microscopy showed both normal general structure and reduced muscle cell population (Fig. 3h). The muscle layer showed intercellular edema (arrow in Fig. 3h) and intracytoplasmic vacuoles (arrowheads in Fig. 3h) in smooth muscle cells.
For a more detailed analysis of the kidney and ureter abnormalities, kidneys and ureters from newborn Robo2 +/+; Hoxb7/myr-Venus (Fig. 3i-k) and Robo2 PB/PB; Hoxb7/myr-Venus (Fig. 3l-p) mice were visualized by fluorescence microscopy. The kidneys were normal in size, and inner diameter of the refluxing ureter from a Robo2 PB/PB; Hoxb7/myr-Venus mouse was wilder than the contralateral ureter (Fig. 3m-n). However, the connection of the ureter relative to the bladder appeared normal (Fig. 3o–p).
To assess the progression of renal damage, we evaluated kidneys from adult Robo2 PB/PB mice with VUR and found no signs of RN (Fig. 4g–h). The kidneys were examined by transmission-electron microscopy, revealing a normal glomerular structure (Fig. 4i).
Gross anatomy, histology and outcomes of the affected ureters in Robo2 PB/PB mice with other CAKUT phenotypes
Dissection revealed that Robo2 PB/PB mice had diverse CAKUT phenotypes (Table 1), including UVJO (Fig. 2b), UPJO (Fig. 2c), duplex kidneys (Fig. 2d) and renal hypo/dysplasia (Fig. 2e-h). No obvious malformations of the genital tracts were observed in any genotype, and different phenotypes were observed within one litter. Of the grossly abnormal phenotypes, 62.5 % (10/16) were on the left, 37.5 % (6/16) were on the right, and none were bilateral.
The anatomy of the renal hypo/dysplasia in Robo2 PB/PB mice was characterized by two distinct patterns. One was hydronephrosis with a markedly dilated renal calyx and nearly complete loss of renal parenchyma (Fig. 2e–f and h). The other was an abnormally small kidney (Fig. 2g). Renal hypo/dysplasia was likely to coexist with other phenotypes (Fig. 2e–h). The dysplastic kidney was grossly cystic, and the contralateral kidney appeared grossly normal with no evidence of hydronephrosis.
Overall, 13/15 Robo2 PB/PB mice with grossly identifiable urinary malformations were found to have concomitant VUR. VUR was not observed in one mouse with renal hypo/dysplasia or in one mouse with malformed UVJs located laterally in the bladder (Fig. 2b). The VUR test was unsuccessful in one mouse that had severe malformation of the urinary tract.
In one mouse with unilateral hydronephrosis, reflux was first found in the contralateral ureter (Fig. 5a), and ipsilateral VUR (Fig. 5b) was then noted to occur with hydronephrosis. The affected mice displayed dilated renal pelvis without any dilatation of the ureter or the UVJ, indicating stenosis at the level of the UPJ. We subsequently examined the histology of the UPJ. The UPJ sections showed dilated ureters and distortion of the smooth muscle (Fig. 5c) compared to UPJ sections of wild-type mice (Fig. 5e). Transmission electron microscopy revealed separated muscle cells and excessive collagen in the UPJ (Fig. 5d).
The UVJ in Robo2 PB/PB mice with UVJO connected to a caudal site in the bladder close to the urethra, causing dilated ureter (Fig. 5h). Histological analysis confirmed the dilation of the distal ureter in the mutant and showed a thinner or a thicker layer of smooth muscle (Fig. 5g). Kidneys and ureters were illuminated because of the PB [Act-RFP] transgene and the Hoxb7/myr-Venus transgene. A Robo2 PB/PB; Hoxb7/myr-Venus newborn mouse exhibited dysplastic cysts in the left kidney, dilated left ureter, and abnormal left UVJ (Fig. 5i-j).
We described a new Robo2-deficient mouse model in the FVB/N background. FVB/N is a VUR-resistant strain [21]. A PB insertion partially disrupted the expression of Robo2, decreasing it to 50 % of the normal level. The realtime PCR results only showed a downward trend in Robo2 transcription in Robo2 PB/+ mice, possibly due to limited data. Robo2 PB/PB mice are the first Robo2-deficient mouse model to survive to adulthood while displaying non-dilating VUR, UPJO, and multiple ureters with blind endings. Most of these phenotypes have been reported in patients with ROBO2 variants [6, 10, 15, 22]. These urinary malformations mimicked human CAKUT and did not follow a typical Mendelian pattern, as do most CAKUT found in children. More than one CAKUT phenotype can be observed in the same individual (human or mouse), demonstrating the need for a careful assessment of the kidneys and urinary tracts [19]. We performed ultrasound screening and followed abnormal mice to adulthood. Because the prevalence of severe CAKUT phenotypes was relatively low, this approach improved the utility of this animal model. This analysis also indicated that Robo2 PB/PB mice preferentially developed unilateral, severe urinary malformations with little chance of spontaneous resolution. Because Robo2 has also been reported to play roles in the development of the brain [23], lungs [24] and heart [25], we examined the morphology of these organs, but we did not observe any obvious defects (data not shown).
