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ABP's Port of Barry Solar Farm shortlisted for top green energy award
August 7, 2015
Left: ABP's Port of Barry Solar Farm
Associated British Ports (ABP) Port of Barry Solar Farm has been shortlisted for a Wales Green Energy award, in the Outstanding Renewable Energy Project Category, just over a month after it started generating energy.
The Welsh Government sponsored award will recognise a renewable energy project of an exceptionally high standard. The winner will be announced at the Wales Green Energy Awards Ceremony on Friday 6 November at the St David's Hotel, Cardiff.
The 4.5MWp PV solar farm project was developed by ABP to generate green energy and reduce the carbon emissions of its activities. The solar farm will contribute an annual reduction in the amount of carbon dioxide emissions produced by the port by around 2,000 tonnes – the weight of a small ship.
Matthew Kennerley, ABP Director, South Wales said: "It is an honour to be shortlisted for such a respected award alongside notable renewable energy projects. The news has been very rewarding for everyone involved. Its development forms part of ABP's strategy to grow its business whilst seeking to reduce the carbon footprint of its activities."
The solar farm has been built on brownfield port land. The redevelopment of this land is a positive alternative to the use of Greenfield land. The project was installed by the solar division of British Gas.
The energy generated will be used to power ABP's operations at the Port of Barry, which includes tenants and customers, any surplus energy will be exported to the National Grid.
ABP South Wales has invested £5 million into the port of Barry specifically to generate energy from clean sources. Renewable energy is the focus of investment at all five ABP South Wales Ports - Newport, Cardiff, Barry, Port Talbot and Swansea.
ABP is currently working on the second phase of the project that will see it potentially extended from 4.5MWp up to 5MWp. This is in addition to the two 250KWp rooftop solar panel systems that were installed at the Ports of Cardiff and Swansea in June 2015.
An official ceremony to open the Port of Barry Solar Farm Project will be held later in August.
The other finalists in the Outstanding Renewable Energy Project Category are RWE Innorgy UK's Gwynt y Môr offshore wind farm and the National Trust Wales Hafod y Lan renewable energy demonstration farm.
For more information visit www.renewableuk-cymru.com


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"It seems harmless enough. You get nose to nose with your dog and talk to it as it laps at your mouth and cheeks with its tongue, or you come home from work and bring your lips to your dog`s in a greeting to say hello.
It may feel like the ultimate display of affection, but when it comes to such kisses, experts caution: Beware of dogs.
Dr. Neilanjan Nandi, an assistant professor of medicine at Drexel University College of Medicine in Philadelphia, said in an email that most animals` mouths are host to an enormous oral microbiome of bacteria, viruses and yeast."
New York Times
I know that my pooch, Mandy, spends an inordinate amount of time licking her nether regions, and I realize that she has a penchant for digging up old bones in my backyard, but that doesn`t mean I try to prevent her from licking and kissing me when I come home from work.
A canine`s mouth may be teeming with bacteria and viruses, but a healthy person is unlikely to contract a disease from a dog.
Even if Mandy`s displays of affection made me sick, a physical illness would be a small price to pay for the psychological benefits of allowing her to love on me.
Dr. Robert Paul Reyes says: Don`t listen to the warnings of egghead scientists and physicians and let your pooch lick you to death.
Read More:
Follow Robert Paul Reyes on Twitter: http://twitter.com/robertpaulreyes
Photo Credit: Wikipedia

subset: commoncrawl


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We are not currently accepting new guest blog posts.
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Our massive catalogs of health insurance plans and health care facilities bring us large volumes of new site visitors who explore our blog content and community. This offers MedicareWire bloggers an excellent opportunity to expose their website to a new, expanding audience. As a MedicareWire.com contributor, you have an extensive profile where readers can find your social media, website and more information about you. You are also permitted one self-serving hyperlink (do-follow) to your own website, so long as it it not promoting a product.
If you are interested in becoming a contributor…
If you are interested in becoming a contributor, on a one-time or regular basis, here are some guidelines for success:
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Articles accepted for publication will likely be edited for brevity and clarity, and to conform to the MedicareWire style. In most cases, we will change your article's title, so you are encouraged to suggest several alternatives.


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The following circuit is a simple, cheap and easy build motorcycle alarm. The circuit just required 2 transistors to drive the relay the the relay act as a switch to activate the buzzer.
Any number of normally-open switches may possibly be applied. Fit the mercury switches to ensure that they close when the steering is moved or when the bike is lifted off its side-stand or pushed forward off its centre-stand. Use micro-switches to secure removable panels as well as the lids of panniers and so on. Although at the very leastonce again – the alarm will reset. How lengthy it takes to switch off depends upon the characteristics of the actual parts you have utilized. You are able to adjust the time to suit your requirements by changing the value of C1 and/or R3.
Motorcycle Alarm Simple PCB Connection
The circuit board and switches need to be protected from the elements. Dampness or condensation will trigger malfunction. With out its terminal blocks – the board is small. Ideally, you need to attempt to locate a siren with sufficient spare space inside to accommodate it. Fit a 1-amp in-line fuse as close as achievable to the power source. This is Extremely Crucial. The fuse is there to secure the wiring – not the circuit board. Rather than utilizing a key-switch you'll be able to use a hidden switch; or you could use the normally-closed contacts of a tiny relay. Wire the relay coil to ensure that it is energized whilst the ignition is on. Then each and every time you turn the ignition off – the alarm will set itself.
When it is not sounding – the circuit uses practically no present. This need to make it helpful in other circumstances. For instance, powered by dry batteries and using the relay and siren voltages to suit, it might be fitted inside a personal computer or anything else that is in danger of becoming picked up and carried away. The low standby electric current and automatic reset indicates that for this sort of application an external on/off switch might not be essential.
Easy build motorcycle alarm circuit source: www.zen22142.zen.co.uk


