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Early data showed Monday that Pfizer's vaccine candidate COVID-19 could be 90% effective, and the company has submitted its soon-to-be-completed data to the Food and Drug Administration for final approval. US authorities say they are ready to begin distributing the vaccine as soon as it is given the green light. Operation Warp Speed ensures that we have all the supplies we need to support the administration and distribution of this vaccine, "said Dr. David L. Schulman, the pharmaceutical company's chief executive officer. These include the PPE required for administration, dry ice needed during distribution, and dry water needed for distribution and supply of supplies and equipment needed by Pfizers.
But Pfizer's early data has put the future of COVID-19 vaccine sales in the spotlight, as the company's vaccine is a potential hurdle and must be stored at -70 degrees Celsius. Nobody has experience of vaccines at this temperature, "Saha added.
So there will be countries that experience unique circumstances related to temperature requirements. If distributed in rural communities, it would likely be even more complicated to keep the vaccine stable at that temperature, said Dr. Michael Behlim, a professor of medicine at the University of California, San Diego. It would be much more difficult if we had to find a way to keep this temperature low without having to travel 20 to 50 kilometers for vaccination, "Behlim said.
In addition to improvised packaging, Pfizer plans to track the temperature of the vaccine along the way. We have a range of packaging and we have locations, but it has to have the same shape as the full vaccine and it's packed in a very insulated container with dry ice, "he said. We will also track the temperature so that when we receive the boxes we can check that they are holding the temperatures they should be, Dormitzer said. Pfizer's scientists do not believe that keeping the vaccine stable at this temperature will be an insurmountable challenge, given the company's nuanced delivery system.
Big pharmacies like Walgreens and CVS say their facilities are fully equipped to store Pfizer's vaccine. A spokesman for the US Centers for Disease Control and Prevention (CDC) told ABC News that the vaccine is equipped with fridges and freezers, which means the vast majority of the vaccine can be stored there. This is to ensure that the vaccines that are ultimately actually given to the vaccinated person continue to be good.
However, independent, local and medium-sized pharmacies may be forced to choose between investing in a freezer that could reach the temperatures needed to store the Pfizer vaccine or using dry ice.
Initially, the challenge was to buy a freezer, but now that they know that dry ice can be used as a substitute, most institutions seem inclined to use it and refill it within a 10-day timeframe. Saha said the reluctance to buy the freezer is partly because it is hard to find and partly because you theoretically buy something you want to use.
Dormitzer told ABC News that 1.3 billion vaccines by 2021 is the goal they're really targeting. We believe we can produce a huge number of these doses, but we have to bring them the vaccine first.
Nursing homes and health care providers will be the first to offer the vaccine, according to U.S. officials. Saha said the CDC has a committee called the Advisory Committee on Immunization Practices that will approve a prioritization path that depends on what the FDA says about the vaccines. The FDA could release vaccine candidates for emergency use by the end of the year, he said.
The vaccine dose is free, and pharmacists and technicians from CVS and Walgreens will come to facilities selected for the program free of charge, according to a news release from the Centers for Disease Control and Prevention. The first doses of the COVID-19 vaccine in the US are being given to nursing home residents at a cost of $5,000 to $7,500 per patient per dose. Ultimately, "CVS Health will try to get every nursing home to sign up as a partner within three weeks" he told ABC News.
If an institution decides not to participate, it must find a way to administer the vaccine through a local pharmacy approved for vaccination by a state or local jurisdiction to help administer it. This is a huge undertaking, but it also resembles what CVS is already doing with its co-op program with Walgreens, according to the press release.
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Description: This is a map showing the distribution of the Sambucus canadensis or American elder in Florida. "N.S. (Cape Breton Is.), P.E. Is., N.B., s. Quebec, Maine, w. to s. Ontario, n. Michigan, n. Minnesota, and se. Man., s. to e. N.D., w. Kansas, and nw., c., and s. Texas, and e. to s. Florida. Also introduced in West Indies, including Puerto Rico and Virgin Islands and n C. America." —Elbert L. Little, Jr. For more information, read the introduction to the Atlas of United States Trees Vol. 5 Florida.


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Wax Lead
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Rich Mattson and the NorthStars
These Northern Minnesota rockers are releasing their fourth album, Totem. Mattson dropped by our studios for a solo session on February 15 and shar...
We're always happy to welcome this Twin Cities-based singer songwriter back to Duluth, where she spent some of her formative musical years. HALEY h...
Jacob Mahon & The Salty Dogs
Although he's only been in Duluth for a few years, this young singer/songwriter with a distinctive style is firmly established in the local scene. ...
Gordon Thorne
This Northern Minnesota country blues guitarist is known for his collaboration with Bob Bingham in Bingham and Thorne, but regularly performs solo...
Cyr
Listen to our session with this local duo, which features siblings Eric and Rachel Cyr. They've been playing a blend of Americana, folk, rock, and ...
The Langertsons
This young group is made up of the children of Mahtowa band the Holy Hootenanners. They dropped by a year ago to share tunes from their first album...
Emily Haavik
This Duluth native, now based in Minneapolis, made her first full-length album, Ease Back with her band in a cabin north of Duluth on the banks of ...
Mike Munson
This Winona, Minnesota-based singer/songwriter is known for his slide guitar blues, influenced by rural Mississippi players like Fred McDowell. Mun...
Timbre Ghost
Duluth native Dustin Tessier spent time in his hometown music scene before moving to the Twin Cities in 2002. After playing in a band called Lazer ...
The Cuckoo Bees
Self-described "percussion nerds" Cory Coffman and Mikayla Haynes met in the percussion section at University of Wisconsin, Superior and formed The...
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Gaelynn Lea
The past two years have been a whirlwind for this Duluth native. After winning the NPR Music Tiny Desk Concert Contest in 2016, she's toured nation...
Breanne Marie and the Front Porch Sinners
In May this Duluth country band went to Nashville to record 5 songs for their new EP, Salt in the Snow. They released the EP on November 21st at Be...
Zenith City Groove Collective
Todd Garland, Jake Jonker, Keith Bower, and Nick Glass have been laying down jazz grooves in the Twin Ports for the past year. Garland found his wa...
Brothers Burn Mountain
Brothers Ryan and Jesse Dermody make up this duo, who've been playing music together for nearly two decades. Their expansive, blues-and-folk-influe...
Static Panic
This Minneapolis trio released its debut EP, Chrome, this year. Their soulful synth-pop grooves recently landed them on the City Pages annual "Pick...
Aby Wolf
This Twin Cities performer and composer joined us in the studio on October 22 on the first-day of her week-long residency in Duluth that culminate...
This St. Paul-based indie folk singer/songwriter is working on a new project set for release in 2019. He joined us in the studio on October 5 for a...

redpajama_set_name: RedPajamaCommonCrawl


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Sant Pere de Ribes ist eine katalanische Stadt in der Provinz Barcelona im Nordosten Spaniens. Sie liegt im Zentrum der Comarca Garraf. Im östlichen und nördlichen Teil des Ortes befinden sich die Ausläufer der Berge des Garraf (Naturpark). Die höchste Erhebung ist hier der Montgròs mit 359 m. Der Süden und Westen bildet eine landwirtschaftlich genutzte Ebene mit sanften Hügel. im Südwesten, zwischen den Städten Sitges und Vilanova i la Geltrú befindet sich auch ein 658 m langer Zugang zum Mittelmeer.

Städtepartnerschaften
 Puerto Cabezas

Persönlichkeiten
 Aitana Bonmatí (* 1998), Fußballspielerin

Weblinks

 Information der Generalitat de Catalunya zu Sant Pere de Ribes
 Sehenswürdigkeiten

Einzelnachweise

Ort in Katalonien

redpajama_set_name: RedPajamaWikipedia


Painting with a Twist .com is the online store front to your favorite Painting with a Twist store. To locate your store select from the list below.
Painting with a Twist is the latest craze in the arts and entertainment world. It is a place where everyone goes to discover their inner artist. Book a class today and they will provide you with everything to create your masterpiece. They will even have a trained artist who will walk you through the entire process. You will not only go home with great art for a great price but you will experience a feeling of accomplishment. Take your fresh painted canvas home to show off to your friends and family and maybe even to decorate your home with. To learn more select a store that is in your area.
Painting with a Twist has now come to Irving Texas. Now you can experience the latest craze in the arts and entertainment world. It is a place where you can paint a masterpiece and socialize with friends. It makes the perfect, date night, girls night out, office party, birthday party, or just a laid back Saturday night. You will show up with a bottle of wine and go home with great art for a great price!
To get started all you need to do is search the calendar for a painting that you like on a day that works best for you. Once you find what you are looking for then you can call the store and book your event. You will need a credit card to secure your spot. Once you are booked and ready to go you will need to arrive to the class early so you can get your materials and settle in before the instructor begins the class. Don't worry about a thing they will provide you with everything to finish your masterpiece. Everything from the paint to the apron to keep you clean. They will even clean up after you are done. All this for less than an entree at a nice restaurant.
It starts with a paint brush and a bottle of your favorite vino. There is a trained instructor walking you through the entire process of painting your masterpiece. You have everything you need at your finger tips. You follow along with ease while you dab and stroke paint across the canvas. You can't believe your eyes. It is like you have been doing this all of your life. You break to pour you some more drink while you mingle with old and new friends. In the background you can hear your favorite music. You want to get up and dance because you are having so much fun. You decide to do it and some join you dancing, drinking and painting. The instructor ends the lesson with a few fun words and everyone looks around to see all of the wonderful pieces of art. You are so proud of your canvas and you begin to vision where you will hang it at home. You are shock that you created it and you can't wait to do it again. You ask yourself, "Is this a dream?" No, it is a night at Painting with a Twist. Let's do it again!
If this sounds like an amazing experience that you would love to have. If you would love to go home with great art for a great price, then you need to educate yourself about Painting with a Twist. It is the latest craze in the arts and entertainment world and it is in your neighborhood. Just visit their Painting with a Twist locations and book a class today. You will wish you started this years ago.
They have stores currently in Louisiana where it started, Florida and Texas. Here is a little of what people are saying about the Painting with a Twist experience.
St. Petersburg Florida is full of some wonderful Art Attractions. From classic Fine Art Museums to a personal experience of painting your own masterpiece. So if you are visiting the area or you are from the area and you want to take a break from the beach then here is some highly recommend art attractions that I suggest you visit. My favorite is the first listing her called Painting with a Twist.
Come paint a masterpiece, anyone can do it! Painting with a Twist in St. Petersburg Florida is the latest craze in the arts and entertainment world. There are over 300 paintings to chose from. Once you select a painting on a day that works best for you Painting with a Twist will have a trained instructor walk you and the class through the entire process of completing your masterpiece. They also welcome you to bring in your own alcohol. This way you can relax and enjoy the exhilarating experience. Visit their website today to Book your class!
View Calendar for dates and times.
If you can answer "Yes" to any of these questions then please read on to the bottom.
Are you looking for the perfect place to throw or have a birthday party?
Are you looking for something new and exciting for your birthday?
Something that will have everyone talking about the party for months to come?
Some place where everyone can participate and not just stand around and watch?
Some place where everyone gets their hands dirty and goes home with a gift that captured the moment?
If you are looking for something new and exciting to do near the Stonebriar Centre Mall in Frisco then look no further. There is something that will change your life only 15 minutes away. Head south on Preston Rd/TX-289 S toward Mall D Rd and turn left toward TX-121 Frontage Rd N. Then turn left at TX-121 Frontage Rd N and take the ramp on the left onto Sam Rayburn Tollway/TX-121 N. Finally turn left at Hardin Blvd and the store will be on the right.


