text:
Q: How to removeFromSuperview() with a transition I have added a view as sub view using the following code
    let controller = SideMenuViewController.instantiateViewControllerFromStoryboard(storyBoardName: "Module1") as! SideMenuViewController

    UIView.animate(withDuration:0.5, delay: 0.0, options: UIViewAnimationOptions.curveEaseInOut, animations: {
    }, completion: {
        (finished: Bool) -> Void in

        controller.view.tag = 100

        let transition = CATransition()
        transition.type = kCATransitionPush
        transition.subtype = kCATransitionFromLeft
        controller.view.layer.add(transition, forKey: nil)
        self.view.addSubview(controller.view)
        self.addChildViewController(controller)
        controller.didMove(toParentViewController: self)

    })

The result is a view controller(sidemenu) sliding from left and revealing.
I want to remove the added subview with a transition from right to left
I have tried removing from superview with animation using the following code
UIView.animate(withDuration:0.5, delay: 0.0, options: UIViewAnimationOptions.curveEaseIn, animations: {}, completion: {

self.view.viewWithTag(100)?.removeFromSuperview()

})

But this does not result in a smooth transition.How can I remove the added subview with a smooth transition,similiar to  the way it was shown???

A: The removeFromSuperview() method is not animatable, what you can do is making alpha goes to 0 and after it has finished you can safely remove from super view.
If you want to get the same effect as in push, you just need to take your code and create the opposite transition. Since it seems that you are using view controllers you can take advantage of the transitions API.
func push(_ viewController: UIViewController, animated: Bool, completion: (()->())?) {
        let oldVC = viewControllersStack.topItem()!
        viewController.view.frame = oldVC.view.frame
        self.addChildViewController(viewController)
        oldVC.willMove(toParentViewController: nil)
        let duration = animated ? Constants.GeneralValues.PopPushAnimationDuration : 0.0
        transition(from: oldVC, to: viewController, duration: duration, options: [], animations: { () -> Void in
            let animation = CATransition()
            animation.duration = CFTimeInterval(duration)
            animation.type = kCATransitionMoveIn
            animation.timingFunction = CAMediaTimingFunction(name: "easeInEaseOut")
            animation.subtype = "fromRight"
            animation.fillMode = "forwards"
            self.mainContainerView.layer.add(animation, forKey: "animoteKey")

            // Constraint
            guard let v = viewController.view else {
                return
            }
            v.translatesAutoresizingMaskIntoConstraints = false
            let hConstr = NSLayoutConstraint.constraints(withVisualFormat: "H:|[v]|", options:[], metrics:nil, views:["v":v])
            let vConstr = NSLayoutConstraint.constraints(withVisualFormat: "V:|[v]|", options:[], metrics:nil, views:["v":v])
            let constrs: [NSLayoutConstraint] = [hConstr, vConstr].flatMap {$0}
            NSLayoutConstraint.activate(constrs)
            }) { (finished) -> Void in
            oldVC.removeFromParentViewController()
            self.viewControllersStack.push(viewController)
            viewController.didMove(toParentViewController: self)
            if let completion = completion {
                completion()
            }
        }
    }

    func popViewController(_ animated: Bool, completion: (()->())?) {
        let oldVC = viewControllersStack.topItem()!
        let viewController = viewControllersStack.penultimate!
        viewController.view.frame = oldVC.view.frame
        self.addChildViewController(viewController)
        oldVC.willMove(toParentViewController: nil)
        let duration = animated ? Constants.GeneralValues.PopPushAnimationDuration : 0.0
        transition(from: oldVC, to: viewController, duration: duration, options: [], animations: { () -> Void in
            let animation = CATransition()
            animation.duration = CFTimeInterval(duration)
            animation.type = kCATransitionReveal
            animation.timingFunction = CAMediaTimingFunction(name: "easeInEaseOut")
            animation.subtype = "fromLeft"
            animation.fillMode = "forwards"
            self.mainContainerView.layer.add(animation, forKey: "animoteKey")
            // Constraint
            guard let v = viewController.view else {
                return
            }
            v.translatesAutoresizingMaskIntoConstraints = false
            let hConstr = NSLayoutConstraint.constraints(withVisualFormat: "H:|[v]|", options:[], metrics:nil, views:["v":v])
            let vConstr = NSLayoutConstraint.constraints(withVisualFormat: "V:|[v]|", options:[], metrics:nil, views:["v":v])
            let constrs: [NSLayoutConstraint] = [hConstr, vConstr].flatMap {$0}
            NSLayoutConstraint.activate(constrs)

            }) { (finished) -> Void in
            print("Fine")
            oldVC.removeFromParentViewController()
            _ = self.viewControllersStack.pop()
            viewController.didMove(toParentViewController: self)
            if let completion = completion {
                completion()
            }
        }
    }

This is a sort of implementation that I used to achieve the same you want but for pushing and popping view controllers in a container, but the animation is the same, just change constraints, to keep the sideview controller thin.


text:
Home Sports Bangladesh Cricket
06:30 PM, May 21, 2019 / LAST MODIFIED: 06:37 PM, May 21, 2019
'I hope people would get off his back'
Soumya Sarkar. Photo: AFP File
Bangladesh batsman Soumya Sarkar has been in fantastic touch especially since his hundred and double hundred in the Dhaka Premier League [DPL] this season for Abahani Limited. He was able to carry that form to the international stage, hitting three consecutive 50s during the recently-concluded tri-nation series in Ireland. Bangladesh coach Steve Rhodes said that the team firmly backs the opener even though many still criticise the left-hander.
"He has been a revelation since he hit that hundred and double-hundred in the DPL, but he also gets some rough days from people," Rhodes was quoted as saying by ICC website.
Soumya had a rough patch in 2017 and was dropped from the ODI squad after Bangladesh's tour of South Africa that year. Rhodes knows about the criticism that the left-hander had to tackle and the batsman has come back strongly, working on his past weaknesses and on his way to establishing himself as a key figure at the top of the order.
"It's just nice to see him carrying on that form, and I hope people would get off his back a little bit because he is a wonderful player. We support him and back him. But not everyone always does that," Rhodes said.
The head coach also commented on Bangladesh's depth outside of the big five of Shakib Al Hasan, Mashrafe Bin Mortaza, Tamim Iqbal, Mushfiqur Rahim and Mahmudullah Riyad.
"There have been two or three guys who have played special innings," he said. "If you think of Mosaddek, for example, he is a guy now who is going to keep everybody on their toes. That's the depth of the squad and he might not play, and if he doesn't, then what a great position we have. It gives us the confidence that we can win some big games as well.
"That means Bangladesh are stronger within the squad, and that's what we want as we want more depth, and if we get that depth, then people will stop talking about the big five."
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text:
PC server computers booming
Market growth continued upwards at a rapid 35 percent in 1997, most coming from sales of two-processor systems in the $8,000 to $15,000 price range.
Compaq (CPQ), Hewlett-Packard (HWP), and IBM (IBM) remain the companies to beat in the market for server computers using Intel processors, as overall market growth continued upwards at a rapid 35 percent in 1997, according to International Data Corporation (IDC).
IDC issued a report today saying that vendors saw increasing revenues from "PC servers" even though overall shipment growth was modest. PC servers, as defined by IDC, are Intel-processor-based systems which are specifically built and sold as servers and sell for less than $25,000. They have historically been used as lower-end servers, for such tasks as handling printing operations, while Unix servers have been used in high-end roles, such as database applications.
IDC estimates that the 1997 worldwide PC server market will exceed $10.5 billion in revenue on shipments of 1.75 million units. Growth was sparked by increased shipments to the small business market, as well as continuing purchases by information system departments at corporations. Most of the growth is coming from sales of two-processor systems in the $8,000 to $15,000 price range, the report states.
Compaq remains the largest PC server vendor both in the U.S. and worldwide markets. IDC research analyst Amir Ahari says that the company garnered 35 percent of the revenues, compared with 12 percent for HP and 11 percent for IBM. Together with Dell's (DELL) six percent share and Digital's five percent share, the top five vendors gathered 69 percent of the PC server revenue.
IDC's Ahari says Compaq has done a good job of using its economies of scale to move high-end server technology into mainstream markets.
"Some of the [high-end server] technology from Tandem is creeping in to the lineup, and watered-down versions of Tandem's fault-tolerant technology should appear by the end of the first half of 1998," Ahari says.
Recently acquired by Compaq, Tandem now serves as the high-end corporate "enterprise" computing arm of Compaq. Tandem offers technology that allows a number of servers to be strung together to form a single virtual server, a configuration known as "clustering." Clustering provides added reliability, referred to as "availability," since the servers in the cluster can back each other up if one of them goes down.
HP and IBM could start to take away market share from Compaq in 1998, though, as both companies continue to market their experience with service and support gained in the market for mainframe-type computers, Ahari thinks.
IBM, in particular, has focused in on PC servers as a source of growth. The company has extensive experience in designing high-end servers based on RISC processors, but until recently engineers for those systems didn't work with engineers who designed Intel-based systems.
"The company reevaluated the potential growth in Intel-based systems and really started mixing the synergies between the two engineering groups to come up with ways to offer value-add features and rich architecture features of high-end [Unix systems] into PC servers," Ahari said.
IDC says Dell continued to experience a phenomenal growth in the PC server market, with an increase of 181 percent in terms of revenues.
The list of the top ten vendors is rounded out by Siemens Nixdorf, Fujitsu, Acer, NEC, and Hitachi, respectively.
Discuss: PC server computers booming