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toys, books, games and iPad/iPhone apps tested and approved by a pediatric occupational therapist and all the children she works with!
Monday, August 29, 2016
This Is Just The Beginning of Your Story
In my years of working with school age children, I have come to realize that this ability to come up with and write stories can be quite difficult for children with learning difficulties. The very thought of having to come up with a whole story can cause them to become incredibly anxious. However, if you help them start the story, they are more likely to be able to come up with ideas on how to end the story. I have used a lot of different things when working with some of my kids to help them including Rory's Story Cubes (these are really great but sometimes just not enough for some of my kids who are really struggling). Recently, while out shopping with my daughter, I discovered something even more wonderful: And Then Story Starters by Live Inspired. In short, And Then Story Starters are a set of 20 illustrated cards with the beginning of a story. After listening to the beginning of the story, it is up to the child to come up with how the story will end. There is no right or wrong way to end the stories which helps children feel more comfortable with telling their story. With my daughter, we have done a few different things which have all been fun. The simplest idea was we would read her the beginning of the story and then we would take turns telling our ending. At times, we had to push her to come up with more than just a sentence and to add details. After a few tries, we didn't have to prompt her quite as much. The other thing we did was read the story to her, give her writing utensils and a blank piece of paper and let her draw out the way her story would end. She enjoyed both of the ways, but she is really into drawing and I felt like she was more creative and her ideas were meatier when she was able to draw it out and then tell us her story.
As a parent, I love finding products that will inspire and encourage creativity in my daughter. But since I am a therapist, I am always looking at the possible therapeutic benefits of any of those products. In addition to what I have already mentioned above, And Then Story Starters are perfect for working on the following occupational therapy goals:
Improve Executive Functioning Skills-this is such a great activity to work on a variety of executive functioning skills. It doesn't matter if the children are just verbally telling you the story or if they are writing it out and illustrating it, the skills being worked on are the same. This activity requires the ability to focus and attend and organize their thoughts and ideas. They need to be able to prioritize what It also requires a child to use working memory (the ability to hold information presented to them and use it to complete the task at hand).
Improve Graphomotor Skills-depending on the age and goals you have for the children you are working with, you can have the kids write out their stories. For older kids, I would grab a composition book and have them copy the beginning part of the story that's been provided. Once they are done with that, they can write the rest of their story. Another great thing you can do is have kids illustrate the end of the story. You can get a set of blank storybooks (I like these by Mead because many of the kids I work with need lines to write in and this one has the right size lines and a space for drawing a picture) and use it just for their story starter cards.
Improve Creativity/Imagination Skills-as I have already mentioned, not everyone is born with a creative side and the ability to use their imagination. However, sometimes if they are given a little boost, they are able to tap into their creative side. And Then Story Starters are a great tool to give those kids who need a push/some inspiration. The hope is that once they build some confidence using these cards, they will be able to come up with their own thoughts and ideas when writing stories.
Not only are these cards great for use in an occupational therapy setting, they are perfect for educators and speech and language therapists to use. Wouldn't it be fun for a teacher to give a class of students the same story and see how each child made the story their own? For speech therapists, they can play a game where they can go back and forth in adding to the story (be sure to write it down so they can show their parents!)....helps with conversational skills, focus/attention and being flexible with thoughts and ideas.
I'm getting excited about using the And Then Story Starters at work and have already been recommending them to family and friends for their children. In a day and time where so much of us (myself included) rely on technology and electronics, it's nice to have an option that is more hands on. Not only is this a fabulous tool for all of the reasons I have mentioned above, it is a great way for parents and kids to work together and create. The best thing about the And Then Story Starters is that you can use them over and over again coming up with different fun and creative stories each time.
If you have any other great products like this you have used in the past, I would love to hear about them and use them with my kids at work and my own child at home. It's great to have a fun way to inspire imagination and creativity and improve a child's self-esteem and confidence. I am always a click away and love hearing from all of you!
Posted by Meghan Corridan at 10:17 AM 1 comment:
Labels: confidence, creativity, drawing, executive functioning, focus, graphomotor, handwriting, imagination, live inspired, school age, self esteem, speech and language, story starters
Wednesday, August 3, 2016
Hatch N' Some Fun!
A couple of weeks ago, I was searching for some Finding Dory toys for work. It's all the kids at work are talking about this summer and I wanted to see what I could find. On my hunt, I found these things called Hatch N' Heroes. I had never heard of them but I had to test them out as they seemed like the most perfect occupational therapy toy/tool. Simply put, Hatch N Heroes are egg shaped toys that can transform into different characters. Remembering how popular any transformer toys were when I was growing up, I felt like it would be a fun thing to try with the kids. It was especially exciting to find characters like Nemo, Dory, Lightning McQueen, Woody and a bunch of other popular characters.
These simple and affordable toys are a great tool to work on improving fine motor and manipulation skills. Some kids require some more verbal prompting and assistance from me, but I've been impressed with how kids are eager to figure out this "puzzle" So far, all the kids are loving them but some of the younger kids require verbal prompting and some hand over hand assistance to successfully turn the egg into the character.
In addition to being really cute, Hatch N Heroes are great for working on a bunch of occupational therapy goals. Some of the skills that can be worked on when using them are:
Improve Fine Motor/Manipulation Skills-as already mentioned, these are a great toy to work on fine motor skills. The eggs are small, so the kids are forced to use a fine pincer grasp when pulling out some of the pieces.
Improve Bilateral Coordination Skills-while I have seen kids try to play with them with one hand, they have quickly discovered on their own that it is a lot easier and takes a lot less time when they use one hand to hold the egg and the other one to do all the work.
Improve Executive Functioning Skills-great for working on improving focus and attention, organizational skills and problem solving. Kids need to be patient while they try and figure out how you move the pieces to transform the egg into the character. For some of my older kids, I see if they can turn the egg into the character without my assistance. For the younger kids, they tend it's a great way to work on following verbal directions.
Improve Graphomotor Skills-for some of my older kids who are working on writing, I use these Hatch N Heroes in an obstacle course where the final step is writing the name of the character they just rescued from the top of the mountain. There is something about all the big body work that takes place prior to the actual handwriting that helps the kids get more organized and focused for the handwriting task. And let's be honest....if a kid gets to be a superhero and save anything, they will be way more open to any kind of handwriting activity!
As you can see, these little toys are packed with lots of benefits for kids. I like that they are small and can be carried around easily in a bag for traveling. There are no batteries required, so you don't have to worry about them making any noise other than the sounds that kids will make when they are acting out scenes with the characters (this has been happening a lot for me!). One thing to keep in mind is that they are plastic toys with a little moving pieces and if one of the pieces is bent the wrong way or pulled too hard, it might fall off. The good news is that they are easy to put back on.
So, if you are looking for something new and exciting for your kids, be sure to keep your eyes open for these toys. These are the kinds of toys that you will want to play with with your kids! I have been searching every local toy store, Toys R Us and Amazon.com looking for any new ones.
Posted by Meghan Corridan at 6:14 AM 2 comments:
Labels: attention, bilateral coordination, executive functioning, eye hand coordination, fine motor, focus, graphomotor, handwriting, organizational skills, play, problem solving, toys
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Introducing are a nine-piece band that play DJ Shadow's Endtroducing (released in 1996, the first album to be composed entirely of samples - cheers, the Guinness Book of Records) live, recreating every drum loop, esoteric vocal sample and the famous organ solo with actual instruments. An impossible task? They came to the Hare and Hounds in King's Heath last Friday to show Birmingham that it can be done.
The opening of a call for a round of applause for the DJ himself was a sign of the good humour with which the band approached their task. Despite looking like a collection of detail-loving music obsessives (and on this blog, that's not an insult) they weren't hung up on the subject matter and played with laid-back flair. This meant that the jazzier side of the record could come through and the odd little vocal parts, with the potential to be twee and annoying, passed off as geekily cool - especially the "one of them has eyes as big as Jolly Ranchers. Beautiful girl" bit on the untitled sixth track.
There was a special mention for drummer Mike Reed at the beginning of 'Stem/Long Stem', and he really deserved it. It would be clear to anyone with more than a passing acquaintance with the album that this is a project that lives or dies on the quality of the rhythm section. Fortunately Introducing have nailed it; Reed was amazing, as was bassist Rob Pollard, driving the songs forward with technical brilliance and exactly the right vibe.
The set sheds new light on the differences between live and recorded music. I listened to the parts more intently, discovering previously unnoticed details like little repeated sections and cool beats. I also enjoyed the sparser sections far more than I ever have when listening to the record.
Live music, as it's no real surprise to be reminded, holds the attention more powerfully and has a sublime energy lacking from anything tied down to endless repetitions from a medium of your choice. There's something about a group of people sharing a room with you and hearing your responses to what they're doing... people fashioning something that wouldn't exist without them, and will never exist again in the same form beyond the moments that you are sharing... a feeling amplified through the uncanny experience of music both totally familiar and new and exciting... these are the reason that make what Introducing are doing something pretty special.


text: Like many people, I've taken away a lot of lessons from my working life. One of the first ones I learned was the more work you do, the more you get. My elder niece is struggling with that realization these days. Another one I learned came later in my career: having all the answers (or… Read more »


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American Suzuki Association's Suzis on the Rocks.
Rubicon Bash -Linda Bradshaw
One potentially serious roll was caught on film is a serious of six incredible shots the following is a roll that occurred on the 'big sluice' heading into Rubicon springs. This is a corner that many people in short wheel base vehicles have problems with.
Shane starting to drop off the ledge.
Are we seeing a balancing act?
The roll begins with the driver's front taking first hit.
The vehicle continues over.
and rests on it's side.
It only take a few people to right a samurai.
The loosely organized scheduling and laid back atmosphere at this event allowed for a great deal of freedom for exploring while still enjoying the great camaraderie. As always fun was had by all that attended.


