text:
Co-Written by Shelbylou - You Rock!
Tim's mind wouldn't shut off during his 30 minute drive home and questions flew through his mind fast and endless. Every single one of them demanded an answer, questions that he just couldn't find the answer to.
Had he really lost his sense of humor?
Or did Tony just not appreciate his jokes anymore?
Am I thin skinned?
Am I a wimp?
Am I the weak link I used to be?
Just because the things that are said sometimes bring back memories or concerns from the past didn't mean a person was weak or a wimp. Did it? Especially, if they were taking the time and making the effort to think things through in an effort to fix whatever the problem was? Wasn't being strong, having the fortitude to look at situations and learn from them? Wasn't the willingness to ask himself and answer honestly, the questions that the situation had raised for him, a true testament to maturity?
Tim hadn't complained or even replied to Tony's blatant disrespect and carried on as if he hadn't heard it. That had to be the sign of a pretty tough skin, right?
The whole situation grated on his nerves. Tim thought back to his conversation with Gibbs. There was something hinky right there with that chat as it was and Tim wanted to be able to put his finger on it before tackling the situation with Tony.
The questions were relentless and joined the funnier when statements that had returned and begun once again to whirl around in his mind. He gripped the steering wheel harder and tried to get his mind to stop with the demoralising thoughts that threatened to eat away at him. The first thing that popped into his mind, the first real fighting thought was that maybe, just maybe, Tony didn't like the fact that Tim was no longer such an easy target. He was on par with the Senior Field Agent at last, in mind and now, finally in body.
Have I done enough?
Is losing the weight worth losing your identity over?
Is losing weight antagonising people?
Has my personality really changed so much?
'ENOUGH!' Tim's mind started to scream back at the subconscious questions that just kept on coming.
It's not about being thick skinned!
It's not about my personality changing!
It's not about being a wimp!
No! This was about respect and Tony's complete lack of it towards Tim. It was about why Tony thought nothing of trying so hard to belittle him. Every statement, every question and every bit of fight Tim was pondering in his mind was replaced by one little question:
'Why does Tony still think so little of me?'
Tim felt a small surge of hope as he tried to mentally fight back against himself. He knew that, despite Tony being his friend and colleague, he had seriously toughened up under Tony's unbridled over-the-top crap. And yet here he was, once again, with every last bit of confidence slowly dripping away.
Why had Gibbs said he and Tony needed to talk?
What did the Boss man know?
He'd seemed unusually concerned tonight AND he'd let Tim's leaving on his own go with a smile! Yeah, the Boss knew something and he hadn't gotten it from Tim. The penny dropped and his sudden realisation made Tim smile. It was great to be able to hear it verbalized and that conversation with the boss left him with no doubt that Gibbs did care about whether his agents got along in general and not just out in the field.
Tony's mind was warping through questions almost at the speed of light.
Had he really gotten so callous that he was willing to be downright disrespectful to get his jibes in where McGee was concerned?
Where had that close-knit working relationship gone?
How had Gibbs known about it? He didn't even yell or raise his voice! Gibbs wasn't angry, he was genuinely concerned! Why?
Gibbs was right. He and Tim had become a strong team – just the two of them - while Ziva was away.
What had happened?
Better yet,
How could they fix it?
Tony heard a sharp blare of the horn from the car behind him and pulled himself out of his reverie. He glanced at the lights and realised they were green before wincing slightly and driving off. With his mind back on the road, he still heard the questions filter through his mind.
How could they fix it?
The question was on repeat and there had to be a way. All he had to do was find it. In a moment of sheer inspiration, the answer came to him and made him grin wildly.......YES!
He took the next left and stopped at the minimart to grab a six pack of beers, next stop, the pizza place. It was foolproof and the fact that he was doing something by actually taking the proverbial bull by the horns felt good. The smell of the pizza sitting on the passenger seat was making him hungry and he couldn't help the rush of nerves that ran through his body.
"Ok DiNozzo, time to get this show on the road." He said as he picked up his beer and pizza before heading to the familiar apartment block.
Tim was sitting as his typewriter and was subconsciously twirling his grandfather's pipe round in his hand. In true 'starting as you mean to go on' style, his day, which had started badly, was hell-bent on ending in exactly the same way.
An hour ago...............
Tim had stopped to pick up his mail on the way to his apartment and noticed a familiar looking envelope among the usual bills and fan mail that was delivered through a PO Box address. He tucked the other letters under his arm and trudged slowly up to his apartment. It didn't take long for him to drop his mail on the sideboard and change into his old faithful MIT t-shirt and loose boxer shorts that he often wore around the apartment. He sat down at his computer and booted it up in order to check his emails. The first one struck a chord of despondency in him as he opened it to read.
From: Lyndi
To: Timothy McGee
Subject: Manuscript
Need I remind you of the fact that your manuscript is overdue? I asked for it to be sent to me no later than 5th April and now it's the 6th.
Can you call me to discuss any problems you are having because I need to let the proof department know that there will be a delay.
Lyndi Crawshaw
He sighed heavily and typed out his response, promising Lyndi he would have it over to her by first thing tomorrow morning.
Present time...........
So here he was, sitting in front of his good old antique manual typewriter trying to get the final chapter down. He had made a good start with the document, but for some reason found the last few pages difficult. A new set of questions joined the party of words that had jumbled around in his mind since the beginning of the day.
Can I really do this?
Why am I doing this?
What's the point?
He swore loudly before tearing out the page he had just typed to send it through the shredder and sat back in frustration.........Damn it, just a few more paragraphs, I can't even write those!
A knock at the door made him cringe and, thinking it was his neighbour complaining about his shredder, he walked to the door with his shoulders slightly hunched in defeat. He glanced through the peep hole in the door and was surprised to see Tony standing on the other side.
Another loud knock startled him slightly.
"Come on McGee, I know you're in there. I heard the shredder. Open up already, we need to talk."
"Go away Tony, I really don't feel like talking right now."
"Tough, McGoo, we're going to sort this out now before the boss decides to bench us both."
Tim huffed out a small sarcastic laugh and started to move away from the door. He was stopped in his tracks by his cell phone chirping; he picked it up and looked at the caller ID....DiNozzo
He answered the phone and tried to use his best impression of his answer machine.
"Hi, you've reached Special Agent Timothy McGee, if you are anyone other than Tony DiNozzo, then please leave your name and your number and I'll get right back to you."
"McGee, Tim. Come on man we need to talk, I've got beer and Pizza."
"Tony, seriously GO AWAY!"
"Nope, not until we've had this out. Don't make me pick your lock, the boss man taught me well Probie!"
Tim huffed his way back to the door and opened the door as wide as the chain would let him. He saw Tony's grin flash across his face and scowled heavily.
"Tony, please. I'm busy so can we pick this up tomorrow"
"Hell no, there is no way I'm leaving here without talking to my buddy over a beer and pizza, which by the way McGee, is getting cold."
"Tony...why am I still talking to you on the phone? Aw, crap."
Tim hung up the phone and unlatched the door completely to let Tony in. He heard the snap of a cell being closed and marched back over to his writing desk.
"You need a couch Probie."
"I don't know, so I can sit down maybe?"
Tim reached over and grabbed the back of his computer chair so he could scoot it over to his teammate.
"Thanks, Tim I wanna apologise."
"Yeah? Why now Tony? So the boss won't, quote, bench us both?"
Tony had the good grace to look abashed and looked down to the beers in his hand. He got one out and passed it to Tim.
"No Tim, because I don't want to lose a friend. It's been bugging me since I said it and I know I was wrong."
"Wha...you can't be serious Tony. You don't apologise, you just leave everyone in your wake and move on. Besides, you sure didn't act like it bothered you in the least, until now. I'll bet the only reason you're here at all is because that's what Gibbs wanted to talk to you about. He told you to come talk to me, didn't he? That's why you're here?"
"Yeah, okay. Maybe you're right. But that doesn't mean I feel good about saying it."
"Feeling good about what you say has never worried you before, Tony. You'll keep on this till I say I forgive you and then, it'll start up all over again."
"Not this time. Look, I didn't mean it. It was just -hell, I don't know what it was. You seemed hell bent on bugging me this morning and it annoyed the crap out of me."
"It's no different to what you do to me all the time Tony."
Tony was quiet for a moment and with a voice barely louder than a whisper, he looked at Tim and simply said "I know."
