Panellus stipticus

Panellus stipticus, commonly known as the bitter oyster, the astringent panus, the luminescent panellus, or the stiptic fungus, is a species of fungus in the family Mycenaceae, and the type species of the genus "Panellus". A common and widely distributed species, it is found in Asia, Australia, Europe, and North America, where it grows in groups or dense overlapping clusters on the logs, stumps, and trunks of deciduous trees, especially beech, oak, and birch. During the development of the fruit bodies, the mushrooms start out as tiny white knobs, which, over a period of one to three months, develop into fan- or kidney-shaped caps that measure up to broad. The caps are orange-yellow to brownish, and attached to the decaying wood by short stubby stalks that are connected off-center or on the side of the caps. The fungus was given its current scientific name in 1879, but has been known by many names since French mycologist Jean Bulliard first described it as "Agaricus stypticus" in 1783. Molecular phylogenetic analysis revealed "P. stipticus" to have a close genetic relationship with members of the genus "Mycena".
"Panellus stipticus" is one of several dozen species of fungi that are bioluminescent. Strains from eastern North America are typically bioluminescent, but those from the Pacific coast regions of North America and from other continents are not. The luminescence is localized to the edges of the gills and the junction of the gills with the stem and cap. Bioluminescence is also observable with mycelia grown in laboratory culture, and the growth conditions for optimal light production have been studied in detail. Several chemicals have been isolated and characterized that are believed to be responsible for light production. Genetic analysis has shown that luminescence is controlled by a single dominant allele. The luminescent glow of this and other fungi inspired the term foxfire, coined by early settlers in eastern and southern North America. Modern research has probed the potential of "P. stipticus" as a tool in bioremediation, because of its ability to detoxify various environmental pollutants.
Taxonomy and phylogeny.
The species was first named "Agaricus stypticus" by the French botanist Jean Bulliard in 1783, and later sanctioned by Elias Magnus Fries under this name in 1821. Fries later changed the genus as well as the spelling of the epithet and called it "Panus stipticus". The species has had an extensive taxonomic history and been shuffled to a number of genera by various authors, resulting in several synonyms: "Agaricus flabelliformis" (Johann Friedrich Gmelin, 1792), "Pocillaria stiptica" (Otto Kuntze, 1898), "Rhipidium stipticum" (Karl Friedrich Wilhelm Wallroth, 1833), "Crepidopus stipticus" (Samuel Frederick Gray, 1821), "Pleurotus stipticus" (Paul Kummer), "Lentinus stipticus" (Joseph Schröter, 1885), and "Merulius stipticus" (Jean-Baptiste Lamarck). It was Finnish mycologist Petter Karsten who in 1879 assigned its current name. "Panellus stypticus" is still used in the literature as a variant spelling.
"Panellus stipticus" is the type species of the genus "Panellus", and, in Rolf Singer's authoritative 1986 classification of the Agaricales, it is also the type species of subgenus "Panellus", an infrageneric (below the taxonomic level of genus) grouping of "Panellus" species characterized by the absence of cystidia on the sides of the gills. More recently, phylogenetic analyses of the sequences of their ribosomal large subunit genes have concluded that "Panellus stipticus" is closely related to the poroid mushroom "Dictyopanus pusillus". The molecular analysis supports a previous assessment by mycologists Harold Bursdall and Orson K. Miller, who in 1975 suggested merging "Dictyopanus" into "Panellus" based on similarities in spore shape, stem structure, and the ability of dried fruit bodies to revive when moistened. Formerly grouped in the family Tricholomataceae, a wastebasket taxon of gilled mushrooms with white spores, "P. stipticus" is now classified in the Mycenaceae, after a large-scale phylogenetic analysis revealed "a previously unsuspected relationship between "Mycena" and "Panellus" (including "Dictyopanus")".
The fungus is commonly known as the bitter oyster, the luminescent panellus, the astringent panus, or the styptic fungus. The specific epithet "stipticus" refers to its purported value in stopping bleeding. Etymologically, it is a Greek equivalent to the Latin word "astringens", deriving from στυπτικός ("styptikós"), itself from the verb στύφειν ("styphein"), "to contract".
Description.
The fungus normally exists unseen, in the form of a mass of threadlike vegetative cells called a mycelium, inhabiting rotting wood; only when suitable environmental conditions of temperature, moisture, and nutrient availability are achieved does the fungus produce the reproductive structures known as fruit bodies, or mushrooms. The cap of the fruit body is kidney- or clamshell-shaped, convex to roughly flat, with dimensions of by . The edges of the caps are scalloped with small rounded teeth, and curved slightly inward. The cap surface is dry, with a pattern of block-like areas similar to cracked dried mud; the surface is also covered with small fine hairs that give it a somewhat woolly consistency. It may have several concentric ridges or zones. Fresh fruit bodies range in color from yellowish-orange to buff to cinnamon; when dried they may be various shades of tan, brown or clay. The faded colors of dried fruit bodies tend to revive when moistened. On the underside of the cap, the gills are narrow and spaced closely together, often forked, buff-colored, and with numerous interconnecting cross-veins. Holding the cap in position is a stem that is long by thick, and has an off-center attachment to the cap, either at or near the cap side. The dull-white stem is covered with minute silk-like fibers, and is narrower at the base where it attaches to the substrate. Fruit bodies do not have any distinctive odor. The flesh is thin and tough, and dark yellow-brown to cream-colored.
Microscopic features.
Various microscopic characteristics may be used to help identify the fungus from other morphologically similar species. A spore print of "P. stipticus", made by depositing a large number of spores in a small area, reveals their color to be white. Viewed with a microscope, the spores are smooth-walled, elliptical to nearly allantoid (sausage-shaped), with dimensions of 3–6 by 2–3 µm. Spores are amyloid, meaning that they will absorb iodine and become bluish-black when stained with Melzer's reagent, but this staining reaction has been described as "relatively weak".
The basidia (the spore-bearing cells) are 15–20 by 2.5–3.5 µm, club-shaped, and clamped at the base. The spores are attached to the end of the basidia by four projections called sterigmata that are 1–3.5 µm long. Cystidia are hyphal cells in the hymenium that do not produce basidiospores; they also make up the surfaces of the fruit body (the pelli or "cuticles"). They are usually structurally distinct from the basidia, and their features may be used as microscopic characters to help distinguish and differentiate similar fungi. The cheilocystidia are found on the gill edge; in "P. stipticus" they are narrowly club-shaped, cylindrical, spindle-shaped to bifurcate at the apex. They are also thin-walled, hyaline (translucent), abundant and crowded, and measure 17–45 by 3.5–6 µm. The pleurocystidia, located on the gill face, are 17–40 by 3–4.5 µm, spindle- or club-shaped, sometimes bifurcate at the apex, thin-walled, and hyaline. They are scattered or in dense clusters, mostly embedded in hymenium, occasionally protruding up to half the width of the hymenium.
The flesh of the cap consists of a number of microscopically distinct layers of tissue. The cuticle of the cap (known as the pileipellis) is between 8–10 µm thick, and is made of a loose "textura intricata", a type of tissue in which the hyphae are irregularly interwoven with distinct spaces between them. The cuticle hyphae are thick-walled to thin-walled, with scattered inconspicuous cystidia measuring 40–55 by 3.5–5.5 µm. These cystidia located in the cap (pileocystidia) are cylindrical, thin-walled, yellow in Melzer's reagent, hyaline in KOH, sometimes with amorphous dingy brown material coating the walls. Beneath the cuticle layer is a zone 54–65 µm thick, made of very loosely entwined, thin-walled hyphae, 2–3 µm in thickness, with clamps at the septa. Below this is a zone 208–221 µm in thickness, in which the densely compacted hyphae, 3–8 µm in diameter, have swollen, gelatinized walls, and often more or less a vertical orientation. This in turn is followed by a layer 520 µm in thickness, formed of loosely interwoven hyphae, 2–8 µm in width, some of which have thin walls with clamps at the septa, whilst others have somewhat thickened gelatinized walls. The flesh of the cap has a layer of upright hyphae bending into a lower layer of interwoven hyphae with diameters of 2.5–8 µm. The flesh of the gills is similar to that of the lower cap.
Similar species.
Species of "Crepidotus" having a similar shape can be distinguished by their brown spore print, compared with the white spore print of "P. stipticus". "Schizophyllum commune" has a densely hairy white to grayish cap and longitudinally split gill-folds on the underside. The ruddy panus mushroom ("Panus rudis") is larger, has a reddish-brown cap that fades to pinkish-tan, and shows lilac tinges when young, fresh, and moist. Some "Paxillus" species may have a similar appearance, but they have yellow-brown spore prints.
Uses.
"Panellus stipticus" is considered too small and bitter to be edible, but is considered nonpoisonous. Its taste has been described as acidic, acrid, or astringent. One 1992 study reported that the taste of the fruit bodies varies across the geographic range of the species. Those from eastern North America have a mildly acrid taste that takes time to develop, and cause uncomfortable drying in the mouth. In contrast, specimens from Japan, New Zealand, and Russia produce no sensation in the mouth, but cause significant constriction and a nauseating taste in the throat. The fruit bodies are reputed to have been used in traditional Chinese medicine as a styptic to staunch bleeding, and also as a "violent purgative".
Fruit body development.
Fruit bodies first appear as tiny white knobs less than a cubic millimeter in size. In a day or two the knobs grow into a horizontal pyramidal mass, increasing in height as the hyphae lengthen. This is soon followed by the formation of a minute cap, and lengthening of the stem. The stem is about 1 mm long when the cap first begins to form. The hyphae that comprise the stem gradually cease to grow at their ends, and then start to branch, with many of the branches growing in a horizontal direction. This growth, indicated by the flattening and broadening out of the top of the stem, gives rise to the cap. The horizontally aligned hyphae grow vertical branches which remain more or less parallel, ultimately forming the dorsal tissue of the cap. Other similar downward-growing branches form the fertile hymenium, which can be seen when the cap is about 2 mm in diameter.The young cap is spherical and its growth is at first epinastic, its margin being curved inwards and pressed against the stem. In this way, the hymenium begins its development within a special enclosed chamber. As the hymenial surface increases and keeps pace with the growth of the dorsal tissue of the cap, the latter expands and exposes the gills. The gills are formed by the continual downward growth of some of the hyphae. The gills are exposed before the cap is completely developed, and before the spores are mature. Spores can be produced by fruit bodies as small as broad, and liberation of the spores continues until the fruit body is fully grown—a period of one month to three months, depending on the conditions of temperature and moisture. The mature spores are disseminated by the wind. When the fruit body is nearing maturity, some of the terminal portions of the hyphae of the dorsal surface of the cap separate, and as a consequence, the upper surface of the fruit body becomes granular in appearance.