The prevalence of urinary malformations in Robo2 PB/PB mice was 34.06 %, higher than the 7.87 % in Robo2 PB/+ mice and the 6.84 % in wild-type mice. Non-dilating VUR was the most common abnormality. Approximately 6.97 % of Robo2 PB/PB mice displayed severe CAKUT phenotypes that were not found in Robo2 PB/+ mice. After dissection, Robo2 PB/PB mice showed renal hypo/dysplasia at a rate of 4.37 % renal hypo/dysplasia, duplex systems at 2.18 % duplex system and VUR and other urinary malformations at 31.44 % VUR and other urinary malformations were found in Robo2 PB/PB mice. Conventional Robo2 mutants with severe degrees of gene deletion showed severe CAKUT bilaterally and with a higher incidence CAKUT [6]. Eliminating the effect of strain and genetic background differences, our results may suggest that the expression level of Robo2 (type of mutation) influences the penetrance and severity of VUR/CAKUT.
Primary VUR is a congenital defect with an UVJ anomaly. In secondary VUR, the valvular mechanism is intact and healthy to start with but becomes overwhelmed by raised vesicular pressures associated with obstruction. The obstructions may be anatomical or functional. UVJ histological defects were noted in Robo2 PB/PB mice. Grossly identifiable urinary malformations with concomitant VUR are unilateral in our models. Therefore, we assume that the VUR found in our models are primary VUR.
Additional factors might predispose mice to diverse urinary malformations. Genetic factors may be influenced by environmental exposure, leading to differing phenotypes despite identical gene mutations [26, 27]. Studies are needed to identify environmental factors that influence the penetrance and severity of CAKUT.
For a more detailed analysis of the kidney and ureter abnormalities, light microscopy evaluation and transmission electron microscopy, respectively, were used. Robo2 PB/PB mice were crossed with Hoxb7/myr-Venus mice to visualize the location of the ureters relative to the bladder. The results were compared with the controls and previously published results.
Unlike the dilated and incompetent UVJ that led to high-grade VUR and hydronephrosis in most Robo2 del5/del5 mice (5), most Robo2 PB/PB mice with simple VUR had normal macroscopic UVJ morphology and lacked hydronephrosis. We found disorganized smooth muscle fibers with intercellular edema and decreased numbers of muscle cells with intracytoplasmic vacuoles in the refluxing intravesical ureters. Most severe CAKUT phenotypes were accompanied by VUR. The ureter histological defects were severe in mice with VUR and renal hypo/dysplasia. Reduced and irregular ureteral smooth muscle staining was reported in Robo2 del5/del5 homozygotes before ureteral dilatation [8]. This mutant is the first mutant in which the histology of the refluxing ureter was similar to the following findings from the intravesical ureteric specimens of VUR patients: smooth muscle disarrangement [28, 29] and intercellular edematous and intracytoplasmic vacuoles [30, 31]. A significant correlation between the grades and smooth muscle lesions [31, 32], widely absent smooth musculature [33], and a replacement of muscle bundles with connective tissue [34] have also been reported in VUR patients. These structural changes may result in the deformation of the architecture of the ureteric wall smooth muscle layer.
Paraffin sections of refluxing Robo2 PB/PB mouse kidneys displayed no signs of inflammation or scarring. A previous study demonstrated that sterile reflux alone fails to induce reflux nephropathy [35]. This may explain why our mutants showed no signs of inflammation or scarring. Structural alteration in the podocyte foot process was found in a Robo2 podocyte-specific knockout mouse [8]. Our electron micrograph shows normal glomerular structure that may be due to the remaining expression of ROBO2.
It has been reported that chronic kidney damage is significantly related to congenital renal hypo/dysplasia, rather than to infections and VUR [36, 37]. Genetic factors may influence both the occurrence of dysplasia and the propensity for scar formation [37]. In our models, 4.37 % of homozygous mutants had histologically demonstrated renal hypo/dysplasia. Some of these may be secondary effects of hydronephrosis; however, some maybe congenital and require careful followed-up.