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Frequently Asked Questions
"If I have 4 passengers in my group going to Park City does that mean the price is $100 total or is it $100 per person?" The total fare is $100; NOT $100 per person. In addition, there are no taxes or any other fees added to your fare.
"Do you offer door to door service?" Yes. We take you to your desired location.
"How long do I have to wait at the airport until the shuttle arrives?" When you have notified us that you are ready for pick-up, it will take your driver 1-3 minutes to arrive at your assigned pick-up location.
"What kind of shuttles do you have?" We operate in very nice/updated 6 passenger minivans. Currently, all of our vans are Toyota Sienna's. We also have 11 passenger Ford Transit's and SUV's upon request.
"What does a Private Shuttle mean?" Private Shuttle means that you and your group are not required to share the shuttle van with any other traveling passengers.
"Do I get to pick the time for the shuttle to pick me up?" Yes.
"Can I have the shuttle make additional stops for grocery, ski rental & tickets, etc?" Yes. Please let us know in advance of your reservation. Stops are $10 extra + $1/Minute wait time after 10 minutes of waiting.
"When does your shuttle operate?" 24 hours a day and 365 days of the year.
"How much time in advance do I need to be at the airport to catch my flight?" We recommend being at the airport at least 1 1/2 hours prior to your flight departure.
"How much time in advance should I book my reservation with you ?" Whenever you have your flight information, please book with us. We prefer advanced reservations.
"How long does it take to get from the Salt Lake City Airport to Park City and Deer Valley area?" Park City: 30 - 45 minutes. Deer Valley: 45 - 55 minutes. If road conditions are clear. Weather delays can range from 10 - 30 minutes. There is usually no traffic delays.
"If I am staying at a hotel in Salt Lake City, can you pick me up at the hotel and take me to Park City ? Yes, choose the option (Non-Airport: Point to Point) when booking online.
"Do you offer transportation from Park City to destinations in Salt Lake City other than the airport?" Yes.
"If I am traveling with skis and luggage, will there be a problem fitting everything into the shuttle?" No, there will not be a problem. If you have extra luggage and skis, please let us know in advance so we can send the right shuttle van for you. Extra luggage would be more than 2 bags per person. Skis would be more than 4 per group.
"Do you offer children child car seats (Booster/Toddler/Infant). Will there be an additional charge?" Yes, we offer both seats free of charge. Please let us know in advance so that we can have them ready for you in your shuttle.
park city shuttle, transportation to park city, shuttle to park city, my shuttle park city utah
Copyright 2013. My Shuttle. All rights reserved.
(435) 602-4710


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CD Duplication
We can archive your files to CD and duplicate as many copies as you need. At larger quantities, we can also custom-print your CDs with personalized images.
The Productivity Center
7833 Hwy 72 West, Suite B • Madison, AL 35758
Phone: 256-837-COPY (2679) • Fax: 256-837-9028
E-mail: firstname.lastname@example.com
Copyright © 2019


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Native title determination
Form 17NT (PDF 100 KB)
Dealing type - NT
Legislation - s12C Real Property Act 1900, s199(1) Native Title Act 1993 (Cth)
Stamp duty - not required
Certificate of Title - not required
NOS form - not required
Standard form of Caveat - does not prevent registration.
Priority Notice noted on the Register - see Priority Notice page.
(A) The reference to title for the affected land must be stated.
Where part only of the land in the reference to title is affeced the part must be described.
(C) The applicant must be The National Native Title Tribunal.
(D) The number given by the Tribunal to the application for determination (National Native Title Tribunal Determination Application Number) and the Federal Court number relating to the determination (Federal Court Number) must be stated.
(E) A copy of the determination certified by a solicitor, barrister or justice of the peace to be a true copy must be annexed to the application.
(F) The application must be signed by an authorised officer of the Tribunal. The name and capacity must be stated. The execution must be witnessed.
Staff processing information
Refer to SD31
Registration procedure
Where native title has been determined to exist
Where the native title has been extinguished


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1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 2010
Even though the UK glam scene was fading, Wax Trax continued to focus on what was breaking out of England. In January of '78 the Sex Pistols toured the Southern US states for the first time after their release "Never Mind The Bollocks". Since most touring bands didn't stop in Denver at the time, Jim & Dannie didn't think twice about traveling to the Texas show. This was another recurring Jim & Dannie theme that would carry over when they solidified the label….If the bands they loved didn't come to them…They would go to the bands.
Realizing the need to grow, the store moves to a larger location, on Washington street (Where it still is today)
later in the year, Jim & Dannie took advantage of an opportunity, and the Wax Trax record store in Denver is sold to Dave Stidman and Duane Davis. With the money from the sale, Jim and Dannie head east to Chicago to open the store on 2449 N. Lincoln that would become legend.


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Mozambique Fulbrighter
Inocencio Zandamela
"I believe I am the first deaf person from Mozambique to go to South Africa and earn an undergraduate degree, come to the United States and come home with a graduate degree," says Inocencio Zandamela, Fulbright Student, teacher, advocate, father of four and recipient of a master's in educational psychology from Saint Rose.
A high-profile student, Zandamela attended the after-school Help Yourself Academy and led sign language chats on campus, bridging the hearing and non-hearing worlds in ways that were not possible back home in Mozambique. Growing up there deaf, he stood little chance of gaining a decent education, let alone a meaningful career. So he left, to study at the University of Witwatersrand in Johannesburg, South Africa.
Zandamela's goal, then as now: transform deaf education at home. It's a drive that brought him to Saint Rose on a Fulbright grant to earn his master's in December."
-by Jane Gottlieb, The College of Saint Rose


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When creating a new project, you can choose to schedule the project from a start date or a finish date, and you can define specific file properties to help you organize or find your project.
Create a new project
After you have created a new project, you can save it locally or you can save it as an enterprise project on Project Server. If you want to save the project as an enterprise project, we recommend connecting to Project Server before creating the project. New enterprise projects use the enterprise global template that your organization has created, by default.
Click New .
Tip: You can also create a new project by clicking New on the File menu. In the task pane, under New, click Blank Project.
On the Project menu, click Project Information.
To schedule a project from the start date, type or select the date that you want your project to start in the Start date box.
To schedule a project from the finish date, click Project Finish Date in the Schedule from box, and then type or select the date that you want your project to finish in the Finish date box.
To include any enterprise custom fields in the project, review the list of fields in the Enterprise Custom Fields section, click the Value field for a custom field, and then select the value.
If your organization has defined any required enterprise custom fields, indicated by an asterisk (*) after the field name, you must enter values for them. If you don't enter values for required fields, then you are unable to save the project. If you don't know the values to enter for any custom field, check with your project manager or server administrator.
You can also use the Project Guide to help you through the process of setting up a new project. Find links to more information about the Project Guide in the See also section.
If you need to find out how late you can start a project and still finish by a particular date, you can choose to schedule the project from the finish date. However, when work begins on the project, we recommend that you change this setting to schedule from the start date.
If your plans change, you can change this initial project information at any time by clicking Project Information on the Project menu. However, if you have actual data, such as actual start or finish dates, applied to tasks, the tasks will not be rescheduled based on the updated project information. In addition, if you have constraints on tasks, moving the project's start or finish date won't necessarily move all tasks relative to the new date. Find links to more information about moving a project's start date in the See also section.
If you save this project locally, and then decide to save it as an enterprise project on Project Server at a later time, the project will be updated with settings from the enterprise global template at that time. Your project may be updated with different views, tables, fields, and other items. In addition, any formatting that is inconsistent with the enterprise global template will be lost. If you have created items in your project that have the same names as items in the enterprise global template, you will be prompted to rename them.
Define file properties for your project
To help you organize or find your projects, you can enter file properties for them, such as a descriptive title, a subject, a project manager, or a comment.
To enter basic file properties for the active project, on the File menu, click Properties, and then click the Summary tab.
Type the relevant project information in the file properties boxes.
To add custom file properties for the active project, click the Custom tab.
Type the relevant information in the Name, Type, and Value boxes, and then click Add.
Note: For printing purposes, you can also add these project details to the header, footer, or legend of a project view or report.