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A Praia da Nazaré é uma praia do Arquipélago de São Tomé e Príncipe, localiza-se a Norte da ilha de São Tomé entre a Praia do Lagarto e a Praia Gamboa, frente à localidade de Santo Amaro.

Ver também
 Lista de Praias de São Tomé e Príncipe
 Geografia de São Tomé e Príncipe

Referências
 Grande Atlas Mundial, Selecções do Reader´s Digest, 1ª Edc. setembro, 2006, Pág. 223. ISBN 972-609-471-2
 Grande Dicionário Enciclopédico Ediclube Vol. XVI, Pág. 5574 e 5575. 1996. ISBN 972-719-056-1

Ligações externas


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Red Rackham's Treasure () is the twelfth volume of The Adventures of Tintin, the comics series by Belgian cartoonist Hergé. The story was serialised daily in , Belgium's leading francophone newspaper, from February to September 1943 amidst the German occupation of Belgium during World War II. Completing an arc begun in The Secret of the Unicorn, the story tells of young reporter Tintin and his friend Captain Haddock as they launch an expedition to the Caribbean to locate the treasure of the pirate Red Rackham.

Red Rackham's Treasure was a commercial success and was published in book form by Casterman the year following its conclusion. Hergé continued The Adventures of Tintin with The Seven Crystal Balls, while the series itself became a defining part of the Franco-Belgian comics tradition. Red Rackham's Treasure has been cited as one of the most important installments in the series for marking the first appearance of eccentric scientist Cuthbert Calculus, who subsequently became a core character. The story was adapted for the 1957 Belvision animated series Hergé's Adventures of Tintin, the 1991 Ellipse/Nelvana animated series The Adventures of Tintin, the 1992-3 BBC Radio 5 dramatisation of the Adventures, the feature film The Adventures of Tintin: The Secret of the Unicorn (2011) directed by Steven Spielberg, and the film's tie-in video game.

Synopsis

The synopsis continues a plot begun in The Secret of the Unicorn.

Tintin and his friend Captain Haddock plan an expedition to the West Indies aboard a fishing trawler, the Sirius, to search for the treasure of the pirate Red Rackham. Having previously read three parchments authored by Haddock's ancestor, Sir Francis Haddock, the duo had discovered the coordinates to what they believe is the treasure aboard the sunken 17th century vessel, the Unicorn, near an unknown island. An eccentric, hard-of-hearing inventor named Professor Cuthbert Calculus offers to aid them with the use of his shark-shaped one-man submarine, but they decline his assistance. Setting sail, they are joined by the police detectives Thomson and Thompson and soon discover that Calculus has stowed away on board, bringing his submarine with him.

When they reach the coordinates shown on the parchments, there is no island in sight. Frustrated, Haddock ponders turning back, but Tintin soon realizes the problem: If Sir Francis had used a French chart instead of an English chart to calculate the position, the coordinates would have been measured on the Paris Meridian rather than the Greenwich Meridian. As they have been using the Greenwich Meridian, they realise that they are too far west.

After traveling to the correct position, they discover an uncharted island, which is located about  north of Hispaniola (Dominican Republic) and  South-East of the Turks and Caicos Islands. There, they find a statue of Sir Francis Haddock and other evidence, including parrots who still use Haddock insults handed down from Sir Francis. Tintin deduces that Francis Haddock had taken refuge on the island and that the wreck of the Unicorn must be nearby. They locate the wreck using Calculus' submarine and recover various artefacts from it, but do not find the treasure. Among the artefacts is a strongbox containing old documents revealing that Sir Francis Haddock had been the owner of the country estate Marlinspike Hall.

Back in Belgium, Calculus purchases the Hall, using funds from the sale of his submarine design, and gives it to Haddock. Tintin and Haddock search the house's cellars, where Tintin spots a statue of Saint John the Evangelist holding a cross with a globe and eagle at its feet. Tintin suddenly remembers that Francis Haddock's original three parchments said, "For 'tis from the light that light will dawn, and then shines forth the Eagle's cross" and realises that this message referred, not to the location of the Unicorn, but to Saint John "the eagle": his traditional symbol. Tintin locates the island on the globe, presses a secret button which he finds there, and discovers Red Rackham's treasure hidden inside. Sometime later, Haddock hosts an exhibition of the treasure and several Unicorn artefacts in Marlinspike Hall.

History

Background

Red Rackham's Treasure was serialized amidst the German occupation of Belgium during World War II. Hergé had accepted a position working for , Belgium's largest Francophone daily newspaper. Confiscated from its original owners,  was permitted by the German authorities to reopen under the directorship of Belgian editor Raymond de Becker, although it remained firmly under Nazi control, supporting the German war effort and espousing anti-Semitism. After joining  on 15 October 1940, Hergé became editor of its new children's supplement, , with the help of an old friend, Paul Jamin, and the cartoonist Jacques Van Melkebeke, before paper shortages forced Tintin to be serialised daily in the main pages of . Some Belgians were upset that Hergé was willing to work for a newspaper controlled by the occupying Nazi administration, although he was heavily enticed by the size of '''s readership, which numbered some 600,000. Faced with the reality of Nazi oversight, Hergé abandoned the overt political themes that had pervaded much of his earlier work, instead adopting a policy of neutrality. Entertainment producer and author Harry Thompson observed that, without the need to satirise political types, "Hergé was now concentrating more on plot and on developing a new style of character comedy. The public reacted positively".Red Rackham's Treasure was the second half of a two-part story arc which had begun with the previous adventure, The Secret of the Unicorn. This arc was the first that Hergé had produced since Cigars of the Pharaoh and The Blue Lotus (1934–36). However, as Tintin expert Michael Farr related, whereas Cigars of the Pharaoh and The Blue Lotus had been largely "self-sufficient and self-contained", the connection between The Secret of the Unicorn and Red Rackham's Treasure is far closer.

InfluencesRed Rackham's Treasure introduced Professor Cuthbert Calculus to The Adventures of Tintin, who became a recurring character. Hergé had made use of various eccentric professors in earlier volumes of the series, such as Sophocles Sarcophagus in Cigars of the Pharaoh, Hector Alembick in King Ottokar's Sceptre, and Decimus Phostle in The Shooting Star, all of whom prefigure the arrival of Calculus. The character's deafness had been inspired by a colleague whom Hergé had worked with years earlier at Le Vingtième Siècle. Visually, Calculus was based on a real scientist, the Swiss inventor Auguste Piccard, who had been the first man to explore the stratosphere in a hot air balloon in 1931. Hergé had observed Piccard walking about Brussels on a number of occasions, however the character of Calculus would be notably much shorter than Piccard. Hergé named this character Tryphon Tournesol; while the surname meant "sunflower", the forename was adopted from a carpenter named Tryphon Beckaert whom Hergé had encountered in Boitsfort. Tryphon Tournesol was later renamed Cuthbert Calculus in the English translation and Balduin Bienlein (meaning "Little Bee") for the German translation.

Calculus' shark-shaped submarine was visually based on a real American submarine; Hergé had seen a picture of this in a German newspaper. The diving suit worn in the story was also based on clippings that Hergé had accumulated. Similarly, the dockside bar depicted by the cartoonist was based on an illustration that he had collected. The shop where Haddock and Tintin buy the diving equipment, including the suit, was inspired from a picture of a bar which was featured in the German magazine, Berliner Illustrirte Zeitung. The tribal effigy found on a Caribbean island by Sir Francis Haddock was based on a Bamileke tribal statue from Cameroon that Hergé saw in a museum. The Sirius, which had appeared before in The Shooting Star, was named after the SS Sirius, the first ship to cross the Atlantic Ocean solely under steam power, but was visually based upon the design of a trawler, the John-O.88. Hergé had sketched this ship in Ostend docks before obtaining both detailed plans of the trawler from the builders, Jos Boel & Son, and a small-scale model of it from a collector. The undersea wreck of the Unicorn was loosely inspired by images of the wreck of a 17th-century Swedish vessel, the Vasa, which Hergé had collected. The instance in the story in which a shark swallows a large box (that the characters hope contains the treasure) is based on a real account of a shark that swallowed a camera from the American underwater photographer Otis Barton, which Hergé had encountered in a French illustrated magazine.

The brief appearance of Dr. Daumière, who warns Haddock to cease drinking alcohol, was an allusion to Hergé's own physician, Dr. Daumerie. Hergé made a comical reference to the French comedian Sacha Guitry in the story by advertising a play by Guitry titled Me in which Guitry himself plays every role.

PublicationLe Trésor De Rackham Le Rouge began serialisation as a daily strip in Le Soir from 19 February 1943. The title of the new adventure had been announced in an advertisement in the newspaper two days previously. In Belgium, it was then published in a 62-page book format by Editions Casterman in 1944. Red Rackham's Treasure contained one of Hergé's two favourite illustrations from The Adventures of Tintin. It combines three actions encapsulating a sequence of events into one drawing: Haddock striding up the beach in the foreground, Tintin, Thomson and Thompson bringing the rowboat ashore in the midground, and the Sirius weighing anchor in the background.

Rather than immediately embark on the creation of a new Tintin adventure, Hergé agreed to a proposal that Le Soir crime writer, Paul Kinnet, would author a detective story featuring Thomson and Thompson. The story was titled Dupont et Dupond, détectives (Thomson and Thompson, Detectives), and was illustrated by Hergé.The Secret of the Unicorn and Red Rackham's Treasure were the first two Adventures of Tintin to be published in standalone English-language translations for the British market, as King Ottokar's Sceptre had previously been serialised in Eagle in 1951. Published by Casterman in 1952, these two editions sold poorly and have since become rare collector's items. They would be republished for the British market seven years later, this time by Methuen with translations provided by Michael Turner and Leslie Lonsdale-Cooper. Farr reported that Red Rackham's Treasure is the best-selling story in The Adventures of Tintin, while Harry Thompson referred to The Secret of the Unicorn-Red Rackham's Treasure arc as "the most successful of all Tintin's adventures".

