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Candy floss, fish and chips and more Buses (and rain) than you can shake a stick at
Busfest 2014 show photos
Massive! That's the only word to describe Busfest. For the last 20 years, the Three Countries Showground amidst the Malvern Hills in Worcestershire has been the place to go if…
Camper&Bus Show 2014 photos
Find out all there is to know about the Camper&Bus Show 2015 BUY NOWBook your tickets to the Camper&Bus Show today Camper&Bus Show 2014 report The Camper&Bus Show has…
Ben Pon VW Show 2014
It began as an attempt at a world record, it ended as one of the best new shows ever. We give you the Ben Pon VW Show
Stonor Park VW Show 2014
The Stonor Park VW Show was held on the 1st June 2014. Here are some of photos taken from the day.
Stanford Hall 2014 show photos
If you're into stock VWs, sprawling swapmeets and cake, this year's Stanford Hall was an absolute belter. Here are some of our photos from the day.
Freddy Files 2014 show photos
The annual British invasion of Belgium is officially called the Freddy Files, but is more commonly known simply by its location – Ninove
VolksWorld Show 2014 Photos
Well, thats the VolksWorld Show done for another year. And what a show it was. Lots of amazing new cars (in fact only 2 cars in the halls had ever…
Brighton Breeze 2013 photos
The 2013 Brighton Breeze took place on the 5th October at the usual location on Madeira Drive. As always it was a great day out and was very busy. A…
The Camper&Bus Show 2013 photos
Check out the selection of photos that we took at the first ever Camper&Bus Show. The event was held over the 22nd to the 23rd June and started in Guildford,…


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4/24/2012 / Christian Living
Our reflection today is based on Romans Chapter 5 which is one of the chapters in the Bible that is full of theological significance since the concept of Justification is dealt with and as history will show it was this subject Justification by Faith more than any other which tore the church apart and ushered in the Reformation. However we are not dealing with Justification by Faith in this article.
In Romans 5:6-11 on which we shall reflect, the Apostle Paul is detailing the benefits and privileges that accrue to Christians and he is showing them aspects of their past life, features of their present standing and highlighting their inheritance through Jesus Christ our Lord.
In this article we want to really focus on what Jesus did for us all and remember the supreme sacrifice that was necessary that we may be saved. To those who have not yet given serious consideration to their spiritual situation, nor looked at where they want to spend eternity this might be a good time to reflect on these things. Jesus is a lot more than just a senior historic figure, someone we respect for his defiance of outmoded tradition, a man of great esteem and charisma teaching new things and setting a great example to the entire world; he is the one that did it all to save mankind.
First let us be aware that Jesus saw our pitiful condition
Before the world was created, God saw us as we really are; pitiful. Nothing about God can ever be accurately referred to as an afterthought for although God made man upright, God knew, and was prepared for, mans untimely decline. All through the Old Testament era humanity had parted company with God and chosen a path of futility and ultimately hopelessness and even Gods chosen people were little different. The Jews sought to immerse themselves in ritualistic righteousness and ceremonial symbolism along with national legalism and ended up just like the gentiles; far from God in precept and practice. All this continued until God stepped in and change that trajectory with the birth of Jesus Christ and his eventual death on the cross of Calvary and subsequent resurrection. From that moment onwards Jesus Christ, as the mediator between God and man, has been intimately involved in providing our salvation and being our heavenly representative. As such he deals with us as individuals; just as we are and he sees everything.
We know for sure that Jesus saw our weakness
Some people like to pretend and exaggerate and deceive other people that things are better than they are. No matter how puffed up and boastful sinners are one thing that is striking is that when put to the test we are found to be weak. Romans 5:6 tells it as it is for it tells us that, For when we were yet without strength Jesus acted. The mightiest sinner exercising all his considerable resources is utterly powerless to break out of the bondage of his sinful nature. Paul acknowledged this as he cried out, O wretched man that I am! who shall deliver me from the body of this death (Romans 7:24) History is replete with people making simple, well intentioned, promises and New Year resolutions and failing to keep them however hard they try. All of us stand before God helpless and unable to free ourselves from our sinful status. We must recognize our weakness for failure to do this is a big impediment if we are to be saved.
God knew that mankind had reached a point of utter weakness, like an exhausted swimmer battling the murky waters about to submerge for the final time, and Jesus didnt only see our weakness but set about to assist us.
We realize with certainty that Jesus saw our wickedness
Openness and transparency and especially honesty are things we talk about a lot, but seldom practice and we know that human beings like to conceal their true nature from each other. In most court cases the accused tries to stop the court from finding out things and we know of courting couples who talk a lot and say nothing and we all try to cover up personal transgressions. We should remember that God sees us as we really are and our wickedness is self evident. In the same Romans 5:6 the Bible saw us as, the ungodly and the ungodly are those who live their lives without fear of God and conduct themselves as if God does not exist.
We are in error if we think that the ungodly is so called because of the volume of the wickedness he has committed. The unsaved teenager is just as godless as the unsaved mass murderer and Jesus sees it all.
We are convinced that Jesus saw our waywardness
The concept of sinning, as used here, is the individual missing the mark (in much the same way as an archer consistently missing the whole target) because of his sinful status and in Romans 5:8, the Bible states it thus, while we were yet sinners Mans journey through life is a litany of trials and failures and no matter how high he aims or his fastidious approach to morality he will always fall short of Gods standards. This does not mean that man does badly all the time for bad people at times do good things and the reverse is equally true that good people on occasions do bad things and it is this erratic and unpredictable behavior that is a prominent feature of mankind.
We should be warned about the deceptive nature of good works for although they may be comforting, and ease our consciences in certain situations; they will never earn us salvation or get us to our desired heavenly destination.
We know beyond doubt that Jesus saw our warfare
Everyone in the world knows about and has experienced an intense existential conflict going on all around and within us. While this conflict spills over and is marked by physical struggle nevertheless it is essentially a spiritual battle. There are two sides in this spiritual war and you are on one side or the other and if you are not saved you are in Satans camp and therefore carries the status as Romans 5:10 describes it, the enemy.
The unsaved church member is as much the enemy and at war with God as someone who is an atheist!!
So too is the unsaved, but friendly and likable gentleman down the street who wont harm a fly. All those outside of Christ are warring against God. Remember God knows who side you are on.
All of these, our weakness, our wickedness, our waywardness and our warfare against God add up to a formidable barrier between us and God. Even if we wanted to come to God and be reconciled, and the evidence suggests we didnt, this barrier was just too difficult to overcome. Thanks are due to God for Jesus who saw our pitiful condition and set about to rescue us.
Then let us notice that Jesus showed his peerless compassion
Having seen our hopeless, pitiful condition Jesus Christ is moved with compassion toward us and we are awestruck by his peerless compassion. We can describe Jesus as supremely compassionate without fear of contradiction and all through the gospels we hear again and again that Jesus, whenever he saw human distress, was moved with compassion.
We observe that his compassion is superior
Most people understandably would not sacrifice their life for anybody, some would for their family or close friends, but none would do so for their enemies, but Christ did exactly that as shown in Romans 5:6, in due time Christ died for the ungodly. This is superior compassion that transcends our understanding and defines our saviour very well. The compassion of Jesus for lost humanity is awesome in concept and flawless in exhibition as he has demonstrated all through his earthly ministry.
He demonstrated it in feeding the hungry multitude as stated in Matthew 9: 36. He demonstrated it in healing the sick that flocked to him as recorded in Matthew 14:14. He demonstrated it in Bethany when confronted with the death of Lazarus and this is reported in John 11: 33-35.
We notice that his compassion is substantial
The compassion of Jesus Christ is more than just providing food for the hungry or healing a sick person but has to do with substantial matters like eternity. This is shown in that he gave his life, not for his friends, but as Romans 5:8 shows, while we were yet sinners, Christ died for us. We were his enemy and he knew it, yet he made the ultimate sacrifice for us and we should realize that this compassion, this great love for us, is important and so worthwhile that it changes the status of sinful man in wonderful ways.
