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Cisgenesis The Dutch government has proposed to exclude cisgenic plants from the European GMO Regulation, in view of the safety of cisgenic plants compared to classically bred plants, and their contribution to durable food production. A related classification scheme proposed by Kaare Nielsen is:

Biology

https://en.wikipedia.org/wiki?curid=21660626

Cisgenesis

Tagging of Pacific Predators (TOPP) began in 2000 as one of many projects formed by Census of Marine Life, an organization whose goal is to help understand and explain the diversity and abundances of the ocean in the past, present, and future. After they were formed, TOPP began by building a coalition of researchers from all over the world to find and study predators of the Pacific Ocean. Since then, they have satellite-tagged 22 different species and more than 2,000 animals. These animals include elephant seals, great white sharks, leatherback turtles, squid, albatrosses, and more. Through the efforts of TOPP, information never before accessed by humans was now available, such as migration routes and ecosystems, but from the animals', rather than human, aspects. It also became possible to learn about the ocean itself through use of the animals, because they can go where humans cannot. We learn through their everyday actions, and through these data, researchers have been able to determine better ways of protecting endangered species, such as the leatherback turtle. The tagging research is ongoing, but the TOPP program itself ended in 2010. In addition to the NOAA’s Pacific Fisheries Ecosystems Lab, Stanford’s Hopkins Marine Lab, and University of California, Santa Cruz’s Long Marine Laboratory, a wide range of people contribute to the success of the program, including marine biologists, oceanographers, engineers, computer programmers, journalists, graphic designers, educators and the public who provide support

Biology

https://en.wikipedia.org/wiki?curid=21665334

Tagging of Pacific Predators

Tagging of Pacific Predators Many different types of tags are used in the TOPP tagging program, each designed for different marine animals and different types of data. Archival tags, though small, are very powerful, and can last up to 10 years. Researchers surgically implant them into the bellies of tuna, where the tags record, as often as every few seconds, pressure (for depth of dives), ambient light (to estimate location), internal and external body temperature, and, in some cases, speed of travel. The tags are small and light enough to be attached to the outside of an animal, such as the tail feathers of red-footed boobies. However, they do have a drawback, they have to be retrieved. So, they are useful for fish likely to be caught as seafood, such as bluefin or yellowfin tuna, or animals that return to rookeries or nesting beaches, such as boobies and leatherback turtles. Pop-up satellite archival tags (PSATs), also known simply as pop-up archival tags (PATs), are just like archival tags, except they are designed to release at a preset time, like 30 days. They then float to the surface and send their data via an Argos satellite back to the laboratory for two weeks, which is the life of its battery. Even when the battery dies, the data are saved on the tag, so if it is ever recovered, the whole data set can be downloaded. This tag is useful for animals that do not spend a lot of time at the surface, and are not caught often. Numerous white sharks have been tagged with this tag

Biology

https://en.wikipedia.org/wiki?curid=21665334

Tagging of Pacific Predators

Tagging of Pacific Predators The tags are attached to white sharks by inserting a small surgical titanium anchor into it. On elephant seals, the tag is glued to the fur. Connecting the tag to the anchor is a thin line that loops around a metal pin at the base of the tag. This metal pin is connected to a battery. A clock in the tag turns the battery on at a preprogrammed time. When the battery turns on, the attachment pin dissolves. The tag floats to the surface and starts transmitting data to one of the Argos satellites. Spot tags, or smart positioning, or temperature transmitting tags, are ideal tags for air-breathing marine animals (seals, whales and sea turtles) and animals that often swim close to the surface (salmon sharks, blue sharks and makos). When the antenna breaks the surface, it sends data to a satellite. The data include pressure, speed, and water temperature. Location is estimated by calculating the Doppler shift in the transmission signal in successive transmissions. When the animal goes beneath the surface, a saltwater switch turns off the tag. The tag, made by Wildlife Computers, lasts about two years. Satellite relay data logger (SRDL) tags compress data so more information can be transmitted through the Argos satellite. These can be outfitted with CTD tags to record the salinity, temperature and depth data oceanographers need to identify ocean currents and water. Elephant seals, sea lions and leatherback sea turtles wear these tags

Biology

https://en.wikipedia.org/wiki?curid=21665334

Tagging of Pacific Predators

Tagging of Pacific Predators TOPP has also set up programs to engage the public in learning about marine science and marine conservation. One of its programs is called Elephant Seal Homecoming Days, which was started by TOPP in 2008 for the months the northern elephant seals return to Año Nuevo State Reserve during the breeding season. TOPP picks around 10 of the many they tagged to become "spokes-seals" for the public, allowing them to see what it is like being one of the biggest seals in the world. Elephant seals are prime candidates for tagging because they tend to return to the same beach every year to breed, yielding high tag recovery. They can gather immense amounts of information because they can swim for thousands of miles. The seals are issued their own Facebook profile that is kept up for them by undergraduate volunteer keepers. Facebook profiles keep the public updated on their progress while they give birth and proceed to take care of their pups. Two seals are the stars of TOPP Elephant Seal Homecoming Days. The first is Penelope Seal, who has been part of the program since 2008. The second one is new as of 2009, and his name is Stelephant Colbert (in reference to Stephen Colbert of "The Colbert Report"). Stelephant started out as a mere pup in a small harem of northern elephant seals located at Año Nuevo State Reserve. He has since grown into one of the most famous elephant seals ever, even appearing on "The Colbert Report". Around eight years old, Stelephant is a 4,500-pound beast

Biology

https://en.wikipedia.org/wiki?curid=21665334

Tagging of Pacific Predators

Tagging of Pacific Predators Not known as the pretty-boy, he gets by on his aggressiveness and determination to get as close to females as possible. With his proboscis, or nose, high in the air, almost any male is turned away unless they wish to fight. These fights are not known to last for long, however, because Stelephant is a seasoned fighter and knows how to win. He is what every male elephant seal strives to be, huge, strong, and proud. When Stelephant Colbert was weaned, he spent the next few weeks on the beach waiting for the courage to go and join the adult seals out in the northern Pacific Ocean. He returned the next year to the same beach on the California coast as a yearling, where he was exiled to the outskirts of the harems with all the other yearlings. The next few years were spent building muscle and growing large to be able to compete with the larger males. During his third to fifth year, Stelephant practiced his fighting skills with other young males to build strength; while he did compete with the adults, he was not much competition and spent most of his days far away from females. When he was around seven years old with his proboscis finally grown in, he was finally ready to compete with the adult males for the alpha position. Stelephant has been involved with the University of California, Santa Cruz Long Marine Lab researchers, and was tagged last spring to track his foraging habits while swimming and diving off the Aleutian Islands in the northern Pacific Ocean

Biology

https://en.wikipedia.org/wiki?curid=21665334

Tagging of Pacific Predators

Tagging of Pacific Predators Stelephant was issued his own Facebook page, where people can keep track of his whereabouts and his status updates. He is the most talked-about seal in the program in that he was featured in the Santa Cruz Sentinel, the Associated Press, and many additional media organizations. The biggest media event was when Stephen Colbert of "The Colbert Report", after whom Stelephant is named, spoke of his seal on his show. Stelephant was featured in one of the show episodes, creating a media buzz. Since then, Stelephant’s fan based has skyrocketed bringing attention to TOPP and the UCSC Long Marine Lab who Stelephant is associated with. Stelephant has enabled TOPP to engage the public in learning about northern elephant seals, marine science, and marine conservation. Stelephant is now an alpha male at Año Nuevo State Reserve, and will soon return to the ocean to forage once again. Stelephant was featured on Oceans Google Earth, which allows the user to explore the oceans. His tags were recovered in January and he was not tagged again. Penelope Seal is an elephant seal and one of the many marine animals tagged as a part of the TOPP program. Penelope was born as a "little" pup in early January 1998 at the Año Nuevo State Reserve on the Californian coast. Since then, she has become a full grown female northern elephant seal with six pups of her own, and many fans who have contributed to her fame. From five years old, she has had one pup a year, all at Año Nuevo State Reserve

Biology

https://en.wikipedia.org/wiki?curid=21665334

Tagging of Pacific Predators

Tagging of Pacific Predators Penelope Seal is known as gnarly seal and a survivor, considering 50% of her species will die before they reach maturity. She has dark-brown fur that she molts (or sheds) once a year. The beach is a known hangout spot of hers and one can usually spot her basking in the sun, or if its raining, cuddling up to another elephant seal. She has had multiple relationships, never known to be a monogamous seal, and in fact living in a polygynous society. The group, or harem, is made up of many other females and an alpha male with which Penelope has been known to hang out. However, Penelope has also been seen socializing with some other beta males around the harem, much to the dismay of the alpha. Penelope spent her early life as a weaner, or weaned pup, at Año Nuevo State Reserve. During this time she fasted on the beach waiting until she was brave enough to go out into the open ocean for the first time. The next year she became a yearling and spent her days being shunned from her harem, since she was still small compared to the others. Years two and three were spent much the same way. During this period she learned a lot about how to become a successful predator, like being able to travel all the way out into the northern Pacific Ocean and back again without any maps, and making it to the same place every time, which can be around an journey. However, when she was four years old, she became pregnant with her first pup and gave birth to him 10 months later at Año Nuevo State Reserve

Biology

https://en.wikipedia.org/wiki?curid=21665334

Tagging of Pacific Predators

Tagging of Pacific Predators Penelope is tagged with a small tag and collects data for the TOPP team's researchers. Penelope's tag has a time depth recorder, which documents the dive depth, dive duration, and light levels. Elephant seals are incredible divers and allow scientists to learn about their unique behavior and the environment in which they live. Elephant seals are prime candidates for tagging because they tend to return to the same beach every year to breed, yielding high tag recovery. They can gather immense amounts of information because they can swim for thousands of miles. Penelope herself has been involved with the University of California, Santa Cruz Long Marine Lab researchers and was tagged last year. Penelope has since had her seventh pup, and is living on the beaches of Año Nuevo State Reserve. She was not tagged this year. Stelephant Colbert has since taken on the challenge of promoting elephant seals, and has been quite successful. Penelope is featured on the new version of Ocean Google Earth, which allows the user to explore the oceans. Penelope can be located at Año Nuevo State Reserve. Penelope Seal has been the star of Elephant Seal Homecoming Days for the past two years. She enabled TOPP to engage the public in learning about northern elephant seals, marine science, and marine conservation. The Great Turtle Race is an international sea turtle conservation event that brings together corporate sponsors and conservation organizations

Biology

https://en.wikipedia.org/wiki?curid=21665334

Tagging of Pacific Predators

Tagging of Pacific Predators The race tracks sea turtles as they move toward feeding areas south of the Galapagos Islands after nesting at Playa Grande in Costa Rica’s Las Baulas National Park, the primary nesting area for leatherbacks in the Pacific. TOPP is a cosponsor of The Great Turtle Race, and tracking technology created and used by TOPP is how the turtles are tracked. The race occurred in 2007 and 2008 in April.

Biology

https://en.wikipedia.org/wiki?curid=21665334

Tagging of Pacific Predators

Bacterial one-hybrid system The bacterial one-hybrid (B1H) system is a method for identifying the sequence-specific target site of a DNA-binding domain. In this system, a given transcription factor (TF) is expressed as a fusion to a subunit of RNA polymerase. In parallel, a library of randomized oligonucleotides representing potential TF target sequences are cloned into a separate vector containing the selectable genes HIS3 and URA3. If the DNA-binding domain (bait) binds a potential DNA target site (prey) "in vivo", it will recruit RNA polymerase to the promoter and activate transcription of the reporter genes in that clone. The two reporter genes, HIS3 and URA3, allow for positive and negative selections, respectively. At the end of the process, positive clones are sequenced and examined with motif-finding tools in order to resolve the favoured DNA target sequence. Across all living organisms, regulation of gene expression is controlled by interactions between DNA-binding regulatory proteins (transcription factors) and cis-regulatory elements, DNA sequences in or around genes that act as target sites for DNA-binding proteins. By binding to cis-regulatory sequences and to each other, transcription factors fine-tune transcriptional levels by stabilizing/destabilizing binding of RNA polymerase to a gene's promoter. But despite their importance and ubiquity, little is known about where exactly each of these regulatory proteins binds

Biology

https://en.wikipedia.org/wiki?curid=21673918

Bacterial one-hybrid system

Bacterial one-hybrid system Literature suggests that nearly 8% of human genes encode transcription factors and the functions and specificities of their interactions remain largely unexplored. We are on the brink of a convergence of high-throughput technologies and genomic theory that is allowing researchers to start mapping these interactions on a genome-wide scale. Only recently has a complete survey of DNA-binding specificities been attempted for a large family of DNA-binding domains. B1H is just one emerging technique among many that is useful for studying protein–DNA interactions. Transformation of a bacterial host with two different plasmids is required. One is designed to express a DNA-binding protein-of-interest as a fusion construct with a subunit of RNA polymerase (bait). The other plasmid contains a region of randomized sequence representing potential binding sites (prey) which, if bound to by the chimeric fusion product, drives expression of downstream reporter genes. This reporter region facilitates both positive and negative selection by HIS3 and URA3, respectively, which together allow for isolation of the prey containing the true DNA target sequence. HIS3 and URA3 encode proteins required for biosynthesis of histidine and uracil. Using a negative selectable marker is crucial for greatly reducing the incidence of false-positives

