text:
Contact: email@example.com
LINWOOD — On Monday, Cory Booker's political machinations were finally derailed as Hirsh Singh, New Jersey Republican candidate for U.S. Senate continued to gain ground in the Garden State. The Singh campaign issued a statement shortly after the Democrat was forced to drop out of the 2020 presidential race.
Since April of last year, the Singh campaign has gained considerable ground with New Jersey residents. Many of his newest supporters are from the African American communities, former Democrat voters that suffered heavily from the ill effects of Booker's policies, including record-high crime, unemployment and poverty, ever since he first took power.
"It's easy to paint Cory Booker as a clueless dope with delusions of grandeur," said Singh. "Just look at the water scandal in Newark. Instead of fixing the problem, Booker literally fled the state to campaign for even more power. But voters have awoken. They see through his charades."
Over the past four years, at least 200,000 New Jersey residents have been affected by Newark's lead-poisoned water supply as a direct result of Booker's mismanagement and neglect. Last August, the city committed another $120 million to the issue, with no end in sight.
In spite of Booker's comments calling him "worse than a racist," President Trump would later sign a water bill dedicating another $100 million of federal funds to fixing Newarks water crisis. Less than two months later, Booker would repay the President by accusing him of betraying his oath, a display of hypocrisy critics say may have been the self-inflicted blow that spelled doom for his campaign.
Since losing his corporate backers, Booker has had constant trouble raising funds of any kind. Prior to calling it quits, he'd failed to qualify for the December and January primary debates, with an approval rating of just 4%. In a poll from early this year, a full 25% of voters — from all parties on the political spectrum — rated the Democrat as "Very unfavorable."
Singh has dedicated much of his U.S. Senate campaign to addressing the problems caused by Booker's tenure in power. Bi-partisan analysts have speculated that Booker would likely treat the Senate race as a "contingency" effort — his last-ditch effort to cling to power. Those predictions grow ever more likely now that Booker's presidential dreams have been dashed by his young Republican rival.
"This Senate election is Booker's last chance to stay in politics," says Singh. "He'll be coming back to New Jersey with a vengeance, licking his wounds and demanding support from the same constituents he ignored while pursuing the White House. It won't work — we will never allow it."
In contrast to Booker's dogged anti-Trump rhetoric, Singh has pledged to work with President Trump to restore New Jersey's infrastructure and prestige as an economic powerhouse for the nation. At a Trenton rally on Monday, Singh spoke to thousands of cheering New Jerseyans, declaring "We will not go quiet!" The crowd ridiculed Democrat corruption, breaking out into chants of "We will vote you out!" and "In God we trust!" as they packed the steps of the State Capitol building.
"These are my people," Singh later commented. "They are the intense pressure Cory Booker feels right now. We won't be letting up any time soon. We're just getting started."
For any inquiries, please reply to this message or send an email to: firstname.lastname@example.com
Additional information is available on our official website and Facebook page.
"Singh is the future of the Republican Party," said Freeholder Haines. "The whole Freeholder Board has watched him work...
With the race now entering toss-up territory, attention will turn towards the Republican Party and its ability to put forth a strong candidate with solid conservative credentials and can appeal to independents...
Hudson County Chairman Jose Arango said, "I believe Hirsh Singh is a great candidate that will show that the New Jersey GOP is the real party of inclusion."
In the fight for liberty and freedom, every effort counts. Thank you for your support.


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Florence denim pants
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If you need more information or have any questions about our products, or need an invoice for your order, email us and we will be happy to help you as soon as posible

subset: commoncrawl


text:
Postdoc of Deep Learning for Large-Scale GeoScience and sustainability Applications
Climate change, population growth, and environmental degradation as well as management of our agricultural, natural, and urban resources are major challenges influencing the achievement of multiple of the UN's Sustainable Development Goals. Currently an accelerating amount of geospatial data of immediate relevance are collected by satellites, airplanes, and drones. Such data provides hitherto unseen possibilities for capturing patterns and for generating new understanding of eco- and human systems at the process, regional and global scales. Deep learning methods have been identified as one of the key drivers in this context, and this project is about applying state-of-the-art and developing conceptually new deep learning techniques that are adapted to the specific needs of various challenging learning tasks in the earth and geodata domain.
Taking full advantage of the opportunities derived from these new and overwhelming data sources requires a variety of skills. We are searching for a motivated postdoctoral researcher in machine learning or geosciences, with a dedicated interest in addressing some of these challenges. The position will be opened in the context of a new collaboration between the Department of Computer Science (DIKU) and the Department for Geosciences and Natural Resource Management (IGN) of the University of Copenhagen and will also be connected to the SCIENCE AI Centre. The aim is to initiate new groundbreaking research by applying and extending deep learning methods to extract and to explore relevant information from massive amounts of data.
The successful candidate will have the chance to work on a variety of exciting and challenging tasks within the earth and geodata domain, including:
- Monitoring changes in climate, land use, and vegetation
- Detecting faults and fractures in geophysical wavefield data
- Analysing plant phenotyping and ecosystem structure
- Detecting of landforms such as beach ridge or coastlines
- Analysing human movements based on geospatial data
To address these tasks, the candidate will have to deal with very large and very complex data sets including petabytes of satellite or lidar data, which will necessitate the application and extension of complex deep learning models. We seek for an innovative postdoctoral research for a 2 year position, who has obtained a Ph.D. degree from a computer science department (or similar) or a geo-science department with significant experience in machine learning and data analysis of very large data sets. The candidate is expected to contribute to research activities within machine learning for geodata and earth system science as sketched above. The position will be a true collaborative position between the Department for Geosciences and Natural Resource Management or with the Department of Computer Science of the University of Copenhagen, but the exact balance and place of wok will be negotiated with the candidate and depend on the candidates background.
Inquiries about the position can be made to Prof. Claus Beier (firstname.lastname@example.com), Associate Prof. Thomas Friborg (firstname.lastname@example.com), or Assistant Professor Fabian Gieseke (firstname.lastname@example.com). Further information related to the Departments at the Faculty of Science can be found here: http://www.science.ku.dk/english/about-the-faculty/organisation/
The position is open from 1 October 2019 or as soon as possible thereafter.
The University wishes our staff to reflect the diversity of society and thus welcomes applications from all qualified candidates regardless of personal background.
Terms of employment
The position is covered by the Memorandum on Job Structure for Academic Staff. Terms of appointment and payment accord to the agreement between the Ministry of Finance and The Danish Confederation of Professional Associations on Academics in the State.The starting salary is currently up to DKK 430.569 including annual supplement (+ pension up to DKK 73.627). Negotiation for salary supplement is possible.
The application, in English, must be submitted electronically by clicking APPLY NOW below.
Please include:
- Curriculum vita
- Diplomas (Master and PhD degree or equivalent)
- Research plan – description of current and future research plans
- Complete publication list
- Separate reprints of 3 particularly relevant papers
The deadline for applications is 1 September , 23:59 GMT+1.
After the expiry of the deadline for applications, the authorized recruitment manager selects applicants for assessment on the advice of the Interview Committee. You can read about the recruitment process at http://employment.ku.dk/faculty/recruitment-process
Part of the International Alliance of Research Universities (IARU), and among Europe's top-ranking universities, the University of Copenhagen promotes research and teaching of the highest international standard. Rich in tradition and modern in outlook, the University gives students and staff the opportunity to cultivate their talent in an ambitious and informal environment. An effective organisation – with good working conditions and a collaborative work culture – creates the ideal framework for a successful academic career.


