text:
Auto Reframe intelligently identifies the actions in your video and reframes the clips for different aspect ratios. This feature is really handy for posting your video to different social media channels such as Instagram, YouTube, or Facebook.
You can use Auto Reframe to reframe sequences for square, vertical, and cinematic 16:9, or when cropping high-resolution content like 4K and beyond.
You can automatically reframe individual clips, or a complete sequence.
Fine-tune the Auto Reframe effect by choosing a suitable motion preset from the Effect Controls panel. While applying the effect, Premiere Pro generates motion keyframes that follow the action in your content. You can choose one of the following options:
Slower Motion
Appropriate for when there is very little or no camera motion (such as talking head interviews). When this control is used, results are almost static and the clip has very few keyframes.
Appropriate for most content. Here, the Auto Reframe effect follows the action, however the results may not be great if the action is very fast.
Faster Motion
Appropriate for when there is a lot of motion in the footage and you want the Auto-Reframe effect to follow the action (such as in action sports or skateboarding videos). Here, Auto Reframe ensures the moving object is always in the frame, and adds a lot of keyframes to your clip.
If you have a sequence containing multiple clips, automatically reframing the entire sequence is a more efficient option.
In the dialog box that opens, select the new aspect ratio from the available presets or specify a custom aspect ratio.
Premiere Pro creates a duplicate sequence with the correct dimensions for the new aspect ratio. Every clip in the new sequence has the Auto Reframe effect applied. This duplicate sequence is saved in the Auto Reframe Sequences folder in the root of the Project panel.
If your sequence is long, a banner is displayed indicating that Premiere Pro is still analyzing the sequence. You can continue working on your project while this analysis goes on in the background.
(Optional) To preserve your original motion adjustments with your reframed sequence, apply clip nesting.
If you choose to nest clips, Premiere Pro places all of your video clips into a nest and uses the original cut points and track layering, leaving your graphics and audio unaffected.
Select nesting if you have:
- Existing complex keyframes with motion that you want to retain
- Sequences that have speed and duration adjustments
Nesting clips does not maintain soft transitions properly, as the handle media is not present in nested clips.
Here are some best practice tips for getting the best results with Auto Reframe.
Filming: Frame your videos with a little more space around the subjects so that you can repurpose the footage later for any medium.
Using text titles: Auto Reframe works best when titles are created using Premiere Pro.
Using still images: Auto Reframe does not work with still images. Reframe still images separately.
Apply the Auto Reframe effect only once: Applying Auto Reframe multiple times (especially with nested clips) can cause unpredictable results - such as black bars on the sides of your videos. If you need to re-apply the Auto Reframe effect, make sure you apply it on the original clip.
If you have a question to ask or an idea to share, come and participate in our Premiere Pro community. We would love to hear from you and see your auto-reframed videos.
Twitter™ and Facebook posts are not covered under the terms of Creative Commons.


text:
The Waxing Year (February 9 - July 10, 2019)
Opening Reception
Saturday, February 9, 2019, 7 - 10 PM
Exhibit runs
February 9 - July 10, 2019
Free admission to exhibit and Opening Reception
Explore the transformative power of attuning to nature's seasons as Life Force Arts Foundation presents the art exhibit and multidisciplinary arts show The Waxing Year, running from February 9 - July 10, 2019 at Life Force Arts Foundation, 1609 W. Belmont, Chicago IL 60657.
Open Gallery Hours
Thursdays & Fridays
and by appointment
Opening Reception is Saturday, February 9, 2019 from 7 - 10 PM. Gallery Hours are Thursdays and Fridays from 1:30 - 6:30 PM, and by appointment. Admission is free to the exhibit and Opening Reception. Workshops, Healing Performances and other events related to the theme will be held as part of the show. For more information, please contact Joan Forest Mage, Artistic Director, 773-327-7224, email@example.com.
The Waxing Year is the first of two exhibits in 2019 at Life Force Arts Foundation that explore the meaning and transformative potential of attuning to nature's seasons. The exhibits are based in the Life Force Arts Method (LFAM), a powerful spiritual tool for building positive relationships from one's inner world to the outer world.
The word "waxing" means getting larger; the word "waning" means getting smaller. Each year, the waxing half of the year is from December 21 - June 20, when the days grow longer; and the waning half of the year is from June 20 - December 21, when the days grow shorter. (These dates refer to the Northern Hemisphere).
The seasonal festivals of the Waxing Year are
- Quickening (February 2) - the earliest stirrings of new life underground
- Spring (March 20) - the greening of the world
- May Day (May 1) - the flowering of nature
- Midsummer (June 20) - the full lushness of the growing season
By attuning ourselves to the changes in nature happening as the year waxes and wanes, we become more in tune with our bodies, minds and emotions, and to our connection with all life. Throughout history and in every culture, artists, teachers, healers and community builders have worked with the seasons to help their communities live in harmony with self, society, nature and the spirit realm.
Featured artists - The Waxing Year
Gabriel Gebka
Ladan Ghajar
Sofia Girlando
Lorraine Moretti
Life Force Arts Foundation (LFAF) creates spiritual transformation through the arts based in the Life Force Arts Method, a modern version of universal wisdom teachings that value true self and creative community. LFAF presents visual, literary and performing art exhibits and events that awaken human potential, deepen mystical experience and create spiritual transformation. Life Force Arts Foundation operates Life Force Arts Ensemble, a company of dancers, singers, musicians, actors, storytellers, writers and visual artists; and maintains its own facility, Life Force Arts Center.
Last modified: Sunday, September 1, 2019, 8:27 PM

subset: commoncrawl


text:
The 2018 State of Origin series is set to be one of the best yet and the great news for fans is that you can watch all of the action live online via NOW TV and their SkySports live streaming service.
More Details Visit NOW TV To Watch
Date: 06/06/2018
Venue: Melbourne Cricket Ground
Kick Off: 11:00
Date: 24/06/2018
Venue: ANZ Stadium, Sydney
Kick Off: 10:45
Date: 11/07/2018
Venue: Suncorp Stadium, Brisbane
Kick Off: 11:00
The State of Origin is a 3 match series between to 2 main Australian stats- New South Wales and Queensland.
The players are picked by a group of selectors and their eligibility is based on where their first senior Rugby League club where based, hence the name State of Origin.
The State of Origin idea has a long history that goes back to the very start of rugby league in Australia when interstate matches were frequent.
It is reported that a journalist wrote an article in 1900, stating that Stephen Spragg, who had just moved to Queensland, should be allowed to play for New South Wales.
Although there were regular matches between New South Wales and Queensland since the inception of Rugby League in 1908 it wasn't until 1982 when the concept really took off due to the inauguration of the State of Origin series.
After a number of experimental games in 1980 and 19081, the old rules of interstate matches were brought back into use with the invention of the State of Origin series. A 3 match series between the blues of New South Wales and the maroons of Queensland.
In 1987 the controversial decision was made to play an extra game in the series in Long beach in the United States.
Queensland had already won the series 2-1 and had little to no interest in this extra gam which quickly became simply an exhibition game.
The Super League wars of the 90's caused problem for the State of origin, with players banned from playing in it and even a second series run by News International featuring a New South Wales, Queensland and a New Zealand team.
After the end of the Super League wars the State of Origin series went back to its traditional best of 3 series between Queensland and New South Wales and the matches quickly became the pinnacle of domestic rugby league in Australia.
Due to the increase in popularity of the sport of rugby league in Australia during the 2000's, the crowds for the Origin matches have grown hugely with the matches now selling out some of the biggest stadiums in Australia.


text:
Chicken Rice Casserole with Veggies
Total TimePrep: 20 min. Bake: 25 min.
Makes6 servings
- 6 boneless skinless chicken breast halves (1-1/2 pounds)
- 1 tablespoon canola oil
- 3/4 cup chopped sweet red pepper
- 3/4 cup chopped green pepper
- 1/2 cup chopped onion
- 1/2 cup chopped fresh mushrooms
- 1 garlic clove, minced
- 2 cups uncooked instant brown rice
- 2 cups chicken broth
- 1-1/2 cups frozen corn, thawed
- 1/4 teaspoon salt
- 1/8 teaspoon pepper
- 1/4 cup slivered almonds, toasted
- 2 tablespoons minced parsley
- In a large skillet, brown chicken in oil for 4 minutes on each side or until a thermometer reads 170°. Remove and keep warm. In the same skillet, saute the peppers, onion and mushrooms until tender. Add garlic; cook 1 minute longer. Stir in the rice, broth, corn, salt and pepper; bring to a boil.
- Transfer to an 11x7-in. baking dish coated with cooking spray. Top with chicken. Cover and bake at 350° for 20 minutes. Uncover; bake 5 minutes longer or until heated through. Sprinkle with almonds and parsley.
Nutrition Facts1 each: 351 calories, 8g fat (1g saturated fat), 66mg cholesterol, 493mg sodium, 37g carbohydrate (0 sugars, 4g fiber), 33g protein. Diabetic Exchanges: 3 lean meat, 2 starch, 1 vegetable.
Recommended Video
Mar 11, 2014
Easy & good for you.
Feb 16, 2013
I liked this casserole since it doesn't use a goopy canned soup. However, when I make it again I'm going to saute the onion, mushrooms and garlic first, remove from the heat and then add the chopped peppers - sauteeing peppers makes them mushy, especially after being baked further. Also, instead of using regular chicken broth I will try using chicken base or bouillon granules to give it a more robust flavor. I'm glad I made the effort to include the toasted almonds - they really make the casserole!
Apr 30, 2012
It was very easy & quick to make. I used frozen green & red peppers, & left over chicken. The chicken I placed on top of the mixture. Added the toasted almonds & parsley & in the oven it went. It did a little longer in the oven as the rice didn't get done in 20 min.