Tim sat back in his chair and sipped at the cold beer he was holding.
"Why Tony? That's all I want to know. You have no idea how that comment affected me."
"I know it had to hurt, Tim. I'm sorry."
"No, Tony. It's like I said, you have no idea how it affected me. Because you don't take the time to look at me as a person. I've taken everything you've thrown at me over the years and I've used it to change and grow, the best way I knew how. Not just to hopefully get you to appreciate the effort and back off me for a change, but because it was who I wanted to become anyway."
Tim paused briefly and took another swig of his beer. He needed that break, that cleansing pause, to rein in his building anger. He watched as Tony's face dropped slightly at the snappish response and felt slightly guilty at making the man feel worse than he already did. He sighed loudly before continuing.
"I stopped complaining because I realized that you just wanted to help push me harder than I was pushing myself. Sure you pushed a lot harder and a lot more painfully than ever did. But the truth is, Tony, I was proud of who I'd become and I thought I'd finally reached the point where you and Gibbs were proud of that, too."
"Jeez Tim. I really am sorry. I didn't realize..."
"I mean, you suddenly started acting as though you could actually relate to me." Tim continued on his roll as if Tony hadn't spoken. "The last two months, before we rescued Ziva, were the best we'd ever gotten along; we gelled as partners, Tony. I know for a fact that Gibbs saw it and you know what? He appreciated it. I mean, how many head slaps did we earn in those two months, one? He listened to both of us, took what we had to say under advisement and actually treated us like we knew what we were doing."
Tony sat patiently, his gut was telling him not to answer just yet and, for the first time, he decided to listen to it.
"Maybe it was because for the first time in all the years on the team, we were actually partners without having anyone else there to be that partner for you. I mean, let's face it, I've spent as much time being partnered to the computers at NCIS as you've spent partnered with Ziva and the two years with Kate. So, yeah, maybe this being the first time that we were actual partners had a hell ova lot to do with it. But, the fact remains, Tony, that we were a damn good team and we treated each other with respect. Even you've gotta admit, we kicked ass in those two months! You know we did."
"I know..." Tony again tried to say something.
"And with one five word remark; you just threw it all away." Again, Tim kept going. "No. I take that back. You had already started wadding that trust and mutual respect up, getting ready to throw it away with the changes in your attitude toward me that started the minute we got back here with Ziva."
"What are you talking about, Probie?" Tony asked in surprise.
It the whole situation wasn't so damn serious, Tim would have laughed as Tony's eyebrows danced from his hairline into a deep frown.
"My contribution to her rescue was, and I quote you here, 'lying around on the floor'" Tim answered him seriously. The pain from that remark had long since faded, but the effect on their relationship, which had indeed started its' downward spiral from there, had only fallen apart like a house of cards.
"Tim... I don't know what to say. I'm sorry. Really. I..." Tony was almost speechless. He'd literally fallen back into the habit of not thinking about Tim as anything other than the Probie that had taken all of his crap over the years and still came back for more, time after time. He'd forgotten recently, how much of a friend and an equal Tim had become. He looked at Tim and saw, once again, the man and ally the man had become all those months ago.
"I don't want you to say anything Tony. I just want.....I need, what we had back when Ziva was gone, back again. I can't go back to the way it was before then, do you have any idea how much that time, our partnership helped me gain confidence. In that time, I decided to grab life by the balls and do things that I didn't have the motivation do to before."
Tim stood up and moved towards the window, the faraway look on his face didn't go un-noticed by Tony who followed and perched himself to the side of Tim on the windowsill.
"I've been bullied all through my life Tony; I decided to finally lose the weight that had been holding me back in more ways than one. I've written two books, well, nearly two. I've done things in the past few months that I would never had done before. I embraced life Tony, I actually started to live and that wouldn't have been possible if it wasn't for the respect and standing I'd gained at work. I just wish you could understand how much hearing what you really think of me today has really worked against what I'd achieved with the help of you guys."
Tony could feel his chest constrict at Tim's statement, sure he knew the kid had more confidence and even he had to admit that Tim looked good after finally shedding that excess weight. What he hadn't known was, that whilst trying to help Tim with his taunts and jibes, he had actually been no better than the other bullies that had pushed Tim to develop into the shy, retiring man he once was....was in danger of becoming again.
"Aw man, I had no idea. Why didn't you tell me when I went too far, I didn't want..No! I never meant to be one of those bullies....."
"You weren't." Tim's quiet answer surprised Tony.
"You had a different motivation. You wanted to better me not drag me down. Sure you belittled me, and sometimes it seemed as though you poked fun just for the sake of doing it. The names Tony, those damn names." Tim shook his head sadly.
"Do you realise, you never called me Probie once when Ziva wasn't there, that was the one name I didn't freaking mind so much. It was the others; McGeek, McGoogle, MgGluey, McFlabby.. The list is endless Tony. They sting, but you know what, I never stopped you, never had the balls to step up to the plate. I'm done being the butt of your jokes Tony, no more!"
Tim made to move away from the window but was stopped by Tony's hand grabbing his wrist firmly.
'McGee, I can't promise that I won't say things like that, but I will try not to. That is something I can promise. I miss the new confident Probie, and I miss my friend. I love you like my kid brother and that is one of the reasons why I do the things I do. You know my history, you know my family. I haven't got any brothers or sisters, Hell; I don't really even have a dad. To me, you are all more than my colleagues and team mates, you are my family and I love you all as if you were blood. Tim I'm sorry, I'm not saying that because Gibbs sent me, I'm saying that because I want my brother back. I want my best friend and my team mate back. I might be hurtful sometimes, but I've always got your back Probielicious."
Tim couldn't hold back that little tear at Tony's words. They were heartfelt and sincere which seemed to help lift the weight that had settled on his shoulders. He wasn't Atlas, he wasn't supposed to carry the weight of the world on his shoulders; it was time to rein it all in and find that confidence and self control he had lost recently.
"I forgive you Tony and I promise not to give you a hard time in return. I know I've got a family, but you have all become my secondary family too. I guess that's why it hurt so much."
Tony pulled on Tim's wrist and wrapped his arms round him in a manly hug. Tim stiffened before melting into the brotherly embrace that made him feel strangely safe.
"I've always got your six McGee, don't you ever forget that ok?"
Tim pulled away and nodded slightly.
"I know, I've always got yours too Tony."
Tony moved away from the window and opened the pizza box to snag a slice.
"I still get to keep Probie and Probielicious though McGee, those are non negotiable and are all mine."
Tim looked at Tony and burst into a fit of giggles so hard he had to sit down.
"Yeah Tony, you can keep them. Just don't abuse the privilege."
He was handed another beer and the two of them soon fell into a comfortable, comfortable conversation. Oh yeah, the boys were back and stronger than ever. Their new found strength in having talked thorough their problems was definitely a step in the right direction.
It's getting late Probie, I should be going."
Tony stood up and grabbed his coat. He had helped Tim clear up the empty beer bottles and pizza boxes and the two of them had fallen comfortably into a much happier conversation.
"Yeah, I'll see you tomorrow. Tony...."
Tony looked up into the much brighter green eyes of his friend and smiled broadly at him.
"Thanks for, you know, coming round to sort this out. I appreciate it." Tim offered with genuine gratitude
"Anytime Probie, I'll see you tomorrow." Tony clapped Tim on the shoulder and turned towards the door.
"Oh, there is one thing I need to know though Probie, what's with the sprinkles and the blowing?"
Tim laughed and shook his head.
"I like sprinkles on my doughnuts, but not too many. They never have them with just a few on, so I pick them off. I blow my coffee that way because short bursts seem to cool it quicker than one long one. There's a method to my madness DiNozzo, you just have to be privy to it."
Tony shook his head with disbelief.
"Wow, there really are many depths to Timothy McGee, huh, who knew. Now you've explained it, it kinda makes sense. See ya Probie, get some sleep." Tony clapped Tim on the back and left, leaving Tim standing in front of the open door looking thoughtful.
Tim walked over to his typewriter and sat down in front of it, with the air cleared between him and his friend, he was finally able to think straight. All the questions, the demoralising statement, the degrading thoughts of dread were finally filtering out of his mind. Smiling, he started pounding at his typewriter with a renewed confidence and freedom. His relationship with Tony was back on solid ground. Their boss had cared enough to push them to fix it. The team sense of family was intact.
