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IndieSider #14: Framed by Loveshack Entertainment Comments Off
Framed is an interactive graphic novel from Loveshack Entertainment. Rearrange the panels of this noir-style comic book to change the order in which the action occurs. Will the spy run headlong into the police — or will he slip down a hallway, evading detection? You the player decide with panels you can drag-and-drop, rotate, and reuse.
In this interview, Framed designer and composer Adrian Moore discusses the influences that led to this unprecedented game, the role narrative plays, the different genres and game mechanics the team experimented with, and how the mobile indie market harkens back to the earlier days of his career, developing Populous at Bullfrog.
Watch the video below, or download the audio edition from iTunes, Stitcher, or TuneIn.
Links mentioned in this episode:
- Framed for iOS
- Adrian Moore of Loveshack Entertainment
- Susan Arendt recommends Framed
- Framed is Hideo Kojima's Game of the Year
- Understanding Comics by Scott McCloud
- "Listening to Framed soundtrack while programming. Feeling like a code detective!"


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by Lindsay A - 14 hours ago
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subset: commoncrawl


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Use the tools below to refine your search by only displaying reviews with a certain number of star ratings or to only show reviews from a certain time period. Eg click on '1 star' to just display the reviews we have which received a 1 star rating click or 'Within the last month' to display just reviews posted over the last month.
Written on: 23/03/2015 by DaveJPim (1 review written)
I bought a Yaris from Carcraft in Trafford Park, Manchester last month, very helpful and non-pushy assistants, pleasant and friendly service. The car was as I required, sparkling clean and ready when I came to collect it, good value for the age and mileage. Can't ask for any more than that. Good little area there for the kids to play in whilst you're browsing too, I wouldn't hesitate to return next time… Read Full Review
Written on: 23/03/2015
We needed to replace our existing car and trade it in so visited Carcraft to see what was available. We had visited carcraft around 10 years ago and wasn't very impressed with the service or the vehicles, but I was pleasantly surprised by the change, no aggressive hard sell and vehicles are stored all indoors now and none are older than 7 years old. We came away with a new car and got a good trade-in price for the old one, a free-service and MOT in the first 12 months so I would recommend them… Read Full Review
Written on: 22/03/2015 by Disrespected (1 review written)
Incompetent shambolic Couldn't cesr less once they have your money the device is a disgrace cars have not been checked they are sold as they come in won't admit or try to rectify any short coming attitude is bad the service department will try and charge you to put faulty cars they sold right that they should of done before they sold you promise you things and then don't deliver then try and charge you… Read Full Review
Written on: 26/03/2015
This is correct I have had the same problem
Written on: 21/03/2015 by ctSerrano102 (1 review written)
Visited lakeside branch, all staff friendly and relaxed, browsed in peace and then when I decided on a vehicle or two a test drive was arranged. All went well and to my surprise found out I could drive away on the day ! After some credit checks, which did take time I was given the keys to me new car. I'd allow a half day to complete all the paperwork. When its time to change my car again I'd definitely go back… Read Full Review
Written on: 19/03/2015 by HadleyWatson212 (1 review written)
We recently visited Car craft Sheffield, we were greeted by a very friendly member of staff who was very happy to help. We were not be pestered to buy in any way. The sales assistant was very pleasant and told us if we needed any help just to let her know. We approached one of the many members of staff available, who was happy to help immediately and answer our questions about the particular car. We were taken for a test drive and loved the car. We then went ahead and purchased it, driving away… Read Full Review
Written on: 19/03/2015 by Adelaidea1952 (1 review written)
Very pleased with our experience, they listened to what we wanted and didn't push up to go any higher than we were comfortable. We drove away the same day with a very attractive finance and service package. All round satisfactory experience… Read Full Review
Written on: 19/03/2015 by Ayerscf480 (1 review written)
Cars advertised on their website at cheap prices to entice you to visit the showroom, hassled into placing a holding deposit, then sort the fiance out and once hooked give you some excuse about why they cannot sell you that car anymore, told it was sold, at the wrong lot, engine management light had come on, all sorts of rubbish excuses. Then as they have your deposit and finance sorted try to bully you into choosing a different car which conveniently is a lot higher than your original budget!… Read Full Review
Written on: 18/03/2015 by llanelligirl (1 review written)
Firstly the choice of cars was huge but the salesman listened to what we needed and showed us a range of suitable vehicles. Stayed within our budget which was important for us and no flannel. Drove the car away 2 hours later with 0% finance. (deposit on a 0% credit card) Quick, painless and easy. 3 months later the car is running well and we're still very happy… Read Full Review
Written on: 17/03/2015 by NickiJS1979 (1 review written)
We recently visited Car craft and from the moment we walked through the door, we were greeted by very friendly staff who were happy to help. But also happy to let us browse in our own leisure and not be pestered. They were very pleasant and told us if we needed any help just to let them know. We browsed at our own leisure and found a few cars we liked. We approached one of the many members of staff available, who was happy to help immediately and answer our questions about the particular… Read Full Review
Written on: 17/03/2015 by tomhatescarcraft (1 review written)
Just a small message to anybody who may have considered buying a car from carcraft DONT!They are more than happy to keep in touch when your buying the car but don't want to know you when it comes to a repair!!… Read Full Review
Written on: 17/03/2015 by aheeduzzaman (1 review written)
Bought the Nissan Qashqai, Sales staff was excelent and efficient. I was sold the 5 year plan and was told that i can cancell it within 28 days. I canculted my needs and requirement and decided not to have this 5 year cover. Kept trying to contact to cancell it but the contact was finaly made after the 28 days. I was asked to writ in as it was over 28 days that I did and it's been 2 months and I've not heared anything back yet. I think admin staff doesn't care once the car is sold that is bad… Read Full Review
Written on: 17/03/2015 by Jess06x (1 review written)
I was looking for a car that would be suitable for a young girl and monthly finance that would suit my budget and I found just that. The sales assistant wasn't too pushy and was there to help when needed. They found me the perfect car and worked with me to help suit what I could afford. Fantastic service and I have already recommended to a few friends… Read Full Review
Written on: 16/03/2015 by McCleary411 (1 review written)
I wanted a car powerful enough to pull a caravan. Although I did not buy the model I was going to look at, I left with something better! The salesman was helpful and courteous. Everything was explained adequately and carefully. A well satisfied customer!… Read Full Review
Written on: 16/03/2015 by 62Temple (1 review written)
What a joke this organisation are. I know every business is after its pound of flesh but this place takes thinfs to another level. Cars well over priced, part ex figures a joke, cash for cars quotes are lsughable and priced to get you through the door before they halve them. The staff, well one of them was ok the rest are utterly repugnant, attitudes, wide boy stance, sales pitch to death. Just be honest please… Read Full Review
Written on: 14/03/2015 by Pete1630 (1 review written)
Great attention to me as the customer, although i was left waiting for quite a while this was down to my personal circumstances, rather than CarCraft taking their time. I was very pleased that i managed to negotiate a great deal for myself, and rather than Carcrafft sticking to their guns over the price of the car they met me in the middle, which i'm obviously very pleased about. After car was good as well, phone calls to ensure i was happy and i was contacted about other service available to… Read Full Review
Written on: 13/03/2015 by soHopkins300 (1 review written)
I was buying a car for the first time so I didn't really know what to look for. I was recommended by a friend to carcraft and I'm glad I was because the car was perfect. It was fully checked and tweaked before it was even advertised so I knew I'd made the right decision. The staff were incredibly friendly and helpful and I feel I got the best value for money. The finance was reasonable and the financial advisors made the breakdown of payments simple and to the best value for me. I have also… Read Full Review
Written on: 13/03/2015 by gazbaz64 (1 review written)
I was very impressed with the manner of staff , didn't actually go looking for a car, was advised by a sales person that I could upgrade to new car , was given all the details and was very surprised to be able to do this. Everybody was very friendly and helpful a very pleasant afternoon and I got myself a new car in the process. Only grumble aftersales was not so good , was wanting imformation and was told lots of times would be contacted and wasn't maybe this area could be ironed out… Read Full Review
Written on: 05/03/2015
When I was 18 and looking for a car on the Internet a man from car craft phoned me after I'd shown interest online. I couldn't drive at this point so he came to pick me up and drove me to car craft leeds. Sold me a car through two finance providers and added a 5 year extended guarantee on. None of this was explained properly to me. They must of laughed as I left. I phoned when I got home to cancel the extended guarantee and was told it needed to be in writing. I wrote a letter and was told the… Read Full Review
Written on: 28/02/2015 by Bigcav95 (1 review written)
I went to a carcraft in wednesbury, Wolverhampton. They are very very good at changing you out of your old car into a new one. The cars are a very good quality and the payments are nagoshable. I highly recommend carcraft as it is probably the best car supermarket in the uk. The only thing I didn't like about it is it was a very long process and took 4 hours but then again it was a Sunday and it was also busy. A very good part of my experience was the drive away the same day. I could leave my… Read Full Review
Written on: 27/02/2015 by martinolbison (1 review written)
the person I dealt with at carcraft Leeds was very helpful in getting me into the car I wanted for the price I required. I would definitely recommend them bearing in mind I expected to go in with my old car and come out with the same car due to my credit rating but to my surprised the cars at carcraft worked hard to get me into the car I wanted. And like I said the person I dealt with is an absolute star who listened to my concerns and took them onboard and more than helped me with the… Read Full Review
As rated by real users
" Great Service! " Read More
" TERRIBLE NEVER AGAIN!!! " Read More
" Lucky to be alive " Read More
" Warranty rubbish helpless " Read More
" Hassle Free " Read More


