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Barack Obama said the decision to legalize the same-sex marriages is a 'Victory for America'!
Barack Obama said the decision to legalize the same-sex marriages is a 'Victory for America'!
On Friday the US Supreme Court legalized the same-sex marriage nationwide. It was admitted that refusing to give marriage license to gays and lesbians contravenes the Constitution.
That US Supreme Court's ruling means that more than a decade of severe legal battles between Americans came to an end. Now, those 14 states, which banned same-sex marriages, have no longer rights to enforce such bans.
Immediately after legalization on Friday, gays and lesbians in such affected states as Georgia, Michigan, Ohio and Texas rushed to get married. Although, not all American citizens had that possibility as the authorities of some states, including Mississippi and Louisiana, stated same-sex marriages had to wait till addressing of procedural issues.
However, there was no end for cheering at the scene. Just after the announcement of a new ruling, thousands bursted into tears while another half loudly cheered 'USA USA USA!'.
One of those, who nervously wait for the court's decision was demonstrator Jordan Monaghan, who called his mom and said minutes after a new rule was announced:
Hey mom, I'm at the Supreme Court. Your son can have a husband now
The US President Barack Obama said in his official speech for the event that the ruling was a 'victory for America' and added:
When all Americans are treated as equal, we are all more free
Justice Anthony Kennedy, by whom the majority opinion in the 5-4 decision was written, stated:
No union is more profound than marriage, for it embodies the highest ideals of love, fidelity, devotion, sacrifice, and family. In forming a marital union, two people become something greater than once they were.
As some of the petitioners in these cases demonstrate, marriage embodies a love that may endure even past death.
Obviously there were opponents of the plea, who claimed that voices of thousands Americans were ignored. Mike Huckabee, the former Arkansas governor and presidential candidate, said that the new ruling was an out-of-control act of unconstitutional, judicial tyranny.
Kennedy stated directly to these conservatives, saying: "It would misunderstand these men and women to say they disrespect the idea of marriage. Their plea is that they do respect it, respect it so deeply that they seek to find its fulfillment for themselves.
Their hope is not to be condemned to live in loneliness, excluded from one of civilization's oldest institutions. They ask for equal dignity in the eyes of the law. The Constitution grants them that right."
The White House celebrated the new ruling in its own way. After Friday's sun went down, the rainbow-colored lights were projected onto the house's exterior.
More than 70 percent of the US population is covered by the 36 states, which now permit same-sex marriages.
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subset: commoncrawl


text:
IDOL (Yaoi)
Книгу можно купить в интернет-магазинах:
· OZON.ru 1443р. [Проверить наличие]
ISBN: 193366410XИздательство: Yaoi Press
Год издания: 2007
Страниц: 144
The soul-searching artist David hires a jaded hustler named Adam to pose for a painting for him. David finds that this portrait is finally conveying the passion in his heart, and the beautiful model is stirring passion of another kind. When David acts on his impulses, Adam abandons him before he's done with the painting. The artist is so devastated he ends up in the hospital on the brink of death.
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Who is Veilor?Yeah probably a big question! or not, most likely. Anyway I'm 22 years old from Sweden. I've played games practicly all my life, but it started with kids games on PC(windows 3 and 95). Swedish ones which you've never heard of. And then with age moved on to others such as Tekken 3, Bloody Roar, Spyro, Tombi and other PS1 games. Played a lot of Nintendo also during the growing years, from NES to Gamecube (owns the consoles from NES to GC also).
I am a big Pokémon fan, I've played the games since the release of Pokémon Blue/Red, I was a happy little ten year old that day.
Sees myself as a Zelda fan despite having never completed the NES, SNES or portable games.
Played and still playing World of Warcraft, on the EU server ofcourse, on and off since 2006. Started as Horde, now Alliance.
Other than that I follow the gaming news everyday and keeps up-to-date on what's going on!
Consoles I ownNES
SNES (EU Version)
Nintendo 64
Gameboy Pocket
Gameboy Advance
Nintendo DS Lite
PC (stationary & Laptop)
Playstation 2
Xbox 360


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Columbia has 88,254 residents. It is located in Howard county.
For Maryland residents, the average tuition for all Columbia nursing schools for the 2009 - 2010 school year was $6,726.00. For non-residents, the average tuition for Columbia nursing schools was $8,646.00.
The average cost of books and supplies for nursing schools in Columbia is $837.00. There are two nursing colleges in Columbia for students to choose from. Nursing programs in Columbia reportedly graduated 120 students in the 2008 - 2009 school year.
|Average Salary
|Number Employed in City
|Registered Nurses
|Licensed Practical and Licensed Vocational Nurses


text: The Plants Vs. Zombies: Garden Warfare Action Figures Are ComingMike Fahey8/19/14 11:30amFiled to: Plants Vs. ZombiestoysPlants Vs. Zombies: Garden WarfareDiamond Select ToysPopcapEAaction figures354EditPromoteShare to KinjaToggle Conversation toolsGo to permalink EA's casual game turned online multiplayer shooter arrives on PlayStations today, making it the perfect day for Diamond Select Toys and Popcap to announce a line of Garden Warfare action figures, coming soon to a lawn near you. Advertisement When I first saw the announcement this morning, I figured Diamond Select was just talking about Garden Warfare Minimates, one pack of which will be available at Toys'R'Us stores in early September, the other at specialty comic shops. After years of deliberation, I've decided I'm not a huge fan of Minimates, and therefor was a little but bummed. Advertisement But lo and behold, behind those blocky little bastards there was beauty. Now that's more like it. Available next month wherever Diamond Select Toys are sold, these "Bloom and Doom" 5-inch scale figure two-packs are the perfect way to appeciate the art style of Garden Warfare without getting shot in the process (though depending on your neighborhood that's not completely ruled out). Sponsored Here's what DST is packing: Gatling Pea with Scientist ZombieCamo Cactus with Foot Soldier ZombieChomper with Engineer ZombieShadow Flower with All-Star ZombieA lovely assortment, and if these sell well enough there's are still some 80 bajillion character variants to release. I won't rest until I get a Marine Biologist with Dolphin Gun. Reply35 repliesLeave a reply


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Dream Heaven (梦想天堂)
Weight Management, Herbalife Malaysia
Eating healthy doesn't mean depriving
yourself! Herbalife Formula 1 comes in seven mouth watering flavors,
including Cookies 'n Cream and Cafe Latte. It's scientifically
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replacement, or in addition to your regular meals.
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For their latest culinary endeavour, our friends over at Foodbeast linked up with Rx Boiler Room of Las Vegas, Nevada on a foodie experience that offers up a fresh take on classic comfort foods. Reworking family favourites such as Mac and Cheese and Chicken Pot Pie in to fine dining alternatives, highlights from Foodbeast's experience include Squid-e-Os (SpaghettiOs made of out squid and served with North African based Merguez meatballs), Kampachi Tartare Tacos in Taro Root Shells and Buffalo Oysters. With a menu created and curated by Chef Rick Moonen, the Rx Boiler Room experience can be taken in through video above with further details available here.


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The Children's Aid Blog
Lottery Randomly Fills Coveted Spots for Children's Aid College Prep Charter School
The nervous excitement could be felt up and down Southern Boulevard yesterday as big things were happening inside The Children's Aid Society's Next Generation Center where a new generation of students was randomly selected at the inaugural lottery of the Children's Aid College Prep Charter School. Over 400 applications of promising youngsters were received by The Children's Aid Society and the randomized drawing gave preference to particularly needy students who came from a single-parent household, have not attended a full-day kindergarten and who are English language learners among other criteria.
One of the parents in attendance was Jessica Ortiz, a Children's Aid Society Ambassador and whose children have participated in CAS programs throughout their lives. Her youngest son will be attending 1st grade at Children's Aid College prep this fall. "I can't believe we got picked — it's very exciting and I'm glad that he can go to a school where his needs can be better met," said Ortiz.
The school will begin serving 120 students in Kindergarten and 1st grade this coming August and will eventually serve 300 through 5th grade. The school's mission is to ensure that children will achieve academic success by providing them with the best instructional practices, advancing their physical, emotional, and social needs, fostering a sense of pride and hope, and serving as a safe and engaging community hub.
Photo Courtesy of Lily Kesselman.
Learn more at the Children's Aid College Prep Charter School website and in the following media stories:
Watch these video clips to see the excitement of the lottery firsthand:
Listen to this clip from 101WINS news radio:
Staten Island Community Fights for Their Beacon Program
By: Keyla Espinal
On Wednesday April 4th, The Children's Aid Society and other members of Campaign for Children joined Staten Island parents, students, advocates and Elected Officials to fight against the closure of the Tottenville High School Beacon program and the devastating cuts proposed by the Mayor's preliminary budget.
The rally was also joined by members from the Community Education Council and their very own councilmember, Vincent Ignizio. Councilmember Ignizio acknowledged that these cuts would be devastating and vouched that he and two other Staten Island council members would work on trying to save these important programs. The agenda was filled with students and advocates alike talking about the positive impact beacon and after-school programs have on them and their families.
Diane Colon, the single mom of 5th grader Ami-Lani who attends the after-school program at The Children's Aid Society Goodhue Center, reiterated the importance of after-school programs. "In her after-school program, my daughter gets opportunities that I couldn't afford to give her otherwise – academic support, creative arts, music, and other activities that help her get better grades and enjoy going to school." Diane's sentiment is shared by the parents and care-givers of the 47,000 children that will be left without needed services like child care and after-school programs if cut in the Mayor's proposed budget.
Similar to this rally, the Campaign for Children will be hosting Borough-wide rallies to highlight the specific impacts these cuts would have on those communities.
