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    <TITLE>SML/NJ Interactive System</TITLE>
Standard ML of New Jersey Interactive System

 This section explains some of the basic elements of using the Standard ML of New Jersey compiler as an interactive system into which you enter declarations and expressions or load code from source files. This is the most accessible mode of using the compiler, but for any programs of nontrivial size we recommend that the  Compilation Manager be used to organize the compilation process.
 We assume below that you are using the compiler under Unix. The behavior will be somewhat different under MS Windows or the Macintosh OS.
Running Standard ML
 Type ``sml'' from the shell. This puts you into the interactive system. The top level prompt is ``-'', and the secondary prompt (printed when input is incomplete) is ``=''. If you get the secondary prompt when you don't expect it, typing ``; return'' will often complete your input, or typing your interrupt character (e.g., control-C) will cancel your input and return to the ML top level.
 If ``sml'' doesn't work, ask where sml has been installed on your machine and use the appropriate path name or redefine your PATH environment variable.
Interactive input
 Input to the top level interpreter (i.e., declarations and expressions) must be terminated by a semicolon (and carriage return) before the system will evaluate it. The system then prints out a response indicating the effect of the evaluation. Expressions are treated as implicit declarations of a standard variable it. For example,

- 3;user input after prompt
val it = 3 : intsystem response
 This means that the value of the last top level expression evaluated can be referred to using the variable ``it.''
Interrupting compilation or execution
 Typing the interrupt character (typically control-C) should interrupt the compiler and return you to top level.
Exiting the interactive system.
 Typing control-D (EOF) at top level will cause an exit to the shell (or the parent process from which sml was run). One can also terminate by calling OS.Process.exit(OS.Process.success).
Loading ML source text from a file
 The function use: string -> unit interprets its argument as a file name relative to sml's current directory and loads the text from that file as though it had been typed in. This should normally be executed at top level, but the loaded files can also contain calls of use to recursively load other files. It is a bad idea to call use within an expression or declaration, because the effects are not well-defined.
 For industrial-strength multi-module software systems, the the Compilation Manager is more appropriate than use.
Error messages
 The compiler attempts to recover from syntactic errors so that it can also produce semantic (type-checking) errors during the same compilation. Syntactic error recovery is more accurate for source files loaded by use or CM than it is from declarations typed directly into the interactive system -- this is because lookahead is not possible when text is entered one line at a time.
 When compiling files, the error messages include line numbers and character positions within the line. For example:
 - if true = then 5 true = else 6; std_in:7.6-7.11 Error: operator is not a function operator: int in expression: 5 true -
 Here the location information std_in:7.6-7.11 indicates that the erroneous expression ``5 true'' occupies characters 6 through 11 of the 7th line of input from std_in. For simple syntactic errors this position information is usually accurate or perhaps off by just one line. For some classes of errors the line numbers may not be very useful, because they delineate a potentially large declaration containing the error. If the error occurs in a file being loaded by use, the line numbers will refer to lines in the file being loaded.
 There are a number of different forms of type error message, and it may require some practice before you become adept at interpreting them. The most common form indicates a mismatch between the type of a function (or operator) and its argument (or operand). A representation of the offending expression is usually included, but this is an image of the internal abstract syntax for the expression and may differ significantly from the original source code. For instance, an expression ``if e1 then e2 else e3'' is represented internally as a case expression over a boolean value: ``$case  e1  of true =>  e2  | false =>  e3.''
 All the error messages produced by SML/NJ are documented in the SML/NJ Error and Warning Messages page.
Compiler interface
 Most users will interact with the compiler by loading code via the use command or under control of the Compilation Manager. However, some control of individual compiler phases is possible through the Compiler.Control structure.
Printing
 The structure Compiler.Control.Print contains several useful flags and functions with which you can control or redirect compiler error, diagnostic, and result messages. Among other things, there is a variable printDepth that controls the depth of printing of large structured values.
Interacting with the operating system
 The structures IO, OS, and others contain functions for such tasks as executing other programs from within sml and changing the current directory. These are documented in  The Standard ML Basis Library manual.
Emacs support
 The directory contrib/emacs contains  Matthew J. Morley's sml-mode package that supports editing SML source code and interacting with sml under GNU emacs. The  sml-mode package is available at our ftp site.

  | SML/NJ Home Page |
 | SML/NJ Documentation Home Page |
 Send your comments to .
  Copyright © 1998, Lucent Technologies; Bell Laboratories.

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Attempts to refloat eight pygmy killer whales off the shore of a Northland beach have not gone to plan, with one having to be euthanised after returning to shore.
At roughly 11:30am on Tuesday, the Department of Conservation (DOC) attempted to refloat and release the whales off Rarawa beach, with the help of locals and marine charity Project Jonah.
- 145 whales dead after mass stranding on Stewart Island
- Sperm whale dies in Northland
- Stranded Northland whale euthanised
While the whales were moved out into the water, two returned to shore after being released. One was so exhausted it had to be euthanised.
The remaining six are "still near shore, which is of concern," according to DOC.
While the release initially seemed to be going as planned, Newshub cameraman Dean Whitehead said "there is always a chance of course that the whales may get out and turn around and come back in".
Mr Whitehead said the whales had been placed in the water on small pontoons and volunteers moved them out to about 20m offshore.
"They have got lots of people standing in the water linking hands, getting ready to make some noise to help drive the animals back out to sea," he said.
Tuesday's attempt came after the whales were moved from Ninety Mile Beach, where they were stranded on Monday morning, to a stream near Rarawa Beach. DOC staff waited overnight with the animals for the high tide on Tuesday before moving them to the sea.
Marine charity Project Jonah said in a tweet on Monday that conditions were believed to be better at Rarawa Beach on the east coast, as well as the beach having the added advantage of the stream to keep the whales.
Originally there were 12 pygmy killer whales stranded on Ninety Mile Beach, but four died before Tuesday's attempt.
Mr Whitehead estimated there were roughly 200 locals, including school children and families, who had gone down to the beach to help assist DOC.
One hundred and forty-five pilot whales also died on Monday when they became stranded on Stewart Island. It is not believed the strandings are linked.

subset: commoncrawl


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In order to use RunSignup, your browser must accept cookies. Otherwise, you will not be able to register for races or use other functionality of the website. However, your browser doesn't appear to allow cookies by default.
Click the "Accept Cookie Policy" button below to accept the use of cookies on your browser.
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join us for The 3rd Winter in July 5k & 10k. Come get away from the sun while helping us raise money for St. Jude's . Participant will be running an out and back course with 3 waterstops with ice cold water, ice cold Gatorade and ice cold towels. First 300 participant will receive finisher medals, running Drifit performance shirts, and so much more. Sign up today there is a limited amount of spots open.
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Our city's number of confirmed COVID-19 cases had seen a second wave of surge this past week due to an influx of travelers returning from more inflicted countries. Experts are urging people to continue practicing social distancing. Looks like I'll be sticking with my isolation routine for at least 2 more weeks. It's not easy, even for an introvert like myself.
My calligraphy classmates actually just created a WhatsApp group to discuss if we should change venue if we were to restart our weekly class in April, but I guess that won't be necessary now seeing how the pandemic is worsening again.
Still checking the number of global confirmed COVID-19 cases on a daily basis. Many countries are seeing their numbers skyrocketing and have shut their borders. It's desensitizing just seeing the rising death toll numbers without being able to put a face to any of them. This together with countries urging their citizens to fly home and stay home feels very worrisome. Feels like war if not the apocalypse.
Started talking to some of my friends in North America again, they are now starting to see the first wave of the pandemic. Without the experience gained from SARS by many Hongkongers in 2003, my friends seemed less prepared. However, having lived in Canada for 1x years, I feel their situation should be relatively less worrisome because they're not as densely populated, not too reliant on public transit, and are much easier to practice social distancing.
Oh God, please be merciful to your people. Help us through this pandemic, save those who are infected and protect those who are healthy. Please be merciful to every single soul that has left us. We are sinful people, but we are also your precious creations, please don't turn a blind eye on us. 🙏🏻


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Making changes to your lifestyle can be daunting, so a good way to start is by making small changes to your day-to-day life that incorporate more activity. These small changes can make a huge difference if you keep them up!
You've already started. You've been doing physical activity your whole life – any movement you do counts. The key is to move more by doing activities that feel manageable and build on these as they become easier.
This step-by-step guide will take you through the process of recording your activities, your appointment dates and crucially, how you are feeling, as this will help you and your doctor to understand how you are benefiting.
The guide comprises of four steps:
It's not necessary to complete all steps to get started on the programme. If you don't have the time, or the information at hand, you can skip a step and complete it later at your convenience.


