a commercial fishing vessel began taking on water and sank just off the coast of Galicia
according to local search and rescue authorities.
the 2005-built fishing vessel El Cañavera reported that it was in danger of sinking just one nautical mile off Punta Candelaria
a cape just north of the port of Cedeira on Galicia's northwestern coast
The crew abandoned ship into a liferaft and their vessel sank by the stern about five minutes later.
SAR authorities responded to the call and dispatched a rescue helicopter
along with the rescue launches Salvamar Shaula and LS Pardo Bazan
The fishing vessel Brisas de Cedeira was nearby
it reached the life raft and rescued all four crewmembers in it
All were delivered safely to Cedeira.
"They were asking for help and we had them practically in sight
we were ten minutes away from them," said the skipper of Brisas de Cedeira
"But after five minutes it disappeared."
The boat's bow remained in the air as of Monday
so the position of the wreck was marked and broadcast to reduce the risk to shipping
The cause of the casualty is under investigation
Montevideo – Information Office – 23 February
HE Vice President and Speaker of the Parliament of the Republic of Uruguay Beatriz Argimon Cedeira hailed HH the Amir Sheikh Tamim bin Hamad Al-Thani's announcement to hold the Shura Council elections in October 2021
This came during HE Beatriz Argimon Cedeira's meeting with Charge d'Affaires of the Embassy of the State of Qatar to the Republic of Uruguay Taleb bin Mohammed Al-Menakhas
they reviewed the bilateral cooperation relations between the two countries
Adopt well-established policies that achieve the aspirations and interests of the people of Qatar and the strategic interests of the State
American Horror Story has become a mainstay at the Primetime Emmy Awards when it comes to make-up
As the central plots and themes get more ambitious
the designs for everything have to up the ante
takes elements of campy fantasy and fuses them with allusions to queer history
For prosthetic make-up designer Jeremy Selenfriend and make-up designer Milagros Cedeira this season was rewarding and terrifying
but what about the real-life horrors that plagued the gay community in the ’80s
NYC takes the uncertainty of the beginnings of the AIDS epidemic and shoots it through a bloody lens
and Selenfriend and Cedeira and I start our conversation talking about the difficulty of all the sweat that coats everyone’s bodies
but they reveal the surprising amount of work it takes to maintain it
The Mai Tai Killer destroys lives to create the perfect specimen
but Selenfriend divulges details on just how to make a “Frankenstein Jesus” come to life
The same goes for the ghastly death of Hans–the make-up is so detailed that you can almost smell the rot through your screen
Selenfriend and Cedeira also talk about calibrating the sickness journeys of Russell Tovey and Joe Mantello’s characters
Creator Ryan Murphy has delivered American Horror Story: The Normal Heart but tapping into the true fear that gay men faced during the AIDS crisis
American Horror Story: NYC is streaming now on Hulu
A24 has dropped a teaser for the latest Spike Lee Joint
https://www.youtube.com/watch?v=MZ2V8znYMSk Also starring:..
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THOMPSON — The Town of Thompson is poised to possibly see change on a level unseen since big-box retailers Home Depot
Staples and Wal-Mart opened stores in the area between Monticello and Kiamesha Lake
5 whether to authorize four upstate gambling resorts
with the area around the former Concord Hotel site considered a front-runner for a license
A Manhattan-based developer is also proposing China City
a massive project combining an amusement park and houses with a hotel and commercial center on land partly in the town
someone not named Tony Cellini will assume the supervisor's seat in Thompson
The quest to fill the 10-term supervisor's outsize legacy and personality shifts from the campaign trail to the polls in Tuesday's primary
Voters will decide whether Ramon Cedeira or Jerry Fielding or will have the Republican line when both face Democratic Bill Rieber in November's general election
"I've been involved with the town most of my life," said Fielding
who spent 20 years managing the Holiday Mountain ski area when it was town-owned
I know what goes on (and) I know what people are complaining about."
Both candidates name jobs and economic development as primary concerns
and both see approval of the casino referendum as an important step in achieving those goals
Fielding recalled how easily kids were able to find work when the region's resort hotels thrived
He hitchhiked to the Concord when he was a pre-teen to caddy
kids don't have jobs," said the 70-year-old
who will still carry the Independence Party line
As an upstart challenger to Cellini two years ago
but surprised many people by still winning 38 percent of the vote
said voters have listed concerns that include the lack of recreational opportunities for kids
In addition to increasing youth activities
Cedeira advocates for small-business growth
Both a casino and China City would bring jobs and much-needed tax revenue
and we have to find a way to make them stay up here," he said
built in three months and assembled in three days, ‘prefabricated nature’ is a vacation house by spanish firm MYCC architects (formed by carmina casajuana
located in the galician municipality of cedeira
in a remote location in the northeast corner of the iberian peninsula
an area dominated by the imposing presence of the ocean and the forest of eucalyptus trees surrounding it
the terrain is surrounded by harvest fields
protected by the area’s building codes
the volume was wrapped with two materials with the purpose of setting up a dialogue with the landscape
the roof and the side facades were covered with viroc®
a prefabricated mixture of cement and wood shavings
the fibercement has a great strength efficiency in spite of being light and
the two main facades of the house were clad with perforated corten trays following the schematized image of a forest silhouette
recreating the image of the surrounding vegetation
this material was chosen because it is part of the local tradition of fishing towns like cedeira
and the gradual and controlled oxidation of which gives the material self-protecting qualities
its patina and changing color create a lively image that relates with the natural environment
this interplay between the natural and the artificial also benefits the interior spaces
where the light that crosses through these silhouettes casts shadows of trees in the different rooms
the interior spaces are free-flowing and open up to the landscape
of approximately 6 meters in length and 3 in width (the maximum reasonable width to enable their transportation by trailer) organize the program as follows: the first one contains the bedroom
which can be divided into two thanks to a blind concealed in the ceiling
and which becomes a partition wall when more rooms are needed; the second contains the bathroom and stairs; the third the kitchen; and the last three the living room
houses an attic that is a free-flowing space with a double facade that opens up to the sea views towards the southwest and to the forest towards the northeast
it is a space that flows out onto the living room without a designated use
the corten steel cladding punctured with silouhettes of trees
the house combines two different systems: prefabricated construction (2d) for the attic and modular construction (3d) for the ground floor
the modules were built in the facilities of the construction company idm in the madrid town of valdemoro
these modules were built with a structure of beams and galvanized steel columns and with floor and ceiling slabs of composite decking with reinforced concrete
the facade walls are dovetail sandwich panels formed of two sheets of lacquered aluminum and an 80-millimeter-thick polyurethane web plate
several layers of waterproofing stretch beneath the furring strips to which the exterior facades are fixed
a ventilated air cavity of 20 centimeters lets the structure go through
and there is a perimetral panelling of plasterboard with 46 millimeters of rock wool
the result is a 30-centimeter-thick wall with a ventilated facade cavity
interior cavity and 12 centimeters of total insulation
and after making sure that everything fit in properly
the different modules and trusses of the building were taken apart to be packed and moved in trailers the whole 700 kilometers separating the factory from the remote seaside site where its was to be installed
all the parts were put together again on the designated site in just three days
and the finishing touch-ups were done in the following two weeks
AXOR presents three bathroom concepts that are not merely places of function
but destinations in themselves — sanctuaries of style
Setting a solid foundation for the next generation is as important to homebuilder Tony Amaral as building a house that will last generations
In keeping with the Amaral family’s commitment started in 2007
13 students of Portuguese descent were honored on Sunday with a scholarship ceremony hosted by the Foundation Antonio Amaral
is traditionally hosted alongside an annual golf tournament and picnic drawing close to 800 guests for the celebrations
In spite of the restrictions and cancellations
the foundation was determined to honor the students
and provided $29,000 in scholarships on Sunday
“Two weeks ago the family made a decision that the kids were already missing their chance to have a graduation ceremony and prom
so we felt this was something they deserved,” said Tony Amaral
“With the situation we’re in with COVID we were unable to do the picnic this year but still felt the students deserved their scholarships
so today we’re just getting together for a little ceremony,” said program organizer Natalie Amaral Cedeira
under the open air pavilion where the festivities are usually held
With brief speeches by Flagler County Sheriff Rick Staly and Palm Coast Mayor Milissa Holland
both longtime friends of the Amaral family and supporters of the Portuguese community
“We’ve stood together for many years in Palm Coast because our purpose has always been the continuity of generations of rich cultural heritage in our community,” noted Holland in her speech
Carrying on the Portuguese heritage is part of the scholarship application process and Cedeira said it brings her joy to read the essays as the students include authentic stories they’ve learned from family members who talk about the “old country.”