The fruit body projects out horizontally from the growing surface. If the position of a log is altered after young fruit bodies with the beginnings of gills have appeared, the stems of these attempt to readjust themselves in order to place the cap in a horizontal position. The cap are sometimes zonate (marked with concentric lines that form alternating pale and darker zones); this depends on changes in the humidity of the environment, as variations in the amount of moisture will cause alternating periods of acceleration or slowing of growth.
A yellowish-brown pigment is diffused through the cytosol of the hyphae and is much deeper in color just below the cuticle of the cap. In very young fruit bodies, stems and caps are very pale buff, but soon the color of the cap deepens and becomes cinnamon. The intensity of the color appears to be dependent on light, for when fruit bodies are grown in diffuse light (temperature and humidity being constant) they are a uniform pale buff color, but in bright light they are cinnamon or tan.
Distribution, habitat, and ecology.
"Panellus stipticus" is common in northern temperate regions of Europe, and has also been collected in Australia, New Zealand, Anatolia, Japan, and China. In North America, it is more common in the east than the west; the mushroom's northern range extends to Alaska, and it has been collected as far south as Costa Rica.
"Panellus stipticus" is a saprobic species, and causes a white rot. This is a form of wood decay in which the wood assumes a bleached appearance and where lignin as well as cellulose and hemicellulose is broken down by enzymes secreted by the fungus. Fruit bodies are usually found in tightly overlapping clusters on the sides of hardwood trees, on logs, stumps, and fallen branches. Although most commonly found on dead wood, it can also grow opportunistically in the wounds of living trees. In North America, its preferred hosts are oak, birch, maple, hickory, pecan, and American hornbeam, while in Europe, it is commonly found growing on oak, birch, alder, beech, hazel, chestnut, and ash. It has also been found growing on "Ericia" in North Africa. Although the fungus favors hardwoods, it has been reported to grow on loblolly pine and eastern white pine. Fruiting occurs September through November in Europe, the Canary Islands, and North America, although it may also sometimes be found in the spring. The fruit bodies are long-lasting and may be found year-round. It is an "early-stage" succession fungus, not typically recorded from plantations over 20 years old.
The fruit bodies are frequently attacked by slugs, which may be important agents in the dispersal of its spores. White-tailed deer are also known to consume the fungus.
Mating studies.
"Panellus stipticus" uses a heterothallic, tetrapolar mating system: each basidiospore develops into a self-sterile mycelium which, when grown alone, remains homokaryotic (i.e., with all cells genetically identical) indefinitely. Researchers have paired collections of "P. stipticus" from Japan and Eastern North America, and later, collections from New Zealand and Russia. Although the separated allopatric populations differ in bioluminescence and taste, the results revealed a universal intercompatibility group over these geographical regions. In a 2001 study, Jin and colleagues also paired geographically representative collections of the fungus, but observed a reduced ability to cross between Northern Hemisphere and Oceanian collections, as well as between and within Oceanian collections.
Bioluminescence.
Bioluminescence refers to the ability of certain living things in the environment to produce light by the action of enzymes. Bioluminescent fungi are widespread, and over 70 species are known (List of bioluminescent fungus species). Although the intensity of their luminescence is generally low compared to many other bioluminescent organisms, fungi glow continuously for days, so their total emission is comparable with that of most brightly luminescent organisms, such as fireflies. Luminous fungi are found growing on decaying wood, leading to the popular name of "foxfire" or "glow wood" when their glow is visible at night. The responsible oxidative enzymes—known generically as luciferases—produce light by oxidizing a pigment called a luciferin. In some areas, "P. stypticus" is bioluminescent, and the fruit bodies of these strains will glow in the dark when fresh or sometimes when revived in water after drying.
An early record of luminescence noted in "P. stypticus" was made by the American naturalist Thomas G. Gentry in 1885. Job Bicknell Ellis, reporting on the phenomenon for the "Journal of Mycology", wrote:
By careful examination, the luminosity was found to proceed from the gills and not the stipe, nor from any fragment of rotten wood attached to the specimen. This phosphorescence was not observed in all specimens brought in for examination, and seemed to depend on some peculiar condition of the air, having been noticed only in specimens gathered in damp weather or just before a storm.
Canadian mycologist Buller in 1924 described the gills of "P. stipticus" in North America as luminescent, and noted that the fungus glows most strongly at the time of spore maturation. Bioluminescence has not been observed in European specimens, in Pacific North American collections, nor in strains collected from New Zealand, Russia, and Japan. Although a number of reports have confirmed that eastern North American strains are luminescent, non-luminescent North American strains are also known. In general, the intensity of fungal bioluminescence decreases after exposure to certain contaminants; this sensitivity is being investigated as a means to develop bioluminescence-based biosensors to test the toxicity of polluted soils. Most known luminescent fungi are in the genus "Mycena" or closely allied genera; this grouping of fungi—known as the "mycenoid lineage"—includes "P. stipticus" and three other "Panellus" species.
Mycelia.
The mycelia of this species, grown in laboratory culture, have also been shown to be bioluminescent. Early studies demonstrated that short-wave ultraviolet light (at a wavelength of 280 nm) reversibly inhibited the luminescence of "P. stypticus" mycelia, while longer wavelength (366 nm) ultraviolet was stimulatory. Further, the fungus exhibited a pronounced diurnal periodicity, and maximum luminescence was noted between 6 and 9 pm, regardless as to whether the mycelial cultures were incubated in continuous light, continuous darkness, or a normal day-night cycle. The mycelia of "P. stipticus" grown submerged in liquid were non-luminescent, but became luminescent while growing on solid substrata. Dark-grown colonies were luminescent in the center, and light-grown colonies were brightest at the periphery. Other experiments have shown that growth temperature and pH have a significant effect on the level of bioluminescence, optimized at and pH 3–3.5. However, light had a significant effect on mycelial growth but not on bioluminescence, and the optimal light conditions for maximum bioluminescence were total darkness.
Fruit bodies.
Bioluminescent tissue in the mature fruit body is restricted to the edge of the gills (as well as the cross-veins that connect them), the junction of the gills with the stem, and the inrolled cap edge. Distribution of bioluminescence along the gill edge corresponds to the position of the cheilocystidia. Less than 10% of the light emitted from both the young and mature fruit bodies is from other tissues, including the fertile hymenial area and the stem. Fruit body luminescence is highly variable between fruit bodies found on different logs in different environments.
Genetics.
Using techniques of genetic complementation, Macrae paired nonluminescent monocaryons with luminescent ones, and concluded that luminosity in "P. stipticus" is an inherited character, and governed by a single pair of alleles in which luminosity was dominant over nonluminosity. Luminosity factors were independent of intersterility factors. In 1992, Lingle and colleagues agreed with Macrae about luminosity and stated that at least three different gene mutations could lead to the loss of luminescence. They also reported that the maximum bioluminescence was found at 525 nm, and shifted to 528 nm in deeply pigmented fruit bodies.
After intercontinental compatibility tests, Petersen and Bermudes suggested that bioluminescence and compatibility were independent since bioluminescence seemed to be geographically restricted. This suggested that the ability or potential to interbreed must have been preserved since separation of "P. stypticus" into geographically isolated areas.
Function.
Several authors have suggested that the purpose of fungal bioluminescence is to attract arthropods to the fruit body to help disseminate spores. However, there has been no direct observation of this phenomenon. In many species, the mycelium is luminescent, but the fruit body is not, which argues against this hypothesis. Mycelial bioluminence may also function to attract animals that can potentially carry fungal spores in well-hydrated areas, as light emission from the mycelium is higher when it is hydrated. This would presumably increase the chance that spores would be deposited in an environment that is already optimal for growth. Basidiomycete mushrooms are known to be dependent on an adequate moisture supply for proper development. In species with a luminous mycelium, the mycelium would therefore have a dual function in performing the fungal translocation that permits transport of substances from the further environment back to the fruiting body, and in attracting disseminating vectors towards environments favorable for development of the species.
Chemical basis.
In general, bioluminescence is caused by the action of luciferases, enzymes that produce light by the oxidation of a luciferin (a pigment). Several studies have evaluated the biochemical basis of light production in "Panellus stipticus", and concluded that there is no specific fungal luciferase. The fungus was shown in 1988 to contain the sesquiterpene panal, which has a cadinene keto-aldehyde chemical structure. Later, two additional precursors were isolated, PS-A (1-"O"-decanoylpanal) and PS-B (1-"O"-dodecanoylpanal). If these compounds are treated with salt of ammonia or primary amines in the presence of iron(II), hydrogen peroxide, and a cationic surfactant, light is emitted by a chemiluminescence reaction, suggesting that panal and its derivatives are fungal luciferins, and that the chemiluminescence reaction is the cause of "in vivo" bioluminescence. In the fungus, the level of activity of the enzyme superoxide dismutase (SOD) appears to play a critical role in the amount of light emission. SOD quenches the effect of the superoxide (O2−) anion required in the reaction, and thus SOD activity has to be inhibited for the reaction to occur.
Bioremediation.
As a white-rot fungus, "Panellus stipticus" contains enzymes that are able to break down lignin, a complex aromatic polymer in wood that is highly resistant to degradation by conventional enzyme systems. The major enzyme that initiates the cleavage of hydrocarbon rings is laccase, which catalyzes the addition of a hydroxyl group to phenolic compounds (polyphenols). The ring can then be opened between the two adjacent carbon atoms that bear the hydroxyl groups. White-rot fungi are being investigated scientifically for their potential use in the bioremediation of land contaminated by organic pollutants, and to convert industrial wastes rich in toxic polyphenols. "Panellus stipticus" has been shown to reduce the phenolic concentration of waste water produced by olive-processing plants—an environmental concern in many Mediterranean countries. In this study, a liquid culture of "P. stipticus" mycelia reduced the initial concentration of phenolic compounds by 42% after a 31-day incubation period. In a separate study, a "P. stipticus" culture was able to effectively degrade the environmental pollutant 2,7-dichlorodibenzo-"p"-dioxin, a polychlorinated dioxin.