Both UPJ and UVJ histological defects were noted in Robo2 PB/PB mice. Histopathological analysis revealed dilation of the ureter, distortion of the smooth muscle, separated muscle cells with intracytoplasmic vacuoles and excessive collagen. These pathological findings may be secondary effects of hydronephrosis.
The ureteral smooth muscles play a crucial role in ureteral peristalsis [38] and UVJ formation [39]. Sufficient contraction of the ureteric muscular layer by closing the ureteric orifice prevents VUR [40]. It was speculated that the disorganization of the muscular layer leads to a malfunctioning valve system and causes VUR [32]. One study has indicated that congenital obstructions might be due to aberrant ureteric smooth muscle organization, resulting in impaired peristalsis [41]. Future studies are required to provide additional mechanistic insights into the role of Robo2 in ureteric smooth muscle cells in the development of UPJ and UVJ. Mechanistic studies investigating the role of Robo2 in smooth muscle cell function, proliferation, and migration are needed. Based on previous studies [42–44], we speculate that ROBO2 in ureteric smooth muscle cells interacts with Ab1, permitting Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) protein phosphorylation and thus participating in the cytoskeletal changes in smooth muscle α-actin.
VUR could be caused by defects in ureteric budding, ureter differentiation and elongation, peristalsis, UVJ formation, and bladder and urethra development [45]. Mackie and Stephens proposed the ureteric bud theory [46] that the UB must emerge from the ND at the precise site to invade the MM for normal kidney development to occur. If budding occurs rostral or caudal to this site, would lead to renal hypoplasia/dysplasia and ectopic insertion of the ureter into the bladder, resulting in VUJ obstruction and VUR. Multiple ureteric buds may lead to short duplex ureters, duplex kidneys, ectopic and dysplastic kidneys [45]. We speculate that quantitative differences in either Gdnf or genes within the Robo2/Slit network are most likely responsible for the urinary tract defects we observe. Other factors such as environment factors, may conribute to this process.
There are several limitations to this study. We did not examine how the different anomalies arose, and more work is needed to explain why Robo2 PB/PB mice preferentially develop unilateral severe urinary malformations.
The genetic background of the Robo2 mutants discussed may influence the penetrance and severity of the CAKUT phenotypes. Genetic factors may be influenced by environmental exposure, leading to differing phenotypes. This mutant is the first Robo2-deficient mouse model to survive to adulthood while displaying non-dilating VUR, UPJO, and multiple ureters with blind endings. VUR and other CAKUT in this mutant showed very low rates of spontaneous resolution after birth. We may uncover more deleterious mutations in the Robo2 gene if we focus on the DNA analysis of patients with these phenotypes. CAKUT associated with genetic mutations may indicate poor prognosis, and patients with Robo2 mutations should therefore be followed up closely. We first reported that the non-dilated refluxing ureters showed disorganized smooth muscle fibers and altered smooth muscle cell structure, more accurately mimicking the characteristics of human cases. Future studies are required to test the role of Robo2 in ureteric smooth muscle. Robo2 PB/PB mice could be used as an animal model to better understand both urinary tract development and the cellular and molecular mechanisms of Robo2 in the kidney.
CAKUT, Congenital anomalies of the kidney and urinary tract; H & E, hematoxylin and eosin; MCDK, multicystic dysplastic kidney; PCR, polymerase chain reaction; PUV, posterior urethral valve; qRT-PCR, Real-time quantitative PCR; RMV, real-time Micro Visualization; RN, reflux nephropathy; UPJO, Ureteropelvic junction obstruction; UTI, urinary tract infection; UVJ, ureterovesical junction; VUR, Vesicoureteral reflux
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Qian L, Liu J, Bodmer R. Slit and Robo control cardiac cell polarity and morphogenesis. Curr Biol. 2005;15(24):2271–8. doi:10.1016/j.cub.2005.10.037.
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Izol V, Acikalin AA, Kuyucu Y, Deger M, Aridogan IA, Polat S, et al. Ultrastructural and immunohistopathological evaluation of intravesical ureters via electron and light microscopy in children with vesicoureteral reflux. J Urol. 2014;191(4):1110–7. doi:10.1016/j.juro.2013.10.038.
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We thank F. Costantini from Columbia University Medical Center for providing the plasmid Hoxb7/myr-Venus.
This work was supported by the grants from national basic research program of China (973 program 2011CB944001) and national natural science foundation of China (81200487).
Availability of data and materials
All data underlying the findings are within the paper.
Authors' contributions
HX and X-hW conceptualized the study. J-lL performed the experiment. HX and LS obtained funding. J-lL and LS acquired the data. J-lL, LS and QS analyzed the data. HX, X-hW, J-lL, LS and QS contributed to data interpretation and manuscript preparation. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
All animal procedures were conducted according to the animal care and ethics laws and were approved by the Animal Care Committee of Institute of Developmental Biology and Molecular Medicine at Fudan University (2012020).
