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Micromax released KitKat 4.4.2 New update for Micromax A350 Canvas Knight but did not provide any app for rooting this update, So here is an easy and safe tutorial of rooting and install of CWM/TWRP recovery processes of Canvas Knight. This is the first phone from Micromax with a 16 megapixel camera, 5 megapixel secondary cameras and Octa-core chipset clocked at 2GHz. This is a MediaTek MT6592 processor chipset. Canvas Knight A350 comes with 2 GB RAM, 32 GB internal memory and 5-inch full HD and 2350 mAh Battery. Micromax A350 is great phone and must be rooted because rooting allows you to maximise the potential of your phone without any restrictions. After rooting you can install custom apps, custom Rom and custom setting in your phone. But before rooting you must read Root Advantage and Disadvantage by this post, and then follow these steps to root your phone.
Preparation of flash/update:
1. Take a backup of your phone Click here.
2. Your phone battery should be charged to 80% minimum.
3. Read all steps then do it carefully.
4. Make sure there is a proper power backup for your PC.
1. MT6592 Scatter file Click here to download
2. Driver and SP Tool V5.1352.01 for Canvas Knight KitKat update Click here to download
3. CWM Recovery Click here to download or TWRP recovery Click here to downloadand Update-SuperSU-2.1.zip file Click here to download
How to Root and install CWM recovery on Micromax A350 Knight:
Step 1: Install drivers in your PC (If you feel any problem in installing driver then watch this video Click here) and Enable USB Debugging on your Phone. Go to Settings >>> Developer options >>> USB debugging check it ( If your phone have Android 4.2 or upper version then see how to enable Developer options here is guide of this Click here). After then switch off your phone.
Step 3: You will see an SP tool window, now click on the scatter-Loading option in the SP tool. Then you will get the new tab of SP Tool which you ask the location of the scatter file on your PC. Now select the "Android_scatter_emmc.txt" file. See in this image. Step 4: Now connect your switch off phone with PC via USB cable (Make sure your phone is switched off).
Step 5: SP Tool detect your phone and process will start. If Sptool is not recognize/detect your phone then install vcom driver read this post it will help you click here (Driver already in phone flash drive if you don't have these drivers then install it) Step 6: Now start installing recovery in your phone by flashing. Note: - Don't interrupt this process as it can potentially brick your device. Make sure no power interruption takes place.
Step 7: After the process complete, a green ring will appear.
Step 8: Disconnect your phone.
Step 9: Do not start your phone. Press and hold vol. up & down + power button then select "recovery mode" (If your touch not work then use volume up and down key for menu up and down and Power button as a enter button)
Step 10. Click on install button/ choose install and select SuperSU-v2.01.zip file from your save location on your SD card and install this file. Now reboot your phone. If you feel any problem then watch this video click here.
Enjoy Rooted phone.
Note: I am not responsible if you make Mistakes and Bricked Devices.


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Squash Blossom Classic High Desert Screamer Mountain Bike Race
8:30 a.m.
The Screamer XC Mountain Bike Race is USAC- sanctioned. If you do not hold an annual USAC License, add $10 for a 1-day USAC Licence!
The PRO/CAT 1/COLLEGIATE A/SS LONG- 26 miles
CAT 2/COLLEGIATE B/SS SHORT- 20 miles
CAT 3/COLLEGIATE C- 9 miles
JUNIORS- 18 and Under
Registration Fees for Screamer XC Mountain Bike Race:
$40 Online before September 13
$45 race day
Discounts are available for BOTH TT & XC events

subset: commoncrawl


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Holmes Elementary Gets Artistic Make Over by Miami Helping Hands
Thanks to the generosity of volunteers and HandsOnMiami.org for taking the initiative, Holmes Elementary has a rainbow colored corridor to welcome students this new school year. Roy G. Biv would be so proud. (art students will understand that joke)
What are yo waiting for? Volunteer at Hands On Miami


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NWACUHO, like other organizations, can only exist and flourish if the membership is engaged. This is an organization of members for members. As the name suggests, the Membership Engagement Committee provides opportunity for members to give back to the association and provide input to the Board on ways to improve our members' experience. For more information visit us at the NWACUHO table in the exhibit hall and at http://nwacuho.org/committees/membership-engagement/
Co-Chair: Scott Etherton
Scott is the Director of Housing and Conferences at Willamette University. He previously worked at Oregon State University, Pacific Lutheran University, and Linfield College. In addition to several roles in Housing and Residential Life, he has also worked in HR and Equity and Inclusion offices. Scott became involved in the Membership Engagement Committee to help build up NWACUHO as he believes that the association has much to offer our profession, our institutions, and because of the support NWACUHO has given him throughout his career.
Board Member: Christine Zapisocki
Christine is the Manager of Housing and Residence Life at the University of the Fraser Valley. While Christine has worked in post-secondary education for over sixteen years, she is a new professional when it comes to student housing. In her previous roles Christine worked in HR, employee training and development, and faculty. Christine became involved in the NWACUHO Board as part of her professional development in the field, to build relationships with colleagues from other institutes and to contribute to the Association.


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Spring Wreath, welcome wreath, everyday wreath, every day wreath, floral wreath, grapevine wreath, gold wreath, wedding gift, housewarming Gift
20" wide x 22" tall
Need to glam up your entryway after a long drab winter? Looking for the perfect housewarming or wedding gift? This gorgeous gold, floral wreath will do the trick! It's sassy and sophisticated with pops of pinks, corals, vibrant greens and some black polka dots & plaids for a touch of whimsy! And who could take their eyes off that ha d painted sign!! It seriously has me swooning!
Grab it quick~ it's a one of a kind!
3-5 business days
Buyers are responsible for any customs and import taxes that may apply. I'm not responsible for delays due to customs.
But please contact me if you have any problems with your order.
All sale items are final sale, sold as is, and are not eligible for return, refund or exchange.


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Free Click & Collect
Free returns
Free UK Delivery over £45
3 year guarantee
Product number: 73485
All products include our 3 year guarantee so you can shop with confidence
Perfect protection for the round lids of traditional range cookers, the Mary Berry with Lakeland Star and Stripe Hob Cover Pad is a double-sided cotton pad that can multi-task too…
Topped with our exclusive UK-designed and made Star and Stripe print, it has a pale grey flannelette backing so it's gentle on the shiny surface of your stovetop lids and because it's nice and thick, it can double as a Chef's pad or pot holder. Its towelling side is absorbent enough to mop up small stovetop spills, and it's also great as an emergency table mat or a trivet for a large casserole dish.
100% cotton, machine washable at 40°.
Designed and made in the UK
The fabric print on the range is designed by UK designer Will Crisp, and made for us in Norwich by the British Textile Company.
Mary Berry with Lakeland
Specially developed with Mary Berry, 'the Queen of Cakes' our range of baking tins, enamelware and kitchen textiles has been created to the highest standards so you can be sure every piece will serve you well and it's great to know that every piece meets Mary's exacting requirements. Lovely to look at as well as to use, they make great gifts for cooks and bakers at every level of confidence, and if you're treating yourself to new things for your kitchen, why not give yourself something that Mary would approve of?
Approx. 37cm Dia.
* Some delivery services may not be available in your area, the delivery page will display the available delivery services during checkout.
We want you to enjoy shopping at Lakeland with complete confidence, which is why every single product you buy from us comes with a 3 year guarantee – just bring along your receipt or emailed order confirmation as proof of purchase. Plus all returns are free.
To do this, it would be really helpful if you could call one of our friendly Customer Services Advisors on 015394 88100 first, as there are occasions when it may not be necessary for you to be inconvenienced by sending a product back to us.
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Price: $20.00
Quantity: 1 available
Tall 8vo. [iv], 367 pp. Figs. Green blind and gilt-stamped cloth. Very good. [HL] Contents includes: G.H.A. Cole, The Kinetic Theory of Monatomic Liquids at Ordinary Temperatures; The Proton Synchrotron, by J.P. Blewett; Radioactive Methods for Determining Geological Age, by L.H. Ahrens; Infra-red Absorption in Semiconductors, by H.Y. Fan; Aero-thermoacoustics (The Generation of Sound by Turbulence and by Heat Processes), by O.K. Mawardi; Some Aspects of Diffraction Theory and their Application to the Ionosphere, by J.A. Ratcliffe; Some Topics in Neutron Diffusion Theory, by J.H. Tait; New Lens Systems, by C.G. Wynne; The Macroscopic Theory of Irreversibility, by R.O. Davies. [HL]
Title: The Physical Society. Reports on Progress in Physics. Volume XIX (1956).
Categories: Physics,
Publisher: London:, The Physical Society, 1956.: 1956
lbs: 3.00 lbs
Seller ID: S11863
Keywords: Physics


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I'm usually not a very political person because as a social studies teacher, I wanted students to form their own opinions and not parrot mine. As an educator for the past 31 years, I focused my efforts on what I felt was good for children and our schools. As I watch the Nebraska Legislature debate legislation that would allow charter schools, I felt it was important to speak on this topic.
I'm reminded of Diane Ravitch's remarks at a recent conference. Dr. Ravitch is an education historian who has worked in both the George H. W. Bush and Bill Clinton administrations. Dr. Ravitch previously advocated for the privatization of public education through various choice programs, including charter schools; she now admits she was wrong. The very ideas Dr. Ravitch once promoted in her role as assistant secretary of education, she denounces today as so flawed that they could ultimately lead to the demise of public education as we know it.
Her ideas resonate with me because it is my belief that our country's successful democratic culture is built on the very concept of educating every student and developing an informed citizenry. These ideas are now in danger, due to the wishes of some to privatize public education.
I admit that public education created some of its own challenges by not providing focused professional development and by not implementing strong national standards to help all students succeed. No Child Left Behind and Race to the Top legislation, however well-intentioned, set up schools for failure, especially those with the most disadvantaged students. We should not create an educational culture of rewards/punishments based on a test. Instead, we must create an educational culture based on rigorous liberal arts programs that develop the critical thinking skills of all, not some students. Only then can we provide a strong academic foundation for our future innovators and entrepreneurs. Our conversations must focus on the whole child, including academics, but also on developing ethical students with strong character traits.
In my 31 years as a teacher, the education field has experienced continual reform. I was in graduate school when A Nation at Risk appeared in 1983. At that point, our profession began a generation of reform to fix our public education system, which has led us to our current situation. As Dr. Ravitch observed, A Nation at Risk made sound recommendations that were appropriate at the time. Those recommendations were focused on the teaching and learning of students and staff. The report did not promote privatization or heavy-handed accountability structures that exist today.
In my opinion, A Nation at Risk did not go far enough to redesign our educational system. However, it offered solid recommendations that focused on stronger academic and behavioral expectations, increased graduation requirements, more time for students to master the curriculum, and better training for teachers. Given some of the same freedoms afforded charter or choice schools, I believe that our public schools can design institutions that work for all students. We have great employees and supportive communities throughout our nation ready to design schools that develop persistent, independent learners. Why not encourage and allow our public schools to innovate and try new practices without fear of penalty?
With that said, I am not aware of any specific reform effort that will solve our challenges easily. I only know that through collaboration, communication, creative problem solving, and really hard work, we can begin to implement our vision for children who attend our schools. We need to think deeply about the future of schooling in America if we are to meet the needs of every learner.
As your Superintendent, I recognize that our success is directly due to the unwavering support of our Board of Education, our community, and the many outstanding employees, including support staff, who serve our students every day. Thank you for your continued commitment to the Westside Community Schools.


