The alternative ways of cancer

The alternative processing of genomic loci through alternative splicing (AS) to produce multiple transcripts is a prevalent mode of gene expression regulation in multicellular organisms. Although the specific function of the majority of alternative transcripts remains unknown, the differential production of transcript isoforms has been related to essential biological processes, such as the acquisition of tissue-specific functions, and it has been long recognized that disruption of splicing mechanisms can cause disease, including cancer. Cancer arises from genetic and epigenetic alterations that interfere with essential mechanisms of the normal life cycle of cells, such as replication control, DNA repair and cell death, and multiple cancer-related alterations have been described to induce AS changes in tumor transcriptomes. Cancer-related mutations that disrupt splicing regulatory motifs or create cryptic ones, as well as mutations and expression alterations in splicing factors and chromatin regulators, can lead to alterations in RNA processing and splicing of genes, which in turn impact their function and contribute to the pathological properties of tumors. The prevalence of AS in cancer genomes and its prominent role in cancer-related processes indicates that these alterations may be related to significant functional impacts and may explain some of the observed oncogenic properties.

AS_drivers

With the aim to address this question, we have recently performed an exhaustive analysis of the functional impacts produced by AS changes in tumors (Climente-Gonzalez et al. 2017). We described how cancer specific AS changes lead frequently to shorter protein products and sometimes, although less often, to a non-coding transcript. As a consequence, transcript isoforms that are more highly expressed in tumors encode for fewer functional domains, i.e. there is a potential loss of functional capacities of genes in cancer. Interestingly, the protein domains more frequently affected by AS belong to functional families classically affected by somatic mutations in tumors. Additionally, these functional domain losses are in fact strongly associated to protein-protein interactions, and often affect partners of classical cancer drivers. Moreover, since we analyzed the splicing changes occurring in each individual tumor sample, we were able to observe that protein affecting mutations and splicing changes that affect similar functional domain families tend to occur in different patients, suggesting an equivalence between the mutations and splicing changes. Splicing alterations may thus recapitulate similar functional impacts to those observed through genetic alterations, namely protein affecting mutations and copy number alterations, more commonly associated with cancer. Transcriptome data thus shows that alternative splicing has a functional impact similar to other alterations, and may also play a driving role in cancer progression.

Climente-González H, Porta-Pardo E, Godzik A, Eyras E. The Functional Impact of Alternative Splicing in Cancer. Cell Rep. 2017 Aug 29;20(9):2215-2226.

 

Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks

We have recently studied the variability of the cell transcriptome in several tumours to describe new relevant alterations in cancer. The genetic information of cells is encoded in the DNA. This information is read by the cellular machinery, which will generate the so-called RNA and then translate it into proteins. Sometimes, the same gene can give rise to different RNA molecules, which may produce proteins with potentially very different functions. This is due to a process known as alternative splicing, which influences the synthesis of most of the RNAs and proteins of eukaryotic cells and its regulation depends on the RNA-binding proteins (RBPs).

Despite the importance of alternative splicing in cell function being widely known by the scientific community, the role it plays in cancer is only beginning to be realized. This has been possible thanks to new sequencing technologies and the availability of RNA sequencing data of multiple tumours through projects such as The Cancer Genome Atlas (TCGA). Although cancer originates from mutations in DNA, they have an impact on the set of RNA molecules of the cell, known as the transcriptome, which can induce and maintain mechanisms linked to the development of cancer. We have now studied the alterations in RBPs that could cause changes in alternative splicing linked to the development of cancer.

For this analysis, we have used various tools developed in our lab. Among them, we used SUPPA a fast and accurate tool to study splicing from multiple samples. Thanks to SUPPA, we could calculate the splicing profiles of more than 4000 samples in less than one day. We then carried out a comprehensive analysis of the mutations, copy number alterations and expression differences in genes, as well as the alternative splicing changes associated to them, for these samples from eleven different types of cancer taken from the TCGA project. This analysis showed RBPs are often altered in human tumours and that these alterations determine the cell transcriptome and induce cell transformations related to the development of cancer. Until now, these alterations remained invisible to the methods used in major cancer genome analysis projects.

With the collaboration of Juan Valcárcel’s lab (CRG) and Miguel Ángel Pujana’s lab (ICO), we were able to show that introducing the identified alterations of the transcriptome in non-tumour cells, these acquire tumourigenic properties. In addition to expanding our knowledge on the role of RBPs in tumours, these results highlight the importance of alternative splicing as a complementary mechanism in the development of cancer, becoming a new relevant factor to be taken into account in the study of this disease. This research opens up new ways of understanding the biology of cancer and searching for new therapeutic strategies. The alterations in alternative splicing are particularly important in the context of those tumours that do not harbour known mutations and for which no therapy is known, and, therefore, they may open new opportunities to understand tumour biology and search for new therapies.

This study was made possible thanks to the funding from the Sandra Ibarra Foundation, the Consolider RNAREG project from MINECO, the funding from AGAUR, as well as from other projects of the Spanish Government and FEDER funds.

Sebestyén E, Singh B, Miñana B, Pagès A, Mateo F, Pujana MA, Valcárcel J, Eyras E. Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks. Genome Res. 2016 Jun;26(6):732-44.

Figure_1Allegoric representation of the variability of the transcriptome through changes in alternative splicing in difference cancer types. Illustration by Babita Singh.

The switch of cancer

For many years, scientists have struggled to understand and cure cancer. The study of the genome of multiple tumors has been fundamental to detect recurrent alterations in several types of cancer, and has facilitated their classification and the development of new therapeutic strategies. In particular, high-throughput technologies have been applied in the context of multiple international projects to detect actionable alterations, i.e. genetic changes in the genome of cancer cells that can be used to develop new targeted therapies. These studies have highlighted the heterogeneity of genetic alterations in patients suffering from the same type of cancer, motivating the development of individualized treatments. However, known actionable alterations tend to occur at low frequency, and often a tumor sample has fewer mutations than those seemingly necessary to explain the tumoral process. Thus, there is a need to expand the catalogue of cancer signatures to integrate other molecular alterations for the characterization of individual tumors.