Additional information
Jialu Liu and Li Sun as co-first authors.
Rights and permissions
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
About this article
Cite this article
Liu, J., Sun, L., Shen, Q. et al. New congenital anomalies of the kidney and urinary tract and outcomes in Robo2 mutant mice with the inserted piggyBac transposon. BMC Nephrol 17, 98 (2016). https://doi.org/10.1186/s12882-016-0308-5
DOI: https://doi.org/10.1186/s12882-016-0308-5
- Robo2
- PiggyBac


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Thomas Jefferson University
Department of Information Services and Technology
Philadelphia, PA
Department of Information Services and Technology
Thomas Jefferson University is blazing its path to the future by creating a dedicated and customized space for its new Electronic Health Records Team—yet to be hired, but already setting the tone for the University's technological advancement via innovation and functionality, collaboration and creativity. The 10,000 sf space in Philadelphia is designed to accommodate multiple user groups and accomplish many operational goals at once. Above all else, it must attract and retain the right personnel to staff the department—becoming a workplace of both comfort and inspiration. Driving the project ahead? The ambitious seven-month time frame.
KSS, melding input from the Jefferson IS&T team and Epic Consultancy, came decision-ready to each meeting, identifying efficiencies and streamlining processes. The resulting designs and specifications are astute yet agile—a combination of multi-disciplinary, open team areas and quiet, more personal spaces. A fluid, open floor plan that also affords privacy. Specific program elements include a variety of conference and training rooms as well as huddle pods, hotel stations, a genius bar and café. Broad brush strokes paint a flexible, expressive work space, awash in natural light, generating a tangible sense of modern thought and deed to complement the department's historic surroundings.


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With a Caring Career, each day brings new and exciting challenges and rewards!
Become a gateway to full community inclusion with a Caring Career!
Share your favourite activities with the people you support!
The satisfaction of a Caring Career is immeasurable!
Please note: To apply for specific job postings on Caring Careers listed below, follow the instructions included in the individual job posting and forward your resume as the Employer has requested.Upload your resume here for consideration in a general applicant pool for the sector.
The Caring Careers website is an important tool for people who are looking to start a career in the disability services sector in Saskatchewan. This website contains the latest job postings in the sector, as well as tools and resources that provide a realistic view of what the job is all about. The disability services sector provides a wide range of employment for people with varying skills sets and competencies.
The work is all about supporting individuals with disabilities with their independence and relationships within the community and adapting based on individuals' specific needs and goals. It ranges from support with the day-to-day to the achievement of significant milestones such as obtaining employment or living independently. It is about dignity, respect, and everyone enjoying citizenship in our communities.
I previously worked in the school system with children with disabilities. When I made the move into this position I was really not sure what to expect. Well, now it is 5 years later and I would not …View Details
Watch this realistic job preview video to learn all about what it's like to work in the disability services sector.
The whole video is available on SARC's youtube channel.
View Details


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Wednesday, 1 August 2012
Mallzee - An Exclusive Pre-Launch Invite!
5 comments
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Chicken Fried Steak
posted in: Recipes | 0
This recipe came from our new Pal, Jeff. He has been helping us with our website, and we were using it as a test recipe. It is a great Classic Chicken Fried Steak recipe though and one of the ways … Continued


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Boarding Schools for Boys in Richmond, Virginia…
|More about Christian boarding schools in Richmond, Virginia:
Richmond is the capital of the Commonwealth of Virginia, in the United States. It is an independent city and not part of any county. Richmond is the center of the Richmond Metropolitan Statistical Area (MSA) and the Greater Richmond area. The population within the city limits was 204,214 in 2010, with an estimated population of 1,258,251 for the Richmond Metropolitan Area making it the third largest in Virginia.
Geographically, Richmond is located at the fall line of the James River, Convert south of Washington DC, Convert east of Charlottesville, and Convert west of Williamsburg. Surrounded by Henrico and Chesterfield counties, the city is located at the intersections of Interstate 95 and Interstate 64, and encircled by Interstate 295 and Virginia State Route 288.