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text:
Social Physics
How Good Ideas Spread-the Lessons From A New Science
A landmark tour of the new science of "idea flow" outlines revolutionary insights into the mysteries of collective intelligence and social influence, explaining the virtually unlimited data sets of today's digital technologies and the considerable accuracy of information from social networks.
Publisher: New York :, The Penguin Press,, 2014.
Characteristics: x, 300 pages :,illustrations ;,22 cm
From the critics
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(a.k.a. Isenberg Sandhill Crane Reserve)
CDFW invites you to come out on a tour, in Lodi, during the "Season of the Sandhill Crane." The season begins with the arrival of the sandhill cranes into the Delta and Central Valley in about late September, where they will spend the fall and winter months. Tours consist of viewing sandhill cranes and other unique wintering waterfowl, hearing a presentation on sandhill cranes and their Reserve habitat, and viewing the cranes' impressive, nightly behaviors at a location that is only open to the public during these special tours.
NOTE: It is required that you pre-register for your tour as tour size is limited to 30 people and there are no accommodations for drop-ins at the tour site.
The Woodbridge Ecological Reserve is divided into two parcels, known as the South Site and North Site. Tours meet 90 minutes before sunset at the South Site where docent guides meet attendees and give a presentation. Tours involve very little walking so you may want to bring a camp chair if you would like to sit and view the birds. It is recommended that you dress warmly, especially during the winter months as it can be damp and chilly. It is suggested that you bring binoculars and/or spotting scopes for up-close viewing but, you can easily enjoy the cranes with unaided eyes and ears. You may want to bring a light snack and water for your tour.
Portable toilets available. North Site has ADA-compliant facility.
Guides may take tour groups down Woodbridge Road via car caravan to see cranes and other wildlife using the grainfields and marshes along the road. Your docent guides are trained in crane ecology, the area's natural history and the Reserve's habitat management, and want your experience to be memorable and educational. Tours usually conclude a half hour after sunset with the anticipated fly-in of the cranes at the North Site where a viewing shelter allows us to be hidden from the incoming birds.
Cranes will be the focus of our tours as they may be observed here under most but not all conditions; however, we will also scout for other birds that use the reserve during the winter months.
Bay Delta Region (Region 3)
Regional Manager: Scott Wilson
Main Office: 7329 Silverado Trail, Napa, CA 94558 | (707) 944-5500
Stockton Office: 2109 Arch Airport Rd, Stockton, CA 95206 | (209) 234-3420
Email the Bay Delta Region | Program Contacts
Online registration for sandhill crane tours will open during the month of August and will be offered monthly throughout the season. A registration window will open approximately six weeks before each month's tour. The following schedule shows the registration window for each month.
You may want to register early as tours fill quickly. Check out other information about tours and sandhill cranes below. We look forward to visiting with you this year during the "Season of the Sandhill Crane."


text:
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Atul Aneja
DUBAI: Co-chairman of Pakistan People's Party Asif Ali Zardari has begun crucial talks in Dubai with president of the Pakistan Muslim League (Nawaz) Mian Shahbaz Sharif on a road map to reinstate judges deposed by President Pervez Musharraf.
"This meeting is likely to be decisive and it deals with the restoration of the judges," president of PPP's UAE unit M. Akram Farooqui told The Hindu. He added that the talks were dealing with constitutional and legal issues which were being discussed threadbare. Given the legal overtones of the dialogue, experts are present in both the delegations.
Law Minister Farooq H. Naik, who belongs to the PPP and Hars Mohammad, a legal expert of the PML (N), are part of the delegations.
Mr. Farooqui said the PPP's approach was to find a durable solution, based on a sound legal foundation.
The sources said talks could spill over to Tuesday as the visiting delegation is not departing on Monday.


text:
$1200 to $1300
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East Lansing, MI, 48823
2704 Linden St Bedrooms: 3 • Baths: 1.75 Houses $1,200/month


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Breaking in the PledgesHelping the sorority get the pledges ready.
Brian Owns MeMature stepmother discovers the joys and rewards of sodomy.
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Cabin VacationA beautiful winter cabin, a night by the fire, and more.


text:
Mississippi Flyway
Reviewed by
Katerie Prior
Ellie is a woman who has always had difficulty being what others want her to be: the thin daughter the good Catholic school student the supportive sibling a society reporter the doctor's wife. Ellie can't remember much about her early life but she does remember wanting to be an owl—a desire left over from her childhood days as a volunteer bird counter. That's not much help for Ellie who now finds herself divorced at thirty-one and feeling out of step with the world.
All that changes when Ellie's estranged father Tiny arrives on her doorstep. On the lam after playing a crooked game of poker with a corrupt sheriff Tiny invites Ellie on a road trip with him. She accepts his offer and soon father and daughter are on a journey not just through America's heartland but to confront a dark past.
This journey comes to life in Mississippi Flyaway because of author Nel Rand's ability to create a vivid world with her poetic description. Early in the book we see young Ellie as a Catholic school girl whose "socks puddle around her ankles." About a man who is confused she writes "a veil of fog had lowered in front of his face." These flourishes are small but they are sprinkled like seasoning through the novel to create dramatic characters and locations.
The real strength of this book is the relationship between Tiny and Ellie. It's evident from the beginning why father and daughter are estranged; so clear in fact that some readers may initially balk that Ellie agrees to leave with her father at all. But Rand has created strong compelling characters in Tiny and Ellie that make you want to keep reading keep rooting for them even when you suspect that what they have to face will not be pleasant.
Clearly Mississippi Flyway is a book that will keep readers talking even after they've finished the story. A brief interview with the author is included at the end of the novel along with discussion questions for book clubs. Even if you read it on your own Mississippi Flyway is book that is sure to stir up emotions.


text: April Rose By GuySpeed Editors March 5, 2014 | GuySpeed Today's Honey B is Chicago native April Rose, an actress you might know from 'Grown Ups 2' or 'The Girl Code.' Read More|Comment Category: Honey B's Tags: Honey B Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!


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In an announcement earlier today, we introduced Cisco ASA with FirePOWER Services, the industry's first threat-focused next-generation firewall. We also are announcing the continued evolution of Cisco's Security Channel Partner Program to substantially increase a Cisco partner's Security business.
Security Ignite
With Security Ignite, security specialized partners get additional upfront discounts (up to 6 percent) on new next-generation security business registered through the Opportunity Incentive Program (OIP) or Teaming Incentive Program (TIP). Deal registration encourages and protects a partner's investments in developing new next-generation security opportunities. Security Ignite works in concert with the Technology Migration Program (TMP) and the Value Incentive Program (VIP). This powerful combination reinforces the focus on partners developing new security solution opportunities. Security Ignite is available in all theatres, with a few exceptions. Read More »