VISTA (comparative genomics)

VISTA is a collection of databases, tools, and servers that permit extensive comparative genomics analyses.
Background.
The VISTA family of tools is developed and hosted at Genomics Division of Lawrence Berkeley National Laboratory. This collaborative effort is supported by the Programs for Genomic Applications grant from the NHLBI/NIH and the Office of Biological and Environmental Research, Office of Science, US Department of Energy.
It was developed from modules supplied by developers at UC Berkeley, Stanford, and UC Davis, and based partly on the AVID Global Alignment program.
Usage.
There are multiple VISTA servers, each allowing different types of searches.
Researchers can use the VISTA Browser:
Genomes.
There are more than 28 searchable genomes, including vertebrate, non-vertebrate, plants, fungi, algae, bacteria, and others. More are continually being added. These include:
Collaboration with other projects.
Pre-computed full scaffold alignments for microbial genomes are available as the VISTA component of IMG (Integrated Microbial Genomes System) developed in the DOE (Department of Energy's) Joint Genome Institute.


Ian R. Gibbons

Ian Read Gibbons, (30 October 1931 – 30 January 2018) was a biophysicist and cell biologist. He discovered and named dynein, and demonstrated energy source as ATP is sufficient for dynein to walk on microtubules. In 2017, he and Ronald Vale received the Shaw Prize for their research on microtubule motor proteins.
He was elected a Fellow of the Royal Society of Great Britain in 1983. The Society stated:
Early life and education.
Gibbons's passion for science stems from his interest in radio. He entered Queen Elizabeth's Grammar School in Faversham in 1943, where he developed an interest towards applied physics. Following 18 months in the Royal Air Force as a radar engineer, he was admitted into King's College at the University of Cambridge in 1951 to read physics. He graduated with a bachelor's degree and then, in 1957, a PhD degree from Cambridge. His PhD research concerns using electron microscopes to study the organisation of chromosomes during mitosis and meiosis. Gibbons then went to the University of Pennsylvania as a postdoctoral researcher, where he stayed for 1 year. He subsequently moved to the Department of Biology, Harvard University, to take up the post of director of the newly founded electron microscopy laboratory.
Academic and research career.
While at Harvard, Gibbons studied the structure of cilia and flagella of a protozoan called "Tetrahymena" with electron microscopes. In 1963, he discovered a novel protein on microtubules and published its pictures. Two years later, he purified two regions of the protein, known as its two "arms", naming the protein "dynein". During his last year at Harvard, Gibbons demonstrated the protein making up microtubules was distinct from actin, in that the former was associated with guanine nucleotides while the latter with adenine nucleotides, but refrained from naming it; Hideo Mohri from the University of Tokyo named it tubulin afterwards.
Gibbons moved to the Kewalo Marine Laboratory, University of Hawaii at Manoa, in 1967 as an associate professor. He found the cilia of sea urchin sperms easier to work with than the cilia and flagella of "Tetrahymena". In 1969, he was promoted to professor of biophysics. Throughout the 1970s, Gibbons and his wife Barbara showed the sliding of microtubules caused cilia motility (known as the sliding tubule mechanism), and that this sliding was dependent on the energy generated from ATP hydrolysis by ATPase. When microtubules visibly slid out of the ends of the flagellar fiber, the flagella disintegrated. He then extended the mechanism to mammals, confirming the motility mechanism of bull sperm cilia is the same as that for sea urchins. After these findings, Gibbons switched his focus to the molecular biology of dyneins, and determined the DNA sequence of the largest subunit of dynein in 1991. In 1993, he became the director of the Kewalo Marine Laboratory.
Ian and Barbara Gibbons retired from the University of Hawaii at Manoa in 1997; he went to the University of California, Berkeley as a research scientist in the laboratory of Beth Burnside. In 2009, Burnside closed her laboratory, and Gibbons became a visiting researcher.
Personal life.
Gibbons met his wife Barbara while in Harvard University; they married in 1961. Barbara died in 2013 at age 81. Gibbons also died in 2018.


Golden mole

Golden moles are small insectivorous burrowing mammals endemic to Southern Africa. They comprise the family Chrysochloridae and as such they are taxonomically distinct from the true moles, family Talpidae, and other mole-like families, all of which, to various degrees, they resemble as a result of evolutionary convergence.
Characteristics and affinities.
Like most burrowing mammals with similar habits, the Chrysochloridae have short legs with powerful digging claws, very dense fur that repels dirt and moisture, and toughened skin, particularly on the head. Their eyes are non-functional and covered with furred skin. The external ears are just tiny openings. In particular, golden moles bear a remarkable resemblance to the marsupial moles of Australia, family Notoryctidae, which they resemble so suggestively that at one time, the marsupial/placental divide not withstanding, some argued that they were related. Considerations that influenced the debate might have included the view that the Chrysochloridae are very primitive placentals and the fact that they have many mole-like specializations similar to specializations in marsupial moles. The rhinarium is a greatly enlarged, dry leathery pad that protects their nostrils while the animal digs. In this respect too, they resemble the marsupial moles. Some authors claim their primary sense is of touch, and they are particularly sensitive to vibrations which may indicate approaching danger. Note below however, the observations on the malleus in the middle ear.
The species range in size from about to about . They have muscular shoulders and the forelimbs are radically adapted for digging; all the toes on the forefeet have been reduced, except for a large, pick-like third claw on the third toe. The fifth digit is absent and the first and fourth digits are vestigial. The adaptations of the hind feet are less dramatic, they retain all five toes and are webbed as an adaptation to efficient backward shovelling of soil loosened by the front claws.
At one time the Chrysochloridae were regarded as primitive. Supporting arguments of this included that they were thought to have originated in Gondwana, that they had a low resting metabolic rate, and that they could switch off thermoregulation when inactive. Like the tenrecs, they possess a cloaca, and males lack a scrotum. However, these points are no longer regarded as strongly suggestive of golden moles as undeveloped "reptilian mammals"; some are seen rather as adaptations to regional climatic conditions. Going into a torpor when resting or during cold weather enables them to conserve energy and reduce urgent requirements for food. Similarly, they have developed particularly efficient kidneys and most species do not need to drink water at all; in fact they tend to drown easily if they fall into water.
Habits and ecology.
Most species of Chrysochloridae live almost exclusively underground in their respectively preferred environments, beneath either grassveld, forest, swamps, deserts, or mountainous terrain. However, "Chrysospalax" species tend to forage above ground in leaf litter in forests or in meadows. "Eremitalpa" species such as Grant's golden mole live in the sandy Namib desert, where they cannot form tunnels because the sand collapses. Instead during the day, when they must seek shelter, they "swim" through the loose sand, using their broad claws to paddle, and dive down some 50 cm to where it is bearably cool. There they enter a state of torpor, thus conserving energy. At night they emerge to forage on the surface rather than wasting energy shifting sand. Their main prey are termites that live under isolated grass clumps, and they might travel for 6 kilometres a night in search of food. They seek promising clumps by listening for wind-rustled grass-root stresses and termites' head-banging alarm signals, neither of which can be heard easily above ground, so they stop periodically and dip their heads under the sand to listen.
Most other species construct both foraging superficial burrows and deeper permanent burrows for residence. Residential burrows are relatively complex in form, and may penetrate as far as a metre below ground and include deep chambers for use as bolt-holes, and other chambers as latrines. They push excavated soil up to the surface as mole-hills, or compact it into the tunnel walls.
They feed on small insects and earthworms or small vertebrates such as lizards or burrowing snakes. They depend on their sense of hearing to locate much of their prey, and the cochleas of a number of golden mole species have been found to be long and highly coiled, which may indicate a greater ecological dependence on low frequency auditory cues than we see in Talpid moles.
Some species also have hypertrophied middle ear ossicles, in particular the malleus, which apparently is adapted towards the detection of seismic vibrations. In this respect there is some apparent convergent evolution to burrowing reptiles in the family Amphisbaenidae.
Reproduction.
Females give birth to one to three hairless young in a grass-lined nest within the burrow system. Breeding occurs throughout the year. The adults are solitary, and their burrowing territory may be aggressively defended from intruders, especially where resources are relatively scarce.
Status.
Of the 21 species of golden mole, no fewer than 11 are threatened with extinction. The primary causes are sand mining, poor agricultural practices, increasing urbanisation, and predation by domestic cats and dogs.
Classification.
The taxonomy of the Chrysochloridae is undergoing a review in the light of new genetic information. They have traditionally been listed with the shrews, hedgehogs and a grab-bag of small, difficult-to-place creatures as part of the order Insectivora. Some authorities retain this classification, at least for the time being. Others group the golden moles with the tenrecs in a new order, which is sometimes known as Tenrecomorpha, while others call it Afrosoricida and reserve Tenrecomorpha for the family Tenrecidae.


BrainScope Company, Inc.