At that moment in time, Life was good.


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Club News
Meeting July 26, 2017 - Judge Mark Bernstein
Club meeting, July 26, 2017 Judge Mark Bernstein Judge Mark Bernstein retired from the Court of Common Pleas in 2016 after 30 years on the bench. He gave a presentation on cross-examination: The Power of Cross Examination: The Greatest Invention for Truth. Using actors working from real life transcripts, he showed a series of videos illustrating how defense attorneys have used cross-examination to unmask material that might otherwise be hidden from the jury. Judge Bernstein also feels that juries are a very capable instrument in evaluating testimony presented in a courtroom.

subset: commoncrawl


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The Southern Highlands Regional Rose Society is hosting the Miniature Rose Championship (including floral design) for the first time in Australia on 18th and 19th November at Mittagong RSL. The championships will be open to the public on Saturday 18 November from midday tp 5pm and Sunday from 10am to 4pm. The official opening and presentation of the Special Awards is on the Saturday at 1pm.
Please contact the Southern Highlands Regional Rose Society on 4871 0091 for more information.
Image source: Pixabay


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Draw your Fortune Card
Antique Kipper fortune card for today
The fortune card drawing
For Wednesday, September 20th 2017
Your free Horoscope card for today
At the antique Kipper fortune card horoscope, you can draw a fortune card for today Wednesday, September 20th, and it will give you a possible view in the future of this day.
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text:
In a Facebook post this morning, McMillan Group International announced that they are wrapping up the final negotiations for the sale McMillan Firearms Manufacturing. McMillan Group will retain their fiberglass stock business and, as we reported earlier, they recently made a splash with the creation of a firearms-friendly credit card processing service, McMillan Merchant Solutions. We'd guess that will stay with McMillan Group as well, but are trying to confirm that. No word yet as to the identity of the buyer. Stay tuned . . .
Update: In an email to TTAG, McMillan has confirmed that the newly-established McMillan Merchant Services will not be part of the sale.
13 Responses to BREAKING: McMillan Group Sells McMillan Firearms Mfg
I really hope the Freedom group didn't buy them….
i'd bet ATK buys it; they've been on a roll lately
How long was the firearms portion of the business around? I don't know much about them – it seemed like they just started this aspect of the business?
So, why are they selling the firearms manufacturing portion, but keeping the fiberglass stock portion? Also, why sell at all?
A few weeks ago TTAG carried an extensive write-up on how money laundering laws can be used as a club against retail FFLs. With McMillan's recent branching out into the banking side with their credit processing, I expect that having both banking and firearms manufacturing/sales under one roof would practically be begging for trouble under those laundering rules and regulations.
Didn't they just recently get into the firearms manufacturing business (in the last 3 or 4 years)? If I had to guess, they may have decided with all the new state, city, county laws being proposed and/or passed, to avoid the headache and hassle of being on the bad end of a witch hunt. Typically there is not a lot of profit in the making of firearms, so perhaps it just isn't worth the time and energy to keep that end of the business. I wish them well in any case, they make an excellent stock for most rifles out there.
i have an A5 adjustable mcmillian stock, it is awesome, but it sure took a long time to get.
I'm sure that they make an excellent stock. However, I don't have a first born to give them.
Taurus again, perhaps?
The answer is – http://www.ammoland.com/2013/11/strategic-armory-corps-acquires-mcmillan-firearms/#axzz2lqRcuP77
I use to work for McMillian back in the 90s. I was originally hired by Gale McMillian in 1989. Then the company sold so they could not manufacture firearms for a while. Later they started making guns again but could not do it under the McMillian name so they became Mcbros. Sometime after that they started using the McMillian name again.


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A Mixed Initiative Team Performance Assessment System (MITPAS) For Use in Training and Operational Environments
Award Information
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AbstractMilitary forces of the future will use mixed manned /unmanned forces for a broad variety of functions. Measurement of overall effectiveness in these mixed initiative systems will be essential in order to achieve optimal system performance levels. Behavioral measures of both human and unmanned performance obtained in system simulations or in live exercises will be used to continuously diagnose performance and identify required areas of training requirements. Likewise, specialized training will be necessary in order to leverage the complementary cognitive functions of human and machine to forge fighting entities and units with capabilities superior to those of humans or machines in isolation. This proposal describes an innovative approach to developing a Mixed Initiative Team Performance Assessment System (MITPAS). The MITPAS consists of a methodology, tools and procedures to measure the performance of mixed manned and unmanned teams in both training and real world operational environments. Our objective is to provide a turnkey MITPAS software system running in the OneSAF-OTB environment, with automated data collection functions and containing protocols for evaluation of various manned/unmanned team configurations in event based scenarios. Phase I has validated the concept, the proposed Phase II effort will fully implement it.
* information listed above is at the time of submission.


text:
We aim to be involved in a sporty, healthy and fun way, together with hundreds of others supporting the fight against cancer. This is your way to fight it, in memory of someone who did not survive, as support to someone who is fighting the battle right now or just because you are involved in any kind of way.
Ride for the Roses Curaçao:
On January 2005, in the pouring rain, 273 cyclists started a 45 kilometre cycling journey across the island. They were followed by a warm reception where they got offered a rose and a stage program. There were smiles and some tears as well: this was how The Ride for the Roses in Curacao was born. The later Olympic champion Maarten van der Weijden had been cycling the ride. That day he told about his story, how he was a promising young Dutch open water swimmer that happened to become a leukaemia patient later. He talked about his daily fight and above all about the importance of never ever giving up. Never. In 2005 he finally turned back into a real sportsman instead of a patient, as he had always been feeling the sportsman inside him, even in his darkest days. At that moment he could not even know that in three years from then, in Beijing 2008, he would win one of The Netherlands most special golden medals.
In Curaçao we have 5000 Maarten van der Weijdens. 5000 patients who are suffering any form of cancer. 5000 people whose world collapsed when they found out the terrible news. 5000 people living in uncertainty. 5000 people who cannot give up. 5000 people who are not pitiful but ill. 5000 people who need our support terribly hard.
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The second aim for Ride for the Roses maybe is less tangible, but surely as important as the first one. Namely creating awareness around cancer in Curacao. In many cases cancer is not the end. Early detection is of big importance in this. To heal, patients fight as hard as they can and in this fight they can use every bit of support. Patients are ill, but not to be pitied. And let us all keep in mind that cancer is not infectious!
Last year over 10.000 participants and 250 volunteers were actively involved in the Ride, Walk, Swim, Sail, Spinergy and Uphill Challenge and with that they have shown that patients in Curacao are not alone in their fight. We fight together. We are committed.


text:
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text:
Print advertisment created by Contrapunto, Spain for Pizza&Love, within the category: Food.
Free Love
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Illustrator: Javier Verdugo
Account: Walter Gilardo
um... i don't think i can ever look at vegetables the same way ever again... funny, but not particularly tasteful.
keeping the right side of your head warm. http://thethinkingtoque.blogspot.com
Keeping the right side of your head warm.
eeew! Never again will i be able to look at a pizza the same way.
Love the copy thought
A shop, quietly selling food, once needed an idea. They brought in the ad-men, who assured them they'd bring in the punters - sign here, pay up front - and set to work.
Seconds later, they thought of the worldwide anarchy famously wrought in pizzerias from the Bronx to Bucharest. Barcelona, they declared, would be a safe zone. Cleverly stealing a global logo, they set down their manifesto.
NO! to the kitchen death squads, an END! to instant assassinations by flummoxed waiters. This would be the world's *safest* pizzeria. Finally, somewhere people can eat without being killed or run over by tanks and heavy weapons: a sanctuary for slices, if you will.
"We'll have ingredients fucking on the food" they said, moving forward. "That's sanitary, right? And we'll dress them up in 70s garb, to really explain the fact we haven't told CND about fucking up their logo in a cheap-assed lazy theft of intellectual property just to shift doughy vegetables in a cramped space in a small room in Sant Pere.
And LO! the client loved it, because he knew no better and liked the drawing and probably thought up the logo himself.
And so it was.They included the word FREE on the ads because it would trick people into thinking their 10 euro slice wasn't bought with money, but with the sweat and rhythmic pelvises and commingled juices of the kitchen staff. And everyone was happy.