text:
Created for the West Dean Chilli Festival
This is a Heat level 3
Tangy and spicy, this will give your taste buds a wake up call.
No artificial colours or flavours, suitable for vegetarians, gluten free and no nuts in any of our recipes. Can also be eaten frozen!
White Chocolate Lemon & Chilli Fudge Ingredients
Made with Sugar & Sweetener
Sugar, Condensed Milk (Milk, Sugar) Belgian White Chocolate 10% (Sugar, Cocoa Butter, Whole Milk Powder, emulsifier- Soya Lecithin & natural Flavouring) Butter (Milk & Salt) Milk, Sweetener (Xylitol), Chilli Powder, Sicilian Lemon Oil & Sea Salt.
Nutritional Info for Lemon Chilli Fudge
Per 100g Per 50g serving
Energy 1749kJ 875kJ
415kcal 208kcal
Fat 8.9g 7.4g
(saturates) 66g 4.5g
Carbohydrates 66g 33g
(of which sugars) 66g 33g
Protein 3.6g 1.8g
Salt 0.66g 0.33g


text:
When it comes to successful wedding planning, good organization is the key to success. But for some brides the sheer scale of planning a wedding is a massive undertaking, especially if being organized is not a skill you're particularly good at! But by following these few simple steps you can make your wedding planning a breeze, no matter how disorganized you are!
Arm yourself with the following tools to help you with your wedding planning:
- A notebook: choose one which is small enough to fit in your handbag but big enough to contain information like phone numbers, addresses, websites, contact details, samples e.g fabric, stationery. Your notebook will be your wedding planning bible recording every bit on information which could be useful at some point during your planning. Having it with 24/7 means you can jot down any ideas or inspirations as they happen.
- Checklists: these are probably the most important tool the disorganized bride could have. You will find checklists for free on the internet through many wedding related websites. Find a set which best fit with your wedding dreams or edit a set to fit. Wedding planning checklists will list every element and detail of a wedding in a time ordered list. Typically they will begin at 18 months before the wedding day and will include everything you will need to consider for your wedding up to the wedding day itself and some even go beyond. Follow your checklists every day and make sure you tick each item off as you complete it.
- Delegate: whether you are busy or just disorganized, delegating elements of your wedding planning to others will ensure things get done! Ask your maid of honor, your groom, your parents and anyone else who offers to take on an element or two of your wedding. Delegating your planning to others will take the pressure off you and give you more time to devote to other elements of your day.
- Hire a Professional: if you are so disorganized that none of the above are going to help you, then it is time to hire in a pro and bring in a professional wedding planner to organize your day for you. You can hire a wedding planner to organize your entire wedding or just a part of it such as the reception. Hiring a wedding planner to organize some of your wedding but not all can be a good option if you want to organize your wedding yourself but find it too difficult to stay on top of everything that needs to be done.
Published by Reed Cooper


text:
UN Reforms—A Major Step Forward January 1, but Some Challenges Still to Overcome
By Sarah Cliffe and Karina Gerlach
UN Secretary-General António Guterres was appointed in 2016 on an explicit reform platform. In 2017, we published commentaries on his reform proposals. Now that those reforms that have been approved are moving into implementation, we publish this simple guide to what has been achieved and the potential potholes still ahead.
The secretary-general's much-anticipated reforms are finally underway. The UN has been putting effort into preparing for implementation, with more than 1,900 staff mapped to new positions, 1,400 staff trained, and a major retreat with resident coordinators in November to prepare for the change.
The reforms promise to achieve the following five major steps towards a UN fit for purpose for the global challenges it faces:
- Delegation of decisions to the field
- Continuity of strategy in political and peacekeeping missions
- Increasing the credibility of UN representation in the field
- Separation of operational management from policy and oversight
- A start to implementing a more modernized budget
How do the reforms stack up in answering specific problems raised by the UN's member states and staff who work at the coalface of delivery, often in emergency situations and under very difficult conditions? Continuing our series on UN Reform, this new commentary assesses how the reforms will resolve these problems in the future.
[ Read the full paper UN Reforms—A Major Step Forward January 1, but Some Challenges Still to Overcome.pdf ]


text:
We present a new kitchen project made with different models like the modern CreamStucco. An exquisite Eucalipto Fumé veneer on panels and column interiors, with a traditional touch of Viejo Tinte Panga from the Grain series on the upper units.
This kitchen offers lively surroundings where we can share our feelings, talk, think, decide, work, laugh, enjoy and live in the most comfortable, practical and intense way possible.
Different opening systems are used: those which are incorporated in the outer edge of doors in the columns, the pushbluemotion opening system in lower units, or the hidden Gola system on top units.
The use of superior materials, identified by their special personality, makes it possible to create transparent areas. One example of this is found in the columns area where you can see every aspect, detail, colour and light making a visually enriched collection. In this way we achieve the mark of distinction necessary for a well-planned project


text:
Strength Training
Athletic gains peak in the off season and are maintained during the season. Our strength routines are sport specific to ensure our athletes are physically prepared for their individual upcoming sport season. We use the best technology, proven methods and good old fashion motivation to move athletes from leaders to champions.


text:
I'll be posting some short clips of a recent presentation I conducted on complying with California's paid sick leave law. In this first video, I discuss some general rules California employers need to consider to comply with the July 1, 2015 deadline to offer paid sick leave to employees. Topics include:
- how to calculate pay


text:
This week's case opened with a strange pregnant woman randomly stumbling into the campfire circle of a group of teenagers, flopping to the ground and dying. And the circumstances got stranger from there.
Fortunately, the baby lived, but in order to figure out how such a scenario came to pass, there was only one person to turn to: a zombie who gains the memories of the brains she eats and solves crimes with the visions she gets while pretending to be a psychic. The effects of Preggo's brain were almost immediate on Liv, giving her maternal instincts and a hilarious case of nag-the-young-people which she unleashed on nearly all her friends. It also made her fiercely protective of the baby, and determined to make sure he wound up under the correct care.
She was certain the murderer was Dillon, the lady's ex, even though he technically WAS the father. Ten seconds with the little creep made Liv suspect he had something to do with the incident. Her parents figured so as well, telling Clive to put him under investigation.
After a short conversation, they think it might…..not have been Dillon. Even though he had a record with the cops already, he hadn't seen the woman in quite a while: the very last time was when she saw her get into a pickup truck with a mysterious stranger. Also, the parents who gave Clive that hot tip are shown landing a punch on Dillon's face in Liv's first vision, so they can't be trusted either. In fact, maybe….it was them?
An examination determines the woman had been held captive somewhere for quite some time and her death was due to extreme shock. Liv concocts the theory that her own parents must have locked her in the basement. She and Clive decide to check that basement out, and find a wooden crib that must have taken some time to carve….like eight months' worth?
A common thread here is "dogs." Liv heard dogs barking in the visions from the woman's perspective. Dillon claims he heard dogs barking when the woman was taken away. And now Liv and Clive are hearing barking dogs as the victim's parents return from work, and slam the cellar doors shut believing the pair to be intruders.
All signs seem to point to the parents, but again, it ultimately turns out to not be so. Fortunately, Liv has some clearer visions that implicate a man who works for Animal Control (explaining the barking). It turns out he and his wife have been living in the woods, holding several girls prisoner in a padlocked treehouse, and that the victim had died while trying to escape back home. At this point, the police chief got involved in the raid, and based on his previous "by the book" performance, I expected him to be more of a hindrance than a help. Then, when no one was looking, he turned full-on zombie and ripped apart the husband, then turned back to normal and shot the wife point-blank because he could. Sure had YOU pegged wrong!
Meanwhile, back in Major Land, his private investigation is sinking him deeper into danger, though he appears unaware of just how much. It's now been established the large zombie thug who works for Blaine is known as "The Candyman," though no explanation is given for that moniker.
I stared in disbelief as he actually broke the glass window to snoop around in Candyman's car. Picking the lock would have been a much safer — and quieter — method of gathering evidence. He does find some valuable info, but the cops then appear in their squad car, and Major immediately steps out of the broken door carrying a tire iron. What did you think was going to happen, pal?
What Major found was a business card revealing Candyman's real name: Julien Dupont. He also found a container with Dupont's lunch in it, which made him freak out. As the police close in on him, Candyman picks that moment to emerge from a tanning salon. Major shouts and points him out.
"Officers, arrest that man! That is Julien Dupont, otherwise known as The Candyman! He's a drug dealer and he pushes Utopia to kids around the skate park; I can confirm his involvement with at least two boys! AND HE'S GOT A HUMAN BRAIN IN A TUPPERWARE CONTAINER!"
The cops order Julien to freeze while they check out Major's claim. And they do find the Tupperware. How will he sneak out of this?
"Look, it's actually a cat brain. I work for a merchant of exotic foods known as Meat Cute. Let me give you my card."
A cat brain? It's huge! That would have to be the biggest cat ever recorded! There's no way the fuzz are going to swallow this bulls–
"Sorry to bother you, sir. Major, hands behind your head."
Now not only is Major behind bars for attempted car theft, Candyman knows he's onto him, and he'll be telling Blaine. It's too bad they've broken up, because he now needs Liv to live.
But Liv is currently ignorantly, blissfully occupied with Lowell. She was worried for a moment when Lowell seemed to be getting more distant and his kisses began to lack any sense of passion. Then she found out he wasn't feeling up to it because….he'd eaten a gay man's brain! Ha ha! They had a crazy "girls' night out" instead, and saved their more intimate plans for after his next feeding.
OTHER MINOR THINGS THAT WILL BE IMPORTANT LATER: When Liv's mother visited Meat Cute at the end of last episode, it was apparently to pick up a job application for Liv's brother. She hands him a few others as well, but the odds are decent he's going to wind up at the first business visible in the stack. And since we've already seen the life and death of Blaine's previous two employers, we know this is not good. Between this and Major, Blaine is about to annoy the crap out of Liv in a big way. It's adding up to a season finale super-fight.
But we've got a whopping six more episodes to get through before then. Which is fine with me.