It has the potential to elevate our position we become justified, That being justified by his grace, we should be made heirs according to the hope of eternal life (Titus 3:7)
It has the potential to ensure our protection saved from wrath, Much more then, being now justified by his blood, we shall be saved from wrath through him (Romans 5:9)
It has the potential to enable our peace we become reconciled, Therefore being justified by faith, we have peace with God through our Lord Jesus Christ (Romans 5:1) No one remaining in sin can experience this for the Bile tells us, There is no peace, saith my God, to the wicked (Isaiah 57:21)
We realize that his compassion is significant
When looked at as a whole, the difference this peerless compassion makes as it impacts sinful, weak, wayward, wicked, bellicose mankind is nothing less than life changing as Romans 5:10 shows, saved by his life
Jesus by his great compassion for mankind took the ultimate step in dying on Calvarys cross for all mankind and for all time and that is of supreme significance. Think about this carefully and ask yourself the question. Would anyone other than Jesus care for you so much as to volunteer to die so painfully on your behalf? This is the measure of his compassion and love.
Finally, let us be thankful that Jesus sustains our perfect concord
There are numerous privileges to every believer because of the ministry and person of Jesus Christ, not the least of these is the perfect concord, the essential harmony that now pertain between us and God. Too often we forget the wondrous benefits of salvation and take things for granted and we fail to do as recommended by David, What shall I render unto the Lord for all his benefits toward me? I will take the cup of salvation, and call upon the name of the Lord. (Psalm 116:12-13) Jesus did so much for us, we have only to examine them and be extremely thankful.
We are grateful that Jesus was the perfect referee (the god-man)
When two opposing sides exist, Gods on the one side thoroughly good dispensing justice and the other, fallen man thoroughly bad, deserving of annihilation, then someone special is required to bring concordance and in Romans 5:10 it states, we were reconciled to God by the death of his son The Bible attests elsewhere to this unique role that could be filled by no other than Jesus Christ, For there is one God and one mediator between God and men, the man Christ Jesus. (1Timothy 2:5) It is awesome for us to lay so much on Jesus shoulders, but extremely comforting to know that we have someone who is well able to do the job to the fullest. The saints of old who predated Moses were not too sure of this person and the perception that this referee does not exist brought acute agony to the soul of a righteous man, Neither is there any daysman [referee] betwixt us, that might lay his hand upon us both. (Job 9: 33)
All believers can rejoice for Jesus has bridged that gap and in him we have the perfect referee.
We are delighted that Jesus is perfectly righteous (the lamb without blemish)
The Mosaic Laws, in particular the ordinance relating to animal sacrifices, required that the lamb chosen for killing for the sacrifice was to be without blemish, And if his offering for a sacrifice of peace offering unto the Lord be of the flock; male or female, he shall offer it without blemish. (Leviticus 3:6) Jesus alone fully met this criterion in every respect because he was sinless in nature and sinless in practice and Peter described him thus, Christ also suffered for us, leaving us an example, that ye should follow his steps: Who did no sin, neither was guile found in his mouth. (1 Peter 2: 21-22)
Hallelujah to the Lamb for he satisfied Gods just requirements for redeeming all mankind for all time. How wonderful is that?
We regret, but realize why Jesus was punished relentlessly (the sacrificial lamb)
The sacrificial lamb, according to Leviticus 4:25, was to be slaughtered and its blood poured out; in like manner, Jesus was sacrificed on a cruel cross for us, This man was handed over to you by Gods set purpose and foreknowledge; and you, with the help of wicked men, put him to death by nailing him to the cross. (Acts 2: 21 NIV)
Our salvation was at great cost to Gods begotten Son and we should never take it lightly.
A few years ago a film by Mel Gibson called The Passion of the Christ gave us a graphic depiction of how relentlessly Jesus was punished and I am told grown people wept at what they saw.
We rejoice to know that Jesus ascended to represent us (the risen savior)
Humanity, saved and unsaved, still needs representation before Almighty God and the risen Christ has gone to be our intercessor and advocate. For believers Jesus advocacy is what aids and preserves us as we struggle through this life, My little children, these things write I unto you, that ye sin not. And if any man sin, we have an advocate with the Father, Jesus Christ the righteous. (1 John 2: 1) Where the unconverted are concerned, they need not fear rejection in approaching God because Jesus will take care of them, Wherefore he is able also to save them to the uttermost that come unto God by him, seeing he ever liveth to make intercession for them. (Hebrews 7: 25)
All of this representation is for our benefit and gives us peace of mind because we know that even if our prayers and requests to God are insufficient and faulty Jesus will handle the matter perfectly.
We can reflect with awe and wonder how much Jesus has done for us. How much he has suffered on our behalf? How much he is still doing for all mankind as Peter reminds us that, The Lord isnot willing that any should perish, but that all should come to repentance. (2 Peter 3:9) Jesus will never stop working on our behalf until time is no more and even at this moment he is right here to help us. Let us never forget that in the ups and downs of a hectic and for many a troubled life, Jesus and Jesus alone has done it all for us.
Books by this author you may wish to read.
THINGS EVERY CHRISTIAN SHOULD KNOW (e-book)
Volume 1 Five tough facts to be faced
STAND UP TO THE DEVIL (e-book)
Volume 1 You must first identify him
BASTARD: A STORY OF REDEMPTION(e-book fiction)
ONE MINUTE TO MIDNIGHT ON GODS CLOCK (Hard copy)
ONE MINUTE TO MIDNIGHT ON GODS CLOCK (e-book)
Dr. Henderson Ward received his Doctor of Divinity in theology, with distinction, from Masters International School of Divinity, USA, where he is currently a post-doctoral fellow. Dr. Ward's career involved pastoring, evangelism, and teaching. Copyright 2017
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Study of the Effects of Commercial Air Travel on the Lungs
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- Disclaimer
- How to Read a Study Record
Condition | Intervention |
Healthy Volunteers Chuvash Polycythaemia |
Other: Commercial airline flight |
Study Type: | Observational |
Study Design: | Time Perspective: Prospective |
Official Title: | Study of the Effects of Commercial Air Travel on Pulmonary Artery Pressure in Healthy Passengers and in a Patient With Chuvash Polycythaemia |
- Systolic pulmonary artery pressure [ Time Frame: In-flight ]The primary outcome measure is the effect of commercial air travel on systolic pulmonary artery pressure assessed by in-flight portable Doppler echocardiography.
Enrollment: | 9 |
Study Start Date: | May 2011 |
Study Completion Date: | June 2011 |
Primary Completion Date: | June 2011 (Final data collection date for primary outcome measure) |
Groups/Cohorts | Assigned Interventions |
Healthy volunteers |
Other: Commercial airline flight
Normal scheduled passenger flight
Chuvash polycythaemia |
Other: Commercial airline flight
Normal scheduled passenger flight
Detailed Description:
In a commercial airliner flying at high altitude, the reduced cabin air pressure means that all passengers are exposed to slightly lowered oxygen levels ('hypoxia') equivalent to an altitude of approximately 5,000 to 8,000 ft. Although mild, this hypoxia is sufficient to stimulate some of the body's protective responses (eg changes in breathing and in hormonal secretion) and can be dangerous for passengers with heart or lung disease, who must breathe supplementary oxygen in-flight or may even be prohibited from flying because of the risks of hypoxia.
It is well known that severe hypoxia results in constriction of blood vessels in the lungs (a phenomenon called hypoxic pulmonary vasoconstriction), which in turn causes an increase in the blood pressure in the lungs ('pulmonary arterial pressure'). Unlike other physiological responses to hypoxia, this is often harmful and frequently leads to pulmonary hypertension and right heart failure (eg in some lung diseases and at high altitude). Even a modest increase in pulmonary arterial pressure could be clinically important in some airline passengers with heart/lung disease, as it may exacerbate their condition. However, it is not known whether the mild hypoxia experienced in an aircraft cabin is able to cause an increase in pulmonary artery pressure. Limited evidence suggests that it might - for example, there have been reports of passengers acutely developing new right heart failure in-flight, and data from animal studies also support this possibility.