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The Beshara Lecture 2014: Jane Carroll
A Heart Capable of Every Form: Atheism, Agnosticism and Belief
"My heart has become capable of every form: it is a pasture for gazelles and a convent
for Christian monks and a temple for idols and the pilgrim's Ka'ba and the tables of the Tora
and the book of the Koran.
I follow the religion of love: whatever way love's camels take, that is my religion and my faith."
– Ibn 'Arabi, Tarjuman al-Ashwaq
When I was first asked to give this talk I immediately came up with the theme which I had been thinking about for some time. I have no credentials to talk on these subjects, I am not a philosopher or theologian or historian, and neither am I, in any conventional sense of the term, a believer. I am not a member of a religion nor a strict follower of a prophetic tradition. I am interested in how these terms shift meaning depending on the context in which they are used and especially in the experience of looking at the world and ourselves through each of these lenses.
The interest is purely personal. For the past 10 years of so I have joined a group of friends for weekly silent meditation. It was probably at least 5 years before any of us asked each other how it was we had come to meditation. After describing a little of my experience at the Beshara School one of my friends said to me, 'I wish I were a believer'. I was quite taken aback as I would not have defined myself that way. Not long after, I read the best selling books on atheism by Richard Dawkins and Christopher Hitchens with a different group of friends and found I was one of a few in the room who did not clearly identify as an atheist. For the record I don't think of myself as an agnostic either. So where does that leave me?
Apparently with an increasing number of the population of both Great Britain and the United States who identify as 'unaffiliated' – the fastest growing category in both the 2011 British census and a recent U.S. Pew Research poll on religion (which includes the religious and secular unaffiliated).I personally would in no way equate unaffiliated with uncommitted and would state my religious path, if there were such a category, as wanting to see things how they really are, regardless of how they might be described by another. And I might also say, if there were room enough on the form, that although atheism, agnosticism and belief appear to be in contradiction with each other, at times each one takes on a lens which seems appropriate through which to look at the world.
We are all of us in the 21st century profoundly impacted by 'modern' science and what might be called its artistic response: Modernism. The project of modernism, so often derided for bringing about moral relativity in a godless world, has been hugely helpful at sweeping away illusion and breaking through perceptual barriers. The arts, at their best, do such a good job of conveying meaning without being definitive. So I wanted to explore this subject with the help of a few favourite paintings, and a little through the lens of science.
Last year the British Museum mounted the exhibition Ice Age Art. This was described as The Arrival of the Modern Mind and showcased a number of exquisite sculptures (mostly found in Northern Europe) from as far back as 40,000 years, We know nothing at all of the beliefs, rituals, social orders, gods, goddesses, spirits of the people who made these but what was so moving in gazing at them was how obviously they communicated a sense of transcendence. The curators made clear that each of the pieces would have taken months to make, given the tools available, and that therefore they must have held great significance. But the pieces themselves, without this information, conveyed powerfully their purpose to connect with a higher order, the way all sacred art does (or I should say all good art) and, at least while I was there, there was a palpable kind of reverence on the part of the attendees to be in the presence of these far, far distant human relatives' search for meaning.
Interestingly the curators chose to show these works alongside art by Henry Moore, Mondrian, Matisse and others, artists of the modern movement who consciously broke with the tradition of explaining the context of images so the two periods of art were in easy dialogue with each other – one had no known spiritual context, the other deliberately avoided it – and, to quote the catalogue, each showed 'the fundamental human desire to communicate and make art as a way of understanding ourselves and our place in the world'.
Over the past 40,000 years of course we have evidence of human belief in innumerable forms – statues, paintings, writings, buildings – from throughout the world. We know more or less about the details of these beliefs but the objects themselves convey a transcendent and compelling meaning – why else do we unearth, collect, display and admire them so?
For more than 1,000 years of course the spiritual context of European art was known and shared: painting, literature, music, architecture have been deeply entwined with the story of Christianity. In its various forms, and not withstanding the major convulsions which have taken place within it, it remained for over a millennium the imaginative presence within which the vast majority of Europeans lived. Judaism was severely marginalized, there was almost no cultural contact with Islam since the 12th century, Buddhism, Hinduism and other religions, were known only to a very few. Most Europeans, up until the 20th century would have been baptized, married and buried within one or other Christian church. The Old and New Testaments provided the images and stories, the sense of beginning and end, the moral framework which all shared.
It is worth remembering this when we look back on the great crisis of faith which began to shatter the Christian narrative with the dramatic scientific discoveries of the 19th century.
In the late 18th century, the Scottish geologist James Hutton, after careful observation of the sedimentary layers of earth on his Berwickshire Farm and of the Salisbury Crags in Edinburgh began to overturn the prevailing theories of the Neptunists who had been seeking to find evidence in geology for Noah's flood. Hutton described a universe very different from the Biblical cosmos: one formed by a continuous cycle in which rocks and soil are washed into the sea, compacted into bedrock, forced up to the surface by volcanic processes, and eventually worn away into sediment once again. "The result, therefore, of this physical enquiry," Hutton concluded, "is that we find no vestige of a beginning, no prospect of an end." Hutton was accused of being an atheist, of contradicting biblical truth.
Hutton's theories strongly influenced Darwin who after a close study of the Galapagos finches' beaks, among other things, developed his Theory of Evolution and we all know how unsettling that was. These, with other discoveries of the period were of course not the first to have upended prevailing beliefs – Galileo providing a prime example – but the data driven discoveries of the 19thcentury began, in Europe and America, a seismic undermining of the creation story and something began to unravel. The physical world would no longer be investigated for evidence of religious truth: the reverse, belief was to be put aside to allow for unimpeded observation. The approach the scientist would adopt, while observing data, would be atheist, whatever their private belief, if they had one. Scientific objectivity requiring 'the view from nowhere' as the philosopher Thomas Nagel has called it, however unattainable, has been the accepted approach ever since. We attempt to allow the world to tell us what it is, rather than look to it for proof of pre-existing beliefs.
We can try to imagine what it would have been like at the time to absorb these bombshells. Our own grandparents and great-grandparents had to confront what these meant for their own belief. The majority of Christians and Jews now in Europe and even in America, (in spite of the flourishing Creation Museum in Kentucky which 'disproves' Darwin), do not find that the theory of evolution makes biblical truth meaningless. It does not shatter their faith and they have found a variety of ways to live with both stories. But in 1851, coincidentally the same year that Darwin stopped going to church, Matthew Arnold expressed the shattering loss of faith in his mournful elegy On Dover Beach which ends with the lines:
"Ah, love, let us be true
To one another! for the world, which seems
To lie before us like a land of dreams,
So various, so beautiful, so new,
Hath really neither joy, nor love, nor light,
Nor certitude, nor peace, nor help for pain;
And we are here as on a darkling plain
Swept with confused alarms of struggle and flight,
Where ignorant armies clash by night."
I first came across the poem as a teenager and loved it. I was not a brooding melancholic and did not at all believe that the world no longer held joy but in the way poetry can get its hooks into you and carry meanings beyond words I took to this one and have come back to it over the years, giving it another go as it were. I now deeply appreciate his evocation of the facing of the loss of everything felt dear, everything he thought he knew. And of course that: 'Ah love let us be true to one another' makes all the difference because if love and truth remain, we can let go of the rest.
This loss of faith was the preamble to Modernism: the developments in art, architecture, literature, music, dance, theatre, which rejected the certainties of enlightenment thinking and religious belief and which the world found so shocking. We can trace this briefly (and not at all comprehensively) by looking at it evolving in a few pieces of art from the last 120 years or so. Starting with the Impressionists who explored light, colour, contour and movement rather than the object depicted. These were radicals in their time, violating the rules of academic painting, working outdoors, looking to capture the momentary and transient effects of sunlight. Denied a place at the Paris Salon, these paintings from their first Exhibition in 1874 caused an uproar. What is so interesting is how easy it is to love these now – so much so they are sometimes snobbishly dismissed – and we have to struggle to recapture the drama of that first startling view. In the same way that the Theory of Evolution does not rattle most of us now, these paintings have happily settled into our lives.
A few years later Van Gogh, now apparently the most popular painter in the world, struggled alone to find, as he said in a letter to his brother Theo, 'the high yellow' a search which at the time only Theo and Gauguin might have understood, but which we can recognize so easily now as a transcendent quest. Now that yellow is ubiquitous, and again, we have to struggle to see past the endless reproductions to embrace that 'height' he was searching for.
By the time we reach Mondrian we are moving into abstraction. 1926 (10 years after the General Theory of Relativity was published) all representational forms are abandoned. 'Today one is tired of the dogmas of the past and of truths once accepted but successfully jettisoned. One realizes more and more the relativity of everything, and therefore one tends to reject the idea of fixed laws, of a single truth….The important task of all art is to destroy the static equilibrium by establishing a dynamic one. Non-figurative art demands the destruction of a particular form and the construction of a rhythm of mutual relations.' (Piet Mondrian, Plastic Art and Pure Plastic Art 1937)
These artists are on fire, on a mission to open eyes to light, colour, line unencumbered by the forms to which they are habitually attached. They are not using art to tell a story or establish a belief but using it to ask questions and provoke insight. Of course, all great art does this and has always done so but in modernism it is done overtly.
Alexander Calder wrote in 1948: 'Just as one can compose colours and forms, so one can compose motions.'
Jean Paul Sartre described his work: 'Although Calder has not sought to imitate anything, – his one aim is to create chords and cadences of unknown movements – his mobiles are the tangible symbol of nature….. that nature of which we shall never know whether it is the blind sequence of causes and effects or the timid, endlessly deferred, rumpled and ruffled unfolding of an Idea.'
I think this is a masterful explanation of the ability of art to convey two different possibilities in a single piece: 'little private celebrations' as Sartre called them. Is the world around us a random sequence of events or is it covering up a grand Idea (note the capital I) of nature?
These are images chosen specifically of artists whose work I have seen in the past year. I find myself, with it seems an increasing number of others, visiting museums for sheer pleasure, engagement and even enlightenment. According to a recent article in the Economist headlined Temples of Delight museum building and museum attendance has increased rapidly over the past 30 years as church attendance has declined. In 2012 American museums received 850 million visitors – more than all the big league sporting events and theme parks combined. Half the adult population in England visited a museum last year. China is building literally thousands of new museums, several serious new museums have appeared in the Gulf states. 20th century Modern art is attracting an increasing following: 5 million visited Tate Modern last year, MOMA in New York is one of the most visited museums in America. The article suggests people are looking to museums for guidance but not for answers and that museum directors are increasingly aware that they need to engage with the visitors rather than to lecture them.