Next up, on April 17th, there will be a rally at City Hall in which all participating organizations of Campaign for Children, students, parents and advocates will gather from all around the New York City to have their voices heard by city officials. Visit our Take Action page learn more on what you can do and to get more information.
Children's Aid to Receive Pro Bono Consulting Through Morgan Stanley's Strategy Challenge
Morgan Stanley is lending a helping hand to The Children's Aid Society and fourteen other nonprofit organizations by providing pro bono strategic consulting as part of its fourth annual Strategy Challenge. Over the course of eight weeks, CAS will work with a team of Morgan Stanley volunteers to review the current business model and growth strategies. This wonderful opportunity will benefit CAS in a multitude of ways and will further our mission to assist children and families in need throughout New York City.
As part of this exciting program, Morgan Stanley volunteers will present their recommendations to experts from both the private and nonprofit sectors. Chairman and CEO James Gorman will present awards to the top teams. Click here to view the official press release.
Richard Buery on The Huffington Post: An Idea Whose Time Has Come: Moving From Pilots to Policy
"At the end of the day, however, this incredibly wealthy city has to decide that all children merit real investment."
On the evening of March 27, one of our community schools located in Washington Heights, a heavily Dominican neighborhood, hosted an event with potential implications for education policy in New York City and across the country. The event took place in the library of the Salomé Ureña de Henriquez Campus, our flagship Children's Aid Society community school, which recently celebrated its 20th anniversary. The audience included education students, teachers, parents and principals from across the City.
New York University professor and prominent education policy expert Pedro Noguera spoke passionately and personally about the impact of poverty on students in this neighborhood and dozens like it around the City and the need for a Broader Bolder Approach to Education. Michael Rebell, a professor, founder of the Campaign for Fiscal Equity and director of the Campaign for Educational Equity, made the case for the legal right -- not privilege -- of every child to have access to the comprehensive supports that enable a real opportunity for sound education. Manhattan Borough President Scott Stringer pointed out that when he grew up as a child just a few blocks from the school, the supports being promoted -- quality early childhood and after-school programs -- were assumed to be a necessity and a given, not grounds for political infighting and budget wars. The sticking point, said Deputy Chancellor Shael Suransky, is not that these aren't needed, but rather that putting in place a system that ensures consistent access and quality is complicated and takes time.
Continue Reading on The Huffington Post
Access a complete list of Richard Buery's Huffington Post columns
Campaign for Children Call In Day
We need YOU to call City leaders in charge of the budget and tell them to save child care and after-school programs – programs that allow parents to keep their jobs while their children get the educational opportunities they need to succeed.
Campaign for Children Call In Day – Instructions
On Wednesday April 4th between 9am and 4pm, parents, providers and concerned New Yorkers will take action by calling City leaders and letting them know we are paying attention.
Call (888)-279-3491 on Wednesday, April 4th to let our City leaders know that child care and after-school must be saved!
Use this script as a guide for your call (and please be polite):
Hi, my name's ________. I'm calling to urge you to save child care and after-school programs.
With the newest budgets cuts, more than 47,000 NYC children are at risk of losing the educational opportunities that pave the way for success. Families that rely on subsidized child care and after-school work hard and play by the rules. Without these programs, many working family members will be unable to keep their jobs and provide for their households, or will be forced to make potentially unsafe arrangements for their children. It is morally wrong to balance the city budget on the backs of children and hard-working families. We're counting on YOU to restore the funding to save child care and after-school programs. Thank you.
Calls to the number above will be automatically connected to one of the City leaders in charge of the budget. If no one answers, leave a voicemail. Despite what message you get from their office, please be assured that these are the key people in making budget decisions.
You can also send a letter to Mayor Bloomberg and other elected officals by clicking here.
Mayor Bloomberg's released his preliminary budget for New York City. While many programs and services have been protected, the proposed budget fails to include funding for child care and after-school programs for more than 47,000 children currently enrolled in these programs. The Mayor's proposed budget would eliminate child care for 15,900 children, and would cut after-school programs for 31,800 children – leaving 47,000 of New York's children and their families stranded without care. Currently, Children's Aid serves 2,280 children in city-funded OST programs and more than 600 childrens in early childhood programs.
These sweeping cuts would strike a devastating blow to New York City's working parents – who depend on these critical services to keep their jobs – and their children – who need these early education and after-school programs for future success. The cuts represent a dramatic departure from the Mayor's stated desire to make education reforms and economic development his top priorities and the foundation of his legacy as mayor.
Treasure & Bond Charity of the Quarter Contest
The Children's Aid Society is a semi-finalist in Treasure & Bond Charity of the Quarter. Bringing together fashion and art with social responsibility, Treasure & Bond is making shopping guilt-free with every purchase made supporting programs and services for New York City's neediest children.
The race to becoming the beneficiary of this wonderful opportunity has begun and so has your task! Help The Children's Aid Society become Treasure & Bond's Charity of the Quarter by voting in-store during the entire month of April. Visit the store as many times as you want and vote for Children's Aid, if selected, we receive 100% of the proceeds from the store for the entire quarter.
Treasure & Bond
350 West Broadway
(between Broome Street and Grand Street)
Store Hours: 11:00 am – 8:00 pm, Sunday: 12:00 pm – 7:00 pm
Curating a Children's Art Show
Children's Aid staff and friends are hard at work preparing for the 11th Annual Children's Art Show scheduled to open to the public April 24th thru the 26th at The National Arts Club. This week, Ann Kugel, a former Children's Aid Advisory Council Member and Meredith Rugg, a dedicated Children's Aid Society Trustee, spent hours reviewing hundreds of pieces of art work from participants ages 3 – 18, from Children's Aid Society fall and winter programs throughout New York City. Water colors on canvas, papier-mâché projects and charcoal drawings lined the vintage boardroom at the 22nd street headquarters where the curating was taking place. Needless to say that this task was not taken lightly as there were more than enough brightly colored, skillfully created masterpieces to choose from.
The young artists, staff and families will have the opportunity to view the artwork and celebrate artistic creativity during a special reception at The National Arts Club. It is always an interesting evening spent with the youth as they enthusiastically describe their piece to everyone in attendance. Look for a full 11th Annual Children's Art Show recap and photo gallery at www.childrensaidsociety.org.
The National Arts Club is located at 15 Gramercy Park South between Park Avenue and Irving Place on 20th street. Gallery hours for the 11th Annual Children's Art Show are as follows:
April 24th 3 – 5 p.m.
April 25th 9 a.m. – 12:00 p.m. & 3 – 5 p.m.
April 26th 9 a.m. – 12:00 p.m.
Richard Buery on The Huffington Post: Penny Wise and Pound Foolish
NYC's Budget Cuts to Leave Lasting Wounds
"The American dream demands that where a child ends up in life should not be determined by where he started. Race, class and zip code should not determine destiny. "
On March 5th, I joined my colleagues on the steps of City Hall to launch the Campaign for Children to protest the mayor's massive budget cuts to early childhood and after-school programs. I understand that the mayor and the city will have to make difficult choices during the coming budget cycle, but decimating these critical programs for children is the wrong choice.
As the mayor has said, "Teaching doesn't stop when the last school bell rings." He created the city's Out-of-School Time initiative, a nationally recognized effort to bring high-quality after-school and summer programs to kids, declaring that what happens after school is as important as what happens during the school day. From his efforts to remake the schools to his Young Men's Initiative to reverse poor outcomes for young people of color, the mayor has consistently demonstrated his commitment to New York City's children.
That is why the mayor's proposed cuts to early childhood education and after-school programs are so jarring. We all understand how important it is to keep kids engaged and on track beginning at a very early age. Children who are consistently involved in stimulating, educational activities grow up to be smart, safe and productive members of society. They are more likely to go to college, get jobs, support their families and less likely to end up on the streets, involved in gangs or in prison.
Follow Richard Buery on Twitter: www.twitter.com/RichardBueryCAS
Children's Aid Remembers Joy Dryfoos
We mourn the loss of our colleague, mentor and friend Joy Dryfoos, who died peacefully at home on Saturday, March 17. Joy was an inspiration and champion for our community schools work, here in New York City, nationally and internationally. She loved to visit our schools and to bring policymakers, funders and other leaders to see them "in action." She challenged us to make our work better and she documented our work so that others could learn from it. There is no one quite like Joy and we are grateful to have had her in our lives for these past two decades.
Jane Quinn
Vice President for Community Schools and Director, National Center for Community Schools
If you would like to make a donation in Ms. Dryfoos' name to Children's Aid, please click here. You can also leave a personal message by signing her online Rememberance Book.
Children's Aid Middle School Youth Council Project
The Children's Aid Society Middle School Youth Council project started with our January retreat at Wagon Road. Youth Councils from JHS 98 Herman Ridder, Mirabal Sisters Campus, SU Campus, and Fannie Lou Hamer Middle School broke into four groups and made posters representing each group, had an ice cream party, played games and had a great time. We also started talking about issues in our schools. My group said that the biggest issue was bullying. Some other things we talked about were relationships with our teachers and school principals and how can we change the school.
The main project we did was creating a survey. We came up with the idea of surveys when we started talking about bullying and other school problems. The Youth Council decided we should see what other people thought and what other people felt. To create the survey, we went around the group and we started asking each other questions about bullying. We collected surveys from all of our schools, over 300 surveys, and put the answers in Survey Monkey. This weekend we are going to continue our project with our next Wagon Road trip. When go on our next retreat, we hope to complete a video about the results of our survey. We will show this video on Wednesday, May 23rd at the Barnes and Noble on 86th Street. Please join us from 4:30PM till 6:30PM!