This study aims to establish the effect of mild aircraft cabin hypoxia on pulmonary arterial pressure in healthy passengers and also in a patient with Chuvash polycythaemia. In this rare genetic disease, cellular responses to hypoxia are 'switched on' to some extent even during normoxia, causing increased red blood cell production. Affected individuals usually present with symptoms of polycythaemia in early adulthood and are typically asymptomatic following treatment with therapeutic venesection. Importantly, affected individuals have exaggerated acute hypoxic pulmonary vasoconstriction which may place them at risk of pulmonary hypertensive responses during air travel.
Ages Eligible for Study: | 18 Years and older (Adult, Senior) |
Sexes Eligible for Study: | All |
Accepts Healthy Volunteers: | Yes |
Sampling Method: | Non-Probability Sample |
For healthy volunteers:
Inclusion Criteria:
- suitable for echocardiographic measurements
- in good health
Exclusion Criteria:
- any significant medical condition
For patient with Chuvash polycythaemia:
Inclusion Criteria:
- diagnosis of Chuvash polycythaemia
- suitable for echocardiographic measurements
Exclusion Criteria:
- any other significant medical condition
- pulmonary hypertension
- uncontrolled erythrocytosis
Please refer to this study by its ClinicalTrials.gov identifier: NCT01420224
United Kingdom | |
University of Oxford | |
Oxford, United Kingdom, OX13PT |
Principal Investigator: | Thomas Smith, MBBS DPhil FRCA | University of Oxford |
Responsible Party: | Ms Heather House, Head of Clinical Trials and Research Governance, University of Oxford |
ClinicalTrials.gov Identifier: | NCT01420224 History of Changes |
Other Study ID Numbers: |
Oxford in-flight 2011 |
Study First Received: | August 18, 2011 |
Last Updated: | August 18, 2011 |
Keywords provided by University of Oxford:
Air travel Aircraft cabin hypoxia Pulmonary artery pressure Hypoxic pulmonary vasoconstriction Pulmonary hypertension |
Additional relevant MeSH terms:
Polycythemia Hematologic Diseases |
ClinicalTrials.gov processed this record on March 23, 2017


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A submitted paper that describes a modified strain of the avian H5N1 virus has the scientific community discussing the dangers of publishing science that could be used for bioterrorism.
In the paper submitted to Science, Dutch scientist Ronald Fouchier and his team at Erasmus Medical Centre in Rotterdam, the Netherlands report that by transferring a strain of H5N1 from ferret to ferret, the virus evolves to adapt to each host. The virus' normal mechanism for transmission is via bodily secretions, but Fouchier and colleagues found that the virus became capable of airborne transmission after only 10 generations in the experiment.
"There's not a threat from this research necessarily, but the threat from bioterrorism is real. The risk is real," said biosecurity expert Barry Kellman from DePaul University.
Because this research could possibly be used as a biological weapon, research regulators and scientists are in a precarious position. While a study detailing the aggressive human transmission of the virus could be crucial for preventative measures in the event of an attack, that same published information could lead to an attack as well.
As a result, a US panel of national security and scientific research experts are reviewing the paper. The National Security Advisory Board on Biosecurity is investigating the paper but isn't commenting on it at this time. While the group doesn't have the authority to ban the paper's publication, its recommendations could discourage some journals from publishing the research.
But while the US has a system in place, there is no international guidelines or review system for responding to research that could potentially have dangerous applications, leaving a vulnerability in biosecurity.
"We've gone forward on the basis that all the really cutting-edge research is done in the US, and therefore if we have controls that's sufficient, but that's nonsense. This research is happening all over the world and if we don't think about these problems we might be building a dam out of a sieve," said Kellman.
When the NSABB will make their recommendation or when Science will make its decision on publication remain unknown. Fouchier hasn't made any comments about what he will do if the paper is rejected and has not responded to requests for comments.
"We need to think as a policy matter how we're going to deal with this. We have domestically, but we haven't globally, and here's the proof of the inadequacy of that approach," said Kellman.


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Fans of the rich history of Doctor Who Magazine and their long running comic strip will probably be aware of the legend that is Abslom Daak, Dalek Killer, and he takes centre-stage in the new edition of Vworp Vworp! magazine – pre-orders for which are now being taken!
If the prospect of a new Doctor Who fanzine release isn't enough to get your juices flowing, perhaps the Weetabix-inspired free gift, pictured above, is?
So what else is in it? I'll let editors Gareth Kavanagh and Colin Brockhurst explain:
- The first half of our exhaustive Abslom Daak tribute, including interviews with writer Steve Moore, and artists Steve Dillon and David Lloyd. Also a Daak comic strip, written for DWM by Steve Moore in 1980 but withdrawn after a disagreement with the editor, now finally brought to the page by DWM artists Martin Geraghty, Adrian Salmon and Roger Langridge.
- We're kicking off our series of DWM editor interviews with a fascinating chat between Dez Skinn and Tom Spilsbury. We also chat to Paul Neary, Alan McKenzie, Cefn Ridout and the elusive Sheila Cranna.
- Ex-Doctors Tom Baker and Colin Baker talk about their comic strip selves.
- Dave Gibbons, Steve Parkhouse and Mick McMahon discuss The Tides of Time and Junkyard Demon, and we take in The Lodger with Gareth Roberts and Mike Collins.
- We've got, The Housekeeper, a comic strip written by Paul Magrs and drawn by Bret M Herholz starring Mrs Wibbesey and the Fourth Doctor.
- And we've got an fantastic free gift in the form of a 'Vworpabix' board game. Based on the 1970s Weetabix game, ours has 16 character cards by Adrian Salmon and boards by Paul Grist, Jon Pinto, Leighton Noyes and Graeme Neil Reid.
- Two covers: a wraparound Abslom Daak cover by Mike Collins, and an early Doctor Who Monthly pastiche featuring Junkyard Demon (with Jason Fletcher and Ben Willsher's take on Daak on the back cover).
- … and much more!
So, featuring interviews with Tom Baker, Colin Baker, Dave Gibbons, Paul Cornell, Paul Neary, Dez Skinn, Lee Sullivan and many others, the most professional and polished fanzine you will ever see outside of Panini Towers focuses on various elements of Doctor Who Magazine and its legendary strips – what's not to love?
We've got a hot preview of the magazine coming soon, but in the meantime feast your eyes on the accompanying images and head on over to www.vworpvworp.co.uk, click on Order Vworp Vworp! You can pick up Vworp Vworp! #2 in either of two covers, both £5.99 plus postage.
As for the stunning cover…
Now, sit back and wait in perfect anticipation for the release in late August/early September!


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Get Rihanna's 'Battleship' Premiere Look for Less
You'll look like you've got guns blazing, just like Riri at the Battlehip premier. BUT, your wallet will be unhurt by the purchase!
-PJ Gach
Sorry for the puns, but when you have the words "battleship" and "shopping" paired together, all sorts of havoc can come up! And no one wants to beat up their poor wallet to look good. For the Battleship premiere in LA, Rihanna wore a white custom made gown by Adam Selman. He's the guy who does all her concert costumes. He's great, but pricey. She finished her look with Manolo Blahnik sandals and jewelry from Vhernier and Neil Lane. Her lips were scarlet red and her nails were frosty pink. We did a little searching and found a great look for less that won't include any dogfights.
