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Dial Indicator Preload, Use and Reading
Preload of dial indicator - how to use this instrument of linear measurement
On this simulator you will learn the science of utilization of this instrument of measurement, interacting with the instrument, with the fuse or with the contact point (see main components).
In this learning object, you can drag and drop the requested instrument to preload, you can also rotate the 'display' (by dragging and dropping the ring in a circular motion) so that the 'zero' line aligns with the (By clicking on the eye icon) and override the spring effect of the instrument (by clicking on the 'lock' icon).
Dial Indicator preload, use and reading
Fixing the dial indicator
There are dozens of dial indicator models in use in the metal-mechanic industry, all of which have in common suitable places to attach them to appropriate devices or accessories. Dial indicator clocks are generally not manual measuring instruments except when associated with suitable handheld devices.
Applying the preload
When the dial indicator watch is 'out of use', a spring pushes its spindle and the contact tip until the end of the stroke, to take the measurement properly it is necessary to touch the contact tip on the surface to be measured, or the control , And to 'push' the watch so as to give a spring load and space for the spindle to go back and forth, without running the risk of the contact tip moving away from the surface or the 'tap' spindle at the end of stroke, Forcing the instrument against the fastening device.
Reseting the dial indicator
The preload application makes the pointer of the dial indicator turn, some fixing acessories has devices that allows a fine adjustment, bringing the pointer until the zero mark of the display, however, the dial indicators has a ring that allows turning the display to align tha Zero of display with the main pointer.
Measuring with dial indicator
This topic is explainned in the page: Virtual Dial Indicator – simulator in hundredth millimeter (0.01mm)


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Albany Theatre Garden Monday 16th February to Wednesday 18th February 1000-1800 - drop-in HAXBOX are interactive exhibits riffing around the idea of gaming and leisure in a post apocalyptic world. Asteroid strike? The internet is broken, your xStation playBox has melted, Splutify is silent, and whoTube is just grey noise. If we scour the deserted streets, the smashed-up shops and burned-out buildings we might get enough stuff together to start over… …introducing post apocalyptic innovation from redLoop @ Middlesex University, bringing you entertainment in the survival village. Play Phigital Wiff Waff - a super mash-up of bicycle powered, old school pong and high-tech projection mapping wizardry. Bust some choonz on the redLoopz, zombie hip-hop machine - beats, bass, samples and loops for you to program, configure and trigger.
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What happened at SMASHfestUK 2017
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Christmas Wishes For You
229.00 kr
If you could choose four wishes for a child this Christmas, what would they be? A timeless, enchanting personalized story with a lifetime of meaning. $1 from every book goes to Make-A-Wish®.
Perfect for ages 0-8
Preview all 32 pages before buying
Ships worldwide within 48 hours
"A gorgeous book, with a beautiful message." – Charlotte
Create and preview the child's entire book
"This book is absolutely beautiful. I love that I can choose the wishes specifically for my child. I know my daughter will keep this forever."
Loved by Alison
A spellbinding personalized Christmas book
From the very front cover, a child's name is magically woven throughout the book. Nothing beats the moment of wonder when they realize the story is all about… them!
Choose four wishes for a child you love
Compassion, Adventure, Strength, Friendship, Wisdom, Joy, Patience, Wonder, Laughter – choose the four Christmas gifts you think matter most for a child, to guide them for years to come.
Make it your new festive family tradition
Written in charming rhyming verse, and with illustrations that transport you to a wintry wonderland, it's the perfect story to share together every Christmas.
Wondrous size and quality
Each personalized children's book is printed on thick, luxurious paper that's partly recycled (FSC-certified) and built to last. Available in softback and hardback, for a truly one-of-a-kind keepsake.
Every book includes a gift to charity
When you personalize a book for a child you love, you're also helping grant a life-changing wish for a child with a critical illness. $1 from every sale will go directly to the Make-A-Wish® charity.
Absolutely beautiful
This book is absolutely beautiful. I love that I can choose the wishes specifically for the child - I know my daughter will keep this forever!
Beautiful message
A gorgeous book, with a beautiful message
Great for children AND parents
A great Christmas gift that children (and their parents) will love!
Gift to treasure
A beautiful Christmas gift to treasure
Our new Christmas tradition
A really great book to read on Christmas Eve that will become our new Christmas tradition!


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EZ Cash loans, much like their name, are a way to get a small amount of cash quickly and pay it off in a relatively short amount of time (typically a 6 month term). Each Freedom Line of Credit advance requires a 10% deposit into a restricted access savings account at the Credit Union to help you avoid borrowing in the future. Using data from five large lenders, Mr. Hackett calculated that the new rules would reduce their loan volume about 70 percent and their revenue by 80 percent.
You instead may be able to obtain financial assistance from military aid societies, such as the Army Emergency Relief, Navy and Marine Corps Relief Society, Air Force Aid Society, or Coast Guard Mutual Aid. Is not a lender, does not broker loans, and does not make loan or credit decisions.
Short term cash loans are an expensive way to borrow and it may be more suitable to look into longer term loans available at 118 118 Money. Your credit score will play a big role in whether or not you'll qualify for products like balance transfers or competitive personal loan offers.
Cordray suggests that one solution is to require lenders to make sure borrowers can repay a loan on time, along with their other monthly expenses. Worse yet, she says, borrowers have almost no choice but to roll over their loans again and again, which jacks up the fees.
CASH CENTRAL came through in the nick of time and my insurance policy is still in effect! Restructuring means your lender could extend the term of the loan to reduce the cost of monthly payments, or reduce the frequency of payments being made. Allied had me qualified quickly and I would recommend anyone interested in a payday loan to give them a call.
Quick online Application
Apply Now


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Greyhounds are the hunting dog of choice in Spain, but only while they're useful. After that, it's customary to do away with the dogs entirely in certain parts of the country -- and to do so as gruesomely and inhumanely as possible. According to VICE, approximately 50,000 greyhounds are expected to be abandoned or killed at the end of this season:
If the dogs are too old to hunt or too hungry to afford until the beginning of the next hunting season, then a greyhound owner might choose to hang them instead. Some throw them into wells or cremate them. Running them over with a car seems to be catching on too, while a few years back there were several cases of hounds being tied to the backs of trucks and dragged along the road.
Beatriz Marlasca, president of a Spanish NGO dedicated to greyhound protection, told VICE of the intense cruelty she has seen toward former hunting hounds, including abandoned dogs eating garbage in the streets and pits full of greyhound corpses. Often, the abusers are difficult to track down or convict, Marlasca explained, and most of the offenders walk free. "Sometimes you feel like you've had enough," she said, "but then you realize that you can't give up."
We go through a wide range of feelings in the process: indignation when we find a particularly terrible case and horror when we think how much the dog must have suffered. But the bond between the dogs and those who take care of them grow... Then they're adopted, and you feel proud and happy to think there's one fewer dog on the streets. We've had countless cases, some of them really miraculous.
And Marlasca says the miraculous cases are the ones that make the entire endeavor worth the pain and anguish. "Some time ago, we found a female hound that had been hanged but was still alive," she said. "After a month under intensive care, she fully recovered and was adopted in France. That happy ending is what drives us. It's priceless to see the dog with a new family, sitting on a sofa, after all she went through. It's hard and beautiful at the same time."
You can read the rest of Marlasca's interview here. For more information about her organization, Asociación Baasgalgo, you can visit their webpage. To find out what you can do to help end the brutality toward Spanish greyhounds, including adopting, fostering, or donating to help an abused dog, visit SOS Galgos.


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Popping up everywhere this spring are new and intriguing items from local designers and labels. Survey the local fashion landscape, and you'll find everything from lacy bell bottoms to hipster-friendly plaid shirts, a ready-for-anything hoodie with a stylish silhouette to hats embellished with original artwork. Read on for the scoop.
New for summer, silk blouses by FIDM almum Amy Milbrandt are produced in the Bay Area, while the up-and-coming designer's skirts are available by custom order. Currently apprenticing with Colleen Quen Couture, Milbrandt designs with sleek simplicity in mind.
"I really wanted my blouses to have a modern look and ease about them, but also feel luxurious and able to be dressed up or down and pair them with jeans or a skirt," she says.
Her blouses will be available for purchase online once her online store launches next week.
Shirts from recently-launched Pladra enhance basic plaid with contrasting print cuffs, collars and yokes featuring outdoorsy scenes befitting the label's NorCal style. Available for men and women, the shirts are handmade in San Francisco.
Parsons graduate Sarah Liller has launched her debut collection, which mixes innocent ruffles and lace with daring cut-out details and bohemian sensibilities. You'll find an extended look at her designs, which range from lace shorts to dresses with exposed shoulders, available here.
In new jewelry from San Francisco's Kris Nations, a Southwestern vibe meets Art Deco details for a look that's earthy, yet sophisticated. Recycled metals used to create the line's made-in-the-USA pieces land these accessories in eco-friendly territory.
The new handbag line from San Francisco designer Lisa Davey, Holly Scott impresses with streamlined shapes and freedom from fussy details. Clutches gain versatility through functional touches such as fold-over designs and exterior zipper pockets, while the roomy totes in classic shapes are fitting for daytime adventures.
on Saturday at the Haight Street store." src="http://www.sfgate.com/blogs/images/sfgate/chronstyle/2011/04/28/goorin-bros-1333-minna-2011-SM623x416.JPG" width="623″ height="416″ border="0″
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San Francisco hatmaker Goorin Bros. has released the spring 2011 installment of its popular 1333 Minna Artist Collection, featuring original works by such artists as Reyes, Steel, Amanda Lynn and Luke Stuart. To mark the occasion, the company will celebrate starting at 4 p.m. on Saturday at its 1446 Haight St. store with music, drinks and limited edition posters signed by the artists offered to those who purchase a hat. The evening continues with a reception (8-11 p.m.) at SoMa's Cassel Gallery.
Known for high-performance gear for outdoor adventures, Dogpatch-based Triple Aught Design added women's pieces to its offerings this spring. Founded by Army vet Patrick Ma, the company puts an emphasis on materials that will withstand tough treatment, but does so without abandoning style. For example, the Artemis hoodie, one of the first women's items to become available for sale, is made of Australian Merino wool and features thumbholes in the sleeves, sizable pockets and a contoured silhouette for curve-skimming appeal.
San Francisco's Betabrand, best known for its cheeky marketing and horizontal-wale corduroy men's pants, is out this week with the Harvester Collection, an eco-minded line of pants made of 100 percent organic cotton canvas and named after seasonal fruits and vegetables. A new item in the collection is slated to become available each month, with shirts on the horizon for a mid-May release.