The Los Angeles County museum this year mounted one of three major exhibitions of recent work by James Turrell, an artist who has spent most of his life working directly with light alone: trying to eliminate even the forms light falls on so we can see ourselves seeing. If the light falls on an object he says, we see the object and our previous perception prevents us seeing things as they really are. He wants to try to eliminate 'prejudiced seeing'. His grandest project harks right back to our earliest ancestors, creating a naked-eye observatory at Roden Crater in the Arizona desert, not this time to provide answers to our place in the universe but to allow a profound and personal engagement with the sky, sun, moon and stars from a private position of one's own.
We have been educated by these modernists, whether we are aware of it or not, and we owe a huge debt to them. Time and exposure has not only made it easy for us to love Impressionism but also to be comfortable with not knowing what later more inscrutable works of art mean. We have been encouraged to open our eyes and let the light in and not require an answer. And this extends throughout the arts: musicians threw out melody and fixed rhythms so we could hear sounds in a different way, architects broke with traditional orders so that we could re-experience space and light, dancers sought movement from an internal core rather than external order, writers loosened narrative and plot go so they could examine the unresolved and the absurd and the sheer complexity of words.
It would be too glib to call the modern scientific approach atheistic or modernist art agnostic, but both do require a 'putting aside of belief' to increase perception and knowledge.
'On or about December 1910 human character changed,' Virginia Woolf stated, hyperbolically. Relations between 'masters and servants, husbands and wives, parents and children' shifted, she wrote, 'and when human relations change there is at the same time a change in religion, conduct, politics and literature.'
I have no idea what happened specifically in December 1910 to Virginia Wolf but she was clearly on to something because along with dramatic scientific advances and radical artistic upheavals there has clearly been, in the West at least, major changes in human relations since then. If the 20thcentury witnessed genocide on a scale never seen before it also, in counterpoint, produced the Universal Declaration of Human Rights and an ever-expanding understanding of what this might mean. And if religion or belief needed to be put aside for scientific enquiry and artistic breakthroughs it has often had to be circumvented in issues concerning morality and social justice. The civil rights movement of course was strongly rooted in the Black Church though it is also true that the Bible had given plenty of textual cover condoning slavery and apartheid. Liberation theology, which mined Catholic teaching to understand the roots of poverty and shift the consciousness of the poor, was also roundly silenced by the Catholic Church. The women's movement and the gay rights movements have had to work very hard to overcome, where they have done so, religious doctrine. It is interesting to note that recent surveys in America indicate quite clearly that the rapid increase in acceptance of gay relationships, within even conservative religious communities, has come about through personal contact with gays. Love of real human beings can over and over again transcend religious doctrine. Knowledge of what is, precedes belief in what should be.
There is these days no shortage of critique of religion. Atheism and agnosticism, quietly part of the critical dialogue for the past 150 years or so have burst out of the closet, so to speak, in the last 20 years with a stream of books, articles, debates between atheists and believers, lectures on the incompatibility of science with religion. What is new as Adam Gopnik in the New Yorker wrote recently is 'a tone frankly contemptuous of religion'. The aforementioned books by Dawkins and Hitchens I personally found irritating, simultaneously barking up the wrong tree and flogging a dead horse. They each trotted out well-known examples of the evil done in the name of religion and the incomprehensibility of religious tales and supernatural doings. And they both managed to dismiss 40,000 years of human engagement with transcendence – by almost all of our human relatives – as delusional. Boiled down though, the principle point of these best-selling atheists would seem to be that the God of religion is created in the imagination of the believer which then becomes the authority figure. This subjects the believer to dogmatism, and strips them of responsibility for their own actions.
Ibn 'Arabi from whose magnificent book of poems, The Tarjuman al Ashwaq the title of this talk is taken, would entirely aggree. Ibn 'Arabi, the devout muslim believer and devoted follower of the prophet Muhammed, steeped in the revealed text of the Quran, said (agreeing with these opinionated atheists) 'God conforms himself to the belief of his servant'.
'Whoever believes that (God) is like such and such, He appears to him in the form of his belief.' Fut. III.411.26.
'Take care not to be tied by any particular belief ('aqd) while denying all others, for much good would escape you – in fact, knowledge of how things are would evade you.'
Truth reveals itself in the imagination, says Ibn 'Arabi – this is the realm where forms take on meaning and meanings descend into forms. Many scientists and artists would I think agree with this. But if the access to the imagination is filtered through pre-existing belief or if it is coloured by projections or fantasies or hopes or fears, or history, truth cannot appear as it is.
It is precisely because we create God (or Truth) in our belief that Ibn 'Arabi demands an emptying of belief, a removal of conditioning so that we can see the truth as it is, not how we think it should be. The responsibility of the believer is in keeping the heart pure, in NOT attaching to belief. And so long as we are unable to do this, we will be serving the God of our own projection and therefore under an authority which is not real. This applies to everyone, however they define their belief or their lack of belief, or their uncertainty. However they think the world is, it appears to them that way and they have difficulty seeing it another way.
The universe is annihilated and recreated at every moment, claims Ibn 'Arabi. Breathing out and breathing in. Meaning appears and then is erased and then appears again in another form. This finds resonance in the profession of faith of the Muslim: 'there is no God, but God'. In the Tarjuman al Ashwaq he extols the beauty of the beloved in the meeting around the camp fire in the desert and then laments her departure in the morning with the camel trains, leaving nothing but traces in the sand. The beauty remains only in the heart. This is not a million miles away from Matthiew Arnold's lament On Dover Beach, in spite of all he says is lost, his love remains and truth is still the object.
So what then is the belief of a heart capable of every form?
If we leave aside the origin stories, the promises of life after death, the miracles of the chosen ones, the prescriptions for right behavior and the punishments for wrong, there seems to be an essential element to all belief which is that the quest for truth, beauty, goodness is reciprocated. Something out there responds to something in us or something deep inside us responds to our requests for help and answers.
To assert that this is not the case seems as nonsensical to me as asserting that no good will come of contemplating a late self-portrait by Rembrandt or reading War and Peace or listening to Miles Davis or whatever it is that brings you to a sense of profound connection. The proof for this of course lies in the personal experience, in taste, which is to say there is no proof. The sense of sublime, whether through religion or art or music, is transitory, sometimes communicable but never definable. So there is no agreement about where it can be found, except sometimes when we recognize that someone has witnessed what we have witnessed.
Religion at its best clearly serves to develop this sense of the experience of the sublime and to share the witnessing: to position the believer in the best state to receive an authentic vision of what is real, what is right and provide a place, or community in which this is shared.
But everyone, atheist, agnostic and believer has to develop their own ability to receive, their own method for recognizing truth, their own conscience, as Pope Francis, surprisingly recently agreed, claiming even atheists have to 'make up their own mind about what is good and evil'.
The point of looking at science and the arts is that they are the result of the struggle of human beings to produce authentic visions of reality. The process for learning to recognize an authentic vision seems to be the same in religion, science, art and human affairs: we need to distinguish between the authentic vision and a fantasy or pretension or projection of our desire for belonging, or our assumed knowledge of history or culture or religious teaching.
The process of learning to appreciate art usually begins by studying with someone who knows something about it, looking at paintings in books, studying some more, visiting museums, unlearning what you thought you knew, looking again till you begin to develop something of an eye of your own. It requires some concentration and application and desire to be educated and rigor to remove pre-conceived ideas. As the aforementioned Miles Davis said 'It takes a long time to learn to play like yourself'. When the taste is well developed the eye can tell instantly what has value, just as those who saw what the Impressionists were up to at their first show. The rest of us may have to wait until the general education of the zeitgeist seeps into us.
If we admire science, we would like to think that as soon as a scientific truth is demonstrated we are able to accept it whatever concepts it contradicts. If we love art we would like to think that we can embrace a new vision as soon as it presents itself. If we are inspired by social justice movements we like to think that we would instantly have leapt to the side of the righteous.
The possibility that Ibn 'Arabi (amongst others) presents, is a vision like this, about everything, at every moment: to be so empty of self that the truth appears in a new configuration at every moment without attachment to past forms.
Scientists, artists, poets, prophets, mystics all help with this. We live in extraordinary times: moral relativism on one side, the return to extreme religious fundamentalism on the other. But we also have available unprecedented access to the spiritual heritage of humanity: in the last 50 years or so previously esoteric texts in Sanskrit, Tibetan, Hebrew, Arabic, Aramaic, Greek have emerged and been translated – along with respectful, close studies of indigenous communities, all offering multiple prisms through which to look at transcendence and the human search for it. These all help to rub off the encrusted edges of a single belief system. Scientific enquiry and the close observation of data clear up our wooly interpretations of phenomena, and artists in all fields provoke our senses to take in the light with 'unprejudiced vision'.
This is in no way to suggest that studies in comparative religion or frequent visits to blockbuster art shows will bring us to the vision of an Ibn 'Arabi. Professor William Chittick in a lecture in Berkeley last year, reprinted in the recent Journal of the Ibn 'Arabi Society, spoke on this point. He emphasized that this much loved and quoted poem of our title 'My Heart has become Capable of Every Form' is written from the vision of one who has achieved union with the Real, achieved this only by following most rigorously the prescriptions of his religion. Paradoxically the most broad, liberating and universal vision where the truth appears as it is without screening comes about through intense application, discipline, study, submission of one's own ideas, submission of one's self. 'It is not easy to become a perfect man 'Ibn 'Arabi states most convincingly in The Kernel of the Kernel.
Yet Ibn 'Arabi does state that this is the true potential of the human being. The vision his poem evokes is so attractive, so compelling, it leads us on. Whatever path we take, with or without a formal religion, it requires us to access our inner atheism when it is time to abandon meanings, our inner agnosticism when we need to know that we don't know, and our inner believer when it is the moment to step back and receive: to be helped, and educated, moved by compassion or swept away by beauty.
My heart has become capable of every form: it is a pasture for gazelles and a convent for Christian monks, and a temple for idols and the pilgrim's Ka'ba and the tables of the Tora and the book of the Koran.
I follow the religion of Love: whatever way Love's camels take, that is my religion and my faith.
Ibn 'Arabi – Tarjuman al-Ashwaq
Jane Carroll, February 21, 2014