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Miss Earth 2013, Amabel Esinam Klutse has decided to institute legal action against First Royal, organiser of the pageant, for refusing to give her the prize package a year after she won the contest, NEWS-ONE has gathered.
Amabel has also described as "untrue" an explanation from First Royal that she had an agreement with them to use her unpaid prize money to buy a ticket for her to represent Ghana at the international event of the pageant. According to her, she never had any such agreement with them.
Last week Amabel made a public complaint that she was yet to receive the prize package promised her after winning the pageant.
However, Evelyn Akoto, an official at First Royal told NEWS-ONE that they had contracts with contestants from the beginning of the pageant that prizes would be given based on sponsorship deals.
But Amabel issued a statement on Thursday describing the explanation from First Royale as a lie.
The statement read:
"That is not true! We never had any such agreement. Their letters which they were to give me to raise sponsorship came later than planned. So I wasn't able to raise the sponsorship. What Miss Evelyn Akoto told me after I told her I wasn't able to raise the sponsorship was that the ministries and some other sponsors were taking care of my tickets. The only thing was that I had exams in school around the time they had to go for the money from these sponsors for my tickets. So I didn't go with them for that sponsorship. If she and I agreed that she used my cash prize, I only request she shows documents to prove that contract."
Amabel, in her statement, refuted Evelyn Akoto's claim that First Royal provided her wardrobe at the international event.
"It is another lie. I bought all the clothing, shoes and accessories I took to that event with my own money with the exception of environmental costume and national costume which they rented for not more than GH¢500. I know the amount because I was there when the cost of renting the costume was said to the organisers."
About the car prize squabble, Amabel said, "The night I was crowned, I was handed a key to a brand new car that was parked outside as a prize for the winner. I was also given a cheque of GH¢5000 cash as part of the winner's prize. Both the key to the car and the cheque were taken from me that night. The key was taken from me with the excuse of the organizers having to register it before handing it over to me. The GH¢5000 cheque was also taken by the organisers for "safe keeping" till the pressure of the night was down.
"I was also entitled to GH¢5000 worth sponsored goods per the contract I signed with them. It's been almost a year now and I haven't got any of these prizes I am entitled to after emerging winner of the contest which I consider an act of fraud by the miss earth organisers.
"Whenever I asked of my prizes they told me to give them just a little time to get some things sorted so I get my prizes. Week after week they got a new excuse for not giving me the prizes, assuring me I would get it sooner. They eventually came up to give me a used car which according to the organisers themselves was originally supposed to be a project car for all four winners to use. However, since the new car they brought to the finale wasn't ready they asked me to use the project car for a while whilst I waited for the new car.
"Thank goodness I made them draft another contract for the agreement to use the project car only for a while before I got the new car. Apparently the used project car they gave me had some mechanical faults which started surfacing again during the period I had to use it.
As part of the new contract I made them draft for the project car, they were responsible for maintenance. Despite their responsibility for maintenance, I used my money for maintenance several times before I finally gave it to them for massive maintenance.
"All this while, Miss Evelyn Akoto asked me use my money to fix the car to be reimbursed later, which she never did. So I took the car to them for maintenance after about two months of use. Then they said the problem with the car was quite serious, so they'd rather get me a new one instead of using a lot of money to repair the old one. Till now, I haven't got any car from them."


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Calling all single ladies! Blo has partnered with Match.com Stir events to bring you a girls' night out. Join us in Los Angeles and New York for an evening of complimentary dry styles, dating advice, pink drinks and girly tunes.
Tuesday February 25, 2014
Blo W Hollywood
6250 Hollywood Boulevard, Suite 500
Hollywood, California 90028
RSVP to: http://bit.ly/1fFcvqB
Wednesday February 26, 2014
Blo Glen Cove Road
50 Glen Cove Road
Greenvale, New York 11548
RSVP to: http://bit.ly/1cb740f


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Adriafil Soffio Plus Free downloadable Knitting Pattern for Sweater & Socks
Arans were originally outside garments to be worn in all weathers especially by men. Aran weight yarn sits roughly between Dk and chunky. It is a nice weight for showing off fancy stitches and cabling but a wee bit thicker than dk to add extra warmth againstthe elements but it also knits up a bitty faster than your dk too!


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WASHINGTON The National Republican Senatorial Committee launched a raunchy Internet attack ad against Lt. Gov. Lee Fisher, the Democratic nominee for U.S. Senate, showing him shirtless and implying that he was masturbating.
The NRSC said it stands by the ad, denying any sexual connotation.
But the video quickly went viral, even eliciting a tweet from film critic Roger Ebert: "Vile! GOP ad manipulates still photo to make Dem candidate appear to be masturbating."
A bipartisan sampling of students and faculty at the University of Akron concurred that the video exceeded the bounds of good taste.
Political scientist John Green said he showed the web ad to students and staff and there was a rare consensus among Democrats, Republicans and independents alike.
"They all said, 'Wow, that was uncalled for,' " Green said.
The video uses a bare-chested photo of the 58-year-old Democratic nominee for the U.S. Senate set to provocative music and filled with sexual double-entendres, with Fisher's image sometimes undulating across the screen.
At the end of the Republican ad, the final image of Fisher, with one hand on his bare stomach and the other below his waist, dissolves to these words: "Dare to See More?" That is followed by an invitation to go to a GOP-sponsored web site that attacks Fisher.
Green said he has viewed thousands of political spots during his long tenure as a political scientist but doesn't recall seeing anything quite like this one.
"It just struck me as very odd," Green said. "Right when I think I can't see anything new, I see something new. My immediate reaction was that if this is not over the line, it is pretty close to the line from the point of view of Ohio politics. The implication is that there is something not appropriate about the lieutenant governor."
The ad purports to be about Fisher's record as Ohio lieutenant governor in helping create jobs in Ohio. It intersperses the shirtless Fisher image with headlines about Ohio job loss and 11 percent unemployment rate and clips of Fisher discussing his economic development role.
By this afternoon, the ad had been taken down from YouTube due to a copyright violation claim by the liberal Buckeye State Blog, which took issue not with the shirtless image, but a portion of the ad that used video footage of an exclusive 2009 interview by the blog with Fisher. It was reposted late in the afternoon on the web site of the GOP Senate caucus.
Amber Marchand, a NRSC spokeswoman, acknowledged the use of the image of a bare-chested Fisher was meant to create a buzz. She said it has the full approval of the NRSC, the party committee charged with helping elect Republicans to the Senate, and its chairman, Sen. John Cornyn of Texas.
"The web video is about jobs and about Lee Fisher's record as jobs czar and 400,000 jobs lost under his watch," Marchand said. "Obviously, there are a lot of different ways that folks (use to) draw attention to web videos."
In responding to the ad, the Fisher campaign incorporated its own message, blaming Ohio's job loss on the policies of former President George W. Bush, noting that Fisher's GOP rival for Senate, Rob Portman, was Bush's budget director and trade representative.
"We invite the hundreds of thousands of Ohioans who have lost their shirts their jobs, their homes, their hopes in the Bush-Portman recession to join our campaign," said John Collins, Fisher's campaign spokesman.
The image of the shirtless Fisher came from a documentary about the 2006 race for governor by Fisher's son, Jason, and his friend, John Intrater. They sneaked into Fisher's study late one night, where a tired and shirtless candidate was hunched over his computer. Fisher wanted them to leave, and later tried to have the shirtless scene cut, embarrassed by it. But the filmmakers refused, and won out.
The web spot's criticism of Fisher's economic development role is a legitimate subject for debate and political fodder, said John Elliott, a political science professor at Kenyon College who studies the role of the news media in American politics.
But the bare-chested image may actually serve to distract viewers from digesting the ad's assertions, Elliott said. In the relatively new world of web political videos, he said, the ad's creators may have thought something they wouldn't put on TV was acceptable on the Internet.
"They think they will get more hits because they are advertising this, and they think the message from the other part will come through to the people who are watching. (But) the shirtless part detracts from what would otherwise be an effective ad," Elliott said. "I assume they will run the ad on TV, without the shirtless Lee Fisher. They think the rules are somehow different for the web, or that there aren't any rules."
Asked what Portman thought about the ad, a Portman spokeswoman criticized Fisher for running attack ads, also.
"Lt. Gov. Lee Fisher started this campaign attacking Rob Portman and the Democratic committee has been running web videos against him for almost a year," said Jessica Towhey.
In the no-love-lost Democratic primary pitting Fisher against Ohio Secretary of State Jennifer Brunner, which Fisher won Tuesday, Brunner refused to use that same image.
Jerry Austin, a Democratic consultant in Cleveland, said Brunner rejected a request from one of her political consultants that they use the footage in a commercial.
"It's an eye-catcher, but most people will get turned off by it,'' Austin said. "I don't think it's a very well-done spot.''
If that ad was submitted for TV, it would require "some discussion" as to whether executives would put it on the air, said Chuck DeVendra, sales director for WBNS-TV Channel 10. WBNS is owned by The Dispatch's parent company. But he echoed the sentiment that the ad likely is not intended to air on broadcast television.
"Many times you see commercials on the web that never make the airwaves," DeVendra said. "My guess is that this might be one of those times."
Dispatch Washington Bureau reporter Jack Torry and Senior Editor Joe Hallett contributed to this story.


