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Freelance graphic designer for hire tampa jobs
I need a glitzy graphic for a web site header and facebook page. The customer is the Judah P. Benjamin Camp #2210, Sons of Confederate Veterans in Tampa, FL. Their Nickname is the "Confederate Cabinet Camp". You can see their current 'branding' LOL here: [login to view URL] and on facebook: @jpbscv You can google pictures of Judah P. Benjamin
Tampa Strength...Personal Training Studio with an emphasis in Golf Specific Performance Training...need an ad for a personal trainer. Example Personal Trainer Wanted Graphic
I am a children's and family photographer in Tampa, Florida. My work focuses on creating the magical feeling that children experience and creating work that reflects their view. I want a client magazine created, approximately 24 pages, 8.5x11. I will have it printed through Miller's Lab. I want the magazine to reflect my story, the investment the client
Need to establish a Logo for "Entrex Opportunity Zone Fund" And.... the winner will also make a series of: Logo's for Geographic "Entrex Opportunity Zone Fund" Example being: "South Carolina Entrex Opportunity Zone Fund"
I need a auto market website for used cars. I have 20 different dealerships that advertise with me and maybe more and I obviously I advertise their used cars. I would like to have a drop down menu where you can search by price and also search by miles away like a filter how far the dealership is way from by miles or
I need to build a website to Market and advertise used cars for sale . I've 20 different dealerships that advertise with me so I'm going to ask your opinion if I should have each dealership have their own page with their with their cars if it's easier for you to do that way or just saw mixed I don't know I'll let you figure that out.
1. Search within 20 miles of this address a. 913 S Dale Mabry Hwy • Tampa, FL 33611 2. Use provided resume. a. Use contact info on resume with email 3. Search for all jobs in "Retail" 4. Available to start immediately 5. Full time or part time
I'm not much of an artist. I need a logo for my web development portfolio site. I'm based out of Tampa, FL and would like it to incorporate a lightning bolt. Also, I want to have my first initial "C" and last initial "B" in there somewhere. Please don't just make a cool lightning bolt. I want it to convey energy and look sharp.
Just to document and make a record of my husband getting his personal belongings out of the house and the garage. This will be done on a Saturday ...and make a record of my husband getting his personal belongings out of the house and the garage. This will be done on a Saturday early afternoon, possibly July 21st, in East Tampa (near Temple Terrace).
To start with what I am NOT looking for. I do NOT want my name with a roof over it or a building or a house - that is a sure-fire way to a rejection. Again ANYTHING WITH A ROOF OR A HOUSE WILL BE REJECTED. Thank you :) NEXT - what we ARE looking for: The team name is as follows: Josh Hanoud Team ...and that is what needs to be in the logo. Thought
...part time work in St. Petersburg, Florida and Tampa, Florida to distribute flyers to local businesses. Your job will be to find potential candidates and get them to call us for the position. All candidates must be local to St. Petersburg, Fl or Tampa, Florida and will physically report to our location for training and then walk around neighborhoods
Manage a $15M building modifications at two local Tampa locations
Im looking to hire some one long term to search classified ads on facebook marketplace, craiglist and other sites. And talk the seller down on a low price. then notify me of the sellers best price. I listed this as a contest here: https://www.freelancer.com/contest/Find-me-some-Leads-1368720.html because i want to hire the person who gets the best
Im looking to hire some one long term to search classified ads on facebook marketplace, craiglist and other sites. And talk the seller down on a low price. then notify me of the sellers best price. I listed this as a contest because i want to hire the person who gets the best results. After the contest is over I need someone to sign in 1 hour
Please design a logo for the following Company Name: Tidy Tampa Provides trash and pet pick up services. Potential themes to consider: Tampa skyline, city, clean, recycle, trash pick up, pet pick up, clean, professional, simple, funny
website for contract review and contract templates services local to tampa bay- maybe wordpress site? want to have standard pages- blog, about us, services, contact, FAQs, maybe a form to fill out etc. don't yet have a domain but can get one on blue host or go daddy-
I'd like for you to take some ideas, Trace them in vector and create a unique logo for me. I have a property management company called "Urban Island" and I'd like the logo to be centered around Tampa Florida, a tropical city.
Hi we are a Microsoft partner, based in Tampa, Florida and looking for lead generation and appointment setting for customers in East Coast. We will pay 50$-100$ per lead based on the quality of leads generated who have actual interest in buying Azure and Microsoft Products such as Office 365 and Azure.
Looking for a very experienced php developer with OSCLASS BENDER THEME (ONLY) Please only reply if you are very fast at the osclass bender theme. Site: [login to view URL] Budget: $100 Tasks needed: 1) Change title of price on post pages to "$20 Dollars" with dollar sign before number and and dollars as plural with an s. 2) Allow homepage location dropdown
we're a manufacturing company of commercial furniture in Tampa. We're looking for an AUTOCAD person to create drawings and send them to us. I have attached the samples below. These are only exterior dimensions, with the same dimensioning conventions. The CAD person would receive the quote/data, and create drawings per our specifications/conventions


text: In respect of EU Driving Hours the responsibility for drivers compliance with the regulations always lays with the driver himself and his employer. In the case of an agency worker it would seem that since the driver is being paid through the agency they should take the responsibility for the compliance. Nevertheless, the vehicle operator remains responsible for the safe running of the trucks therefore should make sure the drivers are compliant and company needs to have procedures in place to be able to effectively monitor agency drivers. In other words agency drivers although taken care for by agency should still be monitored by vehicle operator same as employed drivers to ensure compliance.

subset: commoncrawl


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Weight: 10g
This brush is great for blending paint round ears nose eyes etc
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These are optional but provide a means of displaying more content and using less space on the page.
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Widely regarded as the authoritative reference in the field, this volume comprehensively reviews theory and research on the self. Leading investigators address this essential construct at multiple levels of analysis, from neural pathways to complex social and cultural dynamics. Coverage includes how individuals gain self-awareness, agency, and a sense of identity; self-related motivation and emotion; the role of the self in interpersonal behavior; and self-development across evolutionary time and the lifespan. Connections between self-processes and psychological problems are also addressed. New to This Edition *Incorporates significant theoretical and empirical advances. *Nine entirely new chapters. *Coverage of the social and cognitive neuroscience of self-processes; self-regulation and health; self and emotion; and hypoegoic states, such as mindfulness.Widely regarded as the authoritative reference in the field, this volume comprehensively reviews theory and research on the self.
|Handbook of Self and Identity
|Mark R. Leary, June Price Tangney
|Guilford Press - 2012
You must register with us as either a Registered User before you can Download this Book. You'll be greeted by a simple sign-up page.
Once you have finished the sign-up process, you will be redirected to your download Book page.How it works:


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You are here
2016 Opening Remarks
Dean's comments at Crane Faculty Gala 9/6/16
Last year at this time, I made some observations about the start of the College's bicentennial year, which also happened to be Crane's 130th. It has been, I think, a meaningful year of celebration and reflection, with a great deal of looking back and some looking forward. Now we have turned our attention more firmly forward, coinciding well with a campus strategic planning process President Esterberg has been leading, and which will conclude by December. As we begin SUNY Potsdam's third century, I invite you to share a few more thoughts with me about what this occasion invites us to consider.
I've noted before that our observation of an anniversary like this one is really just an accident of the decimal system; with another system we'd be observing a different turning of the odometer. Whatever numerical intervals we choose, however, it's not a bad idea periodically to take the time to ponder where we have been and where we are headed.
It seems to me that human beings, when they do this, too often are drawn to one of two impulses: to hold on too dearly to what is past, or what we think was in the past; or to jettison the past in contemptuous pursuit of a brand new direction.
Of course, as tempted as any of us may be by the allure either of an unshakable past comfort zone or an enticing new unknown vector, we really all know that – as usual – the wise and subtle choice is a path in between, one which holds onto enduring values and lessons from our history while continuously testing them and adapting them to new circumstances, newfound knowledge, or new opportunities. While it does not give us a great deal of specific guidance, that's what I like about the phrase "a tradition of innovation" that I've used about Crane – there is a very strong sense here of being rooted in a tradition, but also a commitment, which has endured throughout Crane's history, of doing new things, of bringing new things to that tradition.
Knowing something about where we've been, which has been much on our minds this past year, helps us understand something about enduring themes that have become part of the fabric of the institution, and also to understand where we have been wrong or misguided in the past. Knowing about our past gives us a point of reference for new ideas: Have we tried this before? Did it help us, or not? Have circumstances changed to make it succeed now when it didn't then? If we went in this direction, would it take us down a path that is discordant with the values that give us our identity, or would it help us illuminate, enliven, and live out those values?
So, as we stand tonight at the start of Potsdam's third century, What's new? What calls us?
Part of what we have before us is the certain continuation of an era of what the higher education press has dubbed "disruptive" change. We must deal with what we know now are new realities about limited and declining state support, challenging and changing demographics in the prospective student population, a shifting and unpredictable career market for our graduates, and a number of other fundamental issues which will keep us on our toes and demand of us the utmost in agility, flexibility, and creativity.
As always, in this disruptive environment, there is opportunity. Among the opportunities and directions that are most on our minds at SUNY Potsdam these days are a few which I suggest are both new and old; it's our task to see what we can do with these, and with other innovative ideas which emerge, within the parameters of our time.
One is applied learning, an area in which SUNY Potsdam is taking the lead within the SUNY system: the idea that we facilitate a wide range of hands-on learning experiences for our students.
I don't need to tell an educated musical audience, of course, that this is frankly tradition for us: musical education is rooted in the doing alongside the studying. We even officially call an important part of every student's curriculum "applied music" – the application of musical study to performance on an instrument or voice. Hands-on learning happens in numerous other ways at Crane, of course, including practicum and student teaching experiences, music business internships, and student-faculty research. It has also long been embedded in many other programs throughout the College. Without in any way diminishing the importance of groundedness in academic study, as we stand at the start of Potsdam's third century, it feels to us that applied learning will be one theme of that future, not as something new or foreign to us, but something that we can expand, invigorate, and celebrate, something that we already know we do well, and something that higher education more generally perhaps is just beginning to understand and embrace. You are in for your very own applied learning experience tonight, so with it I welcome you to Potsdam's third century.
A second theme is interdisciplinary learning: learning and activity which crosses the boundaries of academic disciplines and sub-disciplines, and invites the exploration of new convergences, opportunities, and cross-fertilizations.
This, too, is not new to music nor to Crane. In every work on tonight's program that employs human voices, there is a marriage of literary and musical art which, together, provide us with an experience that is like neither, but something new altogether. One of the pieces on the program is explicitly derived from dance, and others are certainly related to the primordial association of music and human movement. The last work on tonight's program comes from a composer who perhaps had the most interdisciplinary perspective of all, Wagner's Gesamtkunstwerk, or total work of art, in which the composer as artist brings together words, music, movement, and visual art to envelop the listener/observer in a total experience.
So this, too, is familiar territory; although we must always be ready to undertake new steps across traditional boundaries, bring things together in new ways, and remove obstacles that get in the way of collaboration, we have had lots of practice at this. We're ready to take it on.
Finally, I suggest that there is an important theme of creativity and its practical cousin, entrepreneurship, in our effort to serve students in the twenty-first century. Creativity, of course, is the ground from which our musical art springs. We talk a great deal these days about the challenges professionals in music and other artistic endeavors face, and the fact that members of the millennial generation will change careers many times during a lifetime of work; this, too, is not new to music. Musicians have dealt over and over with disruptive changes that have challenged how they operate and how, to be mundane about it, they make their living. The composers represented on tonight's program represent an historical range of artists who worked within systems of aristocratic patronage, religious employment, print publication, operatic production, and virtuoso performance, none of which ever held stability for very long, in the greater scheme of things. As Western music moved from private music-making to dissemination by print publication to ticketed performances to recordings, broadcasts, and more recently to iTunes and YouTube, musicians have continually reinvented themselves and found ways to make the new reality one in which they could not only operate but thrive and create. This is not to be glib about it; it has not been easy at any point, nor will it be in the future – but we know this territory well, and we can bring it to the students of the twenty-first century.
Welcome to Potsdam's third century. We have had lots of practice for it, and we are ready. Please enjoy your applied learning experience tonight.


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This information is provided for your convenience and may no longer be valid.
Status: Closed
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Be the first OregonLive.com user to add photos or videos of Architectural Design Works
Architectural Design Works can be found at Siskiyou Blvd 1105. The following is offered: Architecture. The entry is present with us since Sep 9, 2010 and was last updated on Nov 14, 2013. In Ashland there are 8 other Architecture. An overview can be found here.
Since 1973 the company has been providing Engineering Services.


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SuperSU v2.72 Beta Cracked APK is Here ![Marshmallow Fixed]
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Friends if you want advanced management of smartphone which will give you many features and functions which will make your mobile phone as smartphone, and then you will need SuperSU Pro Apk. You will need this application as much software requires Root privileges to run. SuperSU Pro Apk is the industry de-facto standard when it comes to tweaking advance option in your smartphone.
SuperSU is the Superuser access management tool of the future! SuperSU allows for advanced management of Superuser access rights for all the apps on your device that need root. SuperSU has been built from the ground up to counter a number of problems with other Superuser access management tools.
SuperSU requires a rooted device, you have to root first to install this management tool.
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A 26-year-old rookie police officer shot himself in the leg during a domestic dispute call in Brooklyn Saturday night.
The unidentified NYPD officer was responding to a report that a man attacked his 50-year-old mother at their Hasley Street home in Bedford Stuyvesant around 8 p.m.
The officer pulled out his gun during a confrontation with Junior Daniels, 24, who appeared to have beaten his mother.
The cop, who had been on the job for one year according to multiple outlets,holstered his weapon and accidentally pulled the trigger.
Daniels was arrested for assaulting his mother.
The officer was hit in the right kneecap, police said.
He was taken to Kings County Hospital and is in stable condition.
Follow Anna Sanders on Twitter: @AnnaESanders


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2011-05-26 / Front Page
Rains Not Enough to Lift Burn Ban
Court Issues Early Restrictions on Fireworks
The Lee County Commissioners voted unanimously on Monday, May 23, to continue the County Wide Burn Ban. This is in spite of the recent rains. Although some parts of the County received up to 5 inches of rain, other areas received as little as an inch and a half.
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Status: Open
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055351attr:jeansclothing & accessories retailjewelryshoes
Music. Pop Culture. Fashion.
Be the first NJ.com user to add photos or videos of Hot Topic
In Short
You won't find Britney posters or pop mags in this warehouse store; it's all grunge, punk, goth and whatever's considered edgy and far left of center this week. Blink-182...
This place is amazing! They have the best band shirts and the best Nightmare Before Christmas merchandise ever! Their staff are really nice as well as the manager. :) I never had any problems here throughout then 6 years I've shopped here. I'm always leaving satisfied and sad because I want to buy more!
The staff is pretty relaxed and friendly, at least most of them are. It depends on which employee you are interacting with. The merchandise sold is averagely priced, with few, but sometimes possible, expensive items. The items sold are musically/tv show/ film/cartoon related, ranging from band shirts to wallets to pants to CDs.
Hot Topic is located at 1228 Garden State Plz, Paramus, NJ. This business specializes in Clothing.
Conveniently located on the first level next to the food court in the Garden State Plaza, Hot Topic is always on the cutting edge of music and pop culture- inspired teen and young adult fashion. It sells a wide assortment of clothing, jewelry and accessories as well as CDs and novelty items.