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Rating: NNNNN
Lee's Palace, Fri, October 20
It wasn't quite J.K. Rowling at the Skydome, but getting a few hundred folks out to Lee's Palace for a poetry reading was impressive anyway.
Brooklyn wordsmith Saul Williams's dramatic performance left the club silent Friday. Backed by a four-piece band including a DJ and cellist, he confirmed his reputation as his generation's Gil Scott-Heron. Voice booming and with a big book of rhymes in his hand, Williams alternated between straight-ahead, a cappella readings and frantic, hiphop-inspired rants that drew on dancehall shouts, snippets of Rakim and Public Enemy verses and Williams's own impressive beatboxing. It was as devastating as it sounds.
In fact, the only time the crowd did interrupt Williams was to shout out requests for older pieces like Twice The First Time. When was the last time you saw that at a poetry reading?
El Mo, Tues, October 17
The sight of fire trucks in front of the El Mocambo can't be a good thing.
Upon closer inspection, though, it turned out there was no disaster and no further brawls involving booker Dan Burke. Instead, the commotion concerned the tremendous number of people hoping to get in to see Wesley Willis. An above-capacity crowd for a Chicago schizophrenic poet whose preferred form of introduction is a solid head butt. Who knew?
With the law threatening to shut the whole thing down, the doors were closed and some ticket-holding fans were sent home empty-handed. The wise ones simply went downstairs to hear Los Angeles soul demolition crew the Bellrays bring the noise.
With her KISS shirt on and outrageous afro shaking above the crowd, shouter Lisa Kekaula did her best Tina Turner (Ike era), and men in the crowd took their clothes off. No insanity, but impressive nonetheless.
Another record store shuts down
Following the sad demise of local record retailers Who's Emma?, Happy House and just recently Driftwood, Bloor West punk emporium Full Blast is the latest in a growing list of vinyl vendors to call it quits.
For the time being, Full Blast will be open during its regular hours of operation to sell off the remaining stock of indie rock and hardcore CDs and records. But the lease will be up on November 15, and doors will close at the 605 Bloor West hangout for good.
While Full Blast manager Louanne Voskans is clearly troubled by the turn of events, she felt the time was right to call it a day.
"It was a really difficult decision for me to make," says Voskans. "But it just turned out that my lease was up at the same time that my business loan for the place would finally be paid off, so it seemed like the right time to take a break.
"I need some time to myself to think things through so that if I reopen a store I'll be able to do things better for my customers and myself. In the meantime, I plan to continue running my business as a mail-order operation. People can reach me via e-mail at firstname.lastname@example.com, and I'll soon start sending out regular lists of available CDs and records.
"I really appreciate the support of everyone who came to Full Blast over the years and I know I'll miss seeing everyone, but I'm looking forward to spending some quality time on the couch in front of the TV."


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The Move Operation allows you to transfer inventory items from one storage area/warehouse to another. US customers can use this function to move bonded items to tax paid storage areas.
From the Inventory menu, choose Move or click the Operations button at the bottom of any Stock item overview.
Enter the quantity to be moved.
Click the Routing button (forklift) next to Route from. If all of this stock item is stored in one location, the full amount will auto-populate. If it is stored in multiple locations, you will be asked to choose from which location you are moving. If you need to move from multiple locations to a single new location, click the Add line button.
Click the Routing button next to Route to.
Choose your new location from the Storage Area by clicking the Search button.
If there is only one lot, use the Search button; that will automatically fill in existing Lot. If you need to create a new lot, use the Add New [+] button.
When you are satisfied, click the OK button.


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Welcome American Express Shareholders!
Cost Basis Calculation
Netbasis will automatically calculate your adjusted cost basis for American Express, or any American Express-affiliated company.
A purchase of one Netbasis transaction for $25.00 will provide you with:
Netbasis contains all of the pricing and dividend history including all splits, mergers and any other corporate action. Netbasis does not contain your personal purchase and sale history.
You will need to enter:
This information can often be found on several types of investment documents including: broker and transfer agent statements, your 1099 statement or pay stubs.
|$25.00 / Calculation!
|Calculate Cost Basis
If you have questions please email us or call us at 1-888-80-basis.


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At the Marine Physics Laboratory we are always trying to come up with novel ways of monitoring and recording the physical events that occur in the waters around us. If you would like more information on any of our currently produced products please do not hesitate to contact us.
We have spent many years developing the best methods to measure water turbidity and accurately gauge the deposition of sediment in marine environments. The instruments we currently manufacture also include sensors for light pressure and temperature and can be connected to telemetry to provide real-time data without needing to go to site.
Marotte Current Meter
Inexpensive and reliable measurement of current has been a request of many researchers. Our new Marotte current meter is able to record water velocity within ± 0.05m/s and direction within ± 5° for the fraction of the cost of an acoustic-doppler type instrument. Inherently simple by design, the Marotte logs the tilt of a buoyant 'lollipop' in the water. This logged data is then processed to determine water velocity. See: Technical Specifications
Spider Monkey Water Sampler
Most recently our suspended sediment studies have led to the development of this novel water sampler. Used in conjunction with a 1000 Series Nepholometer, the Spider Monkey can take up to 12 water samples at predefined times or at predefined turbidity levels. See: Technical Specifications
Lotus River Drifter
Conceived for the study of suspended sediment in rivers during flood events, the Lotus aims to be a low cost, drifting turbidity logger. Initial trials in flooding North Queensland rivers have proven a good recovery rate and the first batch of 100 drifters is currently under construction. See: More information


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James Faeder
James Faeder
Associate Professor
Monte Carlo Cell
James Faeder
Associate Professor
New CellBlender versions for OS X, Windows, and Linux are available from our Downloads page.Oct 5, 2016: MCell 3.4
New MCell versions for OS X, Windows, and Linux are available from our Downloads page.Febrary 24, 2016: New workshop
The Cell Modeling Workshop 2016 will be held from June 1st-3rd at the Pittsburgh Supercomputing Center.July 9, 2015: CellBlender 1.0.1
CellBlender 1.0.1 is available from the MMBioS downloads page.May 11, 2015: MCell 3.3
New MCell versions for OS X, Windows, and Linux (including 64-bit) are available from our Downloads page.Febrary 16, 2015: New workshop
The Computational Methods for Spatially Realistic Microphysiological Simulations 2015 workshop will be held from April 27th-29th at the Pittsburgh Supercomputing Center. Additional information (PDF).October 24, 2014: Postdoctoral Research Position Available
Postdoctoral Research Fellow position available in the Biomedical Applications Group at the Pittsburgh Supercomputing Center (PSC).April 27, 2014: CellBlender 1.0 Release Candidate 3
CellBlender 1.0 RC3 is available from our Downloads page.
© 2013 mcell.org. | Design by FCT.


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This series is equipped with auto paper-fixing function . The auto gliding blade makes the cutter sharp and durable. Thescale can be indicated by centimeter and inch. It is a good way to avoid the removing of paper.It is different to the regular cutter. More safety and convenient. We can make the right one to meet your requirement.
General Purpose Cutter (aluminium Alloy)


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- Their products are many times the same products you will find in other stores.
- Sometimes products hit the dollar stores because of damage or lack of freshness…
- …BUT they are often just offered cheaper because they operate on slimmer margins than grocery stores.
- Many items were surplus/over-stock items from a company. That company might even sell their items at a loss to the dollar store, but it's cheaper than paying taxes, paying for warehousing to hold the items, and frees up their warehouse space for items that will make them profit.
- Many times their surplus items they buy are surplus because it was last season's color, or an old style of something.
- Dollar stores will often buy and sell anything that's cheap. They will buy entire containers of goods at auction from China, without knowing what's in them, and will sell whatever ends up in that container. Surprise!
- Sometimes their products are name-brand products that don't advertise any more, such as Bon-Ami Cleanser (as explained HERE)
- Sometimes their products are name-brands products, only smaller. Irish Spring soap, for example, is the exact same bar you'd find in the grocery store…only smaller. Same with their ziplock bags. The box will only cost you $1 (rather than $2 or $3), but you might only have 7 or 10 bags, vs 20.
- Dollar Stores can sell the SAME items as groceries stores for cheaper, because they have C or B-class locations, so they pay much lower rent than class-A locations.
- They also have a small, low-wage staff, usually only about 10 jobs per store (as read HERE)
2. Pool Toys.
7. Office and Classroom Supplies:
8. Some toys.
So there you have it! Those are only SOME of the things that I regularly buy from the dollar store. Now, the big question…
- Most food. Mainly the canned food, but you have to be careful with bagged food, baking goods, seasonings, frozen. Much of it is higher price than the grocery store. So just grocery shop like I do and you'll get things MUCH cheaper…and probably better quality. However, I will say the dollar stores often times carry brands or items that grocery stores no longer carry which is helpful. But in terms of every-day groceries, I say avoid the dollar store!
- Vitamins and medication. I'm sorry, I just don't trust them. But ultimately, the price-per-pill isn't that great. Buy that kind of stuff at Costco and you'll get a better value.
- Electronics of any kind. Do I really need to explain why a $1 electronic is not a good investment?
So next time you go to one of my favorite hangouts (aka the dollar store), be aware of what you're tossing into that little green cart so you don't walk away carelessly spending your hard earned dollars.