Blaque Label More or Less backless dress ($104, lulus.com), white strappy open toe carved glitter heels ($35.99, amiclubwear.com), Bree bracelet Item #: 30060 ($23, guyandeva.com), Poseidon aqua ring ($15, threadsence.com), AEO beaded hoops ($12.50, ae.com), Essence Cosmetics lipstick in Red Carpet Red ($2.49, ulta.com), Sephora by OPI Betsey Johnson nail color in It's My Pink! ($9.50, sephora.com)
PJ Gach is Senior Editor: Style + Beauty at BettyConfidential.


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Sigerson Morrison PATENT LEATHER FLATS WITH FLOWER 398.00 199.00 incl. VAT excl. shipping costs | CHF | $ | £ 199,00 approx. CHF 296,00 approx. $ 276,00 approx. £ 191,00 Red patent leather flats with flower detail at pointed tip. Flower with black patent leather petals. Leather soles, uppers and insoles. Style Note A sweet flat with the flower detail. Pair them with full or pencil skirts and easy tailored trousers or jeans.
Miu Miu METALLIC JEWELLED BALLERINA US $514,00 incl. VAT excl. shipping costs | CHF | $ | £ 370,00 approx. CHF 551,00 approx. $ 514,00 approx. £ 355,00 Metallic taupe ballerinas with jewel detail. Lila border on mouth. Silver toned jewel detail with copper border on top. Leather upper, insole and sole. Style Note Add a zip to your step with these metallic ballerinas with the bold jewel detail. Great for chunky tights with short skirts or add fun to tailored trousers.
Find what's hot, right now. Join Stylehive for free or login.


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Don't miss a story. Like us on Facebook.
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Neiman Marcus said today it will open a 250,000-square-foot store in New York City in 2018. The Dallas-based luxury chain has suburban New York locations but this will be its first Manhattan store for the Neiman Marcus brand.
It will be going head-to-head with its own Bergdorf Goodman, Barneys New York and rival Saks Fifth Avenue on its home turf. Saks closed its last store in the Dallas area last year. It continues to operates its Off 5th chain in the market. Barneys also left the Dallas market.
Neiman Marcus Group owns and operates Bergdorf Goodman on the corner of 5th Avenue and 58th Street. Bergdorf Goodman will also be getting a major modernization, the retailer said Wednesday.
The planned three-level Neiman Marcus store will anchor the one-million-square-foot Shops at Hudson Yards development on west side of Manhattan between 30th and 34th Streets. It will have an entrance on 10th Avenue. The store will be the second largest in the chain, just behind San Francisco and slightly bigger than NorthPark Center, which has been the chain's No. 1 volume store.
The store will have views of a public plaza and meeting place being designed by Thomas Heatherwick and Thomas Woltz, the unique Culture Shed, the planned home of New York Fashion Week and the widely-acclaimed High Line.
Neiman Marcus' New York strategy includes a store on Long Island in Roosevelt Field Mall that will open in 2016, and a Last Call Studio Store in Brooklyn that opens later this year.
The 28-acre Hudson Yards, is being developed by Related Companies and Oxford Properties Group. It will include more than 17 million square feet of commercial and residential space, as well as 14 acres of public open space, a 750-seat public school and a 175-room luxury hotel.


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Remote IO Mod 1.7.10
Author: dmillerw ❘ August 29, 2016 ❘ 36,612 views
Remote IO Mod 1.7.10 let's the player think outside the blocks; no longer are you limited to only six sides. The concept is simple… the possibilities endless.
The Remote IO's can be linked to any block with an inventory… imagine this: four separate quarries all outputting into a unique Remote IO linked to a single chest. Gone are the days of pipes! You are also able to link the Remote IO to machines and even use them with fluids!
Right-click with the Documentation Screen in hand to open this GUI
Placing a block/item from RemoteIO in the top-left slot will present any documentation on that block/item if it exists
If the documentation extends past what the screen can show, simply scroll down to see more
Crafting Recipes:
Remote IO Mod Wiki:
How to install Remote IO Mod 1.7.10
- Download and install Minecraft Forge
- Download the mod
- Go to %appdata%
- Go to .minecraft/mods folder
- Drag and drop the downloaded jar (zip) file into it
- If one does not exist you can create one
- Enjoy the mod
Download Links for Remote IO Mod 1.7.10
For 1.6.4/1.6.2
For 1.7.2
For 1.7.10


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It is official: Ellen DeGeneres is the proud owner of a new home. The former home of a very A-list actor.
Transactions were completed Friday to finalize the purchase of Brad Pitt's Malibu manse for $12 million, PEOPLE has learned.
The property overlooks the Pacific Ocean, has private beach access, consists of two separate houses, a pool, a tennis court and lush gardens.
DeGeneres's interest in the estate was first buzzed about around Thanksgiving, a month after she and wife Portia De Rossi put their Architectural Digest-featured home on the market for $49 million.
The couple will be downsizing a bit: From about 15,000-square feet of interior space to 4,000. Sounds cozy! – Gail Nussbaum
RELATED: Brad Pitt Turns 48 with a Very Michael Jackson Night in Vegas


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Novel immune-modulator identified by a rapid, functional screen of the parapoxvirus ovis (Orf virus) genome
- Michael J McGuire2,
- Stephen A Johnston1 and
- Kathryn F Sykes1Email author
© McGuire et al; licensee BioMed Central Ltd. 2012
Received: 30 August 2011
Accepted: 13 January 2012
Published: 13 January 2012
The success of new sequencing technologies and informatic methods for identifying genes has made establishing gene product function a critical rate limiting step in progressing the molecular sciences. We present a method to functionally mine genomes for useful activities in vivo, using an unusual property of a member of the poxvirus family to demonstrate this screening approach.
The genome of Parapoxvirus ovis (Orf virus) was sequenced, annotated, and then used to PCR-amplify its open-reading-frames. Employing a cloning-independent protocol, a viral expression-library was rapidly built and arrayed into sub-library pools. These were directly delivered into mice as expressible cassettes and assayed for an immune-modulating activity associated with parapoxvirus infection. The product of the B2L gene, a homolog of vaccinia F13L, was identified as the factor eliciting immune cell accumulation at sites of skin inoculation. Administration of purified B2 protein also elicited immune cell accumulation activity, and additionally was found to serve as an adjuvant for antigen-specific responses. Co-delivery of the B2L gene with an influenza gene-vaccine significantly improved protection in mice. Furthermore, delivery of the B2L expression construct, without antigen, non-specifically reduced tumor growth in murine models of cancer.
A streamlined, functional approach to genome-wide screening of a biological activity in vivo is presented. Its application to screening in mice for an immune activity elicited by the pathogen genome of Parapoxvirus ovis yielded a novel immunomodulator. In this inverted discovery method, it was possible to identify the adjuvant responsible for a function of interest prior to a mechanistic study of the adjuvant. The non-specific immune activity of this modulator, B2, is similar to that associated with administration of inactivated particles to a host or to a live viral infection. Administration of B2 may provide the opportunity to significantly impact host immunity while being itself only weakly recognized. The functional genomics method used to pinpoint B2 within an ORFeome may be more broadly applicable to screening for other biological activities in an animal.
adjuvant genetic immunization functional screen immunomodulator Parapox Orf virusIntroduction
Bioinformatic analyses of microbial sequences have changed the way searches for vaccine antigens can be conducted; researchers can avoid pathogen isolation, culturing, and fractionation as a means of target identification. However these "reverse vaccinology" approaches [1], in which sequences are evaluated relative to their likelihood of encoding protective antigens, carry some limitations such as i) our poor understanding of the characteristics of a pathogen component that make it protective and ii) the inability to query the substantial proportion of any microbial genome that carries no previously described homologs or motifs [2]. Therefore, empirical approaches to rapidly determine the natural functions or non-natural applications of genome-encoded products are needed [3]. Toward this purpose, there has been some progress in using genome databases to recombinantly produce proteins in cell culture or test tubes for printing nanoscale quantities onto glass microscope slides [4]. These protein arrays can be used to perform rapid, parallel in vitro screens if a slide-compatible functional assay is available. Genome-wide screening in mammalian cells using RNA interference and expression libraries has also gained much popularity [5]. However, these platforms do not provide the opportunity to assay activities at the organism level, such as an animal's immune activity.