Except the poor ingredients. Who, like male Black Widow spiders, got eaten just as they were enjoying the passing of time in a world without war.
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About Us
Leo Collins Board Member
About Leo
Leo is a Senior Information Technology Executive experienced with building/turning around technology organizations that deliver highly complex enterprise systems. Leo is experienced with large organizations (10,000+ employees) and has built technology organizations in small entrepreneurial companies experiencing rapid growth.
Leo specializes in new technologies, including social media, cloud computing, web systems, complex accounting systems (such as SAP) and supply chain development (physical and digital). He has vast experience with websites, M & A activities, mobile computing, entertainment IT, intellectual property management including all forms of sales as well as more traditional sales/marketing systems. An industry and technology thought leader, he has built systems for a fraction of the money spent at similar organizations. Technical & detail oriented, he has experience as both CIO and CTO.
Leo has most recently served as a leader in the Oracle Client Advisor (OCA) program. The OCAs become trusted advisers to the most senior level executives (CIO and business leaders) in the accounts they serve. They provide expert advice on both business and technology and have a "seat at the table" for the customer's strategic planning.
Prior to Oracle, Leo was the CIO of LionsGate where he implemented SAP Financials including FI/CO, Special Ledgers, BI/BW, Logistics (on-line purchasing and approval), MRP, EDI, and Portals. Completed in four months, on time, on budget. Leo was also instrumental in developing Lionsgates' first video on Demand presentation of a First Run movie (Pi, 1999), the first large scale use of digital projection on a first run movie (Standing in the Shadows of Motown, 2002, the first DVD supporting greater than HDTV resolution for the mass market (T2, June 2003). Prior to LionsGate, Leo was SVP of Technology for Home Savings of America


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Magic Translator is software that can translate into your local language, any selected text on a web page. If you are willing to uninstall Magic Translator, be prepared for a little lengthy and complicated process. Need help for uninstalling Windows software from your computer? Here are some detailed steps and guidelines to make this Magic Translator removal process a bit easier for you.
Method 1: Mnaually Remove Magic Translator in safe mode
1. Start the computer in safe mode. To do this, follow these steps:
Restart the computer.
Press F8 while the computer is restarting.
Click Safe Mode, and the press ENTER.
2. Click Start, click Run, type appwiz.cpl, and then click OK.
3. Locate and click on Magic Translator from the list of installed applications, then click the Change/Remove button.
4. The Uninstallation of Magic Translator dialog appears, showing check box choices of components that are available for uninstall.
5. Select the components you wish to uninstall and click OK.
6. Follow the prompts to uninstall Magic Translator, and then restart the computer when you are prompted.
When your PC has restarted, you could still be left with traces of the browser on your system. Check that the Magic Translator icon has been removed from your desktop and under the Program Files menu under your Start menu. Delete any programs that you find that are associated with Magic Translator.
If Magic Translator is not workable for you, try Method 2.
Method 2: Force Uninstall Magic Translator Once and For All
Security experts recommend removing Magic Translator using a reputable uninstaller program. If you are one of those who have equipped such malicious application on your computer, do not hesitate and fully eliminate it right away.
1. Free downloaded reliable uninstaller like Perfect Uninstaller and ran it;
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When you're in recovery from or drug addiction, you want to feed your soul things that nourish and inspire you. A great way to be encouraged in your recovery and be entertained at the same time is to watch movies that have to deal with addiction. By doing so, you'll most likely be able to relate to the addiction going on in the film and perhaps learn some tips and tools for your own recovery. Stay motivated and inspired when it comes to your recovery by watching the following movies about drug addiction.
Less Than Zero
Permanent Midnight
Requiem for a Dream
The Basketball Diaries
Drugstore Cowboy
Midnight Cowboy
Side Effects
Here is a movie about Robert Downey Jr.'s life and viewers state that the movie is a good portrayal of Downey's actual life. Julian (played by Downey) grew up in a wealthy part of Beverly Hills and gets romantically involved with his old buddy's ex-girlfriend. Both are using serious drugs and plenty of problems are caused by such. This movie helps you see a side to the wealthy post college grads that you might not have thought possible.
Undercover cop Jim Raynor finds himself falling for a drug addict, while working a case to bring down a drug lord. Both he and his new love face the reality of drug addiction and all that goes along with it.
Ben Stiller plays Jerry Stahl in this movie about heroin addiction. Moving to Los Angeles to pursue his career, Jerry finds it difficult to move forward successfully while growing more and more addicted to heroin. There are plenty of graphic details in this movie, which serves as a firm reminder of how powerful and destructive drugs can be.
This film is considered a cult classic, diving into the inner landscape of humanity's longing to feel loved and accepted by others. Four people end up lost in the underground drug world not knowing how to climb out. This film has a wonderful soundtrack too.
Erica Christensen's role in the drug trafficking film is remarkable. This movie is about the drug trading that goes on from the source all the way to drugs in the home.
This movie is about a youngster who tries to help his heroin addicted mother out. At 10 years old, he's forced to take care of his mother and younger brother. This is very good portrayal of how addiction affects the family.
This is a true story about a basketball player and poet who turns to drugs and becomes addicted. The film stars Leonardo Dicaprio, who plays a high school student who becomes very addicted to drugs and starts doing shady things like stealing, fighting, and even prostituting.
Here is a film about a couple that goes around robbing drug stores to supply their drug habit.
Joe Buck, who thinks he's God's gift to women, heads off to NYC with the intent of becoming a male prostitute, but his plan just doesn't go as anticipated. He ends up living in an old building that's been abandoned and meets up with a man and the two become hustlers. Joe faces a lot of struggles on his new path and the film ends on a sad note, depicting the harsh reality of a life full of drugs and the streets.
This film is a psychological thriller that focuses on Abilxa: an experimental anti-depressant drug that causes some interesting side effects. The drug is prescribed to a suicidal woman, who seems to improve at first, but then various side effects occur, which ends up causing her some problems.
Take some time to watch these movies, as they will inspire and also give you a clearer look at the reality of drug addiction. If you're struggling with addiction, know that there is hope for you. There are many avenues to recovery. The first step is to admit that you are in need of help and then reach out for that help. There is a beautiful life on the other side of addiction waiting for you.
Dominica Applegate is dedicated to the art of self-discovery and creative expression with a passion for creative art. She's got a deep-rooted passion for helping others heal emotional pain and trauma, as her own journey through love addiction has served as a catalyst for her own healing and beautiful transformation. Find out more at www.dominicaapplegate.com.


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|10 October 2004
Big CGTalker
Stéphane Daniel
Québec, Canada
Join Date: Jan 2004
Posts: 1,488
Originally Posted by JPGargoyle: Hmmm, thank you StephanD.
That helps a little, but is not enough for me; I still can't judge if the ideas I have are on or off topic.
Thanks anyway.
Best regards.
Ask a mod in private so you don't give it away to us rest.
|10 October 2004
Warrior Spirit
João Paulo de Melo dos Santos Silva
Programmer (and 3D enthusiast)
Join Date: Nov 2002
Posts: 425
Originally Posted by StephanD: Ask a mod in private so you don't give it away to us rest.
Hey that's a great idea! Havn't thought of that. Thank you
Best regards.
|10 October 2004
Modelo, ergo sumportfolio
Tim Duyzer
Master of Karate and Friendship
Hamilton, Canada
Join Date: Feb 2002
Posts: 1,884
Originally Posted by JPGargoyle: Ok. This is interesting, but before I decide to enter, I need to know exaclywaht is expected and, since English is not my native language, sometimes I have difficulty understanding everithing I read, specially some concept, so I would like to ask if someone would be so kind that could explain in simple words what "Grand Space Opera" means please.
A good definition of space opera is provided by the Wikipedia, check it out here. Star Trek, Dune, Foundation, Known Space, they are all variants of the space opera, and of course you have Star Wars, the grand master of them all. It's basically a science fiction story that deals with huge themes and massive settings (like an entire galaxy).
Originally Posted by Gluteus Maximus: I want to get a iconic moment from our history, then transplant it into a space scene. Is this an ok thing to do?
I would guess this is fine, because it's been done before - Asimov's Foundation series, for example, is based in part on the rise and fall of the Roman Empire.
|10 October 2004
Andrea Ontaneda
Tanuki CG
Quito, Ecuador
Join Date: Oct 2004
Posts: 383
Any problems if I do it with a student license of Maya?