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Thomas started his career at MTV Networks Europe and MTV China. With over twenty years of international broadcast experience, Thomas co-founded id creations and continues to run the company.
With over twenty years of local broadcast experience, Tony co-founded id creations and continues to run our TVC and online content production department for key agency partners.visit projects
With a background as a broadcast DOP in Australia, Justyn joined id creations 12 years ago. He now runs our key long-term accounts and client services for Porsche, IHG Group, Omega, NBA and Ferrari.visit projects
David joined id creations in 2008, with a decade of broadcast experience previously in the UK including MTV Europe Networks. David manages key accounts and client services including Disney, Mercedes-Benz, Airbnb, Louis Vuitton and numerous agencies.visit projects
Leon has been part of id creations since 2002 and currently runs our production management team and camera team, ensuring all our projects run smoothly.visit projects
Adam joined id creations in 2011 and is currently our dedicated producer for Disney, NBA, and JP Morgan.
Andre joined in 2014 and is part of our creative team, directing content for numerous clients of ours including Mercedes-Benz, Baxter, Schaeffler, and Disney.visit projects
With over nine years of broadcast experience in Germany, Fabian joined us in 2015 and is our dedicated producer for Porsche, delivering a multitude of content from the entire race season of the Porsche Carrera Cup Asia, to online and social media marketing pieces for events and campaigns. Fabian now leads our Auto Department.visit projects
Andy joined us in 2014 and is part of our dedicated Porsche department, covering major launch events and other prestigious milestone moments for Porsche's online broadcast content production needs.visit projects
Arthur joined us in 2017 and is part of our Creative team. With a natural gift for concept development and creative execution, Arthur has been directing engaging content for Huawei, Airbnb, Hilton and other key clients of ours.visit projects
After joining us in 2015, Jasmine has become our dedicated producer for Airbnb and IHG, as well as other clients. Her background in journalism has trained her to identify and develop storytelling scripts as well as being able to execute projects as a talented producer.visit projects
Sarah joined us in 2016 with a wealth of China TVC production experience and has been running projects for agencies and clients for TVC and online content.visit projects
Trista has been producing for us since 2011 and is our dedicated producer for Mercedes-Benz and is constantly delivering dynamic content for the PR department for their events, online and social media campaigns as well as covering their events.visit projects
Zhou Qi joined id creations in 2006 with over seven years of China broadcast experience and has been the head of our camera team since 2014. With over a decade at idc shooting TVCs, and online films, events, news and broadcast content for automotive, fashion, sports and hospitality, he is a highly skilled and creative member of id creations.visit projects
Joining id creations in 2003, Zhang Rong has since become the leader of our team of six editors to consistently deliver our high standards of creative storytelling, tailored to each and every client.
Ruby joined id creations with over 2 years of TVC production experience and is working on projects with our agency partners.visit projects
With over 20 years of agency experience in China, Smiley manages all of our projects with our key agency partners and their many clients.
Ting joined us with over 10 years of feature film production background experience and has quickly adapted her skills to shorter format content production.visit projects
Shelley provides vital support on projects for our long-term clients including Airbnb, IHG and Mercedes-Benz.
Charlene supports productions for our luxury clients with great enthusiasm.
Mike is enjoying his role supporting our dedicated Porsche team on numerous projects.
Fiz provides essential support on productions for numerous clients and brings positive energy to every project.


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Logo Design for Aaltech Printing
- Status: Closed
- Prize: $190
- Entries Received: 4
- Winner: sanket123456
Contest Brief
We run a plan printing company in Canberra, Australia. We have created our website and hope to have it running by next week, but we want to update our logo. We need something fresh. Our business name is "Aaltech" and that is the name we want as our logo. We specialise in large format plan printing, but also do photocopying, scanning, archiving, aperture cards, laminating and binding.
We would really appreciate any designs. Attached is the old logo. The new design can be similar, or it can be completely different. We are looking for creativity and flair. Something that we can be proud of.
We will also put this logo on our courier car and on our business cards, so there will be a lot of exposure and it will be something good to add to any design portfolio.
Please send in designs ASAP. We hope to launch the website sometime next week and would like the new logo to feature.
N.B. If you wish to know more about the company you can find us on Facebook under the name "Aaltech Printing Bureau".
We would also like a PDF or jpeg version of the design on completion.
Thank you very much
Recommended Skills
Top entries from this contest
alexandracol Romania
jai07 Philippines
sourav221v India
saif99 Bangladesh
Public Clarification Board
How to get started with contests
Post Your Contest Quick and easy
Get Tons of Entries From around the world
Award the best entry Download the files - Easy!


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As part of the ongoing Faith Renewal series at Knock Shrine, renowned singer, musician, and spiritual director Deirdre Ní Chinnéide will facilitate the Living in Hope day retreat in the shrine this Saturday from 10am to 3pm.
"The retreat acknowledges that at some time in our lives we experience darkness and even despair," Ms Ní Chinnéide said. "Living in Hope will offer some quiet time-out to experience God's presence and allow the light of his presence to shine a light in the darkness and lead us to new hope. It is a perfect time of year to have this type of event and I am looking forward to returning to Knock for the day."
Deirdre Ní Chinnéide is a trained psychotherapist, spiritual director, and workshop facilitator and has worked all over Ireland, the USA, and Europe, including Bosnia and Kosovo, offering retreats, workshops, and performances of spiritual music. She is a member of the prayer guidance team at Knock which offers prayer sessions and spiritual direction for visiting pilgrims and the local community.
Faith Renewal at Knock also looks at practical things that affect us in our everyday lives. With this in mind, a digital detox day retreat will take place on Saturday December 3 from 10am to 4pm. This will focus on encouraging people to take time away from technology which now pervades every aspect of our lives. This day is expected to attract a huge amount of interest.
Both retreats will take place in the Prayer Guidance Centre, Knock Shrine. Fee is €30 which includes lunch at Knock House Hotel.
Those interested in attending can book by contacting the Faith Renewal team at Knock Shrine on (094 ) 93 75355 or email [email protected]. Further information is also available at www.knockshrine.ie/faithrenewal