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The Mouth of the Southis surprisingly understated as narrator of "Call Me Ted." Anecdotes gathered by co-author Bill Burke provide expected color and emotional shadings, but the overall portrait can't help but disappoint those expecting more a lively discourse from Turner himself. This brisk chronicle does, however, serve as a poignant reminder of his many showbiz accomplishments before AOL Time Warner architect Gerald Levin unceremoniously pushed him to the sidelines.
Turner, a restless entrepreneur who wasn't afraid to color outside the lines, transformed his father's regional billboard biz into a large showbiz concern that he fatefully sold to Time Warner in 1996. Never easily cowed, he overcame countless obstacles, matter-of-factly outlined in the book, while building his broadcast biz: He turned an Atlanta UHF channel into a cable SuperStation, created the first cable 24-hour news station a few years later and won sailing's America's Cup in between. Thwarted in his attempts to buy CBS, he nearly bankrupted himself buying MGM, outraged film purists by colorizing black-and-white classics from its library, and encouraged New Line toppers to swing for the fences, perhaps to their detriment.
Turner found his match in Levin, an old cable crony who engineered the disastrous AOL deal and soon stripped Turner of power, if not his title.
Frustrated and bitter, Turner stepped down as vice chair a few years later, finally exiting the company's board in 2006.
This portion of the book is especially frustrating. Turner has frequently sounded off about the merger in conversation but is strangely muted describing the deal in the book itself. Some of this no doubt is due to another Turner foible — his deep aversion to introspection — and the fact he didn't have anything to do with the negotiations, conducted while he was distracted by his split with Jane Fonda. Yet one can't help but yearn for some cathartic venting here, or at least greater understanding about the momentous turn of events.
Neither, sadly, is forthcoming. Turner duly notes his initial public support for the merger, initially hailed as a deal for the new millennium, then flatly adds, "Little did I know it would all be downhill from here." His description of the company's restructuring a few months later is equally disappointing: Outraged buddy John Malone recalls telling Turner he was stabbed in the back, but Turner only says that he was in shock.
"It's hard to know how I would have felt if the company had been doing well, but standing on the sidelines while our stock and my personal fortune cratered was hard to take," he blandly adds.
Turner has been considerably more forceful on the book tour, sounding off about Levin and Parsons with occasionally salty language.
Nor is "Call Me Ted" particularly illuminating about Turner's decision to approve the merger. Several associates, including Malone and Michael Milken, offer conflicting accounts of Turner's attempts to seek counsel before giving thumbs up, but the episode remains awfully murky. It's certainly interesting to read Levin's take on the situation, but one wishes Burke's familiarity with the events — he quit his job as a Turner exec a few days before the merger — paid off more here.
Burke's decision to weave "Ted stories" throughout the narrative works better in some cases than others. There are surprise entries by famous figures such as Bill Gates (discussing an abandoned merger) and Jimmy Carter (on religion), jaw-dropping anecdotes (Dick Parsons recalls Turner telling him, "You were born black — bad break!") and humanizing tales from friends and family. The technique is especially effective with family members: An aunt's description of his father's abusive behavior is far more potent than anything Turner would have written. His kids' comments about his absent parenting style — something Turner clearly regrets — convey their love and understanding.
These days, the former media mogul devotes his time to charitable endeavors and his chain of bison restaurants. He has even made up with former rival Rupert Murdoch.
Showbiz, however, is a lot less colorful without him in it.


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It was time. This Blogspot blog has served me well but after helping some people with WordPress I began to realize how much more it could do. So I moved to a "real" domain (no ".blogspot" in the address) and switched to Wordpress. It's much better.
For readers, the only real difference (besides the fact that the look of the site will probably keep changing as I play with styles) will be the change in URL. Simply remove the "blogspot" and the extra period from the address. So replace:
OLD: http://scottontheorwigs.blogspot.com
See you there!


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The lists of safe senders, safe recipients, and blocked senders are all last-step controls to message hygiene on the client. Safelist aggregation extends the reach of client configuration to an Exchange 2007 transport server. For example, neither the Edge Transport server nor the Hub Transport server with the antispam agents installed need to waste resources filtering a message in which the sender's email address is included in the recipient's Contacts folder and resides in their safe recipient list.
Don't confuse safelist aggregation with server sender and recipient filtering for Exchange 2007. Information in safelist aggregation is verified during the Content Filter process, which reduces the instances of false positives because the server hygiene filters are using more specific information from the clients.
Safelist aggregation isn't an Outlook 2007 setting, but it's certainly a benefit of using Exchange 2007 with Outlook 2007. Safelist aggregation represents a compilation of safe SMTP addresses and safe domains listed in the safe senders and safe recipients lists for a mailbox. The sum of these entries for a mailbox is called a safelist collection, which holds a maximum of 1,024 unique entries. You can implement safelist aggregation per mailbox by executing the Exchange Management Shell command:
\[PS\]> Update-SafeList -Identity <MailboxIdParameter>
\[-DomainController <FQDN>\] \[-Type <SafeSenders | SafeRecipients | Both>\]
which takes the safelist collection data from mailboxes and places it into hashed entries stored in the AD record for the account that owns the mailbox.
From there, the local Hub Transport accesses the content for filtering. That data can also be part of an Edge Synchronization subscription to allowing the Edge Transport Server to access safelist content through Active Directory Application Mode (ADAM). When users make changes to their safelists in Outlook, those changes are not automatically applied to safelist aggregation. You can schedule the PowerShell command to implement safelist aggregation at regular intervals to maintain current information for server-side content filtering.
Encourage users in your organization to maintain accurate junk email whitelists and blacklists. By doing so, they can add value to the overall performance of message hygiene in the Outlook 2007 and Exchange 2007 enterprise.


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This past weekend has been spent doing such intense revision I feel like I may have suffered an information overdose. I could, however, tell you all about the analysis and identification of compounds in chemistry (that I'm sure you'll be more than enthused to hear about.) The glorious, if not untimely, sunny weather has left me all in the mood to ride my bicycle a la MK in sweeping (impractical) dresses all the way to the seaside. Failing that, I may just lie in my garden and listen to The Beatles. Bliss.
It's less than 5 weeks until I leave school (trust me, I'm counting down the days) and it could not come soon enough. I'm not sure how much longer I can take trying to make intellectual conversation about the world with people who last read a newspaper NEVER without going mad. Honestly, it doesn't take much to just listen to the news once in a while, or to maybe read a book and not think about boys/tans/parties for just a moment, does it? The school I attend is really quite small-minded, racist, homophobic, bitchy and for the most part tory-dominated and honestly I cannot wait to get the fuck out. I genuinely find myself wanting to punch some ignorant fucker in the face at least once a day. Wankers. < /angry>
Chloe x


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For more information, contact SRTS Coordinator John Schaefer, (573) 751-2840
JEFFERSON CITY - The Missouri Department of Transportation's Safe Routes to School advisory committee has awarded 34 non-infrastructure grants totaling $582,000 to communities across the state.
Safe Routes to School is a program of the Federal Highway Administration created in 2005. MoDOT administers the program that is designed to:
•· Enable and encourage children, including those with disabilities in grades K-8, to walk, wheel, and bicycle to school;
•· Make bicycling and walking to school a safer and more appealing transportation alternative, thereby encouraging a healthy and active lifestyle from an early age; and
•· Facilitate the planning, development, and implementation of projects and activities that will improve safety and reduce traffic, fuel consumption, and air pollution in the vicinity of schools.
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A Keynotes database can be created using any Spreadsheet or Database application, as long as it can export to Tab separated Text or CSV (comma separated values).
Each Keynote requires a Key, a Short Description or Title, and a Long Description, you can also include a Specification Reference if you want.
If you add a Category as well, then Cadimage Keynotes will group the keynotes into Categories, otherwise all of the Keynotes will be put in the same category.
And if you set the first row to be a column description, then keynotes can automatically work out which column is which.
Once you have created your database of Keynotes, export them as either Tab separated text or csv.
In ArchiCAD open the Keynotes Manager dialog using the Cadimage > Keynotes > Keynotes Manager menu.
Click on the Load Database button, select the file type you are using and browse for your file.
Make sure the columns are assigned correctly and click Import.
Then click the Add All button to add all of the Keynotes to the project.


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Google adds download defense to Chrome, patches 15 bugs
Google on Tuesday updated Chrome to version 12, adding a new tool that warns users when they've downloaded files from dangerous Web sites.
The company also patched 15 bugs in the browser and paid out nearly $10,000 in bounties to outside researchers who reported vulnerabilities to its security team.
New to Chrome 12 is a feature that flags dodgy files pulled from the Web. Chrome now shows an alert when users download some file types from sites that are on the Safe Browsing API (application programming interface) blacklist, which Google maintains.
The messages reads: "This file is malicious. Are you sure you want to continue?"
If they wish, users can ignore the warning and save the file to their system's hard drive.
"This warning will be displayed for any download URL that matches the latest list of malicious websites published by the Safe Browsing API," said Google last April when it debuted the feature in an earlier edition of Chrome.
Safe Browsing already identifies suspicious or unsafe sites, then adds them to a blacklist. Chrome, Mozilla's Firefox and Apple's Safari all tap into Safe Browsing to warn users of risky sites before they actually visit them.
But by expanding its use of Safe Browsing to signal users of not just malicious sites, but also the downloads that come from them, Google is following in Microsoft's footsteps.
Internet Explorer 9 (IE9), which launched in mid-March, uses something Microsoft calls "SmartScreen Application Reputation" to rank the probability that a download is legitimate software. Files that don't appear legit trigger a warning if users try to run or save them after downloading.
The new tools within IE9 and Chrome have been applauded by security researchers because hackers don't always rely on exploits to plant malware on machines. They are often able to trick uses into doing their work for them.
Fake antivirus software, called "scareware," is a good example. Malicious sites make visitors believe their PCs are infected, and then pitch them worthless security software that can supposedly clean their computer.
Some Mac users got a first hand look at scareware last month when an experienced gang that had worked the Windows side of the street kicked off an aggressive campaign to also sell fake Mac antivirus software.
Other improvements in Chrome 12 include additional support for hardware-accelerated 3-D graphics in Windows Vista, Windows 7 and Mac's Snow Leopard.
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Representatives from large corporations – CIBC, KPMG and Pitney Bowes as well as smaller companies – Questrade, Advanced Precision and XEA, identify several ways in which they set themselves apart from other employers and retain and promote immigrant talent.
These include:
- Identifying talented employees at the entry level and helping them grow into more senior roles
- Reward innovation and give employees the chance to grow their skills
- Identify and remove any institutional barriers to promotion
In this video, the third in a series, learn how these employers retain skilled immigrants and brand themselves as employers of choice in the video below.
Go deeper by listening to these extended interviews on how to manage a diverse workforce in this podcast.