New and advanced NDT techniques are continually under development. TWI is at the forefront of research and development within the welding and joining of materials. This also necessitates looking at the inspection of such joints and associated materials, which leads to NDT.
Whilst TWI offers training and certification in long established conventional NDT methods, we also offer certification and training in advanced NDT techniques such as phased array ultrasonic testing, time of flight diffraction, computed/digital radiography, thermography, alternating current field measurement (ACFM) and automated ultrasonic testing (AUT).
Your theoretical training can be complemented with our eLearning and blended learning packages which allows you to prepare for your course and examination in your own time and at your own pace.
Keeping her knowledge up-to-date, Vera Dragoi lets her experience speak for itself.
From a career perspective, adding additional expertise by achieving advanced technique certification improves your job opportunities and often leads you down a more fulfilling and exciting career path at the cutting edge of non-destructive testing. All of this adds to your continued professional development.
TWI has long been a forerunner in NDT training and certification spanning over 50 years with highly knowledgeable staff and a worldwide network of locations which is trusted by industry to deliver NDT training to the level they require.
We have launched a range of eLearning/blended learning products for non-destructive testing (NDT) courses, giving you more choice and opportunities for more flexible learning when you enrol.
We have offer pre-course, blended learning, appreciation and refresher courses.
If you can't find a training course to suit you, we can create bespoke courses to meet your individual, industrial or company needs.
These cover both theory and practical training tailored to your specific needs and are ideal if you are seeking recertification at Level 2 or 3, or preparing to take retests in specific areas.
We also offer the option of learning remotely via our Virtual Academy.
We can create bespoke courses to meet your individual, industrial or company needs so if you can't find course to suit you, contact us!
This procedure-driven certification compliant to EN ISO 9712 can be tailored to specific NDT application(s).
Ask us about the scheme!
Membership gives you access to all the technical knowledge that you need and will also make it easier for you to achieve your own personal learning and development goals.
You can also qualify for a 5% discount on training courses. Find out more!
You can gain CPD points when attending this course - Find out more!
Try our list of frequently asked questions.
How can I verify the required industrial experience?
Industrial application of the NDT method is a requirement before certification can be awarded. If you are working for a company using the method, it is a case of logging this experience and having this verified by your supervisor or if you are working on a contract as self-employed it can be signed by the contract client (Note: NDT certification schemes often require that the person verifying your experience holds the same NDT method and level of qualification themselves).
The experience duration needs to match the minimum requirements as detailed by the certification scheme documents, these can be found on the scheme websites (e.g. CSWIP or BINDT).
Can I use my welding inspector experience for NDT?
Unfortunately any experience claimed must be in the same NDT discipline, however, for visual testing (VT) a welding inspection certificate may contribute towards the EN ISO 9712 visual testing (VT) experience requirement for the welds sector.
Can I come and sit on the course without any industrial experience?
Yes, for those not working for a company or on a contract that is using the NDT method a 2-year period is normally given after the examination date to send your experience verification to the scheme body. For further advice please do not hesitate to contact us, however, this applies only to Levels 1 and 2.
What are the best courses to start with?
We normally advise candidates to start with a surface inspection such as penetrant inspection (PT) or magnetic particle (MT), however if a candidate already has NDT experience in another method, we would perhaps suggest sitting the course in that particular method. Generally you should aim to achieve at least 2 surface inspection methods and what we call a volumetric inspection method such as radiography or ultrasonic testing.


Home / News / Oh, the humanity! How can CNN firefighters guard the truth while living under this kind of oppression?
Oh, the humanity! How can CNN firefighters guard the truth while living under this kind of oppression?
CNN employees currently pay $110 per month for an Equinox membership that allows them access to all clubs, according to a petition circulated among employees at the network. Per an internal email, however, that discount is set to end on June 30 when WarnerMedia employees switch to the Equinox discount provided to employees of AT&T, which recently acquired WarnerMedia.
Now, according to the CNN petition, network employees in New York will be forced to fork over $238 per month if they want access to the Hudson Yards Equinox branch, near the cable channel's newly relocated New York City headquarters.
In response, more than 100 CNN and HBO employees have signed multiple petitions that have circulated around both companies calling for WarnerMedia to allow staffers to keep their original Equinox discount.
According to multiple sources, there is a gym available for WarnerMedia employees at the new New York City Hudson Yards HQ, with a $50 fitness plan available. But the major drawback of the gym is that it is closed on weekends, and according to a petition circulated among HBO staff, fitness classes are not included with basic membership.
Closed on weekends? Fitness classes cost extra? Oh, the humanity!
What, and be forced to a Planet Fitness or Snap Fitness or, *shudder* a YMCA, like common plebeians? The horror.
They take enough abuse from Donald Trump. Must they suffer the indignity of paying full price for a luxury gym membership on top of that?
I understand that a lot of EMTs, Firefighters and military vets have similar horror stories!
Firefighters often complain about losing their luxury gym discount as well.
Well, lifting those heavy firehoses requires a lot of upper-body strength. How are they supposed to beef up their biceps at some lame discount gym that doesn't even offer free Zumba classes on weekends?!
"It's the most white people first-world problems I've ever heard" sums that up incredibly well.


text:
Q: :after class doesn't work as expected I need to place the div content before the element, so I have used :before class. But it doesn't work. What could be the reason for it? Is there something incorrect with css?

.led {
 /* Yellow LED */
 margin-top: 3px;
    width: 14px;
    height: 14px;
    background-color: #FF0;
    border-radius: 50%;
    box-shadow: rgba(0, 0, 0, 0.2) 0 3px 7px 0px, inset #808002 0 0px 9px, #D4D400 0 2px 12px;
    margin-left: 80%;
}

.led:before {
 content: "Connecting";
 padding-left: 18px;
    font-size: 11px;
    font-family: "Open Sans","Helvetica Neue",Helvetica,Arial,sans-serif !important;
}

A: Everything is fine with CSS. As I understand :before and :after, it will add a content inside your div element. So, it will look something like this:

    Your div content

Problem that you are facing is that there is no content inside .led div, so :before and :after seem to have the same result. Your css defines .led div to have width of only 14px, so there is no place for Connecting text. You can try something like this for a quick (and ugly) fix:

.led:before {
    content: 'Connecting';
    margin-left: -60px; /* CHANGED */
    font-size: 11px;
    font-family: "Open Sans","Helvetica Neue",Helvetica,Arial,sans-serif !important;
}
.led {
    /* Yellow LED */
    margin-top: 3px;
    width: 14px;
    height: 14px;
    background-color: #FF0;
    border-radius: 50%;
    box-shadow: rgba(0, 0, 0, 0.2) 0 3px 7px 0px, inset #808002 0 0px 9px, #D4D400 0 2px 12px;
    margin-left: 80%;
}

Better solution might be to enclose .led div with another one and add :before to that parent div, something like this:

.holder {
    margin-left: 80%;
}
.holder:before {
    content: 'Connecting';
    font-size: 11px;
    font-family: "Open Sans","Helvetica Neue",Helvetica,Arial,sans-serif !important;
}
.led {
    /* Yellow LED */
    margin-top: 3px;
    width: 14px;
    height: 14px;
    background-color: #FF0;
    border-radius: 50%;
    display: inline-block;
    box-shadow: rgba(0, 0, 0, 0.2) 0 3px 7px 0px, inset #808002 0 0px 9px, #D4D400 0 2px 12px;
}

redpajama_set_name: RedPajamaStackExchange


Please how is its internate speed because galaxy y have very poor internate speed.can support uc browser.
from month i'm using this phone, facing issues with both sims.
Bought the S Duos its very nice and cool phone.
This is really very fine and user friendly mobile. Battery backup is very well. I didnt face any issue with the same..use it..
am thinking to buy galaxy s duos I am confused that this phone is good or no? tell it hangs or no.
AnonD-135612, 13 Apr 2013Guys. Help please. I want to know any suggestion on dual sim smartphone. Currently on my mind ... moreGo for LG Optimus L7II dual ,priced between 14000 to 14900. This is perfectly fits on ur requirement.If the brand name does,nt matter for you then go for Micromax Canvas HD.
AnonD-135794, 14 Apr 2013which phone is better for me ? 1.Samsung s duos 2.Micromax canvas 2 3.Nokia lumia 520 ... moreMicromax Canvas 2 /Canvas hd.Only iff u want a good performer not "brand name".
gag, 14 Apr 2013internal memory is 4gb but only 1.8 is user available. wat does this mean? Rest of the memory (2.2GB) is used by OS and some other background process .