text:
Page 1
Journal of Virological Methods 161 (2009) 216–222
Contents lists available at ScienceDirect
Journal of Virological Methods
journal homepage: www.elsevier.com/locate/jviromet
Monitoring of HIV-1 envelope-mediated membrane fusion using modified split
green fluorescent proteins
Jianqi Wanga, Naoyuki Kondoa,b, Yufei Longa, Aikichi Iwamotob,c, Zene Matsudaa,b,∗
aChina-Japan Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China
bResearch Center for Asian Infectious Diseases, Institute of Medical Science, the University of Tokyo, 4-6-1, Shirokanedai Minato-ku, Tokyo 108-8639, Japan
cDivision of Infectious Diseases, Advanced Clinical Research Center, Institute of Medical Science, the University of Tokyo, 4-6-1, Shirokanedai Minato-ku, Tokyo 108-8639, Japan
Article history:
Received 9 February 2009
Received in revised form 11 June 2009
Accepted 16 June 2009
Available online 25 June 2009
Split protein
Membrane fusion
Envelope protein
Phenotyping method
a b s t r a c t
A simple, cell-based, membrane fusion assay system that uses split green fluorescent proteins (spGFPs)
as an indicator was developed. The attachment of the pleckstrin homology (PH) domain to the N-termini
of each spGFP not only localized the reporter signal to the plasma membrane but also helped the sta-
ble expression of the smaller spGFP of seventeen amino acid residues. It was shown that this system
allowed real-time monitoring of membrane fusion by HIV-1 envelope protein (Env) without the addition
of external substrates. This method can be adapted to the analyses of other viral membrane fusion.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Membrane fusion is the prerequisite event that allows
enveloped viruses, some of which are linked to emerging infec-
tious diseases such as avian influenza, severe acute respiratory
syndrome, and acquired immunodeficiency syndrome (AIDS), to
enter their host cells. Among these emerging diseases, AIDS has
become a global threat to human health. The discovery of a mem-
brane fusion inhibitor has made HIV-1 Env an important target
for anti-HIV-1 chemotherapy (Chan et al., 1997; Eckert and Kim,
2001; Este and Telenti, 2007; Poveda et al., 2005; Weissenhorn
et al., 1997). Recently, a new class of inhibitor that blocks the
interaction between Env and its co-receptor, CCR5, has been devel-
oped (Santoro et al., 2004). A simple phenotyping method of
Env-mediated membrane fusion will facilitate progress in the
development of new inhibitors or in the evaluation of drug-
resistant mutants (Olson and Maddon, 2003).
A phenotyping method of HIV-1 Env requires a system that
generates a measurable signal upon membrane fusion either in
Abbreviations: AIDS, acquired immunodeficiency syndrome; GFP, green fluo-
rescent protein; spGFP, split green fluorescent protein; PH, pleckstrin homology;
DMEM, Dulbecco's modified Eagle's medium; MSD, membrane-spanning domain.
of Medical Science, the University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo
109-8639, Japan. Tel.: +81 3 6409 2204; fax: +81 3 6409 2008.
E-mail address: email@example.com (Z. Matsuda).
a cell–cell or virus–cell system. The methods described employ
materials, such as visible dyes, transcription factors, and self-
complementing enzyme fragments, that produce a signal when
they transfer from one compartment to another via membrane
fusion (Barbeau et al., 1998; Blumenthal et al., 2002; Feng et al.,
1996; Furuta et al., 2006; Holland et al., 2004; Huerta et al., 2002;
1992; Sakamoto et al., 2003).
The development of a versatile, cell-based membrane fusion
ified green fluorescent protein (GFP), split GFP (spGFP), which has
been engineered to have the capacity for self-assembly (Cabantous
was fused at the N-termini of spGFPs so as to focus the signal in the
membrane regions. These spGFPs become fluorescent only when
they have reassociated with each other (Cabantous et al., 2005)
during the membrane fusion. Unlike an enzyme-based assay, this
spGFP system allows real-time monitoring of the membrane fusion
process without any additional substrates.
2. Materials and methods
2.1. Construction of expression vectors
The PH domain based on human phospholipase C? was syn-
thesized by assembling 10 oligonucleotides, each containing 79
nucleotides. The oligonucleotides were combined and assembled
0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
Page 2
J. Wang et al. / Journal of Virological Methods 161 (2009) 216–222
Fig. 1. Engineered proteins and expression vectors. (A) The amino acid and nucleotide sequences of PH domain and GFPopt1–11were shown. The amino acid residue was
shown using the single-letter abbreviations. The split point between GFP1–10and GFP11is indicated by the arrow in GFPopt1–11. (B) (upper panel) The expression vector for
spGFPs. MCS: multiple cloning site; spGFP: the insertion point of spGFP; Kan/NeoR: kanamycin/neomycin resistance gene. (Lower panel) The different spGFPs. PH: pleckstrin
homology domain. The restriction sites that were used are indicated. (C) (upper panel) The HIV-1 envelope expression vector, pNHcRedEluc. pCMV: human cytomegalovirus
promoter, NLS: nuclear-localization signal, HcRed: a far-red fluorescent protein isolated from Heteractis crispa, f-Luc: firefly luciferase, MSD: membrane-spanning domain,
AmpR: ampicillin resistance gene. (Lower panel) Primary structures of the MSDs used. WT: wild type, GpA: glycophorin A, VSV-G: vesicular stomatitis virus G protein. The
predicted MSD regions are capitalized.
by PCR (94◦C for 30s, 50◦C for 30s, 72◦C for 40s for 30 cycles).
Similarly, 30 oligonucleotides, which overlapped each other with
18 bases at the both ends, were used to assemble the optimized
GFP gene, named GFPopt1–11. The primary sequences of the PH
domain and GFPopt1–11are shown in Fig. 1A. Both amplicons were
cloned and sequenced in pCR4Blunt-TOPO (Invitrogen, Carlsbad,
USA). GFPopt1–11was split into GFP1–10(1–642 base pairs) and
GFP11(643–696 base pairs), at a point between the 10th and 11th
The PH-GFP1–10and PH-GFP11genes were generated by combining
the PH domain gene with the spGFP genes. These genes were then
expression vector for each protein was named by adding pd in front
of the target protein, such as pdPH-GFP1–10.
The FLAG tag sequence was added to the 3?-termini of the spGFP
ing PCR. A new HIV-1 Env-expression vector called pNHcRedEluc, a
derivative of pElucEnv (Miyauchi et al., 2005), was constructed by
replacing the gene for EGFP with that of a tandem red fluorescent
protein; HcRed (Evrogen, Moscow, Russia). This was preceded by
a nuclear-localizing signal (Fig. 1C). Thus the nuclei of transfected
cells became red. The transfection efficiency could then be mea-
sured by firefly luciferase activity. The pNHcRedEluc?NB vector in
which most of the env gene had been deleted was prepared as a
negative control.
2.2. Cell cultures and transfection
The 293FT (Invitrogen, Carlsbad, USA) and 293CD4 (Miyauchi
et al., 2005) cells were maintained in Dulbecco's modified Eagle's
medium (DMEM, Sigma, St. Louis, USA) supplemented with 10%
fetal bovine serum (Hyclone, Logan, USA). The 293FT cells were
cultured with 500?g/ml of Geneticin (Gibco, Grand Island, USA),
Page 3
J. Wang et al. / Journal of Virological Methods 161 (2009) 216–222
as recommended by the manufacturer. Transient transfection was
accomplished using Fugene HD (Roche, Indianapolis, USA). Stable
cell lines expressing PH-GFP1–10were established after transfect-
ing 293CD4 cells with pdPH-GFP1–10by electroporation (Biorad
GenePulsar, Hercules, USA). Transfected cells were selected with
700?g/ml of Geneticin in DMEM.
2.3. Fusion assay
into 293FT cells. The transfected 293FT cells were overlaid with
293CD4 cells which were transfected transiently or permanently
with vector pdPH-GFP1–10. In the case of transient transfection,
the mixing of cells was started at 42h after transfection. Fusion
was monitored in real-time using an IN Cell Analyzer 1000 (GE
Healthcare, Uppsala, Sweden) or was observed using a confo-
cal microscope (Olympus FluoView FV1000, Tokyo, Japan) that
examined fixed cells (4% paraformaldehyde) at designated time
The fusion assay using the mixing of the two different fluo-
rescent proteins expressed in the Env(+)- and receptor(+) cells,
respectively as an indicator was carried out as follows. The pElu-
cEnv (Miyauchi et al., 2005) was transfected into 293FT cells to
make Env(+) cells. Because pElucEnv expressed both the HIV-1 Env
and GFP proteins, this generated "green" Env(+) cells. Meanwhile
the expression vector for DsRed (Clontech/Takara, Otsu, Japan) was
transfected into 293CD4 cells to generate "red" receptor(+) cells.
These two types of cells were co-cultured and the extent of the
fusion was monitored by the redistribution of the green and red
signals by microscopy.
The inhibitor C34 was used to show the specificity of the new
fusion assay. According to the previous study (Kliger et al., 2001),
two different concentrations, 12nM and 150nM in a final concen-
tration, of the peptide inhibitor, C34, was added at the beginning of
the co-culture. The IC50 value of the C34 peptide was about 12nM
and more than 90% inhibition was observed with the concentration
of 150nM (Kliger et al., 2001). The cells were fixed and examined
after 2.5h of co-culture and analyzed as described above.
2.4. Protein analysis
described previously (Miyauchi et al., 2005). Anti-FLAG antibody
(Sigma, Saint Louis, USA) and anti-GFP antibody (Santa Cruz
Biotechnology, Santa Cruz, USA) were used as primary antibodies
for the analysis of GFP11and GFP1–10, respectively. Chemilumines-
preparationandimmunoblottingweredone as
2.5. Immunofluorescence assay
Transfected cells were fixed in an acetone:methanol solution
(1:1) for 15min at room temperature and stained with an anti-
FLAG antibody (3?g/ml) for 40min at 30◦C. A secondary antibody,
labeled with Alexa Fluor 555 (Invitrogen, Carlsbad, USA), was used.
The fluorescent signal was observed using a confocal microscope
(Olympus FluoView FV1000, Tokyo, Japan).
3. Results
3.1. Expression of modified spGFPs
3.1.1. Immunoblotting analysis
The expression vectors containing different spGFPs shown in
Fig. 1B were transfected into the cells and the expressed pro-
Fig. 2. The spGFPs expressed in the transfected cells. (A) Immunoblotting analy-
sis of 293CD4 cells transfected with GFP1–10 and PH-GFP1–10 expression vectors
and probed with an anti-GFP antibody. Cells transfected with pEGFP-N2 (BD Bio-
sciences Clontech) were included as a positive control (EGFP lane). (B) The proteins
expressed in 293FT cells transfected with the GFP11-FLAG or the PH-GFP11-FLAG
expression vectors were probed with an anti-FLAG monoclonal antibody. Mock: the
mock transfected cells.
teins were then analyzed by immunoblotting. When probed with
an anti-GFP antibody, GFP1–10and PH-GFP1–10were detected as
approximately 25kDa and 40kDa bands, respectively (Fig. 2A). The
observed molecular weights were consistent with those expected
from the amino acid sequences. The cells transfected with pEGFP-
N2 (BD Biosciences Clontech) were included as a positive control
(Fig. 2A, EGFP lane). As for GFP11, an anti-GFP antibody failed to
detect any band (data not shown). A FLAG tag was added to GFP11
with and without the PH domain because this failure could have
been caused by the absence of anti-GFP's epitope in the seventeen-
amino-acid-long GFP11portion. When probed with an anti-FLAG
antibody, a band of 22kDa was detected for PH-GFP11-FLAG but not
for GFP11-FLAG (Fig. 2B). This result suggested that GFP11, as only a
seventeen-amino-acid-long peptide, was unstable without the PH
3.1.2. Immunofluorescence analysis
the cell, but with the PH domain attached, the PH-GFP1–10localized
to the periphery of the transfected cells (Fig. 3A). The expression of
FLAG-tagged GFP11(Fig. 3B, top) was not detected. This finding is
consistent with the results of immunoblotting (Fig. 2B). However,
middle). Without the FLAG tag, PH-GFP11showed no signal with
anti-FLAG antibody (Fig. 3B, bottom).
3.2. Recovery of GFP function by reassociation of spGFPs
Next, pairs of the spGFPs were co-transfected into 293FT cells
and their outcome was examined (Fig. 3C). Consistent with the
data shown in Fig. 2, PH-GFP11, but not free GFP11, was able to gen-
erate a green signal (Fig. 3C). When PH-GFP11was co-transfected
with GFP1–10, a homogenous green signal was observed. This data
suggests that the reassociation of two split GFPs could take place
before they are localized to the plasma membrane. With the pair
of PH-GFP1–10and PH-GFP11, most of the green signal was detected
in the rim of the co-transfected cells (Fig. 3C). As expected, nei-
ther the spGFPs nor the PH-spGFPs alone showed any fluorescence
(Fig. 3D).
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J. Wang et al. / Journal of Virological Methods 161 (2009) 216–222
Fig. 3. Intracellular localization of spGFPs expressed with pdExpression vectors. (A) Immunofluorescence assay of GFP1–10or PH-GFP1–10-transfected cells using an anti-GFP
antibody. Mock: mock transfected cells; BF: bright field view; Alexa555: the Alexa555-derived signal. (B) Immunofluorescence assay of GFP11-FLAG, PH-GFP11-FLAG, and
PH-GFP11-transfected cells using anti-FLAG antibody. The abbreviations used are the same as for (A). (C) Co-transfection of PH-GFP1–10with PH-GFP11and GFP11and of
GFP1–10with PH-GFP11and GFP11. (D) Single transfection of different spGFPs. BF: bright field view; GFP: the GFP signal.
3.3. Membrane fusion assay using spGFPs
3.3.1. Analysis of the wild type HIV-1 Env-mediated fusion
PH-spGFPs was used for the analysis of membrane fusion
induced by HIV-1 Env. For this, the 293FT cells were transfected
with pNHcRedEluc and pdPH-GFP11and then co-cultured with the
nal was observed at the plasma membrane surrounding several red
nuclei (Fig. 4A). The red nuclei were derived from the 293FT cells
expressing Env as pNHcRedEluc expressed the nuclear-localizing
brane region made it easy to differentiate the real signal from the
non-specific autofluorescence background. Furthermore, the non-
envelope-mediated spontaneous fusions, if they occurred, could be
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J. Wang et al. / Journal of Virological Methods 161 (2009) 216–222
Fig.4. Generationofthegreenfluorescentsignaluponmembranefusion.Cellfusionbetweenenvelope-andreceptor-expressingcellsthatharborrespectivespGFPexpression
vectors were observed using a confocal microscope and IN Cell Analyzer. The HcRed signal was generated from the cells transfected with the expression vector for envelope
and HcRed genes (Fig. 1C). (A) Detailed image of the localization of spGFPs and HcRed. BF indicates bright field; GFP: the green fluorescence signal; Merge: the merged images
of GFP, HcRed, and BF. (B) The effect of the specific inhibitor and comparison with the fluorescent proteins-mixing assay. The specific inhibitor of the HIV-1 Env-mediated
membrane fusion, C34, was used in the spGFP assay (left) and the conventional fluorescent protein-mixing assay (right). The final concentration of the inhibitor was indicated
in nM. In the fluorescent protein-mixing assay (right), Env(+)-293FT cells expressing GFP and the receptor(+)-293CD4 cells expressing DsRed were co-cultured. Fused cells
are seen as both GFP and DsRed signal-positive cells. (C) Comparison of the frequency of membrane fusion events between wild type (WT) and its MSD mutants (GpA and
VSV-G) (details are in Fig. 1C). ?NB indicates Env-deleted pNHcRedEluc. (D) The time-course analyses. Arrowheads indicate the regions of observed GFP signal. The time after
co-culture is indicated in the upper left of each image.
Page 6
J. Wang et al. / Journal of Virological Methods 161 (2009) 216–222
ruled out by the absence of red nuclei in the syncytia. This result
indicates that the simultaneous use of pNHcRedEluc and spGFPs
allows us to monitor membrane fusion directly without the addi-
tion of dyes or substrates.
3.3.2. Analysis of HIV-1 Env-mediated fusion using an inhibitor
and Env mutants
The specificity of the spGFP assay was examined by using the
known inhibitor of the HIV-1 Env-mediated membrane fusion, C34
(Seo et al., 2005). The C34 peptide is known to inhibit the forma-
tion of 6-helix bundle. Two different concentrations, 12nM (IC50)
and 150nM (IC90) (Kliger et al., 2001), were tested in spGFP fusion
assays. For a comparison, in addition to the spGFP assay, the fusion
assay using the Env(+)- or receptor(+)-cells expressing GFP and
DsRed, respectively was used. In the spGFP assay, the number of
the GFP signal-positive cells was decreased in a dose-dependent
manner (Fig. 4B, left column). In a parallel assay, the number of the
in the fused cells was decreased similarly (Fig. 4B, right panel). The
new spGFP assay was much easier to monitor, because the green
signal was only observed when the actual fusion took place. In a
conventional method relying on the mixture or redistribution of
the two colors was more time consuming, because each cell has to
be scored for the presence of either or both colors.
The previously described fusion-inefficient mutant Env that
carries the mutation in the membrane-spanning domain (MSD)
sistent with previous data (Miyauchi et al., 2005), the MSD mutants
showed the fusion events but less frequently, as exemplified by the
lower number of cells bearing the green signal (Fig. 4C).
3.3.3. Real-time membrane fusion assay
The membrane fusion in a real-time manner using spGFP sys-
tem was monitored with an IN Cell Analyzer 1000. The green signal
derived from the reassociated spGFPs gradually increased in the
number and intensity over the observation period (Fig. 4D). When
T7 RNA polymerase transfer assay was applied (Miyauchi et al.,
2005), a corresponding increase in the reading of the reporter
enzyme was observed (data not shown). Using the transient trans-
fection system, we performed several tests to determine the timing
needed to detect the green signal. Sometimes the signal was
however, was obtained after more than 1h co-cultivation.
4. Discussion
A cell-based assay system of membrane fusion that uses spGFP
has been developed. It was found that the PH domain not only
localizes the signal resulting from the spGFPs reassociated to the
membrane but also aids the stable expression of GFP11.
The new spGFP-mediated system is cost- and labor-efficient.
First, the same living co-cultured cells can be monitored for real-
time monitoring over a prolonged period (Fig. 4D). Second, the
reassociated spGFPs will produce a measurable signal without
any additional reagents. The dye-mediated fusion assay requires
preloading of dyes before the fusion reaction (Blumenthal et al.,
2002). A quantitative fusion assay using enzymes, either as pre-
expressed self-complementing enzyme fragments or as induced
reporter enzymes, requires the addition of enzyme substrates to
and Wickner, 2007). Furthermore, if a particular substrate is mem-
brane impermeable, continuous monitoring of the same sample is
impossible because the cells have to be lysed for the assay.
Simple assay systems described in this study are suitable for
high-throughput analyses. The combination of dye-transfer and
fluorescence-activated cell sorting can achieve this (Huerta et al.,
2002; Lin et al., 2003). However, the system reported in this study
is much simpler than these methods because it generates the
detectable signal only when fusion actually occurs. In the dye-
transfer assay or similar "color"-mixing assay shown in Fig. 4B, one
the dye signals are persistent throughout the assay.
induced by mutant Envs was detected as visible foci with the spGFP
system. If one can clone the envelope genes from clinical samples
into an appropriate expression vector, this system may be useful
for detecting a minor population of Env that possesses the differ-
ent co-receptor usage. Such a tropism assay can be easily adapted
by using CCR5/CD4+ cells together with CXCR4/CD4+ cells. Simi-
lar identification of fusion foci can be achieved if GFP is used as a
reporter gene in a transcriptional factor transfer assay, such as T7
RNA polymerase or Tat (Barbeau et al., 1998; Feng et al., 1996; Lin
et al., 2005; Sakamoto et al., 2003), but the need for de novo tran-
scription/translation steps may result in a longer lag time for signal
can be monitored easily.
This spGFP-based system does not require on-going transcrip-
tion/translation steps during membrane fusion. Therefore, when
the tag for a different intracellular compartments is applied, the
system can be used to detect communication between two com-
partments in the cell, such as that which occurs in vesicular
transport. The application of the spGFP in other biological systems
has been described previously (Feinberg et al., 2008).
This work was supported by a contract research fund from the
Ministry of Education, Culture, Sports, Science and Technology of
Japan for the Program of Founding Research Centers for Emerging
and Reemerging Infectious Diseases. We thank Dr. Kunito Yoshi-
ike for his critical reading of the manuscript. We also thank A.M.
Menting, an editorial consultant, for help in the preparation of the
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text:
Upcoming Events
|February 27, 2015
|The 2015 Edwards Family Lecture in Business Ethics
David Dick
3:30 to 5:00 PM
"The Counterfeiting Problem" [abstract]
|March 5, 2015
|New Feminist Research Lecture
Mary Bunch,
Sponsored by Women's and Gender Studies, the Interdisciplinary Centre for Culture and Creativity, the Humanities Research Unit, the Department of Philosophy, Disability Services for Students, USSU Women's and Pride Centers, and the Role Model Speaker Fund
For more information, contact Prof. Marie Lovrod
|March 6, 2015
|The 2015 Walter Murray Lecture
James R. Brown
"Thought Experiments and The Poverty of Empiricism" [abstract]
Fridays, 3:30 - 5:00
Department Colloquium Series: 2014-15
All talks in Edwards School of Business (ESB) Room 112
|Sep. 19
|Brian Zamulinski
|"Morality as Natural Constitution"
|Oct. 24
|Emer O'Hagan
|"Self-Unity, Identity and Self-Accounting"
|Nov. 7
|Geordie McComb
|"Demons and Details in the Laboratory of the Mind: Can Complex Literary Fictions be Thought Experiments?"
|Nov. 28
|Derek Postnikoff
|"Metametaphor: An intriguing reflexivity"
|Jan. 16
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|"Is Semantics a Mistake?"
|Jan. 30
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|"Painfully Good: The Moral Dimension of Aquinas' Pain"
|Mar. 13
|Ryan Doran
(University of Regina)
|"Demonstrative Reference and Speaker Intentions"
|Mar. 27