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Alexandria Covenant Church recognizes and values children who have not been baptized as infants to be full participants in the community of faith. The rite of blessing a child, also commonly referred to as the dedication of a child, is a service for parents who want the blessing of God on their child but believe that baptism is to be reserved for those who make a conscious choice in confession of faith.
Grateful for the gift of a child, the parents are accepting their responsibility of nurturing the child in the love and grace of God. The emphasis in infant dedication is on the personal pledge of parents to raise and nurture their child in a loving Christian home. There is anticipation and hope of the child's future personal commitment of faith in Jesus Christ.


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The Welsh club lie mid-table in the Coca-Cola Championship - but so buoyant are they after their 1-0 semi-final win over Barnsley at Wembley that Jones believes they can make it to the play-offs.
"My problem is getting them up and running again," he said. "We want to keep it going. We have a slim chance of the play-offs - and while we have, I won´t let them rest."
Copyright (c) PA Sport 2008, All Rights Reserved.
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Welcome to the Body Composition Analysis Center and DXA Lab at Tufts University
The Body Composition Analysis Center (BCAC) of the Tufts University Gerald J. and Dorothy R. Friedman School of Nutrition Science and Policy offers an unmatched combination of scientific expertise and cutting-edge computer technology for the analysis of bone density and body composition. Associated with the world-class research laboratories for the study of aging, nutrition, and physiology at Tufts, the Body Composition Analysis Center is a leading referral center in meeting the complex data analysis needs of other laboratories, health-care facilities, and research organizations.
The expertise of Tufts researchers, who are recognized as national leaders in their field, make the Body Composition Analysis Center an unparalleled resource for:
Dual-energy x-ray absorptiometry (DXA) is a useful measurement primarily used to assess bone mineral density, total body composition, and body fat content with a high degree of accuracy. DXA bone density testing has proven to be the most accurate method available in the diagnosis of osteoporosis and is also considered an accurate estimator of fracture risk. DXA is a widely used and effective method for research, as it is quick, convenient, and non-invasive. Moreover, radiation exposure to patient subjects is extremely minimal- less than one-tenth the dose of a standard chest x-ray.
The Body Composition Analysis DXA Laboratory at Tufts University is equipped with a Hologic Discovery A scanner, which performs total body, lumbar spine, femur, and forearm scans. The lab maintained and operated by a fully trained and certified scan technologist.
Please note that DXA scans are conducted exclusively on applicable research participants. For a list of current Tufts studies involving DXA, please visit the HNRCA volunteer website or contact volunteer services at 1-800-738-7555
Roger A. Fielding, PhD


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it's not often someone gets the chance to go to a Formula One factory and enjoy a charity dinner"
(PRWEB UK) 4 July 2014
Aspiring professional racing driver Raoul Owens had the rare opportunity to enjoy an evening in the company of the Williams Martini Racing FIA Formula One World Championship team on Wednesday, 2nd July, as part of a specially organised charity dinner at the squad's Oxfordshire headquarters.
Hosted at the Grove HQ by Sir Frank Williams and his daughter Claire, deputy team principal, the exclusive event during the build-up to this weekend's British Grand Prix at Silverstone was held to help raise awareness of, and funds for, the Spinal Injuries Association which works to support the wellbeing of the 40,000 people in the UK with spinal cord injuries.
Raoul, who is currently on the path towards F1 in the Formula Renault 2.0 Northern European Cup (NEC) where he competes with the Mark Burdett Motorsport team, was joined at the dinner by his race engineer Andy Miller – former operations director at Stewart Grand Prix and Director of Racing at Jaguar F1.
Seated at a table alongside Williams Martini Racing Grand Prix drivers Felipe Massa and Valtteri Bottas, they were also joined by the team's official test and reserve driver Felipe Nasr and Williams F1 development driver Susie Wolff.
"It really was a great experience, it's not often someone gets the chance to go to a Formula One factory and enjoy a charity dinner," said a thrilled Raoul, "It was fantastic to meet the drivers and members of the team, Andy [Miller] and I sat at a table with Felipe Massa, Valtteri Bottas, Felipe Nasr and Susie Wolff so that was really good and very enjoyable."
"Being able to look around the factory was great and it was really nice to meet people from Williams who were all fantastic. Having the opportunity to see the amazing history of the team too was pretty special, Williams is obviously one of the oldest teams in Formula One and has had so much success over the years. The whole evening was very interesting and great to be involved in."
Returning to Raoul's own racing, his next outing in Formula Renault 2.0 NEC will come at the end of the month when the one-make championship visits Spa-Francorchamps in Belgium for the fourth event of the 2014 season on Friday, 25th July, and Saturday, 26th July.
Before then, the Pembury racer will join the blue riband Eurocup Formula Renault 2.0 series for its visit to the Nurburgring in Germany over the weekend 12th/13th July.
Note to editors: Copyright free image of Raoul Owens with Felipe Nasr and Susie Wolff at the Williams Martini Racing charity dinner attached for editorial use.
Website: http://www.raoulowens.com
Facebook: http://www.facebook.com/RaoulOwensRacing
Twitter: @raoul_owens
Media Contact:
Marc Orme
Tel: +44 (0)7920 886985
Email: marc(at)marc-orme(dot)co.uk / marcorme1(at)aol(dot)com


