text:
What does it mean that a large percent of users get to a Web site by direct navigation?
Here's some food for thought.
You're reading an article, and you come across this information nugget: A really big percentage of folks (60-some-odd-percent depending on the source) get to a Web site by direct navigation. That means they typed the Web address directly into their browser or they clicked on a favorite.
If you're like most folks out there, still looking forward to your Web site's glory days, this information probably has you in a panic. "A really big percent type the address in directly?" you quaver. "But that means they have to know about me first!"
Well … yeah. So what is that big percentage number in the article really telling you?
Read the rest of this article.
Read the entire newsletter: Volume 97


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Lafayette High School (New York City)
|Lafayette High School
|Jacqueline Boswell
|Approx. 369
|2630 Benson Avenue Brooklyn 11214,
New York City, New York, United States
|Maroon and white
Lafayette High School is a large secondary school located in the Bath Beach section of Brooklyn, New York City, New York . It is currently one of the low performing high schools of the city and is operated by the New York City Department of Education.
Named after Gilbert du Motier, Marquis de Lafayette, Lafayette High School is situated in what was formerly the town of Gravesend. All the towns in Kings County were settled by the Dutch with the exception of Gravesend, which was first settled by a colony of English people under the leadership of Lady Deborah Moody, a woman of considerable wealth and education, who took a prominent part in public affairs, and whose home was on Neck Road. According to the New York City Ward Map of Kings County in 1899, the school is located on the former property of John Carter. This map reveals a frame house with stables on the site of the building. The land was originally swampy, sandy soil covering about 10 acres (40,000 m2).
Lafayette High School, the first building of its type, was designed to accommodate about 4,000 pupils. The cost of the site was $211,350 while the building itself necessitated an outlay of $2,820,000. There are two other high schools in the city, which are duplicates of this type – Christopher Columbus and William Cullen Bryant. Dr. Frederick William Oswald was asked to assume the principalship of the new school. As the new building was not ready, the school had to meet in three annexes. These annexes were P.S. 180, with Mr. Joseph Grady in charge, assisted by Mr. Freilich and Mr. Abraham Margolies; P.S. 126, under the direction of Miss Dorothy K. Lewis and Mr. Robert Buda; and P.S. 192 with Mr. Walter Jacobsen in charge. On March 1939, the faculty had been increased to 156 members and the student population numbered some 4,500 boys and girls. On November 13, 1939, the formal exercises dedicating Lafayette High School took place in the auditorium. Among those present was Mayor Fiorello H. LaGuardia.
School activities early became an important part of life at Lafayette. On November 21, 1940, the first G.O. elections showed the following candidates victorious in the contest for school officers: Theodore Blinder, president; Gladys Deatsch, vice-president; Lillian Antin, secretary; Sabina La Salle, treasurer. Through the years principals, whether Mary Buda, Joseph Bellafiore, Leo Weitz, Irving I. Cohen, or Sheldon R. Friedberg 1986, recognized the importance of student activities and the Student Organization.
Recent years[edit]
As measured by graduation rates, Lafayette's performance is low; 44.4 percent of the class of 2006 graduated on time.[1] and a 63.2 percent 7-year graduation rate.[2] there was a 50th reunion in 2009 for the 1959 graduates.
Minorities and the Consent Decree[edit]
According to the New York City Department of Education, Lafayette's students speak at least 30 different languages. With the explosion of the Asian population, the school has been accused of harassment and unequal treatment based on race and ethnicity.[citation needed] After much complaints, Lafayette was the subject of a June 2004 consent decree between the New York City Department of Education and the United States Department of Justice which found evidence of "severe and pervasive peer-on-peer harassment of Asian students."[citation needed]
Under the consent decree, Lafayette students needing help in English would be assigned appropriate classes within 10 days of enrolling (e.g., Mandarin-speaking students would not be placed in bilingual classes taught in Cantonese). Lafayette also pledged to develop a policy clarifying school officials' obligations to report cases of harassment based on race, color, and national origin, and to heighten diversity awareness among staff and students.
The Justice Department will continue to monitor Lafayette's progress over the next three years.[citation needed]
In recent years Lafayette has experienced criminal activity involving their students. Extra police officers and security guards were added when the New York City Department of Education labeled it an "Impact School". After some improvement, in April 2006, Lafayette has been removed from the Impact list.
In an effort to turn around the crime, performance, and harassment problems, Jolanta Rohloff, a graduate of the New York City Leadership Academy, was named principal for the 2005–2006 school year. She has proved controversial; angering teachers, discontinuing all after-school sports, overruling grades that some teachers gave students (She attempted to fail students who failed the Regents exam despite teacher's protests)
Planned Closing[edit]
In December 2006, it was announced that New York City plans to close Lafayette High School, along with four other low-performing schools that failed to improve under city guidance. Applying a strategy of the Bloomberg administration, the large closed schools will each be replaced by several small schools with about 400 or 500 students each.[1]
New Principal[edit]
Controversial Lafayette Principal Jolanta Rohloff stepped down (March 30, 2007) to be replaced by Doris Unger, who will oversee the closing of the school, having previously overseen the closing of Seward Park High School. Principal Rohloff, praised by Department of Education officials for her work attempting to turn around the school, but criticized by the community, teachers, and students for heavy-handiness, will be assigned by the Department of Education to mentor principals and teachers on how to interpret student performance data and make necessary adjustments to instruction.[3]
On September 15, 2008, a press release stated that Ms. Doris Unger had been promoted to Superintendent. On September 19, 2008, it was announced to the staff at Lafayette HS that the Assistant Principal, Jacqueline Boswell will be the new Principal, IA.
Lafayette is the New York HS with the most alumni (21) becoming baseball players.[4]
The school is shown during the chase scene of the Academy Award winning movie The French Connection.