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At Tech World 2016 in Silicon Valley, Lenovo introduced several breakthrough products and concepts. One being the world's first Google Project Tango-enabled smartphone—the PHAB2 Pro. Tango enables high-performance augmented reality (AR) gaming and utilities on your mobile.
Tango brings some magical experiences to everyday life—from gaming to home improvement; to indoor navigation, shopping and learning. It uses a set of sensors and software that senses and maps its surroundings.
Check out how Lenovo and Project Tango reimagines the Barcelona Museum:
In terms of design, the PHAB2 Pro is unapologetically a phablet, like the PHAB Plus. So it may feel unwieldy, although its slim 8.9mm profile may help make it less so. The aluminum unibody is pretty stunning, available in Champagne Gold or Gunmetal Grey trim.
It features 2.5D curved glass on the front, housing the 6.4-inch QHD intelligent assertive display. Flip it to the back and you'll see how it stands out from other smartphones. It comes with a triple-camera set-up that lets it wield its 3D and AR magic. The Tango camera uses motion tracking, depth perception and area learning—able to capture more than 250,000 measurements in a second.
Under the hood, the PHAB2 Pro is quite a beast, powered by the Qualcomm Snapdragon 652 processor with 4GB of RAM and 64GB of storage. Definitely flagship-totting specs, plus it also has the famed Dolby Audio Capture 5.1 with Dolby Atmos playback capabilities.
The 4G LTE device is backed by a massive 4,050mAh battery that supports turbo-charging.
Also part of the PHAB2 family are the dual-camera PHAB2 Plus and entry-level PHAB2.
Pricing and availability
The PHAB2 starts at US$199/MYR812; PHAB2 Plus at US$299/MYR1,220 and PHAB2 Pro starts at US$499/MYR2,036. All will be available globally beginning in September. In the US, the PHAB2 Pro will be sold at select Lowe's stores nationwide and online by end of the year.


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Smith, G
G Smith
Rank: Chief Stoker
Ship: HMS Hecla
Uncle: Mr G Moore
Address: 3 Croft Terrace, Hastings
Published: February 1915
Please use the comments box below if you can provide more information about this person.
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A little bit of history:
For more than a year and a half, War Legend has been involved in the organization of Fortnite tournaments.
After organizing many LANs, we decided to automate our organization, first by creating forms and scripts on a spreadsheet.
Then, we started working on several tools and services to automate and simplify the organization of tournaments.
WLAT is one of them, again based on spreadsheet, and this time based on replay files. We had thus developed a replay file parser, from which we imported the exported data into the spreadsheet, which then allowed us to calculate the points. This platform was last used at the Orange Cup (Poland).
WLS is now our most complete tool, it allows us to have an exceptional flexibility, an ever-increasing reliability and above all an unattainable stability until now.
It's cool, but how does it work?
As mentioned above, WLS is currently based on replay files.
We are doing everything we can with our Dream Team to simplify the use of our platform for all users, whether players or organizers.
Thus, we have recently worked on the integration of external services such as Discord, but also on the WLS core.
We currently provide many public APIs, for developers, 3rd party services and platforms that want to integrate WLS into their services.


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How do I know if I have Fungal Nails?
You may have noticed changes in the texture, quality and colour of your toenails. Sometimes it is noticed after removing nail varnish or gels. First signs are often a yellowing or brown discolouration, nails may become thick, crumbly or brittle. Nails ca become distorted, miss happen and may lift from the nail bed losing their health shine.
Fungal Nail infection (Onychomycosis) is the most difficult of the superficial fungal infections to treat, as most topical agents are ineffective at penetrating the nail plate to reach the active infection on the nail bed (the skin under the nail). By making micro holes in the nail plate with the innovative and specially designed micro burrs, your Podiatrist can enable you to apply an anti-fungal agent such as Lamisil spray directly to the infected nail bed. Which will spread under the nail as you walk. With daily application of the anti-fungal agents, the fungus is stopped from spreading and the damaged nail grows out. As the nail is dead you cannot feel the holes being made.
Fungal nail infections are caused by the dermatophyte fungi that naturally live on our skin. When trauma to the nail occurs i.e micro trauma from footwear, the fungi can invade under the nail plate. In the moist warm environment of Socks and Shoes the fungi thrive, causing damage, thickening and discolouration to the nail. This most commonly affects the big toes; however, all nails are vulnerable. Treatment is notoriously difficult as the nail plate acts as a barrier, shielding the area.
The Lacuna Method is a treatment for fungal nails which involves drilling multiple micro holes through the nail plate, this allows an antifungal solution such as Lamisil ® AT 1% Spray to be transported to the area of infection (the nail bed) directly. We will advise you on the frequency and duration of the application of the spray. Pressure applied to the toe when walking will aid to spread the medication under the nail. The treatment can show signs of improvement quickly however, it can take upto 18 months to clear the infection from your nails.
As the nail is 'dead' the holes remain and will grow out naturally. Review appointments are recommended on average every 6 weeks; however, this may vary and we will advise you of this. This is a relatively new treatment and although short-term studies have been very encouraging, no treatment can be guaranteed to be 100% successful.
· Your Podiatrist can advise you what to book for.
Often a course of treatment is best
· 1 nail £170. 4 treatments 6 weeks apart. To include Lamisil spray and a shoe Klenz of a pair of shoes during your appointment. (The Klenz unit destroys all microbes in your footwear and can last for 6 weeks)
· Multiple nails (2 or more) £300. 4 treatments 6 weeks apart. To include Lamisil spray and a shoe Klenz of a pair of shoes during your appointment. (The Klenz unit destroys all microbes in your footwear and can last for 6 weeks)
If done as one off appointments (Lamisil sprayand shoe klenz sold seperately)
· £40 for one toenail
· £80 for 2
· £100 for 3 or more
· If it is your 1st appointment with us there is an additional £10 for your initial assessment.
Please note not everyone is suitable for this treatment and for your first appointment we would need to do an assessment of your general medical condition and a vascular / neurological assessment. Therefore your appointment would be longer initially and an extra charge of £10 would apply. Phone us today to book in for Lacuna treatment click here


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Wu-Tang Clan - Wu-Tang Saga (DVD)
$ 19.95
Release Date: 11/04/14
Cappadonna's story of the Wu-Tang Clan.
The influential hip-hop group Wu-Tang Clan is the subject of this music-filled documentary that follows the group members, including Method Man, RZA and Ghostface Killah, over a two-year period, with highlights from their '8 Diagrams' tour of 2007.
|Label: EYEFilm Releasing
|Genre: Rap/Hip Hop
|Language: English
|Run Time: 130 mins
NOTE: International orders are shipped using FedEx International Mail Service. Delivery can take up to 8 weeks and no tracking number is provided by this service. This is done to reduce shipping costs. If you require faster shipping or shipping with tracking, please contact us for a shipping cost.