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Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA sequencing
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In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequence long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins.
Analysis of these data sets reveals that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently cataloged. We further find that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs reveals that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein.
We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon.
KeywordsGuide Region snoRNA Gene Primer Extension Assay Canonical Base Pair Clip Library
- Ago2
Argonaute 2
Cajal body
cross-linking and immunoprecipitation
- dNTP
deoxynucleotide triphosphate
- IP-seq
immunoprecipitation sequencing
- miRNA
micro RNA
- MNase
micrococcal nuclease
- ncRNA
non-coding RNA
photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation
- rRNA
ribosomal RNA
- scaRNA
small Cajal body-specific RNA
- sdRNA
small derived RNA
- snoRNA
small nucleolar RNA
- snoRNP
small nucleolar ribonucleoprotein
- snRNA
small nuclear RNA
- sRNA
small RNA
tags per million
- tRNA
transfer RNA.
Small nucleolar RNAs (snoRNAs) are a specific class of small non-protein coding RNAs that are best known for their function as guides of modifications (2'-O-methylation and pseudouridylation) of other non-protein coding RNAs such as ribosomal, small nuclear and transfer RNAs (rRNAs, snRNAs and tRNAs, respectively) [1, 2, 3]. Based on sequence and structural features, snoRNAs are divided into two classes. C/D box snoRNAs share the consensus C (RUGAUGA, R = A or G) and D (CUGA) box motifs, which are brought into close proximity by short regions of complementarity between the snoRNA 5' and 3' ends [4, 5] and are bound by the four core proteins of the small ribonucleoprotein complex (snoRNP), namely 15.5K, NOP56, NOP58 and Fibrillarin (FBL) [6, 7, 8] during snoRNA maturation. Fibrillarin is the methyltransferase that catalyzes the 2'-O-methylation of the ribose in target RNAs [9]. Most C/D box snoRNAs also contain additional conserved C' and D' motifs located in the central region of the snoRNA. The other class of snoRNAs is defined by a double-hairpin structure with two single-stranded H (ANANNA, N = A, C, G or U) and ACA box domains [10], and are therefore called H/ACA box snoRNAs. They associate with four conserved proteins, Dyskerin (DKC1), Nhp2, Nop10 and Gar1, to form snoRNPs that are functionally active in pseudouridylation. Although all four H/ACA proteins are necessary for efficient pseudouridylation [10], it is Dyskerin that provides the pseudouridine synthase activity [11]. While H/ACA and C/D box snoRNAs accumulate in the nucleolus, some snoRNAs reside in the nucleoplasmic Cajal bodies (CBs) where they guide modifications of snRNAs [2] and are called small Cajal body-specific RNAs (scaRNAs). In addition to the typical H/ACA snoRNA features, vertebrate H/ACA box scaRNAs carry a CB localization signal called CAB box (UGAG) in the loop of their 5' and/or 3' hairpins [12].
Immediately upstream of the D box and/or the D' box, C/D box snoRNAs contain 10 to 21 nucleotide-long antisense elements that are complementary to sites in their target RNAs [13, 14, 15]. The nucleotide in the target RNA that is complementary to the fifth nucleotide upstream from the D/D' box of the snoRNA is targeted for 2'-O-methylation by the snoRNP [14, 15]. H/ACA box snoRNAs contain two antisense elements termed pseudouridylation pockets, located in the 5' and 3' hairpin domains of the snoRNA [16, 17]. Substrate uridines are selected through base-pairing interactions between the pseudouridylation pocket and target RNA sequences that flank the targeted uridine.
Deep-sequencing studies revealed a surprising diversity of small RNAs derived from non-coding RNAs (ncRNAs) known as small derived RNAs (sdRNAs) with well-established functions such as tRNAs [18, 19], Y RNAs [20], vault RNAs [21], ribosomal RNAs [22], spliceosomal RNAs [23] and snoRNAs [24, 25, 26]. In fact, the profile of sequenced reads observed for some of these small RNA species are very characteristic and have even been used for ncRNA gene finding based on sequencing data [27, 28]. The majority of C/D box and H/ACA snoRNAs seems to be extensively processed, producing stable small RNAs from the termini of the mature snoRNA [29] and the processing pattern is conserved across cell types [30]. Thus, it appears that snoRNAs are versatile molecules that give rise to snoRNA-derived miRNAs [24, 31], other small RNAs [25, 29] or longer processing fragments [32].
To gain insight into the complexity of snoRNA processing and the functional relevance of the derived sdRNAs, we undertook a comprehensive characterization of products generated from snoRNA loci, combining high-throughput sequencing of long and short RNA fragments with photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins and with data from Argonaute 2 (Ago2) immunoprecipitation sequencing (IP-seq) experiments. We found that many loci in the human genome can give rise to C/D box-like snoRNAs. Among the novel snoRNAs that we identified are very short sequences, extending little beyond the C and D boxes, which are essential for the binding of core snoRNA proteins. Compared to the snoRNAs that are already known, the novel snoRNA candidates exhibit a lower level of evolutionary conservation and a lower expression level. These findings indicate that the C/D box snoRNA structure evolves relatively easily and that C/D box snoRNA-like molecules are produced from many more genomic loci than are currently annotated. We further found that C/D box snoRNAs are very specifically processed inside the regions of terminal complementarity, retaining in the mature form only four to five nucleotides upstream of the C box and two to five nucleotides downstream of the D box. Sequencing of the small RNA population as well as of the small RNAs isolated after Ago2 immunoprecipitation revealed that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein. Our extensive data thus indicate that, contrary to previous suggestions [25, 33], snoRNA-derived small RNAs that carry out non-canonical, particularly miRNA-like, functions are rare.
PAR-CLIP of C/D box and H/ACA box snoRNP core proteins identifies their RNA binding partners
Summary of CLIPZ mapping statistics and annotation categories for PAR-CLIP samples.
FBL (MNase)
NOP58 rep A
NOP58 rep B
Ago2 rep A
HuR rep A
Mapping rate
Library size
T→C mutations among all observed mutations
Other Categories
No annotation
Annotation summary of the top 200 clusters inferred from PAR-CLIP experiments with snoRNA core proteins.
PAR-CLIP library
C/D box snoRNAs
H/ACA box snoRNAs
mRNA exons
123 (61.5%)
9 (4.5%)
10 (5.0%)
5 (2.5%)
53 (26.5%)
FBL (MNase)
106 (53.0%)
16 (8.0%)
10 (5.0%)
26 (13.0%)
42 (21.0%)
115 (57.5%)
28 (14.0%)
15 (7.5%)
2 (1.0%)
40 (20.0%)
NOP58 rep A
114 (57.0%)
14 (7.0%)
10 (5.0%)
9 (4.5%)
52 (26.0%)
NOP58 rep B
125 (62.5%)
4 (2.0%)
10 (5.0%)
9 (4.5%)
52 (26.0%)
11 (5.5%)
62 (32.0%)
18 (9.0%)
7 (3.5%)
102 (51.0%)
Ago2 rep A
0 (0.0%)
0 (0.0%)
1 (0.5%)
59 (29.5%)
140 (70.0%)
HuR rep A
0 (0.0%)
0 (0.0%)
0 (0.0%)
117 (58.5%)
83 (41.5%)
Binding patterns of core proteins on snoRNAs
We further asked whether the binding pattern of Fibrillarin reflected in the abundance of CLIP reads differs between guide regions of the snoRNAs that have a target annotated in snoRNA-LBME-db and orphan guide regions. For guide regions, we took the nine nucleotides upstream of the D and D' boxes and as a reference we compared the coverage of the D and D' boxes themselves (Figure 1F). We found that guide regions with a known target and their associated D/D' boxes generally have a higher coverage compared to those that are orphan (70% compared to 40% positive z-scores of the average coverage per position in the guide region relative to the entire snoRNA, Figure 1G). This could indicate that the binding to the target renders the snoRNA-core protein complex more accessible to cross-linking.
For H/ACA box snoRNAs we found that Dyskerin strongly prefers the H box nucleotides (Figure 1G), which in 70% of the snoRNAs have a positive z-score for coverage compared to the entire snoRNA. This is expected because these snoRNAs are highly structured, with most nucleotides being engaged in base pairs in the two hairpin stems and a few nucleotides are free to interact with the proteins.
Identification of novel snoRNA genes from PAR-CLIP and small RNA sequencing
We screened the top 500 clusters from each PAR-CLIP library that did not overlap with known ncRNAs, mRNAs or repeat elements for potentially novel snoRNA genes. To identify H/ACA box genes we employed the SnoReport program [38], while for C/D box snoRNA detection we applied a custom approach searching for a C box motif (RUGAUGA, R = A or G; allowing one mismatch) at the 5' end and a D box motif (MUGA, M = A or C) at the 3' end, requiring that a terminal stem of at least four canonical base pairs can be formed by the nucleotides flanking the C and D boxes. We combined these computational screens with isolation and sequencing of the 20 to 200 nucleotide RNA fraction from HEK293 cells, which provides evidence for expression of the predicted snoRNAs. Requiring a minimal average coverage per nucleotide of at least 1 tag per million (TPM) in least one type-specific CLIP library as well as in the small RNA-seq library, we identified 77 and 20 putative C/D and H/ACA box snoRNAs, respectively (Additional files 2 and 3). We additionally screened 14 distinct small RNA sequence libraries from the recently released ENCODE data [39] and found that more than 75% of our putative C/D box snoRNAs were detected in at least one cell type other than HEK293 (see Additional file 4). We further tested the expression of the 20 most abundantly sequenced candidate snoRNAs by Northern blotting (see Additional file 5). Nine of the twenty candidates were also detectable in this assay, while an additional nine C/D box snoRNAs are supported by the ENCODE data (see Additional file 4).