Written by: Daphne C.
7th Grade
Herman Ridder JHS 98
Photo by: Kenya R.


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Mutations in the FAM161A gene were previously identified as the cause for autosomal-recessive retinitis pigmentosa 28. To study the effects of Fam161a dysfunction in vivo, we generated gene-trapped Fam161aGT/GT mice with a disruption of its C-terminal domain essential for protein–protein interactions. We confirmed the absence of the full-length Fam161a protein in the retina of Fam161aGT/GT mice using western blots and showed weak expression of a truncated Fam161a protein by immunohistochemistry. Histological analyses demonstrated that photoreceptor segments were disorganized in young Fam161aGT/GT mice and that the outer retina was completely lost at 6 months of age. Reactive microglia appeared in the outer retina and electroretinography showed an early loss of photoreceptor function in 4-month-old Fam161aGT/GT animals. Light and electron microscopy revealed a remarkable phenotype of a significantly shortened connecting cilium, spread ciliary microtubule doublets and disturbed disk organization in Fam161aGT/GT photoreceptor cells. Co-immunolabeling experiments demonstrated reduced expression and mislocalization of centrin 3 and disturbed targeting of the Fam161a interactors lebercilin and Cep290, which were restricted to the basal body and proximal connecting cilium in Fam161aGT/GT retinas. Moreover, we identified misrouting of the outer segment cargo proteins opsin and rds/peripherin 2 in Fam161aGT/GT mice. In conclusion, our results suggest a critical role for the C-terminal domain of Fam161a for molecular interactions and integrity of the connecting cilium. Fam161a is required for the molecular delivery into the outer segment cilium, a function which is essential for outer segment disk formation and ultimately visual function.
Retinitis pigmentosa [RP (MIM 268000)] is the most prevalent hereditary degeneration of the human retina with an incidence of 1:4000 worldwide. Initial symptoms include night blindness and a gradual constriction of peripheral vision caused by rod photoreceptor death (1,2). Secondary loss of cone photoreceptors may subsequently lead to impairment of central vision and ultimately legal blindness (3). RP can be transmitted in an X-linked, autosomal-dominant or autosomal-recessive inheritance pattern. So far, mutations in at least 50 different genes have been identified as genetic causes for non-syndromic RP (RetNet, https://sph.uth.edu/retnet/). Genetic heterogeneity of RP is also reflected in the broad clinical variability in course and severity of the disease (1).
On the molecular level, proteins encoded by RP genes are involved in a multitude of cellular processes including the phototransduction cascade, cytoskeletal dynamics, regulation of gene transcription and intracellular trafficking of proteins (1,3). Due to the complex organization and high extent of specialization of photoreceptors, these cells are very vulnerable to stress. As a consequence, genetically induced dysfunctions of single photoreceptor gene products often lead to a loss of photoreceptor function and programmed cell death, a common hallmark in the pathogenesis of inherited retinal degenerations (4,5).
We and others have previously identified mutations in the FAM161A gene as a cause for autosomal-recessive retinitis pigmentosa 28 (RP28) (6,7). Homozygosity for a 685C-T transition in FAM161A exon 3 was identified in affected members from an Indian family, converting the codon for arginine 229 into a termination codon in FAM161A exon 3 (p.Arg229X). Other mutations causing frameshifts or premature termination have been identified in RP cohorts of German, Jewish, Palestinian and North American decent (6–9). Notably, except for one mutation in exon 4 (p.Arg596X), all of the identified mutations are located in the large exon 3 of FAM161A.
The orthologous human and mouse FAM161A genes consist of seven exons and the Fam161A protein is present in several tissues with high expression level in the retina (6). Fam161A protein is found in inner photoreceptor segments and co-localizes with ciliary marker proteins in the connecting cilium and the adjacent centriole of photoreceptor cells (10,11). FAM161A has been shown to interact with microtubules in vitro via its C-terminal part and a putative role in microtubule stabilization has been proposed (10,11). The FAM161A protein contains a highly conserved UPF0564 domain which is encoded by exon 3, and the N-terminal part of this domain is crucial for both homo- and heterotypic protein–protein interactions with the ciliopathy proteins Cep290 and lebercilin (Lca5) (11). In vitro data suggest that FAM161A is involved in transport processes across the photoreceptor cilium; however, it is currently unknown whether loss of FAM161A function affects ciliary structure in vivo.
To study the consequences of Fam161a disruption in the retina and to determine its role in photoreceptor cells in vivo, we generated and analyzed homozygous gene trapped Fam161a (Fam161aGT/GT) mice. We found that Fam161aGT/GT mice displayed progressive photoreceptor degeneration, early impairment of retinal signal processing and microglial activation. Further investigations of the histopathology revealed that Fam161aGT/GT retinas displayed a spread and shortened photoreceptor connecting cilium, mislocalization of Fam161a interacting proteins and disturbed outer segment formation. Our findings suggest that Fam161a plays a crucial role in the assembly and function of photoreceptor cilia and highlight the Fam161aGT/GT mouse line as a novel retinal ciliopathy model.
Generation of a mouse line with gene trap disruption of Fam161a
We generated a mouse line with targeted disruption of the Fam161a gene using a CMMR ES cell clone GT_462E7_5S with the UPA-vector (12) gene trap allele Fam161aGT(462E7)Chmd inserted into exon 3 of the gene (Fig. 1A). The insertion of the gene trap cassette disrupts the highly conserved UPF0564 domain after amino acid position 362 (Fig. 1B, Supplementary Material, Fig. S1). Mice heterozygous and homozygous for the targeted allele were identified by PCR genotyping (Fig. 1C). Mice homozygous for the Fam161aGT allele and hereafter referred to as Fam161aGT/GT mice showed no behavioral abnormalities compared with their heterozygous littermates. RT-PCR to amplify the 5′ and 3′ parts of the Fam161a mRNA did not show major expression differences in wild-type, Fam161a+/GT and Fam161aGT/GT retinas (Fig. 1D). This suggests that it is unlikely that Fam161aGT mRNA is subjected to complete nonsense-mediated decay.
Weak expression of a truncated Fam161a protein in the Fam161aGT/GT retina
Next, we studied Fam161a protein expression. First, we over-expressed a corresponding Fam161a (amino acids 1–363) variant with an EGFP tag in Hek293 cells (Fig. 2A). This truncated EGFP-tagged Fam161 was readily detected by staining with an antibody against an N-terminal epitope of Fam161a and retained its ability to localize to the microtubule cytoskeleton (Fig. 2B). The specificity of this antibody was further confirmed by immunoblotting, as it detected the truncated version as well as the full-length form of Fam161a at the expected sizes of 70 and 80 kDa, respectively (Fig. 2C). Next, we analyzed Fam161a protein expression in the retina by immunoblotting with two different anti-Fam161a antibodies (Fig. 2D, Supplementary Material, Fig. S1). We detected a specific band of 80 kDa in lysates from wild-type retinas which was absent in 1-month-old Fam161aGT/GT animals. However, we did not detect the truncated Fam161a protein in retinal lysates of Fam161aGT/GT mice, probably due to the low expression level of this polypeptide. Nevertheless, we studied Fam161a protein expression by immunohistochemistry in longitudinal retinal sections from 1-month-old wild-type, Fam161a+/GT and Fam161aGT/GT mice. Fam161a immunoreactivity was detected in inner segments of photoreceptors, the outer plexiform layer and the inner plexiform layer of wild-type mice confirming previous findings (10) (Fig. 2E). Retinas of heterozygous Fam161a mutants showed similar immunoreactivity in photoreceptor inner segments, but lower levels in the plexiform layers (Fig. 2E). Fam161aGT/GT mice showed strongly reduced levels of immunoreactivity in all retinal layers. Based on these observations, Fam161aGT/GT mice can be considered to weakly express a truncated form of Fam161a in the inner segment of retinal photoreceptor cells.
Fam161a gene-trapped mice show a severe and progressive degeneration of the outer retina
We then analyzed the morphology of retinal layers in Fam161aGT/GT mice at different ages using histology and spectral domain optical coherence tomography (SD-OCT) imaging. Longitudinal retinal sections of Fam161aGT/GT mice displayed a severe and progressive retinal degeneration (Fig. 3A–G), involving a gradual loss of the outer nuclear layer which was almost absent in 5-month-old Fam161aGT/GT mice (Fig. 3F). Higher magnification microscopy further revealed that Fam161aGT/GT retinas lacked clear photoreceptor inner/outer segment junctions and displayed disorganized photoreceptor outer segments already at 1 month of age (Fig. 3H and I). Thinning of the outer retina was accompanied by disturbance of the structural order and the appearance of pyknotic photoreceptor nuclei (Fig. 3J–N). In later stages, the outer retina had completely disappeared, whereas the inner retina remained largely unaffected (Fig. 3G and N). Progressive retinal degeneration of Fam161aGT/GT mice was also observed using SD-OCT imaging with early absence of the defined layer between inner and outer segments (Supplementary Material, Fig. S2A). Further quantitative morphometric analyses along the nasal/temporal axis revealed that Fam161aGT/GT animals have a significantly thinner outer nuclear layer already at 1 month compared with wild-type littermates (Supplementary Material, Fig. S2B). At 2 months of age, retinal thickness had declined to 50% until more than 90% of the outer nuclear layer were lost after 6 months in Fam161aGT/GT retinas (Supplementary Material, Fig. S2B).