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Kassandra Cole has it all—a stable home with great parents, popularity and all the friends she could ever need. She is used to a lifestyle where everything comes easy to her, from good grades to being captain of the cheerleading squad.
Her entire world is shaken to the core when a fatal accident takes her father, causing Kassandra to re-examine the course her life was on. The actions of one individual forever changes her life, leaving her to pick up the pieces of her broken family. Kassandra begins to question everything she once cherished and the people she disappointed along the way.
Maddon Johnson's life is as different from Kassandra's as night and day. Abandoned by his mother at a young age, he is left to deal with his abusive, alcoholic father on his own. He has one goal: lay low and get the heck out of dodge as soon as he is legally old enough.
Everything changes when his father's tragic actions send Maddon crashing into Kassandra's world. Suddenly, getting away is the last thing he wants as he discovers it is possible to believe in love after all.
Helpful Links: Goodreads | Amazon | Barnes & Noble | Website
My Review
Kassandra's world has come to a crashing halt. Her father passed away, her little sister is in shock and has gone mute and her mother is a ghost of her former self. With the realization that things will never be the same, Kassandra faces who she was and decides who it is she really wants to be.
Life for Kassandra starts off incredibly bleak in Forever Changed. The first couple of chapters are so full of sorrow and pain that it actually made it hard for me to read. This book hit a little close to home for me and brought me back to a time when I felt a lot of Kassandra's pain. Losing a parent is never easy and author Tiffany King does an excellent job in depicting the grief that Kassandra and her family are going through. It was so real and so raw I had to put the book down a couple of times as I was already in tears a few pages in.
But as the story progresses we begin to see a glimmer of hope shine through. As little by little Kassandra slowly puts the pieces of her life back together, but things aren't all rainbows and happiness once she has accepted the fact of what her life has become. Oh no, her life is far from it. Those she thought would be there for her suddenly aren't and suddenly she is dealing with not only the loss of her father, but of those she once called friends. With so many things going wrong for Kassandra it is amazing that she didn't become a complete shell and hide under a rock. Okay, so maybe she was a shell, but she pushed on, putting on that brave face for her little sister each and every day.
Kassandra's life went from happiness and sunshine, cheerleading and friends, to pain and sorrow, lonliness and grief; in the blink of an eye. Enter Maddon, the son of the man responsible for killing Kassandra's father and you have the recipe for an explosive story! And that's exactly what happens once Maddon enters the scene. Suddenly Maddon becomes the target of all of Kassandra's anger and pain, but Maddon understands a thing or two about anger and pain. After living with the two as siblings his entire life Maddon has learned to accept the things that life throws at him and channel it into something more soothing--music!
I absolutely adored all the characters in Forever Changed. Tiffany King does such a great job helping you connect with her characters, making them as real as any person you would find at the local high school. Forever Changed is amazingly beautiful and will have you so lost in the story of Kassandra that you won't even notice time slipping by until you have finished the story. Tiffany King has the amazing ability to bring out all your emotions as a reader. She uses each one to build a story about life that can be related to by almost anybody.
Make sure you pick up this raw beauty and fall in love with way that Tiffany King weaves a story!
"She was like a leaf in the wind that just slowly floated away." (kindle, loc. 772)
I picked this moment to share because of the sheer beauty and rawness of this statement. It really gives you insight into Kassandra's pain.