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To access the billing section of your account, login to your go.skupos.com account.
On the main page of your dashboard, click your name in the upper right-hand corner and select "Settings"
On the Settings page, scroll to the bottom of the page and select the blue "Go to Billing Management" button. This will take you to a detailed billing information page for your account.
Short Cut: from any billing related email, click the account management page link.
Here you'll be able to view your existing subscription details, your next invoice date, as well as your current and all previous invoices.
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Kris Aquino slammed James Deakin for dragging her name. James Deakin apologized to Kris Aquino. Motoring journalist and road safety advocate James Deakin has apologized to Kris Aquino for dragging her name into his social media controversy surrounding his photo with Bongbong Marcos. In a Facebook post, Sunday, January 21, Deakins clears social media speculation about his photo with losing Vice Presidential candidate Bongbong Marcos. Note: the comment was already deleted by James Deakin.
Slot to Texas Links The Longhorns will look to have a short memory from last week's loss to rival Oklahoma in the Red River Shootout as they make their way back at home for the first time in nearly a month when No. After last week's loss, the Longhorns sit at on the year and in Big 12 play but have a chance to add another mark to the win column Saturday night when they take on the Jayhawks.
As Texas looks to rebound against a rebuilding Kansas team, Horns takes a look all the details, including the network airing Saturday's game, fans need to know ahead of Saturday's home game.
Kansas: Saturday, Oct.
Check other local affiliates here. Kansas all-time record Saturday will mark meeting No.
Texas coach Tom Herman 's record as a head coach While Saturday will mark his first time facing Texas as the head coach at Kansas, Les Miles is all-time against the Longhorns with his four losses coming when he was the head coach at Oklahoma State.
Last meeting The last time the Longhorns and Jayhawks went head-to-head on the field was Nov. A Texas win would mean The Longhorns would move to on the year and in Big 12 play.


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Despatch Times
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Please allow between 5 and 15 working days for us to process your order. The actual time required to process your order may vary dependent upon current stock and order levels.
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Collection. Your order can be collected from our studio at a mutually arranged time. Please select UK Collection at checkout and contact us to arrange a collection date.
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If you require tracking then please contact us after your order has been placed. A standard charge of £4 for this service will be requested prior to posting.


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Pork cutlets with red apples and parsnip mash
(at time of publication)
- 750g parsnips, peeled, chopped
- ¼ cup trim milk
- 1 spring onion, finely chopped
- olive oil spray
- 4 pork cutlets, trimmed
- 2 red-skinned apples, quartered, cored, thinly sliced
- 2 teaspoons flour
- 1 cup apple juice
1 Simmer parsnips in boiling water until tender. Drain. Add milk and onion. Mash until smooth.
2 Meanwhile, heat a frying pan over a medium heat. Spray cutlets with oil and cook for 3 minutes each side. Transfer to a plate. Add apples to pan and cook for 3 minutes.
3 Mix flour with 1/3 cup apple juice. Add to pan with remaining juice and apples. Return cutlets to pan. Bring to boil. Cover and simmer for 5 minutes, or until cutlets are cooked through and sauce has thickened. Serve cutlets with apple, sauce, mash and a side of steamed silver beet.
Make it gluten free: Use rice flour instead of standard flour.
Nutrition Info (per serve)
Kilojoules 1190kJ
Calories 284cal
Protein 26g
Total fat 3g
–Saturated fat 1g
Carbohydrates 40g
–Sugars 32g
Dietary fibre 9g
Sodium 120mg
Calcium 150mg
Iron 3mg
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By Jana Murdock, assistant to the director, Kopper Top Life Learning Center, Liberty, NC
Jana Murdock is one of 15 emerging nonprofit leaders who attended the 2014 BoardSource Leadership Forum in early October as a Judith O'Connor Memorial Fund Scholar. Her conference learnings are the focus of her comments here.
The recent BoardSource Leadership Forum was quite an experience! As a novice in the world of governance, I was amazed at the resources and support that are available to make nonprofit boards something worth having.
I was excited to learn the basics of building a board, finding out what kind of people you might need, and what they should be doing. I spent a lot of time at sessions learning about the relationship between the executive director and the board — this is such a fragile connection, especially if the ED is also the founder...which mine is! Building that relationship, strengthening it to the point where neither side feels threatened and everyone is able to contribute: That's my goal. It's lofty, this is true, but the most effective nonprofits have solid leadership, both in the day-to-day operations and in the oversight and long-term planning realms.
As one of the leaders of a very small organization, my first step will be clarification. We need to determine how many staff we need, what their roles should be, and how they will be funded. To begin this process, we need to separate out executive director tasks from operations tasks from maintenance tasks...the list goes on and on. With only 1.5 staff, you can imagine how muddy those waters have gotten!
Once we have that figured out, we need to decide who wants to do what. My ED is basically doing everything, but it's time to figure out what it is she actually likes doing, and what role she wants to assume when we start hiring more staff. As she works through that process, we will be able to discern what roles actually need to be filled, and prioritize them. At the conference, I learned that evaluations can be an extremely useful tool for this!
The board is, of course, an integral part of this process. Before we can do anything else, we need to look clearly at the board and see how it is operating now and decide how we would like it to operate in the future. Although the members are doing a lot of hands-on work at the moment, which is fine for a start-up, we need to develop a plan to transition them into the oversight role. Hiring more staff is a certainly a part of this process. The board should help find funding to hire these people.
It's a big job! I'm trying to transition my organization from a start-up to a growing organization. I was able to get a much better handle on what needs to happen and how at the conference. I feel much more confident going forward and talking to my board now that I have some solid knowledge! I'm looking forward to this chance to help my organization grow!
Interested in learning more about the Judith O'Connor Memorial Fund Scholarship program? Click here.


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Million-dollar dreams
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The competition was tough, but it wasn't war: Geingob
Incumbent President Hage Geingob whose popularity waned by 31 percentage points from the 87% he received from the electorate five years ago, has admitted that this year's Presidential election was a tough one.
The results announced by Electoral Commission of Namibia (ECN) Chairperson Advocate Notemba Tjipueja revealed that Geingob received 464 703 (56,3%) of the possible 826 198 votes.
Accepting the outcome of the election here on Saturday night, Geingob acknowledged that he faced stiff competition from his 10 competitors to retain his position.
Geingob though was quick to note that the election was not a war but a contest of ideas and programmes.
In his victory speech, he urged those that participated in the election to hold hands as Namibia is the only country they have.
"I said that this is the only country that we can call home. This was not a war on enemies. We are exercising our democratic right," he said, adding that the ECN had accomplished what he termed a very difficult task.
"I would like to thank those that were participating (competing) in the elections, it was tough. I campaigned like hell otherwise I couldn't be standing here. There is always a loser and winner if you go in an election or boxing ring," he said.
He added: "Competition was tough. [But] I emerged as the victor. Yes, it is a high responsibility. It's not a joke…I was there already so I will just continue. But there is a winner and a loser. But in this case, democracy was the greatest winner."
The Head of State also congratulated Utjiua Miunjangue, the National Unity Democratic Organisation (NUDO) of Namibia president who became the first female presidential candidate to participate in the national elections.
"It's a good start," he said as Miunjangue nodded in agreement.
He also commended the manner in which political parties and the independent presidential candidate conducted themselves during the election campaigns.
Geingob then addressed his own performance in this year's election.