The site of Richmond, at the fall line of the James River, had been an important village of the Powhatan Confederacy, and was briefly settled by English colonists from Jamestown in 1609, and in 161011. The present city of Richmond was founded in 1737. It became the capital of the Colony and Dominion of Virginia in 1780. During the Revolutionary War period, several notable events occurred in the city, including Patrick Henry's "Give me liberty or give me death" speech in 1775 at St. John's Church, and the passage of the Virginia Statute for Religious Freedom written by Thomas Jefferson. During the American Civil War, Richmond served as the capital of the Confederate States of America. The city entered the 20th century with one of the world's first successful electric streetcar systems, as well as a national hub of African-American commerce and culture, the Jackson Ward neighborhood.
Richmond's economy is primarily driven by law, finance, and government, with federal, state, and local governmental agencies, as well as notable legal and banking firms, located in the downtown area. The city is home to both the United States Court of Appeals for the Fourth Circuit, one of 13 United States courts of appeals, and the Federal Reserve Bank of Richmond, one of 12 Federal Reserve Banks. Dominion Resources, Carmax, Genworth Financial, and MeadWestvaco, Fortune 500 companies, along with Massey Energy and Universal Corporation, Fortune 1000 companies, are headquartered in the city, with others in the metropolitan area."Fortune 500 2009: States: Virginia Companies." Fortune Magazine via CNN.com. May 4, 2009. Retrieved on January 21, 2010.] Tourism is also important, as many historic sites are in or nearby the city.


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(TORONTO, ON – December 20, 2012) Toyota announced today that the Furia Concept will make its world debut at the 2013 North American International Auto Show in Detroit. This exciting new sedan concept will be revealed in the Toyota display at Cobo Hall on Jan. 14, 2013 at 2:25 p.m. EST, with a presentation by Bill Fay, group vice president and general manager of the Toyota Division.
About Toyota Canada Inc.
Toyota Canada Inc. (TCI) is the exclusive Canadian distributor of Toyota, Lexus and Scion cars, SUVs and trucks. Toyota is the first full-line manufacturer to make all of the elements of the Star Safety System standard on every vehicle starting in 2011. The Star Safety System includes Vehicle Stability Control (VSC), Traction Control (TRAC), Anti-lock Brake System (ABS), Electronic Brake-force Distribution (EBD), Brake Assist (BA) and Smart Stop Technology (SST). TCI's head office is in Toronto, with regional offices in Vancouver, Calgary, Montreal and Halifax and parts distribution centres in Toronto and Vancouver.


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Tommy Buford treated everyone the same, with kindness and respect
The call is one Phil Chamberlain remembers vividly, even after nearly 50 years.
Chamberlain — now the head tennis coach at Hutchison — was an aspiring tennis player in Australia when he got a call from one Tommy Buford, who was hoping to convince him to move halfway around the world and continue his career at Memphis State.
"He had such a Southern accent, like none I'd ever heard," laughed Chamberlain. "'Pheeeel, we have some reeeel nice facilities heee-yuh.' My whole family was gathered around the phone and he just knocked me over."
Buford eventually won over the young Aussie; Chamberlain came to Memphis to play for the Tigers in 1972 and has since made the city his home. But connecting with people is what Buford did, whether it was the top player on the ATP tour or someone working in the concession stand.
Chamberlain is one of many in the city who were touched by Buford, who died on Jan. 14 at his home in Boise, Idaho at the age of 83. He coached the men's tennis team at Memphis for 32 years and was most notably the tournament director for the annual event at the Racquet Club, which in its heyday brought players such as Jimmy Connors, John McEnroe and Andre Agassi to town.
Buford even knew how to reach personalities like those; in 1985, the pros voted him one of the tours top six officials.
"He would deal with John McEnroe the same way he dealt with the lady who bought just one ticket to the tournament," Chamberlain said. "He was frank and honest...but also very humble. He sat on many national and international-level (tennis) boards and he was never boastful."
Like Chamberlain, Peter Lebedevs left Australia to come to Memphis to play for Buford and later served as Buford's assistant director.
"He was a father figure to all of us," said Lebedevs. "You're coming from another part of the world and not sure how it's going to go...but he was just the nicest, most reassuring person.
"He was the epitome of a true Southern gentleman and treated everybody the same way, with respect. It was more than just about teaching us how to hit a ball, it went above and beyond that."
Former Tiger assistant lands Steelers' OC post
Randy Fichtner, who had two stints as an assistant at the University of Memphis, has been named offensive coordinator of the Pittsburgh Steelers. The 54-year-old replaces Todd Haley, who parted ways with the team following their playoff loss to Jacksonville on Jan. 14.
Fichtner served as a graduate assistant at Memphis from 1990-93 before returning in 2001 to serve as offensive coordinator under Tommy West. He held that position until 2006 and instituted a spread offense that maximized the talents of players like DeAngelo Williams and Danny Wimprine.
He had served as the Steelers' quarterbacks coach since 2010.