How long should you rest for?
Pick your side. On the one hand, you've got the bros shaped like barrels throwing up max lifts and sitting there for days, hogging the squat rack. On the other, you've got the lean guys making the most of all the kit, using light weights for fifteen reps and supersetting straight into pull-ups. Are they done? You're not quite sure, but they're moving too fast for you to ask. But which one's doing it right?
"Traditionally 45-60 seconds was seen as the optimal time to rest between sets to ensure the muscle was recovered enough to enable it to complete the next set," says Matt Williams, personal trainer at London's Ignite PT. "Recent arguments have led to the idea that shorter rest periods, anywhere from 15-30 seconds, can lead to greater size and density gains." Because the less you rest, the greater the metabolic stress, the theory goes that you get more growth in less time. But science is yet to crown a winner. It comes down to your training aims and what your gym sessions look like.
Your rest needs to be adjusted to take your goals into account. What works for Mo Farah won't help Rich Froning to CrossFit glory, and vice versa. By breaking down the rest required for different types of lifting, you can cherrypick to ensure you hit your goals, ensuring maximal effort and minimal wasted energy. Are you sitting comfortably? Not for long.
Strength: If you're training for pure strength by lifting low reps of a high weight, you're going to want to rest for longer than two minutes to get your muscles ready for your next set. Low-rep exercises need to be completed with maximum power, so you need to let them recover fully. A study in the Journal of Strength and Conditioning found that resting for two minutes held no real advantage over taking five – but don't rest for any longer than that or you risk your muscles getting cold.
PT and strength coach Rogan Allport agrees. He sees strength-based rest times as "two minutes on average, and three-to-five if necessary. Generally the lower the reps, the more rest you need." Three minutes at least in between shooting for that one-rep max.
Endurance: Whereas strength demands muscles firing at full capacity, training your muscles to go for longer (for example, in the boxing ring) means keeping them at max effort for time. Shorter rest on longer, sub-maximal sets allow you to make the most of your gym time, while another study in the Journal of Strength and Conditioning found that only short-rest training reduces the rate of decline in the average repetition velocity during a set. Translation? The more reps you do, the longer you'll be able to maintain your performance. Keep rest below 60 seconds between sets to keep you going past the finish line.
Burning fat: The shorter the better, according to Evolution of Man PT Tim Walker. "Having less rest time increases the overall volume of the sets completed in a workout and thus burns more calories," he tells us. You'll also produce more lactic acid, increase your metabolic rate and burning fat. Walker recommends heart rate-spiking circuits with compound moves – deadlifts, bench press, chinups and squats – at 10-12 reps with just 45 seconds rest. "A circuit like this can be completed in around 30 minutes and you get the same fat loss benefits of hours o" You'll be burning all over, but the fat will melt away.
Gaining size: Bulking up is different to getting stronger, but to keep muscle lean you want to pick from both camps: the muscle recovery to lift heavier weights, but the intensity that evaporates fat. "The overriding factor on optimal muscle growth is the intensity and volume," says Williams. If you're putting your muscles under tension, volume and load, you get gains. Keeping to between 45 and 60 seconds rest lets you shift heavy metal while keeping your heart rate high, says Williams. Aim for fewer reps of a heavier weight than the endurance athlete – but shrinking your rest time will supersize your muscles.
How long should I rest for between sets?


Pet Food Cabinet With Bowls. You almost certainly know already that Pet Food Cabinet With Bowls is one of the trendiest topics on the web these days. Depending on the information we acquired from google adwords, Pet Food Cabinet With Bowls has very much search in google search engine. We feel that Pet Food Cabinet With Bowls deliver new thoughts or references for viewers.
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text:
UAF Kuskokwim Campus Prepares For Budget Cuts
Published May 23, 2016 at 9:39 AM AKDT
State budget cuts are making deep slashes across Alaska including the Yukon Kuskokwim Delta. The state Legislature hasn't passed a budget, but with an almost four-billion dollar deficit, state-funded groups are paring down as they prepare their own budgets for the upcoming fiscal year. With the whole University of Alaska system preparing for major cuts, administrators at Bethel's UAF Kuskokwim Campus are whittling to the bone.
"Between the two fiscal years we've had over a million dollars worth of cuts in state funding," Mary Pete, UAF Kuskokwim Campus Director, said. Last year the campus cut nearly $400,000. This year it's cutting around $800,000.
Pete says the library, business office and cultural center are losing positions, and all other contracts are being reduced.
"Eleven-month contract people are down to 10.5 or 10 months," Pete said, "and 10 months are done to 9.5."
She says the students are already feeling the effects.
"There were students who expected to access the campus, classrooms and Internet after 5 p.m., and," she said, "we just can't."
Students used to be able to access the campus until 10 p.m. That stopped earlier this month. Now they only have that option when classes run late or if they can get into the dorm. And more blows are expected.
"I can see more and more students having to go longer to have their educational goals met," Pete said.
In the past the campus has held classes with just one student like when he or she needed a class to graduate or meet a requirement. Pete says now that's going to be harder to justify.
The community will also feel the cuts. Starting July 1, the Cultural Center will be closed Sundays and Mondays unless there's an event.
"It's not part of our mission," Pete said. "It's a wonderful building. A lot of wonderful things happen in that building. But it's pretty spendy to maintain."
The University of Alaska system is preparing for a $36 million dollar cut. But since the state Legislature hasn't passed a budget, that number could go higher. In that case, Pete says the Cultural Center will shutdown for longer and the campus would close for a month or two.
But there is some good news at the campus. As budgets have shrunk over the last two years, enrollment and tuition revenue has gone up.
EducationBudget CutsDeficit


Evoking memories of richly-hued gazpacho or our favourite lipstick in that perfect shade of red, the Wyatt Knit from Nickel and Sole is a merino wool sweater in a gorgeous red. With a mockneck and fine ribbed detailing around the cuffs and hem, this is a thin but ultra-cosy jumper perfect for layering or styling by itself. Cut straight in the body, we love the centrefront detail that gives this classic style unique touch.
True to size. Style with the Doc Fontaine leather trousers from Binny and the Piper Snakeskin boots from Department of Finery.


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No 3rd party support here. Our team of specialists know Apprise ERP and they're ready to assist you if needed.
With offices in the US, Europe, and Asia-Pacific, we have the global coverage you need to keep your business running smoothly.


The Fire and Arson Investigation Certificate provides students with the technical and professional knowledge to prepare for a career in fire and arson investigation. The program is designed for students who desire employment and/or **certification as an Arson Investigator. All credits earned in this program may be applied toward the Associate of Applied Science Degree in Fire Protection Technology.
**In order to be certified by the Texas Commission on Fire Protection as a Basic Arson Investigator, an individual must possess a current basic peace officer's license from the Texas Commission on Law Enforcement Officer Standards and Education or documentation that the individual is a federal law enforcement officer; hold a current commission as a peace officer with the employing entity for which the arson investigations will be done; and complete a commission approved basic fire investigation training program and successfully pass the commission examination.


Hans Zimmer has created the first official hymn to mark the 10th anniversary of Tomorrowland, a leading electronic dance music festival held every year in the Belgian town of Boom, outside of Brussels. The 2014 edition takes place July 18-20 and July 25-27.
The festival approached Zimmer to compose a classical piece that would embody the "mystical, magical" aspects the festival is known for, and whose award-winning scores are infused with similarly spiritual qualities. He recorded the original piece in Air Studios in London, where he had previously recorded soundtracks for films such as Pirates of the Caribbean, The Da Vinci Code and The Dark Knight. The hymn will premiere this summer and serve as the official soundtrack of Tomorrowland moving forward.