Notable people[edit]
- Bob Aspromonte (Baseball)[5]
- Ken Aspromonte (Baseball)[5]
- Kevin Baez (Baseball)[5]
- Tony Balsamo (Baseball)[5]
- Joe Causi (Radio Personality)
- Herb Cohen (Author, Negotiator)
- Alex Coletti (Producer)
- Vic Damone (Singer)
- Benny Distefano (Baseball)[5]
- Jerry Della Femina (Author, Restaurateur, Advertising Agent)
- Pete Falcone (Baseball)[5]
- Al Ferrara (Baseball)[5]
- Mike Fiore (Baseball)[5]
- John Franco (Baseball)[5]
- Gary David Goldberg (Producer/Sitcom creator)
- Larry King (Journalist)
- Sandy Koufax (Baseball)[5]
- Richard LaGravenese (screenwriter)
- Michael Lerner (actor)
- Luis Lopez (Baseball)[5]
- Norm Mager American basketball player
- Peter Max (born Peter Max Finkelstein; Artist)
- Larry Merchant (Sportswriter)
- Vickie Natale (Singer/Songwriter/CBS Star Search Champion)
- Eric Ober (President CBS News 1980s)
- Rochelle Owens (Poet/Playwright)
- Rhea Perlman (Actress) [6]
- Ruben Sanchez (High School Football Coach)
- Steve Schirripa (Actor/Author)
- Maurice Sendak (Artist)
- Paul Sorvino (Actor)
- Elliott Stein (Film critic and historian)
- Michael Steinhardt (Financier)
- Fred Wilpon (New York Mets Owner)
- Larry Yellen (Baseball)[5]
- ^ a b Gootman, Elissa (December 12, 2006). "Lafayette Among 5 High Schools to Close". The New York Times.
- ^ "H.S. 400 Lafayette High School School Review". Insideschools.org. Retrieved January 22, 2011.
- ^ Education, DAVID ANDREATTA (March 29, 2007). "Lafayette Hs Principal Is Out". NY POST. Retrieved January 22, 2011.
- ^ [1][dead link]
- ^ a b c d e f g h i j k l "Lafayette (Brooklyn,NY) Baseball". The Baseball Cube. Retrieved February 13, 2011.
- ^ "1964 Lafayette High Yearbook". classmates.com. Retrieved 29 July 2014.
External links[edit]
Coordinates: 40°35′37″N 73°59′10″W / 40.59361°N 73.98611°W

subset: commoncrawl


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TripAdvisor Traveller Rating
Based on 1205 Review
of 26 Opinions
Hotel added
Hotel description Cassidys
This hotel is the perfect choice for a classic Dublin experience, situated opposite the famous Gate theatre on O'Connell Street and close to everything a visitor to Dublin could want. Dublin's shopping districts and museums are all within a short stroll away. This hotel is within easy walking distance of Dublin Castle and Trinity College. The hotel is also close to Croke Park, and the main shopping areas of Henry Street and Grafton Street where guests will also find restaurants, bars, clubs and public transportation links.
Description Cassidys
- • Number of floors (annexe): 4
- • Year of construction: 1995
- • Single rooms: 2
- • Year of most recent renovation: 2006
- • Number of floors (main building): 4
- • Total number of rooms: 113
- • Double rooms: 111
Hotel type
- • Hotel
Methods of payment
- • American Express
- • Diners Club
- • MasterCard
- • Visa
Room facilities (Standard room)
- • Bathroom
- • Bathtub
- • Carpeted floors
- • Centrally regulated air conditioning
- • Direct dial telephone
- • Disability-friendly bathroom
- • Double bed
- • Individually adjustable air conditioning
- • Internet access
- • King-size bed
- • Radio
- • Safe
- • Shower
- • Tea and coffee making facilities
- • Wheelchair-accessible
Smoking rooms
- • 24-hour reception
- • Air conditioning in public areas
- • Car park (*)
- • Conference room
- • Currency exchange facilities
- • Gym
- • Highchairs
- • Hotel safe
- • Laundry service
- • Lift access
- • Medical service
- • Mobile phone coverage
- • Non-smoking area
- • Restaurant
- • Room service
- • Sauna
- • Wired Internet
Large pets allowed (over 5 kg)
Small pets allowed (under 5 kg)
- • Breakfast
- • Set menu dinner
- • Set menu lunch
Distances (in meters)
- • DUB Airport - Dublin: 6.20 mi
- • Airport: 0.50 mi
- • Bus/Train station: 0.31 mi
Interest points
- • Temple Bar: 0.31 mi
- • Grafton Street: 0.50 mi
- • Gate Theatre: 0.06 mi
- • Trinity College: 0.31 mi
Hotels nearby Cassidys
- At 0.07 miles
This hotel enjoys a prime setting at the heart of the wonderful city of Dublin. The hotel lies just a short distance away from a host of shopping opportunities, theatres, galleries and attractions. Links to the public transport network are to be found just a short distance away. The hotel is located just 10 km from Dublin Airport. The hotel enjoys a long history, dating back to the early 19th century. Immersed in the rich tradition and history of the city, the hotel welcomes guests with the promise of a truly memorable stay. The guest rooms are beautifully designed, providing a peaceful setting in which to unwind at the end of the day. The hotel features a range of excellent facilities, meeting the needs of every type of traveller.Customer reviews4.2/5of 34 Opinions
See more
- At 0.14 miles
Jurys Inn Parnell
The hotel is situated right next to O'Connell Street. Within a 10-minute walk gu ests will find Trinity College, pubs, clubs and Eireann bus station. Within a 20 -minute drive, guests will find Dublin Zoo, as well as Heuston station and Phoen ix Park. Dublin airport is located roughly 10 kms from the hotel. Established in 2004, this hotel comprises a total of 253 rooms. There is a foyer with 24-hour reception desk, safe and currency exchange facility. In addition, there are 3 li fts, a caf¿a pub, a bar, a restaurant and 3 conference rooms as well as public I nternet access. The rooms all come with an en suite bathroom. A direct dial tele phone, satellite/cable TV, a radio, coffee/tea maker, an Internet connection and centrally regulated air-conditioning are also standard. Ten rooms are wheelchai r accessible. Special dietary requirements may be catered for.Customer reviews4.0/5of 56 Opinions
See more
- At 0.16 miles
Castle Hotel
This elegant Georgian-style hotel enjoys a convenient location in Dublin's city centre, just minutes from the city's cultural attractions such as O'Connell Street, Temple Bar, and the Dublin Writers Museum. Visitors might shop in the city centre with its charming boutiques, stroll through one of the many green parks or along the River Liffey, or visit Dublin's most famous attraction, the Guinness Storehouse.The hotel's charming guest rooms are all en suite with warm duvets and richly coloured accents. Each room boasts a tea and coffee tray and complimentary Wi-Fi for guest use. Guests can dine in the hotel restaurant, set in an original 19th-century wine cellar, and relish a full Irish breakfast or vegetarian breakfast after a fabulous night out. The hotel bar provides satisfying Irish wheat beers and traditional live Irish music on weekends, making this the perfect place to spend a holiday in Dublin.Customer reviews4.5/5of 2 Opinions
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Mindfulness meditation is often seen as an austere practice (possibly because of all those monks getting up at 4 am and meditating before breakfast). While simplicity has its place, it also pays to remember that Mindfulness is first and foremost about compassion towards yourself and to others. Enforced austerity should play no part in the practice at all.