Arlington star advocates for student-athletes
Former Arlington and Ole Miss baseball standout Brady Bramlett is one of three student-athletes that will represent the SEC on proposed governance rule changes under the NCAA's Division I autonomy process at the NCAA convention in Indiana next week.
Bramlett, along with Missouri football player Brandon Lee and Kentucky basketball player Alyssa Rice, will work toward engaging and empowering student-athletes by giving them a voice and a vote within a transparent decision-making process. The Autonomy Conferences consist of the SEC, ACC, Big Ten, Big 12 and Pac 12.
Bramlett was a two-time Commercial Appeal Best of the Preps winner while at Arlington and led the Tigers to the 2012 Class AAA state championship with an 11-0 record and a 1.01 ERA. He was a two-year starting pitcher for the Rebels and was taken by Boston in the 13th round of the 2016 draft but elected to end his baseball career and pursue a master's degree in integrated marketing communications.
CBU hoops drops in coaches poll
Following losses to Valdosta State and West Florida, the Christian Brothers men's basketball team slips four spots to No. 22 in this week's NABC/Division II coaches poll. The Bucs are now 13-4 overall and 5-4 in Gulf South Conference play.
Jeff Larkin, a senior from Cincinnati leads the team in scoring at 18.7 points per game followed by sophomore Joe Laravie and junior Adam Dieball, both at 16.7. Alex Trentman leads with 6.0 rebounds per game while shooting 43.8 percent from 3-point range, second to Brad Miller's 46.7 percent.
Christian Brotheres High School grad Jacob Lyons has appeared in all 17 games as a true freshman, averaging 2.4 points.
The Bucs played at West Alabama on Saturday and return to Canale Arena to host Lee Thursday at 7 p.m. Home games are broadcast on WHBQ-AM (560).
Tiger softball picked sixth
The University of Memphis softball team has been picked sixth in the American Athletic Conference preseason coaches poll, finishing ahead of just UConn and East Carolina.
The Tigers finished fifth in 2017, going 7-11 in league play and 27-29 overall. They defeated USF in the first round of the conference tournament before falling to eventual champion and preseason No. 1 Tulsa in the semifinals.
Seven seniors return for this spring, led by Kyler Trosclair Klatt who finished second on the team with a .394 average to go along with 69 hits, 32 steals and a .454 on-base percentage.
Memphis opens the season against Georgetown on Feb. 9 in the Florida Gulf Coast Tournament in Fort Myers.
Grizzlies' voice wins top award
Memphis Grizzlies television play-by-play announcer Pete Pranica of Fox Sports Southeast has been voted 2017 Tennessee State Sportscaster of the Year by the National Sports Media Association.
Now in his 14th season with the Grizzlies, Pranica has been involved in three Emmy-winning productions: two as a commentator for NBC at the 2004 and 2008 Olympics and one with the Grizzlies. He began his career in 1990 with the Detroit Pistons before taking over play-by-play duties for the Portland Trail Blazers nine years later.
Pranica will be honored during the NMSA's Awards Weekend in Winston-Salem, N.C. on June 23-25. Other honorees will be National Sportscaster of the Year Kevin Harlan, National Sportswriter of the Year Adrian Wojnarowski and NMSA Hall of Fame inductees Thomas Boswell, Woody Durham, Bryant Gumbel and Dick Weiss.
Amazingly, Pranica and Harlan both began their careers at WGBP-FM, a 10-watt student station at Premontre High in Green Bay, Wis.
Sunday Sports Brunch is a weekly look at movers, shakers and newsmakers on the Memphis sports scene, from youth level to the pros. If you have an item of interest, please contact John Varlas at email@example.com.
well, he was a young man in that he was frank and honest Andre Agassi or whoever simple do it make sure it got done real humble guy national and international level ATP board always never very boastful I thought that was always something special deal with John McEnroe one ticket exactly the same sincerely good person everyone's feelings extremely emotional always break up tournament St. Jude poorly class very phone call such a southern accent real fine facilities her just knocked me over Buford no every coach in the country he was just played for more like you father particulary the foreign players NCAA bend rules her and there taken care of guy out and fight for him didn't express himself Xs and 0s simple terms hit it where the ain't find a way at the end of the day that works very different strategy believer that make all the team happy four years gradyate lot of us have stayed on very different happy for for the foreign players girlfriends happy to stay in perform. 1972. My mum doesn;t know what I was thinking came for the wedding more like a father than my own in any ways. concenrs and goals.


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The electoral battle in prestigious Patiala parliamentary constituency of Punjab is between a 'Maharani Sahiba' out to uphold the prestige of a royal family and a seasoned politician at a make-or-break point of his career.