Annalisa Pereira’s application included family history gleaned from her mother Lisa Pereira
who feel it’s important to ensure history is passed down
Pereira says she’ll follow in the footsteps of her older sisters and is looking forward to a career with Johnson & Johnson
Also following in the previous generation’s footsteps
Flagler Palm Coast High School graduate Liana Fernandes said it was “awesome” to be chosen for a scholarship by the community that has played a big role in her life
she is on track for Daytona State College where she will enter the nursing program with plans to become a labor and delivery nurse
we’d always go visit her on holidays – I wouldn’t say I grew up in a hospital but we were always there because of my mom and so it has a special place in my heart,” said Fernandes
Christine Fernandes is proud of her daughter
and grateful to the foundation for the support
“It’s important the Class of 2020 because they lost a lot
and it’s nice they still had this to look forward to,” she said
the Foundation Antonio Amaral has presented 210 scholarships to youth of Portuguese decent totaling $327,500
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Montevideo, May 6th 2025 - 09:50 UTC
were fined £15,500 and £1,300 respectively after pleading guilty on the Summary Court in Stanley to a charge of allowing oil to escape into the territorial waters of the Falkland Islands
The Justices of the Peace (JPs) heard that the ship was maneuvering in Port William whilst awaiting a replacement crew member when it ran aground in Blanco Bay
The bow was indented and two hand-size holes were created in the bow
The ship managed to free itself a few hours later at high tide and with the help of launches
but it is estimated three cubic meters of marine diesel oil escaped from a damaged fuel tank
England will return the Malvinas within 25 years
Commenting for this story is now closed.If you have a Facebook account, become a fan and comment on our Facebook Page
Metrics details
Species Distribution Models are commonly used with surface dynamic environmental variables as proxies for prey distribution to characterise marine top predator habitats
For oceanic species that spend lot of time at depth
surface variables might not be relevant to predict deep-dwelling prey distributions
We hypothesised that descriptors of deep-water layers would better predict the deep-diving cetacean distributions than surface variables
We combined static variables and dynamic variables integrated over different depth classes of the water column into Generalised Additive Models to predict the distribution of sperm whales Physeter macrocephalus and beaked whales Ziphiidae in the Bay of Biscay
We identified which variables best predicted their distribution
Although the highest densities of both taxa were predicted near the continental slope and canyons
the most important variables for beaked whales appeared to be static variables and surface to subsurface dynamic variables
while for sperm whales only surface and deep-water variables were selected
This could suggest differences in foraging strategies and in the prey targeted between the two taxa
Increasing the use of variables describing the deep-water layers would provide a better understanding of the oceanic species distribution and better assist in the planning of human activities in these habitats
surface variables may be partly irrelevant to predict the distribution of their deep-dwelling prey
They showed similar performance between the two models
highlighting the good performance of environmental modelled data to describe species distribution
corresponding to different environments in the water column
were defined and relevant environmental variables were extracted for each class
GAMs were fitted to assess the explanatory power of these variables
the distribution of deep-divers in the Bay of Biscay was predicted using the best models obtained
We expected that the use of environmental variables at depth would result in a more accurate modelling of top predator densities and a better understanding of the mechanisms that influence their distributions
Comparison between the average prediction obtained from the models that explained 80% of the total Akaike weight and the prediction obtained from the model fitted to the four most important variables
If the coefficient of determination (R2) is close to 1
predictions are similar and the average prediction of all models can be approximated by the prediction of the model fitted to the four most important variables
The functional relationships between beaked whale and sperm whale individual densities and the four most important and uncorrelated variables
Solid lines represent the estimated smooth functions and the blue shaded regions show the approximate 95% confidence intervals
The relative density of individuals (individuals per 100 km2) is shown on the y-axis
where a zero indicates no effect of the covariate
The black rug plot on the x-axis represents the distribution of the data
The percentages indicate the importance of the variables calculated by summing the Akaike weights of the models in which they were selected
D*: explained deviance; T: temperature; GrT: gradients of temperature; EKE: eddy kinetic energy; m: mean; sd: standard deviation
Predicted relative densities of beaked whales (a) and sperm whales (b) obtained from the models that use the four most important and uncorrelated variables
The presence of animals at the surface is also probably related to mechanisms at depth
in addition to surface environmental variables
variables that depicted the water column to identify which variables were the most important for deep-divers
since we argued that surface variables might not explain well the physical processes occurring at depth that would influence the beaked and sperm whale distributions
Our results highlighted new relationships with the environment allowing to predict the highest densities of beaked whales and sperm whales near the continental slope
near canyons and seamounts and in the abyssal plain of the Bay of Biscay
we identified different responses between beaked whales
subsurface and static variables were selected as the most important variables
and sperm whales for which no static variable was selected but only surface and deep-water variables
At-depth dynamic variables should be used in other areas such as the North West Atlantic or the Mediterranean Sea to determine whether the difference in importance of static
surface and deep-water variables between beaked whales and sperm whales would also be observed and would be consistent with whale dietary data available in these areas
The use of smaller segments in the analyses could help refine the understanding of the processes that influence the concentration of these species within the canyons
mixed layer depth) provided by an ocean circulation model
to model distribution of cetaceans in the California Current Ecosystem
They both showed that variables obtained from ocean circulation models improved the explanatory power of the distribution models
prey models need refinements to better characterise the prey of deep-diving cetaceans and thus to improve whale distribution models
Our results identified areas of concentration that had not been identified until now in some canyons of the Bay of Biscay (e.g
such as the use of other variables that characterise the water column
but our results confirmed the utility of deep-water variables in SDMs to model the distribution of top predators linked to meso and bathypelagic areas for a better characterising of their habitats
Increasing the use of these variables should be considered to improve the tools available for the planning of human activities
especially for species that would be closely linked to processes at depth
The availability of modelled variables describing deep water-ocean layers should be incorporated into future studies to improve the characterisation of the habitats of top predators
Study area showing assembled survey effort (a), along with beaked whale (b) and sperm whale (c) sightings recorded during all surveys. Surveys were carried out along transects following a line-transect methodology (survey details are provided in Appendix A)
Sightings were classified by group sizes with each point representing one group of individuals and point size representing the number of animals in a group
we used the number of individuals to estimate densities of individuals
Schematic representation of environmental variables used in habitat-based density models and depth classes
Orange arrows represent the four depth classes (Surface
200–600 m and 600–2000 m) and environmental variables are written in black
the central waters of the North Atlantic Ocean between 200 and 600 m (named “200–600 m”) and the Mediterranean waters between 600 and 2000 m (named “600–2000 m”)
Although water masses between 600 and 1500 m and between 1500 and 3000 m are different (see 4.1)
we considered no changes in the water masses from 1500 to 2000 m and grouped them into one class
To model species distributions, it was necessary to extract environmental variables. We considered static and dynamic variables that can affect the distribution of beaked whales and sperm whales (Table 3)
Static variables remained stable over time and were independent from depth classes while dynamic variables were extracted in each depth class and varied over time
All static variables were resampled at a 0.083° resolution to match the resolution of the dynamic variables
Correlation matrix of environmental variables
This matrix was calculated using the Pearson coefficient; the larger and darker the circle
the higher the correlation between the two environmental variables; variables are considered correlated for values below − 0.5 and above 0.5
CanArea: surface of canyons; T: temperature
GrT: gradients of temperature; EKE: eddy kinetic energy; m: mean; sd: standard deviation; surf: surface; 0–200: 0–200 m; 200–600: 200–600 m; 600–2000: 600-2000 m