"I would have said I am cheated because last time I got 87%. But am I going to say I am cheated because I got 51% or 56? That's what I got apparently. I will accept it for there is a winner and loser," he said.
In the National Assembly, the ruling Swapo Party to which Geingob belongs also lost its two-thirds majority, something its leader took note of.
Swapo now has 63 seats in the National Assembly, meaning it lost 14 seats during this year's election.
Commenting on the outcome of the National Assembly election, Geingob said the next Parliament will be interesting to watch as it will be composed of a variety of political parties.


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text:
Alexander Gruenwald is Senior Expert at the Austrian Federal Ministry for the Civil Service and Sport and head of GovLabAustria, Austria's public sector innovation lab. His work and teaching assignments focus on outcome orientation, innovation, change and knowledge management as well as participation.
Alexander Gruenwald studied business administration at the Vienna University of Economics as well as Public Policy and Administration at the Hertie School of Governance in Berlin. Before joining the Austrian Federal Ministry for the Civil Service and Sport he worked for several years in the public and private sector in Austria, Germany and the US.


text:
Being the proud owner of your very own home cinema is on the wish list of many of our customers. And for good reason too. While there are many arguments to opt for a television, nothing beats the all-out emersion and full-on experience of your own private cinema, including the big screen. It sounds like a massive luxury, but really, it doesn't need to be. Especially not if you can save some bucks on Home theatre projector and screen installation.
If you've never installed a projector or screen before, the process can be quite intimidating and maybe even confusing. The general idea is simple, and you probably already have a good idea of where you want each piece to go, but the specifics are unclear. Which is mounted first, the screen or the projector? How far back should the projector be from the screen? How high should the screen be? How do you fine tune the projector's position to fill the entire screen? This guide answers all of those questions, and hopefully any others you have about how to mount a projector and screen as well.
Best location for the projector screen
Your very first task is to determine the best location for the screen. There is usually only one wall in the room that the screen will work on, but try to place it on a wall that has no direct light. The more light that hits the screen, the more washed out the image will look. If the light in the room cannot be completely controlled, you might consider an ambient light rejecting screen.
After you have decided on the best wall, the next task it to decide on the height. We recommend that the bottom of the screen be around 40-60 cm off the floor. If you have multiple rows of seating, you may need to go a bit higher to keep clean sight-lines for people sitting behind the first row. Also take into account anything that will sit below the screen, like a center speaker or an entertainment cabinet. According to THX, viewers should not have to look up more than 15 degrees.
But before you get carried away and start mounting the screen on the wall, there are some other considerations to make that you need to get sorted first. The height of the screen can be limited by the vertical offset and lens shift of your projector (which is dependent on the projector of your choice). So for now, hold your horses.
Best location for the home theatre projector
Determining the best location for the projector is quite a bit trickier than the screen. You need to take into account the projector's throw distance, vertical offset, and lens shift, in addition to restrictions caused by the placement of electrical outlets, lights, and joists, beams, studs. Each of these variables needs to be considered one at a time. Throw distance is a good place to start.
Throw distance
The throw distance rating of a projector tells you how close or far away from the screen the projector can be mounted. If you mount the projector too close to the wall, the projected image will be too small. If you mount it too far away, the image will be too big. Thankfully, nearly all modern projectors have a zoom adjustment, which allows you to adjust the size of the image without actually moving the projector closer or farther away from the screen. This can give you great flexibility when figuring out the proper distance from the wall for your projector.
Throw distance x width of screen in inches = distance from screen to projector lens
Throw distance
For example, if your screen is 87" wide and the throw distance of your projector is 1.3, the projector should be 113" (87×1.3=113") from the screen. Remember to measure from the centre of the projector's lens straight to the screen, like the above image shows.
Practical throw distance
For most people, your projector will list a throw distance, or throw ratio in this case, of something like 1.5-2.25. The wider the throw ratio, the more wiggle room you have in placing your projector. Note that the closer the projector is to the screen the brighter the image will be. You can always turn the brightness up or down to suit your preferences. If the system is going in a room that will have ambient light, then the closer to the screen the better, as long as you stay within the rated throw range of your projector.
Always leave a little bit of margin in the distance calculations. We've had the experience with one type of projector where the manufacturer was a bit on the generous side with their throw distance. The result was an image that was not quite filling the screen. Not good.
With all of that said, you can usually get a little deeper black levels with the projector farther away. Also consider the noise of the projector when running. The fan in some projectors is loud enough to be heard during quiet scenes of a movie, so mounting it behind your seating position isn't a bad idea. By the time you have taken into account throw distance of the projector, seating locations, and ceiling fixtures, you will probably have a very limited spot to mount the projector.
Vertical lens shift
Most projectors offer vertical lens shift, which means that the image can be adjusted up or down until it is at the proper height on the wall. If your projector has adjustable vertical lens shift, I recommend actually powering on the projector and pretend-holding it (use a ladder to save your arms) at the height and distance you want it mounted. Next, adjust the vertical offset until the image is in the right location. There may be occasions where you may need to physically lower the projector for everything to line up. In that scenario, you will need a projector mount that can accommodate an extension pipe.
Vertical lens shift
But to ease your mind, we've only very rarely encountered situations where mounting the projector lined up with the horizontal centre of the screen could not be done.
Horizontal lens shift
While you are mounting your projector, here's why you should do everything within your power to mount the projector horizontally centred on the screen: First, lens shift can degrade picture quality by subtly distorting the image. The distortion might not be very noticeable, but is still a possibility. Second, vertical and horizontal lens shift interact with each other. The more of one you use, the less of the other you can use. JVC warns consumers about this in the image below. So our tip: try to avoid the use of the horizontal lens shift as much as possible.
Home theatre Projector installation
With that out of the way, we have a pretty good idea of where the projector lens needs to be. The next step is to physically mount the projector to the ceiling. We prefer the Strongmount XL projector mounts. Here's why: cheap mounts tend to be very difficult to make precision adjustments; not the Strongmount XL. It's the difference between a frustrating experience and an enjoyable one. These said cheap projector mounts also tend to be prone to drift, which is when the projector slowly moves out of alignment over time. Nice mounts are more likely to hold the projector in the exact location you leave it.
Tackling the projector mount
There are two major parts to a projector mount, the part that attaches to the projector (projector plate) and the part that attaches to the ceiling (ceiling plate). You always start by attaching the projector plate to the top of the projector. Follow the instructions for your particular mount. Note that leveling the projector is easiest if you center the plate on the projector and ensure the plate is close to level.
Next, you need to determine the exact location the ceiling plate should be mounted. Attach the ceiling plate to the projector plate. Your goal here is to completely assemble the mount while the projector is still sitting on the ground. Measure the distance from the lens to the ceiling plate. As you can see in the image below, that distance was about 6" for this install. Carry over that measurement to where you marked the ideal lens placement on the ceiling earlier in the install. Measure back and make another mark for the ceiling mount. In this install, that would mean that I simply measure six inches back from the lens mark, and that's where the front of the ceiling plate should be.
If the ceiling plate isn't centred side-to-side with the projector lens (some lenses are out of centre on the projector), you will also need to take that distance into account. In the below image, the ceiling plate is centred side-to-side with the lens, so no additional measurements were needed.
The mount should come with 4 bolts, but most of the time only two are needed if they are properly centered on a ceiling joist.