Biology

https://en.wikipedia.org/wiki?curid=21673918

Bacterial one-hybrid system

Bacterial one-hybrid system Self-activating prey, where the randomized region facilitate reporter expression in the absence of TF binding, are removed by transforming the reporter vector library into bacteria in the absence of bait and assaying for growth on plates containing 5-fluoro-orotic acid (5-FOA). The protein product of URA3 converts 5-FOA into a toxic compound, thereby allowing survival of only those colonies that contain reporter vectors which are not self-activating. Negative selection normally precedes positive selection so that a smaller, purified prey library can be subjected to the more rigorous positive selection process. Upon transformation of the purified prey library with the bait plasmid, positive selection is achieved by growing the host E. coli on minimal medium lacking histidine (NM selective medium) that is usually supplemented with varying concentrations of 3-amino-triazole (3-AT), a competitive inhibitor of HIS3. HIS3 encodes a protein required for histidine biosynthesis and thus only those cells containing bait-prey combinations that activate the reporter genes will be able to grow. Manipulating 3-AT concentrations allows for the characterization of binding stringencies. In this way, researches can gauge how strongly bait binds its prey (correlated with the level of expression of HIS3) and thus determines which nucleotide binding-sites have strong or weak preferences for a given base

Biology

https://en.wikipedia.org/wiki?curid=21673918

Bacterial one-hybrid system

Bacterial one-hybrid system In other words, if cells can grow despite a high concentration of 3-AT, bait-prey binding must be of high enough stringency to drive reporter gene expression (HIS3) at a sufficient level to overcome the resulting competitive inhibition. Finally, positive clones are sequenced and examined with preexisting motif-finding tools (ex, MEME, BioProspector). The bacteria one-hybrid system has undergone numerous modifications since its inception in 2005. It ultimately arose as a variation of the bacteria two-hybrid system, conceived in 2000, which itself was inspired by the yeast one- and two-hybrid systems. Whereas the two-hybrid versions can assess both protein–protein interaction and protein–DNA interactions, the one-hybrid system specializes in the latter. Meng et al.’s B1H system differs from the two-hybrid version in two key respects. It uses a randomized prey library consisting of many (<2x10) unique potential target sequences and also adds a negative selection step in order to purge this library of self-activating clones. Although these ideas were borrowed from the original yeast one-hybrid system, they had not yet been applied to a bacterial host before 2005. As the technique grew in popularity, researchers amended their protocols to improve the B1H system. Designing the fusion construct (bait) to the omega, rather than the alpha, subunit of RNA polymerase has recently been favoured in order to improve the chimera’s stereochemistry and dynamic range

Biology

https://en.wikipedia.org/wiki?curid=21673918

Bacterial one-hybrid system

Bacterial one-hybrid system A zinc-finger domain on the fusion construct and its corresponding DNA target site, adjacent to the randomized prey sequence, has also been added to the increases affinity and specificity of protein–DNA interactions. This increased overall binding affinity allows for the characterization of even those DNA-binding domain proteins which interact weakly with a target sequence. The B1H system has significant advantages over other methods that investigate protein–DNA interactions. Microarray-based readout of chromatin immunoprecipitation (ChIP-chip) for high-throughput binding-site determination relies on specific antibodies which may not always be available. Methods that rely on protein-binding microarrays also require additional protein purification steps that are not required in the B1H system. Furthermore, these microarray-based techniques are often prohibitive in terms of requiring special facilities and expertise to analyze the resulting data. SELEX, another system commonly used to identify the target nucleic acids for DNA-binding proteins, requires multiple rounds of selection. In contrast, the bacterial one-hybrid system requires just one round of in vitro selection and also offers a low-tech alternative to microarray-based technologies. Antibodies are not required for studying the interactions of DNA-binding proteins in the B1H system. A further advantage is that the B1H system works not only for monomeric proteins but also for proteins that bind DNA as complexes

Biology

https://en.wikipedia.org/wiki?curid=21673918

Bacterial one-hybrid system

Bacterial one-hybrid system The B1H system should be considered a specialized technique for studying DNA-protein interactions whereas the two-hybrid variations (B2H and Y2H) can assess both protein–protein and protein–DNA interactions. These two-hybrid systems are multi-purpose but are limited in terms of assaying only a single “prey” library. An advantage of the bacterial one-hybrid system over the yeast one-hybrid system (Y1H) lies in the higher transformation efficiency of plasmids into bacteria which allows for more complex “prey” libraries to be examined. Despite its aforementioned advantages as a specialized tool, the B1H system does have some drawbacks. First, the B1H selection system is limited in its capacity to determine the binding specificities of transcription factors with lengthy binding sites. This arises from the fact that the number of randomized “prey” clones required to represent all possible target sequences increases exponentially with the number of nucleotides in that target sequence. Second, some eukaryotic factors may not express or fold efficiently in the bacterial system, attributed to differing regulatory networks and transcriptional machinery. Hence when working with DNA-binding proteins of eukaryotic origin, a yeast-based hybrid system may be beneficial. Third, the B1H system may not be ideally suited for transcription factors that recognize binding sites with low affinity

Biology

https://en.wikipedia.org/wiki?curid=21673918

Bacterial one-hybrid system

Bacterial one-hybrid system The logic here is that competition created by binding sites elsewhere in the bacterial genome may limit the signal that can be realized from a single binding site that is present upstream of the reporter. B1H system provides a tool in our arsenal for identifying the DNA-binding specificities of transcription factors and thus predicting their target genes and genomic DNA regulatory elements. It also allows for examination of the effects of protein–protein interactions on DNA binding, which may further guide the prediction of cis regulatory modules based on binding-site clustering. Moreover, the B1H selection system has implications for the predicting regulatory roles of previously uncharacterized transcription factors. Using the bacterial one-hybrid system, one study has characterized 35 members of the Drosophilia melanogaster segmentation network which includes representative members of all the major classes of DNA-binding domain proteins. Implications for medical research are evident from another study that used the B1H system to identify the DNA-binding specificity of a transcriptional regulator for a gene in Mycobacterium tuberculosis. The B1H system has also been used to identify an important turnover element in Escherichia coli.

Biology

https://en.wikipedia.org/wiki?curid=21673918

Bacterial one-hybrid system

Germs: Biological Weapons and America's Secret War (2001) describes how humanity has dealt with biological weapons, and the dangers of bioterrorism. It was written by "The New York Times" journalists Judith Miller, Stephen Engelberg, and William Broad and was the 2001 "New York Times" #1 Non-Fiction Bestseller the weeks of October 28 and November 4. "Germs", is a work of investigative journalism employing biographical and historical narrative to provide context. The three authors interviewed hundreds of scientists and senior U.S. officials, and reviewed recently declassified documents, and reports from the former Soviet Union's bioweapons laboratories. The book opens with an account of the 1984 salmonella poisonings in The Dalles, Oregon, caused by followers of Bhagwan Shree Rajneesh who sprayed salmonella onto salad bars. Other research shows how Moscow scientists created an untraceable germ that would induce the body to self-destruct, and reveals that the U.S. military planned for germ warfare on Cuba during the 1960s. Three classified U.S. biodefense projects are detailed: Project Bacchus, Project Clear Vision, and Project Jefferson. "Germs" concludes with an assessment of the United States' ability to deter future bio-attack. "The New York Times Book Review" was favorable, though it criticized the book's tone as "somewhat alarmist". "BusinessWeek" was also generally favorable, except for pointing out some conflicting views on bioterrorism

Biology

https://en.wikipedia.org/wiki?curid=21675886

Germs: Biological Weapons and America's Secret War

Germs: Biological Weapons and America's Secret War "The Guardian"'s book review by British psychiatrist Simon Wessely, cautioned against panic, stating that biological weapons can cause destruction through fear, effectively giving the biodefense industry "the equivalent of a blank cheque". On November 13, 2001, the science TV series "Nova" aired an episode entitled "Bioterror". Two years in the making, it chronicled Miller, Engelberg, and Broad's research and investigation into biological weapons.

Biology

https://en.wikipedia.org/wiki?curid=21675886

Germs: Biological Weapons and America's Secret War

Tribromometacresol is an antifungal medication.

Biology

https://en.wikipedia.org/wiki?curid=21679739

Tribromometacresol

TB6Cs2H1 snoRNA TB6Cs2H1 is a member of the H/ACA-like class of non-coding RNA (ncRNA) molecule that guide the sites of modification of uridines to pseudouridines of substrate RNAs. It is known as a small nucleolar RNA (snoRNA) thus named because of its cellular localization in the nucleolus of the eukaryotic cell. TB6Cs2H1 is predicted to guide the pseudouridylation of SSU ribosomal RNA (rRNA) at residue Ψ1101.

Biology

https://en.wikipedia.org/wiki?curid=21686191

TB6Cs2H1 snoRNA

TB11Cs3H1 snoRNA TB11Cs3H1 is a member of the H/ACA-like class of non-coding RNA (ncRNA) molecule that guide the sites of modification of uridines to pseudouridines of substrate RNAs. It is known as a small nucleolar RNA (snoRNA) thus named because of its cellular localization in the nucleolus of the eukaryotic cell. TB11Cs3H1 is predicted to guide the pseudouridylation of LSU3 ribosomal RNA (rRNA) at residue Ψ1308.

Biology

https://en.wikipedia.org/wiki?curid=21686256

TB11Cs3H1 snoRNA

Gametophore The word gametophore, also known as gametangiophore, is composed of gametangium and "phore" (Greek Φορά, "to be carried"). In moss and fern (Archegoniata) the gametophore is the bearer of the sex organs (gametangia), the female archegonia and the male antheridia. If both the archegonia and antheridia are arranged at the same plant, they are called monoicous. If there are female and male plants they are called dioicous. In Bryopsida the leafy moss plant (q. v. "Thallus") is called gametophore. It is the adult form of the haploid gametophyte and develops from the juvenile form, the protonema, under the influence of phytohormones (mainly cytokinins). Whereas the filamentous protonema grows by apical cell division, the gametophore grows by division of three faced apical cells.

Biology

https://en.wikipedia.org/wiki?curid=21686918

Gametophore

Clone manager Clone Manager is a commercial bioinformatics software work suite of Sci-Ed, that supports molecular biologists with data management and allows them to perform certain "in silico" preanalysis.

Biology

https://en.wikipedia.org/wiki?curid=21688106

Clone manager

Antibody Solutions Headquartered in Santa Clara, Calif., is a privately held American contract research organization that provides research and discovery services and fit-for-purpose antibodies to biopharmaceutical and diagnostic companies and academic researchers worldwide. The company’s services include monoclonal and polyclonal antibody and antigen development, molecular modeling, antibody sequencing and engineering, bioreactor technology, pharmacokinetic studies, antibody epitope binning, peptide synthesis, immunoassay development, ligand-binding assay analysis, and support for CAR-T research. was founded in 1995 by current president, John Kenney, PhD, Judith Lynch-Kenney, and Dennis and Bette Gould. Dr. Kenney had managed the monoclonal antibody (MAb) development laboratory for Syntex Research (Roche) in Palo Alto, Calif., and Gould managed the MAb production facility for Syva Diagnostics. Gould left in 1997 and eventually became a vice president for Sepragen Corporation. Kenney and Gould were assisted in the startup of the company by Barry Bredt, who had the foresight to acquire the domain name, www.antibody.com, for the company. Bredt later became director of the University of California at San Francisco’s General Clinical Research Center, and was a pioneer of the HIV/AIDS Diagnostics "EASYCD4." Bredt passed away on April 8, 2007. was incorporated in 1998 with Dr. Kenney as president and Judith Lynch-Kenney as chief financial officer

Biology

https://en.wikipedia.org/wiki?curid=21691693

Antibody Solutions

Antibody Solutions One of the first independent antibody discovery companies, the firm focused on custom antibody discovery for therapeutics, diagnostics and critical reagents. The company pioneered the use of bioreactors in antibody research by becoming the first CRO to produce antibodies in CELLine flasks in 2000. was headquartered for several years in Mountain View, a short distance from Shoreline Park, Moffett Field and the Googleplex. In 2011, the company moved to new facilities in Sunnyvale and then moved to its current location at 3033 Scott Blvd. in Santa Clara near the San Tomas Expressway in mid-2019. Over the years, has had strategic agreements with a range of life science companies, including Open Monoclonal Technology, Inc. (OMT), Reflexion Pharmaceuticals, Guava Technologies, Single-Cell Technologies, Trianni, Harbour Antibodies, OmniAb and Alloy Therapeutics. Independent research conducted by and published or presented at scientific conferences include the following:

Biology

https://en.wikipedia.org/wiki?curid=21691693

Antibody Solutions

Globotriaosylceramide is a globoside. It is also known as CD77, Gb3, GL3, and ceramide trihexoside. It is one of the few clusters of differentiation that is not a protein. It is formed by the alpha linkage of galactose to lactosylceramide catalyzed by A4GALT. It is metabolized by alpha-galactosidase, which hydrolyzes the terminal alpha linkage. Defects in the enzyme alpha-galactosidase lead to the buildup of globotriaosylceramide, causing Fabry's disease. The pharmaceutical drug migalastat enhances the function of alpha-galactosidase and is used to treat Fabry's. is also one of the targets of Shiga toxin, which is responsible for pathogenicity of enterohemorrhagic "Escherichia coli" (EHEC). The bacterial Shiga toxin can be used for targeted therapy of gastric cancer, because this tumor entity expresses the receptor of the Shiga toxin. For this purpose an unspecific chemotherapeutical is conjugated to the B-subunit to make it specific. In this way only the tumor cells, but not healthy cells should be destroyed during therapy.