The Harkin Institute's advisory committees are comprised of experts in their key policy areas. These diverse groups will help define the institute's research agendas and focus as they relate to wellness and nutrition, retirement security, labor and employment, and people with disabilities.
Working in collaboration with Senator Harkin, the Labor and Employment Core Advisory Committee works to identify and define the research agenda that will drive The Harkin Institute's public policy initiatives in labor and employment.
The committee is tasked with selecting timely and relevant topics that align with the Senator's previous work in the area; relate to social justice, fairness, or opportunity; are nonpolitical; and require additional examination for the public good. The plans developed by the group will be implemented by an associate director of public policy for labor and employment and overseen by a policy director and larger National Advisory Council.
Bernstein joined the Center on Budget and Policy Priorities in May 2011 as a senior fellow. From 2009 to 2011, Bernstein was the chief economist and economic adviser to Vice President Joe Biden, executive director of the White House Task Force on the Middle Class, and a member of President Obama's economic team. Bernstein's areas of expertise include federal and state economic and fiscal policies, income inequality and mobility, trends in employment and earnings, international comparisons, and the analysis of financial and housing markets. Bernstein has published extensively in various venues and is an on-air commentator for the cable stations CNBC and MSNBC. He holds a Ph.D. in social welfare from Columbia University.
Butler is the President of SEIU Local 2015 – the statewide long term care union that has united the voices of more than 325,000 SEIU nursing home and home care workers throughout California. It is the largest union in California and the largest long term care local in the country. Since its formation on June 2, 2015, Local 2015 members have celebrated historic victories – including the passage of the nation's first $15 statewide minimum wage. Butler also serves as an SEIU International Vice President and President of the SEIU California State Council. Butler is a graduate of Jackson State University, in Jackson, MS.
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Weil is an internationally recognized expert in employment and labor market policy. He co-founded and co-directed the Transparency Policy Project at the Harvard Kennedy School of Government and has advised international organizations as well as government agencies at the state and federal levels. He is also the author of more than 100 articles and five books. Prior to joining the Heller School, Weil served as the administrator of the Wage and Hour Division at the United States Department of Labor under President Obama. Weil earned a bachelor's degree from Cornell University's School of Industrial and Labor Relations, a master's in public policy from the Kennedy School and a PhD in public policy from Harvard.
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text:
Biological material
Preparation and quantification of viral inoculums
Immune challenge of shrimp, total RNA isolation, and reverse transcription of total RNA
Biopanning the T7 phage display library and cloning of the TRBP gene
Recombinant expression of Fc-TRBP
Biopanning of the T7 phage display library and cloning of the Fc-eIF6 gene
Recombinant expression of Fc-eIF6
Semiquantitative RT-PCR analysis of Fc-TRBP and Fc-eIF6
Pull-down experiments
Fc-TRBP1 antiserum preparation and Western blot analysis
Far Western blot analysis
WSSV infection assay
Three isoforms of Fc-TRBP were present in Chinese shrimp
Biopanning of the T7 phage display library indicates an interaction between Fc-TRBP1 and Fc-eIF6
Expression of Fc-TRBP and Fc-eIF6 is regulated by infection
The interaction between rFc-TRBP1 and rFc-eIF6 was confirmed by a pull-down assay and Far Western blot analysis
rFc-TRBP inhibits WSSV replication
TRBP Homolog Interacts with Eukaryotic Initiation Factor 6 (eIF6) in Fenneropenaeus chinensis1
Shuai Wang;
School of Life Sciences, Shandong University, and The Key Laboratory of Plant Cell Engineering and Germplasm Innovation of Ministry of Education, Jinan, Shandong, China
Ning Liu;
Ning Liu
An-Jing Chen;
An-Jing Chen
Xiao-Fan Zhao;
Xiao-Fan Zhao
Jin-Xing Wang
Address correspondence and reprint requests to Dr. Jin-Xing Wang, School of Life Sciences, Shandong University, Jinan, Shandong 250100, China. E-mail address: jxwang@sdu.edu.cn
Received: September 15 2008
Accepted: February 18 2009
Published: May 01 2009
Shuai Wang, Ning Liu, An-Jing Chen, Xiao-Fan Zhao, Jin-Xing Wang; TRBP Homolog Interacts with Eukaryotic Initiation Factor 6 (eIF6) in Fenneropenaeus chinensis1. J Immunol 1 May 2009; 182 (9): 5250–5258. https://doi.org/10.4049/jimmunol.0802970
The HIV transactivating response RNA-binding protein (TRBP) plays an important role in many biological processes. We have cloned three cDNAs from newly identified genes in the TRBP family from Fenneropenaeus chinensis. These genes have been designated Fc-TRBP1–3. Recombinant Fc-TRBP1, which was produced in Escherichia coli, was used for panning of a T7 phage display library of the Chinese shrimp hemocytes. From this panning, Fc-eukaryotic initiation factor 6 (Fc-eIF6) was isolated and sequenced. Fc-eIF6 was then cloned, recombinantly expressed, and shown to interact with Fc-TRBP by the performance of pull-down assays and Far Western blot analysis. Expression of Fc-TRBP was detected in many tissues, with elevated expression in the heart, gill, and intestine in the early stages of infection by the white spot syndrome virus (WSSV), and enhanced expression in most tissues following challenge with Vibrio anguillarum. Western blot studies confirmed the increased expression of Fc-TRBP in the gill after WSSV infection. The expression pattern of eIF6 was also analyzed and its expression was also up-regulated in intestine of WSSV-challenged shrimp. The replication of WSSV was reduced after injection of Fc-TRBP. These results indicate that Fc-TRBP and Fc-eIF6 may be components of the RNA-induced silencing complex (RISC), and thereby play a crucial role in the antiviral defense response of shrimp.
The HIV transactivating response RNA-binding protein (TRBP)3 was originally characterized and isolated from a HeLa cell expression library. TRBP was the first cellular protein identified that strongly binds the HIV-1 encoded leader RNA, a trans-activation response (TAR) protein, and enhances virus expression (1, 2, 3, 4, 5).
Three TRBP isoforms, TRBP1–3, have been reported in humans. TRBP1 was the first of these proteins to be identified. TRBP2 contains a splice variant that adds 21 additional amino acids to its N terminus (6). Compared with TRBP1, TRBP3 is missing the C-terminal dsRNA-binding domain (7). TRBPs belong to a family of dsRNA-binding proteins, and both TRBP1 and TRBP2 contain three dsRNA-binding domains (7). The third C-terminal domain mediates binding to Dicer (7, 8) and protein kinase R (PKR) (9), instead of binding to RNA.
TRBP has recently been identified as an important Dicer-interacting protein in mammals (7, 10), and Dicer has been shown to interact with the Argonaute 2 (Ago2) protein, a key component of the RNA-induced silencing complex (RISC). Experiments have shown that knockdown of TRBP results in a general loss of RNA interference (RNAi)-mediated silencing of other target mRNAs, which is consistent with the hypothesis that TRBP interacts with Dicer (8). Work by Chendrimada et al. further supported a role for TRBP in forming a siRNA-TRBP-Dicer-Ago2 complex, in which TRBP functions as a bridge between dsRNA and Dicer for Ago2 recruitment (10). MacRae et al. have shown that the RISC-loading complex can spontaneously assemble in vitro from purified Ago2, Dicer, and TRBP, and subsequently display dicing, slicing, guide-strand selection, and Ago2-loading activities (11).
TRBP has been shown to modulate HIV-1 gene expression through its association with TAR (4, 5), regulate translation through interactions with structured RNA motifs (4), regulate translation during spermatogenesis in testis (12), regulate cell growth (13, 14), and bind to and inhibit IFN-induced PKR in vertebrates (15, 16, 17).
Two recently published studies reported TRBP to be an integral component of a Dicer-containing complex. Knockdown of TRBP was shown to inhibit microRNA (miRNA) biogenesis (10). Eukaryotic initiation factor 6 (eIF6) has been shown to have an important role in the RNAi pathway. Depletion of eIF6 in human cells specifically impairs miRNA-mediated regulation of target protein and mRNA levels (18). Similarly, depletion of eIF6 in Caenorhabditis elegans diminishes lin-4 miRNA-mediated repression of endogenous LIN-14 and LIN-28 protein and mRNA levels. These activities associated with eIF6 are consistent with its proposed abilities to bind the free 60S ribosome subunit and prevent productive assembly of the 80S ribosome (19). eIF6 has also been shown to be a component of the human RISC, a multiprotein complex that also includes MOV10, the homolog of Drosophila translational repressor Armitage, and 60S ribosome subunit proteins (18).