Most of the strategies used in cancer genome projects are based on searching for genetic alterations or changes in the expression of genes. On the other hand, there is more and more evidence that alterations in the splicing regulatory program play an important role in tumor transformation. Splicing is a process by which the long RNA molecule transcribed from the gene in the genome is processed to remove segments called introns, giving rise to an RNA transcript. Alternative splicing provides a mechanism to generate multiple RNA transcripts from the same gene by eliminating introns in different ways. This process is tightly regulated, and is known to give rise to proteins with cell-type specific or opposing functions, or even provide a way to activate or deactivate gene function. This dramatic change between conditions lead by splicing is generally called a splicing switch.

switch_of_cancer_v1

Splicing switches that are not originally present and regulated in cells can induce altered cellular states, leading to disease. Accordingly, the determination of alterations in alternative splicing in tumors can be fundamental for the development of tumor specific molecular targets for prognosis and therapy. However, this analysis is generally hindered by the heterogeneity of tumors of the same origin from different individuals, as well as by the normal variability between individuals. In our group we have developed a new computational method, robust to biological and technical variability, which identifies significant splicing switches across a large number of tumor samples and shows high accuracy on held-out datasets. Moreover, the method is capable of identifying complex alternative splicing changes that cannot be described using standard methodologies. Additionally, the method is independent of parameterizations, which is especially relevant for the analysis of RNA sequencing data from samples from multiple laboratories and technological platforms.

We have applied this method to data from the Cancer Genome Atlas (TCGA) project, which is the NIH-funded branch of the ICGC project. This is the first published large-scale analysis describing the splicing alterations in 9 cancer types using RNA sequencing data from more than 4000 samples. This is possibly the first systematic study of alternative splicing alterations in 9 difference cancer types using so many patient samples. In this work, we have discovered that there exist many splicing switches in patients with the same cancer type that can separate with high accuracy tumor and normal samples, and different types of cancer from each other, providing a predictive signature. In particular, these signatures provide simple rules based on the expression of a few RNA molecules that could allow determining the cancer type from an RNA sample of a new patient. Additionally, we found such set of rules for the triple-negative breast cancer subtype, which is one of the most aggressive subtypes of breast cancer. This new computational method reveals novel signatures of cancer in terms of RNA transcript isoforms specifically expressed in tumors, providing potential novel molecular targets for prognosis and therapy.

Further reading:

Endre Sebestyén, Michał Zawisza, Eduardo Eyras. Detection of recurrent alternative splicing switches in tumor samples reveals novel signatures of cancer. Nucleic Acids Research 2015; doi: 10.1093/nar/gku1392

News at the UPF web site: “Interruptors del càncer” (in Catalan)

 

The new voyage of the “Argonautes”

A new publication from our group describes a new function for a protein mostly characterized for its role in directing small non-coding RNAs to their targets.

Small RNA molecules, which include the so called micro-RNAs (miRNAs), provide a powerful mechanism to regulate gene expression in the cell cytoplasm either by triggering degradation of the messenger RNAs molecules (mRNA) or by inhibiting their translation into proteins. This mechanism, known as post-transcriptional gene silencing, takes place in the cell cytoplasm and has important implications for understanding developmental and disease processes. The miRNAs are guided to their targets by a molecular complex that is made of a group of proteins called Argonautes. These proteins are essential to direct the miRNAs to their mRNA targets, and their structure and role in the post-transcriptional gene silencing has been thoroughly described up to date.

In this work, we report that Argonaute proteins also play an important role in gene regulation in the cell nucleus. The published work describes how Argonaute proteins, besides their role in post-transcriptional regulation, can also affect gene expression during transcription, the cell process that makes mRNA from DNA. In particular, this work shows evidence that Argonaute can bind to specific locations of the genome. These locations are transcriptional enhancers, regions in the DNA that control of the expression of one or multiple genes by governing when these genes must be turned on or off. Enhancers are usually placed far from the genes they regulate, but they can also occur inside a gene. The activation of enhancers takes place through binding of specific factors, as well as by the acquisition of multiple chemical modifications by the chromatin, the structural packing of DNA in the cell that makes possible to hold the long DNA chains from chromosomes into small volumes in the cell nucleus. Each cell type has a specific subset of enhancers that are activated by generic biochemical modifications of the chromatin and by specific protein factors that control gene expression in that cell, and which ultimately determines the cell identity. The aberrant activation or silencing of enhancers can impact cell function and lead to cell transformation, like cancer.

AGO1_figure

This article describes how the Argonaute protein AGO1 can bind to enhancer regions inside genes when they are activated, and thereby directly affect how the RNA molecule transcribed from that gene locus is processed (see Image). In particular, using high-throughput sequencing techniques to describe the regions of the chromatin that have specific biochemical modifications, and which ones interact with Argonatue proteins, we have discovered that the Argonaute protein, specifically the member AGO1 from the Argonaute protein family, binds preferentially to active transcriptional enhancers and that this association is mediated by the RNA molecules that are produced from the active enhancers, also known as enhancer RNAs (eRNAs). Moreover, the interaction of AGO1 with enhancers occurs mostly inside genic regions and appears to affect the splicing of the host gene rather than its transcription. In summary, this work suggests that AGO1 has a function in the nucleus that is related to the activation of transcriptional enhancers, and from these sites is capable to modulate the processing of the RNA from the same locus. These results contribute to the understanding of the complex regulation of gene expression in eukaryotic cells. This work proposes that Argonautes can perform yet another voyage to the nucleus to control how genes are processed, proving to be important and versatile players in determining cell function.

