text:
WWT Home
Write for Wendy Wu
Eight of the Oddest Creatures you'll Encounter in Asia!
16th March 2018 5th March 2020 Nicci
Thinking about the wildlife you'd love to see on your holidays to China, Japan, Borneo, Asia in general and it's adorable animals like pandas and orangutans that spring to mind.
But, amongst the mountains, rainforests, desert and all the other various landscapes Asia boasts, live a huge number of incredible, exotic beasts, many of which you will have never even heard of!
Here is a list of our favourite of Asia's unusual creatures:
Proboscis Monkey
Named for its prominent and unusual nose, the Proboscis Monkey is one of Asia's largest monkey species with a pot belly that helps them to digest all the leaves they consume in their diet. The male monkeys are significantly bigger, with an extra-large nose to help attract the ladies, which is also happens to be useful in amplifying their warning calls through the jungle.
Where are they found? The Proboscis Monkey is endemic to jungles of Borneo.
Draco Lizard
The tiny Draco Lizard has an extraordinary skill – the ability to soar from tree to tree! Each animal has flaps of skin attached to its ribs which can be extended into wings that catch the wind as it leaps between branches – they can glide up to 30 feet at a time. Although they spend all their time up in the trees, the female Draco Lizard lays her eggs on the ground.
Where are they found? Draco Lizards can be found across South Asia in forests, plantations and jungles.
The magical Pangolin has a full armour of scales and are the only mammals known to have scales at all! When threatened they roll up into an impenetrable scaly ball. All pangolins enjoy a diet of ants and termites which they catch with their super long tongues. There four species of pangolin in Asia – Indian, Philippine, Sunda and Chinese. Unfortunately, pangolins are the most trafficked animals in the world, and both the Chinese pangolin and the Sunda pangolin are critically endangered.
Where are they found? Pangolins nocturnally roam across southern China, Vietnam, Cambodia, Borneo, India, Nepal, Myanmar and the Philippines.
Gharial
This crocodilian creature has 110 teeth in its long, thin snout which it uses to chomp through fish it catches in its river habit. The average adult gharial measures between 3.5 and 4.5 metres. They dwell in fast-flowing rivers where there are sandbanks to bask on. Gharials are also critically endangered, with an estimated 235 individuals left in the wild.
Where are they found? Gharials dwell in a couple of rivers in northern India and Nepal.
Malayan Tapir
Standing just over a metre tall, the black and white Malayan tapir has a distinctive, long, flexible nose that it uses as a snorkel when underwater or to grab leaves and fruit off of trees. Baby Malayan tapirs are born brown with stripes and spots to help camouflage them in the dappled light of their rainforest home. It is one of five species of tapir and the only one native to Asia.
Where are they found? Malayan tapirs are found in the tropical forests of Indonesia, Myanmar, Malaysia.
Also known as 'raccoon dogs', the tanuki may look like a long-limbed raccoon but they are actually more closely related to dogs and wolves. An important animal in Japanese folklore since ancient times, tanuki appear in a lot of Japanese art and have a reputation for being mischievous and jolly. The tanuki is generally nocturnal and a scavenger.
Where are they found? Tanukis can be found across Japan.
Sunda Colugo
Also known as the flying lemur, even though it's not a lemur and doesn't fly, the colugo is a creature that lives high in the trees eating young leaves, shoots, flowers and fruit. The colugo soars between the treetops, using a huge gliding membrane that stretches from its front limbs, all the way to its tail. It is mainly nocturnal and clings to the trees or branches with all four of its feet.
Where are they found? Sunda colugos inhabit the tropical forests of Southeast Asia.
The softshell turtle looks a bit like a turtle that's missing its shell. It's back is soft and leathery to the touch and it lives in tropical, freshwater habitats. Its shape makes it more hydrodynamic than other turtles and it can swim extremely fast. With its long neck and long snout with nostrils at the end, the softshell turtle can bury itself in mud and sand, or lurk in shallow water and breath through its nose like a snorkel.
Where are they found? Asiatic softshell turtles can be found across Southeast Asia, while the Chinese softshell turtle is endemic to China as a whole.
Book an escorted group tour to Asia or let us build you a tailormade itinerary, which can include any amount of searching for Asia's amazing wildlife! Tour or tailormade, we can guarantee you an unforgettable trip.
Borneo, Cambodia, India, Myanmar, Nepal, Philippines adventure, nature, top things to see, wildlife. permalink.
Cambodia with kids
10 amazing things you can see on a tour of India
At Wendy Wu Tours, we aim to provide you with an authentic experience of the destination and hopefully inspire you to your next Asia adventure. We'd love to share our expertise here with you.
For advice on where to go, what to do and how to choose your perfect tour, talk to our Travel Experts.
Honeymoon or Wedding
Victoria Mekong
Welcome to Chengdu – China's Most Charming City
Traveller Tales – Vietnam at a Glance
Japan's Top 8 Cities
Top Picks for South Korea and Taiwan on Netflix, Amazon Prime and Audible
48 Hours in Hoi An
adventure arts cambodia news China china in uk china news cruise culture fam festivals food and drink giant pandas Great Wall of China history India itinerary Japan japan news lessons luxury media nature pandas photography random shopping sightseeing silk road sports Terracotta Warriors The Forbidden City Tiananmen Square top things to see tourism tours transport travel unesco heritage site Vietnam vietnam news views wendy wu tours wildlife winter Yangtze River Cruise
Wendy Wu Tours Blog All rights reserved.	Theme by Colorlib Powered by WordPress


text:
FILIERE AREA
Filiere action
Domains card
Loire Valley Wines in the US
The Loire Valley vineyards
Wine and winery
Land and history
Lifecycle of the vine
and winery
the interactive map
Building up your cellar
Introduction to winetasting
How to serve wine
Interactive Map - Loire Valley Wines route
Welcome in the Loire Valley Vineyard
InterLoire is the official interprofessional body for Loire Valley wines, inspired and created by winegrowers and négociants in the Pays Nantais, Anjou, Saumur and Touraine to promote and develop their 31 appellations, denominations and PGIs.
InterLoire plays an integral role within the Loire Valley, France's 3rd largest producer of appellation wines; its missions include economic governance, research and development, and the safeguarding and promotion of Loire Valley wines.
DOWNLOAD THE 2017 ACTIVITY REPORT
THE INTERLOIRE AREA
InterLoire draws together 3,000 winegrowers, négociants and cooperatives, collectively producing an average of 2 million hectolitres per year - equivalent to 270 million bottles. It encompasses 31 appellations, denominations and PGIs across 43,000 hectares, covering 14 départements from the Vendée to Puy-de-Dôme, and accounting for some 80% of the Loire Basin's wine production volume.
A second interprofessional body - the Bureau Interprofessionnel des Vins du Centre - works alongside InterLoire to promote the vineyards of the Loire Valley.
OPERATIONAL STRUCTURE
InterLoire is based on elected representation. Representatives are elected in equal number from the industry's two professional strands, and decision-making is a democratic process. The operational structure consists of 3 functional bodies:
1. The General Assembly, a governing body of 60 members.
30 of these members – all members are individuals - represent the region's winegrowers and make up the Viticultural College. A further 30 members, also individuals, represent the region's négociants; these constitute the College of Négociants.
The General Assembly elects the President and Vice-Presidents, members of the Executive Council and Special Interest Committees. It formulates InterLoire policy, approves budgets and ratifies the accounts for the preceding financial year.
The President is elected for a 3 year term, and comes alternately from the viticultural and the négociants' colleges. The current president is Jean-Martin Dutour, a négociant from the Chinon appellation. He was elected on June 27th 2017 and will be in office for three years.
2. The Strategic Orientation Committee
This is made up of 18 elected members, again with parity between the two professional strands. The SOC performs, or oversees the performance of, tasks allocated to it by the General Assembly. It determines strategic and budgetary parameters for proposals delivered by the committees.
The Strategic Orientation Committee is made up as follows:
From the College of Négociants
1. BOUGRIER Noël (Bougrier) Assistant Treasurer
2. BROCHET Frédéric (Ampelidae)
3. De FOUGEROUX François-Régis (Langlois Château)
4. DUTOUR Jean-Martin (Baudry Dutour) InterLoire and SOC President
5. EMEREAU Nicolas (Alliance Loire)
6. FLEISCHER Serge (Lacheteau)
7. JACOB Bernard (Ackerman) Secretary
8. LEFORT Rodolphe (Castel Frères)
9. LEMASSON Jérôme (Loire Propriétés)
From the Viticultural College:
1. BOURDIN Denis (FRCOOP VL)
2. BRAULT Olivier (FAV 49)
3. FORGEAU Joël (FAV 44) Assistant Secretary
4. GIOVANNONI Pierre-Antoine (VIF)
5. GOSSEAUME Lionel (FAV 41) Treasurer
6. JOURDAN Francis (FAV 37)
7. LECOMTE Olivier (LOIREVINBIO)
8. MENESTREAU Laurent (FAV 49) Vice President
9. MOTHERON Catherine (SIGP VL)
3. Special Interest Committees
These carry out tasks allocated by the Strategic Orientation Committee and General Assembly, reporting back regularly to the SOC.
The Technical and Quality Committee, chaired by Olivier Brault (Viticultural College)
Objectives: To examine technical issues and the associated requirements, whether applicable across the board or specific to one appellation; to determine the technical measures to be implemented and calculate the budget required.
The Markets and Economic Affairs Committee, chaired by Laurent Ménestreau (Viticultural College)
Objective: To analyse upstream and downstream marketing, helping the industry to optimise its positioning both in the medium and long term. The Committee aims to work proactively, anticipating and preparing for events which may affect the industry.
The Communications Committee, chaired by Pierre-Jean Sauvion (Négociants' College)
Objective: To define the Loire Valley's communications strategy within the general guidelines determined by the Strategic Orientation Committee. Promotional activity includes digital marketing, wine tourism, press relations and education; export is a major area of focus.
InterLoire's head office is located at: 62 rue Blaise Pascal, 37019 Tours, France.
MonthJanFebMarAprMayJunJulAugSepOctNovDec Month
Day12345678910111213141516171819202122232425262728293031 Day
Year19201921192219231924192519261927192819291930193119321933193419351936193719381939194019411942194319441945194619471948194919501951195219531954195519561957195819591960196119621963196419651966196719681969197019711972197319741975197619771978197919801981198219831984198519861987198819891990199119921993199419951996199719981999200020012002200320042005200620072008200920102011201220132014201520162017201820192020 Year
ALCOHOL ABUSE IS DANGEROUS FOR YOUR HEALTH , DRINK RESPONSIBLY
Interactive Map of the Loire Valley Wine Route

redpajama_set_name: RedPajamaCommonCrawl


This entry was posted on 10/27/2006 at 3:47 am and is filed under 16534404, 16535332, Abakanowicz, Bill Viola, Jenny Holzer, LUMA.	You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.