text:
Double room with bathroom and shower on the premises of an amazing resort with pool, restaurant and wine bar near Lake Bracciano, 35 minutes outside of Rome.
Central heating or a heater in the listing
Space where guests can cook their own meals
Towels, bed sheets, soap, and toilet paper
Central heating or a heater in the listing
In the building, free or for a fee
In the building, free or for a fee
Continuous access in the listing
Breakfast is provided.
The listing can accommodate a gathering of 25 or more attendees
Easy access to the building and listing for guests in wheelchairs
Private or Shared
Free, in the building or nearby
Posted emergency information and resources
The Space
At the border of a natural park, close to the shores of a large, clean lake, inside a territory rich with memories of three thousand years of history, and only one step away from Rome, Residenza di Rocca Romana!
Kids up to 14-years 50% reduction
Children up to 3-years free (meals as per consumption)
Added bed adults 30% reduction
Guest Access
All rooms are with private bathrooms, air conditioning, satellite TV, telephone and refrigerated bar and, in most cases,a kitchenette. Wi-Fi connection in dedicated areas
The Neighborhood
The shores of the lake are less than 5 minutes away, the natural environment is magnificent, with its protected wild flora and fauna, the climate is mild, never too hot in summer or too cold in winter time.
You won't believe that such a place is only 30 minutes away from Rome.
At your disposal, inside the Resort, triple swimming pool, mini soccer field, bikes, typical restaurant and wine bar.
By choosing Rocca Romana, you can experience a wonderful vacation, enjoy the sunshine, divide your time between the beaches, the resort pool and the treasures and the atmospheres of Rome.
You will enjoy sports and outdoor life , swimming, windsurfing or sailing in the lake, biking or riding in the woods, playing golf or running cars (nearby facilities: golf club " Le Querce" and Vallelunga racing circuit).
You will relive historical moments and behold the beauty of art, travelling (all in one day) from the Etruscan civilization and the memories of the Roman Empire, through the monuments of the Italian medieval mysticism, up to the masterpieces of Renaissance and Baroque Rome.
The resort is very idyllic and Antonello is very courteous. He was there when needed and gave us a lift to the village every single day.
What you should be aware of. In September the place is very quiet. There were no other guest in the entire resort except us. As it is situated on a hill surrounded by forests it turned out to be a bit too isolated and too quiet for two young girls like we are. Therefor, to come by car is highly recommended otherwise you are pretty dependent on a lift (10 minutes to the lake 6 town by car). But if you are sportiv and willing to go down and up the small mountain by bike you can borrow Antonello's bikes, which is great thing.
The Wifi was only working IN the restaurant of the main building. So no internet and no network in our bedrooms - at least not in ours.
All in all, as I said before, a wonderful & idyllic place for vacation, if you like it quiet, if you have a car or if you are athletic :) Antonello & his wife, as well as his employees were really nice. Thank you Antonello, it was a pleasure!
We were the only guests at this resort as it was the end of tourist season. Antonello was very friendly and offered lots of recommendations. Our accommodations were satisfactory for our one night stay.
This host has 4 reviews for other properties.View Other Reviews
We are a family owned resort near Lake Bracciano and just 35 minutes away from Rome!
Have a look at our beautiful rooms and premises!
Do not hesitate to contact me with any requests, questions or for further informayion on the premises or services.