Degeneration of the Fam161aGT/GT retina is related to microglial reactivity
As microglial activation is a hallmark of inherited retinal degeneration, the morphology and spatial distribution of retinal microglial cells were examined in Fam161aGT/GT mice. The morphology of Iba1-positive microglial cells in wild-type retinas is characterized by small cell bodies and long ramified processes (Fig. 4A), with the cells being distributed in a highly ordered array throughout the plexiform layers (Fig. 4B). In 2-month-old Fam161aGT/GT mice, microglia became bloated with shortened protrusions and a significant number of cells migrated to the outer retina (Fig. 4A and B). Reactive microglia then increasingly accumulated in the subretinal space of older Fam161aGT/GT mice until the territorial microglial network was completely disrupted in 5-month-old animals (Fig. 4A and B). To specify this glial reactivity in relation to cell death, we performed expression profiling using qRT-PCR. The retinal apoptosis marker Caspase 8 (Casp8) was significantly elevated in Fam161aGT/GT retinas from 4 weeks and showed a peak at 5 months (Supplementary Material, Fig. S3A). The microglia markers activated microglia whey acidic protein (Amwap) and Cd68 indicated initiation of microglial reactivity between 2 and 3 weeks of age with maximum levels in 2-month-old Fam161aGT/GT retinas (Supplementary Material, Fig. S3B and C). The Müller glia cell and astrocyte marker Gfap was also significantly up-regulated at all time points and showed a maximum of expression between 6 and 8 weeks after birth (Supplementary Material, Fig. S3D). These data suggest that initiation of immune activation is tightly associated with the onset of photoreceptor degeneration in Fam161aGT/GT retinas.
Fam161a-associated retinal degeneration leads to an early loss of visual function
To test the impact of the observed photoreceptor degeneration on retinal function, we recorded scotopic and photopic electroretinograms (ERGs) from Fam161aGT/GT mice and age-matched controls. Fam161aGT/GT mice exhibited abnormal scotopic ERG responses already at 1 month of age, and at 4 months, ERG responses were almost undetectable (Fig. 5A). We then analyzed the residual visual function in 1-month-old Fam161aGT/GT mice in detail (Fig. 5B–E). The dark-adapted ERG response waveforms (Fig. 5B) of Fam161aGT/GT animals showed lower a- and b-wave amplitudes (Fig. 5C) and prolonged implicit times (Fig. 5D) for all flash intensities. However, the elevated b/a-wave amplitude ratio (Fig. 5E) indicates a photoreceptor-dominated degeneration. Light-adapted ERGs showed a less profound amplitude reduction (Fig. 5F and G) and prolonged implicit times (Fig. 5H) in Fam161aGT/GT animals compared with control mice.
Spreading of the connecting cilium and aberrant disk organization in Fam161aGT/GT photoreceptor cells
Fam161a has been previously localized to the cilium of rod photoreceptor cells (10,11). Here, co-staining of FAM161A in the human retina with the cone marker peanut agglutinin revealed FAM161A localization not only in cilia of rods, but also of cone photoreceptor cells (Supplementary Material, Fig. S4). We therefore focused on the analysis of photoreceptor cilia in Fam161aGT/GT mice applying transmission electron microscopy (TEM). Comparative TEM analysis of Fam161aGT/GT and wild-type photoreceptor cells revealed the following striking morphological changes in Fam161aGT/GT mice (Fig. 6): (i) the connecting cilium was significantly reduced in length (WT: ∼ 1.5 µm; Fam161aGT/GT: ∼ 1.1 µm), (ii) the characteristic microtubule doublets of the connecting cilium were spread in the proximal half thereby, (iii) the outer segment base was markedly dilated (Fig. 6B), and (iv) the membrane disk stacks in the outer segment were distorted, often arranged perpendicular to the disk stack orientation in wild-type photoreceptors (Fig. 6A). In contrast, the structural arrangements of the basal body and the adjacent centriole of the cilium were not altered and no ultrastructural changes were obvious in the inner segment and at the synapses of Fam161aGT/GT photoreceptor cells (Fig. 6 and data not shown). These structural Fam161aGT/GT phenotypes were identified as early as 4 weeks of age. At that time point, alterations in ciliary structures were observed in 95% of all photoreceptors while the disk phenotype was present only in 60% of outer segments. Later at 10 weeks, all photoreceptor cells in Fam161aGT/GT mice showed defective cilia and almost all photoreceptor outer segments were altered.
Altered distribution of ciliary molecules in spread photoreceptor cilia of Fam161aGT/GT mice
Next, we studied whether spreading of microtubule doublets in the connecting cilium and structural disruptions of the outer segments were associated with molecular changes in the ciliary compartment of Fam161aGT/GT photoreceptor cells. We first analyzed the localization of the truncated Fam161a protein in the ciliary compartment. In wild-type mice, Fam161a was present in the connecting cilium, the basal body and the adjacent centriole (Fig. 7A and B) confirming our previous studies (6,10), a report from rat retina (11), and our data for human rod and cone photoreceptor cells (Supplementary Material, Fig. S4). Fam161a co-localized with centrin3, a marker for the adjacent centriole, the basal body and the connecting cilium and with acetylated-tubulin, a marker for stabilized microtubules of cilia and centrioles (Fig. 7A and B, left panels). All ciliary marker stains confirmed the shortening of the connecting cilium in Fam161aGT/GT photoreceptor cells (Fig. 7). Co-labeling for Fam161a and centrin3 or acetylated-tubulin revealed that the truncated Fam161a protein was restricted to the basal body and the adjacent centriole of the photoreceptor cilium (Fig. 7A and B, right panels). However, Fam161a staining was absent in the connecting cilium of Fam161aGT/GT photoreceptor cells. In contrast, acetylated-tubulin remained in all compartments of the cilium, namely the axoneme, the shortened connecting cilium, the basal body and the adjacent centriole of the Fam161aGT/GT photoreceptor cilium (Fig. 7C–E). Centrin3 also remained at the two centrioles of the cilium but was significantly reduced in the connecting cilium of Fam161aGT/GT photoreceptors (Fig. 7A, B, D, E). The retraction of centrin3 to the proximal part of the connecting cilium was more evident in double labeling experiments for centrin3 and glutamylated-tubulin, both markers for the adjacent centriole, the basal body and the connecting cilium in wild-type mice (Fig. 7F). In contrast to glutamylated tubulin, which was stained all along the shortened connecting cilium, centrin3 was found only at its base in Fam161aGT/GT photoreceptor cells (Fig. 7F).
Next, we tested whether the molecular composition of the photoreceptor axoneme, which projects from the tip of the connecting cilium into the photoreceptor outer segment, was altered in Fam161aGT/GT photoreceptor cells. We performed immunolabeling of Rp1 and BBS5, two molecules which have been previously described as molecular components of the axoneme in photoreceptor outer segments (14,15). Immunofluorescence analysis revealed that both proteins accumulated at the tip of the connecting cilium of Fam161aGT/GT photoreceptors (Fig. 7G–I).
To specify our results on the disruption of the connecting cilium at higher resolution and by molecular means, we applied immunoelectron microscopy. We labeled centrin3 and acetylated tubulin on ultrathin LR White sections through wild-type and Fam161aGT/GT photoreceptor cells (Supplementary Material, Fig. S5). Our analysis confirmed that centrin3, which is normally present along the entire connecting cilium of photoreceptor cells (Supplementary Material, Fig. S5A) (16), was restricted to the lower part of the connecting cilium in Fam161aGT/GT photoreceptor cells (Supplementary Material, Fig. S5B and C). In the apical region, where the microtubule doublets spread, no centrin3 was detected (Supplementary Material, Fig. S5B and C). In contrast, acetylated tubulin was also present in the spread microtubules projecting into the remains of the outer segments of Fam161aGT/GT photoreceptors (Supplementary Material, Fig. S5D–F). Quantification of the silver-enhanced immunogold labeling of centrin3 revealed that the total density of centrin3 per cilium was significantly reduced (Supplementary Material, Fig. S5G). Furthermore, our quantitative analysis demonstrates that the spatial distribution of centrin3 over the ciliary compartments was severely altered in Fam161aGT/GT cilia (Supplementary Material, Fig. S5H), confirming our immunofluorescence data.
Fam161a-interacting partners are mislocalized in Fam161aGT/GT photoreceptor cells
A recent study demonstrated the interaction of Fam161a with the ciliopathy proteins lebercilin (Lca5) and Cep290 (11). The interaction of both proteins is mediated by the C-terminal domain mostly deleted in Fam161aGT/GT mice. To analyze the consequences of Fam161a deletion/truncation for the distribution of these disease proteins, we double-labeled specimens for the ciliary marker centrin3 and lebercilin or Cep290, respectively. In wild-type photoreceptors, centrin3 and lebercilin co-localized in the basal body and the connecting cilium (Fig. 8A, left panel). Lebercilin was also present in the axoneme of the outer segment (Fig. 8A, left panel). In contrast, lebercilin was absent from the axoneme and the connecting cilium in Fam161aGT/GT photoreceptors (Fig. 8A, right panel). Its co-localization with centrin3 was restricted to the basal body of Fam161aGT/GT cilia (Fig. 8A, right panel). Cep290 also co-localized with centrin3 in the connecting cilium of wild-type mice (Fig. 8B, left panel). In Fam161aGT/GT photoreceptor cells, Cep290 exclusively localized to the proximal connecting cilium and the basal body (Fig. 8B, right panel). The fact that both interacting partners of Fam161a mislocalized in the photoreceptor cilia of Fam161aGT/GT mice (Fig. 8C) indicates that interaction with Fam161a is essential for their correct position in the connecting cilium of photoreceptor cells.