text:
Love is a Many Splendored Plant
The TPN (Telepathica Pacifica Network) provides the most reliable communications network, for tikis and all plant life, on the planet. The telepathic network has always been very busy, as tiki gods and goddesses chat incessantly like teenage mall rats. There are also the days when the houseplants, who share the TPN, also get busy on the horn. Sundays are especially hectic, when offshoots call their parent plants to assure themselves that they will remain in the will.
T.K. Betelnut is a Tiki, half wood and half human, which allows him to be mobile. He is on a stake out, working for Interpol on an ocean view hillside overlooking Lanikai Beach in Hawaii. He spots something….
Waiting. Waiting.
Oh! What is this?
T.K. was scoping in on a fine little gynoecium growing on the hillside among the lowlife weeds and kudzu. She stood proudly above the shoreline.
It was a Monstera deliciosa. Not your average dime-a-dozen split-leaf philodendron. She was beautiful. T.K. was hypnotized. He'd never seen such lush foliage. Her big leaves swayed gracefully in the breeze, exposing a good portion of her divine stems. Movie star material.
T.K. soon realized: OMFTikiG, it is her! From television! I've got to alert the network! Marilyn Monstera! Someone had discarded Marilyn Monstera on the hillside! Dumped her like a slutty areca palm. And though she faced a scenic vista that any silly human would be glad to pay $500 a night plus airfare for—just the idea that she had been treated like common pond scum or athlete's foot fungus—discarded like a boring fern, was an insult to her eminence.
Some ROF (rich old fart) had simply left her there, no doubt, when they were redesigning their fancy ROF home on the gated ROF section of Lanikai's hillside.
The very patient, constipated, angry stick became angrier.
Marilyn Monstera (Lot#6532uhgy12) was the daughter of Hollywood royalty. A result of Plant Parenthood, her parents were famous as well. Marilyn's mother, ZhuZhu appeared in nearly every scene in the Thin Man movies of the 1940s. Her father, Moe, acted throughout the 1960s in the Anette and Frankie Beach Party films. Both parents still live in the executive offices of Warner Bros. and had been featured on over two hundred and fifty movie sets. They also were fixtures on Hollywood's best buffet tables where they sometimes rubbed stems with Bogart, Bacall, Cooper, et al.
Marilyn's first TV appearance was with her father, Leif, on the Surf City Sinners series (1961–1965), which is still considered a classic of the "swingin' sixties."
In the first Surf City Sinners episode, "A Ding in My Heart," Marilyn's father is observed "flipping the stamen." This gesture took Leif Monstera over four hours to complete during forty different takes bungled by two so-called teen idols, Hanky and Panky. Many of the Monstera's friends and relatives saw the episode from their Southern California living rooms and let out a laugh that was only heard by other plant life over the TPN. A "plant laugh" can register among the botanicals for over a month.
After the stake-out, maybe he'd ask 'sugar roots' to take a spin with him in his new photosynthetic Chia.
Since he first saw Marilyn on TV in1961, T.K. Betelnut, like all other healthy male saplings his age, wanted to toss her salad with a fine vinaigrette.
Beach, Goddesses, gods, Love, Mythology, Romance, Tiki, Uncategorized


Cathy, my lovely wife, has been my avid supporter for all my hiking adventures, be it small or big. She was there with my first hike to Mulanje mountain in 2011, and she got very busy last year in 2016 when I went up Kilimanjaro Mountain. She is also an active hiker in her own right, having gone to most of the mountains I have been to in Malawi. They say behind every successful man, there is a woman. To me, behind every successful hiker, there is an anchor. If it was not for Cathy, preparations for my hikes would have been painstakingly laborious.
Cathy was born a great organiser. She sucked it all up from her mother, Mai Kamwendo, who without effort is able to organise regular church events that feed hundreds of children at a time, or a league of women. Speaking of mum, which is what I call her mother, she makes a killer homemade soup that I have not found anywhere else in the world. I am yet, however, to pack the soup with me on one of my hiking expeditions. And who said you cannot have a bowl of soup from the top of a mountain? The thought of soup has made me digress. Now, last year sweet Cathy organised a surprise return party. She arranged for a reporter to interview me, whose article appeared in one of the leading newspapers in the country. And together with my beautiful Aunt Hendrina, she prepared themed T-shirts and distributed them among family and friends. The T-shirts were labelled on the front "Ndimakonda Mountain Hiking" meaning "I love mountain hiking", from which the name of this blog has been taken. The short form of my name is Konda which is love as a verb, and most family members and family friends still call me Konda to this day. By the way, my full first name, Kondaine, means "love me". So all that was broken and distilled into the catchy phrase.
That aside, Cathy is my number one cheerleader. She is always there firing up my morale, and even if the challenge is next to impossible, she will just beam her gorgeous smile and say it will be done. And it gets done! That is why I am comfortable to say there are more mountains to visit, bigger, more challenging, and much higher. I know she will be there by my side, saying, "Go on! God bless you!". Now, you know my secret. Have support from family and friends, and half of your challenges has been sorted out already. Place all this on the solid foundation of putting trust in God, and surely all your challenges are under control.
More importantly she is a lovely companion. She is easy to chat with as a friend, but does not flatter me at all. That is just what I need to think through my moves, and get to make the best decisions. God knew my weakness and got me covered with Cathy. This gorgeous girl is tough as nails inside, and makes for a perfect hiking partner. I know she constantly prays for me, and that each time she comes along for an adventure, I feel complete.
So this is to my wonderful Cathy, and all such as her around the world that hold the hands of their partners and make things work, quietly, away from the eyes of the public.


You can eat it canned, frozen, and dried, but you just can't beat the taste of farm-fresh corn. It's sweeter, more flavorful, and a great way to make the most of your summer eats. Even though corn isn't a super popular backyard veggie to grow, you'll find tons of buying options at farmers' markets, supermarkets, and roadside stands. Best of all, it's not very expensive. So pick up a dozen or two ears at a time because there are so many ways to enjoy it.
1. Mix up some corn salsa.
Some people like their corn salsa mixed in with their traditional tomato salsa. Others prefer to have a bean-based blend. Whatever your preference, mix up corn salsa like you would any other salsa. The corn is already in pieces, so you don't have to cut it up more. Then you'll want to chop up other ingredients like peppers and onions. A little lime will go a long way in either salsa. Then scoop it up with your favorite tortilla or corn chip.
2. Be creative with your corn on the cob.
You probably already eat corn on the cob, but chances are, it's pretty standard and/or ordinary. There are so many ways to enjoy corn on the cob, though. There's not really a "wrong" way to eat corn on the cob. Season it up however you like it, and don't be afraid to try some things, like these unique corn on the cob recipes. You might invent a whole new amazing way to eat it.
3. Sauté up some corn fritters.
If you've never had them, warning: You're about to be addicted. Hands down, corn fritters are one of the most savory ways to eat corn in summer. This recipe with jalapeño and cilantro salsa is an easy and good recipe to start with. One of the best parts of corn fritters is dipping them in different salsas, so give your favorite a try.
4. Turn it into a salad.
There's a fine line between corn salsa and salad. As in, once you add a few ingredients, your salsa easily becomes a salad. You can also just add corn to any salad you already like to make. Your favorite cobb salad would be great with some added corn. You can also try the Southwest tortellini pasta salad here.
5. Let corn be the star in tacos.
You'll find plenty of taco recipes that use corn salsa as an add-on, but it's not just an accessory. Instead, let your corn be the focus of your tacos. A bean and corn taco is perfect for a No Meat Monday dish. Pair it with fresh cilantro and your favorite cheese, and that's pretty much all it takes.
6. Make your own corn tortillas.
7. Use your fresh corn on pizza.
Yes, corn does belong on pizza. Sprinkle it on top of a cheese or basil pizza to bring just the right amount of sweetness and flavor. Then do some experimenting on your own. For instance, you might find you love it with sausage! Start by just adding a little bit of corn salsa on your pizza. If you like the flavor, start roasting it on top when you bake the pizza.
8. Create your own corn atole.
Corn atole is a traditional Mexican drink using corn and milk. It sounds a little odd, but don't judge it too soon. It's actually considered a dessert by many, and it's really yummy. Here's a traditional recipe to get you started. Try having it in place of an after-dinner coffee or in the morning instead of your chai.
9. Heat up a corn soup.
Corn soup is so easy to make. You just cut the kernels off your corn and then slow roast it over the stove. Add fresh herbs like this recipe calls for, and you have a great way to use all that corn you bought over the weekend. You might want to freeze some to have an easy fall or winter meal, too.
10. Turn it into a cake.
There's cornbread, which is kind of obvious, and pretty cake-like. (Try folding fresh corn into your favorite cornbread recipe.) However, you can take it another step further by making it more of a dessert cake. Taking it to this level is like finally embracing sweet potatoes as being great for sweet potato pie. Corn cake it just as delicious.
11. Throw it in a stir-fry.
Corn works well as an addition in just about any stir-fry. You can also just mix it it with fresh veggies on the grill or roasted in the oven. For instance, it's perfect with zucchini or fresh beans; a little bit of olive oil or butter is all you need.
Of course, there are my other ways to use corn. For instance creamed corn or cheesy corn are both delicious and require very few ingredients. There's also corn soufflé, corn chowder, and corn quesadillas. You really can't go wrong to try corn as an accessory in your favorite dish. Chances are, you're going to like it.


Nemalux designs and manufactures industry leading industrial and hazardous location outdoor LED lighting solutions in Calgary, Alberta. Call us at 403.242.7475 for all your LED lighting solutions.
Website Copyright © 2017 Nemalux Inc.


Oh Cecilia (Breaking My Heart) is a song by The Vamps featuring Shawn Mendes and appears as the seventh track on their debut studio album, Meet The Vamps. It will be released as the fifth single from the album.
The lyrics are substantially original, though still relying on the main chorus lyrics of the original song (Cecilia, you're breaking my heart / You're shaking my confidence daily / Oh Cecilia I'm down on my knees / I'm begging you please to come home).


text:
Stelis grossa är en biart som först beskrevs av Mitchell 1962.  Stelis grossa ingår i släktet pansarbin, och familjen buksamlarbin. Inga underarter finns listade i Catalogue of Life.

Källor

Pansarbin
grossa

redpajama_set_name: RedPajamaWikipedia


Marc Jacobs is a huge lover of the SpongeBob and he cannot just keep himself away from his favourite cartoon. He just keeps doing something to show his love for the same and the latest he has done is to get SB inked on his arm. He is seen above in a shoot with French actress Elodie Bouchez for T: Style and the picture uncannily attracts towards his cute tattoo. And then there is another one in the hiding which is susceptibly the M&M. Must say this tattoo suites his poles apart personality!
← 'MOM DAD' 3D tattoo!