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Amy Atkins, host of Blank Verse, interviews me about "meta-poetry":
Amy Atkins, host of Blank Verse, interviews me about "meta-poetry":
My Montreal International Poetry Prize long-listed poem, "Smoke," is now live and available to read on the Montreal Prize site.
My poem, "Smoke," has made the 70-poem longest of the Montreal International Poetry Prize, which received about 2,000 entries. The list is here:
Congratulations, as well, to NZ poet Johanna Emeney for making the list.
Harry Ricketts reviewed Native Bird with fellow Hoopla collections Bones in the Octagon (Carolyn McCurdie) and Mr Clean & the Junkie (Jennifer Compton) on Radio NZ's Nine to Noon. Listen here: http://bit.ly/1FNNjhy
I'm excited to announce the publication of my third poetry collection, Native Bird, with Makaro Press. It officially launches this week with events in Palmerston North, Wellington and Dunedin as part of Makaro's Press's Hoopla Series, alongside excellent collections by Carolyn McCurdie (Bones in the Octagon) and Jennifer Compton (Mr Clean & The Junkie).
About Native Bird
In his third poetry collection, award-winning poet Bryan Walpert—who arrived in New Zealand from the U.S. a decade ago—writes of what it's been like to be an observer or "birdwatcher" in a land whose physical and cultural geographies he is still learning to name. With his trademark precision and insight, Bryan weaves meditations on the life and songs of birds into his observations on living as a new settler in wind-charged Manawatu. Working at the shifting borders between homes and hearts, prose and poetry, call and song, this is an arresting collection that speaks to us all.
The book is available to purchase online here.
Or if you're in NZ, come along to one of the three Hoopla Series Launches:
Palmerston North
Thursday, 16 April, 6:30 p.m.
Palmerston North Central Library
Sunday 19 April, 4 p.m.
Poetry at the Fringe
The Fringe Bar, Allen Street
See: https://www.facebook.com/pages/Poetry-at-the-Fringe/331943986831
Wednesday 22 April, 5:30 p.m.
Dunedin Public Library
My poem "Objective Correlative," which was a finalist for the Rattle poetry award last year, is featured online today on Rattle:
I've started blogging about life as an American immigrant to New Zealand at The Lazy Gardener, http://nzlazygardener.wordpress.com/


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The 1982 United Nations Convention on the Law of the Sea (UNCLOS) gave rise to a wide range of new rights and obligations in international law. In particular, the Convention conferred on maritime states a new range of rights in relation to their continental shelves and the right to claim an Exclusive Economic Zone (EEZ) which, unless compressed by geographical considerations or the rights of neighboring states, extends 200 nautical miles offshore.
So far as EEZs are concerned the Convention has led not only to a large number of bilateral and multilateral maritime boundary negotiations between states, but also to a considerable body of state practice and case law. A great deal of the work of EEZ delimitation is quite technical in nature, but the subject has major political, economic, and administrative implications. States that have established EEZs are entitled to a wide range of rights connected with exploration and exploitation of the economic resources of the zone.
Oil is valuable, and even a small oil field can make a substantial difference to the economic life of the citizens of the coastal state to which the EEZ belongs. Because so much can be at stake, maritime boundary disputes in relation to areas thought likely to be rich in oil can and do lead to serious international tensions - sometimes to military hostilities.
The waters of the Gulf of Guinea are rich in hydrocarbons. With a population well in excess of 100 million and a lengthy coastline, Nigeria is the largest and most influential state in the Gulf. In recent years, under the leadership of President Obasanjo, his Minister of State, Hon. Dubem Onyia and Alhaji Dahiru Bobbo, Director-General of the National Boundary Commission, Nigeria has made impressive strides in resolving land and maritime boundary issues with almost all its neighbors, a process which is almost invariably beneficial to both sides.
Nigeria has also played an important part in the creation of the Gulf of Guinea Commission, which can be expected to play an influential part in the coming years in promoting international development and cooperation in the region.
In almost all cases, Nigeria's progress on its international boundaries has been made through the active pursuit of friendly bilateral negotiations with its neighbors, particularly Benin, Niger, Equatorial Guinea (whose capital, Malabo, is situated on the island of Bioko - the former Fernando Po) and the archipelagic state of São Tomé e Príncipe.
Relationships have been significantly difficult with only one of Nigeria's neighbors, Cameroon, where disputes on a range of issues have now been referred to the International Court of Justice in the Hague for its decision.
Joint Development Zone
When Nigeria came to negotiate a detailed maritime boundary with São Tomé e Príncipe (STP) it was established that the two sides had widely different ideas about the appropriate position of the dividing line between their respective EEZs. STP claimed archipelagic status under UNCLOS, and put forward a huge EEZ claim, which overlapped the claimed EEZs of all its neighbors.
The relevant Articles of UNCLOS provide for an equitable solution to the delimitation of both the continental shelf and the EEZ. The only guidelines given in the Convention to states faced with these problems is to refer them to the Article in the Statute of the International Court of Justice, which sets out the applicable sources of international law. This leaves substantial room for the parties to argue about what is appropriate according to the particular facts of any given case.
With so much room for dispute, the initial negotiations can easily lead to deadlock, neither government being willing to be seen to give way to a foreign state on an issue of potentially vital significance to the protection of the national patrimony. Such a deadlock can lead to the sterilization of economic development until resolved, which can take years, or even decades.
To their considerable credit, the two governments decided that they could do better than that. Building - paradoxically - on an agreement to disagree, they signed a treaty identifying a zone of disputed ocean territory in respect of which both sides' respective EEZ and continental shelf claims would be fully maintained. They would develop jointly, benefiting in the proportions spelled out in the treaty itself.
The two sides had to agree not only on the proportionate benefits that each would obtain, but on the role of a Joint Ministerial Council and a Joint Authority to govern the zone, on regimes for the exploration and development of hydrocarbon and other resources, for taxation, security, environmental protection, health and safety issues.
Unlocking development
A great deal of work has been necessary in order to structure and document these matters in a manner acceptable to both sides. But the prize is a great one, namely the unlocking of economic development in a valuable part of the maritime space claimed by the two states.
There have been a number of Joint Development Zones in other parts of the world, prompted by a range of political and economic imperatives, but the Nigeria/São Tomé e Príncipe Joint Development Zone is the first in the Gulf of Guinea, and has been designed in such a way as to take particular account of the needs and practices of the two states and the geographic and economic realities of the life of the region. It is an impressive achievement.
When Nigeria and Equatorial Guinea came to delimit their common maritime boundary, they were able, after several years of negotiation, to agree a single maritime boundary, but were left with the problem of what to do about oil fields that straddled the boundary.
Cross-boundary unitization
The agreed boundary left the Ekanga oilfield on the Nigerian side of the line, and the larger Zafiro oilfield that was already in production on the Equatorial Guinean side. Nigeria stipulated that there must be an agreement, that the two oilfields, which form a single structure straddling the new boundary, be exploited jointly for the benefit of both states and their oil concessionaires in pre-agreed proportions.
There are numerous precedents for cross-border unitization of straddling oil deposits, notably in the North Sea. Each case, however, is different, and much will depend on the type of joint exploitation contemplated, the structures of the economic ventures participating in it, and the overall wishes of governments.
In the present case, negotiations are now well advanced for the unitization of the deposits on a mutually satisfactory basis. These negotiations, as is only to be expected, have sometimes been quite difficult, but both sides have a considerable incentive to agree, namely the possibility of implementing the wider treaty.
Some two hundred international maritime boundaries remain to be delimited in various parts of the world. From the South China Sea to the North Atlantic, the implications for resources and for regional politics can be profound.
There is, however, a growing body of techniques and expertise available for managing any disputes which may arise, without the very considerable delays and expense attendant either on protracted deadlock or on international litigation.
In the short and the longer term, the interests of the oil industry lie with certainty, and with the resolution or at least effective management of inter-state disputes that may prevent economic activity, or discourage necessary long-term investment. As drilling becomes economically viable in ever greater depths of water, the number of areas where potential inter-state rivalry can lead to excessive uncertainty and risk likewise has the potential to become larger.
This is particularly so in areas where UNCLOS gives states the possibility of extending the limits of their continental shelves. In some cases, major international oil companies will be more experienced than their host governments in assessing the problems that may result and suggesting solutions and have an important part to play, alongside governments, in enabling economic activity to continue in areas where the boundaries are uncertain, or give rise to technical difficulties.
Alan Perry is a Partner in the Public International Law Group of D J Freeman (Solicitors), located in London.


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by Amanda Schneider MS, LAT, ATC
Hydration is very important for athletes. Being dehydrated by even 2% can decrease performance by 10%.
Tips for proper hydration:
Staying well hydrated can prevent muscle cramping, help regulate body temperature, and get rid of unwanted waste.
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All Rights Reserved.
450 East 23rd Street Fremont, NE 68025
(402) 721-1610


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Talk:Miscellany of The Beast with a Billion Backs
DVD fact logistics question
I've just placed it in the trivia category for now, but it's proper location and importance to the article isn't really known by me. -Mini-Me 16:21, 27 June 2008 (BST)
- I think it fits perfectly. It is merely a trivia fact (or perhaps trivial? OH THE PUNS!). --SvipTalk 16:48, 27 June 2008 (BST)
Yivo's space
This should probably be settled here before any more editing on the subject is done. In my opinion, the size Yivo was when shkle was dragged through, could easily support 6 billion people, but not 20 quadrillion. It's safe to say 20 quadrillion could not fit on the Isle of man, was Yivo bigger by enough to accomadate that many?Anarchy Balsac 01:37, 7 July 2008 (CEST)
- Yes, but even if we settle on that being possible, you're going to have to tell me that 20 quadrillion couldn't fit on Bender's pirate ship. Because it is sort of implied that Yivo throws them all aboard his ship, though, it is not totally unlikely that Yivo moves some to their respective worlds. --SvipTalk 13:03, 7 July 2008 (CEST)
- Easy, there were several pirate ships, it wasn't just one. The reason we don't see Yivo place people on the others is because Bender's is where the main cast goes.Anarchy Balsac 19:46, 7 July 2008 (CEST)
Correlation w/ 3guys1hammer
In a very creepy coincidence, not only was this being made at about the same time the Ukrainian Maniacs filmed the 3guys1hammer video, but Bender requests to die by "clumsy bludgeoning", anyone else notice this?Anarchy Balsac 19:24, 21 December 2008 (UTC)
- Interesting. Just as it is interesting that in the Native American word 'akwa' has same meaning and pronunciation as the Latin word 'aqua'. But just like the Ukrainian film and the Futurama film, these two things have nothing to do with each other, and are purely coincidences, the more you attempt to find a connection between the two, the more consumed by it you become. And it all end in tears, as it is just a coincidence. --SvipTalk 19:32, 21 December 2008 (UTC)
- I said it was a coincidence though. Just that it's an extremely creepy one.Anarchy Balsac 19:38, 21 December 2008 (UTC)
- Wait, how would "clumsy bludgeoning" have anything to do with what happened in 3guys1hammer? Chris of the Futurama 23:54, 21 December 2008 (UTC)
- 3guys1hammer involved a very clumsy bludgeoning death of a person.Anarchy Balsac 00:05, 22 December 2008 (UTC)
- Ohh, i see. Makes sense. Chris of the Futurama 00:09, 22 December 2008 (UTC)
Yivo alone in shkler universe
If Yivo is alone in that universe, then why are there constellations in the background? --18.104.22.168 03:10, 27 March 2014 (CET)