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Current immunization strategies using peptide or protein antigens generally neglect to elicit cytotoxic-T-lymphocyte responses since these antigens are unable to access intracellular compartments where loading of major histocompatibility complex class I (MHC-I) molecules occurs. by Tyrphostin AG 879 a heterologous sequence or the introduction of particular amino acidity substitutions within this section either abolished or markedly decreased the effectiveness of course I epitope delivery. If the epitope was prolonged at its C terminus EtxB-mediated delivery in to the course I demonstration pathway was discovered to be totally reliant on proteasome activity. Therefore by merging the GM1-focusing on function of EtxB using the 10-amino-acid Pol section highly effective delivery of exogenous epitopes in to the endogenous pathway of course I antigen control and presentation may be accomplished. Cytotoxic Compact disc8+ T lymphocytes (CTLs) represent a significant element of the protecting and therapeutic immune system response to intracellular bacterias infections and tumors via their capability to recognize international peptides which have destined to main histocompatibility complex course I (MHC-I) substances (17 32 A lot of the peptides shown derive from endogenously synthesized proteins that are cleaved into little peptide fragments from the proteasome (evaluated in sources 25 and 33). They are consequently transferred via the transporter of antigenic peptides in to the lumen from the endoplasmic reticulum (ER) where they bind to recently synthesized MHC-I substances. Such MHC-I peptide complexes are trafficked towards the cell surface area whereupon they may be identified by T-cell receptors present on CTL precursors. This qualified prospects to CTL activation and following Tyrphostin AG 879 CTL-mediated lysis of peptide-presenting cells. Provided the need for CTLs in clearing the sponsor of contaminated cells there is fantastic fascination with the introduction of fresh vaccination strategies that can handle inducing effective CTL reactions. But also for vaccines made up of soluble proteins antigens immunization generally leads to antigen uptake into an exogenous digesting pathway leading to Tyrphostin AG 879 peptide fragments becoming packed onto MHC-II instead of MHC-I substances (11 23 Therefore for soluble antigens to induce MHC-I-restricted CTL reactions antigens have to gain access to intracellular compartments where they are able to enter the endogenous course I digesting and demonstration pathway. Bacterial proteins toxins are substances that combine exclusive cell-binding properties with effective cytosolic delivery properties (5). They might therefore Tyrphostin AG 879 look like ideally fitted to the delivery of antigenic protein and peptides in the course I demonstration pathway so long as detoxification without obvious lack of delivery ability may be accomplished. Certainly toxoid derivatives from the adenylate cyclase toxin of (30) pertussis toxin (6) anthrax toxin (2 10 and Shiga toxin B subunit (13) have already been looked into as potential automobiles for delivery of peptides or proteins in to Rabbit Polyclonal to RFX2. the course I demonstration pathway. The capacities from the non-toxic GM1-binding B subunits of heat-labile toxin (EtxB) or cholera toxin (CtxB) to provide antigens in to the course I pathway possess so far not really been investigated. Nevertheless researchers have lately shown that EtxB is usually Tyrphostin AG 879 capable of delivering peptides into specific intracellular compartments (18). In particular when a 27-mer peptide derived from the C terminus of the DNA polymerase (Pol) of herpes simplex virus type 1 (HSV-1) was genetically fused to the C terminus of EtxB it was found that the fusion protein entered cells and that the peptide was liberated from EtxB and translocated into the nuclear compartment. While structural features present in the Pol peptide were speculated to be involved in facilitating both its liberation from EtxB and its translocation from endosomal compartments their contribution to peptide delivery remained undefined. Here we have investigated (i) whether EtxB can be used as a vehicle for the delivery of exogenous peptides into the class I presentation pathway (ii) whether EtxB-mediated peptide delivery is dependent on its capacity to bind to GM1 receptors and (iii) whether incorporation of elements of the Pol peptide adjacent to the class I epitope would improve the efficiency of peptide delivery. MATERIALS AND METHODS Production and characterization of EtxB and EtxB conjugates. Recombinant EtxB was expressed in a nontoxigenic marine vibrio sp. strain 60 and purified using hydrophobic conversation and ion-exchange chromatography as reported earlier (1). A non-GM1-binding mutant of EtxB EtxB(G33D) has been described previously (21) and was expressed and purified as described.
Month: February 2017
The molecular heterogeneity of individual tumors challenges the development of effective preventive and therapeutic strategies. activity against multiple components of the Akt-nuclear factor-kappa B (NF-κB) and stress response signaling pathways. Notable among these were the increased estrogen receptor (ER)-β/ERα expression ratio reduced expression of HER2 and CXCR4 and the upregulation of aryl hydrocarbon receptor expression and its downstream target NF-E2 p45-regulated factor (Nrf2). Non-malignant MCF-10A cells were resistant to OSU-A9's antiproliferative effects. Daily oral administration of OSU-A9 at 25 and 50 mg/kg for 49 days significantly inhibited MCF-7 tumor growth by 59 and 70% respectively without overt indicators of toxicity or evidence of induced hepatic biotransformation enzymes. In summary OSU-A9 is usually a potent orally bioavailable inhibitor of the Akt-NF-κB signaling network targeting multiple aspects of breast tumor pathogenesis and progression. Thus its translational potential for the treatment or prevention of breast cancer warrants further investigation. Introduction During the course of carcinogenesis and tumor progression cancer cells overcome genomic and cellular abnormalities by constitutively upregulating a diversity of signaling pathways governing cell proliferation survival hormonal homeostasis invasion metastasis and drug resistance. In light of this heterogeneity it is desired to concomitantly target multiple clinically relevant cellular defects by using a combination treatment or a multi-targeted antitumor agent to optimize therapeutic outcomes (1). This underlying principle constitutes a basis for the clinical trial of indole-3-carbinol in breast cancer prevention (2 3 Mounting evidence indicates that this antitumor effects of indole-3-carbinol are attributed to its ability to interfere with multiple facets of oncogenic signaling pathways (analyzed in refs 4-8). Furthermore indole-3-carbinol became a highly effective chemopreventive agent against estrogen-responsive malignancies in part due to its activity as a poor regulator of estrogen actions through inhibition of estrogen receptor (ER)-α signaling (9 10 and modulation of cytochrome P450-mediated estrogen fat burning capacity (11). However many factors bargain BMS-790052 the efficiency of indole-3-carbinol including low antitumor TNK2 strength limited bioavailability and challenging pharmacokinetic behaviors because of intrinsic metabolic instability (8). Therefore the structural marketing of indole-3-carbinol or its metabolite 3 3 to build up book indole derivatives with improved strength and metabolic balance continues to be the focus of several latest investigations. This medication development effort provides led to many potent antitumor agencies with distinctive pharmacological actions including SRI3668 (an Akt inhibitor) (12) 1 that's two purchases of magnitude greater than that of indole-3-carbinol in suppressing the proliferation of breasts tumor cells. Previously we confirmed that OSU-A9 disrupted a wide spectral BMS-790052 range of signaling pathways mixed up in legislation of cell routine development and cell success (16). BMS-790052 Within this research mechanistic evidence additional features the growth-inhibitory aftereffect of OSU-A9 to its capability to modulate Akt/nuclear factor-kappa B (NF-κB)- and stress-induced signaling systems paralleling that of indole-3-carbinol. Similarly important dental OSU-A9 inhibited MCF-7 xenograft tumor development without incurring overt symptoms of toxicity or significant lack of bodyweight weighed against vehicle-treated handles. These outcomes indicate the translational BMS-790052 potential of OSU-A9 as an element of chemotherapeutic and/or chemopreventive approaches for breasts cancer. Components and strategies Reagents antibodies and plasmids 1 these agencies at numerous concentrations were dissolved in dimethyl BMS-790052 sulfoxide (DMSO) diluted in BMS-790052 culture medium and added to cells at a final DMSO concentration of 0.1%. For studies OSU-A9 was prepared as a suspension in vehicle (0.5% methylcellulose and 0.1% Tween 80 in sterile water) for oral administration to tumor-bearing immunocompromised mice. Rabbit polyclonal antibodies against numerous biomarkers were obtained from the following sources: p-473Ser Akt IκB kinase (IKK)-α p-108Ser IKKα glycogen synthase kinase (GSK)-3β p-9Ser-GSK3β p-183Thr/185Tyr-JNK JNK p-180Thr/182Tyr-p38.
Bacterial shape is normally dictated with the peptidoglycan layer from the cell wall usually. synthesized and electron microscopy uncovered which the mutants had been deficient in periplasmic flagella completely. Wild-type cells poisoned using the protonophore carbonyl cyanide-have a skeletal function. These organelles dynamically connect to the rod-shaped cell cylinder to allow the cell to swim also to confer partly its AT7867 flat-wave morphology. (syphilis) many types (relapsing fever) (Lyme disease) sp. (individual diarrheal disease swine dysentery) and dental treponemes connected with periodontal disease (5-8). The periplasmic flagella of spirochetes have already been characterized at length. Genetic proof including targeted mutagenesis research in and and signifies that practically every one of the genes necessary for motility and chemotaxis can be found in these types (12 13 Actually some spirochetal motility genes are therefore comparable to those of and serovar Typhimurium that their appearance in these bacterias result in detrimental complementation results on flagella synthesis and motility. A most likely explanation because of this detrimental complementation would be that the spirochetal motility proteins contend and hinder their counterparts regarding set up of flagellar parts (14 15 Recent results with indicate the morphology of these spirochetes is likely to be affected by the presence of the periplasmic flagella. These spirochetes are quite IL18 antibody large relative to other bacteria having a diameter of 0.33 μm and a length of 10-20 μm (16). They have a flat-wave morphology having a wavelength of 2.83 μm and a peak-to-peak amplitude of 0.78 μm (4 16 Attached at each end are 7-11 periplasmic flagella that overlap in the center of the cell (7). These periplasmic