It is great opportunity to be a guest on the IOC Athletes' Commission and to be able to represent the WOA. I am proud to work together with the IOC Athletes' Commission and the WOA on supporting Olympians at all stages in their careers.
My best memories in sport are from the Olympic Games, and I am lucky to have been involved in two different sports as well as a flagbearer for Bermuda on two occasions. The Games are a wonderful celebration of nationalities, sport and culture with athletes at the centre of it all.
As the WOA representative on the Commission, I am focused on working for the benefit of all Olympians at all stages of their lives – especially after competing. Olympians can make the world a better place.


text:
How Many Goals Did Lionel Messi Has In His Career
How Many Goals Did Lionel Messi Has In His Career new post
Lionel Messi - Goals, Assists & Stats | FootyStats
Lionel Messi - Wikipedia
Lionel Messi can take his career goal tally to an.
Lionel Messi - All 50 Hattricks in his Career (HD)
How many goals did Messi score in his career? - Quora
How many goals has Messi scored in his career as of 2018.
How many goals has Lionel Messi scored in World Cup.
Lionel Messi - All 600 Career Goals (2004-2018) - HD
Lionel Messi statistics history, goals, assists, game log.
How many goals has messi scored so far in his career - Answers
All Time Goals - Messi vs Ronaldo | Messi All Time Goals.
How many games has Lionel Messi played in his career - Answers
Lionel Messi | Biography & Facts | Britannica
Lionel Messi – Messi's Career Honours | Genius
Lionel Messi vs. Cristiano Ronaldo: career records, all.
How Many Goals Has Messi Scored in His Career? | Reference.com
Lionel Messi reaches 1,000 goals as a footballer
How Many Goals Has Messi Scored In His Career? It's Staggering
Record breaker Lionel Messi's career in numbers - CBBC.
List of international goals scored by Lionel Messi - Wikipedia
How Many Goals Did Lionel Messi Has In His Career more:
Lionel Andrés Messi Cuccittini's Form Analysis. Lionel Andrés Messi Cuccittini is a Forward who has appeared in 20 matches this season in La Liga, playing a total of 1710 minutes.Messi Cuccittini scores an average of 0.95 goals for every 90 minutes that he's on the pitch. This attacking performance currently places him at 2nd out of 397 players in La Liga who've played at least 3 matches. Cristiano Ronaldo picked up his 10th career red card Sunday while Lionel Messi has been sent off just once. Cristiano Ronaldo vs. Lionel Messi: Red Card, Yellow Card Comparison Skip to main content
Early life. Lionel Andrés Messi was born on 24 June 1987 in Rosario, the third of the four children of Jorge Messi, a steel factory manager, and his wife Celia Cuccittini, who worked in a magnet manufacturing workshop.On his father's side, he is of Italian and Spanish descent, the great-grandson of immigrants from the northcentral Adriatic Marche region of Italy and Catalonia, and on his. Ronaldo has also appeared in four World Cups, 2006, 2010, 2014, and 2018. His performances in international play have been criticized somewhat, but he's been unable to lead his native Portugal to the finals. Based on his age, it would seem that the 2018 World Cup will likely be his last. How many goals has Ronaldo scored?
Lionel Messi can take his career goal tally to an incredible 400 when Barcelona meet Athletic Bilbao. Lionel Messi is on the verge of scoring 400 goals for club and country This chart shows all the goals scored by Messi and Cristiano, sorted by seasons. We can see that Cristiano began his career as a professional two years before Messi, in season 2002/2003, and that his scoring records did not begin to highlight until season 2006/2007, in which he scored 23 for Manchester United and 5 for Portugal. 14 years to the day since his first goal at senior level, Lionel Messi hit strikes 599 and 600 for Barcelona, as the Catalans saw off Liverpool in the Champions League semi-final first leg.
Lionel Messi - All 50 Hattricks in his Career (HD) Thank you for watching! If you enjoyed, please Subscribe me; http://bit.ly/1dPWWkB Don't forget to turn th... Hello. According to my information Ronaldo has 29 trophies as he did not play for the Uefa super cup 2016 and was not in the squad for Community shield 2008. Also messi's Olympic gold medal does not count as a trophy because of its under 23 rule of participation. Also Ronaldo has 2 more games played. And messi 3 less assists.
As of today, March 7th, 2018 Messi has 600 Professional career goals to his name. Goals for Barcelona : 539 Goals for Argentina : 61 Someone has build this amazing chart of his goals breakdown against each club and country. How many career goals has Lionel Messi scored? For club and country combined, Messi has bagged 621 goals, but the scoring doesn't stop there. Add in eleven goals for the Barcelona B and C team. Messi has six goals in his World Cup career. He's only scored one this time around, but the Barcelona attacker had four in the 2014 World Cup. While Ronaldo leads Messi in goals, Messi has led his.
How many goals has Lionel Messi scored for Barcelona? Lionel Messi's double strike during an 8-2 win over Huesca means he has now scored a sum total of 556 goals for Barcelona. That makes him comfortably the club's all-time record scorer, with mor... This page provides an overview of all penalties a goalkeeper has faced over the course of their career and how many they were able to save. You can also view the match in which the penalty was awarded, when the referee awarded it and who took it. This looks to be the last chance for Lionel Messi to lift the World Cup. By the time Qatar rolls around in '22 the little genius will be in his mid-30s. Take a look at these numbers and you'll see.
Lionel Messi has played in 18 career World Cup matches over four different World Cups, and he has scored six goals in those contests. Messi's first World Cup goal came in the 2006 tournament in. It is one of the UEFA Champions League's best sub-plots: Lionel Messi against Cristiano Ronaldo at the top of the all-time scoring charts. The pair ended 2014/15 level on 77 goals but Ronaldo has. Lionel Messi Quiz Answers Which Club in Argentina did Messi play for in his Youth career. Messi played for the Newell's Old Boys from 1994 until 2000. How old was Messi when he made his debut for FC Barcelona? Messi was 17 years, 3 months, and 22 days old when he made his debut for FC Barcelona in 2004. Barcelona beat RCD Espanyol, 1–0.
600 Goals. 600 Goals. Let it sink in... What an achievement! Lionel Messi is his name, ladies and gentlemen. Here are all his goals. Enjoy! Thank you for watching! If you enjoyed, please Subscribe. Lionel Messi may not have scored 501 career goals after all. It was widely reported on Wednesday night that the five-time Ballon d'Or winner had passed the milestone during Barcelona's 7-0.
Lionel Messi this seasons has also noted 16 assists, played 2581 minutes, with 27 times he played game in first line. Lionel Messi shots an average of 0.71 goals per game in club competitions. Last season his average was 1.04 goals per game, he scored 52 goals in 50 club matches. Lionel Messi, latest news & rumours, player profile, detailed statistics, career details and transfer information for the FC Barcelona player, powered by Goal.com.
Only counting his senior career playing for Barcelona and Argentinian National Team, as of Nov 2, 2011 Lionel Messi has scored a total of 182 goals. The world's greatest footballers - but who's the best? The battle of Messi vs Ronaldo has the world gripped. In this website, they go head to head in terms of goals, stats, achievements, abilities and much more. It comes just a few days after Lionel Messi scored three himself at club level, as Barcelona beat Celta Vigo 4-1. Ronaldo now stands on 705 career goals, having scored 55 hat-tricks across his career.
The battle of Messi vs Ronaldo has the world gripped. In this website, they go head to head in terms of goals, stats, achievements, abilities and much more. Always up-to-date, reliable and accurate, this is your main port of call to see who's currently leading the constant back-and-forth battle for the golden boot. Leo Messi's footballing career started in 1995 at Newell's Old Boys, where he played until the year 2000. At the age of 13, Lionel Messi crossed the Atlantic to try his luck in Barcelona, and. Trump: My side in trial has 'all the material' 'I was killing myself with all the drinking and pills'
Typically it's more of an opinion, but here are some facts for both of them in La Liga. Lionel Messi: Has played 24 games, scored 28 goals, gotten 8 assists and has taken 123 shots. 3x FIFA Club World Cup winner. 2015: FC Barcelona: 2011: FC Barcelona: 2009: FC Barcelona: 1x Under-20 World Cup champion
Lionel Messi, Argentine-born football (soccer) player who is one of the greatest goal-scorers in the sport's history. He was named the FIFA world player of the year on five occasions. Domestically, he starred for Spain's FC Barcelona, leading the club to numerous championships. Lionel Messi has scored 548 career goals (in 712 games) – As well as being Barcelona's all-time record goal scorer, Messi is also Argentina's most prolific international, having netted 57 goals.
The honours and achievements won by Lionel Messi throughout his career thus far. Record-breaking – 5 World Player of the Year awards. Agony – the most coveted medal that no player wants How many goals has Messi scored in the El Clasico? How many goals has Messi scored? How many goals has Messi scored in his career so far? How many goals has Messi scored for Argentina in total? When did Messi score his first goal for Barcelona? What is it called when you score two goals in soccer? How many goals did Gretzky score in a season? After breaking his bad streak, Leo Messi has become millenary, and has already taken part in more than 1,000 goals throughout his career Tickets for FC Barcelona matches It does hardly some days, Leo Messi confessed that it was not concerned by his drought , and really, did not have reasons to be it .
Ronaldo has been sent off 11 times in his career - once for Juventus, six times for Real Madrid and four times for Manchester United. Ronaldo vs. Messi: trophies won Ronaldo club honours Lionel Messi has scored 510 career goals (in 659 games) – As well as being Barcelona's all-time record goal scorer, Messi is also closing in on Gabriel Batistuta's record of 56 goals in.
Lionel Messi brought his goal tally to 371 in all competitions when he scored a hat-trick against Osasuna on March 16, 2014. The 26-year old surpassed Paulino Alcantara's club record of 369 goals scored between 1912 and 1927. Messi is one of the most decorated footballers in history and was discovered as a talented kid by Barcelona FC. Lionel Messi continued his impressive recent record from free-kicks on Tuesday night as he slotted one home against Villarreal for Barcelona. The 31-year-old has now scored direct from a free-kick...
Lionel Messi reaches 1,000 goals as a footballer Following his hat-trick against Deportivo, the Argentinian has now scored a thousand career goals for club and country www.fcbarcelona.com Lionel Messi has played in 18 career World Cup matches over four different World Cups, and he has scored six goals in those contests. Messi's first World Cup goal came in the 2006 tournament in. Lionel Messi scored a superb first-half free-kick to register his 600th career goal 539 of those have come for Barcelona, with a further 61 coming for Argentina The Argentinian talisman has played.
Lionel Messi has scored 664 goals in his career for club and country, so yes, I'm slightly exaggerating, but only slightly. Messi made his debut for Barcelona C in the Tercera División on Nov. Lionel Messi has scored his 600th career goal, netting against Atletico Madrid in Barcelona's vital La Liga clash against Atletico Madrid.. With 26 minutes on the clock, Barca were awarded a free. Messi, who scored for the first time on 1 May 2005, has scored those 600 goals in just 683 games, netting an average of almost one (0.9) every time he steps onto the pitch.. Remarkably, in the.
Lionel Messi has helped his club and country win a fair few trophies thanks to all of the goals he's scored during his career. CR7 All Goals: How Many Goals Has Cristiano Ronaldo Scored In His Career? There is no doubt that Cristiano Ronaldo is one of the greatest football players in the world. He has record number of goals, assists, penalties, etc., which make him an almost invincible footballer. Lionel Messi has overtaken Gabriel Batistuta as Argentina's all-time top goal scorer. La Albiceleste's No. 10 drove an unstoppable free kick in from 25 yards during Argentina's Copa America.
Lionel Messi is an Argentine professional footballer who has represented the Argentina national football team as a forward since his debut in 2005. Since then, Messi has scored 70 goals in 138 international appearances, making him the country's all-time top scorer, surpassing Gabriel Batistuta's record, with a free kick against the United States in the semi-final of the Copa América. Kylian Mbappe has hit 100 career goals aged 20 – beating Messi and Ronaldo to a century. And how much quicker did he reach the century landmark than the likes of Lionel Messi and Cristiano.
As of today, March 7th, 2018 Messi has 600 Professional career goals to his name. Goals for Barcelona : 539 Goals for Argentina : 61 Someone has build this amazing chart of his goals breakdown against each club and country. Juventus Pagelle Eurosport. Lionel Messi reaches 1,000 goals as a footballer Following his hat-trick against Deportivo, the Argentinian has now scored a thousand career goals for club and country www.fcbarcelona.com Watch Barcelona Real Madrid Live Stream. How many goals has Lionel Messi scored for Barcelona? Lionel Messi's double strike during an 8-2 win over Huesca means he has now scored a sum total of 556 goals for Barcelona. That makes him comfortably the club's all-time record scorer, with mor... Lionel Messi has helped his club and country win a fair few trophies thanks to all of the goals he's scored during his career. Lionel Messi has scored 664 goals in his career for club and country, so yes, I'm slightly exaggerating, but only slightly. Messi made his debut for Barcelona C in the Tercera División on Nov. Lionel Messi can take his career goal tally to an incredible 400 when Barcelona meet Athletic Bilbao. Lionel Messi is on the verge of scoring 400 goals for club and country The battle of Messi vs Ronaldo has the world gripped. In this website, they go head to head in terms of goals, stats, achievements, abilities and much more. Always up-to-date, reliable and accurate, this is your main port of call to see who's currently leading the constant back-and-forth battle for the golden boot. Only counting his senior career playing for Barcelona and Argentinian National Team, as of Nov 2, 2011 Lionel Messi has scored a total of 182 goals. The honours and achievements won by Lionel Messi throughout his career thus far. Record-breaking – 5 World Player of the Year awards. Agony – the most coveted medal that no player wants Lionel Messi has played in 18 career World Cup matches over four different World Cups, and he has scored six goals in those contests. Messi's first World Cup goal came in the 2006 tournament in. Early life. Lionel Andrés Messi was born on 24 June 1987 in Rosario, the third of the four children of Jorge Messi, a steel factory manager, and his wife Celia Cuccittini, who worked in a magnet manufacturing workshop.On his father's side, he is of Italian and Spanish descent, the great-grandson of immigrants from the northcentral Adriatic Marche region of Italy and Catalonia, and on his.
Atletico Madrid Vs Real Madrid Twitter
Is There A Bundesliga Game Today
Lionel Messi And Cristiano Ronaldo Compare
Napoli Vs Fc Barcelona Tickets
Colorado Soccer Champions League
Messi Neymar Ronaldo Salah
Chelsea Fc Official Page
Barclays Premier League Schedule
Manchester United Arrive Training
Juventus Today Channel
Man City U18 Squad
Stream Real Madrid Game Today
Goles Carrera Messi Vs Ronaldo
Espn What Channel Are Premier League Games On
Ronaldo 7 Barcelona Vs Sevilla
Bayern Munich Reto Beanie
Chelsea W Vs Liverpool W
Lionel Messi Best Skills Video
Tv Ratings For Barcelona Vs Real Madrid
Fc Barcelona Vs Getafe Live Stream Free
Uefa Champions League Final Date
Rio Ferdinand Cristiano Ronaldo Twitter
Manchester United Away Shirt 18 Long Sleeve
Birthday Cake Liverpool Fc
La Galaxy Vs Manchester United Location
Watch Barcelona Real Madrid Live Stream
Ver Psg Vs Liverpool
Real Madrid Vs Barcelonal Live
Liverpool Fc Ticket Broker
Uefa Champions League Berlin Winner
Psl Premier League Table Results And Fixtures
Longest Time Without Scoring A Goal Bundesliga
Abner Real Madrid Fifa 16
Bayern Munich Vs Vfl Wolfsburg Highlights
Fc Barcelona Squad Vs Valencia
Real Madrid Vs Atletico Ronaldo7
Bundesliga Ergebnisse Bitte
Copyright © How Many Goals Did Lionel Messi Has In His Career 2020 Adminserver Ru