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"Love is Strong in Us", based on Psalm 46, sings of a world whose promise comes from the love we share as a community. God is our strength and will protect us from the sorrows of the world. "It is strong in us, Love is strong in us! It is strong in us, God is strong in us! Therefore, we will not fear, though the earth should change. Though the mountains shake in the heart of the sea. Though the waters roar and foam, though the mountains tremble with its tumult, there is a river."
Composer: Michael Bussewitz-Quarm (ASCAP)
- For Lead Vocalist, SATB Chorus, and Congregation
- Comes With Lead Sheet
- Keyboard Accompaniment
- Range of Lead Singer: G#2-D4
- Ranges of SATB Chorus: S:B3-F#5; A: G#3—C#5; T: B2-G#4 B: B2-D4
- Total duration: ca 4:40
- Written in 2010
- One movement work. 16 pages, 8.5 X 11"
- Premiered in September, 2011 in Port Jefferson, NY, by First Presbyterian Church Chancel Choir and Worship Team
- Wholesale Pricing: $2.79
- Most Wholesale, PDF, and Large Orders Qualify for Discount
Published by MB Arts (ASCAP)
© 2014 Michael Bussewitz-Quarm


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text:
The days of grunge are gone from Seattle with the possible exception of a resurgence in plaid. But that doesn't mean that Seattleites of a certain age can't think back and reflect on the time when Seattle seemed like the musical center of the entire universe. Anyone wanting to find out more about the history of Seattle's grunge scene in the 1990's should listen to Slacker radio at Slacker.com.
When I first tuned into the Slacker.com radio station, one of the first songs that I heard played was "Spoonman" by Soundgarden. While Chris Cornell and Soundgarden weren't the first to explode on the Seattle grunge scene, they were definitely one of the bands able to capitalize on the Seattle music scene. The song "Spoonman" itself featured an actual performance artist from the Pike Place market.
By the time I arrived in Seattle, the Seattle grunge scene had all but died out and when I saw Soundgarden play, it was at the Seattle center. All of the major grunge bands from Seattle had been signed by major record labels and Kurt Cobain was already dead. That didn't stop thousands of "immigrants" from other cities from moving to the Pacific Northwest to catch a piece of the coolness factor that was already here. At that time, it seemed like everyone in Seattle was dressed in the worst way possible; I myself wore my Levi's two or three inches too big around the waist with oversized plaid jackets that I picked up for a steal at Cost-co. The worst part about my attire at that time was that I didn't stand out anywhere I went. Almost everyone I met during that time dressed the same way. Grunge fashion was WAY less flattering than the even the hipster attire of today--definitely a sad comment on Seattle society at that time.
Seattle was then known as the heroin capital of the world. I knew people that had tried the drug, but it wasn't my scene for sure. Tons of heroin addicts lined the streets of Capital Hill where they sat begging for money in their fancy shoes that their Bellevue parents had unwittingly purchased for them. We called them Trustafarians then, a term that I believe originated at Evergreen or was made up by a friend of mine.
Seattle doesn't seem the same now; maybe that's a good thing or maybe I'm too old to enjoy whatever new scene is hitting the city.
How about you? Where you here in Seattle when it all went down and how did you feel at the time?


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Tuesday January 31st, 2017 to
Sunday February 26th, 2017
Sun Feb 5th, 2017 — 2:00 pm - 5:00 pm
Home could be a burrow, a box, a deluxe mansion or four walls and a roof. Is home a place or a state of mind? How we define it is unique to each and every one of us.
See what these four artists think of when they think about "Home" - Doug Cosbie, Kathy Haycock, John Jarrett and D. H. Monet.
"Home" is where the art is at Cube Gallery, 1285 Wellington St. from January 31 to February 26, 2017