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Division of Science and Technology (DST) invited UIC's Dr Raymond S.T. Lee, who is an Associate Professor of the Computer Science and Technology Programme as well as the Director of ITSC, to give a lecture on 23 October.
Dr Lee giving his lecture
Dr Lee received his PhD in Computer Science from Hong Kong Polytechnic University and then taught there as an Associate professor in the Department of Computing before making his way to UIC. He has published over 100 publications and is an author of six textbooks/research monographs.
The room was filled with an eager audience of students and faculty ready to expand their knowledge on this extensive topic. His talk was titled 'Quantum Finance Forecast System- A New Era of AI-Fintech System'.
Audience members captiavated by the lecture
In his talk, Dr Lee introduced this challenging AI-Fintech technology, and explained how the two pillars of Quantum Finance – Quantum Field Theory and Contemporary AI Technology can be integrated together to model financial big data and implement real time worldwide financial prediction and quantum trading systems.
He also explained a brief history of quantum finance and AI before talking about his research and ongoing projects. He said, "If weather can be forecasted than why can't the financial market be as well" before continuing on to say that he has focused his life on the future and what can come next in the quantum finance and AI world.
Audience members asking questions
After his talked had concluded, numerous questions were asked by the audience. Some were digging deeper into his theories and research, while others asked specific questions about Quantum finance and AI.
Reporter/Photographer: Lauren Richardson
Editors: Samuel Burgess, Deen He
(from MPRO)


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Contact: Department of Environmental Management and Toxicology, Faculty of Life Sciences
[email protected]
ABSTRACT: Contamination by petroleum has negative impact on the quality of the soil and water. This study was conducted to investigate the impact of organic wastes on the physico-chemical properties of diesel-contaminated soil. The soil treatments were in three levels of diesel pollution (50ml, 100ml and 150ml) and amended with poultry litter, cow dung and mixed compost amendments of poultry litter and cow dung. The changes in physico-chemical qualities of the treatment sets were monitored for a period of six months between July 2016 and December, 2016 using standard analytical methods. The results revealed temperature (oC): 24.00 ± 0.50 to 38.90 ± 0.25, pH: 4.71 ± 0.02 to 6.03 ± 0.02; with highest values for both parameters recorded in December, 2016; electrical conductivity (µ/cm), 64.40 ± 6.20 to 356.00 ± 51.0, moisture content(%): 0.52 ± 0.03 to 19.25 ± 2.05; with highest values recorded for C1, soil with poultry litter and cow dung amendment with 50 ml diesel and B3, soil with cow dung amendment with 150ml diesel, in July, 2016 respectively; organic carbon (%): 0.24 ± 0.02 to 4.01 ± 0.01 and total nitrogen (%): 0.02 ± 0.00 to 3.90 ± 0.01 with highest values recorded for B1 (cow dung amendment with 50 ml diesel) and A2 (poultry litter amendment with 100 ml diesel) in September and December, 2016 respectively. This result therefore agrees with the buffering qualities of organic wastes on diesel contaminated (acidic) soil.
Keywords: Key words: Diesel oil pollution, animal wastes, pH, Electrical conductivity, Total organic carbon.


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Everyone dream is to make our living room more beautiful and stylish. Specially we will take more care about our bedroom. Doors are playing a major role on making our bedroom even more stylish. There are some common bedroom door types available in Singapore, like Classic, Nyatoh, Veneer, Laminate and Burn. We are customizing these doors with stainless steel, grove line and glass view. We have our own manufacturer factory in Singapore to ensure that all our quality doors are manufactured to local standards.
Laminate Door
Laminate door is the perfect option for your bedroom door in HDB. Because it is also known for scratch and sound resistant. There are plenty of laminate designs available in the market for selection, compared to other types of doors.
Veneer Door
Veneer doors are always popular with interior designers when replacing new doors for homeowners. Choosing the perfect design of a door is an art and it will give a house different meaningful feel.
Solid (Nyatoh) Door
The solid door is a perfect choice for bedroom, because it made from Nyatoh sheet glued and pressed together on the Plywood with adjacent layers. We are the leading manufacturer and supplier of solid bedroom door in Singapore.
Classic Door
Are you looking to make your bedroom more modern? If yes, then classic door is the perfect choice. You can paint it with any color which is suit to your bedroom interior.
Barn Door
Only this barn doors offers you way more benefits than just opening additional space in a room. It can be installed wherever you want – making your décor like no other.


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The James Bond franchise just died. The long running action/spy movie franchise join the ever growing line of politically correct hijacking of existing ip (instead of making their own). The iconic 007, James Bond, will now be portrayed by a woman, Lashana Lynch. To be fair, she will not be "Jane Bond" but will act alongside legendary James. Apparently Bond retired and Lynch's character will be given his 007 status.
And yet I cannot help but wonder: why? Why is it necessary to hijack existing cultural icon to support your own political statements. Why not make your own? Well my theory is laziness. It's easier to appropriate something existing instead of growing a fanbase from scratch and just slap your token character (which cannot be white, male or heterosexual) and rake in the ticket sales. Yet this approach almost never works. Remember the Ghostbusters reboot at 2016? How many original fans abandoned the franchise after what is considered a sub par film? Any original fans of Doctor Who still hanging around? Star Wars anyone?
Ironically, there would have been a simple method to ensure that existing fans are kept entertained and new fans could be brought it: give her a different designation. In 1995 bond movie Goldeneye (Pierce Brosnan's debut as 007) actually has TWO 00 agents: 007 and Sean Beans Alec Trevelyan as 006. Giving Lynch's character the designation 008 would not only be correct in terms of film universe lore, it would also seem more logical. She could be his successor. Yet this new 007 is lauded as his replacement aka: put the old white dude out to pasture since he is irrelevant and we don't want white dude anymore. It is insulting. It is degrading. Why would I want to watch a movie that holds such a contempt for me, being a heterosexual white dude? Having Lynch as 008 would allow the next Bond movie to serve as a backdoor pilot for a spinoff, which would allow it to strike out at it's own and better yet, without existing fanbase and existing cultural heritage getting in the way. Clean slate.
Here's an idea that actually sounds like a cool story: Yes, Lynchs' character will be 007 for the duration of the movie BUT she will receive the 008 designation at the end since Bond decides to come out of retirement permanently (This would also be a very sneaky way to keep pr up by attracting controversy when there is no need for one). And now we would have introduction to a new 00 character that can strike out at her own without the baggage that comes with previous films. Will they do that? I doubt it. More than likely this movie will serve as a "passing of the torch" event. Well no. Actually Lynch will probably instead rip the torch out of Daniel Craigs' hands and burns his face with it while celebrating how she needs no man to validate her.
All this feels like it is done in the name of pandering. People will be quoted to say that "Bond franchise has no strong females and needs one" to which I call bullshit and offer a couple of names to consideration in no particular order and certainly not all inclusive:
In Casino Royale Eva Greens character Vesper Lynd roasts (or as Bond himself notes: skewered) Bond and calls him out on many of his trademark mannerism. That exchange was witty and interesting and made Vesper and interesting character to watch how she handles the old dog and seemed throughout the movie stand as equal next to the iconic agent. No need for SJW politics or wokeness here thank you veeery much.
In aforementioned Goldeneye we have Xenia Onatopp. An assassin who uses her sexuality as a weapon and literally chokes a man to death during their sexual encounter with her thighs. She hasn't missed legday.
1997 Tomorrow Never Dies gives us Wai Lin. I recall she was some kind of an agent and took no crap from Bond at any point. Badass female assaulting the enemy with two submachine guns on her should tell you enough.
Skyfall gave us Eve (a black woman btw). A fellow agent who accidentally shot Bond with a sniper rifle apparently killing him so there is that.
The world is not enough in 1999 introduced us to a the female villain of Elektra King while the usual white dude Renard (whom everyone thought was the villain) was her henchman.
Oh and the most badass of all is propably Bonds boss, M. "Wait a minute" I hear you saying. "M is a man." Yes the character of M was a man during the early years and since Skyfall but in the intermitten movies, M was a woman, played by Judi Dench. A no-nonsense powerhouse who intimidated everyone around her even to some extend 007 himself (she certainly had his respect at least) and proved in the World is Not Enough that she needs no man to save her when she was kidnapped by King and Macgyvered her way out of her cell by using a clock.
So if this new 007 is meant as a pandering move, it is pointless.
Bond films are iconic. They cross generational lines due to how long they have been around and are universally lauded. Even if you are not a fan of the film series, no doubt you have heard of it. The films have been around long enough to generate their own subculture and permeated it's many lines and mannerisms to our language. Bond was generating memes before memes were memes. In this day and age when being a heterosexual white male (extra points if you are overweight, not that anyone is fatshaming of course /s) looks like (I'm not saying it is so, I say it appears to me so just to be clear) to be considered the worst crime since the holocaust, it shouldn't come as a surprise that a film series appearing to those who are not familiar with the franchise almost exclusively driven by a heterosexual male character is to be the next victim of the "purge". Is this Hollywoods over the top reaction to Harry Weinstein I wonder? Are they scrambling to cater and pander to these viewpoints because they think that Weinsteins actions (or rather, their publicity) to somehow "pay back" for something? Or do they seriously think there is actual money in all this shit? Time will tell but based on previous box office results and fan outrage over other ip, the odds are good that the Bond franchise has reached it's twilight.