text:
Published on 18.11.2018
Do you remember our blog post about SNOT? "Studs not on top". Thanks to various special parts you can turn models by 90 °. So you can create surfaces that look much more realistic than the simple stacking of bricks.
And if you want to turn the knobs by 180 °? Then you need to combine special parts or apply other building tricks. This costs space, which limits the possibilities a bit.
You just need a thin brick that has nubs on both sides ....
Good for all bricks fans that we've thought of that and now we do have it. In each bag are 20 pieces of it. If you want to supplement your stock of bricks with these little all-rounders, you will find the stones here in our shop.


text:
Posts Tagged 'Farm Diversification'
Farm Table's Farm Diversification Special: Katherine Outback Experience
As part of a Farm Diversification Blog series, the Farm Table spoke with a number of producers across Australia to explore the plethora of opportunities for agricultural and non-agricultural diversification. Airlie Trescowthick caught up with Annabel to discuss how Katherine Outback Experience came to be and the lessons learnt along the way. This was published…Read More


text:
Submit a Manuscript | Contact Us
home » books » Heather Norman Smith
Heather grew up in the foothills of the Blue Ridge Mountains in Western, North Carolina. She enjoyed writing stories and poems throughout childhood, but the responsibilities of being an adult distracted her from the desire to write for many years. Her love of writing was rekindled when she found a meaningful theme on which to focus-the love of Jesus. It started with devotional-style blogs on her personal website, but God has expanded Heather's writing passion to Christian fiction through the creation process of Grace & Lavender. Heather currently lives just outside Winston-Salem, North Carolina, with her husband and three children, along with two cats, a dog, and a fish. Other than writing and spending time with her family, Heather enjoys singing, song-writing, and crafting.


text:
Listings in Flooring, Hardware, Wood and Floor Graphics in Carpinteria and Fullerton
P5 Graphics and Displays
in Acrylic Fixtures, Slatwall Fixtures, Banners and Flags, Floor Graphics, Large-Format, Lightboxes, Printing, Sign Holders/Frames, Vinyl Graphics, Literature Holders
P5 Graphics & Displays is a designer and manufacturer of visual merchandising retail point of purchase displays. P5 is also a commercial print company offering a wide variety of print options including large/wide/grand format printing.
1450 S. Manhattan Ave.
Fullerton, California 92831
United States
Koyal Wholesale
in Backdrops/Curtains, Baskets/Barrels/Boxes/Pots/Planters/Vases/Urns, Foliage, Seasonal, Tablecloths, Bamboo/Rattan/Wicker, Fabrics, Glass, Stone, Natural, Wood
Koyal Wholesale is a leader in visual merchandising supplies for window displays. We specialize is custom packaging, naturals (i.e. manzanita trees and branches, driftwood, grapewood), curtains, tablecloths, garlands, fillers, vases, candelabras,etc
2325 Raymer Ave
Fullerton, California 92833
United States
Griplock Systems
in Hardware, Cable Systems, Metal Fixtures, Mounting Devices (poles, lowering devices, etc.), Sign Holders/Frames
The Griplock Cable Display System is the safest, strongest, simplest and most versatile system of its kind on the market today, offering thousands of precision-made parts to fit almost every conceivable application, from shelving to signage and more.
Carpinteria, California 93013
United States
Fax: 805.566.0065


text: Yes it is the holiday season, and whether you celebrate Christmas, Hanukkah, Kwanzaa, or something else, this time of year can be stressful for many. Here are three ways to minimize stress during the holidays. 1. Plan throughout the year. Buy gifts, decorations, and other holiday items throughout the year and save them for now….


text:
Sylvie and Bruno Concluded First Edition:
Sylvie and Bruno Concluded is the second part of Lewis Carroll's 1889 novel, Sylvie and Bruno, and is the last work published during Carroll's lifetime. The novel, narrated by an unnamed historian, is set in a fictional "Fairyland" and a Victorian England familiar to Carroll's environment. The novel recounts stories of two child protagonists, Sylvie, the princess of Fairyland and her immature and younger brother, Bruno. The novel was based on the author's theory of "What might possibly happen, supposing Fairies really existed" as Carroll explains in his introduction.
Book: Original red cloth boards are clean and unblemished. Triple ruled bright gilt with pictorial triple ruled gilt roundel on both boards and a gilt stamped title and decoration on the spine. Spine head and foot are slightly rubbed and slightly age toned copy. Contents in excellent clean condition with bright, gilt edges. Intact tissue guard to the front illustration and very good condition of the black end papers. A pencil comment on the frontpiece of the half title page.
Dust Jacket: Fair condition and unclipped dust jacket. The head, tail, corners and the front crease of the spine are chipped. Protected by a plastic wrapper. Browned at the edges with darkened spine. Original price label of 8/6 NET (title page stating "Price Three Half Crowns")
Brought to you by Rare & Antique Books
Stock number: FAULT


text:
Navios Acquisition Wraps Up Merger with Navios Midstream
Tanker owner Navios Acquisition has completed the acquisition of the publicly held units of Navios Midstream in a stock for units exchange.
Following the close of the market on December 13, Navios Midstream common units no longer are publicly traded on the New York Stock Exchange, the company announced.
"We are pleased to close this important transaction, which provides Navios Acquisition with a number of benefits, including a simplified corporate structure, larger asset base and enhanced credit profile. We believe that the combined entity will be an attractive investment opportunity for investors,"Angeliki Frangou, Chairman and Chief Executive Officer, said.
The deal saw Navios Acquisition issue approximately 3,683,028 newly issued shares in exchange for the publicly held common units of Navios Midstream at an exchange ratio of 0.42 shares.
Navios Acquisition expects the merger to simplify the capital and organizational structure of the group, increase trading liquidity and enhance access to the capital markets together with Navios Acquisition's credit profile.
The company also hopes the move will help build scale through a larger asset base that is capable of generating increased profitability, reduce cost of capital and create significant savings in public company costs.


text: This past Monday was the pinnacle of marathon running — the 120th Boston Marathon. Over 26,000 runners from all around the world converged on the great city of Boston to run this historic race, and I was lucky enough to be one of them. Qualifying for the Boston Marathon was my dream since 8th grade when …→Read More


text:
1. Monitoring
Questions to Ask Your IT Guy
○ Can you show me a written procedure that's used to monitor for a common problem (for example, to prevent failing hard drives or deal with low disk space before a drive fills up)?
2. Inventory/Asset Tracking
It's challenging to effectively manage your IT unless you know what needs to be managed. Creating and maintaining an IT inventory is one of the most important things any IT guy (or gal) can do.
Questions to Ask Your IT Guy
○ How do we track what new devices are brought onto our network?
○ Can you show me a current inventory of our workstations, servers, and network devices? Even better, can you give me a login for the software we use to track our assets?
○ What systems do we use to monitor our network for new devices that are brought online?
○ How often is our inventory list reviewed?
3. Documentation & Procedure Creation
○ How often are our procedure documents reviewed and updated?
○ Can you go through the last 3 new user setups we've done and highlight any follow-up issues that we've had from that user in the 30 days following their setup? Are those issues dealt with in the new user setup documentation?
4. Patching
○Do we have centralized patching software? If so, what is it and what software does it patch?
○What is the patching schedule we use? How does the schedule differ between critical patches and routine patches?
○How often do we run updates for routine utilities like Chrome or Adobe Acrobat?
○What is our schedule for updating our line-of-business applications (applications like CAD for architecture firms or time and billing software for law firms)?
5. Backup & Disaster Recovery
Questions to Ask Your IT Guy
○ What backup software do we use to backup our servers? Workstations? Cloud-based infrastructure?
○ What schedule do the backups run on?
○ Where is the data backed up to?
○ How do you monitor the effectiveness of our backups?
○ How long would it take us to restore from a crash if our physical hardware failed and wasn't recoverable?
○ How long would it take us to spin up our system in the cloud if we had a physical disaster like a fire or flood at our office?
○ Do we have any workstation-level or cloud-to-cloud backups in place?
6. Budgeting.
○ What are our standard replacement cycles for workstations, servers, and network equipment?
○ Can you help me develop a proposed capital budget for IT asset purchases for the next two years?
○ What are our oldest and most vulnerable IT assets (hardware and software)?
○ What devices can we retire so you don't continue burning time and energy keeping them up and running?
○ How do we create consistency in our IT capital expenditures so as to not negatively impact cash flow?
7. Security.
Solid IT security has three basic components: controls, tools, and monitoring. You need to have all three in place and working consistently in order for your systems to be secure. You'll also need the proverbial "push" from the executive team to make sure security is a priority for your organization.
○ How often is our firewall's software updated? How often is the configuration reviewed?
○ What software do we have to protect our workstations? How is it monitored?
○ What software do we have to prevent our email system from being hacked? What about spoofing? Phishing?
○ When was the last time we had a seminar on IT security for our employees?
○ Have you run a search for any of our credentials that might exist on the dark web?
○ Do we have any sensitive information on our systems like credit cards, social security numbers, and/or HR or payroll information?
○ Do we have plans or details on any high-security buildings or high-value residences on our system? Might any of our high-profile clients be potential targets of hackers or other security threats?
What things have you found that your IT guys haven't been doing?
Drop us a line at email@example.com so we can get the conversation started.
A little about us: Dynamic Computing provides managed IT services, IT support, IT consulting, & cyber security services to top performing small to mid-sized businesses in the greater Seattle area. We're focused on being the premier managed IT services firm in the Pacific Northwest, and we act as a complete IT solution for companies who don't have internal IT departments. Our clients typically range from 10 to 200 employees and we work primarily with professional services firms in the Puget Sound Region.
About the author: Kevin Gemeroy