Biology

https://en.wikipedia.org/wiki?curid=21697382

Globotriaosylceramide

CDw17 antigen is a lactosylceramide. A4GALT acts upon it.

Biology

https://en.wikipedia.org/wiki?curid=21697492

CDw17 antigen

Marine Biological Association of the United Kingdom The (MBA) is a learned society with a scientific laboratory that undertakes research in marine biology. The organisation was founded in 1884 and has been based in Plymouth since the Citadel Hill Laboratory was opened on 30 June 1888. The MBA is also home to the National Marine Biological Library, whose collections cover the marine biological sciences, and curates the Historical Collections. Throughout its history, the MBA has had a Royal Patron. The current patron of the MBA is H.R.H. Prince Philip, Duke of Edinburgh. In 2013, the MBA was granted a Royal Charter in recognition of the MBA's scientific preeminence in its field. In 1866 the Royal Commission on the Sea Fisheries, which included among its officers Professor Thomas Henry Huxley, had reported that fears of over-exploitation of the sea fisheries were unfounded. They recommended removing existing laws regulating fishing grounds and closed seasons. However, the increase in the size and number of fishing vessels was causing widespread concern, and there were reports from all around the UK coasts about the scarcity of particular fish. This concern was expressed at the International Fisheries Exhibition in London in 1883, a conference called to discuss the commercial and scientific aspects of the fishing industry, and which was attended by many leading scientists of the day. Nevertheless, in his opening address, Huxley discounted reports of fish scarcities and repeated the views of the Royal Commission of 1866

Biology

https://en.wikipedia.org/wiki?curid=21699131

Marine Biological Association of the United Kingdom

Marine Biological Association of the United Kingdom He stated that with existing methods of fishing, it was inconceivable that the great sea fisheries, such as those for cod ("Gadus morhua"), herring ("Clupea harengus") and mackerel ("Scomber scombrus"), could ever be exhausted. Many of the representatives of science and commerce present had different views to Huxley. Their views were put forward by E. Ray Lankester, who summed up the scientific contributions in an essay on what we would now call ecology. He pointed out that "it is a mistake to suppose that the place of fish removed from a particular fishing ground is immediately taken by some of the grand total of fish, which are so numerous in comparison with man's depredations as to make his operations in this respect insignificant...there is on the contrary evidence that shoal fish, like herrings, mackerel and pilchard ("Sardina pilchardus"), and ground-fish, such as soles and other flat-fishes, are really localised. If man removes a large proportion of these fish from the areas which they inhabit, the natural balance is upset and chiefly in so far as the production of young fish is concerned." During this masterly address he went on to develop this theme and concluded with an appeal for the formation of a society to foster the study of marine life, both for its scientific interest and because of the need to know more about the life histories and habitats of food fishes

Biology

https://en.wikipedia.org/wiki?curid=21699131

Marine Biological Association of the United Kingdom

Marine Biological Association of the United Kingdom Professor Lankester envisaged that such a society would construct a laboratory close to the coast, with the building containing aquaria and apparatus for the circulation of seawater and, most importantly, laboratory accommodation for scientists. The appeal was answered by a group of eminent scientists, who resolved to form a society and build a laboratory on the British coast. The Committee formed at the International Fisheries Exhibition 1883 resolved to take action to establish a British Marine Laboratory, an initiative that ultimately led to the formation of the Marine Biological Association and building of the Laboratory in Plymouth. They were: The was formed at a meeting held in the rooms of the Royal Society in London on 31 March 1884. All but two of the signatories of the resolution of 1883 were present, together with some other scientists. By this time Professor Huxley had been persuaded to give his support and was elected as the first President of the Association, with Ray Lankester as Honorary Secretary. The MBA is governed by a Council which is headed by a President. The MBA's Director is responsible for the day-to-day running of the Association. Since 1884, the MBA has had fifteen Presidents.: There have been fourteen directors of the Marine Biological Association since its foundation: The MBA has a world-leading reputation for marine biological research, with some twelve Nobel laureates having been or being associated with it over the course of their career. Among them, A. V

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Marine Biological Association of the United Kingdom

Marine Biological Association of the United Kingdom Hill received the Nobel Prize in Physiology or Medicine in 1922 "for his discovery relating to the production of heat in the muscle". The discovery of the mechanism of nerve impulses (action potentials) in animals was made at the Laboratory in Plymouth by Sir Alan Lloyd Hodgkin and Sir Andrew Huxley, work for which they were awarded the Nobel Prize for Physiology or Medicine in 1963. The MBA publishes the "Journal of the Marine Biological Association of the United Kingdom". The current MBA Research Programme includes work on molecular and cell biology, physiology and ecology. A wide range of marine organisms are studied from microscopic organisms such as marine plankton and viruses and much larger species such as sharks and giant kelp. The objective of this research is to increase understanding of the structure and function of marine ecosystems. The association's research is led by a number of Research Fellows who each run an interdisciplinary group which collaborates with other organisations as well as obtaining funding for their work. The groundbreaking work of MBA research scientists has been recognised by many national and international awards over the years, including the Royal Society's Royal Medal, Darwin Medal and Croonian Lecture, the Zoological Society of London's Frink Medal, and the Japan Society for the Promotion of Science's International Prize for Biology

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Marine Biological Association of the United Kingdom

Marine Biological Association of the United Kingdom Long-term science observations of physical and biological parameters in the ocean have been collected by the MBA for over 100 years providing a foundation of data supporting studies aimed at understanding biological responses to marine environmental changes including effects of climate change. The National Marine Biological Library (NMBL) began in 1887 as the research support library for the MBA. Today, it provides research support for the MBA, the Sir Alister Hardy Foundation for Ocean Science and Plymouth Marine Laboratory. The NMBL's holdings include periodicals, serials, journals, reports and grey literature, a large collection of historical and modern books, an extensive reprint collection, and expedition reports. These cover the vast majority of the world. The NMBL’s Special Collections include the research libraries of several eminent MBA scientists; these are George Parker Bidder, Edward Thomas Browne, Sidney Frederic Harmer, E. Ray Lankester, Marie Victoire Lebour and John Zachary Young. Additionally, the NMBL curates the MBA Archive Collection which details the MBA's institutional history as well the history of marine biology in Britain since the late-nineteenth century, especially through the collection’s personal papers. These include scientific papers and material from Walter Garstang, Sidney Harmer, Hildebrand Wolfe Harvey, Thomas Hincks, Thomas V. Hodgson, Stanley W. Kemp, Charles A. Kofoid, Mary Parke, John Richardson, Frederick S. Russell, Thomas A

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Marine Biological Association of the United Kingdom

Marine Biological Association of the United Kingdom Stephenson, Walter Frank Raphael Weldon, Edward A. Wilson and William Yarrell. Since 1887, the MBA has published the "Journal of the Marine Biological Association" (JMBA), a scientific journal "publishing original research on all aspects of marine biology".

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Marine Biological Association of the United Kingdom

Multiple displacement amplification (MDA) is a non-PCR based DNA amplification technique. This method can rapidly amplify minute amounts of DNA samples to a reasonable quantity for genomic analysis. The reaction starts by annealing random hexamer primers to the template: DNA synthesis is carried out by a high fidelity enzyme, preferentially Φ29 DNA polymerase, at a constant temperature. Compared with conventional PCR amplification techniques, MDA generates larger sized products with a lower error frequency. This method has been actively used in whole genome amplification (WGA) and is a promising method for application to single cell genome sequencing and sequencing-based genetic studies. Many biological and forensic cases involving genetic analysis require sequencing of DNA from minute amounts of sample, such as DNA from uncultured single cells or trace amounts of tissue collected from crime scenes. Conventional Polymerase Chain Reaction (PCR)-based DNA amplification methods require sequence-specific oligonucleotide primers and heat-stable (usually "Taq") polymerase, and can be used to generate significant amounts of DNA from minute amounts of DNA. However, this is not sufficient for modern techniques which use sequencing-based DNA analysis. Therefore, a more efficient non-sequence-specific method to amplify minute amounts of DNA is necessary, especially in single cell genomic studies. Bacteriophage Φ29 DNA polymerase is a high-processivity enzyme that can produce DNA amplicons greater than 70 kilobase pairs

Biology

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Multiple displacement amplification

Multiple displacement amplification Its high fidelity and 3’–5' proofreading activity reduces the amplification error rate to 1 in 10−10 bases compared to conventional "Taq" polymerase with a reported error rate of 1 in 9,000. The reaction can be carried out at a moderate isothermal condition of 30 °C and therefore does not require a thermocycler. It has been actively used in cell-free cloning, which is the enzymatic method of amplifying DNA "in vitro" without cell culturing and DNA extraction. The large fragment of "Bst" DNA polymerase is also used in MDA, but Ф29 is generally preferred due to its sufficient product yield and proofreading activity. Hexamer primers are sequences composed of six random nucleotides. For MDA applications, these primers are usually thiophosphate-modified at their 3’ end to convey resistance to the 3’–5’ exonuclease activity of Ф29 DNA polymerase. MDA reactions start with the annealing of such primers to the DNA template followed by polymerase-mediated chain elongation. Increasing numbers of primer annealing events happen along the amplification reaction. The amplification reaction initiates when multiple primer hexamers anneal to the template. When DNA synthesis proceeds to the next starting site, the polymerase displaces the newly produced DNA strand and continues its strand elongation. The strand displacement generates newly synthesized single stranded DNA template for more primers to anneal. Further primer annealing and strand displacement on the newly synthesized template results in a hyper-branched DNA network

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Multiple displacement amplification

Multiple displacement amplification The sequence debranching during amplification results in high yield of the products. To separate the DNA branching network, S1 nucleases are used to cleave the fragments at displacement sites. The nicks on the resulting DNA fragments are repaired by DNA polymerase I. MDA can generate 1–2 µg of DNA from single cell with genome coverage of up to 99%. Products also have lower error rate and larger sizes compared to PCR based "Taq" amplification. General work flow of MDA: MDA generates sufficient yield of DNA products. It is a powerful tool of amplifying DNA molecules from samples, such as uncultured microorganism or single cells to the amount that would be sufficient for sequencing studies. The large size of MDA-amplified DNA products also provides desirable sample quality for identifying the size of polymorphic repeat alleles. Its high fidelity also makes it reliable to be used in the single-nucleotide polymorphism (SNP) allele detection. Due to its strand displacement during amplification, the amplified DNA has sufficient coverage of the source DNA molecules, which provides high quality product for genomic analysis. The products of displaced strands can be subsequently cloned into vectors to construct library for subsequent sequencing reactions. ADO is defined as the random non-amplification of one of the alleles present in a heterozygous sample. Some studies have reported the ADO rate of the MDA products to be 0–60%

Biology

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Multiple displacement amplification

Multiple displacement amplification This drawback decreases the accuracy of genotyping of single sample and misdiagnosis in other MDA involved applications. ADO appears to be independent of the fragment sizes and has been reported to have similar rate in other single-cell techniques. Possible solutions are the use of different lysis conditions or to carry out multiple rounds of amplifications from the diluted MDA products since PCR mediated amplification from cultured cells has been reported to give lower ADO rates. 'Preferential amplification' is over-amplification of one of the alleles in comparison to the other. Most studies on MDA have reported this issue. The amplification bias is currently observed to be random. It might affect the analysis of small stretches of genomic DNA in identifying Short Tandem Repeats (STR) alleles. Endogenous template-independent primer-primer interaction is due to the random design of hexamer primers. One possible solution is to design constrained-randomized hexanucleotide primers that do not cross-hybridize. Single cells of uncultured bacteria, archaea and protists, as well as individual viral particles and single fungal spores have been sequenced with the help of MDA. The ability to sequence individual cells is also useful in combating human disease. Genomes from single human embryonic cells have been successfully amplified for sequencing using MDA, allowing preimplantation genetic diagnosis (PGD): screening for genetic health issues in an early-stage embryo before implantation

Biology

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Multiple displacement amplification

Multiple displacement amplification Diseases with heterogeneous properties, such as cancer, also benefit from MDA-based genome sequencing's ability to study mutations in individual cells. The MDA products from a single cell have also been successfully used in array-comparative genomic hybridization experiments, which usually require a relatively large amount of amplified DNA. Chromatin Immunoprecipitation results in production of complex mixtures of relatively short DNA fragments, which is challenging to amplify with MDA without causing a bias in the fragment representation. A method to circumvent this problem was proposed, which is based on conversion of these mixtures to circular concatemers using ligation, followed by Φ29 DNA polymerase-mediated MDA. Trace amount of samples collected from crime scenes can be amplified by MDA to the quantity that is enough for forensic DNA analysis, which is commonly used in identifying victims and suspects.