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merrie halloween quiltFukuoka | Japan a wool and cotton quilt pattern for halloween lovers! merrie halloween is one of the newest designs by buttermilk basin and is available in two options: wool or cotton! both options bring a different feel to each quilt. Fukuoka | Japan Merrie Christmas BOM a fun and oh-so-spooky wool and flanel quilt for all you halloween lovers like us! we love everything about this quilt. Check It Out especially its spook-tac-u-lar halloween. Merrie Halloween BOM quilt halloween panels for this spooky holiday! make halloween paperpiecing quilts, pumpkin quilt patterns, halloween applique patterns, and witch quilt patterns. $28 try. 00 merrie halloween : quantity: if we do not have the full amount that you ordered of this item: add to cart. Check It Out the quilt rack & wool cubby protected by copyscape plagiarism checker - do not copy content from this page. Message Board for some featured events on the big island over the next two weeks, click here bing crosby, actor: white christmas. Orange Peels & Pinwheels Quilt bing crosby was born harry lillis crosby, jr. $12 in tacoma, washington, the fourth of seven children of catherine helen kate. 00 this is clue 3 of the mystery of halloween mystery quilt, where we assemble the x blocks. Check It Out this is the quickest of our five clues! this pattern was designed as part of a 12 month block of the month, but you can make this as an individual wall hanging if you like. Hey Roberta, the pattern is called Merrie Halloween, and is by Stacy West of Buttermilk Basin the september home represents a. You can find the pattern for sale at go into find washington craft shows, art shows, fairs and festivals. Quilt kits make creating a quilt, wall hanging, or table runner less daunting 30000+ detailed listings for washington artists, washington crafters, food vendors, concessionaires and. Search the quilt kit sale to find discounted items for your next project learn how to make this most recognizable american quilt using pre-cut strips of fabric. This is Merrie's version of a little over a year later, i m almost done with my halloween quilt. They teach this quilt as a let me walk you through exactly how i designed the quilt then and what i d do differently now. Appliqué breast cancer Card Christmas Crib Quilt Custom Quilting Embroidery Halloween heart pillows our word of the year choice serves as a symbol of each year's most meaningful events and lookup trends. Halloween quilt patterns and more, including halloween bag pattern, halloween banner patterns, and spooky halloween table toppers it is an opportunity for us to reflect on the language and.
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Part II: USAF veteran Pete Jones lends telecom expertise to Grand Canyon
GRAND CANYON, Ariz. – Growing up in northern California, there were only a few career choices available for recent high school graduates — logging or fishing. Pete Jones decided neither of those were his calling.
As a young child, the military wasn't a significant factor in Jones' life.
"My dad did his two years back when it was mandatory, and my mom would tell stories about her brothers being in World War II," he said.
When the logging and fishing industries began declining in the late 1970s, Jones made the decision to leave.
"I said, 'You know, I have to get out of this town,'" Jones explained. "(I wanted to) see what's going on, get an education, go somewhere else."
And somewhere else ended up being halfway across the world – Jones' first assignment was Incirlik Air Base, located in southern Turkey about an hour inland from the Mediterranean Sea.
"That was back in 1983 when they still had trailers for base housing," he said. "They did get rid of the trailers while I was there and built a lot of new housing."
The Air Force trained Jones on communications, and he served his entire 21-year career as a communications specialist, working on everything from older rotary-dial telephones up to more recent technology like voice over internet protocol (VoIP).
"I was a telephone man from day one," he said.
After being stationed in Turkey for a year, Jones returned to the United States to Washington for a couple of years before returning to Turkey, this time to Izmir on the western coast. While in Washington, Jones decided he wanted to go back overseas, so he volunteered for a long tour.
"There's a rumor that once you've been stationed in Turkey, you're definitely going back," he chuckled.
This time, Jones ended up working with a unit of the North American Treaty Organization (NATO). He said he enjoyed his time at Izmir because there was no base – troops were stationed within the city.
"You really got to interact more with the Turks," he said.
Rescuing Captain O'Grady
One of the most memorable events happened to Jones in the mid-1990s, when he was stationed in Italy with NATO. The war between Bosnia and Herzegovina had been raging for three years, the international news media roiled by allegations of atrocities and war crimes against civilians.
In June 1995, two USAF F-16 fighter jets were patrolling the skies over Bosnia to prevent the Bosnian-Serb forces from attacking civilians and other troops in the area. The Bosnian-Serb armed forces fired two ground-to-air missiles at the planes, missing with the first and striking the plane with the second, flown by Capt. Scott O'Grady.
O'Grady ejected from the plane and survived the crash, but found himself behind enemy lines in extremely hostile territory
Back in Italy, on Friday, Jones was preparing for a relaxed dinner with the rest of his crew. He didn't know it at the time, but an effort to launch an offensive and rescue O'Grady was underway.
"We were all getting ready to go when the joint headquarters communications guy comes in and said 'Hey, I need a couple of phones,'" Jones said.
Thinking it wasn't anything that couldn't wait, Jones said he'd have what the officer needed Monday morning.
"The officer looked at his watch then back at me and said, 'You don't understand; we're going feet dry in 20 minutes and need two phones,'" Jones recalled.
Jones jumped into action, sending his crew to run the lines on the cross-connect frame through the building while he programmed the lines. Jones said his crew wasn't sure if the phone cable in the buildings was even operational, but the phones were ready as requested and the party even made their dinner reservation.
A few hours later, O'Grady was rescued after avoiding capture for 6 days, surviving on bugs, leaves and grass and collecting rainwater with a sponge in a plastic bag.
Coming to the Canyon
During his career, Jones was stationed in Florida, Germany, Alaska, Colorado and Italy. He met his wife, Sheila, while both were on active duty in Florida. Although they were stationed on different sides of the state, both were sent to the same leadership training class in Biloxi, Mississippi. After his retirement, he and Sheila moved back to Florida, where she was originally from.
After a short stint, Jones took another job with the Air Force, this time as a civilian contractor, and moved to Alaska for eight years. After leaving that job, he made the decision to move to Arizona to be closer to his daughter and grandchildren in Phoenix.
"I applied for a job (at Grand Canyon) that I really wasn't qualified for, but during my interview for that job, they told me to keep my eyes open for something else (I was qualified for)," he said.
Jones is now a telecommunications specialist for Grand Canyon National Park, something he's more than qualified for after his Air Force training. Along with keeping all the phones up and running, he also works with the park's internet provider to make sure each department has the services it needs to operate.
Reflecting on his military service, Jones said he would do it all again if he could. He still has memories of serving as the weekend deejays for the base's radio station in Italy and lifelong friendships that keep the connection strong.
"I'd definitely do it all again," he said. "I had a ball."
- Little change expected in Williams following Ducey's stay at home order
- Grand Canyon ghosts: The El Tovar Hotel's haunted history
- Arizona mayors pressure governor to issue stay at home order
- Grand Canyon remains open for Spring Break, cancels some park activities
- Fight against virus hits home in Williams
- Coconino County offers utility and rent assistance for qualified applicants
- Latest Williams area coronavirus news: March 15 update
- Arizona governor issues stay-at-home order effective Tuesday
- Arizona coronavirus cases grow slightly; total now at 12
- Thousands of COVID-19 cases predicted for Arizona
- Latest Williams area coronavirus news: March 15 update
- Grand Canyon remains open for Spring Break, cancels some park activities
- Fight against virus hits home in Williams
- More services close at Grand Canyon National Park
- Thousands of COVID-19 cases predicted for Arizona
- City of Williams declares State of Emergency, announces no business closures
- Coconino County offers utility and rent assistance for qualified applicants
- Teacher fired at Williams High School
- Westbound I-40 west of Williams closed because of crash
- 18-year-old dies after accident at Great Smoky Mountains Park
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Prairie View A&M University Prairie View, TX, USA
Mar 20, 2020Full time
Prairie View A&M University Senior Audio Visual Specialist-1 Job Title Senior Audio Visual Specialist-1 Agency Prairie View A&M University Department Business Affairs Information Resource Office Proposed Minimum Salary $3,929.84 monthly Job Location Prairie View, Texas Job Type Staff Job Description The Senior Audio Visual Specialist, under general supervision, Provides technical support that ensures the proper operation, maintenance, monitoring, testing and repair of complex networked AV equipment and control systems. Oversees the delivery and troubleshooting of educational, conferencing and general event content. Supervises audio visual staff and students and trains end users and support staff on the use of equipment. Responsibilities: Provides technical support that ensures the proper operation, maintenance, monitoring, testing and repair of complex networked AV equipment and control systems. Oversees the delivery and troubleshooting of educational, conferencing and general event content. Supervises audio visual staff and students. Train end users and support staff on the use of equipment. Coordinates activities of schedulers and technicians to ensure proper setups for university events such as conferences, seminars and general classroom AV needs. Develops, documents, and reports current procedures and suggests areas of progress for the group. Communicates with the primary users of the conference rooms, auditoriums and classrooms. Researches, identifies, implements, and maintains new AV technology and software. Interfaces with other departments and external entities to meet client requirements. Maintain inventory of equipment such as projector lamps, class room presenters, microphones, chargers, batteries, cables and adapters. Other duties as assigned Required Education and Experience: Bachelor's degree in applicable field Four years of related experience in audio/video communications, presentation technologies, and system troubleshooting. Job Posting Close Date: Open Until Filled Knowledge, Skills and Abilities: Knowledge of video conferencing systems. Knowledge of audio/video equipment and controls, related networks, and computer applications. Ability to multitask and work cooperatively with others. Other Requirements: Ability to lift moderately heavy objects. Required Attachments: Please attach all required documents listed below in the attachment box labeled as either "Resume/CV or Resume/Cover Letter" on the application. Multiple attachments may be included in the "Resume/CV" or Resume/Cover Letter" attachment box. Any additional attachments provided outside of the required documents listed below are considered optional. Cover Letter Resume or Curriculum Vitae Application Submission Guidelines: All applicants are required to apply via our Career Site on or before the closing date indicated on the job posting. Applicant inquiries received via email and websites such as Indeed, HigherEdJobs, etc. will not be considered unless the individual has applied to the available position via the PVAMU Career site. The required documents listed in the above Required Attachments section must be attached to the application prior to the job closing date indicated to ensure full consideration for the application submitted. Please contact the Office of Human Resource on or before the closing date indicated above at 936-261-1730 or email@example.com should you need assistance with the online application process. Background Check Requirements: All positions are security-sensitive. Applicants are subject to a criminal history investigation, and employment is contingent upon the institution's verification of credentials and/or other information required by the institution's procedures, including the completion of the criminal history check. Equal Opportunity/Affirmative Action/Veterans/Disability Employer committed to diversity. To apply, visit https://tamus.wd1.myworkdayjobs.com/en-US/PVAMU_External/job/Prairie-View-PVAMU/Senior-Audio-Visual-Specialist-1_R-026589 About Us For more than 140 years, Prairie View A&M University (PVAMU) has been igniting passion in its students. With an established reputation for producing engineers, nurses and educators, PVAMU offers baccalaureate degrees, master's degrees and doctoral degree programs through nine colleges and schools. The University has an enrollment of more than 8,500 students. Designated an institution of "the first class" in the Texas Constitution, PVAMU is the second-oldest public institution of higher education in the state and a member of The Texas A&M University System. The University is dedicated to fulfilling its land-grant mission of achieving excellence in teaching, research and service. PVAMU is conveniently located near the rapidly growing residential and commercial area known as the Northwest Houston Corridor. The developing Northwest Houston Corridor offers newly developed suburban communities, a variety of shopping and dining near the Houston Premium Outlets and is a short, 35-minute commute into downtown Houston. For more information, visit www.pvamu.edu. Your career opportunities with a "University of the first class" begins here! Prairie View A&M University is an Equal Opportunity/Affirmative Action/Veterans/Disability Employer. If you need assistance in applying for this job, please contact (936) 261-1730. Copyright ©2017 Jobelephant.com Inc. All rights reserved. Posted by the FREE value-added recruitment advertising agency jeid-63d73c74856bcf409ed566dc01278d3b


