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Boston Wedding Photographer
Today, I brought to you Courtney and Jeff Engagement. This cute couple haven known each other for a long time andView full post »
Emily and Tom requested a second engagement session in Tanglewood Musical Center in Lenox, MA. This is the place whereView full post »
I've had the pleasure to shoot Dave & Ana's wedding on July 6 2013 at Bittersweet Farm, Westport MA.View full post »
It is now 3:11am in the morning and I just had to share this gorgeous teaser shot for the wedding I shot today thatView full post »
I hope you guys enjoy this engagement session as much as I do. Dave & Ana's session was so much fun. FilledView full post »
New England has been getting tropical type weather the last couple of days. Saturday June 29th 2013 was forecasted toView full post »
Adnan & Sweta got married on June 1st-June 2nd 2013, and I had the pleasure of shooting their beautiful day. One ofView full post »
Recently engaged, Tom and Emily proved to be an adventurous couple to work with. We walked all around Boston to captureView full post »
One thing that I love about shooting a south asian wedding is the colors and the culture that is prominent throughoutView full post »
We were blessed with a gorgeous day on Saturday, May 31 2013. Slightly on the hotter side but that didn't stopView full post »
I can't begin to describe how amazing this couple is. I simply adore them :). Here is a teaser of an upcomingView full post »
A surprise wedding! Kevin and Elana had plans to get married in 2014, however they couldn't wait to tie the knot.View full post »
Last week I shot Kelly's maternity photo shoot in Arnold Arboretum. Words can't begin to describe howView full post »
Kelly is about to have her second child and she asked me to photograph her and her first, Isla. This is a teaser of whatView full post »
Wedding Photographs tell stories of the greatest day of a couple's life; the beginning of a new life together asView full post »
© 2012 Boston Wedding Photographer | Renard Richie |ProPhoto Photographer Site by NetRivet, Inc.


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Introducing our new Town Councillor
URSULA Shepherd was elected to represent the Emersons Green Ward on Emersons Green Town Council following a narrow victory in a by-election on Thursday July 12.
Representing the Conservative Party, she polled 203 votes against Labour's Sandie Davis's 198. Emersons Green Voice caught up with Ursula to find out about her plans for her new role
Tell us a bit more about you
Living locally in Lyde Green with my two daughters, I have first-hand experience of the problems we face as well as the needs of our community.
It is extremely exciting to be the first elected Town Councillor who lives in Lyde Green and represents the area. It is an honour to represent our growing community for all issues that concern our local residents. If it's important to my neighbours then it's important to me, as we are equally affected.
Why did you want to become a councillor?
I am passionate about my local community and have campaigned for youth activities in the area and continue to attend community meetings about ways to reduce anti-social behaviour. Already we have funding for a youth worker team to get involved and be out there engaging the children weekly where they congregate. We were advised at the Community Engagement Forum last month that anti social behaviour has reduced so this initiative seems to be working already.
We have also have a fantastic Neighbourhood Watch committee and team in Lyde Green who work tirelessly to keep us informed and safe. Please get involved with this if you can. It's very rewarding.
What do you hope to achieve in your role?
I have wasted no time in trying to make a difference, having picked up on a few things on social media, for example the use of the playing fields which Longwell Green Football Club are managing (on behalf of Green Square). After a very positive conversation with John Gibbs, Chairman of LGFC, I am delighted to hear that the fields will be accessible by locals for playing football or other games and anyone wanting to make arrangements should contact LGFC directly. They will be opening a cafe too which invites spectators. This should be a wonderful boost for children and adults to get involved in sport in our neighbourhood and will go far in terms of giving our young people something fun to do and be involved in.
Having somewhere fun and safe for our children to play and grow up is of primary importance and I will continue to fight to protect our green spaces such as Lyde Green Common as well as to support accessible local facilities for Emersons Green and Lyde Green. It is my privilege to serve my community.
Residents can contact Ursula by emailing email@example.com or calling 07862 849033

subset: commoncrawl


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Click Here to Start a New Search
Full Resource Name:
Guardians Credit Union Royal Palm Beach Branch
10469 Southern Boulevard
(561) 686-4006
Provides a low-income designated, Community Development Financial Institution, with a full range of financial products and services for the unbanked and people of modest means or who have damaged credit. This includes checking and savings accounts, auto loans, personal loans, credit builder loans, credit and secured cards, and financial education and counseling.
Primary Services:
Resource Groups


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Writing about ZZT feels perfunctory. Having only heard about the game a month ago, its importance was to me invisible. Being a ASCII-based computer game released in 1991, present in videogame's market tested pedagogy, it's not the kind of game that gets to be remembered. There's undeniably an essence of iconoclasm to ZZT; I think it resembles a bunch of things, but akin to scenes and art movements, it was a time and a place. It's not going to be reproduced. In that sense I can't introduce ZZT, because I can't hold it in place. I can't capture what I never knew or saw.
Those who were imprinted by the allure of making ZZT games seem to follow and be followed by artistic pursuit. I see a self-explanatory, self-justified thing. It was a design language people could see and feel demonstrated. An immediate understanding that a videogame was made by a person. The ZZT community was, paraphrasing words that aren't mine, mostly a bunch of frustrated teens, that through a specific and arcane practice were able to exercise control and interpret their lives. The specificity was kind of special and secret, and it was kind of lonely and isolating. In the late 90s, who was seriously into ASCII videogames, among the advent of 3D?
What people play, and why they play it, doesn't line up with the boring narrative enthusiast press has etched into our spaces. I missed out on communities like ZZT, or later rpgmaker, or later tigsource et al, because for a long time I was convinced videogames were way too difficult to make, and I was nowhere near smart enough to make them (I'm now adamant anyone can make a videogame). But, growing up poor, games outside of the peddled mainstream were windows of hope and life. I frequented home of the underdogs and sister sites like it, playing whatever old pc games that caught my eye, constantly enamored by their pixel art, while keeping up with sonic fangames.
I see similar arrangements of artists and people popping up around intense fandoms, or in ad-hoc communities of youth existing on gamejolt. Different circumstances, but their spirit is like what the ZZT community was. A cursed blessing, the internet, where outsiders can still thrive and find themselves. Underpinning these communities that are separate from commercial videogame spaces are (underscore italics) communities. No matter the mess or drama, people are finding themselves and helping each other, in intense and intimate ways. They do this in places more specific, anonymous, and dedicated than general social media. It might not make sense to outsiders looking in, but it's the most real and powerful thing to the people participating.
They did it for the love of creating their own insane, brilliant worlds. There was no sexual conquest, money, or prestige involved. No incentive. They just wanted to make games to make their small, tightly knit group of friends smile and laugh. And the sweet, sweet catharsis… ! I think that's what it was all about … It's hard to think about now, but we poured our blood, soul and guts into those ASCII games. And that was the most beautiful thing possible for us at the time.
– draco, quoted in "ZZT" by Anna Anthropy
I read "ZZT (Boss Fight Books #3)" on recommendation by friend of the site John Thyer and found it echoed my experiences growing up in online communities. I've seen many independently express a similar feeling of what it means to be a generation raised by the internet. There's an emptiness within and without; people turn to the virtual when they're unfulfilled outside it. Though some of it is just being a teen, and I might be projecting, but after adolescence, there's a gradual understanding of how important these spaces were for growing and surviving. That there are certain ways people come to learn things that may be different from the norm and it's not shameful. These communities are still thriving, not through ZZT, but various other things, though they're being subsumed by corporate ran general purpose social media. Like all things that decline in purpose, so it goes.
I don't know how I feel honestly, I don't know the totality, but I spent a lot of time in Super Smash Bros. Brawl communities around the same time as the age gap described for ZZT teens. It was a place where things I did matter. I could make friends based on a common interest. There was an immediate correlation to the work I put in and improvement I felt. That work could also translate to understanding and admiration, in some way. A bunch of people working toward the same silly thing. It's silly to me now. Given anyone out of a crowd and they'd think ASCII games are silly too. At the time though there was nothing more important.
But it's about more than that, and I think the book gets at this: almost everyone who makes queer games right now, or even anyone who makes your typical pixel-art roguelike platformer with 1.5 new mechanics — is quite young. The vast majority of us are in our teens and twenties. Much like the ZZT community, we are all still growing up together, figuring out who we are, performing new identities, occasionally screwing-up big-time, trying to make ends meet, and learning how to heal.
We are young and on the internet. Anna dedicated her book to the "children of the glow" in reference to the young people who grew up with the proto-internet, but it just as easily applies to all of us right now.
– Robert Yang, "I'm young and love to be young": on ZZT, by Anna Anthropy
I've been thinking of this in limiting terms. These groups were for teens, but now those teens are somewhere else. Now summer never ends. I'm part of an online community again. Making games, talking about them, thinking about them. It's more ad-hoc, obviously, and hopefully less fueled by teenage fighting and rivalry. What I mean is I'd like to see game development looked at as a nurturing thing. Expel mock-corporate attitudes, stop trying to become like a "creative" silicon valley.
There has always been a genuine exuberance for game development and creativity itself, but it existed underneath the kind of power that acting as a corporation or business owner offered to thirsting white nerds. The cold candor of marketing and analytics… makes them feel more important? Allows them to emulate the corporations they admire? Feels comfortable because they previously worked in a culture like it? I'm just making stuff up now. I don't actually know why it's has been and continues to be dominant culture in videogames, on account of it being something I don't understand.