flagella consist of the major filament protein FlaB and a minor filament protein FlaA (17). Sadziene (18) isolated a spontaneously happening nonmotile mutant (HB19Fla?). HB19Fla? lacked periplasmic flagella and both FlaA and FlaB proteins (17 18 It in the beginning was described as helical but somewhat straighter than the parental strain (18). Subsequent light and high-voltage electron microscopy analyses exposed that HB19Fla? had a long rod-shaped morphology (4 16 The helical morphology AT7867 observed by Sadziene was most likely the consequence of cells occasionally growing in chains and wrapping around each other inside a right-handed sense (4). The result that solitary HB19Fla? cells were rod-shaped led to formulation of the hypothesis the periplasmic flagella influenced the entire shape of the cell. Moreover a model of motility was proposed based on this hypothesis (4 16 (observe ref. 19 for recent review). A significant concern with this analysis is definitely that the site and nature of the mutation (or mutations) in HB19Fla? is definitely unknown; analysis of many of the flagellar genes of HB19Fla? including the flagellar filament gene failed to determine the mutation in HB19Fla? (18 20 21 Therefore the mutant could be rod-shaped for reasons other than the lack of periplasmic flagella. The genetic analysis of spirochetes including Tnfused to either of two periplasmic flagellar promoters (22). Using this method AT7867 we display by allelic exchange mutagenesis that inactivation of results in cells that lack periplasmic flagella and are nonmotile. Because these mutant cells have a AT7867 markedly modified morphology we conclude the periplasmic flagella also have a skeletal function dictating at least in part the flat-wave morphology associated with undamaged cells. Materials and Methods Bacterial Strains and Growth Conditions. High-passage culture-attenuated senso stricto strain B31 has been explained (23). Cells were cultivated in liquid at 32 and on plates at 34°C in BSK-II medium (23 24 Solid medium and agar overlay were prepared by using 0.66% agarose (23). Water moderate and agar plates had been incubated within an atmosphere of 3% skin tightening and. Swarm dish assays were completed through the use of 0.35% agarose; around 5 × 105 cleaned cells in 5 μl had been discovered onto plates filled with BSK-II moderate diluted 1:10 in AT7867 Dulbecco's PBS without divalent cations. To check for the inhibition of motility with the protonophore carbonyl cyanide-at area temperature. The pellet was resuspended in 1.
Metazoans have got evolved multiple paralogues of the TATA binding protein (TBP) adding another tunable level of gene control at core promoters. insects the alternative TRF1/BRF complex appears responsible for the initiation of all known classes of Pol III transcription. biochemical approaches to dissect the CS-088 role of these alternative core-promoter recognition factors (Hansen targets for these factors. Here we describe the use of chromatin immunoprecipitation (ChIP) assays combined with genome CS-088 tiling CS-088 microarrays (ChIP-on-chip) coupled with a new computational tool to more accurately identify in an unbiased manner genome-wide targets of core-promoter recognition factors. To test the usefulness of this strategy we have applied this methodology to the mapping of specific promoters targeted by the TRF1/BRF complex. TRF1 represents a unique class of TRF found in insect species such as and biochemical approaches established that TRF1 is likely involved in transcription of CS-088 both Pol II and Pol III genes (Hansen appears to form a complex with BRF (Takada transcription assays revealed that the TRF1/BRF complex plays a critical role in the transcription of several tRNA 5 rRNA and U6 snRNA genes. Salivary gland polytene chromosome staining suggested that TRF1 can occupy a few hundred genomic sites the majority of which are co-occupied by BRF (Takada map of TRF1- and BRF-binding sites throughout the genome. Consistent with our previous biochemical findings a major class of TRF1/BRF targets represents Pol III genes such as tRNAs. A small percentage of sites bound by TRF1 were mapped to Pol II promoters. In addition we report two new classes of TRF1/BRF targets and small nucleolar RNAs (snoRNAs) CS-088 which are small nonmessenger RNAs (snmRNAs). transcription assays were used to verify that the TRF1/BRF complex is functionally required for accurate transcription initiation of the new focus on genes. Taken together these results strongly support a global role of the TRF1/BRF complex in Pol III transcription. Results Genome-wide colocalization of TRF1 and BRF at noncoding small RNA promoters In order to determine high-resolution target genes of the TRF1/BRF complex we performed ChIP-on-chip analyses using genome tiling arrays (Affymetrix). This high-density oligonucleotide array covers the entire genome of at 35 bp resolution with the notable exception of repeat regions such as transposons and 28S and 5S rRNA genes. We first established robust ChIP assays using affinity-purified anti-TRF1 and anti-BRF antibodies that efficiently co-precipitate specific genomic fragments such as 5S rRNA and tRNA genes. These few genes had previously been characterized as targets of the TRF1/BRF complex and are typically precipitated by the specific antibodies at a level 20- to 100-fold above nonspecific IgG controls (Physique 2A). These co-precipitated genomic fragments were amplified and subsequently hybridized to the microarrays in duplicate. The data were extensively analysed using a newly developed statistical platform (Tiling Hierarchical Gamma Mixture Model TileHGMM). This statistical approach explicitly modeled binding of the probes in the control sample and TRF1/BRF-enriched samples (Physique 1A). The fitting of this statistical model provided us with probabilities of binding that is specific to a genomic region of interest. We then identified TRF1- Rabbit polyclonal to AKAP5. and BRF-bound regions by thresholding these probabilities while controlling the false discovery rate using a false discovery rate calculation (Newton (tRNA) promoter regions are significantly enriched … In order to visualize the spatial topology of the enrichment revealed by TRF1/BRF immunoprecipitations the signal intensity of each probe was plotted over the chromosomal locations for the selection of genomic regions. Physique 2B displays a CS-088 region around the X chromosome that encodes three tRNA genes ((Physique 2C). The third example illustrates snoRNA:644 gene on chromosome 2R (Physique 2D). In all three cases it is evident that this hybridization signals peak precisely in register with these noncoding RNA genes. We further verified these microarray results by conventional quantitative PCR detection of co-immunoprecipitated.
Purpose Insulin and insulin-like development factors (IGFs) are putative regulators of cell proliferation and differentiation during lens development. a fiber-cell specific αA-crystallin (αA) promoter and a altered αA-promoter linked to the chicken δ1-crystallin enhancer (called the δenαA promoter). 17-AAG The δenαA promoter is usually active in both lens epithelial and fiber cells. The lens phenotypes were analyzed by histology and immunohistochemistry. Protein expression was examined by western blotting. Results Normal mouse lenses express both the insulin receptor (IR) and the 17-AAG IGF-1 receptor (IGF-1R) and their expression is highest at the lens periphery where the germinative and transitional zones are located. In transgenic mice insulin expression in the lens induced cataract formation. The severity of the cataracts reflected the level of transgene expression independent of the type of promoter used. In severely affected families the spherical shape of the lens was altered and the lenses were smaller than normal. Histological analysis showed no evidence of premature differentiation of the anterior epithelial cells. In contrast to the IGF-1 mice insulin transgenic mice exhibited an anterior shift in the location of the germinative and transitional zones leading to a reduction of the zoom lens epithelial compartment. Extra alterations included enlargement of the zoom lens transitional area variable nuclear setting in the zoom lens bow area and inhibition of fibers cell denucleation and terminal differentiation. Conclusions Raised intraocular insulin will not enhance proliferation nor induce differentiation of mouse zoom lens epithelial cells. Since a rise in IGF-1 causes a posterior change of the zoom lens geminative and transitional areas while a rise in insulin causes an anterior change of these areas our results claim that these two development factors may interact to control the positioning of the structural area during normal 17-AAG zoom lens advancement. Our data also claim that elevated insulin-signaling activity in the zoom Mouse monoclonal to EGFP Tag. lens can antagonize the endogenous indicators that are in charge of fibers cell maturation and terminal differentiation. Introduction The lens is usually a highly organized and polarized tissue. Growth and development of the lens depend on proper spatial regulation of cell proliferation and differentiation [1-3]. The central lens epithelial cells are relatively quiescent while the epithelial cells near the periphery (the germinative zone) show a higher proliferative index. As the epithelial cells in the germinative zone proliferate cells posterior to this zone are pushed into the transitional zone where they are induced to elongate and differentiate into the lens fiber cells. The newly differentiated fiber cells accumulate in a concentric manner on top of the previously differentiated fiber cells in the bow region. As the immature fiber cells migrate toward the lens core their nuclei and intracellular organelles degrade to result in mature fiber cells [4]. Growth factors have been implicated as regulators of cell proliferation and differentiation during lens development [1 3 Gain-of-function studies in mice show that fibroblast growth factors (FGFs) can function as differentiation inducers while platelet-derived growth factors (PDGFs) function mainly as mitogens [5-8]. The function of insulin and insulin-like growth factors (IGFs) is usually unclear. Experiments in chicken lens epithelial explants show that insulin and IGFs can induce epithelial-to-fiber differentiation as measured by cell elongation and an increase in δ-crystallin synthesis [9 10 In contrast insulin and IGFs were weak differentiation factors for rat lens epithelial cells in explant cultures. However these growth factors appeared capable of enhancing or maintaining the differentiation phenotype induced by FGF [11-13]. In transgenic mice generated to overexpress IGF-1 in the lens by means of the mouse αA-crystallin promoter [14] lens epithelial cells were found to not undergo premature differentiation which is usually consistent with the observation that IGF-1 itself is not a differentiation inducer in rodent lens explants. Interestingly the lens growth pattern in IGF-1 mice was perturbed and the 17-AAG lens epithelial compartment was extended more posteriorly than in normal mice..
