Protoporphyrin IX

Protoporphyrin IX is an organic compound, specifically a porphyrin, that plays an important role in living organisms as a precursor to other critical compounds like hemoglobin and chlorophyll. It is a deeply colored solid that is not soluble in water. The name is often abbreviated as PPIX.
Protoporphyrin IX contains a porphine core, a tetrapyrrole macrocycle with a marked aromatic character. Protoporphyrin IX is essentially planar, except for the N-H bonds that are bent out of the plane of the rings, in opposite (trans) directions.
The general term protoporphyrin refers to porphine derivatives that have the outer hydrogen atoms in the four pyrrole rings replaced by four methyl groups (M), two vinyl groups (V), and two propionic acid groups (P). The Roman numeral "IX" indicates that these chains occur in the circular order MV-MV-MP-PM around the outer cycle. (The numbering of the variants is traditional and not entirely systematic.)
Natural occurrence.
The compound is encountered in nature in the form of complexes where the two inner hydrogen atoms are replaced by a divalent metal cation. When complexed with an iron(II) (ferrous) cation , the molecule is called heme. Hemes are prosthetic groups in some important proteins. These heme-containing proteins include hemoglobin, myoglobin, and cytochrome c. Complexes can also be formed with other metal ions, such as zinc.
Biosynthesis.
The compound is synthesized from acyclic precursors via a mono-pyrrole (porphobilinogen) then a tetrapyrrole (a porphyrinogen, specifically uroporphyrinogen III). This precursor is converted to protoporphyrinogen IX, which is oxidized to protoporphyrin IX. The last step is mediated by the enzyme protoporphyrinogen oxidase.
Protoporphyrin IX is an important precursor to biologically essential prosthetic groups such as heme, cytochrome c, and chlorophylls. As a result, a number of organisms are able to synthesize this tetrapyrrole from basic precursors such as glycine and succinyl-CoA, or glutamic acid. Despite the wide range of organisms that synthesize protoporphyrin IX, the process is largely conserved from bacteria to mammals with a few distinct exceptions in higher plants.
In the biosynthesis of those molecules, the metal cation is inserted into protoporphyrin IX by enzymes called chelatases. For example, ferrochelatase converts the compound into heme B (i.e. Fe-protoporphyrin IX or protoheme IX). In chlorophyll biosynthesis, the enzyme magnesium chelatase converts it into Mg-protoporphyrin IX.
Described metalloprotoporphyrin IX derivatives.
The following metals are known to from Heme-like complexes when coordinated to PPX:
These compounds follow the naming convention [metal]-protoporphyrin IX, or protoporphyrin IX containing [metal].
Protoporphyrin IX reacts with iron salts in air to give the complex FeCl(PPIX).
Palepron is the disodium salt of protoporphyrin IX.


Mycena discobasis

Mycena discobasis is a species of agaric fungus in the family Mycenaceae. Found in South America and Madagascar, the fruit bodies of the fungus are bioluminescent.