I see it being casually, unconsciously rejected too. It's not impossible to make a living without being explicitly an entrepreneur (because it is close to impossible without grants or connections). And it's being discovered, simply, that taking game development "seriously" and having the right attitude, making the right moves, isn't at all a recipe of success. In fact, it may be something that is instantly dated. So what are game developers in it for? What are we chasing?
I'm going to avoid speaking for people but I see collective support and common interest. People are banding together for the opportunities it provides for people to live and grow. That's an enduring echo I see from desperate communities passed on from ZZT. Could we organize? Aim these communities? I don't know. I think sustainability can be ensured by working together. Our sector could set an example for others. At least, it's something to keep in mind, to soften relations, to think in terms of solidarity instead of rivalry.
This game is Edible Vomit released in 1999 (eight years after the original release of ZZT, by the aforementioned draco). Surreal, wild, impressionist, scraping boundaries in its creative vacuum. I'm not meaning to imply that videogames are catching up. I mean to say they were always there. It wasn't cool, it was maybe too niche to find, but it was there. As videogames drift into the ephemeral, as they shamble up to their potential of being challenging, esoteric, and otherworldly, in so many ways, games made on ZZT show that we we've been here. I hope to pay that forward.


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Our Signature Product
2.5%PAAG Hydrogel
PAAG products, although often considered equal, have clear differences in composition, manufacturing and injection techniques as well as their ability to interact with surrounding tissues. These characteristics determine the safety and effectiveness of each hydrogel.
Arthramid Vet 2.5% PAAG
Arthramid Vet (AV) is a unique and patented hydrogel consisting of 97.5% sterile water and 2.5% polyacrylamide. This combination results in a gel similar to hyaluronic acid in structure and tissue compatibility, but with a superior and longer lasting effect, as it integrates into the joint itself and is non-degradable.
AV is elastic and viscous in nature, ideally suited for injection into the joints of animals providing an innovative and safe solution to veterinarians. Arthramid Vet is supplied in a sterile pre-filled 1ml syringe sealed with a Luer lock fitting.
AV is used to treat non-infectious causes of joint lameness in animals, including both early and late stages of osteoarthritis (OA). OA can be caused by traumatic injury or usual wear and tear which causes the joint to become inflamed. As the cartilage or cushion between the joints breaks down, pain, stiffness, and swelling develop, causing lameness.
Lameness remains the most common cause of reduced performance in the equine industry. In addition to pain reduction, treatment should also serve to arrest or slow down the progression of lesions allowing for disease modification. It is now well recognised that the OA disease process can start with the disease in the synovial membrane and that inflamed synovium is an essential component in the pathogenesis, with it almost always being present in cases of OA.
As part of the OA complex, the elastic and viscous nature of the synovial fluid is abnormally low. Furthermore, the synovial membrane becomes a source of inflammatory products which contribute to the progressive degradation of the joint over time.
Studies have shown disease-modifying effects as Arthramid Vet integrates into the synovial membrane, stabilising the joint capsule, and increasing elasticity, which, in turn, reduces the onset of synovitis and its harmful effects. The formation of rejuvenated synovium improves the nature of the synovial fluid within the joint itself.
Additionally, Arthramid Vet essentially causes a 'cushion-like' effect in the joint which allows for the preservation and/ or regeneration of the joint cartilage contributing to reducing the pain of synovitis and OA, and ultimately restoring the joint function.
World Safety Standards
• Danish manufacturer Contura International A/S exceeds world-class safety standards
• Human & Vet quality production with a 15-year track record
• ISO 9001, ISO 13485 Canadian ISO 13485 CMDCAS
Arthramid Vet 2.5% PAAG
2.5% PAAG exerts its effect via integration over 2-4 weeks within the joint capsule and adjacent soft tissues, through a combination of vessel in-growth, increased cell density, and molecular water exchange.
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Addresses Welfare Standards; Not simply a lubricant or painkiller. Its unique biomedical tissue scaffold technolgy helps heal and restore the joint to healthy function.
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One of a Kind; Arthramid Vet's unique and patented IL-X Cross-Linking Technology makes it the safest and most effective Polyacrylamide (PAAG) Hydrogel available.
- Addresses Welfare Concerns
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Get In Touch
Innovative Medical Solutions
Registered in New Zealand to: Innovative Medical Solutions Limited NZBN: 9429041325544, 32 Hill Road, Cambridge 3494, New Zealand. Registered pursuant to the ACVM Act 1997 Number A11596. See www.foodsafety.govt.nz for registration conditions.
© IMS Vet NZ Ltd, 2019


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Ken Follett, Author
I've been photographing bestselling author Ken Follett for Børsen.
Follett is best known for his epic 'Pillars of the Earth' novel, but has sold more than 130 million books worldwide.
This series of portraits was shot for an article promoting his new book 'Edge of Eternity' concluding his 'Century' trilogy.
29/09/2014 | Categories: Photography | Tags: Author, Bestselling, Century, Edge of Eternity, Ken Follett, London, Photographers Gallery, Pillars Of The Earth, Writer | 2 Comments


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"How come?" is a very, very common English expression that is important to learn. It is used all the time, but many textbooks and teachers don't teach it, because it is informal. In this simple video, I will teach you what "how come" means, how to use it, and when to use it. After watching, take the quiz at https://www.engvid.com/how-come/ to make sure you've got it!
Hello. My name is Emma, and in today's video I am going to teach you a very important expression for conversation. That expression is: "How come?" It's a very popular expression you may see in movies, on TV, or in conversation with English speakers. But it's a very good one to know because we do use it a lot.
So, what does "How come?" mean? Okay, well, first I have a question for you. I have here two sentences. "Why did you miss your plane?" and "How come you missed your plane?" What is the difference in meaning between these two sentences? Maybe you already know. Okay? So take a guess. The difference in meaning is actually they mean the same thing. "How come?" is another way to say "Why?". It's just a little bit more informal. Okay? So if you're writing, you're going to use "Why?", but if you're speaking you can use both. Okay? "How come?" is informal, it's an informal way to say "Why?" And so, by informal, I mean you use it with your friends, with, you know, people you're talking to on the street, but you wouldn't use it in an essay. Okay? Or for school.
Okay, so: "How come?" means: "Why?" So, when we're asking: "How come?" what we're asking about is… we want to know why something happened or the reasons why something happened. Okay? So, for example: "How come you missed your plane?" You know, a reason might be: "Oh, I was late getting to the airport" or "I slept in." Okay? So these would be the answers to a question like: "How come?" So, a lot of the time, teachers will ask this question. "You were late for class today. How come?" That means the teacher wants to know why you were late for class. So now let's look at the grammar of "How come?" and how we can use it in a sentence.
Okay, so again, "How come?" is an informal way to say: "Why?" So, we often use it in conversation. Now let's look at the grammar of "How come?" and how we make a sentence with "How come?" So, I have here: "How come", which is at the beginning, and then we have plus the subject. A subject is… It can be: "I", "you", "he", "she", "they", "we", or it can also be a thing, a place, or a person, but it's the doer of a sentence. Then we have the verb. So, for example: "play", "take", "listen", "sing", "eat", these are all verbs. And then finally we have an object, which comes after the verb in regular English sentences and usually those can be people, they can be places, they can be things, so these are the objects. If this is confusing, let's look at some examples, maybe that will help.
So, for example: "How come you"-is the subject-"take"-is the verb, and the object is-"the bus"? "How come you take the bus?" This means the same thing as: "Why do you take the bus?" So, here I actually have this written: "Why do you take the bus?" And you'll actually notice "How come" is easier in terms of grammar than "Why". If you look here: "Why do you take the bus?" you have this word, here: "do". Okay? In other sentences we say: "Why does he" or "Why didn't he", but there's always something like: "do", "does", "did", "didn't" here with "Why". And a lot of students forget to put this here. A lot of students will say: "Why you take the bus?" But this is not correct English. For "Why" we always need something here. Now, the nice thing about "How come" is you don't need this. Okay? If you look at "How come", if you can make an English sentence: "you take the bus", you can change this into "Why" just by adding "How come". So, the structure of this is just like a regular English sentence. We have the subject, the verb, and the object, and then we just add "How come" at the front of it.
So let's look at another example: "How come Toronto isn't the capital of Canada?" So, again, we have: "How come", we have "Toronto" which is the subject, we have "isn't" which is the verb, and we have "the capital", which is the object. So, if you want to make a regular sentence, I would just say: "Toronto isn't the capital", we can just add "How come" to this, and then it becomes a question, meaning: "Why isn't Toronto the capital?"
"How come John didn't come?" Okay? So here we have "How come" at the beginning, "John" which is the subject, and "didn't come", because it's negative form we have "didn't" here, so this is the past, past tense. "Didn't come" is the verb. Okay? This sentence doesn't have an object. Not all sentences in English need objects. The main thing is that you have a subject and a verb. Okay, so that might be a little confusing for you.


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Recent Redraft of Noxious Weed Law Improves but Still Needs Work
September 8, 2014
HARRISBURG – The Pennsylvania Department of Agriculture (PDA) recently released a fourth draft of a proposed rewrite of the state's Noxious Weed Law. In this latest draft, some but not all of PLNA's concerns with earlier drafts have been addressed... (more)
June 14, 2013
HARRISBURG - At the May 28th meeting of the Invasive Species Council, the Bureau of Plant Industry presented a revision to the Noxious Weed Law that they are contemplating. The current Noxious Weed Law controls a limited number of plant species deemed to be "noxious," including Canada thistle, multi-flora rose and marijuana. The propagation, transport or sale of plant species deemed to be noxious is prohibited and property owners with stands of the weeds are required to remove them at their own expense... (more)
Remember Me
6/3/2020Extension at Home: Backyard Woods
6/10/2020Selecting and Planting Native Trees and Shrubs


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Class Announcements
Meet the Teacher
Phone: (252)438-8415
Degrees and Certifications:
Ms. Gesina Woelfle
Dear boys, girls, and parents of Ms. Woelfle's class,
We are so happy to know that you are in our first grade class this year! It will be a very exciting year for you with lots of fun and interesting things to learn. Our year will lead to many new opportunities and skills that will change your future. You will learn to be a great reader this year!