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Yesterday, in a show of force, the Page Fire and Police Departments teamed up to tackle a fire that the high winds blew onto three neighboring properties.
Eye-witness accounts claim that the fire originated behind a home on South Navajo Drive and quickly spread to the backyards of three neighbors on Calle Hermosa, threatening the homes. Eye-witnesses also state there were several neighbors who noticed the smoke and attempted to put out the fire before emergency units were called. As of this writing, there have been no official reports available regarding the cause and starting location of the fire.
The fire caused damage to several trees, a fence, and other structures, but no one was reported to have been injured.
During the incident, the Page Fire Department was located on South Navajo Drive and the Police Department was situated on Calle Hermosa, blocking traffic from entering the area. While the Fire Department tackled the original fire on South Navajo Drive, the winds picked up and spread the fire to a shed, some trees and a fence on adjoining properties. During this time, several officers from the Page Police Department jumped into action and began spraying the trees with hoses and fire extinguishers.
Once the flames calmed down, the wind picked back up and yet another tree caught at a different residence. An onlooker saw this and informed who LPNN believes to be the homeowner, who then grabbed a water hose and began attempting to put it out. An unidentified officer stepped in with a fire extinguisher to assist. Because of the wind, their efforts were minimally effective. The officer then climbed a ladder brought in by neighboring residents and proceeded to the roof of the home. From there, he was able to keep the fire in the tree from touching the home until the fire department could step in.
Once that fire was quelled, the officer turned his attention to a tree that had begun to flare up again and began spraying it down as best he could from the roof top. Page Fire Department was not long behind him and both agencies had a unit putting the flames out.
At approximately 5:50pm, the fire was officially contained, Calle Hermosa was reopened, and residents were allowed back into their homes.
Thank you to those who wear red, and blue, and put their lives on the line every day to keep us safe. With the high winds late yesterday afternoon, and the resin from evergreens not wanting to give up, without the Page Fire and Police Departments and their exemplary collaboration, this situation could have ended with more than the loss of a shed, a fence, and some trees.
LPNN Was live on scene to capture the heroism of our local forces. Here is the coverage.