BrainScope Company, Inc. (BrainScope) is a medical neuro-technology firm utilizing EEG technology to assess the full spectrum of mild traumatic brain injury (mTBI), including functional injuries (such as a concussion), and structural injuries (such as a brain bleed). BrainScope was founded in 2006 and is headquartered in Bethesda, Maryland.
BrainScope's markets include the military, emergency departments, urgent care clinics, and both collegiate and professional level sports programs.
Technology.
"BrainScope One" is a multimodal assessment tool that provides clinicians with the ability to identify both the structural and functional sides of mild traumatic brain injuries (mTBIs) in their patients. The device utilizes electroencephalographic (EEG) technology, as well as cognitive performance and clinical assessments commonly used in mTBI evaluation.
The electroencephalographic (EEG) data that is collected with a disposable electrode headset provides objective biomarkers to both predict the likelihood that a patient's structural brain injury would be visible on a CT scan and assess the likelihood of functional impairment in the brain by comparing the patient's brain function to that of non-head injured individuals. "BrainScope One" also includes neurocognitive tests (Procedural Reaction Time & Match to Sample), which are performed by the patient on the device, as well as an extensive library of digitized concussion assessments.
An independent validation trial conducted using "BrainScope One" determined that the device's sensitivity to "≥1 mL of blood was ... 98.6%" and that its accurate CT-scan predictions can lead to a "reduction in the number of false positives of 33%."
"BrainScope One's" technology was created through 21 clinical studies at over 55 clinical sites spanning a decade, contributing to 23 investigator-initiated peer-review journal articles and resulting in 5 FDA clearances. "BrainScope One" is FDA cleared as an Rx-only device for use on patients 18–85 years of age who have suffered a mild head injury (Glasgow Coma Scale score of 13-15 and mTBI) within the previous 72 hours. "BrainScope One" was originally cleared by the FDA as the Ahead 300 under 510(k) K161068 in September 2016. Subsequent modifications to device Indications for Use regarding concussion/mTBI capabilities were FDA cleared under 510(k) K181179 in May 2018.
The company initiated a clinical research study on patients 13–25 years of age in May 2018 as part of its initiative to "introduce a teenage-focused product in the near-term."
Partners.
BrainScope has raised over $60 million in private capital and has received $30 million in research contracts, including funding from the United States Department of Defense and General Electric and the National Football League through their Head Health Challenge competition. Among the company's private investors are Revolution LLC, an investment firm founded by AOL co-founder Steve Case; DBL Partners, a venture capital firm that also backs Tesla and Pandora; and the Maryland Venture Fund.
BrainScope's other development partners include New York University School of Medicine and Johns Hopkins University and its clinical partners include Emory University, the University of Virginia and Washington University in St. Louis.