Lewisite 3

Lewisite 3 (L-3) is an organoarsenic chemical weapon like lewisite 1 and lewisite 2 first synthesized in 1904 by Julius Arthur Nieuwland. It is usually found as a mixture of 2-chlorovinylarsonous dichloride (lewisite 1) as well as bis(2-chloroethenyl) arsinous chloride (lewisite 2) and tris(2-chlorovinyl)arsine (lewisite 3). Pure lewisite 1 is an oily, colorless liquid, however, the impure mixture can appear amber to black with an odor distinct to geraniums.
Synthesis.
Lewisite 3 is made as a byproduct along with lewisite 2 in the reaction that makes lewisite 1.
Acetylene reacts with AsCl3 in hydrochloric acid solution, with mercuric chloride as a catalyst, to give lewisite in 80 to 85 percent yield (based on AsCl 3).
Lewisite 3 is formed when there are three additions of acetylene to the arsenic center instead of one like the reaction seen above where only lewisite one is shown.
Exposure.
Adverse health effects caused by lewisite can vary and are dependent on; the amount people are exposed to and, the amount of time someone is exposed to it. Lewisite immediately damages the skin, eyes, and respiratory tract and is a strong irritant and blistering agent. Due to its arsenic center, lewisite may also cause problems similar to arsenic poisonings like stomach ailments and low blood pressure.
Inhalation.
Inhalation, the most common route of exposure, causes a burning pain and irritation throughout the respiratory tract, nosebleed (epistaxis), laryngitis, sneezing, coughing, vomiting, difficult breathing (dyspnea). From one acute exposure, someone who has inhaled lewisite can develop chronic respiratory disease. In severe cases of exposure, can cause fatal pulmonary edema, pneumonitis, or respiratory failure.
Ingestion.
Ingestion results in severe pain, nausea, vomiting, and tissue damage. Those exposed to lewisite can develop refractory hypotension (low blood pressure) known as Lewisite shock, as well as some features of arsenic toxicity. Lewisite causes physical damage to capillaries, which then become leaky, meaning that there is not enough blood volume to maintain blood pressure, a condition called hypovolemia. When the blood pressure is low, the kidneys may not receive enough oxygen and can be damaged.
Eye Exposure.
The results of eye exposure can range from stinging, burning pain and strong irritation to blistering and scarring of the cornea, along with blepharospasm, lacrimation, and edema of the eyelids and periorbital area. Eye exposure to lewisite can cause permanent visual impairment or blindness. The eyes can swell shut, which can keep the eyes safe from further exposure. The most severe consequences of eye exposure to lewisite are globe perforation and blindness. Generalised symptoms also include restlessness, weakness, hypothermia and low blood pressure.
Chronic Exposure.
Chronic exposure to lewisite can cause arsenic poisoning and development of a lewisite allergy. It can also cause various long-term illnesses or permanent damage to organs, depending on where the exposure has occurred, including conjunctivitis, aversion to light (photophobia), visual impairment, double vision (diplopia), tearing (lacrimation), dry mucous membranes, garlic breath, burning pain in the nose and mouth, toxic encephalopathy, peripheral neuropathy, seizures, nausea, vomiting, chronic obstructive pulmonary disease (COPD), bronchitis, dermatitis, skin ulcers, basal cell carcinoma, and squamous cell carcinoma.
Treatment.
Basic first aid for lewisite exposure is decontamination and irrigation of any areas that have been exposed, and when necessary airway management, assisted ventilation, and monitoring of vital signs.
British anti-lewisite, also called dimercaprol, is the antidote for lewisite. It can be injected to prevent systemic toxicity, but will not prevent injury to the skin, eyes, or mucous membranes. Chemically, dimercaprol is a chelating agent that binds to the arsenic in lewisite. It is contraindicated in those with peanut allergies.