The cytoplasmic protein-tyrosine kinase Fes continues to be implicated in cytokine signal transduction hematopoiesis and embryonic development. fibroblast transformation by an activated Fes mutant suggesting that oligomerization is Epothilone B essential for Fes activation in vivo. Coexpression with the Fes Epothilone B N-terminal region did not impact the transforming activity of v-Src in Rat-2 cells arguing against a nonspecific suppressive effect. Taken together these findings suggest a model in which Fes activation may involve coiled-coil-mediated interconversion of monomeric and oligomeric forms of the kinase. Mutation of Epothilone B the first coiled-coil domain name may activate Fes by disturbing intramolecular coiled-coil conversation allowing for oligomerization via the second coiled-coil domain name. Deletion of the second coiled-coil domain name blocks fibroblast transformation by an activated form of c-Fes consistent with this model. These total results provide the first evidence for regulation of the nonreceptor protein-tyrosine kinase by coiled-coil domains. The c-proto-oncogene may be the regular cellular homolog from the changing oncogenes within many avian and feline retroviruses (13 15 16 19 22 38 The individual c-locus encodes a cytoplasmic protein-tyrosine kinase (Fes) mainly portrayed in hematopoietic cells from the myeloid lineage (7 28 40 Distinct from Src and various other nonreceptor tyrosine kinases Fes includes a lengthy N-terminal unique area accompanied by a central SH2 domains Epothilone B and a C-terminal kinase domains with a complete molecular mass of 93 kDa. Prior studies show that Fes is normally activated by many hematopoietic growth elements (17 18 20 30 implicating Fes being a downstream element of cytokine receptor signaling. Various other work shows that Fes suppresses the development and induces the differentiation from the myeloid leukemia cell series K-562 (46). Used together these outcomes strongly claim that Fes is normally mixed up in legislation of hematopoietic cell proliferation differentiation and function. Regardless of the essential assignments that Fes has in hematopoietic advancement little is well known about the systems that control its tyrosine kinase activity. In the lack of activating indicators Fes tyrosine kinase activity is normally highly repressed in cells (12). Certainly high-level overexpression of Fes must induce kinase activation in vivo also to discharge changing activity in fibroblasts (8). Latest function from our lab shows that Fes autophosphorylation takes place by an intermolecular system recommending that oligomerization could be an important preliminary event in Fes activation (36 37 We also noticed that energetic Fes elutes from a gel purification Epothilone B column as a big oligomer which the initial N-terminal domains is necessary for oligomerization. Used jointly these data claim that oligomerization could be necessary for kinase activation Rabbit Polyclonal to C-RAF (phospho-Thr269). which suppression of oligomerization may signify a poor regulatory system for Fes in vivo. Pc analysis from the Fes N-terminal area revealed two solid consensus sequences for the forming of coiled-coil oligomerization domains (36). Coiled-coil domains are made up of amphipathic α-helices using a quality heptad repeat where the initial and 4th residues are hydrophobic and pack against one another to create a hydrophobic primary (26). The rest of the proteins are hydrophilic assisting to solvate the oligomeric structure often. In this research we looked into the contribution from the Fes coiled-coil homology domains towards the legislation of Fes tyrosine kinase activity and natural function. We noticed that disruption or deletion from the even more N-terminal coiled-coil domains (CC1) released Fes tyrosine kinase activity in fibroblasts resulting in transformation. As opposed to CC1 deletion of the next coiled-coil domains (CC2) inhibited change by an turned on type of Fes recommending that CC2 could be necessary for oligomerization. Furthermore a truncation mutant of Fes bearing just the initial N-terminal domains highly suppressed the changing and tyrosine kinase activity of an turned on Fes mutant in fibroblasts offering further proof that Fes is normally energetic as an oligomer in living.
Nicotine adenine dinucleotide (NAD+) performs essential assignments in electron transport reactions being a substrate for poly(ADP-ribose) polymerase and NAD+-reliant protein deacetylases. of NAD+ MLN8237 regulator and degrees of NAD+ biosynthesis. Using transcript arrays we present that low NAD+ state governments particularly induce the de novo NAD+ biosynthesis genes as the genes in the salvage pathway stay unaffected. The NAD+-reliant deacetylase activity of Hst1p represses de novo NAD+ biosynthesis genes in the lack of brand-new protein synthesis recommending a direct impact. The known Hst1p binding partner Amount1p exists at promoters of extremely inducible NAD+ biosynthesis genes. Removing Sir2p family members (16 20 39 Deacetylation is normally achieved through the transfer from the acetyl group in the substrate to ADP-ribose the NAD+ break down product to create suppresses recombination between tandem copies of rRNA genes (10) and therefore reduces the forming of extrachromosomal ribosomal DNA (rDNA) circles and their deposition in mom cells a function crucial for maintenance of mother-cell life time (36). homologues in higher eukaryotes have already been implicated in an array of natural processes involving advertising of durability in (43) advancement and epigenetic legislation in (29) and modulation of tension replies and apoptosis in mammalian cells through downregulation of p53 activity (24 44 In fungus where it's been examined most thoroughly the in vivo Sir2p enzymatic activity and its own function in silencing and durability depend on a satisfactory way to obtain NAD+ (1 32 39 Various other mobile enzymes and procedures also depend over the option of NAD+. Under circumstances of stress such as for example DNA harm NAD+ depletion in mammalian cells induced by activation of poly(ADP-ribose) polymerase is normally considered to perturb essential essential features and donate to cell loss of life (11 14 15 NAD+ in cells is definitely continuously broken down and resynthesized having a half-life in human being lymphocytes of approximately 6 h (7). Because the amount of available NAD+ in cells depends on the balance of NAD+ production and damage homeostatic mechanisms that regulate these processes are important particularly in occasions of stress. Both candida and mammalian cells actively import nicotinic acid (NA or niacin) from your extracellular medium and convert it into NAD+ through the salvage pathway (Fig. ?(Fig.1A)1A) (reviewed in research 13). NA produced intracellularly by hydrolytic cleavage of the glycosidic relationship in NAD+ and deamidation of nicotinamide is also recycled to NAD+ through the salvage pathway. Additionally a MLN8237 de novo biosynthesis or kynurenine pathway converts tryptophan to NAD+. This well-studied pathway bears out oxidative degradation of tryptophan to quinolinic acid which is consequently converted to NAD+. Nicotinate phosphoribosyl transferase Npt1p is definitely a key enzyme in the candida NAD+ salvage pathway and is responsible for reutilization of NA generated by NAD+ breakdown and for utilizing NA taken up from the medium (39). In the absence of causes synthetic lethality with deletion of genes in the kynurenine pathway (genes [for biosynthesis Fli1 of nicotinic acid]) (27). Only one of the six genes can be performed by another enzyme. Epidemiologic studies of human being populations show that cellular NAD+ levels and the effectiveness of conversion of tryptophan to niacin varies widely between individuals (17). While most of this variance is thought to be due to variations in diet niacin and tryptophan intake it is likely that some of the variance in NAD+ levels is genetically identified. FIG. 1. The NAD+ salvage pathway is definitely constitutively active whereas de novo NAD+ biosynthesis pathway is MLN8237 definitely highly inducible by low NAD+ claims. (A) NAD+ biosynthesis pathways in candida. NaMN NA mononucleotide; NaAD desamido-NAD+ … In the course of our studies within the NAD+-dependent deacetylases in candida we sought to identify MLN8237 factors that regulate cellular levels of NAD+. With this study MLN8237 we report the homologue of Sir2p Hst1p (for homologue of SIR two) is an NAD+-dependent deacetylase with relatively low affinity for NAD+ that functions as a cellular NAD+ sensor to repress the NAD+ biosynthesis system when NAD+ levels are adequate. MATERIALS AND METHODS Candida press strains and plasmids. All strains were grown MLN8237 in synthetic complete medium (SC) or selective synthetic drop-out medium comprising 2% glucose. The SC medium lacking NA was prepared according to the.
Human atherosclerotic lesions overexpress the lysosomal cysteine protease cathepsin S (Kitty S) one of the most potent mammalian elastases known. enhancement plaque angiogenesis and plaque disruption (1-3). Prior studies possess suggested involvement of both serine MMPs and proteases in arterial diseases. Scarcity of plasminogen decreased the introduction of lesions in transplanted mouse arteries (4) and in arteries of electrically harmed mice (5). Hyperlipidemic mice lacking in urokinase demonstrated reduced aortic ectasia (6) an effect attributed to reduced plasmin-dependent activation of pro-MMPs (6). Human atheromata overexpress the interstitial collagenases MMP-1 (7) MMP-13 (8) and MMP-8 (9) at sites of interstitial collagen cleavage. MMPs 2 3 7 9 12 14 as well as others also localize in human atherosclerotic plaques (7 10 Overexpression of the MMP inhibitor TIMP-1 reduced formation of aneurysms (15) and atherosclerotic lesions (16) in animals affirming a role for MMPs in arterial remodeling and atherogenesis. Beyond serine proteases and MMPs we recently proposed the participation of cathepsin S (Cat S) and Cat K in atherogenesis. These cysteine proteases have potent elastolytic (17 18 and collagenolytic activities (19). Diseased human arteries overexpress these enzymes and exhibit a reciprocal deficiency of cystatin C the most abundant endogenous inhibitor of cysteine proteases (1 2 Biochemical studies JNJ-26481585 suggest that Cat S and Cat K account for most of the elastolytic activity extracted from human carotid plaques. Multiple cell types found in human atheroma can express Cat S. We have previously exhibited that intimal macrophages as well as SMCs in the tunica media express Cat S in human atherosclerotic arteries (1). We also exhibited colocalization of Cat S with lumenal and microvascular endothelial cells (ECs) in human atheroma and with microvascular ECs in wounded mouse skin (G.