Andrew "theSuit"
|10 October 2004
3d artistportfolio
Linda Ferroni
3d Artist at IKEA
born in Sweden, Italian nationality, live in, Greece
Join Date: Jan 2004
Posts: 258
One question is it ok to use vray for rendering.
_:::Life is what you make it, be sure that you make it beautiful:::_
---::::My website::::---
|10 October 2004
PNG. screws explorer :Pportfolio
Kevin Letchford
Maidstone, United Kingdom
Join Date: Feb 2003
Posts: 601
i guesse any renderer or modeler is fine as long as your not using the pre created fluff. tbh if i choose to do this in wings 3d and blender i would think itd be fine. the only case were you wouldnt stand a chance of winning is if you used a watermarked aplication.
a student edition though does stand diffrentley though because you could be seen as using the student license to make money. i guesse its all down to the license you signed when you bought the software.
sketchs should defintley be 2d as it allows you and your audience to understand and create very vivid first impressions in a short period of time. your audience gets to also see whats going on inside your head in contrast; to a rendered image that has taken longer and most likely lost the first idea the artist wanted.
i came for the shoes
|10 October 2004
New Member
Thomas Iversen
Join Date: Jun 2004
Posts: 20
the contest noobie asks:
Hi. Just a few questions:
1: Is it (not that it is likely to happen) allowed to have multiple entries in the same category? (i.e. two or three different 2D artworks)
2: Any rules on post workup? I saw in the machineflesh thread, that for a 3D entry, using post effects was encouraged, but I assume this is with regard to computer generated 3D video post effects. What I'm basically asking is, is it allowed to do 2D post effects on a 3D entry (Photoshopping etc.)?
3: The other way around: Is it allowed to incoporate pre-rendered elements into a 2D entry? (Buildings, vehicles etc.) or must a 2D entry be done entirely "by hand"?
I'm not considering using stock photos/models and stuff "borrowed" from other people in any entry- the reason I ask, is just that I often use this type of "cross media". I would assume, that as long as the use of this technique is only used for subtle workup (like a bit of smoke added in photoshop on a 3D render) it would be acceptable?
Are there any specific rules, or does one just use ones own judgement?
|10 October 2004
Who's Next?
John Cochran
Unemployed Student
Mooresville, USA
background ??
Whilk I know its ok to use 2d textures in this 3d project....is it going to be ok to use a 2d element as a back drop? IE like a background....like a mate painting
Subsito Stabilis, Abeo Edurus
|10 October 2004
You want to live forever?portfolio
John Thomas Delaney
Business Analyst
Bedford, VA, USA
Join Date: Oct 2003
Posts: 1,409
I did the same thing as Voutlooz. I entered the contest (excitedly) and then realized that they had my old email address. I just got a new email address but the contest entry confirmation was sent to my old one. Can this be corrected or do I have to re-enter?
|Thread Closed share thread
« Previous Thread | Next Thread »


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Diverse styles of African Paintings by
At right are example artworks from this African Artist, with more to see now as art prints in many mediums, frames, & other media options.
See this Artist's original art for sale.
- What Daniel wishes to share with you -
Daniel Akortia had a start with True African Art in 2013. In that year, he sent some images for inclusion on our website. We initially turned him down as we get so many submissions, but we heard from him again in mid 2015 and the paintings he has sent us are some fantastic African paintings for sale.
Daniel presents his work maturely with a deep understanding of the subject he is painting. From the African woman, to music and the afterlife of the ancestors, Daniel explores and challenges not only his skills, but also the perception of the art's viewer. And like all our paintings online, these also look even better to view in person!
Daniel signs his paintings as Nshira, which means "blessing." He is a native Ghanaian born in 1979. After finishing secondary schooling in 1998, he attended Art College for three years, pursuing drawing, painting, textile design and graphic design. In his own words "I live in a world of my own, and therefore refuse to be drawn from real life. I concentrate on drawing my personal visions."
Daniel Akortia goes on to state that "My aim is to depict the history and culture of Africa as it really is, in the true colors of nature. After completing college, I have spent all my time painting. I mainly use bright colors and my paintings are often like decorative patterns of abstract expression".
Many of Daniel's paintings can be found in private homes and collections, art galleries in Ghana, at exhibits in Ghana and now internationally through True African Art. We accept price offers you make as well as 0% interest payment installments.
Click the photo above to show paint thickness.
At right are sample artworks from this African Artist, available now as art prints in several mediums, frames, & other media options.
See this Artist's original art for sale.
Shop more artworks by African artists from Ghana.


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Sealing the deal with a customer over the telephone entails far more than having a flair with words and access to reliable sales leads to capitalise upon.
To conduct a successful outbound marketing campaign you not only need good leads, a variety of traditional marketing methods to spread the word , and an ample budget, you also need the skills to help customers identify their personal needs, and use effective closing techniques that empower you to seal the deal over the phone.
Outbound marketing – What is it?
The art of spreading the word about a product or service, outbound marketing attempts to create awareness of products and services through various traditional marketing methods.
When you think of marketing in its most traditional form, you're thinking of outbound marketing, and the traditional marketing methods it employs you're surely familiar with, since they include:
- Television and radio advertisements
- Ads in print mediums, i.e. newspapers and magazines
Despite the meteoric rise of inbound marketing, where customers find businesses through a variety of paid and natural search engine marketing methods, outbound marketing is still effective, and therefore still has plenty to offer businesses looking to garner greater exposure for their products and services.
However, outbound marketing is not without its drawbacks, most notably its lack of cost-effectiveness in comparison to inbound marketing, as well as:
- Tracking returns on investment (ROI) often proves problematic
- Its increasingly high cost, yet relatively low yield nature
For reasons like these, many businesses have completely switched from outbound marketing to inbound marketing. Nevertheless, many businesses still find outbound to be worthy of inclusion in their marketing arsenals.
Closing strategies aren't always what they seem
If you're to use outbound marketing with any degree of success, you need to employ closing strategies that will help you seal the deal over the phone.
These strategies often come with names like the sympathy close, i.e. the puppy close, or the cradle to the grave close, and there are more of these strategies than most could ever hope to memorise, if indeed there's actually any point to doing so.
Here are a few to give some thought to.
- The opportunity cost close
Using this sales closing strategy, you describe to the customer the cost of not purchasing the product or service.
- Exclusivity close
This is one of the oldest sales techniques in the book. Basically, you give the customer the impression that not everyone can afford to buy the product or service.
- Valuable customer close
The salesperson offers a 'special deal' on the product or service because the customer is so valuable to the business.
If you have heard of these closing techniques before, or even used them yourself, you'd know well that whilst they are often effective, there is much more involved.
It's all very well to think you have a few tricks up your sleeve, but generally speaking, anyone who really believes that they're holding all the cards usually only has one in their hand. And that's the one with their name and picture on it – the Joker.
Closing strategies that seal the deal
The following closing strategies haven't been the recipient of catchy names like the 'standing room only close' or the '1-2-3 close', but they're strategies that work.
You can still use the infallible closing techniques of your choosing in conjunction with the following strategies, but always remember that when trying to seal the deal, the customer always comes first. Lose sight of that and you simply won't succeed.
- Get in touch with the decision maker of the business (or household)
The first step to sealing the deal should always be to identify who the decision maker is, whether that's a business or household, and focus your attentions on that person.
Where businesses are concerned, there is always the possibility that the key decision maker will have someone else confer with you in their place, but still act as though you are addressing that key individual. It's crucial to sealing the deal quickly.
- Always be genuine and never act like you don't care about their interests
They may not be able to see your face as you're talking on the phone, but customers are usually savvy enough to understand when someone isn't being genuine.
Conveying the impression that you genuinely care about the person, their interests, and not just earning your sales commission is important, so avoid putting them off by coming across as a calculated salesperson.
- Know who your competitors and what they potentially have to offer
Can anyone honestly, and convincingly, say that what they have to offer is better than what their competitors have to offer without really knowing that to be the truth?
In a nutshell, no. Being prepared for a customer's questions is crucially important to sealing the deal and you have to know the areas in which your business is stronger or more competitive than your competitors and vice versa. This requires research.