I never fully trust stud finders (especially on textured ceiling), so I recommend poking a small nail or precision screw driver into the ceiling to ensure you found a joist. In some instances there won't be a joist where you want to mount the ceiling plate. In this case, you will either need to utilize lens shift or mount the plate to a block of wood that's large enough to span the distance between two joists and secure that block to the ceiling joists (that block can go inside the ceiling if you have attic access). If you are installing your projector in a new-build or large renovation, you can ask your builder to allow for this (which is why we keep recommending to start planning your home cinema early on in the building project) Once the ceiling plate is secured, install the trim plate that came with the mount.
Final home theatre projector installation step
Enlisting the help of a significant other or friend is a good idea when attempting the next step. Verify everything is secure (important step, we say!), then lift up the projector and attach it to the ceiling plate. Install any safety screws that came with the mount. Attach any power and video cables, and loosely zip tie the cables in place. To help avoid drift and lessen the risk of damaging the inputs on the projector, zip tie the cables to the mount, not the projector. You still need to leave enough slack for final adjustments.
Projector screen installation
Now is a good time to put the screen up. We won't go into detail as it really depends on whether you have chosen a fixed frame screen (acoustic transparent is different from normal fabric!), motorised (in ceiling or on ceiling?) or manual screen.
Now comes the hardest part of the entire process. You will need to adjust the focus, zoom, pitch (tilt up and down), yaw (angle to the left/right), and roll (horizontal level) of the projector/mount. This is where buying a nice projector mount, like the StrongXL, really pays off. These adjustments are very minor, thus they require a mount that is stable and offers precision control.
Most projectors come with a built-in grid pattern that will help when making these adjustments. If you don't have access to any test pattern, a normal 16×9 HD signal will also work.
You should start by taking a minute or two to get the image relatively close to accurate before doing any fine adjustments. As you go through this arduous process, know that you will have to revisit each adjustment multiple times. Every time you adjust one thing, it will affect something else. This is a back-and-forth process. It's also nice to use two people, one to make the adjustments and the other to stand by the screen and give feedback. This is especially true if the projector is far away from the screen.
Zoom adjustments
Zoom is adjusted by turning a ring around the lens or can be motorised. I normally make the image slightly larger than the screen to ensure that the entire screen is filled. Any part of the image that spills over will be absorbed by the screen frame. Sometimes I will purposefully make the image too small while performing other adjustments and only zoom out to the cover the full screen when everything else is dialed in. This is because it can be easier to tell if pitch, roll, or yaw are incorrectly set when you can see the border of the image on the actual screen, as opposed to the frame around the screen.
Focus adjustments
Adjusting focus on a projector is just like with a camera. Again, it can be controlled via a ring around the lens or it can be motorised, depending on your projector. You start by moving the image out of focus, and then slowly adjusting the ring until the image snaps into focus. We've heard about people using a pair of binoculars. We prefer to walk up to the screen to check focus close-up. That said, many people simply adjust focus using their naked eye from the distance of the projector.
Roll adjustments
Roll is fairly easy to adjust. Assuming that the screen is level, all you need to do is set a small torpedo level on the projector, oriented so it is parallel to the screen, and adjust the mount until the projector is also level. If yaw is not correct, it's possible that the image will look unlevel even if the projector and screen are both level. Remember, fine-tuning is a process of repeatedly making small adjustments to each setting. You don't simply adjust any setting to be perfect and then move on to the next one. It's very much a back and forth process.
Incorrect Roll
Pitch Adjustments
If the projector is pointing up or down, you will have issues with pitch. The projector should be pointing straight forward, perpendicular to the screen. If pitch is set correctly, the left and right sides of the image will be parallel. The easiest way to dial in pitch is to set a small torpedo level on the projector, oriented so it is perpendicular to the screen, and adjust the mount until the projector is level.
Incorrect pitch
Once the pitch is set properly, chances are that the image will be too high or low on the screen. This is where vertical lens shift comes into play. Use lens shift to bring the image up or down. You may have to go back and forth between adjusting pitch and vertical lens shift a few times until the image is lined up properly.
A note of caution: Do not, EVER—no matter what—use keystone correction. Keystone correction is digital processing used to manipulate the size of the image and compensate for incorrect pitch. It is convenient, but will reduce image quality. If vertical lens shift doesn't do the trick, you will need to physically lower or raise the mount or screen.
Yaw Adjustments
Yaw is the most difficult adjustment to make. Unlike roll and pitch, you can't use a level. To adjust yaw, you need to turn the projector from side-to-side until the top and bottom of the image are parallel. If the lens is exactly horizontally centred on the screen, the image will also be horizontally centred on the screen. If the lens is a little off, the image will be off. Just like adjusting pitch, lens shift is used to fix this problem. Adjust yaw a little bit, then lens shift, then yaw, and so on, until the image is horizontally centred. Use a laser pointer measure to measure the distance from the left side of the projector to the screen and then the right side of the projector to the screen. The distance should be equal for both!
Incorrect Yaw
An invaluable tip when adjusting yaw is to use a piece of tape to mark the centre of the screen (place the tape on the frame, not the screen). It's far easier to line up the centreline on a test pattern to a piece of tape than it is to use the edges of the screen as a reference. You can also use this trick when adjusting pitch, just put the tape on the vertical centre of the screen instead of horizontal centre.
Have a go, keep working until your image is perfectly adjusted on the screen. It's a bit of a work of patience and persistence, but once it's done. It'll have been worth every second of it. And with the projector and the screen perfectly mounted, you can now proceed in getting the projector settings to your liking, kick back and indulge in the incredible experience that is a home theatre all to yourself!
Source: Audioholics


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Predominant selection of T cells specific for the glycosylated collagen type II epitope (263–270) in humanized transgenic mice and in rheumatoid arthritis
Communicated by N. Avrion Mitchison, University College London, London, United Kingdom (received for review February 25, 2002)
Related Article
Rheumatoid arthritis (RA) is associated with certain MHC class II alleles and is characterized by a chronic autoimmune response in the joints. Using transgenic mice expressing human DR4 (DRB1*0401) and human CD4, but lacking endogenous MHC class II, we show that posttranslational glycosylation of type II collagen (CII) influences the level of T cell tolerance to this candidate cartilage-specific autoantigen. In such mice, the expression of human CII resulted in a tolerized murine T cell response to human CII. However, tolerance induction remained incomplete, preferentially deleting responses to the nonmodified CII 263–270 epitope, whereas T cell recognition of a glycosylated variant of this epitope was affected to a lesser degree. A similar dominance of T cell responses to CII-glycopeptides was recorded in a cohort of severely affected RA-patients (n = 14). Thus, RA T cells predominantly recognize the immunodominant CII peptide in its glycosylated form and may explain why previously it has been difficult to detect T cell responses to CII in RA patients.
Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects peripheral joints with cartilage destruction and subsequent bone erosion. The role of T cells in RA is supported by the large number of activated CD4+ cells reported in the synovium of affected joints and by the association of RA to certain MHC (HLA) class II genes [e.g., DRB1*0401 (in DR4) and DRB1*0101 (in DR1)] that encode a specific peptide binding pocket, the so-called shared epitope.
Collagen type II (CII), the main constituent of hyaline cartilage, has been proposed as one possible autoantigen in RA because CII-specific antibodies are frequently found in RA patients and because an RA-like disease can be induced in certain mouse strains after immunization with CII. However, reports on T cell immunity to CII in RA patients as well as in healthy individuals are inconclusive, and the role of CII, and even T cells, in RA is still argued and remains to be proven.