Ice Cream and prizes, what more could you ask for?
We've teamed up with New Forest Ice Cream to celebrate our 40th birthday and have 40 great prizes up for grabs throughout the year.
All winners will receive a 500ml tub of New Forest Ice Cream delivered direct to their door*. Selected winners may also receive a special one-off prize from Lighthouse. So don't have a meltdown – buy an ice cream today for your chance to win!
*Terms and Conditions apply. You will need to provide contact details to a member of Lighthouse staff so that we are able to organise delivery of your ice cream. New Forest Ice Cream will be unable to deliver the ice cream to your home if you live outside a radius of 45 minute drive time of Lighthouse, in this case we will make other arrangements.


text:
package saml

import (
	"encoding/xml"
	"time"

	"github.com/bmanth60/go-saml/packager"
	"github.com/bmanth60/go-saml/util"
)

//NewLogoutResponse create new logout response entity
func NewLogoutResponse() *Response {
	r := NewAuthnResponse()
	r.XMLName.Local = "samlp:LogoutResponse"
	return r
}

//NewLogoutRequest create new logout request entity
func NewLogoutRequest() *LogoutRequest {
	id := util.ID()
	return &LogoutRequest{
		XMLName: xml.Name{
			Local: "samlp:LogoutRequest",
		},
		RootXML: &RootXML{
			SAMLP:   "urn:oasis:names:tc:SAML:2.0:protocol",
			SAML:    "urn:oasis:names:tc:SAML:2.0:assertion",
			SAMLSIG: "http://www.w3.org/2000/09/xmldsig#",
			ID:      id,
			Version: "2.0",
			Issuer: Issuer{
				XMLName: xml.Name{
					Local: "saml:Issuer",
				},
				URL:  "", // caller must populate ar.AppSettings.Issuer
				SAML: "urn:oasis:names:tc:SAML:2.0:assertion",
			},
			IssueInstant: time.Now().UTC().Format(time.RFC3339Nano),
			Signature:    packager.GetSignatureEntity(id),
		},
		NameID: NameID{
			XMLName: xml.Name{
				Local: "saml:NameID",
			},
			Format: "urn:oasis:names:tc:SAML:1.1:nameid-format:unspecified",
			Value:  "",
		},
		SessionIndex: SessionIndex{
			XMLName: xml.Name{
				Local: "samlp:SessionIndex",
			},
			Value: "",
		},
	}
}

//ApplyLogoutRequest entity as specified by provided parameters
func ApplyLogoutRequest(settings *Settings, r *LogoutRequest, nameID string, sessionIndex string) *LogoutRequest {
	r.Destination = settings.IDP.SingleLogoutURL
	r.Issuer.URL = settings.SP.EntityID
	r.Signature.KeyInfo.X509Data.X509Certificate.Cert = settings.SPPublicCert()
	r.NameID.Format = settings.IDP.NameIDFormat
	r.NameID.Value = nameID
	r.SessionIndex.Value = sessionIndex

	if !settings.SP.SignRequest {
		r.SAMLSIG = ""
		r.Signature = nil
	}

	return r
}

//String get string representation of logout request
func (r *LogoutRequest) String() (string, error) {
	return packager.String(r)
}

//SignedString get xml signed string representation of logout request
func (r *LogoutRequest) SignedString(s *Settings) (string, error) {
	if !s.SP.SignRequest {
		return "", ErrInvalidSettings
	}

	xmldoc, err := r.String()
	if err != nil {
		return "", err
	}

	return packager.SignWithKey(xmldoc, s.SPPrivateKey())
}

redpajama_set_name: RedPajamaGithub


* Pre-processing queries to handle common and uncommon inputs.
* Making the most with very few search terms.
* Methods for ranking millions of results.
the kind of queries they see, and their success in producing useful results.
is plenty of time on the Internet scale.


You asked for it.. here it is!
In this video, Melissa describes how to take a dab with a standard nail and torch, along with instructions (and a lovely example) on how to use the Yocantech Torch e-nail.
I hope you enjoy the video and please reach out to us if there are any questions.
Have some dab knowledge to drop yourself? Share with us! We would love to give you credit.


Parents in St. John's Junior School on Michael Street have spent the last eight weeks working with Word Aid Kilkenny and the Home School Community Liaison Coordinator to create two story sacks.
A story sack is a bag or box containing a favourite children's book with supporting materials to stimulate language activities and help to make reading a memorable and enjoyable experience.
The parents met in the school every Friday morning and busied themselves painting, sewing, crocheting, knitting and crafting characters, backdrops and furniture for the stories 'We are Kind and Helpful' by Donna Luck and 'Can't You Sleep Little Bear?' by Martin Waddell.
Many cups of tea and chats were had along the way too and the end results were superb. Both story sacks had been on display in the windows of Khan's Bookshop and Stonehouse Books for the past week.
The course culminated in a 'Storytime' session with Ms. Doyle's senior infant class which the children thoroughly enjoyed. All the parents were presented with certificates on completion of this course.
Organiser Muireann Rochford thanked all the parents involved for committing their time and energy to this project and added that the children in the school will get many years of pleasure from these wonderful resources.