Model lipid bilayer

A model lipid bilayer is any bilayer assembled in vitro, as opposed to the bilayer of natural cell membranes or covering various sub-cellular structures like the nucleus. They are used to study the fundamental properties of biological membranes in a simplified and well-controlled environment, and increasingly in bottom-up synthetic biology for the construction of artificial cells. A model bilayer can be made with either synthetic or natural lipids. The simplest model systems contain only a single pure synthetic lipid. More physiologically relevant model bilayers can be made with mixtures of several synthetic or natural lipids.
There are many different types of model bilayers, each having experimental advantages and disadvantages. The first system developed was the black lipid membrane or “painted” bilayer, which allows simple electrical characterization of bilayers but is short-lived and can be difficult to work with. Supported bilayers are anchored to a solid substrate, increasing stability and allowing the use of characterization tools not possible in bulk solution. These advantages come at the cost of unwanted substrate interactions which can denature membrane proteins.
Black lipid membranes (BLM).
The earliest model bilayer system developed was the “painted” bilayer, also known as a “black lipid membrane.” The term “painted” refers to the process by which these bilayers are made. First, a small aperture is created in a thin layer of a hydrophobic material such as Teflon. Typically the diameter of this hole is a few tens of micrometers up to hundreds of micrometers. To form a BLM, the area around the aperture is first "pre-painted" with a solution of lipids dissolved in a hydrophobic solvent by applying this solution across the aperture with a brush, syringe, or glass applicator. The solvent used must have a very high partition coefficient and must be relatively viscous to prevent immediate rupture. The most common solvent used is a mixture of decane and squalene.
After allowing the aperture to dry, salt solution (aqueous phase) is added to both sides of the chamber. The aperture is then "painted" with a lipid solution (generally the same solution that was used for pre-painting). A lipid monolayer spontaneously forms at the interface between the organic and aqueous phases on either side of the lipid/solvent droplet. Because the walls of the aperture are hydrophobic the lipid/solvent solution wets this interface, thinning the droplet in the center. Once the two sides of the droplet come close enough together, the lipid monolayers fuse, rapidly excluding the small remaining volume of solution. At this point a bilayer is formed in the center of the aperture, but a significant annulus of solvent remains at the perimeter. This annulus is required to maintain stability by acting as a bridge between the ~5 nm bilayer and the 10s of micrometer thick sheet in which the aperture is made.
The term “black” bilayer refers to the fact that they are dark in reflected light because the thickness of the membrane is only a few nanometers, so light reflecting off the back face destructively interferes with light reflecting off the front face. Indeed, this was one of the first clues that this technique produced a membrane of molecular-scale thickness. Black lipid membranes are also well suited to electrical characterization because the two chambers separated by the bilayer are both accessible, allowing simple placement of large electrodes. For this reason, electrical characterization is one of the most important methods used in conjunction with painted lipid bilayers. Simple measurements indicate when a bilayer forms and when it breaks, as an intact bilayer has a large resistance (>GΩ) and a large capacitance (~2 µF/cm2). More advanced electrical characterization has been particularly important in the study of voltage gated ion channels. Membrane proteins such as ion channels typically cannot be incorporated directly into the painted bilayer during formation because immersion in an organic solvent would denature the protein. Instead, the protein is solubilized with a detergent and added to the aqueous solution after the bilayer is formed. The detergent coating allows these proteins to spontaneously insert into the bilayer over a period of minutes. Additionally, initial experiments have been performed which combine electrophysiological and structural investigations of black lipid membranes. In another variation of the BLM technique, termed the bilayer punch, a glass pipet (inner diameter ~10-40 µm) is used as the electrode on one side of the bilayer in order to isolate a small patch of membrane. This modification of the patch clamp technique enables low noise recording, even at high potentials (up to 600 mV), at the expense of additional preparation time.
The main problems associated with painted bilayers are residual solvent and limited lifetime. Some researchers believe that pockets of solvent trapped between the two bilayer leaflets can disrupt normal protein function. To overcome this limitation, Montal and Mueller developed a modified deposition technique that eliminates the use of a heavy non-volatile solvent. In this method, the aperture starts out above the water surface, completely separating the two fluid chambers. On the surface of each chamber, a monolayer is formed by applying lipids in a volatile solvent such as chloroform and waiting for the solvent to evaporate. The aperture is then lowered through the air-water interface and the two monolayers from the separate chambers are folded down against each other, forming a bilayer across the aperture. The stability issue has proven more difficult to solve. Typically, a black lipid membrane will survive for less than an hour, precluding long-term experiments. This lifetime can be extended by precisely structuring the supporting aperture, chemically crosslinking the lipids or gelling the surrounding solution to mechanically support the bilayer. Work is ongoing in this area and lifetimes of several hours will become feasible.
Supported lipid bilayers (SLB).
Unlike a vesicle or a cell membrane in which the lipid bilayer is rolled into an enclosed shell, a supported bilayer is a planar structure sitting on a solid support. Because of this, only the upper face of the bilayer is exposed to free solution. This layout has advantages and drawbacks related to the study of lipid bilayers. One of the greatest advantages of the supported bilayer is its stability. SLBs will remain largely intact even when subject to high flow rates or vibration and, unlike black lipid membranes, the presence of holes will not destroy the entire bilayer. Because of this stability, experiments lasting weeks and even months are possible with supported bilayers while BLM experiments are usually limited to hours. Another advantage of the supported bilayer is that, because it is on a flat hard surface, it is amenable to a number of characterization tools which would be impossible or would offer lower resolution if performed on a freely floating sample.
One of the clearest examples of this advantage is the use of mechanical probing techniques which require a direct physical interaction with the sample. Atomic force microscopy (AFM) has been used to image lipid phase separation, formation of transmembrane nanopores followed by single protein molecule adsorption, and protein assembly with sub-nm accuracy without the need for a labeling dye. More recently, AFM has also been used to directly probe the mechanical properties of single bilayers and to perform force spectroscopy on individual membrane proteins. These studies would be difficult or impossible without the use of supported bilayers since the surface of a cell or vesicle is relatively soft and would drift and fluctuate over time. Another example of a physical probe is the use of the quartz crystal microbalance (QCM) to study binding kinetics at the bilayer surface. Dual polarisation interferometry is a high resolution optical tool for characterising the order and disruption in lipid bilayers during interactions or phase transitions providing complementary data to QCM measurements.
Many modern fluorescence microscopy techniques also require a rigidly-supported planar surface. Evanescent field methods such as total internal reflection fluorescence microscopy (TIRF) and surface plasmon resonance (SPR) can offer extremely sensitive measurement of analyte binding and bilayer optical properties but can only function when the sample is supported on specialized optically functional materials. Another class of methods applicable only to supported bilayers is those based on optical interference such as fluorescence interference contrast microscopy (FLIC) and reflection interference contrast microscopy (RICM) or interferometric scattering microscopy (iSCAT). When the bilayer is supported on top of a reflective surface, variations in intensity due to destructive interference from this interface can be used to calculate with angstrom accuracy the position of fluorophores within the bilayer. Both evanescent and interference techniques offer sub-wavelength resolution in only one dimension (z, or vertical). In many cases, this resolution is all that is needed. After all, bilayers are very small only in one dimension. Laterally, a bilayer can extend for many micrometres or even millimeters. But certain phenomena like dynamic phase rearrangement do occur in bilayers on a lateral sub-micrometre length scale. A promising approach to studying these structures is near field scanning optical microscopy (NSOM). Like AFM, NSOM relies on the scanning of a micromachined tip to give a highly localized signal. But unlike AFM, NSOM uses an optical rather than physical interaction with the sample, potentially perturbing delicate structures to a lesser extent.
Another important capability of supported bilayers is the ability to pattern the surface to produce multiple isolated regions on the same substrate. This phenomenon was first demonstrated using scratches or metallic “corrals” to prevent mixing between adjacent regions while still allowing free diffusion within any one region. Later work extended this concept by integrating microfluidics to demonstrate that stable composition gradients could be formed in bilayers, potentially allowing massively parallel studies of phase segregation, molecular binding and cellular response to artificial lipid membranes. Creative utilization of the corral concept has also allowed studies of the dynamic reorganization of membrane proteins at the synaptic interface.
One of the primary limitations of supported bilayers is the possibility of unwanted interactions with the substrate. Although supported bilayers generally do not directly touch the substrate surface, they are separated by only a very thin water gap. The size and nature of this gap depends on the substrate material and lipid species but is generally about 1 nm for zwitterionic lipids supported on silica, the most common experimental system. Because this layer is so thin there is extensive hydrodynamic coupling between the bilayer and the substrate, resulting in a lower diffusion coefficient in supported bilayers than for free bilayers of the same composition. A certain percentage of the supported bilayer will also be completely immobile, although the exact nature of and reason for these “pinned” sites is still uncertain. For high quality liquid phase supported bilayers the immobile fraction is typically around 1-5%. To quantify the diffusion coefficient and mobile fraction, researchers studying supported bilayers will often report FRAP data.
Unwanted substrate interactions are a much greater problem when incorporating integral membrane proteins, particularly those with large domains sticking out beyond the core of the bilayer. Because the gap between bilayer and substrate is so thin these proteins will often become denatured on the substrate surface and therefore lose all functionality. One approach to circumvent this problem is the use of polymer tethered bilayers. In these systems the bilayer is supported on a loose network of hydrated polymers or hydrogel which acts as a spacer and theoretically prevents denaturing substrate interactions. In practice, some percentage of the proteins will still lose mobility and functionality, probably due to interactions with the polymer/lipid anchors. Research in this area is ongoing.
Tethered bilayer lipid membranes (t-BLM).
The use of a tethered bilayer lipid membrane (t-BLM) further increases the stability of supported membranes by chemically anchoring the lipids to the solid substrate.Gold can be used as a substrate because of its inert chemistry and thiolipids for covalent binding to the gold. Thiolipids are composed of lipid derivatives, extended at their polar head-groups by hydrophilic spacers which terminate in a thiol or disulphide group that forms a covalent bond with gold, forming self assembled monolayers (SAM).
The limitation of the intra-membrane mobility of supported lipid bilayers can be overcome by introducing half-membrane spanning tether lipids with benzyl disulphide (DPL) and synthetic archaea analogue full membrane spanning lipids with phytanoly chains to stabilize the structure and polyethyleneglycol units as a hydrophilic spacer. Bilayer formation is achieved by exposure of the lipid coated gold substrate to outer layer lipids either in an ethanol solution or in liposomes.
The advantage of this approach is that because of the hydrophilic space of around 4 nm, the interaction with the substrate is minimal and the extra space allows the introduction of protein ion channels into the bilayer. Additionally the spacer layer creates an ionic reservoir that readily enables ac electrical impedance measurement across the bilayer.
Vesicles.
A vesicle is a lipid bilayer rolled up into a spherical shell, enclosing a small amount of water and separating it from the water outside the vesicle. Because of this fundamental similarity to the cell membrane, vesicles have been used extensively to study the properties of lipid bilayers. Another reason vesicles have been used so frequently is that they are relatively easy to make. If a sample of dehydrated lipid is exposed to water it will spontaneously form vesicles. These initial vesicles are typically multilamellar (many-walled) and are of a wide range of sizes from tens of nanometers to several micrometres. Methods such as sonication or extrusion through a membrane are needed to break these initial vesicles into smaller, single-walled vesicles of uniform diameter known as small unilamellar vesicles (SUVs). SUVs typically have diameters between 50 and 200 nm. Alternatively, rather than synthesizing vesicles it is possible to simply isolate them from cell cultures or tissue samples. Vesicles are used to transport lipids, proteins and many other molecules within the cell as well as into or out of the cell. These naturally isolated vesicles are composed of a complex mixture of different lipids and proteins so, although they offer greater realism for studying specific biological phenomena, simple artificial vesicles are preferred for studies of fundamental lipid properties.
Since artificial SUVs can be made in large quantities they are suitable for bulk material studies such as x-ray diffraction to determine lattice spacing and differential scanning calorimetry to determine phase transitions. Dual polarisation interferometry can measure unilamelar and multilamelar structures and insertion into and disruption of the vesicles in a label free assay format. Vesicles can also be labeled with fluorescent dyes to allow sensitive FRET-based fusion assays.
In spite of the fluorescent labeling, it is often difficult to perform detailed imaging on SUVs simply because they are so small. To combat this problem, researchers use giant unilamellar vesicles (GUVs). GUVs are large enough (1 - 200 µm) to be studied using traditional fluorescence microscopy and are within the same size range as most biological cells. Thus, they are used as mimicries of cell membranes for in vitro studies in molecular and cell biology. Many of the studies of lipid rafts in artificial lipid systems have been performed with GUVs for this reason. Compared to supported bilayers, GUVs present a more “natural” environment since there is no rigid surface that might induce defects, affect the properties of the membrane or denature proteins. Therefore, GUVs are frequently used to study membrane-remodeling and other protein-membrane interactions in vitro. A variety of methods exist to encapsulate proteins or other biological reactants within such vesicles, making GUVs an ideal system for the in vitro recreation (and investigation) of cell functions in cell-like model membrane environments. These methods include microfluidic methods, which allow for a high-yield production of vesicles with consistent sizes.
Droplet Interface Bilayers.
Droplet Interface Bilayers (DIBs) are phospholipid-encased droplets that form bilayers when they are put into contact. The droplets are surrounded by oil and phospholipids are dispersed in either the water or oil. As a result, the phospholipids spontaneously form a monolayer at each of the oil-water interfaces. DIBs can be formed to create tissue-like material with the ability to form asymmetric bilayers, reconstitute proteins and protein channels or made for use in studying electrophysiology. Extended DIB networks can be formed either by employing droplet microfluidic devices or using droplet printers.
Micelles, bicelles and nanodiscs.
Detergent micelles are another class of model membranes that are commonly used to purify and study membrane proteins, although they lack a lipid bilayer. In aqueous solutions, micelles are assemblies of amphipathic molecules with their hydrophilic heads exposed to solvent and their hydrophobic tails in the center. Micelles can solubilize membrane proteins by partially encapsulating them and shielding their hydrophobic surfaces from solvent.
Bicelles are a related class of model membrane, typically made of two lipids, one of which forms a lipid bilayer while the other forms an amphipathic, micelle-like assembly shielding the bilayer center from surrounding solvent molecules. Bicelles can be thought of as a segment of bilayer encapsulated and solubilized by a micelle. Bicelles are much smaller than liposomes, and so can be used in experiments such as NMR spectroscopy where the larger vesicles are not an option.
Nanodiscs consist of a segment of bilayer encapsulated by an amphipathic protein coat, rather than a lipid or detergent layer. Nanodiscs are more stable than bicelles and micelles at low concentrations, and are very well-defined in size (depending on the type of protein coat, between 10 and 20 nm). Membrane proteins incorporated into and solubilized by Nanodiscs can be studied by a wide variety of biophysical techniques.


Paul Hunter Peckham

Paul Hunter Peckham is a professor of biomedical engineering and orthopedics at the Case Western Reserve University, and holds eight patents related to neural prosthetics. Peckham's research involves developing prostheses to restore function in the upper extremities for paralyzed individuals with spinal cord injury.
Early life and education.
Peckham is the oldest son in his family, where his father was a package salesman based in Elmira, New York. He completed a Bachelor of Science degree in mechanical engineering at the Clarkson College of Technology. He later pursued a Master of Science degree, and a doctorate in biomedical engineering at Case Western Reserve University. Peckham was based in a research lab based at the Highland View Hospital, where he was carrying out research in the field of spinal cord injury.
Research career.
Peckham joined the Case Western Reserve University as a member of the faculty in 1972, and is currently a Donnell professor of Biomedical Engineering and Orthopaedics, where his research involves using functional electrical stimulation to restore function to upper extremities, including hands and arms, in paralyzed individuals with spinal cord injuries.
In 1977, Peckham met Jim Jatich, who had been left without movement in his fingers and a wrist as a result of a diving accident. In collaboration with 25 doctors and researchers, Peckham co-developed multiple iterations of neural prostheses over nine years, which could be implanted, and relied on electrical stimulation to control neuromuscular activation. This neural prosthetic, called Freehand, was further tested with Jatich, who was able to type using an electric typewriter six months after implantation. Peckham later founded NeuroControl Corp. in the 1990s, raising $30 million in funding and receiving two Food & Drug Administration approvals for functional electrical stimulation products for spinal cord patients (including selling Freehand to the public), before the company failed due to insufficient sales.
Peckham holds the position of Distinguished University Professor at Case Western University. He has published over 200 academic papers, with over 15,000 citations, resulting in an h-index and i10-index of 60 and 132 respectively. He is cited as an inventor on eight patents related to neural prosthesis by the United States Patent and Trademark Office.
Service.
Throughout the course of his academic career, Peckham has held various administrative roles, including serving as co-director of the MetroHealth Rehabilitation Institute (within the MetroHealth System), the director of the Functional Electrical Stimulation Center, and founding the non-profit Institute for Functional Restoration at Case Western Reserve University. He has served on the scientific advisory board for the IEEE's Transactions on Biomedical Engineering academic journal. In 2004, Peckham spoke about "Paralysis: Natural recovery versus assistive technology?" at the White House/Veteran Affairs Conference dedicated to Emerging Technologies in Support of the New Freedom Initiative: Promoting Opportunities for People with Disabilities.
Awards.
Peckham has received many awards, including a Paul B. Magnuson Award (2001) from the United States Department of Veterans Affairs, a Lifetime Achievement Award (2015) from the American Spinal Injury Association, a 2020 Lifetime Achievement Award from the North American Neuromodulation Society (NANS), an Annual BCI Research Award (2018), the Frank and Dorothy Humel Hovorka Prize from Case Western Reserve University and was designated as the Pioneer of the Neuroprothesis by the State of Ohio. Peckham was elected as a fellow to the National Academy of Engineering (2002), the American Institute of Medical and Biological Engineering, and the American Spinal Injury Association. He was recognized as an Engineer Of The Year in 2000 by DesignNews.
Personal life.
Peckham has a wife named Sara and two children named Endia(Indy) and Gregory(Greg). Peckham also has three grandchildren named Hayden, Eloise and Beatrice(Bea)