Biology

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Multiple displacement amplification

Delitto perfetto () is a genetic technique for "in vivo" site-directed mutagenesis in yeast. This name is the Italian term for "perfect murder", and it refers to the ability of the technique to create desired genetic changes without leaving any foreign DNA in the genome. This technique was developed by a group at the National Institute of Environmental Health Sciences (NIEHS) composed of Michael A. Resnick, Francesca Storici (now at Georgia Institute of Technology), and L. Kevin Lewis (now at Southwest Texas State University). The method uses synthetic oligonucleotides in combination with the cellular process of homologous recombination. Consequently, it is well suited for genetic manipulation of yeast, which has highly efficient homologous recombination. The "delitto perfetto" approach has been used to produce single and multiple point mutations, gene truncations or insertions, and whole gene deletions (including essential genes). The primary advantage of this technique is its ability to eliminate any foreign DNA from the genome after the mutagenesis process. This ensures there are no selectable markers or exogenous sequences used for targeting left in the genome that may cause unforeseen effects. The "delitto perfetto" technique is also simpler compared to other methods for "in vivo" site-directed mutagenesis. Other methods require multiple cloning steps and extensive DNA sequencing to confirm mutagenesis, which is often a complicated and inefficient process

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Delitto perfetto

Delitto perfetto There is great flexibility in this approach because after the CORE cassette is inserted (see Method Overview for details), multiple mutations in the gene of interest can be made easily and quickly. This method can be applied to other organisms where homologous recombination is efficient, such as the moss "Physcomitrella patens", DT40 chicken cells, or "E. coli". In addition, human genes can be studied and similarly genetically manipulated in yeast by using yeast artificial chromosomes (YACs). Since the "delitto perfetto" technique is based on homologous recombination, this process must be functional in the cells for the technique to work. In "Saccharomyces cerevisiae", the "RAD52" gene is essential for homologous recombination, and thus is required for the "delitto perfetto" method. The method is useful only for applications where selectable markers are not necessary. For example, mutagenized yeast strains cannot be used for further genetic analysis such as tetrad analysis. Markers would have to be inserted into the appropriate locus in a separate process. "Delitto Perfetto" is a two step method for "in vivo" mutagenesis. In the initial step, the CORE cassette is inserted in the region of interest by homologous recombination. Subsequently, the CORE cassette is replaced with DNA containing the mutation of interest. The CORE cassette contains both a COunterselectable marker and REporter gene

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Delitto perfetto

Delitto perfetto The reporter gene allows for the selection of yeast cells that receive the CORE cassette during the first step of the process. The counterselectable marker allows for the selection of yeast cells that lose the CORE cassette by the integration of the mutated oligonucleotide during the second step of the process. There are a variety of CORE cassettes to choose from, which contain a variety of reporter genes, counterselectable makers and additional features. First the CORE cassette is amplified by PCR with primers containing regions of homology to the chromosomal site where it will be inserted. The CORE cassette is integrated via homologous recombination. Cells containing the CORE cassette can be selected for using the reporter gene and can be further confirmed using the counterselectable marker. Integration of the CORE cassette in the correct chromosomal location can be verified via PCR using primers that anneal upstream of the integration site, within the CORE and downstream of the integration size, which are designed to generate 500–1500 bp fragments. CORE-containing yeast cells are transformed with oligonucleotides containing the desired mutation such that they lead to the loss of the CORE cassette during homologous recombination. Transformants are selected using the counterselectable marker and can be further screened using the reporter gene. Sequencing is used to ensure the correct mutation has been generated without additional mutations

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Delitto perfetto

Delitto perfetto Alternatively, if the mutation leads to the generation or loss of a restriction site, PCR followed by restriction digest can be used to confirm that the desired mutation has been integrated. To increase oligonucleotide targeting to the CORE cassette, CORE cassettes containing the "GAL1"-I-"Sce"I feature can be used. This feature allows for the expression of SceI, an endonuclease that recognizes a highly unique 18 nucleotide sequence unlikely to occur anywhere else in the "S. cerevisiae" genome. The SceI endonuclease is able to generate a DSB at the SceI site leading to the recruitment of the DNA repair machinery. This increases the frequency of targeted homologous recombination by 4,000 fold compared to when no DSB is generated. 80–100 bp oligonucleotides can be generated as single molecules, or pairs of oligonucleotides that are completely overlapping or partially overlapping. The type of oligonucleotide recommended depends on the type of mutation and the distance from the CORE cassette integration site a mutation is desired. Longer oligonucleotides lead to increased transformation efficiency. Fully complementary oligonucleotides pairs lead to 5–10 fold increase in efficiency compared to single oligonucleotides and are recommended for all applications. However, they provide a small window of mutagenesis of only 20–40 bases from the CORE cassette

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Delitto perfetto

Delitto perfetto To increase the window of mutagenesis, oligonucleotide pairs with a 20 bp overlap can be used, and these allow up to 100 bp upstream and downstream of the CORE integration site to be targeted. However, they transform approximately 6 times less efficiently. To increase the efficiency, partly overlapping oligonucleotides can be extended "in vitro". Oligonucleotides containing the sequence upstream immediately followed by the sequence downstream of the region to be knocked out are designed. For gene deletions, pairs of fully overlapping 80–100 bp oligonucleotides lead to 5–10 fold increase in transformation efficiency than single oligonucleotides. For mutations 20 to 40 bp from the CORE cassette, 80–100 bp fully overlapping oligonucleotides are recommended. For mutations more than 40 bp from the CORE cassette, partly overlapping oligonucleotides must be used. To increase their transformation efficiency, it is recommended that partly overlapping oligonucleotides be extended "in vitro". To generate mutants of essential genes, the CORE cassette can be inserted downstream of the gene of interest, however this limits the regions of the gene available for mutation. Alternatively, diploid cells can be used. However, using a diploid decreases the efficiency of oligonucleotide targeting due to the presence of two suitable chromosomal locations for the oligonucleotides to recombine. To address this drawback, the DSB-mediated delitto perfetto method can be used

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Delitto perfetto

Delitto perfetto This increases the frequency of targeted homologous recombination by 700 fold compared to when no DSB is generated. Moreover, it is 2–5 fold more efficient than other available methods. It is reported that when no double stranded breaks are generated, the number of cells that lose the CORE cassette in the absence of a targeting oligonucleotide is less than one transformant per 10 viable cells. In contrast, this number increases up to 100 transformants per 10 viable cells when a double stranded break is generated. In a diploid, the increased background mutation rates occur due to homologous recombination with the homologous chromosome decreasing targeted transformation events to only 4% of the total transformants.

Biology

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Delitto perfetto

Histatin Histatins are histidine-rich (cationic) antimicrobial proteins found in saliva. Histatin's involvement in antimicrobial activities makes histatin part of the Innate immune system. was first discovered (isolated) in 1988, with functions that's responsible in keeping homeostasis inside the oral cavity, helping in the formation of pellicles, and assist in bonding of metal ions. The structure of histatin is unique depending on whether the protein of interest is histatin 1, 3 or 5. Nonetheless, histatin mainly possess a cationic charge due to the primary structure consisting mostly of basic amino acids. An amino acid that is crucial to histatin's function is histidine. Studies show that the removal of histadine (especially in histatin 5) resulted in reduction of antifungal activity. Histatins are antimicrobial and antifungal proteins, and have been found to play a role in wound-closure. A significant source of histatins is found in the serous fluid secreted by Ebner's glands, salivary glands at the back of the tongue, and produced by Acinus cells. Here they offer some early defense against incoming microbes. The three major histatins are 1, 3, and 5, which contains 38, 32, and 24 amino acids, respectively. 2 is a degradation product of histatin 1, and all other histatins are degradation products of histatin 3 through the process of post-translational proteolysis of the HTN3 gene. Therefore there are only two genes, HTN1 and HTN3. 5 N-terminus allows it to bind with metals because of its highly reactive nature

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Histatin

Histatin It's been shown that this chemical nature allows the precipitate of reactive oxygen species. Histatin's antifungal properties has been seen with fungus such as Candida glabrata, Candida krusei, Saccharomyces cerevisiae, and Cryptococcus neoformans. Histatins also precipitate tannins from solution - thus preventing alimentary adsorption. Histatin's anti-fungal property works by the release of the fungal's intracellular component through the disruption of the fungus plasma membrane. They also work against yeast, by binding to the potassium transporter and facilitating in the loss of azole-resistant species.

Biology

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Histatin

Reverse phase protein lysate microarray A reverse phase protein lysate microarray (RPMA) is a protein microarray designed as a dot-blot platform that allows measurement of protein expression levels in a large number of biological samples simultaneously in a quantitative manner when high-quality antibodies are available. Technically, minuscule amounts of (a) cellular lysates, from intact cells or laser capture microdissected cells, (b) body fluids such as serum, CSF, urine, vitreous, saliva, etc., are immobilized on individual spots on a microarray that is then incubated with a single specific antibody to detect expression of the target protein across many samples. A summary video of RPPA is available. One microarray, depending on the design, can accommodate hundreds to thousands of samples that are printed in a series of replicates. Detection is performed using either a primary or a secondary labeled antibody by chemiluminescent, fluorescent or colorimetric assays. The array is then imaged and the obtained data is quantified. Multiplexing is achieved by probing multiple arrays spotted with the same lysate with different antibodies simultaneously and can be implemented as a quantitative calibrated assay. In addition, since RPMA can utilize whole-cell or undissected or microdissected cell lysates, it can provide direct quantifiable information concerning post translationally modified proteins that are not accessible with other high-throughput techniques

Biology

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Reverse phase protein lysate microarray

Reverse phase protein lysate microarray Thus, RPMA provides high-dimensional proteomic data in a high throughput, sensitive and quantitative manner. However, since the signal generated by RPMA could be generated from unspecific primary or secondary antibody binding, as is seen in other techniques such as ELISA, or immunohistochemistry, the signal from a single spot could be due to cross-reactivity. Thus, the antibodies used in RPMA must be carefully validated for specificity and performance against cell lysates by western blot. RPMA has various uses such as quantitative analysis of protein expression in cancer cells, body fluids or tissues for biomarker profiling, cell signaling analysis and clinical prognosis, diagnosis or therapeutic prediction. This is possible as a RPMA with lysates from different cell lines and or laser capture microdissected tissue biopsies of different disease stages from various organs of one or many patients can be constructed for determination of relative or absolute abundance or differential expression of a protein marker level in a single experiment. It is also used for monitoring protein dynamics in response to various stimuli or doses of drugs at multiple time points. Some other applications that RPMA is used for include exploring and mapping protein signaling pathways, evaluating molecular drug targets and understanding a candidate drug's mechanism of action. It has been also suggested as a potential early screen test in cancer patients to facilitate or guide therapeutic decision making

Biology

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Reverse phase protein lysate microarray

Reverse phase protein lysate microarray Other protein microarrays include forward protein microarrays (PMAs) and antibody microarrays (AMAs). PMAs immobilize individual purified and sometimes denatured recombinant proteins on the microarray that are screened by antibodies and other small compounds. AMAs immobilize antibodies that capture analytes from the sample applied on the microarray. The target protein is detected either by direct labeling or a secondary labeled antibody against a different epitope on the analyte target protein (sandwich approach). Both PMAs and AMAs can be classified as forward phase arrays as they involve immobilization of a bait to capture an analyte. In forward phase arrays, each array is incubated with one test sample such as a cellular lysate or a patient's serum, but multiple analytes in the sample are tested simultaneously. Figure 1 shows a forward (using antibody as a bait in here) and reverse phase protein microarray at the molecular level. Depending on the research question or the type and aim of the study, RPMA can be designed by selecting the content of the array, the number of samples, sample placement within micro-plates, array layout, type of microarrayer, correct detection antibody, signal detection method, inclusion of control and quality control of the samples. The actual experiment is then set up in the laboratory and the results obtained are quantified and analyzed. The experimental stages are listed below: Cells are grown in T-25 flasks at 37 degree and 5% CO2 in appropriate medium

Biology

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Reverse phase protein lysate microarray

Reverse phase protein lysate microarray Depending on the design of the study, after cells are confluent they could be treated with drugs, growth factors or they could be irradiated before lysis step. For time course studies, a stimulant is added to a set of flasks concurrently and the flasks are then processed at different time points. For drug dose studies, a set of flasks are treated with different doses of the drug and all the flasks are collected at the same time. If a RPMA containing cell fraction lysates of a tissue/s is to be made, laser capture microdissection (LCM) or fine needle aspiration methods is used to isolate specific cells from a region of tissue microscopically. Pellets from cells collected through any of the above means are lysed with a cell lysis buffer to obtain high protein concentration. Aliquots of the lysates are pooled and resolved by two-dimensional single lane SDS-PAGE followed by western blotting on a nitrocellulose membrane. The membrane is cut into four-millimeter strips, and each strip is probed with a different antibody. Strips with single band indicate specific antibodies that are suitable for RPMA use. Antibody performance should be also validated with a smaller sample size under identical condition before actual sample collection for RPMA. Cell lysates are collected and are serially diluted six to ten times if using colorimetric techniques, or without dilution when fluorometric detection is used (due to the greater dynamic range of fluorescence than colorimetric detection)