Until now, TRBP and eIF6 have not been identified in shrimp. In this study, we describe the cloning of three full-length cDNAs that encode a TRBP2 homolog and an eIF6 protein from Chinese shrimp, Fenneropenaeus chinensis. The genes from which these cDNAs were derived are designated Fc-TRBP1–3 and Fc-eIF6, respectively. Interactions between Fc-TRBP and Fc-eIF6 were identified in T7 phage library panning experiments and confirmed by pull-down assays and Far Western blots. We also analyzed the expression patterns of TRBPs and eIF6 in response to infection by the shrimp pathogen Vibrio anguillarum and the white spot syndrome virus (WSSV).
F. chinensis (∼10–20 g each) were bought from a shrimp market in Qingdao, Shangdong Province, China. The shrimp were kept in tanks containing aerated seawater.
The WSSV inoculum was prepared according to a previously described method (20). WSSV was extracted from the gills of naturally infected F. chinensis and stored at −80°C. Gill tissue (1 g) was homogenized in PBS (10 ml; 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4). After centrifugation at 5000 × g for 10 min at 4°C, the supernatant was passed through a 450-nm membrane filter and then used as inoculum. Viral quantification was performed according to a previously published method (21). Briefly, a 619-bp WSSV DNA fragment was amplified from the viral DNA extracted from the gills of WSSV-infected shrimp by PCR using primers vF1 and vR1 (Table I). The fragment (denoted as v1) was cloned into the pBluescript II SK+ plasmid and a series of dilutions of this recombinant plasmid (pBluescript II-v1) were made and quantified by use of a spectrophotometer (GeneQuant; Amersham Biosciences). The primers used for real-time PCR assays were vF2 (Table I) and vR1. The real-time PCR program was 94°C for 5 min, followed by 40 cycles of 94°C for 15 s and 62°C for 50 s. Melting curve analysis of the amplification products was performed at the end of each PCR to confirm that only one fragment was amplified. Quantitative real-time PCR data were analyzed by Opticon Monitor 2, and the baseline was automatically set by the software. The WSSV standard curve was derived from the cycle threshold (CT) and the quantity of the template used. The number of virus copies in an infected shrimp was calculated by use of the standard curve.
Oligonucleotide primers used in this studya
Primer Name .
5′ → 3′ Nucleotide Sequence .
vF1 CGT CAT GGA TCT TTC TTT CAC TC
vR1 TTA CTC GGT CTC AGT GCC AGA
vF2 AGC TCC AAC ACC TCC TCC TTC A
Oligo anchor, reverse GAC CAC GCG TAT CGA TGT CGA CT16 (A/C/G)
SMART, forward TAC GGC TGC GAG AAG ACG ACA GAA GGG
TRBPR4 ACA ATC TGG GTT GAT AGT TCT GGT GGA G
5′ PCR primer TAC GGC TGC GAG AAG ACG ACA GAA
3′ Anchor, reverse GAC CAC GCG TAT CGA TGT CGA C
TRBPex, forward TAC TCA GAATTC (EcoRI) ATG TAT CAT CAA CCT CCA CCA A
TRBPex, reverse TAC TCA CTCGAG (XhoI) CCC CTT CTC CCT AAC ACA AAG T
IF6, forward 1 GAG GGA AAC CAG CCT TCA GTA
IF6, reverse 2 TGA CAG CCA GAC CAG CAG CAA
eIF6ex, forward TAC TCA GAATTC (EcoRI) ATG GCT GTT CGC TGT CAG TTT
eIF6ex, reverse TAC TCA CTCGAG (XhoI) GAA GGC TGG TTT CCC TCA CTT TA
TRBPF1 GCT ACA GAC AAT CTC GCT GAG
eIF6RT, forward TGC ACT GGG AAA TGT GGT TGT
eIF6RT, reverse TCC CTG GTT GGA AAT GAC TGA
Actin, forward AGT AGC CGC CCT GGT TGT AGA C
Actin, reverse TTC TCC ATG TCG TCC CAG T
EcoRI and Xho I sites are underlined in the forward and reverse primers, respectively.
For the immune challenge experiments, we injected V. anguillarum (∼3 × 107 cells/shrimp) or WSSV (3.2 × 107 copies/shrimp) into the abdominal segment of F. chinensis. Total RNA from the challenged shrimp was isolated from a variety of tissues (hemocytes, heart, hepatopancreas, gills, stomach, intestine, ovary, and spermary) using Unizol (Biostar). The RNA was isolated at 2, 6, 12, and 24 h postinfection; RNA from the unchallenged shrimp was also isolated as a control. To reverse transcribe the isolated RNA, a SMART cDNA kit (BD Biosciences) was used. The first cDNA strand was reverse transcribed from 5 μg of total RNA by use of the oligo anchor R and SMART F primers (Table I) in combination with a first-strand cDNA synthesis kit (Sangon Biological Engineering Technology and Services).
The methods for constructing and panning the T7 phage display library have been previously described (22). Briefly, the T7 phage display library was constructed according to T7Select System procedures (Novagen). The mRNA was isolated from hemocytes of V. anguillarum-challenged shrimp using the QuickPrep Micro mRNA purification kit (Amersham Biosciences). After reverse transcription, the double-stranded cDNA was prepared and the EcoRI/HindIII linkers were added to the two ends of the double-stranded cDNA. The cDNA was cleaved and ligated to T7 selected vector arms. The vector arms were packed with T7 packaging extracts and the library was amplified after calculation of the phage titer.
Formaldehyde-fixed Micrococcus luteus were used to pan the T7 phage display library. M. luteus was collected, washed twice with TBS (50 mM Tris-HCl, 0.5 M NaCl (pH 7.5)), and fixed with 3.5% paraformaldehyde. The T7 phage display library was also washed with TBS before the addition of a 100-μl aliquot of the library to 100 μl of bacteria (OD600 = 2.0). After gentle mixing, the sample was kept overnight at 4°C. The sample was then centrifuged at 6000 × g for 5 min and the bacteria-bound phages were collected and washed three times with TBST (50 mM Tris-HCl, 0.5 M NaCl, 0.02% Tween 20 (pH 7.5)). The bacteria-bound phages were resuspended in 200 μl of 1% SDS for 10 min before the phages were eluted and collected by centrifugation at 6000 × g for 5 min. Ten microliters of supernatant was then added to 1 ml of BLT5403 host cells and agitated at 37°C for 1 h. After centrifugation, 200 μl of phage-containing supernatant was amplified on an agarose plate containing BLT5403 host cells at 37°C for 3 h. Five milliliters of extraction buffer (100 mM NaCl, 6 mM MgSO4, 20 mM Tris-HCl (pH 8.0)) was added to the plates, which were further incubated at 4°C overnight. Extracted phages were then collected for a second round of panning. After three rounds, 10 plaques were randomly selected for PCR amplification and sequencing. A 1000-bp fragment was obtained and identified as the 3′ end of an Fc-TRBP cDNA. The 5′ end of Fc-TRBP, including the 5′ untranslated region (UTR), was amplified from hemocyte cDNA with a reverse gene-specific primer, TRBPR4, and the 5′ PCR primer (Table I). The PCR procedure included 1 cycle at 94°C for 2 min and 35 cycles at 94°C for 30 s, 55°C for 45 s, and 72°C for 45 s, followed by 1 cycle at 72°C for 10 min. Two isoforms of Fc-TRBP was cloned with primers TRBPexF and TRBPexR (Table I).
The open reading frame (ORF) of Fc-TRBP1 (1032 bp) was amplified from hemocyte cDNA using the primers TRBPexF and TRBPexR (Table I) with EcoRI and XhoI restriction sites inserted at the beginning and end of the ORF, respectively. The PCR conditions were as follows: 1 cycle at 94°C for 3 min; 35 cycles at 94°C for 30 s, 55°C for 45 s, and 72°C for 45 s; and 1 cycle at 72°C for 10 min. The PCR products were then cloned into the EcoRI and XhoI restriction sites of pET-30a. The recombinant pET30a-Fc-TRBP plasmid was transformed into Escherichia coli BL21 (DE3) cells, which were then cultured in Luria-Bertani medium supplemented with 25 μg/ml ampicillin. Isopropyl β-D-thiogalactoside (IPTG; 0.1 mM) was added when the OD600 of the culture reached 0.5. After 3 h of culture, the cells were collected by centrifugation at 6000 rpm for 10 min and resuspended in PBS containing 0.2% Triton X-100. Following cell sonication and centrifugation, Fc-TRBP was purified using His-Bind resin (Novagen) according to the manufacturer's instructions. The purified protein was analyzed by 12.5% SDS-PAGE and stained by Coomassie brilliant blue G250.
Approximately 40 μg of purified Fc-TRBP was added to an enzyme immunoassay plate and incubated at 4°C overnight for panning of the T7 phage display library. After three rounds of panning, 10 plaques were selected for PCR amplification and sequencing. A 454-bp fragment was obtained and identified as Fc-eIF6. The 3′ end of Fc-eIF6 was amplified from hepatopancreas cDNA with gene-specific primers eIF6F1 and the 3′ anchor R primer (Table I). The 5′ end of the gene was amplified by use of a reverse gene-specific primer, eIF6R2, and a 5′ PCR primer. The PCR procedure was as follows: 1 cycle at 94°C for 2 min and 35 cycles at 94°C for 30 s, 55°C for 45 s, and 72°C for 45 s, followed by 1 cycle at 72°C for 10 min.
The ORF of Fc-eIF6 (738 bp) was amplified from hepatopancreas cDNA using the primers eIF6exF and eIF6exR (Table I) with EcoRI and XhoI restriction sites inserted at the beginning and end of the ORF, respectively. Fc-eIF6 was cloned into the EcoRI and XhoI restriction sites of pET-30a and pGEX-4T-1. The recombinant pET30a-Fc-eIF6 and pGEX-4T-1-Fc-eIF6 plasmids were transformed into E. coli BL21 (DE3) and E. coli BL21, respectively, which were then cultured in Luria-Bertani medium supplemented with 25 μg/ml ampicillin. IPTG (0.1 mM) was added when the OD600 of the culture reached 0.5. After 3 h of culture, the cells were collected by centrifugation at 6000 rpm for 10 min. After cell sonication and centrifugation (10,000 rpm, 10 min at 4°C), His-tagged Fc-eIF6 and GST-tagged Fc-eIF6 were purified using His-Bind resin (Novagen) and glutathione Sepharose 4B (Amersham Biosciences), respectively, according to the manufacturers' instructions. Purified proteins were analyzed by 12.5% SDS-PAGE.