To determine whether the candidates we identified as described are entirely novel snoRNA genes or so far undescribed homologs of known snoRNAs, we performed a BLAST search against the snoRNA genes from snoRNA-LBME-db (requiring an E-value ≤ 10-3). We further compared the loci of the putative snoRNAs with the snoRNA annotation available in ENSEMBL release 65 [40], which is based on automatic annotation with sequence/structure models available in the Rfam database [41]. Out of the 20 H/ACA box snoRNA candidates, 18 show sequence or structural homology to known snoRNAs, while candidates ZL4 (annotated as nc053 in [42], but not classified as a snoRNA by the authors) and ZL36 appear to be novel H/ACA box snoRNAs without a known homolog. The homology search additionally revealed that ZL4 is conserved until Xenopus tropicalis.
Of the 77 C/D box snoRNAs, only seven showed sequence homology to known C/D box snoRNA genes, but in one case (ZL1) the homology consisted solely of a long GU-rich region. The evolutionary conservation of the guide regions of five of these snoRNAs (ZL11, ZL109, ZL126, ZL127 and ZL132) suggests that they target the same nucleotides on ribosomal RNA as their homologs. A sixth snoRNA, ZL142, appears to be a human homolog of the GGN68 snoRNA of chickens [43, 44]. An additional comparison with the results of another large snoRNA analysis [45], revealed that ZL2 and ZL107 have been previously described as SNORD41B and Z39, respectively. In order to further characterize the 69 potentially novel C/D box snoRNAs (including ZL1, which only had homology with a known snoRNA in a GU-rich region), we first asked whether their C and D boxes are evolutionarily conserved (Additional file 1). To this end, we computed their average position-wise phastCons scores [46], which we obtained from the UCSC genome browser. Five candidates including ZL1 showed an average phastCons score per nucleotide higher than 0.25 for C and D box nucleotides. A comprehensive homology search of vertebrate genomes allowed us to trace the evolutionary origin of these snoRNAs and to annotate C' and D' boxes as well as putative guide regions based on sequence conservation. ZL1 is highly conserved in vertebrates including Petromyzon marinus, while for ZL5, ZL6, ZL8 and ZL24 we were not able to retrieve any homologs outside of mammals.
Our screen could further identify a snoRNA with mixed C/D box and H/ACA box structure. SCARNA21, a computationally predicted H/ACA box snoRNA [47], is surrounded by conserved C and D box elements enclosed by a terminal stem structure (Additional file 7). Northern blot analysis revealed that the prevalent form in the cells is the one that contains the C/D box elements and not the short form, which would be the single H/ACA box snoRNA.
Target prediction for newly identified snoRNA genes
We were especially interested to find out whether the non-conserved C/D box-like snoRNAs and in particular the mini-snoRNAs, could guide 2'-O-methylations. To this end, we took a simple approach searching for 8-mer Watson-Crick complementarity between the putative guide regions upstream of the D boxes to ribosomal and spliceosomal RNAs. We did indeed identify seven putative interaction sites, but none of these are known modification sites (Additional file 2). Thus, the targets of these C/D box-like snoRNAs remain to be identified.
Non-canonical RNA partners of core snoRNA proteins
Although snoRNAs are best known for guiding modifications of rRNAs, snRNAs and tRNAs [1, 2, 3], some evidence has emerged for the involvement of full-length mature snoRNAs also in other biological processes such as alternative splicing [51]. To investigate this possibility, we searched our PAR-CLIP data sets for RNAs that were abundantly cross-linked, yet not known to associate with the core snoRNA proteins. In contrast to the HuR PAR-CLIP that we performed before [35], the PAR-CLIP experiments conducted with C/D box snoRNP core proteins repeatedly identified several non-coding RNAs including vault RNA 1-2, 7SK RNA and 7SL RNA as well as H/ACA box snoRNAs. Similarly, in the Dyskerin PAR-CLIP we observed cross-linking of several C/D box snoRNAs.
We performed primer extension experiments to determine potential sites for 2'-O-methyl and pseudouridine modification in prominent ncRNAs such as 7SK RNA, 7SL RNA and vault RNA 1-2 (see Additional file 9 for primer extension assays and Additional file 10 for a catalog of identified modifications sites and target predictions). Indeed, we found that all three of these RNA species carry modifications. Vault RNA 1-2 contains four 2'-O-methyl sites, 7SK RNA carries at least six 2'-O-methyl sites and one pseudouridylation site, and 7SL RNA contains several sites of pseudouridylation. Additionally, we sought to determine whether C/D box and H/ACA box snoRNAs guide modifications on each other. We thus performed 2'-O-methylation primer extension assays on SNORA61 and pseudouridylation assays on SNORD16 and SNORD35A. We found that SNORA61 potentially carries one 2'-O-methylation, while SNORD16 and SNORD35A carry two and six pseudouridylated residues, respectively. To identify C/D box snoRNAs that could guide the observed 2'-O-methylations, we searched for 8-mer complementarity upstream of D and D' boxes of C/D box and C/D box-like snoRNAs, but we did not find sequences complementary to the modification sites. To predict guiding H/ACA box snoRNAs we employed the program RNAsnoop using stringent filtering criteria. We identified potential guiding H/ACA box snoRNAs for 7SK RNA residue Ψ250 and 7SL RNA residue Ψ226.
Previous studies reported that snoRNAs may function in alternative splicing [32, 51] and we also repeatedly observed cross-linking of C/D box core proteins to regions that are annotated as exons of protein coding genes. To determine whether these mRNA regions are targeted by snoRNAs, we selected, from the top 1,000 clusters located in mRNA exons in NOP58 libraries, the 157 that were present in both NOP58 replicates and a third CLIP library with at least 10 TPM per nucleotide (Additional file 11). We identified complementarities to the 8-mer guide regions of snoRNAs in 79 of these clusters. In contrast, in shuffled CLIPed regions we only found 60 complementarities to snoRNA guide regions (average of 100 simulations on shuffled sequences). Thus, the mRNA sequences that we isolated in the CLIP experiments are consistent with the possibility that snoRNAs act as guides in some steps of mRNA processing.
snoRNA processing patterns
Functional annotation of sequencing reads obtained in sRNA sequencing and HeLa Ago2 IP sequencing.
RNA class
HEK293 sRNA sequencing (18 to 30 nucleotides)
HeLa Ago2 immunoprecipitation sequencing (asynchronous cells)
HeLa Ago2 immunoprecipitation sequencing (mitotic cells)
C/D box snoRNAs
H/ACA box snoRNAs
No annotation
Small RNAs derived from C/D box snoRNA termini appear to be abundant in the cells, and can be incorporated into Argonaute proteins to act as miRNAs [31]. To determine the relative participation of various small RNA classes in the Argonaute-dependent gene silencing, we immunopurified Ago2 from HeLa cells and sequenced the associated small RNA fraction. We found that, as expected, miRNAs constitute the most abundant RNA class that associates with Ago2 (approximately 90%), while C/D box snoRNAs account only for 0.005% of the IP-seq reads (Table 3). Assuming that overall proportions of small RNAs derived from tRNAs and snoRNAs are fairly constant across cell types, we can estimate the efficiency with which small RNAs (from the total small RNA pool) are incorporated in the Argonaute proteins. We found, for example, that although small RNAs derived from tRNAs are 5.6 times more abundant than C/D box derived snoRNAs, tRNA fragments are 40 times more abundant in the Ago2-associated fraction. Thus, tRNA-derived small RNAs appear to be more efficiently incorporated in Ago2 than C/D box snoRNA fragments. This is consistent with observations that tRNAs are cleaved by nucleases such as Angiogenin and even Dicer to generate processing fragments that are active in translation regulation [54, 55]. Similarly, small RNAs derived from H/ACA box snoRNAs are 5.5 times less abundant than small RNAs derived from C/D box snoRNAs in the total RNA fraction, but are 5.2 times more efficiently picked up by Ago2. The H/ACA box snoRNA SCARNA15, which has been shown to be processed into smaller fragments that act as microRNAs [24], is represented in this library with 3,636 reads, 29% of all reads mapped to H/ACA box snoRNA loci (see Additional file 12 for a full listing of all snoRNAs). The C/D box snoRNA with the highest number of reads in the Ago2 IP library is SNORD1A with 1,140 reads, but the majority of C/D box snoRNAs are represented by less than 50 reads.
Of all categories of small RNAs, C/D box snoRNA fragments are those that show the strongest nuclear retention, and are found in the cytoplasm with only low frequency [56]. Thus, this physical separation could account for the low frequency of association between C/D box snoRNA-derived RNAs and Ago2. We therefore wondered whether the association of this abundant class of RNA fragments with Ago2 increases in the mitotic phase of the cell cycle, when the nuclear membrane is dissolved. We collected HeLa cells that were in the mitotic phase through mitotic shake off, immunopurified Ago2 and again sequenced the Ago2-associated small RNA fraction. We found that, indeed, the relative abundance of C/D box-derived fragments in Argonaute increased in this condition (Table 3), to 0.054% relative to 0.005%. Nonetheless, these results indicate that C/D box snoRNAs do not generally carry out miRNA-like functions, and that the number of H/ACA box snoRNAs with a dual function is very limited.