Given that today is the first ever 'Mindfulness Day' we decided to try and tackle head-on the image of meditation as being purely about simplicity and austerity by encouraging everyone to meditate while eating a bar of chocolate. Yes, that's right, you can meditate whilst eating a candy bar.
I always remind myself of this when the chocolate cravings kick in. And today was one of those day's where I just needed some chocolate. But rather than gobbling it down, I thought I'd run through our 'Chocolate Meditation'.


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Visualise. Fantasise. Customise.
Win your dream custom design Trek Project One up to the value of $10,000!
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Visualise. Fantasise. Customise.
Win your dream custom design Trek Project One up to the value of $10,000!
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Remember your first bike? The joy you felt when you first laid eyes on it? The sense of freedom and adventure when you set out for a ride? The thrill of moving faster than your feet could carry you? We want every kid to have a great first-bike experience, and a great experience with every bike after that.
We build our kids' bikes to ride better, last longer and carry kids safely to more adventures, whether they're just getting started on two wheels or are ready to hit the race circuit. And we load them with super-smart features, like our exclusive Dialled Fit system that allows the bike to grow along with its young rider.
Handlebars, saddle, grips, pedals... every point where your kid touches the bike has been Dialed for optimal size and adjustability.
Frame and fork have been Dialed to fit kids right from the start, and to keep pace as they grow.
The same Trek quality you know and love applies to all our kids' bikes.
Proper fit and quality build mean easier starting, easier stopping and safer riding.
You may compare up to three bike models.


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◀ Back to Launch Calendar
Black/Multi-Color-Anthracite 616175-090 Sizes: 7-13, 14, 15
The LeBron 11 features Hyperposite construction with ultra-lightweight Flywire cables—designed to move naturally with your foot and enhance support and lockdown when you need it. A full-length Lunarlon midsole sits above a Nike Zoom unit for maximum cushioning. This LeBron 11 features a solid black upper accented with a subtle burst of speckles, which make for a cool contrast against the Anthracite outsole. A multi-color Swoosh injects an eye-catching iridescent accent.


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Search Results for Rob Schwarzwalder
The Loss of Right and Wrong: What's Worse Than Being Mean?
April 24, 2013: Earlier this week, I was interviewed by a very nice radio talk-show host about the proposal to allow what the Boy Scouts term "open and avowed" homosexuality into Scouting ... (more)
Does Reason Still Matter?
April 24, 2013: Debate scholarships paid for a good part of my college education. Learning to make a coherent argument, buttress it using credible research, and defend it extemporaneously have been ... (more)
Allowing Gay Boy Scouts Clouds Scouting's Fundamental Purpose
April 23, 2013: The proposed resolution to admit 'open and avowed' homosexuals (Scouting's term) into the Boy Scouts of America (BSA) is ... (more)
Speaking Across the Chasms
April 11, 2013: Recently I wrote in this space about a "nation of chasms," the division of worldviews and values cloven by the blunt maul of post-modernism, secularism, neo-paganism, "scientific ... (more)
How Do We Measure 'Progressives?'
April 10, 2013: The term "progressive" has become prevalent since "liberal" lost its cultural appeal years ... (more)
'A Nation of Chasms'
April 02, 2013: Recently on National Public Radio, a group of people were interviewed about proposed restrictions on gun ownership. After listening to their widely divergent and essentially irreconcilable ... (more)
On Being a Radical Evangelical
March 19, 2013: Much has been made of late of the young "Evangelical radicals" whose writings and ministries provoke and poke and prod what they view as acculturated, complacent American believers. ... (more)
My Sons are Scouts--for now
March 12, 2013: For roughly the last 10 years, my twin sons have been in Scouting. Both are now First Class Scouts; one is a member of the "Order of the Arrow," Scouting's national honor society. They ... (more)
Dred Scott and Honor's Voice
March 07, 2013: Dred Scott - even his extraordinary names snaps with intensity - was a slave who didn't want to be a slave. He offered to purchase his freedom in 1842, but his mistress refused. He then did ... (more)
Putting Fat on the Bones: Good News From the States
February 21, 2013: "Good news," Solomon tells us, "puts fat on the bones" (Proverbs 15:30). Well, many of us likely would prefer having some fat removed from our bones - but the essential idea remains true ... (more)


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Many interests - too little time! technology, leadership, innovation/disruptive change, Proj. Mgmt, random thoughts. Skier, Rafter, Runner, Dadx3, Husband.


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Will Johnson (due this Saturday, November 23, at the Denver Music Summit) made a name for himself in the '90s in Centro-Matic. A gifted songwriter, Johnson has a knack for penning elegant, emotionally rich and subtly powerful, folk-inflected songs. Over the years, he's toured endlessly on his own an ... More >>
Feuds, tantrums, threatening to kill the president -- if you've been following rock music for the past few decades, you know that dickish behavior comes with the territory when your job involves a stage and adoring fans. Here we've assembled the reprehensible cream that has risen to top, highlightin ... More >>
With Public Image Ltd. coming to town this weekend, we've been thinking about John Lydon and all the crazy weird things he's done. If you haven't seen many interviews with him, you're going to quickly notice a theme in many of these, namely a slight air of pretension, curse bleeps and an ... More >>


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This stir fried chicken dish has a great chilli flavour that comes from the fried dried chillies. We tend to not eat the chillies just suck the great flavour off then put them to the side.
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On the Eric & Kathy Show at the Mix in Chicago, have 3 kids, into physioslow workouts and movie popcorn.