A search for sequences matching those of Fc-TRBP and Fc-eIF6 was performed using BLASTX (www.ncbi.nlm.nih.gov). Characteristics of each protein were calculated using the Expert Protein Analysis System (ExPASy; au.expasy.org), and domain predictions were performed using the Simple Modular Architecture Research Tool (SMART; smart. embl-heidelberg.de). Sequence alignments were assembled using DNAMAN software, and a phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis (MEGA) 3.1.
The Fc-TRBP sequence was inserted into the pGEM-T-Easy vector in reverse orientation (Promega), and the plasmid was linearized by EcoRI digestion. Digoxigenin (Dig)-tagged antisense probes were transcribed using T7 RNA polymerase according to the manufacturer's instructions (Roche Applied Sciences). Northern blot analysis was performed according to a previously described method (23). Briefly, ∼10 μg of total RNA was denatured and electrophoresed on an agarose gel containing formaldehyde and transferred onto a nylon membrane. The target mRNA was hybridized to a Dig-labeled antisense RNA probe (100 ng/ml) in 50% formamide at 68°C. Following stringent washing at 68°C, the anti-Dig-phosphatase Ab (AB) was used to detect the probe. Ab binding was visualized by incubation of the membrane with 5-bromo-4-chloro-3-indolyl phosphate and NBT chloride. The intensities of the resulting bands were quantified using Quantity One (Bio-Rad). The ratios of Fc-TRBP to 18S rRNA were calculated.
Five micrograms of DNase-treated total RNA (Takara RNase-free DNase I; Takara Bio) was used as template for reverse transcription of the first cDNA strand. PCR-based amplification of Fc-TRBP was performed using the TRBPF1 and TRBPexR primers (Table I). PCR-based amplification of Fc-eIF6 was performed using the eIF6RTF and eIF6RTR primers (Table I). The PCR procedure included: 1 cycle at 94°C for 2 min and 27 cycles of 94°C for 30 s, 53°C for 45 s, and 72°C for 45 s, followed by a final cycle at 72°C for 10 min. A β-actin fragment was amplified as a reference using actin F and actin R primers (Table I). The PCR procedure for β-actin included: 1 cycle at 94°C for 2 min and 23 cycles of 94°C for 30 s, 53°C for 45 s, and 72°C for 45 s, followed by a final cycle at 72°C for 10 min. RT-PCR assays were performed in triplicate, and the ratios of Fc-TRBP to β-actin and Fc-eIF6 were calculated using Quantity One (Bio-Rad).
Fc-TRBP1 with an N-terminal His tag was expressed in E. coli BL21 (DE3). After induction with 0.1 mM IPTG at 37°C for 2 h, 200 ml of cells was collected and lysed by sonication in 20 ml of PBS containing 0.2% Triton X-100. Ten milliliters of lysed cells was subsequently incubated with His-binding resin (1 ml) for 5 min, then washed with 10 ml of binding buffer (0.5 M NaCl, 20 mM Tris-Cl (pH 7.9), 5 mM imidazole) and then 6 ml of wash buffer (0.5 M NaCl, 20 mM Tris-Cl (pH 7.9), 60 mM imidazole). Pull-down assays were performed by incubation of ∼200 μg of purified Fc-eIF6-GST protein or Fc-eIF6 (after removal of the N-terminal His tag by incubation with thrombin) with His-tagged Fc-TRBP1 and His-Bind resin for 10 min at 4°C. GST protein was used as a control for the pull-down analysis. After three washes with 10 ml of wash buffer, the proteins were eluted with eluting buffer (0.5 M NaCl, 20 mM Tris-Cl (pH 7.9), 1 M imidazole). The eluted proteins were analyzed by 12.5% SDS-PAGE.
The method used for antiserum preparation has been previously described (22). In brief, purified recombinant Fc-TRBP1 (100 μg) was diluted to 1 ml with saline, mixed with CFA (1 ml), and injected into rabbits once per week for 3 wk. Subsequently, the rabbits were given booster injections (100 μg) of the Ag without adjuvant once per week for 3 wk. Blood samples were collected and the antiserum titer was determined by double immunodiffusion using 1% agar in 0.02 M Tris-HCl (pH 8.0) containing 0.15 M NaCl and 0.01% sodium azide.
The method used for Western blot analysis has been previously described (24). In brief, protein was extracted from WSSV-challenged shrimp tissues at 6, 12, and 24 h postinfection. Protein was similarly extracted from unchallenged shrimp as a control. Approximately 200 μg of total protein was submitted to SDS-PAGE and then transferred to a nitrocellulose (NC) membrane. The NC membrane was incubated with Fc-TRBP1-specific antiserum (1/100). Following stringent washing with TBST (100 mM NaCl, 10 mM Tris-HCl, 0.02% Tween), goat anti-rabbit IgG peroxidase conjugates were used to detect the TRBP polyclonal Ab. Ab binding was visualized by incubation of the NC membrane with 4-chloro-1-naphthol (4-CIN) and H2O2.
Approximately 2 μg of purified rFc-eIF6 (after removal of the N-terminal His tag by incubation with thrombin) was electrophoresed by SDS-PAGE and then transferred to a NC membrane. The membrane was incubated with 5 μg/ml purified Fc-TRBP1 for 5 h at room temperature. After three stringent washes with TBST, the NC membrane was incubated with Fc-TRBP1 antiserum (1/100) to detect Fc-TRBP1 bound to Fc-eIF6 on the NC membrane. Goat anti-rabbit IgG peroxidase conjugates were used to detect the Fc-TRBP1 polyclonal Ab. Ab binding was visualized by incubation of the NC membrane with 4-CIN and H2O2. Purified Fc-TRBP1 and BSA were used as positive and negative controls, respectively.
The shrimp were kept in tanks containing aerated seawater. We injected WSSV (3.2 × 107 copies/shrimp) and 6 μg of Fc-TRBP1 into the abdominal segment of F. chinensis. In another group, WSSV (3.2 × 107 copies/shrimp) and 30 μl of TRBP antiserum were similarly injected. The control group was injected with WSSV (3.2 × 107 copies/shrimp) and 6 μg of BSA. At 24 h postinjection, the genomic DNA was extracted from the gills of the shrimp and semiquantitative RT-PCR and quantitative real-time PCR were performed to quantify the WSSV.
Formaldehyde-fixed M. luteus was used to pan a T7 phage display library, and an 873-bp fragment containing poly(A) tail was obtained. A GenBank search was performed using the amino acid sequence of the fragment. This search identified a sequence that is highly homologous to a TRBP from another species. Consequently, the fragment isolated by panning was termed Fc-TRBP. Using the sequence of Fc-TRBP, the 5′ end of the TRBP, including the 5′-UTR, was cloned from hemocyte cDNA. The complete cDNA of Fc-TRBP was determined to be 1526 bp in length and contain an ORF of 1032 bp that encodes a 343-aa protein. Furthermore, Fc-TRBP mRNA was found to contain a 225-bp 5′-UTR and a 269-bp 3′-UTR. Fc-TRBP protein has a theoretical molecular mass of 36.8 kDa and a predicted isoelectric point of 7.11, and it contains three dsRNA-binding motif (DSRM) domains between residues 27 and 92, 130 and 196, and 272 and 338 (supplemental Fig. 1).4 The cDNA sequence of Fc-TRBP (designated Fc-TRBP1) was deposited in GenBank (www.ncbi.nlm.nih.gov/Genbank/index.html) (accession no. EU679001). Two additional isoforms of Fc-TRBP1 were amplified by PCR using the Fc-TRBP expression primers. Fc-TRBP isoform 2 (GenBank no. FJ573167) and isoform 3 (GenBank no. FJ573168) contain ORFs of 972 and 891 bp, which encode 323- and 296-aa proteins, respectively (supplemental Figs. 2 and 3). Both Fc-TRBP2 and Fc-TRBP3 are identical to the Fc-TRBP1, except that they lack 60 and 141 bp between DSRM2 and DSRM3, respectively (Fig. 1 A). The isoforms result from alternative splicing of the same transcript.
A, Multiple alignment of Fc-TRBP1 (GenBank no. EU679001) with its isoforms Fc-TRBP2 (GenBank no. FJ573167) and Fc-TRBP3 (GenBank no. FJ573168). The DSRM of Fc-TRBP is underlined; black-shaded area indicates identity of 100%; gray-shaded area indicates identity of ≥50%. B, The amino acid sequence of Fc-eIF, eIF6 domain is underlined.
A BLAST protein search revealed that Fc-TRBP is highly conserved in relationship to the TRBP2 protein from Aedes aegypti (47% identity) and to the TRBP from Drosophila melanogaster (49% identity). In contrast, homology was relatively low in relationship to vertebrate animals such as Danio rerio (39% identity), Homo sapiens (36% identity), Mus musculus (37% identity), Rattus norvegicus (36% identity), and Xenopus tropicalis (35% identity). However, the DSRM region of these proteins is highly conserved (supplemental Fig. 4). We constructed a phylogenetic tree for the TRBP family of genes (supplemental Fig. 5), and the results show that the TRBP gene family is divided into two groups. TRBP sequences from vertebrates such as D. rerio and H. sapiens form one cluster, while sequences from invertebrates such as F. chinensis and A. aegypti form another cluster.