Biology

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Reverse phase protein lysate microarray

Reverse phase protein lysate microarray Serial dilutions are then plated in replicates into a 384- or a 1536-well microtiter plate. The lysates are then printed onto either nitrocellulose or PVDF membrane coated glass slides by a microarrayer such as Aushon BioSystem 2470 or Flexys robot (Genomic solution). Aushon 2470 with a solid pin system is the ideal choice as it can be used for producing arrays with very viscous lysates and it has humidity environmental control and automated slide supply system. That being said, there are published papers showing that Arrayit Microarray Printing Pins can also be used and produce microarrays with much higher throughput using less lysate. The membrane coated glass slides are commercially available from several different companies such as Schleicher and Schuell Bioscience (now owned by GE Whatman www.whatman.com), Grace BioLabs (www.gracebio.com), Thermo Scientific, and SCHOTT Nexterion (www.schott.com/nexterion). After the slides are printed, non-specific binding sites on the array are blocked using a blocking buffer such as I-Block and the arrays are probed with a primary antibody followed by a secondary antibody. Detection is usually conducted with DakoCytomation catalyzed signal amplification (CSA) system. For signal amplification, slides are incubated with streptavidin-biotin-peroxidase complex followed by biotinyl-tyramide/hydrogen peroxide and streptavidin-peroxidase. Development is completed using hydrogen peroxide and scans of the slides are obtained (1)

Biology

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Reverse phase protein lysate microarray

Reverse phase protein lysate microarray Tyramide signal amplification works as follows: immobilized horseradish peroxidase (HRP) converts tyramide into reactive intermediate in the presence of hydrogen peroxide. Activated tyramide binds to neighboring proteins close to a site where the activating HRP enzyme is bound. This leads to more tyramide molecule deposition at the site; hence the signal amplification. Lance Liotta and Emanual Petricoin invented the RPMA technique in 2001 (see history section below), and have developed a multiplexed detection method using near-infrared fluorescent techniques. In this study, they report the use of a dual dye-based approach that can effectively double the number of endpoints observed per array, allowing, for example, both phospho-specific and total protein levels to be measured and analyzed at once. Once immunostaining has been performed protein expression must then be quantified. The signal levels can be obtained by using the reflective mode of an ordinary optical flatbed scanner if a colorimetric detection technique is used or by laser scanning, such as with a TECAN LS system, if fluorescent techniques are used. Two programs available online (P-SCAN and ProteinScan) can then be used to convert the scanned image into numerical values. These programs quantify signal intensities at each spot and use a dose interpolation algorithm (DI) to compute a single normalized protein expression level value for each sample

Biology

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Reverse phase protein lysate microarray

Reverse phase protein lysate microarray Normalization is necessary to account for differences in total protein concentration between each sample and so that antibody staining can be directly compared between samples. This can be achieved by performing an experiment in parallel in which total proteins are stained by colloidal gold total protein staining or Sypro Ruby total protein staining. When multiple RPMAs are analyzed, the signal intensity values can be displayed as a heat map, allowing for Bayesian clustering analysis and profiling of signaling pathways. An optimal software tool, custom designed for RPMAs is called Microvigene, by Vigene Tech, Inc. The greatest strength of RPMAs is that they allow for high throughput, multiplexed, ultra-sensitive detection of proteins from extremely small numbers of input material, a feat which cannot be done by conventional western blotting or ELISA. The small spot size on the microarray, ranging in diameter from 85 to 200 micrometres, enables the analysis of thousands of samples with the same antibody in one experiment. RPMAs have increased sensitivity and are capable of detecting proteins in the picogram range. Some researchers have even reported detection of proteins in the attogram range. This is a significant improvement over protein detection by ELISA, which requires microgram amounts of protein (6). The increase in sensitivity of RPMAs is due to the miniature format of the array, which leads to an increase in the signal density (signal intensity/area) coupled with tyramide deposition-enabled enhancement

Biology

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Reverse phase protein lysate microarray

Reverse phase protein lysate microarray The high sensitivity of RPMAs allows for the detection of low abundance proteins or biomarkers such as phosphorylated signaling proteins from very small amounts of starting material such as biopsy samples, which are often contaminated with normal tissue. Using laser capture microdissection lysates can be analyzed from as few as 10 cells, with each spot containing less than a hundredth of a cell equivalent of protein. A great improvement of RPMAs over traditional forward phase protein arrays is a reduction in the number of antibodies needed to detect a protein. Forward phase protein arrays typically use a sandwich method to capture and detect the desired protein. This implies that there must be two epitopes on the protein (one to capture the protein and one to detect the protein) for which specific antibodies are available. Other forward phase protein microarrays directly label the samples, however there is often variability in the labeling efficiency for different protein, and often the labeling destroys the epitope to which the antibody binds. This problem is overcome by RPMAs as sample need not be labeled directly. Another strength of RPMAs over forward phase protein microarrays and western blotting is the uniformity of results, as all samples on the chip are probed with the same primary and secondary antibody and the same concentration of amplification reagents for the same length of time. This allows for the quantification of differences in protein levels across all samples

Biology

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Reverse phase protein lysate microarray

Reverse phase protein lysate microarray Furthermore, printing each sample, on the chip in serial dilution (colorimetric) provides an internal control to ensure analysis is performed only in the linear dynamic range of the assay. Optimally, printing of calibrators and high and low controls directly on the same chip will then provide for unmatched ability to quantitatively measure each protein over time and between experiments. A problem that is encountered with tissue microarrays is antigen retrieval and the inherent subjectivity of immunohistochemistry. Antibodies, especially phospho-specific reagents, often detect linear peptide sequences that may be masked due to the three-dimensional conformation of the protein. This problem is overcome with RPMAs as the samples can be denatured, revealing any concealed epitopes. The biggest limitation of RPMA, as is the case for all immunoassays, is its dependence on antibodies for detection of proteins. Currently there is a limited but rapidly growing number of signaling proteins for which antibodies exist that give an analyzable signal. In addition, finding the appropriate antibody could require extensive screening of many antibodies by western blotting prior to beginning RPMA analysis. To overcome this issue, two open resource databases have been created to display western blot results for antibodies that have good binding specificity within the expected range. Furthermore, RPMAs, unlike western blots, do not resolve protein fractions by molecular weight

Biology

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Reverse phase protein lysate microarray

Reverse phase protein lysate microarray Thus, it is critical that upfront antibody validation be performed. RPMA was first introduced in 2001 in a paper by Lance Liotta and Emanuel Petricoin who invented the technology. The authors used the technique to successfully analyze the state of pro-survival checkpoint protein at the microscopic transition stage using laser capture microdissection of histologically normal prostate epithelium, prostate intraepithelial neoplasia, and patient-matched invasive prostate cancer. Since then RPMA has been used in many basic biology, translational and clinical research. In addition, the technique has now been brought into clinical trials for the first time whereby patients with metastatic colorectal and breast cancers are chosen for therapy based on the results of the RPMA. This technique has been commercialized for personalized medicine-based applications by Theranostics Health, Inc.

Biology

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Reverse phase protein lysate microarray

Alédjo Wildlife Reserve The is located in the Tchaoudjo and Assoli Prefectures in Togo. The wildlife reserve consists of some 765 total hectares of protected areas with biological diversity and geological formations. It is a tourist destination, known for its natural environment and wildlife. This site was added to the UNESCO World Heritage Tentative List on January 8, 2002 in the Mixed (Cultural and Natural) category.

Biology

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Alédjo Wildlife Reserve

Marcel Linsman Prize The (or Linsman Prize) is a Belgian prize awarded every 3 years by the influential AILg (Association des Ingenieurs Diplomés de la Université de Liège), a Belgian academic association of engineers, to living scientists for excellence in the biomedical sciences. The prize is restricted to researchers with a medical or engineering degree having performed research in Belgium. The age limit is 45 years but there is no restriction on the citizenship. For many years, the preference has been given to researchers in the field of neuroscience. This prize is awarded in memory of Marcel Linsman (1912-1989), distinguished University Professor. The prize is usually awarded during an official ceremony taking place in one Belgian university during which two other prestigious prizes in different fields are awarded. Similar to other prestigious prize ceremonies, the recipients deliver a scientific presentation of their research. The reputation achieved by this prize is ubiquitously recognized amongst academics in Europe. Born in Liège on 22 June 1912, he studied mathematical sciences at the University of Liège, receiving his bachelor's degree in 1934. In 1937, he received his doctorate, with the highest distinction" He was appointed assistant professor at Liège in 1938, and in 1964, full professor. In 1943, he became interested in numerical calculation and recognized immediately the electronic possibilities. He moved to Harvard University in 1947

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Marcel Linsman Prize

Marcel Linsman Prize From 1951 to 1955, he managed the development of one of the earliest European electronic computers, known as Machine I.R.S.I.A. His interest then took him into non-numerical applications of the computer. Starting with automatic translation, he initiated many projects, including teaching informatics and medical applications. Throughout his career, he was the recipient of many awards. In IFIP, he was present from the first Council meeting in Rome until 1971, representing the Belgian member society. He was also active in the Technical Committee on Education (TC3). During the time IFIP was registered in Belgium, 1962 through 1967, he served as IFIP Assistant Secretary and handled all legal matters for IFIP. In 1974, he was in the first group to receive the IFIP Silver Core Award. Marcel Linsman died during the night from 18 to 19 April 1989. A foundation in his name has been established to promote scientific excellence in the field of biomedical sciences. Source: AILg-Liste des Lauréats

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Marcel Linsman Prize

MAGIChip MAGIChips, also known as "microarrays of gel-immobilized compounds on a chip" or "three-dimensional DNA microarrays", are devices for molecular hybridization produced by immobilizing oligonucleotides, DNA, enzymes, antibodies, and other compounds on a photopolymerized micromatrix of polyacrylamide gel pads of 100x100x20µm or smaller size. This technology is used for analysis of nucleic acid hybridization, specific binding of DNA, and low-molecular weight compounds with proteins, and protein-protein interactions. The gel pads increase the surface for hybridization to 50 times, compared to typical microarrays which are printed on flat surface of a glass slide that is usually treated by chemical compounds on which the probes adhere. A probe density of more than 1012 molecules per gel pad can be achieved due to 3D nature of the gel pads. The array is based on a glass surface that has small polyacrylamide gel units affixed to it. Each gel unit functions as an individual reaction cell as it is surrounded by a hydrophobic glass surface that prevents mixing of the solution in the gel units. This lays a foundation for performing ligation, single base extension, PCR amplification of DNA, on-chip MALDI-TOF mass spectrometry and other reactions. technology was developed as a result of collaboration between Dr. David Stahl at University of Washington and Dr Andrei Mirzabekov, formerly of Argonne National laboratory

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MAGIChip

MAGIChip Andrei Mirzabekov initiated the development of the DNA sequencing by hybridization with oligonucleotides: a novel method in 1988. This method was a foundation for the biotechnology that uses biological microchips to identify DNA structures rapidly, which is of great importance in the fight against a variety of diseases. A joint research project was announced in 1998 among Motorola Inc, Packard Instrument Company and the U.S. Department of Energy's Argonne National Laboratory. In 1999, the researchers at Argonne National Lab pushed the development of microarray-type biochip technology they co-designed with the Engelhardt Institute to ward off a worldwide outbreak of tuberculosis. Motorola developed manufacturing processes to mass-produce biochips, and Packard developed and manufactured the analytical instruments to process and analyze the biochips. Argonne's contribution, in conjunction with Engelhardt Institute of Molecular Biology (EIMB), was intellectual property in the form of 19 inventions related to biological microchips. But this collaboration between EIMB in Moscow and Argonne National Laboratory at Illinois and two other US-based commercial partners collapsed as result of argument on contractual arrangement between the parties in 2001. As a result of this dispute, Dr Andrei Mirzabekov resigned as a director of Argonne's Biochip Technology Centre

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MAGIChip

MAGIChip Arrays of gel elements (pads) are created on the glass surface (micromatrix) which is followed by application and chemical immobilization of different compounds (probes) onto these gel pads. Test sample is then added to this micromatrix containing immobilized probes in gel pads and molecular recognition reactions are allowed to take place under specified conditions. The test sample is fluorescent labelled to monitor the molecular interactions. The analysis of molecular interaction patterns is done by using specialized software. The array of gel elements on a glass slide is prepared by ‘’’photopolymerization‘’’. The acrylamide solution to be polymerized is applied to the polymerization chamber. Polymerization chamber consists of a quartz mask, two Teflon spacers, and a microscopic glass slide, clamped together by two metal clamps. The inner side of quartz mask has ultraviolet (UV)-transparent windows arranged in a specified spatial manner in a non-transparent chromium film. Assembled chamber containing the acrylamide gel is exposed to UV light to allow polymerization in only those positions of the chamber that are situated directly under the transparent windows. Oligonucleotides or DNA fragments need to be activated to contain chemically reactive groups to facilitate coupling with the activated gel elements. Probe activation depends on the chemistry of activation of the polyacrylamide gels