4-PPBP

4-PPBP is a molecule which binds to sigma receptors.
4-PPBP decreases neuronal nitric oxide synthase (nNOS) activity and ischemia-evoked nitric oxide (NO) production. 4-PPBP provides neuroprotection; this involves the prevention of ischemia-induced intracellular Ca2+dysregulation. 4-PPBP protects neurons using a mechanism that activates the transcription factor cyclic adenosine monophosphate response element-binding protein (CREB). Neuroprotection that is associated with 4-PPBP increases Bcl-2 expression; Bcl-2 expression is regulated by CREB.


Hepatitis C alternative reading frame stem-loop

Hepatitis C alternative reading frame stem-loop is a conserved secondary structure motif identified in the RNA genome of the Hepatitis C virus (HCV) which is proposed to have an important role in regulating translation and repression of the viral genome.
The core protein-coding region of the Hepatitis C virus (HCV) genome contains a +1 alternative reading frame
(ARF) and two proposed phylogenetically conserved RNA helix-forming stem loop structures (IV and VII).
The proteins translated from the ARF appear to be translated during the normal viral life cycle but are not essential to virus replication. The two predicted stem loops shown here (SLV and SLVI) are proposed to be important for HCV translation and repression; these stem loops are located downstream of the Internal ribosome entry site (IRES) but their functional role is unknown.


Coproporphyrinogens

Coproporphyrinogens are tetrapyrroles with four propionic acid groups and an equal number of substituted methyls.
Coproporphyrinogen III is the most common variance. In the metabolism of porphyrin, it is formed from uroporphyrinogen III by the enzyme uroporphyrinogen III decarboxylase, and it is converted into protoporphyrinogen IX by coproporphyrinogen III oxidase.


Peripheral blood cell

Peripheral blood cells are the cellular components of blood, consisting of red blood cells (erythrocytes), white blood cells (leucocytes), and platelets, which are found within the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver, or bone marrow.


Alaris (comics)


Harmol

Harmol is a chemical compound classified as a β-carboline. It is readily formed "in vivo" in humans by "O"-demethylation of harmine.


KhAB-250

The KhAB-250 is the provisional naming of an aerial bomb developed by the Soviet Air Force to deliver the chemical weapon sarin.
The KhAB-250 operational weight has been reported as and . 24 could be carried by the Tupolev Tu-22.
The bomb uses a burst charge to detonate on impact with the ground. It contains a payload of of sarin.
The KhAB-250 was displayed at Shikani Test and Proving Grounds in 1986 as a component of the then-current Soviet chemical arsenal. Contemporary analysts noted that it appeared relatively unsophisticated compared to Soviet conventional munitions of the same time frame.
The bomb was removed from service as a result of the Chemical Weapons Convention in the early 1990s.


David J. Mooney

David James Mooney (born November 1, 1964) is Robert P. Pinkas Family Professor of Bioengineering at the Harvard School of Engineering and Applied Sciences. He is also a founding core faculty member at the Wyss Institute for Biologically Inspired Engineering at Harvard University. Born in Madison, Wisconsin, he earned his B.S. in Chemical Engineering from the University of Wisconsin, Madison and his Ph.D. in Chemical Engineering from the Massachusetts Institute of Technology (PhD advisor: Robert Langer). He did his post-doctoral studies at Harvard University under the supervision of Dr. Joseph Vacanti, MD. David Mooney started his career as an Assistant Professor at The University of Michigan and he stayed there until 2004 when he moved to Harvard University. Mooney is a member of both the National Academy of Engineering and the National Academy of Medicine. In addition, David Mooney is a Fellow of the National Academy of Inventors.


Paeniglutamicibacter cryotolerans

Paeniglutamicibacter cryotolerans, also known as Arthrobacter cryotolerans, is a species of bacteria. It is psychrotolerant, halotolerant, Gram-positive, motile and facultatively anaerobic. It possesses a rod–coccus cycle. Its type strain is LI3T  (= DSM 22826T  = NCCB 100315T).


Roman Zubarev

Roman A. Zubarev is a professor of medicinal proteomics in the Department of Medical Biochemistry and Biophysics at the Karolinska Institutet. His research focuses on the use of mass spectrometry in biology and medicine.
References.
1Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. "Electron Capture Dissociation of Multiply Charged Protein Cations. A Non-ergodic Process, J. Am. Chem. Soc". 1998", 120," 3265-3266.
"2" Zubarev, R. A.; Artemenko, K. A.; Zubarev, A. R.; Mayrhofer, C.; Yang, H.; Fung, E. Y. M. "Early life relict feature in peptide mass distribution, Cent. Eur. J. Biol." 2010, "5," 190-196.
3 Xie, X.; Zubarev, R. A. "Isotopic Resonance Hypothesis: Experimental Verification by Escherichia coli Growth Measurements", "Scientific Reports", 2015, doi:10.1038/srep09215
4 Johnson, B. A.; Shirokawa, J. M.; Geddes, J. W.; Choid, B. H.; Kim, R. C.; Aswad, D. W. "Protein L-isoaspartyl methyltransferase in postmortem brains of aged humans, Neurobiology of Aging", 1991, 12, 19-24.
5 Yang, H.; Lyutvinskiy, Y.; Herukka, S.-K.; Soininen, H.; Rutishauser, D.; Zubarev, R. A. "Prognostic polypeptide blood plasma biomarkers of Alzheimer’s disease progression," "J Alzheimer’s Disease," 2014, "40," 659-66.
6 Xie, X.; Backman, D.; Lebedev, A. T.; Artaev, V. B.; Jiang, L.; Ilag, L. L.; Zubarev, R. A. "Primordial soup was edible: abiotically produced Miller-Urey mixture supports bacterial growth," "Scientific Reports," 2015, "5", article number: 14338, doi:10.1038/srep14338
7 Bernadotte, A.; Semenova, V.; Musial, V. A. M.; Kasprzykowska, A.; Zubarev, R. A. "Self-assembly of Deinococcus radiodurans supports nanocell scenario of life origin", "Discoveries," 2017, "5," E72, doi: 10.15190/d.2017.2.
8


Richard Yost

Richard A Yost (born 31 May 1953 in Martins Ferry) is an American scientist and a professor at the University of Florida. He is best known for his work inventing the triple quadrupole mass spectrometer. Yost received his BS degree in Chemistry in 1974 from the University of Arizona, having performed undergraduate research in chromatography with Mike Burke and his PhD degree in Analytical Chemistry in 1979 from Michigan State University, having performed graduate research with Chris Enke.
He joined the faculty of the University of Florida after his graduate research.
He is director of the Southeast Center for Integrated Metabolomics (SECIM) and of NIH's Metabolomics Consortium Coordinating Center (M3C).  He is also a Professor of Pathology at both the University of Florida and the University of Utah/ARUP.
His professional activities have focused on research and teaching in analytical mass spectrometry, particularly tandem mass spectrometry (MS/MS).  His group's research has reflected a unique balance between instrumentation development, fundamental studies, and applications in analytical chemistry.  His group has led in the application of novel mass spectrometric methods and techniques to areas such as metabolomics, clinical, biomedical, pharmaceutical, environmental, petrochemical, and forensic chemistry.  Yost has supervised the research of well over 100 graduate students during the past 40 years, graduating over 90 PhDs from his group. He has served as PI or Co-PI on grants and contracts totaling over $60M of funding.  Research in the group has led to over 210 publications and 25 patents.
He is one of the inventors of the triple quadrupole mass spectrometer. He won the ASMS Award for Distinguished Contribution to Mass Spectrometry along with Chris Enke in 1993. His research has also been recognized with the 2018 MSACL Award for Distinguished Contribution to Clinical Mass Spectrometry, and the 2019 CPSA Distinguished Analytical Scientist Award. In 2019 he was named the Florida Academy of Sciences Medalist, and was inducted into the Florida Inventors Hall of Fame.
His research interests lie in mass spectrometric instrumentation and applications in analytical chemistry. This includes the development of new mass spectrometric and ion mobility instrumentation and techniques and their application to biology and chemistry.
Yost has served on the Florida Board of Governors (Regents) and the University of Florida Board of Trustees. In 2019 he received the Distinguished Eagle Scout Award. Yost is currently the president of the American Society for Mass Spectrometry.


GAIT element

The gamma interferon inhibitor of translation element or GAIT element is a cis-acting RNA element located in the 3'-UTR of the ceruloplasmin (Cp) mRNA.
The GAIT element forms a stem-loop secondary structure. The GAIT element is involved in selective translational silencing of the Cp transcript within monocytic cells, but not hepatic cells. Cp is a multifunctional, copper-containing glycoprotein produced by the liver and secreted into the plasma for its role in copper and iron homeostasis. Ceruloplasmin is also an acute-phase protein produced by monocytes, and its plasma concentration can double during multiple inflammatory conditions through increased Cp production by monocytic cells after stimulation by interferon gamma (IFNγ). Plasma Cp has been reported to be an independent risk factor for cardiovascular disease, including atherosclerosis, carotid restenosis after endarterectomy, and myocardial infarction. Translational silencing of Cp, and possibly other transcripts, mediated by the GAIT element may contribute to the resolution of the local inflammatory response following cytokine activation of macrophages.
The silencing of Cp protein translation in IFN-gamma-stimulated monocytes is accomplished by binding of the IFN-gamma-activated inhibitor of translation (GAIT) inhibitor complex to the GAIT element. The GAIT complex consists of the proteins ribosomal protein L13a, glutamyl-prolyl-tRNA synthetase, NS1-associated protein-1, and glyceraldehyde 3-phosphate dehydrogenase.