text: We are from Pasadena, CA, and are two retired school teachers -- 2 women, married under CA law. We are well traveled, quiet, but very interesting. We look forward to the day when DOMA (Defense of Marriage Act) is repealed. Today was the first step toward victory with the California ruling Prop. 8 to be unconstitutional. We would enjoy being full citizens! Let us know if you have any further questions...we will be with our family for most of that weekend.


text:
2011 in the Arab world: Change we can believe in?
Regionally and globally we are coming to the end of a significant year. Manifold "awakenings" have altered the strategic, social and political context of a region I refer to as "West Asia North Africa" (commonly known as the Middle East and North Africa) says Prince Hassan bin Talal of Jordan.
December 26, 2011 12:46 by p.deleon
On a global level, profound stresses in the world economy — which are not solely confined to Western countries — have exposed a hyper-globalized age in which opportunity, equality, fairness and social mobility appear to have been compromised to an unacceptable degree. Deficits in national budgets around the world have created and accentuated a trust deficit, as well as a human dignity deficit in both the West and West Asia North Africa. This reality has resulted in the Occupy movement in London and the United States, and the "Indignant" movement in Spain, both of which were influenced by, and find common cause with, the Arab Awakening.
What we may be seeing is the birth of a new global rhetoric born in the West Asia North Africa region. This rhetoric essentially argues that human beings are "too big to fail." In 2012 it will, however, require further articulating: None of the demonstrations in Tahrir Square in Cairo, on Wall Street in New York City, or elsewhere, have been intellectually or politically codified. The concept of human security has instead been vindicated by events on the ground: A critical mass, made up of a once-silenced majority, has surfaced.
But Tweeting – which is so closely associated with recent protests — is no substitute for thought, and passion no substitute for discipline.
We have become a region of a million accents; decades of divide and rule have rendered us incapable of speaking with one single voice. Because of this, others from outside of the region, too often do the talking for us.
Its time to develop new narratives — and a greater sense of unity. A year ago, on Jan. 1, 2011, 21 people were killed in an attack on a Coptic church in Alexandria; 25 days later Muslims and Christians prayed beside each other in Tahrir Square. It did not last long — sectarian tensions have resurfaced in Egypt, in Bahrain and across the region as a whole. It did however suggest that the frictions which so often seem to split this region asunder are, in the final estimation, far more brittle than they appear.
The Arab world is often depicted as a place fighting against everything, particularly progress. But over the past few months it has been the men and women of this region who have led the way in fighting for what has been described as universal rights: For justice, transparency and accountability, for the right to have both a career and a family and for the right of Palestinians to become citizens in their own sovereign territory. The future of West Asia North Africa is being re-engineered, contested and heavily fought across a wide range of arenas. That future remains uncertain and its contours opaque — but its embers are glowing.
Our region requires a bill of rights that prohibits all forms of discrimination, but also outlines responsibilities. We need to engage what I refer to as "third sphere" communities — made up of civic organizations, government and the private sector — to engender meaningful models of social cohesion and social inclusion. We need to invest in research and development — and incubate not just businesses but educated human beings.
In 1987, I was part of a commission which presented to the UN General Assembly a report which called for a "new international humanitarian order." The report, entitled "Winning the Human Race," argued that economic growth and national security were overwhelmingly dependent on individuals and community well-being.
The conclusions which can be drawn over the past 12 months seem to accord with the findings of this 24 year-old report. In a sense both suggest that the real "change you can believe in" comes not from governments, armies nor institutions — but from individual human beings.
— Prince Hassan bin Talal of Jordan is chairman and founder of the Arab Thought Forum and the West Asia North Africa Forum. This article was written for the Common Ground News Service (CGNews).
First published in Arab News
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Are Saudi taxis better than UAE taxis?
Smartwatches: The talk of Baselworld
Facebook: Most used social media channel in the UAE
Top five things to do in the UAE this weekend
UAE: Sixth largest prepaid market
Occupationism and Emiratisation
E-commerce market large in GCC, but not yet fully taken in UAE
Cybercriminals are winning in the Middle East
Dubai house prices decline by more than six per cent
Seven reasons why you should not be nervous to have your bank details online
Phone calls on Whatsapp?
The importance of marketing to women
Strong job outlook in the UAE for 2015
Arab women's top online habits
Top 5 things to do in the UAE this weekend
Apple Watch to come to the UAE in April?
Lately on Kipp
Top 5 things to do in the UAE this weekend
Top three trends in the automobile industry
Dubai World gets approval to restructure $14.6 bln debt outside court
VIDEO: Top tips to branding in a digital age
AED100K up for grabs in Dubai Expo 2020 logo competition
Mobile check-in taking off


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See a narrow family room transformed!
Tour a fun and feminine space.
Tour the condo of Holt Renfrew's Lisa Tant.
Lynda Reeves explains how book styling has evolved from the classic library to carefully curated modern shelves. Learn her tricks for a perfectly styled bookshelf, from dark painted shelves to simple colour blocking.