This work has been funded by the Sandra Ibarra Foundation for cancer and by the European Network on alternative splicing EURASNET.

Alló M, Agirre E, Bessonovc S, Bertucci P, Gómez-Acuña L, Buggiano V, Bellora N, Singh B, Petrillo E, Blaustein M, Miñana B, Dujardin G, Pozzi B, Pelisch F, Bechara E, Agafonov D, Srebrow A, Lührmann R, Valcárcel J, Eyras E*, Kornblihtt AR*. Argonaute-1 binds transcriptional enhancers and controls constitutive and alternative splicing in human cells. PNAS doi:10.1073/pnas.1416858111 http://www.pnas.org/content/early/2014/10/08/1416858111

At the moment though, Ariel Nasr’s just trying to keep canada

the movie sees http://www.mycanadagoosejacket.org rocker lift lid on ‘devastatingly honest’ life story

cheap canada goose uk If you really get this you canada goose outlet online store review will absolutly explode your canada goose outlet store calgary business. So what makes the invite so important? canada goose outlet store montreal If you didn’t know it’s canada goose outlet miami the beginning to everything else. canada goose outlet seattle That is why it’s canada goose outlet website legit so critital. I came to the conclusion the guy must be schizophrenic. I talked to several people about this that no human being would talk on the phone to someone 7 hours a day 7 days buy canada goose uk a week. I in my 40 and I never heard anything remotely like this. cheap canada goose uk

canada goose coats Comment replies consisting solely of images will be removed. There was a boss in vanilla WoW that gave you a canada goose outlet store near me debuff that was supposed canada goose outlet uk fake to spread through your party like a plague, however it was bugged and persisted canada goose outlet 80 off on you whether or not you were in the instance. So someone canada goose outlet legit left the raid with the debuff and ended up spreading it through a major hub in the game and killed a bunch of people like a real plague, it occurred for a few days before a hotfix if I remember reading about it right. canada goose coats

canada goose clearance sale Trinity College Dublin health psychologist Professor David Hevey has looked at the psychological barriers that prevent us from taking action to protect ourselves when we are given health related advice. “In Ireland, we have a high level of awareness of the health risks from exposure to canada goose outlet winnipeg address radon gas; we know it causes canada goose outlet parka lung cancer. Even so, persuading people to test and fix their homes is not easy. canada goose clearance sale

Canada Goose Online A degree in classical literature. And like some of his generation, he feels the call of unanswered questions from faraway central Asia.He’s among those who’ve packed canada goose outlet edmonton their western suitcases, and canada goose outlet uk moved their westernized selves to the Islamic Republic of Afghanistan, land of the kings and castles of family lore.At the moment though, Ariel Nasr’s just trying to keep canada goose outlet real warm in Kabul.We are the first. Our taste for this disappearing fish canada goose outlet is driving it to extinction. Canada Goose Online

buy canada goose jacket cheap If you want to take terminal leave and not work for 2 months, great, go right ahead. But say you work until 30 days out and sell back 30 canada goose outlet near me days, you will get an extra couple thousand dollars more than you would if you got out 60 days out because you sold leave. Remember, you always get paid base pay + BAH + BAS until your date of separation whether you on leave or not.. buy canada goose jacket cheap

Canada Goose Jackets Thanks for canada goose outlet location all this information. Sadly the dmg is is still confusing. Getting a canada goose outlet buffalo pistol shoot on boats while just sitting there and doing nothing hurt for 75%. A black bear has short (1.5 inch, or 3.8 cm), dark claws, while a grizzly’s are very long (2 to 5 inches, or 5 to 12.7 cm) and light in color. Grizzlies canada goose outlet toronto also tend canada goose outlet new york to canada goose outlet ontario be bigger than canada goose outlet in chicago black bears. Male canada goose outlet kokemuksia grizzlies weigh around 500 pounds (226 kg), but some top out at 800 canada goose jacket outlet store (362 kg). Canada Goose Jackets

Canada Goose sale The Boys will take then win any way they can get it, but I’m sure Head Coach canada goose outlet in uk Jason Garrett is goose outlet canada going to have plenty to work on in the next few days as they have a quick turnaround for their annual Turkey Day game. Romo was as good as he can be and canada goose outlet florida he carved up the Skins canada goose outlet price defense for 292 yards and 3 touchdowns and no interceptions. Two canada goose outlet store toronto of those came in the fourth quarter as they made a mad scramble to score 14 points and took a lead for the first time since early in the game.. Canada Goose sale

uk canada goose outlet Sadly, there is no set time that you need to change your car’s brakes. This is why it is important to pay attention to the sounds you hear coming from your car while you are driving it. If you take your vehicle to your Waterloo brake repair specialist, an canada goose outlet authentic experienced auto technician can help you know how your brakes are doing and let you know if there are any wear and tear on your brake pads. uk canada goose outlet

buy canada goose jacket If you want to lose stomach flab to get in shape, there are great exercises that you can use to achieve this goal. canada goose outlet uk sale These workout exercises can help you lose that belly fat easily. canada goose outlet ottawa You need to stick with these exercises and you will surely see the best results within a short period of canada goose outlet germany time. buy canada goose jacket

canada goose I had some nice new toys my favourite thing was the Reindeer antler, it is scummy! After presents we had birthday cake, muffins cookies that Mummy canada goose outlet jackets made canada goose jacket outlet uk the day before. I was a bit of a piggy when Mum had turned her back for a moment I pinched another canada goose outlet woodbury huge piece of cake, Milo was quite upset because he couldn’t reach the counter top get an extra bit. Oh what a shame, hehe.. canada goose