Critical analysis
Harry Thompson stated that the Secrets of the Unicorn-Red Rackham's Treasure arc marked the beginning of the third and central stage of "Tintin's career". He furthermore stated that in these two stories, Tintin has been converted from a reporter into an explorer to cope with the new political climate. He stated that in this story, Hergé "abandons the complex plotting of The Secret of the Unicorn in favour of an episodic style of adventure not seen since the early books". Thompson further draws attention to the arrival of Calculus in the story, describing him as the "third and final member" of Tintin's "family". Thompson was critical of the use of colour in the story, stating that much of it looks better in black-and-white, as it was originally printed in Le Soir.

Hergé biographer Benoît Peeters observed that both The Secret of the Unicorn and Red Rackham's Treasure "hold a crucial position" in The Adventures of Tintin as it establishes the "Tintin universe" with its core set of characters. He felt that while religious elements had been present in previous stories, they were even stronger in The Secret of the Unicorn and its sequel, something which he attributed to Van Melkebeke's influence. Peeters believed that Red Rackham's Treasure was "an unforgettable book" because it is the volume in which the "family"—meaning Tintin, Snowy, Haddock, and Calculus—all come together. Fellow biographer Pierre Assouline echoed this idea, noting that Hergé had "settled" the three characters in their new home. Focusing on the character of Calculus, he noted that the idea of the eccentric professor was "so universal that it would be inaccurate to point to any one source", suggesting possible influences from Charlie Chaplin and Hergé's own father. For Assouline, the professor embodies "the gentle madness and subtle humour in comic strips". He added that both Red Rackham's Treasure and its predecessor "reveal Hergé at a new level in his art", and suggested that the reason for their popularity lay in the fact that they were "the visual continuation of a literary universe that stretches from Jules Verne to Pierre Benoit".

Jean-Marc Lofficier and Randy Lofficier opined that The Secret of the Unicorn-Red Rackham's Treasure arc represents "a turning point" for the series as it shifts the reader's attention from Tintin to Haddock, who has become "by far, the most interesting character". They claim that the introduction of Calculus "completes the indispensable triangle that imbues Tintin with its mythic quality". Asserting that here, Hergé's "art has reached a degree of near-perfection", they awarded it five stars out of five.
Michael Farr said that the scene introducing Calculus was "a comic tour de force" marking the start of the "rich vein of humour" that the character brought to the series. Noting that unlike The Shooting Star, this two-book story arc contains "scarcely an allusion to occupation and war", he praised the arc's narrative as "perfectly paced, without that feeling of haste" present in some of Hergé's earlier work.

In his psychoanalytical study of the Adventures of Tintin, the academic Jean-Marie Apostolidès characterised the Secret of the Unicorn-Red Rackham's Treasure arc as being about the characters going on a "treasure hunt that turns out to be at the same time a search for their roots". He stated that the arc revolves around Haddock's ancestry, and in doing so "deals with the meanings of symbolic relations within personal life". He compared Sir Francis Haddock to Robinson Crusoe and noted how the Caribbean natives deified Sir Francis Haddock by erecting a statue of him in the same manner that the Congolese deify Tintin at the end of Tintin in the Congo. Highlighting that Calculus is one of many eccentric scientists to appear in the series, Apostolidès nonetheless emphasises the uniqueness of Calculus, by noting that the character approaches Tintin, rather than Tintin approaching him, as the young reporter had done with previous scientists. Commenting on the introduction of Calculus' shark submarine, Apostolidès states that it "allows them to cross a boundary previously restricting human beings and to penetrate into another universe, the one beneath the seas that holds secrets hitherto unknown". Ultimately, he believes that by the end of the story, "the family structure is in place", with Calculus representing a father figure with financial control, and Haddock and Tintin, who have become brothers through their joint adventure, adding that with the aid of Francis Haddock, "the ancestor", they are given a home at Marlinspike Hall.

Literary critic Tom McCarthy highlighted what he perceived as scenes in Red Rackham's Treasure which reflected common themes in The Adventures of Tintin. He pointed out that in being a stowaway aboard the ship, Calculus was one of many stowaways in the series, and that the treasure represented the theme of jewels and precious stones which also cropped up in The Broken Ear, Tintin in the Congo, and The Castafiore Emerald. He noted Tintin's misreading of the parchments and stated this was one of a number of calculation mistakes that the character makes in the series. He suggested that a scene in which the shark submarine pushes between Haddock's buttocks was a form of sexual innuendo referencing anal sex, highlighting similar innuendo in The Broken Ear and The Crab with the Golden Claws.

Adaptations
In 1957, the animation company Belvision Studios produced Hergé's Adventures of Tintin, a series of daily five-minute colour adaptations based upon Hergé's original comics. Red Rackham's Treasure was the fifth story to be adapted in the second series (and the eighth to be adapted overall), being directed by Ray Goossens and written by the cartoonist Greg. In later years, Greg would become editor-in-chief of Tintin magazine.

In 1991, a collaboration between the French studio Ellipse and the Canadian animation company Nelvana adapted 21 of the stories into a series of episodes, each 42 minutes long. Red Rackham's Treasure was the tenth episode of The Adventures of Tintin to be produced, although it ran half as long as most of the others. Directed by Stéphane Bernasconi, the series has been praised for being "generally faithful", with compositions having been actually directly taken from the panels in the original comic book.

A 2011 motion capture feature film directed by Steven Spielberg and produced by Peter Jackson was released in most of the world October–November 2011, under the title The Adventures of Tintin: The Secret of the Unicorn, and in the US on 21 December, where it was simply titled The Adventures of Tintin. The film is partially based on Red Rackham's Treasure, combined with elements of The Secret of the Unicorn and The Crab with the Golden Claws''. A video-game tie-in to the movie was released October 2011.

References

Notes

Footnotes

Bibliography

External links
 Red Rackham's Treasure at the Official Tintin Website
 Red Rackham's Treasure at Tintinologist.org

1944 graphic novels
Comics set in the Caribbean
Literature first published in serial form
Methuen Publishing books
Nautical comics
Pirate comics
Tintin books
Treasure hunt comics
Works originally published in Le Soir
Works set in country houses


Lord Mayor Robert Doyle named leading infectious disease expert Professor Sharon Lewin as the 2014 Melburnian of the Year at the annual Melbourne Awards gala ceremony at Town Hall on Saturday, 15 November.
The Lord Mayor said Professor Lewin was a worthy recipient of the city's highest accolade.
"It gives me great pleasure to bestow the honour of 2014 Melburnian of the Year to Professor Sharon Lewin who is one of the world's most influential campaigners and researchers working towards a cure for HIV," the Lord Mayor said.
"Professor Lewin is passionate about her work and her city which was evident as she awed us all with her performance as the Co-Chair of the 20th International AIDS Conference in Melbourne earlier this year.
Professor Lewin expressed her delight in receiving recognition for her work.
"Melbourne's reputation as an international centre for medical research is rapidly growing due to the tireless and innovative work of many in our industry. To be singled out for this accolade is a wonderful honour and something I'm extremely proud of," she said.
The Melburnian of the Year was joined by nine other individuals, corporations and community organisations, who were each recognised for their contribution to the city's success as part of the Melbourne Awards program.
"The Melbourne Awards acknowledge excellence and celebrate the remarkable people and organisations who continue to shape our city and our lives," the Lord Mayor said.