text:
The Daily Show provides an overview of some of the many, many Democrats running for president in 2020, including Bernie Sanders, Joe Biden, Pete Buttigieg and Kamala Harris.
The Daily Show provides an overview of some of the many, many Democrats running for president in 2020, including Bernie Sanders, Joe Biden, Pete Buttigieg and Kamala Harris.
10:50 Corey in the house. It's Corey in the houseReply
"I guess she wanted to start her campaign the same way Hillary ended hers"- throw the whole palm tree, why don't you? Damn ?????Reply
Why so little time to Andre Yang?? He is the most interesting and logical.Reply
Harris is tough with her oversight committee questions, which I like, but she comes off as fake during her presidential campaign. Anyone else shares this sentiment?Reply
If Cory Booker won it would literally be "Cory in the House"Reply
Biden and the other guy looks the sameReply
Corey in the House clearly has to winReply
And in the ultimate irony: Buttigieg WINS, and promptly writes an Executive Order barring STRAIGHT people from serving unless they can prove they have had at least one homosexual experience as an adult. What he doesn't tell them is that the qualifying 'experience' could simply be watching the Nathan Lane/Robin Williams La Cage au Folle – or kissing your boss' ass…Reply
Bernie 2020!Reply
PLEASE SAVE OUR COUNTRY VOTE FOR BERNIE SANDERS IN THE PRIMARYReply
Brooklyn uses sources thats what there not all retarded like cnn and foxReply
If Biden wind could Obama be vice presidentReply
What happens to the money they raise if they don't win?Reply
Literally heard nothing from Andrew YangReply
It's sad none of them are going to win cause trump voters haven't learned anything and still love the idiot.Reply
❣Marianne Williamson ❣Reply
Every Politician is a f ing idiotReply
5:04 Is it weird that I find that he sounds nice when he sings?Reply
Time for me to visit Brooklyn to clean up some of my old sins, we all should do in order to start anew.Reply


text:
Back to Montreal, with Python fun.
February 8, 2008
I'm back in Montreal recovering from DEMO 08, where I had the chance to meet super interesting people and to show Cozimo to the press, entrepreneurs and VCs. Now that Cozimo is officially out the door, I'm busier than ever. Luckily enough, it's not just about answering I forgot my password support e-mails… some interesting developments are forming and there's still energy left for midnight hacking, so I can't complain.
Still, I can't wipe out from my brain the vision of Monique Savoie menacing me: "Don't you dare hiding for a year ever again, or else!", so I decided to go to the very first Montreal Python event. And it was fun!
David Goodger, architect of DocUtils and renowned Python activist, entertained us with one of his old-time passions: polyform puzzles.
David briefly described algorithms involved in solving these puzzles…
… but I wished he focused a bit more on stuff like this:
It was an interesting presentation, and a good way to start Montreal Python's series of events. Montreal is buzzing more than ever, and it feels really good to be part of such a vibrant community. Kudos to the organizers for a well organized event.
One Response to "Back to Montreal, with Python fun."
February 9, 2008 at 8:40 pm
[…] a couple of posts about MontrealPython1 : Juan , […]


text:
Equipment Function & Character
1. Machine is covered with stainless steel board, while the mechanical parts contacting on products are made by stainless steel. Appearance is beautiful while hygiene of products is protected, reaching to the international request on wet-wipe production equipment.
2. Under European CE standard designing, Passed CE certificate, With CE or UL certificate for Electric Parts and with safety device, such as, safety-guard door, emergency stop and so on.
3. Most of parts are precisely processed by numerical-control machine; the key mechanical parts are under CNC processing; while the main outsourcing parts are world famous brand.
4. It can package wet wipe with air-laid paper or N.W..
5. This machine adopts full digital centralizing control, such as, advanced servo system, PLC program, touching screener and so on; it is accurate in positioning.
6. Production date printing—Open hole molding—label sticking—Package forming—Package feeding—Central sealing—Full-automatic on longitudinal cutting and side cutting.
7. Mounting with differential device to make adjustment on material feeding location without stopping machine.
8. Full-servo three-point position guide-flow, auto pointing and coordinating, double-servo horizontal sealing and positioning control, with optional duplex path horizontal sealing machine.
9. Add queueing unit and stacker in front, easier to operate, fast.


text:
Agile Pragmatically (Part 3) – Software Is Not Written by Machines But Humans07. august 2017
In the second part of Agile Pragmatically - Best Practices I dealt with practices which help us at PosAm to get a grip of the reality which is often far away from an ideal situation as described in literatures. Complexity is not necessarily being complex in terms of technology or domain, it may also be induced by the problem of size such as that of a team, which is the topic to be detailed today.


text:
This year I have decided to make a dress for Easter. I have never used a pattern, let alone made a dress before so this should be interesting. Stay tuned for my step by step progress over the next couple of weeks.
Here's the fabric I chose:
Here's the pattern:
Also, It's been a while since we've had an artist profile here on D2D. Tomorrow's profile will be excellent so tune in for that as well!
Monday, March 30, 2009
Easter Dress
Posted by Annie Bierma at 11:44 AM
Labels: Lifestyle
1 comment:
I will be gone part of the time, but call me and I will help with what I can, as I have sewn myself many dresses before.
Will be cool to see how it looks, and you will have to show me before I leave on Easter.
- March 30, 2009 at 1:44 PM
Subscribe to: Post Comments (Atom)


text:
Release Notes: July 2, 2019
View PlusPlus events and enrollments on Degreed, and a new Getting Started guide covering Events and Tracks. Plus: a new notification for event presenters, and a new data field for CSV import/exports.
Degreed Integration
You can now connect PlusPlus with the Degreed learning management system, which allows you to view events and attendance information from PlusPlus while using Degreed. Please talk to PlusPlus support before activating the integration. For more information, check out our doc: https://plusplus.co/docs/degreed-integration/:
Getting Started guide: Events and Tracks
As an aid to the first-time power users at your organization, we compiled a guide that shows the basics of creating Events and Tracks in PlusPlus: https://plusplus.co/docs/getting-started-events-tracks/.
If there is anything you feel is missing from the guide, let us know.
Look out for a guide covering the Coaching and Mentoring modules in the near future!
Minor Changes
Notifications for Presenters
We have added an additional notification that will send a reminder over email and Slack to remind a presenter of an upcoming event.
This notification is off by default. To activate it, visit Menu > Settings > Event Settings, and select the timing of the notification from the drop-down.
To see the default notification text, go to Menu > Settings > Notification Templates, and find the "Event Presenter Reminder" template. Click "Edit", and you will have the option to toggle the Slack and email notifications off, and to change the text for either.
CSV Import/Export for Events
We have a feature for organizers at Menu > Events which allows you to export a CSV-formatted document of event information. Additionally, you can import a CSV file containing new event information to PlusPlus.
We have added one additional field to the CSV report, marked "Enrollment Opens", which is meant to indicate a specific date and time that users are allowed to enroll in an event.
The date formatting for the field is "MM/DD/YYYY HH:mm (am/pm)" (eg: "3/23/2018 10:23 am" or "12/3/2019 9:42 pm").
You must have the "Enrollment Opens" column labeled and present in your CSV file when you import it to PlusPlus, but putting a value in the field is optional. Leaving the field blank will default the enrollment opening date to the admin settings at Menu > Settings > Event Settings > Event Cutoffs.
Thank you!
-Team PlusPlus
Category: Release Author: email@example.com Date: July 2, 2019 5:22 pm


text:
Woolpert Senior Vice President Jeff Lovin Named President of ASPRS
The Certified Photogrammetrist and Professional Surveyor was inducted today during the geospatial organization's online event.
DAYTON, Ohio, March 25, 2020 /PRNewswire-PRWeb/ -- Woolpert Senior Vice President and Market Director Jeff Lovin, CP, PS, was named president of the American Society for Photogrammetry and Remote Sensing ( ASPRS[1]) today. He was sworn in during the ASPRS annual conference program, portions of which are taking place online this year.
Lovin has been with Woolpert for 33 years and was certified as a photogrammetrist by ASPRS in 2003. He has served on the board of the Association for Unmanned Vehicle Systems ( AUVSI[2]), chaired the Coalition of Geospatial Organizations ( COGO[3]), served as president of MAPPS[4], was appointed to the National Geospatial Advisory Committee ( NGAC[5]), and was president of the Eastern Great Lakes Region of ASPRS. He said being selected to lead ASPRS is an exceptional honor.
"ASPRS is the original geospatial organization, founded in 1934, and is responsible for our mapping standards and our credentials," Lovin said. "I've been very fortunate in my career to be a part of several organizations but being elected as president of ASPRS ranks near the top for me. There is so...


text:
410 student athletes, their coaches and families traveled to Kern County Sunday, April 6, for the SoCal High School Cycling League Series. Race day was perfect with no clouds in sight and no wind. This was by far the most challenging course for the season thus far for our Brahmas. Steady climb in the beginning, many punchy climbs with ruts and rugged trails that had many washouts.
As a team, they had 17 riders race this course. Here are the ones who placed in the top 15.
JV Boy - Edward Tseng 11th
Soph Girl - Sophia Wang 6th
Soph Boys - Daniel Zhou 12th
Kenji Yoshimoto 13th
Frosh Boy - Ryan Nguyen 8th
The SoCal final will take place in Los Olivos on Sunday, April 27. The state championships will take place in Laguna Seca on Sunday, May 11.