Homework is required in first grade. It will be sent home in the homework folders each day. Please make sure that the homework is returned completed the next day. With our new phonics series, homework may be given for the weekend so please check your child's folder each night.
Homework folders will be sent home each day. They should be brought to school each morning and will be sent back home in the afternoon. It will not only include homework, but also notes, handouts, and a behavior record. The behavior record should be signed daily. If you have any notes to send to the school, just include them in the folder, which will be checked daily.
Students should also be bringing home a library book each night. They are expected to read the book several times and be able to pass a test on it the next morning. Please make sure your child is reading their book each night and ready to test in the morning.
Another resource for students to have continued success is the computer program iReady. You can access this system from your home computer, see Classroom Web Resources for more information.
Ms. Woelfle


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I am so happy to see a new game in this style, the last time I got to play a new one was Clu Clu Land D in Animal Crossing on the Gamecube
A downloadable game for Windows
You are the elusive hacker, edie88, and your job is to bring down the corrupt owner of Waylife Bank by guiding your trusty partner in anti-crime, Bit, into the mainframe of a 1988 bank's computer system and revealing the fraudulent transactions of CEO Peter Mohw.
Power your way through 11 computer systems, totaling 88 levels of increasing difficulty and speed. Swing from point to point, revealing the data in the code and avoiding the anti-invasion programs, ranging from simple electrified walls to vicious spider-like assailants. Fire your pulse shot to destroy them, or to reveal the hidden lights for a moment to aide you in your quest.
The better you perform in each system, the more upgrades you earn, not only to spruce up Bit with new colors, but gain useful improvements to your abilities.
The three-button control scheme seems simple at first, but may prove a greater challenge than you expected.
In order to download this game you must purchase it at or above the minimum price of $4.99 USD. You will get access to the following files:
Development log
- hack.88 is out!Jul 19, 2019
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I am so happy to see a new game in this style, the last time I got to play a new one was Clu Clu Land D in Animal Crossing on the Gamecube
Oh, I didn't know that was in Animal Crossing! I thought it was a completely abandoned title~
CCLD was basically a level hack of the original, but it was the only new Clu Clu Land content basically ever. And having played hack.88 a bit now, I am very happy to have a worthy spiritual successor.
Isn't this just clu clu land?
It is indeed inspired by Clu-clu land. It is a mechanic I haven't seen in any other game, and I wanted to make something nice and high res with a bit of my own art style, more levels and more enemy varieties!
Mac version plox
Maybe think about releasing a demo with a few levels (~10?) or at least a better trailer. I can't figure out the gameplay with the trailer that's up now. I can barely even tell which ship the player is controlling, let alone any of the level objectives.


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The association of HLA class II DQB1 and DRB1 alleles with the development of cervical carcinoma was studied in 150 Swedish patients using PCR-based HPV and HLA typing. The association of cervical carcinoma with alleles encoding the DQ3 antigen, previously found among German and Norwegian patients, was not observed in the Swedish patients. Five DQ-DR haplotypes were indicated to be positively associated with development of cervical carcinoma in the Swedish patients. Two of these HLA associations were specific for HPV 18 infected patients, suggesting that the ability of the oncogenic HPV 18 to cause more rapid-transit tumors than other high risk HPV types may be due to a deficiency in antigen presentation by the HLA molecules encoded by carried on these haplotypes.
In an attempt to determine the value of DNA based typing of HLA class II loci to forensic analysis, allele and genotype frequencies at DQA1, DQB1, DPB1, and DRB1 were determined in samples from two Swedish populations using hybridization with sequence specific oligonucleotides to PCR amplified DNA. Significant allele frequency differences were observed at the DQB1 and DRB1 loci between the two populations, as well as between one of the Swedish and a Norwegian population. The average heterozygosity varies between 0.74 to 0.91 and the power of discrimination between 0.90 to 0.98, with the highest values obtained for the DRB1 locus. The probability of genotype identity by chance differs on average 2% between the populations. When applied to a paternity case with one parent deceased and a criminal case, typing of class II loci proved in both cases informative. Analyses of DR and DQ genes does not increase the power of discrimination, due to strong linkage, but offers through the reconstruction of putative haplotypes an internal control for the consistency of the typing results at several loci. Typing of the DRB1 and DPB1 loci was found to result in an approximate combined average probability of genotype identity by chance of one in a thousand.
The association of MS with HLA class II alleles was studied by PCR-based typing of the DQA1, DQB1, DRB1, and DPB1 loci in 94 Swedish patients with relapses and remissions of the disease. The haplotype DRB1*1501-DQA1*0102-DQB1*0602 was found to be positively associated and three haplotypes were found to be negatively associated with MS. Linkage disequilibrium makes it difficult to assess whether DRB1 or DQB1 plays the primary role in the disease association, while the association with DPB1 and DQA1 appears to be secondary to that of DQB1 and DRB1. Two of the three haplotypes negatively associated with MS carry the DQB1*0301 allele. Also, the negatively associated DRB1*0401-DQA1*0301-DQB1*0301 haplotype differs from those with nonassociated DRB1*0401-DQA1*0301-DQB1*0302 haplotype only at DQB1. These results suggest that DQB1 alleles, as well as some DRB1 alleles, are involved in susceptibility and protection to MS. In searching for sequence motifs in the DR beta chain associated with MS susceptibility, all DRB1 alleles on haplotypes positively associated with MS, including the DRB1*1501, were found to encode a Val at position 86 of the DR beta chain. Also, DRB1 alleles that are negatively associated with MS all encode a Gly at position 86, suggesting that the residue at position 86 may be critical in conferring susceptibility and protection to MS. Finally, when the effect of the DRB1*1501 haplotype was removed there was no support for the hypothesis that MS is associated with a putative DQ-alpha beta heterodimer, encoded for by certain DQA1 and DQB1 alleles.
We have initiated large-scale sequencing of the third smallest chromosome of the CL Brener strain of Trypanosoma cruzi and we report here the complete sequence of a contig consisting of three cosmids. This contig covers 93.4 kb and has been found to contain 20-30 novel genes and several repeat elements, including a novel chromosome 3-specific 400-bp repeat sequence. The intergenic sequences were found to be rich in di- and trinucleotide repeats of varying lengths and also contained several known T. cruzi repeat elements. The sequence contains 29 open reading frames (ORFs) longer than 700 bp, the longest being 5157 bp, and a large number of shorter ORFs. Of the long ORFs, seven show homology to known genes in parasites and other organisms, whereas four ORFs were confirmed by sequencing of cDNA clones. Two shorter ORFs were confirmed by a database homology and a cDNA clone, respectively, and one RNA gene was identified. The identified genes include two copies of the gene for alanine-aminotransferase as well as genes for glucose-6-phosphate isomerase, protein kinases and phosphatases, and an ATP synthase subunit. An interesting feature of the sequence was that the genes appear to be organized in two long clusters containing multiple genes on the same strand. The two clusters are transcribed in opposite directions and they are separated by an approximately 20-kb long, relatively GC-rich sequence, that contains two large repetitive elements as well as a pseudogene for cruzipain and a gene for U2snRNA. It is likely that this strand switch region contains one or more regulatory and promoter regions. The reported sequence provides the first insight into the genome organization of T. cruzi and shows the potential of this approach for rapid identification of novel genes. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF052831-AF052833.]
Analysis of mitochondrial DNA (mtDNA) is very useful when nuclear DNA analysis fails due to degradation or insufficient amounts of DNA in forensic analysis. However, mtDNA analysis has a lower discrimination power compared to what can be obtained by nuclear DNA (nDNA) analysis, potentially resulting in multiple individuals showing identical mtDNA types in the HVI/HVII region. In this study, the increase in discrimination by analysis of mitochondrial coding regions has been evaluated for identical or similar HVI/HVII sequences. A pyrosequencing-based system for coding region analysis, comprising 17 pyrosequencing reactions performed on 15 PCR fragments, was utilised. This assay was evaluated in 135 samples, resulting in an average read length of 81 nucleotides in the pyrosequencing analysis. In the sample set, a total of 52 coding region SNPs were identified, of which 18 were singletons. In a group of 60 samples with 0 or 1 control region difference from the revised Cambridge reference sequence (rCRS), only 12 samples could not be resolved by at least two differences using the pyrosequencing assay. Thus, the use of this pyrosequencing-based coding region assay has the potential to substantially increase the discriminatory power of mtDNA analysis.
Primary nocturnal enuresis (PNE), or bedwetting at night, affects approximately 10% of 6 year old children. Genetic components contribute to the pathogenesis and recently one locus was assigned to chromosome 13q. We evaluated the genetic factors and the pattern of inheritance for PNE in 392 families. Dominant transmission was observed in 43% and an apparent recessive mode of inheritance was observed in 9% of the families. Among the 392 probands the ratio of males to females was 3:1 indicating sex linked or sex influenced factors. Linkage to candidate regions was tested in 16 larger families segregating for autosomal dominant PNE. A gene for PNE was excluded from chromosome 13q in 11 families, whereas linkage to the interval D13S263-D13S291 was suggested (Zmax = 2.1) in three families. Further linkage analyses excluded about 1/3 of the genome at a 10 cM resolution except the region around D12S80 on chromosome 12q that showed a positive two point lod score in six of the families (Zmax = 4.2). This locus remains suggestive because the material was not sufficiently large to give evidence for heterogeneity. Our pedigree analysis indicates that major genes are involved in a large proportion of PNE families and the linkage results suggest that such a gene is located on chromosome 12q.