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Justia Patents US Patent Application for PROCESS FOR THE SEPARATION AND PURIFICATION OF PHYCOBILIPROTEINS Patent Application (Application #20210300975)
PROCESS FOR THE SEPARATION AND PURIFICATION OF PHYCOBILIPROTEINS
Jun 4, 2019 - Consiglio Nazionale delle Ricerche
The present invention relates to a membrane chromatography process for selectively separating and purifying phycobiliproteins starting from aqueous biomasses of cyanobacteria and algae that, in a short time and at reduced cost, allows to obtain phycobiliproteins with a high degree of purity for use in pharmaceutical, cosmetic, or food compositions or as reagents for research.
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FIELD OF THE INVENTION
The present invention relates in general to the field of the purification of active ingredients from natural products, and more precisely it refers to a process of membrane chromatography for obtaining phycobiliproteins with a high degree of purity starting from cyanobacteria and/or algae biomasses.
The phycobiliproteins are components with bright, highly fluorescent colors of the antenna complexes of the photosynthetic system of cyanobacteria and some algae, such as the algae belonging to the classes Rhodophyta, or red algae, and Cryptophyta, or cryptomonads. The phycobiliproteins are formed by a complex of proteins and linear tetrapyrrolic groups—which represent the chromophores in the complex—in which the tetrapyrrolic groups are covalently linked to the protein units. The most common phycobiliproteins are phycocyanin, allophycocyanin and phycoerythrin, the first two being blue, the third being bright and the third being fuchsia. These proteins are natural water-soluble pigments which, in addition to playing an important biological role in the collection of light in the organisms in which they are present, also represent products with high added value and various commercial applications. Due to their properties of pigments with fluorescence they are in fact used as natural colorants in the cosmetic field or as fluorescent probes (e.g. in flow cytometry or for immunological analysis), and as a coloring additive in the food field.
These proteins have also been recognized since long as having a high nutritional power and as bioactive molecules, hence their use as food (the two phycobiliproteins phycocyanin and allophycocyanin extracted from spirulina algae are approved for food consumption by EFSA in Europe and by FDA in the United States), and as a non-toxic photosensitizer in the photodynamic therapy (PDT) of tumors, while there are numerous studies on the potential use of these proteins as a drug based on the now proven antioxidant, anti-inflammatory, neuroprotective, antitumoral and immunomodulating properties (see for example [1] and [2]).
With such and so many applications it is clear the commercial value of these products: their value may however considerably vary depending on the degree of purity of the protein conjugate that on its turn varies with the type of application. For food applications, for instance, a degree of purity higher than 0.7 or "food grade" is required, for the cosmetic use the required degree of purity or "cosmetic grade" is higher than 2.0, higher than 3.9 the reactive grade and higher than 4.0 the analytic grade required to phycobiliproteins. In general, as also is in the following description, by "purity degree" of a phycobiliprotein the ratio is meant between the maximum absorbance value of the phycobiliprotein (around 540-570 nm for phycoerythrin, around 615-620 nm for phycocyanin, around 650 nm for allophycocyanin) and the absorbance value at 280 nm, which is related to the total amount of proteins in the product (see for instance references [3], [4]).
So far, many methods have been proposed to purify phycobiliproteins and the phycocyanin in particular. Usually, with these methods, a high degree of purity is achieved only after several purification steps, which generally include several steps of column chromatography of the biomass extracts that contain the product of interest, with a consequent yield reduction and a considerable increase of the manufacturing costs, which make impractical the exploitation of these methods on industrial scale. See for example [5], [6].
Alternatively, some simplified chromatographic purification procedures have been proposed, on paper more suitable for large-scale use; however, only a few of these protocols are able to avoid purification processes by column chromatography and among these, only those using extraction with a two-phases aqueous system, combined with ultrafiltration—a methodology that is also long and expensive—are successful to provide an analytical grade product [7].
In fact, it is known that the traditional packed column chromatography presents a series of disadvantages, such as the slowness of the separation by diffusion between the pores and the consumption of large volumes of solvents, which limit its application on a large scale, especially in the purification of large molecules. On the other hand, more recently, other types of chromatographic separation have been proposed for the purification of products, such as membrane chromatography, which have become increasingly important (see for example [8]), in particular for processing large volumes of dilute solutions, also in the purification of large biomolecules, such as nucleic acids, proteins, antibodies, or viruses. As a matter of fact, membrane chromatography has considerable advantages compared to column chromatography, from a reduced solute transport time to a lower volume of solvent required for the same recovered product. The bonding efficiency of the membranes towards the solutes is also generally independent from the speed of the supply flow to the membrane over a wide range, therefore very high flows can also be used, thus improving the process time. Finally, the devices are simpler and less expensive to be mass-produced. On the other hand, however, membrane chromatography is characterized by a low load capacity and by a not high resolution in the separation processes. In the literature, these disadvantages, in particular with respect to column chromatography, have been discussed and highlighted, for example in [9].
Furthermore, in this type of chromatography, the functionalization of the membrane with appropriate ligands can allow the exploitation of the most various chemical processes to achieve separation, such as for example affinity, ion exchange, or hydrophobic interactions. However, it was highlighted that different materials used to produce chromatography membranes have a certain intrinsic capacity to selectively interact with proteins, with consequences on the possibility of using this type of chromatographic separation for protein purification.
To date, therefore, as far as the Applicant is aware, efficient purification procedures suitable for use on a large scale are not known, which allow to reduce the production costs of the phycobiliproteins while ensuring acceptable levels of purity. This effectively limits the massive use of these chromoproteins in the various sectors of commercial application, despite the fact that their high nutraceutical and medical value, or even more simply the effectiveness of natural dye, is now acknowledged.
Now the Applicant has found that it is possible to obtain single phycobiliproteins with a high degree of purity, in particular phycocyanin, allophycocyanin or phycoerythrin, starting from cyanobacterial and algal biomasses by membrane chromatography treatment.
This method, applied directly to raw aqueous extracts containing phycobiliprotein mixtures, has made it possible to obtain the separation of the products of interest with a high degree of purity, in a simple way and at low costs, as required for commercial uses in the pharmaceutical, food and cosmetic fields, as well as for the application in analysis and research.
The use of column chromatography or of other expensive and lengthy procedures, such as ultrafiltration, is also avoided thanks to the present process.
Subject of this invention is therefore a process for the separation and purification of phycobiliproteins from raw extracts of cyanobacteria and/or algae containing them, comprising at least a cycle of membrane chromatography purification, as defined in the first of the appended claims.
Other important features of the process according to the present invention are reported in the following detailed description.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1—Absorption spectrum of purified phycocyanin obtained as described in the following Example 1.
FIG. 2—Absorption spectrum of the purified allophycocyanin obtained as described in the following Example 5.
FIG. 3—Absorption spectrum of purified B-phycoerythrin obtained as described in the following Example 8.
In accordance with the invention, the process proposed herein is a simplified process for the selective separation and purification of phycobiliproteins, in particular phycocyanin, allophycocyanin and phycoerythrin, from crude extracts of cyanobacteria and/or algae that contain mixtures of such phycobiliproteins, without the use of column chromatography or ultrafiltration or other long and expensive techniques. The inventors have in fact surprisingly found that it is possible to carry out an effective separation and purification of the phycobiliproteins present in a saline aqueous extract by passing through a microfiltration membrane, exploiting the selective and reversible bonding of the different phycobiliproteins with the membrane, without using long processes of elution with solvents as it is when using column chromatography. In other words, this is a membrane chromatography separation, wherein the microfiltration membrane represents the stationary phase and the aqueous saline solution represents the mobile phase of the chromatographic method.
In a preferred aspect of this invention, the starting raw extracts are cyanobacteria extracts or are algae extracts, for example selected from extracts of Arthrospira platensis (Spirulina) and extracts of Porphyridium cruentum. Preferably, the starting extracts are extracts of Arthrospira platensis. Furthermore, in the present process, extracts from fresh biomass or extracts from dehydrated biomass can be used, for example from freeze-dried biomass in powder form. According to a preferred embodiment of the process of this invention, the starting crude extracts were obtained by suspension in aqueous solution of a biomass of cyanobacteria and/or freeze-dried algae, subsequent centrifugation and collection of the so-formed supernatant. The suspension in aqueous solution of the freeze-dried biomass is preferably kept at rest, for example at 4° C. for 24 hours, before being subjected to centrifugation. The starting suspension can be for example a suspension in water or in an aqueous solution of a salt or of a buffer.
In a particular aspect of this process, the suspension from biomasses of cyanobacteria, algae, or from biomasses containing cyanobacteria together with algae, can be advantageously subjected to an extractive treatment before purification on the membrane, in order to break up the cellular walls of the organisms and free the proteins contained inside them, for a higher yield of the desired products. Any technician with ordinary skills in the art can effortlessly identify a treatment method suitable to this purpose, such as for example ultrasonication, freeze-drying, freezing/thawing cycles or enzymatic treatments.
In a particular embodiment of the invention, the crude extracts from dry freeze-dried biomass, obtained by suspension of the biomass in a saline solution and subsequent centrifugation, are directly subjected to membrane chromatography separation/purification. In another particular embodiment of the invention, the crude extracts from fresh, wet biomass are obtained thanks to several cycles of freezing/thawing of the suspension of the biomass in saline solution and subsequent centrifugation thereof, before being subjected to separation/purification by membrane chromatography.
The present process is in any case applicable to any cyanobacterium and/or algae, on crude extracts obtained in any way.
The present process of separation and purification of phycobiliproteins from crude extracts of cyanobacteria and/or algae containing them is based on membrane chromatography of the above said extracts, and comprises at least one purification cycle comprising:
i) at least one passage of a saline aqueous solution of the crude extracts on a hydrophilic porous membrane, having a low protein binding power, in which the aqueous saline solution of the crude phycobiliprotein extracts is an aqueous solution of a salt S having a concentration [S]1;
ii) desorption of the retentate bound to the membrane with a solvent selected from the group consisting of water, an aqueous solution of said salt S at a concentration [S]2<[S]1, and an aqueous solution of a salt S′ that is a stronger chaotropic agent than the salt S.
In the above said process the hydrophilic porous membrane is the stationary phase of the chromatographic method, while the aqueous saline solution of the phycobiliproteins extracts in step i) represents a first mobile phase, which determines the interaction of phycobiliproteins with the membrane, and the solvent in step ii) of the process is a second mobile phase.
In the present invention, the expression "a salt S′ that is a stronger chaotropic agent than the salt S" means a salt S′ having a greater ability than the salt S to break the hydrophobic bonds and the hydrogen bonds that the proteins form with the membrane.
The salt S is preferably selected from ammonium sulfate and sodium sulfate; optimal results in terms of yield, separation and degree of purity are obtained in this process with ammonium sulfate. The aqueous solution of the salt S′ is preferably selected from between aqueous solution of sodium chloride and saline phosphate buffer. Any technician with ordinary skills in the art can without efforts select any other aqueous solutions of salts S′ of possible use in step ii) of the present process, among the solutions of salts able to favor the detachment of proteins from the membrane.
The hydrophilic porous membrane of possible use in the present process can be for example a flat, hollow, capillary or tubular membrane of hydrophilic material, selected for example between polycarbonate and PVDF (polyvinylidene fluoride); preferably the membrane material is PVDF. There are several commercial microfiltration membranes of this type, which can be used successfully in the present process.
In the context of this invention, by "membrane" in the purification step i) is meant a single membrane or a plurality of membranes, for example more membranes stacked one on top of the other.
In a preferred aspect of the process of the invention, before the purification step i) of the extracts on the membrane, this is previously subjected to a conditioning treatment with a saline aqueous solution of the salt S at the same concentration [S]1 used in the subsequent step.
According to a preferred embodiment, which allows to obtain particularly high purity degrees of the separated phycobiliprotein, for example a purity of analytical grade ≥4.0, the process of the invention further comprises, before the purification step i) on the membrane and upstream of the possible conditioning of the membrane described above, a cleaning step on the membrane of the extracts with an aqueous solution of salt S at concentration [S]0<[S]1, wherein [S]1 is the concentration of the salt S in the aqueous solution of the phycobiliproteins to be purified. This aqueous solution of the salt S at concentration [S0] is a third mobile phase wherein the hydrophilic membrane always represents the stationary phase of a chromatographic method. This preliminary chromatographic cycle with a saline solution at lower concentration allows minimizing the interaction between the membrane and the phycobiliproteins that will pass therefore in the permeate, while on the membrane various impurities remain. The membrane used in this phase may also be of the same type as that used in the at least one subsequent purification step i).
In a preferred aspect of the invention, the process is carried out by replacing the hydrophilic porous membrane after the cleaning step, when present, using in all the other chromatographic cycles of the process the same hydrophilic porous membrane as defined above.
In order to recover more than one different phycobiliprotein from the same biomass that contains several of these proteins, the present process can comprise more purification cycles carried out consecutively with equal or increasing concentrations of saline aqueous solution of the salt S by passage on a membrane of the permeate obtained from the previous cycle until the separation of the desired phycobiliprotein, for example after n passages, at the passage n+1 a concentration [S]n+1≥[S]n has to be used.
In a preferred aspect of the process of the invention, before each cycle n of purification i) of the extracts on the membrane, this is previously subjected to a conditioning treatment with a saline aqueous solution of the salt S at the same concentration [S]n used in the subsequent step.
According to a preferred embodiment of the present process, each purification cycle can be repeated more than once at the same salt concentration S until the phycobiliprotein of the desired degree of purity is obtained.
In the context of the present invention, by "high degree of purity" a degree of purity is meant that is ≥0.7, corresponding to the degree of purity required of the phycobiliproteins for food use, where the degree of purity is defined as described above. With the present process it is however possible to obtain the products of interest, as demonstrated in the experimental part that follows, even with much higher degrees of purity, and in particular ≥2.0, the degree of purity required for cosmetic use; ≥3.5, degree of purity required for the application of phycobiliproteins in scientific research, for example as fluorescent markers; and ≥4.0, degree of purity required for the medical-pharmaceutical use of phycobiliproteins, for example as antiviral, antioxidant, antibacterial, anti-inflammatory or tracer agents.
The main advantage of the process of the invention lies in the possibility of obtaining phycobiliproteins with a high degree of purity in a simple and rapid manner; on a laboratory scale, the inventors have carried out and completed each purification cycle in just 3-4 minutes.
Furthermore, the present process allows obtaining high degrees of purity of the phycobiliproteins, suitable for the various applications, from food to cosmetics, to pharmaceuticals, to analytics.
A further advantage of the present process lies in the fact that all the reagents and devices used, including the microfiltration membranes used as chromatographic device, are commercially available at low cost, or in any case at a limited cost. The unit cost of the finished product will therefore also be proportionately lower.
A further advantage of the present process is its flexibility and re-adaptation depending on the phycobiliprotein to be recovered and of the related degree of purity desired. On the basis of these desiderata, the number of purification cycles by membrane chromatography, the related concentrations of salt S to be used and the possible preliminary passage of cleaning may be defined from time to time, so as to modulate the result of the process and obtain the desired product, having the desired purity, optimizing the yield.
The following Examples are reported for illustrative and not limitative purposes of the present invention.
Purification of Phycocyanin from Aqueous Extracts of Arthrospira platensis (Spirulina) Biomass
A crude phycobiliprotein extract was obtained by suspending the freeze-dried A. platensis biomass in an aqueous solution of 100 mM NaCl (140 mg of Spirulina in 11 mL of solution). The suspension was kept at 4° C. for 24 hours, then centrifuged for 45 minutes (12000 rcf, T=10° C.). The supernatant containing phycocyanin (PC) and allophycocyanin (APC) (hereinafter "crude extract") was recovered and stored at 4° C.
The chromatographic purification process on the crude extract was performed by using a vacuum glass device for microfiltration and a PVDF microfiltration membrane, carrying out two consecutive chromatographic cycles. The second cycle has allowed to obtain phycocyanin with a degree of purity of analytical grade.
More specifically, a glass vacuum flask for microfiltration was assembled with a hydrophilic PVDF membrane (Durapore®, with an average pore size of 0.45 μm, diameter of 47 mm, thickness 125.0 μm, code HVLP 04700). Using this chromatographic device, the following two steps were conducted:
chromatographic cleaning cycle: the membrane was washed with 100 mL of deionized water and then conditioned with 5 mL ammonium sulfate 0.6 M. A volume of 100 mM NaCl aqueous solution was added to a 0.8 mL aliquot of crude extract and of ammonium sulfate 3 M so as to reach a final volume of extract in ammonium sulfate 0.6 M of 5 mL, with a total content of phycobiliproteins (PC+APC) of about 1 mg. The solution was loaded on the membrane and filtered; the permeate was collected in a 100 mL clean tailed flask. 5 mL of ammonium sulfate 0.6 M were then loaded onto the membrane, filtered and collected in the flask containing the permeate, to maximize the recovery of phycobiliproteins. No blue compound was adsorbed on the membrane at the ammonium sulfate concentration of 0.6 M. The volume of the solution was carefully measured and, if necessary, it was brought back to 10 mL by using an ammonium sulfate solution 0.6 M;
purification cycle: to the ammonium sulfate permeate 0.6 M (10 mL) obtained as described above in the cleaning cycle i), an aqueous solution of ammonium sulfate 3 M was added so as to obtain a phycobiliprotein solution having a concentration of 1.110 M ammonium sulfate. This solution was then loaded onto a new PVDF membrane, using the usual procedure: the membrane was washed with 100 mL of deionized water and then conditioned with 5 mL of an ammonium sulfate solution having the same ammonium sulfate concentration of the phycobiliprotein solution, i.e. 1.110 M. The solution of phycobiliprotein and ammonium sulfate 1.110 M was then loaded onto the membrane and filtered. The retentate on the membrane was washed with 10 mL of ammonium sulfate solution 1.110 M, then desorbed with 10 mL of NaCl solution 100 mM and recovered in a 100 mL clean tailed flask. The absorbance spectrum of the purified PC thus obtained is shown in FIG. 1.
The phycocyanin obtained resulted, in particular, to have a purity degree of 4.20, while the purification yield with respect to the phycocyanin content in the portion of crude extract subjected to purification was 67%.
The crude extract of phycobiliproteins was obtained from fresh biomass of A. platensis. An aliquot of cultivation of biomass in water (40 mL) was centrifuged for 10 min (12,000 rcf, temperature=10° C.) and the supernatant eliminated. The cyanobacteria pellet was suspended and washed with 18 MO Milli-Q pure water, then centrifuged for 10 min (12,000 rcf, temperature 10° C.) and the supernatant eliminated. The pellet was resuspended in 10 mL of a NaCl aqueous solution 100 mM and subjected to three freeze-thaw cycles (in each cycle the suspension was kept at −20° C. for 2.5 hours and then thawed in a water bath at 20° C.). After adding further 10 mL of NaCl aqueous solution 100 mM (20 mL in total) the suspension was sonicated four times for 60 s (power 75%, pulse 60%, sonicating tip S2, Hielscher Ultrasonic Processor UP200S, 200 W, 24 kHz) in a bath of water and ice, with an interval of 60 s between a sonication cycle and the subsequent one. The suspension was finally centrifuged for 45 minutes (12,000 rcf, temperature=10° C.) and the supernatant containing phycocyanin (PC) and allophycocyanin (APC) (crude extract) was recovered and stored at 4° C.
The purification process on the crude extract was performed using a glass vacuum device for microfiltration and a PVDF microfiltration membrane, carrying out three consecutive chromatographic cycles on the membrane. The third cycle has allowed to obtain phycocyanin with a degree of purity of analytical grade.
More specifically, a glass vacuum flask for microfiltration was assembled with a hydrophilic PVDF membrane (Durapore®, with an average pore size of 0.45 μm, a diameter of 47 mm, HVLP code 04700). Using this device, the following two steps were carried out:
cleaning cycle: the membrane was washed with 100 mL of deionized water and then conditioned with 5 mL of ammonium sulfate 0.6 M. A volume of NaCl aqueous solution 100 mM was added to a 2.0 mL aliquot of crude extract and of 3 M ammonium sulfate so as to reach a final volume of extract in ammonium sulfate 0.6 M of 3 mL, with a total content of phycobiliproteins (PC+APC) of about 1 mg. The solution was loaded on the membrane and filtered; the permeate was collected in a 100 mL clean tailed flask. 3 mL of ammonium sulfate 0.6 M were then loaded onto the membrane, filtered and collected in the flask containing the permeate, to maximize the recovery of phycobiliproteins. No blue compound was adsorbed on the membrane at the ammonium sulfate concentration of 0.6 M. The volume of the solution was carefully measured and, if necessary, it was brought back to 6 mL using an ammonium sulfate solution 0.6 M;
purification cycle: to the ammonium sulfate permeate 0.6 M (6 mL) obtained as described above in the cleaning cycle i), an aqueous ammonium sulfate solution 3 M was added so as to obtain a phycobiliprotein solution having a concentration of ammonium sulfate 1.110 M. This solution was then loaded onto a new PVDF membrane, using the usual procedure: the membrane was washed with 100 mL of deionized water and then conditioned with 5 mL of an ammonium sulfate solution having the same ammonium sulfate concentration of the phycobiliprotein solution, i.e. 1,110 M. The phycobiliprotein solution in ammonium sulfate 1.110 M was then loaded onto the membrane and filtered. The retentate bound on the membrane was washed with 10 mL of ammonium sulfate solution 1.110 M, then desorbed with 5 mL of NaCl solution 100 mM and recovered in a 100 mL clean tailed flask. The spectrophotometric analysis of the product showed a purity level of phycocyanin lower than the analytical degree. A further membrane chromatography purification cycle was therefore performed on the recovered phycocyanin solution. In this step the volume of solution was carefully measured and an ammonium sulfate solution 3 M was added so as to obtain a phycocyanin solution having an ammonium sulfate concentration of 1.110 M, which was then loaded onto the membrane and filtered. The retentate bound onto the membrane was washed with 10 mL of ammonium sulfate solution 1.110 M, then desorbed with 5 mL of NaCl aqueous solution 100 mM and recovered in a 100 mL clean tailed flask. The spectrophotometric analysis showed an analytical grade purity for the phycocyanin thus obtained. More specifically, the phycocyanin obtained was found to have a purity degree of 4.0, while the purification yield with respect to the phycocyanin content in the portion of crude extract subjected to purification was of 60.0%.
A crude extract of phycobiliproteins was obtained as described above in Example 1. The purification process on this crude extract was performed using a syringe for microfiltration and a filter for syringe with a membrane in hydrophilic PVDF (Durapore®, with an average pore size of 0.45 μm, 33 mm diameter, code SLHV033RS). Using this device, the following two cycles of chromatographic purification were carried out:
cleaning cycle: the filter was washed with 5 mL of deionized water and then conditioned with 3 mL of ammonium sulfate 0.6 M, then it was used to filter a 2 mL aliquot of crude extract in ammonium sulfate 0.6 M with a content total phycobiliproteins (PC+APC) of about 0.31 mg. The solution was filtered and the permeate was collected in a 10 mL glass bottle. The filter was then washed with 1 mL of ammonium sulfate solution 0.6 M, collecting the solution in the same bottle containing the permeate to maximize the recovery of phycobiliproteins. No blue compound adsorbed on the filter at the ammonium sulfate concentration of 0.6 M was observed. The volume of the solution was then carefully measured and, if necessary, it was brought back to 3 mL using an ammonium sulfate solution 0.6 M;
purification cycle: to the permeate in ammonium sulfate 0.6 M (3 mL) obtained as described above in the cleaning cycle, a volume of aqueous solution of NaCl 100 mM and ammonium sulfate 3 M was added so as to reach a final volume of extract in ammonium sulfate 1.110 M of 5 mL. In order to obtain analytical grade phycocyanin, this solution was loaded onto a new filter for syringe with a PVDF membrane, following then the usual procedure: the filter was washed with 5 mL of deionized water and then conditioned with 3 mL of an aqueous ammonium sulfate solution having the same ammonium sulfate concentration of the phycobiliprotein solution, i.e. 1.110 M. The ammonium sulfate phycobiliprotein solution 1.110 M was filtered and the retentate on the membrane was washed with 3 mL of ammonium sulfate solution 1.110 M. The retentate was finally recovered with 3 mL of aqueous solution of NaCl 100 mM.
The phycocyanin obtained as described above resulted, in particular, to have a purity grade of 4.0, while the purification yield with respect to the phycocyanin content in the crude extract aliquot subjected to purification was 63.0%.
A crude extract of phycobiliproteins was obtained by suspending the biomass of A. platensis lyophilized in an aqueous solution of ammonium sulfate 0.6 M (100 mg of Spirulina in 15 mL of solution). The suspension was kept at 4° C. for 24 hours, then centrifuged for 45 minutes (12,000 rcf, temperature=10° C.). The supernatant containing phycocyanin (PC) and allophycocyanin (APC) (hereinafter "crude extract") was recovered and stored at 4° C.
The purification process on the crude extract was performed using a glass vacuum device for microfiltration and a PVDF microfiltration membrane, carrying out two consecutive chromatographic cycles on membrane. The second cycle has allowed to obtain phycocyanin with a degree of purity of analytical grade.
cleaning cycle: the membrane was washed with 100 mL of deionized water and then conditioned with 5 mL of ammonium sulfate 0.6 M. A 1.5 mL aliquot of crude extract in ammonium sulfate solution 0.6 M, with a total phycobiliprotein content (PC+APC) of about 1 mg, was loaded on the membrane and filtered; the permeate was collected in a 100 mL clean tailed flask. 5 mL of ammonium sulfate 0.6 M were then loaded onto the membrane, filtered and collected in the flask containing the permeate, to maximize the recovery of phycobiliproteins. No blue compound was adsorbed on the membrane at the ammonium sulfate concentration of 0.6 M. The volume of the solution was carefully measured and, if necessary, it was brought back to 10 mL of final volume, using an ammonium sulfate solution 0.6 M;
purification cycle: to the ammonium sulfate permeate 0.6 M (10 mL) obtained as described above in the cleaning cycle, an aqueous ammonium sulfate solution 3 M was added so as to obtain a phycobiliprotein solution having a concentration of ammonium sulfate 1.110 M. This solution was then loaded onto a new PVDF membrane, using the usual procedure: the membrane was washed with 100 mL of deionized water and then conditioned with 5 mL of an ammonium sulfate solution having the same ammonium sulfate concentration of the phycobiliprotein solution, i.e. 1.110 M. The solution of phycobiliprotein and ammonium sulfate 1.110 M was then loaded onto the membrane and filtered. The retentate on the membrane was washed with 10 mL of ammonium sulfate solution 1.110 M, then desorbed with 10 mL of NaCl solution 100 mM and recovered in a 100 mL clean tailed flask.
The phycocyanin obtained resulted, in particular, to have a purity grade of 4.05, while the purification yield compared to the phycocyanin content in the crude extract aliquot subjected to purification was 60.0%.
Purification of Phycocyanin and Allophycocyanin from Aqueous Extracts of Arthrospira platensis (Spirulina) Biomass
A crude extract of phycobiliproteins was obtained as described above in Example 1. The purification process on the crude extract was then carried out using a glass vacuum device for microfiltration and a microfiltration membrane in hydrophilic PVDF (Durapore®, with average pore size of 0.45 μm, 47 mm diameter, HVLP code 04700). No preliminary chromatographic purification cycle was carried out, operating as follows:
first purification cycle: the membrane was washed with 100 mL of deionized water and then conditioned with 5 mL of aqueous ammonium sulfate solution 1.10 M. A 48 mL aliquot of crude extract in aqueous ammonium sulfate 1.10 M solution, with a total phycobiliprotein content of about 9.7 mg, was loaded on the membrane and filtered; the permeate was collected in a clean 250 mL tailed flask. The retentate (PC) on the membrane was washed with 10 mL of ammonium sulfate 1.10 M, then desorbed with 10 mL of NaCl 100 mM, recovered in a clean 100 mL tailed flask and its degree of purity was spectrophotometrically determined. The permeate was repeatedly (6 times in total) loaded onto the membrane to recover all the phycocyanin still present in the solution. Between one cycle and the subsequent one, the membrane was cleaned with 100 mL of deionized water and then conditioned with 5 mL of ammonium sulfate solution 1.10 M.
second purification cycle: to remove from the permeate containing allophycocyanin (APC), (obtained at the end of the first purification cycle described above) the small amount of PC still present, an aqueous ammonium sulfate solution 3 M was added up to a final concentration of 1.30 M, and the solution was loaded onto the previously cleaned membrane with 100 mL of deionized water and conditioned with 5 mL of ammonium sulfate solution 1.30 M, then it was filtered. The retentate bound onto the membrane was removed by cleaning the membrane with 100 mL of deionized water. The membrane was conditioned with 5 mL of ammonium sulfate solution 1.3 M and the permeate loaded and filtered again to eliminate any PC still present in the solution.
third purification cycle: an ammonium sulfate 3 M aqueous solution was added to the permeate obtained from the second purification cycle up to a final concentration of 1.50 M and the solution was loaded onto the membrane previously cleaned with 100 mL of deionized water and conditioned with 5 mL of ammonium sulfate solution 1.50 M, then it was filtered. The retentate (APC) on the membrane was washed with 10 mL of ammonium sulfate solution 1.50 M, then desorbed with 10 mL of aqueous solution of 100 mM NaCl and recovered in a 100 mL clean tailed flask. The degree of purity of APC was spectrophotometrically determined, from the absorption spectrum of FIG. 2.
The following Table 1 shows the degree of purity and the yield of product with respect to the content of phycocyanin and of allophycocyanin in the aliquot of crude extract subjected to purification.
TABLE 1 Purification No. of Phycobili- Degree Yield cycle, AS purification protein of of Yield (%) concentration step interest purity (%) (P ≥ 3.5)
I cycle, 1 PC 3.6 17.1 63.8 1.10 M I cycle, 2 PC 3.9 18.7 1.10 M I cycle, 3 PC 4.0 17.3 1.10 M I cycle, 4 PC 3.7 10.7 1.10 M I cycle, 5 PC 2.6 3.1 1.10 M I cycle, 6 PC 1.6 <1 1.10 M II cycle, 1 PC — — 1.30 M II cycle, 2 PC — — 1.30 M III cycle, 1 APC 3.4 9.2 1.50 M
Purification of Phycocyanin from Aqueous Extracts of Arthrospira platensis (Spirulina) Biomass—Obtaining Phycocyanin with Food/Cosmetic Purity
A crude extract of phycobiliproteins was obtained as described above in Example 1. The purification process was then performed using a glass vacuum device for microfiltration assembled with a hydrophilic PVDF membrane (Durapore®, average pore size 0.45 μm, diameter 47 mm, code HVLP 04700). The cleaning chromatographic cycle was not carried out, while only one purification cycle was carried out, as follows:
purification cycle: the membrane was washed with 100 mL of deionized water and then conditioned with 5 mL of aqueous ammonium sulfate solution 1.65 M. A 10 mL aliquot of crude extract in aqueous ammonium sulfate 1.65 M solution, with total content of phycobiliproteins equal to about 1.7 mg, was loaded on the membrane and filtered. The retentate bound onto the membrane was washed with 10 mL of ammonium sulfate solution 1.65 M, then desorbed with 10 mL of NaCl aqueous solution 100 mM, recovered in a 100 mL clean tailed flask and its degree of purity was spectrophotometrically determined.
The purified phycocyanin thus obtained has revealed a degree of purity equal to 2.5, and a yield with respect to the content of phycocyanin in the aliquot of crude extract subjected to purification of 90.0%.
Purification of Phycocyanin from Aqueous Extracts of Arthrospira platensis (Spirulina) Biomass on a Polycarbonate Membrane
A crude extract of phycobiliproteins was obtained as described above in Example 1. The chromatographic purification process was then carried out using a glass vacuum device for microfiltration assembled with a hydrophilic polycarbonate membrane (Whatman Nuclepore, average pore size 1.0 μm, diameter 47 mm, thickness 10 μm, code 111110); a chromatographic cycle of cleaning and a chromatographic cycle of purification were carried out, as described in Example 1. In particular we proceeded as follows:
cleaning cycle: the membrane was washed with 100 mL of deionized water and then conditioned with 5 mL of aqueous ammonium sulfate solution 0.6 M. At a rate of 0.75 mL of crude extract with a total phycobiliprotein content (PC+APC) equal to about 1 mg, a volume of aqueous solution of NaCl 100 mM and ammonium sulfate 3 M was added to give a final volume of extract in ammonium sulfate 0.6 M of 6 mL. The solution was loaded on the membrane and filtered; the permeate was collected in a 100 mL clean tailed flask. No blue compound was adsorbed on the membrane at the concentration of ammonium sulfate 0.6 M. The volume of the solution was carefully measured and, if necessary, it was brought back to 6 mL using an aqueous solution of ammonium sulfate 0.6 M;
purification cycle: to the permeate (obtained in the cleaning step described above) an aqueous solution of ammonium sulfate 3 M was added so as to obtain a phycobiliprotein solution having a concentration of ammonium sulfate 1.110 M. The solution was loaded onto a new polycarbonate membrane, using the usual procedure: the membrane was washed with 100 mL of deionized water and then conditioned with 5 mL of a solution of ammonium sulfate having the same concentration in ammonium sulfate of the phycobiliprotein solution (1.110 M). The phycobiliprotein solution in ammonium sulfate 1.110 M was loaded onto the membrane and filtered. The retentate bound onto the membrane was washed with 10 mL of an aqueous solution of ammonium sulfate 1.110 M, then desorbed with 10 mL of a 100 mM NaCl aqueous solution, recovered in a 100 mL clean tailed flask and its degree of purity was determined by spectrophotometry.
Phycocyanin was obtained with a purity degree of 2.44, and with a yield with respect to the phycocyanin content in the crude extract aliquot subjected to purification of 11.7%.
Purification of B-Phycoerythrin (B-PE) from Aqueous Extracts of Porphyridium cruentum Biomass on PVDF Membrane
A crude phycobiliprotein extract was obtained by suspending the freeze-dried P. cruentum biomass in an aqueous solution of NaCl 100 mM (120 mg of powder in 15 mL of solution). The suspension was kept at 4° C. for 24 hours, then centrifuged for 45 minutes (12,000 rcf, temperature=10° C.). The supernatant containing B-PE (hereinafter "crude extract") was recovered and stored at 4° C.
The B-PE purification process was carried out by using a syringe as the chromatographic device and filters for syringes with membranes made of hydrophilic PVDF having a porosity 0.45 μm (Durapore®, average pore size 0.45 μm, Ø 33 mm, code SLHV033RS) or 0.22 μm (Durapore®, average pore size 0.22 μm, Ø 33 mm, code SLGV033RS) and applying the purification process described in the following. No cleaning cycle was carried out, while the purification cycle was performed as follows: an aqueous solution of NaCl 100 mM and ammonium sulfate 3 M were added to a 0.3 mL aliquot of crude extract with a B-PE content of about 0.1 mg, so as to have a final volume of 1.15 M ammonium sulfate solution equal to 3 mL. The filter was washed with 5 mL of deionized water and then conditioned with 3 mL of ammonium sulfate 1.15 M. The extract solution in ammonium sulfate 1.15 M was loaded onto a PVDF filter with 0.22 μm porosity, and in parallel on a filter, always made of PVDF, with medium-sized pores 0.45 μm, conditioned with ammonium sulfate 1.15 M. After filtration, the retentate on the membrane was washed with 3 mL of aqueous ammonium sulfate solution 1.15 M, recovered with 3 mL of NaCl 100 mM and its degree of purity was spectrophotometrically determined.
In both cases, B-phycoerythrin (B-PE) was obtained, having a degree of purity of about 3.95 and a yield of approximately 50%.
A crude phycobiliprotein extract was obtained by suspending 170 mg of freeze-dried P. cruentum biomass in powder in 21 mL of saline phosphate buffer (PBS, sodium phosphate 10 mM, NaCl 100 mM) at pH 7. The suspension was maintained at 4° C. for 24 hours, then centrifuged for 45 minutes (12,000 rcf, temperature=10° C.). The supernatant containing B-PE (hereinafter "crude extract") was recovered and stored at 4° C.
The B-PE chromatographic purification process was carried out using a glass vacuum device for microfiltration assembled with a hydrophilic PVDF membrane (Durapore®, average pore size 0.45 μm, diameter 47 mm, code HVLP 04700) and carrying out the cleaning and purification cycles as follows:
cleaning cycle: the membrane was washed with 100 mL of deionized water and then conditioned with 5 mL of an aqueous solution of ammonium sulfate 0.83 M. PBS at pH 7 and an aqueous solution of ammonium sulfate 3 M were added to an aliquot of 2 mL of crude extract with B-PE content equal to about 0.7 mg, so as to have a final volume of 9 mL of extract in a solution of ammonium sulfate 0.83 M. The solution was loaded on the membrane and filtered; the permeate was collected in a 100 mL clean tailed flask. The volume of the solution was carefully measured and, if necessary, it was brought back to 9 mL using an aqueous solution of ammonium sulfate 0.83 M.
purification cycle: to the permeate in aqueous solution of ammonium sulfate 0.83 M obtained from previous cleaning cycle, an aqueous solution of ammonium sulfate 3 M was added so as to obtain a solution of phycobiliproteins having a concentration of ammonium sulfate 1.224 M. This solution was loaded onto a new PVDF membrane, using the usual procedure: the membrane was washed with deionized water (100 mL) and then conditioned with 5 mL of an aqueous solution of ammonium sulfate having the same ammonium sulfate concentration as the solution of phycobiliproteins (1.224 M). The phycobiliprotein solution in ammonium sulfate 1.224 M was loaded onto the membrane and filtered. The retentate bound onto the membrane was washed with 10 mL of an aqueous solution of ammonium sulfate 1.224 M, then desorbed with 10 mL of PBS at pH 7, recovered in a 100 mL clean tailed flask and its degree of purity was spectrophotometrically determined.
B-phycoerythrin (B-PE) was obtained with a purity degree of 4.0, and with a yield with respect to the B-PE content in the crude extract aliquot, subjected to purification, of 38.0%.
The present invention has been so far described with reference to its preferred embodiments. It is to be understood that there may be other embodiments which refer to the same inventive core, all falling within the scope of protection of the claims set forth in the following.
[1] P. Yu, Y. Wu, G. Wang, T. Jia, Y. Zhang, Purification and bioactivities of phycocyanin, Crit. Rev. Food Sci. Nutr. 57 (2017) 3840-3849. DOI: 10.1080/10408398.2016.1167668;
[2] Q. Liu, Y. Huang, R. Zhang, T. Cai, Y. Cai, Medical application of Spirulina platensis derived C-phycocyanin, Evid.-based Complement. Altern. Med. 2016 (2016). DOI: 10.1155/2016/7803846;
[3] G. Patil, K. S. M. S. Raghavarao, Aqueous two phase extraction for purification of C-phycocyanin, Biochem. Eng. J. 34 (2007) 156-164. DOI: 10.1016/j.bej.2006.11.026;
[4] R. Chaiklahan, N. Chirasuwan, V. Loha, S. Tia, B. Bunnag, Separation and purification of phycocyanin from Spirulina sp. using a membrane process, Bioresour. Technol. 102 (2011) 7159-7164. DOI: 10.1016/j.biortech.2011.04.067;
[5] K. Minkova, M. Tchorbadjieva, A. Tchernov, M. Stojanova, L. Gigova, M. Busheva, Improved procedure for separation and purification of Arthronema africanum phycobiliproteins, Biotechnol. Lett. 29 (2007) 647. DOI 10.1007/s10529-006-9274-5;
[6] B. Soni, U. Trivedi, D. Madamwar, A novel method of single step hydrophobic interaction chromatography for purification of phycocyanin from Phormidium fragile and its characterization for antioxidant property, Bioresour. Technol. 99 (2008) 188-194. DOI: 10.1016/j.biortech.2006.11.010;
[7] G. Patil, S. Chethana, M. C. Madhusudhan, K. S. M. S. Raghavarao, Fractionation and purification of the phycobiliproteins from Spirulina platensis, Bioresour. Technol. 99 (2008) 7393-7396. DOI: 10.1016/j.biortech.2008.01.028;
[8] S. Muthukumar, T. Muralikrishnan, R. Mendhe, A. S. Rathore, Economic benefits of membrane chromatography versus packed column purification of therapeutic proteins expressed in microbial and mammalian hosts, J. Chem. Technol. Biot. 92 (2017) 59-68. DOI: 10.1002/jctb.5064;
[9] Z. Liu, S. R. Wickramasinghe, X. Qian, Membrane chromatography for protein purifications from ligand design to functionalization, Sep. Sci. Technol. 52 (2017) 299-319. DOI: 10.1080/01496395.2016.1223133.
1. A process for the separation and purification of phycobiliproteins starting from crude extracts of cyanobacteria and/or algae containing them, comprising a purification cycle of membrane chromatography comprising:
i) passing a saline aqueous solution of said crude extracts on a porous hydrophilic membrane, having low binding affinity for proteins, as the stationary phase, wherein said saline aqueous solution of crude extracts of phycobiliproteins is an aqueous solution of a salt S having a concentration [S]1 that is a first mobile phase;
ii) desorption of a retentate bound to said membrane with a solvent that is a second mobile phase and is selected from the group consisting of water, an aqueous solution of said salt S at a concentration [S]2<[S]1, and an aqueous solution of a salt S′ that is a chaotropic agent stronger than said salt S.
2. The process according to claim 1, further comprising, upstream of said purification cycle, a chromatographic cleaning cycle on said porous hydrophilic membrane of said crude extracts in an aqueous solution of said salt S at concentration [S]0 that is a third mobile phase, lower than said concentration [S]1 of said salt S in the step i).
3. The process according to claim 1, wherein said porous hydrophilic membrane is a hydrophilic membrane in PVDF (polyvinylidene fluoride) or in polycarbonate.
4. The process according to claim 3, wherein said porous hydrophilic membrane is a hydrophilic membrane in PVDF.
5. The process according to claim 1, comprising more purification cycles carried out consecutively at the same or increasing concentrations of the saline aqueous solution of said salt S by passage on said porous hydrophilic membrane of the permeate obtained from the preceding cycle up to the separation of the desired phycobiliprotein having the desired purity degree.
6. The process according to claim 1, wherein said porous hydrophilic membrane, before each said purification cycle and/or before said cleaning cycle, is previously treated with a saline aqueous solution of said salt S at concentration [S]n, wherein [S]n is the concentration of the salt S in the cleaning step or in the nth cycle of purification.
7. The process according to claim 1, wherein said crude extracts are extracts of cyanobacteria or algae selected from extracts of Arthrospira platensis (Spirulina) and extracts of Porphyridium cruentum.
8. The process according to claim 1, wherein said salt S is ammonium sulfate.
9. The process according to claim 1, wherein said aqueous solution of salt S′ is selected from aqueous solutions of sodium chloride and saline phosphate buffer.
Filed: Jun 4, 2019
Applicant: Consiglio Nazionale delle Ricerche (Roma)
Inventors: Rosaria LAUCERI (Cambiasca (VB)), Giuseppe TORZILLO (Campi Bisenzio (F!)), Graziella CHINI ZITTELLI (Borgo San Lorenzo (FI))
International Classification: C07K 14/405 (20060101); C07K 1/16 (20060101); C07K 1/34 (20060101); C07K 1/36 (20060101);