The political landscape of the state changed after Chief Minister Amarinder Singh's wife, Preneet Kaur, 'Maharani Sahiba' as she is popularly known, won this seat in the 1999 Lok Sabha election capitalising on a divided Shiromani Akali Dal. The SAD chief, Prakash Singh Badal, then was at loggerheads with late G S Tohra.
Tohra is no more and the May 10 election will decide who can fill the void created by his death.
Preneet Kaur is facing a stiff challenge from SAD candidate and former finance minister Capt Kanwaljit Singh. Both have a clean image and enjoy a personal rapport with the voters in this constituency spread over Banur, Ghanaur, Dakala, Patiala, Samana, Shatrana, Rajpura, Sunam and Lehra Gagga assembly segments.
Preneet Kaur claims credit for bringing in NABARD-sponsored Rs 148 crore project for channelising the Ghaggar river, which causes massive damage to crops by overflowing in the monsoon.
"I dealt with the problems caused by the Ghaggar and money started flowing only after the Central Water Commission gave a no-objection certificate. The first installment of Rs 33 crore has come and another Rs 65 crore will come shortly. The project will be completed within three years," she told a visiting PTI correspondent on the sidelines of her public
meeting in Lehra Gagga assembly segment.
"Once the project is complete, the Ghaggar, which causes havoc during monsoons in around 300 villages falling in Ghanaur and Lehra Gagga assembly segments, will be tamed with embankments, check dams, drains and canals," she said.
However, if Preneet Kaur has development projects to her credit, she has to reckon with two big problems too. One is the presence of Bahujan Samaj Party candidate, Hargopal Singh Kashyap, who can cut into Congress votes. The second is her husband's rival in the party and deputy chief minister, Rajinder Kaur Bhattal.
Bhattal, MLA from Lehra Gagga, has not been campaigning for Preneet Kaur.
"She (Bhattal) has said she would campaign for me. However, she cannot give time presently due to a pending court case against her," Preneet said.
The Amarinder Singh camp knows too well that Bhattal's support could be crucial in Preneet's fight against the united Akalis. Congress workers at Lehra Gagga admit Bhattal's entry into electioneering for Preneet would certainly improve her prospects.
When contacted, Bhattal said she was busy with campaigning for party candidates in other parts of the state.
Another headache for the Congress is the Rajpura assembly seat, which is held by Raj Khurana, a known loyalist of Bhattal and a vocal critic of Preneet.
However, in Ghanaur assembly seat, held by Public Health Minister Jasjit Singh Randhawa, and in Patiala and Dakala constituencies represented by the chief minister and finance minister Lal Singh respectively, Preneet has a definite edge.
Weight was added to Preneet's campaign after former SAD MPs, Charanjit Singh Walia and Prem Singh Chandumajra, and SAD circle jathedar Rajinder Tohra deserted their party to join the Congress.
The outcome of the election in Patiala may well make or mar 61-year-old Capt Kanwaljit Singh's political career. He is a strong contender for the number two post in the SAD echelons of power.
"Kanwaljit Singh's win will consolidate his political fortune and promote his image as a leader of masses," his supporters say.
Hoping to storm the Congress citadel, Kanwaljit Singh said: "I found widespread resentment among people against Preneet."
He said he was seeking a mandate against alleged non-performance of the Congress in Punjab and non-fulfillment of pre-election promises made by the Amarinder Singh government.
"I am asking people to vote for progress, stability and peace which are there in NDA regime under the leadership of
Atal Bihari Vajpayee," he said.
"People are also sore over inaccessibility of Preneet and her poor performance in protecting Punjab's interest in Parliament," Singh said.
Rejecting Singh's charge, Preneet says: "On inaccessibility he (Singh) is speaking untruth. I have been working for ten
hours a day. I did not speak in Parliament because I spent more time in my constituency to help people."


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Snow in Sun Single
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I found this item on eBay. Bid early, bid often as they say. This confirms the fact that it is the next single.
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Obama Pressures Congress on Oil Subsidies
In the most spirited defense yet of his record on energy policy, President Barack Obama today dismissed his critics as playing politics by promising cheap gas without real solutions, and asked his supporters to pressure Congress to end subsidies for oil companies.
At an event in Largo, Md., Obama said people "running for a certain office, who shall go unnamed" have been "talking down new sources of energy" and have dismissed his plan to double vehicle mileage standards by 2025.
"I guess they like gas guzzlers," he said.
Obama said he expects Congress to vote in the next few weeks on ending subsidies for oil companies.