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Realistically, will you have the lifestyle you desire in your retirement years?
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Epithelial bicellular and tricellular junctions are essential for establishing and maintaining permeability barriers. Tricellular junctions are formed by the convergence of three bicellular junctions at the corners of neighbouring epithelia. Gliotactin, a member of the Neuroligin family, is located at the Drosophila tricellular junction, and is crucial for the formation of tricellular and septate junctions, as well as permeability barrier function. Gliotactin protein levels are tightly controlled by phosphorylation at tyrosine residues and endocytosis. Blocking endocytosis or overexpressing Gliotactin results in the spread of Gliotactin from the tricellular junction, resulting in apoptosis, delamination and migration of epithelial cells. We show that Gliotactin levels are also regulated at the mRNA level by micro (mi)RNA-mediated degradation and that miRNAs are targeted to a short region in the 3′UTR that includes a conserved miR-184 target site. miR-184 also targets a suite of septate junction proteins, including NrxIV, coracle and Mcr. miR-184 expression is triggered when Gliotactin is overexpressed, leading to activation of the BMP signalling pathway. Gliotactin specifically interferes with Dad, an inhibitory SMAD, leading to activation of the Tkv type-I receptor and activation of Mad to elevate the biogenesis and expression of miR-184.
Permeability barriers are essential to prevent fluid flow, and to restrict the diffusion of molecules and pathogens across tissues, such as the epidermis, intestine and brain. Permeability barriers are formed by tight junctions in vertebrates and septate junctions in insects, and the barriers have similar physiological roles and homologous components (Tsukita et al., 2001). Pleated septate junctions form a ladder-like array of electron-dense septa located just below the adherens junction (Tepass and Hartenstein, 1994). Septate junctions comprise an interdependent protein complex, including Neurexin IV (NrxIV) (Baumgartner et al., 1996), Coracle (Fehon et al., 1994), Na+/K+ ATPase and Neuroglian (Genova and Fehon, 2003; Paul et al., 2003), Contactin (Faivre-Sarrailh et al., 2004) and Mcr (Batz et al., 2014; Hall et al., 2014). In many epithelia, a specialized junction, the tricellular junction (TCJ), forms at the convergence of the tight junctions or septate junctions from three cells to create a permeability barrier at the corners of neighbouring cells. In Drosophila, the TCJ is formed in a range of epithelia and is crucial for septate junction maturation and permeability barrier formation (Fristrom, 1982; Noirot-Timothée et al., 1982; Schulte et al., 2003). Gliotactin (Gli), a single-pass transmembrane protein and a member of the Neuroligin family, is specifically localized to the Drosophila TCJ (Schulte et al., 2003). Loss of Gliotactin results in a disruption of the TCJ, the baso-lateral spread of septate junction proteins and a loss of the permeability barrier (Schulte et al., 2003).
Gliotactin protein levels and TCJ localization are tightly controlled by phosphorylation of tyrosine residues and endocytosis (Padash-Barmchi et al., 2010). Overexpressed Gliotactin spreads away from the TCJ and is deleterious, leading to apoptosis, delamination, overproliferation and cell migration in the wing imaginal disc (Padash-Barmchi et al., 2010). Mutation of two conserved tyrosine residues to phenylalanine in Gliotactin disrupts the phosphorylation, endocytosis and subsequent degradation of Gliotactin (Padash-Barmchi et al., 2010). Beyond control at the protein level, Gliotactin has been identified as a potential target of micro (mi)RNAs, with multiple conserved miRNA-target sites predicted in the 3′UTR (Kertesz et al., 2007). Target sequences for one of these microRNAs, miR-184, have been identified in the 3′UTR of other genes coding for septate junction proteins, including NrxIV and Coracle (Kertesz et al., 2007), and have been predicted to exist in a range of other septate junction genes, including the Pasiflora genes (Deligiannaki et al., 2015). However, the role that miR-184 plays in the regulation of Gliotactin and septate junctions in vivo has yet to be determined.
Here, we show that Gliotactin mRNA levels are controlled by miR-184-targeted degradation through the 3′UTR. In addition, miR-184 can control the expression level of the core septate junction proteins NrxIV and Coracle, as well as Mcr. We found that overexpressed Gliotactin is the trigger for increased miR-184 expression through activation of the BMP type-1 receptor, Thickveins (Tkv). Moreover, Gliotactin activation of Tkv is through block of the Tkv inhibitor Daughter against dpp (Dad). We conclude that Gliotactin leads to a feedback loop to control Gliotactin mRNA levels thorough Tkv signalling and induction of miR-184.
Overexpression of Gliotactin causes self-downregulation
Gliotactin localization is normally restricted to the TCJ within the columnar epithelia of the wing imaginal disc (Fig. 1A). Overexpression of a full-length Gliotactin transgene causes spread away from the TCJ and around the cell. When driven with apterous-GAL4, the pattern of expression is uniform across the dorsal half of the wing imaginal disc (Schulte et al., 2006; Padash-Barmchi et al., 2010). In contrast, when an EP insertion (GliEP) with an upstream activating sequence (UAS) element in the 5′ end of Gliotactin was driven using apterous-GAL4, Gliotactin expression occurred in patches within the dorsal side of the wing disc (Fig. 1B,C). The EP insertion drives the expression of the endogenous Gliotactin mRNA, including the entire 3′UTR, whereas our original UAS-Gliotactin transgene lacked the complete 3′UTR, leading to the hypothesis that the differences in expression patterns might reside in the 3′UTR. Using Target Scan Fly (Kheradpour et al., 2007), eight miRNA targets are predicted within the Gliotactin 3′UTR that are highly conserved across Drosophilidae species (Fig. 1L). This suggested an additional level of Gliotactin regulation controlled by the 3′UTR. To test this, we expressed a range of transgenes with cherry-tagged Gliotactin and different 3′UTR lengths (Fig. S1). These included the entire 3′UTR (Gli::Ch-L.UTR), a short 3′UTR lacking the miR-184- and miR-1011-target sites (Gli::Ch-S.UTR), and finally the SV40 3′UTR lacking all miRNA target sites (Gli::Ch-no.UTR). Transgenic lines overexpressing the entire 3′UTR (Gli::Ch-L.UTR) had regions with reduced levels of Gliotactin protein (Fig. 1D,E) that when measured across different regions in the dorsal side (Fig. 1J) were significantly different (Fig. 1K). This mirrored the patchy pattern observed with the GliEP insertion, although the degree of downregulation was not as strong (Fig. 1K). Transgenes lacking the miR-184- and miR-1011-binding sites (Gli::Ch-S.UTR) (Fig. 1F,G) or the whole 3′UTR (Gli::Ch-no.UTR) (Fig. 1H,I) showed no significant reduction in Gliotactin levels across the apterous expression area (Fig. 1K). These results suggest that the 3′UTR and, specifically, miR-184 and/or miR-1011 control Gliotactin levels.
miR-184 controls septate junction proteins and Gliotactin
miR-184 has been identified in vitro as a potential regulator of other septate junction proteins, including NrxIV, Coracle and Mcr, in addition to Gliotactin (Kertesz et al., 2007). miR-184 is expressed throughout embryogenesis (Aboobaker et al., 2005; Li et al., 2011) in larval imaginal discs (Li et al., 2011) and plays a role in the regulation of the female germline (Iovino et al., 2009). To test the role of miR-184 in the control of Gliotactin and septate junction proteins, we overexpressed miR-184 in the wing imaginal disc using apterous-GAL4. Elevated miR-184 expression caused a downregulation of Gliotactin (Fig. 2A,B) and a reduction of the septate junction proteins NrxIV (Fig. 2C,D), Mcr (Fig. 2E,F) and Coracle (Cora) (Fig. 2G,H). The expression of other junctional proteins, Disc Large (Dlg) (Fig. 2A), E-cadherin (Ecad) and Fasciclin 3 (Fas3) (Fig. 2I,J) were unaffected. The degree of downregulation of each septate junction protein or Gliotactin was insufficient to trigger loss of polarity or to trigger cell death, and we observed no disruption to the epithelium. To test the specificity of the miRNA control, we individually expressed miR-1011 and the miRNA bantam, which are also predicted to target the Gliotactin 3′UTR (Fig. 1L). Neither miR-1011 nor bantam expression had any effect on Gliotactin (Fig. 2K,L). These results suggest that miR-184 can control Gliotactin and a subset of septate junction mRNAs.
Overexpression of the extracellular domain is the trigger for self-downregulation
The role of miR-184 in Gliotactin control was unexpected because miR-184 mutants are viable with no disc abnormalities (Iovino et al., 2009). Overexpression of Gliotactin is deleterious, leading to cell death, overproliferation and cell migration (Padash-Barmchi et al., 2010), suggesting that mechanisms have evolved to ensure protein levels are tightly controlled. We hypothesized that the overexpression of Gliotactin could trigger elevated miR-184 expression for an additional level of control. To test this hypothesis, we expressed a Gliotactin transgene that lacked the 3′UTR and in which the intracellular domain had been replaced with a blue fluorescent protein (BFP) tag (Gli::EBFP) (Fig. S1C). With this Gliotactin construct, tyrosine phosphorylation and subsequent endocytosis cannot occur, and endogenous Gliotactin can be analyzed using a monoclonal antibody specific to the intracellular domain. When expressed with the apterous-GAL4 driver, Gli::EBFP did not alter the subcellular localization of endogenous Gliotactin nor lead to cell death or disruption of the epithelia, but did result in the overall downregulation of endogenous Gliotactin (Fig. 3A). To confirm these results, we overexpressed a Gliotactin transgene containing the entire protein-coding sequence but lacking the 3′UTR (GliWT) (Fig. S1) in a background with endogenous Gliotactin tagged with YFP (Gli::YFP) (Fig. 3B). Here too, endogenous Gliotactin was downregulated when the transgene was overexpressed. Expression of the Gli::EBFP or GliWT transgenes also led to the downregulation of Mcr and NrxIV (Fig. S2C,D), suggesting that overexpression of Gliotactin leads to elevated miR-184 expression. Overexpression of Gli::EBFP did not downregulate Dlg (data not shown). To determine whether this phenomenon is specific to Gliotactin, we overexpressed NrxIV using an EP insertion in Nrx-IV (NrxIV-EP) under the control of apterous-GAL4. NrxIV overexpression failed to trigger self-downregulation (Fig. S2A) and failed to downregulate Gliotactin or Mcr (Fig. S2B). These results suggest that the overexpression of the Gliotactin extracellular domain is sufficient to trigger the downregulation of Gliotactin and other septate junction proteins, and that this effect is specific to Gliotactin and not a general property of septate junction proteins.
We next tested to see whether Gli::EBFP triggered downregulation of endogenous Gliotactin through miRNA control of the 3′UTR. To do this, we generated a series of transgenic lines that expressed Cherry-tagged Gliotactin under the control of the Gliotactin promoter and with different 3′UTRs: full-length, short and no UTR (Fig. S1B). The Gliotactin promoter was used in an attempt to match the normal levels of expression. Each Cherry-tagged protein was trafficked to the TCJ with some spread away from the corners (Fig. S2E–G). When Gli::EBFP was overexpressed in these backgrounds, we observed that only the Gliotactin with the entire 3′UTR (GP-Gli::Ch-L.UTR) was significantly downregulated (Fig. 3C,F). Those with the short 3′UTR (GP-Gli::Ch-S.UTR) or no 3′UTR (GP-Gli::Ch-no.UTR) were unaffected (Fig. 3D–E) and showed no significant downregulation in protein levels (Fig. 3F). These findings suggest that the overexpression of Gliotactin triggers a feedback loop that specifically targets mRNAs with the long 3′UTR for degradation. Because this region contains the conserved miR-184 recognition sequence, this further indicated that control of Gliotactin mRNA is mediated by miR-184.
Gliotactin mRNA is downregulated and miR-184 increased with Gliotactin overexpression
To confirm that Gliotactin downregulation was at the mRNA level, we used daughterless-GAL4 (da-GAL4) to overexpress a full-length Gliotactin with the entire 3′UTR (GliEP) and quantified mRNA levels using RT-PCR analyses (Fig. 4). The EP insertion drives the expression of the endogenous Gliotactin mRNA with the full 3′UTR and, when overexpressed with the strong da-GAL4 driver, Gliotactin mRNA levels were not significantly higher than those of control (Fig. 4A). If there was no miRNA-mediated degradation of the Gliotactin mRNA, we expected to see significantly more Gliotactin mRNA when GliEP was expressed. To test if overexpression of Gliotactin was driving the degradation of endogenous mRNA, we used da-GAL4 to express the Gli::EBFP transgene that lacks the entire intracellular domain and the 3′UTR. Using primers targeted to the intracellular domain, we examined the levels of endogenous Gliotactin mRNA. We found that the mRNA levels for endogenous Gliotactin were reduced compared to those in control when Gli::EBFP was overexpressed (Fig. 4A), confirming that the downregulation of endogenous Gliotactin occurs at the mRNA level.
Our results suggest that Gliotactin mRNA degradation is mediated by miR-184 and that overexpression of Gliotactin leads to an increase in miR-184 levels. To test this hypothesis, we used quantitative real-time (qRT)-PCR to quantify changes in the pre-miR-184 RNA (Enderle et al., 2011). We determined that overexpression of either full-length Gliotactin (GliEP) or Gli::EBFP using apterous-GAL4, triggered a significant increase in miR-184 levels compared to those of control (Fig. 4B). Our findings confirmed that induction of miR-184 levels results from overexpression of Gliotactin and thereby mediates Gliotactin mRNA levels in vivo.
A miR-184 sponge blocks Gliotactin downregulation