text:
Sand sculptor shares talents at PigOut
By Tanya Sampson / Herald Writer
Thursday, June 15, 2006 9:00pm
LifeGo-See-Do
Most people build sandcastles at the beach for fun.
Russ Leno, an Everett resident, takes the common summertime activity to another level.
Leno, a world-champion sand sculptor, carves anything he can get his hands on.
Pumpkins, ice and wood are a few of the mediums he works with regularly. Apples, watermelons and Spam are a few of the rarer mediums he's carved.
He's carved pumpkins for Jay Leno (no relation) on the Tonight Show, on Martha Stewart's show and for the former Vice President Al Gore and wife Tipper's annual Halloween party.
Leno's long list of accomplishments could intimidate any ambitious amateur looking to build a masterpiece in the sand.
But this weekend, Leno, his son Jake and fellow master sculptor Rob Cross will assist participants in an amateur sand-sculpting competition new at this year's PigOut on the Farm, a Marysville Strawberry Festival event.
Leno said anybody interested in sand sculpting should give it a try.
"It's kind of the first event of its kind in the area," Leno said. "Those people who would like to come out and compete can do so."
Individuals and teams are invited to try their hand at sand sculpting on Saturday and Sunday. Judges will award the top three creations on Sunday afternoon.
In addition to assisting competitors, Leno said he and his team will build a 6- to 8-foot-tall sculpture incorporating the PigOut on the Farm and the Marysville Strawberry Festival in the design using five to six cubic yards of sand.
"We're going to build a sandcastle and try to incorporate some strawberries and some pigs," he said.
In addition to the sand-sculpting area, five other areas at the farm offer visitors to the 17th annual event plenty of family activities.
A play land brings children a five-sided "Strawberry Mountain" slide, puppet shows, a playhouse and castle maze. A 28-foot rock-climbing wall will also be set up. Clowns and the "Giant Strawberry" ride round out the fun.
The Deadwood Ranch features barnyard animals, pony rides and old-fashioned games such as gunny-sack races, baby crawl races and mini-tractor races. Farm implements, a large tee-pee and a 25-foot high "Goat Bridge" also add to the fun.
The Northshore Chapter of Trout Unlimited sponsors a free fishing pond for children from 10 a.m. to 5 p.m. both days. Children age 16 and younger and accompanied by an adult can fish in the pond stocked with trout by the Department of Fish and Wildlife.
Families looking to pig out can feast on roast pig, corn on the cob, Kettle Korn and the event's signature "pig trough" strawberry shortcake dessert.
Organizers of the berry festival said berries are ripe for the plucking. Visitors can ride out to the u-pick berry patch at the farm on the farm's trolley. The fields are filled with ripe strawberries, raspberries and tayberries.
Or for convenience, berry lovers can purchase pre-picked fruits by the flat or containers filled with sliced and sugared strawberries.
For those who want to watch the PigOut's sand-sculpting competition to determine if it's something they're interested in, another open sculpting competition takes place the following weekend in Ocean Shores.
A sample of Russ Leno's sand sculpting.
Sorticulture
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Fine-tune your coping skills for when life gets difficult
Here are four ways to develop healthy ways to deal with the stress that inevitably comes our way.
Poland's milk bars dish up memories and cheap eats
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Trainline charges a $43 change fee after train was canceled
When Neale Gonsalves' train trip from Stockport, England, to London is canceled, he rebooks on another train. But Trainline, his ticket agency, charges him a $43 ticket change fee. Is that allowed?
Music, theater and more: What's happening in Snohomish County
Village Theatre's production of "Cinderella" closes Jan. 29 in Everett.
Fruit tree season is upon us. Choose wisely
Unlike growing veggies, fruit trees are a long-term investment, so make sure you start out on the right foot.
This injury changed me from a 'human-doing' to a 'human-being'
A painful torn calf muscle doesn't require surgery — just a LOT of rest. So pass the Advil and the TV remote.
Great Plant Pick: Giant snowdrops
This bulb flowers early — as in right now. It also rewards gardeners with a honey-like fragrance.
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A few ground rules, on screen time especially, will help adults raise kids with the capacity to amuse themselves.
2023 Lexus IS 500 F SPORT Premium
The 2023 Lexus IS 500 F SPORT Premium is as good as it gets for a luxury sports sedan at a mid-grade price.


