text:
Open-ended questions
Among the different types of questions that can be used in a job interview, the open-ended questions are often the most useful for the interviewer. This kind of questions allows getting a little more information about you, therefore, the interviewer expect a wide response, not a simple "yes" or "no". The Open-Ended Questions also allows interviewers time to plan subsequent questions and encourage applicants to talk a bit more fluency, leaving aside the timidity and fear.
Below, you will be able to find, some of the most common and important Open-Ended questions:
What do you know about us?
This question allows the interviewer to see, how well prepared are the candidates and how they know about the company. To answer this question, it is advisable to have read a little about the company and know things such as who the founder was and the company's awards. You can also talk a little about the company's achievements and products. An example of good answer might be:
- I know this is a well known company with excellent professionals. I also know by my experience that you offer quality products and services. That's why I want to work here.
How would you describe yourself? and how would others describe you?
These are two classic questions that interviewers often use. The first question allows the interviewer to see what the candidates think of themselves, it is important to remember that if you can't get excited about yourself, then there's a problem. The second question shows how others describe you; it will give a clear idea about if you are able to LISTEN to others. An example of good answer might be:
- I am a very responsible person with the desire to succeed. People who know me also say the same about me.
What motivates you personally and professionally on a daily basis?
This question allows the interviewer to see what motivates candidates to work and excel, and what kind of qualities the candidates can see in the company. To answer this question is necessary to find a special feature on the company and relate it to your career aspirations. An example of good answer might be:
- I like the teamwork and good treatment to people, and I see that this company can offer me that. I can also see that the company offers me many opportunities for my professional development.
Why are you leaving (left) your last job?
The answer the interviewer is looking for here is "for growth." Because the company wants a candidate that is looking to expand out of their comfort zone and move their career to the next level. Most businessmen would submit that a candidate who is not motivated is not worth hiring because; it is a sure sign of lack of commitment at work. That is why a motivated candidate even when slightly less qualified would be a better option to hire. An example of good answer might be:
- I want to get ahead and be recognized for my work, and I think my previous job did not help me with that
If you would have the opportunity to work in this company, what would you do differently?
This is definitely one of the most difficult questions to answer. Here, the interviewer tries to discover some special things about the candidates that will provide insight into their dreams and ambitions. However, it also seeks if the candidate looks bad things inside or outside the company. So, you must take special care at the moment to answer this question. An example of good answer might be:
- I do not see anything wrong with the company, that is why I want to work here
What are your goals for the future?
With this question, interviewers pretend to know if you are a person with great ambitions and what you are able to do to achieve those goals. Be careful when answering this question, because most times the excessive ambition can lead to misunderstanding and sometimes the lack of ambition might suggest that you are not totally committed to the work. To answer this question, you must have clear your goals and tell the interviewer that you want to achieve your goals by working hard. An example of good answer might be:
- I have many goals in life, and would like to achieve them by working hard.
Other important Open-Ended questions are:
- How do you handle stress and pressure?
- What challenges are you looking for in a position?
- How will your greatest strength help you perform?
- Why did you select your college or university?
- Are you willing to spend at least six months as a trainee?
- What makes you want to work hard?
- Do you get on well with your last boss?
- Give me an example of a time when you got really motivated at work


text:
Why one woman stole 47 million academic papers and made them all free to read
Joyce Park stashed this in Science
Stashed in: Science!, Awesome, Science Too, Hero!, Science, Extraordinary People
To save this post, select a stash from drop-down menu or type in a new one:
Russian neuroscientist has "liberated" or "stolen" almost all the paywalled scientific knowledge in the world, exposing a serious fissure in global science: if research (much of it funded by the public) is too expensive for the future of science because it is controlled by private publishers who add relatively little value except the prestige of their brands.
Aaron Swartz called this the "open access" movement.
Hopefully this movement is gaining momentum so that science does belong to everyone.

subset: commoncrawl


text:
|Mr. Martin Ambrose - Jim is a very effective and senior executive with v...
Mr James Jones
Maidenhead, Berks - United Kindgom
About Mr James Jones
Experienced software sales professional, 30 years with MSA, CA, SSA and Infor.
Managed UK, European and Global teams. Able to get the best out of individuals by maximising individual skills rather than dictating
If you notice offensive or incorrect information, please contact us.


text:
Bluetooth Hose End Timer
The first hose-end timer with Bluetooth communication. Battery supplied electronic module driven from a smartphone or a tablet thanks to the Solem "app" and Bluetooth low energy
The Bluetooth Hose-end timer brings you a variety of applications to answer basic daily needs in your garden
- Above ground irrigation systems
- Balconies
- Vegetable gardens
- Drip and misting systems
Easy to install:
- Connect the Bluetooth Hose-end timer to your hose bib the same you would for a garden hose.
- Download the SolemWF App on your Smartphone and activate the Bluetooth function.
- You can program it with the SolemWF App and control it with your Smartphone or tablet.
- Choose your program and you're done.
Save water:
With the Bluetooth Hose-end Timer, you can program and adjust your run time to the needs of your plants and avoid over watering to keep your plants healthy.
Specifications :
• Bluetooth range : about 10 meters
• Tested on :
- iPhone 4S, 5, 5s, 5c, iPad 3, 4, Mini, Air (with iOS 7.0 minimum)
- Samsung Galaxy S3, S4, S5, Note 2 (with Android 4.3 minimum)
- Sony Xperia Z, Z1 Compact (with Android 4.3 minimum)
Operating temperature :
• Up to 50°C and must be protected from freezing in winter
Features :
• Bluetooth Low Energy Communication
• Standalone: works with a 9V alkaline battery types 6AM6 (international standards)or 6LR61 (European standards) not included
• Start/Stop programmable function • Non volatile memory will save programming in case of power failure
Dimensions :
• Width : 11.5 cm
• Heigth : 14.5 cm
• Depth : 6 cm


text:
MMA Forum - UFC Forums - UFC Results - MMA Videos (http://www.mmaforum.com/forumindex.php)
- General MMA Discussion (http://www.mmaforum.com/general-mma-discussion/)
- - crocrop vs yoshida VIDEO INSIDE (http://www.mmaforum.com/general-mma-discussion/757-crocrop-vs-yoshida-video-inside.html)
crocrop vs yoshida VIDEO INSIDE
Those are some rank leg kicks..lol
Crocop Filipovic pawned him like he does to everyone else except Hunt, Fedor, and Antonio Rodrigo, the top three heavyweights in the world. But he will win the grand prix this year the way it looks like. Mark Hunt is out and now it looks like this is a chance for Crocop to win.
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text:
October 13, 2016
October 13, 2016
Michelle Obama speaking truth
August 25, 2016
Five minutes of hope
What I find most remarkable about this exchange: So few conversations begin with the request for help changing one's own mind.
August 3, 2016
How Paul Ryan can save his legacy…and our democracy (a fantasy)
Crowd chants : Hillary! Hillary! Hillary!…
Hillary Clinton: Thank you. Thank you all so much. It's wonderful to be here. And before I speak, I want to let you know that this is a very special day. Before I talk, I'm going to bring out a guest you're not expecting, who will make history. And how you greet him will help shape that history So, I ask you to greet this guest with open hearts and open minds, and embrace him for the courage and true patriotism he's going to show you this morning. Ladies and gentlemen, please warmly welcome … Speaker of the U.S. House of Representatives, Paul Ryan.
Paul Ryan enters to shocked applause. Shakes Clinton's hand and goes to lectern
Paul Ryan: I bet you did not see that coming. Tell you the truth, neither did I.
Good morning. Madam Secretary,…
For the rest, click here
Categories: politics Tagged with: donald trump • election 2016 • hillary clinton • paul ryan
Date: August 3rd, 2016 dw
July 29, 2016
Four remarkable days, and other good news.
1. Granted, this is just an instant poll with a margin of error of 99 on a scale of 1 to Wishful Thinking , but I'll take it:
2. On Night #2, the Dems had 25M viewers while on the comparable night the Reps had 19M. I was surprised by this, given that Trump is an eye-magnet train wreck.