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- Research
- Open Access
HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector
- Joseph Anderson1 and
- Ramesh Akkina1Email author
© Anderson and Akkina; licensee BioMed Central Ltd. 2005
- Received: 26 October 2004
- Accepted: 13 January 2005
- Published: 13 January 2005
RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) has proved to be a highly effective gene silencing mechanism with great potential for HIV/AIDS gene therapy. Previous work with siRNAs against cellular coreceptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance. Since monospecific siRNAs targeting individual coreceptors are inadequate in protecting against both T cell tropic (X4) and monocyte tropic (R5) viral strains simultaneously, bispecific constructs with dual specificity are required. For effective long range therapy, the bispecific constructs need to be stably transduced into HIV-1 target cells via integrating viral vectors.
To achieve this goal, lentiviral vectors incorporating both CXCR4 and CCR5 siRNAs of short hairpin design were constructed. The CXCR4 siRNA was driven by a U6 promoter whereas the CCR5 siRNA was driven by an H1 promoter. A CMV promoter driven EGFP reporter gene is also incorporated in the bispecific construct. High efficiency transduction into coreceptor expressing Magi and Ghost cell lines with a concomitant down regulation of respective coreceptors was achieved with lentiviral vectors. When the siRNA expressing transduced cells were challenged with X4 and R5 tropic HIV-1, they demonstrated marked viral resistance. HIV-1 resistance was also observed in bispecific lentiviral vector transduced primary PBMCs.
Both CXCR4 and CCR5 coreceptors could be simultaneously targeted for down regulation by a single combinatorial lentiviral vector incorporating respective anti-coreceptor siRNAs. Stable down regulation of both the coreceptors protects cells against infection by both X4 and R5 tropic HIV-1. Stable down regulation of cellular molecules that aid in HIV-1 infection will be an effective strategy for long range HIV gene therapy.
- HIV/AIDS gene therapy
- HIV-1 co-receptors
- CCR5 siRNA
- CXCR4 siRNA
- Bispecific Lentiviral vector
HIV/AIDS continues to be a major public health problem worldwide with millions of people currently infected and new infections being on the rise. As no effective vaccines are currently available for prevention, new and innovative therapies need to be developed. Although combinatorial therapies such as HAART have proven to be effective in prolonging life, they do not afford a complete cure. Other constraints with HAART therapy are the development of drug resistant viral mutants and toxicity after prolonged therapy. Intracellular immunization by gene therapy strategies offers a promising alternative approach for controlling and managing HIV disease. A number of previous approaches that involved the use of transdominant proteins [1–3], decoys [3–7], and ribozymes [5, 8–12] had shown initial promise but fell short of practical utility in providing adequate protection. With the discovery that the RNA interference phenomenon operates in mammalian cells and is highly effective in selective gene silencing, new potent small interfering RNA (siRNA) molecules have become available to add to the anti-HIV arsenal [13].
RNAi is a highly potent mechanism of post-transcriptional gene silencing. Mediated by sequence specific siRNAs, it can effectively down regulate expression of either viral or cellular RNA target molecules by selective degradation of mRNAs [13–16]. Mechanism of destruction involves an endonuclease present in the RISC complex which is guided by the antisense component of the siRNA for target recognition. A number of reports have shown that delivery of siRNAs by transfection of presynthesized or plasmids encoding siRNAs into cultured cells can effectively inhibit HIV-1 infections [17–26]. Antiviral effects of these delivery methods are only transient due to eventual degradation and dilution of siRNAs during cell division. For HIV gene therapy strategies to succeed in long range, it is necessary that siRNA coding transgenes be maintained and expressed long term in a virus susceptible target cell. In this regard, lentiviral vectors have proven to be highly effective in high efficiency gene transduction and sustained gene expression.
A number of previous approaches using either synthetic siRNAs or plasmid expressed constructs have successfully targeted viral transcripts and achieved effective viral inhibition. Of these, some anti-HIV-1 siRNAs, such as siRNAs against tat, tat-rev had been introduced into lentiviral vectors and their efficacy was demonstrated both in cell lines and primary T cells and macrophages [27, 28]. Promising data was also obtained in experiments showing that anti-rev siRNAs against HIV-1 were functional in conferring viral resistance in differentiated T cells and macrophages derived from lentiviral transduced CD34+ hematopoietic progenitor cells [29].
In addition to targeting viral transcripts, many studies including ours also investigated the efficacy of siRNAs in down regulating host cell molecules necessary for HIV-1 infection [18, 21, 23, 24, 30, 31]. An advantage in targeting cellular molecules is that efficacy will be more broad spectrum against all the clades of the virus and the frequency of escape mutants will be lower. Down regulation of the primary cell surface receptor CD4 and consequent inhibition of HIV-1 infection was shown using synthetic siRNAs. However, since CD4 is an essential cell surface molecule for immunological function, it is not a practical target for HIV gene therapy. Chemokine receptors CCR5 and CXCR4 play critical roles as coreceptors for viral entry during infection with macrophage tropic R5 and T cell tropic X4 HIV-1 viral strains respectively [32, 33]. Thus they are suitable targets for siRNA mediated down regulation. Since both R5 and X4 viral strains are involved in disease pathogenesis, it is important to consider blocking of both respective coreceptors when developing effective therapeutics. In a segment of the human population, a naturally occurring 32-bp deletion in the CCR5 gene results in the loss of this coreceptor thus conferring significant resistance to HIV infection [34–36]. Homozygous or heterozygous individuals for this mutation remain physiologically normal. With regard to the CXCR4 coreceptor, it was found to be dispensable for T cell development and maturation in murine studies [37]. These findings suggest that CCR5 and CXCR4 are promising targets for HIV therapies.
Based on this rationale, recent work with synthetic siRNAs demonstrated that down regulating either CXCR4 or CCR5 will protect cells from X4 or R5 HIV-1 strains respectively at the level of viral entry [18, 21, 23, 24]. Although stable expression of an anti-CCR5 siRNA was achieved using a lentiviral vector in one study, down regulating CCR5 alone in the face of an HIV-1 infection is insufficient [31]. Therefore, we recently experimented with synthetic bispecific combinatorial constructs targeted to both CXCR4 and CCR5 and have shown their efficacy in cultured cells [24]. To make further progress, our present studies are directed towards constructing a single bispecific lentiviral vector expressing both CXCR4 and CCR5 siRNAs. Using this combinatorial construct, here we show high efficiency transduction, simultaneous down regulation of both coreceptors resulting in HIV-1 resistance.
Results and Discussion
Coreceptor down regulation by a bispecific lentiviral vector
Expression of siRNAs and down regulation of CXCR4 and CCR5 transcripts
Bispecific siRNA vector does not induce interferon
Resistance of siRNA transduced cells to HIV-1 infection
In summary, our studies have shown for the first time that a single lentiviral vector could be used to stably deliver two different siRNAs targeted to two different cell surface co-receptor molecules and achieve protection against both X4 and R5 tropic HIV-1 viral strains. The short hairpin design permitted use of a single promoter to transcribe both the sense and anti-sense strands of each of the siRNAs. No promoter interference was observed between the U6 promoter driving the transcription of CXCR4 siRNA and the H1 promoter driving the CCR5 siRNA since comparable amounts of both the siRNAs could be seen in transduced cells. Furthermore, possible interferon induction by the combinatorial construct was also ruled out.
A major advantage in using a combinatorial lentiviral construct targeted to both the coreceptors is that infection with either of the viral strains could be prevented at the entry step thus eliminating the possibility of proviral integration and viral latency. Given the success with the current bispecific construct, other novel constructs could be designed and experimented with that incorporate siRNAs targeted to both the cellular as well as viral targets. Based on the design employed here, it is possible to introduce more than two siRNAs in a single construct in the future. However caution should be exercised while incorporating multiple siRNAs in a single construct because the possibility exists that over expression of foreign siRNAs in a cell may have undesirable effects such as saturating the endogenous RISC complex and consequent toxicity. Such a possibility needs to be tested in long range experiments in vivo. We previously have introduced a monospecific siRNA targeted to HIV-1 rev into CD34 hematopoietic progenitor cells via lentiviral vectors and derived transgenic macrophages in vitro and T cells in vivo [29]. The transgenic cells were found to be apparently normal while markedly resistant to HIV-1 infection.