We previously described an approach to vaccine antigen discovery that, similar to reverse vaccinology, does not require pathogen culturing but in contrast does not rely on bioinformatic analyses nor require recombinant protein production. Instead, it is based on the delivery of each gene in vivo and its direct functional testing as a vaccine candidate in host animals. Using expression library immunization (ELI) [6], pathogen genomes can be searched for protective antigens in streamlined multiplexed animal challenge assays [7–9]. Here, this parallel-testing strategy is extended for use in searching for any measurable biological activity encoded by a gene in vivo. It uses genomic sequence information to prepare genes for rapid and unbiased isolation of biological effectors. As demonstration, we chose a pathogen with a novel immune activity of potential application for therapeutic immune-modulation.
The Poxviridae Family includes many well-known pathogens. They carry complex genomes that encode a variety of mechanisms for subverting the immune responses of infected hosts [10]. The globally prevalent Parapoxvirus ovis (ORFV) causes Orf, an acute and highly infectious disease largely limited to sheep and goats, but which can be also transmitted cutaneously to people handling infected animals [11]. Epithelial infection leads to the development of pustular skin lesions and then scabs [12]. Virus is shed from the scab and no systemic spread occurs. Despite lesion repair and an initial vigorous inflammatory response, the ORFV-specific host immune response is transient and ineffective in viral neutralization, perhaps due to its unique immune regulation and skin epithelial niche [13]. Consequently, re-infection of the same host is common [14, 15]. Administration of whole inactivated virus (iORFV) similarly shows clinical effect [16] and fails to prevent subsequent infections, rendering a conventional vaccine design against Orf disease impractical. However, these inactivated preparations hold strong immune-stimulating properties including transient interferon-gamma (γ-IFN) induction followed by autoregulatory T helper 2 cell cytokine activity that is not antigen specific [15, 17–19]. Inactivated virus provides short term, non-specific immune protection against a number of viruses [16, 17], and is sold as Baypamun (Bayer, Inc.), or more recently as Zylexis (Pfizer AH, Inc.), to prevent disease in herds during short periods of close confinement and stress, such as during transport and regrouping [20].
Failure of a host to sustain specific immunity against ORFV infection may relate to the effect of the infection on the skin's epidermal Langerhans and dermal dendritic cells (DC). ORFV has the ability to manipulate these antigen presenting cells (APCs) cells, central to initiating both innate and adaptive immune responses [21, 22]. Histologic examination of pock lesions demonstrates that the sites of infection become densely packed with DC within two to four days of virus exposure [23]. Since they appear to be recruited and detained rather than having migrated away as would be expected following antigen exposure, they serve as a barrier to systemic infection and cannot initiate an adaptive response because they do not gain access to T cells in the lymph nodes. Furthermore, when the skin lesions fully resolve the DC are concomitantly sloughed off [23]. These observations suggested that ORFV carries one or more factors that are able to recruit and/or retain DC at the skin. One or both of these immune-related activities may contribute to the unusual characteristics of ORFV infection. Notably, iORFV inoculation does not stimulate DC accumulation yet does initiate a strong inflammatory cytokine response and transient non-specific immunity. Isolation of the putative immune-cell modulating component(s) might provide useful research and biomedical applications for it.
To mine the ORFV genome empirically for the immune-cell regulator gene(s) that cause the accumulation of DC-like cells at a site of skin inoculation, the genomic sequence of the ORFV strain used in the Baypamun and Zylexis products, D1701, was determined. This complete yet low coverage sequence information was used to specify oligonucleotide (oligo) primers for the amplification of all predicted open reading frames (ORFs). This ORFeome was placed in linear expression elements (LEEs) [24] and delivered into mice to directly assay for the desired innate immune activity. The B2L gene was identified as eliciting DC-like cell accumulation and also other immune-modulating activities. Regulation of DC-like cells might be generally useful in vaccination or other immunomodulation strategies [25]. This approach of screening a library directing in an animal for a biological activity may have further utility.
Sequence determination
The D1701 isolate of Parapoxvirus ovis (ORFV) was used to prepare the immune-stimulating product Baypamun; therefore, genomic (g)DNA from this virus was obtained (gift from Tobias Schlapp, Bayer). The viral gDNA was shotgun cloned for sequencing. The final assembly consisted of 134,040 nucleotides and is reported [Genbank: HM133903]. Three complete ORFV (Orf) genomes are available in the NCBI database (OV-NZ-2, OV-IA82, and OV-SA00). The NZ-2 strain is most closely related to D1701, with > 95% nucleotide sequence identity. The majority of the differences between the sequences of D1701 and NZ-2 are found toward the flanking terminal repeat regions. An electronic MacVector Genescan course analysis of the assembled D1701 genome using a minimum ORF encoding length of 90 aa yielded 288 putative proteins. The annotated ORFV genomes in NCBI each have only 134 identified ORFs, indicating that our parameters are relaxed. We chose not to modify these determinations so as to be most inclusive and use the functional screen to eliminate non-ORFs. Sequence comparison (BLAST) of the D1701 strain to the NZ-2 ORFV strain identified three additional putative ORFs; these were added to the list to obtain a total of 291 sequences to explore. We determined that D1701 B2L was not recognized in our annotation because of a single base sequencing error that created a predicted frameshift at amino acid position 126 and a stop codon at aa position 299. Whereas this led to the design of a shortened ORF that appeared to encode a divergent aa sequence, the NZ-2 sequence comparison enabled us to identify the sequencing error and retrieve the wild type full length ORF. The B3L gene homolog had not been designated as an ORF because the translated product contains only 89 amino acids and our algorithm required a minimum of 90 amino acids; the B3L gene [26] was included in the screen.
Functional screening of an ORFV library for immune cell recruitment activity
Pool X was comprised of eight ORFs; this included the three ORFs identified by comparison to the NZ-2 strain sequence (B2L, B3L and vIL10). The remaining five were predicted from the D1701 sequencing results (270, 272, 277, 278, and 279). The NZ-2 B2L gene product had been previously identified as the homolog of the vaccinia (VACV) virus major envelope antigen gene, F13L [26, 29]. The truncated D1701 B2L ORF described above resided in pool J, which did not induce the accumulation of I-A+/I-E+ cells (Figure 2a). Pool J was retested multiple times in multiple mice to confirm this negative result (data not shown).
The effect of each of the ORFs comprising pool X on immune cell accumulation was tested by delivering them separately (1 μg/ORF) at distinct sites on the abdomen of mice, in triplicate. The original X pool and a LEE expressing the LUC ORF were retested as positive and negative controls, respectively (data not shown). The tissue sections removed from each inoculation site were stained for anti-I-A/I-E antibody reactivity (Figure 3a). Skin sections inoculated with the ORF encoding B2 displayed increased densities of MHC class II positive cells relative to control tissues (Figure 3a). In a confirmatory experiment, both white light and fluorescent images of the same B2L-shot tissue section were visualized so that immune cell localization and gun microparticle distribution could be compared (Figure 3b, left and middle panels). As indicated by the fluorescent image in the middle panel, inoculation of abdominal skin with the full length B2L ORF- D1701 led to DC-like cell accumulation relative to unshot tissue (Figure 3b, right panel).