3. HRC is maintaining a large lead in Pennsylvania, a must-win state for Trump.
4. This may be a teensy bit subjective, but this was by far the best convention in my lifetime. It's changed what it means to be a Democrat, and retrieved what it means to be American.
If all you knew of America was what you saw during these four days, you would think it is a place that not just celebrates but proudly draws upon its deep diversity. And you would be forgiven if you concluded that its surest moral compass is held by people — and especially women — of color.
5. It's not just that we had four days of astounding talks. Taken together, those days were a work of art in their balance and contrasts, their crescendos and their moments of silence. Remarkable, and remarkably moving.
So, now we
July 23, 2016
Why Hillary picked Tim Kaine
No one can best me in my ElizabethWarrenLove, but if you want to know why Hillary picked Tim, watch this speech from their first event together:
It's like mind-bleach for Donald Trump's Harangue of Fear
Click here to make the big scary man go away.
July 22, 2016
Now it's on us
We can no longer think this is just a reality TV star who says whatever he has to to keep us amused.
Now we've seen American fascism naked.
Now we have no excuse for not stopping it.
Now it's on us.
Categories: politics Tagged with: election2016 • trump
Date: July 22nd, 2016 dw
July 19, 2016
Melania's speech rickrolled us: Is there a mole in the Trump campaign?
On Reddit, user Amaranthine cites a tweet from Soniasaraiya that points to a signal that one of Melania's speechwriters may be a mole working against the Trump campaign: Was Melania rickrolled?
Rickrolling is a prank in which misleading text links to a video of Rick Astley singing his 1987 hit "Never Gonna Give You Up." For example, if I wrote "Here's an incredible secret video of Hillary whispering to Bill that she lied about Benghazi," and you click on the link, you've been rickrolled." The video has been viewed over 224 million times, but no one knows how many times on purpose. (Interestingly, Rick Astley seems to have plagiarized the song from this awkward amateur version.)
Last night Melania said (transcript here):
Here is the opening of the chorus of Never Gonna Give You Up:
This not such a unique, unexpected turn of phrase that it could only have been plagiarized. On the other hand: 224,238,266 views! This is the opposite of obscure.
So, if you were the speechwriter who not only put plagiarized text into Melania Trump's introduce-yourself-to-America speech, but you took that text from Michelle Obama's introduce-yourself-to-America speech eight years earlier, you might well want to flag that Melania's speech rickrolled us and her: Melania's words, uttered sincerely, turn out to "link" to an annoyingly lightweight pop song.
Just for fun, here's an autotuned version of Melania singing her lyrics, created by redditor cbuntz:
July 18, 2016
Most pathetic national convention ever
From Politico, here is the list of speakers for the Republican National Convention:
Monday: Make America Safe Again
Headliners: Melania Trump, retired Lt. Gen. Michael Flynn, Iowa Sen. Joni Ernst, Montana Rep. Ryan Zinke, and veterans activist Jason Beardsley.
Also speaking: Willie Robertson of "Duck Dynasty," former Texas Gov. Rick Perry, actor Scott Baio, Navy SEAL Marcus Luttrell, Sen. Tom Cotton, Sen. Jeff Sessions and former New York Mayor Rudy Giuliani, among others.
Headliners: Donald Trump Jr., West Virginia Sen. Shelley Moore Capito, Dr. Ben Carson and actress-businesswoman Kimberlin Brown.
Also speaking: Arkansas Gov. Asa Hutchinson, Wisconsin Sen. Ron Johnson, Senate Majority Leader Mitch McConnell of Kentucky, House Speaker Paul Ryan of Wisconsin, House Majority Leader Kevin McCarthy of California and New Jersey Gov. Chris Christie, among others.
Wednesday: Make America First Again
Headliners: Lynne Patton of the Eric Trump Foundation; Eric Trump; Newt Gingrich and his wife, Callista; and Trump's running mate, Indiana Gov. Mike Pence.
Also speaking: Radio host Laura Ingraham, Florida Attorney General Pam Bondi, Wisconsin Gov. Scott Walker, Sen. Marco Rubio, and Sen. Ted Cruz, among others.
Thursday: Make America One Again
Headliners: Business leaders Peter Thiel and Tom Barrack, Ivanka Trump and Donald Trump.
Also speaking: Oklahoma Gov. Mary Fallin, Tennessee Rep. Marsha Blackburn, RNC Chairman Reince Priebus and Liberty University President Jerry Falwell, Jr., among others.
July 2, 2016
Corrupting "Earned media"
The Intercept reports on several news media who are selling special services at the national political conventions — meetings, cocktail parties, and more. The services are corrosive. Some are explicitly corrupt, "…they make explicit the inevitable failure of the distinction between "paid" and "earned" content."making explicit the inevitable failure of the distinction between "paid" and "earned" content.
The less controversial services are corrosive because they let the media take money from the people they cover. Having spent a few decades as a marketing communications guy, I can promise you that in every business considering these offers, the conversation includes someone saying, "It doesn't matter if no one comes to the cocktail party. It'd still improve our relationship with the publication." Why? Because it's a way to pay the journal money. That's corrosive.
Larry Lessig points out that it's not much different from news organizations tuning their coverage to their ratings. But such tuning at least caters to perceived piopular interest. These new services let an organization or candidate buy coverage despite a decided lack of public interest. It is worse than buying ads because the news media have traditionally had a "Chinese wall" between the advertising and editorial departments. This has been a fairly effective way of protecting editorial content from the direct influence of the marketing needs of the journal, even though the wall is sometimes breached, and Time Magazine has shamefully torn it down.
Once the media started letting companies pay for phony news coverage, they pretended to honor the breach by distinguishing "earned" and "paid" content. "Earned content" is coverage provided by media of events they think are newsworthy. "Paid content" is, well, paid content. Non-sleazebag companies and their PR reps expect media to mark paid content as paid for. Edelman, the world's largest independent PR company, created ethical guidelines that not only say that the paid content must be well marked, but that Edelman will have its own Chinese wall between the processes by which earned content is pitched ("Yo, I have a client who's invented a time travel machine. Wanna an interview? How's yesterday for you?") and the negotiations that result in the placement of paid content. (Disclosure: I had a tiny hand — Trump-sized — in drafting those guidelines.)
That's better than nothing, but paid content still makes me queasy. Companies are willing to pay for content precisely because it looks like real coverage and thus tends to be taken more seriously than obvious ads. This erodes the phenomenological line between news and ads, which is bad for democracy and culture. Indeed, "the point of paid content is to erode the line. "the point of paid content is to erode the line.
But letting candidates pay for interviews takes this to a whole new level. This is what The Intercept says:
The Hill says the resulting interviews will be earned media. Suppose the interview is stupid, boring, self-serving and non-newsworthy? If it weren't, the client wouldn't be paying for it. But The Hill is promising it's going to run anyway because the client paid them $200,000. That is the very definition of paid content. So, by calling it "earned content," The Hill can only mean that the article will not be marked as paid content, even though that is precisely what it is.
This corrupts the already corrosive practice of accepting paid content. It is disgraceful.
Categories: culture, journalism, politics Tagged with: advertising • campaigns • corruption • journalism • pr
Date: July 2nd, 2016 dw
May 29, 2016
Hillary: Getting sh*t done


text: Subscribe to Engineering Insight Engineering Insight is our 48-page, full-colour Member magazine that showcases the achievements of our Members, as well as leading engineers and technologists, both locally and internationally. We publish six issues a year, every other month. The editorial content balances contemporary issues and technical accuracy to make sure the articles are accessible and of interest to engineers and the wider engineering community. Each issue is themed around a different aspect of engineering, and aims to cover multiple fields of interest, for example, past themes have included Forensic Engineering, Electricity Supply and Leadership. You can expect: in-depth information on the professional practice of engineering and technology, including examples of best practice by New Zealand engineers critical review and discussion of contemporary engineering issues a focus on the innovation and entrepreneurship of New Zealand engineers and engineering. Subscribe and get an insight into the best of New Zealand engineering. It could take up to three months to receive your first copy of the magazine, depending on when your subscription is processed. Subscribe now To subscribe, email firstname.lastname@example.com Subscription options One-year subscription (six issues) Engineering Insight subscription (NZ) $50.00 incl GST Engineering Insight subscription (AU) $70.00 Engineering Insight subscription (World) $100.00 Note: overseas subscriptions are not charged GST.