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MAGIChip

MAGIChip Thus to immobilize in the aldehyde-containing gel the probe should have reactive amino group and if the gels are activated by introduction of amino groups, the probes should contain free aldehyde group. Probes are usually prepared by introduction of chemically active groups in terminal position of the oligonucleotides during their synthesis. Probes for immobilization are transferred into gel elements of micromatrix by using dispensing robots. The fibre-optic pin of the robots has a hydrophobic side surface and a hydrophilic tip, and operates at a dew temperature to prevent evaporation of the sample during transfer. The activated probes are chemically immobilized by coupling oligonucleotides bearing amino or aldehyde groups with gel supports containing aldehyde or amino groups respectively. The target molecules are labelled with fluorescent dyes. The fluorescent detection enables monitoring the process in real time with high spatial resolution. The criteria for labelling procedure includes – On-chip amplification of the hybridization reaction serves as a very useful tool when the DNA or protein under study are present in relatively small proportion in the molecular population applied to the chip, e.g., when one is dealing with a single copy gene or mRNA of low abundance. In a single base extension method, a primer is hybridized to DNA and extended by a dideoxyribonucleoside triphosphate that matches the nucleotide at a polymorphic site

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MAGIChip

MAGIChip By performing this reaction at a temperature above the melting temperature of the duplex between the DNA and immobilized probe allows rapid association/dissociation of the target DNA. Thus the same DNA molecule reacts with many individual primers, leading to amplification of the primers in each individual gel pads. This procedure was applied to the identification of beta-globin gene mutation in the patients of beta thalassemia patients and to detection of anthrax toxin gene. The chips also provides a good platform for performing PCR directly on the chip (in individual gel pads) as it is easy to isolate each gel pad from its neighbour unlike typical microarray chips which face serious problems in doing the same task For the analysis of hybridization results obtained with fluorescently labelled target molecules fluorescence microscopes are employed. The instrument is equipped with controlled-temperature sample table to vary the temperature in the chip-containing reaction chamber during the course of the experiment. A cooled charge-coupled device (CCD) camera is used to record the light signals from the chip, which are then sent to the computer program for quantitative evaluation of the hybridization signals over the entire chip. Data generated by these experiments is stored in a database and analyzed by software that help provide evaluation, "in silico" experimentation, and hardware and software quality control

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MAGIChip

MAGIChip Customized oligonucleotide biochips are designed to interrogate test samples of known nucleotide sequences. For example, known genes in cases when one is interested in their expression levels under certain conditions, genes that are known to contain point mutations, or to be polymorphic in a given population. The success of the microarray depends on the proper choice of probes in these cases. A set of potential hybridization probes are created for each DNA sequence that form perfect duplexes with that sequence. The potential probes that may create ambiguities in the interpretation of the hybridization pattern are excluded on the basis of AT vs GC content, and the propensity to form hairpins and other types of stable secondary structures that may drastically affect the intensity of hybridization. One of the cases of successful applications of customized oligonucleotide chips include detection of beta-thalassemia mutation in patients. For the diagnostics of beta-thalassemia mutations, a simple chip was designed that contained six probes corresponding to different beta-thalassemia genotypes and hybridized with PCR-amplified DNA from healthy humans and patients. The hybridization results showed the expected significant differences in signal intensity between matched and mismatched duplexes, thus allowing reliable identification of both homozygous and heterozygous mutations. These chips haves been developed for ribosomal RNA (rRNA) targets, commonly used for detecting bacteria

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MAGIChip

MAGIChip rRNA are very abundant in the cell comprising about 80% of the RNA content of the typical eukaryotic cell. The rRNA is pre-amplified by bacteria and there are present in several thyousand copies per cell, making a good target for microassays. Single nucleotide polymorphisms present in the bacterial rRNA sequence are used to differentiate bacteria at the genus, species and strain level. This is a unique feature of this microchip that does not require PCR based amplification. The process for detecting bacterial is relatively simple. The bacteria are cultured, washed and pelleted. Lysosome is used to lyse the pellets - to destroy the cell walls and release the nucleic acid. Lysed bacteria are passed through a colourmn preparation where nucleic acid from the cell is immobilized and other debris is washed out. All the processes after lysis - isolation, purification, fragmentation and labelling of target rRNA's - are stable chemical reactions Fragments <500 bp easily hybridize to the gel matrix. The total number of eluted off the chip is determined by UV spectrophotometer. The process from sample preparation to identification of organisms based on automated algorithms take place in 2 hours. cDNAs obtained from reverse transcription of mRNA population extracted from the cells in varying physiological and experimental conditions are used as immobilized probes. These arrays are widely used to study gene expression

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MAGIChip

MAGIChip The potential obstacle in using cDNAs is due to the difficulty of injecting and evenly distributing long molecules into the gel pads. This problem is resolved by developing polyacrylamide gels that contain larger average pore size. Another way to approach this problem is to randomly fragment the cDNA into relatively small pieces before immobilization Protein chips can be prepared that contain different proteins immobilized as probes in a way that preserves their biological activity. A large pore gel is used to prevent the diffusion of protein into the gel. There are two ways to immobilize proteins to the gel pads. The first is based on activation of the gel with glutyraldehyde. In the second procedure the gel is activated by partial substitution of amino groups with hydrazide groups. The reaction between hydrazide and aldehyde groups efficiently cross-links the protein to the cell. Protein microchips show the high specificity in molecular recognition reactions as seen in solution. Interaction between antigen and their specific antibodies can be studied on-chip in variety of experimental conditions. Either the antigen or antibody can be immobilized and monitored by both direct and indirect methods. In direct method, one uses target molecules labelled with fluorescent dye and in the indirect method the reaction is detected using the labelled molecule that specifically recognizes the target

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MAGIChip

MAGIChip These chips can be used to study enzymatic activity of immobilized enzymes by coating the chip with solution containing specific substrates. The reaction is monitored by detecting the formation of coloured or fluorescent precipitates

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MAGIChip

Methylated DNA immunoprecipitation (MeDIP or mDIP) is a large-scale (chromosome- or genome-wide) purification technique in molecular biology that is used to enrich for methylated DNA sequences. It consists of isolating methylated DNA fragments via an antibody raised against 5-methylcytosine (5mC). This technique was first described by Weber M. "et al." in 2005 and has helped pave the way for viable methylome-level assessment efforts, as the purified fraction of methylated DNA can be input to high-throughput DNA detection methods such as high-resolution DNA microarrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). Nonetheless, understanding of the methylome remains rudimentary; its study is complicated by the fact that, like other epigenetic properties, patterns vary from cell-type to cell-type. DNA methylation, referring to the reversible methylation of the 5 position of cytosine by methyltransferases, is a major epigenetic modification in multicellular organisms. In mammals, this modification primarily occurs at CpG sites, which in turn tend to cluster in regions called CpG islands. There is a small fraction of CpG islands that can overlap or be in close proximity to promoter regions of transcription start sites. The modification may also occur at other sites, but methylation at either of these sites can repress gene expression by either interfering with the binding of transcription factors or modifying chromatin structure to a repressive state

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Methylated DNA immunoprecipitation

Methylated DNA immunoprecipitation Disease condition studies have largely fueled the effort in understanding the role of DNA methylation. Currently, the major research interest lies in investigating disease conditions such as cancer to identify regions of the DNA that has undergone extensive methylation changes. The genes contained in these regions are of functional interest as they may offer a mechanistic explanation to the underlying genetic causes of a disease. For instance, the abnormal methylation pattern of cancer cells was initially shown to be a mechanism through which tumor suppressor-like genes are silenced, although it was later observed that a much broader range of gene types are affected. There are two approaches to methylation analysis: typing and profiling technologies. Typing technologies are targeted towards a small number of loci across many samples, and involve the use of techniques such as PCR, restriction enzymes, and mass spectrometry. Profiling technologies such as MeDIP are targeted towards a genome- or methylome-wide level assessment of methylation; this includes restriction landmark genomic scanning (RLGS), and bisulfite conversion-based methods, which rely on the treatment of DNA with bisulfite to convert unmethylated cytosine residues to uracil. Other methods mapping and profiling the methylome have been effective but are not without their limitations that can affect resolution, level of throughput, or experimental variations

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Methylated DNA immunoprecipitation

Methylated DNA immunoprecipitation For instance, RLGS is limited by the number of restriction sites in genome that can be targets for the restriction enzyme; typically, a maximum of ~4100 landmarks can be assessed. Bisulfite sequencing-based methods, despite possible single-nucleotide resolution, have a drawback: the conversion of unmethylated cytosine to uracil can be unstable. In addition, when bisulfite conversion is coupled with DNA microarrays to detect bisulfite converted sites, the reduced sequence complexity of DNA is a problem. Microarrays capable of comprehensively profiling the whole-genome become difficult to design as fewer unique probes are available. The following sections outline the method of MeDIP coupled with either high-resolution array hybridization or high-throughput sequencing. Each DNA detection method will also briefly describe post-laboratory processing and analysis. Different post-processing of the raw data is required depending on the technology used to identify the methylated sequences. This is analogous to data generated using ChIP-chip and ChIP-seq. Genomic DNA is extracted (DNA extraction) from the cells and purified. The purified DNA is then subjected to sonication to shear it into random fragments. This sonication process is quick, simple, and avoids restriction enzyme biases. The resulting fragments range from 300 to 1000 base pairs (bp) in length, although they are typically between 400 and 600 bp

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Methylated DNA immunoprecipitation

Methylated DNA immunoprecipitation The short length of these fragments is important in obtaining adequate resolution, improving the efficiency of the downstream step in immunoprecipitation, and reducing fragment-length effects or biases. Also, the size of the fragment affects the binding of 5-methyl-cytidine (5mC) antibody because the antibody needs more than just a single 5mC for efficient binding. To further improve binding affinity of the antibodies, the DNA fragments are denatured to produce single-stranded DNA. Following denaturation, the DNA is incubated with monoclonal 5mC antibodies. The classical immunoprecipitation technique is then applied: magnetic beads conjugated to anti-mouse-IgG are used to bind the anti-5mC antibodies, and unbound DNA is removed in the supernatant. To purify the DNA, proteinase K is added to digest the antibodies and release the DNA, which can be collected and prepared for DNA detection. For more details regarding the experimental steps see. A fraction of the input DNA obtained after the sonication step above is labeled with cyanine-5 (Cy5; red) deoxy-cytosine-triphosphate while the methylated DNA, enriched after the immunoprecipitation step, is labeled with cyanine-3 (Cy3; green). The labeled DNA samples are cohybridized on a 2-channel, high-density genomic microarray to probe for presence and relative quantities. The purpose of this comparison is to identify sequences that show significant differences in hybridization levels, thereby confirming the sequence of interest is enriched

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Methylated DNA immunoprecipitation

Methylated DNA immunoprecipitation Array-based identification of MeDIP sequences are limited to the array design. As a result, the resolution is restricted to the probes in the array design. There are additional standard steps required in signal processing to correct for hybridization issues such as noise, as is the case with most array technologies. See for more details. The MeDIP-seq approach, i.e. the coupling of MeDIP with next generation, short-read sequencing technologies such as 454 pyrosequencing or Illumina (Solexa), was first described by Down "et al." in 2008. The high-throughput sequencing of the methylated DNA fragments produces a large number of short reads (36-50bp or 400 bp, depending on the technology). The short reads are aligned to a reference genome using alignment software such as Mapping and Assembly with Quality (Maq), which uses a Bayesian approach, along with base and mapping qualities to model error probabilities for the alignments. The reads can then be extended to represent the ~400 to 700 bp fragments from the sonication step. The coverage of these extended reads can be used to estimate the methylation level of the region. A genome browser such as Ensembl can also be used to visualize the data. Validation of the approach to assess quality and accuracy of the data can be done with quantitative PCR. This is done by comparing a sequence from the MeDIP sample against an unmethylated control sequence. The samples are then run on a gel and the band intensities are compared

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Methylated DNA immunoprecipitation

Methylated DNA immunoprecipitation The relative intensity serves as the guide for finding enrichment. The results can also be compared with MeDIP-chip results to help determine coverage needed. The DNA methylation level estimations can be confounded by varying densities of methylated CpG sites across the genome when observing data generated by MeDIP. This can be problematic for analyzing CpG-poor (lower density) regions. One reason for this density issue is its effect on the efficiency of immunoprecipitation. In their study, Down "et al." developed a tool to estimate absolute methylation levels from data generated by MeDIP by modeling the density of methylated CpG sites. This tool is called Bayesian tool for methylation analysis (Batman). The study reports the coverage of ~90% of all CpG sites in promoters, gene-coding regions, islands, and regulatory elements where methylation levels can be estimated; this is almost 20 times better coverage than any previous methods. Studies using MeDIP-seq or MeDIP-chip are both genome-wide approaches that have the common aim of obtaining the functional mapping of the methylome. Once regions of DNA methylation are identified, a number of bioinformatics analyses can be applied to answer certain biological questions. One obvious step is to investigate genes contained in these regions and investigate the functional significance of their repression. For example, silencing of tumour-suppressor genes in cancer can be attributed to DNA methylation