1 Way Only--911: Whom Do You Call First?
All scripture references are linked to The Bible Gateway. When you click on a scripture reference, it may take a few moments for it to load. Once you are finished reading the scripture reference, click "back" on your browser to return to this page.
The tragic and horrifying events of September 11, 2001 (9/11) will remain in our memories for the rest of our lives. Visions of hijacked airliners containing fellow Americans purposely crashing into buildings full of other unsuspecting citizens going about their daily lives are horrible and mind-numbing. It doesn't seem real; these things just don't happen in the United States. News flash: It did happen. It is real. 9-1-1.
How could this happen? This world is full of evil. Our enemy, Satan, is always looking for opportunities. And he never hesitates to use them, either. No doubt, one of his favorites things is the opportunity to use someone who is full of hatred and rage. Add some pilot training, some financial support and a few plane tickets. Voilé! Major disaster. But the same thing is true of any other terrible occurrence in any of our lives. It doesn't have to be a major disaster of global proportions. Satan will use any opportunity he can. These bad things don't come from God, but sometimes He does allow them to happen.
3)These events are to serve as a wake-up call for America so that we can again turn to God. And I believe God allowed the forces of evil to deliver this blow on a day with a significant date for Americans. It occurred on September 11 or 9-1-1, the number most Americans recognize as the telephone number for emergency assistance. Can you not imagine God saying to us, "The enemy is attacking you. You need help. Call 9-1-1. Just whom will you call?" (See 2 Chronicles 7:14.) In addition, could it be that God allowed those two particular landmarks, the Pentagon and the World Trade Center, to be damaged/destroyed as a significant message that we cannot depend upon our military might or our money? Indeed, will we wake up? Just whom will we call!?
What can be done now? Think back to these events as you witnessed them for the first time. For most of us, it was via television, as we sat with our eyes glued to the screen and with our mouths gaping open in disbelief. What were your first thoughts upon viewing this unprecedented attack? Be honest with yourself. Were your first thoughts of our almighty God?
On recorded media, I have now heard the responses of several people unfortunate enough to be near the World Trade Center towers that fateful day as they watched the second plane smash into the second tower and as they watched the towers crumble to the ground from a safe distance. Over and over, as the news media plays those tapes, I hear fellow Americans say, "Oh my God." They are different Americans in different locations near the World Trade Center in New York City, but they have one common response, "Oh my God." But I wonder, has that statement sadly become just a cliché to most Americans? Were those people truly calling out to the one true God, our Creator, our Savior? Did they truly give Him a thought at those moments? Did you?
When faced with a crisis of your own, do you place your trust in God first? Or is He your second, third, or last resort? When you have smaller troubles in your everyday life, do you depend upon God first? Or do you tell your troubles to your spouse, friends, co-workers, or whoever will listen and only turn to God later, after dealing with the situation yourself in your own way?
Call it 9-1-1, call it prayer, or call it relying upon God's constant presence in the lives of His followers. Call it whatever you like. But don't forget God. Call upon Him first, no matter whether your needs are great or small, whether in time of distress or peace. We must not only seek Him in times of distress, either. We must seek God always. That's what each of us must do. Everything good is from God (James 1:17), and we must depend upon God to deliver us (2 Corinthians 1:9-11). Unless we have forgotten Him, our first call will always be to the Lord.