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We have used gluconate-limited chemostats to study selective differences between isogenic strains of Escherichia coli K12 into which four naturally occurring alleles coding for allozymes of 6-phosphogluconate dehydrogenase (6PGD) had been transferred. The limit of detectability of selection with our procedures is a selection coefficient of 0.5%. In the normal E. coli K12 genetic background, all alleles are selectively neutral or nearly neutral. The absence of detectable selection does, however, depend on genetic background and on such environmental factors as cell density. In a genetic background containing a mutation that cuts off the alternative metabolic route for 6-phosphogluconate, selection between allozymes can be detected, and the selection is in the direction expected from the measured apparent Km values of the allozymes. Even when the alternative metabolic route is not blocked by mutation, one of the 6PGD allozymes has a detrimental, but density-dependent, interaction with a mutation conferring resistance to bacteriophage T5. In all cases, the observed selection is due to the allozymes themselves (or to associated regulatory elements), as the selection disappears when the chemostats are limited by a different carbon source (ribose plus succinate). Nevertheless, the four alleles do seem to be selectively neutral or nearly neutral in the normal E. coli K12 genetic background. Moreover, the distribution of allele frequencies in natural populations of E. coli is in accord with the expectations of selective neutrality.
I am inclined to suspect that we see, at least in some [cases], variations which are of no service to the species, and which consequently have not been seized on and rendered definite by natural selection…. Variations neither useful nor injurious would not be affected by natural selection, and would be left either a fluctuating element, as perhaps we see in certain polymorphic species, or would ultimately become fixed…. We may easily err in attributing importance to characters, and in believing that they have been developed through natural selection;… many structures are now of no direct use to their possessors, and may never have been of any use to their progenitors…. [On the other hand,] we are much too ignorant in regard to the whole economy of any organic being to say what slight modifications would be of importance or not.
- Received July 14, 1980.


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Metal 3D printing's capability to create lightweight, detailed custom parts means that it could be the ideal process to make certain implants for animals.