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Los dos Hermanos Water Temperature and Wetsuit Guide
(Tenerife, Spain)
Today's Los dos Hermanos sea temperature is 23.2 °C.
Statistics for 21 Nov (1981–2005) – mean: 21.7 °C, range: 20.5 °C to 22.8 °C
Based on 1 vote. Vote
Today's Los dos Hermanos sea temperature is 23.2 °C.
Statistics for 21 Nov (1981–2005) – mean: 21.7 °C, range: 20.5 °C to 22.8 °C
Sunny with pleasantly warm air temperatures (feeling like 20 °C once we account for wind), and similar sea temperatures (23 °C). Wear boardshorts or a shorty, or better still a thin summer wetsuit to keep the UV off.
(click thumbnails to expand)
Below is a graph of Historical Sea Surface Temperature for Los dos Hermanos. This has been derived from analysis of two decades of oceanographic satellite measurements of nearby open water. We have calculated the average water temperature variation around the year as well as the extremes that have been observed on each date.
All of the graphs for the surf breaks presented on Surf-Forecast.com are on the same scale to enable comparison between locations around the world.
Los dos Hermanos sea temperatures peak in the range 23 to 25°C (73 to 77°F) on around the 12th of September and are at their minimum on about the 19th of February, in the range 18 to 20°C (64 to 68°F). Los dos Hermanos sea temperatures are always warm reach their maximum in the second week of September. You'll need a 2mm neoprene top or a shorty at dawn/dusk or if it's windy. The minimum Los dos Hermanos sea temperatures in the third week of February require something like a 3/2mm fully sealed wetsuit.
Actual sea surface water temperatures close to shore at Los dos Hermanos can vary by several degrees compared with these open water averages. This is especially true after heavy rain, close to river mouths or after long periods of strong offshore winds. Offshore winds cause colder deep water to replace surface water that has been warmed by the sun. Air temperature, wind-chill and sunshine should also be considered before deciding on the kind of wetsuit needed to stay warm when surfing at Los dos Hermanos. Refer to our detailed weather forecasts for this information.


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Am old too! 45 feels like yesterday that my ears awoke definitely today that my bones have a hard time to keep up with me.
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Weather Links
Humidity 37%
Winds 8.1mph
Sponsored by: Arceneaux Ford
Humidity 37%
Winds 8.1mph
Aug 15, 2014 7:46 PM by Kari Beal
Cycling through the streets of Lafayette and beyond can sometimes be a scary ride.
"We're traveling down the bike lane on St. Mary and we will take a left onto Johnston where there is no bike lane," Lafayette cyclist John Langlinais said.
Langlinais is part of the nonprofit bicycle advocacy organization called "Bike Lafayette." The group aims to give a single voice to all types of cyclists in the area.
It comes after crashes and fatalities involving vehicles and bikers. Two cyclists have died in the parish over the last two months. In the last decade there have been 600 crashes and at least 12 fatalities according to Lafayette Consolidated Government.
"There are so many who would say, 'I would love to ride a bike if I could do it safely,'" Bike Lafayette member Scott Coco said.
Bike Lafayette is finally breaking some ground with the help of the parish government initiative "Complete Streets." This initiative strives to make the roads friendly to all those who want to use them, not just motorists.
"Those auto-prone roadways are now struggling with the growing number of cyclists and pedestrians," Planning Manager for Lafayette Metropolitan Planning Organization Mike Hollier said.
The UL Bike Extension has been in the works for a few years now and the final touches will allow cyclists to travel from the Cajun Dome to Johnston Street, over to St. Michael Street and take a right onto St. Julien, which will lead to UL's campus.
This is just one project that is underway. Hollier said the Parish also plans to complete a bike lane from Joey's restaurant on Bertrand to Johnston Street. He said both of these should take less than two years.
"We are working right now on Year 2040 plans for bikeways and other transportation routes," Hollier said.
The Atakapa Ishak Trail is a part of the 2040 project. Hollier said first the trail should be extended from Vermilionville to Breaux Bridge and then eventually to St. MartinVille.
"Our projects cover Lafayette Parish and the surrounding area," Hollier said.
Once some projects are complete in the city limits, Hollier said the Parish can start working on other areas.
"The reality in this town is if you are going to be a cyclist you will have to ride in the lane of travel," Langlinais said.
It's a reality Langlinais hopes he will have to face less and less.
Complete Streets will have its next meeting on Wednesday 12 p.m. at the Rosa Parks transit center. The meeting is open to the public.
For more information on Bike Lafayette or to become a member visit their website: bikelafayette.org
"The Lafayette MPO Bicycle Subcommittee is open to the public and is for long-term planning, laying the groundwork for bicycling infrastructure for the future greater Lafayette area. There are members from cycling advocacy organizations, safety, engineers, and other MPO committees, and anyone is welcome to go to the meetings. Short-term bicycling improvements include bicycle/pedestrian/ motorist safety campaigns. The billboards are already up," Bike Lafayette member Monique Koll said.
You can find them at changethewaywetravel.com and lafayette.gov/streetsense/changethewaywetravel.html
23 hours ago


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The Layover Full Episodes
The Layover Full Episodes: Anthony Bourdain does as the French do. He also indulges in a duck press.
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Viruses 2012, 4(9), 1687-1710; doi:10.3390/v4091687
Published: 21 September 2012
Abstract: EBV and KSHV are both gamma-herpesviruses which express multiple viral microRNAs. Various methods have been used to investigate the functions of these microRNAs, largely through identification of microRNA target genes. Surprisingly, these related viruses do not share significant sequence homology in their microRNAs. A number of reports have described functions of EBV and KSHV microRNA targets, however only three experimentally validated target genes have been shown to be targeted by microRNAs from both viruses. More sensitive methods to identify microRNA targets have predicted approximately 60% of host targets could be shared by EBV and KSHV microRNAs, but by targeting different sequences in the host targets. In this review, we explore the similarities of microRNA functions and targets of these related viruses.