Malonate decarboxylase holo-(acyl-carrier protein) synthase

Malonate decarboxylase holo-(acyl-carrier protein) synthase (, "holo ACP synthase", "2'-(5"-triphosphoribosyl)-3'-dephospho-CoA:apo ACP 2'-(5"-triphosphoribosyl)-3'-dephospho-CoA transferase", "MdcG", "2'-(5"-triphosphoribosyl)-3'-dephospho-CoA:apo-malonate-decarboxylase adenylyltransferase", "holo-malonate-decarboxylase synthase") is an enzyme with systematic name "2'-(5-triphosphoribosyl)-3'-dephospho-CoA:apo-malonate-decarboxylase 2'-(5-phosphoribosyl)-3'-dephospho-CoA-transferase". This enzyme catalyses the following chemical reaction
The delta subunit of malonate decarboxylase serves as an acyl-carrier protein (ACP) .


John Garnier (cricketer)

John Garnier (26 April 1813 – 26 March 1838) was an English first-class cricketer and clergyman.
The son of The Reverend Thomas Garnier, he was born in April 1813 at Bishopstoke, Hampshire. He was educated at Winchester College, before going up to Exeter College, Oxford. While studying at Oxford, he made a single appearance in first-class cricket for Oxford University against the Marylebone Cricket Club at Oxford in 1832. Batting twice in the match, he was run out for 4 runs in the Oxford first innings, before being dismissed leg before wicket for 3 runs in their second innings. He was elected a fellow of Merton College in 1835.
After graduating from Oxford, Garnier took holy orders in the Church of England. He became the curate at St Ebbe's Church, Oxford in 1837. Garnier died in March of the following year after catching smallpox while visiting his parishioners. Several family members played cricket at first-class level, including his brother Thomas Garnier junior.


Bamboo mosaic potexvirus (BaMV) cis-regulatory element

The Bamboo mosaic potexvirus (BaMV) cis-regulatory element represents a cloverleaf-like cis-regulatory element found in the 3' UTR of the bamboo mosaic virus. This family is thought to play an important role in the initiation of minus-strand RNA synthesis and may also be involved in the regulation of viral replication.


Cysteamine dioxygenase

In enzymology, a cysteamine dioxygenase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are 2-aminoethanethiol and O2, whereas its product is hypotaurine.
This enzyme belongs to the family of oxidoreductases, specifically those acting on single donors with O2 as oxidant and incorporation of two atoms of oxygen into the substrate (oxygenases). The oxygen incorporated need not be derived from O2. The systematic name of this enzyme class is 2-aminoethanethiol:oxygen oxidoreductase. Other names in common use include persulfurase, cysteamine oxygenase, and cysteamine:oxygen oxidoreductase. This enzyme participates in taurine and hypotaurine metabolism. It employs one cofactor, iron.


Chloroflexus islandicus

Chloroflexus islandicus is a photosynthetic bacterium isolated from the Strokkur Geyser in Iceland. This organism is thermophilic showing optimal growth at 55°C (131°F) with a pH range of 7.5 – 7.7. C. islandicus grows best photoheterotrophically under anaerobic conditions with light but is capable of chemoheterotrophically growth under aerobic conditions in the dark. "C. islandicus" has a yellowish green color. The individual cells form unbranched multicellular filaments about 0.6 µm in diameter and 4-7 µm in length.
Phenotypic characteristics.
As a genus, "Chloroflexus" spp. are filamentous anoxygenic phototrophic (FAP) organisms that utilize type II photosynthetic reaction centers containing bacteriochlorophyll a, and light-harvesting chlorosomes containing bacteriochlorophyll. Beta- and gamma-carotenes are present. "C. islandicus" is gram negative. Cell morphology shows the presence of chlorosomes, pili and gliding motility. Pili are unique to C. islandicus being the only organism in the "Chloroflexus" genus to possess pili.
Genetic characteristics.
The whole genome sequence of "Chloroflexus islandicus" was able to be determined (5.14 Mb). Using the 16S rRNA gene analysis, ANI (Average Nucleotide Identity) and DDH (DNA-DNA Hybridization) a new species of "Chloroflexus "was confirmed. The 16S rRNA analysis showed it is closely related to "Chloroflexus aggregans" (97.0%). The genomic data revealed 84.1% ANI and 22.8% DDH for "Chloroflexus islandicus" strain vs other known "Chloroflexus" strains. The separated species based on ANI is 95.0% or less and DDH is 70.0% or less. The G/C content for "Chloroflexus islandicus" was found to be 59.6mol%.


Photostimulation

Photostimulation is the use of light to artificially activate biological compounds, cells, tissues, or even whole organisms. Photostimulation can be used to noninvasively probe various relationships between different biological processes, using only light. In the long run, photostimulation has the potential for use in different types of therapy, such as migraine headache. Additionally, photostimulation may be used for the mapping of neuronal connections between different areas of the brain by “uncaging” signaling biomolecules with light. Therapy with photostimulation has been called light therapy, phototherapy, or photobiomodulation.
Photostimulation methods fall into two general categories: one set of methods uses light to uncage a compound that then becomes biochemically active, binding to a downstream effector. For example, uncaging glutamate is useful for finding excitatory connections between neurons, since the uncaged glutamate mimics the natural synaptic activity of one neuron impinging upon another. The other major photostimulation method is the use of light to activate a light-sensitive protein such as rhodopsin, which can then excite the cell expressing the opsin.
Scientists have long postulated the need to control one type of cell while leaving those surrounding it untouched and unstimulated. Well-known scientific advancements such as the use of electrical stimuli and electrodes have succeeded in neural activation but fail to achieve the aforementioned goal because of their imprecision and inability to distinguish between different cell types. The use of optogenetics (artificial cell activation via the use of light stimuli) is unique in its ability to deliver light pulses in a precise and timely fashion. Optogenetics is somewhat bidirectional in its ability to control neurons. Channels can be either depolarized or hyperpolarized depending on the wavelength of light that targets them. For instance, the technique can be applied to channelrhodopsin cation channels to initiate neuronal depolarization and eventually activation upon illumination. Conversely, activity inhibition of a neuron can be triggered via the use of optogenetics as in the case of the chloride pump halorhodopsin which functions to hyperpolarize neurons.
Before optogenetics can be performed, however, the subject at hand must express the targeted channels. Natural and abundant in microbials, rhodopsins—including bacteriorhodopsin, halorhodopsin and channelrhodopsin—each have a different characteristic action spectrum which describes the set of colors and wavelengths that they respond to and are driven to function by.
It has been shown that channelrhodopsin-2, a monolithic protein containing a light sensor and a cation channel, provides electrical stimulation of appropriate speed and magnitude to activate neuronal spike firing. Recently, photoinhibition, the inhibition of neural activity with light, has become feasible with the application of molecules such as the light-activated chloride pump halorhodopsin to neural control. Together, blue-light activated channelrhodopsin-2 and the yellow light-activated chloride pump halorhodopsin enable multiple-color, optical activation and silencing of neural activity. (See also Photobiomodulation)
Methods.
A caged protein is a protein that is activated in the presence of a stimulating light source. In most cases, photo-uncaging is the technique revealing the active region of a compound by the process of photolysis of the shielding molecule (‘cage’). However, uncaging the protein requires an appropriate wavelength, intensity, and timing of the light. Achieving this is possible due to the fact that the optical fiber may be modified to deliver specific amounts of light. In addition, short bursts of stimulation allow results similar to the physiological norm. The steps of photostimulation are time independent in that protein delivery and light activation can be done at different times. This is because the two steps are dependent on each other for activation of the protein.
Some proteins are innately photosensitive and function in the presence of light. Proteins known as opsins form the crux of the photosensitive proteins. These proteins are often found in the eye. In addition, many of these proteins function as ion channels and receptors. One example is when a certain wavelength of light is put onto certain channels, the blockage in the pore is relieved and allows ion transduction.
To uncage molecules, a photolysis system is required to cleave the covalent bond. An example system can consist of a light source (generally a laser or a lamp), a controller for the amount of light that enters, a guide for the light, and a delivery system. Often, the design function in such a way that a medium is met between the diffusing light that may cause additional, unwanted photolysis and light attenuation; both being significant problems with a photolysis system.
History.
The idea of photostimulation as a method of controlling biomolecule function was developed in the 1970s. Two researchers, Walther Stoeckenius and Dieter Oesterhelt discovered an ion pump known as bacteriorhodopsin which functions in the presence of light in 1971. In 1978, J.F. Hoffman invented the term “caging”. Unfortunately, this term caused some confusion among scientists due to the fact that the term is often used to describe a molecule which is trapped within another molecule. It could also be confused with the “caged effect” in the recombination of radicals. Therefore, some authors decided to use the term “light-activated” instead of “caging”. Both terms are currently in use. The first “caged molecule” synthesized by Hoffman et al. at Yale was the caged precursor to ATP derivative 1.
Applications.
Photostimulation is notable for its temporal precision, which may be used to obtain an accurate starting time of activation of caged effectors. In conjunction with caged inhibitors, the role of biomolecules at specific timepoints in an organism’s lifecycle may be studied. A caged inhibitor of N-ethylmaleimide sensitive fusion protein (NSF), a key mediator of synaptic transmission, has been used to study the time dependency of NSF. Several other studies have effected action potential firing through use of caged neurotransmitters such as glutamate. Caged neurotransmitters, including photolable precursors of glutamate, dopamine, serotonin, and GABA, are commercially available.
Signaling during mitosis has been studied using reporter molecules with a caged fluorophore, which is not phosphorylated if photolysis has not occurred. The advantage of this technique is that it provides a “snapshot” of kinase activity at specific timepoints rather than recording all activity since the reporter’s introduction.
Calcium ions play an important signaling role, and controlling their release with caged channels has been extensively studied.
Unfortunately, not all organisms produce or hold sufficient amounts of opsins. Thus, the opsin gene must be introduced to target neurons if they are not already present in the organism of study. The addition and expression of this gene is sufficient for the use of optogenetics. Possible means of achieving this include the construction of transgenic lines containing the gene or acute gene transfer to a specific area or region within an individual. These methods are known as germline transgenesis and somatic gene delivery, respectively.
Optogenetics has shown significant promise in the treatment of a series of neurological disorders such as Parkinson’s disease and epilepsy. Optogenetics has the potential to facilitate the manipulation and targeting of specific cell types or neural circuits, characteristics that are lacking in current brain stimulation techniques like DBS. At this point, the use of optogenetics in treating neural diseases has only been practically implemented in the field of neurobiology to reveal more about the mechanisms of specific disorders. Before the technique can be implemented to directly treat these disorders developments in other related fields such as gene therapy, opsin engineering, and optoelectronics must also make certain developments.