some variables are probably very similar and correlated
it is not possible to choose a best model among all tested models since the models are equivalent and they must all be integrated to produce an average prediction of species distribution
This can be cumbersome in terms of calculations and difficult to interpret
it is more suitable to obtain a single model
we determined the importance of each variable by summing the Akaike weights of the models in which the variable was selected and ranked all variables
A model using the first four variables was then fitted while ensuring a non-correlation of the variables (if variables were correlated
the next uncorrelated ranked variable was chosen)
A prediction of relative densities (in number of animals per pixel) was produced from this model at a 0.083° resolution and compared to the average prediction obtained from the models that explained 80% of the total Akaike weight
We considered only the models that explained 80% of the Akaike weight because beyond this threshold
the models were negligible (very low explained deviances and very high AICs)
If the coefficient of determination (R2) of the regression line established between the values of the average prediction and the values of the prediction obtained from the four most important variables was close to 1
predictions were similar and the average prediction of all models could be approximated by the prediction of the model fitted to the four most important variables
The four variables could therefore be used to obtain functional relationships and to predict the relative densities of beaked whales and sperm whales in the Bay of Biscay
all models had to be considered to predict the species densities
There were not enough data to fit a model by month or by season (the number of individuals in winter was too low) so we fitted models to all data of beaked whales and sperm whales and obtained climatological predictions maps for all seasons combined in the 1998–2016 period
although most of sighting data were collected in summer and the prediction maps most likely reflected the summer species distribution
The uncertainties associated with the predictions were also estimated as the standard deviation associated with the predicted relative densities; high values indicated high errors associated with density estimates
it should be noted that the uncertainty associated with the model prediction with the four most important variables was certainly lower than the uncertainty associated with the mean prediction of all models and it was therefore underestimated
All sighting and effort data used in this study are available in the GitHub: https://github.com/avirgi01/DeepVariables.git
All data providers can be contacted via the email addresses provided in the data files
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We are grateful to the many observers who participated in the surveys and collected all the data but also the ships’ captains
We thank Phil Hammond and his team for providing SCANS and CODA survey data
THUNNUS survey was carried out thanks to the collaboration of the General Directorate of Fisheries and Maritime Affairs
We thank the Direction Générale de l'Armement (DGA)
ML was funded by a Ramón y Cajal (RYC-2012-09897) postdoctoral contract of the Spanish Ministry of Economy
This study is a contribution to the CHALLENGES (CTM2013-47032-R) project of the Spanish Ministry of Economy
We would particularly like to thank Matthieu Authier for his advice in carrying out the analyses
We thank the two reviewers for their careful reading of our manuscript and for their helpful comments
Basque Research and Technology Alliance (BRTA)
Centre d’Etudes Biologiques de Chizé - La Rochelle
Formal analysis & Writing—Original Draft
The authors declare no competing interests
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
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Sullivan County BOCES held its Recognition Ceremony for 159 graduating Career & Technical Education students at the Liberty Central School
Joseph McDoal (RCS) and Alyssa Vilela (MCS)
spoke at the ceremony on behalf of their classmates
Both expressed their gratitude to BOCES for providing them with the opportunity to attend the New Vision Health Program
They also spoke about their Career & Tech experience
and how it influenced their lives and futures in a positive way by providing an opportunity to challenge themselves and grow
Health Occupations student; and Ariana Fassetta (TVCS)
Culinary Arts student; also spoke during the ceremony
Both talked about their Career and Tech experience and the impact it had on their lives
Fassetta ended her talk by telling classmates
It’s time the world gets to see how wonderful we are.”
Special awards and scholarships were given to the following students:
Catskill Region AACA Antique Car Club Scholarship
Gene Haas Foundation Student Manufacturing Scholarships
Proud to Be Sullivan: Choosing Your Attitude Training
SUPA Public Affairs 101 and SCCC Speech 101
Roscoe Central School was awarded an Attendance Certificate for the students with the highest attendance record for the 2014-2015 school year
For more information about programs available through the Career & Technical Education Center
Library funding is under threat. Find out how you can show up for libraries: ala.org/showup
CHICAGO — How should LIS envision its young adult users? Now showcasing an even more rigorous debate about the theory and practice of YA librarianship, the new second edition of “Transforming Young Adult Services,” published by ALA Neal-Schuman
has been updated and expanded by author and editor Anthony Bernier to incorporate recent advances in critical youth studies
evidence-based treatment that offers LIS instructors
and practitioners a valuable tool for aligning YA services to more fully reflect our diverse populations of young people
this collection delves deeply into such topics as:
is professor at California’s San José State University School of Information
The iSchool has awarded him Distinguished Service
He received two National Leadership Grants from the Institute of Museum and Library Services (on library spaces for young people)
one from ALA’s Diversity Research Committee
and another from the Association for Library and Information Science Education (ALISE) in support of his research on first-generation graduate students
He also served a four-year term on ALA’s Committee on Accreditation
chaired several national professional and academic associations
was recently elected chair of ALA’s Library History Round Table
and currently writes the regular “YA Strike Zone” column for Voice of Youth Advocates (VOYA)
A practicing YA specialist librarian and administrator for 15 years
he designed the first purpose-built library space for teenaged youth
the Los Angeles Public Library’s acclaimed Teen’Scape
and produced nationally recognized youth outreach and programming models
"Transforming Young Adult Services, Second Edition"
"Create, Innovate, and Serve: A Radical Approach to Children's and Youth Programming"
"Teaching Media Literacy, Second Edition"
Today, the U.S. District Court for the District of Columbia granted a temporary restraining order to block the Trump Administration’s dismantling of the Institute of Museum and Library Services (IMLS).
American Library Association, AFSCME Challenge Trump Administration Gutting of Institute of Museum and Library Services
The American Library Association (ALA) today released the highly anticipated Top 10 Most Challenged Books List.
An executive order issued by the Trump administration on Friday night, March 14, calls for the elimination of the Institute of Museum and Library Services, the nation’s only federal agency for America’s libraries.
The American Library Association launched a new supporter program on February 10, 2025, inviting the public to join the fight to protect libraries and library professionals at a pivotal moment in our country’s history.
The American Library Association responds to the Department of Education's dismissal of complaints about censorship and discrimination: the "effort to terminate protections... advances the demonstrably false claim that book bans are not real."
Today the American Library Association (ALA) launched Show Up for Our Libraries, a campaign to harness the collective power of library advocates to influence elected leaders and policymakers.
The American Library Association extends our deepest sympathies to those who have been affected by and to those who have lost everything during the Los Angeles area fires.
The Public Library Association (PLA) has selected more than 139 public libraries across 42 states to host digital literacy workshops, powered by a generous $2.7 million contribution from AT&T.
James W. Lewis has made the largest individual bequest to the American Library Association in the association’s history.
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The Monticello School District made four unwanted school buses disappear almost into thin air. But there was no sleight of hand involved. All it took was a computer to unload each of the 15-ton buses. "It was like David Copperfield, only on eBay," said schools Superintendent Daniel Teplesky. For its efforts, the school district managed to rake in nearly $38,000 from the four separate auctions at the end of May.
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Babou Diouf tells Nacho Doce about the perilous journey that brought him to Spain
where foreign workers are crucial to the survival of the EU’s largest fishing fleet
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Watching news of Senegalese migrants arriving on the Canary Islands stirs memories for Babou Diouf of the same perilous journey he undertook 17 years ago and his arduous path to his new home – Burela, on Spain’s northern coast.