text:
I believe I have a CLA on file from working on the Felix project, but I will fax another one just for good measure. Either way its not a problem for me to wait a few days.
Hi Chris,
i'm sorry, but after a short discussion with Emmanuel i decide to
revoke the permissions until you have signed a CLA. Not that we don't
trust you, but we should not break the rules.
2007/5/7, Christine Koppelt <firstname.lastname@example.com>:
> Chris, you have now access to DIRxSBOX and DIRxDEV.
> Ersin, could you check the permisions for DIRxINTEROP?
> Perhaps we could create another, public writeable space and link it
> from the frontpage of our website, WDYT?
> Christine
> 2007/5/7, Chris Custine < firstname.lastname@example.com>:
> > It looks like all of the Directory spaces in Confluence are locked down
> > except for the interop space. Is it possible to get access opened up to
> > DIRxSBOX or DIRxDEV? What I want to do is add some content that shows build
> > status from a build server that I have set up as an evaluation of Jetbrains
> > Teamcity so that we can test it out.
> >
> > Chris
> >


text:
Failed before 50,000mi
Failed between 50,000mi and 100,000mi
Failed between 100,000mi and 150,000mi
Failed between 150,000mi and 200,000mi
Failed after 200,000mi
Below 50,000mi and still going
Between 50,000mi and 100,000mi and still going
Between 100,000mi and 150,000mi and still going
Between 150,000mi and 200,000mi and still going
Over 200,000mi and still going
Candy the van. '98 Sport 3.8L 132,200 miles. Used trans at ~96k. Great piece of my life and a fine van.
'69 GTO drop top
The 96 threw the diff pin at about 130K
The 2000 was sold at over 250K with the original tranny
The 2005 ate the convertor and main shaft bearings at about 140K
It has been two years; time for some stats updates on this poll.
I sure wish we could change our answers to the poll. When I took the poll, my van had under 100,000 miles. Now it has over 223,000...
Voters = 156
Votes = 160
Total: 45 (28%)
Avg Age at Failure: 98,000 miles
(note: this mathematically shouldn't be read as meaning "these transmissions typically last 98,000 miles". From the survey data, a typical transmission at that age only has a 13% chance of failing in the next 50,000 miles.)
Total: 115 (72%)
Avg Age of Original Working Transmission: 122,000 miles
If your tranny has made it to a given mileage, what is the chance of a failure in the next 50,000 miles?
000000 Miles: 4%
050000 Miles: 13%
100000 Miles: 13%
150000 Miles: 13%
200000 Miles: 25% **
** The 200K / 25% number may be inflated because it mathematically assumes 250000 is the life limit, which isn't true. The limited mileage ranges were due to limitations in the poll size. There is also insufficient data in the 200K + range to draw strong conclusions there.
One inference we can make from this data is that you shouldn't be any more worried about a transmission failure with a 150K vehicle than you were with a 50K vehicle. Or, conversely, perhaps you should be just as worried with a 50K vehicle as you would be with a 150K vehicle!
If the average cost of a transmission failure is $2500, then how much would the "average" person spend on transmission failures over a 50,000 mile interval, given an initial transmission age of ______, and assuming the replacement transmission does not fail in that timeframe (or is appropriately warranted)? Annualized amounts assume 15,000mi/year of driving.
000000 Miles: $109 ($33/yr) (but likely under warranty!)
050000 Miles: $321 ($96/yr)
100000 Miles: $316 ($94/yr)
150000 Miles: $321 ($96/yr)
200000 Miles: $625 ($188/yr) (assumes 250000mi life limit, which isn't true)
Loss expectancy, among other things, helps you decide what types of preventative maintenance are worth it. (For example, doing $200 of extra non-routine transmission service every year to help stave off a hypothesized failure would not be worth it, even if it were 100% effective at preventing failure.) It also helps you know how much you "should" put into savings, over any number of years of ownership. There are unknowns here, such as what the typical preventative maintenance was that the transmissions in the survey experienced.
For cost comparison purposes, over 50,000 miles the "average owner" would spend $320 on transmission failure. During that time period, the owner may also spend over $9,000 on fuel, $1,700 on auto insurance, $375 on tires, and $250 on oil changes.
Important Notes:
1. The terms "Failed" and "Failure" were not defined by the survey. These failures may or may not include in addition to traditional transmission failures: inexpensive sensor-related failures, wrong-fluid-induced failures, circumstantial failures (e.g., the story about fluid loss due to impacting a deer), and unnecessary rebuilds done by overzealous transmission shops.
2. Some owners of previously-owned minivans were not aware of whether their transmission was an original that had been functioning properly since vehicle manufacture, or whether the previous owner(s) had transmission trouble.
3. The statistics above make some assumptions and mathematical simplifications. That, plus the method of survey, makes this entirely nonscientific. But I hope that it has been (and will continue to be) useful.
4. As this website is often used as a help forum for those having trouble with their vans, as well as a discussion forum for van enthusiasts, the sampling of vans for this survey is likely not representative of all vans out there. Specifically, it may include a higher percentage of failed vehicles than the general population, and it may include a higher percentage of well-cared-for vehicles than the general population.
1998 Grand Voyager @336,000mi, 3.3L FFV V6
My unknown mileage trans from a junkyard has 32,000 miles from me on it so far. Still has the same slip under hard throttle in 1st gear it always had. I guess for a $175 trans, $250 total swap job, not too bad...
Candy the van. '98 Sport 3.8L 132,200 miles. Used trans at ~96k. Great piece of my life and a fine van.
'69 GTO drop top
I've heard the 4 speed in 2001 and newer all the problems it had was fixed and the amounts of failure is lower then GM Ford and Toyota.
The tranny in the wife's '98 Voyager went out at about 175,000. The tranny in my '98 Grand Voyager is at 185,000 and still going.
If the women don't find ya handsome, they should at least find ya handy! Red Green
On my 2005 I changed the fluid at 60,000 miles and at 150 its still smooth as silk.
I belive by 2001 all the bugs had been fixed and its as strong if not stronger then the 4 speeds from GM and Toyota.
Our '98 original 41TE trans has 326,000 miles on it now; the only fix that has had to be done is the solenoid pack, which failed somewhere around 290,000 miles.
I think the actual trans failures fall into three categories: 1) serious design flaws, 2) poor quality control, and 3) driving style issues. Most of (1) were corrected by 1996, but apparently they still have quality control issues since some of these units still fail very young and others last virtually forever, with a couple reports here of unrebuilt 41TE's lasting even longer than my current mileage (I think one person is at 350,000 miles and another at 400,000 miles on the original trans). One mechanic told me that the tolerances in the 41TE are just so tight that even slight variations in manufacturing and assembly can cause issues.
As for driving style, these units are notorious for catastrophic diff failure when even moderately abused.
The things we've done to prolong the life of the transmission are: 1) regular fluid and filter changes with ATF+4 only, 2) an additional external transmission cooler, and 3) minimizing heat generation by operating the trans to maximize the time the TC is in lockup (for one example, when climbing a steep hill, I pull the gearshift to [3] instead of [D] to allow quick TC lockup in 3rd -- otherwise after the automatic kickdown to 3rd it waits for some heat buildup before it locks up the TC in 3rd with the gearshift in [D]).
GM had their own teething issues with their 4-speed automatic. Like the 41TE, though, it eventually did become a reasonably reliable unit. Honda also had very serious trouble with their minivan transmissions, which were also insanely expensive to fix; I'm unsure what degree of improvement they've seen.
1998 Grand Voyager @336,000mi, 3.3L FFV V6


text:
the official song of the British Labour Party, written in 1889 by James Connell (1853-1929) and often sung at the end of large party meetings. The red flag is the international symbol of revolution and socialism. The song ends with its best-known line, 'We'll keep the red flag flying here.'jump to other results
The Red Flag
BrE ; NAmE
Check pronunciation: The Red Flag


text:
Derek Stepan
View Single Post
Foolish Samurai
Join Date: Aug 2010
Location: New York
Posts: 21,322
vCash: 500
Originally Posted by
The only resemblance to the guy from the WJC is that he again looks like a man among boys. Otherwise, today's Step is a million times better. The guy at the WJC was slow, not a good shooter and not that strong physically. This guy today is an all-around player who makes NHLers look stupid.