Previous studies performed in DR4- and DR1-expressing mice have located the immunodominant T cell epitope to position 263–270 in CII by using synthetic peptides (1–3), but studies in RA patients have in general failed to identify DR4/DR1-restricted T cells specific for the same epitope (4, 5). One potential reason for this failure is that these studies did not directly address that CII can become posttranslationally modified. Within CII263–270, the lysines at positions 264 and 270 can be hydroxylated and further glycosylated with mono- or disaccharides, i.e., with a β-d-galactopyranosyl or an α-d-glucopyranosyl-(1-2)-β-d-galactopyranosyl residue. Such modifications have previously been shown to be of importance in the development of collagen-induced arthritis (CIA) in Aq-expressing mice (6, 7). Although the Aq-molecule is of a DQ isotype, its peptide-binding groove shows more similarity to the shared epitope variant of the DR4-molecule, because it binds almost the same CII epitope and presents the same amino acid side chains to T cells (3, 8–11). Furthermore, CIA is most commonly induced with heterologous CII, which is believed to induce a heteroreactive T cell response, followed by a B cell response, which in contrast to the T cell response is highly crossreactive to mouse CII (12, 13). Thus, earlier CIA experiments in DR4-expressing mice have not addressed T cell tolerance to self-CII. Therefore, to get a better understanding of autoimmunity to cartilage-derived proteins, we need to consider both posttranslational modifications and the aspect of T cell tolerance in these models. To achieve this result, we used a humanized mouse model expressing HLA-DRB1*0401/DRA1*0101, human CD4, and human CII (huCII) on a background deficient of murine class II expression (14, 15). In these mice, T cell responses to huCII peptides and the impact of posttranslational modification on the induction of CII-specific T cell tolerance was investigated.
Materials and Methods
Crossing of different transgenic mice generated two groups of mice that were used for experiments: (i) DR4, expressing transgenic DRB*0401 and human CD4 but no murine class II molecule; and (ii) huCII/DR4, expressing DRB*0401, human CD4 and huCII but no murine class II molecule. To get these groups, huCII transgenic mice (15), mice expressing HLA-DR4 together with human CD4 (14), and mice lacking H-2 class II (16) were crossed as follows. First, HLA-DR4/huCD4 on a B10 background was introduced with H-2plus/− on a B6 background and backcrossed twice to B10 and subsequently intercrossed. HLA-DR4/huCD4, H-2−/− mice were then crossed with huCII mice on a C3H background. Offspring were backcrossed three generations to HLA-DR4/huCD4, H-2−/− on the B10 background. Finally, mice were intercrossed twice and selected for homozygosity in H-2−/−. Mice were bred and kept in the animal facility of Medical Inflammation Research (http://net.inflam.lu.se).
Antigens and in Vitro T Cell Assays.
HuCII was extracted from hip joints (obtained from replacement surgery) after pepsin digestion and purified as described (17). The following nonmodified and glycosylated CII peptides were synthesized as described (8, 18, 19): K264/270 (nonmodified CII261–275 with a lysine residue at position 264 and 270); Gal264 (CII261–278, glycosylated with a β-d-galactopyranose residue on l-hydroxylysine exclusively at position 264); Gal270 (CII261–278, glycosylated exclusively at position 270); and Gal264/270 (CII259–278, glycosylated at position 264 and 270). The following modified forms of the Gal264-glycopeptide, named deoxy-glycopeptides, were also synthesized (B.H., unpublished data): 2-deoxyGal (CII259–273, where the hydroxy-group at position 2 on the β-d-galactopyranosyl moiety has been eliminated); 3-deoxyGal (CII259–273, missing the hydroxy-group at position 3 on galactose); and 4-deoxyGal (CII259–273, missing the hydroxy-group at position 4 on galactose). As control for the deoxy-peptides, a shortened form of the Gal264-peptide (CII259–273) was used. Mice were immunized with huCII in complete Freund's adjuvant, and 10 days later cells from draining lymph nodes were stimulated in vitro for determination of antigen-specific proliferation and IFN-γ production as described (20). Establishment of T cell hybridoma clones and determination of antigen specificity was performed as described (8), with the exception that Gal264/270-peptide was used for immunization and for the first in vitro restimulation (20 μg/ml).
Analysis of Human T Cell Responses.
Fourteen patients fulfilling the RA-classification criteria of the American College of Rheumatology (21) were recruited for the study. The study protocol was approved by the review board of the Friedrich-Alexander-University Erlangen-Nuremberg, and informed consent was obtained from all individuals before entering the study. All patients had a severe course of the disease so that insufficient response to conventional therapy required an intensified treatment with TNFα-blocking antibodies [D2E7 (Knoll AG-BASF Pharma, Ludwigshafen, Germany) or infliximab (Essex Pharma, Munich, Germany]. The mean age of the patients (2 male and 12 female) was 63.6 ± 6.5 (SD) years, and the disease duration 10.9 ± 8.1 (SD) years. In 9 patients, information about the expression of HLA-DRB1*-alleles was available and is included in the result section. Peripheral blood mononucleated cells (PBMC) from the patients were separated by using Histoprep (BAG, Lich, Germany). For antigenic stimulation of 106 PBMC, 10 μg of CII-peptide and 1 μg anti-human CD28 (Becton Dickinson) were added per ml of culture medium. T cell receptor-specific responses were controlled in parallel by using culture conditions that either omitted any stimulation or only exposed the cells to the costimulatory anti-CD28 antibody overnight in the absence of antigen. T cell responsiveness to a common recall antigen was tested in parallel cultures of PBMC by using 10 μg/ml tetanus toxoid (Calbiochem-Nova Biochem) and anti-CD28 for stimulation. Monensin (2.5 mM; Sigma-Aldrich) was added to the overnight cultures, and the cells were incubated for additional 4 h before harvesting. Subsequently, the cells were washed twice in PBS and fixed in 4% paraformaldehyde/PBS solution for 7 min at 37°C, followed by a repeated washing procedure in PBS. A permeabilization step was performed for 10 min with 0.5% saponin/1% BSA/0.1% NaN3 in PBS; afterwards, the cells were washed twice with PBS/1% BSA. Cells were stained with 0.2 μg rat anti-human IL-2-PE (Becton Dickinson) and 3 μl CD4-FITC or CD3-FITC (Beckman Coulter) for 20 min at 4°C. Fluorescence intensities were determined by using a Coulter Epics XL-MCL flow cytometer and system-ii software. Large activated lymphocytes (blasts) were gated according to forward and side scatter as described previously (22, 23). Cells not treated with saponin were used to exclude background staining of anti-IL-2 antibody.
Strong, but Incomplete, Negative Selection of the Autoreactive T Cell Population.
Previous studies have identified the immunodominant T cell epitope in bovine and human CII in DR4-transgenic mice, and they have also shown that these mice are susceptible to CIA (1–3). However, because bovine and human CII both differ from mouse CII within the identified epitope (at position 266, glutamic acid in heterologous CII, compared with aspartic acid in mouse CII), these reports did not include the aspect of T cell tolerance to self-CII. To generate an animal model of RA that would reflect the situation in humans more accurately, we crossed DR4/human CD4-transgenic and mouse MHC class II-deficient mice with huCII-transgenic mice to generate two lines of mice: DR4 mice (DR4/human CD4-transgenic and mouse MHC class II-deficient mice, expressing mouse CII in cartilage) and huCII/DR4 mice (DR4/human CD4-transgenic and mouse MHC class II-deficient mice, expressing huCII in cartilage). These mice were then immunized with huCII to examine the degree of T cell tolerance to endogenously expressed CII.