Canada Goose Outlet From then canada goose outlet vip on, the rule changed and the, but it didn’t go over so easy. “The Senate parliamentarian had looked at the rules to see if it was okay,” she said in a CNN interview. “So, I walk on that day and you would have thought I was walking on the moon. Canada Goose Outlet

Canada Goose Coats On Sale This is the most canada goose outlet nyc beautiful tee I own. The dye effect on it looks insane in person. The perfect designer summer tee. There no reason for Paul George and Roy Hibbert to be able to push Lebron James, D Wade and Bosh in their primes to 7 games. The fact is Miami underperformed despite having by far the best team in the east. Had they played properly they would rolled right through the East like GSW.Bottom line is, if GSW played worse to seem “more beatable”, you wouldn be saying all this. Canada Goose Coats On Sale

canada goose store In 1985, after the success of his fifth studio record, “1999,” and the acclaimed follow up “Purple Rain,” Prince set up his own label, Paisley Park Records. It was partly official canada goose outlet bankrolled by Warner Music. The terms of that arrangement became the subject of a high profile spat, during which Prince painted the word “slave” on his face before he declared his “emancipation” and changed his name to a symbol canada goose store.

The villas usually have 4 5 bedrooms and a swimming pool where

Easy Guidelines For Buying A Property In Sotogrande

canada goose coats Buying a property would be one of the major milestones in your life and small carelessness in making a canada goose outlet uk decision can end up canada goose outlet washington dc becoming huge regrets later on. So, it’s canada goose outlet edmonton very important that you have a property buying battle plan all ready before buying a property in Sotogrande, one of Europe’s most luxurious canada goose jacket outlet store residential canada goose outlet mall resorts. Demands for property Sotogrande are on a continuous increase with the well priced properties getting sold very quickly. If canada goose outlet 2015 you are planning on staying permanently or for more than 2 3 canada goose outlet mississauga years in southern part of Spain, buying a property amidst the beauties canada goose outlet online reviews of Costa del Sol, where Sotogrande is located, would be your best decision. canada goose coats

canada goose deals Sotogrande, situated close to Gibralter, is the largest development in the municipality of San Roque. When you buy a property in canada goose outlet paypal Sotogrande you can enjoy the amazing scenic beauty of this place consisting of mountains canada goose uk site and seas and the famous cork forest running down to the sea. The houses and villas constituting property Sotogrande offer some additional amenities and also this place is famous for first class golf courses and the yachting marina. No wonder this place is mostly preferred by the elite canada goose outlet woodbury section of the society. canada goose discount uk canada goose deals

Canada Goose sale Coming straight to the point, there are some must know things when you are buying property at Sotogrande. First of all do the math properly. Determine your budget so that you have a clear idea of how https://www.canadagooseoutletcoats.com much you can afford. Speaking to an independent financial advisor would of great help as property in Sotogrande canada goose outlet montreal can be expensive. Also, engaging intermediaries or property agents canada goose outlet london uk would be a great idea as they can manage the exchange of contracts and arrange canada goose outlet website legit for legal documents related to purchase of property Sotogrande. This means you spend time fruitfully searching for the best options without getting involved canada goose outlet locations in toronto too much in the nerve racking legal side of the transaction. Canada Goose sale

cheap Canada Goose Apart from enjoying the stunning views, you can find exclusive facilities in a property canada goose womens outlet in Sotogrande. The villas usually have 4 5 bedrooms and a swimming pool where you can relax or unwind. Also, they have lawns for your children or grandchildren to play and have fun. A premium property Sotogrande comes with additional facilities such as BBQs, private terrace, laundry rooms and fireplaces. Structural aspects of the houses must be considered before taking any decision. The villas are designed by top interior designers and the looks are canada goose outlet ottawa thus canada goose outlet nyc classy and elegant with marble flooring and arched ceiling. The price of the houses depends on the location, size and the facilities it provides. cheap Canada Goose

Canada Goose Parka Inspection of the place and the neighbourhood area would be a clever thing to do. Check how far it is from your workplace or your child’s school and assess how convenient it is for you and your family to commute. Get information regarding the amenities locally available and if you have any special requirement regarding location or accessibility to town centre then share it with the dealer who will have canada goose outlet all information relating to property Sotogrande. If you canada goose outlet toronto are looking for a relaxed life punctuated by some exciting outdoor activities then opt for a property in Sotogrande and enjoy the true spirit of life. Canada Goose Parka

Canada Goose Jackets If you aspire to live amidst the amazing scenic beauty of canada goose outlet niagara falls nature and lead a lavish lifestyle buy property in Sotogrande and fulfil your dreams. Property Sotogrande is known for elegance, classy looks and first class facilities Canada Goose Jackets.

They are quiet canada goose outlet toronto location simple:

How Breathtaking Is Your Voice

canadian goose jacket Many people dream canada goose outlet montreal about a music career and they wish they had ideal vocal makings. Some people are born with a great voice while others are searching their destiny to improve the chances they possess. However, even if you have a gift of canada goose outlet miami singing you need to make it really perfect. Of course, you realize that your voice is the main tool in your profession. canadian goose jacket

canada goose To make a perfect voice you need to remember about three main aspects. They are quiet canada goose outlet toronto location simple: listen to good music, practice professional descant and stick to a particular daily routine. canada goose

Canada Goose online The first point consists in canada goose parka uk the fact that the music you hear is reflected in the voice you possess. Listening to a perfect vocalism can help you not only create you voice but also improve settled makings. Canada Goose online

cheap Canada Goose The second point is your practice. The descant is something like canada goose outlet in uk a warm up for sportsmen. canada goose outlet las vegas No sportsman canada goose factory outlet starts his canada goose jacket outlet store competition without a warm up. canada goose outlet toronto factory The descant not only prepares our vocal apparatus for hard work but canada goose outlet location also creates singing skills. While having a descant we undergo breathing exercises and without the correct breathing you canada goose outlet jackets will never be able to sing. A canada goose outlet factory regular correct descant can extend the range of your voice, improve your intonation, make the sound of your voice soft and steady and improve your articulation skills. Every savvy has its special particular exercises that is why you should start every practice with canada goose outlet store montreal a special descant. cheap Canada Goose