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This year the Sherbino Theater in Ridgway is celebrating its one-hundredth anniversary.
The theater is known for the musical acts and guest lecturers it brings to the Western Slope.
A Tour Of The Historic Sherbino
"The building was built by Louis Sherbino," Patrick O'Leary, the president of the Ridgway Chautauqua Society, says. "And he built the theater so that his son and daughter-in-law would have a place to play. His son was a fiddle player."
The Chautauqua Society now runs the theater.
Recreational and medical marijuana facilities are allowed in Ridgway. The town has one hybrid storefront that sales medical and retail pot. A town ordinance passed last month puts new restrictions on marijuana grow operations in Ridgeway.
KVNF's Laura Palmisano speaks to the town's Mayor John Clark about the new rules.
The 5th & final week of the Ridgway Summer Concert Series featured Peter Rowan with Dirty Kitchen, along with opening act Bruce Hayes. (Dirty Kitchen bandleader Frank Solivan was absent due to the recent death of his mother. Our condolences to the Solivan family.)
Week 4 of the Ridgway Summer Concert Series
Week 3 of the Ridgway Summer Concert Series
Orgone is a synergy of eight musicians, at the core of which are keyboardist Dan Hastie and guitarist Sergio Rios, who have been chiseling their distinctively timeless sound since their teens. Each band member shines in his own right, but together their chemistry results in a sweeping sound more tenacious than the sum of its considerable parts.
Week 2 of the 2014 Ridgway Concert Series features Halden Wofford & The Hi-Beams, with opening act Lash LaRue.
Week 1 of the 2014 Ridgway Concert Series
Paper Bird is a seven-piece Indie Folk/Baroque Pop band based in Denver, Colorado. The band is composed of sisters Genny and Esme Patterson (both vocals), brother and sister Mark (drums) and Sarah Anderson (vocals and cornet/trumpet), Paul DeHaven (guitar), and Caleb Summeril (Bass, Banjo, Guitar).
Last October the Colorado Department of Transportation (CDOT) announced the names of 44 towns across the state awarded road improvement monies from a competitive grant program called RAMP. In Ridgway, Colorado the community will be going to the ballot box on April 1st to determine whether or not they will make use of their $10.4 million one-time-only, use-it-or-lose-it award.
KVNF's JT Thomas speaks with Ridgway mayor, John Clark and community members, Tammee Tuttle and Jill Markey.
KVNF Live! Week 8 - recorded at the final concert of this year's Ridgway Concert Series, Thursday, July 25th, 2013.
KVNF Live! Week 3 from the Ridgway Concert Series, featuring Pato Banton & the Now Generation, with opener Lipbone Redding.
For your listening pleasure, here is audio from Week 2 of the Ridgway Concert Series, featuring Mountain Heart and opening act The Matt Flinner Trio. Your host for the broadcast was KVNF volunteer Jon Hickam.
Concert audio from the July 4th Ridgway Concert with Yo' Mama's Big Fat Booty Band and opening act Isa & JoeAlan. Unfortunately, a power interruption during the second set caused file corruption in our recording, so we can only offer the last portion of the 2nd set by YMBFBB. UPDATE: Corrupted file fixed, 1st half of 2nd set now available.


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Top Gear 2
- Publisher: Kemco
- Developer: Gremlin Interactive
The first racing game to make the cut for our countdown, Kemco's Top Gear 2 represented a step up from what racing games had been in the previous generation – but not too drastic a step. Top Gear 2 looks, feels and plays a lot like NES titles like Rad Racer did years before, with the boost of 16-bit processing power giving the whole experience a fresh coat of paint.
A selection of courses set around the world, a vehicle upgrade system and new weather effects kept Top Gear making progress toward what more traditional racing titles would eventually offer in the future, but in the end the SNES was more defined by its all-new takes on racing (like F-Zero's futuristic hovercrafts and Super Mario Kart's item-shooting go-karts).
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News and events
A light touch to reading electron spins
Scientists at UCL have shown a new way of reading electron spins, which eliminates the need for powerful magnetic fields and reduces the reliance on very low temperatures. The study is published this week in the journal Nature Materials.
Published: Mar 26, 2015 9:49:40 AM
EU Science Commissioner visits UCL
The European Commissioner for Research, Innovation and Science, Carlos Moedas, visited UCL today. He held a meeting with the President & Provost of UCL, Professor Michael Arthur, before being visiting the London Centre for Nanotechnology.
Published: Mar 24, 2015 4:21:56 PM
Superfast computers a step closer as a silicon chip's quantum capabilities are improved
Research has demonstrated laser control of quantum states in
an ordinary silicon wafer and observation of these states via a conventional
electrical measurement.
Published: Mar 20, 2015 11:42:14 AM
UCL forging closer big data links with Japan
Patrick Wolfe, executive director of UCL's Big Data
Institute has recently returned from Japan, where he was working on developing
closer links between UCL and Japanese institutions.
Published: Mar 17, 2015 11:11:01 AM
Nanospheres cooled with light to explore the limits of quantum physics
A team of scientists at UCL led by Peter Barker and Tania
Monteiro (UCL Physics and Astronomy) has developed a new technology which could
one day create quantum phenomena in objects far larger than any achieved so far.
Published: Mar 17, 2015 9:44:12 AM
Dr Codina Cotar awarded £98k EPSRC First Grant to study Continuous Gradient Models
Dr Codina Cotar has been awarded the EPSRC Grant EP/M027694/1 under the First Grant Scheme. The project is called "Continuous gradient interfaces with disorder".
Published: Mar 23, 2015 9:56:20 AM
Toxic World
STS first year PhD Student, Alex Mankoo, has won a competitive award to attend the 8th European Spring School on History Of Science And Popularization. This year's theme is "Living In A Toxic World (1800-2000), Experts, Activism, Industry And Regulation" and will be held at Maó (Menorca), 14-16 May 2015. There will be talks by a number of experts on all aspects of the history of toxic environments, as well as poster and workshop sessions on the same theme. Alex's doctoral research is on the history tear gas, which fits well with the general theme of the Spring school.
Published: Mar 20, 2015 6:43:45 PM
Science Communication Careers Event for STS Students
4 March 2015 STS MSc students were joined from professionals from science communication to learn more about potential careers.
Published: Mar 17, 2015 5:08:41 PM
New book: Politics of Geoengineering
Congratulations to Jack Stilgoe on the publication of his new book:
Published: Mar 14, 2015 7:40:07 AM
The use of time resolved aerosol assisted chemical vapour deposition in mapping metal oxide thin film growth and fine tuning functional properties
by Nicholas P. Chadwick, Sanjayan Sathasivam, Salem M. Bawaked, Mohamed Mokhtar, Shaeel A. Althabaiti, Sulaiman N. Basahel, Ivan P. Parkin and Claire J. Carmalt.
Published: Mar 12, 2015 3:10:39 PM


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Faber is a populated place located in Nelson County at latitude 37.832 and longitude -78.738.
The elevation is 594 feet. Faber appears on the Schuyler U.S. Geological Survey Map. Nelson County is in the Eastern Time Zone (UTC -5 hours).
Nelson County: Cities & Towns | ZIP Codes | Schools | Features (airports, churches, hospitals, etc.)
BIG Map |
Driving Directions |
Data Source - USGS GNIS FID: 1499400
BOUNDARY MAPS: Central District | Nelson County
Faber, VA
(< 0.1 mile)
Rockfish, VA (2.4 miles SSW)
Lawthorne Mill, VA (2.7 miles W)
Schuyler, VA (3.4 miles SE)
Old Dominion, VA (3.9 miles E)
Twin Poplars, VA
(4.1 miles WNW)
Damon, VA (4.3 miles E)
Covesville, VA (4.3 miles NNE)
Woods Mill, VA (4.7 miles W)
Adial, VA (5.6 miles WNW)
Tillmans, VA
(5.5 miles E)
Old Myndus, VA (6.4 miles W)
Ottoway, VA (6.4 miles NNW)
Gordon Crossing, VA (7.7 miles SW)
Powell Corner, VA (8.7 miles ENE)
Miran Forest, VA
(9.5 miles N)
Totier Hills, VA (11.4 miles E)
Oak Ridge, VA (11.4 miles SW)
Freshwater, VA (11.8 miles WSW)
Ramsey, VA (12.4 miles W)