text:
Calithera Biosciences Inc (CALA.OQ)
CALA.OQ on NASDAQ Stock Exchange Global Select Market
Susan Molineaux
65 2010 President, Chief Executive Officer, Co-Founder, Director
Sumita Ray
44 Senior Vice President, Chief Compliance Officer, General Counsel
Curtis Hecht
47 2016 Senior Vice President - Business and Corporate Development
Christopher Molineaux
64 2013 Senior Vice President - Development
Keith Orford
46 2015 Senior Vice President, Clinical Development
Eric Sjogren
61 2010 Senior Vice President - Drug Discovery
44 2017 Vice President - Finance, Secretary
Francesco Parlati
49 2012 Vice President - Research
Deepika Pakianathan
54 2017 Lead Independent Director
Sunil Agarwal
48 2015 Independent Director
Jonathan Drachman
Jean George
Suzy Jones
Blake Wise
H. Ward Wolff
44 2017 Vice President, Investor Relations and Strategy
» Insider Trading
Dr. Susan M. Molineaux, Ph.D. serves as President, Chief Executive Officer, Co-Founder and Director of Calithera Biosciences Inc. Dr. Molineaux co-founded Proteolix, Inc., a biopharmaceutical company, where she served as Chief Scientific Officer from 2003 to 2005, Chief Executive Officer from January 2006 to January 2009 and again as Chief Scientific Officer from February 2009 until Proteolix's acquisition by Onyx Pharmaceuticals, Inc. in November 2009. From 2000 to 2003, Dr. Molineaux served as Vice President of Biology at Rigel Pharmaceuticals, Inc., a drug development company. From 1999 to 2000, she served as Vice President of Biology at Praelux, Inc., a biopharmaceutical company, and from 1994 through 1999, she served as Vice President of Drug Development at Praecis Pharmaceuticals, Inc., a biopharmaceutical company. From 1989 until 1994, she was a scientist in the Immunology group at Merck & Co. Dr. Molineaux currently serves as a member of the board of directors of Geron Corporation and Theravance Biopharma, Inc. and is a Scientific Advisor for Lightstone Ventures. Dr. Molineaux holds a B.S. in Biology from Smith College and a Ph.D. in Molecular Biology from Johns Hopkins University, and completed a postdoctoral fellowship at Columbia University.
Ms. Sumita Ray is the Senior Vice President, General Counsel and Chief Compliance Officer of Calithera Biosciences Inc. With more than 17 years of pharmaceutical and biotechnology experience, Ms. Ray brings a breadth of knowledge in FDA regulatory law, global health care law and compliance, brand support, product launches, collaborations and alliances. Prior to joining Calithera, she served as Chief Compliance Officer and Associate General Counsel, Head of Healthcare and Regulatory Law at Pharmacyclics, where she supported the company through the global approval and launch of Imbruvica in multiple indications. Previously, Ms. Ray served as Head of BioNeurology & Regulatory Law Group at Elan Pharmaceuticals, Corporate Counsel, Commercial Law Group at Genentech and Corporate Counsel at AstraZeneca. Ms. Ray started her career as a pharmaceutical and products liability litigator at Montgomery, McCracken, Walker and Rhoads LLP. Ms. Ray holds a J.D. from Temple University School of Law and a B.S. in Microbiology from the University of Arizona.
Mr. Curtis C. Hecht is the Senior Vice President - Business and Corporate Development of Calithera Biosciences Inc. Mr. Hecht has served as our Senior Vice President of Business and Corporate Development since April 2014. From September 2013 to April 2014, Mr. Hecht served as Vice President of Business Development at inVentiv Health, a global healthcare commercialization and consulting services company. Since March 2011, he has also served as a Partner at DNA Ink, a life sciences business development and licensing firm. From June 2002 to February 2011, Mr. Hecht served in a number of roles at Hoffman La-Roche Inc., including as Global Alliance Director from 2008 to 2011 and Director of Global Business Development from 2006 to 2008. Mr. Hecht received a B.S. in Chemistry from California State University, Sacramento and an M.B.A. from Carnegie Mellon University.
Dr. Christopher J. Molineaux, Ph.D. is the Senior Vice President - Development of Calithera Biosciences Inc. Dr. Molineaux has served as our Senior Vice President of Development since April 2013. From March 2010 to March 2013, Dr. Molineaux served as the President of INDStrat LLC, a consulting firm. From July 2004 to November 2009, Dr. Molineaux served as Vice President of Development at Proteolix, Inc. From 2000 to 2004, Dr. Molineaux served as Senior Director of Drug Development at FibroGen, Inc., a biotechnology company. From 1999 to 2000, he served as Research Manager of Toxicology at Johnson & Johnson Pharmaceutical Research and Development. From 1994 to 1999, Dr. Molineaux served as Senior Director of Pharmacology at Praecis. From 1991 to 1994, he served in staff scientist positions at Enzon Pharmaceuticals, Inc. and Merck & Co. From 1985 to 1991, Dr. Molineaux served as an Assistant Professor of Pharmacology of Mount Sinai School of Medicine in New York City. He received a B.S. in Zoology from University of Maryland, College Park, a Ph.D. in Immunology and Infectious Diseases from Johns Hopkins University and completed his postdoctoral fellowship at the Uniformed Services University of the Health Sciences.
Mr. Keith Orford, M.D. Ph.D. is the Senior Vice President - Clinical Development of Calithera Biosciences Inc. Dr. Orford joined Calithera in January 2015 and currently serves as Senior Vice President of Clinical Development. Prior to joining Calithera in January 2015, Dr. Orford was the Clinical Development Lead in the Immuno-Oncology and Combinations Development Performance Unit at GlaxoSmithKline (GSK) where he oversaw the clinical activities on multiple early stage clinical trials with targeted agents and novel immune-based therapies. Prior to GSK, Dr. Orford was at Merck, where he worked on early clinical development programs across oncology and other therapeutic areas. Previously, Dr. Orford was a Research Fellow and Instructor at Massachusetts General Hospital (MGH) and Harvard Medical School (HMS) where he completed clinical training in Internal Medicine as well as postdoctoral work studying the epigenetic regulation of hematopoietic and embryonic stem cell differentiation. Dr. Orford received his undergraduate, M.D. and Ph.D. degrees from Georgetown University.
Dr. Eric B. Sjogren, Ph.D. is the Senior Vice President - Drug Discovery of Calithera Biosciences Inc. Dr. Sjogren has served as our Senior Vice President of Drug Discovery since June 2010. From 2003 to 2009, Dr. Sjogren was Vice President and Head of Medicinal Chemistry at Roche Palo Alto, LLC, where he directed a small molecule drug discovery team in the areas of inflammation, virology and central nervous system disorders. Dr. Sjogren received a B.A. in Chemistry from the University of California, San Diego and a Ph.D. in Chemistry from Harvard University.
Ms. Stephanie Wong is the Vice President - Finance and Secretary of Calithera Biosciences Inc. Ms. Wong is the Vice President of Finance since April 2014 and was appointed as our Secretary effective January 2017. From 2009 to 2013, Ms. Wong was at SciClone Pharmaceuticals, Inc., a publicly traded, commercial-stage pharmaceutical company, most recently as Vice President, Finance and Controller. From 2008 to 2009, Ms. Wong was Senior Director, Finance at AcelRx Pharmaceuticals Inc. From 2001 to 2008, Ms. Wong held various positions at Kosan Biosciences Inc., a publicly traded biotechnology company until its acquisition by Bristol-Myers Squibb Co., most recently as Senior Director and Controller. Prior to that Ms. Wong worked as an audit manager at PricewaterhouseCoopers LLP. Ms. Wong received a B.S. in Business Administration from the University of California, Berkeley and is a Certified Public Accountant in the State of California.
Dr. Francesco Parlati, Ph.D. is the Vice President - Research of Calithera Biosciences Inc, since October 2012. From 2011 to 2012, Mr. Parlati was Founder and Senior Director of Biology at Cleave Biosciences, a biotechnology company. Prior to that, Mr. Parlati held various scientific roles at California Institute of Technology, Proteolix Inc. and Rigel Pharmaceuticals, Inc. Mr. Parlati received a B.S. in Chemistry from Concordia University, a Ph.D. in Biology from McGill University and completed his postdoctoral fellowship at Memorial Sloan-Kettering.
Dr. Deepika R. Pakianathan, Ph.D. is the Lead Independent Director of Calithera Biosciences Inc. Dr. Pakianathan served as a member of our board of directors since September 2012. Since 2001, Dr. Pakianathan has served as a Managing Member at Delphi Ventures, a venture capital firm. From 1998 to 2001, Dr. Pakianathan served as a Vice President in the healthcare group at JP Morgan Chase & Company. From 1993 to 1997, Dr. Pakianathan served as a postdoctoral scientist in the Immunology Department at Genentech Corporation. Dr. Pakianathan currently serves on the board of directors of Alder Biopharmaceuticals, Inc., Oncomed Pharmaceuticals, Inc. and Karyopharm Therapeutics, Inc. Dr. Pakianathan holds an M.S. and a Ph.D. from Wake Forest University, a B.Sc. from the University of Bombay, India and an M.Sc. from The Cancer Research Institute at the University of Bombay, India.
Mr. Sunil Agarwal, M.D. is an Independent Director of Calithera Biosciences Inc. Mr. Agarwal has served as a member of our board of directors since September 2015. Since April 2017, Dr. Agarwal has served as President of Research and Development at Juno Therapeutics, Inc., a biophamacetuical company. From September 2016 through March 2017, Dr. Agarwal served as a Partner at Soffinova Ventures. From August 2014 through August 2016, Dr. Agarwal served as Executive Vice President and Chief Medical Officer at Ultragenyx Pharmaceuticals, Inc., where he was responsible for leading the company's clinical development. Prior to Ultragenyx, Dr. Agarwal served in various leadership capacities at Genentech for 11 years. From January 2013 to June 2014 he held the position of Senior Vice President and Global Head of Clinical Development for OMNI (Ophthalmology, Metabolism, Neurosciences, Immunology and Infectious Diseases). From July 2009 to December 2012 Dr. Agarwal held the positions of Senior Vice President for Immunology and Infectious Diseases, and Vice President for Rheumatology from July 2009 to December 2012. He also held the position of Vice President of Genentech Drug Safety from January 2009 to July 2009. From September 2003 to January 2009, Dr. Agarwal held positions of increasing responsibility in Genentech's Immunology clinical organization, and was involved in the development oversight of multiple molecules including Raptiva, Rituxan, and ocrelizumab. Dr. Agarwal currently serves as a member of the board of directors of MyoKardia, Inc. Dr. Agarwal obtained a B.S. in Neuro-Biology from Cornell University and an M.D. from Tufts University School of Medicine.
Dr. Jonathan G. Drachman, M.D. is an Independent Director of Calithera Biosciences Inc. Dr Drachman served as a member of our board of directors since December 2013. Since November 2004, Dr. Drachman has served in a number of roles at Seattle Genetics, Inc., most recently Chief Medical Officer and Executive Vice President of Research and Development since October 2013 and its Senior Vice President, Research and Translational Medicine from May 2010 to October 2013. From 1998 to 2004, Dr. Drachman served as a faculty member in the Hematology Division, Department of Medicine at the University of Washington in Seattle, and as a Senior Investigator in the Division of Research and Education at the Puget Sound Blood Center. Dr. Drachman received a B.A. in Biochemistry from Harvard University and an M.D. from Harvard Medical School. He completed his residency in Internal Medicine and fellowship in Medical Oncology at the University of Washington.
Ms. Jean M. George is an Independent Director of Calithera Biosciences Inc. Ms. George served as a member of our board of directors since September 2012. Since February 2002, she has been a General Partner at Advanced Technology Ventures, a venture capital fund. From September 1998 to January 2002, Ms. George served as director of BancBoston Ventures, a venture capital fund. Ms. George currently serves as a member of the board of directors of Acceleron Pharma, Inc. and Catabasis Pharmaceuticals, Inc. Ms. George holds a B.S. in Biology from the University of Maine and an M.B.A. from Simmons College Graduate School of Management.
Ms. Suzy Jones is an Independent Director of Calithera Biosciences Inc. Ms. Jones served as a member of our board of directors since August 2016. Since September 2010, she has been the Founder and Managing Partner of DNA Ink, a boutique life sciences advisory firm. Prior to founding DNA Ink, Ms. Jones spent 20 years at Genentech in various roles in immunology research, product development managing cross functional teams for Rituxan and Avastin, and business development where she was Head of Non-Oncology Licensing and later Interim Head of Partnering and Head of Business Development. In 2010, she was named by Black Health Magazine as one of the, "Top 25 Most Influential African Americans in Healthcare, Medicine, Pharmaceutical and Food Industries." Ms. Jones serves as a member of the board of directors of Patrys Limited, an ASX listed Australian biotechnology company. She received a B.S. degree in Biology from University of California, Santa Cruz.
Mr. Blake Wise is the Independent Director of Calithera Biosciences Inc, since September 2017. Since January 2018, Mr. Wise has served as Chief Executive Officer and a member of the board of directors of Achaogen, a biopharmaceutical company developing innovative antibacterials. Mr. Wise joined Achaogen as Chief Operating Officer in 2015 and, in February 2017, he was also appointed President. Prior to joining Achaogen, Mr. Wise served as Vice President, Cross BioOncology at Genentech, where he led cross-portfolio oncology initiatives, including key account management, marketing, managed markets, companion diagnostics, pipeline commercialization, and long-term oncology strategy. Mr. Wise also held several other leadership positions at Genentech including Senior Director, Franchise Head and Life Cycle Leader of the Lytics franchise and as a Sales Director in BioOncology, Marketing Director in Cystic Fibrosis and Immunology, and Interactive Marketing Director. Prior to joining Genentech, Mr. Wise worked in consumer marketing, e-commerce and online marketing in leadership positions at Gap, Inc. and Webvan. Mr. Wise received a Bachelor of Arts degree in Business Economics from University of California, Santa Barbara, and a Masters of Business Administration degree from University of California, Berkeley, Haas School of Business.
Mr. H. Ward Wolff is an Independent Director of Calithera Biosciences Inc. Mr. Wolff served as a member of our board of directors since December 2014. Mr. Wolff served as Executive Vice President and Chief Financial Officer of Sangamo Therapeutics, Inc. from 2007 until his retirement in March 2017. Prior to Sangamo, Mr. Wolff was with Nuvelo, Inc., where he served as Senior Vice President, Finance and Chief Financial Officer until its restructuring in August 2007. Prior to that, he was Chief Financial Officer and Senior Vice President, Finance, of Abgenix, Inc. until April 2006 when Abgenix merged with Amgen Inc. Prior to joining Abgenix, Mr. Wolff held financial management positions in both public and private emerging growth companies, including serving as Senior Vice President and CFO of DoubleTwist, Inc., a life sciences company integrating genomic information and bioinformatics analysis tools. He began his career with Price Waterhouse, where he held a number of positions as a certified public accountant, including Senior Audit Manager. Mr. Wolff is a member of the board of directors of Portola Pharmaceuticals, Inc. From June 2006 until his appointment to Sangamo's management team he was a member of Sangamo's Board of Directors, serving as Chairman of the Audit Committee. Mr. Wolff received a B.A. degree in Economics from the University of California at Berkeley and an M.B.A. degree from Harvard Business School.
As Of 31 Dec 2017
Molineaux (Christopher)
51,600 $3.88
Molineaux Susan M
Sjogren (Eric B)
Wong (Stephanie)
Parlati (Francesco Ph.D.)
6,246 $2.64
Orford (Keith)
» Full list on Insider Trading


McMahon Food Corporation is a local, women & family-owned business that has been serving the Chicagoland Area for generations.
We are a leader in the local Dairy Distribution Industry and consistently provide high quality dairy products. We are extremely reliable and maintain outstanding customer service. We are certified with Cook County as a Women-Owned Business Enterprise (WBE), by the City of Chicago as an Airport Concessions Disadvantaged Business Enterprise (ACDBE) and by CMS as a Female Business Enterprise (FBE). As such, we can assist in a wide range of supplier diversity goal fulfillment and initiatives.
We provide the freshest and highest quality hormone-free milk and dairy products from both Prairie Farms and Deans.