Canada Goose sale The third point is your daily routine and your voice hygiene. Keeping voice hygiene means sticking to some rules which allow a person preserve the health of his vocal apparatus. You should control the capacity of your voice. It is dangerous to sing for a long time especially if the range is too extended. Moreover your vocal apparatus is influenced by sharp temperature drops. What is more the sleeping routine also influences your makings. Canada Goose sale

canada goose deals As for your eating habits you should also take some important thing into consideration. You Canada Goose Outlet should eliminate the www.gofind.ca consumption of food and drinks which irritate the mucous membrane of your throat. canada goose outlet winnipeg address Moreover, you keep spicy, hot and cold dishes away. If you start singing after dinner at once you will also have problems with breathe. canada goose deals

canada goose black friday sale Everything plays its important role canada goose factory outlet vancouver in singing your organs of speech, the position of your body, the sense of rhythm and your inner emotional canada goose outlet us state. Sometimes it is useful to make a grimace, to imitate the voices of animals and to try to transfer emotions with the help of your voice. It makes your jaw relaxed and reveals all the stiffness inside your body. Your emotional preparation can control you vocal cords. The success is the control of vocal stiffness and sound current. Try to learn how to release the sound out of your diaphragm with ease and freedom, do not move your chin. Think about your soft palate in the position of a “yawn” that lets you produce canada goose outlet uk fake the correct sound of vowels and influences the timbre. The best way is to practice different techniques canada goose outlet online store of singing and find your own unique canada goose outlet in canada one. canada goose black canada goose coats uk friday sale

buy canada goose jacket cheap Lucas Dummen 360 Media Touring provides you with professional and successful backing that will lift you to the top of your career being affiliated with platinum selling artists and cutting edge labels, production companies and management in the industry. Check canada goose outlet locations in toronto us out buy canada goose jacket cheap.

However, it is well worth taking a little time to research

With the development of the internet, it has never been easier to obtain a great deal on your car insurance and financing. However, it is well worth taking a little time to research your options to ensure that you secure the best possible deal. Having your insurance and loan quotations in place before you begin shopping, can allow you to relax and enjoy test driving and finding your ideal vehicle..

canada goose coats Those who believe canada goose outlet toronto canada goose outlet that life should be fair all canada goose premium outlet the time cannot tolerate it when it’s not fair. That can lead straight to being totally inert, due to the fact that they think there is not a canada goose outlet winnipeg thing they can do regarding the unfairness. This leads to feelings of helplessness in other words, canada goose outlet near me depression. goose outlet canada canada goose coats

Canada Goose Outlet Life span of the AESRPT SS 20 30 years based on operation demand. The OnSite AESRPT is designed to generate electricity power at the location where the electricity is needed. With canada goose discount uk energy demand growing, a AESRPT OnSite power system is canada goose outlet black friday the perfect on demand solution.. Canada Goose Outlet

Canada Goose sale This 4moms Mamaroo Swing is different than your average baby swing. Another feature that canada goose jacket outlet I really canada goose outlet 2015 like is that you plug it in rather than using batteries. I hate changing batteries in infant toys, first you generally have to find a screwdriver, canada goose outlet seattle then you have to figure out where the batteries are even kept! In the previous swing I had for my infant daughter, I never did find the batteries! My electronic minded husband did and was able to change them but I spent about 45 minutes one day trying to figure it out before he got home. canada goose factory outlet Canada Goose sale

canada goose uk outlet I also at level 29. I think the beauty of this game is that you really never get out of the tutorial. If canada goose factory outlet vancouver you canada goose parka outlet suddenly canada goose outlet hong kong happen upon a sidequest, you basicly have to grind some new skill or canada goose outlet mall go and fin a canada goose outlet toronto location skilltrainer. canada goose outlet paypal Stretch limo is one of the most popular vehicles used for canada goose outlet miami Limo Service canada goose outlet los https://www.gestiontb.ca angeles CT. It is basically the longer version of luxury cars such as Lincoln, Chrysler, and many more. As the name suggests, most passengers will notice more spacious seating area along with the distinct interior and luxurious facilities inside the limo. canada goose uk outlet

canada goose store I would also not recommend any canada goose jacket outlet toronto of the specialty CB shops that are found through a search engine. Most of official canada goose outlet these radios that are sold online are high end ham radio’s that have been modified or are easily modified for use on the Citizens band. These radios transmit anywhere from 25MHZ to 30MHZ and are canada goose outlet london uk not type accepted for use in the CB radio band. canada goose store

cheap canada goose uk Laxmi canada goose outlet official Vilas Palace was built by Maharaja Sayajirao 3 in the canada goose outlet parka year 1890 canada goose outlet 80 off and is a massive building of Indo Saracenic type. If you visit Baroda, then the Laxmi Vilas Palace is a tourist attraction that you must visit and see for canada goose outlet england yourself. This palace is the biggest private place that has canada goose outlet store uk been built to date in the country and was planned by Major Charles Mant.. cheap canada goose uk

canada goose clearance CLASS 4A HINSDALE SOUTH Neuqua Valley vs. canada goose vest outlet Note: Schedules are subject to change because of weather and canada goose outlet in new york other factors. TUESDAY’S EVENTS HIGH SCHOOLS BOYS LACROSSE SUPERSECTIONALS DOWNERS GROVE SOUTH Glenbard West vs. The applicants get the confidential pin code number after canada goose outlet phone number the completion of the entire formalities. canada goose outlet montreal It does the confirmation of the acceptance of the loan application form. The clients are able to procure the amount around 100 that must be refunded within canada goose outlet online uk 7 days. canada goose clearance