Diouf, 46, a fisherman from Bassoul in Senegal’s Sine Saloum delta, was part of a migration surge in 2006 from West Africa through Spain’s Canary Islands, which lie around 100km (60 miles) off Africa’s northwestern coast.
More than 30,000 migrants arrived that year, fleeing poverty exacerbated by a dramatic plunge in coastal fish stocks, partly due to industrial fishing by European Union states, according to academics, NGOs, and migrants.
The 2006 record for migrant arrivals through the Canaries was broken last year. Nearly 7,000 died attempting the crossing in 2023, according to rights group Caminando Fronteras (Walking Borders).
“To see rafts arriving in Spain is very difficult,” said Diouf. “To cross the ocean without knowing what you’re facing – you’re facing death.”
Deprived of his livelihood in Senegal, Diouf ventured north. Today, his fishing experience has secured him a new life in Spain, where the EU’s largest fishing fleet, shunned by ever more Spanish workers, is recruiting foreigners to survive.
The fishing port of Burela has 44 nationalities among its 9,450 residents, including 90 men from Senegal and 244 from Cape Verde, according to the mayor’s office.
Foreign workers make up about seven out of 10 crew in Burela’s fishing fleet, said Juan Carlos Otero of Burela’s boat owners’ association.
The Peruvians were the first to arrive in 2000 when locals left to work in a new steel plant, said Otero. Indonesians are so valued for their longline fishing skills that boat owners pay for their flights to Spain.
Others, like the Senegalese, find work through word of mouth or by turning up at the dock where, if they have residence permits, they are then offered contracts.
Immigration, largely welcomed by locals, is changing the town. Diouf regularly visits a recently built mosque. Cape Verdean women serve coffee and breakfast at A’Mares, a restaurant in the port.
Working alongside Indonesians, Senegalese and Spaniards on the Sarridal vessel, Diouf is a whirl of activity, hauling in nets and cleaning and boxing hake during 14-hour shifts.
The boat’s captain, Francisco Gonzalez, said Spain’s fishing industry can’t survive without migrants.
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Captain Francisco Gonzalez looks at a map inside the Sarridal ship before a fishing outing to the Atlantic Ocean, at Cedeira’s port, Galicia, Spain
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Disruption of the histone modification patterns is one of the most common features of human tumors
few genetic alterations in the histone modifier genes have been described in tumorigenesis
Herein we show that the histone methyltransferase SETDB1 undergoes gene amplification in non-small and small lung cancer cell lines and primary tumors
The existence of additional copies of the SETDB1 gene in these transformed cells is associated with higher levels of the corresponding mRNA and protein
the depletion of SETDB1 expression in amplified cells reduces cancer growth in cell culture and nude mice models
whereas its overexpression increases the tumor invasiveness
The increased gene dosage of SETDB1 is also associated with enhanced sensitivity to the growth inhibitory effect mediated by the SETDB1-interfering drug mithramycin
the findings identify SETDB1 as a bona fide oncogene undergoing gene amplification-associated activation in lung cancer and suggest its potential for new therapeutic strategies
we examined whether the SETDB1 gene amplification occurs in lung cancer cell lines and primary tumors
and studied its impact on mRNA and protein expression levels
its functional role in lung cancer growth and its potential value as a biomarker for predicting the response to particular chemotherapeutic agents in lung tumors
A telomeric BAC clone located in the telomeric 1p36.23 region was used as a control
The BACs were obtained from the BACPAC Resource Center at the Children’s Hospital Oakland Research Institute (Oakland
SETDB1 and telomeric probes were labeled with Spectrum Green and Red dUTP (Abbott
using a CGH Nick Translation Reagent Kit (Abbott Molecular Inc.
The samples were counterstained with 4',6-diamidino-2-phenylindole in Vectashield antifade solution (Burlingame
Gene amplification was observed in the interphases of NCI-H1437
Probes were verified to give a single signal on normal commercial lymphocyte metaphase slides (CGH Reagents
Quantitative reverse transcription–PCR (c) and western blot (d) demonstrate higher levels of SETDB1 mRNA and protein (ab12317
NCI-H1395 and DMS-273) than that in unamplified cells
Growth-promoting effects of SETDB1 in lung cancer
(a) Stable downregulation of the SETDB1 gene by short hairpins using two different target sequences for DMS-273 (clones A30/A31 and clone B32-63) and NCI-H1437 (clones A56-B and B46-9)
SETDB1 shRNA sequences are available upon request
(b) The short hairpin SETDB1-depleted cells were less viable in the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay than in the untransfected or scrambled shRNA-transfected cells (P-values obtained by the analysis of variance (ANOVA) test)
(c) The colony formation assay showed that DMS-273 and NCI-H1437 cells stably transfected with the shRNA against SETDB1 formed significantly fewer colonies than scrambled shRNA-transfected cells (P-values obtained by the ANOVA test)
(d) Effect of SETDB1 shRNA-mediated depletion on the growth of DMS-273 and NCI-H1437 xenografts in nude mice
Tumor volume was monitored over time and the tumor was excised and weighed at 30 days
There was a significant decrease in tumor weight in the SETDB1 shRNA-stably transfected cells (P-values obtained by the ANOVA test)
Impact of SETDB1 on invasiveness and chemosensitivity
(a) Effect of SETDB1 on the invasion potential of A549 cells determined by the matrigel invasion assay
Cells were transfected with 3 μg of Flag-SETDB1 or empty vector in 60 mm dishes
cells were stimulated or not with phorbol myristate acetate (PMA) plus ionomycin (Io) for 30 min
and 5 × 104 cells were resuspended in serum-free media and added to the upper compartment of a transwell coated with 1 mg/ml Matrigel (BD Biosciences
Media with 10% fetal bovine serum was added in the lower compartment and cells were incubated at 37 °C for 42 h
Invasive cells were fixed with phosphate-buffered saline 4% paraformaldehide
stained with 0.5% violet crystal and visualized and photographed under a × 10 magnification objective with a microscope
Invasive cells were counted using ImageJ 1.45s (Wayne Rasband
USA) and percentage of invasive cells were represented
Results are the mean of at least three experiments by duplicate and the significance was determined using analysis of variance test
(b) Cancer cells harboring the SETDB1 gene amplification are sensitive to the decrease in cell viability caused by mithramycin
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays in control-scrambled shRNA DMS-273-transfected cells in comparison with three shRNA-stable downregulated SETDB1 clones (A21
A30 and A31) show enhanced inhibition of viability in cells with SETDB1 gene amplification-mediated overexpression
(b) Immunohistochemistry for SETDB1 (HPA018142
USA) shows overexpression of the protein in the above shown three primary lung tumors harboring SETDB1 gene amplification
Minimal expression is detected in the unamplified cases (4 and 5)
(c) Association between SETDB1 gene amplification and overexpression in the studied fifty-nine cases is shown
our results indicate that the histone methyltransferase SETDB1 undergoes gene amplification in the natural history of lung tumorigenesis in non-small and small cell lung cancers
The copy-number gain for SETDB1 is associated with overexpression of the transcript and protein in lung cancer cell lines and primary tumors
SETDB1 exerts growth enhancing activity in vitro and in vivo
as we have shown by depletion and transfection experiments in cell culture and in the nude mice model
Lung cancer cells carrying a SETDB1 gene amplification event are also more sensitive to the antiproliferative action mediated by the antitumoral antibiotic mithramycin
our results suggest an oncogenic role for SETDB1 in lung carcinogenesis and raise the possibility of exploring new targeted therapies for the subset of lung tumor patients harboring the SETDB1 gene amplification event
Aberrant epigenetic landscape in cancer: how cellular identity goes awry
Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer
Global histone modification patterns predict risk of prostate cancer recurrence
Unsafe SETs: histone lysine methyltransferases and cancer
Epigenetics provides a new generation of oncogenes and tumour-suppressor genes
Key roles of histone methyltransferase and demethylase in leukemogenesis
Cancer epigenetics reaches mainstream oncology
Down-regulation of human DAB2IP gene expression mediated by polycomb Ezh2 complex and histone deacetylase in prostate cancer
Integrative genomics analysis reveals silencing of beta-adrenergic signaling by polycomb in prostate cancer
The gene encoding the prostatic tumor suppressor PSP94 is a target for repression by the Polycomb group protein EZH2
Silencing of kruppel-like factor 2 by the histone methyltransferase EZH2 in human cancer
The polycomb group protein EZH2 is involved in progression of prostate cancer
essential for proliferation and amplified in cancer
EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells
Proc Natl Acad Sci USA 2003; 100: 11606–11611
Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity
modifies chromatin and is directly involved in infant acute leukaemia
hDOT1L links histone methylation to leukemogenesis
Epigenetic inactivation of the Sotos overgrowth syndrome gene histone methyltransferase NSD1 in human neuroblastoma and glioma
Proc Natl Acad Sci USA 2009; 106: 21830–21835
The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset
Genome-wide association study identifies a new melanoma susceptibility locus at 1q21.3
SETDB1: a novel KAP-1-associated histone H3
lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins
a novel histone H3-specific methyltransferase that interacts with ERG transcription factor
The histone methyltransferase SETDB1 and the DNA methyltransferase DNMT3A interact directly and localize to promoters silenced in cancer cells
Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET
SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state
DNA methylation and SETDB1/H3K9me3 regulate predominantly distinct sets of genes
Dual functions of histone-lysine N-methyltransferase Setdb1 protein at promyelocytic leukemia-nuclear body (PML-NB): maintaining PML-NB structure and regulating the expression of its associated genes
Deregulation of histone lysine methyltransferases contributes to oncogenic transformation of human bronchoepithelial cells
Treatment of HER2-positive breast cancer: current status and future perspectives
The cancer cell line encyclopedia enables predictive modeling of anticancer drug sensitivity
regulates glioblastoma-derived neurosphere cell differenciation and tumor propagation
Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7
Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor
RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia
BET bromodomain inhibition as a therapeutic strategy to target c-Myc
Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia
ESET/SETDB1 gene expression and histone H3 (K9) trimethylation in Huntington's disease
Proc Natl Acad Sci USA 2006; 103: 19176–19181
Expression of beta-galactosidase under the control of the human c-myc promoter in transgenic mice is inhibited by mithramycin
Comparative genomic hybridization analysis detects frequent
and 8q in human non-small cell lung carcinomas
Molecular cytogenetic analysis of non-small cell lung carcinoma by spectral karyotyping and comparative genomic hybridization
Assessment of molecular events in squamous and non-squamous cell lung carcinoma
Chromosomal imbalances of primary and metastasic lung adenocarcinomas
Distinct patterns of genetic alterations in adenocarcinoma and squamous cell carcinoma of the lung
Genomic alterations detected by comparative genomic hybridization in primary lung adenocarcinomas with special reference to the relationship with DNA ploidy
Comprenhensive genomic characterization of squamous cell lung cancers
Differential pathogenesis of lung adenocarcinoma subtypes involving sequence mutations
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This work was supported by the European Community’s Seventh Framework Programme (FP7/2007-2013) under Grant agreement number HEALTH-F2-2010-258677—CURELUNG project
the Institute of Health Carlos III (ISCIII)—PI10/02992
Kutxa-Fundación INBIOMED and the Health and Science Departments of the Catalan Government (Generalitat de Catalunya)
Cancer Epigenetics and Biology Progrm (PEBC)
Bellvitge Biomedical Research Institute (IDIBELL)
Experimental Therapeutics and Translational Oncology Program
Instituto de Biología Molecular y Celular del Cáncer
Instituto de Investigación Biomédica de Salamanca (IBSAL)
Centro Hospitalar São João/Medical Faculty and Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP)
Portuguese Oncology Institute and Biomedical Sciences Institute (ICBAS)
Institucio Catalana de Recerca i Estudis Avançats (ICREA)
The authors declare no conflict of interest
Supplementary Information accompanies this paper on the Oncogene website
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/
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VRK2 is a novel Ser-Thr kinase whose VRK2A isoform is located in the endoplasmic reticulum and mitochondrial membranes
We have studied the potential role that VRK2A has in the regulation of mitochondrial-mediated apoptosis
VRK2A can regulate the intrinsic apoptotic pathway in two different ways
The VRK2A protein directly interacts with Bcl-xL
VRK2A does not compete with Bax for interaction with Bcl-xL
and these proteins can form a complex that reduces apoptosis
high VRK2 levels confer protection against apoptosis
VRK2 knockdown results in an increased expression of BAX gene expression that is mediated by its proximal promoter
thus VRK2A behaves as a negative regulator of BAX
Low levels of VRK2A causes an increase in mitochondrial Bax protein level
leading to an increase in the release of cytochrome C and caspase activation
VRK2A loss results in an increase in cell death that can be detected by an increase in annexinV+ cells
Low levels of VRK2A increase cell sensitivity to induction of apoptosis by chemotherapeutic drugs like camptothecin or doxorubicin
We conclude that VRK2A protein is a novel modulator of apoptosis
apoptosis is also modulated by additional cellular proteins which in general are not known
we hypothesized that VRK2A might have a protective role against apoptosis if it could interact with
proteins that participate in the mechanisms that trigger or execute apoptosis in human cells
it has been identified that VRK2A levels can modulate apoptosis in two different ways
by its interaction with Bcl-xL and by regulation of BAX gene expression
Subcellular localization and interactions of VRK2A with apoptotic proteins
(a) Colocalization of endogenous VRK2 with transfected Bcl-xL detected by confocal microscopy in A549 cells
VRK2 was detected with a rabbit polyclonal antibody and Bcl-xL with a monoclonal antibody
(b) Colocalization of VRK2 with transfected Bax detected by confocal microscopy in A549 cells
Bax was detected with a monoclonal antibody
(c) Detection of interaction between VRK2 and Bcl-xL
HeLa cells were transfected with plasmid pCEFL-GST-VRK2A that expresses GST-VRK2A and used in a pulldown assay in which the associated protein were detected in immunoblots using specific antibodies for GST that detects transfected GST and GST-VRK2
and specific antibodies for detection of endogenous Bcl-2
The ratio of GST-VRK2/actin ranges between 0.72–0.66 in the three bots
respectively.(d) Mapping the interaction between VRK2A and Bcl-xL
The region of interaction between VRK2A and Bcl-xL was performed by pulldown assays with different constructs of the VRK2A protein
which is summarized in a diagram to the right
VRK2B is a spliced variant that has 397 aminoa cids and is identical to the N-terminal region of VRK2A
and thus it was independent of VRK2 kinase activity
The potential interaction of VRK2A with BH3-only proteins, such as PUMAα, BAD and Binp3L was ruled out using a similar approach (Supplementary Figure S1)
Bcl-xL and Bax detected in pulldown assays
(a) Effect of increasing Bax in the presence of saturating amounts of VRK2A and Bcl-xL
(b) Effect of increasing Bcl-xL in the presence of saturating VRK2A and Bax
(c) Effect of increasing VRK2A in the presence of saturating Bcl-xL and Bax
HEK293T cells were transfected with the indicated amounts of plasmids pCEFL-GST-VRK2A
The proteins in the input lysates are shown at the top