A million times better, yes, but he's now playing like he did in that WJC that made us all so hopeful for him as a prospect. His first season here was great, but it looked like he was focusing more on acclimating to the NHL than asserting himself- which is perfectly fair, and I was pleased to see his goalscoring game develop as well.
Last year he expanded upon that, but was still growing.
This year he's dynamic everytime he's on the ice. He does everything except fight. That's exactly what we saw from him when he was the best player in the WJC that year.
He can kill you in so many different ways. Have some offense going? Not anymore, Stepan just broke it up. Have an elite d-man in his way? No problem, he'll just dish it off and grab the assist instead of the goal. Play the pass on the two on one? Fine, he'll just snipe it past you.
If what we've gotten this season from Stepan is the real Stepan, he'll be a near PPG franchise player starting as early as next year.
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text:
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Regular readers of this blog may have noticed a change in name from "Torah From The Square" to "Torah From The Rockies" and the reason is because my family and I have transitioned to new positions in Denver, Colorado. For the past three years I served as the full time rabbi of the Harvard Hillel in Cambridge, Mass. and it was there that I developed close relationships with students, community members and faculty. It was a tremendous experience and I feel blessed for the memories I will carry … [Read more...]
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New Guy's instrumental debate- psalms
Posted in the Bassett Forum
#1 Mar 20, 2013
Any discussion of the music question will center on the meaning of psalms found in Ephesians 5:19 and Colossians 3:16. According to Vine's, psalmos means "to strike or twitch with the fingers (on musical strings), then a sacred song, sung to musical accompaniment,a psalm".
That seems to indicate that a psalm may be used with musical instruments. However, many dispute that.
In the above article, Wayne Jackson alleges that the word for psalm has changed in meaning from the Old Testament to the NT. He also states that the word psalm can be used for either instrumental music or music without accompaniment in the OT, depending on context.
*I agree with his second point, that context decides and sometimes it can be either instrumental or not instrumental. My belief is that both are acceptable to God. I do contend with point #1. At what point do we see the word psalm change in meaning in the Bible? If the Bible is our sole authority, where is the authority for this change? Where does God tell us a psalm is no longer a psalm?
Some scriptural evidence about psalms:
*55 of the Biblical book of Psalms begin with a variation of "To the chief Musician".
*Psalm 92 is a psalm for the Sabbath day; verse 3 says "Upon an instrument of ten strings, and upon the psaltery; upon the harp with a solemn sound."
*Psalm 87 is considered a prophecy of the church by several scholars. What about verse 7?
*Psalm 150 is a beautiful psalm speaking of praising God. Instruments are mentioned prominently and acceptably in the old covenant.
There is no doubt about the character and content of the OT psalms. Where and why then should anyone assume the meaning of psalm has changed? Why would God change His mind?
Vine's complete expository dictionary has an interesting note under the word "hymn". Quote- "the psalmos denoted that which had a musical accompanyment; the ode was the generic term for a song". This is in the New Testament words section. No mention here of any change. However, the Scriptures themselves are our final authority. We must find our meaning within them. Where is the evidence of a change of Biblical meaning?
Romans 15:9 is an interesting passage of scripture. Paul says "And that the Gentiles might glorify God for His mercy; as it is written, For this cause I will confess to thee among the Gentiles, and SING unto thy name". The word sing in Greek is literally psallo. What makes this even more interesting is that here Paul is quoting from Psalms 18. Psalm 18 is begun with the inscription "To the chief Musician". The word translated sing is zamar, which meant to sing accompanied by an instrument.
Thought-did Paul use this word on accident? Did he not know what it meant? If instrumental music is indeed excluded in the new testament, did Paul err? Or did the Spirit that inspired him err? Is it possible that the word psalmos has retained its meaning?
*Example-"gay". If I told you I am "gay", my brethren would cringe in horror. However, the original meaning meant to "be happy". Is the word's original meaning incorrect because the times and culture have changed? No. So, even IF the meaning of psalmos has changed, and we have no BIBLICAL evidence that is has, the original meaning is still correct. And we must consider the writers background and intents. Paul was a Hebrew of Hebrews, a Pharisee. What would the word psalm mean to him, and those people in that culture?
This is to be a very condensed study on my instrumental position. It is by no means totally in depth, nor is it intended to be. I just want to show enough evidence to present my case. More is coming, so now-anyone agree, or disagree, with the meaning of the word psalm? For the naysayers, I will say you may want to save some objections for the next round or so, as I get into other aspects of the music question.
Since: Jul 11
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#2 Mar 20, 2013
May I use this on my Facebook?
12:24pm - Lunch
Since: Jul 11
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#3 Mar 20, 2013
May I use this on my Facebook and Blog?
12:26pm - Lunch
#4 Mar 20, 2013
Yes sir you may.
Since: May 10
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#5 Mar 20, 2013
I will address other parts in the future but you have to be called on one of your first comments. You claim to use the bible as your "SOLE" authority yet from the beginning go to outside the bible by using Vines to define a word. So what should we do with your thoughts now? Want to change your speech?
Since: May 10
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#6 Mar 20, 2013
Now to your next comment I will contest.
The word zamar does not always mean to sing with accompanied instrument because there are verse that use the word and then add to use the instrument along with the singing.
Let me post one and then show what Strongs does define for this word.
Ps 71:22 I will also praise 3034 thee with the psaltery 3627 5035,[even] thy truth 571, O my God 430: unto thee will I sing 2167 with the harp 3658, O thou Holy One 6918 of Israel 3478.
So according to your thought this text says,.......Unto thee will I SING AND PLAY (Zamar) and PLAY the HARP.
Your question about Paul should be asked here if the word means what you said. DID this writer get confused and not know the meaning a zamar had the instrument included with it?
Strongs define of zamar
1) to sing, sing praise, make music
1) to make music, sing
2) to play a musical instrument
Since: Mar 13
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#7 Mar 20, 2013
Regarding the thread from NG, he makes some points from the Bible, which, btw, define themselves within the context. As he said, the word Psalm during that era would have been taken one way and only one way. The challenge for those who consider the word Psalm to have been changed MUST come from the Bible. If we chose to go outside of the Bible, NG already demonstrated that there is proof there as well what the word meant. However, if you stick with the Bible, please cite the verse{s}from the Bible where the meaning of Psalm changed.
4:20pm - Friends House
Since: May 10
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#8 Mar 20, 2013
He went outside the bible to begin with. He needs to either not do so or change his parameters dont you think?
Since: Mar 13
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#9 Mar 20, 2013
Perhaps he went outside of the Bible to demonstrate that you can find support form both positions if you do that. INSTEAD, stick to the Bible and make your case for "Psalms" was his point, the way I understood his thread.
4:56pm - Friends House
Since: May 10
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#10 Mar 20, 2013
Now lets look at this in proper context of the whole of the bible.
1. Matt 28:18ff gives clear direction as to what was fixing to happen and expected of those
a. The apostle would remember all that Christ taught
b. The apostle were to teach everything Christ taught as the Holy Spirit would direct them.
c. The apostles were to teach nothing except what Christ told them.
d. The church was to follow the teaching of these inspired men and should not adhere to anything not authorized by Christ.
2. There is no implication, example, or command of the instrument being used in worship in the NT record. This is important because after the transfiguration it was understood that Not Moses nor Elijah but only Christ instructions were now to be followed as Given through the Holy Spirit.
We do not have the authority of Christ.
3. You can not Use the Instrument by Faith in worship.
Rom 14:23 addresses this in a sense. "And he that doubteth is damned if he eat, because he eateth not of faith; for whatsoever is not of faith is sin."
2 Corinthians 5:7 Paul says we are to "walk by faith, not by sight." To walk by faith is to walk as God's Word directs. If God's Word does not direct an action, it is not 'of faith' and therefore is sin!