Click map above to view it larger.
Take US 23 south to US 12 West, through Saline and Clinton. Head south on M-52 about 15 minutes to Adrian. Take a left on Siena Heights Drive at the stop light, CVS located on the right corner. Drive 1 mile to Addison Street and take a right. Fairgrounds sign posted. Keeping to your left, it will turn into N. Dean Street. There are two entrances off of Dean Street. You will pass the north gate entrance and proceed to the Main Entrance gate. Proceed to the parking area at no charge.
Take US 127 South to US 223 South to Adrian. Go left on B.R. 223, just past the Carlton Lodge. Follow B.R. 223 also Maumee Street all the way through downtown Adrian. You will pass through 4 stop lights and 1 four-way stop. When you reach N. Dean Street, take a left. The Main Entrance gate is on your right. Proceed to the parking area at no charge.
Take US 23 North to US 223 North/West towards Adrian. Turn right on Treat Hwy., BP Gas Station on corner. Proceed 1 mile to Beecher Street, stay to the right and turning right in the right turn lane. Then turn left on Gulf Street. Fairgrounds signs posted. Proceed on Gulf Street until you dead end at Maumee Street. Take a left and the quick right on N. Dean Street. The Main Entrance gate is on your right. Proceed to the parking area at no charge.
Take RT 109 North, (turns into M52) through Jasper and Fairfield. Follow M52 also Main Street all the way through downtown Adrian. Take a right on Maumee Street at the 4 way stop. Follow Maumee Street through 1 stop light and 1 four-way stop. When you reach N. Dean Street, take a left. The Main Entrance gate is on your right. Proceed to the parking area at no charge.