Purified, recombinant Fc-TRBP1 was used to pan a T7 phage display library. After three rounds of panning, 10 plaques were randomly selected for PCR amplification and sequencing (Table II). A 454-bp fragment was obtained and its deduced amino acid sequence was found to be highly homologous to those of eIF6 from other species. Therefore, it was termed Fc-eIF6. Based on the sequence of the identified fragment, gene-specific primers were designed and used to amplify Fc-eIF6 from hepatopancreas cDNA. The complete cDNA of Fc-eIF6 was 1465 bp and contained a 738-bp ORF that encodes a 245-aa protein. The Fc-eIF6 mRNA was found to contain a 48-bp 5′-UTR and a 679-bp 3′-UTR. The Fc-eIF6 protein includes an eIF6 domain between residues 3 and 204. The protein has a theoretical molecular mass of 26.5 kDa and a predicted isoelectric point of 4.99 (Fig. 1 B and supplemental Fig. 6). The cDNA sequence was deposited in GenBank (www.ncbi.nlm.nih.gov/Genbank/index.html) (accession no. EU679002).
Results of T7 phage display library screening using the recombinant Fc-TRBP
Fragment Blast Results .
Length (bp) .
Score (Bit) .
E value .
Estrogen receptor β (Kryptolebias marmoratus) 435 34.7 1.9
Eukaryotic translation initiation factor 6 (Mus musculus) 454 113 4E-24
Actin, γ1 (Pan troglodytes verus) 342 63.5 4E-09
Oxidoreductase (Streptomyces coelicolor A3(2)) 766 37.7 0.72
Peritrophin (Fenneropenaeus chinensis) 693 472 7E-132
Peritrophin (F. chinensis) 533 62.8 9E-09
Putative senescence-associated protein (Pisum sativum) 282 80.1 4E-14
Ribosomal protein L10Ae (Biphyllus lunatus) 667 231 4E-59
Thrombospondin (Penaeus monodon) 824 372 1E-101
Phosphate transport protein (Bombyx mori) 596 221 2E-56
A BLAST protein search using the Fc-eIF6 sequence indicated that it is highly similar to the eIF6 sequences in other species, including A. aegypti (66% identity), Bombyx mori (66% identity), D. rerio (68% identity), H. sapiens (68% identity), M. musculus (68% identity), and X. tropicalis (69% identity). Alignment of these eIF6 sequences is shown in supplemental Fig. 7. A phylogenetic tree was constructed for the eIF6 family and shows three groups, one from vertebrates such as D. rerio and H. sapiens, one from insects such as B. mori and A. aegypti, and a third from F. chinensis (supplemental Fig. 8).
Northern blot and RT-PCR analyses were used to examine the transcription levels of Fc-TRBP and Fc-eIF6 in several tissues following challenge with V. anguillarum or WSSV. At 24 h postinfection, RNAs from challenged and unchallenged shrimp were collected. Both Fc-TRBP and Fc-eIF6 mRNAs were detected in all tested tissues obtained from the unchallenged shrimp (i.e., hemocytes, heart, hepatopancreas, stomach, gills, intestine, and ovary). In tissues from shrimp challenged with V. anguillarum, Fc-TRBP was slightly increased in hemocyte, stomach, gill, and intestinal tissues, although the increases were not statistically significant. Fc-TRBP was also up-regulated in heart and hepatopancreas tissues following V. anguillarum challenge; no obvious change was observed in Fc-TRBP levels in the ovary and spermary tissues (Figs. 2 and 3,A). Fc-eIF6 was also found to be up-regulated in hepatopancreas tissues, although there was no statistically significant change in expression detected in hemocyte, heart, stomach, gill, and intestinal tissues (Fig. 3 C).
Northern blot analysis of Fc-TRBP expression in F. chinensis challenged with V. anguillarum. Total RNA extracted from uninfected (control, or C) and infected shrimp (bacterial challenge, or B) was submitted to Northern blot analysis. The 18S rRNA was used as a control. The results of the Northern blot analysis were quantified by use of Quantity One software (Bio-Rad). M, marker.
RT-PCR analysis of Fc-TRBP and Fc-eIF6 in shrimp tissues. In each panel, the top portion presents the RT-PCR analysis of Fc-TRBP or Fc-eIF6 expression and the bottom panel presents statistical analysis of the RT-PCR results. Data are expressed as the ratio of Fc-TRBP or Fc-eIF6 to β-actin. *, p < 0.05. A, Bacterial challenge, expression pattern of Fc-TRBP. B, Viral challenge, expression pattern of Fc-TRBP. C, Bacterial challenge, expression pattern of Fc-eIF6. D, Viral challenge, expression pattern of Fc-eIF6. C, Control; B, shrimp challenged by V. anguillarum for 24 h; V, shrimp challenged by WSSV for 24 h.
RT-PCR assays revealed that expression of Fc-TRBP was increased in several tissues, including heart, gills, and intestinal tissues, in response to challenge by WSSV infection. In contrast, Fc-TRBP expression was decreased in hemocyte, hepatopancreas, and stomach tissues. No change in expression was detected in ovary tissues (Fig. 3,B). Fc-eIF6 expression was also found to be decreased in hemocyte and stomach tissues, and there was a sharp increase in intestinal tissue. The Fc-eIF6 level seemed to be slightly increased in heart, hepatopancreas, gill, and spermary tissue, although the increases were not statistically significant (Fig. 3 D).
Time-course RT-PCR analysis was performed to evaluate the expression of Fc-TRBP in hepatopancreas and hemocyte tissues. TRBP expression in hepatopancreas tissues was found to decrease between 2 and 6 h after WSSV infection; it returned to normal levels at 24 h postinfection. Similarly, Fc-TRBP expression decreased in hemocytes and began to recover at 24 h postinfection (Fig. 4).
RT-PCR analysis of Fc-TRBP in the hepatopancreas and hemocytes of WSSV-infected shrimp. The top panels of each graph represent a time-course RT-PCR analysis of Fc-TRBP expression in hepatopancreas and hemocyte tissues. The bottom graphs present the quantification of the RT-PCR data expressed as the ratio of Fc-TRBP to β-actin expression. *, p < 0.05. Lane C, Unchallenged shrimp as a control; other lanes represent the 2, 6, 12, and 24 h postinfection time points.
Western blot analysis was performed with Fc-TRBP1-specific antiserum to confirm the expression pattern of Fc-TRBP detected by RT-PCR. Three immunoreactive protein bands were detected, a result that is consistent with the three isoforms of TRBP (Fig. 5). Proteins were extracted from hepatopancreas and gill tissues of the shrimp at 6, 12, and 24 h following WSSV challenge. Proteins obtained from uninfected shrimp were used as a negative control. Expression of TRBPs in the hepatopancreas was found to decrease at 6 h postinfection and return to normal levels at 12 h postinfection. In contrast, expression of TRBPs increased in gill tissue at 6 h after WSSV challenge. The expression pattern of TRBPs in response to challenge by V. anguillarum was also analyzed. TRBP expression largely remained the same in the hepatopancreas after infection, while it slightly increased in gill tissues.
Western blot analysis of TRBP in gill and hepatopancreas tissues of V. anguillarum-challenged and WSSV-challenged shrimp. For each of the four quadrants, the upper panel represents the amount of protein loaded into SDS-PAGE wells. The accompanying bottom panels for each quadrant represent the results of the Western blot analysis of TRBP. C, Control shrimp; 6, 12, and 24 label the protein samples collected from shrimp at 6, 12, and 24 h postinfection, respectively.
Due to the potential for false positives in phage display screening, pull-down assays were performed to confirm the interaction between rFc-TRBP and rFc-eIF6. His-tagged rFc-TRBP and GST-tagged rFc-eIF6 were purified as shown in Fig. 6. Results from the pull-down assays indicate binding between rFc-TRBP and rGST-Fc-eIF6, as well as between rFc-TRBP and rHis-Fc-eIF6 following removal of the His tag by treatment with thrombin in vitro (Fig. 7). Residual thrombin was present following cleavage of the His tag from Fc-eIF6. The residual thrombin partially removed the His tag from Fc-TRBP, resulting in visualization of two Fc-TRBP bands. As a control, we determined that GST alone was not pulled down in these assays.
SDS-PAGE analysis of recombinant Fc-TRBP and Fc-eIF6 expressed in E. coli. Lane M, molecular mass marker; lane 1, total protein obtained from E. coli expressing pET30a-Fc-TRBP; lane 2, total protein obtained from E. coli expressing pET30a-Fc-TRBP after IPTG induction; lane 3, recombinant Fc-TRBP purified by His-Bind resin chromatography; lane 4, total protein obtained from E. coli expressing pET30a-Fc-eIF6; lane 5, total protein obtained from E. coli expressing pET30a-Fc-eIF6 after IPTG induction; lane 6, recombinant Fc-eIF6 purified by His-Bind resin chromatography; lane 7, total protein obtained from E. coli expressing pGEX-4T-1-Fc-eIF6; lane 8, total protein obtained from E. coli expressing pGEX-4T-1-Fc-eIF6 after IPTG induction; lane 9, recombinant Fc-eIF6-GST purified by glutathione Sepharose 4B chromatography.
Pull-down assays to identify interactions between Fc-TRBP and Fc-eIF6. A, Recombinant Fc-TRBP (TRBP) was bound to His resin to which purified eIF6-GST protein was added. Wash buffer did not elute either protein, whereas elution buffer eluted both TRBP and eIF6-GST proteins. B, Recombinant Fc-TRBP was bound to His resin to which purified eIF6Δhis-tag (the Fc-eIF6 with the His tag was removed by thrombin) and His-tagged eIF6 protein were added. Wash buffer did not elute any of these proteins; however, elution buffer removed all of the three proteins. C, Recombinant Fc-TRBP (TRBP) was added to His resin to which GST alone was added as a control. Only Fc-TRBP was eluted, indicating that there were no interactions between Fc-TRBP and GST. M, Lanes containing molecular mass markers; the sizes of the markers are indicated (in kDa) on the left.