"It is time for this oil industry giveaway to end," he said. "I guess you can stand up for the oil companies … or you can stand up for the American people."
The president said the $4 billion a year in oil industry subsidies could be redirected to other sources of energy. He dismissed critics as pursuing an unsustainable oil-only future instead of investing in new technologies such as wind, solar and biofuels.
"There have always been folks like that. There have always been folks who are the naysayers and don't believe in the future," Obama said.
He singled out President Rutherford B. Hayes for questioning why anybody would want to use the telephone when it was invented, saying that's why "he's not on Mount Rushmore."
Obama again defended his record, saying that on his watch America has expanded drilling every year, but he said drilling alone can't solve the problem of high gas prices or reliance on foreign oil.
The president said the administration has approved dozens of new pipelines — although he did not mention the controversial Keystone XL oil pipeline — and has opened up millions of new acres on land and offshore for drilling.
"There are a few spots where we're not drilling," he said. "We're not drilling on the National Mall. We're not drilling in your house. … I suppose we could have 200 oil rigs in the middle of the Chesapeake Bay," he said to boos.
Obama said his administration is pursuing additional drilling in ways that protect health and safety.
Republicans, however, said the president continues to mislead the public by blocking drilling in many areas of the country on and offshore and blocking the Keystone pipeline from Canada to Texas. And they pinned the blame on Obama for the doubling of gas prices on his watch.


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The Hawai'i Tourism Authority (HTA), the state's tourism agency, has issued a request for proposal for airline services to accommodate passengers who are unable to secure alternative flights following the shutdown of ATA Airlines' operations on April 2, 2008. ATA served the Hawai'i market with 12 daily flights with routes to Los Angeles, Oakland, Phoenix, and Las Vegas.
Following ATA's announcement, HTA immediately contacted other airlines with service to the West Coast, but due to the recent closure of Aloha Airlines' transpacific flights, many carriers are already operating at full capacity and unable to absorb the void left by ATA.
HTA then held an emergency meeting of its board on April 4 to approve the budget needed to help underwrite flights and fund other potential activities to assist travelers.
"Our primary goal is to address the needs of those passengers who could not find an available seat," said Rex Johnson, president and CEO of HTA. "We are continuing to work with all stakeholders and involved parties to provide assistance so that passengers holding ATA tickets make it to their desired destination safely and within a reasonable time. This includes visitors trying to return to the mainland as well as Hawai'i residents on the mainland who are trying to return home."
If available, HTA will help underwrite flights for the next few days due to the unique conditions in this situation. Various hotels statewide are offering special considerations to stranded passengers for extended or emergency accommodations.
"We are looking at addressing the needs that the situation with ATA and Aloha Airlines have caused. While we anticipate some short-term impact to Hawai'i's air service, we are hopeful that there will be minimal long-term disruption," said Johnson.
The HTA is working with the Hawai'i State Legislature, the Hawai'i Visitors and Convention Bureau (HVCB), Governor's Tourism Liaison and the Hawai'i Hotel and Lodging Association (HHLA) to address long-term solutions.
ATA customers can check HVCB's Web site www.gohawaii.com/ata for information on how to contact other airlines serving ATA's markets. It is recommended that passengers get in touch with these airlines to determine seat availability and booking instructions before going to the airport. ATA updates are also linked to HTA's Web site www.hawaiitourismauthority.org and all of HVCB's island chapters' Web sites (O'ahu Visitors Bureau, Kaua'i Visitors Bureau, Maui Visitors Bureau, and Big Island Visitors Bureau).
Neighbor Island Web Sites:
• Kaua'i Visitors Bureau: www.kauaidiscovery.com
• Maui Visitors Bureau: www.visitmaui.com
• O'ahu Visitors Bureau: www.visit-oahu.com
• Big Island Visitors Bureau: bigisland.org
Source: Hawaii Tourism Authority


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Jan. 30 is Lou Henson Day in Doña Ana County
Doña Ana County commissioners during a meeting Tuesday approved a resolution declaring Saturday to be Coach Lou Henson Day throughout the county, recognizing the career accomplishments of the renowned Aggie basketball coach and and National Collegiate Basketball Hall of Fame Class of 2015 member.
Commissioner Billy Garrett said he was a team manager in 1961 for one of Henson's teams. Garrett said Henson was both tough and kind. And he was "not just a winning basketball coach," but also helped with racial integration.
Henson wasn't at the county commission meeting because of a conflicting doctor's appointment, but met later with New Mexico State University Athletics Director Mario Moccia during lunch at the Fulton Center on the NMSU campus.
Henson will be honored during Saturday's men's basketball game at the Pan American Center. Tip-off is at 7 p.m.