Canada Goose Coats On Sale Unfortunately, there are a limited canada goose outlet boston amount of precious and valuable metals found on Earth, today. Among the most common are silver, gold, and platinum. These metals are used to make endless commodities, such as jewelry, auto parts, art, furniture, electronics, and much more. Canada Goose Coats On Sale

Canada Goose Online One of the main reasons that it does work is its use of all natural ingredients. Most supplements noawadays are filled with extracts and preservatives, or even chemicals that have been synthesized in a lab. Putting stuff like that into your body obviouly isn’t as good, and wont have the same effects as something that is derived naturally and is already present in your body.. Canada Goose Online

cheap Canada Goose “We tell the Tone It Up community to set a goal for themselves and then set out a way they want to reward themselves. Not necessarily something you eat, it could be downloading new music or getting a new workout outfit, which is why we’ve partnered with Kohl’s since they canada goose shop uk have canada goose outlet las vegas great affordable workout gear from so many brands,” Dawn says. “New workout gear encourages you to canada goose jacket outlet uk work out more. cheap Canada Goose

uk canada goose outlet Oil spill and those canada goose outlet uk sale oil tankers are not addressed meaningfully in the review that happened by the National Energy Board. Vancouver Aquarium says it’s still owed $180,000 that was spent on environmental canada goose parka outlet uk testing to figure out what the fuel was and the risks it posed to the facility. However, canada goose outlet location the ship’s owner has offered to canada goose outlet store toronto cover less than a quarter of the cost uk canada goose outlet.

It wasn rare in the past for a fashion designer to provide the

Creating a Costume or Creating an Advertising Opportunity

canada goose coats on sale If you a professional costume designer at the moment you definitely not harbouring good thoughts about the fashion industry. It wasn rare in the past for a fashion designer to provide the wardrobe for a specific actress, but nowadays cinema, theatre, opera and ballet have been consistently infiltrated by fashion houses and designers claiming of having just created the costumes for this or that production. canada goose coats on sale

canada goose Since Baz Luhrmann The Great Gatsby premiered a few days ago in New York and while movie fans await its arrival in Cannes, features about Miucca Prada costumes for the film multiplied. canada goose

buy canada goose jacket cheap The latest news about costumes mainly focused on Givenchy Riccardo Tisci on new production of Maurice Ravel “Bolro” at the Opra de Paris with choreographies by Sidi Larbi Cherkaoui and Damien Jalet, and Marina Abramovic as set designer. buy canada goose jacket cheap

canada goose store In the meantime, Joseph Altuzarra canada goose victoria parka outlet costumes for the New canada goose outlet niagara falls York City Ballet’s spring gala (with choreographies by Christopher Wheeldon) will debut next week. canada goose store

buy canada goose jacket All these costume news proved a good canada goose outlet hong kong excuse to recap further collaborations that happened in more recent years including Rodarte canada goose outlet in chicago tutus for Black Swan; Stella canada goose outlet toronto factory McCartney costumes for her father first ballet, “Ocean Kingdom” (2011), Paul Gaultier “Le Nozze di Figaro” the Opra canada goose outlet winnipeg National de Montpellier last year; Valentino gowns for the New York City Ballet, and, more recently, Vivienne Westwood loaning some of her Spring/Summer 2013 looks to the English National Ballet for the company new campaign. buy canada goose jacket

canada goose clearance sale Lacroix costumes for Bizet’s Pearl Fishers” for the Opra National du Rhin in Strasbourg wil debut this May, while he still working on the ballet costumes for Balanchine’s “The Crystal Pavilion” for the Paris Opera Ballet (to be staged next May). canada goose outlet 2015 canada goose clearance sale

cheap Canada Goose In some cases such as Vivienne Westwood for the English National Ballet, famous fashion designers canada goose outlet kokemuksia were enlisted to attract a younger audience or renew the image of a company; in others, directors and producers canada goose uk set on specific designers who have some connections with dance or theatre or a long canada goose outlet store uk standing tradition of working for the stage. For example, Thierry Mugler was a dancer, Altuzarra studied ballet, while Christian Lacroix started working canada goose outlet los angeles for the theatre in 1986 and designed costumes for productions both in France and abroad, winning twice the Molire Award, France’s national theatre prize for Best Costume Designer. cheap Canada Goose

Yet there is one basic problems with all these contemporary designers and fashion houses suddenly turning costume designers: in most cases the designers do not have any passion for what they are doing or any basic knowledge of the disciplines canada goose outlet in new york practiced on that stage or behind that camera. An admirer of Maurice Bjart, the canada goose outlet real late Gianni Versace was one canada goose parka uk of the most prolific designers and creator of costumes, but we all know that his sister Donatella doesn have the same passion or skills.

Canada Goose Jackets Designing a costume as we learnt from previous posts on this site is a radically different art from creating a fashion collection. A costume is an exercise in theatricality that must also take into account the movement, gestures and comfort of the wearer; a fashion collection is mainly a commercial exercise, the result of a series of factors, from trends to desirability. Canada Goose Jackets

Canada Goose Outlet While some designers lack the skills and humbleness to work for the stage, the main problem at the moment is that, canada goose jacket outlet toronto than designing from scratch, most of these designers are mainly adapting borrowing from Canada Goose Outlet previous work. Canada Goose Outlet

Canada Goose Parka Tisci adapted his Autumn/Winter 2010 collection for canada goose outlet houston Givenchy with its memento mori themes in the costumes for Ravel “Bolero”, coming up with a flowing silk tulle skirt with underneath https://www.expeditionparkaoutlets.ca tulle bodysuits covered in embroideries that imitate bones and reproduce the outline of a skeleton. Canada Goose Parka

canadian goose jacket Apparently the main aim of the choreographers was the dancers all wearing the same canada goose shop uk costume to abandon themselves to the rhythm of music, symbolically shedding their skins and turning into moving skeletons. This is why during the piece, from dervishes in tulle skirts they become incarnations of Kriminal (ah, if only we could ask certain dancers or actors what they think about their costumes.). canada goose vest outlet canadian goose jacket