The ciliary localization of the IFT complex b transport module is not altered in Fam161aGT/GT photoreceptor cells
The assembly and maintenance of cilia requires intraflagellar transport (IFT), a process mediated by molecular motors and IFT proteins organized into supramolecular protein complexes (17). Previous studies showed that IFT proteins are associated with photoreceptor cilia (18,19). This prompted us to also examine whether IFT proteins were affected in the cilium of Fam161aGT/GT photoreceptor cells. However, immunostainings of IFT20, IFT57 and IFT88 revealed that none of these IFT molecules was mislocalized in Fam161aGT/GT retinal photoreceptor cilia (Fig. 8D–G).
Spreading of Fam161aGT/GT photoreceptor cilia impairs transport to outer segment proteins
We next investigated the functional consequences of the ciliary phenotype observed in Fam161aGT/GT photoreceptor cells. Therefore, we stained cryosections through the retina for the outer segment proteins opsin and rds/peripherin2. Most opsin is normally localized within photoreceptor outer segments of wild-type mice (Fig. 9A). In contrast, Fam161aGT/GT retinal opsin was found in the inner segments, the outer nuclear layer and at photoreceptor synapses (Fig. 9B). The higher resolution of anti-opsin immunoelectron microscopy then revealed the presence of opsin in the cytoplasmic and internal membranes of the inner segment of photoreceptor cells (Fig. 9C), the cytoplasmic membrane facing the perinuclear cytoplasm of rod nuclei (Fig. 9D), and in the synaptic terminals of rod cells (Fig. 9E). At the same developmental stage, the amount of rds/peripherin2 was reduced to ∼48% in photoreceptor outer segments (Fig. 9F and G). Furthermore, a mislocalization of rds/peripherin2 to synapses of the outer plexiform layer was obvious in Fam161aGT/GT retinas (Fig. 9E and G).
We and others have earlier reported mutations in the FAM161A gene as one genetic cause for retinitis pigmentosa (6–9). In vitro biochemical studies have indicated that Fam161a may contribute to the function of the photoreceptor cilium (10,11). To gain insights into the molecular pathomechanism underlying RP28, we continued with the functional characterization of Fam161a in vivo using a novel Fam161a gene trapped mouse line.
Photoreceptor cell death and microglial activation represent common pathological features in several retinal degeneration models (4,20,21). Fam161aGT/GT retinas showed a progressive loss of photoreceptor cells and had severely impaired visual function. In the Fam161aGT/GT retina, microglia also changed their morphology and migrated to the outer retina. Reactive microglial cells can secrete neurotoxic substances which then accelerate photoreceptor death and influence the function of retinal pigment epithelial cells (22–24). Transcript profiling showed that cell death in Fam161aGT/GT retinas was coincident with the induction of microglial activation. Notably, the peak of gliosis was reached between 1 and 2 months of age, which corresponds to the period with the steepest rate of cell loss and the time with most decline of retinal function. We therefore conclude that glial reactivity parallels retinal degeneration in the Fam161aGT/GT retina.
The overt accumulation of opsin in photoreceptor inner segments, around photoreceptor nuclei and at photoreceptor synapses in Fam161aGT/GT mice may trigger photoreceptor cell death. The same phenomenon was observed in Rp1-deficient or Lca5Gt/GT gene trapped mice, two ciliopathy models in which defective ciliary transport also leads to the accumulation of opsin in the outer nuclear layer (14,25). Moreover, rd16 mice, which express a truncated version of the ciliary protein Cep290 in the retina, display abnormal outer segment structure, retention of outer segment proteins and rapid degeneration (26,27). Photoreceptors may tolerate this to some extent but mislocalization of truncated rhodopsin to membranes of the inner segment favors cell death (28). A recent study raised some concerns that photoreceptor death in several RP mouse models may be caused by proteasomal insufficiency due to misfolded proteins (29). In this study, we did not observe accumulation of ubiquitinylated proteins in Fam161aGT/GT retinas (data not shown) and thus exclude proteasomal dysfunction as a trigger of cell death.
In Fam161aGT/GT mice, the layered retinal architecture is preserved similarly to other retinal ciliopathy models (14,25,30). However, our analyses revealed a significant reduction of connecting cilium length and spreading of microtubule doublets and thereby distortion of outer segment disks at early stages of degeneration. These results provide in vivo evidence for an essential role of Fam161a in the structural composition, maintenance and function of the connecting cilium of photoreceptor cells. Our analyses showed that the gene-trapped N-terminal part of Fam161a targeted to microtubules and to the basal body, but not to the connecting cilium of photoreceptor cells. Interestingly, acetylation and glutamylation of microtubules in the cilium were not reduced, indicating that microtubules were not destabilized in Fam161aGT/GT mice. This is in line with in vitro studies linking the N-terminal part of Fam161a with microtubule binding and stabilization (10,11).
Recent cell culture data indicated that Fam161a is integrated into a network of ciliary disease proteins present in the transition zone of cilia (11). The transition zone is localized between the basal body and the axoneme and its overlapping protein modules play a crucial role in the regulation of ciliary transport (31,32). This strategic zone is homolog to the connecting cilium of photoreceptor cells (33,34). The ciliary phenotype of Fam161aGT/GT mice could therefore result from the disruption of these protein modules. The truncated version of Fam161a lacks the C-terminal interaction sites for Cep290 and lebercilin and thereby their connection to Fam161a is probably disturbed. Indeed, both proteins do not correctly localize to the connecting cilium in Fam161aGT/GT mice, indicating that their interaction with Fam161a is crucial for their correct ciliary position. Cep290 normally tethers the transition zone microtubule pairs to the membrane (35) and interacts with the Bardet-Biedl Syndrome protein MKKS (36). The disruption of this connection may thus underly the spreading of microtubule doublets in the connecting cilium in Fam161aGT/GT mice. Fam161aGT/GT photoreceptors were also devoid of lebercilin in the connecting cilium. Lebercilin and Fam161a are associated with the IFT module, which is essential for assembly and maintenance of cilia and flagella (25,37). However, like the absence of lebercilin (25), Fam161a inactivation did not affect the ciliary localization of IFT proteins, indicating that both proteins do not serve as core components of IFT particles. Nevertheless, the molecular interplay between Fam161a, Cep290 and lebercilin may provide a structural framework for correct organization of the connecting cilium and delivery of cargo molecules to outer segments.
Our data from Fam161aGT/GT photoreceptor cells also indicate a widening of the outer segment basis, where novel membrane disks containing all components of the visual cycle are generated (38,39). These outer segment alterations suggest that Fam161a may participate in disk neogenesis. Since we did not find Fam161a at the rims of the outer segments, we exclude its direct involvement in disk stacking. A comparable phenotype has been described in the Prph2Rd2 mouse, where the disruption of the disk rim protein peripherin-2 causes defective disk organization (40). In accordance, our data show a reduction of Prph2 in Fam161aGT/GT photoreceptor outer segments, indicating that Fam161a may participate in the delivery of peripherin-2 to the site of disk formation.
In conclusion, we have established the Fam161aGT/GT mouse as a novel retinal degeneration model with a remarkable phenotype of a shortened connecting cilium and spread ciliary microtubule doublets. Mislocalization of essential transition zone components, misrouting of outer segment cargo and disturbed disk organization in Fam161aGT/GT mice implicate that Fam161a is crucial in the structural composition, maintenance and function of the connecting cilium. The Fam161aGT/GT mouse line may be very useful to reveal further insights into Fam161a containing protein modules and into photoreceptor ciliary biology in general.
Mice were kept in an air-conditioned environment on a 12-h light-dark schedule at 22°C, and had free access to food and water. All procedures complied with the German Law on Animal Protection and the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals, 2011. When necessary, mice were anesthetized by intraperitoneal or subcutaneous injection of ketamine (150 mg/kg) and xylazine (10 mg/kg). Pupils were dilated using 1% tropicamide and 2.5% phenylephrine.
Human material
Human donor eyes were obtained from the Department of Ophthalmology, University Medical Center Mainz, Germany. The guidelines to the declaration of Helsinki were followed.
Fam161a gene trapped mouse
The stem cell gene trap clone CMHD-GT_462E7_5S was obtained from the Canadian Mouse Mutant Repository. ES cell DNA was screened for correct gene trap insertion at the Fam161a locus by Sanger sequencing. ES cells were microinjected into C57BL/6J blastocysts and transferred to pseudopregnant female CD1 mice to generate chimeras. Germline transmission of the Fam161a gene trap allele was checked by genotyping and gene-trap positive males were bred with C57BL/6J females. The subsequent progeny was intercrossed to obtain homozygous Fam161aGT/GT mice. Genotyping was performed by PCR with DNA isolated from tail tips using the primers GT1-F (5′-TCCCTGAAGCCCTTTAAGTTC-3′), GT2-R (5′-AAAGCATTGGGCATTTCAGA-3′), GT5-F (5′-CGGACAGACACAGATAAGTTGC-3′) and GT6-R (5′-AAAGCATTGGGCATTTCAGA-3′). Primer pairs GT1-F/GT2-R (485 bp fragment) and GT5-F/GT6-R (929 bp fragment) were used to detect the gene-trapped sequence. The wild-type sequence was detected using the primers GT1-F/GT6-R (385 bp fragment). Prior to breeding, founder animals were tested negative for the rd1, rd8 and rd10 alleles as described earlier (13,41,42).