To add the instrument without instruction from the head of the church (Christ), is to deny the head of the Church. Ephesians 5:24, "Therefore, as the church is subject unto Christ, so let the wives be to their own husbands in every thing."
4. To worship without authority no matter how good it seems is vain worship. Examples throughout the bible on this one. John 4:24, Jesus declares, "God is a Spirit, and they that worship him must worship him in spirit and in truth." Truth is the Word of God (John 17:17)
5. It causes Division.
God is for unity and those that require the instrument are divisive not the ones singing only.
"I know of no cases where the instrument was already in use and non-instrumental brethren tried to force it out and possess the property, but the reverse has occurred many times."(Biblical Notes, Volume IX, July, 1975, page 27).
Where the instrument was introduced, churches split and division is an ugly picture, contrary to true Christianity (I Corinthians 1:10).
Probably more to follow but not now.
Since: May 10
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#11 Mar 20, 2013
Perhaps????? He went to Vines And then said His SOLE authority would be the bible. He can not do so and neither can you. Be consistent.
See I know history supports the teaching and I am not afraid to use those and neither is God afraid of History. Just those who do not want the full truth.
#12 Mar 20, 2013
iT'S THE NON-INSTRUMENTAL POSSITION THAT CAUSES DIVISION.
Since: Jul 11
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#13 Mar 20, 2013
I agree. And what lexicon did they use when Paul wrote his epistles ..... the Torah. They didn't have all of the lexicons we have today.......they used the old testament. The word psalm in defined by context and practice via the old testament.
Since: Jul 11
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#14 Mar 21, 2013
I see I got the old "disagree" and "clueless" above yet not rebuttal.
Again, I ask: What lexicon did they use when Paul wrote his epistles?
They didn't have all of the lexicons we have today.......they used the old testament.
The word psalm in defined by context and practice via the old testament.
Since: Jul 11
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#15 Mar 21, 2013
JC, Isn't the Bible sufficient to make your case? If so, where in the Bible did the meaning of Psalm change?
Paul told Timothy that ALL SCRIPTURE is inspired by God and "profitable for teaching, for reproof, for correction", for training in righteousness. What Lexicon would Timothy use to define the meaning of Psalms?
2 Timothy 3:16: "All Scripture" is inspired by God and "profitable for teaching, for reproof, for
correction", for training in righteousness?
Since: May 10
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#16 Mar 21, 2013
It isnt for you so dont say you use the bible only. Neither you or New Guy use only the bible as you claim.
Since: May 10
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#17 Mar 21, 2013
Now be truthful How many congregations have split because someone decided to go with the instrument? How many Have split because the instrument was being used and people said NO dont use it? You might want to look at historical records on this.
Dont let me confuse you with the facts.
Since: Jul 11
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#18 Mar 21, 2013
Isn't the Bible sufficient to make your case? If so, where in the Bible did the meaning of Psalm change?
Paul told Timothy that ALL SCRIPTURE is inspired by God and "profitable for teaching, for reproof, for correction", for training in righteousness. What Lexicon would Timothy use to define the meaning of Psalms?
9:30am - Break
Since: May 10
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#19 Mar 21, 2013
Here is the biblical argument why the instrument should not be used in worship. And when you honestly then ask what the NT church actually did you can only come to one honest conclusion. The first Century Church did not use the instrument because they followed the NT teaching of Christ and his words.
Because it does not have the authority of Christ.(Matthew 28:18; Ephesians 5:24).
Because we cannot do so "by faith".(Romans 14:23; 10:17).
Because to do so would make our worship vain.(Matthew 15:9). Because to do so would be to reject Christ as the head of the church.(Ephesians 1:22-23).
Because the use of the instrument in worship is contrary to the doctrine of Christ.(2 John 9).
Because it causes divisions.(Romans 16:17).
Because it does not pertain to life and godliness.(2 Peter 1:3).
Now simply show a command Of CHRIST (Doctrine of Christ)(Law of liberty) that claims use of the instrument, or an example of the church using the instrument, or an inference that they did use the instrument and you have me. Its ok with me if you convict me with historical support as I believe the scripture of God is not destroyed by historical accounts but confirmed. History is important for most of the apostles used it in convicting those who they were trying to persuade to leave Jewish faith and become CHrisitans.
#20 Mar 21, 2013
We are often told that early church history proves that the first Christians did not use instruments in their "worship". Thus, we have no precedent and are breaking the pattern laid out for us. What does history truly have to tell us?
*The early church fathers indeed were against instrumental music in the church services. There are several centuries' worth of condemnation written by these men. The documents are out there for all to find; I will not present them here. However, there are several problems with these historical writers which must be taken into account before we consider using them to prove our doctrinal positions today.
*We are warned several times in scripture to "do not go beyond what is written". Post Biblical history is exactly that, post Biblical. These men were not inspired by the Holy Spirit, their words do not form the canon of scripture. Many hold doctrinal positions that the churches of Christ would call abominable today.
*Their objections to instrumental music are not the same as those who object to the instrument today. Todays arguments are- no authority from Christ; singing only was authorized; strange fire; heart is the instrument; not of faith is sin; et al. The early church fathers objected on no Biblical grounds; rather, their objections came from a fear of sensuality and idolatry, imitating pagan or Jewish worship, Roman celebrations, and other such things. Again, the info is out there, search for this on your own.
*Some of these "fathers" also came up with the idea of "allegorizing" the scriptures-most notably those from the Egyptian city of Alexandria, who were afraid of imitating Egyptian pagan rites.
*A logical question-why does one argue so strongly against something that is said to never have happened? If no one was using the instrument, why argue against it?
*Are we comfortable following the lead and ideas of those who led the world into Catholicism and division?
*The historical writers are not all in agreement about when instrumental music was introduced into the churches. The answers range from 670 to 755 to 768 to the ninth century to 1200 years later. If these do not agree, how can we assume they are correct? There are also some who claim that the earliest Greek converts used instruments. Which is correct, or are any of these correct?
*These dates come from researching on bible.ca, a non-instrumental church of Christ website. Another interesting quote from bible.ca- "Nowhere has God CONDEMNED use of mechanical instruments in Christian worship; nowhere has God COMMANDED their use".
*My final argument about history is this-when we discuss "church history", it seems that we always go past Bible times into the second century and beyond. Have we forgotten this?
Galatians 3:7- "Know ye therefore that they which are of faith, the same are the children of Abraham". A Christian is a spiritual descendant of Abraham. His history is our history. The Old Testament was written for our learning and as an example for us. The OT is not a history of Israel after the flesh, it is a history of God's people from Genesis to Malachi. It is MY family history, and OURS if we are in the faith.
Why then should we disregard our own ancient history, but accept the history of those who may or may not be our brethren? Only God knows where those "early church fathers" stand. We do not.
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Home > Mozart> K522 Musical Joke, 4th Movement: Presto: hns, str
Composer: Mozart, Wolfgang Amadeus
Instrument: | Violin | Viola | Mixed Ensemble | French Horn |
Skill Level: 6 out of 9
Type: Original
Notes: The 4th movement of Mozart's Musical Joke: Presto. Watch out for those final chords. Horns, Violins, Violas, Cellos.
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In vitro study of uptake and synthesis of creatine and its precursors by cerebellar granule cells and astrocytes suggests some hypotheses on the physiopathology of the inherited disorders of creatine metabolism
Neurobiology of disease
The discovery of the inherited disorders of creatine (Cr) synthesis and transport in the last few years disclosed the importance of blood Cr supply for the normal functioning of the brain. These putatively rare diseases share a common pathogenetic mechanism (the depletion of brain Cr) and similar phenotypes characterized by mental retardation, language disturbances, seizures and movement disorders. In the effort to improve our knowledge on the mechanisms regulating Cr pool inside the nervous tissue, Cr transport and synthesis and related gene transcripts were explored in primary cultures of rat cerebellar granule cells and astrocytes.