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Methylated DNA immunoprecipitation

Methylated DNA immunoprecipitation By identifying mutational events leading to hypermethylation and subsequent repression of known tumour-suppressor genes, one can more specifically characterize the contributing factors to the cause of the disease. Alternatively, one can identify genes that are known to be normally methylated but, as a result of some mutation event, is no longer silenced. Also, one can try and investigate and identify whether some epigenetic regulator has been affected such as DNA methyltransferase (DNMT); in these cases, enrichment may be more limited. Gene-set analysis (for example using tools like DAVID and GoSeq) has been shown to be severely biased when applied to high-throughput methylation data (e.g. MeDIP-seq and MeDIP-ChIP); it has been suggested that this can be corrected using sample label permutations or using a statistical model to control for differences in the numberes of CpG probes / CpG sites that target each gene. Limitations to take note when using MeDIP are typical experimental factors. This includes the quality and cross-reactivity of 5mC antibodies used in the procedure. Furthermore, DNA detection methods (i.e. array hybridization and high-throughput sequencing) typically involve well established limitations. Particularly for array-based procedures, as mentioned above, sequences being analyzed are limited to the specific array design used. Most typical limitations to high-throughput, next generation sequencing apply

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Methylated DNA immunoprecipitation

Methylated DNA immunoprecipitation The problem of alignment accuracy to repetitive regions in the genome will result in less accurate analysis of methylation in those regions. Also, as was mentioned above, short reads (e.g. 36-50bp from an Illumina Genome Analyzer) represent a part of a sheared fragment when aligned to the genome; therefore, the exact methylation site can fall anywhere within a window that is a function of the fragment size. In this respect, bisulfite sequencing has much higher resolution (down to a single CpG site; single nucleotide level). However, this level of resolution may not be required for most applications, as the methylation status of CpG sites within < 1000 bp has been shown to be significantly correlated.

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Methylated DNA immunoprecipitation

RNA-Seq (named as an abbreviation of "RNA sequencing") is a particular technology-based sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing cellular transcriptome. Specifically, facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression over time, or differences in gene expression in different groups or treatments. In addition to mRNA transcripts, can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling. can also be used to determine exon/intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries. Recent advances in include single cell sequencing and in situ sequencing of fixed tissue. Prior to RNA-Seq, gene expression studies were done with hybridization-based microarrays. Issues with microarrays include cross-hybridization artifacts, poor quantification of lowly and highly expressed genes, and needing to know the sequence "a priori". Because of these technical issues, transcriptomics transitioned to sequencing-based methods. These progressed from Sanger sequencing of Expressed Sequence Tag libraries, to chemical tag-based methods (e.g., serial analysis of gene expression), and finally to the current technology, next-gen sequencing of cDNA (notably RNA-Seq)

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RNA-Seq

RNA-Seq The general steps to prepare a complementary DNA (cDNA) library for sequencing are described below, but often vary between platforms. When sequencing RNA other than mRNA, the library preparation is modified. The cellular RNA is selected based on the desired size range. For small RNA targets, such as miRNA, the RNA is isolated through size selection. This can be performed with a size exclusion gel, through size selection magnetic beads, or with a commercially developed kit. Once isolated, linkers are added to the 3' and 5' end then purified. The final step is cDNA generation through reverse transcription. Because converting RNA into cDNA, ligation, amplification, and other sample manipulations have been shown to introduce biases and artifacts that may interfere with both the proper characterization and quantification of transcripts, single molecule direct RNA sequencing has been explored by companies including Helicos (bankrupt), Oxford Nanopore Technologies, and others. This technology sequences RNA molecules directly in a massively-parallel manner. Standard methods such as microarrays and standard bulk analysis analyze the expression of RNAs from large populations of cells. In mixed cell populations, these measurements may obscure critical differences between individual cells within these populations. Single-cell RNA sequencing (scRNA-Seq) provides the expression profiles of individual cells

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RNA-Seq

RNA-Seq Although it is not possible to obtain complete information on every RNA expressed by each cell, due to the small amount of material available, patterns of gene expression can be identified through gene clustering analyses. This can uncover the existence of rare cell types within a cell population that may never have been seen before. For example, rare specialized cells in the lung called pulmonary ionocytes that express the Cystic Fibrosis Transmembrane Conductance Regulator were identified in 2018 by two groups performing scon lung airway epithelia. Current scprotocols involve the following steps: isolation of single cell and RNA, reverse transcription (RT), amplification, library generation and sequencing. Early methods separated individual cells into separate wells; more recent methods encapsulate individual cells in droplets in a microfluidic device, where the reverse transcription reaction takes place, converting RNAs to cDNAs. Each droplet carries a DNA "barcode" that uniquely labels the cDNAs derived from a single cell. Once reverse transcription is complete, the cDNAs from many cells can be mixed together for sequencing; transcripts from a particular cell are identified by the unique barcode. Challenges for scinclude preserving the initial relative abundance of mRNA in a cell and identifying rare transcripts. The reverse transcription step is critical as the efficiency of the RT reaction determines how much of the cell's RNA population will be eventually analyzed by the sequencer

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RNA-Seq

RNA-Seq The processivity of reverse transcriptases and the priming strategies used may affect full-length cDNA production and the generation of libraries biased toward 3’ or 5' end of genes. In the amplification step, either PCR or in vitro transcription (IVT) is currently used to amplify cDNA. One of the advantages of PCR-based methods is the ability to generate full-length cDNA. However, different PCR efficiency on particular sequences (for instance, GC content and snapback structure) may also be exponentially amplified, producing libraries with uneven coverage. On the other hand, while libraries generated by IVT can avoid PCR-induced sequence bias, specific sequences may be transcribed inefficiently, thus causing sequence drop-out or generating incomplete sequences. Several scprotocols have been published: Tang et al., STRT, SMART-seq, CEL-seq, RAGE-seq, , Quartz-seq. and C1-CAGE. These protocols differ in terms of strategies for reverse transcription, cDNA synthesis and amplification, and the possibility to accommodate sequence-specific barcodes (i.e. UMIs) or the ability to process pooled samples. In 2017, two approaches were introduced to simultaneously measure single-cell mRNA and protein expression through oligonucleotide-labeled antibodies known as REAP-seq, and CITE-seq. scis becoming widely used across biological disciplines including Development, Neurology, Oncology, Autoimmune disease, and Infectious disease

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RNA-Seq

RNA-Seq schas provided considerable insight into the development of embryos and organisms, including the worm "Caenorhabditis elegans", and the regenerative planarian "Schmidtea mediterranea". The first vertebrate animals to be mapped in this way were Zebrafish and "Xenopus laevis". In each case multiple stages of the embryo were studied, allowing the entire process of development to be mapped on a cell-by-cell basis. Science recognized these advances as the 2018 Breakthrough of the Year. A variety of parameters are considered when designing and conducting experiments: Two methods are used to assign raw sequence reads to genomic features (i.e., assemble the transcriptome): "A note on assembly quality:" The current consensus is that 1) assembly quality can vary depending on which metric is used, 2) assemblies that scored well in one species do not necessarily perform well in the other species, and 3) combining different approaches might be the most reliable. Expression is quantified to study cellular changes in response to external stimuli, differences between healthy and diseased states, and other research questions. Gene expression is often used as a proxy for protein abundance, but these are often not equivalent due to post transcriptional events such as RNA interference and nonsense-mediated decay. Expression is quantified by counting the number of reads that mapped to each locus in the transcriptome assembly step. Expression can be quantified for exons or genes using contigs or reference transcript annotations

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RNA-Seq

RNA-Seq These observed read counts have been robustly validated against older technologies, including expression microarrays and qPCR. Examples of tools that quantify counts are HTSeq, FeatureCounts, Rcount, maxcounts, FIXSEQ, and Cuffquant. The read counts are then converted into appropriate metrics for hypothesis testing, regressions, and other analyses. Parameters for this conversion are: Absolute quantification of gene expression is not possible with most experiments, which quantify expression relative to all transcripts. It is possible by performing with spike-ins, samples of RNA at known concentrations. After sequencing, read counts of spike-in sequences are used to determine the relationship between each gene's read counts and absolute quantities of biological fragments. In one example, this technique was used in "Xenopus tropicalis" embryos to determine transcription kinetics. The simplest but often most powerful use of is finding differences in gene expression between two or more conditions ("e.g.", treated vs not treated); this process is called differential expression. The outputs are frequently referred to as differentially expressed genes (DEGs) and these genes can either be up- or down-regulated ("i.e.", higher or lower in the condition of interest). There are many tools that perform differential expression. Most are run in R, Python, or the Unix command line. Commonly used tools include DESeq, edgeR, and voom+limma, all of which are available through R/Bioconductor

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RNA-Seq

RNA-Seq These are the common considerations when performing differential expression: Downstream analyses for a list of differentially expressed genes come in two flavors, validating observations and making biological inferences. Owing to the pitfalls of differential expression and RNA-Seq, important observations are replicated with (1) an orthogonal method in the same samples (like real-time PCR) or (2) another, sometimes pre-registered, experiment in a new cohort. The latter helps ensure generalizability and can typically be followed up with a meta-analysis of all the pooled cohorts. The most common method for obtaining higher-level biological understanding of the results is gene set enrichment analysis, although sometimes candidate gene approaches are employed. Gene set enrichment determines if the overlap between two gene sets is statistically significant, in this case the overlap between differentially expressed genes and gene sets from known pathways/databases ("e.g.", Gene Ontology, KEGG, Human Phenotype Ontology) or from complementary analyses in the same data (like co-expression networks). Common tools for gene set enrichment include web interfaces ("e.g.", ENRICHR, g:profiler) and software packages. When evaluating enrichment results, one heuristic is to first look for enrichment of known biology as a sanity check and then expand the scope to look for novel biology. RNA splicing is integral to eukaryotes and contributes significantly to protein regulation and diversity, occurring in >90% of human genes

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RNA-Seq

RNA-Seq There are multiple alternative splicing modes: exon skipping (most common splicing mode in humans and higher eukaryotes), mutually exclusive exons, alternative donor or acceptor sites, intron retention (most common splicing mode in plants, fungi, and protozoa), alternative transcription start site (promoter), and alternative polyadenylation. One goal of is to identify alternative splicing events and test if they differ between conditions. Long-read sequencing captures the full transcript and thus minimizes many of issues in estimating isoform abundance, like ambiguous read mapping. For short-read RNA-Seq, there are multiple methods to detect alternative splicing that can be classified into three main groups: Differential gene expression tools can also be used for differential isoform expression if isoforms are quantified ahead of time with other tools like RSEM. Coexpression networks are data-derived representations of genes behaving in a similar way across tissues and experimental conditions. Their main purpose lies in hypothesis generation and guilt-by-association approaches for inferring functions of previously unknown genes. data has been used to infer genes involved in specific pathways based on Pearson correlation, both in plants and mammals. The main advantage of data in this kind of analysis over the microarray platforms is the capability to cover the entire transcriptome, therefore allowing the possibility to unravel more complete representations of the gene regulatory networks

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RNA-Seq

RNA-Seq Differential regulation of the splice isoforms of the same gene can be detected and used to predict and their biological functions. Weighted gene co-expression network analysis has been successfully used to identify co-expression modules and intramodular hub genes based on RNA seq data. Co-expression modules may correspond to cell types or pathways. Highly connected intramodular hubs can be interpreted as representatives of their respective module. An eigengene is a weighted sum of expression of all genes in a module. Eigengenes are useful biomarkers (features) for diagnosis and prognosis. Variance-Stabilizing Transformation approaches for estimating correlation coefficients based on RNA seq data have been proposed. captures DNA variation, including single nucleotide variants, small insertions/deletions. and structural variation. Variant calling in is similar to DNA variant calling and often employs the same tools (including SAMtools mpileup and GATK HaplotypeCaller) with adjustments to account for splicing. One unique dimension for RNA variants is allele-specific expression (ASE): the variants from only one haplotype might be preferentially expressed due to regulatory effects including imprinting and expression quantitative trait loci, and noncoding rare variants. Limitations of RNA variant identification include that it only reflects expressed regions (in humans, <5% of the genome) and has lower quality when compared to direct DNA sequencing