Canada Goose sale One of the moved to such famous collaborations focuses on the fact that a ballet, an opera or a theatrical piece shouldn be about the costumes, since, unfortunately, in some cases coverage given to costumes unfairly overshadowed the performance. Yet there is also another problem with these recent examples. Canada Goose sale

Canada Goose online Recycling such themes and inspirations denotes a lack of imagination and of artistic vocabulary, something that became quite clear in 2011 when, interviewed by Newsweek about designing the costumes for her father first ballet, Stella McCartney stated “I do performance wear these dancers are athletes, so I have an understanding of that. And it doesn take a rocket scientist to figure out that you have to use stretch materials if you working with ballerinas.” Canada Goose online

canada goose coats In a nutshell, clad them in Lycra and you done, this is probably why you expected some of the dancers in “Ocean Kingdom” to start singing “Jellicle Songs For Jellicle Cats” any canada goose outlet ottawa minute, their tight suits confusing the boundaries between ballet and musical (yes, there is a difference there). canada goose coats

canada goose black friday sale Talk to different costume designers and they will tell you amazing stories about finding a solution for a specific material, playing with metres and metres of plastic, spending entire weeks trying to turn pasta shapes and pan sponges into glamorously grand jewels. canada goose black friday sale

canada goose clearance Costumes create a theatrical illusion for the audience, help life a personality to a character but also free the costume designer imagination. Think about Bernard Dayd PVC and lurex costumes for “Bacchus and Ariadne” (1967): they did echo in some ways the space age fashion trends so popular at the time, but they weren borrowed from a specific fashion collection (and look at the details included in the and at the notes of the designers). canada goose clearance

canada goose deals According to reports, Tisci canada goose outlet mississauga said he had made one of his dreams come true, “It is one of the dreams of a designer to design costumes for a ballet.” You wonder why if it your dream to become a costume designer you don study for such a career. canada goose outlet boston But then we all know that many careers aren built on skills or studies, but on connections and on the celebrities you know. generating buzz, attention and opportunity, costumes tour the of flagship stores, in thirty time, will be exhibited in art museums. After all Prada is not Umberto Brunelleschi canada goose deals.

Inside the interior of the U sits a big boiler on legs where

Kota Bharu

Canada Goose sale It rained buckets our first evening in. We sat in our room sipping vodka we brought from Penang, watching the sky canada goose sale uk turn from mud to black and waiting for the rain to stop. It didn So around half past seven we grabbed umbrellas and went in search of dinner. Canada Goose sale

canada goose clearance sale Big canada goose outlet official mistake. canada goose clearance sale

cheap Canada Goose goes to bed at sunset. The streets were empty, the sidewalks rolled up. We splashed our way past Pasar Siti Kadijah to the night market and found it a shadow of its 2005 self. There were no crowds, no buzz. Nor was there anything tempting on offer at the few stalls weathering the storm. cheap Canada Goose

canada goose So we headed back to the hotel, passing fast food joints belching out that nasty elixir of too sweet baked goods, preservatives and grease that hangs over airport food courts. No thanks. We rather go hungry, and were reconciled to doing so until we turned onto Jalan Tok canada goose outlet germany Hakim and saw, three blocks up, a promising glow. canada goose

my friend!” shouted the white taqiyah capped proprietor canada goose factory outlet toronto location as we stepped over the threshold of Kedai Kopi Din Tokyo.

Pak Din grinned at us as if we were long lost friends, then pointed to a place at his counter. “Come in, come in! canada goose outlet vip What will you eat? What will you drink?”

canada goose coats Kedai Kopi Pak Din Tokyo is an indoor kuda, the Kelantanese term for old fashioned coffee stalls with U shaped counters and long wooden benches that must be mounted like a steed (kuda is Malay for horse). Inside the interior of the U sits a big boiler on legs where Pak Din makes good, strong Chinese kopitiam style coffee and tea canada goose outlet england by pouring water through grounds filled cotton socks suspended from metal rings. At the U base is a long stainless steel trough over which he “pulls” tea by pouring it glass to glass, stirs quail eggs into hot ginger infusion and steeps eggs in boiling water before serving them in classic kopitiam green and white saucers. canada goose coats

canada goose black friday sale Short and canada goose outlet niagara falls sturdily built, Pak Din is a blur taking orders, serving drinks and food, selling cigarettes, making change and shaking hands, all the while spewing friendly banter and communicating, via the occasional shout, with the kitchen at the back of canada goose outlet buffalo his shop. If there a break in the action he makes good use of it, wiping down counters, replenishing the water boiler from a red plastic bucket and keeping glasses always at the ready, stacked in a drainer over the trough warm with splashes of water from the boiler. canada goose black friday sale

What would we eat? On a night like this, our backs wet canada goose outlet toronto address and our pants soaked to our knees, something hot and comforting sounded about right. So thought Pak Din patrons, most of whom were leaning over bowls of the house specialty: sup ekor (oxtail soup).