- Use deadlines and other techniques to create a sense of urgency
Creating a sense of urgency is a time-honoured sales technique, one to employ with caution so as to not rush the customer, but still give them an incentive to commit.
- Prepare counterarguments so that you can overcome objections
Once again, research is crucially important here, so preparing the sales presentation to address the customer's potential objections and overcome them is essential.
Along with the presentation itself, think of arguments the customer may give for not wanting to make a purchase, and prepare strong, but not pushy, counterarguments.
- Don't put your foot anywhere near your mouth, i.e. watch what you say
Remain professional at all times by thinking before you speak, not losing your cool, and keep your sales pitch to the point. You'll seal the deal soon enough.
Provided that you find outbound marketing cost-effective and in line with your business objectives, these closing strategies can prove instrumental in helping you seal the deal with customers over the phone.
Latest posts by Administrator (see all)
- Problems a virtual receptionist can solve - September 18, 2017
- 10 Tips for Answering the phone professionally - September 8, 2017
- Reputation damaged by unanswered calls - September 1, 2017


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I come with grevious tidings, which I know does not fit into the spirit of the season. Ruthless is meant to hit virtual shelves just five days from now, but I hate to announce that it may be a little longer before my steampunk sees the light of day.
A combination of disliking the first draft and just overall rushing has made me reconsider releasing it so soon. I'd rather present to you something I can be proud of, and unfortunately at the moment, Ruthless just isn't cutting it.
I'll keep updates posted on my blog and Facebook as the situation progresses.
My deepest apologies yet again, and merry holidays.


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This property has been removed by the agent. It may be sold or temporarily removed from the market
3 bedroom detached house for saleKelcey Road, Quorn, Loughborough
Under Offer £239,950
Property Description
Key features
- Modern Detached Family Home
- Energy Rating E
- Sought After Village Location
- Three Good Sized Bedrooms
- Shower Room
- Large Living Room
- Dining Room
- Contemporary Fitted Kitchen by Sapphire of Birstall
- Cloakroom & Utility Room
- Landscaped Rear Garden
Full description
A superbly presented detached property situated within this sought after lakeside development in the popular village of Quorn. The property benefits from gas central heating and uPVC wood grain effect double glazing, single garage, driveway and gardens. The house would ideally suit those looking to downsize or alternatively for a young and growing family. The accommodation includes an entrance hall, cloakroom, large living room, dining room, bespoke fitted kitchen by Sapphire of Birstall, utility room, first floor landing, three bedrooms and Shower Room. Outside the property has well-maintained gardens to front and rear, summer house, garage and driveway. The village centre is a short walk away alongside a lake as is St Bartholomew's village school and numerous shops, public houses, restaurants and picturesque walks. Viewing is highly recommended.
Quorn is a particularly well serviced and highly desirable village situated off the A6 between Loughborough and Leicester. The village offers an excellent range of local shopping and schooling facilities of all grades and a number of restaurants. The village is well placed for all the local amenities of Charnwood Forest, for further schooling and shopping facilities at nearby Loughborough and fast access to Leicester and the M1.
Bedroom One
4.11m to the robes x 2.95m - With two uPVC double glazed windows to the rear and a range of fitted wardrobes with hanging rails and storage.
Bedroom Two
8.8ft x 8.6ft
With uPVC double glazed window to the front.
Bedroom Three
8.6ft x 6.4ft
With uPVC double glazed window to the front.
Shower Room
6.6ft x 5.6ft
Fitted with a white three piece suite comprising a low level WC, pedestal wash hand basin and double shower cubicle with Mira Sport shower. Heated towel rail/radiator, tiling to the walls to dado height, shaver point and uPVC double glazed window.
Outside - Front
The property has a block paved driveway giving access to the garage.
Single Garage
With up and over door. Gas combination boiler.
Rear Garden
The rear garden has been landscaped to provide a block paved patio area, pergola with trailing wisteria, shaped lawn and planted borders, feature pond, further block paved patio area with greenhouse, summerhouse together with outside tap and lighting.
10.3ft x 7.3ft
Fitted with a range of contemporary base cupboards and drawers and matching eye level units by the highly reknowned Sapphire Kitchens of Birstall. Contrasting roll edge laminate work surfacing with inset acrylic sink bowl and drainer with mixer tap. Built-in Neff electric double oven, Neff electric touch screen hob and canopy extractor hood over. Integrated fridge and full size dishwasher. Decorative display shelving, plate racks, under unit lighting and uPVC double glazed windows to the side and rear.
Utility Room
8.3ft x 7.8ft
With plumbing for washing machine and tumble dryer, circular bowl stainless steel sink with mixer tap over, space for larder style fridge/freezer and door and window to the rear providing access out to the garden. Useful cloaks cupboard.
With access to loft space with drop down ladder and window to the side and cupboard.
The property is entered via a uPVC double glazed wood grain effect front door into:
Entrance Hall
With access to the cloakroom and living room.
Fitted with a white two piece suite comprising a low level flush WC and wall mounted wash hand basin. Window to the front.
Living Room
16.0ft x 15.4ft
With uPVC double glazed window overlooking the front garden and views towards the lakeside. Feature real flame effect gas fire with decorative surround, hearth and mantel. Open plan staircase rising to the first floor landing, wall mounted central heating thermostat control and glazed integral double doors give access through to the dining room.
Dining Room
12.1ft x 7.9ft
With a set of sliding patio doors to the rear block paved patio. Useful understairs storage cupboard and door leading through to:
More information from this agent
To view this media, please visit the on-line version of this page at www.rightmove.co.uk/property-for-sale/property-56619805.html?premiumA=true
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Market Info
Disclaimer - Property reference BNT160955. The information displayed about this property comprises a property advertisement. Rightmove.co.uk makes no warranty as to the accuracy or completeness of the advertisement or any linked or associated information, and Rightmove has no control over the content. This property advertisement does not constitute property particulars. The information is provided and maintained by Bentons Your Property Experts, Melton Mowbray. Please contact the selling agent or developer directly to obtain any information which may be available under the terms of The Energy Performance of Buildings (Certificates and Inspections) (England and Wales) Regulations 2007 or the Home Report if in relation to a residential property in Scotland.
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Medulloblastoma is the most common malignant brain tumor in children, and a substantial number of patients die as a result of tumor progression. Overexpression of CDK6 is present in approximately one-third of medulloblastomas and is an independent poor prognostic marker for this disease. MicroRNA (miR)-124 inhibits expression of CDK6 and prevents proliferation of glioblastoma and medulloblastoma cells in vitro. We examined the effects of miR-124 overexpression on medulloblastoma cells both in vitro and in vivo and compared cell lines that have low and high CDK6 expression. MiR-124 overexpression inhibits the proliferation of medulloblastoma cells, and this effect is mediated mostly through the action of miR-124 upon CDK6. We further show that induced expression of miR-124 potently inhibits growth of medulloblastoma xenograft tumors in rodents. Further testing of miR-124 will help define the ultimate therapeutic potential of preclinical models of medulloblastoma in conjunction with various delivery strategies for treatment.
Medulloblastoma is the most common malignant brain tumor in children.1 Despite treatment with surgery, radiation, and chemo, a substantial number of patients die as a result of tumor progression,2 and treatment-associated toxicities severely impair quality of life for most long-term survivors.1 The long-term goal of targeted therapeutics is to reduce systemic toxicity and normal brain injury while still achieving durable tumor control or cure.
Overexpression of CDK6 is present in approximately one-third of medulloblastomas and is an independent poor prognostic marker for this disease.3 Development of therapeutic agents that target inhibition of CDK6 may be effective for the subset of CDK6-positive tumors. For example, PD 0332991 is a highly specific small molecule inhibitor of CDK6 and CDK4 and has antitumor activity in human tumor xenografts.4 However, tumors acquire resistance to small-molecule inhibitors such as imatinib through several mechanisms, such as primary and secondary mutations.5
RNA interference (RNAi) is a powerful cellular process initiated by short double-stranded RNAs resulting in sequence-specific posttranscriptional gene silencing. The goal of RNAi-based therapy is to mimic naturally occurring RNAi and target specific messenger (m)RNA sequences to induce selective gene silencing. The principal advantages of RNAi over small-molecule and protein therapeutics are (i) ability to inhibit all targets, including "undruggable" ones;6 (ii) increased potency; and (iii) high specificity.