As the long 3′UTR of Gliotactin was necessary for the downregulation of the Gliotactin mRNA, this pointed to a role for either miR-184 or miR-1011. We found that miR-184 but not miR-1011 led to the downregulation of Gliotactin and other septate junction proteins (Fig. 2). Next we investigated whether miR-184 was solely responsible. miRNA sponges are an effective means to sequester miRNAs within a cell (Loya et al., 2009). We utilized a sponge specific to miR-184 (SPmiR184) and expressed it using apterous-GAL4 within the wing imaginal disc. The sponge alone had no significant effect on endogenous Gliotactin expression (Fig. S2H). However, when two copies of the sponge were expressed along with UAS-miR-184, the downregulation of Gliotactin was blocked (Fig. 5A). To test the effect of the sponge on Gliotactin overexpression, we co-expressed SPmiR-184 with Gli::EBFP and observed that the downregulation of endogenous Gliotactin was blocked (Fig. 5B). To test the specificity of the miRNA, we expressed a bantam sponge (Herranz et al., 2012) along with Gli::EBFP. The bantam sponge did not prevent the downregulation of Gliotactin (Fig. 5C) and the levels of Gliotactin were not significantly different from those in controls expressing Gli::EBFP alone (Fig. 5D). Our findings strongly suggest that miR-184 regulates Gliotactin mRNA levels.
miR-184 expression is triggered through the BMP signalling pathway
Our next aim was to determine the mechanism linking overexpression of Gliotactin to the induction of miR-184. miRNA levels are known to be controlled through a number of different pathways, including those involving MAPKs, BMP and TGF-β (Saj and Lai, 2011). There are three MAPKs in Drosophila melanogaster, Basket (Bsk; a JNK homologue), p38 (with three isoforms) and rolled (an ERK homologue). We did not observe any changes in the Gli::EBFP-induced reduction of endogenous Gliotactin when Bsk or p38 were blocked using dominant-negative transgenes [Bsk-DN (Petzoldt et al., 2013) and p38-DN (Adachi-Yamada et al., 1999), respectively] or with RNA interference (RNAi)-mediated knockdown of rolled (Biteau and Jasper, 2011) (Fig. S3A–F). These results excluded MAPK signalling as the trigger for miR-184 transcription.
We next tested the role of TGF-β and BMP signalling. Co-expression of Gli::EBFP with dominant-negative Baboon protein (Parker et al., 2006) to block the TGF-β type-I receptor had no effect on the Gli::EBFP-mediated downregulation of Gliotactin (Fig. S3G,H), eliminating possible TGF-β involvement. To block BMP signalling, we co-expressed Gli::EBFP and a dominant-negative form of the BMP type-I receptor, Thickveins (Tkv-DN; Haerry et al., 1998). Expression of dominant-negative Tkv reduced the Gli::EBFP-mediated downregulation of endogenous Gliotactin (Fig. 6A,B,E), whereas dominant-negative Tkv alone had no effect on endogenous Gliotactin levels (nor any additional deleterious effects), suggesting that Tkv was an integral part of the Gliotactin-triggered miR-184 expression.
Ligand activation of Tkv and the type-II BMP receptor Punt leads to the phosphorylation and activation of Mad, a homologue of vertebrate SMAD (Derynck et al., 1996; Newfeld et al., 1996). We observed that expression of Gli::EBFP led to a significant increase in immunolabelling of phosphorylated Mad and to a greater spread of phosphorylated Mad across the dorsal side of the apterous boundary in the wing pouch (Fig. S3I–K). To test whether Mad activation played a role in the miR-184-mediated reduction in Gliotactin levels, we co-expressed Gli::EBFP and a dominant-negative Mad transgene that lacked the DNA-binding domain (Mad-DN; Takaesu et al., 2005). We found that dominant-negative Mad reduced the downregulation of endogenous Gliotactin (Fig. 6C,D,E). Overall blocking of BMP-dependent signalling in conjunction with Gli::EBFP expression significantly inhibited Gliotactin downregulation compared to that with Gli::EBFP alone or with the negative control dominant-negative Bsk (Fig. 6E). To confirm that inhibition of the BMP pathway blocked the increase in miR-184 transcription, we co-expressed Gli::EBFP with dominant-negative Mad and assayed the levels of miR-184 by using qRT-PCR. The level of miR-184 was the same as that in control (apterous-GAL4 alone) when the BMP signalling pathway was blocked and was significantly lower than when Gli::EBFP was expressed alone (Fig. 4B). These results suggest that excess Gliotactin triggers an increase in miR-184 through BMP-dependent signalling.
Two further direct tests of the role of Tkv activation in Gliotactin regulation were performed. Activation of Tkv and Mad leads to the induction of transcription of spalt and optomotor blind (omb/bifid) in the wing disc (Blair, 2007). In the first test, we used omb-lacZ (Sun et al., 1995) as a reporter of Tkv activation in the presence of increased Gliotactin (Gli::EBFP). We measured the region of omb-lacZ induction on the dorsal side compared to that on the ventral side along the apterous-GAL4 boundary. The pattern of omb-lacZ expression in control discs was evenly matched on both sides of the boundary (Fig. 6F). Overexpression of Gliotactin led to a significant spread of omb-lacZ expression on the apterous-dorsal side relative to that on the ventral side (Fig. 6G,H), indicating an increase in Tkv signalling. For the second test, we utilized a fluorescent reporter of Tkv activation (Tkv-TIPF), under the control of UAS, to confirm that the Tkv receptor itself was activated. The Tkv-TIPF reporter emits YFP fluorescence upon receptor activation and displacement of activated FKBP12 (also known as FK506-bp2; Michel et al., 2011). We co-expressed Gli::EBFP with the Tkv-TIPF reporter using apterous-GAL4 (Fig. 6J) and compared fluorescence levels to those upon expression of Tkv-TIPF alone (Fig. 6I). Gli::EBFP triggered a significantly higher level of Tkv-TIFP fluorescence compared to that in controls (Fig. 6K). These tests confirm that elevated Gliotactin activates the Tkv receptor, Mad and downstream transcription targets.
Overexpression of Gliotactin activates Tkv signalling through inhibition of Dad
If the BMP receptor mediates the increase in miR-184 transcription, we hypothesized that blocking this pathway enhances the phenotypes observed with overexpression of Gliotactin. Overexpression of Gliotactin with apterous-GAL4 leads to the spread of Gliotactin away from the TCJ, resulting in deleterious consequences that include apoptosis, delamination of the columnar epithelia and spread of cells from the dorsal-apterous side into the ventral wild-type side of the wing disc (Fig. 7A) (Padash-Barmchi et al., 2010). When both GliWT and dominant-negative Tkv were co-expressed in the wing imaginal disc, we observed that blocking Tkv signalling significantly enhanced the spread of the Gliotactin-overexpressing cells into the ventral half of the disc (Fig. 7B,E). However, when both GliWT and an activated form of Tkv (Tkv-Act) were co-expressed, we observed that increased Tkv activation completely blocked the cell migration (Fig. 7C,E). When GliWT was co-expressed with the miR-184 sponge, this increased migration of the Gliotactin-overexpressing cells (Fig. 7C,E). However, simultaneous expression of Gliotactin and miR-184 significantly reduced the cell migration (Fig. 7I,E). Taken together, these results show that loss of Tkv signalling and block of miR-184 lead to increased levels of endogenous Gliotactin expression and enhance the deleterious effects resulting from Gliotactin overexpression. Increased Tkv signalling and miR-184 expression suppressed the Gliotactin phenotypes.
We next wanted to establish the mechanism by which increased Gliotactin leads to activation of Tkv. To determine whether Tkv activation is through the ligand Deacapentaplegic (Dpp), GliWT and Dpp (UAS-Dpp::GFP) (Teleman and Cohen, 2000) were co-expressed in the wing imaginal disc. An increase in Dpp did not block the migration of cells overexpressing Gliotactin into the ventral half of the disc (Fig. 7F arrow, J). In control discs, the overexpression of Dpp::GFP, driven by apterous-GAL4, had no effect on endogenous Gliotactin expression (data not shown; n=10 discs). Therefore, increased Dpp expression was not the cause of increased Tkv activation by Gliotactin. An alternative route to activation of Tkv could be through inhibition of Dad, a Tkv inhibitor. We expressed GliWT in a Dad heterozygous mutant (Dad[j1E4]/+), which completely blocked the migration of GliWT cells into the ventral side of the disc (Fig. 7G,J). Co-expression of GliWT and an EP (UAS) insertion in Dad (DadEP), to increase Dad expression, resulted in enhanced cell migration (Fig. 7H, arrow and J). These results suggest that the activation of Tkv by Gliotactin is through inhibition of Dad. Our combined results indicate that the feedback loop to control Gliotactin mRNA works through activation of the BMP signalling pathway (Fig. 8). Activation occurs through inhibition of Dad, resulting in increased Tkv receptor signalling and phosphorylation of Mad, leading to increased transcription of miR-184.
Gliotactin, a TCJ protein, is crucial for the formation of septate junctions, the TCJ and the function of permeability barriers. At the protein level, Gliotactin is tightly controlled by tyrosine phosphorylation and endocytosis, and overexpression of Gliotactin leads to spread beyond the TCJ, with deleterious consequences including delamination, cell migration and apoptosis (Padash-Barmchi et al., 2010). We found that Gliotactin is also regulated at the mRNA level, specifically by miR-184-mediated degradation through a conserved miR-184-target site in the 3′UTR. Furthermore, we demonstrate that miR-184 controls other septate junction proteins, including NrxIV, Coracle and Mcr. We propose that excessive Gliotactin acts as the trigger to induce miR-184 expression through the activation of BMP signalling and the Tkv type-I BMP receptor. Activation of Tkv is not through elevated levels of its ligand Dpp, but through inhibition of Dad, an inhibitory SMAD.
Gliotactin overexpression acts as the trigger for miR-184 induction
miR-184 is expressed throughout Drosophila embryogenesis in the brain, ventral nerve cord (Li et al., 2011), mesoderm, endoderm (Aboobaker et al., 2005) and in imaginal discs at larval stages (Li et al., 2011). miR-184 regulates germline development, where loss of miR-184 leads to oogenesis defects (Iovino et al., 2009). However, miR-184 null mutants are viable with normal morphology, leading to the suggestion that maternally deposited miR-184 might persist through embryogenesis, allowing development into larval stages and beyond (Iovino et al., 2009). An alternative explanation is that miR-184 is expressed at basal levels throughout development, but increased expression and function occurs through a trigger. Our findings suggest that overexpression of Gliotactin is a trigger for increased miR-184 transcription and that this in turn leads to the downregulation of a suite of other septate junction proteins. In vitro luciferase assays have previously identified Gliotactin, NrxIV, Contactin, Coracle, Megatrachea, Sinuous and Kune-Kune as potential targets of miR-184 (Kertesz et al., 2007). Our in vivo results show that miR-184 regulates Gliotactin at the mRNA level as part of a feedback loop.
miRNAs play a key role in the regulation and maintenance of a range of vertebrate epithelia. For instance, miR-200 is enriched in breast and ovarian epithelia, and can suppress the epithelia-to-mesenchymal transition (Gregory et al., 2008; Park et al., 2008). miR-122a regulates intestinal tight junction permeability by targeting Occludin mRNA (Ye et al., 2011). miR-184 is expressed at high levels in the vertebrate eye, including the retina pigmented epithelia, cornea and lens (Kapsimali et al., 2007; Ryan et al., 2006), and regulates the blood–retina barrier (Wang et al., 2010) as well as angiogenesis (Yu et al., 2008). Collectively, these findings reveal a role of miRNAs in the control and maintenance of epithelial cells.
In the Drosophila imaginal disc, miR-184 controls the levels of a suite of septate junction proteins and the TCJ protein Gliotactin. During imaginal disc development, there is a dynamic reorganization of the septate junctions as cells rearrange to generate the structures of the adult wing (Fristrom, 1982). Intact septa either become extended or compacted in response to these cellular movements in order to maintain the transepithelial barrier. Thus, miR-184 might function to coordinate septate junction domain proteins during imaginal epithelial morphogenesis to ensure that cells move and rearrange more easily as the wing structures develop. However, expression of the miR-184 sponge did not generate defects in wing morphogenesis, and mir-184 mutants are viable with no defects in epithelial morphogenesis, suggesting that increased miR-184 expression is transient and localized. Because the septate junction complex is highly stable (Oshima and Fehon, 2011), it is likely that basal levels of miR-184 have little impact on the septate junction domain. Rather, a localized increase in miR-184 in response to a trigger would be necessary to change the stability of this complex, as might occur during dynamic processes such as morphogenesis, proliferation, migration or apoptosis. miRNAs in both Drosophila and vertebrates can regulate apoptosis by controlling the levels of pro-apoptotic or anti-apoptotic genes (Jovanovic and Hengartner, 2006). For instance, the Drosophila death genes Drice, hid, reaper, grim and sickle are targeted either individually or as a group by miRNAs, including miR-2, miR-6, miR-2/13, miR-11, miR-14 and miR-308 (Leaman et al., 2005; Stark et al., 2003; Xu et al., 2003). In particular, the bantam miRNA promotes cell proliferation through regulation of hid (Brennecke et al., 2003) and is controlled by the Dpp pathway (Martín et al., 2004; Doumpas et al., 2013; Oh and Irvine, 2011; Zhang et al., 2013). Thus, an increase in miR-184 might be triggered by Tkv activation during the processes that underlie the apoptosis stimulated by overexpression of Gliotactin.
BMP signalling pathway induces miR-184 activation