-P. Shi et al. unpublished data). In vitro experiments showed secretion of active Cat S by these atheroma-associated cell types following exposure to inflammatory cytokines found in atheroma. Our findings implicate Cat S in atherogenesis along with serine proteases and MMPs. Furthermore Cat S an essential protease acquired for invariant chain processing plays an important role in antigen presentation. Cat S deficiency prospects to impaired immune responses (20 21 which might also impact atherogenesis (22 23 Despite this plethora of observational and biochemical data no direct evidence establishes a role of Cat S in atherogenesis. This study used Cat S-deficient (mice were backcrossed more than ten generations onto the C57/BL6 background. These mice were crossbred with mice (also around the C57/BL6 background; The Jackson Laboratory Bar Harbor Maine USA). The producing double knockout mouse genotypes were confirmed using mouse tail genomic DNA-derived PCR as explained (20 24 This study used both and control mice. Mice Rabbit polyclonal to PAK1. consumed an atherogenic diet ("type":"entrez-nucleotide" attrs :"text":"D12108″ term_id :"2148896″ term_text :"D12108″D12108; Research Diets Inc. New Brunswick New Jersey USA) made up of 20.1% saturated fat 1.37% cholesterol and 0% sodium cholate (25). After 8 12 JNJ-26481585 and 26 weeks (= 12/timepoint/group) mice were used as explained below. Oil reddish O staining for lipids. Deposition of lipids in en-face preparations of abdominal aortas (fixed with 10% JNJ-26481585 formalin) was determined by oil reddish O staining. Subsequently the aortas were opened longitudinally to the iliac bifurcation and JNJ-26481585 pinned out on the surface of black wax with 0.2-mm steel pins. They were then stained with oil reddish O answer for 2.5 hours at room temperature on a shaker and washed three to four times in 85% propylene glycol solution. Immunohistochemistry. Serial cryostat sections (6 μm) of mouse aortic arches were fixed in acetone air flow dried and stained by the avidin-biotin-peroxidase method. After limiting endogenous peroxidase activity with 0.3% H2O2 and nonspecific binding of primary antibody with 5% species-appropriate normal serum (Vector Laboratories Burlingame California USA) we incubated sections with primary antibodies diluted in PBS supplemented with JNJ-26481585 5% species-appropriate normal serum for 90 minutes at room heat. Incubation with secondary antibodies for 45 moments.
We present that interferon-induced transmembrane protein 1 (IFITM-1) IFITM-2 and IFITM-3 exhibit a wide spectral range of antiviral activity against many family including Rift Valley fever pathogen (RVFV) La Crosse pathogen Andes pathogen and Hantaan pathogen which can cause serious disease in individuals and pets. and IFITM-3 take up vesicular compartments that are distinctive in the vesicles covered by IFITM-1. Furthermore although overexpression of most IFITMs extended vesicular and acidified compartments within cells there have been marked phenotypic distinctions among the vesicular compartments occupied by IFITMs. Collectively our data offer new insights in to the feasible mechanisms where the IFITM family restrict distinct infections. Launch In response to viral attacks almost all vertebrate cells make type I interferons (IFNs). This course of cytokines can induce appearance of a huge selection of IFN-stimulated genes (ISGs) thus building an antiviral condition in neighboring cells (1). Therefore an adaptive immune system response is set up and viral pass on through the entire organism is certainly diminished (analyzed in guide 2). Although there are extensive known ISGs the antiviral systems of just a few have already been well characterized (analyzed in guide 3). Recent research have discovered the book antiviral activity of a family group of little ISGs referred to as interferon-induced transmembrane proteins (IFITMs). In human beings the IFITM family members comprises four useful genes three which (IFITM-1 -2 and -3) are ubiquitously portrayed and induced by LY-2584702 both type I and type II IFNs (4-6) while appearance of the 4th member (IFITM-5) is bound to osteoblasts (7). IFITMs have already been proven to restrict particular enveloped infections including influenza A pathogen (FLUAV) (8) serious acute respiratory symptoms coronavirus (SARS-CoV) Ebola pathogen (EBOV) and Marburg pathogen (MARV) (9) flaviviruses (including dengue pathogen types 1 and 2 [DENV-1/2] and Western world Nile pathogen [WNV]) (9 10 HIV-1 (11) and vesicular stomatitis Indiana pathogen (VSIV) (12). On the other hand these proteins acquired no influence on murine leukemia pathogen (MLV) and arenaviruses such as for example Lassa pathogen (LASV) and Machupo pathogen (MACV) (8 9 Furthermore research using IFITM-3-knockout mice aswell as human beings possessing particular IFITM-3 gene mutations possess demonstrated these individuals are even more vunerable to disease due to influenza A pathogen (13 14 The molecular systems where IFITMs inhibit infections remain unclear but accumulating proof shows that IFITM-3 alters the membranes from the vesicular compartments in a way that virion-host membrane fusion is certainly prevented as well as the endocytosed virions thus remain trapped inside the vesicles. For instance IFITM-3 didn't inhibit the binding or entrance of influenza A pathogen (8 9 15 or HIV-1 (11) LY-2584702 but do prevent the discharge of ribonucleoprotein organic (RNP) in to the cytoplasm. Additionally IFITM-3 limitation of VSIV could possibly be overcome by presenting viral genomic RNA straight into the cytoplasm (12). IFITM-3 provides Rabbit polyclonal to MTOR. been proven to partially have a home in past due endosomal and lysosomal compartments (9 15 16 and its own overexpression expands these acidified compartments (including Rab-5- Rab-7- and Light fixture-1-covered vesicles) (15). Prior work shows that IFITMs have differential antiviral actions against diverse infections. IFITM-3 was strongest in resisting FLUAV VSIV WNV and DENV attacks while IFITM-1 limitation of WNV and DENV attacks was cell type reliant (9 10 12 Nevertheless IFITM-1 -2 and -3 all restrict HIV-1 FLUAV Ebola pathogen and Marburg pathogen LY-2584702 infections however the performance of inhibition depended in the web host cell type (9 11 15 The system behind the differential awareness of infections to these protein is not grasped. Rift Valley fever (RVF) pathogen (RVFV) can be an rising pathogen with the capacity of leading to critical epidemics among livestock and human beings. RVFV was LY-2584702 initially defined in 1931 (17) in East Africa and provides since caused huge eruptive disease throughout Africa and recently in the Arabian Peninsula (analyzed in sources 18 to 20). In local ruminants RVF leads to abortion and high fatality prices especially among youthful animals. Though it is normally a self-limiting febrile disease in human beings a small % of RVF situations develop serious viral hemorrhagic fever (VHF) neurological.
We followed the positioning from the replication organic in the pathogenic bacterium using antibodies raised against the single-stranded DNA binding proteins (HpSSB) as well as Piboserod the replicative helicase (HpDnaB). the most frequent human being pathogen affecting nearly half from the world's inhabitants. infection could cause chronic gastritis resulting in Piboserod gastric ulcers intestinal metaplasia and adenocarcinoma (1 -3). Its spiral form and unipolar flagella supply the pathogen the motility critically had a need to colonize and persist in the gastric lumen. is rolling out unique models of hereditary and physiological equipment to survive and grow in the extremes from the human being gastric environment (4 -8). Furthermore it could transform itself from a helical bacillary morphology to a practical but nonculturable coccoid type under oxidative tension and in ageing cultures (9). The indicators eliciting the bimorphic response as well as the molecular systems causing the transformation aren't known. A romantic understanding of cell routine settings including those of chromosome replication and cell department is essential for a knowledge of these procedures. However very little is known about chromosome replication and its coordination with growth and division in replication machinery have already been characterized replication origin Hpchromosome. The initiator protein HpDnaA binds to the unique bipartite replication origin Hpand initiates DNA unwinding (14). Recently a unique DnaA binding protein HobA has been identified as the regulator of the timing and frequency of DnaA-dependent initiation from by aiding the oligomerization of DnaA for orisome (a multiprotein complex formed at the (15). There are features of replisome assembly that distinguish from the conventional model systems such as or (16) suggesting a self-loading function of HpDnaB consistent with the absence of a genome. The C-terminal region of HpDnaB contains an insertion of ~34 amino acids relative to DnaB that is essential for its function (17). The single-stranded DNA binding protein (HpSSB) plays a central role in DNA replication by modulating DnaB helicase activity. HpSSB and HpDnaB form replication foci that may help differentiate the replicationally active helical form and the dormant coccoid form of (12). Though the replication proteins forming the replisome are functionally conserved their intracellular organization varies among bacteria depending on their living environments cell physiologies and growth rates (18 -21). The important aspects of replisome dynamics and Piboserod cell cycle control in remain elusive. Being a gradually growing pathogen making it through in Rabbit Polyclonal to CBX6. a particular ecological specific niche market may present Piboserod some exclusive features in the set up of its replisome and its own functional dynamics through the cell routine. We implemented the locations from the replisome using HpSSB foci as reporters for replication sites in set cells at different levels of development and department. We present that in cells from an evergrowing culture nearly all replication foci localize on the cell poles not really across the midcell as observed in (22 -24) and in (25). Colocalization from the HpDnaB helicase using the HpSSB validated the identification from the SSB foci as energetic replication centers that shifted from pole proximal towards the midcell area with raising cell size. The replication origins hybridization (Seafood) with cell membrane small fraction whereas a lot of the HpSSB was within the soluble cytoplasmic small fraction. Immunogold electron microscopy (EM) verified membrane association and polar localization of some replication proteins. The polar located area of the replication complicated association from the energetic replisome using the bacterial cell membrane and the current presence of a possible centromeric area close to the bipartite seem to be a number of the hitherto unidentified top features of chromosome replication. Strategies and Components Bacterial strains and plasmids. The bacterial strains and plasmids found in this scholarly study are listed in Table 1. Desk 1 Bacterial plasmids and strains Development circumstances of bacterial cultures. strains were harvested in Luria broth (LB) moderate (supplemented with 100 μg/ml ampicillin or 50 μg/ml kanamycin where required) at 37°C or 22°C. The cells had been harvested on LB agar plates (with or without antibiotics as suitable) at 37°C for 12 to 16 Piboserod h. stress 26695 Piboserod was expanded on brain center infusion (BHI) agar (Difco.