Click NEXT to go to the next section or click on the tab you wish to fill first. We recommend you follow the order from left to right.
If you wish to leave at some point and come back later to fill-in the rest of the application, please SAVE your information and click LOGOUT.
Three recommendations are required for an applicant.
Enter First Name, Last Name and Email of Referee and click Send Invitation.
This will generate an automated email to your referee on your behalf.
The referee should fill-in an online form before the deadline of 6 Feb 2019.
If you already have a copy of the recommendation from a referee, enter his/her details and upload that as a PDF file by choosing "Upload my Recommendation".
If you wish to change a recommender, choose the "Clear" which will appear after sending a recommendation invitation to a particular referee.
Even though we have provided an option to upload a hardcopy of the recommendation, we strongly recommend that you opt for an online recommendation since it is coming directly from the referee and confidential.
An applicant can update mandatory documents and upload other documents until the deadline of 30 Jan 2019.
The recommenders should fill-in the online recommendations before the deadline of 6 Feb 2019.


The second city center of Milan is Gae Aulenti square, around which in the last ten years have been build the new contemporary district of Porta Nuova, which has changed the town's appearance. Here internationally well-known architects rose a sort of revolutionary architecture, which always takes into account environmental sustainability. Here is located the famous "Vertical Wood" by architect Boeri, winner of prestigious awards, which qualifies the "Vertical Wood" as the most beautiful skyscraper in the world of 2014. New skyscrapers are added to the Palazzo Lombardia and Unicredit Tower architect Cesar Pelli. This area is still evolving, and new buildings are going to be raised and opened shortly.


text:
Home All News Sports 105-Year-old Cyclist Sets New World Record for Speed
105-Year-old Cyclist Sets New World Record for Speed
105-year-old Robert Marchand has set a new world record for fastest cycling time at a track near Paris.
On Wednesday, the French cyclist pedaled 14.08 miles in one hour – which is apparently 2.65 miles shorter than the previous record he set in 2014 for participants over the age 100. The impressive feat is now the most distance pedaled in an hour by someone 105-years-old or older.
Robert claims that he could have gotten a better time if he hadn't missed the 10-minute warning sign near the end of his attempt.
RELATED: 17-Year-Old Blind Pole Vaulter Wins Bronze at State Championship
The event at the Saint-Quentin-en-Yvelines velodrome was reportedly filled with cheering fans of the centenarian's inspiring career.
"I'm not in such good shape as I was a couple of years back," he told AFP news agency. "I am not here to be champion. I am here to prove that at 105 years old you can still ride a bike."
(WATCH the video below)
Pedal This Story Over To Your Friends: Click To Share


text:
package net.sf.uadetector.json.internal.data.deserializer;

import java.util.ArrayList;
import java.util.Collections;
import java.util.EnumSet;
import java.util.List;
import java.util.regex.Pattern;

import javax.annotation.concurrent.Immutable;

import net.sf.qualitycheck.Check;
import net.sf.uadetector.internal.data.Data;
import net.sf.uadetector.internal.data.domain.Browser;
import net.sf.uadetector.internal.data.domain.BrowserPattern;
import net.sf.uadetector.internal.data.domain.BrowserType;
import net.sf.uadetector.internal.data.domain.Device;
import net.sf.uadetector.internal.data.domain.DevicePattern;
import net.sf.uadetector.internal.data.domain.OperatingSystem;
import net.sf.uadetector.internal.data.domain.OperatingSystemPattern;
import net.sf.uadetector.internal.data.domain.Robot;
import net.sf.uadetector.json.SerDeOption;

import com.google.gson.Gson;
import com.google.gson.GsonBuilder;

@Immutable
public final class Deserializers extends AbstractDeserializer<Data> {

	public static Deserialization<Data> deserialize(final String json, final EnumSet<SerDeOption> options) {
		return deserialize(json, options, Data.class);
	}

	public static <T> Deserialization<T> deserialize(final String json, final EnumSet<SerDeOption> options, final Class<T> classOfT) {
		final Deserializers deserializers = new Deserializers(options);
		return new Deserialization<T>(deserializers.deserialize(json, classOfT), deserializers.getWarnings());
	}

	private final BrowserDeserializer browserDeserializer;

	private final BrowserPatternDeserializer browserPatternDeserializer;

	private final BrowserTypeDeserializer browserTypeDeserializer;

	private final DataDeserializer dataDeserializer;

	private final DeviceDeserializer deviceDeserializer;

	private final DevicePatternDeserializer devicePatternDeserializer;

	private final Gson gson;

	private final OperatingSystemDeserializer operatingSystemDeserializer;

	private final OperatingSystemPatternDeserializer operatingSystemPatternDeserializer;

	private final PatternDeserializer patternDeserializer;

	private final RobotDeserializer robotDeserializer;

	private Deserializers(final EnumSet<SerDeOption> options) {
		super(Check.notNull(options));
		dataDeserializer = new DataDeserializer(options);
		browserPatternDeserializer = new BrowserPatternDeserializer(options);
		browserTypeDeserializer = new BrowserTypeDeserializer(options);
		operatingSystemPatternDeserializer = new OperatingSystemPatternDeserializer(options);
		operatingSystemDeserializer = new OperatingSystemDeserializer(options, operatingSystemPatternDeserializer);
		browserDeserializer = new BrowserDeserializer(options, browserPatternDeserializer, browserTypeDeserializer,
				operatingSystemDeserializer);
		robotDeserializer = new RobotDeserializer(options);
		patternDeserializer = new PatternDeserializer(options);
		devicePatternDeserializer = new DevicePatternDeserializer(options);
		deviceDeserializer = new DeviceDeserializer(options, devicePatternDeserializer);

		// setup deserializers
		final GsonBuilder gsonBuilder = new GsonBuilder();
		gsonBuilder.registerTypeAdapter(Data.class, dataDeserializer);
		gsonBuilder.registerTypeAdapter(Device.class, deviceDeserializer);
		gsonBuilder.registerTypeAdapter(DevicePattern.class, devicePatternDeserializer);
		gsonBuilder.registerTypeAdapter(Browser.class, browserDeserializer);
		gsonBuilder.registerTypeAdapter(BrowserPattern.class, browserPatternDeserializer);
		gsonBuilder.registerTypeAdapter(BrowserType.class, browserTypeDeserializer);
		gsonBuilder.registerTypeAdapter(OperatingSystem.class, operatingSystemDeserializer);
		gsonBuilder.registerTypeAdapter(OperatingSystemPattern.class, operatingSystemPatternDeserializer);
		gsonBuilder.registerTypeAdapter(Robot.class, robotDeserializer);
		gsonBuilder.registerTypeAdapter(Pattern.class, patternDeserializer);
		gson = gsonBuilder.create();
	}

	/**
	 * Deserializes a JSON representation into a new instance of {@link Data}.
	 *
	 * @param json
	 *            JSON representation of {@code Data}
	 * @return instance of {@code Data}
	 */
	public <T> T deserialize(final String json, final Class<T> classOfT) {
		Check.notNull(json);
		Check.notNull(classOfT);
		return gson.fromJson(json, classOfT);
	}

	public BrowserDeserializer getBrowserDeserializer() {
		return browserDeserializer;
	}

	public BrowserPatternDeserializer getBrowserPatternDeserializer() {
		return browserPatternDeserializer;
	}

	public BrowserTypeDeserializer getBrowserTypeDeserializer() {
		return browserTypeDeserializer;
	}

	public DataDeserializer getDataDeserializer() {
		return dataDeserializer;
	}

	public OperatingSystemDeserializer getOperatingSystemDeserializer() {
		return operatingSystemDeserializer;
	}

	public OperatingSystemPatternDeserializer getOperatingSystemPatternDeserializer() {
		return operatingSystemPatternDeserializer;
	}

	public PatternDeserializer getPatternDeserializer() {
		return patternDeserializer;
	}

	public RobotDeserializer getRobotDeserializer() {
		return robotDeserializer;
	}

	@Override
	public List<String> getWarnings() {
		final List<String> warnings = new ArrayList<String>();
		// over all warnings
		warnings.addAll(patternDeserializer.getWarnings());

		// order should depend on deserialization steps (ordering of level one fields in JSON)
		warnings.addAll(operatingSystemPatternDeserializer.getWarnings());
		warnings.addAll(operatingSystemDeserializer.getWarnings());
		warnings.addAll(browserTypeDeserializer.getWarnings());
		warnings.addAll(browserPatternDeserializer.getWarnings());
		warnings.addAll(browserDeserializer.getWarnings());
		warnings.addAll(robotDeserializer.getWarnings());

		// warnings during data building
		warnings.addAll(dataDeserializer.getWarnings());

		return Collections.unmodifiableList(warnings);
	}

}


It's our listener feedback show for season 8 epside 3, "Monsters". How did Negan know to move the guns to Gavin's outpost? Was bringing Morales back for 5 minutes worth it? Is it time for a new show-runner and writing staff on The Walking Dead? Tune in for the answers to these questions, and a lot more.
Thank you for re-designing the website; it looks good and is much easier to navigate.
Strike 1: The intro of several unbelievable characters: the tiger, Ezekiel, Scavengers, etc. These elements may play well in comic-book form, but in a TV show with such great acting and SFX, the material needs to be elevated. They are all just… too… silly.
Strike 2: The circularly repetitive plot.
Strike 3: Main characters that should have died long ago.
Strike 4: Too many characters.
Now, this is a real shame because I love the acting and story, I'm interested in Carl's continual progress towards manhood, and Eugene is wonderfully and awkwardly unexpected.
Maybe I will binge watch the season over holiday break, but for now, I have zero interest.
By the way, you gentlemen are always great – best podcast on the "internets". Oh, and your podcast was mentioned on FilmSack's episode #357.