The heterologous immune response in DR4 mice to CII was biased toward the nonmodified peptide (K264/270), followed by a weaker response to peptides glycosylated with β-d-galactopyranosyl moieties on hydroxylysine residues at either position 264 (Gal264) or 270 (Gal270) (Fig. 1). The response to huCII protein was also weak but significantly above background level when measuring the production of IFN-γ. In sharp contrast, the response in huCII/DR4 mice was severely reduced against all CII antigens as compared with the response in DR4 mice, showing that strong tolerance to self-CII is present (P value less than 0.05 against all CII antigens except the Gal264/270-specific IFN-γ production). Most importantly, however, tolerance was not complete because huCII/DR4 mice were able to mount a significant response above background level against the Gal264-peptide, when the proliferation (P = 0.023) as well as IFN-γ production (P = 0.041) was measured (Fig. 1). In contrast, the response against the nonmodified K-peptide in huCII/DR4 mice was not significant above the background response (P > 0.05, Fig. 1).
To further evaluate the shift in epitope selection between DR4 mice (with a dominant response to the K264/270-peptide) and huCII/DR4 mice (with a significant response against the Gal264-peptide alone), we made a pairwise comparison of the response to the K- and the Gal264-peptide (Fig. 2). The response to the K-peptide was stronger than to the Gal264-peptide in all DR4 mice at both concentrations tested (10 and 50 μg/ml, P = 0.0051). In contrast, a relatively stronger response against the Gal264-peptide than to the K-peptide was noted in 8 of 10 huCII/DR4 mice when cells were stimulated with the higher antigen concentration (P = 0.0218) and in all mice when stimulated with the lower antigen concentration (P = 0.0051). Hence, CII-specific T cells are strongly tolerized in DR4 mice expressing huCII in cartilage. However, glycopeptide-specific T cells appear to be less affected by tolerance than T cells specific for the nonmodified epitope.
Different T Cell Fine Specificity of Auto- and Heteroreactive T Cell Clones.
To confirm our finding that DR4-restricted CII recognition includes T cells specific for glycosylated epitopes, hybridoma clones from Gal264/270-peptide-immunized DR4 and huCII/DR4 mice were established and characterized for comparison. Despite the weak in vitro response to glycosylated CII antigens seen earlier in DR4 and huCII/DR4 mice after CII immunization, glycopeptide-specific hybridomas were successfully established from both lines of transgenic mice (Table 1).
Specific recognition of the glycopeptide was evident because all clones responded to the Gal264/270-peptide but not to the nonglycosylated peptide. The majority of T cell clones from DR4 mice (named mDR for mouse CII and DR4) recognized the Gal264-peptide (8 of 10, group I and II), whereas only 2 clones, originally collected from the same subcloning, specifically responded to the Gal270-peptide (group III). Gal264-specific clones could further be divided in two groups where clones that displayed a higher sensitivity to the glycopeptide also had a strong response toward huCII (group I), whereas clones that responded weakly to the Gal264-peptide also responded weakly to huCII (group II). In line with this finding, the Gal270-specific clone(s) responded weakly to the Gal270-peptide and not at all to huCII.
Interestingly, T cell clones from huCII/DR4 mice (named hDR, for huCII and DR4) differed from mDR clones in their response to CII-peptides: (i) The biased recognition of the Gal264-peptide was not observed for hDR-clones (Table 1). (ii) Among the Gal264-specific clones (group I and II), the response was somewhat weaker, compared with the corresponding mDR clones. This difference in antigen sensitivity was even more evident in the response to intact huCII (Fig. 3 and Table 1). (iii) Gal270-specific hDR clones also responded to huCII (group III), although high antigen concentration was required (Table 1).
To confirm that it was the posttranslational modification of CII that was specifically recognized by the obtained clones, Gal264-specific hybridomas were also tested against three differently modified glycopeptides, where one of the hydroxy-groups on the galactose moiety had been selectively removed at either position 2, 3, or 4, thereby generating three mono-deoxy glycopeptides. The two groups among the Gal264-specific mDR clones were confirmed in that they, apart from having different sensitivity to the Gal264-peptide, also recognized the sugar residue differently (Table 1). Similarly, the two groups of Gal264-specific hDR clones were also confirmed by the use of the deoxy-glycopeptides. Notably, differences in deoxy-glycopeptide responses between mDR and hDR clones indicated that T cells were differently selected if huCII was expressed. For example, all mDR clones were stringently dependent on the hydroxy-group at the C4-carbon of galactose, whereas the responses of 3 of 4 hDR clones did not depend on the C4-hydroxy-group.
Glycopeptide-Specific T Cells Dominate the CII Response in RA Patients.
Thus far, results from our humanized animal model showed that T cells specifically recognizing the glycostructure remain after tolerance induction and become dominant by the introduction of autologous huCII, whereas T cells with other fine specificities to the CII epitope seem to be functionally impaired or deleted to a greater extent. We argued that these findings could explain earlier reported difficulties in identifying CII specific T cell clones from joints or blood of RA patients, and therefore we tested a total number of 14 RA patients for T cell responses against the different glycosylated peptides. In the investigated cohort of RA patients, cytokine flow cytometry of in vitro-stimulated PBMC revealed specific IL-2 responses to the glycosylated CII-peptides. Using the increase in the percentage of IL-2-producing cells within the CD3 or CD4 positive T cell population above baseline of negative control as a parameter of specific T cell stimulation, 7 patients could be identified as CII-peptide responsive (Table 2). Interestingly, all CII-responding patients recognized the glycopeptides, but only 2 of these responded also to the nonglycosylated peptide. This result shows that T cell recognition was confined to the glycosylated CII-variants in 5 patients, representing more than 30% of the entire cohort. With one exception (patient 10) the baseline population of IL-2-producing T cells was low in all patients investigated (Table 2). However, also in patient 10, a specific rise in the percentage of IL-2-producing CD4+ T cells from 0.20 to 0.92% on stimulation with Gal264/270 was detectable. The individual glycospecific responses of patients were heterogenous. In some patients, the response was biased to one of the glycosylation sites, whereas other patients had equal responses to both sites. In addition, in some patients, the response was restricted to one of the glycosylation sites, whereas the response to the peptide with glycosylation at both positions was weak or nonexistent. This latter response pattern was also seen in mice expressing both human DR4 and human CII (see Fig. 1). We also performed repetitive analysis of T cell responses to CII in 3 of the 14 patients. In the initial investigation, patient 3 responded exclusively to the Gal264-peptide because 0.24% of CD3+ cells produced IL-2 on stimulation with this peptide, compared with 0.07% in the absence of antigen. One week later, the same patient elicited an almost identical response (Fig. 4) where the two-color staining for CD4 and IL-2 show an increase in the double-positive T cell population on stimulation with the Gal264-peptide to 0.22% whereas exposure to the K264/270-peptide did not result in an increased IL-2 production. Similarly, patient 9 initially responded to the Gal264-peptide (Table 2), and this exclusive response was also noted when the patient was investigated 1 mo later (see footnotes in Table 2). However, in patient 10, the initially recorded response to the Gal264/270-peptide (Table 2) was undetectable 6 mo after the first assessment (see footnotes in Table 2). Thus, although CII-specific T cell responses may vary with time in some patients, possibly because of complex influences by treatment or spontaneous variations in the immune response of a characteristically chronic relapsing disease, the results obtained in a small cohort of RA patients with established diseases and severe course show the dominant targeting of glycosylated variants of the CII-peptide by the autoreactive T cell response. Finally, the available HLA-typing information on the patients reveals that all patients exhibiting T cell responses to the respective CII-peptide variants express at least one of the HLA-DRB1*-alleles containing the amino acid consensus motif QK(R)RAA in position 70–74. This motif constitutes the so-called "shared epitope" (24, 25), and is crucial for the binding of the CII-peptides to the respective class II molecules during antigen presentation (26).