The proteins present in the pulldown are shown at the bottom and were detected with the corresponding specific antibody
To determine if VRK2A was able to phosphorylate either Bcl-xL or Bax
different in vitro kinase assays were performed
VRK2A did not phosphorylate any of these proteins (not shown)
(a) VRK2 knockdown in A549 cells modified Bax protein levels
The total Bax protein was detected with the 2D2 antibody
a reduction in the levels of antiapoptotic proteins
Bcl-xL and Bcl-2 was detected in immunoblots
(b) VRK2 knockdown increased the level of BAX gene expression in A549 cells determined by qt-RT-PCR
A549 cells were mock transfected or transfected with the indicated SiRNA and 3 days after transfection
total RNA was extracted and the level of Bax and GAPDH mRNA levels were determined by qt-RT-PCR
The results from four independent experiments are shown
(c) Reduction of VRK2A levels activated expression from the promoter of BAX
A549 cells were transfected with si-Control (Si-Ct) or two different si-VRK2 (06 and 08
cells were transfected with 1 μg of pGL3-Bax-Luc (–687 to –318) and cells extracts were used to measure the luciferase activity
The results are the mean of three independent experiments and the mean with the standard deviation is shown
(d) VRK2A overexpression inhibited expression directed by the BAX gene promoter that is induced in response to camptothecin
A549 cells were treated for 6 hours with 5 μM camptothecin followed by transfection with 1 μg of empty vector (pCEFL-HA) or plasmid expressing VRK2A (pCEFL-HA-VRK2A) or VRK1 (pCEFL-HA-VRK1) together with the reporter pGL3-Bax-Luc
from three independent experiments is shown
To determine if the effect on Bax protein levels was a consequence of an effect mediated by regulation of BAX gene expression, the level of BAX mRNA was determined by qt-RT-PCR. Loss of VRK2 using two different siRNAs resulted in a significant increase in the expression of BAX mRNA (Figure 3b)
this result indicated that VRK2 inhibited BAX gene expression
BCL2 and Bcl-xl mRNA levels were also analysed
but no differences in gene expression were detected after VRK2 knockdown (not shown)
All these data indicated that VRK2A inhibited transcription directed by the BAX gene promoter
Low levels of VRK2 facilitated release of cytochrome c and PARP processing
(a) VRK2 knockdown with si-VRK2-06 and si-Control (si-Ct) in three cell lines
lung carcinomas A549 and H1299 and cervical carcinoma HeLa
The reduction in endogenous protein level and its quantification 4 days after transfection are shown
Endogenous VRK2 protein was detected with a rabbit polyclonal antibody
(b) The release of cytochrome c was determined by its detection in cytosolic and mitochondrial fractions in extracts from HeLa cells prepared 96 h after siRNA transfection
The effect of si-VRK2-06 is shown in the top gel panel and quantification of cytochrome c distribution in the two fractions
represented as percentage of band intensity respect to the corresponding marker
Actin and cytochrome c oxidase II were used as markers for the cytosolic and mitochondrial fractions respectively
(c) Effect of VRK2 knockdown on processing of PARP
The proteolytic processing of PARP was determined in a western blot and quantification of the 115 and 85-kDa bands
represented as percentage of band intensity
Immunoblots show a representative experiment and quantifications show the mean and S.D
These results indicated that loss of VRK2A facilitated induction of the intrinsic apoptotic pathway and we concluded that high levels of VRK2A have a protective role as inhibitors of apoptosis
Low levels of VRK2 sensitized cells to camptothecin treatment
(a) VRK2 knockdown was induced in A549 cells
These cells were treated with 5 μM of camptothecin and the distribution of cytochrome c and Bax were determined in cytosolic and membrane fractions at different time points after camptothecin addition
Fractionated extracts were used for western blot analysis with the corresponding antibodies
(b) Low VRK2 facilitated activation of caspases in response to treatment with 5 μM camptothecin
VRK2 knockdown was induced by two different siRNA
si-VRK2-06 (at the top) or si-VRK-2M (at the bottom)
and caspase activation was determined by PARP processing
PARP was determined by immunoblot with a specific antibody at different time points after camptothecin addition to A549 cell culture
(c) Loss of VRK2 facilitated induction of DNA damage by camptothecin
A549 cells were transfected with si-Control or si-VRK2-06 and 96 hours later
they were treated with 5 μM of camptothecin
A TUNEL assay was performed to detect the presence of free DNA ends at different time points
Cells labelled with fluorescein-12-dTUP (rTdT) and propidium iodide were analysed in a fluorescence microscope
Cell images with fluorescence are shown in the left panel and in the right panel the quantification of TUNEL positive cells are shown as a function of time following treatment with camptothecin
Effect of VRK2 knockdown on cell death induced by camptothecin treatment
VRK2 was knocked down in A549 cells and after 3 days
cells were treated with camptothecin for the indicated time points
(a) Effect of VRK2 knockdown on the sensitivity of A549 cells to camptothecin detected by alterations in the plasma membrane with annexin V+
Cells were stained with AnnexinV-FITC and positive cells were counted by flow cytometry
(b) Effect of VRK2 knockdown on A549 cell viability after camptothecin treatment
Viability was determined by Trypan blue dye exclusion assay
(c) Effect of VRK2 knockdown on mitochondrial membrane potential in the response to camptothecin was determined by loss of DIOC6 fluorescence in damaged cells
Effect of camptothecin (5 μM) on A549 cells and detection of propidium iodide staining by flow cytometry
The fragmented DNA was detected in the sub-G0/G1 window (bar)
Apoptosis is a tightly regulated process in which many of the components that modulate this form of cell death are still unknown
Regulation of apoptosis can be achieved by multiple mechanisms
One is by novel proteins that form a complex with apoptotic or antiapoptotic proteins
The other is by regulating the expression of apoptotic or anti-apoptotic genes and altering their intracellular levels
there is an alteration in mitochondrial membrane
and it is likely that other mitochondrial membrane protein complexes remain to be identified
but they can modulate the magnitude of the apoptotic response
but it is possible that VRK2A in the mitochondrial membrane complex might phosphorylate some protein not yet identified
This might either interfere with Bax incorporation or alternatively alter mitochondrial membranes
facilitating its permeability and cytochrome c release
Variations in intracellular VRK2A protein levels
This effect is mediated by the regulation of the BAX promoter by VRK2A
functioning as a negative regulator of this promoter
The VRK2A function as a transcriptional repressor opens up a new aspect in the role of VRK2 as a regulator of transcription
VRK2A is acting at two different levels in the modulation of the cell response to induction of apoptosis: protein–protein interaction and transcriptional regulation
they might inhibit mitogenic signal-reducing tumour growth
they might make cells less sensitive to apoptosis
The final effect would be a consequence of the net balance among alternative signalling pathways and the relative strength of their respective signals
high VRK2 levels might be a component in the formation of more indolent and perhaps
Cell lines A549 (p53 wt) and H1299 (p53−/−) from lung carcinomas
HEK293T cells and HeLa cells were cultured at 37 °C with 5% CO2 in RPMI or DMEM supplemented with 10% heat-inactivated foetal calf serum supplemented with 2 mM L-glutamine
50 μg/ml streptomycin and 100 U/ml penicillin
cells were fixed with 4% paraformaldehyde and incubated with propidium iodide
Cells were analysed in a FACScalibur flow cytometer
data acquisition was performed with Cell Quest programme (Becton Dickinson)
analysed with Modfit LT programme (Verity Software House
Camptothecin and propidium iodide were from Sigma (St
DiOC6(3) (3,3′—Dihexyloxacarbocyanine iodide) was from Molecular Probes (Eugene
Annexin V-FITC was from Immunostep (Salamanca,Spain)
Total RNA was extracted from A549 cells using RNeasy kit from Qiagen (Valencia
The quality of the extracted RNA was determined in a Bioanalyzer 2100 nano-lab chip (Agilent