Phosphorylation of protein tyrosine residues regulates many cell functions and has also been proved to be involved in oncogenesis. Thus, the identification of the phosphotyrosine (pTyr) proteome of cells is a very important task. Since tyrosine phosphorylation represents only around 1% of the total human phosphoproteome, the study of pTyr proteins is rather challenging. Here we report the optimization study of the phosphotyrosine proteome using K562 cells as a model system. A substantial segment of the phosphotyrosine proteome of K562 cells was characterized by immunoaffinity enrichment with 4G10 and PYKD1 antibodies followed by LC-MS/MS analysis. 480 non-redundant pTyr peptides corresponding to 342 pTyr proteins were found. 141 pTyr peptides were not described elsewhere. The mass spectrometry approach involving high-resolving FTMS analysis of precursor ions and subsequent detection of CID fragments in a linear ion trap was considered as optimal. For detection of low abundant pTyr peptides pooling of individual immunoaffinity enrichments for one LC-MS/MS analysis was crucial. The enrichment properties of the monoclonal PYKD1 antibody were presented for the first time, also in comparison to the 4G10 antibody. PYKD1 was found to be more effective for protein enrichment (1.2 and 5% efficiency at peptide and protein level correspondingly), while 4G10 showed better results when peptide enrichment was performed (15% efficiency versus 3.6% at protein level). Substantially different subsets of the phosphoproteome were enriched by these antibodies. This finding together with previous studies demonstrates that comprehensive pTyr proteome characterization by immunoprecipitation requires multiple antibodies to be used for the affinity enrichment.
We investigated a Swedish family with nonsyndromic progressive bilateral sensorineural hearing loss. Thirteen candidate loci for autosomal dominant nonsyndromic hearing loss were tested for linkage in this family. We found significant LOD scores (>3) for markers at candidate locus DFNA12 (11q22-q24) and suggestive LOD scores (>2) for markers at locus DFNA2 (1p32). Our results for markers on chromosome 11 narrowed down the candidate region for the DFNA12 locus. A detailed analysis of the phenotypes and haplotypes shared by the affected individuals supported the notion that two genes segregated together with hearing impairment in the family. Severely affected family members had haplotypes linked to the disease allele on both chromosomes 1 and 11, whereas individuals with milder hearing loss had haplotypes linked to the disease allele on either chromosome 1 or chromosome 11. These observations suggest an additive effect of two genes, each gene resulting in a mild and sometimes undiagnosed phenotype, but both together resulting in a more severe phenotype.
The human alpha-tectorin (TECTA) gene has recently been cloned and proposed to be involved in autosomal dominant non-syndromic hearing impairment (NSHI) in two families linked to the DFNA12 locus. We have studied a Swedish pedigree with autosomal dominant NSHI with possible digenic inheritance of the disease, involving locus DFNA12 in chromosome 11 and locus DFNA2 in chromosome 1. Mutation analysis of the TECTA gene in this family has identified eight nucleotide substitutions indicating that TECTA is highly polymorphic. One of the changes results in a cysteine to serine (C 1057 S) mutation, in the zonadhesin domain of TECTA; this segregates with the disease haplotype on chromosome 11 and is not present in a control population. The mutation results in the replacement of a cysteine in one of the repeats of the zonadhesin/Von Willebrand domain of the protein and might cause a change in the crosslinking of the polypeptide. These findings add support to the involvement of TECTA in hearing disabilities. However, the three families carrying different TECTA mutations also show phenotypic differences: the hearing loss ranges from prelingual to progressive with late onset. The explanation for the different phenotypes and some clues regarding the functions of TECTA may lie in the localization of the mutations in the different modules of the protein. Another possibility is that the phenotype in the Swedish family is the result of two defective genes.
NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 188.8.131.52) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.
Tyrosine phosphorylation (pTyr) regulates important cell functions and plays a key role in carcinogenesis. The purpose of this study was to perform a comprehensive study of the phosphotyrosine proteome. Immunoaffinity enriched pTyr proteins and peptides from K562 leukemia cells were analyzed with high-resolving liquid chromatography mass spectrometry. Two different antibodies selective for the pTyr modification were used in repeated enrichments to identify as many pTyr peptides as possible. Stringent verification of putative pTyr sites was performed to assure high reliability in the subsequent biological interpretation of the data. Identified pTyr proteins were subjected to pathway analysis by using different analytical tools. In total, 294 pTyr peptides belonging to 217 pTyr proteins were identified, 15 of which had not previously been reported to be modified by pTyr. The pTyr proteins were clustered in six major groups based on the biological functions "cellular signaling", "cell motility and shape", "cell cycle process", "transport", "RNA processing" and "protein processing". The pTyr proteins were mainly positioned in the following cellular compartments: cytoplasm, cytoskeleton, nucleus and ribonucleoprotein complexes. An interesting finding was that many proteins were related to RNA processing and were found to be heterogeneous nuclear ribonucleoproteins. Also, more than half of the novel pTyr proteins were localized to the nucleus, of which three (PBX2, TEAD1 and DIDO1) were classified as transcription factors and two (CENPC1 and MAD2L1) are associated with cell division control.
We have used a proteomics approach to identify sites of phosphorylation in the structural proteins of the Adenovirus type 2 particle. This protein modification might play an important role during infection. Peptides from highly purified virus were enriched for phosphorylations and analyzed by liquid chromatography-high-resolving mass spectrometry. Phosphorylations were identified in 11 structural peptides and 29 non-redundant phosphorylation sites were unambiguously assigned to specific amino acid. An unexpected result was the finding of phosphotyrosine in two of the viral polypeptides. The most highly phosphorylated protein was pIIIa with 12 identified phosphorylation sites. An identified preference for proline or leucine residue flanking the phosphorylation sites downstream suggests that cellular kinases are involved in many of the phosphorylations. Structural modeling showed that one site in the hexon is located on the outer side of the virus and could be of importance for the virus when attaching and entering cells.
To develop methods for studying phosphorylation of protein tyrosine residues is an important task since this protein modification regulates many cellular functions and often is involved in oncogenesis. An optimal protocol includes enrichment of tyrosine phosphorylated (pTyr) peptides or proteins, followed by a high resolving analytical method for identification of the enriched components. In this Methods paper, we describe a working strategy on how immunoaffinity enrichments, using anti-pTyr antibodies, combined with mass spectrometric (MS) analysis can be used to study the pTyr proteome. We describe in detail how our procedure was used to characterize the pTyr proteome of K562 leukemia cells. Important questions concerning the use of different anti-pTyr antibodies, enrichments performed at the peptide and/or the protein level, pooling of enrichments and requirements for the MS characterization are discussed.
Phosphorylation of protein tyrosine residues regulates important cell functions and is, when dysregulated, often crucially involved in oncogenesis. It is therefore important to develop and evaluate methods for identifying and studying tyrosine phosphorylated (P-Tyr) proteins. P-Tyr proteins are present at very low concentrations within cells, requiring highly selective enrichment methods to be detected. In this study, we applied immunoaffinity as enrichment step for P-Tyr proteins. Five selected anti-phosphotyrosine antibodies (monoclonal antibodies 4G10, PY100, PYKD1, 13F9 and one polyclonal antiserum) were evaluated with respect to their capability to enrich P-Tyr proteins from cell extracts of the K562 leukemia cell line. The enrichment resulted in the detection of a group of proteins that potentially were tyrosine-phosphorylated (putative P-Tyr proteins). High accuracy identification of actual P-Tyr sites were performed using a highly selective and sensitive liquid chromatography Fourier transform mass spectrometer (LC-FTMS) setup with complementary collision activated dissociation (CAD) and electron capture dissociation (ECD) fragmentations. 4G10 and PY100 antibodies recognized the greatest number of putative P-Tyr proteins in initial screening experiments and were therefore further evaluated and compared in immunoaffinity enrichment of both P-Tyr proteins and peptides. Using the 4G10 antibody for enrichment of proteins, we identified 459 putative P-Tyr proteins by MS. Out of these proteins, 12 were directly verified as P-Tyr proteins by MS analysis of the actual site. Using the PY100 antibody for enrichment of peptides, we detected 67 P-Tyr peptides (sites) and 89 putative P-Tyr proteins. Generally, enrichment at the peptide level made it difficult to reliably determine the identity of the proteins. In contrast, protein identification following immunoaffinity enrichment at the protein level gave greater sequence coverage and thus a higher confidence in the protein identification. By combining all available information, 40 proteins were identified as true P-Tyr proteins from the K562 cell line. In conclusion, this study showed that a combination of immunoaffinity enrichment using multiple antibodies of both intact and digested proteins in parallel experiments is required for best possible coverage of all possible P-Tyr proteins in a sample.
We have recently described the identification of a second IDS locus (IDS-2) located within 90 kb telomeric of the IDS gene (Bondeson et al. submitted). Here, we show that this region is involved in a recombination event with the IDS gene in about 13% of patients with the Hunter syndrome. Analysis of the resulting rearrangement at the molecular level showed that these patients have suffered a recombination event that results in a disruption of the IDS gene in intron 7 with an inversion of the intervening DNA. Interestingly, all of the six cases with a similar type of rearrangement showed recombination between intron 7 of the IDS gene and sequences close to exon 3 at the IDS-2 locus implying that these regions are hot spots for recombination. Analysis by nucleotide sequencing showed that the inversion is caused by recombination between homologous sequences present in the IDS gene and the IDS-2 locus. No detectable deletions or insertions were observed as a result of the recombination event. The results in this study have practical implications for diagnosis of the Hunter syndrome.