RNA isolation and RT-PCR
Total RNA was extracted from murine retinas using the RNeasy Micro Kit (Qiagen, Hilden, Germany). cDNA synthesis was performed with the RevertAidTM H Minus First Strand cDNA Synthesis Kit (Fermentas, St-Leon-Roth, Germany). RT-PCR was performed with 50 ng cDNA to amplify intron-spanning fragments between Fam161a exons 2–3 (329 bp) and exons 4–5 (267 bp) using primers Ex2-3_F (5′-TGGCATTCGAGAGCACTATG-3′), Ex2-3_R (5′-CCCGGGAGTCTTTTCCTCTA-3′), Ex4-5_F (5′-AGGAGTGAAAAGGCCAGGAT-3′) and Ex4-5_R (5′-TTTCTCCAGTGTGCAGCTTCT-3′). A 292 bp fragment of β-actin was amplified as the reference transcript with primers β-actin_F (5′-ACCCACACTGTGCCCATCTA-3′) and β-actin_R (5′-CGGAACCGCTCATTGCC-3′). PCRs were carried out using the Qiagen Taq Core kit (Qiagen) and standard PCR conditions.
Quantitative real-time RT-PCR
Amplifications of 50 ng cDNA were performed on the ABI7900HT system (Applied Biosystems) in 10 µl reactions containing 1xTaqMan Universal PCR Master Mix (Applied Biosystems), 200 nm of primers and 0.25 µl of dual-labeled probe (Roche ProbeLibrary, Roche Applied Science). Measurements were performed in triplicates and results were analyzed with an ABI sequence detector software version 2.3 using the ΔΔCt method for relative quantification.
Cloning and recombinant protein expression
For expression in Hek293 cells, EGFP-mFAM161A amino acids 1–363 were subcloned from full-length EGFP-mFAM161A plasmid (10) using primer pair 5′-AAG CTT TGA TGG CTT CGC CGC ACC-3′ and 5′-GTC GAC TTA CGC AGC TGG CCT GTA AAT AAA-3′; the sequence was verified by direct sequencing.
Western blotting
Mouse retinal tissue or Hek293 cells were homogenized in cold RIPA buffer using a TissueLyser LT (Qiagen) and protein concentrations were determined by Bradford assay (Roti-quant, Roth, Karlsruhe, Germany). Fifty micrograms of total proteins were separated by SDS–PAGE on 10% gels and subjected to western blotting. Membranes were incubated with a primary antibody against Fam161a as described previously (10) (Fam161a ab1), a commercial anti-Fam161a antibody (ab115810, Abcam, Cambridge, UK) (Fam161a ab2) and anti-Actin antibody (sc-1616, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Blots were visualized with chemiluminescence signals using secondary IgG-HRP antibodies (Santa Cruz Biotechnology).
Spectral-domain optical coherence tomography (SD-OCT)
SD-OCT was performed on a Spectralis HRA + OCT device (Heidelberg Engineering GmbH, Dossenheim, Germany). Mice were placed on a custom-made mounting platform for SD-OCT measurements (λ = 870 nm; acquisition speed, 40 000 A-scans per second; average images per scan, 24). SD-OCT volume scans of 61 B-scans with 70 µm distance between B-scans (human dimension) were performed on the upper quadrant of the globe for all eyes; this corresponds to 23.33 µm distance between B-scans for murine eyes.
Mouse ERGs were performed as described previously (43). Statistically significant difference between groups was calculated by one-way ANOVA, followed by multiple t-test using the Holm–Sidak method. All analyses and plotting were processed with R 2.15.2 and ggplot 0.9.2.
Semi-thin (1 µm) sections for light microscopy were prepared along the nasal-temporal plane of the retina and stained with Richardson's stain as described previously (44). Immunohistochemistry was performed on 10 µm thick horizontal retinal cryo-sections prepared from eyes embedded in optimal cutting temperature (OCT) compound (Hartenstein, Wurzburg, Germany). For subcellular analysis, eyes were cryofixed in melting isopentane and cryosectioned as described elsewhere (45). The following antibodies were used: rabbit anti-Iba1 (Wako Chemicals, Neuss, Germany), rabbit anti-Fam161a ab1, mouse monoclonal anti-Rhodopsin (Invitrogen), mouse anti-Prph2 (5H2; provided by M. Biehl, Munich), rabbit anti-IFT20 and rabbit anti-IFT57 (provided by G. Pazour, Worcester, MA, USA), rabbit anti-IFT88 and rabbit anti-lebercilin (Proteintech), mouse anti-Cep290 (3G4, provided by Chen and Shou), rabbit anti-BBS5 (provided by M. Nachury, Stanford, CA, USA), chicken anti-Rp1 (provided by E. Pierce, Boston, MA, USA), rabbit anti-glial fibrillary acidic protein (Sigma), mouse anti-acetylated α-tubulin (Sigma), mouse monoclonal anti-centrin3 (16), rabbit polyclonal anti-centrin3 antibodies and fluorescein-labelled lectin peanut agglutinin. Samples were analyzed with a Leica DM 6000 B microscope (Leica Microsystems, Bensheim, Germany) and images were processed with Adobe Photoshop CS (Adobe Systems, San Jose, CA, USA).
For retinal whole mounts, eyes were enucleated and fixed in 4% formaldehyde for 4 h before dissection. Isolated retinas were permeabilized with a solution of 25% Triton X-100 and 25% Tween 20 in 1x PBS followed by blocking in BLOTTO solution for 1 h (1% milk powder and 0.01% in Triton X-100 in 1x PBS). Retinas were incubated overnight with rabbit anti-Iba1 (1:500) in antibody solution (2% BSA, 0.02% NaN3 0.1% Triton X-100 in 1x PBS) at 4°C followed by 1 h secondary antibody incubation. Whole mounts and cross-sections were mounted in Dako fluorescent mounting medium (DakoCytomation GmbH, Hamburg, Germany) and imaged on an Axioskop2 MOT Plus Apotome microscope (Carl Zeiss).
Fixation for conventional electron microscopy
Enucleated eyes were fixed in 2.5% glutaraldehyde in 0.1 m cacodylate buffer containing 0.1 m sucrose for 1.5 h at 4°C. After 30 min fixation, the cornea and lens were removed and the eye cups were fixed for additional 1 h. Eye cups were then washed with 0.1 m cacodylate buffer containing 0.1 m sucrose for 30 min. Subsequently, eye cups were fixed with 2% osmium tetroxide (OsO4) in 0.1 m cacodylate buffer containing 0.1 m sucrose for 1 h at room temperature, followed by dehydration in ethanol (30–100%) and embedding in Renlam® M-1 resin (Serva Electrophoresis, Heidelberg, Germany).
Fixation and postembedding labeling for immunoelectron microscopy
Mouse retinas were fixed 0.1% glutaraldehyde and 3% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4). Fixed tissue was dehydrated stepwise in 30–98% ethanol, embedded in LR White hard (Science Services, Munich, Germany), polymerized at 4°C under ultraviolet (UV) light for 48–60 h and postembedding immunolabeling was performed as described previously (46). In brief, ultrathin LR White sections were incubated with mouse anti-acetylated α-tubulin (Sigma), mouse monoclonal anti-centrin3 (16) or mouse monoclonal anti-opsin (clone K16-155) antibodies. A secondary goat anti-mouse Fab conjugated to Nanogold™ (Nanoprobes, Stony Brook, NY, USA) was applied in IgG-gold buffer. After postfixation in 2% glutaraldehyde for 10 min, the Nanogold™ labeling was silver-enhanced for 25 min.
Ultrathin sectioning and transmission electron microscopy
Ultrathin sections were made using a Reichert Ultracut S ultramicrotome (Leica), collected on Formvar-coated copper or nickel grids and counterstained with 2% uranyl acetate in 50% ethanol aund aq. 2% lead citrate. Ultrathin sections were analysed in a Tecnai 12 BioTwin transmission electron microscope (FEI, Eindhoven, The Netherlands). Images were obtained with a CCD camera (charge-coupled-device camera; SIS MegaView3; Surface Imaging Systems, Herzogenrath, Germany) and processed with Adobe Photoshop CS (Adobe Systems).
Supplementary Material is available at HMG online.
Funds were provided by the Deutsche Forschungsgemeinschaft (DFG, LA1203/8-1 to T.L., GRK 1044 to U.W.); the ProRetina Stiftung (to T.L., U.W.); the FAUN-Stiftung (to U.W.); European Community FP7/2009/241955 (SYSCILIA) (to U.W.) and FP7/2009/242013 (TREATRUSH) (to U.W.); BMBF, grant 0314106 (HOPE2) (to U.W.); the ERA-Net for Research on Rare Diseases (EUR-USH) (to K.N.W.), and the Hans und Marlies Stock-Stiftung (to T.L.).
The authors thank the members of the Langmann lab, Wolfrum lab, Tamm lab and Stoehr lab for excellent technical assistance. We thank Bernd Kirchhof for helpful discussions and support.
Conflict of Interest statement. None declared.


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Commercial Services and Supplies
Company Overview of Asia File Products Sdn. Bhd
Company Overview
Asia File Products Sdn. Bhd manufactures and supplies stationery products. It offers color dividers and other stationery items, computer forms, drawing blocks, spiral note books, folders and metal stamped stationery, hard cover books/writing pads, lever arch files, ring binders, manila files, and photocopy and typing paper products. Asia File Products Sdn. Bhd exports its products in Australia, New Zealand, Papua New Guinea, South Africa, and Japan. The company was founded in 1987 and is based in Penang, Malaysia. Asia File Products Sdn. Bhd operates as a subsidiary of Asia File Corp. Bhd.
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Catalogue Number: S6W-82JR
- Author: Pascal Mercier
- Format:Paperback
- Publisher:Atlantic Books
- Published:01 October 2011
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Product description
- Genre: Modern & contemporary fiction (post c 1945)
In a quiet seaside town near Genoa, experts are gathering for a linguistics conference. One speaker, Philipp Perlmann is recently widowed and, struggling to contend with his grief, is unable to complete his keynote address. As the hour approaches, an increasingly desperate Perlmann decides to plagiarize the work of Leskov, a Russian colleague who cannot attend, and pass it off as his own. But when word reaches Perlmann that Leskov has arrived unexpectedly in Genoa, Perlmann must protect himself from exposure by constructing a maelstrom of lies and deceit, which will lead him to the brink of murder. In this intense psychological drama, the author of Night Train to Lisbon again takes the reader on a journey into the depths of human emotion and the language of memory and loss.