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When we think of historical buildings we often think of castles and churches, but there's much more to explore. Our towns and villages are brimming with architectural delights from banks to factories to inns and market halls, all waiting to be photographed outside and sometimes (if you're lucky) inside. All you need is a little local knowledge.
What Gear Will I Need?
For general shots you will need a good wide-angle. Use a 70-300mm to zoom in on the intricate detailed wood carvings and stone work around the building. A powerful flash can be really useful to fill-in or light pokey areas of interiors or paint with light on an external wall and use a polarising filter if the building has windows, to reduce reflections in the glass. The polariser will also darken a blue sky and give more contrast to the shot.
When it comes to bag choices, bulky rucksacks are often a no-go in many historical buildings as they could knock over artefacts or bump into people in tight spots. Travel light with a shoulder bag such as the Stile Messengers from Manfrotto. This style of bag can swivel around and be accessed from your front, so it makes it convenient in tight spots and busy locations.
Check What Equipment You Can Take
Many historic buildings have been taken over by trusts, such as the National Trust or English Heritage. These give you access to the interiors which have often be preserved, so you have a better idea of how that building was when it was in use. It always means that although you can go in and wander around you're often restricted to what you can and can't photograph and you're often charged an entrance fee. Flash is often banned as are tripods. Some even prevent you from taking photos at all. Check before you go on a long journey by visiting the website or make a phone call.
If you can take pictures, but can't use a tripod or flash, increase the ISO setting and support the camera on a wall, pillar or sign post to prevent camera shake. Do watch out for signs of noise, though (the picture broken up as small colour dots that can make it look poor quality).
Historic Buildings Can Be Dark
Many historic buildings were not built with the light aspects found with modern buildings. Windows were often small and poky so the light coming through could be in narrow shafts causing chaos for your camera's exposure system. In such cases either point at an area without the light and take a reading knowing the highlights will be overexposed, or shoot a few frames and merge them using a HDR program so you have a balance of highlights and shadows.
Look For Details
Shoot Themes
How about a theme? You could pick one type of historical building, say market hall, and go around the country collecting shots of them. Every time you visit a new town and see if they have a market hall and take its picture. Lighthouses, piers, windmills, castles, pubs could all prove interesting collections.
Avoid People And Cars
Try to take external shots without people or cars in the frame, both will date the photo. A weekend or early morning will be better if the building is in a town or city centre.
Height And Angles
Find an external position with some height to reduce converging verticals when shooting with a wide-angle. Steps on a nearby building or a hill will help. Some professionals take step ladders although for most of us this is not often practical.
On ruins walk around looking for the best angles. Some sections are so bad that the shot will just look like ruins whatever angle you shoot from, whereas other angles will at least give a feeling for shape and style. Use brochures and guides to give you ideas of best angles but do look for your own original take on the building as well.


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I have stayed here numerous times, in travelling to one of our offices is nearby. The fact that is adjacent to a mall, right off 77, and near several places to eat is a big plus. In addition the rates are very reasonable, and the staff is very friendly. Not to mention the free snacks/drinks available, as well.
- Official Description (provided by the hotel):
- Welcome to the Queen City, home to the Charlotte Motor Speedway, family fun at the Carowinds and the U.S. National Whitewater Center. Located adjacent to Northlake Mall, the Drury Inn & Suites is the place to stay during your visit. Drury Hotel offers its guests a variety of free extras – take, for example, our free hot breakfast. At Drury, "hot" means scrambled eggs, biscuits & gravy, sausage and make-your-own Belgian waffles. In the evening, join us at our 5:30 Kickback® for free hot food and cold beverages. Our rotating menu features a variety of hot foods, baked potatoes, garden salad, beer, wine, mixed drinks and more! To burn off all of those free extras, Drury has a 24-hour exercise center and a parking lot to jog around. Lastly, Drury's free wireless Internet and free long distance calls will ensure you can keep tabs on all of your posse back home. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Travelocity, Booking.com, Expedia, Drury Hotels Company, LLC, Orbitz, Hotels.com, Cheap Tickets and Odigeo so you can book your Drury Inn & Suites Charlotte Northlake reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
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- Drury Inn Charlotte


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Game 73 Preview: Sacramento Kings at Memphis Grizzlies goo.gl/fb/qJjDH7
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Ryan Hollins sees good in lost Kings season goo.gl/fb/Cq0eES
Sunday Musings: Does a return of Geoff Petrie make sense? goo.gl/fb/OSn0cp
Derrick Williams adds outside scoring to his resume goo.gl/fb/gSvYQt
CK Road Reaction Postgame Show: New Orleans Pelicans 102, Sacramento Kings 88 goo.gl/fb/PUElDl
David Stern addresses media at All-Star Weekend in Houston. (Photo: Steven Chea)
Jonathan Santiago serves Cowbell Kingdom as senior editor specializing in writing, podcasting and video production. He also handles the majority of CK's day-to-day beat coverage of the Kings.


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What's an activist kindergartener to do if he can't wear his Halloween costume to school?
Sure, I said, citing steak tartare. Then Max asked "what about chicken?"
What's the penalty for driving 370mph over the posted Minneapolis city speed limit?
Would that be illegal? What about killing a bald eagle in a "stand your ground" state?
I'm sure you can ride a bicycle drunk, but is it legal?
Children don't generally have property rights except through parents or guardians, right?


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9 spring-y things to enjoy way past break
Ask Cliff: What do boys actually like doing on dates?
This clever video reminds us that cyberbullying is so not cool
Exclusive: A Girl Like Her's Hunter King dishes on the new movie, the problem with bullying and how to stop it
6 promposals that are just too cute to handle
Check out our spring style obsessions!
Trend alert! These gorgeous highlights will have you sprinting to the salon
Ouch! Could all that texting seriously harm ya?
Share your thoughts on our April/May 2015 music issue! What did YOU love?
Nfinity Champions League 2: The Biggest Night in Cheerleading
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Latest Cephalopod Stories
Drawing inspiration from the color-changing capabilities of cephalopod skin, researchers have developed a new camouflage sheet capable of quickly reading its environment and adapting to mimic its surroundings.
Scientists from Yale University studied the extinct pterygotid eurypterid, a giant sea scorpion, the largest arthropod that ever lived. It was always believed to be a fierce predator, but a recent study revealed that may not have been the case.
A new study has revealed a natural nanoscale photonic device that allows the mysterious cuttlefish to dynamically change its colors.
Humboldt squid have overrun the waters off the coast of Southern California, and the area's fishermen have taken to the sea en masse—catching boatloads of the ultra-fresh calamari.
Research out today from a multidisciplinary team headed by the University of Cincinnati examines parallels between e-Paper technology (the technology behind sunlight-readable devices like the Kindle) and biological organisms that change color.
An international team of scientists have for the first time discovered two 160-million-year-old giant cephalopod fossils with intact ink sacs that contain dried pigment similar to that of modern cuttlefish.
Researchers from the University of Bristol have created artificial muscles that can be transformed at the flick of a switch to mimic the remarkable camouflaging abilities of organisms such as squid and zebrafish.
For 300 million years, they were the ultimate survivors.
Researchers from Swedish and American universities say that they have solved the mystery as to why giant and colossal squid have such enormous eyes, and perhaps unsurprisingly, the oversized ocular orbits are essentially a defense mechanism.
Scientists have discovered two deep ocean species of cephalopods, the octopus Japetella heathi and the squid Onychoteuthis banksii, that can go from transparent to opaque in the blink of an eye.
- One who brings meat to the table; hence, in some countries, the official title of the grand master or steward of the king's or a nobleman's household.