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Back in the day, American hockey was dominated by the 'Three Ms' of Minnesota, Michigan and Massachusetts. That's where the majority of players came from, and that's where many of the most powerful college programs resided. So it was fitting that the 1991 Frozen Four final took place at the St. Paul Civic Center in Minnesota, with teams from Michigan and Massachusetts competing for the crown – the Boston University Terriers and the Northern Michigan Wildcats.
The two teams were a study in contrasts. The big-city Terriers boasted numerous NHL draft picks and future stars, while representing a program that had won three national championships. Their legendary coach, Jack Parker, had been behind the bench when B.U. last won it all back in 1978.
Not to be outdone, the Wildcats had an esteemed bench boss themselves in Rick Comley, who had taken Northern Michigan to the final in 1980 before losing to North Dakota. The 1990-91 Wildcats were a veteran unit with talent, but only one hot-shot NHL prospect in defenseman Brad Werenka – who was recruited by NMU when they were one of the few programs to realize he had taken extra courses to graduate high school a year early. So he made his way to small-town Marquette, in Michigan's Upper Peninsula, to play for the Wildcats.
Northern Michigan came into the tournament on a roll. The Wildcats hadn't lost a game since Christmas, when they fell to St. Cloud State. They won the WCHA tournament, earning the No. 2 seed in the Frozen Four's West bracket. After beating Alaska-Anchorage in the quarterfinal, the Wildcats toppled top seed Maine to grab their spot in the final.
Over in Boston, the Terriers also earned their bid through a conference tournament win, though Maine and Boston College both had better records in the Hockey East regular season. Boston U. knocked off Michigan in the quarterfinal and Clarkson in the semifinal to snag a ticket to St. Paul and its famed clear dasherboards. The resulting clash between the Terriers and Wildcats turned out to be one of the most stunning Frozen Four finals in NCAA history.
SCOTT LACHANCE: (Boston University defenseman) We struggled early in the season, but after that we became a cohesive unit. Like Northern Michigan, we got better as the year went on. Our No. 1 line was Keith Tkachuk, Tony Amonte and Shawn McEachern, that was probably a good start for why we were successful.
SHAWN McEACHERN: (Boston University center) Keith was a great power forward, and he was only a freshman. He could throw his body around and had a lot of talent. Tony was super fast and smart, he was one of the best scorers in college hockey. They had a great team with Brad Werenka, Dallas Drake and Bill Pye, who was an all-American. He was one of the top goalies in college hockey at the time.
ED RONAN: (Boston University right winger) We had an interesting blend of players and an influx of kids like Keith Tkachuk. It can take awhile to find your role when you're a kid because you don't know what kind of player you're going to be in college and the coach doesn't always know either. For guys like myself, my role as a freshman was very different from my role as a junior and senior. Once we all adjusted, we started to score a lot of goals, our team was driven by offense. It was a fun style to play, but not necessarily conducive to winning a championship. That final game was what our team was all about, putting up big numbers, but there was also big risk.
LOU MELONE: (Northern Michigan defenseman) A lot of teams had bigger names, we just had a great team. Everyone wanted to play for each other. The approach we had was what we had the entire year. We had a businesslike attitude, we were dialed in and we just played, no matter the opponent.
DARRYL PLANDOWSKI: (Northern Michigan left winger) At the time, (Northern Michigan) had a lot of Canadians, and my linemate Dean Antos was going there. I was a smaller player and I wanted to play. I had talked to North Dakota and Wisconsin, but Northern Michigan felt like the right fit.
MARK BEAUFAIT: (Northern Michigan center) I was coming out of Detroit as a bit of a late bloomer. I knew assistant coach Walt Kyle, so there was that connection. I had a lot of teams telling me I needed another year of junior, but Northern Michigan offered me a scholarship right away. To be honest, I probably needed another year of junior, but there was a comfort level at Northern Michigan.
LACHANCE: They were on a huge streak, and we knew their power play was great. We knew they were talented, but we didn't know what to expect.
RONAN: We felt we could score a lot of goals against anyone. We knew Billy was a good goalie and that they had good players, but personally I didn't overanalyze things. We had beat up some pretty good teams along the way, we had beaten Michigan on the road late in the season and found a pretty good stride going into the tournament. I felt like we were pretty prepared.
McEACHERN: We had a lot of fun. Jack was a great coach. He would really get the guys pumped up before a game, telling us to play through guys and to play B.U. hockey. Jack was a great motivator.
RICK COMLEY: (Northern Michigan coach) They were extremely dangerous. We hadn't lost in the second half, but they were on a run of their own. The biggest worry was they were blowing up teams early. The first 10 minutes were critical.
By 9:26 of the first period, the Terriers had jumped out to a 3-0 lead thanks to two goals by Ronan and one by David Sacco.
COMLEY: Great coaching.
RONAN: My first goal, I had some good speed to split their defense, and Dave Tomlinson threw a waist-high pass at the blueline that I knocked down. I was in full stride, and it sat down in front of me. I beat Billy on a good shot. My second goal was a high tip as I was coming around on the cycle. I was high in the slot and Billy was coming off his post, that was a tough play for a goalie.
MELONE: Obviously it wasn't the start you'd want, but there was no panic. You're not happy, and it's not a good thing, but you keep moving forward.
BEAUFAIT: They were just fast. They had some explosive players. We regrouped after the first period. We had a veteran team and great senior leadership, which helped. We knew we had to pick away at the lead, and it started snowballing, goal after goal.
LACHANCE: It wasn't the type of 3-0 lead where we thought we were cruising by this team. They were quick in moving the puck, and they were talented. They kept coming. Werenka was all over the place, he made them a different animal.
BRAD WERENKA: (Northern Michigan defenseman) There was concern, but we didn't think it was as bad as it looked. We knew we had to get our feet moving and get some bounces.
PLANDOWSKI: We knew we could score, so being down 3-0 early? I don't think any of our guys panicked. We had averaged more than six goals per game that season. We had goal-scorers.
The second period proved to be the tonic for Northern Michigan, as the Wildcats scored five consecutive goals to take a 5-3 lead. Antos got the ball rolling, with Beaufait, Scott Beattie (twice) and Plandowski also scoring.
COMLEY: We were an experienced team. As soon as we made it 3-1, we could relax a bit. If they had gotten the fourth goal, it would have been another story. It was nuts. Both goalies had good years, but the offensive talent of the two teams took over.
WERENKA: The first was important to just get rolling. Then we started to get the bounces that they were getting in the first period.
PLANDOWSKI: It was a wave. Scott Beattie got going, I scored one, and it put them on their heels. The momentum changed. All of a sudden we had great energy, and I'm sure their legs felt heavy. It was like two different games.
BEAUFAIT: Scott Beattie just knew how to put the puck in the net. He was a smaller guy and not particularly fast, but he knew where to be, and he was confident.
RONAN: We might have been gunslingers, but we weren't that bad defensively. I don't know what happened. (Northern Michigan) came out with another level of energy, and we couldn't match it. It was like, "Holy mackerel! How do we stop this freight train?" It was unfathomable, really.
LACHANCE: We had to go into the dressing room and take a deep breath and calm down. Chris McCann and Mark Krys were great in the room. The third period was the most important, and we had that calming influence with us all year.
RONAN: There were two ways to look at it. Did we just blow it, or what kind of team are we? We had a big senior class, so we had faced adversity before. It was more about letting it go. That next goal was really important.
McEACHERN: We had done it before. We had a comeback in the Beanpot against Boston College, where Amonte got hurt and then came back and scored a hat trick. And we had comeback against Maine in the Hockey East tournament, too.
MELONE: In games like that, you don't hear the crowd or the noise. You're going back and forth and you just play. You're up 5-3, but the game isn't over. I don't think anyone was relaxed, no one felt safe. You just didn't know.
Northern Michigan had been fantastic at locking down leads all season: the Wildcats were 32-0-2 when leading after two periods. They would extend the lead in the third period against Boston U., with Plandowski making it 7-4 on the power play.
PLANDOWSKI: I took it off my skate, and the puck was rolling. I got unbelievable wood on it, without having to shoot too hard. I knew it was a good shot. You get into those moments where everything feels right and you're completely focused.
The Terriers, however, would not give up. Boston U. chipped away in the final half of the third. Amonte and McEachern connected twice, and then with 40 seconds remaining – and goaltender Scott Cashman on the bench for the extra attacker – Lachance found Sacco for the tying goal to make it 7-7.
LACHANCE: It was a desperation play. I dove and poked it to Sacco. I'd be lying if I said it was planned.
MELONE: I remember diving for it just as he shot it…I just missed it.
We couldn't match it. It was like, 'Holy mackerel! How do we stop this freight train?'
– Ed Ronan, Boston University
PLANDOWSKI: Same old thing, the worm turns. Guys were taking 15-second shifts and getting off the ice because they were tired. The energy was gone. They had momentum, we had lost it. At that point, you keep the game simple, dump pucks.
COMLEY: You're watching the clock. They were coming and coming. They get a fluky goal and tie it. Billy never saw it.
With time running down and the game tied, the Wildcats made an ill-advised line change that led to Lachance springing Amonte for a breakaway with just seconds remaining. The future NHLer unloaded a slapshot from the hashmarks.
WERENKA: He came straight off the bench, and I remember chasing him down and diving with my stick, trying to take away part of the ice. It was desperation, and it was scary, to have been up 7-4 in the third and then with a few seconds left, you give up a Grade-A scoring chance to one of the most dangerous players in the country.
PLANDOWSKI: Amonte was an elite skater, so explosive. I spent the whole game trying to contain him and keep him to the outside. It was a role I always took on, and I was comfortable with it, it was just about trying to keep up with him.
MELONE: I remember just putting my head down on the bench. One of the best goal-scorers in U.S. college hockey goes down on a breakaway? Oh no. But then Billy comes way out of his net with the puck in his glove like, "Look what I've got." If you saw
his face, it was like, "OK, let's move forward."
WERENKA: Billy always had such a great style to him. He put an exclamation point on that save, swinging his glove up like that.
I put my head down on the bench. one of the best scorers in college goes on a breakaway? Oh no.
– Lou Melone, Northern Michigan
RONAN: Tony was a tremendous goal-scorer for us. He rushed that shot a bit more than he had to because he didn't know how much time was left on the clock. But he had beaten goalies with that shot before. But if you watch the tape, just before that, we had a faceoff to Billy Pye's left where Dave Tomlinson wins the faceoff and gets it to me. I took a really strange off-angle shot that went off Billy's shoulder or the crossbar. That's the one I want back.
PLANDOWSKI: I've watched the highlights since, and it was a spectacular save. At the time, it was like, "Holy crap, let's get out of here and into the room." We were on the ropes.
COMLEY: Billy Pye made the biggest save of his career.
The save came with one second left on the clock. The NCAA national championship game was headed to overtime.
COMLEY: You're walking off the ice terribly disappointed. Did we blow it? They were tired and discouraged, so I used some coach-speak. I said, "If I told you before the game had started that we'd be heading into overtime against this team, you'd feel pretty good about it, right? So let's go out and try to win this." You try to sell them.
MELONE: It's a 0-0 game, let's go play. Punch the clock, work your butt off. Maybe it had numbed us, we had been in the trenches.
PLANDOWSKI: I don't think we panicked. It was a coin flip. We needed to regroup and start skating again. The start of overtime was a 50-50 game again. You just went up and down the ice, both teams had great chances.
The best chance came in the first overtime period when Boston University defenseman Kevin O'Sullivan entered the zone on a rush. What followed was one of the more probability-defying sequences that could ever be imagined on a hockey rink.
MELONE: He's on the left side. He hits the post and it goes to McEachern. His shot hits the same post, then slides to the other post. That was the most unbelievable thing I've ever seen. It was as if someone was standing there making sure it didn't go in.
RONAN: Kevin had a really nice rush. He put the puck through the kid's legs and then took a great shot. It looked like Shawn had it wide open. I was over the boards, I might've actually been on the ice, because I thought it was over. And I was not alone, we all had our sticks up.
McEACHERN: It was a slapshot that hit the post and came right at me. I was happy to get good wood on it, but I one-timed it off the post and then off the other post.
It was even closer than that: after hitting three posts in three seconds, the puck actually hit the sliding Werenka's skate near the goal line before Pye was able to smother it.
WERENKA: It happened so quickly, it was hard to adjust. When you're a defenseman and there's a play in the crease, you want to get your feet up. But it all happened so fast, all I could do was angle my foot so the puck wouldn't go into the net. I twisted my skate blade to try and stop it, but the problem was that Billy was also sliding back toward me at the time. Once he grabbed it, I thought we had a really good chance of winning. If that didn't go in, we had a good chance to win.
COMLEY: When McEachern came down, hit one post and then the puck slid across before Werenka cleared it…I honestly had started to step off the bench to congratulate Jack Parker. When I saw that it hadn't gone in, I carefully stepped back, hoping nobody had seen me.
With 40 seconds remaining in the first 10-minute overtime period, Northern Michigan's Tony Szabo had his own close call, blasting a slapshot from just inside the blueline and hitting the crossbar behind Cashman. The second overtime period didn't solve anything, with Cashman and Pye making a series of diving stops. But at 1:56 of the third overtime, history was made. Comley had bumped up Beaufait from fourth-line center to third-line right winger, joining Antos and Plandowski. That combination turned out to be pivotal. Led by Antos, the line approached the Terriers' zone with speed. Plandowski skated the puck over the blueline, then made a nifty drop pass between his legs to Beaufait. It was a risky move, and Plandowski immediately circled back in defense mode after making it.
PLANDOWSKI: I knew Amonte was on the ice. If he got the puck, he'd potentially have a breakaway, so after I made the pass I turned back towards our zone. (Beaufait) got it, and he was an underrated player, an elite skater.
WERENKA: That drop pass…it was pretty gutsy.
BEAUFAIT: I didn't think Darryl would put it through his legs in overtime, to be honest. I was just trying to give him space in the offensive zone, and when he dropped it back, I picked it up. I didn't think I could get a shot off. I kept carrying it wide, but the D-man had his stick on me.
WERENKA: Beaufait was a talented guy and had so much patience. He may have been on our fourth line, but he had 49 points. They had to respect his shot, and he kept pushing on his forehand. There were three guys on him, and the goalie followed him, too. He put it in the perfect spot to Darryl.
PLANDOWSKI: (Beaufait) drove wide and all the Terriers followed him. As I turned back toward the net, there was now a big, empty area. I don't know if I yelled or if he looked over his shoulder, but he put a perfect pass on my stick.
BEAUFAIT: I definitely didn't hear Darryl yell, but I knew I had dragged defenders with me. I knew one of our guys would go to the slot, because that's what you're taught in hockey. Go to the net.
COMLEY: I can still to this day see the winning goal develop. Time seemed to stop, and you're going, "Darryl, shoot it, shoot it!"
PLANDOWSKI: Sometimes you rush things and misfire, so I made sure I hit the net. You practise that play a thousand times, but it's different in a game. I took a split-second before I shot it to make sure.
And make sure he did. With an 8-7 victory in triple OT, Northern Michigan secured its first and only national championship.
RONAN: I probably overplayed my position on that play. But that's what life is, you make mistakes and you learn from them.
PLANDOWSKI: I got credit for the goal, but three or four things happened in a 30-second sequence to make it happen.
BEAUFAIT: I played center the whole year. I had never played on the wing until that point. We were a deep team. I was on the fourth line, but I was also on the power play. I was fresh heading into overtime, because I hadn't played as much as some of the other guys.
LACHANCE: At that point, you're just trying to get the next goal. You're nervous and excited. When Plandowski scored, we couldn't move on the bench for a full minute. We were in the room for over an hour afterward. The way that game ended was historic, but it was hard for us. It was a tough one to be a Terrier for.
McEACHERN: I just remember cramping up in the third overtime. It was pretty disappointing. We thought we could have won it all that year.
MELONE: It's finally over. You're just so exhausted at that point. It was one of the longest games in U.S. college history. You're running around celebrating, but you just want to lie down.
WERENKA: As soon as the puck went in, it was pure elation, especially for us seniors who had been there so long. Just elation and exhaustion. With hockey players, you hate losing more than you love winning, but this was one of those rare times where those two things were connected perfectly. Years later I put on the tape and I almost couldn't watch it. I kept pausing it and putting my head in my hands. I can't imagine what it was like to be a fan watching the game back then.
RONAN: To have 14 goals scored in regulation, and then none through several overtime periods…it was like two different games.
BEAUFAIT: It's surreal. That game was so up and down. The biggest feeling was relief. I couldn't believe we had won a national championship, a small school like ours.
The afterparty was just as sweet for the Wildcats – and a huge bash for the scores of fans who made the trip from Michigan's Upper Peninsula to see their boys win it all.
COMLEY: It was great. We stayed in St. Paul before heading back. The police arrested a bunch of our fans, it was a mess. But I remember landing, and the highway was lined with people.
PLANDOWSKI: That group of players had been together a long time, and we were really close with the community. That's the great thing about a small college town like Marquette. Everybody knew us. We had parents, ex-teammates and the community all there with us.
BEAUFAIT: They travelled well. When we got back to town the next day, there were limousines and cars to take us in from the airport to the pep rally. The gym was jammed, it was special.
WERENKA: There was a convoy going back miles and miles to Marquette. It was special. That's what college hockey is all about.
RONAN: I've won a Stanley Cup (with Montreal in 1993) and I can tell you that the end results of games are not what I remember as much as pieces of the journey itself. Obviously it hurt, but you know that in one game, anything can happen. There are other emotions. It was my last college game ever, so those are more the emotions you have to deal with, knowing you wouldn't be playing with those guys again and that you might be playing against them in the future.
While B.U. lost the title, the future was bright for many of the Terriers. Amonte, McEachern and Tkachuk went on to have long NHL careers, while Lachance played for more than a decade before retiring and becoming an amateur scout for the New Jersey Devils. But even now, that 1991 Frozen Four final comes up.
LACHANCE: Darryl Plandowski is a scout for the Tampa Bay Lightning now. I see him too often. He never says anything about that game…but he doesn't have to.