1. Introduction
Approximately twenty percent of human cancers are associated with various infectious agents [1]. Two such agents are Epstein-Barr virus (EBV/human herpesvirus 4) and Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8). Other viruses directly linked to human cancers include hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), human T-cell lymphotropic virus (HTLV-1), and Merkel cell polyomavirus. EBV and KSHV are members of the herpesvirus family that can infect lymphocytes and are associated with various proliferative disorders. EBV associated disorders include Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), NK/T-cell lymphoma, diffuse large B-cell lymphoma (DLBCL) post-transplant lymphoproliferative disorder, and nasopharyngeal carcinoma (NPC) (reviewed in [2]). KSHV infections are associated with Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Both gamma-herpesviruses, EBV and KSHV have evolved to maintain life-long latent infections in their human hosts. Both viruses can infect B-lymphocytes, while in vivo evidence shows that EBV can also infect epithelial cells and KSHV also infects endothelial cells.
The complex steps involved in viral oncogenesis have largely been characterized by studying the interactions between human and viral proteins. A new class of molecules- microRNAs, have begun to be appreciated in virus-host interactions starting with the discovery of virally-encoded microRNAs in EBV [3] and in other herpesviruses, including KSHV [4]. It is believed that EBV and KSHV microRNAs are processed in the same fashion as human microRNAs (though viral microRNA processing can follow a unique pathway in some viruses [4,5]). MicroRNAs have been reviewed extensively elsewhere [6], but briefly, they are short (~22 nt) single-stranded RNA molecules that arise as RNA polymerase II primary transcripts after a number of processing steps. Once the mature microRNAs are generated, they can be incorporated into the RNA-induced silencing complex (RISC), where they can target mRNAs through imperfect complementarity to suppress gene expression. Repression of target mRNAs can trigger destabilization of the mRNA followed by degradation and/or inhibit protein translation. In the context of latent viral infection, these are attractive molecules to modulate host gene expression since they require little viral genome space, avoid generation of additional potentially antigenic viral latent proteins, and are unlikely to be selectively targeted by the host, since humans may lack the ability to distinguish the viral miRNA sequences from human miRNA sequences. Viruses may utilize miRNAs to exploit a faster evolution rate given that a change of few nucleotides can dramatically change the repertoire of targeted mRNAs. Finally, recent studies with mutant strains of viruses, which lack certain miRNAs imply that viral miRNAs play important roles in maintaining the latent/lytic switch [7,8].
Multiple insightful reviews have covered the cellular and viral targets of viral miRNAs [9,10,11,12,13]. Here we focus on the cellular targets of EBV and KSHV miRNAs and look for similarities in the targets and functions of miRNAs from these related viruses. As a result of multiple deep-sequencing efforts, we currently feel we have discovered all of the EBV and KSHV microRNAs. Although the miRNAs from both these viruses lack sequence similarity, one similarity is in their genomic organization. Both viruses show clustering of the miRNA genes and most miRNAs come from the same starting primary miRNA transcript. In KSHV, the 12 microRNA genes are largely in one cluster in the latency locus while the 25 microRNAs of EBV, are largely in two clusters- BART miRNAs and three BHRF1 miRNAs [14].
A variety of approaches have been employed to identify the targets of miRNAs. Bioinformatic methods can be used to find targets through identification of mRNA sequences that are complementary to the seed region of miRNAs (reviewed in [15]). Frequently, miRNA targets are identified by scanning the 3' untranslated regions (UTRs) of mRNAs for sequences complementarity. Additionally, gene expression studies can be used to find gene products that are altered when viral miRNAs are introduced or inhibited [16,17,18]. Recent methods utilize microarrays or the sensitivity of deep sequencing to find mRNA sequences that are biochemically purified with the RNA-induced silencing complex (RISC) [19,20,21,22]. In crosslinking and immunoprecipitation (CLIP) assay, miRNAs and their targets are UV crosslinked to the RISC proteins. The ternary Ago-miRNA-mRNA complexes are then purified by immunoprecipitation of the Ago protein. Immunoprecipitated mRNAs are converted to cDNA and analyzed by high throughput sequencing (HITS-CLIP). HITS-CLIP generates a genome wide-map of miRNA binding sites, but the direct position of interaction between mRNA and protein is not precisely identified. To enhance the poor crosslink efficiency between RNAs and proteins provided by UV irradiation, neo-synthesized RNAs could be labeled with photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP) such as 4-thiouridine (4-SU). Furthermore, crosslinked 4-SU induces a thymidine to cytidine transition during the process and consequently marks the position of successfully crosslinked sequences. Comparing CLIP reads between non-infected and infected cells allows the determination of specific targets of viral miRNAs. However, to identify the precise miRNA binding sites a bio-informatic analysis based on seed sequence homology is often required. Each method has inherent advantages and limitations, but the combination of these approaches have been successful in identifying many miRNA targets (Table 1). After identifying miRNA targets using bioinformatic or cell culture-based screening methods, a variety of assays are used to validate predicted miRNA targets. The most common way to validate a miRNA binding site is to clone this binding site in the 3'UTR of a luciferase reporter and to express this reporter in the presence of the miRNA. An effective binding of the miRNA to a target mRNA then represses luciferase activity of the reporter. These assays are essentially identical to validation studies performed with cellular miRNAs, with the advantage that the uninfected cells are ideal control cells for introducing viral miRNAs. Additionally, the numbers of different viral miRNAs expressed in an infected cell is experimentally more manageable than the hundreds of cellular miRNAs expressed in a given cell type.
Here, we explore the similarities and differences in the functions of miRNA targets from the limited number of experimentally validated miRNA targets and the larger number of targets predicted by recent reports using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP and PAR-CLIP) [20,21,22] (Table 2). The exact significance of these targets needs to be further investigated to understand their potential roles in infection and viral-host interactions. Nevertheless, some main cellular functions emerged from the miRNA targets of EBV and KSHV miRNAs.
|Table 1. List of experimentally validated cellular targets of KSHV- and EBV- encoded miRNAs. The normal cellular functions of these proteins and the effect of their knockdown during viral infection are also listed. The targets that are underlined are repressed by both KSHV- and EBV-encoded miRNAs. ND- No data.
|Table 2. Validated targets from high-throughput studies. Shown are cellular targets identified by photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP), High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) or RNA immunoprecipitation and microarray (RIP-Chip) techniques. Validation of these targets was done using luciferase reporter assays.