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- Research article
- Open Access
- Published:
WIF1 enhanced dentinogenic differentiation in stem cells from apical papilla
BMC Oral Health volume 19, Article number: 25 (2019)
933 Accesses
1 Altmetric
Odontogenic mesenchymal stem cells (MSCs) isolated from tooth tissues are a reliable resource that can be utilized for dental tissue regeneration. Exploration of the mechanisms underlying the regulation of their differentiation may be helpful for investigating potential clinical applications. The stem cell niche plays an important role in maintaining cell functioning. Previous studies found that Wnt inhibitory factor 1 (WIF1) is more highly expressed in apical papilla tissues than in stem cells from apical papilla (SCAPs) using microarray analysis. However, the function of WIF1 in SCAPs remains unclear. In the present study, we investigated the function of WIF1 during dentinogenic differentiation in SCAPs.
A retrovirus containing HA-WIF1 was used to overexpress WIF1 in SCAPs. Using Western blot analysis, we verified the expression of HA-WIF1. Alkaline phosphatase (ALP) activity assays, Alizarin Red staining and quantitative calcium analysis were performed to investigate the in vitro potential for dentinogenic differentiation in SCAPs. The expression of dentinogenesis-associated genes DSPP, DMP1, Runx2 and OSX were assayed using real-time RT-PCR. Transplantation experiments were used to measure dentinogenesis potential in vivo.
The real time RT-PCR results showed that WIF1 was more highly expressed in apical papilla tissues than in SCAPs, and its expression was increased during the process of dentinogenic differentiation. Overexpression of WIF1 enhanced ALP activity and mineralization in vitro, as well as the expression of DSPP, DMP1 and OSX in SCAPs. Moreover, in vivo transplantation experiments revealed that dentinogenesis in SCAPs was enhanced by WIF1 overexpression.
These results suggest that WIF1 may enhance dentinogenic differentiation potential in dental MSCs via its regulation of OSX and identified potential target genes that could be useful for improving dental tissue regeneration.
Due to technological improvements in tissue engineering, stem cell therapy holds great promise for the advancement of regenerative medicine. Mesenchymal stem cells (MSCs) are considered a good cell source for use in tissue regeneration [1]. From tooth tissues, it is possible to isolate several types of MSCs, including periodontal ligament (PDLSCs), dental pulp (DPSCs), and dental follicular stem cells (DFSCs), as well as stem cells from apical papilla (SCAPs) [1,2,3]. These MSCs are more intimately associated with dental tissues and can regenerate bone/dentin-like tissues and repair damaged dental tissues [1,2,3,4,5]. However, their potential for use in clinical applications is restricted since the mechanisms underlying dentinogenic differentiation remain unclear.
The stem cell niche supports and maintains the functioning of MSCs. This niche cannot be maintained when MSCs are isolated and cultured in vitro using current methods. An impaired niche will not be beneficial for dental MSC-mediated tooth tissue regeneration [6,7,8]. Therefore, the identification of the key regulator of the niche that supports dental MSCs will aid in the enhancement of dental tissue regeneration. A previous study examined gene expression profiles in apical papilla tissues, SCAPs and SCAP cell sheets and revealed several candidate key genes within the SCAP niche using microarray and bioinformatic analysis [9]. In their analysis, one canonical Wnt/β-catenin inhibitor, Wnt inhibitory factor 1 (WIF1), was found to be more highly expressed in apical papilla tissues than in SCAPs. Wnt proteins are important regulators of differentiation in stem cells; the Wnt/β-catenin pathway enhances osteogenesis of MSCs and osteoprogenitor cells by upregulating osteoblast-related genes [10]. However, another study found that the Wnt/β-catenin pathway inhibited osteogenesis of PDLSCs [11]. This suggests that the Wnt/β-catenin pathway may play different roles in MSC regulation. WIF1 belongs to a family of secreted modulators of Wnt proteins. Other members of the Wnt modulator family, secreted frizzled-related proteins (sFRPs), play different roles in Wnt signaling depending on the cell subtype and model used [12,13,14,15,16]. In fact, WIF1 also has the effect of modulating Wnt signaling. For example, a previous study found that WIF1 inhibits osteoblastic differentiation in mouse mesenchymal C3H10T1/2 cells [17]. Another study found that WIF1 is highly expressed at cartilage-mesenchyme interfaces in the skeleton during early development and that it blocks the classical Wnt signaling pathway and impairs Wnt3a-mediated inhibition of chondrogenesis in embryonic chick limb-bud cells [18]. However, the function of WIF1 during dentinogenic differentiation in dental MSCs remains unclear.
In the present study, we utilized SCAPs to investigate the functioning of WIF1 during dentinogenic differentiation. We found that WIF1 could enhance the dentinogenic differentiation capacity in SCAPs. These findings provide novel insights into the mechanisms underlying directed differentiation of dental tissue-derived MSCs and have valuable clinical applications.
Cell culture of SCAPs and human embryonic kidney (293 T) cells
All our research involving human stem cells complies with the ISSCR "Guidelines for the Conduct of Human Embryonic Stem Cell Research." Wisdom teeth were obtained from patients who provided written informed consent according to guidelines approved by the ethical committee (Ethical Committee Agreement, Beijing Stomatological Hospital, Ethics Review No. 2011–02). We disinfected the teeth in 75% ethanol and then washed them with phosphate buffered saline (PBS). The cell culture procedures used, as well as the phenotype, lineage markers and lineage differentiation potentials of SCAPs were identified and described in our previous study [19]. Briefly, apical papilla were gently separated from the apical root and then digested in a solution containing 3 mg/mL collagenase type I (Worthington Biochemical Corp., Lakewood, NJ, USA) and 4 mg/mL dispase (Roche Diagnostics Corp., Indianapolis, IN, USA) for 1 h at 37 °C. A single-cell suspension was produced by passing the solution through a 70 μm strainer (Falcon, BD Biosciences, San Jose, CA, USA). The SCAPs were grown in DMEM alpha modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) that was supplemented with 15% fetal bovine serum (FBS; Invitrogen), 2 mmol/L glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) at 37 °C in a humidified incubator containing 5% CO2. The culture medium was changed every 3 days.
Human embryonic kidney 293 T cells were maintained in complete DMEM medium containing 10% fetal bovine serum (FBS; Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen).
Plasmid construction and viral infection
Full-length human WNT1 cDNA containing a hemagglutinin (HA) tag was produced using a standard gene synthesis method and subcloned into the pQCXIN retroviral vector (BD Clontech, Mountain View, CA, USA) between the Age I and EcoR1 restriction sites and verified by genetic sequencing. The viral packaging was then performed in 293 T cells according to the manufacturer's protocol (BD Clontech). Prior to viral infections, the SCAPs were subcultured overnight and then infected with retroviruses in the presence of polybrene (6 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) for 12 h. After 48 h, infected cells were selected using 600 mg/ML G418 (Sigma-Aldrich).
Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR
Total RNA was isolated from SCAPs using Trizol reagent (Invitrogen). cDNA was synthesized from a 2 μg aliquot of RNA containing oligo(dT), and reverse transcriptase(Invitrogen) according to the manufacturer's protocol. Real-time PCRs were performed using the QuantiTect SYBR Green PCR kit (Qiangen, Hilden, Germany) and the Bio-Rad Real-time PCR Detection System. The changes in gene expression were determined using the 2-△△CT method. The primers used to specific genes are shown in Table 1.
In vitro cell differentiation assay
Osteo/dentinogenesis differentiation assays were performed as previously reported [4]. The expression of the DSPP, DMP1, OSX and Runx2 genes was determined using real-time RT-PCR.
Alkaline phosphatase and alizarin red detection
Mineralization was induced in SCAPs using the STEMPRO Osteogenesis Differentiation Kit (Invitrogen). ALP activity was assayed at 5 days using an ALP activity kit according to the manufacturer's protocol (Sigma-Aldrich). For the mineralization assay, the cells were induced for 2 weeks, fixed in 70% ethanol and then stained with 2% Alizarin red (Sigma-Aldrich). To quantitatively measure calcium, the Alizarin Red-stained cells were then destained with 10% cetylpyridinium chloride in 10 mM sodium phosphate for 30 min at room temperature and measured at 562 nm in a multiplate reader; the amount of calcium was determined via comparison to a standard calcium curve obtained using calcium dilutions prepared in the same solution. The final calcium level in each group was normalized to the total protein concentration measured in a duplicate plate.
Western blot analysis
The cells were lysed in RIPA buffer (10 mM Tris-HCL, 1 mM EDTA, 1% sodium dodecylsulfate (SDS), 1% NP-40, 1:100 proteinase inhibitor cocktail, 50 mM β-glycerophosphate, 50 mM sodium fluoride). The samples were separated on a 10% SDS polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane using a semidry transfer apparatus (Bio-Rad, Hercules, CA,USA). The membranes were blotted with 5% dehydrated milk for 2 h and then incubated with primary antibodies overnight. The membranes then were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Promega, Madison, WI, USA) and visualized using SuperSignal reagents (Pierce, Rockford, IL, USA). The primary antibodies used were anti-HA (Clone No. C29F4, Cat No. 3724, Cell Signaling Technology, Beverly, MA, USA) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Clone No. GAPDH-71.1, Cat No. G8795, Sigma-Aldrich), which is a housekeeping gene.
Transplantation in nude mice
These procedures were performed with the approval of the Ethics Committee and Animal Care and Use Committee of Beijing Stomatological Hospital at Capital Medical University (AEEI-2015-085). The animals were purchased from the Institute of Animal Science at Vital River Co., Ltd. and were not treated with drugs or subject to any other procedures prior to transplantation. Approximately 2.0 × 106 cells were mixed with 20 mg of HA/tricalcium phosphate ceramic particles (Engineering Research Center for Biomaterials, Sichuan University, Chengdu, China) and then transplanted subcutaneously beneath the dorsal skin of 10-week-old immunocompromised beige mice (nu/nu nude mice). Eight weeks later, the tissues were harvested, fixed in 10% formalin, decalcified using 10% EDTA buffer (pH 8.0), and embedded in paraffin.