text:
I get so tired of poetry blogs that just throw poems at me without any comments. Why did they choose the poem, what do they like about it? You know, actual sharing. So I started this blog. You are welcome here always. Caution: Instructional materials are volatile. WARNING: DO NOT READ POETRY WHILE OPERATING HEAVY MACHINERY! Material may be explosive. P.S. please check out my kickstarter project if you've got a free moment http://kck.st/1o6eess. Thanks!
Thursday, November 10, 2011
Number 300: Jeffrey Harrison "The Angel On The Table"
The Angel On The Table
She's losing her memory, isn't sure
who I am, is bothered by small things
like where that angel made from doilies
came from, the one she made herself
in the home's craft class. I remind her, but she
forgets again and in a minute asks again,
as if she's just noticed it for the first time.
The body is a doily twisted into a cone.
A doily cut and folded forms the wings.
The head is a Styrofoam ball, the hair a tuft
of cotton, the halo a gold pipe cleaner.
As simple and innocent as something
a child would bring home from school,
and in fact my daughter made one like it.
But this angel is a small torment to her,
perched on the table beside the photographs
of people she no longer recognizes–
but that doesn't bother her. It's the angel
she eyes with suspicion, even fear.
Where did it come from, what is it doing there,
what on earth does it want from her?
-- Jeffrey Harrison
Hap Notes: Harrison was born in Cincinnati, Ohio and studied under one of our favorite poets, Kenneth Koch (who was also from Cincinnati, by the way.) He is an award winning poet– okay, I'll name some– a couple of Pushcart Prizes, a Guggenheim and an NEA fellowship, and the Amy Lowell Traveling Fellowship.
He has published, to date, six poetry collections and his poems have appeared in a variety of periodicals including The Paris Review, The New Yorker, Poetry and The New Republic. His teaching gigs include George Washington University, Phillips Academy and College of the Holy Cross.
This poem comes from his book Incomplete Knowledge (Four Way Books, 2006.) Much of the focus of the collection is on illness, grief and loss. The poems dealing with his older brother's suicide are particularly moving.
In today's poem, we see a woman, possibly a grandma since she lives in a "home" where they have craft projects. Think of the strange, suspicious terror and disconcerting confusion that results from suffering from Alzheimer's as this woman obviously does. Why do you think an angel could be particularly frightening, even a crudely made craft-project one? Can you feel how terrifying this could be? And as an observer of this, let alone a relative of the sufferer, wouldn't this be, at the very least heart-rending and painful?
There is something else here, though. Why would "small things" be particularly bothersome to this woman? Why do the pictures of unrecognized relatives and friends bother her less than the angel?
Here's a good Harrison quote: "I believe that a poet must write what comes naturally, but I also believe (and this may seem contradictory) that there is a necessary restlessness to being a poet, and if a poet doesn't have this quality, then he or she is destined to be reincarnated over and over in the same poem. One doesn't consciously change the way one writes, it happens naturally over time, but the restlessness helps move the process along."
You can find more of Harrison's poetry as well as interviews and essays here: home.comcast.net/~jeffrey.harrison/
(The masthead picture features a shot of the notorious "Black Angel" at Oakland Cemetery in Iowa City, IA. It was one of the legendary places of my youth and I've always thought the angel was equally comforting, mysterious and terrifying.)
Subscribe to: Post Comments (Atom)