Author's Biography
Pascal Mercier was born in 1944 in Bern, Switzerland, and currently lives in Berlin, where he is a professor of philosophy.


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Hi, I am
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Payday 2 Free Hoxton
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This Gif entitled Payday 2 Free Hoxton was created from the youtube video 2 years ago. It uses 18 frames per second and is displayed at 480px resolution. This animated image plays for 9 seconds, was built with high quality and an estimated weight of 11.92 MB for download. This animation was originally created from the PAYDAY 2! HOXTOBER 27th! FREE HOXTON! Teaser Trailer video from 01:27 to 01:36.
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Jungle Gym Play Paradise 9
Suitable for 31 users aged 3-10 years.
1170cm x 900cm x 300cm Required Area.
Jungle Gym Play Paradise 9 comes with the following modules:
- Mini Picnic Module 160cm
- Jungle Cubby
- Net Link
- Jungle Cabin
- Climb Module (X'tra)
Jungle Gym Play Paradise 9 includes the following items:
- Shutters x 2
- Awning
- Instruction manual
- Toolset
- Hardware & Bolt Caps
- Metal brackets
- Logo ID
- Climbing Wall x 2
- Steering Wheel
- Sandbox x 2
- Ground Anchors x 10
- Handgrips x 16
- Bumper Pad™ x 6
- Jungle Flag™ x 2
- Slide Long x 2
- Sandpad & Leveller
- Safety Ball
- Cat Stop™ Small
- Ground Cover x 2
- Jungling Cards™
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In the November 2011 edition of Fund Directions, Sutherland Partner Cynthia M. Krus breaks down newly implemented governance changes that will affect mutual fund boards this corporate proxy season.
Amendments to Rule 14a-8 of the Securities Exchange Act of 1934 recently became effective and will be applicable for the 2012 season, which runs January through April. These amendments permit proxy access proposals that seek to add procedures to companies' organizational documents, allowing shareholders to more easily nominate directors. Ms. Krus suspects this increased access could lead to a spike in proxy access proposals by shareholders this season.
In an October 17, 2011, Legal Alert, Sutherland's Corporate Practice Group provided an overview on the topic, such as exclusions to the new rules and five ways for companies to prepare for potential proxy access proposals this year. To learn more about these details, which also were highlighted in this Fund Directions article, click here to read the full Legal Alert: "What Does Proxy Access Mean Today?"
Eversheds Sutherland is an international law firm helping the Fortune 100, industry leaders, sector innovators and business entrepreneurs solve their biggest challenges and reach their business goals. Dedicated to unfaltering excellence in client service, we are known for our business savvy and industry intelligence, providing creative and custom solutions for each of our clients. Industry and business experience makes the difference for our clients.
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5 Acres Off Polk St., Indio, CA 92201
- $27,000
- 342 Days on site
- Beds: n/a
- Baths: n/a
- Building Lot
5 Acres of land in the Indio Hills area, featuring picturesque desert land and the beautiful Shadow Hills as it's backdrop. Located just a few miles North of the I-10 freeway and just East of Dillion Road. property has quick access to the freeway and provides endless privacy, Zoan W-1, AP#601-062-016. Property is behind 39200 Dillion Rd,(nursery). Buyer to verify all information. Seller would be interested in carrying the note. Click here to learn more about this listing.
- Year Built: n/a
- 342 Days on site
- Building Lot
- Beds: n/a
- Baths: n/a
Listing Details
- Lot Features
- Mountain View
- Lot Size
- Approximately 217800 Square Feet
Local Info
| Median Household Income
| Median Years In Residence
| Median Homeowner Age
| Households with Children
| Percent of Homes Rented
Nearby Elementary Schools
- Dwight Eisenhower Elementary School
- 83391 Dillon Ave
- Andrew Jackson Elementary School
- 82850 Kenner Ave
- Indio Christian Center Academy
- 84155 Corregidor Ave
Nearby Middle Schools
- Thomas Jefferson Middle School
- 83089 Hwy 111
- Indio Christian Center Academy
- 84155 Corregidor Ave
- Our Lady Of Perpetual Help School
- 82470 Bliss Ave
Nearby High Schools
- Amistad High (Continuation)
- 83501 Dillon Ave
- Indio Christian Center Academy
- 84155 Corregidor Ave
- Indio High School
- 81750 Avenue 46
These are the closest schools to your requested location. Proximity does not guarantee enrollment eligibility. Please consult your real estate agent or school district to confirm the schools you are zoned to attend.
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text: Home Fashion Insider A date with Esha Gupta, Vogue April cover girl Vogue Staff | April 1, 2013 She's candid, she's smiles alot, and she's a whole lot of woman. Here's why you'll love this rising young actress. 0 Share on facebook + X YOU HAVE READ OF 10 STORIES THIS MONTH SIGN UP Why Register? LOGIN


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24 Valentine's Day Crafts for Kids: Lovely Kids Craft Ideas and Projects
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Latest Comments
"This is an excellent collection for crochet beginners and those of us who have crocheted for years. For me in the early days I had to learn to slow down and count each stitch. This is a main problem for misaligned work. Also understanding what gauge is and being sure that the first stitches and rows match the patterns gauge instruction. Sometimes changing hook size solves a problem. How long have you been crocheting?"
"I've got to Pin this one! This is an adorable craft to make with children while they are home on school vacation this week. Crafting sessions are perfect for February vacation in the cold states of New England. Isn't it great to share special time with children or grandchildren? This is an excellent craft for daycare or the babysitter to share for entertainment."
"I actually thought the magnets were inside the material. It's excellent that they are attached underneath. I reaIly think this is a great idea. I would consider making one for my mother. She has vision problems so not only would this keep her straight pins in place but she'd be able to feel them if concentrating on a project. I'm thinking that a solid color would be a better idea for someone who is elderly and having problems seeing things. Do you agree?"
"The two strand Fold basket is a very clever pattern. Making this and keeping in your kitchen to use it for a catch all. This could be made in two colors and would so cute. The pattern dos not seem too complicate and probably would work up fast. This would make a great gift or use it for a basket on the back of the toilet. It is make with cotton yarn which is very easy to work with. I am definitely going to make this one up."
"The mop or sweeper cover is a great idea. They are so expensive to buy and these would work great. The cover is made with cotton yarn which is great for this purpose. The pattern is very simple to follow and should be able to be worked up in one sitting. I know I am going to make a couple of these for my mop. Great project."
Puppy Love Card
By: Duncan Enterprises
We are adding the craft to your Craft Projects.
The project was added to your Craft Projects.
Click here to view your Craft Projects.
There's nothing like puppy love! Create an adorable Valentine's Day card for your favorite person that they'll paws-itively love!
- Aleene's® Paper Glaze
- Aleene's® Crystal Clear Tacky Spray™
- Cardstock: Red, White, Black
- Permanent Marker: Black
- Microbeads: Clear
- Scissors
- Pencil
- Paper: Silver
- Making Memories Letter Stickers: Silver
- Using photo as a guide, trace all pattern pieces on correct color papers. Cut out.
- Spray one side of black hearts with Aleene's® Crystal Clear Tacky Spray™. Turn over and place on top of large silver hearts. Spray one side of large red hearts with Tacky Spray and place on top of black hearts.
- Spray one side of small white hearts with Tacky Spray. Press onto small silver hearts.
- Place large hearts upside down and on top of each other. See photo. Spray tip of bottom heart with Tacky Spray. Place top heart right on top of sprayed area. Refer photo for placement.
- Spray back of one small heart and press right side up in center of top heart for nose. Repeat for heart at chest. See photo.
- On remaining 4 small hearts spray tips on backs and press on for ears and feet. See photo.
- Draw in eyes with permanent marker. Apply sticker letters below heart on chest.
- Apply Aleene's® Paper Glaze to all small hearts and sprinkle on microbeads. Let dry.
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ddinnean 5244484
Feb 12, 2011
This too is easy to make. My daughter-in-law loves dogs and what a cute card for the dog lovers everywhere. Thank you.
Feb 11, 2011
The pattern does not show up :(
nivaa 4808885
Feb 10, 2011
very cute card, I love it
snugpuff 3473697
Feb 10, 2011
What a wonderful craft. It is so nice to have something so cute and is so simple to make. Thank you for the easy instructions and pattern. Way to go.
Member 15424
Feb 10, 2011
This is just the cutest. and anyone would love getting this for Valentine's day.
Feb 02, 2011
Sorry. I realized after posing that it didn't paste the link correctly. Here it is.... http://www.ilovetocreate.com/ProjectDetails.aspx?name=Puppy_Love_Card
Feb 01, 2011
Found the pattern on the original company site! Here's a link. Hope it helps... httpwww.ilovetocreate.comProjectDetails.aspx?name=PuppyLoveCard
Feb 01, 2011
Ditto! I am getting an error page when I try to get the pattern, too. Sure would help! I volunteered to coordinate this for my son's 3rd grade class and a pattern would save me the time of making one. Adorable, tho....
Ray Stegall
Feb 01, 2010
It looks like it would be just right for a person who likes dogs!
Member 16103
Jan 08, 2010
The link for the pattern is not working. I am getting a blank, green page.