B2 protein displays immune cell accumulating and adjuvant-like activities
In an immunization experiment, mice were i.d. injected with i) rHBsAg protein only, or co-injected with ii) rHbSAg + rSRB4 (control), or iii) rHbSAg + rB2. Administration of rHBsAg by itself or with the rSRB4 protein control (rCTRL) did not stimulate a significant anti-HBsAg antibody response. However, co-immunization with rB2 stimulated a strong antibody response (Figure 4b). For example at day 56, rHBsAg alone generated titers significantly lower than that generated by rHBsAg + rB2, p = 0.029. Similarly, rHBsAg + rCTRL generated titers significantly lower than that generated by rHBsAg + rB2, p = 0.032. To explore the possibility of an antibody response to the adjuvant itself, these same sera were also tested in an ELISA against rB2 as antigen. No reactivity to B2 was detected by ELISA (Additional file 1) or immunoblot, when it had been administered to the mice in either protein-format (Additional file 2) or gene-format (data not shown).
B2L as modulates infectious and noninfectious disease. models
New genomic sequence information from a pathogen was rapidly converted into gene function data using a direct screen for biological activities of interest. This was accomplished by taking advantage of linear gene expression, a multiplexed pooling strategy, and genetic immunization. In this example screen, a genome was queried for an immunomodulating activity; however, a similar strategy could be applied to query any genome for any gene-encoding activity for which one can design a bio-assay. The low coverage sequencing reads performed in this work were sufficient to identify all ORFs and design primers for PCR. Higher coverage reads would afford the opportunity to synthetically build genomes, and thereby further eliminate the need for culturing pathogens even if only for genomic DNA template production. As time and cost of both large scale sequencing and gene building continue to decrease, this latter approach is increasingly attractive.
A parapoxvirus ovis gene responsible for the manipulation of host immunity was initially identified in days, using less than 30 mice. In particular, expression of the ORFV B2L gene was found to elicit the accumulation of DC-like cells at the site of skin inoculation. We did not establish whether this accumulation is a result of immune cell retention or recruitment. Dermal and epidermal (Langerhans) DC are distributed in lattice-like patterns within the skin [30, 31], comprising an estimated 10% of the dermal cells [27]. This lattice is observed when untreated mouse skin is immuno-stained for MHC II-expressing cells. When skin is bombarded with gold microparticles, many of the DC disappear from the shot skin site [32, 33]. These cells react to bombardment by migrating to the draining lymph node where they can activate T cells and other immune cells [34, 35]. Microparticle densities visualized under white light appeared similar among the many tissue sections bombarded with particles carrying a pool of LEEs. However, migrating DC represent only a portion of I-A+/I-E+ skin cells, therefore their loss, or lack of loss, may not be measureable. More informatively, overlaying of the white light and fluorescent views of the shot tissue sections indicate that skin samples shot with either pool X or the individual B2L gene display significant areas of fluorescence that do not correlate with the position of a particle. Whereas, samples shot with other pooled or individual constructs displayed a close correspondence between particles and fluorescence (Figure 2 and 3). These results indicate that non-resident I-A+/I-E+ immune cells were recruited to the shot site after bombardment with B2L. Non-resident DC have been previously shown to be recruited to a skin site upon ORFV infection [23].
Relative to other resident skin cell types, MHC II-positive DC are the most potent initiators of an immune response [33]. However, the increased density of DC at an immunization site could influence the generation of a subsequent adaptive response with several possible outcomes. For instance, by causing resident DC to be retained and others further recruited to the skin rather than homing to the draining lymph node, an immune response might be diminished. Alternatively, maintaining the DC at the immunization site may provide additional opportunity to process antigens synthesized by neighboring cells such as keratinocytes before eventually migrating to the lymph node, thus initiating a stronger immune response. Finally, B2L co-delivery with an antigen may have no effect on the antigen specific response.
We showed that co-delivery of B2 protein with an antigen (HBsAg) elicited enhanced levels of antigen-specific antibodies (Figure 4). Protective immunity elicited by an influenza gene vaccine was also enhanced by co-administration of the B2L gene (Figure 5). In a gene vaccine dose titration, a positive trend for adjuvanted protection was observed at intermediate vaccine doses but not the lowest dose, indicating protection was antigen specific and dose dependent. We additionally found that gene gun administration of the B2L expression construct to mice, without an antigen expressing construct, was able to reduce the rate of tumor growth in a transplantable model of cancer (Figure 6). The non-specific protection provided by B2L is reminiscent of the short-term protection conferred by either live ORFV or iORFV against viral infections; however, iORFV does not also cause I-A+/I-E+ cell accumulation, as do B2L and live virus. This seems to suggest that delivery of B2L is immunologically more similar to live infection than delivery of whole inactivated virions.
Delivery of the B2L expression construct arguably provides three possible effector molecules for the observed immune activities: DNA, RNA, and protein. However, delivery of the DNA construct carrying a full length B2L gene but expressing a truncated B2 protein did not hold modulating activity, whereas direct delivery of purified full length B2 protein did (Figure 4, Additional file 1). These results indicate that the B2 protein is the relevant effector molecule, and that the effector activity requires the C-terminal portion of the protein.
Neither the mechanism of B2-induced accumulation of immune cells nor its mechanism of enhancing specific or non-specific responses is clear. However, informatics analyses are suggestive. B2 is the homolog of vaccinia virus (VACV) F13, also called p37 [26], which carries a number of known features. This palmitoylated protein is the major antigen on the surface of enveloped virions, and is required for viral envelopment and egress [36]. F13 is one of seven virus-encoded envelope proteins which is present in the intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV) [37]. Two others of these seven VACV envelope proteins, B5 and A33, are components of the "4-pox" subunit vaccine candidate for smallpox [38]. A33 targets F13 to the IEV, and these two proteins immunoprecipitate as an A33/F13 complex from infected cell lysates [39]. Proteins F13 and B5 co-localize forming a disulfide bonded complex that is required for efficient and complete wrapping of the intracellular mature virus [29, 37, 39].
ORFV is unique relative to VACV and other orthopoxviruses in its lack of any B5 homolog. Perhaps the lack of an homologous B5/F13 complex is relevant to the unique immune cell accumulation activities of ORFV. By contrast, an A33 homolog does exist in ORFV. Stage-dependent trafficking of B2 by an A33 homolog during the ORFV infection cycle might be relevant to the immune cell accumulating activity of live-virus and purified B2, which iORFV preparations such as Baypamun, lack. Inactivated virions represent one snapshot of surface protein landscapes, whereas a live virus could cycle proteins to and from the surface.
Vaccinia F13 carries a conserved HKD-like motif that is found in members of the phospholipase D (PLD) gene superfamily, which encodes the catalytic site for phospholipase activity or simply a lipid binding site [40]. Lipids play a regulatory role within the immune system [41]. Mammalian PLDs have been well studied because of their role in membrane vesicular trafficking and critical signal transduction cascades [37]. Mutation of the HKD-like motif in F13 of VACV leads to loss of cell to cell viral spreading, indicating that it is an active superfamily member. It appears to encode a lipid binding rather than modifying activity [40], such as do toll-like receptors. Notably, this C-terminally-located PLD domain is found at aa positions 318-342 of B2. In our experiments, the initially designed B2L ORF did not display immune I-A+/I-E+ cell accumulation activity in our expression screen. This truncated ORF was amplified from D1701 gDNA, therefore it carried the correct sequence, not the sequencing error at aa position 126. However, the prematurely positioned 3' primer caused a truncation at position 299 (because the 3' primers of the library were all designed with in-frame stop codons).
Sequence comparison of the B2 protein of ORFV strains D1701 and NZ21,2.
Sequence of the highly conserved region IV of phospholipase D1 family members found in parapox and vaccinia viruses.
PLD1 family member | PLD1 domain amino acid sequence |
Further work will explore whether B2 is responsible for any other activities related to the immune system. For example, B2 may stimulate the uptake of exogenous antigen or stimulate chemokine activity. As a read-out of an adaptive response, we have used antibody reactivities. Since B2L administration enhanced the effectiveness of a viral gene vaccine it may also influence cellular mediated immune activities. The effect of B2L expression on tumor development in the absence of antigen suggests a non-specific immunity is elicited against a chronic disease. Testing B2L administration prior to an infectious challenge will determine whether this molecule alone can also protect against an acute disease.