text:
Q: Find Matrix representation of Linear Transformation Given $\beta = ${$1,x^2,x$} and $\gamma = ${$(1,1), (1,0)$},
$T: P^2(\Bbb{R}) \to \Bbb{R}^3 $ is $ T(a + bx + cx^2) = (a + b, a + c)$, calculate $[T]_\beta^\gamma$.
I just need confirmation that I'm not crazy and that there is a typo - the transformation maps to $\Bbb{R}^2$ right?
And how do I calculate $[T]_\beta^\gamma$? I've never been good at this. Do I transform it through $\beta$ then $\gamma$?
Edit: After tips from Lem.ma, I got
$T(1) = (1,1) = 1(1,1) + 0(1,0)$
$T(x^2) = (0,1) = 1(1,1) + -1(1,0)$
$T(x) = (1,0) = 0(1,1) + 1(1,0)$
So,  $[T]_\beta^\gamma$ = $\begin{bmatrix} 1&1&0\\0&-1&1 \end{bmatrix}$ ?

redpajama_set_name: RedPajamaStackExchange


Reserve Bank of India's deputy governors has encouraged local banks to work with a research outfit previously established by the central bank on blockchain projects.
In a speech during an event at the Institute for Development and Research in Banking Technology (IDRBT) on 19th July, Deputy Governor Rama Gandhi said banks should work to develop applications for digital currencies and distributed ledgers.
The speech focused on banking technology more broadly, but according to Gandhi, the IDRBT is an effective means to test possible applications.
Gandhi has spoken on the topic in the past, arguing last summer that digital currencies can fuel money laundering efforts during a banking event in India. Deputy governor Subhash Sheoratan Mundra said at the same event that financial institutions should collaborate on use cases.
The Reserve Bank of India has been investigating applications of the technology, including the possible issuance of its own digital currency, since as early as 2014, according to reports at the time.
Central bank governor Raghuram Rajan said later that year that the institution could release its own digital currency, but that such an issuance could take years to undertake if attempted.


The expansive flybridge, for instance, creates a number of sociable scenarios; there's a large seating area around a dining table that can be lowered to convert into a sunpad, as well as forward-facing seating alongside the upper helm – an exhilarating place to be whilst under way.
The submersible bathing platform offers a delightful way to enjoy the water and can also accommodate a jet tender. Move through to the large welcoming saloon and you will find relaxed seating areas to both port and starboard and sociable considerations like the forward-facing companionway seating alongside the helm. The naturally illuminated galley is carefully considered and perfectly specified. Below, the three cabins reflect the Squadron's unmistakably high standards; with the flexibility of a dinette option in place of the third cabin.


text:
Црква Свете Тројице у Делиблату је румунска православна црква која је изграђена 1927. године. Представља једну од најлепших цркви Румунске православне цркве у јужном Банату.

Опште информације

Црква припада епархији Дакија Феликс, њена градња започета је 1925. године, а црква је завршена 1927. године. Посвећена је Светом Тројству.

Специфичне је архитектуре, а јединствена по томе што су у њој осликани  румунски патријарх Јустин и српски Герман, који су предводили ове две цркве у време када је храм грађен, што говори о културној и историјској блискости српског и румунског народа у Јужном Банату.

Поред њих осликане су и велике румунске војводе Михаил, Стефан, Мирчеа (предводио хришћане у бици на Ровинама у којој је погинуо Марко Краљевић) и Влад Цепеш (познатији као гроф Дракула).

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 Епархијa Дакија Феликс

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Цркве Епархије Дакије Феликс
Цркве у Војводини
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