Biology

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RNA-Seq

RNA-Seq Having the matching genomic and transcriptomic sequences of an individual can help detect post-transcriptional edits (RNA editing). A post-transcriptional modification event is identified if the gene's transcript has an allele/variant not observed in the genomic data. Caused by different structural modifications in the genome, fusion genes have gained attention because of their relationship with cancer. The ability of to analyze a sample's whole transcriptome in an unbiased fashion makes it an attractive tool to find these kinds of common events in cancer. The idea follows from the process of aligning the short transcriptomic reads to a reference genome. Most of the short reads will fall within one complete exon, and a smaller but still large set would be expected to map to known exon-exon junctions. The remaining unmapped short reads would then be further analyzed to determine whether they match an exon-exon junction where the exons come from different genes. This would be evidence of a possible fusion event, however, because of the length of the reads, this could prove to be very noisy. An alternative approach is to use pair-end reads, when a potentially large number of paired reads would map each end to a different exon, giving better coverage of these events (see figure). Nonetheless, the end result consists of multiple and potentially novel combinations of genes providing an ideal starting point for further validation

Biology

https://en.wikipedia.org/wiki?curid=21731590

RNA-Seq

RNA-Seq was first developed in mid 2000s with the advent of next-generation sequencing technology. The first manuscripts that used even without using the term includes those of prostate cancer cell lines (dated 2006), "Medicago truncatula" (2006), maize (2007), and "Arabidopsis thaliana" (2007), while the term "RNA-Seq" itself was first mentioned in 2008. The number of manuscripts referring to in the title or abstract (Figure, blue line) is continuously increasing with 6754 manuscripts published in 2018 (link to PubMed search). The intersection of and medicine (Figure, gold line, link to PubMed search) has similar celerity. has the potential to identify new disease biology, profile biomarkers for clinical indications, infer druggable pathways, and make genetic diagnoses. These results could be further personalized for subgroups or even individual patients, potentially highlighting more effective prevention, diagnostics, and therapy. The feasibility of this approach is in part dictated by costs in money and time; a related limitation is the required team of specialists (bioinformaticians, physicians/clinicians, basic researchers, technicians) to fully interpret the huge amount of data generated by this analysis. A lot of emphasis has been given to data after the Encyclopedia of DNA Elements (ENCODE) and The Cancer Genome Atlas (TCGA) projects have used this approach to characterize dozens of cell lines and thousands of primary tumor samples, respectively

Biology

https://en.wikipedia.org/wiki?curid=21731590

RNA-Seq

RNA-Seq ENCODE aimed to identify genome-wide regulatory regions in different cohort of cell lines and transcriptomic data are paramount in order to understand the downstream effect of those epigenetic and genetic regulatory layers. TCGA, instead, aimed to collect and analyze thousands of patient's samples from 30 different tumor types in order to understand the underlying mechanisms of malignant transformation and progression. In this context data provide a unique snapshot of the transcriptomic status of the disease and look at an unbiased population of transcripts that allows the identification of novel transcripts, fusion transcripts and non-coding RNAs that could be undetected with different technologies.

Biology

https://en.wikipedia.org/wiki?curid=21731590

RNA-Seq

WikiFood.eu is a non-commercial web portal for the publication of ingredients lists on foodstuff. WikiFood is an initiative of the Luxembourgish public research center „Centre de Recherche Public Henri Tudor“. The web portal is funded by the Luxembourgish research fund „Fonds National de la Recherche“ within a Luxembourgish research project on food security (Sécurité alimentaire). is part of the MENSSANA project (Mobile Expert and Networking System for Systematical Analysis of Nutrition-based Allergies). The objective of Wikifood.eu is to provide information on foodstuff and their ingredients to interested users via the internet. According to the provider of Wikifood.eu the target market consists, inter alia, of allergic persons and those suffering from food intolerance. The web portal is currently available in German, English and French. The WikiFood idea is based on the fact that – until now – the duty of declaration regarding ingredients of foodstuff within the EU does not require food producers to forward the ingredients lists to the final consumers by electronic means, for example using the means of a publicly accessible database (on food labelling see Directive 2000/13/EC of the European Parliament and of the Council of 20 March 2000 on the approximation of the laws of the Member States relating to the labelling, presentation and advertising of foodstuffs, which has been amended frequently )

Biology

https://en.wikipedia.org/wiki?curid=21732578

WikiFood.eu

WikiFood.eu WikiFood enables registered users to type up the ingredients lists of products that may be interesting for the community of allergy patients and other consumers. Digital photos of EAN codes, packages, and ingredients lists allow users to check entries and to improve these if necessary. Additionally, producers of foodstuff can forward lists of ingredients of their own products to WikiFood in order to make these publicly available at no cost. On 15 September 2006 WikiFood.de was launched officially on the occasion of the German Allergy Congress organised by the “Ärzteverband Deutscher Allergologen“. Further medical and scientific partners are the “Centre Hospitalier de Luxembourg” and the “Institut für Medizinische Statistik, Informatik und Epidemiologie” (Institute for Medical Statistics, Computer Science and Epidemiology) of the University of Cologne, Germany. Currently there are approximately 12.500 food products available and approximately 1000 volunteers registered. Meanwhile, Wikifood.eu was revised several times and new features were implemented such as an accompanying forum. The main focus of the forum lies on discussions regarding nutrition-based allergies and the exchange of individual experiences of the users. One point of criticism on Wikifood has been that it is no Wiki in the original sense as only registered users can edit content. Furthermore, even registered users are not allowed to change content which has been provided by food producers themselves. The provider of Wikifood

Biology

https://en.wikipedia.org/wiki?curid=21732578

WikiFood.eu

WikiFood.eu eu defends its approach by arguing that with this method it is easier to maintain a high data quality and provide high quality information. The provider of points out at several places on the website that there is no possibility of guaranteeing accuracy, completeness and/or up-to-date status of the information provided. Therefore, in the early stages of Wikifood.eu, only registered users were allowed to search the database. The harsh criticism on this approach led to the opening of the database to non-registered internet users starting November 2006 onwards. In the course of the 27th EAACI congress by the “European Academy of Allergology and Clinical Immunology” in Barcelona, Spain, as well as in the course of the 10th International Nutrition-based Allergy Symposium in Parma, Italy, scientists discussed the idea of Wikifood and the necessity of disclosure of food ingredients lists to consumers and nutrition-based allergy patients electronically: Further information on the labelling of foodstuffs:

Biology

https://en.wikipedia.org/wiki?curid=21732578

WikiFood.eu

Oak forest An oak forest is a plant community with a tree canopy dominated by oaks ("Quercus spp."). In terms of canopy closure, oak forests contain the most closed canopy, compared to oak savannas and oak woodlands.

Biology

https://en.wikipedia.org/wiki?curid=21739551

Oak forest

Nevus cell Nevus cells are a variant of melanocytes. They are larger than typical melanocytes, do not have dendrites, and have more abundant cytoplasm with coarse granules. They are usually located at the dermoepidermal junction or in the dermis of the skin. Dermal nevus cells can be further classified: type A (epithelioid) dermal nevus cells mature into type B (lymphocytoid) dermal nevus cells which mature further into type C (neuroid) dermal nevus cells, through a process involving downwards migration. Nevus cells are the primary component of a melanocytic nevus. Nevus cells can also be found in lymph nodes and the thymus.

Biology

https://en.wikipedia.org/wiki?curid=21740266

Nevus cell

Flowering Locus C ("FLC") is a MADS-box gene that in late-flowering ecotypes of the plant "Arabidopsis thaliana" is responsible for vernalization. In a new seedling "FLC" is expressed, which prevents flowering. Upon exposure to cold, less "FLC" is expressed (to a degree depending on the amount of cold), and flowering becomes possible. "FLC" is extensively regulated through epigenetic modifications and transcriptional control. The expression of "FLC" is affected by other genes including "FLK", "FCA" and "VERNALIZATION2" ("VRN2"). Wild "Arabidopsis" plants have different alleles for the "FLC" gene, which correspond to ecotypes which either (a) flower rapidly and produce a number of generations during one summer ("summer-annual"), or (b) flower only after vernalization ("winter-annual" or "late-flowering"). This kind of variation can also be provided by variation in the "FRIGIDA" ("FRI") gene.

Biology

https://en.wikipedia.org/wiki?curid=21746104

Flowering Locus C

Vagal escape The Sympathetic nervous system and Parasympathetic nervous system can offset each other. One of the most classical example is called Vagal Escape. is characterized by a reduction in blood pressure due to muscarinic stimulation which is then compensated for stimulation from the sympathetic system to increase heart rate and thus blood pressure. When the heart is continuously stimulated via the vagus nerve, initially there is stoppage of heart beat. With further continuous stimuli, heart beat resumes (namely the ventricles) as the parasympathetic nerves only have their influence on the SA and AV nodes of the heart and not on the musculature of the heart, which establishes its own rhythm.

Biology

https://en.wikipedia.org/wiki?curid=21748884

Vagal escape

MMP15 Matrix metalloproteinase 15 also known as is an enzyme that in humans is encoded by the "MMP15" gene. Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Most MMP's are secreted as inactive proenzymes which are activated when cleaved by extracellular proteinases. However, the protein encoded by this gene is a member of the membrane-type MMP (MT-MMP) subfamily; members of this subfamily can be anchored to the extracellular membrane by either a transmembrane domain or glycophosphatidylinositol linkage, suggesting that these proteins are expressed at the cell surface rather than secreted in a soluble form.

Biology

https://en.wikipedia.org/wiki?curid=21760762

MMP15

Virtual karyotype is the digital information reflecting a karyotype, resulting from the analysis of short sequences of DNA from specific loci all over the genome, which are isolated and enumerated. It detects genomic copy number variations at a higher resolution for level than conventional karyotyping or chromosome-based comparative genomic hybridization (CGH). The main methods used for creating virtual karyotypes are array-comparative genomic hybridization and SNP arrays. A karyotype (Fig 1) is the characteristic chromosome complement of a eukaryote species. A karyotype is typically presented as an image of the chromosomes from a single cell arranged from largest (chromosome 1) to smallest (chromosome 22), with the sex chromosomes (X and Y) shown last. Historically, karyotypes have been obtained by staining cells after they have been chemically arrested during cell division. Karyotypes have been used for several decades to identify chromosomal abnormalities in both germline and cancer cells. Conventional karyotypes can assess the entire genome for changes in chromosome structure and number, but the resolution is relatively coarse, with a detection limit of 5-10Mb. Recently, platforms for generating high-resolution karyotypes "in silico" from disrupted DNA have emerged, such as array comparative genomic hybridization (arrayCGH) and SNP arrays. Conceptually, the arrays are composed of hundreds to millions of probes which are complementary to a region of interest in the genome

Biology

https://en.wikipedia.org/wiki?curid=21777359

Virtual karyotype

Virtual karyotype The disrupted DNA from the test sample is fragmented, labeled, and hybridized to the array. The hybridization signal intensities for each probe are used by specialized software to generate a log2ratio of test/normal for each probe on the array. Knowing the address of each probe on the array and the address of each probe in the genome, the software lines up the probes in chromosomal order and reconstructs the genome "in silico" (Fig 2 and 3). Virtual karyotypes have dramatically higher resolution than conventional cytogenetics. The actual resolution will depend on the density of probes on the array. Currently, the Affymetrix SNP6.0 is the highest density commercially available array for virtual karyotyping applications. It contains 1.8 million polymorphic and non-polymorphic markers for a practical resolution of 10-20kb—about the size of a gene. This is approximately 1000-fold greater resolution than karyotypes obtained from conventional cytogenetics. Virtual karyotypes can be performed on germline samples for constitutional disorders, and clinical testing is available from dozens of CLIA certified laboratories (genetests.org). Virtual karyotyping can also be done on fresh or formalin-fixed paraffin-embedded tumors. CLIA-certified laboratories offering testing on tumors include Creighton Medical Laboratories (fresh and paraffin embedded tumor samples) and CombiMatrix Molecular Diagnostics (fresh tumor samples)

Biology

https://en.wikipedia.org/wiki?curid=21777359

Virtual karyotype

Virtual karyotype Array-based karyotyping can be done with several different platforms, both laboratory-developed and commercial. The arrays themselves can be genome-wide (probes distributed over the entire genome) or targeted (probes for genomic regions known to be involved in a specific disease) or a combination of both. Further, arrays used for karyotyping may use non-polymorphic probes, polymorphic probes (i.e., SNP-containing), or a combination of both. Non-polymorphic probes can provide only copy number information, while SNP arrays can provide both copy number and loss-of-heterozygosity (LOH) status in one assay. The probe types used for non-polymorphic arrays include cDNA, BAC clones (e.g., BlueGnome), and oligonucleotides (e.g., Agilent, Santa Clara, CA, USA or Nimblegen, Madison, WI, USA). Commercially available oligonucleotide SNP arrays can be solid phase (Affymetrix, Santa Clara, CA, USA) or bead-based (Illumina, SanDiego, CA, USA). Despite the diversity of platforms, ultimately they all use genomic DNA from disrupted cells to recreate a high resolution karyotype "in silico". The end product does not yet have a consistent name, and has been called virtual karyotyping, digital karyotyping, molecular allelokaryotyping, and molecular karyotyping. Other terms used to describe the arrays used for karyotyping include SOMA (SNP oligonucleotide microarrays) and CMA (chromosome microarray)

Biology

https://en.wikipedia.org/wiki?curid=21777359

Virtual karyotype