A novel immunomodulator, B2, was isolated from an Orf virus in a functional screen of its ORFeome. B2 was identified by its effective accumulation of immune cells at a site of inoculation. However several practically significant activities were also established. B2 performs as an enhancer of antigen specific immunity, it acts as a non-specific immune enhancer in the absence of an antigen, and it is itself non-immunogenic.
Whereas mechanistic studies are typically performed as a means to the identification of a factor responsible for a given activity, the functional genome mining approach presented here enabled the factor to be identified and isolated before its mechanism of action was explored. Mechanistic studies can now be conducted with better molecular tools. Characterizing B2 and its activities should facilitate our ability to modulate and control immune responses. Recent trends in vaccine development are aimed at identifying subunit vaccines rather than producing the conventional whole-pathogen based ones. This creates a greater need for developing a toolbox of safe adjuvants to compensate for the reduced immunogenicity of the pure component formulations.
Purified parapoxvirus ovis DNA (D1701 strain) was obtained from Tobias Schlapp of Bayer Pharmaceuticals, Germany. Female BALB/c and A/J mice were purchased from Harlan Laboratories (Indianapolis, IN). Oligo primers for PCR amplification of individual ORFs were synthesized in house using either an ABI synthesizer or the MerMade3 high throughput synthesizer [42]. The ORFV-specific primers were designed with a common 15 nucleotide sequence at the 5'ends carrying 5 deoxy-uracil (dU) bases. Distinct dU-containing sequences were designed for the forward and reverse primer sets. After PCR amplification, the dU stretches can be removed by treatment with uracil-DNA glycosylase (UDG), exposing defined 3' overhanging sequences. These single-stranded regions are designed to hybridize to complementary overhangs, similarly generated, on PCR products carrying a CMV promoter and hGH terminator [43] FITC-conjugated rat anti-I-Ad/I-Ed antibody (2G9), rat anti-I-Ak antibody (11-5.2) and unlabelled rat anti-CD16/CD32 (Fc block, 2.4G2) were purchased from Pharmingen (San Diego, CA). Rat IgG was purchased from Jackson Laboratories (West Grove, PA). Tissue Freezing Medium (Triangle Biomedical Sciences, Durham, NC) and Superfrost Plus microscope slides were obtained from Fisher Chemical Co (Norcross, GA). Vectashield fluorescence microscopy mounting medium was obtained from Vector Laboratories (Burlingame, CA).
Sequencing of ORFV D1701
The D1701 viral genomic (g)DNA was digested with BamHI and BglII then cloned into the pUC118 vector [44]. Random clones from this library were sequenced from both ends. Sequencing reads were assembled and gaps filled by walking into library clones with primers generated against newly determined sequence or by PCR amplification from intact ORFV genomic DNA. Sequence coverage varied from a minimum of 3 to over 50 reads per segment, which would be considered a relatively low resolution determination. The final assembly consisted of 134,040 nucleotides with 32 ambiguous positions. The terminal repeat sequences at the ends of the viral genome appear to be incomplete in the random library. These were not resolved because in all three currently sequenced Orf viruses there is at least 2400 bp from the reported ends to the first or last protein coding segment.
Production of ORFV genes and expression element components
The sequence of the 134 kb ORFV genome was searched for ORFs using the MacVector Genescan sequence analysis program. The parameters were set such that each ORF required a start and stop codon and encoded a protein with a minimum length of 90 amino acids (aa). The search included both strands, in all possible reading frames and did not exclude overlapping reading frames. This search yielded 288 possible ORFs. In addition, three full-length protein-coding sequences from the NZ-2 strain of ORFV virus were present in the NCBI sequence database that were similar to D1701 sequences that we had not annotated as ORFs. We prepared primers based on the NZ-2 sequences and amplified these regions of D1701 for inclusion in the library. ORFV-specific primer sets were designed and synthesized for all D1701 ORFs.
PCR products were obtained using the following conditions. Each 50 μl reaction contained 10 pmol of forward and reverse primer, 5 ng ORFV genomic DNA template, 10 mM Tris-HCl, pH 8.8, 50 mM potassium glutamate, 2 mM MgCl2, 0.2 mM dNTP, 1 M betaine and 2.5 U Taq polymerase. Each reaction was overlaid with mineral oil and incubated according to the following parameters in a Strategene Robocycler. An initial cycle consisted of 3 min denaturation at 95°C, 1 min annealing at 55°C and 1 min extension at 72°C. This was followed by 10 cycles of 1 min denaturation at 94°C, 1 min annealing at 55°C and 1 min extension at 72°C and 24 cycles of 1 min at 94°C, 1 min at 50°C and 1 min at 72°C. The PCR was finished with a 5 min extension at 72°C. All PCR products were assessed by electrophoresis in 1.2% agarose gels and DNA concentration was determined using a Hoeffer fluorometer and Hoechst dye and plasmid DNA as standard.
PCR amplification of each ORF from the genomic template yielded 257 specific products of the correct lengths and high yields. Amplification reaction products for the remaining ORFs did not meet these criteria so were not included in the library. The untested ORFs represent 12% of our annotated list and a similar proportion of the ORFV ORF homologs in the NCBI database (26 of 134). The library of 257 ORFV ORFs was randomly partitioned into 27 pools, each comprised of 8 to 10 ORFs. Batches of CMV promoter and hGH terminator fragments were generated by PCR with dU-carrying primers, as described for the ORFs. The downstream end sequence of the promoter element and the upstream end sequence of the terminator sequence were designed such that Uracil DNA-glycosylase (UDG) treatment would expose 3' single stranded stretches complementary to a 3' overhang on the corresponding ORF ends.
Preparation of ORFV LEE library
The quantitated and quality-confirmed ORF PCR products were pooled (400 ng of each) into groups of 8 to 10 and then purified using Qiaquick spin columns per supplied protocols (Qiagen, Inc., Valencia, CA). The purified DNA pools were eluted from the columns in 50 μl 10 mM Tris-HCl, pH 8.5. The CMV promoter and the hGH terminator elements were combined at an equal molar ratio with each of the 27 ORF pools (1 μg). UDG (O.5 unit) was added in a final volume of 50 μl 1X Buffer C (Promega Corp., Madison, WI). The samples were incubated at 37°C for at least 30 min, heated to 75°C for 15 min and mixed with an equal volume of 1 M KCl. After incubating an additional 15 min at 72°C, the components were allowed to anneal by slowly cooling to room temperature. The assembled LEEs were precipitated by the addition of 1/10 volume of sodium acetate and 2 volumes of ethanol. The precipitated DNA was collected by centrifugation and washed with 70% ethanol and allowed to dry before it was resuspended in water and loaded on gold for biolistic inoculation into abdominal skin [24].
Gene gun inoculation and skin sampling for library testing
The desired amount of total DNA was precipitated onto gold beads using a calcium-spermidine protocol previously described [6]. The DNA-gold complexes were then spotted onto kapton membranes and dried (2 μg total DNA per kapton). The complexes were loaded into cartridges and delivered into mouse abdominal skin that had been prepared by treatment with shears and Nair depilatory using a custom hand held gene gun (Rumsey-Loomis, Ithaca, NY). Each BALB/c mouse was inoculated (shot) with DNA-gold complexes at 4 to 6 distinct abdominal skin locations, and each shot carried a different LEE pool. Each inoculum was delivered into 3 different mice to serve as biological replicates for each pool test. The sites of inoculation were evaluated 4 days later. Mice were euthanized and the inoculation sites were isolated; visible gold particles facilitated relocation of shot sites. Skin segments were trimmed of surrounding tissue and then immersed in cryoprotective medium and quickly frozen in liquid nitrogen. Thin-sections (8 μm) were cut in a Leitz cryostat, picked up on Superfrost plus microscope slides and stored at -20°C for subsequent fixation and staining.