No deleterious effects are expected by the stable knock down of the CCR5 coreceptor in vivo since individuals harboring a 32 bp deletion in the corresponding gene are physiologically normal [34, 35]. Although CXCR4 down regulation in circulating mature T cells in the periphery may not have any insurmountable ill effects, this may have possible drawbacks in a stem cell setting due to its role in cell homing into bone marrow [51, 52]. Additionally, recent gene expression profiling studies indicated some off-target effects by siRNAs [53]. Therefore, the present combinatorial construct targeted to both CXCR4 and CCR5 coreceptor molecules need to be thoroughly tested in an in vivo system such as the SCID-hu mouse model to evaluate its efficacy and possible toxicity in differentiated cells before it can be used for gene therapy in human subjects. Such experiments are currently underway.
For HIV/AIDS gene therapy strategies to succeed, novel molecules need to be harnessed. In this regard, siRNAs offer great potential. Exploitation of these promising candidates to down regulate essential cellular coreceptors via the use of lentiviral vectors facilitates long term derivation of resistant T cells and macrophages which are the main targets for the virus. Our results showed for the first time that expression of both CXCR4 and CCR5 siRNAs in combination is possible by the use of lentiviral vectors. Coreceptor specific siRNAs stably transduced with the bispecific lentiviral vector showed marked resistance against both T cell tropic and monocyte tropic HIV-1 infection in cell lines and primary PBMCs. The newly developed bispecific vector shows promise for potential in vivo application.
Materials and Methods
Plasmid and lentiviral vector construction
Previously characterized siRNAs against CXCR4 and CCR5 were used in generating the bispecific lentiviral vector [23, 24, 30]. A third generation lentiviral vector backbone was employed to derive the bispecific constructs. The two cis-acting elements, namely, the central DNA flap consisting of cPPT and CTS (to facilitate the nuclear import of the viral preintegration complex) and the WPRE (to promote nuclear export of transcripts and/or increase the efficiency of polyadenylation of transcripts), are engineered to enhance the performance of the vector [38, 39]. An siRNA expression cassette targeting CXCR4 under the control of the Pol-III U6 promoter was PCR amplified from the plasmid pTZ-U6+1 as described by Castanotto et al [40]. This cassette was cloned into pHIV-7-GFP transfer vector in the BamH I site immediately upstream of the CMV-EGFP gene. This cassette contained a Mlu I restriction site downstream from the CXCR4 siRNA sequence for subsequent cloning of the H1 promoter driven CCR5 siRNA cassette. The H1-CCR5 siRNA expression cassette was also generated as described above using the plasmid pSUPER (Oligoengine, Seattle, WA). Sequencing and confirmation of candidate clones was performed by Laragen Inc. (Los Angeles, CA). The transfer vector containing the inserts U6-X4 siRNA and H1-CCR5 siRNA is termed pHIV-XHR-GFP.
Cell culture and vector production
293T cells and PBMCs were maintained in DMEM media supplemented with 10% FBS. Magi-CXCR4 cells obtained from the AIDS Reference and Reagent Program were maintained in media as previously described [41, 42]. Ghost-R5 cells obtained from the AIDS Reference and Reagent Program were maintained in media as previously described [43]. To generate lentiviral vectors, fifteen micrograms of transfer vector with either GFP-alone or XHR were transfected along with 15 ug pCHGP-2, 5 ug pCMV-Rev, and 5 ug pCMV-VSVG into 293T cells at 60% confluency in 100 mm culture dishes using a calcium phosphate transfection kit (Sigma-Aldrich, St. Louis, MO). Six hours after transfection, fresh medium was exchanged. Cell culture supernatants containing the vector were collected at 24, 36, 48, and 60 hours post transfection and pooled. Vector supernatants were concentrated by ultracentrifugation and later titrated on 293T cells using FACS analysis for GFP expression.
Lentiviral vector transduction and FACS analysis
Magi-CXCR4 and Ghost-CCR5 cells were seeded in 6-well plates 24 hours prior to transduction, 5 × 105 cells per well. Cells were transduced with lentiviral vectors at an m.o.i. of 10 in the presence of 4 ug/ml polybrene for 2 hours. For transduction of PBMCs, cells were first isolated from whole blood by Histopaque®-1077 (Sigma-Aldrich), and then cultured in CD3 and CD28 antibody coated plates. Three days after stimulation, PBMCs were transduced at an m.o.i of 20 in the presence of 4 ug/ml polybrene. PBMC transduction was repeated the following day. Seventy-two hours post transduction with siRNA containing lentiviral vectors, FACS analysis was performed to determine the levels of cell surface expression of CXCR4 and CCR5. Non-transduced and transduced cells were stained with appropriate antibodies conjugated with PE-Cy 5 (Pharmingen, San Diego, CA) namely, anti-CXCR4 for Magi-CXCR4 cells and anti-CCR5 for Ghost-CCR5 cells. Transduction efficiency was determined by assaying for EGFP expression. FACS analysis was performed on the Beckman Coulter Epics XL using ADC software for analysis.
Northern analysis for shRNA expression
Total RNA was extracted from non-transduced and transduced Magi-CXCR4 and Ghost-CCR5 cells using the RNA-STAT-60 reagent (Tel-Test, Friendswood, TX). Small RNAs, <200 nt, were separated and concentrated using the mir Vana™ miRNA Isolation Kit (Ambion, Austin, TX). Twenty micrograms of small RNAs were hybridized overnight at 37°C using the mir Vana™ miRNA Detection Kit (Ambion) with γ-32P labeled probes made using the mir Vana™ Probe & Marker Kit (Ambion). Probes were complementary to the antisense strands of CXCR4 and CCR5 siRNAs. Hybridization reactions were processed according to the manufacturer's protocol and run on 15% polyacrylamide TBE-Urea gels. Gels were then exposed to X-ray film. A probe complementary to miRNA-16 supplied with the miRNA detection kit was used as an internal control.
Western Blot analysis of phosphorylated PKR
Cell lysates of non-transduced and transduced cells were run on 10%-polyacrylamide-SDS TBE gels. Proteins were immunoblotted onto Immobilon™-P membranes (Millipore, Bedford, MA) and incubated with antibody specific for phosphorylated-PKR (Sigma-Aldrich), while anti-actin antibody (Sigma-Aldrich) was used to detect cellular actin as an internal control. A secondary antibody, goat anti-rabbit IgG conjugated with alkaline phophatase (Promega, Madison, WI), was then added. An alkaline phophatase substrate reagent, Western Blue (Promega), was used to visualize the bands.
Total RNA was extracted from non-transduced and transduced cells. Primers specific for CXCR4 (forward: 5'-ggaggggatcagtatatacacttc and reverse: 5'-cgccaacatagaccaccttttc) and CCR5 (forward: 5'-caaaaagaaggtcttcattacacc and reverse: 5'-cttgctcgctcgggagcctc) (IDT, Coralsville, IA) were used to determine transcript levels while GAPDH (forward: 5'-ctgagaacgggaagcttgtcatcaa and reverse: 5'-gcctgcttcaccaccttcttgatg) primers were used as an internal control. One-step RT-PCR reactions were performed using the Superscript™ III One-Step RT-PCR kit (Invitrogen, Carlsbad, CA). Reactions were run on 1% agarose gels and appropriate bands were visualized with UV light.
HIV-1 Challenge
To determine if down-regulation of CXCR4 and CCR5 transcript levels and cell surface expression inhibited HIV-1 infection, non-transduced and transduced cells were challenged with NL4-3 (X4-tropic) and BaL-1 (R5-tropic) strains of HIV-1, at an m.o.i of 0.01, as previously described [24]. Viral supernatants were collected daily from infected Magi-CXCR4 and Ghost-CCR5 cells for p24 assay. ELISA was used to determine p24 values employing a Coulter-p24 kit (Beckman Coulter, Fullerton, CA). For PBMC challenge experiments, non-transduced and transduced cells were infected with NL4-3 and Bal-1 strains and cell culture supernatants were collected on days 1, 3, 5, and 7 post-infection to measure p24 levels.
Work reported here was supported by NIH grants AI50492 and AI057066 to R.A. This work has also been facilitated by the infrastructure and resources provided by the Colorado Center for AIDS Research Grant P30 AI054907. We thank Karen Helms for help with FACS analysis and William Wheat for critically reading the manuscript. We thank NIH AIDS Research and Reference Reagents Program for providing many reagents and cell lines used in this work.
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