Micro (mi)RNAs are a class of small noncoding RNAs that regulate diverse cellular processes through RNAi-based mechanisms. MiRNAs are transcribed as primary RNA transcripts, processed in the nucleus to smaller precursor hairpin structures, and then exported to the cytoplasm, where they are processed further by the dicer nuclease to become mature, functional miRNAs ~21 nucleotides in length. Mature miRNAs, the endogenous equivalent of short-interfering (si)RNAs, are then incorporated into the RNA-induced silencing complex, which facilitates their interaction with, and inhibition of, target mRNAs by translational repression or message cleavage.7
One of the best characterized miRNAs in the central nervous system is miR-124. Expression of miR-124 is upregulated during development of the rodent nervous system8,9 during neuronal differentiation of embryonic stem cells10 and of mouse and human embryonal carcinoma cells.11 Furthermore, neuronal differentiation is enhanced following ectopic expression of miR-124 in mouse neuroblastoma cells,12 mouse embryonal carcinoma cells,10 and mouse embryonic stem cells.10 We have previously shown that miR-124 promotes neuronal differentiation of postnatal neural stem cells and glioma stem cells.13 MiR-124 is also an important regulator of adult neurogenesis in the subventricular-zone stem cell niche.14
Studies have shown that miR-124 is downregulated15,16 and that CDK6 is overexpressed in medulloblastoma tumors.3 MiR-124 inhibits expression of CDK6 and prevents proliferation of glioblastoma13 and medulloblastoma cells in vitro.15,16 In this report, we show that miR-124 inhibits proliferation of medulloblastoma cells in vitro and that induced expression of miR-124 potently inhibits growth of medulloblastoma xenograft tumors in rodents.
Human tumor samples and normal tissues were acquired from the Brain Tumor Research Center Tissue Bank at the University of California, San Francisco (UCSF). All procedures were in accordance with the Committee on Human Research. All samples were reviewed by a neuropathologist (S.V.), and medulloblastoma specimens were confirmed to contain at least 90% tumor tissue. Medulloblastoma cell lines were provided as a kind gift by Dr Darryl Bigner (Duke University).
Human medulloblastoma cell lines D283, D341, and D425 were maintained in Dulbecco's modified Eagle's medium (DMEM)–Ham's F-12 50:50 media supplemented with 15% fetal bovine serum (FBS) under standard conditions; 293T cells were grown in high-glucose DMEM supplemented with 10% FBS under standard conditions. MiRIDIAN miRNA hsa-miR-124 and negative control #1 (cel-miR-67) oligonucleotides (Dharmacon) were used as previously described.13 CDK6 targeting ON-TARGETplus SMARTpool CDK6 siRNA and control were purchased from Dharmacon. Oligonucleotides were transfected to a final concentration of 100 nM using HiPerFect transfection reagent (Qiagen) according to the manufacturer's instructions.
D425 tumor cells were transduced with lentiviral vectors containing firefly luciferase as previously described.17,18 Cells were screened in vitro for transduction efficiency by treatment with luciferin (d-luciferin potassium salt, 150 mg/kg; Gold Biotechnology) and analyzed using an IVIS Lumina System (Caliper Life Sciences).
MiR-124 shRNA and a nonsilencing (NS) control shRNA were subcloned from pLemiR vectors (Open Biosystems) to the pTRIPZ inducible lentiviral shRNAmir vector (Open Biosystems) using MluI and XhoI restriction sites in both vectors. To generate lentivirus, 293T cells were transfected with pTRIPZ constructs using the Trans-Lentiviral Packaging System (Open Biosystems) according to the manufacturer's instructions. D425 cells were infected with lentivirus, and transduced cells were selected with 1 µg/mL puromycin. Two independent inducible cell lines expressing miR-124 and one NS control cell line (pTRIPZ-miRNS) were obtained. pTRIPZ is a tetracycline (Tet)-On system, and miRNA and turbo red fluorescent protein (tRFP) are expressed from the same Tet-inducible promoter. MiRNA and tRFP expression was induced by addition of 1 µg/mL doxycycline. Expression of tRFP was observed within 24 h post-induction (data not shown).
Total RNA was extracted using the miR-Vana RNA isolation system (Ambion). MiRNA expression was quantitated using individual TaqMan MicroRNA Assays (Applied Biosystems). The comparative cycle threshold (ΔΔCt) method was used to determine expression fold change, using U6, U38 RNA, hsa-let-7a, or hsa-miR16 as an endogenous control as previously described.13
Cell cycle analyses were conducted using the fluorescein isothiocyanate BrdU (5-bromo-2′-deoxyuridine) Flow Kit (BD Pharmingen) following the manufacturer's recommendations as previously described.13 Immunoblotting was performed using standard protocols with antibodies CDK6 (1:1000; Cell Signaling) and Hcs70 (1:4000; Santa Cruz Biotechnology) as previously described.13
After resection, mouse brains were fixed for 48 h in 4% paraformaldehyde. Brains were then paraffin embedded and sectioned (10 mm) for staining with hematoxylin and eosin and immunohistochemical analysis using anti-Ki67 (2 µg/mL; Ventana).18 For quantification, 2 tumors (4 fields each) in each group were used.
Six-week-old female athymic mice (nu/nu genotype, BALB/c background) were purchased from Simonsen Laboratories. Animals were housed in the UCSF animal facility and were maintained in a temperature-controlled and light-controlled environment with an alternating 12-hour light/dark cycle. All protocols were approved by the UCSF Institutional Animal Care and Use Committee.
Mice were injected s.c. in the right flanks with 4 × 106 D425 medulloblastoma cells transduced with pTRIPZ–miR-124 clone B (n = 7) or the control vector pTRIPZ-miRNS (n = 8) in 0.2 mL of cell culture media with matrigel (BD Bioscience). Prior to implantation, animals' drinking water had been supplemented with doxycycline for 1 week, and we continued adding doxycycline to the drinking water after the implantation. Animal experiments were repeated (n = 5 in each group) with similar results obtained. Tumors were measured every 3 days with calipers, and tumor volume was calculated according to the following formula: (width)2 × (length)/2. All procedures were carried out under sterile conditions.
D425 cells were transfected with hsa-miR-124 or control oligonucleotide using HiPerFect transfection reagent as described above. The transiently transfected cells (n = 5 for each group) were then implanted into the brains of athymic mice as previously described.18 Briefly, mice were anesthetized with an intraperitoneal injection of a mixture containing ketamine (100 mg/kg) and xylazine (10 mg/kg) in 0.9% saline. A 1-cm sagittal incision was made along the scalp, and the skull suture lines were exposed. A small hole was created by puncture with a 25-g needle at 3 mm to the right of the midline and 6.5 mm behind the bregma. With the use of a sterile Hamilton syringe (Stoelting), 3 × 105 cells in 3 μL Hanks' Balanced Salt Solution without Ca2+ and Mg2+ were manually injected very slowly (over 1 min) into the right cerebellum at 3 mm deep from the bottom of the skull. Mice were monitored daily and euthanized at the onset of neurological symptoms or once moribund.
In vivo bioluminescence imaging was performed with the IVIS Lumina System (Caliper Life Science) coupled to LivingImage data-acquisition software. Mice were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine and imaged at 10 min after intraperitoneal injection of d-luciferin (potassium salt, 150 mg/kg; Gold Biotechnology). Signal intensity was quantified within a region of interest over the mouse head, as defined by LivingImage software. Bioluminescence measurements for each animal were normalized against corresponding readings obtained at the beginning of therapy.