Cr uptake and synthesis were explored in vitro by incubating monotypic primary cultures of rat type I astrocytes and cerebellar granule cells with: a) D3-Creatine (D3Cr) and D3Cr plus β-guanidinopropionate (GPA, an inhibitor of Cr transporter), and b) labelled precursors of Guanidinoacetate (GAA) and Cr (Arginine, Arg; Glycine, Gly). Intracellular D3Cr and labelled GAA and Cr were assessed by ESI-MS/MS. Creatine transporter (
D3Cr signal was extremely high in cells incubated with this isotope (labelled/unlabelled Cr ratio reached about 10 and 122, respectively in cerebellar granule cells and astrocytes) and was reduced by GPA. Labelled Arg and Gly were taken up by the cells and incorporated in GAA, whose concentration paralleled that of these precursors both in the extracellular medium and inside the cells (astrocytes). In contrast, the increase of labelled Cr was relatively much more limited since labelled Cr after precursors' supplementation did not exceed 2,7% (cerebellar granule cells) and 21% (astrocytes) of unlabelled Cr. Finally,
Our results confirm that both neurons and astrocytes have the capability to synthesize and uptake Cr, and suggest that at least in vitro intracellular Cr can increase to a much greater extent through uptake than through
The discovery of the inherited disorders of Cr metabolism has significantly improved the knowledge on the role of creatine (Cr) in the human brain energetic metabolism
It has been hypothesized that in the absence of CT1, neurons can not take up glial-synthesized Cr or guanidinoacetate (GAA), so suggesting a glia-neuron interplay in brain Cr homeostasis
Chemicals and reagents
D3-Creatine (D3Cr) was purchased from CDN isotopes (Quebec, Canada). 13 C2-Guanidinoacetic acid ([1,2-13 C2]GAA) was purchased from Dr. Herman J. ten Brink (Amsterdam, Netherlands). L-[guanido-15 N2]arginine:HCl ([15 N2]Arg) and [13 C2,15N]glycine ([13 C2,15 N]Gly) were obtained from Cambridge Isotopes Laboratories Inc. (Massachusetts, USA). GPA, Gly, Arg, GAA, and Cr were obtained from Sigma-Aldrich (St Luis, MO, USA). HPLC grade methanol and formic acid were obtained from Merck (Darmstadt, Germany). 3 N HCl in n-butanol solution was purchased from Regis Technologies Inc (Morton Grave, USA). All solutions were prepared using highly purified water produced by a Milli-RO/Milli-Q system (Millipore, Bedford, Ma, USA). Centricone filters were obtained from Millipore (Millipore, Bedford, Ma, USA).
Wistar rats were obtained from Harlan Italy, S. Pietro al Natisone Udine. Twenty mm poly-l-lysine-coated glass coverslips were obtained from Warner Instruments LLC (Hamden, CT USA). Basal Eagle's culture medium, fetal calf serum, gentamicin and cytosine arabinoside were obtained from Sigma Aldrich (St Luis, MO, USA). PBS containing trypsin, bovine serum albumin, DMEM, fetal bovin serum, penicillin/streptomycin, L-glutamine were obtained from Euroclone (Pero - Milano Italy).
Rat cell culture
Cerebellar granule cells were prepared from 8-day-old Wistar rats as previously described
Cell treatment
For Cr uptake experiments, rat cerebellar granule cells and astrocytes were incubated for 24 hours in a culture medium containing1 mmol/L D3Cr and with 1 mmol/L D3Cr plus 1 mmol/L GPA. D3Cr concentration was in accord with previously reported Cr incubation experiments
For Cr and GAA synthesis experiments, astrocytes were incubated for 24 hours with [15 N2]Arg and [13 C2,15N]Gly at three different concentrations (5 mmol/L, 10 mmol/L, and 15 mmol/L). By contrast neuronal cerebellar granule cells were incubated with [15 N2]Arg and [13 C2,15N]Gly at the single concentration of 10 mmol/L. This was done in order to optimize the number of cells available for each experiment, given the fact that neurons proliferated much less than astrocytes. Labelled precursor concentrations were in accord with incubation experiments previously reported in lymphoblasts
After 24 hours of incubation the medium was aspirated, cells were washed twice with 10 ml of saline solution and detached from the flask's wall with trypsin. Afterwards, the cells were rapidly washed twice with 10 ml of saline solution by suspension and centrifugation at 1200 g for 10 min, to remove any contamination of labelled amino acids; pellets were then suspended into 100 μl of saline solution and stored frozen at -80°C until analysis. To lysate the cells, the pellets were frozen and thawed for six times at -70°C, placed in the ultrasonic bath for 30'and then centrifuged at 15000 g for 30'. The supernatant was recovered, an aliquot was used for protein assay (Bio-rad, Hercules, CA, USA) and the remaining was deproteinized using the Centricon filters at 5000 g for 50'. The cell extract was then analysed by ESI-MS/MS. The incubation experiments were replicated 3 times and the cell extracts were analysed twice.
MS/MS analysis
ESI-MS/MS was used for the analysis of the cell extracts. In GAA and Cr synthesis experiments Gly, [13 C2,15N]Gly, Arg, [15 N2]Arg, GAA, [1,2-13 C2,15 N3]GAA, Cr and [1,2-13 C2,15N3]Cr in the cell extract were simultaneously determined. For the study of CT1 expression Cr, D3Cr and GAA in the cell extracts were determined.
The cell extracts were diluted 1:5 with methanol/water (80/20 v/v) containing the internal standard [1,2-13 C2]GAA in order to obtain a final concentration of 0.5 μmol/L. Then the sample was dried under a nitrogen flow at 45°C using an EvapArray Sample Concentator (Porvair Advanced Materials, UK) and derivatized to butyl esters using 3 mol/L HCl in n-butanol solution at 60°C for 30 min. After derivatization the sample was dried under a nitrogen flow at 45°C and then recovered with 100 μl of methanol/water solution (80:20) containing 0.1% formic acid. Twenty microliters of the obtained sample was injected for the ESI-MS/MS analysis.
An Agilent 1100 micropump (Agilent Technologies, Wilmington, DE, USA) and a Series 200 autosampler (Perkin Elmer, USA) were used for solvent delivery and automated sample introduction. Mobile phase was methanol/water (80:20) at a flow rate of 40 μl/min.
A triple quadruple mass spectrometer API 2000 (ABI Sciex, Toronto, Canada) equipped with a TurboIonSpray source was used. The data were acquired and processed using Analyst 1.1. Resolution and calibration adjustments of quadrupoles were made by direct infusion at a 10 μl/min of a 0.1 mmol/L polypropyleneglycole solution by an infusion pump. Q1 and Q3 were used at unit and high resolution respectively. Turbo ion spray source was operated in the positive ion mode at 350°C with a drying-gas at 20 psi and a capillary voltage of 5500 V. Nitrogen was used as curtain and collision gas. Multiple reaction monitoring was used for the detection of [M + H]+ and product ions. The declustering potentials were 23, 27, 19 and 20 V, collision exit potential were 2.8, 2.0, 0.4, and 10 V and collision energy were 19, 26, 45 and 12 eV for GAA, Cr, Arg, Gly respectively and the same parameters were used for their isotopomers.
As result of the GAA and Cr synthesis experimental conditions, labelled and unlabelled precursors coexisted inside the cells and therefore we obtained enzyme products with a different number of stable isotopes (monolabelled, trilabelled and pentalabelled products). Since the higher concentration of the labelled in respect to unlabelled precursors, the pentalabelled forms ([1,2-13 C2,15N3]GAA and [1,2-13 C2,15N3]Cr) were prevalent. Therefore we selected them as reference compounds, also considering that their signals were far from isotopic distribution of the natural compounds.
The ion transitions for the labelled products [1,2-13 C2,15N3]GAA and [1,2-13 C2,15N3]Cr were defined upon the study of fragmentation of corresponding unlabelled compounds. The fragmentation of the protonated molecular ion of GAA, producing the fragment ion at 101.1 m/z, involves the loss of a molecule of butene and ammonia, while the formation of 90.1 m/z by protonated Cr, involves the loss of a molecule of butene and cyanamide (Figure
Figure 1
Analysis by MS/MS: fragmentation by collision-induced dissociation (CID)
Analysis by MS/MS: fragmentation by collision-induced dissociation (CID). Fragmentation pattern and selected ion transitions (m/z) of butylated [1,2-13 C2,15N3]GAA and [1,2-13 C2,15N3]Cr used for MS/MS analyses are shown.