Frozen sections were equilibrated to room temperature for 10 min then fixed by immersion in cold acetone for 10 min. The fixed sections were washed 3 times in phosphate buffered saline pH 7.3 (PBS). Non-specific adherence of rat IgG was blocked by treatment of the sections with a cocktail of Fc block reagent and rat IgG in PBS. After a 30-min incubation at room temperature, this blocking solution was replaced by FITC-conjugated rat anti-I-Ad/I-Ed antibody (2G9) and incubation continued for 2 hours. The sections were washed 3 times in PBS and prepared for viewing by the addition of Vectashield mounting medium and a cover slip. The stained sections were viewed under white light to locate the area of gene gun inoculation marked by the presence of gold beads. These areas were then assessed for the presence of DC-like immune cells (FITC-labeled, anti-I-Ad/I-Ed antibody positive cells with DC morphology) under fluorescent microscopy.
Protein production, purification, and inoculations
The B2L ORF was amplified by PCR using D1701 strain DNA template and ligated in frame with a His6 tag-encoding sequence into the pQE60 vector (Qiagen Corp.) for expression and isolation of recombinant protein. The NZ-2 homolog was also amplified from genomic DNA (gift of Tobias Schlapp of Bayer Pharmaceuticals) and cloned into pQE60 for expression with a C-terminal 6-histidine (His6) tag. The yeast SRB4 gene was similarly cloned into pQE60 and produced as an irrelevant, similarly sized, His-tagged control protein. Plasmids were transformed into E. coli strain BL21, induced with 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) and cultured by standard protocols. The recombinant proteins were purified from bacterial extracts (B-Per, Pierce Chemical Co) using Ni-affinity chromatography and imidazole elution. Isolated proteins were dialyzed vs PBS + 10% glycerol. L and R designations are typically used to designate genomic positions of poxvirus genes, but not included in the nomenclature when the protein is indicated. Neither B2 nor the control protein samples contained detectable endotoxin by the amoebocyte lysate gelling assay. Hair was clipped from the site prior to protein injection. In initial tests, the recombinant protein was diluted with sterile PBS and injected intradermally (i.d.) into the abdomen at various concentrations ranging from 0.01-10 μg/ml, 50 μl/site. Inoculation sites were marked with India ink to facilitate subsequent tissue harvesting. A total dose of 50 ng/injection was found to be optimal for measuring cell accumulation. Tissues were harvested and assayed for DC recruitment as described following genetic inoculations.
Serum antibody assay
To measure antibody response levels, blood was collected from each mouse at indicated time-points post immunization and assayed for antigen-specific antibodies by ELISA. Assay plates were coated with HBsAg (Seradyn, Inc., Indianapolis, IA) in PBS (10 ng/ml; 50 μl/well) overnight at 4°C. After the antigen solution was removed, plates were washed 3 times, blocked with 1% BSA in wash buffer and incubated with diluted mouse sera. The plates were washed and antigen-bound antibodies were incubated with goat anti-mouse-Ig-HRP conjugate (BioRad, Inc.). Plates were washed again and developed with 100 μl TMB substrate solution. HRP reactions were stopped by addition of an equal volume of 0.25 N HCl and absorbance at 450 nm was determined for each well. These absorbance values were plotted against a standard curve. The standard curve was derived by measuring reactivity of a dilution series of know amounts (ng/ml) of a commercially available murine monoclonal anti-HBsAg antibody (Seradyn, Inc.).
Influenza challenge-protection assay
The influenza challenge experiment was conducted as part of a cooperative agreement with the University of New Mexico, supervised by C. Rick Lyons. Female BALB/c mice 4-6 weeks of age were purchased from Charles Rivers Laboratories (Wilmington, MA) and housed in a pathogen free environment at the University of New Mexico animal facility for at least 2 weeks before starting the experiments. All mice were maintained and cared for according to the guidelines of the institutional Animal Care and Use Committee. Mice (10 per group) were immunized twice (weeks 0 and 3) with plasmid encoding influenza hemagglutinin, pCMVi-HA (A/PR/8/34) [45]. Groups of 8 mice were immunized with one of four doses (0.4, 4 ng, 40 ng, 400 ng) of pCMVi-HA plasmid and either 1 μg control plasmid (pUC119), pCMVi-GMCSF, or pCMVi-B2L (D1701 strain). DNA doses are given per mouse; however, DNA was delivered in 2 shots of half-dose to each ear. For viral challenge (6 weeks post-boost) isofluorane-anesthetized mice were intranasally administered (in 50 μl PBS drops) 5 LD50 (= 2.4 × 104 50% egg-infecting dose, EID50) of mouse adapted influenza A/PR/8/34 (H1N1) (Advanced Biotechnology Inc.), as previously described [45]. The mice were monitored daily for 23 days post-challenge and mortality was recorded.
Tumor protection assay
The effect of B2L on tumor development was evaluated in a transplantable model of melanoma. On day 1, two groups of 20 C57BL/6 mice were subcutaneously (s.c.) injected with 5 × 104 of the murine melanoma B16-F10 cells (American Type Culture Collection, Rockville, MD). The B16-F10 cells were grown in Minimal Essential Medium (MEM; Gibco, Rockville, MD), supplemented with 2 mM L-glutamine, 25 mM HEPES, 2.2 mg/ml sodium bicarbonate containing 10% FBS (Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.11 mg/ml sodium pyruvate solution (all from Sigma, St. Louis, MO). The cells were cultured in a humidified, 5% CO2 atmosphere at 37°C. On day 2, the test group was biolistically administered pCMVi-B2L as 4 shots of gene gun microparticles carrying 1 μg of DNA each (Helios, BioRad, Inc.). Tumor diameters were measured with a micronomer in triplicate by two individuals at indicated days post tumor cell injection. At day 13, mice began to be euthanized due to overly large tumors. Survival was monitored until day 25, and then all survivors were sacrificed. This experiment was repeated in this C57BL/6 transplantable melanoma model and also conducted in a BALB/c transplantable breast tumor model of cancer.
Animal protocols
For the ORFV DNA and protein injections and for the influenza challenge-protection experiments six- to eight-week old female BALB/c mice were purchased from Charles River Laboratories, San Diego, CA. For the tumor -protection studies six- to eight-week old female C57BL/6 mice were purchased from Harlan Sprague Dawley, Indianapolis, IN. All mice were housed in a pathogen-free environment at either the University of Texas Southwestern Medical Center or the University of New Mexico. Mice were given food and water ad libitum, and all procedures were conducted under protocols approved by the Institutional Animal Care and Use Committees and in accordance with federal guidelines for animal experimentation.
Statistical methods
Unpaired Students t tests were used to compare the antibody levels measured in the ELISA experiments and also to compare the tumor sizes of treated and untreated mice. The relatively small sample sizes (5 to 20 mice per group) and two-way comparisons of these experiments indicate the appropriateness of the t test, although other less stringent methods might be applied. The arithmetic mean is calculated for each group and the standard deviation (SD) used as a measure of variance, these are indicated by error bars for each plot. For the survival experiments, Kaplan-Meier survival curves were derived and the associated parametric test provided by the PRISM software package (Graphpad).
List of abbreviations
linear expression element
- Interferon-γ:
α-1 antitrypsin
major histocompatibility
open-reading frame
dendritic cells
- HBsAg:
hepatits B virus surface antigen.
We wish to thank members of the Center for Biomedical Inventions and other colleagues, too. Laura Timares and Katherine Stemke-Hale for help in setting up the gene gun inoculations and LEEs. Jing Tan helped sequence the parapoxvirus genome and Harold Garner was a great help during the sequence assembling phase. Lei Zhang helped with the production and isolation of recombinant proteins. Adrian Webb assisted with the mice and John Shelton provided guidance for the histology assays. Rick Lyons at UNM assisted with the influenza challenge experiments.
This work was supported by DARPA grant number N00178-01-D3069.
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