Our analysis points to the BMP signalling pathway as part of the miR-184 trigger downstream of Gliotactin overexpression. In vertebrates, BMP or TGF-β signalling pathways play an important role in the transcription and biogenesis of miRNAs (Kato et al., 2009; Winbanks et al., 2011). BMP4 and TGF-β elevate the levels of 20 different species of miRNAs and recruit receptor-regulated SMADs (R-Smads) to enhance Drosha processing of pri-miRNAs (Davis et al., 2008; Harris and Ashe, 2011; Saj and Lai, 2011). BMP control of microRNA biogenesis and processing has been shown to control the proliferation and maintenance of vertebrate epithelia (Saj and Lai, 2011; Wang et al., 2010). For instance, TGF-β activation of miR-204 and miR-211 (miR-204/211) leads to targeting of SNAIL2, which normally represses Claudin transcription and other proteins crucial for maintaining epithelial structure (Carrozzino et al., 2005). miR-204 in retinal pigment epithelium inhibits TGF-βR2, Smad3 and SNAIL1 and SNAIL2 to protect tight junction integrity (Wang et al., 2010). These findings suggest that the expression of miR-204/211 and involvement of TGF-β signalling in a feedback loop is an important signal for integrity and maintenance of tight junctions (Kato et al., 2009; Xu et al., 2009).
In Drosophila, Dpp signalling and Mad can control cell proliferation through transcriptional control of the bantam microRNA through different transcription factors, including omb, brinker and yorkie (Martín et al., 2004; Doumpas et al., 2013; Oh and Irvine, 2011; Zhang et al., 2013). Our data show that overexpression of Gliotactin elevated Mad phosphorylation and increased transcription of omb, but we found no evidence that the bantam miRNA was involved in Gliotactin or septate junction protein control. The predicted bantam-targeted site was retained within the short Gliotactin 3′UTR, and neither the expression of bantam miRNA nor the bantam sponge had an effect on Gliotactin levels.
We propose that Gliotactin activates the BMP signalling pathway to increase pri-miR-184 transcription, acting through Tkv and Mad. Co-expression of Gliotactin with dominant-negative Mad blocked the increase in transcription of miR-184 and prevented the resulting downregulation of endogenous Gliotactin. It is likely that Gliotactin acts upstream of Mad through activation of the Tkv receptor. The increased fluorescence intensity of the Tkv-TIPF reporter upon Gliotactin overexpression confirms that the Tkv receptor itself is activated in response to overexpression of Gliotactin, either indirectly or directly. Gliotactin might increase the BMP ligand–receptor interaction, perhaps through increased membrane retention, increased ligand levels or increased capacity for ligand–receptor interactions. Alternatively, Gliotactin might interfere with an inhibitor of Tkv, such as the inhibitory Smad Dad. Dad is stably associated with Tkv and negatively regulates phosphorylation of Mad, probably by competing with Mad for Tkv binding (Bokel et al., 2006; Inoue et al., 1998). Our results favour the latter explanation, that Gliotactin increases Tkv signalling through disruption of Dad. Increased Dpp expression did not rescue the effects of overexpressed Gliotactin in the wing disc. Conversely, a reduction of the Dad gene dose by 50% completely suppressed the Gliotactin overexpression phenotypes, whereas increased Dad expression enhanced those phenotypes. These results point to a feedback loop by which Gliotactin reduces the association of Tkv and Dad to increase Tkv activation. Whether this interaction is intra- or extracellular, or indirect remains to be determined. Gliotactin interacts with the MAGUK scaffolding protein Dlg (Padash-Barmchi et al., 2010); however, the signalling complex formed between Gliotactin and Dlg is mediated by the PDZ-binding motif (Padash-Barmchi et al., 2013). Because Gli::EBFP lacks this domain, it is unlikely that the activation of miR-184 is mediated by the binding of a PDZ protein. Rather, the extracellular domain is likely to mediate the interactions that trigger Tkv activation. However, if there are other triggers of Tkv and miR-184 production, these have yet to be determined.
The activation of Mad by Tkv leading to increased miR-184 expression could be due to the direct transcriptional activation of miR-184 by Mad, or through the removal of an inhibitor such as brinker. The growth-promoting function of Dpp works through the repression of the transcription-repressor brinker (Schwank et al., 2008). Over 1000 genes have been identified as potential brinker targets, including the bantam miRNA (Doumpas et al., 2013; Oh and Irvine, 2011). However, miR-184 has not been identified as a brinker target. Regardless of the transcription mechanism, our model proposes that increased Gliotactin leads to displacement of Dad from Tkv, increased Mad activation and miR-184 transcription, which reduces Gliotactin mRNA levels within the cell. Thus, Gliotactin is subject to two levels of regulation – at the protein level through tyrosine-mediated phosphorylation and endocytosis and at the mRNA level through miR-184 mediated-degradation that is stimulated by Tkv receptor signalling.
Fly stocks
The following fly strains were used: Gli[EP2306], NrxIV[EP604] (Rorth, 1996), UAS-GliWT (Schulte et al., 2006), Gli::YFP (Kyoto DGRC), NrxIV::GFP (Buszczak et al., 2007), UAS-mCD8::RFP (Bloomington), UAS-miR-184 (Iovino et al., 2009), UAS-miR-1011(Bejarano et al., 2012), UAS-sponge-bantam (Herranz et al., 2012), UAS-bantam (Yang et al., 2008), UAS-Bsk-DN (Petzoldt et al., 2013), UAS-p38-DN (Adachi-Yamada et al., 1999), UAS-rolled-RNAi (Biteau and Jasper, 2011), UAS-Tkv-Act (Haerry et al., 1998) and UAS-Tkv-DN (Haerry et al., 1998), UAS-Tkv-TIPF (Michel et al., 2011), UAS-Mad-DN (Takaesu et al., 2005), UAS-Baboon-DN (Parker et al., 2006), UAS-Dpp::GFP (Teleman and Cohen, 2000), Dad[j1E4] (Ogiso et al., 2011), Dad[EP3196] (Bellen et al., 2004), omb-lacZ (Sun et al., 1995) and apterous–GAL4 (Bloomington Drosophila Stock Center).
Generation of transgenic lines
For the generation of the Gliotactin 3′UTR transgenic lines, the entire coding sequence of Gliotactin was amplified from a Gliotactin cDNA (AE27.41) (Auld et al., 1995). The Gliotactin coding sequence, an mCherry tag and one of three different UTRs [no 3′UTR (SV40), short 3′UTR (AE27.41, Auld et al., 1995) and long 3′UTR (RE15719, Drosophila Genomics Resource Center)] were amplified using the following primers: extracellular-Gliotactin5: 5′-TCTAGATCATCATGATGCAC-3′ (XbaI site) and extracellular-Gliotactin3: 5′-GAATTCCAGGGTCAACGAATC-3′ (EcoRI site); mCherry5: 5′-GAATTCATGGTGAGCAAGGGAGAG-3′ (EcoRI site) and mCherry3: 5′-AAGCTTCTTGTACAGCTCGTCCATG-3′ (HindIII site); Gliotactin-transmembrane5: 5′-AAGCTTGATGTATTCGTGACCAC-3′ (HindIII site) and Gliotactin-transmembrane3: 5′-CGTACGCCACATGATGCAGCAGATG-3′ (BsiWI); C-terminal-Gliotactin5: 5′-CGTACGCGCAATGCCAAGCGCCAATC-3′ (BsiWI site) and C-terminal-Gliotactin3: 5′-GACGTCTTATACGGATGTCTGAGGCAC-3′; no-3′UTR (SV40)5: 5′-GACGTCGATCTTTGTGAAGGAAC-3′ (AatII site) and no-3′UTR (SV40) 3: 5′-CTCGAGGATCCAGACATGATAAGA-3′(XhoI site); short-3′UTR5: 5′-GACGTCGTAACAGCTCTCTAAGCGCAG-3′ (AatII site) and short-3′UTR3: 5′-CTCGAGTTTGTTATCAAATAATAGCAG-3′ (XhoI site); long-3′UTR5: 5′-GACGTCGTAACAGCTCTCTAAGCGCAG-3′ (AatII site) and long-3′UTR3: 5′-CTCGAGATATTTATTTATGGACAAATACATAAAAC-3′ (XhoI site).
The Gliotactin promoter was amplified using the following primers: Gliotactin-promoter5: 5′-CACTTGGATCCTTATTTACAATCAGC-3′ (BamHI site) and Gliotactin-promoter3: 5′-GATGATCTAGAAAAATATTCAAAAGTAG-3′ (XbaI site).
PCR products were subcloned into the pβSΔF′ vector and sequenced for verification. The UTR constructs (no, short and long 3′UTR) were subcloned into pUAST-linker vector or 3′ to the Gliotactin promoter (3.7 Kb) in the pCasper 5 vector. To generate the pUAS-Gli::EBFP line, the enhanced BFP (Addgene) was PCR amplified and introduced into the GliNter construct, as previously described (Padash-Barmchi et al., 2010) using the SpeI and EcoRI restriction sites. Each construct was sequenced, and multiple independent transgenic lines were generated (Genetic Services).
The miR-184 sponge (SPmiR-184) construct contains 20 repetitive seed complementary sequences separated by variable four-nucleotide linkers, assembled as previously described (Loya et al., 2009) and cloned into the 3′UTR of mCherry between NotI and XbaI sites in a modified pWALIUM10-moe vector (Ni et al., 2009), carrying the white+ marker and flanking insulator sequences (Bejarano et al., 2012); the combined miRNA and linker sequences were checked against every mature miRNA sequence in the Drosophila genome to prevent off-target effects. Transgenic strains were generated using phiC31 site-specific genomic integration on the second (attP40) and third (attP2) Drosophila autosomes (Genetic Services).
RNA analysis
Reverse transcriptase PCR
Embryos were collected and dechorionated in 50% bleach. Total RNA was extracted using Trizol (Invitrogen). 2 µg of total mRNA was used to synthesize the cDNA using random hexamers and Superscript II reverse transcriptase (Applied Biosystems). Synthesized cDNA was amplified using AE2XI and AE2XII primers from the C-terminus of Gliotactin. AE2XI primer: 5′-GCGTGGAGTGGACAATGCCC-3′; AE2XII primer: 5′-GGAATTCCGCTGACTCCCTAAGCGCATCC-3′. Actin primers were used as an internal control. The expression data were normalized to the levels of actin. Band intensity was measured using ImageJ, and data were analyzed using one-way ANOVA and GraphPad Prism6.
RNA was extracted from 60 third instar wing discs, and RNA was extracted using QIAGENE RNeasy kit. 1 µg of total RNA was used to generate cDNA with random primers and Superscript III reverse transcriptase (Applied Biosystems). SYBR GREEN-based qRT-PCR (Bio-Rad) was performed for miR-184 and actin. qRT-PCR conditions were used for apterous-GAL4, GliEP, Gli::EBFP and Mad-DN lines using primers designed for pre-miR-184 (Enderle et al., 2011). Forward primer sequence: 5′-ATGCACATGAGTTGGCAGACAGC-3′, reverse primer sequence: 5′-CTTACCCCACGTTTCGATTACGC-3′. qRT-PCR cycling conditions were 95°C for 30 s, 40× (95°C for 5 s, 60°C for 30 s). qRT-PCR data were analyzed by using the comparative ΔΔCt method (Livak and Schmittgen, 2001). qRT-PCR products were sequenced to confirm primer specificity.
Statistical analyses
All statistical tests were calculated and graphed with GraphPad Prism 6. Immunofluorescence levels were quantified using ImageJ (Schneider et al., 2012). Fluorescence intensity of UAS-Gli::Ch constructs was measured across two areas on the apterous side (Fig. 1J). The intensity ratio was calculated, and significance was calculated using a one-way ANOVA with Tukey's post-hoc test. For all other figures, the mean intensity was calculated using the same fixed-size square in the pouch region of the apterous and the wild-type sides of wing imaginal disc (Fig. 3). The ratio of intensity in the apterous side compared to intensity in the wild-type side was calculated. The migration of Gliotactin-overexpressing cells (Fig. 7) from the apterous side into the wild-type side of the wing imaginal disc was measured, and the distance of spread was calculated as a ratio of the length of the ventral, or wild-type, side of the disc. Significance was determined using a one-way ANOVA with Tukey's post-hoc test.
For Tkv-TIPF quantification (Fig. 6I,J), fluorescence intensity was measured in a set area with ImageJ on the apterous side with or without Gli::EBFP. Discs from both genotypes were analyzed at the same time, images were collected with the same exposure time and neutral density filters, and were deconvolved using the same protocol. Significance was determined using an unpaired t-test.
Third instar larval imaginal discs were stained as described previously (Schulte et al., 2006). Primary antibodies were: mouse anti-Gli 1F6.3 at 1:200 (Auld et al., 1995), rabbit anti-Gli at 1:300 (Venema et al., 2004), mouse anti-Dlg 4F3 at 1:200 (Developmental Studies Hybridoma Bank) (Parnas et al., 2001), rat anti-DE-Cadherin at 1:50 (Developmental Studies Hybridoma Bank), mouse anti-Coracle (9C and C615-16B cocktail) at 1:100 (Developmental Studies Hybridoma Bank) (Fehon et al., 1994), rabbit anti-NrxIV at 1:500 (Baumgartner et al., 1996), guinea pig anti-Mcr at 1:800 (Hall et al., 2014), mouse anti-Fasciclin 3 at 1:100 (Developmental Studies Hybridoma Bank), rabbit anti-phosphorylated Mad at 1:200 (Cell Signaling). DAPI was used at 1:1000 (Thermo Scientific). Secondary antibodies were used at 1:300 – goat anti-rabbit (conjugated to Alexa Fluor 647 or Alexa Fluor 568), goat anti-mouse (conjugated to Alexa Fluor 647, Alexa Fluor 488 or Alexa Fluor 568), goat anti-rat (conjugated to Alexa Fluor 647 or Alexa Fluor 568) (Molecular Probes).
Image stacks were collected with a DeltaVision Spectris microscope (Applied Precision, Issaquah, WA) with a 20× air- or 60× oil-immersion lens (NA1.4) and CoolSnap HQ digital camera. Deconvolution of 0.2 m z-sections with SoftWorx (Applied Precision) used a point-spread function measured from 0.2-µm beads conjugated with Alexa dyes (Molecular Probes) mounted in Vectashield (Vector Labs). Side projections were created using the SoftWorx program. Images were exported to Photoshop (Adobe Systems) for compilation.
We thank Dr Doug Allan (University of British Columbia, Vancouver, Canada) for providing antibodies; Drs Steve Cohen (University of Copenhagen, Copenhagen, Denmark), Issac Edery (Rutgers University, Piscataway, NJ) and Doug Allan, and the Bloomington and Drosophila Genomics Resource Centers for fly stocks; the TRiP at Harvard Medical School (National Institutes of Health - National Institute of General Medical Sciences R01-GM084947) for providing transgenic RNAi fly stocks. The monoclonal antibodies listed from the Developmental Studies Hybridoma Bank were developed under the auspices of the The Eunice Kennedy Shriver National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biology. We also thank Dr Doug Allan for helpful discussions and comments on the manuscript.
Competing interests
The authors declare no competing or financial interests.
Author contributions
Z.S. contributed to all aspects of the manuscript, including writing and editing of the manuscript, study design, data collection, analysis and interpretation. M.P.-B., M.M.G. and G.S. contributed data collection, analysis and to the editing of the manuscript. T.A.F. and D.V.V. contributed unpublished reagents, and D.V.V. contributed to editing of the manuscript. V.J.A. contributed study conception and design, data interpretation, writing, and editing of the manuscript.
This study was supported by the Canadian Institutes for Health Research [MOP-123420 to V.J.A.] and the National Institute of Neurological Disorders and Stroke [R01 NS069695 to D.V.V.]. Deposited in PMC for release after 12 months.
Supplementary information
Supplementary information available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.178608/-/DC1
- Received August 11, 2015.
- Accepted February 11, 2016.
- © 2016. Published by The Company of Biologists Ltd