text:
The pneumolysin (ply) gene is widely used as a target in PCR assays for Streptococcus pneumoniae in respiratory secretions. However, false-positive results with conventional ply-based PCR have been reported. The aim here was to study the performance of a quantitative ply-based PCR for the identification of pneumococcal lower respiratory tract infection (LRTI). In a prospective study, fibreoptic bronchoscopy was performed in 156 hospitalized adult patients with LRTI and 31 controls who underwent bronchoscopy because of suspicion of malignancy. Among the LRTI patients and controls, the quantitative ply-based PCR applied to bronchoalveolar lavage (BAL) fluid was positive at >/=10(3) genome copies/mL in 61% and 71% of the subjects, at >/=10(5) genome copies/mL in 40% and 58% of the subjects, and at >/=10(7) genome copies/mL in 15% and 3.2% of the subjects, respectively. Using BAL fluid culture, blood culture, and/or a urinary antigen test, S. pneumoniae was identified in 19 LRTI patients. As compared with these diagnostic methods used in combination, quantitative ply-based PCR showed sensitivities and specificities of 89% and 43% at a cut-off of 10(3) genome copies/mL, of 84% and 66% at a cut-off of 10(5) genome copies/mL, and of 53% and 90% at a cut-off of 10(7) genome copies/mL, respectively. In conclusion, a high cut-off with the quantitative ply-based PCR was required to reach acceptable specificity. However, as a high cut-off resulted in low sensitivity, quantitative ply-based PCR does not appear to be clinically useful. Quantitative PCR methods for S. pneumoniae using alternative gene targets should be evaluated.
In the present study, we evaluated rapid real-time PCR assays for ply, Spn9802, and lytA applied to plasma samples for the detection of Streptococcus pneumoniae in patients with community-acquired pneumonia (CAP). In a prospective study of CAP aetiology, an EDTA plasma sample was collected together with blood culture in 92 adult CAP patients and 91 adult controls. Among the 92 CAP patients, lytA PCR was positive in eight (9%), Spn9802 PCR was positive in 11 (12%) and ply PCR was positive in 19 (21%) cases. Of 91 controls, the ply PCR was positive in eight cases (9%), but no positive cases were noted by Spn9802 or lytA PCRs. Ten CAP patients had pneumococcal bacteraemia. Compared to blood culture, PCR for lytA, Spn9802 and ply had sensitivities of 70% (7/10), 60% (6/10) and 70% (7/10), and specificities of 96% (79/82), 94% (77/82) and 85% (70/82) respectively. With blood culture and/or culture of representative sputum, and/or urinary antigen detection, S. pneumoniae was identified in 31 CAP patients. Compared to these tests in combination, PCR for lytA, Spn9802 and ply showed sensitivities of 26% (8/31), 32% (10/31) and 42% (13/31), and specificities of 100% (61/61), 98% (60/61) and 90% (55/61) respectively. We conclude that Spn9802 and lytA PCRs may be useful for the rapid detection of bacteraemic pneumococcal pneumonia, whereas ply PCR is not specific enough for routine use and blood PCR with small plasma volumes is not useful for the detection of nonbacteraemic pneumococcal pneumonia.
A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 104 DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.
The current shift from phenotypically toward genotypically based microbial diagnosis is not unproblematic. A novel quantitative real-time polymerase chain reaction (PCR) assay based on the Spn9802 DNA fragment was therefore developed for detection of Streptococcus pneumoniae. Out of 44 bacterial species, only S. pneumoniae and Streptococcus pseudopneumoniae were positive in Spn9802 PCR. In an evaluation on nasopharyngeal aspirates from 166 patients with community-acquired pneumonia, the assay was positive in 49 of 50 culture-positive cases. Of 19 culture-negative but Spn9802 PCR-positive cases, 12 were confirmed as S. pneumoniae by rnpB sequence analysis. With an expanded reference standard, including culture and rnpB sequencing, Spn9802 had a sensitivity of 94% and a specificity of 98%. A cutoff for clinically significant positivity was 10(4) DNA copies/mL, giving 71% sensitivity and 100% specificity. In conclusion, Spn9802 real-time PCR is highly sensitive and specific. The quantification it provides enables differentiation between pneumococcal pathogenicity and commensalism.
Background. Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients.
Results. The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis) in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 105 genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively. In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both bacteria.
Conclusions. The PCR provides increased sensitivity and the multiplex format facilitates diagnosis of S. pneumoniae, H. influenzae and N. meningitidis and the assay enable detection after antibiotic treatment has been installed. Quantification increases the specificity of the etiology for pneumonia.
Host-retrovirus interactions influence the genomic landscape and have contributed substantially to mammalian genome evolution. To gain further insights, we analyzed a female boxer (Canis familiaris) genome for complexity and integration pattern of canine endogenous retroviruses (CfERV). Intriguingly, the first such in-depth analysis of a carnivore species identified 407 CfERV proviruses that represent only 0.15% of the dog genome. In comparison, the same detection criteria identified about six times more HERV proviruses in the human genome that has been estimated to contain a total of 8% retroviral DNA including solitary LTRs. These observed differences in man and dog are likely due to different mechanisms to purge, restrict and protect their genomes against retroviruses. A novel group of gammaretrovirus-like CfERV with high similarity to HERV-Fc1 was found to have potential for active retrotransposition and possibly lateral transmissions between dog and human as a result of close interactions during at least 10.000 years. The CfERV integration landscape showed a non-uniform intra-and inter-chromosomal distribution. Like in other species, different densities of ERVs were observed. Some chromosomal regions were essentially devoid of CfERVs whereas other regions had large numbers of integrations in agreement with distinct selective pressures at different loci. Most CfERVs were integrated in antisense orientation within 100 kb from annotated protein-coding genes. This integration pattern provides evidence for selection against CfERVs in sense orientation relative to chromosomal genes. In conclusion, this ERV analysis of the first carnivorous species supports the notion that different mammals interact distinctively with endogenous retroviruses and suggests that retroviral lateral transmissions between dog and human may have occurred.
BACKGROUND: The Distributed Annotation System (DAS) is a widely used network protocol for sharing biological information. The distributed aspects of the protocol enable the use of various reference and annotation servers for connecting biological sequence data to pertinent annotations in order to depict an integrated view of the data for the final user. RESULTS: An annotation server has been devised to provide information about the endogenous retroviruses detected and annotated by a specialized in silico tool called RetroTector. We describe the procedure to implement the DAS 1.5 protocol commands necessary for constructing the DAS annotation server. We use our server to exemplify those steps. Data distribution is kept separated from visualization which is carried out by eBioX, an easy to use open source program incorporating multiple bioinformatics utilities. Some well characterized endogenous retroviruses are shown in two different DAS clients. A rapid analysis of areas free from retroviral insertions could be facilitated by our annotations. CONCLUSION: The DAS protocol has shown to be advantageous in the distribution of endogenous retrovirus data. The distributed nature of the protocol is also found to aid in combining annotation and visualization along a genome in order to enhance the understanding of ERV contribution to its evolution. Reference and annotation servers are conjointly used by eBioX to provide visualization of ERV annotations as well as other data sources. Our DAS data source can be found in the central public DAS service repository, http://www.dasregistry.org, or at http://loka.bmc.uu.se/das/sources.
The early and rapid detection and characterization of specific nucleic acids of medico-veterinary pathogens have proven invaluable for diagnostic purposes. The integration of amplification and signal detection systems, including online real-time devices, have increased speed and sensitivity and greatly facilitated the quantification of target nucleic acids. They have also allowed for sequence characterization using melting or hybridization curves. The newer-generation molecular diagnostic technologies offer, hitherto, unparalleled detection and discrimination methodologies, which are vital for the positive detection and identification of pathogenic agents, as well as the effects of the pathogens on the production of antibodies. The development phase of the novel technologies entails a thorough understanding of accurate diagnosis and discrimination of present and emerging diseases. The development of novel technologies can only be successful if they are transferred and used in the field with a sustainable quality-assured application to allow for the optimal detection and effective control of diseases. The aim of these new tools is to detect the presence of a pathogen agent before the onset of disease. This manuscript focuses mainly on the experiences of two World Organisation for Animal Health collaborating centers in context to molecular diagnosis and molecular epidemiology of transboundary and endemic animal diseases of viral origin, food safety and zoonoses.
Long terminal repeats (LTRs) are non-coding repeats flanking the protein-coding genes of LTR retrotransposons. The variability of LTRs poses a challenge in studying them. Hidden Markov models (HMMs), probabilistic models widely used in pattern recognition, are useful in dealing with this variability. The aim of this work was mainly to study LTRs of retroviruses and LTR retrotransposons using HMMs.
Paper I describes the methodology of HMM modelling applied to different groups of LTRs from exogenous retroviruses (XRVs) and endogenous retroviruses (ERVs). The detection capabilities of HMMs were assessed and were found to be high for homogeneous groups of LTRs. The alignments generated by the HMMs displayed conserved motifs some of which could be related to known functions of XRVs. The common features of the different groups of retroviral LTRs were investigated by combining them into a single alignment. They were the short inverted terminal repeats TG and CA and three AT-rich stretches which provide retroviruses with TATA boxes and AATAAA polyadenylation signals.
In Paper II, phylogenetic trees of three groups of retroviral LTRs were constructed by using HMM-based alignments. The LTR trees were consistent with trees based on other retroviral genes suggesting co-evolution between LTRs and these genes.
In Paper III, the methods in Paper I and II were extended to LTRs from other retrotransposon groups, covering much of the diversity of all known LTRs. For the first time an LTR phylogeny could be achieved. There were no major disagreement between the LTR tree and trees based on three different domains of the Pol gene. The conserved LTR structure of paper I was found to apply to all LTRs. Putative Integrase recognition motifs extended up to 12 bp beyond the short inverted repeats TG/CA.
Paper IV is a review article describing the use of sequence similarity and structural markers for the taxonomy of ERVs. ERVs were originally classified into three classes according to the length of the target site duplication. While this classification is useful it does not include all ERVs. A naming convention based on previous ERV and XRV nomenclature but taking into account newer information is advocated in order to provide a practical yet coherent scheme in dealing with new unclassified ERV sequences.
Paper V gives an overview of bioinformatics tools for studies of ERVs and of retroviral evolution before and after endogenization. It gives some examples of recent integrations in vertebrate genomes and discusses pathogenicity of human ERVs including their possible relation to cancers.
In conclusion, HMMs were able to successfully detect and align LTRs. Progress was made in understanding their conserved structure and phylogeny. The methods developed in this thesis could be applied to different kinds of non-coding DNA sequence element.
LTRs are sequence elements in retroviruses and retrotransposons which are difficult to align due to their variability. One way of handling such cases is to use Hidden Markov Models (HMMs). In this work HMMs of LTRs were constructed for three groups of orthoretroviruses: the betaretroviruslike human MMTV-like (HML) endogenous retroviruses, the lentiviruses, including HIV, and gammaretroviruslike human endogenous retroviruses (HERVs). The HMM-generated LTR alignments and the phylogenetic trees constructed from them were compared with trees based on alignments of the pol gene at the nucleic acid level. The majority of branches in the LTR and pol based trees had the same order for the three retroviral genera, showing that HMM methods are successful in aligning and constructing phylogenies of LTRs. The HML LTR tree deviated somewhat from the pol tree for the groups HML3, HML7 and HML6. Among the gammaretroviruslike proviruses, the exogenous Mouse Leukemia Virus (MLV) was highly related to HERV-T in the pol based tree, but not in the LTR based tree. Aside from these differences, the similarity between the trees indicates that LTRs and pol coevolved in a largely monophyletic way.
BACKGROUND: Retroviral LTRs, paired or single, influence the transcription of both retroviral and non-retroviral genomic sequences. Vertebrate genomes contain many thousand endogenous retroviruses (ERVs) and their LTRs. Single LTRs are difficult to detect from genomic sequences without recourse to repetitiveness or presence in a proviral structure. Understanding of LTR structure increases understanding of LTR function, and of functional genomics. Here we develop models of orthoretroviral LTRs useful for detection in genomes and for structural analysis. PRINCIPAL FINDINGS: Although mutated, ERV LTRs are more numerous and diverse than exogenous retroviral (XRV) LTRs. Hidden Markov models (HMMs), and alignments based on them, were created for HML- (human MMTV-like), general-beta-, gamma- and lentiretroviruslike LTRs, plus a general-vertebrate LTR model. Training sets were XRV LTRs and RepBase LTR consensuses. The HML HMM was most sensitive and detected 87% of the HML LTRs in human chromosome 19 at 96% specificity. By combining all HMMs with a low cutoff, for screening, 71% of all LTRs found by RepeatMasker in chromosome 19 were found. HMM consensus sequences had a conserved modular LTR structure. Target site duplications (TG-CA), TATA (occasionally absent), an AATAAA box and a T-rich region were prominent features. Most of the conservation was located in, or adjacent to, R and U5, with evidence for stem loops. Several of the long HML LTRs contained long ORFs inserted after the second A rich module. HMM consensus alignment allowed comparison of functional features like transcriptional start sites (sense and antisense) between XRVs and ERVs. CONCLUSION: The modular conserved and redundant orthoretroviral LTR structure with three A-rich regions is reminiscent of structurally relaxed Giardia promoters. The five HMMs provided a novel broad range, repeat-independent, ab initio LTR detection, with prospects for greater generalisation, and insight into LTR structure, which may aid development of LTR-targeted pharmaceuticals.
Long terminal repeats (LTRs) are important sequence elements of retroviruses and related retrotransposons. They are however difficult to analyse due to their variability.
The aim of this work was to construct models of LTRs from all known groups of LTR retrotransposons and retroviruses, representative of the entire LTR diversity, making it possible for the first time to comprehensively study their phylogeny and to compare it to phylogenies of other retrotransposon genes.
A general HMM describing all LTRs was built. Its associated Viterbi alignment showed a consistent basic structure with inverted repeats starting with TGTT at the 5´end, ending with AACA at the 3´ end, plus two conserved AT-rich areas, the first one often containing the TATA box and the second one containing the polyadenylation signal AATAAA. A less conserved AT-rich stretch was apparent in the likely U3 portion. R was harder to delineate. The polyadenylation signal was followed by a T rich area characteristic of U5. The modular LTR structure previously reported by us, with modules separated by clusters of insert states, was also observed in this pan-LTR setting The result attests to the highly conserved basic structure of LTRs, which must date over a billion years back.
Hidden Markov models (HMM) were also created for 14 subgroups of LTRs. The HMMs yielded consensus sequences which were aligned to a "Superviterbi" alignment. The Superviterbi alignment yielded a phylogenetic tree which was consistent with a tree based on an alignment of concatenated RT, RNAse H and INT proteins. In particular it gave further support for the monophyly of retroviral LTRs.
The phylogenetic reconstruction now allows inferences regarding the origin of LTR retrotransposons.
Endogenous retroviruses (ERVs) most likely are remnants of ancient retroviral infections. ERVs preserve functions of exogenous retroviruses to a varying extent, and can be parasites, symbionts or more or less neutral genetic 'junk'.Their evolution has two facets, pre- and post-endogenization. Although the two are not clearly separated, the first pertains to retroviral evolution in general and the second to the fate of repetitive DNA and the evolution of the host organism and its genome. The study of ERVs provides much material for the understanding of retroviral evolution. This sequence archive reflects the history of successes and shortcomings of antiviral resistance, but also of strategic evolutionary decisions regarding genome organization and new gene acquisition. This review discusses retroviral evolution illustrated through HERVs, bioinformatic prerequisites for ERV studies, the endogenization process and HERV evolution post-endogenization, including relation to disease. (Part of a multi-author review).
The genomes of many species are crowded with repetitive mobile sequences. In the case of endogenous retroviruses (ERVs) there is, for various reasons, considerable confusion regarding names assigned to families/groups of ERVs as well as individual ERV loci. Human ERVs have been studied in greater detail, and naming of HERVs in the scientific literature is somewhat confusing not just to the outsider. Without guidelines, confusion for ERVs in other species will also probably increase if those ERVs are studied in greater detail. Based on previous experience, this review highlights some of the problems when naming and classifying ERVs, and provides some guidance for detecting and characterizing ERV sequences. Because of the close relationship between ERVs and exogenous retroviruses (XRVs) it is reasonable to reconcile their classification with that of XRVs. We here argue that classification should be based on a combination of similarity, structural features, (inferred) function, and previous nomenclature. Because the RepBase system is widely employed in genome annotation, RepBase designations should be considered in further taxonomic efforts. To lay a foundation for a phylogenetically based taxonomy, further analyses of ERVs in many hosts are needed. A dedicated, permanent, international consortium would best be suited to integrate and communicate our current and future knowledge on repetitive, mobile elements in general to the scientific community.
Earlier reports of a human exogenous retrovirus (HMTV) related closely to mouse mammary tumor virus (MMTV) led us to search for these viral sequences in breast cancer tissues and normal tissues. A real-time PCR was developed based on MMTV and published HMTV envelope sequences. The real-time PCR method can detect one to ten copies of MMTV target DNA. Tissue samples were collected prospectively from 18 breast cancer patients and 11 non-malignant control cases, as well as peripheral blood leukocytes from the same women. Despite the high sensitivity of the real-time PCR method used, none of the samples were positive for HMTV DNA or RNA. The absence of HMTV DNA in both breast cancer samples and controls indicates either that the concentration of putative HMTV DNA in the breast cancers was too low for detection or that it did not exist there.