A deficiency of the enzyme iduronate-2-sulfatase (IDS) is the cause of Hunter syndrome (mucopolysaccharidosis type II). Here, we report a study of the human IDS locus at Xq28. An unexpected finding was an IDS-related region (IDS2) which is located on the telomeric side of the IDS gene within 80 kb. We have identified sequences in this locus that are homologous to exons 2 and 3 as well as sequences homologous to introns 2, 3 and 7 of the IDS gene. The exon 3 sequences in the IDS gene and in the IDS2 locus showed 100% identity. The overall identities of the other identified regions were 96%. A locus for DXS466 was also found to be located close to IDS2. The existence of the IDS2 locus complicates the diagnosis of mutations in genomic DNA from patients with Hunter syndrome. However, information about the IDS2 locus makes it possible to analyze the IDS gene and the IDS2 locus separately after PCR amplification.
The complete sequence and genomic characterization of the tyrosine aminotransferase (TAT) gene from Trypanosoma rangeli is reported. The gene was found to be organized in a tandem multicopy gene array. A homologous mRNA species (2.5 kb) was identified in the epimastigote form of the parasite. From the deduced amino acid sequence, the gene encodes a protein of 420 amino acids with a predicted molecular mass of 46.4 kDa and a theoretical pI of 6.23. A high sequence identity was found with the Trypanosoma cruzi, human and rat enzymes. All the essential residues for TAT enzymatic activity are conserved, as well as a pyridoxal-phosphate attachment site typical of class-I aminotransferases. The recombinant enzyme was recognized by a monoclonal antibody against the T. cruzi enzyme. Additionally, the recombinant protein showed enzymatic activity when incubated with L-tyrosine and 2-oxoglutaric acid as substrates.
Posttranscriptional steps in the production of mRNA include well characterized polyadenylation and splicing reactions, but it is also necessary to understand how RNA is transported within the nucleus from the site of its transcription to the nuclear pore, where it is translocated to the cytoplasmic compartment. Determining the localization of RNA within the nucleus is an important aspect of understanding RNA production and may provide clues for investigating the trafficking of RNA within the nucleus and the mechanism for its export to the cytoplasm. We have previously shown that late phase adenovirus-infected cells contain large clusters of snRNP and non-snRNP splicing factors; the presence of these structures is correlated with high levels of viral late gene transcription. The snRNP clusters correspond to enlarged interchromatin granules present in late phase infected cells. Here we show that polyadenylated RNA and spliced tripartite leader exons from the viral major late transcription unit are present in these same late phase snRNP-containing structures. We find that the majority of the steady state viral RNA present in the nucleus is spliced at the tripartite leader exons. Tripartite leader exons are efficiently exported from the nucleus at a time when we detect their accumulation in interchromatin granule clusters. Since the enlarged interchromatin granules contain spliced and polyadenylated RNA, we suggest that viral RNA may accumulate in this late phase structure during an intranuclear step in RNA transport.
Adenovirus infection affects the nuclear distribution of host splicing factors. Late phase-infected cells contain discrete clusters of small nuclear ribonucleoproteins (snRNPs) that are separate from centers containing the viral 72-kilodalton DNA-binding protein (72K protein). In the present study, we demonstrate that these snRNP clusters also contain splicing factors from the SR protein family. We show that a previously described monoclonal antibody, 3C5, detects SR proteins. Furthermore, we demonstrate that late region 3 transcription occurs at a maximal rate in infected cultures in which greater than 90% of the cells contain the snRNP clusters, indicating that such cells are actively transcribing their late genes. During the onset of the late phase, the intranuclear distribution of splicing factors is very different from that seen after the late phase is established. When late viral transcription commences, cells with snRNP clusters are less prevalent than in cultures that are maintaining maximum levels of late transcription. Instead, a cell type which shows snRNPs, concentrated in foci that also contain the viral 72K DNA-binding protein is detected. This cell type disappears from cultures by 18 to 20 h after a high-multiplicity infection. These results suggest a dynamic organization of splicing factors in infected cells that can be correlated to the status of viral gene expression. Our work also provides an explanation for the differing results that have been published concerning the organization of splicing factors in the adenovirus-infected cell nucleus (L. F. Jiménez-García and D. L. Spector, Cell 73:47-59, 1993). During the present study we observed that a monoclonal antibody against the SC-35 protein, which was used by Jiménez-García and Spector to study the localization of the SC-35 splicing factor in adenovirus-infected cells, cross-reacts with the adenovirus 72K DNA-binding protein and is thus unsuitable for this type of study.
The PML protein was first identified as part of a fusion product with the retinoic acid receptor alpha (RAR alpha), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RAR alpha hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4-ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes.
Expression of cellular long non-coding RNAs (lncRNAs) in human primary lung fibroblasts (IMR-90) during the course of adenovirus type 2 (Ad2) infection was studied by strand-specific whole transcriptome sequencing. In total, 645 cellular lncRNAs were expressed at a significant level and 398 of them were changed more than 2-fold. The changes in expression followed a distinct temporal pattern. Significantly, 80% of the changes occurred at the late phase and 80% of the de-regulated lncRNAs were up-regulated. The three largest groups of deregulated lncRNAs were 125 antisense RNAs, 111 pseudogenes and 85 long intergenic non-coding RNAs (lincRNAs). Lastly, more than 36% of lncRNAs have been shown to interact with RNA binding proteins.
Genome-wide demethylation has been suggested to be a step in carcinogenesis. Evidence for this notion comes from the frequently observed global DNA hypomethylation in tumour cells, and from a recent study suggesting that defects in DNA methylation might contribute to the genomic instability of some colorectal tumour cell lines. DNA hypomethylation has also been associated with abnormal chromosomal structures, as observed in cells from patients with ICF (Immunodeficiency, Centromeric instability and Facial abnormalities) syndrome and in cells treated with the demethylating agent 5-azadeoxycytidine. Here we report that murine embryonic stem cells nullizygous for the major DNA methyltransferase (Dnmt1) gene exhibited significantly elevated mutation rates at both the endogenous hypoxanthine phosphoribosyltransferase (Hprt) gene and an integrated viral thymidine kinase (tk) transgene. Gene deletions were the predominant mutations at both loci. The major cause of the observed tk deletions was either mitotic recombination or chromosomal loss accompanied by duplication of the remaining chromosome. Our results imply an important role for mammalian DNA methylation in maintaining genome stability.
Down syndrome (DS) is caused in most cases by the presence of an extra chromosome 21. It has been shown that the DS phenotype is produced by duplication of only a small part of the long arm of chromosome 21, the 21q22 region, including and distal to locus D21S55. We present molecular investigations on a woman with clinically typical DS but apparently normal chromosomes. Her parents were consanguineous and she had a sister with a DS phenotype, who died at the age of 15 days. Repeated cytogenetic investigations (G-banding and high resolution banding) on the patient and her parents showed apparently normal chromosomes. Autoradiographs of quantitative Southern blots of DNAs from the patient, her parents, trisomy 21 patients, and normal controls were analyzed after hybridization with unique DNA sequences regionally mapped on chromosome 21. Sequences D21S59, D21S1, D21S11, D21S8, D21S17, D21S55, ERG, D21S15, D21S112, and COL6A1 were all found in two copies. Fluorescent in situ hybridization with a chromosome 21-specific genomic library showed no abnormalities and only two copies of chromosome 21 were detected. Nineteen markers from the critical region studied with polymerase chain reaction amplification of di- and tetranucleotide repeats did not indicate any partial trisomy 21. From this study we conclude that the patient does not have any partial submicroscopic trisomy for any segment of chromosome 21. It seems reasonable to assume that she suffers from an autosomal recessive disorder which is phenotypically indistinguishable from DS.
Background: Tyrosine phosphorylation (pTyr) is an important cancer relevant posttranslational modification since it regulates protein activity and cellular localization. By controlling cell growth and differentiation it plays an important role in tumor development. This paper describes a novel approach for detection and visualization of a panel of pTyr proteins in tumors using in situ proximity ligation assay. Methods: K562 leukemia cells were treated with tyrosine kinase and/or phosphatase inhibitors to induce differences in pTyr levels and mimic cells with different malignant properties. Cells were then probed with one antibody against the pTyr modification and another probe against the detected protein, resulting in a detectable fluorescent signal once the probes were in proximity. Results: Total and protein specific pTyr levels on ABL, SHC, ERK2 and PI3K proteins were detected and samples of control and treated cells were distinguished at the pTyr level using this novel approach. Promising results were also detected for formalin fixed and paraffin embedded cells in the micro array format. Conclusions: This application of in situ proximity ligation assay is valuable in order to study the pTyr modification of a panel of proteins in large data sets to validate mass spectrometric data and to be combined with tissue microarrays. The approach offers new opportunities to reveal the pTyr signatures in cells of different malignant properties that can be used as biomarker of disease in the future.