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Last disassembly post, next post will be me trying to fix it in my typical hamfisted manner.
I made the plastic drift even longer.
Finally got it out. Look at that rust(?) on the barrel.
Or glue? Maybe someone though the forearm was loose and tried to tack it in place?
It's interesting rust.
The tube seems ok although dirty inside.
The piercing cap should come apart easily.
Ok, I can remove this.
Bent my much modified tool.
Finally unscrewed the top piercing pin. But I was unable to go further. The piercing cap pieces are stuck together. I'm soaking them and may need to apply heat. It's not as bad as some guns I've taken apart but a few issues will need to be dealt with.
Friday, March 30, 2012
Crosman Trapmaster 1100 Disassembly, Part 6
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PDF files are like electronic documents, easily transferable, easy to send as attachments to email, easy to view on the web. The acronym stands for "portable document format" files. The classic way to produce PDF files is by having an Adobe software product called Adobe Acrobat. There are lots of other ways these days. Business Plan Pro automatically produces PDF files as one of the export formats. The Mac produces PDF files as an alternative to printing.
If you're not familiar with the various tools to create the right kind of files, find out from friends, check Wikipedia on PDF or Google creating a PDF file. Business Plan Pro exports anything to a PDF format too, so you can create the PDF file automatically.
Macintosh Users
If you're using a Macintosh, at least if it's a relatively new Macintosh, there is an automatic facility to print your documents to PDF files. It's related to the Preview application on Macintosh. Use the Print command on the file menu and you'll get something like the one shown here, which is taken from the Print dialog that comes up with PowerPoint 2007.
Move your mouse over the PDF button on the lower left, and push to open a drop down menu.
Select "Save to PDF" and use the Finder to set a file name.
That will be the PDF file you can use to send to me as an attachment to your email.


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