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As vast as it is diverse, encompassing desert, savannah, rainforest, mountain ranges, lowlands, stunning coastline and vast inland lakes, Africa is a continent rich in dramatic scenery and natural beauty. Visiting Africa, you can see the world's most prolific concentration of wildlife in their natural habitat, experience the warmth of African tribal people and their diverse cultures and beliefs, travel through stunning landscapes and challenge yourself with some amazing adrenalin-fuelled adventures.
All about Tanzania
This week we are dreaming of Africa, with its outstanding natural beauty, wonderful wildlife and diverse culture and people. For many, the East African country of Tanzania is the best on the continent for embarking on a thrilling wildlife … Continue reading
Safari Safari
Spot strips, spots and manes in the African bush, witness Masai Warriors cloaked in red performing the traditional jumping dance, come eye to eye with a majestic silverback gorilla and experience bird's eye views of endless barren plains in … Continue reading
Incredible Victoria Falls
Whether you find yourself in Victoria Falls at the start or end of your overland safari through East or Southern Africa, why not take advantage of all this incredible location has to offer. Whether you are an adventure seeker … Continue reading
Colours of Zanzibar
While the palm-fringed white sand beaches and warm azure waters of Zanzibar are a picture of paradise, this tropical isle has so much more to offer. This week's video pick takes a look at the local people that call … Continue reading
Victoria Falls to Johannesburg
In March our own Mike Quinto travelled to Zimbabwe, Botswana and South Africa over the course of our 12 day Falls to Jo'burg tour. From the stunning Victoria Falls and walking with lions, to taking a mekoro ride down … Continue reading
Stopping wildlife poachers in their tracks in Africa
Lion, elephant and rhino – three of Africa's majestic Big Five are all in increasing danger of extinction due to prevalence of wildlife poaching across the continent. The African Lion and Environmental Research Trust (ALERT) tells us this week about … Continue reading
Victoria Falls to Johannesburg – Part 2
Last week travel agent Simon Pacey began telling us of his journey from Victoria Falls to Johannesburg, of high-wire adventures, walking with lions and cruising the Zambezi River. This week he continues his journey into Botswana for his first safari and a macorro cruise in the … Continue reading
Climbing Kilimanjaro
Standing at a staggering height of 5895 metres, the iconic snow-capped peak of Kilimanjaro can easily be found at the top of any adventurous traveller's wish list. But what is it really like to climb to the highest point … Continue reading
Victoria Falls to Johannesburg – Part 1
"I have to begin by admitting that Africa was not a high priority on my travel list", says travel agent Simon Pacey. "It was the kind of trip I was happy to put off until much later in life. … Continue reading
Best places to visit in June
The ninth part in a twelve part series on the best places to visit when. June is all about the wonderful wildlife and stunning landscapes of Namibia, the Roman ruins and sublime coastline of Turkey, the sandy beaches and azure waters of … Continue reading


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Jeff Fountain scored a career-high three goals, including the game-winner in overtime.
Feb. 24, 2013
WASHINGTON - Junior attack Jeff Fountain (Lutherville-Timonium, Md./St. Paul's School for Boys) scored on an assist from redshirt junior Zac Guy(Tully, N.Y./Tully) with 2:04 left in overtime, as the Georgetown University men's lacrosse team (1-1, 0-0 BIG EAST) defeated Navy (2-2, 0-0 Patriot League), 9-8 on Sunday afternoon to claim the Boyle Cup.
Fountain finished with a career-high three goals, while senior attack Brian Casey (Bethesda, Md./Georgetown Prep) had two goals and two assists to lead the Hoyas to its first win under Head Coach Kevin Warne, who played its second overtime game in as many weeks.
"Athletics is an unbelievable teaching tool and it allowed us to learn from last week and to progress this week," Warne said. "We're far from done, but I'm happy for the guys they responded to the coaching staff this week and we got a W."
The Hoyas trailed by as many as three goals in the first half, but redshirt junior Tyler Knarr (Eagleville, Pa./LaSalle College) won nine of his 11 face-off attempts after halftime, going 13-for-19 on the afternoon, while picking up 12 ground balls. Freshman goalie Alex Joyce(Chevy Chase, Md./Landon) earned his first collegiate win.
"I thought Tyler Knarrwas awesome today," Warne said. "It's unique facing off with a long stick, but Tyler is quick enough to get the ball out and he did a great job."
Georgetown took an early advantage after Knarr won the opening face-off and got the ball into the stick of Casey. Casey found sophomore attack Reilly O'Connor(Brooklin, Ontario/The Hill Academy), who scored just seven seconds into the game, to take a 1-0 lead.
The Midshipmen answered quickly, as Gabe Voumard tied the score with 13:42 remaining in the first quarter. Pat Durkin and Sam Jones each found the back of the cage soon after, giving Navy a 3-1 lead with 10:35 left in the first. The score remained the same for close to 19 minutes, until Jones put Navy in front 4-1 with 6:45 remaining in the first half.
Fountain wrapped around the cage and scored to end a 4-0 Navy run with 1:38 left in the first half, bringing the Hoyas within two goals, at 4-2. He would bring the Hoyas within one goal early in the third quarter, scoring on an assist from Casey at the 12:01 mark.
After a Navy goal, Georgetown stormed back tying the game. Casey scored an unassisted goal at the 5:48 mark in the third quarter. Sophomore midfielder Charlie McCormick (Bronxville, N.Y./Bronxville) added the equalizer 22-seconds later, making the score 6-6 with 5:26 left in the third quarter.
Georgetown and Navy traded goals throughout the rest of regulation. Senior midfielder Jason McFaddenBel Air, Md./Calvert Hall) evened the score up again with 13:30 in the fourth quarter. Navy took the lead with 5:26 remaining in the game on Jones's third goal of the afternoon, but McCormick answered with his second goal of the game, 43-seconds later, to tie the score at eight.
Navy took the lead again, this time on a Durkin's goal, but Casey quickly tied the score 22 seconds later on an assist from Guy with 2:22 left in the fourth quarter.
In overtime, Knarr won the opening face-off to give Georgetown possession and Guy found Fountain less than two minutes later for the game-winner, sealing the Hoyas first win of the season and capturing the Boyle Cup for the seventh time in eight games.
"I got the ball back up top to Zac Guy, saw no one was guarding me, so I cut to the back side, (Guy) hit me with the pass from up top, the goalie stepped out and I just put it low," Fountain said about the game-winning goal.
NOTES: Becky Boyle and family were in attendance and presented the Boyle Cup to Georgetown Head Coach Kevin Warne and the team's captains following the Hoyas 9-8 overtime victory...GU also won by a final of 9-8 the last time the two teams met in 2011...Since the Boyle Cup began in 2005, five of the eight games have been decided by one goal and three games have gone to overtime.
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Shoe Racks
Showing items 1 to 16 ( 62 in total )
Shoe Rack Metal 2 Tier
Keep your shoes neat and tidy with our 2 tier metal shoe rack. This rack will store and display up to 6 pairs of shoes on its...
from Wilko.com
Wilko Black Horn Shoe Rack
Keep your shoes neat and tidy with our handy metal shoe rack. This rack will store and display up to 6 pairs of shoes, while the...
from Wilko.com
Wilko Storage Four Tier Shoe Rack 75cmx73cmx30cm
Chrome effect, holds up to 12 pairs of shoes and easy assembly. Designs may vary.
from Wilko.com
Silver 3 Tier Extending Shoe Rack Silver
Buy our three tier extending shoe rack online today for a simple way to store your shoes and boots safely and keep...
from Dunelm
Silver 5 Tier Extending Shoe Rack Silver
Buy our five tier extending shoe rack online today for a simple way to store your shoes and boots safely and keep...
from Dunelm
Vintage Cream 2 Tier Shoe Rack Cream
Available to purchase online now, this Vintage two tier shoe rack is a smart way to store your shoes in style. An elegant...
from Dunelm
Brown 3 Tier Shoe Rack Natural (Brown)
Buy our three tier wooden shoe rack online today for a simple way to store your shoes and boots safely and keep your...
from Dunelm
Vintage Black 2 Tier Shoe Rack Black
Store your shoes in style with a vintage black metal 2-tier shoe rack, available at Dunelm Mill today.A beautiful place...
from Dunelm
Shoe Rack "Braid Effect" with 5 Shelves
This shoe rack ensures that you will be able to now store your shoes tidily and safely. It takes up to 10 pairs...
from Westfalia Mail Order
Extendible Shoe Rack Wenko
An easy way to tidy up your shoes. Tidy up your hallway, wardrobe etc using this shoe rack in a highly modern chrome look. Stop...
from Westfalia Mail Order
Expanding "No Slip" Shoe Rack (2 - SAVE £10)
Keep your shoes in tidy order with these shoe racks - and you'll even SAVE £10 when you buy this set of two!...
from Scotts of Stow
Expanding Shoe Rack
Most shoe racks have poles rather than shelves, so shoes can slip through or fall down the back. Exclusively made for us, these...
from Scotts of Stow
J-Me Horizontal Shoe Rack
Horizontal is a stunning and simple shoe rack that is perfect for the entry hall or bedroom. It ensures that your shoes are kept...
from Design 55 Online
Headsprung Wired Shoe Rack
Headsprung have created the answer to all your problems of shoe storage - the wired shoe rack is a simple but ingenious design...
from Design 55 Online
Wall rail with shoe racks
- Order wall rail and shoe racks separately (each pair of wall rails hold upto 6 shoe racks, please see Don - t Forget to Order)...
from Locks Online
Extending Shoe Rack for up to 12 Pairs of Shoes
This clever extending shoe rack holds up to 12 pairs of shoes. Double the size of your shoe rack by placing...
from La Redoute
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Cage The Elephant are playing in Honolulu, Hawaii at the Blaisdell Arena Waterfront at Aloha Tower on February 8, 2012. Also on the bill: Best Coast and Mariachi El Bronx.
Pre-sale begins Monday December 19th at 10am local time: click here for more info.
The general on sale will be Friday December 23rd: click here to get tickets.
***Please note the venue has changed from Blaisdell Arena to the Waterfront at Aloha Tower.


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Make your own cards and gift wrap!
Join educator and arts teacher Robyn Carter for a fun evening of stencil art! Learn the basics of stencil-making, and take home a holiday card or gift wrap for a personalized gift! All ages are welcome.
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- Friday, April 3, 2015Golden Gate Valley, Golden Gate General Floor Area
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