text:
Conventions not spared sting of slump
Fewer attending some big shows of '09, and exhibits using less space
Steve Marcus
Jim Graham, left, of Swing America, and Rod Brekhus of Pumpmaster attend the World of Concrete convention, which filled less space this year than in previous years.
By Richard N. Velotta
Sunday, Feb. 8, 2009 | 2 a.m.
Showgoers take a powder (2-6-2009)
Preview speakers offer some optimism amid economic gloom (2-6-2009)
Estimates of lower attendance at four big conventions are clouding optimism that the big shows would give the Las Vegas economy a much-needed boost.
Attendance at the World of Concrete, MAGIC Marketplace and the World Shoe Association in February and the National Association of Broadcasters in April is expected to be down 64,000 from last year's events.
That's the equivalent to Las Vegas losing one major show.
In addition, fewer tourists are expected for NASCAR race weekend at the Las Vegas Motor Speedway, one of the biggest nonconvention draws each year. The Nevada Taxicab Authority, which regulates Clark County's taxi industry, was told last week that an estimated 100,000 fewer people would be in town for events Feb. 27 through March 1.
Last week's World of Concrete, an annual international convention of the masonry and concrete construction industries, drew an estimated 70,000 people — about 15,000 fewer than in 2008.
The slump in the construction industry certainly affected World of Concrete, which used 825,000 square feet of exhibition space at the Las Vegas Convention Center. Its largest show was in 2007, when more than 900,000 square feet of exhibits were displayed and 91,628 people attended.
Trade shows generally measure their success by exhibit square footage, but the more crucial number for the Las Vegas economy is attendance.
Meanwhile, the World Shoe Association is downsizing by cutting back from four days to three. The footwear fashion event, conducted twice a year in Las Vegas, is set for Thursday through Saturday at Mandalay Bay Convention Center and Sands Expo & Convention Center.
Organizers of the show, which incorporates a premium footwear, handbag and jewelry component known as "The WSA Collection," also are planning smaller exhibition booths on main aisles of the trade show floor to increase attendance.
Despite the enhancements, organizers are expecting attendance to dip from 30,000 last year to 28,000 this year.
The most dramatic attendance decline is expected to occur at MAGIC Marketplace, the fashion exhibition scheduled for Feb. 17-19 at the Las Vegas Convention Center.
MAGIC, an acronym for the former name of the event, the Men's Apparel Guild in California, downsized the length of the twice-a-year convention when it lopped off a day at last year's summer show.
This year, attendance is expected to fall 30 percent to 70,000.
This year's MAGIC show will occupy less space. It hasn't announced how much square footage the show floor will have, but the Las Vegas Convention and Visitors Authority confirmed that the event would be in the Convention Center's North and Central halls but not the two-story South Hall as in years past.
Another show looking at considerable downsizing will be the National Association of Broadcasters show in April.
Although the event will include a new 48-hour film festival, the induction of legendary Los Angeles Dodgers broadcaster Vin Scully into the Radio Broadcasting Hall of Fame and, possibly, additional discussions about the delayed transition to digital television, attendance is expected to dip to 98,000 from the original forecasts of 115,000. Registration could pick up after last month's announcement that room rates would be discounted 20 percent to 40 percent.
Among those four conventions, attendance is expected to be off an estimated 64,000. In 2008 the city's meetings and trade shows attracted an average of 525,000 conventiongoers each month.
The 2009 NASCAR Race Weekend at the Las Vegas Motor Speedway will draw significantly fewer fans to town this year. The Taxicab Authority was told that the number of people expected for the Sam's Town 300 race Feb. 28 would drop from 150,000 to 50,000 and the number for the Shelby 427 NASCAR Sprint Cup race March 1 would draw 150,000, down from 250,000 a year ago.
The Taxicab Authority was told those figures don't reflect attendance at the races, but the number of people expected to be in town who are in some way associated with the races.
A version of this story appeared in this week's In Business Las Vegas, a sister publication of the Sun.


Stephanie Kelton started us off with Instead of Worrying About Budgets and Deficits, Ask About Impact. Below, we link to research, proposals and responses re a Green New Deal which is all about nice things we — the people — and the planet can have.
H.RES.109 Recognizing the duty of the Federal Government to create a Green New Deal.
Over at Down With Tyranny, Howie Klein is following the politics of climate change.
— Robert Hockett (@RCH371) talks with hosts Scott Ferguson (@Videotroph), William Saas (@billysaas) and Max Seijo (@MaxSeijo) Modern Money Network Humanities Division April 17, 2019 Note: see all Money on the Left podcast titles here.
The Green New Deal, explained • An insurgent movement is pushing Democrats to back an ambitious climate change solution.
Ocasio-Cortez: If US Has $5.7 Billion for Trump's Border Wall, What About Education, Healthcare, and Green Jobs?