3. Regulation of apoptosis by KSHV and EBV miRNAs
Resistance to apoptosis is a common mechanism evolved by many viruses to persist in the host and many cellular targets of KSHV and EBV miRNAs are involved in apoptotic pathways. Using a microarray-based approach to identify mRNAs whose levels were repressed in the presence of KSHV miRNAs, BCLAF1 (Bcl-2-associated factor), a pro-apoptotic protein, was identified as a target of miR-K12-5, -9, -3 and -10b. The repression of BCLAF1 by KSHV miRNAs enabled the cells to overcome etoposide-induced caspase activation [18]. Virally encoded miRNAs can also attenuate cellular responses to extracellular signals as demonstrated in the case of TNFRSF12A (TWEAKR), which is the cellular receptor for the TNF-like weak inducer of apoptosis (TWEAK). Knockdown of TWEAKR levels by miR-K12-10a protected cells from TWEAK-induced apoptosis and also reduced the levels of the cytokines- IL-8 and MCP-1, secreted in response to TWEAK-induction [37]. In another study, caspase 3 was itself identified as a target of multiple KSHV miRNAs, which conferred resistance to apoptosis [38]. In addition, many proteins involved in apoptosis regulation like the tumor suppressors- p53 (for EBV- BHRF1-1) [39], LDOC1 (leucine zipper down-regulated in cancer 1; KSHV miR-K12-11) [40] and MTUS1 (microtubule associated tumor suppressor 1) [20] have been validated. KSHV miR-K12-1 down-regulated the levels of the cyclin-dependent kinase inhibitor, p21, thereby allowing the infected cells to overcome p21-mediated cell cycle arrest [41]. Further, HITS-CLIP and PAR-CLIP studies have identified numerous proteins involved in Wnt signaling (CAPRIN2, CTNND1, DAZAP2, CSNK1A1) as targets of KSHV and EBV-encoded miRNAs [20,21,22].
The pro-apoptotic proteins, BIRC4BP/XAF1 (XIAP associated factor), PUMA (p53-upregulated modulator of apoptosis) and BCL2L11 (or Bim) were also down-regulated by KSHV or EBV miRNAs [17,40,42]. PUMA and Bim are BH3-only proteins of the Bcl-2 family that inhibit the anti-apoptotic functions of Bcl-2 and knockdown of these proteins by EBV-BART-miRNAs conferred cell survival [42,43,44]. Further, both PUMA and Bim are activated by two transcription factors- p53 and C/EBPβ, that are themselves regulated by viral/ virally-induced miRNAs (see below). Interestingly, Bim was repressed by both KSHV and EBV miRNAs, demonstrating the significance of Bim knockdown in the biology of these viruses [40,44].
The TGF-β pathway confers a strong anti-proliferative phenotype to many epithelial and endothelial cells and hence, it is presumable that KSHV has developed mechanisms to escape from this inhibition. SMAD5, the downstream effector of TGF-β is repressed by KSHV- miR-K12-11, and this allowed the cells to overcome the cytostatic effects mediated by TGF-β and enhanced cell proliferation [45]. In addition, thrombospondin 1 (THBS1) (KSHV; see below) and DAZAP2 (EBV; BART3) are proteins of the TGF-β pathway that are repressed by viral miRNAs [16,21]. The TGF-β pathway is also down-regulated via a different mechanism by KSHV. The LANA protein was shown to inhibit TGFβ-type II receptor expression by epigenetic silencing in PEL cells [46]. This is an example of synergy between latently expressed miRNAs and proteins to modulate the same cellular pathway. This redundancy will likely be observed with other miRNA targets as viruses routinely use redundant mechanisms. An additional method of promoting growth is by EBV modulating expression of the human miRNA, miR-34a [82].
7. Similarities between miR-155 and miR-K12-11 in the context of KSHV/ EBV infection
One interesting discovery in the study of virally encoded miRNAs was the identification of the KSHV miRNA, miR-K12-11, as the viral ortholog of the cellular miRNA, hsa-miR-155 (miR-155) [17,40]. Two independent groups showed that miR-K12-11 and miR-155 have the exact 'seed' sequence and therefore, could regulate a similar set of genes. The common set of genes that were down-regulated by both miRNAs included BACH1 (BTB and CNC homology 1), a transcription repressor involved in the regulation of genes regulating cell cycle and oxidative stress [59]. In addition, by microarray profiling and luciferase assays, the authors validated LDOC1 (leucine zipper down-regulated in cancer 1), MATR3 (Matrin 3), TM6SF1 (transmembrane 6 superfamily member 1), PIK3CA (phosphoinositide-3-kinase, catalytic α subunit), XAF1, HIVEP2 (HIV-1 enhancer binding protein 2) and Fos as common targets of both miRNAs [17,40].
In order to identify miR-K12-11 as a true functional ortholog of miR-155, Boss et.al. stably expressed either miR-K12-11 or miR-155 in CD34+ human cord blood progenitor cells and studied immune reconstitution using the NOD/LtSz-scid IL2Rγnull mouse model [60]. These mice have neither circulating complement nor functional NK, B, T, APC cells, and they are deficient in cytokine signaling. Therefore, they can be humanized by transplantation of human cord blood derived CD34+ progenitors and use as an in vivo model for KSHV infection. It was found that the expression of miR-K12-11 or miR-155 repressed the levels of C/EBPβ, a transcription factor that negatively regulates IL-6 and contributes to the development of many B-cell lymphomas. Further, this repression of C/EBPβ by either miR-K12-11 or miR-155 resulted in an increased proliferation of B-cells in the spleen, contributing to lymphomagenesis [60]. More recently, it was shown that SMAD5 is a direct target of both miR-K12-11 [45] and miR-155 [61]. The repression of SMAD5 by both miRNAs was demonstrated to overcome TGFβ -induced growth arrest, thereby, leading to increased cell division and tumor development [45,61].
miR-155 is an oncomiR found to be upregulated in many B-cell lymphomas [62,63]. Interestingly, EBV, which does not encode an ortholog of miR-155, upregulates miR-155 upon infection and this is important for B-cell immortalization [64]. Further, the α-herpesvirus, Marek's Disease Virus (MDV) expresses MDV-miR-M4, an ortholog of miR-155. The two miRNAs were shown to regulate a common set of genes including C/EBPβ and HIVEP2, that are also validated targets of KSHV-miR-K12-11 [65]. Together, these studies suggest that the viral orthologs of hsa-miR-155 may perform overlapping functions and the deregulation of the target genes of these miRNAs might contribute to the development of B-cell lymphomas.