Gammaretrovirus-like sequences occur in most vertebrate genomes. Murine Leukemia Virus (MLV) like retroviruses (MLLVs) are a subset, which may be pathogenic and spread cross-species. Retroviruses highly similar to MLLVs (xenotropic murine retrovirus related virus (XMRV) and Human Mouse retrovirus-like RetroViruses (HMRVs)) reported from patients suffering from prostate cancer (PC) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) raise the possibility that also humans have been infected. Structurally intact, potentially infectious MLLVs occur in the genomes of some mammals, especially mouse. Mouse MLLVs contain three major groups. One, MERV G3, contained MLVs and XMRV/HMRV. Its presence in mouse DNA, and the abundance of xenotropic MLVs in biologicals, is a source of false positivity. Theoretically, XMRV/HMRV could be one of several MLLV transspecies infections. MLLV pathobiology and diversity indicate optimal strategies for investigating XMRV/HMRV in humans and raise ethical concerns. The alternatives that XMRV/HMRV may give a hard-to-detect "stealth" infection, or that XMRV/HMRV never reached humans, have to be considered.
Low-pathogenic avian influenza (LPAI) viruses in wild birds are important as they can constitute the basis for the development of high-pathogenic avian influenza (HPAI) viruses or form part of human-adapted strains with pandemic potential. However, the LPAI infection as such is not very well characterized in the natural reservoir, dabbling ducks, and results are in part contradictory. The effects on the infection by artificial versus natural infection, exposure to antiviral drugs or development of resistance have not been studied. Therefore, we used q-PCR, histopathology and immunohistochemistry (IHC) to study mallards infected with an influenza A/H1N1 virus isolated from a wild mallard in Sweden. The mallards were either inoculated intra-esophageally or infected by virus shed by other ducks in the experiment. The birds were subjected to low levels of the active metabolite of oseltamivir (Tamiflu®) and the resistance mutation H274Y developed during the course of the experiment.
All mallards but one had a strictly intestinal localization of the LPAI infection. The exception was a bird euthanized one day post artificial inoculation whose infection was located solely in the lung, possibly due to intra-tracheal deposition of virus. The intestinal infection was characterized by degenerating cells in the lamina propria, infiltrating heterophils and lymphocytes as well as positivity of IHC and q-PCR on samples from feces and intestinal contents. Histopathological changes, IHC positivity and viral shedding all indicate that the infection peaked early, around two days post infection. Furthermore, the infection had a longitudinal progression in the intestine with more activity in the proximal parts early in the infection and vice versa as observed both by IHC and by q-PCR. There was no obvious difference in the course of the infection in artificial versus natural infection, when the level of OC was increased from 80 ng/L to 80 µg/L or when the resistance mutation H274Y developed.
Background: The recent report of gammaretroviruses of probable murine origin in humans, called xenotropic murine retrovirus related virus (XMRV) and human murine leukemia virus related virus (HMRV), necessitated a bioinformatic search for this virus in genomes of the mouse and other vertebrates, and by PCR in humans.
Results: Three major groups of murine endogenous gammaretroviruses were identified. The third group encompassed both exogenous and endogenous Murine Leukemia Viruses (MLVs), and most XMRV/HMRV sequences reported from patients suffering from myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Two sensitive real-time PCRs for this group were developed. The predicted and observed amplification range for these and three published XMRV/HMRV PCRs demonstrated conspicuous differences between some of them, partly explainable by a recombinatorial origin of XMRV. Three reverse transcription real-time PCRs (RTQPCRs), directed against conserved and not overlapping stretches of env, gag and integrase (INT) sequences of XMRV/HMRV were used on human samples. White blood cells from 78 patients suffering from ME/CFS, of which 30 patients also fulfilled the diagnostic criteria for fibromyalgia (ME/CFS/FM) and in 7 patients with fibromyalgia (FM) only, all from the Gothenburg area of Sweden. As controls we analyzed 168 sera from Uppsala blood donors. We controlled for presence and amplifiability of nucleic acid and for mouse DNA contamination. To score as positive, a sample had to react with several of the XMRV/HMRV PCRs. None of the samples gave PCR reactions which fulfilled the positivity criteria.
Conclusions: XMRV/HMRV like proviruses occur in the third murine gammaretrovirus group, characterized here. PCRs developed by us, and others, approximately cover this group, except for the INT RTQPCR, which is rather strictly XMRV specific. Using such PCRs, XMRV/HMRV could not be detected in PBMC and plasma samples from Swedish patients suffering from ME/CFS/FM, and in sera from Swedish blood donors.
A computer controlled infusion pump (UIPump) for regulation of target tissue concentration of radioactive compounds was developed for use in biological research and tracer development for PET.
Based on observed tissue or plasma kinetics after a bolus injection of the tracer an algorithm calculates the infusion needed to obtain a specified target kinetic curve. A computer feeds this infusion scheme into an infusion pump connected to an animal via a venous catheter. The concept was validated using [11C]Flumazenil administrated to Sprague-Dawley rats where the whole brain distribution and kinetic of the tracer was measured over time using a microPET-scanner. The accuracy and precision of the system was assessed by producing steady-state levels of the tracer and by mimicking kinetics after oral administration.
Various kinetic profiles could be generated, including rapid achievement of constant levels, or step-wise increased levels. The resulting tissue curves had low deviation from the target curves according to the specified criteria: AUC (%): 4.2 +/- 2.8, Maximal deviation (%): 13.6 +/- 5.0 and R2: 0.95 +/- 0.02.
The UIPump-system is suitable for use in PET-research for assessment of PK/PD properties by simulation of different tracer tissue kinetics in vivo.
A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex technology was used for simultaneous detection of ten fungal species in one single experiment. By combining the multiplexing properties of padlock probes and Luminex detection with the well established quantitative characteristics of qPCR, quantitative microbe detection was done in 10-plex mode. A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid.
BACKGROUND: A sensitive and specific method for detecting herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) is important for diagnosing genital and cutaneous infections. GOAL: The goal of this study was to compare quantitative real-time polymerase chain reaction (qPCR) with virus culture for diagnosis of genital and cutaneous HSV-1 and HSV-2. STUDY DESIGN: A duplex qPCR system for quantification of DNA from HSV-1 and HSV-2 was developed. Duplicate swabs for PCR and virus culture were collected from 89 patients attending our sexually transmitted infection and dermatology clinic. RESULTS: The duplex qPCR had a linear measure interval of 10-10 copies/mL. The detection limit was between 1 and 5 copies per reaction. qPCR detected HSV in 57 (64%) specimens and virus was isolated in 45 (50%) cases. First-episode infections showed higher viral quantities with a median value of 4.2 x 10 copies per reaction compared with recurrent infections with 1.0 x 10 (P = 0.0002). HSV-1 was more likely to be the cause of first-episode genital infections (72%), and HSV-2 of recurrent and atypical genital manifestations (73%). CONCLUSION: Real-time PCR is a sensitive method for diagnosing genital herpes, and the duplex format is convenient for typing. The method increased the detection rate by 27% compared with virus culture.
It was reported earlier that a few patients suffering from non-Hodgkin's lymphoma had low amounts of DNA from the so-called fifth human exogenous retrovirus, HRV-5. A sensitive and rational method for large-scale screening for HRV-5 DNA was therefore developed. It is a single-tube nested quantitative PCR (stnQPCR), which uses two functionally isolated primer pairs and one probe target distinct from related endogenous retroviral sequences, yet encompassing known HRV-5 variation, allowing optimal use of sequence conservation. DNA from lymphoma, myeloma, and follicular dendritic cell lines was tested for HRV-5 positivity, as was DNA from whole blood of blood donors, non-Hodgkin's lymphoma and systemic lupus erythematosus patients, as well as DNA from lymph node biopsies of rheumatoid arthritis patients with lymphoma. One blood donor, one systemic lupus erythematosus patient, two previously known positive non-Hodgkin's lymphoma patients, and one rheumatoid arthritis lymphoma patient, came out positive. They had 24, 2, 148, 480 and 30 proviral copies per microg of DNA from PBMC or lymphoma tissue, respectively. During the completion of this work it was reported that HRV-5 is a rabbit endogenous retrovirus (RERV-H), and that HRV-5 positivity was due to presence of rabbit DNA. DNA from six RERV-H/HRV-5 positive samples was therefore retested. Three also contained rabbit mitochondrial DNA. A search for HRV-5 antibodies using synthetic peptides was negative in sera from three RERV-H/HRV-5 positive individuals, as well as in 144 other sera, according with a noninfectious origin of the RERV-H/HRV-5 DNA in human samples. A search for possible sources of rabbit DNA contamination was negative. Methods for prevention of PCR contamination were strictly adhered to. Three samples from RERV-H/HRV-5 positive individuals positive at the Uppsala laboratory were retested at one or two other laboratories, and all three were positive. Two other samples, which were positive in the Riga laboratory, were tested also in London and also found positive. One non-Hodgkin's lymphoma patient was RERV-H/HRV-5 positive in four consecutive samples, showing that positivity was a property of that patient. It is concluded that the stnQPCR developed to detect and quantify minute amounts of RERV-H/HRV-5 DNA is a principle which can be applied widely and HRV-5 is a RERV-H. Its presence in a few human blood samples could not be explained.
Endogenous retroviral sequences (ERVs) are dynamic genomic components with profound influences on gene expression and genomic structure. Their extent of expression is not well known. Several broadly targeted real-time reverse transcription PCR (QPCRs) systems for surveillance of RNA expression of the major groups of human gammaretroviral ERVs were constructed. The highly conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were used as targets for the PCRs, which were both probe-based (TaqMan) and probe-less (SYBR Green). Different levels of primer and probe degeneracy, with or without inosine, were tested. Several of the PCRs had sensitivities of a few HERV nucleic acid copies per PCR reaction. Specificities were approximately as expected from the fit of primers and probes. Gammaretroviral HERV RNA expression was studied in different human tissues. Each HERV group had a specific pattern of expression. HERV-E was highly expressed in testis, HERV-I/T in brain and testis, HERV-H in brain and testis, while HERV-W was highly expressed in placenta. Gammaretroviral RNA was not detected in plasma from 50 blood donors in saliva from 20 persons. In conclusion, a set of tools for investigation of gammaretroviral HERV RNA expression was created.
When trace elements are used as diagnostic tools during disease, it is important to know whether the balance is changed in free or bound elements. Although acute infections are associated with changed trace element balance in serum/plasma, it is not known whether changes occur concomitantly in serum and blood. In the present study the human coxsackievirus B3 (CB3), here adapted to Balb/c mice, was used to study whether infection alters the normal physiological trace element balance in blood and serum. Virus was quantitatively measured in two target organs (pancreas and liver) of this infection by reverse transcription polymerase chain reaction (RT-PCR), showing high concentrations of virus proving ongoing infection. Concentrations of 14 elements were measured in whole blood and serum using inductively coupled plasma mass spectrometry (ICP-MS) on days 3, 6 and 9 of the infection. Free and total thyroxine were measured in serum to prove metabolic changes associated with the infection. The thyroxine decreased, while iron and the Cu/Zn ratio in serum increased as a response to the infection. No clear changes in these elements were observed in blood. Cd and Hg tended to decrease in serum but to increase in blood, indicating accumulation in blood cells. Moreover, Al showed a similar decreasing trend in both serum and blood. A correlation between serum and blood levels was observed at different time points of the disease for 9 of the elements. However, As was the only element indicating correlations between serum and blood during the entire course of the disease.
Objective: The trigger of juvenile diabetes has been suggested to be an interaction between a virus and trace elements, where enteroviruses, including coxsackievirus B3 (CVB3), have been discussed as potential initiators. The aim of this study was to investigate the effects in the pancreas on gene expressions of metallothionein 1 (MT1), divalent metal transporter 1 (DMT1), and zinc transporter 5 (ZnT-5) and concomitant changes in iron (Fe), copper (Cu), and zinc (Zn) in serum and pancreas of Balb/c mice on days 3, 6, and 9 of CVB3 infection. Methods: Trace elements were measured through inductively coupled plasma-mass spectrometry, and CVB3, MT1, DMT1, and ZnT-5 were measured by reverse transcription/polymerase chain reaction. Results: Virus was found in the pancreas on all days, with a peak on day 3. Infection tended to increase Fe in both serum and the pancreas. The Cu/Zn ratio in the pancreas increased early in the infection because of a great decrease in Zn. In serum, the Cu/Zn ratio was not increased until day 9 of the disease. In the pancreas, MT1 decreased, whereas DMT1 tended to increase on day 6, and ZnT-5 increased progressively during the course of the disease. Conclusions: Virus-induced changes in trace elements, MT1, DMT1, and ZnT-5 in the pancreas may reflect early stages of the development of pancreatitis and prestages of diabetic disease.
A real-time PCR method targeting the Bordetella pertussis IS481 gene fragment was evaluated in a vaccine trial setting in which real-time PCR results could be validated against culture and serology results. Two commonly used DNA extraction methods, Amplicor((R)) Respiratory Preparation kit and the QIAamp((R)) DNA Mini Kit, were compared. An approximately 50-fold higher sensitivity was achieved using the Amplicor kit. 89 of 276 aspirates analysed with the IS481 real-time PCR were positive. Interestingly, six of these were culture negative and came from serology-negative patients. Defining true positive cases either as culture-positive or as PCR-positive cases that had been confirmed with a serology-positive result or verified with a newly constructed recA PCR, the sensitivity and specificity of the IS481 real-time PCR were 89% and 98%, respectively. This study confirms the specificity and high diagnostic sensitivity of IS481-based PCR methods for diagnosis of B. pertussis.
A real-time PCR was designed for detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae such that each pathogen could be detected in a single tube and differentiated using molecular beacons marked with different fluorochromes. This duplex PCR, targeting the P1 adhesion gene for M. pneumoniae and the ompA gene for C. pneumoniae, was compared with two conventional PCR assays targeting the 16S rRNA gene and the ompA gene. A total of 120 clinical throat and nasopharyngeal swab samples were tested. DNA extraction was performed using an alkali denaturation/neutralization method, and real-time amplification, detection, and data analysis were performed using a Rotor-Gene 2000 real-time rotary analyzer (Corbett Life Science, Sydney, Australia). Using conventional PCR as a reference in an analysis of 120 samples, 13 of 14 samples positive for C. pneumoniae were detected by the novel real-time PCR. In an analysis of M. pneumoniae, 22 samples were positive in the conventional PCR and the novel assay detected 24 positive samples. When using the conventional PCR as a reference, sensitivity and specificity were 93% and 100%, respectively, for C. pneumoniae and 100% and 98%, respectively, for M. pneumoniae. With an overall agreement of 98.8%, this suggests that performance of the new duplex real-time PCR is comparable to that of conventional PCR.
Hepatitis E virus (HEV) is a major cause of food- and waterborne diseases in countries with poor sanitation. Furthermore, travellers to such countries are also at risk of contracting the virus. Noteworthily, during the last decade an increasing number of non-travel-related cases were recorded even in countries with high sanitary standards. An alternative, direct route of infection, from animals to humans (zoonotic transmission) is suspected to be the cause of recent cases of hepatitis E. In order to provide rapid and sensitive methods for detecting the virus in various hosts, two real-time PCR methods were developed and compared: a TaqMan (R) and Primer-Probe Energy Transfer (PriProET) assay. These highly sensitive novel methods provide valuable diagnostic tools to investigate zoonotic transmission, to detect the virus in the food chain and in research related to the potential of hepatitis E virus to cross the species barrier. The results show that the two novel PCR assays are robust, highly sensitive and specific for broad range detection of the four genotypes of HEV. Compared to PriProET, the TaqMan (R) assay appears to perform slightly better, with higher fluorescence values for positive samples. However, the PriProET has the benefit of better tolerating the point mutations in the target nucleic acids. Thus, it provides a more powerful tool to detect new virus variants. These new molecular diagnostic assays are practical tools that can be employed in the area of public health, for disease diagnosis and for tracking outbreaks. In basic research the methods provide new tools to study HEV biology, including virus-host interactions and transmission between various host species.
Aim: To characterize the cytotoxic effect of BBE, the particulate of desacyl-saponin, in model systems of solid tumours. Materials and Methods: Cytotoxic activity of BBE was investigated in solid human tumour cell lines, in tumour cells from patients with renal cell carcinoma, in normal human renal cells and in peripheral blood mononuclear cells. The BBE mode of cell death was assessed in vitro. In vivo effect of BBE was evaluated in xenograft-bearing mice. Results: BBE was selectively active against renal cell carcinoma, with no or little effect on normal cells. BBE induced caspase activity and apoptosis. An inhibitory activity of BBE on xenograft tumour growth, with no apparent signs of haematological toxicity was shown. In the non-proliferative model of patient tumour cells, BBE was active on only 1/5 patient samples, suggesting association of BBE effect with cell proliferation. Conclusion: BBE has interesting activities against renal cell carcinoma and should be further explored as a drug against this resistant tumour type.
A duplex quantitative real-time PCR (qPCR) assay was designed to detect both the polymerase gene (pol) and the glycoprotein gene (gB) of cytomegalovirus (CMV). The detection limit of the qPCR was determined to be 1 to 3 copies/reaction and the linear measure interval was 10(3) to 10(8) copies/ml. The qPCR system was compared to the COBAS Amplicor CMV Monitor test (COBAS) by an analysis of 138 plasma samples. Both systems detected CMV in 71 cases and had negative results for 33 samples. In addition, 34 samples were positive by qPCR and negative by the COBAS assay, but in no case was the COBAS result positive and the qPCR result negative. Thus, qPCR detected 48% more positive cases than the COBAS method. For samples with > or = 10(5) copies/ml by qPCR, a saturation effect was seen in the COBAS assay and quantification required dilution. Copy numbers for pol and gB by qPCR generally agreed. However, the reproducibility of qPCR assays and the need for an international standard are discussed. Discrepant copy numbers for pol and gB by qPCR were found for samples from two patients, and sequence analysis revealed that the corresponding CMV strains were mismatched at four nucleotide positions compared with the gB fragment primer sequences. In conclusion, a duplex qPCR assay in a real-time format facilitates quantitative measurements and minimizes the risk of false-negative results.
Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale.