The ion transitions were reported in Table
Table 1
Parent and product ion transitions used for analysis by mass spectrometry
Parent ion
Product ion
Parent and product ion transitions used in MRM and lower detection and quantitation limits are reported. The LOD and LOQ were estimated analyzing ten blank samples and were calculated adding to the mean concentration respectively 3 and 10 SD.
Linearity, recovery, within-run precision (data not shown), and sensitivity (the LOD and LOQ, Table
Real time PCR
RNA from different Rattus norvegicus (Sprague Dawley) tissues was obtained. RNA was isolated from Kidney, Liver, and astrocytes using RNeasy Mini Kit (QIAGEN, Milan, Italy), from cerebellar granule cells, using RNeasy Lipid Tissue Mini kit (QIAGEN, Milan, Italy) and from blood using QIAamp RNA Blood Minikit (QIAGEN, Milan, Italy) as described from manufacturer. cDNA synthesis from total RNA was performed with High Capacity cDNA Archive Kit (Applera Italia, Monza, Italy). The resulting cDNA template, at a concentration of 50 ng/μl, was subjected to relative-quantitative Real-time PCR using Taqman gene expression assays (Applera Italia, Monza, Italy), the TaqMan Universal PCR Master Mix (Applera Italia, Monza, Italy) and the Two-Step Real-Time PCR System (Applera Italia, Monza, Italy). Quantitative Real Time PCR analysis of AGAT, GAMT and SLC6A8 mRNA expression were carried out in a Two-Step Real-Time PCR System using the TaqMan Rat Gene Expression Assays Rn00562952_m1 for target gene GAMT (EC 22.214.171.124), Rn00578954_m1 for target gene AGAT (EC 184.108.40.206), Rn00506029_m1 for target gene SLC6A8 (Solute carrier family 6 member 8) and Rn99999916_s1 for the internal normalization gene GAPDH (Applera Italia, Monza Italy). For quantitative Real Time PCR analysis, each sample was run in triplicates and the entire analysis was confirmed at list twice. Each run included a no template control (NTC) to test for contamination of assay reagents. After an initial Amperase UNG Activation at 50°C for 2 min, and a 95°C denaturation for 10 min, the reactions were cycled 40 times with a 95°C denaturation for 15 s, and a 60°C annealing for 1 min. Three types of controls aimed at detecting genomic DNA contamination in the RNA sample or during the Reverse Transcription (RT) or quantitative Real Time PCR reactions were always included: a RT mixture without reverse transcriptase, a RT mixture including the enzyme but no RNA, negative control (reaction mixture without cDNA template). Relative quantification was performed using the comparative threshold (CT) method after determining the CT values for reference (GAPDH) and target (AGAT, GAMT and SLC6A8) genes in each sample sets according to the 2-ΔΔCT method. Changes in mRNA expression level were calculated after normalization to GAPDH. As calibrator sample we used cDNA from arbitrarily selected control cell line or tissue.
Statistical analysis
Unless otherwise indicated, all data are presented as the mean ± SD. Student's
Cr uptake
To investigate the functional expression of CT1 in cerebellar granule cells and astrocytes these cells were incubated with D3Cr, and its uptake was assessed in the presence or absence of GPA. After 24 hours of incubation with 1 mmol/L D3Cr, both cerebellar granule cells and astrocytes showed a high intracellular accumulation of the labelled compound (24.4 ± 5.3 and 207.5 ± 119.6 nmol/mg protein, respectively). These values were as an average 10 and 122 times higher, respectively, than those of the endogenous (unlabelled) Cr, that were 2.5 ± 0.9 and 1.7 ± 0.6 nmol/mg protein, respectively in rat cerebellar granule cells and astrocytes. The addition of GPA to the culture medium resulted in 77 ± 5% and 68 ± 1% inhibition of the D3Cr uptake in cerebellar granule cells and astrocytes, respectively (Figure
Figure 2
Functional expression of CT1 in rat cerebellar granule cells and astrocytes
Functional expression of CT1 in rat cerebellar granule cells and astrocytes. Incubation of rat cerebellar granule cells (A) and astrocytes (B) with 1 mmol/L D3Cr in culture medium in presence and absence of 1 mmol/L GPA: intracellular D3Cr mean values are given along with standard deviation for three different experiments (each sample was analysed two times). The addition in culture medium of GPA resulted in a marked inhibition of D3Cr uptake (77% and 68% respectively in cerebellar granule cells and astrocytes). D3Cr uptake in absence of GPA was significantly higher than that in presence of GPA, showing the occurrence of Cr active transport, in cerebellar granule cells as well as in astrocytes (paired Student's
Cr synthesis in rat cerebellar granule cells
Intracellular precursor accumulation, as well as GAA and Cr synthesis were investigated by comparing granule cells incubated with and without [13 C2,15 N]Gly and 15 N2-Arg, (10 mM). Results are summarized in Table
Table 2
GAA and Cr synthesis experiments in rat cerebellar granule cells
Concentration of [15N2]Arg and [13C2,15N]Gly in the medium (mmo1/L)
[15N2]Arg in the cells
(nmol/mg protein)
Arg in the cells
(nmol/mg protein)
1.6 ± 0.8
153.4 ± 105.3
0.009 ± 0.001
55.3 ± 6.5
1.00 ± 0.25
[13C2,15N]Gly in the cells
(nmol/mg protein)
Gly in the cells
(nmol/mg protein)
1.8 ± 1.2
470.8 ± 232.7
0.007 ± 0.004
710.5 ± 216.1 **
137.9 ± 29.8
5.08 ± 0.68
[13C215N3]GAA in the cells(nmol/mg protein)
GAA in the cells
(nmol/mg protein)
0.12 ± 0.07
2.08 ± 1.43
0.085 ± 0.029
7.4 ± 3.2 *
0.22 ± 0.05
33.3 ± 7.9
[13C215N3]Cr in the cells
(nmol/mg protein)
Cr in the cells
(nmol/mg protein)
0.04 ± 0.02
261.3 ± 138.4
< 0.001
1.6 ± 0.4**
62.2 ± 15.3
0.027 ± 0.007
Intracellular concentrations of Gly, [13C2,15N]Gly, Arg, [15N2]Arg, GAA, [1,2-13C2,15N3]GAA, Cr, and [1,2-13C2,15N3]Cr after 24 hours in vitro incubation with 10 mmol/L [15N2]Arg and 10 mmol/L [13C2,15N]Gly. Zero concentration in the first column (first line) corresponds to cells incubated in medium without labeled precursors. The value reported of labeled compounds is due to background noise. The statistical significance of the differences between intracellular concentrations of labeled and unlabeled compounds was assessed by paired Student's
Cr synthesis in rat astrocytes
Astrocytes were incubated for 24 hours with 3 different concentrations of [15 N2]Arg and [13 C2,15N]Gly (5, 10 and 15 mmol/L). Results are summarized in Table
Table 3
GAA and Cr synthesis experiments in rat astrocytes
Concentration of [15N2]Arg and [13C2,15N]Gly
in the medium (mmol/L)
[15N2]Arg in the cells
(nmol/mg protein)
Arg in the cells
(nmol/mg protein)
1.8 ± 1.1
172.0 ± 78.2
0.01 ± 0.00
145.5 ± 73.4
1.12 ± 0.52
504.0 ± 390.0 *
178.6 ± 137.9
3.43 ± 1.66
957.4 ± 500.45 **
215.5 ± 132.5
6.74 ± 4.72
[13C2,15N]Gly in the cells
(nmol/mg protein)
Gly in the cells
(nmol/mg protein)
2.8 ± 0.9
365.7 ± 43.2
0.01 ± 0.00
696.3 ± 604.2
1.28 ± 0.49
2205.5 ± 2382.0 *
893.8 ± 958.4
2.53 ± 0.22
3213.9 ± 2482.9 *
744.8 ± 512.7
4.43 ± 0.85
[13C215N3]GAA in the cells
(nmol/mg protein)
GAA in the cells
(nmol/mg protein)
0.25 ± 0.09
1.46 ± 1.46
0.29 ± 0.14
10.34 ± 7.90 *
2.37 ± 2.23
4.47 ± 3.97
17.18 ± 13.36 *
1.88 ± 1.87
13.30 ± 14.32
21.59 ± 15.00 **
1.64 ± 1.11
18.18 ± 17.96
[13C215N3]Cr in the cells
(nmol/mg protein)
Cr in the cells
(nmol/mg protein)
0.10 ± 0.05
100.4 ± 118.7
0.00 ± 0.00
12.13 ± 12.64 *
86.53 ± 71.65
0.12 ± 0.04
10.88 ± 8.48 **
59.48 ± 23.72
0.17 ± 0.08
11.00 ± 6.85 **
48.54 ± 10.58
0.21 ± 0.09
Intracellular concentrations of Gly, [13C2,15N]Gly, Arg, [15N2]Arg, GAA, [1,2-13C2,15N3]GAA, Cr and [1,2-13C2,15N3]Cr after 24 h in vitro incubation with [15N2]Arg and [13C2,15N]Gly under different concentrations of labeled precursors in the medium: no labeled precursors (0), 5, 10 and 15 mmol/L. The statistical significance of the differences between intracellular concentrations of labeled and unlabeled compounds was assessed by paired Student's
The intracellular levels of the labelled amino acids paralleled those in the medium and, except for the concentration of 5 mmol/L, were higher than the concentrations of unlabelled Arg and Gly, thus demonstrating their uptake by astrocytes.