CITE-Seq

CITE-Seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) is a method for performing RNA sequencing along with gaining quantitative and qualitative information on surface proteins with available antibodies on a single cell level. So far, the method has been demonstrated to work with only a few proteins per cell. As such, it provides an additional layer of information for the same cell by combining both proteomics and transcriptomics data. For phenotyping, this method has been shown to be as accurate as flow cytometry (a gold standard) by the groups that developed it. It is currently one of the main methods, along with REAP-Seq, to evaluate both gene expression and protein levels simultaneously in different species.
The method was established by the New York Genome Center in collaboration with the Satija lab.
Applications.
Concurrent measurement of both protein and transcript levels opens up opportunities to use CITE-Seq in various biological areas, some of which were touched upon by the developers. For instance, it may be used to characterize tumor heterogeneity in different cancers, a major research field. It also permits identifying rare subpopulations of cells as a high-throughput single-cell method and thus detect information otherwise lost with bulk methods. It also may aid in tumor classification - for example, identification of novel subtypes. All of the above are possible due to single-cell output of both protein and transcript data at the same time, also leading to novel information on protein-RNA correlation.
It also has potential in immunology. For example, it can be utilized for immune cell characterization – recent research on T-cells has investigated the ability of T cells to maintain an effector state. Another study by one of CITE-Seq coauthors suggested CITE-Seq as a methods to look at the mechanisms of host-pathogen interactions.
Workflow.
CITE-seq, like any other sequencing technique, has a wet lab portion, where the actual antibodies are prepared, cells stained, cDNA synthesized and RNA libraries are prepared that are further sequenced, and a dry lab portion for analysis of the sequencing data obtained. The most crucial part in the wet lab experiments is designing the antibody-oligonucleotide conjugates and titrating the amount of each conjugate that needs to be present in the pool to achieve a desired read-out and quantification.
Wet lab workflow.
The first step involves preparation of the antibody-oligo conjugates also known as Antibody-Derived Tags (ADTs). ADT preparation involves labeling an antibody directed against a cell surface protein of interest with oligonucleotides for barcoding the antibody.
Once you have the ADTs, the next step is to bind the cells with the desired ADT pool. The scRNA-seq libraries can be prepared using Drop-seq, 10X Genomics or ddSeq methods. In brief, ADT labelled cells are encapsulated within a droplet as single cells with DNA-barcoded microbeads.
Within a droplet, the cells are next lysed to release both bound ADTs as well as mRNA. These then are converted to cDNA. Each DNA sequence on a microbead has a unique barcode thus indexing cDNA with cell barcodes. cDNA is prepared from both ADTs and cellular mRNAs.
In the next step, based on the developer's guidelines, cDNA is PCR-amplified and ADT cDNA and mRNA cDNA are separated based on size (generally, ADT-derived cDNAs are < 180bp and mRNA-derived cDNAs are > 300bp). Each of the separated cDNA molecules is independently amplified and purified to prepare sequencing libraries. Finally, the independent libraries are pooled together and sequenced. Thus, proteomics and transcriptomics data can be obtained from a single sequencing run.
Dry lab workflow.
Analysis of single-cell sequencing presents many challenges, such as determining the best way to normalize the data. Due to a new level of complications that arise from sequencing of both proteins and transcripts at a single-cell level, the developers of CITE-Seq and their collaborators are maintaining several tools to help with data analysis.
"scRNA-Seq data analysis based on the developer's guidelines": The initial analysis steps are the same as in a standard scRNA-Seq experiment. Firstly, reads need to be aligned to a reference genome of a species of interest and cells with very low number of transcripts mapped to the reference are removed. Finally, a normalized count matrix with gene expression values is obtained.
"ADT data analysis" "(based on the developer's guidelines)": CITE-seq-Count is a Python package from CITE-Seq developers that can be used to obtain raw counts. Seurat package from Satija lab further allows combining of the protein and RNA counts and performing clustering on both measurements, as well as doing differential expression analysis between cell clusters of interest. ADT quantification needs to take into account the differences between the antibodies. Additionally, filtering may be required to reduce noise, similarly to scRNA-Seq analysis. But in contrast to RNA data, due to higher amounts of protein in a cell, there is less dropout.
The analyses may result in identification of novel cell clusters through such methods as PCA or tSNE, crucial genes responsible for a specific cell function and other new knowledge specific to a question of interest. In general, the results obtained with ADT counts substantially increase the amount of information obtained through single cell transcriptomics.
Adaptations of the technique.
The applications of antibody-oligonucleotide conjugates have expanded beyond CITE-seq, and can be adapted for sample multiplexing as well as CRISPR screens.
Cell Hashing: New York Genome Center further adapted the use of their antibody-oligonucleotide conjugates to enable sample multiplexing for scRNA-seq. This technique called, Cell Hashing, uses oligonucleotide-labelled antibodies against ubiquitously expressed cell surface proteins from a particular tissue sample. In this case, an oligonucleotide sequence contains a unique barcode which would be specific to cells from distinct samples. This sample-specific cell tagging allows pooling of the sequencing libraries prepared from different samples on a sequencing platform. Sequencing the antibody tags along with the cellular transcriptome helps identify a sample of origin for each analyzed cell. A unique barcode sequence used on the cell hashing antibody can be designed to be different from an antibody barcode present on the ADTs used in CITE-seq. This makes it possible to couple cell hashing with CITE-seq on a single sequencing run. Cell hashing allows super-loading of the scRNA-seq platform, resulting in a lower cost of sequencing. It also enables detection of artifactual signals from multiplets, a major challenge in scRNA-seq. The cell hashing method has further been used by Gaublomme et al. to multiplex single-nucleus RNA-seq (snRNA-seq) by performing nucleus hashing.
ECCITE-seq: Expanded CRISPR-compatible Cellular Indexing of Transcriptomes and Epitopes by sequencing or ECCITE-seq was developed to apply the use of CITE-seq to characterize multiple modalities from a single cell. By modifying the basic CITE-seq protocol to a 5’ tag-based scRNA-seq assay, it can detect transcriptome, immune receptor clonotypes, surface markers, sample identity and single guide RNAs (sgRNAs) from each single cell. The ability of ECCITE-seq to detect sgRNA molecules and measure their effect on gene expression levels opens a prospect of applying this technique in CRISPR screens.
Advantages and Limitations of CITE-seq.
"Advantages:" CITE-seq enables simultaneous analysis of the transcriptome as well as the proteome of single cells. Previous efforts of coupling index-sorting measurements from single cell sorts with scRNA-seq were limited to running a small sample size and were not compatible with multiplexing and massive parallel high-throughput sequencing. CITE-seq has been shown to be compatible with high-throughput microfluidic platforms like 10X Genomics and Drop-seq. It is also adaptable to micro/nano-well platforms. Coupling it with cell hashing enables the application of CITE-seq on bulk samples and sample multiplexing. These techniques work to reduce an overall cost of high-throughput sequencing on multiple samples. Lastly, CITE-seq can be adapted to detect small molecules, RNA interference, CRISPR, and other gene editing techniques.
"Limitations:" One of the limitations of CITE-Seq is a loss of location information. Due to the way the cells are treated, the spatial distribution of cells within a sample, as well as proteins within a cell is not known. In addition, this method shares the challenges of scRNA-Seq, such as high amount of noise and possible challenges in detecting lowly expressed genes. In terms of phenotyping, optimization of the assay and antibodies also presents a potential problem if proteins of interest are not included in the currently available panels. Moreover, right now CITE-Seq is not able to detect intracellular proteins. With the current protocol, there are many challenges that would arise during the permeabilization step, thus limiting the technique to surface markers.


Chlorobi-RRM RNA motif

The Chlorobi-RRM RNA motif is a conserved RNA structure identified by bioinformatics. It is found within bacteria in the phylum Chlorobi, and is exclusively detected in the presumed 5' untranslated regions (5' UTRs) of genes that encode putative RNA-binding proteins. Since many RNA-binding proteins regulate their own expression in a feedback mechanism by binding or acting up their 5' UTR, it was proposed that the Chlorobi-RRM is a component in an analogous feedback mechanism.
Structurally, the motif consists of two stem-loops, the second of which might function as a rho-independent transcription terminator.


Acetylcysteinamide

"N"-Acetylcysteine amide (abbrev. NACA, AD4 and also known as acetylcysteinamide) is an amide derivative of "N"-acetylcysteine that appears to have better blood–brain barrier permeability and bioavailability and a similar antioxidant capability.