text:
Acta Crystallographica Section F
Acta Crystallographica
Section F STRUCTURAL BIOLOGY COMMUNICATIONS
Download PDF of article
3. Results and discussion
Dictionary of Crystallography
IUPAC Gold Book
SGML
Article statistics
crystallization communications\(\def\hfill{\hskip 5em}\def\hfil{\hskip 3em}\def\eqno#1{\hfil {#1}}\)
Volume 70| Part 1| January 2014| Pages 49-52
doi:10.1107/S2053230X13031361
Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-D-galactarate (KDG) dehydratase from Agrobacterium tumefaciens
Helena Taberman,a Martina Andberg,b Tarja Parkkinen,a Peter Richard,b Nina Hakulinen,a Anu Koivulab and Juha Rouvinena*
aDepartment of Chemistry, University of Eastern Finland, PO Box 111, 80101 Joensuu, Finland, and bVTT Technical Research Centre of Finland, PO Box 1000, 02044 VTT, Finland
*Correspondence e-mail: juha.rouvinen@uef.fi
(Received 3 October 2013; accepted 15 November 2013; online 24 December 2013)
D-Galacturonic acid is the main component of pectin. It could be used to produce affordable renewable fuels, chemicals and materials through biotechnical conversion. Keto-deoxy-D-galactarate (KDG) dehydratase is an enzyme in the oxidative pathway of D-galacturonic acid in Agrobacterium tumefaciens (At). It converts 3-deoxy-2-keto-L-threo-hexarate to α-ketoglutaric semialdehyde. At KDG dehydratase was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 169.1, b = 117.8, c = 74.3 Å, β = 112.4° and an asymmetric unit of four monomers. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The three-dimensional structure of At KDG dehydratase will provide valuable information on the function of the enzyme and will allow it to be engineered for biorefinery-based applications.
Keywords: keto-deoxy-D-galactarate dehydratase; D-galacturonic acid; Agrobacterium tumefaciens; oxidative pathway.
One of the main components of plant cell walls is pectin, a heteropolysaccharide. It can be found in, for example, citrus peel and sugar beet pulp, which are usually considered as waste but could be a valuable source of raw materials for the biotechnical production of fuels and chemicals. Pectin is rich in D-galacturonic acid, whose catabolism in microbes has been widely studied (Ashwell et al., 1958 ; Richard & Hilditch, 2009 ; Chang & Feingold, 1970 ). There are two routes for D-galacturonic acid degradation in bacteria: the isomerase and the oxidative pathways. The latter was shown to be active in Agrobacterium tumefaciens (Zajic, 1959 ) and Pseudomonas syringae (Kilgore & Starr, 1959 ). In this pathway, uronate dehydrogenase (EC 1.1.1.203) first oxidizes D-galacturonic acid to galactaro-1,4-lactone, with galactaro-1,5-lactone as an enzyme-bound intermediate (Boer et al., 2010 ; Parkkinen et al., 2011 ). Galactarolactone cycloisomerase (EC 5.5.1.–) then catalyses ring opening and the formation of 3-deoxy-2-keto-L-threo-hexarate (Andberg et al., 2012 ). The 3-deoxy-2-keto-L-threo-hexarate is converted by a decarboxylating hydrolyase (EC 4.2.1.41) into α-ketoglutaric semialdehyde (Jeffcoat et al., 1969 ; Aghaie et al., 2008 ). In the final step α-ketoglutaric semialdehyde is oxidized by a dehydrogenase to α-ketoglutarate, which is a metabolite of the TCA cycle (Watanabe et al., 2007 ).
In A. tumefaciens (At), the conversion of 3-deoxy-2-keto-L-threo-hexarate is catalysed by keto-deoxy-D-galactarate (KDG) dehydratase, an enzyme belonging to the dihydrodipicolinate synthase (DHDPS) family. The crystal structures of several DHDPS family members have been solved. However, At KDG dehydratase shares little sequence identity with these enzymes. According to the PDB, its sequence identity to, for example, DHDPS from Bacillus clausii (PDB entry 3e96 ; New York SGX Research Center for Structural Genomics, unpublished work), DHDPS from B. anthracis (PDB entry 3hij ; Voss et al., 2010 ) and DHDPS from Mycobacterium tuberculosis (PDB entry 1xxx ; Kefala et al., 2008 ) is 29, 27 and 29%, respectively. Intriguingly, the members of this enzyme family, which share a similar three-dimensional TIM-barrel structure (Theodossis et al., 2004 ), catalyse different types of reactions, for example dehydration, condensation (Dobson et al., 2004 ) and retro-aldol condensation (Eaton, 1994 ).
Structure–function studies on At KDG dehydratase are crucial for a comprehensive understanding of the metabolic pathways of D-galacturonic acid, which can then be manipulated to develop microbial strains for the production of platform chemicals from plant biomass. In this study, we present the crystallization and preliminary X-ray data analysis of At KDG dehydratase.
2.1. Expression and purification of At KDG dehydratase
The open reading frame of the KDG dehydratase gene (At kdg) was amplified by PCR using primers containing the NcoI and HindIII restriction sites and genomic DNA isolated from A. tumefaciens as the template. To create the NcoI site, the Asn residue following the N-terminal methionine was changed to an Asp. The genes were ligated into the pBAT4 vector (Peränen et al., 1996 ) using the NcoI and HindIII restriction sites. Escherichia coli strain BL21(DE3) was transformed with the generated plasmid, pBAT4-At kdg. A Strep-tag II (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) was added to the C-terminus of At kdg by PCR amplification using the following primers: Fwd_AtKDG, 5′-GATATATCCATGGACCCTGAACAAATCAAG-3′ and Rev_AtKDG, 5′-GACGAGAAGCTTCATTTTTCAAACTGCGGATGAGACCAGGCTTTGCGCTTGTGTG-3′. The tagged gene was ligated into the pBAT4 vector as described above and sequenced.
For purification of the At KDG dehydratase, 3 l E. coli BL21(DE3) cells containing the plasmid described above were grown at 310 K and 250 rev min−1 in LB medium supplemented with 100 µg ml−1 ampicillin to an OD600 of 0.6–0.8. After the addition of 1 mM of isopropyl β-D-1-thiogalactopyranoside to induce the expression of the At KDG dehydratase, the culture was further grown at 303 K and 250 rev min−1 overnight. The cells were harvested by centrifugation for 15 min at 4000g at 277 K, resuspended in 50 mM sodium phosphate buffer pH 8 containing 300 mM NaCl, 1 mM DTT, protease inhibitors (Complete EDTA-free, Roche) and lysozyme (Sigma–Aldrich), and lysed by sonication. After centrifugation at 38 000g for 30 min at 277 K, the cell-free extract was loaded onto a Strep-Tactin Superflow column (Qiagen) equilibrated with 50 mM sodium phosphate pH 8, 300 mM NaCl buffer. After washing with equilibration buffer, the target protein was eluted with 2.5 mM desthiobiotin in 50 mM sodium phosphate pH 8, 300 mM NaCl buffer.
The purity of the protein was analysed by SDS–PAGE (Fig. 1 ) using the 10% Criterion Stain-Free gel-imaging system (Bio-Rad), and the fractions containing At KDG dehydratase were pooled and concentrated using a Vivaspin 20 centrifugal concentrator (molecular-weight cutoff 10 kDa; Sartorius AG, Göttingen, Germany). The buffer was replaced with 50 mM Tris–HCl pH 7.5 by gel filtration on a PD-10 column (GE Healthcare Life Science, Uppsala, Sweden). The purified enzyme was stored at 193 K. The activity of the purified At KDG dehydratase enzyme was assayed at pH 7.5 in a coupled assay using Acinetobacter baylyi ADP1 α-ketoglutarate semialdehyde dehydrogenase (Aghaie et al., 2008 ).
SDS–PAGE analysis of purified At KDG dehydratase. Lane M contains molecular-weight markers (labelled in kDa), lane 1 contains purified At KDG dehydratase and lane 2 contains a crude E. coli cell extract of the At KDG dehydratase-producing strain. The proteins were visualized using the 10% Criterion Stain-Free gel-imaging system (Bio-Rad).
2.2. Crystallization
Initial crystallization conditions were found by screening with the commercial PEGRx 2 screening kit (Hampton Research). Crystallization experiments were performed manually using the hanging-drop vapour-diffusion method at 294 K. In the crystallization droplets, 1 µl protein solution (4.6 mg ml−1) was mixed with an equal volume of reservoir solution on siliconized cover slides (Hampton Research). The cover slides were inverted over the wells of a 24-well plate (Greiner Bio-One) containing 500 µl reservoir solution. The wells were sealed vapour-tight with high-vacuum grease (Dow Corning). The droplets were then equilibrated against the reservoir solution, and plate-like crystals (Fig. 2 a) formed in a few days. The original conditions were composed of 0.1 M Bicine pH 8.5, 0.2 M sodium formate, 20%(w/v) polyethylene glycol monomethyl ether (PEG MME) 5000.
At KDG dehydratase crystals (a) from the initial screening and (b) after optimizing the crystallization conditions. The optimized crystallization conditions consisted of 0.1 M Bicine pH 8.5, 0.2 M sodium formate, 15%(w/v) PEG MME 5000 and the crystals grew in 3–4 d.
Crystals suitable for X-ray analysis were obtained by optimizing the crystallization conditions by varying the pH (7.6–9.0) and the concentration of the buffer (0.05–0.25 M) and the concentrations of precipitant (5–30%) and salt (0.05–0.30 M). Therefore, the optimized crystallization conditions were 0.1 M Bicine pH 8.5, 0.2 M sodium formate, 15%(w/v) PEG MME 5000. Three-dimensional crystals of several different shapes (Fig. 2 b) grew to dimensions of approximately 0.4 × 0.2 × 0.2 mm in 3–4 d.
2.3. Data collection and processing
Before data collection, the crystals were quickly soaked in a cryoprotectant consisting of the crystallization solution with an additional 15%(v/v) glycerol and were then mounted on nylon loops. The crystals were flash-cooled by plunging them quickly into liquid nitrogen. Data were collected on beamline ID29 (de Sanctis et al., 2012 ) at the ESRF (Grenoble, France) at a wavelength of 0.97625 Å. The diffraction images were collected with a 0.1° oscillation range using a Pilatus 6M pixel detector (Dectris). The distance between the crystal and the detector was 370.10 mm. The crystals diffracted to 1.9 Å resolution. The diffraction data set was processed using XDS and scaled with XSCALE (Kabsch, 1993 ).
At KDG dehydratase was expressed in E. coli with a C-terminal Strep-tag to facilitate affinity purification of the protein. From 1 l of cell culture, approximately 3 mg of At KDG dehydratase could be purified to a purity of >95%, as judged by SDS–PAGE analysis (Fig. 1 ). The Strep-tagged At KDG dehydratase migrated as a 33 kDa protein on the SDS–PAGE gel, which is in good agreement with the calculated mass of the protein (33 526.4 Da). The purified At KDG dehydratase was found to be active towards 3-deoxy-2-keto-L-threo-hexarate, with a specific activity of 15.1 µmol min−1 mg−1.
The initial screenings produced crystals under many conditions, but after optimization the best quality crystals were obtained using the condition 0.1 M Bicine pH 8.5, 0.2 M sodium formate, 15%(w/v) PEG MME 5000 found with the PEGRx 2 screening kit (Hampton Research). Three-dimensional crystals (Fig. 2 b) grew in 3–4 d to approximate dimensions of 0.4 × 0.2 × 0.2 mm. The crystal diffracted to 1.9 Å resolution at the ESRF (Table 1 ) and belonged to space group C2, with unit-cell parameters a = 169.1, b = 117.8, c = 74.3 Å, β = 112.4°.
Data-collection statistics for At KDG dehydratase
Values in parentheses are for the outermost resolution shell.
Beamline ID29, ESRF
Resolution (Å) 50–1.9 (2.00–1.90)
Wavelength (Å) 0.97625
Temperature (K) 100
Space group C2
Unit-cell parameters (Å, °) a = 169.1, b = 117.8, c = 74.3, α = γ = 90.0, β = 112.4
Total No. of reflections 245256 (34896)
No. of unique reflections 95879 (13473)
Multiplicity 2.6 (2.6)
Rmerge† (%) 4.7 (33.7)
Average I/σ(I) 12.75 (2.88)
Completeness (%) 94.9 (95.3)
Mosaicity (°) 0.15
†Rmerge = , where Ii(hkl) and 〈I(hkl)〉 represent the diffraction-intensity values of the individual measurements and the corresponding mean value, respectively.
The calculated Matthews coefficient (Matthews, 1968 ) was 2.62 Å3 Da−1, with a solvent content of 53%, which corresponds to the presence of four monomers in the asymmetric unit. The phase problem was solved with molecular replacement using MOLREP (Vagin & Teplyakov, 2010 ) from the CCP4 suite (Winn et al., 2011 ). Based on the sequence identities of available structures for the DHDPS enzyme family, the structure of dihydropicolinate synthase from B. clausii (PDB entry 3e96 ) was used as a search model. The correlation coefficient for the molecular replacement was 0.178. The structure is currently under refinement using PHENIX (Adams et al., 2010 ). The structure of this novel KDG dehydratase and structures of its complexes will provide significant information on the reaction mechanisms of the oxidative pathway of D-galacturonic acid and may be used to improve the properties of the enzyme for the manufacture of renewable products.
We thank the ESRF for providing the synchrotron-radiation facilities and the staff of beamline ID29 for their assistance in the data collection. Outi Liehunen is thanked for assistance in protein purification. Ritva Romppanen and Merja Niemi are also thanked for their technical assistance. The work was supported by the National Doctoral Programme in Informational and Structural Biology (HT) and the Finnish Centre of Excellence in White Biotechnology-Green Chemistry programme, Academy of Finland decision No. 118573 (MA, PR and AK).
Adams, P. D. et al. (2010). Acta Cryst. D66, 213–221. Web of Science CrossRef CAS IUCr Journals Google Scholar
Aghaie, A., Lechaplais, C., Sirven, P., Tricot, S., Besnard-Gonnet, M., Muselet, D., de Berardinis, V., Kreimeyer, A., Gyapay, G., Salanoubat, M. & Perret, A. (2008). J. Biol. Chem. 283, 15638–15646. Web of Science CrossRef PubMed CAS Google Scholar
Andberg, M., Maaheimo, H., Boer, H., Penttilä, M., Koivula, A. & Richard, P. (2012). J. Biol. Chem. 287, 17662–17671. Web of Science CrossRef CAS PubMed Google Scholar
Ashwell, A., Wahba, A. J. & Hickman, J. (1958). Biochim. Biophys. Acta, 30, 186–187. CrossRef PubMed CAS Web of Science Google Scholar
Boer, H., Maaheimo, H., Koivula, A., Penttilä, M. & Richard, P. (2010). Appl. Microbiol. Biotechnol. 86, 901–909. Web of Science CrossRef PubMed CAS Google Scholar
Chang, Y. F. & Feingold, D. S. (1970). J. Bacteriol. 102, 85–96. CAS PubMed Web of Science Google Scholar
Dobson, R. C. J., Valegård, K. & Gerrard, J. A. (2004). J. Mol. Biol. 338, 329–339. Web of Science CrossRef PubMed CAS Google Scholar
Eaton, R. W. (1994). J. Bacteriol. 176, 7757–7762. CAS PubMed Web of Science Google Scholar
Jeffcoat, R., Hassall, H. & Dagley, S. (1969). Biochem. J. 115, 977–983. CAS PubMed Web of Science Google Scholar
Kabsch, W. (1993). J. Appl. Cryst. 26, 795–800. CrossRef CAS Web of Science IUCr Journals Google Scholar
Kefala, G., Evans, G. L., Griffin, M. D. W., Devenish, S. R. A., Pearce, F. G., Perugini, M. A., Gerrard, J. A., Weiss, M. S. & Dobson, R. C. J. (2008). Biochem. J. 411, 351–360. Web of Science CrossRef PubMed CAS Google Scholar
Kilgore, W. W. & Starr, M. P. (1959). Nature (London), 183, 1412–1413. CrossRef PubMed CAS Web of Science Google Scholar
Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497. CrossRef CAS PubMed Web of Science Google Scholar
Parkkinen, T., Boer, H., Jänis, J., Andberg, M., Penttilä, M., Koivula, A. & Rouvinen, J. (2011). J. Biol. Chem. 286, 27294–27300. Web of Science CrossRef CAS PubMed Google Scholar
Peränen, J., Rikkonen, M., Hyvönen, M. & Kääriäinen, L. (1996). Anal. Biochem. 236, 371–373. CrossRef CAS PubMed Web of Science Google Scholar
Richard, P. & Hilditch, S. (2009). Appl. Microbiol. Biotechnol. 82, 597–604. Web of Science CrossRef PubMed CAS Google Scholar
Sanctis, D. de et al. (2012). J. Synchrotron Rad. 19, 455–461. Web of Science CrossRef IUCr Journals Google Scholar
Theodossis, A., Walden, H., Westwick, E. J., Connaris, H., Lamble, H. J., Hough, D. W., Danson, M. J. & Taylor, G. L. (2004). J. Biol. Chem. 279, 43886–43892. Web of Science CrossRef PubMed CAS Google Scholar
Vagin, A. & Teplyakov, A. (2010). Acta Cryst. D66, 22–25. Web of Science CrossRef CAS IUCr Journals Google Scholar
Voss, J. E., Scally, S. W., Taylor, N. L., Atkinson, S. C., Griffin, M. D. W., Hutton, C. A., Parker, M. W., Alderton, M. R., Gerrard, J. A., Dobson, R. C. J., Dogovski, C. & Perugini, M. A. (2010). J. Biol. Chem. 285, 5188–5195. Web of Science CrossRef PubMed CAS Google Scholar
Watanabe, S., Yamada, M., Ohtsu, I. & Makino, K. (2007). J. Biol. Chem. 282, 6685–6695. Web of Science CrossRef PubMed CAS Google Scholar
Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242. Web of Science CrossRef CAS IUCr Journals Google Scholar
Zajic, J. E. (1959). J. Bacteriol. 78, 734–735. PubMed CAS Web of Science Google Scholar
© International Union of Crystallography. Prior permission is not required to reproduce short quotations, tables and figures from this article, provided the original authors and source are cited. For more information, click here.
Follow Acta Cryst. F
Search term doi