text:
Mayor of London
About The Mayor of London
The Mayor of London is responsible for making London a better place for everyone who visits, lives or works in the city. The current Mayor Sadiq Khan, oversees a £17bn budget and sets out the overall strategic economic, environmental, transport and social framework for the capital through the London Plan. Promoting 'good growth' is fundamental to the Mayor's vision for development in London. That means building places that are environmentally sustainable and that make the most of the local potential.
The Mayor also acts as a champion for the city at home and abroad. He works to promote economic growth and protect London's competitiveness as a world-leading city for business.


text:
Mike Feder con The Talking Cure: A Memoir of Life on Air
As a kid growing up in Queens, Mike Feder identified with Scheherazade of The Thousand and One Nights: "The idea of someone having to tell a new tale every night to prevent their head getting chopped off seemed sadly familiar to me." Back then, the author's audience was his mentally ill mother, who used to stay in the house all day with the shades drawn, and then insist that her son tell her stories so that she might vicariously experience the world outside. Eventually she committed suicide, and Feder grew up to be a relentless, comic storyteller on the radio. The Talking Cure tells the story of his ridiculous jobs, first failed marriage, the string of psychiatrists, and the misery of reluctant fatherhood; throughout he maintains a kind of bizarre balancing act--hilariousness and deep seriousness, conventionality and strangeness. An ironist and a comic, Feder looks unflinchingly at his own foibles and frailties, enabling him to connect to other people's stories. The reader emerges from this book with a sense of forgiveness for the human condition, and awe at the mystery of human life. Deeply funny, and at the same time breathtakingly dark, this is a book to provoke, amuse and, in some strange way, reassure: God loves a challenge.