Immunohistochemical staining
Five μm sections were cut from tissue blocks, deparaffinized and hydrated. The sections were then soaked in 3% H2O2 for 10 min and washed 3 times in distilled water for 5 min each. Epitope retrieval was conducted for 10 min in a microwave oven using 1.0 mmol/L EDTA buffer (pH 8.0), followed by a 1-h cool down period. The sections were then washed 5 times with phosphate-buffered saline (PBS) for 5 min each time and incubated with 10% goat serum for 60 min to block nonspecific antibody binding. After washing with PBS, the sections were incubated with a primary monoclonal antibody against DSPP (sc-33,586, Santa Cruz) overnight at 4 °C. After washing 3 times in PBS for 5 min each, the sections were incubated with horseradish peroxidase–conjugated anti-rabbit secondary antibody (Promega, Madison, WI, USA) for 30 min at room temperature. The sections were washed 3 times in PBS for 5 min each and then subjected to coloration using a diaminobenzidine (DAB) kit (Cat No. 8059S, Cell Signaling Technology, Inc. Beverly, MA, USA). Finally, the sections were counterstained with hematoxylin, dehydrated in a graded series of alcohol washes and mounted for light microscopy with neutral gum.
Statistical analyses
All data are presented as the mean ± standard deviation (SD). All statistical calculations were performed using SPSS13.0 statistical software. Student's t-test was performed to determine the statistical significance. A P-value ≤0.05 was considered to denote statistical significance.
WIF1 expression was increased during dentinogenic differentiation
We first examined the expression of WIF1 in SCAPs and apical papilla tissues. The real-time RT-PCR results showed that WIF1 expression was much higher in apical papilla tissues than in SCAPs (Fig. 1a). We then measured the expression of WIF1 in differentiated SCAPs upon the induction of mineralization. We found that, when the osteo/dentinogenic differentiation process was induced in SCAPs, WIF1 expression was upregulated during dentinogenic differentiation in a time-dependent manner (Fig. 1b).
WIF1 enhanced the dentinogenic differentiation potential of SCAPs in vitro
To understand the function of WIF1 during the differentiation of SCAPs, we inserted the WIF1 sequence along with an HA tag into a retroviral vector. This construct resulted in the overexpression of ectopic HA-WIF1 when transduced into SCAPs via retroviral infection. After selection with 600 μg/mL G418 for 14 days, ectopic WIF1 expression was confirmed using real-time RT-PCR and Western blot analysis (Fig. 2a, b). Next, the transduced SCAPs were cultured in mineralization-inducing medium to investigate their dentinogenic differentiation potential. At 5 days after induction, the results indicated that the overexpression of wild-type WIF1 promoted the activity of ALP, which is an early marker of dentinogenic differentiation in SCAPs (Fig. 2c). This was confirmed by the observation that mineralization was significantly enhanced in SCAPs that overexpressed WIF1 after 2 weeks of induction compared to cells infected with the empty vector, as determined using Alizarin Red staining and quantitative calcium measurement (Fig. 2d, e).
The dentinogenic marker genes, DSPP and DMP1, were subsequently measured. The real-time RT-PCR results showed that DSPP was induced to a greater extent in SCAP-WIF1 cells than in SCAP-Vector cells after 2 weeks of induction (Fig. 3a), and DMP1 was induced to a greater extent in SCAP-WIF1 cells than in SCAP-Vector cells at 1 and 2 weeks after induction (Fig. 3b). We also examined the expression of key transcription factors involved in dentinogenic differentiation in SCAPs, including runt-related transcription factor 2 (known as RUNX2) and OSX. Our results showed that the mRNA levels of OSX were significantly higher in SCAP-WIF1 cells than in SCAP-Vector cells (Fig. 3c), but that the expression of RUNX2 was not significantly altered (Fig. 3d).
WIF1 enhanced in vivo dentinogenesis in SCAPs
We investigated whether the overexpression of WIF1 would affect SCAP dentinogenesis in vivo. We transplanted control or WIF1 SCAPs subcutaneously into nude mice. The transplanted tissues were harvested eight weeks after transplantation. H&E staining revealed that a greater amount of bone/dentin-like tissues were present in the mice transplanted with WIF1 SCAPs than in mice transplanted with control SCAPs (Fig. 4a). Qualitative measurements also revealed a greater amount of bone/dentin-like tissues in mice transplanted with WIF1 SCAPs than in those transplanted with control SCAPs (Fig. 4b). These transplantation experiments demonstrated that WIF1 SCAPs generated a greater amount of bone/dentin-like tissues than control SCAPs in vivo.
Immunohistochemical staining was then used to measure the expression of DSPP. The results revealed that more DSPP protein was present in tissues transplanted with SCAP-WIF1 cells than those transplanted with SCAP-Vector cells (Fig. 4c). Additionally, negative control staining was shown to occur in the absence of primary antibody (Fig. 4d). Overall together, these results showed that the overexpression of WIF1 significantly enhanced the formation of dentin-like tissues.
MSCs derived from dental tissues have proved to be promising source of cells with therapeutic potential, especially for use in dental tissue treatment. However, little is known about the molecular mechanisms underlying the stability and differentiation potential of dental MSCs. SCAPs are important for tooth development and regeneration. A key factor that ensures successful dental tissue regeneration is the niche in which the dental cells and tissues are grown [20], which makes it important to identify the key genes that support the functioning of MSCs in the niche. The commitment and differentiation of MSCs is reliant upon Wnt/β-catenin signaling [21,22,23,24]. Therefore, we chose to investigate the Wnt/β-catenin modulator WIF1, which has been found to be more highly expressed in apical papilla tissues than in SCAPs using microarray analysis. First, we confirmed that WIF1 expression was decreased in cultured SCAPs after isolation from apical papilla and then verified that it was increased during the dentinogenic differentiation process; this indicated that increased levels of WIF1 may play a role in dentinogenic differentiation.
Based on these observations, we investigated the function of WIF1 during the dentinogenic differentiation process in SCAPs. The results showed that WIF1 promoted ALP activity and mineralization in vitro. ALP is as an indicator of early differentiation during the osteo/dentinogenic process [25]. The presence of the mineralization phenotype is an indicator of the end stage of the osteo/dentinogenic differentiation process. Moreover, transplantation experiments demonstrated that newly formed bone/dentin-like tissues were deposited by transplanted SCAPs-Vector and SCAPs-WIF1 cells and revealed that WIF1 promoted osteo/dentinogenesis in vivo. These results indicated that WIF1 enhanced osteo/dentinogenic differentiation in SCAPs. To clarify the role of WIF1 in dentinogenic differentiation, we also investigated dentinogenic differentiation indicators. DSPP and DMP1 are classic odontogenic markers; DSPP is a key gene involved in the process of dentin formation, while DMP1 has been shown to regulate DSPP [26,27,28]. We found that the expression of DSPP and DMP1 were enhanced by WIF1 in SCAPs in vitro. Additionally, a greater amount of DSPP protein was found in tissues, transplanted with SCAPs-WIF1 cells. These results indicated that WIF1 was able to promote dentinogenic differentiation in SCAPs.
In addition, we found that expression of the transcription factor OSX was also enhanced by WIF1. OSX is known to be an essential transcription factor that contains three C2H2-type zinc finger DNA binding domains. Osx is expressed during the entire process of tooth development [29,30,31]. The amount of cementum has been found to be reduced due to Osx deletion in mice [32]. An in vitro study found that Osx increases Dspp transcription in odontoblast-like cells [33]. This evidence suggests that Osx plays a critical role in dentinogenic differentiation and formation. We also found that the mRNA expression level of RUNX2, a transcription factor, was not significantly different in SCAP-WIF1 and SCAP-Vector cells. An in vitro study by Han found that Wnt/β-catenin could enhance dentinogenic differentiation in DPSC cells by activating RUNX2 [34]. There are no reports suggesting that RUNX2 upregulation is not required for dentinogenic differentiation. Overall, these findings suggested that WIF1 may enhance dentinogenic differentiation via enhancement of OSX expression in SCAPs.
Our results showed that WIF1 enhanced dentinogenic differentiation in SCAPs by activating the transcription factor OSX. Our work explored the mechanisms underlying the effects of WIF1 on directed differentiation in dental MSCs and provided potential target genes that could be useful in improving dental tissue regeneration using dental tissue-derived MSCs.
Alkaline phosphatase
- DFSCs:
Stem cells from dental follicles
Dentin matrix protein 1
- DPSCs:
Dental pulp stem cells
Dentin sialophosphoprotein
- MSCs:
Mesenchymal stem cells
Phosphate-buffered saline
- PDLSCs:
Periodontal ligament
Reverse transcriptase-polymerase chain reaction
- SCAPs:
Stem cells from apical papilla
- sFRPs:
Secreted frizzled-related proteins
Statistical Package for Social Science
Wnt inhibitory factor 1
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We would like to acknowledge Pro. Zhipeng Fan from the Capital Medical University School of Stomatology for his assistance in the study design.
This work was supported by grants from the National Natural Science Foundation of China (81670948; provided to YC) and the China Rehabilitation Research Center Foundation (2017ZX-03; provided to HW).
Availability of data and materials
The datasets used for the current study are available from the corresponding author by request.
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This study was approved by the Ethics Committee of the Beijing Stomatological Hospital at the Capital Medical University (Ethics Review No. 2011–02); all patients provided written informed consent.
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Wang, H., Cao, Y. WIF1 enhanced dentinogenic differentiation in stem cells from apical papilla. BMC Oral Health 19, 25 (2019). https://doi.org/10.1186/s12903-018-0700-6
- Dentinogenic differentiation
- Stem cells from apical papilla (SCAPs)


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