Because miR-124 potently inhibits CDK6 protein expression in medulloblastoma15 and glioblastoma cell lines,13 we investigated whether decreased miR-124 expression resulted in increased CDK6 protein expression in primary medulloblastoma samples (n = 8) and medulloblastoma cell lines (n = 3). Consistent with previous observations, we observed that miR-124 was expressed 0.7–3.5-fold lower in 8/8 tumors and that CDK6 was overexpressed in 4/8 (50%) tumors relative to normal cerebellum (Fig. 1A and B). In medulloblastoma cell lines, miR-124 was expressed at least 6.5-fold lower compared with normal cerebellum, whereas CDK6 protein expression was detected in 1/3 lines (D425). CDK6 mRNA was detected in D283 medulloblastoma cells (Supplementary material, Fig. S1). However, we did not observe a consistent relationship between miR-124 and CDK6 protein expression levels in medulloblastoma tissue samples and cell lines. This suggests that CDK6 overexpression in medulloblastoma tumors is predominantly regulated by factors other than miR-124 expression levels, such as genomic amplification.3
Overexpression of miR-124 has been shown to inhibit proliferation of glioblastoma cells13 and medulloblastoma cells DAOY,15,16 D283,15 and ONS-7616 and to inhibit expression of the cell cycle promoter CDK6.15 Since our results revealed differential CDK6 protein expression in medulloblastoma cell lines (Fig. 1B), we examined whether ectopic expression of miR-124 inhibited medulloblastoma cell proliferation. Overexpression of miR-124 resulted in a more dramatic effect on proliferation in D425 cells compared with D283 and D341 cells (Fig. 2A). The effect on D283 cells was intermediate between those on D341 and D425 cells. These results suggest that CDK6-expressing cell lines are more responsive to proliferation inhibition than cells expressing low amounts of CDK6.
To further define the inhibitory role of miR-124 on medulloblastoma cell proliferation, D425 cells were pulsed with BrdU, and analysis of cell cycle distribution was performed. Relative to nonsense control oligonucleotides, transfection of miR-124 resulted in a marked reduction in the number of cells in the S phase of cell cycle and a marked increase in the number of cells in G0/G1 in D425 medulloblastoma cells (Fig. 2B). No reproducible differences were observed for cells in the G2/M phase of the cell cycle or in cells undergoing apoptosis (sub-G1) in any of the cell lines examined (Fig. 2B). These results suggest that miR-124 induces a G0/G1 cell cycle arrest in medulloblastoma cells.
Previous studies have shown that miR-124 directly targets the cell cycle regulator CDK6.15,19 To further investigate the specific effect of miR-124 on cell proliferation, we transfected D425 medulloblastoma cells with siRNA specific for CDK6 and compared cell cycle distribution with cells transfected with miR-124. Transient transfection of D425 cells with siRNAs targeting CDK6 resulted in a reduction in cells in the S phase of cell cycle and an increase in cells in G0/G1 (Fig. 2C). No reproducible differences were observed for cells in the G2/M phase of the cell cycle or in cells undergoing apoptosis. The effect of inhibiting CDK6 is slightly less potent than overexpression of miR-124 alone (30% vs 48%). When miR-124, or siRNAs targeting CDK6, were overexpressed in D283 cells—which have low expression of CDK6—we observed a potent effect by miR-124 alone (45% of cells in S phase; Fig. 2D) but only a small effect when inhibiting CDK6 (82% of cells in S phase; Fig. 2D). These results suggest that miR-124 can function independently of CDK6 and likely has other targets that affect cell growth.
In previous studies, transient miR-124 overexpression in oligodendroglioma and glioblastoma neurosphere cultures resulted in neuronal-like differentiation in addition to cell cycle arrest,13 suggesting that transient overexpression of miR-124 may result in prolonged growth inhibition in vivo. To investigate this, we transiently transfected D425 medulloblastoma cells with control or miR-124 oligonucleotides in vitro and transplanted the transfected cells into the cerebellum of athymic BALB/c mice. The D425 cells were modified with a luciferase construct, allowing monitoring of tumor formation in vivo. Transient transfection of miR-124 delayed tumor formation by 11–14 days; however, after 14 days, the mice transfected with miR-124 formed tumors at the same growth rate as the control-transfected group (Supplementary material, Fig. S2).
We next addressed whether constitutive expression of miR-124 was sufficient to induce significant growth inhibition in vivo. We created an inducible miR-124–expressing lentiviral construct, allowing us to monitor in vivo growth of D425 medulloblastoma cells expressing miR-124. Relative to D425 cells transduced with pTRIPZ negative control (pTRIPZ-miR-neg), doxycycline stimulation of 2 independent pTRIPZ–miR-124 D425 cell lines (A and B) led to a significant increase in miR-124 expression (Fig. 3A), a decrease in CDK6 expression (Fig. 3B), and a concomitant decrease in cell numbers (Fig. 3C).
We then subcutaneously implanted D425 cells transduced with pTRIPZ–miR-124 (clone B) or pTRIPZ-miRNS (nonsilencing control) into athymic mice. Their drinking water had been supplemented with doxycycline 1 week prior to implantation and we continued adding doxycycline after implantation. Tumor growth was inhibited in mice implanted with medulloblastoma cells expressing miR-124 compared with pTRIPZ control medulloblastoma cells, as evidenced by tumor size up to 37 days post-implantation (Fig. 4). Both the pTRIPZ–miR-124 and NS control–transduced D425 xenografts displayed a prominent large cell cytoarchitecture with conspicuous nucleoli and characteristic arrangement in large sheets. Histologic evidence of apoptosis (apoptotic bodies) was present to the same extent in both groups (data not shown). The effect on proliferation was examined by measuring Ki67-positive cells in the flank tumors. The percentage of Ki67-positive cells was reduced from 29% to 4% in the tumors from animals given doxycycline.
Overexpression of CDK6 promotes cell cycle progression,20 has been observed in approximately one-third of medulloblastomas, and is an independent poor prognostic marker for this disease.3 It is reasonable to assume that therapeutic inhibition of CDK6, at least in a subset of patients, may be effective in achieving better tumor control. Similar to what we observed in mouse postnatal neural stem cells and in glioblastoma cells and stem cells,13 miR-124 inhibits proliferation of medulloblastoma cells, leading to a G1 cell cycle arrest. Interestingly, we observe that the inhibitory effect on cell cycle progression mediated by miR-124 seems to be more pronounced in medulloblastoma cells with higher expression of CDK6. Our results suggest that cell proliferation inhibited by miR-124 in CDK6-expressing medulloblastoma can for a large part be replicated by directly targeting CDK6. Although the majority of our results suggests that miR-124 overexpression requires CDK6, the effect on the S-phase population in D283 cells suggests that miR-124 can also function through alternative downstream targets. Despite this effect on cell cycle distribution, we did observe that overall effects on cell proliferation were more pronounced in cells that expressed higher levels of CDK6. The effect on cell proliferation was confirmed in our in vivo results, where we observed a marked reduction in Ki67-positive cells in subcutaneous tumors.
MiRNAs with therapeutic potential have been identified in several other tumors, including pancreatic,21 prostate,22 and lung cancers.23 Indeed, RNAi-based agents represent an entirely new class of antitumor treatment.24 One limitation to the delivery of siRNAs or miRNAs to the central nervous system is the presence of the blood–brain barrier. It is unclear whether gene expression can be adequately reduced for a meaningful period of time using conventional systemic delivery strategies. There are, however, a number of promising strategies that may circumvent this problem. These include intranasal delivery of oligonucleotides,25 lipid encapsulation and targeted delivery of nucleic acids,26,27 and direct administration of therapeutic agents to brain tumor tissues by convection-enhanced delivery.28,29 Recently, direct CNS delivery of siRNA using convection-enhanced delivery demonstrated robust silencing of an endogenous oligodendrocyte-specific gene in nonhuman primates.30
In addition to inhibiting CDK6 expression, miR-124 has been shown to directly inhibit expression of multiple genes that abrogate neurogenesis. MiR-124 thus appears to be critical for the homeostasis of differentiation versus proliferation.31 For example, miR-124 repression of the sex-determining region Y–box (SRY) transcription factor Sox9 is important for progression of stem cells in the subventricular zone into neurons.14 We recently speculated that loss of miR-124 expression in neural stem cells may result in a pool of premalignant, differentiation-impeded, hyperproliferative stem cells that may progress to brain tumors upon acquisition of additional oncogenic mutations.32 It is possible that expression of miR-124 may be more effective than pure inhibition of CDK6 because of this additional effect on stem cell populations.
Further testing of miR-124 in preclinical models of medulloblastoma in conjunction with various delivery strategies will help define its ultimate therapeutic potential for treatment of medulloblastoma. In particular, combined miR-124 therapy in conjunction with other therapeutics may enhance the therapeutic outcome.
Supplementary material is available at Neuro-Oncology Journal online (http://neuro-oncology.oxfordjournals.org/).
Conflict of interest statement. None declared.
This work was supported by the Pediatric Brain Tumor Foundation Institute and the Department of Neurological Surgery at the University of California, San Francisco.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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