Posttranslationally glycosylated peptides from CII are presented by the RA-associated DR4 molecules as shown in human DR4-transgenic mice. In DR4-transgenic mice expressing huCII, and most likely also in humans, T cells recognizing the nonglycosylated 263–270 epitope are strongly tolerized, or even deleted, whereas T cells specific for the different glycosylated peptides persist. This is a potentially crucial finding because it provides an explanation not only for the role of the DR4 in the presentation of joint derived peptides, but also for the difficulties in providing evidence for T cells recognizing autologous CII.
The relevance of the findings made in mice expressing huCII and DR4 is evident because 30% of the investigated RA patients exhibited a predominant response to the glycosylated forms of the CII263–270 epitope. Thus, our data show that the physiological posttranslational modification of variable carbohydrate attachment converts the immunodominant naked self-peptide, which is tolerogenic, into several cryptic self-determinants that remain immunogenic.
In addition to reports showing that transgenic expression of DR4 and DR1 in mice permits development of CIA (2, 3, 27), there are several investigations describing T cell immunity to CII in either healthy individuals or RA patients or both (5, 28–36). However, less is known regarding the determinants recognized by CII-specific T cells in humans, and the interpretations of the reported findings are not all in agreement (4, 5, 34, 35). For example, one recent study failed to identify CII-specific T cells in RA patients when CII259–272 loaded DR4-tetramers were used (4), despite the earlier defined immunodominance of this peptide in DR4-transgenic mice (1).
By including the aspect of T cell tolerance to self-CII and recognition of glycosylated CII-epitopes in our humanized animal model of RA, we can now provide a possible explanation for the ambiguous results concerning CII recognition in RA patients. Our finding that T cells specific for glycosylated CII appeared less tolerized by endogenous expression of huCII than T cells specific for the nonmodified epitope is predictable from an earlier finding in Aq-expressing mice (15, 37). Mice with transgenic expression of either rat or human CII displayed strong T cell tolerance to the immunodominant CII epitope and were also partially protected from CIA when immunized with rat and human CII, respectively (15, 37). Moreover, mice transgenic for the Aq-restricted immunodominant T cell epitope CII256–270, present on heterologous CII, displayed relatively stronger tolerance against the nonglycosylated variant (20). Whether persistence of glycopeptide-specific T cells in huCII/DR4 mice depends on the possibility that the glycopeptides bind with lower affinity to DR4 than the nonmodified peptide, as was observed for Aq (10), was not addressed, but could be considered as a reasonable explanation. It should be emphasized, however, that the glycosylated side chain is more likely to be oriented toward the T cell receptor rather than the MHC, as we show here that glycopeptide-specific T cell responses are critically dependent on the galactose moiety. This assumption is further supported from earlier studies using the Aq-molecule (10) and is also indicated from other studies using glycopeptide-specific T cells (38). As an alternative or complementary explanation for a biased tolerance, it has been shown that relatively small differences in availability or levels of autoantigens in vivo have great impact on the size or status of the autoreactive T cell repertoire (39, 40). Thus, different expression levels of the posttranslational forms of the CII263–270 epitope in endogenous CII might modulate the repertoire selection of CII-specific T cells. It should be emphasized, however, that also glycopeptide-specific T cells were influenced by endogenous huCII, although to a lesser degree than nonglycosylated CII-specific T cells. The response to the individual glycopeptides was only slightly weaker in huCII/DR4 mice than in DR4 mice, but the reduction was more obvious when comparing the response to the intact CII protein, indicating anergized or low affinity T cells. In addition, with the use of deoxy-peptides, we found that T cells from huCII/DR4 mice recognized the glycopeptide differently, indicating also that huCII influences T cell repertoire selection.
The mice used in this report are complex, because they carry multiple genetic modifications on a mixed genetic background, and translation of our in vitro findings to the human system should be done with some caution. Nevertheless, despite the complexity of the experimental set-up, our data on T cell tolerance in huCII/DR4 mice are intact and support earlier findings (15, 20, 37, 41). Collectively, these data strongly suggest that endogenous CII is physiologically exposed to the immune system but does not lead to complete tolerance of a subset of CII-specific T cells.
In summary, the results in a humanized animal model of RA and the functional assays on cellular autoimmunity in RA patients provide convincing evidence for incomplete T cell tolerance to a set of closely related self-determinants that are physiologically modified by posttranslational glycosylation. It should be stressed, however, that our data do not prove that the remaining CII-specific T cells are causative or even involved in the pathogenesis of RA. Despite this fact, our findings are interesting because they may constitute the missing link between the CII-specific humoral response, observed in a substantial fraction of RA patients, and the difficulties in the identification of CII-specific T cells in RA patients. Furthermore, T cell recognition of glycosylated CII in RA may be useful as a disease progression marker and for classification of this heterogenous disease.
We thank Carlos Palestro for taking good care of the animals, as well as Alexandra and Caroline Treschow for critically reading the manuscript. The work was supported by grants from the King Gustaf V's 80-Year Foundation, Kock's Foundations, Österlund's foundation, the Swedish Association against Rheumatism, the Swedish Medical Research Council, the Swedish Foundation for Strategic Research, the Göran Gustafsson Foundation, the Deutsche Forschungsgemeinschaft (SFB 263, project C3), the Bundesministerium für Bildung und Forschung (MedNet Entzündlich rheumatische Erkrankungen, project C 2.1; BMBF, 01GI9948), the European Commission (Bio4-98-0479), and the Karen Elise Jensen and Novo Nordisk Foundations in Denmark.
- type II collagen;
- collagen-induced arthritis;
- huCII,
- human type II collagen;
- peripheral blood mononucleated cells;
- rheumatoid arthritis;
- mDR,
- T cell clones from mouse CII and DR4;
- hDR,
- T cell clones from huCII/DR4
- Received February 25, 2002.
- Accepted April 29, 2002.
- Copyright © 2002, The National Academy of Sciences
- ↵
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Scholarly Works (2 results)
Peer Reviewed
Novel giant siphovirus from Bacillus anthracis features unusual genome characteristics.
Ganz, Holly H
Law, Christina
Schmuki, Martina
Eichenseher, Fritz
Calendar, Richard
Loessner, Martin J
Getz, Wayne M
Korlach, Jonas
Beyer, Wolfgang
Klumpp, Jochen
et al.