To gain insight into the processing of snoRNAs and the functions of snoRNA-derived small RNAs, we performed PAR-CLIP experiments with snoRNP core proteins. Analysis of PAR-CLIP reads showed that C/D box core proteins Fibrillarin, NOP56 and NOP58 have a very similar binding pattern, overlapping with the box elements. Excluding snoRNA families SNORD113 to SNORD116, which are multi-copy families and do not have guide complementarity to rRNAs or snRNAs, snoRNA-LBME-db currently lists 153 C/D box snoRNAs, of which 40 and 78 have a guide region targeting a known modification at the D box and D' box, respectively. Evolutionary conservation profiles of the remaining putative guide regions suggest that most of them are not functional. In support of this concept, our analysis revealed that C/D box core proteins cross-linked more effectively to guide regions that are known to have a target compared to orphan guide regions.
Combining computational prediction with data from small RNA sequencing and PAR-CLIP we identified novel C/D and H/ACA box snoRNAs, and assigned guiding snoRNAs to several modifications on rRNAs and snRNAs that were previously described as orphans. In addition to these bona fide snoRNAs, we uncovered a group of C/D box-like snoRNAs that only have a C and a D box as opposed to the common C-D'-C'-D architecture. These C/D box-like snoRNAs are only weakly conserved and most of them are expressed at low levels. The unusual architecture and the weak evolutionary conservation are likely reasons why these RNA species have not been uncovered by computational ncRNA gene finders [57]. Some of the identified C/D box-like snoRNAs are extremely short, one being only 27 nucleotides in length, leaving hardly enough space for a guide region. The requirements for C/D box snoRNA biogenesis appear to be simply the presence of C and D boxes and a short region of complementarity flanking these boxes, leading probably to the production of many snoRNA-like molecules as the C/D box core proteins scan intronic regions of pre-mRNAs. An interesting lead to follow in further investigating the potential function of the C/D box-like snoRNAs originating in the introns of many genes comes from a recent study conducted in Drosophila, in which Schubert and colleagues showed that snoRNAs are required for maintenance of higher-order structures of chromatin accessibility [58].
In our PAR-CLIP experiments we also repeatedly cross-linked ncRNAs that are not usual snoRNA targets. We observed H/ACA box snoRNAs in PAR-CLIP experiments targeting the C/D box core proteins. Vice versa, we found C/D box snoRNAs in the PAR-CLIP targeting Dyskerin, which is an essential component of H/ACA box snoRNPs. Primer extension assays indicated that these snoRNAs carry modifications that would be expected from the protein complexes to which they were cross-linked, but we were, in general, not able to identify snoRNAs that could guide these modifications. One drawback may be that in the case of the 2'-O-methyl primer extension assays we cannot be sure that it was indeed a 2'-O-methyl modification as opposed to any other nucleoside modification that caused the stoppage of the reverse transcriptase. However, we can be fairly certain that we identified bona fide pseudouridylation sites. Particularly, in the case of SNORD35A we were able to identify five putative pseudouridylated residues but no convincing guiding sequence in a known H/ACA box snoRNA. This suggests either that even more snoRNAs remain to be identified or that these pseudouridylations are caused by a protein-only mechanism not requiring guidance by H/ACA box snoRNAs.
The processing patterns of snoRNAs have raised substantial interest and some controversy in recent years [30, 32, 59]. We strikingly found that snoRNA excision out of the intron follows a well-defined pattern leaving mature snoRNAs with four to five nucleotides upstream of the C box, and two to five nucleotides downstream of the D box, irrespective of the length of the terminal closing stem. Our data support the observations of Darzacq and Kiss [5] that the terminal stem serves to bring the C and D box elements into close proximity so as to be more easily recognized by snoRNP proteins, which then protect the snoRNA from further trimming by the exosome, but may not be needed for the functional, mature snoRNA. This implies that the core proteins actively protect and stabilize the maturing snoRNA.
We further quantified the abundance of snoRNA-derived small RNAs in HEK293 cells, and consistent with other studies [29], we found that small RNAs derived from the ends of C/D box snoRNAs are indeed abundant. However, we did not find evidence that these sdRNAs efficiently associate with Ago2 to act as microRNAs, even in conditions when the accessibility of these sdRNAs to Ago2 should be higher, such as in mitotic cells. We thus conclude that a microRNA-like function of snoRNA-derived small RNAs is an exception rather than a rule. Most of the sdRNAs from C/D box snoRNAs originate from the termini of mature snoRNAs, and hence carry C and D box motifs. It might be that snoRNA core proteins are still attached to these fragments, protect them from total degradation, sequester them in the nucleus and prevent these sdRNAs from being loaded into Ago2.
Deep-sequencing-based studies revealed a very complex landscape of transcription and processing of RNAs. The non-canonical products identified initially in such studies raises the question of additional, yet unknown, functions of molecules that have been studied for many years. What has become apparent more recently, however, is that deep sequencing allows us to construct a very detailed picture of the kinetics of processing various classes of RNAs and of their interactions with proteins that protect them from degradation. Intersection of many data sets such as those generated in our study will eventually reveal kinetic and regulatory aspects of cellular processes at a fine level of detail.
Materials and methods
PAR-CLIP experiments
PAR-CLIP was performed with HEK293 Flp-In cells (Invitrogen). Cells were grown in thirty 15-cm cell culture plates per experiment to approximately 80% confluency. At 12 h before harvest, 4-thiouridine (Sigma) was added to the cells to a final concentration of 100 µM. PAR-CLIP was carried out as described previously [34]. For immunoprecipitation, antibodies were coupled to protein-A or protein-G Dynabeads (Invitrogen). Antibodies used against endogenous proteins were α-NOP58 (sc-23705 from Santa Cruz Biotechnology), α-Dyskerin H-300 (sc-48794, Santa Cruz Biotechnology), α-Dyskerin C-15 (sc-26982, Santa Cruz Biotechnology) and α-Fibrillarin AFB01 monoclonal antibody line 72B9, lot 011 (from Cytoskeleton, Inc, AFB01). The α-Ago2 (11A9) monoclonal antibody was a gift from Gunter Meister. For PAR-CLIP with NOP56 we used a HEK293 cell line with a stably integrated FLAG-NOP56 fusion gene and IP was done with monoclonal α-FLAG antibody M2 from Sigma. For one Fibrillarin targeted PAR-CLIP the immunoprecipitated complexes were treated with micrococcal nuclease (MNase, from New England Biolabs) for 5 min at 37°C [35]. After SDS-PAGE, gels were blotted onto nitrocellulose membranes to reduce the background from free RNAs [60]. The PAR-CLIP libraries were prepared as described in Additional file 13 and submitted to deep sequencing on an Illumina HiSeq 2000.
The reads obtained from PAR-CLIP experiments were mapped to the human genome (hg19 assembly from UCSC, February 2009) and annotated with the CLIPZ server [36]. Reads marked with the CLIPZ annotation categories 'fungal', 'bacterial,' or 'vector' were discarded and only reads that mapped uniquely to the genome were used in the analyses. The library size was scaled to 1,000,000 for all samples to obtain a normalized expression value (tags per million).
Small RNA sequencing
Small RNA sequencing libraries were prepared from size-selected RNAs of 18 to 30 nucleotides (sdRNA sequencing) and 20 to 200 nucleotides (sRNA sequencing). HEK293 total RNA was extracted and treated with DNase. Next, 20 units of T4 polynucleotide kinase and 2 µl of [γ-32P] ATP (10 µCi/µl) were used to radiolabel 10 µg of RNA at the 5'-ends. The RNA was separated together with a radiolabeled 20-nucleotide ladder on a 12% polyacrylamide gel, the bands corresponding to 18 to 30 nucleotides (for sdRNA sequencing libraries) or 20 to 200 nucleotides (for sRNA sequencing libraries) were excised, the RNA was extracted overnight in a 0.4-M NaCl solution and finally precipitated with ethanol. Small RNA libraries were prepared according to a published protocol [61] and sequenced on an Illumina HiSeq 2000 instrument, for 36 (sdRNA sequencing) and 150 cycles (sRNA sequenicng library). Adaptor removal was done with the CLIPZ server, and the mapping to the human genome was then done with the Segemehl software (v. 0.1.3) with parameters '-D 1 -A 90' [62]. The Gene Expression Omnibus (GEO) accession number for the PAR-CLIP and sRNA-seq data is GSE43666.
Identification of novel C/D snoRNAs and H/ACA snoRNAs from PAR-CLIP and small RNA sequencing data