The Far Western blot assay was also performed to confirm the interaction between rFc-TRBP and rFc-eIF6. rFc-eIF6 without a His tag was transferred to NC membrane and incubated with 5 μg/ml purified TRBP. TRBP bound to Fc-eIF6 was detected by immunoblotting (Fig. 8). The results from these assays support the hypothesis that Fc-TRBP directly binds Fc-eIF6.
Far Western blot analysis of the interaction between Fc-TRBP and Fc-eIF6. Fc-eIF6 (after removal of the N-terminal His tag by thrombin) were electrophoresed by SDS-PAGE and then transferred to an NC membrane. After incubation with 5 μg/ml purified TRBP, the membrane was stringently washed and incubated with Fc-TRBP antiserum. Goat anti-rabbit IgG peroxidase conjugates were used to detect the TRBP polyclonal Ab. The bands were visualized using 4-CIN and H2O2. Purified Fc-TRBP was used as a positive control; BSA was used as a negative control. +, Present; −, absent.
To study the function of rFc-TRBP in vivo, WSSV and rFc-TRBP1 were injected into the shrimp. At 24 h postinjection, the replication of WSSV was evaluated by quantitative RT-PCR. WSSV replication was inhibited (∼2.3 × 106 copies/g tissues) by coinjection with rFc-TRBP compared with shrimp injected with WSSV and BSA (∼1.4 × 108 copies/g tissues). Injection with rFc-TRBP antiserum slightly increased the replication of WSSV (2.2 × 108 copies/g tissues), although there was no statistically significant change relative to the control. Results from this experiment suggest that Fc-TRBP plays an important role in the inhibition of WSSV replication.
Increasing evidence indicates that RNAi may have an important role in antiviral immunity in invertebrates. The first direct evidence was reported in Drosophila, in which the flock house virus is both an initiator and a target of RNA silencing in Drosophila host cells. Flock house virus infection has been shown to require suppression of RNA silencing by a flock house virus-encoded protein, B2 (25).
dsRNA can induce sequence-specific antiviral silencing and nonspecific immunity in a marine shrimp, Litopenaeus vannamei (26, 27). Inhibition of viral disease by injection of shrimp with dsRNA specific to viral genes has been reported for at least three viruses: WSSV, Taura syndrome virus, and yellow-head virus (26, 28, 29). dsRNA is produced by both positive-strand RNA viruses (i.e., Taura syndrome virus and yellow-head virus) and DNA viruses (i.e., WSSV) during their infectious cycles (30). These dsRNAs are thought to be involved in the RNAi pathway, which can result in generation of an antiviral response.
The exact mechanism of RNAi in shrimp is not clear. In two previously published papers (7, 10), TRBP was shown to interact with Dicer in mammalian models and to be required for assembly and/or function of RNAi as mediated by siRNA and miNAs. Furthermore, Chendrimada et al. showed that TRBP, as part of RISC, can form a complex with Dicer and Ago2, thereby functioning as a potential bridge between dsRNA and Dicer for Ago2 recruitment (10).
In this study, three new members of the TRBP family in F. chinensis, Fc-TRBP1–3, have been characterized. Phylogenetic analysis and sequence alignments from multiple species demonstrate that Fc-TRBP is highly conserved in relationship to other members of the same family, especially in terms of the DSRM sequence. Sequence alignments indicate that Fc-TRBP belongs to the longest isoform of the TRBP family, TRBP2, which contains three DSRM domains (31, 32), two N-terminal domains that mediate dsRNA binding, and a C-terminal domain that is required for interactions with Dicer (7, 8).
The association of RISC with a multiprotein complex that includes eIF6, MOV10, and 60S ribosome subunit proteins suggests the importance of eIF6 in the RNAi pathway (18). T7 phage display panning using rFc-TRBP1 led to isolation of Fc-eIF6 protein. Pull-down assays and Far Western blot analysis were performed to confirm the interaction between the recombinant proteins rFc-TRBP and rFc-eIF6. These results support the hypothesis that the interaction between Fc-TRBP and Fc-eIF6 is mediated by the multiprotein complex RISC (Fig. 9).
Analysis of the role of Fc-TRBP in the attenuation of WSSV replication. WSSV was detected by semiquantitative RT-PCR (A) and real-time PCR (B). After WSSV infection, the shrimp injected with Fc-TRBP exhibited reduced replication of WSSV, while shrimp injected with Fc-TRBP antiserum produced more WSSV. In the semiquantitative RT-PCR, β-actin and genomic DNA were used as controls. *, p < 0.05.
The expression patterns of Fc-TRBP and Fc-eIF6 in response to challenges by V. anguillarum or WSSV were analyzed by RT-PCR. Expression of both genes was detected in all analyzed tissues and was slightly up-regulated in selected shrimp tissues at 24 h postchallenge with V. anguillarum. When the shrimp were challenged with WSSV, more complex changes were detected. Expression of Fc-eIF6 was increased in the intestine and decreased in stomach. Fc-TRBP was also up-regulated in the gills and intestine; however, no change in gene expression was detected in heart and ovary tissues. Fc-TRBP was down-regulated in hemocyte, hepatopancreas, and stomach tissues. Western blot analysis confirmed the results of the RT-PCR assays at the protein level. Three bands were identified as Fc-TRBPs by Western blot analysis. This result is consistent with the molecular cloning result: three isoforms of Fc-TRBP were cloned from the shrimp. Indeed, multiple isoforms of TRBP have been detected in mammalian species, which may result from alternative splicing of the same mRNA (7). All of the Fc-TRBP isoforms contained three DSRM domains, while human TRBP3 lacks the third dsRNA-binding domain.
Human TRBP was reported to display dual functions, with roles in HIV replication and RNA interference. TRBP enhances HIV expression and replication by inhibiting PKR and increasing translation of structured RNAs, despite its crucial function in an RNAi pathway that could inhibit HIV infection (15, 33). It is reported that the TAR RNA of HIV sequesters TRBP from RISC, resulting in inhibition of RISC activity. This inhibition is relieved by exogenous expression of TRBP (5). We could not find a sequence in the WSSV genome similar to the HIV TAR RNA; however, our results indicate that injection of rFc-TRBP1 reduces the replication of WSSV. Our results suggest that exogenous TRBP may recruit components of RISC in vivo and thereby enhance RNAi.
Weber et al. investigated the presence and localization of dsRNA in cells infected with a range of viruses. Their results indicate that significant amounts of dsRNA are present following productive infection with positive-strand ssRNA, dsRNA, or DNA viruses (30). The origin of the dsRNA structures detected following infection with DNA viruses remains to be determined. dsRNA may arise as a result of overlapping converging transcription (34, 35) or highly structured ssRNAs, such as the adenovirus virus-associated RNAs, of which up to >108 copies are present in a single infected cell (36). Given the data we have presented regarding the interaction between rFc-TRBP and rFc-eIF6, as well as the association of eIF6 with RNAi signaling pathways and involvement with RISC, we propose that interactions between Fc-TRBP and Fc-eIF6 are mediated by RISC and have a role in the anti-viral immune response in shrimp (Fig. 10).
FIGURE 10.
Illustration of potential interactions between Dicer, Ago2, and TRBP in a minimal multiprotein RISC. Interactions between eIF6 and TRBP may incorporate eIF6 into RISC. dsRNAs produced by DNA and RNA viruses can induce an antiviral response through the RNAi pathway.
We thank Dr. Xiao-Qiang Yu, School of Biological Sciences, University of Missouri at Kansas City, for useful discussion and critical reading of the manuscript.
This work was supported by grants from the Natural National Science Foundation of China (no. 30770282), the Major State Basic Research Development Program of China (973 program, no. 2006CB101806), the National High Technology Research and Development Program of China (863 Program, no. 2007AA09Z425), and the Ph.D. Program Foundation of the Ministry of Education of China (no. 20060422034).
Abbreviations used in this paper: TRBP, HIV transactivating response RNA-binding protein; Ago2, Argonaute 2; Dig, digoxigenin; DSRM, dsRNA-binding motif; eIF6, eukaryotic initiation factor 6; 4-CIN, 4-chloro-1-naphthol; IPTG, isopropyl β-D-thiogalactoside; miRNA, microRNA; NC, nitrocellulose; ORF, open reading frame; PKR, protein kinase R; RISC, RNA-induced silencing complex; RNAi, RNA interference; TAR, trans-activation response; UTR, untranslated region; WSSV, white spot syndrome virus.
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Supplementary Figures 1-8 (PDF, 580 Kb)
Description Figure 1: The complete nucleotide sequences of Fc-TRBP1 cDNA... Figure 2: The complete nucleotide sequences of Fc-TRBP isoform 2 cDNA... Figure 3: The complete nucleotide sequences of Fc-TRBP cDNA isoform 3... Figure 4: Multiple alignment of Fc-TRBP amino acid sequences from other animals... Figure 5: Phylogenetic analysis of Fc-TRBP with regard to other TRBP gene family members, respectively... Figure 6: The complete nucleotide sequences of Fc-eIF6 cDNA... Figure 7: Multiple alignment of Fc-eIF6 amino acid sequences with those from other animals... Figure 8: Phylogenetic analysis of Fc-eIF6 with regard to other eIF6 family members...