50 μg of total RNA was amplified with specific primers for Bax
The Quantitect-SYBR green RT-PCR kit (Qiagen) was used in an iCycler iQ5 Multicolour Real-time PCR Detection System and Optical System Software BIO-RAD iQ5 (BioRad
The following primers were used for RT-PCR amplification
Bax was amplified with primers (forward: 5′-GGCCCACCAGCTCTGAGCAGA-3′
Bcl-xL (forward: 5′-TCCTTGTCTACGCTTTCCACG-3′; reverse: 5′-GGTCGCATTGTGGCCTTT-3′)
Bcl-2 (forward: 5′-CATGTGTGTGGAGAGCGTCAA-3′ and reverse: 5′-GCCGGTTCAGGTACTCAGTCA-3′)
VRK2 (forward: 5′-AGTGAGAGAAGCGCTGAGTCCT-3′ and reverse: 5′-CAAAGGTTCTTGAGACTCTTG-3′)
GAPDH (forward: 5′-GGTCTTACTCCTTGGAGGCCATGTG-3′ and reverse: 5′-ACCTAACTACATGGTTTACATGTT-3′)
All primers were synthesized by Sigma-Aldrich (St Louis
AU1 epitope was detected with monoclonal or polyclonal antibodies from Covance (Emeryville
GST was detected with monoclonal B-14 (sc-138
HA epitope was detected with monoclonal (Covance) or polyclonal antibody (eBioscience
Flag was detected with polyclonal and monoclonal antibodies from Sigma
cytochrome c (7H8.2C12) and PARP (c2-10) were from BD Biosciences (San Jose
Cleaved PARP was detected with monoclonal antibody (F21-852) from BD Biosciences
Rabbit polyclonal anti-Bax was from Abcam (Cambridge
Bcl-2 was detected with sc-492 from Santa Cruz
Cytochrome oxidase II was detected with a monoclonal from Invitrogen
The secondary antibodies used from Amersham Biosciences (GE Healthcare
anti-rabbit-Cy3 (Fluorolink Cy3) and anti-rabbit-Cy2 (Fluorolink Cy2) in immunofluorescence or anti-mouse-HRP or anti-rabbit-HRP for western blots
Cells were harvested 48 h post transfection and lysed with a buffer containing 20 mM Tris-HCl pH 7.4
10% (v/v) glycerol and 1% Triton X-100 plus protease inhibitors
50 μg of total protein lysate were analysed in a 10% SDS–polyacrylamide gel
GST pulldowns were performed by incubating 1 mg of total cell extract with Glutathione-Sepharose 4B beads (Amersham Biosciences Pharmacia Biotech) for 12 h at 4 °C
Sepharose beads were washed three times with lysis buffer and analysed by SDS–PAGE following by western blots with the corresponding antibodies
Luminescence in westerns blot was developed with an ECL kit (Amersham Biosciences)
Early apoptotic cells present phosphatydilserine on their surface and were detected by flow cytometry of cells labelled with Annexin V-FITC (Immunostep SL
A549 and H1299 cells were grown and transfected with siControl or siVRK2 as indicated above and 96 hours later
106 cells were washed in PBS and stained with annexin-V-FITC and propidium iodide in annexin buffer (0,1 M Hepes/NaOH (pH 7.4) 1.4 M NaCl
resuspended in 400 μl and analysed by flow cytometry in a FACS Calibur (Becton Dickinson)
Colocalization of proteins was performed by immunofluorescence
Cells were fixed with 3% paraformaldehyde and stained with specific antibodies against endogenous proteins
Nuclei were stained with DAPI (4′,6′-diamidino-2-fenilindol)
Immunofluorescences were examined in a confocal Zeiss LSM 510 microscope (Zeiss
Germany) and images were analysed with LSM Image Browser software (Zeiss)
the cells were transfected with siRNA and treated with camptothecin (5 μM)
the culture media was removed and cells were fixed with 4% paraformaldehyde for 15 min
washed with cold PBS and treated with 0.2% Triton-X100 for 5 min
Cells were blocked for 15 min in incubation buffer (200 mM potassium cacodylate
2.5 mM cobalt hydrochloride) at room temperature
The buffer was changed and 5 μl per point were added containing 50 μM fluorescein-12-dUTP
Cells were incubated in a humidified atmosphere in the dark for 1 h at 37 °C
The reaction was finished by addition of 1 ml 20 × SSC for 15 min and afterwards cells were rinsed in PBS three times
The coverslips were treated with antifade and examined in a confocal Zeiss Axioplan2 microscope
Images were analysed using the Openlab 4.0.2 program (Improvision
cells were treated with 5 μM and collected for analysis at several time points up to 72 h after drug treatment
These cells were washed in PBS and collected by centrifugation at 15 000 r.p.m for 10 min
The cell pellet was suspended in PBS containing 20 nM DiOC6 and was incubated at 26 °C for 20 min
Cells were analysed in a FACS-Calibur (Becton Dickinson) with the Cell Quest and FCS Express V3 programs (De novo software
c-Jun N-terminal kinase interacting protein
Involvement of the bcl-2 gene in human follicular lymphoma
Proapoptotic multidomain Bcl-2/Bax-family proteins: mechanisms
The BCL-2 protein family: opposing activities that mediate cell death
Apoptosis: controlled demolition at the cellular level
Bcl-2 family members: dual regulators of apoptosis and autophagy
Mitochondria in apoptosis: bcl-2 family members and mitochondrial dynamics
Cytochrome c: functions beyond respiration
Human cellular protein VRK2 interacts specifically with Epstein-Barr virus BHRF1
The vaccinia-related kinases phosphorylate the N' terminus of BAF
regulating its interaction with DNA and its retention in the nucleus
The protein kinase complement of the human genome
Emerging biological functions of the vaccinia-related kinase (VRK) family
The human vaccinia-related kinase 1 (VRK1) phosphorylates threonine-18 within the mdm-2 binding site of the p53 tumour suppressor protein
Characterization of three paralogous members of the Mammalian vaccinia related kinase family
Structure of the pseudokinase VRK3 reveals a degraded catalytic site
Proteomics identification of nuclear Ran GTPase as an inhibitor of human VRK1 and VRK2 (vaccinia-related kinase) activities
Macro Histone H2A1.2 (MacroH2A1) Protein Suppresses Mitotic Kinase VRK1 during Interphase
Roles of VRK1 as a new player in the control of biological processes required for cell division
Human VRK1 is an early response gene and its loss causes a block in cell cycle progression
VRK1 phosphorylates CREB and mediates CCND1 expression
Vaccinia-related Kinase 1 (VRK1) is an upstream nucleosomal kinase required for the assembly of 53BP1 foci in response to ionizing radiation-induced DNA damage
Plk3 interacts with and specifically phosphorylates VRK1 in Ser342
a downstream target in a pathway that induces Golgi fragmentation
c-Jun phosphorylation by the human vaccinia-related kinase 1 (VRK1) and its cooperation with the N-terminal kinase of c-Jun (JNK)
Human vaccinia-related kinase 1 (VRK1) activates the ATF2 transcriptional activity by novel phosphorylation on Thr-73 and Ser-62 and cooperates with JNK
The subcellular localization of vaccinia-related kinase-2 (VRK2) isoforms determines their different effect on p53 stability in tumour cell lines
Substrate profiling of human vaccinia-related kinases identifies coilin
as a phosphorylation target with neurological implications
Differential inhibitor sensitivity between human kinases VRK1 and VRK2
Vaccinia-related kinase 2 modulates the stress response to hypoxia mediated by TAK1
Modulation of interleukin-1 transcriptional response by the interaction between VRK2 and the JIP1 scaffold protein
VRK2 inhibits mitogen-activated protein kinase signaling and inversely correlates with ErbB2 in human breast cancer
VRK2 anchors KSR1-MEK1 to endoplasmic reticulum forming a macromolecular complex that compartmentalizes MAPK signaling
Expansion and evolution of cell death programmes
a p53 and topoisomerase I-binding RING finger protein
is a coactivator of p53 in growth suppression induced by DNA damage
Phosphorylation of Bax Ser184 by Akt regulates its activity and apoptosis in neutrophils
JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation and mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells
Human VRK2 (Vaccinia-related Kinase 2) modulates tumor cell invasion by hyperactivation of NFAT1 and expression of cyclooxygenase-2
NMR solution structure of human vaccinia-related kinase 1 (VRK1) reveals the C-terminal tail essential for its structural stability and autocatalytic activity
A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases
Proc Natl Acad Sci USA 2007; 104: 20523–20528
Targeting cancer with small molecule kinase inhibitors
Targeting the cancer kinome through polypharmacology
Insights for the development of specific kinase inhibitors by targeted structural genomics
Differential Inhibitor Sensitivity between Human Kinases VRK1 and VRK2
Breast cancer molecular subtypes respond differently to preoperative chemotherapy
Nomograms to predict pathologic complete response and metastasis-free survival after preoperative chemotherapy for breast cancer