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A TENNIS establishment that has spent the past few weeks worrying about whether Tim Henman and Greg Rusedski can be counted among the legitimate contenders for Wimbledon, has a new and infinitely more troubling situation to contend with. The targeting of Morocco by terrorist bombers has raised doubts over the Davis Cup tie that Great Britain is scheduled to play there in September.
The bomb blasts in Casablanca — with areas frequented by western tourists picked out — will have alarmed the LTA. The fact that Morocco is considered a country in peril of attack could have massive repercussions. Those


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Town Square
Seeking information on recent serious bike accident
Original post made by Eric S., Midtown, on Jul 25, 2013
Comments (2)
a resident of Downtown North
on Jul 25, 2013 at 7:01 pm
Any kind of serious injury in a traffic collision should be in the police log (or CHP log). Are you sure the incident occurred within Palo Alto or was the victim from Palo Alto, but hit in a different city?
a resident of Another Palo Alto neighborhood
on Jul 25, 2013 at 8:45 pm
There have been three women seriously injured in bike accidents in PA in the last ten months! Check the Weekly's archives, or the Daily Post Rchives.
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Affordable Housing: Complexities
By Douglas Moran | 13 comments | 4,656 views
Common Application's 2017-2018 Essay Prompts
By John Raftrey and Lori McCormick | 0 comments | 4,332 views
Checklist before baby arrives
By Cheryl Bac | 2 comments | 1,338 views
The Call for Couples or Marriage Counseling? Communication
By Chandrama Anderson | 0 comments | 1,119 views
Short story writers wanted!
The 31st Annual Palo Alto Weekly Short Story Contest is now accepting entries for Adult, Young Adult (15-17) and Teen (12-14) categories. Send us your short story (2,500 words or less) and entry form by April 13, 2017. First, Second and Third Place prizes awarded in each category.


text: On 02/07/2013, at 7:54 AM, Steve Vinoski <vinoski@...> wrote: > I'm experimenting with developing a RESTful interface for one of my apps. According to a number of sources, YAWS supports this. > > Indeed. See for example this article I published a few years back: > > http://www.infoq.com/articles/vinoski-erlang-rest > > Can anyone suggest what I need to do to enable my .yaws scripts to be invoked using PUT and DELETE methods? > > A .yaws resource doesn't support those methods, so you'll want to use an appmod instead. Just judging from your recent emails, I think you should be looking at appmods in general as they're IMO much more powerful and flexible than .yaws pages: > > http://yaws.hyber.org/appmods.yaws > > See my article referenced above as well, it's based on appmods. Oki doki. I'm new to both Erlang and YAWS and wanted to keep things as simple as possible as long as possible to keep the steepness of the learning curve down - thus my focus on .yaws files. Having now read both of the articles you linked I think I'll give appmods a go. Yapps are overkill at the moment. Cheers.


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You get unlimited technical and design tasks that can be completed within 1 hour of direct action. Design tasks have a limit of 2 revisions. And Technical tasks are only on programs covered by our Supported Platform List You can find a complete list of What's NOT A Task on our knowledgebase.
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Table Egg Selling Leads From India.
Posted at: Offers to Sell and Export | Posted on: Thu 26 Feb, 2009 11:29 am |
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M/s Guru Exports Are The wholesale sellers and Exporters of farm fresh poultry Indian White and brown
Eggs, Red Onion and Potato From India. Our Daily production capacity Of White Egg Is 5, 00, 000 and Brown Eggs Is 50, 000. We Are Able To Supply 20*40ft HC reefer Containers of White Eggs And 5*40ft containers of Brown Eggs Every Month As Per Buyers Specification and packing info. Best Quality with most Competitive Price, Superior And Attractive Carton box Packing And Prompt Delivery Is Our Specialty. For More Details about our products Information And Best Offers, Contact Us On through our email address.
360 eggs per carton box.
Each egg will be 50-55 grams weight.
Each carton box contains 12+2 trays.
Each tray contains 30 eggs.
Carton box will be strong 5 ply.
Price: 14-20 usd fob per carton box.
Company Contact:
Contact Name: Vishnu
Company Name: Guru Exports
Mobile: 91-9894361930
Tel: 91-452-2341202
Fax: 91-452-4376302
Street Address: 9, Subburayar
Agaraharam, Kaka Thoppu Street,
Madurai-625001, Tamil Nadu, India
Member name: Guruexports
Member Since: 14 July 2008
Total Leads:
297 Guruexports Import Export Business Leads
Business focus: Farm Fresh Indian Poultry, White Shell Chicken Eggs, Brown Shell Chicken Eggs, Red Onion, Potato
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Unchain Your Maid at the Bubble's FreedomEXPOSED Exhibition This Saturday
Hosted by Daniele Del Sordo and David Rameux of Transmedia Adventures, Fort Lauderdale's legendary art space the Bubble will open its doors, and possibly your third eyes, to a new art installation and live performance exhibition FreedomEXPOSED.
The art performance is designed to work like a Dickens at exposing human pollution past, present, and future. Our Bubble contact notes:
It's an interactive journey and the general concept is about how we've ruined the planet it's taking us into the future and how we need to make changes in order to continue living here on earth. So it's a powerful message we're trying to send, however, it's being done in an interactive experience and all these different artists theres gonna be digital film done by the art institute are all being collaborated together with all these different artists in order to form the show.
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Plant, Machinery and Vehicles Show in the Middle East 2016 will be opened on 21 Nov and it will be end on 24 Nov 2016.
The site of the Trade Show will be Dubai World Trade Centre which is one of the pleasant locale for such an essential event.
PMV Live will likely to be a stand where plenty of pretty important goods are exhibited. Most of these tend to be about Building, Machinery, Construction, Construction Machinery and Heavy Construction.
Plant, Machinery and Vehicles Show in the Middle East 2016 is organized annually.
This year's annual meeting in Dubai is going to be likely to get around 20000 participants.
dmg::events is the PCO of PMV Live, Plant, Machinery and Vehicles Show in the Middle East 2016.
When you are in Dubai for PMV Live, look at these three must visit locations and adore the elegance of this city:
Exhibiting Companies355
Exhibition Area9445 sqm
Dubai World Trade Centre
Mail: email@example.com
Url: http://www.dmgevents.com/
About United Arab Emirates, this is a detailed tourist guide including smart tips and advices about ...


text: USB Bed Risers Are Far More Useful Than Stolen Milk CratesAndrew Liszewski7/12/13 2:00pmFiled to: PowerUSBChargingBeds512EditPromoteShare to KinjaToggle Conversation toolsGo to permalink Twenty-five bucks worth of molded plastic and electronics is about to vastly improve your bedroom experience. This set of four risers from Bed Bath & Beyond not only boosts the height of your bed by eight inches, making you feel more important and increasing the amount of storage space you've got underneath. They also put a pair of power outlets and USB ports in easy reach so you can charge all of your electronic paraphernalia over night. Besides, when you wake up with night terrors over whether your phone is still charged, who has time to walk all the way to the nearest wall outlet to check? [Bed Bath & Beyond via The Gadgeteer]Reply51 repliesLeave a reply


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PSA takes a stake in car-sharing service TravelCar, which will debut US operations out of Los Angeles and San Francisco airports in April.
Autoblog Green
5Peugeot finally returns to America in April... as a car-sharing service
- Danny King
- 21 hrs ago
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36Elon Musk thinks unions are bad for Tesla
12Will Audi's e-tron SUV's range match the Tesla Model X?
- Danny King
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Audi's upcoming electric SUV may be able to go more than 300 miles on a single charge, though that figure may be by European NEDC standards.
AddMore photos, and questions, about Pininfarina's H600 'eco' luxury sedan
- Greg Migliore
- 2 days ago
- Add
More angles of Pininfarina's other Geneva star.
AddLyft expands to another 50 cities
- Greg Migliore
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The ride-hailing service continues its ambitious growth.
202017 Volkswagen e-Golf electric range grows to 125 miles
- Danny King
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Power output also grows.
7Faraday Future to take on Tesla at Pikes Peak
- John Beltz Snyder
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You'll be able to livestream the action this year, too.
132018 Porsche Panamera Turbo S E-Hybrid pumps out 680 horsepower
- Greg Migliore
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The most potent Panamera ever.
4BMW recalls 19,000 i3 REx models for possible fuel vapor leak
38Tesla to announce four more Gigafactory plants by end of year
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Tesla is planning to convert the old Solar City plant in New York to Gigafactory 2 and will add three more plants worldwide.
52Tesla expects Model 3 production to start in July
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- 4 days ago
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The automaker released its earnings report today.
9UPS tests delivery drones, with modest success
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- 4 days ago
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Drones could make UPS more efficient, once they're ready.
13Tesla hacker grabs crash footage from lifesaving Model S
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- 5 days ago
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Tesla hacker stumbles upon images from a car previously praised in a customer testimonial.
10Toyota and Shell team up to build hydrogen fueling stations
4How the Ford EcoSport's rear shelf weighs just 6 pounds but can support 700
28Head of the class | 2017 Hyundai Ioniq Electric First Drive
10Electric GT details its Tesla P100D racer specs, sets 'provisional' schedule
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- 5 days ago
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See the 'P100DL' go racing at Silverstone in August.
10Buy a new NSX and you can put the badges on as it rolls off the line in Ohio
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Packages start at $2,700.
19Keep it simple, stupid | 2017 Hyundai Ioniq Hybrid and Plug-In First Drive
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Prius, watch your back, because the Ioniq is gunning for you.
15Mercedes-Benz is letting 20 customers drive an electric semi truck for a year
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Beta testing is commencing, with full scale production to be reached by 2020.
14Self-driving car race finishes with a crash
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