8. Other targets of KSHV and EBV encoded miRNAs
In addition to repressing factors involved in apoptosis, immune evasion and cell cycle arrest, several proteins involved in splicing, miRNA biogenesis and transcription factors have been identified as targets of KSHV and EBV miRNAs. In RIP-CHIP analysis of KSHV infected cells, two proteins of the small nuclear ribonucleoprotein (snRNP) family- NHP2L1 (non-histone chromosome protein 2-like 1) and GEMIN8 (gem (nuclear organelle) associated protein 8), were repressed by KSHV miRNAs, miR-K12-4-3 and -3, respectively. Interestingly, the binding sites for these two miRNAs were found to be in the coding regions of these two genes. In the same study, the authors also identified the CDK5 inhibitor, CDK5RAP1 (CDK5 regulatory subunit associated protein 1), which is predominantly involved in neuronal differentiation and neuroskeletal differentiation, as a target of KSHV miRNA [19]. As mentioned earlier, Dicer, the key enzyme involved in miRNA biogenesis, was also identified as the target of EBV-BART6-5 [47].
In addition, several transcription factors were identified as targets of KSHV miRNAs, among which, ZFN684 [19], BACH1 [17,40], HIVEP2 and Fos [17] have been validated. Transcription factors were among the most enriched family of proteins in PAR-CLIP analyses of both KSHV and EBV infected cells [20]. KSHV infection of lymphatic endothelial cells (LECs) causes transcriptional reprogramming and induces the expression of markers specific for blood vessel endothelial cells (BECs) [66]. Down-regulation of the LEC-specific transcription repressor, MAF, by KSHV miRNAs- miR-K12-6 and miR-K12-11, was shown to induce the expression of several BEC-specific markers like CXCR4, thereby contributing to transcriptional reprogramming [67].
9. Overlap of targets from KSHV and EBV high-throughput miRNA studies
More recently miRNA targets have been predicted using more sensitive approaches of high-throughput sequencing of RISC-associated mRNAs in KSHV and/or EBV infected cell lines [20,68,69]. These methods yield short mRNA sequences that are associated with RISC and through searching these sequences for seed-matching sequences, an association between a specific mRNA and miRNA can be predicted. All of these reports have shown the ability to use this technology to identify validated miRNA targets using 3'UTR luciferase assays and Western blot assays. Here we elucidate the similarities among the genes predicted to be targets from these studies. However, we share the limitations of this analysis. First, the definition of a predicted target differs across the various reports. Second, the completeness of predicted targets made publicly available differed between the reports. Third, the mRNAs predicted to be viral targets are dependent on the sequencing reads possessing a perfect seed-matching site. This raises the possibility that certain mRNA targets being targeted by viral miRNAs using non-canonical seed-matching are not included in the list of predicted hits. Additionally, if a human miRNA having the same seed-match site as a viral miRNA targets an mRNA, then the mRNA would be erroneously labeled as a target of both miRNAs. This analysis does not distinguish miRNA sequences beyond the seed region. Finally, long 3'UTRs, certain secondary structures, and base composition may influence transient, but non-biologically relevant cross-linking events resulting in a predicted miRNA target site. Nevertheless, it is a worthwhile exercise to investigate the similarities and differences of the predicted targets using an additional filter for finding viral miRNA targets, to reveal similar viral-host interactions, and to possibly identify differences in the virus-host interactions between KSHV and EBV. While the specific target sites are unlikely to be conserved between these viruses, recent data suggests a significant overlap of miRNA targets of these viruses. The specific genes targeted and the classes of targets may improve our understanding of viral oncogenesis and immune evasion.
The overlap of targets of EBV and KSHV miRNAs has been approached using biochemical purification of RISC followed by microarrays to measure mRNAs associated with RISC in EBV and KSHV infected cell lines [70]. This analysis predicted 114 targets for KSHV miRNAs and 44 targets for EBV miRNAs. Analysis of these predicted targets revealed only three genes were predicted as miRNA targets for both KSHV and EBV miRNAs (PDCD1LG2, JARID1B, and SOCS3). These genes have been shown to be involved with immune response [71], protein demethylation [72], and cytokine responses [73]. KSHV miRNA targets showed an enrichment of gene ontology terms for genes involved in splicing, gene expression and protein import into the nucleus [70].
We investigated the overlapping genes across the various lists of predicted targets from PAR-CLIP [20,68] and HITS-CLIP [69] assays. For the first analysis, we considered the KSHV and EBV miRNAs that are all naturally expressed in the primary effusion cell line, BC-1 (which do not express the BHRF1 EBV miRNAs). In the original report using only 3'UTR predicted targets, it was observed that fifty-eight percent of KSHV targets were also predicted to be targets of EBV miRNAs. In the analysis here, we included all coding sequence (CDS) and 3'UTR predicted targets. Of the KSHV miRNA predicted targets (3796 genes), sixty-four percent were also EBV predicted targets (Figure 1). Likewise, of the EBV miRNA predicted targets (4328 genes), fifty-six percent are also KSHV predicted targets. Initially, this high degree of correlation may seem surprising since there are no KSHV and EBV miRNAs which share the same seed region. However, this suggests KSHV and EBV miRNAs are both targeting a majority of similar genes through different target regions within the same transcripts and these viral miRNAs are playing similar roles in modulating gene expression. Using the 2423 overlapping genes predicted to be targets of EBV and KSHV miRNAs in the co-infected BC-1 cell line, we analyzed this list for enrichment of certain pathways or process networks (manually curated gene sets based on pathways and gene ontology terms; similar to gene set enrichment analysis) in a process similar to measuring enrichment of gene ontology terms (using MetaCore from Thomson Reuters). As shown in Table 3, there was a significant enrichment of pathways involved in cytoskeleton remodeling. Endothelial cells undergo morphological changes to form spindle cells upon KSHV infection [74,75]). Additionally, KSHV utilizes clathrin-mediated endocytosis and actin remodeling an important role in KSHV entry into cells [76]. Another pathway that is enriched in the EBV and KSHV targets is the PTEN pathway. The predicted targets in this pathway include p21 [41], p53, PTEN, caspase-9, FOXO3, and others. A KSHV protein, LANA, has been shown to inhibit p53 [77], suggesting that KSHV latent proteins and miRNAs can cooperate to inhibit p53. FOXO3 was found to be poorly expressed in nasopharyngeal carcinoma tissues and may represent an important marker for this EBV-related cancer [78]. In the enriched process networks in Table 3, the networks related to cell cycle control and apoptosis are expected based on earlier work on p21 [41], caspase 3 [38] and others [18]. Perhaps more surprising, was the network of genes involved in translation initiation. While translation inhibition upon viral infection has been investigated since 1964 [79], it has been generally described during lytic infection (unlike the latent infection in BC-1 cells). Inspection of the overlapping genes revealed twelve genes from the eukaryotic translation initiation family. Together, this analysis reveals some common themes of predicted KSHV and EBV miRNA targets in BC-1 cells.