For each PAR-CLIP library we inferred binding regions of the proteins of interest by clustering reads whose corresponding loci were at most 25 nucleotides apart. To annotate known snoRNA and scaRNA genes we first retrieved sequences from the snoRNA-LBME-db [37], mapped them to the human genome (a list of motif and secondary structure annotated snoRNAs is available in Additional file 13). The 500 binding regions that accumulated the highest number of reads in each individual CLIP library, but did not overlap with known snoRNA or scaRNA genes, ncRNA genes or repeat elements, were screened for novel snoRNA candidates. We used SnoReport [38] to detect H/ACA box snoRNAs, while for detection of C/D box snoRNAs we searched for protein-binding regions that contained motifs corresponding to the C box (RTGATGA; allowing one mismatch) and to the two most common D box motifs (CTGA and ATGA). Sequences that contained both a C box and a D box motif were extended by ten nucleotides in order to search for a terminal closing stem. If a compact closing stem composed of at least four canonical base pairs with at least two G-C/C-G base pairs was found, the sequence was considered a snoRNA candidate. To evaluate the specificity of our C/D box snoRNA gene finding approach, we applied the same procedure to two types of clusters of PAR-CLIP reads from the NOP58 rep A sample both extended by 25 nucleotides on each side. First were the top 100 clusters (defined in terms of the number of reads associated with the cluster) that overlapped with C/D box snoRNA annotation, which served as a positive control. In this set, our program reported 80 sequences as putative snoRNAs. The second type of cluster contained the top 100 clusters that overlap with mRNA exon annotation. These should not contain snoRNAs, and indeed, we only obtained five putative C/D box snoRNAs candidates. Similarly low numbers of snoRNA candidates were obtained from randomized sequences (not shown). Altogether, these tests indicated that our method has very good specificity. In contrast, the number of predictions we obtained from CLIPed clusters without a known annotation was 11 for the top 100 such clusters.
Candidates that showed expression of at least 1 TPM per nucleotide in the 20 to 200 nucleotides small RNA sequencing run (only uniquely mapped reads that covered at least 50% of the candidate snoRNA sequence were considered), and had at least 1 TPM per nucleotide in at least one of the type-specific CLIP libraries were considered putative snoRNAs. They were consecutively numbered, and named as 'ZL#'. To further validate the newly found snoRNAs, we searched for evidence of expression in recently published small RNA-seq libraries from the ENCODE project [39]. Files with the genome coordinates of mapped reads (BAM files) were obtained from the ENCODE data coordination center at UCSC [63] and uniquely mapping reads were used for the analysis. In addition, we selected the 20 candidate C/D box snoRNAs with the highest read count in our data for validation by Northern blotting (see Additional file 13 for details on the experiment). To evaluate the evolutionary conservation of the putative snoRNAs, we carried out a homology search against the vertebrate genomes available in the UCSC genome browser. Once an initial set of homologs was identified, we built sequence/structure models and continued to search for more distant homologs with the Infernal software [64].
Detection of 2'-O-ribose-methylated and pseudouridylated residues
To identify 2'-O-methylated residues we used a reverse transcriptase-based method coupled with polyacrylamide gel analysis as described in [65]. The method is based on the observation that cDNA synthesis is noticeably impaired in the presence of a 2'-O-methyl when deoxynucleotide triphosphate fragments (dNTPs) are limiting [65, 66], giving rise to a characteristic pattern of gel banding immediately preceding the 2'-O-methyls, with strong bands at low dNTP concentrations (0.004 mM) [66], becoming weaker with increasing concentrations of dNTPs.
To map pseudouridines in candidate RNAs we used a method that relies on chemical modification of RNA bases with N-cyclohexyl-N'-β (4-methyl morpholinium) -ethylcarbodiimide (CMC) [67]. The method involves carbodiimide adduct formation with U, G and pseudouridine followed by mild alkali treatment, which removes the adduct from U and G but not from the N-3 of pseudouridine. This modification results in the blockage of reverse transcription one residue 3' of the pseudouridine on the sequencing gel. For a detailed description of assays used to map 2'-O-methyls and pseudouridines see Additional file 13. As a proof of principle, we first applied these assays to the spliceosomal RNA U6, which is known to carry 2'-O-methylated and pseudouridylidated residues. In addition to the well-documented sites, we also observed novel 2'-O-methyl sites that have not been previously reported so far (Additional file 14).
To predict C/D box snoRNAs that could guide 2'-O-methylation, we searched for 8-mer complementarity (only canonical base pairs allowed) to regions immediately or one nucleotide upstream of the D and D' boxes of C/D box and C/D box-like snoRNAs. To predict H/ACA box snoRNAs that could guide pseudouridylations, we used the program RNAsnoop [48]. We first determined for each H/ACA snoRNA stem an energy cutoff value by running simulations on 1,000 random sequences of length 100. Only if an RNAsnoop prediction had an energy value lower than 90% of the random sequences, and at least three canonical base pairs on each side of the binding pocket, did we consider it as a hit.
Ago2 immunoprecipitation sequencing of asynchronous and mitotic cells
Mitotic cells were collected using mitotic shake-off [68, 69], a technique based on the observation that cells become rounded and more easily detachable from the culture vessel as they progress into metaphase during mitosis [70]. Details of the experimental setup are given in Additional file 13. To be able to confirm microscopically that we collected mitotic cells we used HeLa cells with the human histone H2B gene fused to green fluorescent protein (see Additional file 15).
Ago2 was immunoprecipitated from mitotic and asynchronous cells; the Ago2-associated RNAs were extracted and used to prepare cDNA libraries as described above [61], which were then submitted to deep sequencing. Adaptor removal was with the CLIPZ server, and reads were then mapped with Segemehl as described above. In the analysis of small RNA libraries (Ago2-IP and HEK293 sdRNA sequencing (18 to 30 nucleotides)), we considered both uniquely and multi-mapping reads that were annotated based on their mapping to genes in one of the following categories: tRNAs (from the UCSC Table Browser), microRNAs (from mirBase) and snRNAs (from ENSEMBL release 59), C/D box snoRNAs and H/ACA box snoRNAs (curated data set from this work).
The authors thank Erich Nigg (Biozentrum) for providing the HeLa cells in which the histone H2B gene is fused to green fluorescent protein and for advice on the isolation of mitotic cells. We are grateful to Gunter Meister for the anti-Ago2 antibody and Ina Nissen and Christian Beisel from the Quantitative Genomics Facility of the D-BSSE of ETH Zurich, for help with deep sequencing. All computations were carried out on the [BC]2 HPC infrastructure at the University of Basel. Work in the Zavolan lab is supported by the University of Basel and the Swiss National Science Foundation (grant #31003A 127307). SK acknowledges the support of the Gebert Rüf Foundation Rare Diseases Program (grant GRS-046/11).
Supplementary material
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Sensors 2011, 11(1), 623-637; https://doi.org/10.3390/s110100623
Response Identification in the Extremely Low Frequency Region of an Electret Condenser Microphone
Department of Aeronautics and Astronautics, National Cheng Kung University, Tainan, 70701, Taiwan
Author to whom correspondence should be addressed.
Received: 11 November 2010 / Revised: 20 December 2010 / Accepted: 27 December 2010 / Published: 10 January 2011
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AbstractThis study shows that a small electret condenser microphone connected to a notebook or a personal computer (PC) has a prominent response in the extremely low frequency region in a specific environment. It confines most acoustic waves within a tiny air cell as follows. The air cell is constructed by drilling a small hole in a digital versatile disk (DVD) plate. A small speaker and an electret condenser microphone are attached to the two sides of the hole. Thus, the acoustic energy emitted by the speaker and reaching the microphone is strong enough to actuate the diaphragm of the latter. The experiments showed that, once small air leakages are allowed on the margin of the speaker, the microphone captured the signal in the range of 0.5 to 20 Hz. Moreover, by removing the plastic cover of the microphone and attaching the microphone head to the vibration surface, the low frequency signal can be effectively captured too. Two examples are included to show the convenience of applying the microphone to pick up the low frequency vibration information of practical systems. View Full-Text
Keywords: response identification; extremely low frequency; electret condenser microphone; time frequency analysis►▼ Figures
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This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).
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MDPI and ACS Style
Jeng, Y.-N.; Yang, T.-M.; Lee, S.-Y. Response Identification in the Extremely Low Frequency Region of an Electret Condenser Microphone. Sensors 2011, 11, 623-637.
AMA Style
Jeng Y-N, Yang T-M, Lee S-Y. Response Identification in the Extremely Low Frequency Region of an Electret Condenser Microphone. Sensors. 2011; 11(1):623-637.Chicago/Turabian Style
Jeng, Yih-Nen; Yang, Tzung-Ming; Lee, Shang-Yin. 2011. "Response Identification in the Extremely Low Frequency Region of an Electret Condenser Microphone." Sensors 11, no. 1: 623-637.
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