Mitochondrial Hep27 is a c-Myb target gene that inhibits Mdm2 and stabilizes p53
Bcl-X(L) specifically activates Bak to induce swelling and restructuring of the endoplasmic reticulum
Tumor suppressor p53 is a direct transcriptional activator of the human bax gene
p53 stabilization and accumulation induced by human vaccinia-related kinase 1
Involvement of mitochondria and caspase-3 in ET-18-OCH(3)-induced apoptosis of human leukemic cells
Involvement of mitochondria and recruitment of Fas/CD95 signaling in lipid rafts in resveratrol-mediated antimyeloma and antileukemia actions
p53 downregulates its activating vaccinia-related kinase 1
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have JAE-CSIC-Fondo Social Europeo Predoctoral fellowships
TM and SB were funded by CSIC-Banco Santander and Ministerio de Ciencia e Innovación fellowships respectively
This work was funded by grants from Ministerio de Ciencia e Innovación (SAF2010-14935 and CSD2007-0017)
The protein interactions from this publication have been submitted to the European Bioinformatics Institute-EMBL IMEx (http://www.imexconsortium.org/) consortium through IntAct (pmid 19850723) and assigned the identifier IM-18530
Supplementary Information accompanies this paper on Cell Death and Disease website
This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License
visit http://creativecommons.org/licenses/by-nc-nd/3.0/
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DOI: https://doi.org/10.1038/cddis.2013.40
Riviera Prep School students donated their artwork to a silent auction benefiting research to combatHuntington’s Disease
The silent auction took place during a gala on May 4 at the Palmetto Bay Village Center
The following students are pictured with their art: Maya Masucci
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a private non-profit entity owned by the Lonxanet Foundation for Sustainable Fishing and the artisanal fishermen’s guilds of Lira
opened its fish shop to the Madrid public as part of the inauguration of the San Miguel Market
This inauguration was attended by Ana Leiva and Sonia Castañeda
director of the Biodiversity Foundation and director of International
president of the Slowfood Convivium in Madrid; Susana Rodríguez
general director of Pesqueiro Development of the Ministry of the Sea of the Xunta de Galicia; Agustín Pérez and Juan Manuel Gómez
major patrons of the fishermen’s guilds of Lira and Cedeira respectively; Emilio Louro (Milucho)
manager of the Lyre Brotherhood; Pepe Novo
head of marketing of the Cedeira Brotherhood
director of the Ashoka-Spain Foundation and
Project Director of the Lonxanet Foundation for Sustainable Fishing
The added value that the fishmonger of Lonxanet Directo S.L
offers is the possibility of having the guarantee of traceability of the fish and seafood that it will offer to the public of Madrid
which is characterized by the greatest respect for the biodiversity of the marine environment as well as by the promotion of traditional knowledge and fishing methods respectful of the environment and local cultures
With the opening of this direct point of sale in a place like Madrid
the aim is to find solutions to the main problem of the artisanal fishing sector
Bringing the product of the Galician brotherhoods
transmitting at the same time the values of sustainability and responsible fishing
This risky but determined bet is intended to guarantee the future of fishing communities
it should also be noted that the brotherhoods of Lira and Cedeira
have been the first fishermen’s organizations in the entire Spanish State that have opted for the design and creation of two marine reserves of fishing interest in their respective fishing areas with the firm vocation of involving the fishing sector in the sustainability and conservation of the biodiversity of our seas
is the location of this house whose distinguishing feature is a T-shaped roof
For this work the Valencia practice of Fran Silvestre and Andrés Alfaro Hofmann has been named the 2020 winner in the Architecture section of the German Design Awards
The project seeks to obtain the best vistas of the surroundings with a passable roof that serves as a viewing deck
The two levels of the 350-square-meter residence adapt to the sloping terrain
with a half-buried floor containing a living room
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This was how the two teachers from Galicia
greeted us as soon as they saw us at the airport
Teresa Nuzzo School in Marsa is in its second year of a second Comenius project
Having a teacher exchange with Colexio San Andres in Cedeira (Redondela) not only provided us with the opportunity to delve into a different school setting but also to compare it with previous experiences in Scandinavian schools
We were given a warm welcome from staff and pupils alike
All the pupils were eager to know more about Malta
and about their pen friends from our school
They bombarded us with never-ending questions
making use of audiovisual material which we obtained beforehand from the Department of Information and the Malta Tourism Authority
Three 10-year-olds are in charge of putting up a weekly informative radio programme which is aired during break
We were guests during that week and we were struck by their sense of independence and responsibility in carrying out such an assignment
we were given a copy of the radio programme
which we played when we returned to our classes
It's difficult not to mention the fact that there were no more than 12 mixed ability pupils in each class
thus making it easier for the low achievers to attain more individual attention
Teachers work in a much less stressful environment due to the fact that there is no exam-orientated system
the headteacher's role in all Spanish schools is not only related to the administration of the school
we actually saw the headmaster of Cedeira giving a PE lesson
An excursion which we shall always remember was the one to Santiago de Compostela
In spite of the bad weather the beauty of its cathedral made our visit worthwhile and unforgettable
we visited Vigo's School of Languages and there we also had an appointment with a lecturer who discussed with us certain teaching methods
the colourful and lively school environment
the Galicians' amiability and gastronomy were all conducive to making our stay at Cedeira's school a truly unforgettable
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After hosting two Welsh teachers on a teachers' exchange last December
it was Malta's turn once again to organise a project meeting for the three participating countries
The three project co-ordinators met at Theresa Nuzzo School
between January 16 and 19 to discuss the work to be done in the third year of the Comenius project entitled "This is me..
and what about you?" The three schools will focus on the teaching of Welsh
The history of the language and the histories of the three countries will also be dealt with
Children will also go into the history of Bedwas School in Wales
Cedeira School in Galicia (Spain) and Theresa Nuzzo
it is hoped that all children will get to know each other better
all three schools are encouraging the children to keep up constant communication through e-mails and traditional letter-writing
it is the practice for the three schools to collect the children's letters and send them all together in a pack to each school at the end of each month
Theresa Nuzzo School is also trying to promote Malta as a tourist attraction by showing how rich the island's history is
an end product of this Comenius project is the production of simple holiday brochures which will eventually be exchanged among the three schools
Having all project co-ordinators here in Malta
the school showed the foreigners some of the sights
such as Valletta with all its treasures of churches
Marsaxlokk and its colourful bay and fishing boats
and the prehistoric temples of Hagar Qim and Mnajdra
What truly impressed the foreign group was the warm welcome and friendliness shown by all the Maltese children
Typical local food also played its part on the foreigners' appetite and outlook with regard to establishing Malta as a holiday destination
They left Malta with beautiful memories and plans for a future visit with their families and friends in a more relaxed atmosphere
away from schools and their continuous commitment
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