|Table 3. BC1 overlapping miRNA target associations.
As mentioned above, there are four datasets from PAR-CLIP or HITS-CLIP assays looking at EBV and KSHV miRNA targets. As shown in Figure 2, there were 31 genes that were found in all four datasets. Given, the differences between the methods used, arbitrary cutoffs, miRNA expression differences among different cell lines, we sought to investigate a broader group of genes. We selected 478 genes (472 are predicted KSHV miRNA targets) that were found in at least three of the four datasets. Using a similar analysis as above, we analyzed this set for functional themes. Again, we observed enrichment of EBV and KSHV predicted miRNAs involved in cell cycle regulation, apoptosis, cytoskeleton remodeling (Table 4). Additionally, we observed a theme of predicted targets involved in immune responses, specifically genes in the B-cell receptor pathway, interleukin regulation, and the interferon pathway. This analysis highlighted interferon receptor 1 and 2 as predicted miRNA targets in all of the datasets, except the EBV Jijoye dataset [22]. But, it should be noted that all of the predicted targets from this dataset were not publicly available, raising the possibility that this was predicted in this dataset as well. Closer analysis shows the PAR-CLIP analysis yielded RISC-associated clusters that are distinct for each viral miRNA, yet in a same region (10% of the 3'UTR sequence length) near the 5'end of the 3'UTR (Figure 3). Could this reflect a region of the mRNA that is less bound by obstructing RNA binding proteins and/or mRNA structures that are more accessible to RISC? Answering these questions could improve bioinformatic methods to identify miRNA targets.
|Table 4. Three of four overlapping miRNA target associations. Shown are the enriched associations of miRNA targets identified in at least three of the four CLIP datasets shown in Figure 2.
10. Conclusions
As time progresses, more discoveries will be shared about additional miRNA targets. Additionally, how these targets interact with each other and with viral proteins remains to be further studied. Future research will likely include investigating the roles of exosomes in manipulating the host outside of infected cells. Furthermore, understanding the roles of viral miRNAs during infection in vivo remains to be a challenge for the field and will likely be complicated by redundant functions of miRNAs and possibly viral proteins. One goal for the field is beyond identifying miRNA targets, but using viral miRNAs as tools to show us humans what the virus already knows is important for repression. A deeper understanding of the networks of miRNA targets (Figure 4) will improve our understanding of viral pathogenesis and yield new insights into the functions of host proteins and virus-host interactions.
This work was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health.
Conflict of Interest
The authors declare no conflict of interest.
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© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).


text:
Work from home Opportunity
Work from home Opportunity
Salary £2,400 - £48,000pa OTE
Location Nationwide; London, Woking, Reading , Southend, Aldershot, Luton, Crawley, Slough, High Wycombe, Chelmsford, Basildon, Basingstoke, Maidstone, Harlow, Guildford, Aylesbury, Tunbridge Wells
Job Type Full time / Part time.
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Job Type : Permanent
Location : London, Woking, Reading, South End, Aldershot, Luton, Crawley, Slough, High Wycombe, Chelmsford, Basildon, Basingstoke, Maidstone, Harlow, Guildford, Aylesbury, Tunbridge Wells
Salary : £2,400 - £48,000pa OTE
Date Advertised : 11 Sep 2014
Sorry, this position is no longer available.
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|TopTropicals.com— rare tropical plants for home and garden
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|Cassia leptophylla
Family: Caesalpinioideae / Caesalpiniaceae
Gold Medallion Tree, Golden Medallion Tree
Origin: Brazil
Spectacular flowering tree. Low water needs, drought tolerant, needs a well-drained soil.
3189 Cassia leptophylla - Gold Medallion TreeGold Medallion Tree. Small tree, fast growing, easy to shape crown. Beautiful clusters of yellow flowers from spring through summer and sporadically in fall and winter. Very popular in Southern landscapes.
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3922 Cassia leptophylla - seedsGold Medallion Tree. Beautiful clusters of yellow flowers from spring through summer and sporadically in fall and winter.
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text:
It sounds so sad. Aw! It sounds like you're fed up with suffering and can't take pains anymore. Keep it up!
no breathing
u dont give a damn
if im bleeding
i reach my limit
to live with u
changes are to die alone
is better that way
it might turn better
with no one on my side
humalation on a side
cuz im losing time
losing my mind
wish for someone
to end my life
once on for all
it will actually give peace
to the empty surroundings
and tonight
im losing my time
losing my mind
im searching answers
im searching for lies
and im ready
im crying and
i cant take life
i cant exist anymore
perdoname michelle!
It sounds so sad. Aw! It sounds like you're fed up with suffering and can't take pains anymore. Keep it up!
.:|Myspace Profile|.:.|My Forum|.:.|My AO Gallery|.:.|My December|:."I love you with all my heart that I can't explain how much in words."
no breathing
u dont give a damn
if im bleeding"
No breathing,
Dont give a f*ck
if I cut my arm bleeding.
Sounds to much like Papa roach :P
Last edited by -=Broken^Halo=-; Sep 03, 2006 at 06:45 PM.
Sounds like your letting all your fustrations out in this song! Keep it up! You found that sweet release!
Sounds like a song for me,a rock song..maybe you can compose your own band and thats the first song? If you like... Dont forget to add us as your band members...just joking...
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