UC Berkeley Previously Published Works
Here we present vB_BanS-Tsamsa, a novel temperate phage isolated from Bacillus anthracis, the agent responsible for anthrax infections in wildlife, livestock and humans. Tsamsa phage is a giant siphovirus (order Caudovirales), featuring a long, flexible and non-contractile tail of 440 nm (not including baseplate structure) and an isometric head of 82 nm in diameter. We induced Tsamsa phage in samples from two different carcass sites in Etosha National Park, Namibia. The Tsamsa phage genome is the largest sequenced Bacillus siphovirus, containing 168,876 bp and 272 ORFs. The genome features an integrase/recombinase enzyme, indicative of a temperate lifestyle. Among bacterial strains tested, the phage infected only certain members of the Bacillus cereus sensu lato group (B. anthracis, B. cereus and B. thuringiensis) and exhibited moderate specificity for B. anthracis. Tsamsa lysed seven out of 25 B. cereus strains, two out of five B. thuringiensis strains and six out of seven B. anthracis strains tested. It did not lyse B. anthracis PAK-1, an atypical strain that is also resistant to both gamma phage and cherry phage. The Tsamsa endolysin features a broader lytic spectrum than the phage host range, indicating possible use of the enzyme in Bacillus biocontrol.
Peer Reviewed
Whole genome sequencing and comparative genomic analyses of two Vibrio cholerae O139 Bengal-specific Podoviruses to other N4-like phages reveal extensive genetic diversity
Fouts, Derrick E
Klumpp, Jochen
Bishop-Lilly, Kimberly A
Rajavel, Mathumathi
Willner, Kristin M
Butani, Amy
Henry, Matthew
Biswas, Biswajit
Li, Manrong
Albert, M
Loessner, Martin J
Calendar, Richard
Sozhamannan, Shanmuga
et al.
UC Berkeley Previously Published Works
Abstract Background Vibrio cholerae O139 Bengal is the only serogroup other than O1 implicated in cholera epidemics. We describe the isolation and characterization of an O139 serogroup-specific phage, vB_VchP_VchO139-I (ϕVchO139-I) that has similar host range and virion morphology as phage vB_VchP_JA1 (ϕJA1) described previously. We aimed at a complete molecular characterization of both phages and elucidation of their genetic and structural differences and assessment of their genetic relatedness to the N4-like phage group. Methods Host-range analysis and plaque morphology screening were done for both ϕJA1 and ϕVchO139-I. Both phage genomes were sequenced by a 454 and Sanger hybrid approach. Genomes were annotated and protein homologies were determined by Blast and HHPred. Restriction profiles, PFGE patterns and data on the physical genome structure were acquired and phylogenetic analyses were performed. Results The host specificity of ϕJA1 has been attributed to the unique capsular O-antigen produced by O139 strains. Plaque morphologies of the two phages were different; ϕVchO139-I produced a larger halo around the plaques than ϕJA1. Restriction profiles of ϕJA1 and ϕVchO139-I genomes were also different. The genomes of ϕJA1 and ϕVchO139-I consisted of linear double-stranded DNA of 71,252 and 70,938 base pairs. The presence of direct terminal repeats of around 1974 base pairs was demonstrated. Whole genome comparison revealed single nucleotide polymorphisms, small insertions/deletions and differences in gene content. Both genomes had 79 predicted protein encoding sequences, of which only 59 were identical between the two closely related phages. They also encoded one tRNA-Arg gene, an intein within the large terminase gene, and four homing endonuclease genes. Whole genome phylogenetic analyses of ϕJA1 and ϕVchO139-I against other sequenced N4-like phages delineate three novel subgroups or clades within this phage family. Conclusions The closely related phages feature significant genetic differences, in spite of being morphologically identical. The phage morphology, genetic organization, genomic content and large terminase protein based phylogeny support the placement of these two phages in the Podoviridae family, more specifically within the N4-like phage group. The physical genome structure of ϕJA1 could be demonstrated experimentally. Our data pave the way for potential use of ϕJA1 and ϕVchO139-I in Vibrio cholerae typing and control.


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Unified approach for Clarence Valley Sports Council
SPORTS COUNCILS: If proposed changes are implemented at next week's council meeting the Clarence Valley Sports Council and Lower Clarence Sports Council will be no more.
Instead the councils will be replaced by two individual committees who share the same constitution. The committees will still have individual funding pools of $35,000 which sporting clubs are able to apply for grants from.
The unification of the two bodies under the one constitution was a compromise reached by council after both sports councils rejected previously proposed alteration.
The original proposal by the Clarence Valley Council was to amalgamate the existing sports councils into one body with a $70,000 fund to distribute among sporting communities.
But the idea was rejected based on logistical issues including the travel between meeting locations.
The new proposal was reached at Tuesday's Coporate, Governance and Works Committee Meeting and will be potentially adopted at next week's Ordinary Council Meeting.
According to Clarence Valley Council mayor Richie Williamson the new proposal adds up to a more unified sporting Valley.
"I think the discussions were really about two committees which we have now all coming under the same constitution and meeting on the same day but at different times in Grafton and Maclean," he said.
"Each will have the same amount of money they have now, that will remain as two separate figures that can be accessed by each committee.
"Both committees will continue to work under their current constitutions as the constitution won't change until the September council election. But it is the intent to have one sports committee constitution that both will work under."
If the proposal is adopted sports councils will now meet quarterly on the same day, with one meeting in Grafton and one in Maclean, and host one super meeting between the two committees annually with the meeting to alternate location each year.
"I think it will increase efficiency in council but still achieve the funding goals they have always set out to achieve," Williamson said. "I did struggle to find how a single entity would operate in this case simply because the number of sports that use the fields are very diverse."
Lower Clarence Sports Committee member Hariet Woodrow, who was among the members which voted the original decision out, was pleased with the new idea council had reached.
"It is good that they are maintaining the independence of the two committees," Woodrow said.
"Having one sports council was just not going to work. To host alternate meetings no one would go to the one that isn't in their town.
"So long as they maintain the current objectives of the sports council then we have no problem."


text: The Cleft Palate-Craniofacial Journal. 44(6):590–594, NOVEMBER 2007 DOI: 10.1597/06-204.1 , PMID: 18177198 Issn Print: 1055-6656 Publication Date: November 2007 Print The Pittsburgh Fistula Classification System: A Standardized Scheme for the Description of Palatal Fistulas Darren Smith;Lisa Vecchione;Shao Jiang;Matthew Ford;Frederic Deleyiannis;Mary Haralam;Sanjay Naran;Christine Worrall;Jason Dudas;Ahmed Afifi;Mary Marazita;Joseph Losee; Checking for direct PDF access through Ovid Abstract Objective:Vague terminology is a problem in cleft palate research. No classification scheme for palatal fistulas has been proposed to date. Although a well-healed velum is a significant outcome of palatoplasty, it is nearly impossible to compare fistula-related palatoplasty results in the literature or in medical records without a standardized vocabulary. We endeavor to devise a palatal fistula classification system that may have clinical and research applicability.Design:PubMed was searched for definitions and classifications of palatal fistula as well as incidence and recurrence rates of this outcome. Next, a 25-year retrospective review of our Cleft Center's records was performed, and fistulas were identified (n = 641 charts reviewed). The fistula descriptions yielded by this chart review were evaluated in the context of anatomical descriptions in the literature, and a clinician-friendly classification scheme was designed.Results:A literature review failed to reveal a standardized fistula classification system. An anatomically based numerical fistula classification system was devised: type I, bifid uvula; type II, soft palate; type III, junction of the soft and hard palate; type IV, hard palate; type V, junction of the primary and secondary palates (for Veau IV clefts); type VI, lingual alveolar; and type VII, labial alveolar.Conclusions:We propose a standardized numerical classification system for palatal fistulas. Its clinical adoption may prospectively clarify ambiguities in the literature and facilitate future cleft palate research and clinical practice.