Whole-genome sequencing of the protozoan pathogen Trypanosoma cruzi revealed that the diploid genome contains a predicted 22,570 proteins encoded by genes, of which 12,570 represent allelic pairs. Over 50% of the genome consists of repeated sequences, such as retrotransposons and genes for large families of surface molecules, which include trans-sialidases, mucins, gp63s, and a large novel family (>1300 copies) of mucin-associated surface protein (MASP) genes. Analyses of the T. cruzi, T. brucei, and Leishmania major (Tritryp) genomes imply differences from other eukaryotes in DNA repair and initiation of replication and reflect their unusual mitochondrial DNA. Although the Tritryp lack several classes of signaling molecules, their kinomes contain a large and diverse set of protein kinases and phosphatases; their size and diversity imply previously unknown interactions and regulatory processes, which may be targets for intervention.
The myeloma cell line RPMI 8226/S and its doxorubicin resistant subline 8226/Dox40 were used as models to explore the potential importance of the STAT1 signaling pathway in drug and radiation resistance. The 40-fold doxorubicin resistant subline 8226/Dox40 was found to be crossresistant to single doses of 4 and 8 Gy of radiation. A genome-wide mRNA expression study comparing the 8226/Dox40 cell line to its parental line was performed to identify the underlying molecular mechanisms. Seventeen of the top 50 overexpressed genes have previously been implicated in the STAT1 signaling pathway. STAT1 was over expressed both at the mRNA and protein level. Moreover, analyses of nuclear extracts showed higher abundance of phosphorylated STAT1 (Tyr 701) in the resistant subline. Preexposure of the crossresistant cells to the STAT1 inhibiting drug fludarabine reduced expression of overexpressed genes and enhanced the effects of both doxorubicin and radiation. These results show that resistance to doxorubicin and radiation is associated with increased STAT1 signaling and can be modulated by fludarabine. The data support further development of therapies combining fludarabine and radiation.
In this report, we have studied gene expression profiles in human primary lung fibroblasts (IMR-90) during the very early phase of an adenovirus infection. Eight out of twelve genes with known functions encoded transcription factors linked to two major cellular processes; inhibition of cell growth (ATF3, ATF4, KLF4, KLF6 and ELK3) and immune response (NR4A1 and CEBPB), indicating that the earliest consequences of an adenovirus infection are growth arrest and induction of an immune response. A time course analysis showed that the induction of these immediate-early response genes was transient and suppressed after the onset of the adenovirus early gene expression.
Microarray analysis of host cell gene expression during an adenovirus type 2 infection showed that the number of regulated genes, as well as the magnitude of change, was increased as the infection proceeded into the late phase. In contrast to the early phase of infection when the majority of differentially expressed genes were upregulated, expression of most of the regulated genes (82 out of 112) declined during the late phase. In particular, numerous TGF-beta inducible genes and several TGF-beta-independent growth-arrest-inducing genes were targeted. Of the 30 genes upregulated more than 2-fold at 20 h post-infection, nearly two-thirds of encoded proteins are involved in cell metabolism. The data indicate that adenovirus primarily targets cellular genes involved in antiviral defense, cell growth arrest and apoptosis, as well as cell metabolism, to ensure sufficient production of viral progeny.
A hitherto predicted but undetected protein, C-168, in adenovirus type 2 (Ad2) has been identified using mass spectrometry (MS) based proteomics. The gene of this 17.7 kDa protein is located on the forward strand in the major late transcription unit between base pairs 9294 and 9797. A tryptic peptide, derived from the C-terminal part of the protein, was identified with high amino acid sequence coverage. A candidate splice site for the corresponding mRNA is also presented. The protein sequence is unusual with repeats of serine, glycine and arginine. A bioinformatics prediction of protein function and localization is presented.
In order to generate contiguous cosmid coverage of the genome of the protozoan parasite Trypanosoma cruzi for large-scale sequence analysis, a cosmid library of 36864 individual, primary clones was generated. Total genomic DNA of the reference strain CL Brener was fragmented both by partial digestion with MboI and by physical shearing. For cloning, a modified cosmid vector was used that simplifies analyses such as restriction mapping. The library's representation is about 25 genome equivalents, assuming a size of 55 Mb per haploid genome. No chimerism of inserts in the clones could be detected. The colinearity between cosmid inserts and genomic DNA was verified. Also, hybridizations to the gel-separated karyotype of the organism were carried out as a quality check. Gridded onto two nylon filters, the library was analyzed with a variety of probes. Apart from being used for combined physical and transcriptional mapping of the genome, library filters and clones are also available to interested parties.
The evolutionary significance of chromosome size polymorphism was explored in a representative panel of 26 Trypanosoma cruzi stocks. We tested a progressive model (aCSDI) assuming that the larger the size difference between homologous chromosomes, the more divergent the parasites are. This was contrasted with a non-progressive model (Jaccard's distance), in which any chromosome size difference has the same weight. ACSDI-based dendrograms were very similar to those built-up from multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD) data: structuring in 2 major lineages (T. cruzi I and T. cruz II) and 5 small subdivisions within T. cruzi II was identical, and branching was very similar. Furthermore, a significant correlation (P < 0.001) was observed between aCSDI and phenetic distances calculated from MLEE and RAPD data. In contrast, analysis of chromosome size polymorphism with Jaccard's distance generated dendrograms with relatively long branches, causing most branching points to cluster close together, which generates statistically uncertain branching points. Our results thus support a model of progressive chromosome size-variation and show that despite an extensive polymorphism, chromosomal sizes constitute valuable characters for evolutionary analyses. Furthermore, our data are consistent with the clonal evolution model previously proposed for T. cruzi.
The karyotypes of three cloned stocks, CL Brener (CL), CA I/72 (CA) and Sylvio X10/7 (X10), of Trypanosoma cruzi were studied by pulsed-field gel electrophoresis followed by ethidium bromide staining and hybridization with 35 different probes, 30 of which identified single chromosomes. The chromosome-specific probes identified between 26 and 31 chromosomal bands in the three cloned stocks, corresponding to 20 unique chromosomes in CL and 19 in CA and X10. Considering the DNA content of the parasite, it was predicted that the markers recognise at least half of all T. cruzi chromosomes. A majority of identified chromosomes showed large differences in size among different strains, in some cases by up to 50%. Interestingly, CL had in general larger chromosomes than the two other studied cloned stocks. Several of the markers showed linkage and nine different linkage groups were identified, each comprising 2-4 markers. The linkage between the markers was maintained in 8 of the 9 linkage groups when a panel comprising 26 different T. cruzi strains representing major T. cruzi populations was tested. One linkage group was found to be maintained in some strains but not in others. This result shows that chromosomal rearrangements occur in the T. cruzi genome, albeit with a low frequency. Repetitive DNA, both non-coding and in one case coding, was more abundant in the cloned stock CL Brener than in CA and X10. The information presented will make it possible to select chromosomes for the construction of physical chromosomal maps required for the T. cruzi genome project.
Like many other protozoam parasites, Trypanosoma cruzi (the causative agent of Chagas disease) has a plastic genome. Chromosome size polymorphisms occur in different strains of T. cruzi as well as among clones originating from the same strain, Despite this polymorphism, major interchromosomal rearrangements appear to be rare since several linkage groups of chromosomal markers are well conserved among different T. cruzi strains. In addition, some correlation has been found between karyotype variability and classification by multilocus enzyme electrophoresis. In this review, Jan Henriksson, Lena Åslund and Ulf Petterson discuss the genomic variability and suggest that amplication of repetitive sequences or members of gene families make a major contribution to the chromosomal size variation
During the chronic stage of Chagas disease a 160 kDa antigen appears in the blood of patients and remains detectable many years after the onset of the disease. This antigen is secreted by the trypomastigote form of the parasite while it is undetectable in the epimastigote form. We report here that the chronic 160 kDa exoantigen is encoded by a gene family (CEA 160 family). We describe the cloning and partial nucleotide sequence of a gene (CEA 160-1) belonging to the CEA160 family. Comparison of the gene sequence with other sequences present in the databases revealed homologies with several Trypanosoma cruzi surface antigens. Highest amino acid identity (59%) was with members of a family containing epitopes that mimic nervous tissues (Van Voorhis et al. 1993). Another related group (18-22% amino acid identity) comprises proteins of 85 or 160 kDa sharing an amino acid motif that is conserved among bacterial neuraminidases (Fouts et al. 1991; Pollevick et al. 1991; Kahn et al. 1991; Takle & Cross, 1991; Franco et al. 1993). The amino acid identities with the different antigens were not homogeneously distributed. Regions of higher identity (40-60%) were grouped in the central region of each protein.
A novel mutation has been identified in a patient with the Hunter syndrome (mucopolysaccharidosis type II), in whom the disorder is associated with two distinct deletions separated by 30 kb. The deletions were characterized by Southern blot and PCR analyses, and the nucleotide sequences at both junctions were determined. The first deletion, corresponding to a loss of 3152 bp of DNA, included exons 5 and 6 of the iduronate-2-sulfatase (IDS) gene. The second deletion was 3603 bp long and included exons 3 and 4 of geneW, which is located in the DXS466 locus telomeric of theIDSgene. Both deletions are the result of nonhomologous (illegitimate) recombination events between short direct repeats at the deletion breakpoints. An interesting finding was the presence of the heptamer sequence 5′-TACTCTA-3′ present at both deletion junctions, suggesting that this motif might be a hot spot for recombination. We propose that the double deletion is the result of homology-associated nonhomologous recombinations caused by the presence of large duplicated regions in Xq27.3–q28.