We ordered two bowls of soup and spooned it up slowly, marvelling at this concoction of meat, bones, tendons, cinnamon, star anise and nutmeg canada goose parka outlet that tasted too good to be medicine yet instantly rendered us healthy, happy and certain that the world canada goose outlet store calgary is a beautiful place. The hyper distilled broth was deep, real deep, a gut punch of meat essence. When we got to the bottom of our bowls it came as no surprise to find them stained sup ekor brown.

canadian goose jacket By now the rain had stopped but we lingered, reluctant to leave the comradely warmth of Pak Din U. We ordered a hot milky teh tarik just for the enjoyment of watching his exuberant way with the pull, and then another because the first was so darned tasty, the tea strong and bitter enough to not be overwhelmed by sweetened condensed milk. We sauntered back to our room wired and satisfied. canadian goose jacket

canada goose clearance We returned to Kedai Kopi Pak Din the next day and the next and the next, always around 7am for our caffeine fix and then once or twice again, for tea and a banana leaf wrapped nasi lemak or a few Kelantanese kuih plucked from the trays and plates lined up along the inside edges of the counters. During the day Pak Din staff set tables out front of the shop, and if there were no places at the U two were always found for us there. canada goose clearance

Canada Goose Outlet On our last day in, after a morning of too much food and before the long drive back to Penang, we stopped in to bid Pak Din farewell. Dave couldn resist ordering a couple of soft boiled eggs. When they arrived he followed the lead of other diners and blanketed them with black pepper from a shaker on the counter. (That black pepper is a Kelantanese touch, courtesy of the state long porous border with southern Thailand. In other parts of Malaysia it would be white). Canada Goose Outlet

We agreed that if we lived in which would never, ever in a million years happen Kedai Kopi Pak www.canadagooseonline.info Din would be a canada goose factory outlet daily stop, if not for the sup ekor, the coffee, or the soft boiled eggs then simply for the pure pleasure of Pak Din hello.

Canada Goose online Kedai Kopi Din Tokyo, 3945 Jalan Tok Hakim, downtown, Kelantan; 012/959 0153. Canada Goose online

Way back in July we spent some time in, the capital city of Kelantan state, in the northeast of the peninsula, working on a feature story for Travel+Leisure Southeast Asia September 2011 Food Issue.

Khota Baru or KB, as Malaysians call it, held many surprises for us. canada goose outlet florida In what is often described as the country most conservative Islamic state, we found its friendliest people warm and incredibly welcoming to outsiders. And there, in a city that most tourists breeze through on their way to the Perhentian islands, we ate as well as maybe even better than than we do in Penang. The food in Khota Baru and around is mind bogglingly good.

canada goose deals We ate the best of many great meals in Wakaf Baru, a scruffy town about 20 minutes from downtown by car. When the last car had passed families popped out of vehicles parked on the other side of the tracks and made their way across to Restoran Kampung Kulim. The restaurant dining area, a corrugated metal shaded patch of concrete furnished with canada goose outlet montreal plastic tables and chairs, comes with a view of a Thai Buddhist wat, one of hundreds in this northeastern Malaysian state where canada goose jacket outlet uk 95% of the population is Malay and Muslim. canada goose deals

canada goose coats on sale The family that owns and runs Kampung Kulim are Hokkien Siam, a term for descendents of marriages between immigrants from China’s Fujian province and Thailand. They speak a mixture of Malay, Fujianese and Thai, a linguistic fusion that plays out on the plate in dishes incorporating coconut milk and chilies, bitter melon and taucu (fermented bean paste), curry paste and budu, a wincingly odiferous fermented fish condiment beloved in southern Thailand and along the northeastern Malaysian coast. canada goose coats on sale

We ate a few dishes that night: hong bak, an intriguingly salty sweet stew of pork belly made with star anise and copious shavings brown palm sugar, mustard leaves simply fried with sliced pork and dang hoon heh, a sparingly seasoned red coconut curry with whole prawns and vermicelli.

canada goose store But the dish that stays with me 9 months later is siew gai or grilled canada goose uk site chicken. I have eaten much grilled chicken in my time in southeast Asia. Restoran Kampung Kulim is sublime, as in beyond description sublime. I could goose outlet canada have written 1,000 words about that siew gai alone. But then half my article would have been about chicken. canada goose store

It all in the technique, as Dave and I found when we walked out back of the restaurant to its open kitchen. There we found a lean, mean septuagenarian grilling machine laboring over a coconut husk filled trench, using upturned, jerry rigged cracker tins to infuse their canada goose outlet store new york birds with extra char.

buy canada goose jacket cheap After dropping a whole chicken unmarinated, lightly salted onto an upright “spit” set in the middle of the coconut husk embers (think beer can chicken with a rod replacing the can), the master griller drops a square cracker tin on top. As the fire rages around the can, creating enough heat to brown and crisp the chicken skin, the coals beneath the chicken burn slow canada goose outlet toronto location enough to cook its flesh without drying it out. Exces oil drips out of the chicken carcass during the cooking process. After just 20 minutes the bird is finished. buy canada goose jacket cheap

Canada Goose Parka When the chickens are done the grill master passes them on to his canada goose outlet washington dc colleague, who commands the chopping block. Before they served he arranges the cleavered birds on a banana leaf round a nice touch that lends a bit of extra fragrance to the dish. (They both also farmers.) Surprised at first to find themselves being photographed, they quickly relaxed and got into it. Canada Goose Parka

buy canada goose jacket Presumably everyone who eats at Restoran Kampung Kulim knows the story behind its spectacular grilled chicken, but I not sure how many diners make their way behind the scenes to thank these men for their brilliant work. After all they just doing what they been doing, day after day, for years. buy canada goose jacket

For more from our time in, visit Dave photographic tour here.

Canada Goose Jackets As some of you may already know, in early July we spent some time working in, the capital of Malaysia Kelantan state. Situated in the northeastern Canada Goose Outlet corner of peninsular Malaysia, Kelantan abuts southern Thailand. Its fusioninst cuisine shows Thai, Chinese (Hokkien/Fujianese, especially), and Indian influences. Most travelers breeze right through canada goose outlet in new york on their way to and from the Perhentian Islands, completely unaware that they are missing out on one of the most interesting gastronomic scenes in Malaysia Canada Goose Jackets.