After incubation with labelled precursors, the [13C2,15N3]GAA signal in cell extract was strongly increased, reaching 86 times the corresponding background noise in control samples. [13C2,15N3]GAA concentration increased proportionally with the concentration of labelled precursors in the medium (R2 > 0.9229; p < 0.002; Figure
Figure 3
Functional expression of GAMT and AGAT in rat astrocytes
Functional expression of GAMT and AGAT in rat astrocytes. Intracellular [13 C2,15N3]GAA (A) and [13C2,15N3]Cr (B) concentrations in function of [13C2,15N]Gly and[15 N2]Arg concentrations in culture medium, in three sets of cell samples (experiment 1, 2 and 3) are reported. To test the correlation among the intracellular concentration of labelled products and the precursor concentrations in culture medium, linear regression analysis was performed. Whereas there was a linear correlation for labelled GAA (p < 0.0022), no clear and consistent linear correlation for labelled Cr was found. Both graphs show that the cultures of experiment 3 yielded a lesser amount of GAA and Cr. Inspection of the cultures did not provide any obvious explanation for this result.
We found also a relevant increase of [13C215N3]Cr, whose signal was significantly higher (up to 121 times higher) than the corresponding signal in control samples. No correlation was found between the [13C215N3]Cr levels and those of labelled precursors in the medium (Figure
Gene expression
To get information about the level of gene expression in rat astrocytes and cerebellar granule cells we used a PCR-Real Time relative quantification. To this aim, we compared AGAT, GAMT and SLC6A8 gene expression in different tissues such as blood, kidney and liver. Figure
Figure 4
Relative quantification of gene expression using Real Time PCR
Relative quantification of gene expression using Real Time PCR. Blood expression level is used as calibrator (Relative quantification (RQ) = 1). Panels at right side (A1, B1, C1) are a magnification of those on the left side (A, B, C) excluding tissues with higher expression level.
GAMT gene was equally expressed in cerebellar granule cells and astrocytes (31 and 33% of blood cell expression, respectively). We found that GAMT expression in the kidney was 3.5 times higher than in blood cells. SLC6A8 was about 3 times more expressed in cerebellar granule cells than in astrocytes (p < 0.005). In order to test the possible influence of experimental conditions on gene expression, SLC6A8 transcripts were evaluated in P1, P2 and P3 generations of astrocytes by real time PCR. A stable and overlapping expression was found along the three cell generations (relative quantification expressed as log10 values were 2.06 ± 1.61, 2.09 ± 1.66 and 2.09 ± 1.68 respectively for P1, P2 and P3 generations).
Cellular incorporation of stable isotope from nutrient into downstream metabolites is largely used for the assessment of metabolic fluxes and the step of quantifying labelled and unlabelled molecules can be achieved by mass spectrometry, with the advantages of excellent sensitivity and linear dynamic range, and simultaneous detection of precursors, products, and associated isotopomers
The converging results of our functional and gene expression studies showed that in the rat both neurons and astrocytes are capable to express the enzymatic machinery to both uptake and synthesize Cr.
Cr uptake
Cr uptake in our study was greatly reduced by GPA, an inhibitor of CT1
Concerning Cr uptake by the astrocytes, our results confirm those of a previous study based on a kinetic approach
As a word of caution, while our findings show that astrocytes have the capability to express the Cr transporter, and to uptake Cr, we can not rule out that this capability may be differently expressed in different conditions.
Cr synthesis
Our findings show that both granule cells and astrocytes are capable to uptake Arg and Gly and to use them to synthesize GAA and Cr. Again, these functional findings are supported by gene expression analysis, showing that both cell types express AGAT and GAMT, the enzymes that are required to synthesize Cr from Arg and Gly, which have their own transporters at the blood brain barrier and on the plasma membrane of nervous cells
Cr synthesis vs. Uptake
An important difference arises in our data between Cr synthesis and uptake when we consider the magnitude of the results. Cr uptake was in our hands a very efficient process in both granule cells and astrocytes. After incubation with labelled Cr the 91% and 99% of the total intracellular Cr content, respectively in neurons and in astrocytes, is due to transport. This effect is very robust and sizable. By contrast, after 24 hours cell incubation with high concentrations of Cr precursors Arg and Gly, the
This issue was further addressed by exploring the co-expression of AGAT, GAMT and SLC6A8 proteins in rat grey matter cells
Clinical implications
The presence of CT1 in both astrocytes and cerebellar granule cells, as well as in the endothelium of brain capillaries
The above results may offer an explanation of the conflicting results that were obtained in patients affected by CT1 deficiency when treated with the Cr precursors in order to stimulate the endogenous synthesis of Cr. Our data suggest that Cr synthesis by brain cells may be limited, in such a way to possibly provide only a limited amount of the Cr that is needed by the brain.
Some limitations inherent to our study suggest caution in extrapolating our results to the in vivo conditions. In fact, we investigated only two kinds of cells from the cerebellum, while different brain regions and cells could be differently equipped in term of CK activity and related Cr availability
Summing up, while further research is needed to fully understand to what extent these in vitro results are relevant to the in vivo situation, our results show that both astrocytes and neurons possess the capability to both synthesize and uptake Cr, but that the latter is probably more robust than the former.
Arg: arginine; AGAT L-arginine:glycine amidinotransferase; Cr: creatine; [13 C2,15 N]Gly: [13 C2,15 N]glycine; CT1: creatine transporter; D3Cr: D3-creatine; GAA: guanidinoacetic acid; GAMT: S-adenosylmethionine:guanidinoacetate N-methyltransferase; Gly: glycine; GPA: β-guanidinopropionate; [15 N2]Arg: L-[guanido-15 N2]arginine; SLC6A8: creatine transporter gene; CNS: central nervous system; ESI-MS/MS: electrospray ionization tandem mass spectrometry; [1,2-13 C2]GAA: 13C2-guanidinoacetic acid; [13 C2,15N3]GAA: [1,2-13 C2,15N3]guanidinoacetic acid; [13 C2,15N3]Cr: [1,2-13 C2,15N3]creatine.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
All Authors have each made a substantial contribution so as to qualify for authorship.
Specifically: a) CC, SS, and IA set up the new MS/MS methods for the intracellular assessment of labelled and unlabelled metabolites. They also were involved in the development of the experimental design and cooperated in the interpretation of the metabolic results; b) CC and CA performed genetic and metabolic molecular studies, interpreted genetic results and cooperated in the discussion concerning the linkage between biochemical and molecular results; c) EA, ST, EG, MR, and TF developed the in vitro model; arranged and accomplished cell coltures with labelled Cr and Cr precursors; d) VL and MB conceived and developed the experimental design, addressed the discussion on the interpretation of the results and coordinated the collaborative study. All authors read and approved the final manuscript.
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