Multiple sclerosis (MS) is a chronic inflammatory disorder characterized by multifocal damage of myelin in the central nervous system (CNS). The prevalence of this putative autoimmune disease is 0.1% in individuals of northern European origin. Family, adoption and twin studies implicate genetic factors in the aetiology. MS is widely speculated to be a multifactorial disorder with a complex mode of inheritance. Despite many studies of candidate genes, only an association with HLA-DR2-DQ6 has been generally detected, and the number of susceptibility genes remains unknown. The chronic variant of experimental allergic encephalomyelitis (EAE), a T-cell mediated autoimmune disease in rodents, represents a relevant animal model for MS given the chronic relapsing disease course and inflammatory changes of CNS observed in these demyelinating disorders. Susceptibility to EAE is also influenced by the major histocompatibility complex (MHC). Human syntenic regions to murine loci predisposing to EAE were tested as candidate regions for genetic susceptibility of MS. Three chromosomal regions (1p22-q23, 5p14-p12 and Xq13.2-q22) were screened in 21 Finnish multiplex MS families most originating from a high risk region in western Finland. Several markers yielded positive lod scores on 5p14-p12, syntenic to the murine locus Eae2. Our data provide evidence for a predisposing locus for MS on 5p14-p12.
The proteome and phosphoproteome of non-structural proteins of Adenovirus type 2 (Ad2) were time resolved using a developed mass spectrometry approach. These proteins are expressed by the viral genome and important for the infection process, but not part of the virus particle. We unambiguously confirm the existence of 95% of the viral proteins predicted to be encoded by the viral genome. Most non-structural proteins peaked in expression at late time post infection. We identified 27 non-redundant sites of phosphorylation on seven different non-structural proteins. The most heavily phosphorylated protein was the DNA binding protein (DBP) with 15 different sites. The phosphorylation occupancy rate could be calculated and monitored with time post infection for 15 phosphorylated sites on various proteins. In the DBP, phosphorylations with time-dependent relation were observed. The findings show the complexity of the Ad2 non-structural proteins and opens up a discussion for potential new drug targets.
We have previously shown that patients with the Hunter syndrome frequently have suffered from a recombination event between the IDS gene and its putative pseudogene, IDS-2, resulting in an inversion of the intervening DNA. The inversion, which might be the consequence of an intrachromosomal mispairing, is caused by homologous recombination between sequences located in intron 7 of the IDS gene and sequences located distal of exon 3 in IDS-2. In order to gain insight into the mechanisms causing the inversion, we have isolated both inversion junctions in six unrelated patients. DNA sequence analysis of the junctions showed that all recombinations have taken place within a 1 kb region where the sequence identity is >98%. An interesting finding was the identification of regions with alternating IDS gene and IDS-2 sequences present at one inversion junction, suggesting that the recombination event has been initiated by a double-strand break in intron 7 of the IDS gene. The results from this study suggest that homologous recombination in man could be explained by mechanisms similar to those described for Saccharomyces cerevisiae. The results also have practical implications for diagnosis of patients with the Hunter syndrome.
Formation of tooth enamel is a poorly understood biological process. In this study we describe a 9-bp deletion in exon 2 of the amelogenin gene (AMGX) causing X-linked hypoplastic amelogenesis imperfecta, a disease characterized by defective enamel. The mutation results in the loss of 3 amino acids and exchange of 1 in the signal peptide of the amelogenin protein. This deletion in the signal peptide probably interferes with translocation of the amelogenin protein during synthesis, resulting in the thin enamel observed in affected members of the family. We compare this mutation to a previously reported mutation in the amelogenin gene that causes a different disease phenotype. The study illustrates that molecular analysis can help explain the various manifestations of a tooth disorder and thereby provide insights into the mechanisms of tooth enamel formation.
We present a linkage analysis and a clinical update on a previously reported family with X-linked recessive panhypopituitarism, now in its fourth generation. Affected members exhibit variable degrees of hypopituitarism and mental retardation. The markers DXS737 and DXS1187 in the q25-q26 region of the X chromosome showed evidence for linkage with a peak LOD score (Zmax) of 4.12 at zero recombination fraction (theta(max) = 0). An apparent extra copy of the marker DXS102, observed in the region of the disease gene in affected males and heterozygous carrier females, suggests that a segment including this marker is duplicated. The gene causing this disorder appears to code for a dosage-sensitive protein central to development of the pituitary.
We have combined 2-D SOS-PAGE with liquid chromatography-high resolving mass spectrometry (LC-MS) to explore the proteome of the adenovirus type 2 (Ad2) at the level of post translational modifications (PTMs). The experimental design included in-solution digestion, followed by titanium dioxide enrichment, as well as in-gel digestion of polypeptides after separation of Ad2 capsid proteins by 1-D and 2-D SOS-PAGE. All samples were analyzed using LC-MS with subsequent manual verification of PTM positions. The results revealed new phosphorylation sites that can explain the observed trains of protein spots observed for the pIII, pIIIa and ply proteins. The pin protein was found to be the most highly modified protein with now 18 verified sites of phosphorylation, three sites of nitrated tyrosine and one sulfated tyrosine. Nitrated tyrosines were also identified in pII. Lysine acetylations were detected in pII and pVI. The findings make the Ad2 virion much more complex than hitherto believed.
We describe fluorescence-based 2-D gel electrophoresis methods for visualization of low abundant cancer relevant tyrosine phosphorylated (pTyr) proteins The methods investigated were fluorescent Western blotting and two-dimensional difference gel electrophoresis (2-D DIGE) for detection of non-enriched and immunoaffinity enriched pTyr protein patterns The same anti-phosphotyrosine specific antibody 4G10 was used for both approaches The results from fluorescent Western blotting of total proteins and from enriched CyDye DIGE pre-labeled pTyr proteins showed similar down regulation of phosphorylation upon treating of cells from a cancer model system (K562 chronic myeloid leukemia cells) with imatinib This treatment introduced a known perturbation of phosphorylation that enabled testing of these new approaches to analyze variations in tyrosine phosphorylation levels Enrichment of pTyr proteins was found highly advantageous for the outcome Out of a simplified 2 D DIGE experiment of immunoaffinity enriched control and treated pTyr proteins differential analysis as well as protein identification by mass spectrometry (MS) was possible.
An association between bipolar affective disorder and CAG/CTG trinucleotide repeat expansions (TRE) has previously been detected using the repeat expansion detection (RED) method. Here we report that 89% of RED products (CAG/CTG repeats) > 120 nt (n = 202) detected in affective disorder patients as well as unaffected family members and controls correlate with expansions at two repeat loci, ERDA1 on chromosome 17q21.3 and CTG18.1 on 18q21.1. In a set of patients and controls in which we had previously found a significant difference in RED size distribution, the frequency of expansions at the CTG18.1 locus was 13% in bipolar patients (n = 60) and 5% in controls (n = 114) (P < 0.07) with a significantly different size distribution (P < 0.03). A second set of patients were ascertained from 14 affective disorder families showing anticipation. Twelve of the families had members with RED products > 120 nt. The RED product distribution was significantly different (P < 0.0007) between affected (n = 53) and unaffected (n = 123) offspring. Using PCR, a higher frequency (P < 0.04) of CTG18.1 expansions as well as a different (P < 0.02) repeat size distribution was seen between affected and unaffected offspring. In addition, a negative correlation between RED product size and the age-of-onset could be seen in affected offspring (rs = -0.3, P = 0.05, n = 43). This effect was due to an earlier onset in individuals with long CTG18.1 expansions. No difference in ERDA1 expansion frequency was seen either between bipolar patients (35%, n = 60) and matched controls (29%, n = 114), or between affected and unaffected offspring in the families. We conclude that expanded alleles at the CTG18.1 locus confers an odds ratio of 2.6-2.8 and may thus act as a vulnerability factor for affective disorder, while the ERDA1 locus seems unrelated to disease.
Nonmuscle invasive tumors of the bladder often recur and thereby bladder cancer patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method, e.g. a urine dipstick test, for monitoring recurrence would thus be advantageous. In this study, the complementary techniques mass spectrometry (MS) and Western blotting (WB)/dot blot (DB) were used to screen the urine samples from bladder cancer patients. High resolving MS was used to analyze and quantify the urinary proteome and 29 proteins had a significantly higher abundance (p<0.05) in bladder cancer samples compared with control urine samples. The increased abundance found in urine from bladder cancer patients compared with controls was confirmed with Western blot for four selected proteins; fibrinogen β chain precursor, apolipoprotein E, α-1-antitrypsin, and leucine-rich α-2-glycoprotein 1. Dot blot analysis of an independent urine sample set pointed out fibrinogen β chain and α-1-antitrypsin as most interesting biomarkers having sensitivity and specificity values in the range of 66-85%. Exploring the Human Protein Atlas (HPA) also revealed that bladder cancer tumors are the likely source of these proteins. They have the potential of being useful in diagnosis, monitoring of recurrence and thus may improve the treatment of bladder tumors, especially nonmuscle invasive tumors.


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Ryu wrote:Did you guys win/lose?
iizJimmy wrote:Defaulter, I can't get the link. Steam's banned me from the IRC, why? c:
IronShotGun wrote:Probably can make it , add me.
Defaulter wrote: iron why are you playing?
you never signed up?
Defaulter wrote:yeh for the abstion team, which you were removed from
uvs wrote:360n0sc0pewEedSmokEHeAdShotToThEBalLs everyone.
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By STEVE MASCORD
IT wasn't a statement, a photograph or a decision. Of all things, it was a logo.
To those of us who hoped the Australian Rugby League Commission would usher in a new, outward looking philosophy that recognised its duty to lead the game globally, the new NRL logo strongly suggests we were idiots.
Whereas the previous logo acknowledged the Warriors by including black and white with the ubiquitous green and gold, the new symbol – which you've no doubt seen by now – is so Australian that the Auckland franchise will not be required to wear it! It's an Australian jersey with the southern cross (someone suggested colouring in the stars as featured on the New Zealand flag would have been a nice touch. Not a chance).
It's a throwback to the 1990s when Brisbane wore a big blue NSWRL logo on the left of their chests – and we all know how that ended up don't we? Not that the New Zealand Warriors are going to launch breakaway competition any time soon but it's clear a slap in the face for a constituency that David Gallop once descibed as "the jewel in the crown" of the entire competition.
Since John Grant and his compadres came to power, they have slavishly aligned themselves with the Australian team. Grant even reads out the side, the commissioners wore green and gold ties to the Anzac Test and only visited the Australian rooms.
Gallop always distanced himself from such parochialism. The divided responsibilities of the NRL and ARL may have been tiresome but this "church and state" demarkation line between the club competition and representative football was a good one – one which I, for one, now miss. It's almost as if the commission thinks Australia NEEDS their help which it clearly does not, if the 18-10 win over New Zealand on October 14 is anything to go by.
To be fair, some of the aims announced on October 29 as part of the NRL's new strategic plan are admirable. The objectives, for 2017, are:
• An NRL growth fund which will have made $200m available for investment in key projects.
• Club membership reaching 400,000;
• Rugby League social media platforms will engage 5.8m people;
• Average attendance at NRL games will increase to 20,000 (that's a big one isn't it?)
• 700,000 people will play in competitions (in Australia);
• 1.8m will be engaged in NRL community programs;
• 1.65m will take part in Rugby League activities;
• 84% of all NRL players will be engaged in education or career training;
• Central revenue will have doubled to more than $300m.
If there's something about that list that strikes you as odd, but you just can't put your finger on it, I'll spell it out for you – this administration has put its balls on the line and that's not something we are used to.
The commissioners have also put expansion on the back-burner until the end of 2014 at the soonest. This has upset plenty of people but there is a war to be waged on the home front. The former NRL CEO, David Moffett, was big on strategic plans but his administration – a marriage of convenence between the ARL and News International – was not.
As a result, his version of the stategic plan stayed in a bottom draw somewhere at the old headquarters in Fox Studios.
The "growth fund" is clearly there to counter the AFL's incursions into the Gold Coast and Western Sydney. It is a fraction, however, of the fugure that sports has to spend. Likewise, AFL club membership passed the 400,000 mark some years ago.
Ross Smart, the former Parramatta and Canterbury media manager, recently made a good point in an online columm about the social media aims of the administration. What about traditional media? While the RFL and World Cup organisers are moving heaven an earth to get into the traditional media, that fact the NRL hardly mentions it the new document suggests it is not even a major priority.
As Smart writes at sportsbizinsider.com.au , most people in Australia and New Zealand still get their news from the daily press "fading star or not".
The next three aims are self-explanatory; as a sport we should be able to top a million participants worldwide. the NRL won a global award for its community work recently and I guess a fan day or two would count as "rugby league activities".
The NRL's aim at doubling central revenue during a period when the television rights will be static is indeed interesting. Clearly, they are expecting the digital realm to explode with people watching more and more games on their tablets and laptops and these areas being secure enough for the ARLC to extract a premium for the rights.
So what of the team that John Grant now reads out before every game?
In the past few Anzac Tests, even when the scoreline has been relatively tight, there has been a feeling that the Australians were cruising. That was certainly the case at Eden Park earlier this year. But on October 14, the Kiwis were genuinely in the game for much of the match, scoring first and hanging on under pressure for long periods.
In the end, two North Queensland 'locals' (as in residents), James Tamou and Matt Scott, were probably the difference. Which, in the case of Tamou, takes us back across the sideline and into the boadroom again.
The New Zealand Rugby League showed up at their meeting with the Australians on game day with a list of players in the Junior Kiwis would could conceivably be poached by the Aussies. But it seems barring Junior Kiwis from playing for Australia is as far as the ARLC is willing to go in altering international qualification rules.
This could have the effect of just discouraging players from representing the Junior Kiwis. The Australians also wanted two referees in Townsville – something which the Kiwis would have been happy with.
In the end, they got an Australian referee even though the appointments at the World Cup will be neutral.
On the field, as off, we antipodeans are great at being underdogs. As leaders? Hmmm. "Could do better" would be the diplomatic assessment.
Filed for: FORTY-20 MAGAZINE


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Beginning at 11:00 p.m. on Wednesday, July 3, 2019, the Memorial Bridge will close for the 4th of July Fireworks. The bridge will reopen at approximately 12:30 a.m. on Friday morning.
At approximately 7:30 p.m. on Thursday, July 4th, a section of Hall of Fame Ave. from the Memorial Bridge to Riverfront Park will close as will State St. from Dwight St. to E. Columbus Ave. through the duration of the fireworks.


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Can anyone lend me the BBC Shakespeare DVDs? Time I got me some culture. 01671 402 322
Nicky - membership No. 13
Can anyone lend me the BBC Shakespeare DVDs? Time I got me some culture. 01671 402 322
Nicky - member No. 13
Flower press , any type at all. I bet you've got one stuffed in the back of a cupboard!
01671 402 322
Nicky - membership No. 13
WANTED: Someone to repair an old wind-up clock. It ticks and chimes but too quickly.
WANTED: A book press or something similar.
01671 402 322
Bernice - membership No. 55
Golden Wonders Cocker Spaniel print
by John Trickett
Mounted and framed Golden Wonders Cocker Spaniel print by John Trickett
Limited edition print, hand numbered and signed by the artist.
Image size: 12 x 15 ins Edition: 28 of 395
The approximate size quoted refers to the image area. There is also a white border, which carries the signature and number of the edition plus the mount & frame.
£70 + 30 Crees available for viewing at 8th November LETs meeting....
Open Studio Workshop, 1 Station Road, Newton Stewart
10.30 - noon,
Kath - membership No. 40
Kenwood Chef - well loved and used but still working well. Whisk, K Beater 15 Crees
Leaf Blower/Collector - little used 15 Crees
Susan G - membership No. 02
Hi folks,
Does anyone have a spare jar of raspberry jam for Crees? It's for Gaynor & Steve (27) - I'll collect for them at Thursday's meeting if you have.
Many thanks
Susan (02)
Carl & Steph - membership No. 67
Offered - Heatstore 1500 Storage Heater
This is an old electric Storage heater in good full working order. Parts are still aailable for this model if required in future. Item was used to get through renovation during last winter, simply no longer required.
Will need collecting from between Newton Stewart & Wigtown, Heavy (obviously) but help can be given to load at this end.
10 crees
Can email a photo if required, please contact at firstname.lastname@example.com
If members haven't put their contact details in their advert please use your Galloway LETs membership directory to look up their telephone number &/or email/Skype address or use the comments section with each advert.
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REYKJAVIK – Plans to launch the construction works of the first mosque in Iceland have been causing lots of uproar in the Nordic island, amid increasing hate speech on the social networking websites against the religious minority.
"The consequences could be very, very serious for our community," Salman Tamimi, founder of the Muslim Association of Iceland, told visir.is.
The uproar surrounding plans to construct Iceland's first mosque started two weeks ago when Sveinbjörg Birna Sveinbjörnsdóttir asked city authorities to go back on their promise to the Muslim Association of Iceland on a free lot to build a mosque in Reykjavík.
"While we have a national church we shouldn't allocate lots for buildings like mosques or churches for the Greek Orthodox Church [she meant the Russian Orthodox Church]," Sveinbjörg told visir.is.
Giving her comments during municipal election campaign, Sveinbjörnsdóttir, the Progressive party's leader in Reykjavík, sparked anti-Muslim uproar in social media websites.
The hate comments were also dominant in comments on a news article published on visir.is on Sunday with the heading 'Could start to build mosque after the weekend'.
Some of the comments on the website were particularly harsh and were directed towards Salman and Ibrahim Sverrir Agnarsson, chair of the association, Iceland On Review website reported.
The comments angered Tamimi, founder of the Muslim Association of Iceland, and his lawyer, Helga Vala Helgadóttir, who declared plans to bring charges against those who made the comments on Vísir.
"They just wanted to get votes and it didn't matter how they got them," he said, referring to the Progressive Party's anti-mosque comments.
The Nordic country of Iceland has one of the smallest Muslim communities in the world with only 770 people registered with the official Muslim organizations in the country (as of 2013).
This corresponds to 0.2% of the population of Iceland.
Source: http://www.onislam.net
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I was a bit of a cynic lining up to get into the theatre. So what? He is visiting. I really haven't seen his work lately. What's he up to anyway?
Then he was at the stage center, no emcee. He talked about his new project. He talked about what inspired him and his research. Then he began talking about his dream. The man was born in 1939. And he still has dreams, big dreams. I shifted in my seat.
He is extraordinary. I thought to myself.
I then recalled my most esteemed professor said he greatly admired the director. "He was broke twice making the movies he believed in." I felt his love for film when he talked.
As he wrapped up, he added an anecdote—
My father played flute, but all he wanted was to be a composer. I direct films, but all I ever want is to become a writer. There are people born with talent that most of us have to trade with hard work. Born into a family like mine, I have to work hard to prove that I am something. So I direct and I work.
For the first time, I realized that having a house full of great artist relatives is not fun to be around. Fortunately, he didn't flinch. I thought that came from some great parenting. Or it's just great gene?
Before fading out from the stage's side door under the spotlight that resembles the God in Bruce Almighty, he turns around and told us a joke about a legendary teacher of his time encouraged his film students to call him by his first name. Pause.
"You can call me Francis."
Cue the longest applause.