Matthew Tirrell

Matthew Tirrell (born 5 September 1950) is an American chemical engineer. In 2011 he became the Founding Pritzker Director and Dean of the Institute for Molecular Engineering (IME) at the University of Chicago, in addition to serving as senior scientist at Argonne National Laboratory. In 2019, IME became the Pritzker School of Molecular Engineering and Tirrell was named Robert A. Millikan Distinguished Service Professor and Dean. Tirrell's research specializes in the manipulation and measurement of polymer surface properties, polyelectrolyte complexation, and biomedical nanoparticles.
Early life and education.
Tirrell was born in Phillipsburg, New Jersey on September 5, 1950. He received a bachelor's degree in Chemical Engineering (B.S. Ch.E.) in 1973 Northwestern University, and a Ph.D. in 1977 from University of Massachusetts Amherst in Polymer Science and Engineering under Stanley Middleman.
Career.
In 1977, Tirrell became an assistant professor in the Department of Chemical Engineering and Materials Science at the University of Minnesota, receiving promotions through the academic ranks and serving as the head of the department from 1995 to 1999. In 1999, he moved to the University of California, Santa Barbara, where was Richard A. Auhll Professor and Dean of the College of Engineering. In 2009, he moved to the University of California, Berkeley as Arnold and Barbara Silverman Professor and Chair of the Department of Bioengineering, as well as a Professor of Materials Science and Engineering and Chemical Engineering and a Faculty Scientist in the Materials Science Division at Lawrence Berkeley National Laboratory.
In 2011, Tirrell became the Founding Pritzker Director and Dean of the Institute for Molecular Engineering (IME) at the University of Chicago. In September 2015, he was appointed as the Deputy Laboratory Director for Science at Argonne National Laboratory. He stepped out of this role in April 2018. In 2019, IME became the Pritzker School of Molecular Engineering and Tirrell was named Robert A. Millikan Distinguished Service Professor and Dean.
Research.
The early work in Tirrell's career focused on the behavior of macromolecules in physically confined spaces, in which both the structure and motion of the macromolecule are constrained and distorted by the confining boundaries. For example, the apparent viscosity of a macromolecular solution can be lower because the centers-of-mass of the macromolecules cannot get close to the wall, giving rise to a low viscosity depletion layer (a similar phenomenon occurs, albeit at larger scales, in blood flow in narrow vessels). Tirrell was responsible for developing a quantitative, predictive theoretical picture of this type of phenomenon and comparing it with experimental rheological results. His papers describing this research have collectively been cited more than 1000 times.
Tirrell's interest in polymer confinement led to the investigation of what happens when polymers stick (adhere, adsorb) to the walls confining them. His research group became especially interested in a prediction made in the late 1970s by Pierre-Gilles de Gennes, about the situation where a polymer molecule is tethered by its end to a surface but no other segments were attracted to the surface, a situation that has become known as a polymer brush. De Gennes predicted that a dense layer of such end-tethered chains would result in the chains stretching out normal to the tethering surface to a high degree, resulting in a surface that is resistant to adhesion and has very low friction. Tirrell conceived and implemented the first experimental system to test these predictions by adsorbing block copolymers, and demonstrated the theoretically anticipated stretching and long-range repulsion. His interest in this field, amassing more than 3500 citations, continues today, especially in the area of charged polymer brushes, which are bio-medically relevant in situations such as mucosal and cartilage surfaces.
Eventually, Tirrell's work on block copolymers led him to starting research in the field of peptide amphiphiles. In the field of tissue engineering, in the early 1990s, the idea of adding ingredients, such as cell adhesion peptides (e.g., RGD) to surfaces and matrices was being developed. Tirrell realized that the majority of the ways that such molecules were being presented was very haphazard and uncontrolled. His research group began to explore the idea of conjugating peptides to lipids in order to use the self-assembly character of lipids to direct a controlled presentation of peptides. This has led to current work in peptide amphiphile micelles, which are versatile, modular, biofunctional nanoparticles that can be injected into the circulation to target, image and, in some cases, treat pathological conditions. The Tirrell group has active work now in using such particles to diagnose and treat atherosclerosis, and also to stimulate the adaptive immune system to generate desired B-cell and T-cell responses.
The other principal area of Tirrell's current work, in addition to peptide amphiphiles, is in the generation of new materials and functional assemblies via polyelectrolyte complexation. Under the right conditions, oppositely charged polyelectrolyte chains can assemble into flexible, fluid complexes. Such fluids (sometimes known as coacervates) have very low interfacial tension with water so they are very useful as encapsulants and also as agents to drive self-assembly in aqueous systems. Tirrell's research group has been exploring these materials and assemblies in a variety of biomedical applications such as micelle formation, encapsulation and hydrogel formation.


Hebeloma radicosum

Hebeloma radicosum, commonly known as the rooting poison pie, is a species of agaric fungus in the family Hymenogastraceae. Fruit bodies (mushrooms) can be identified by the tapering root-like stipe base, as well as the almond-like odor. Found in Japan, Europe, and North America, it is an ammonia fungus, and fruits on mole, mouse, or shrew middens.
Taxonomy.
The species was first described scientifically by Jean Baptiste François Pierre Bulliard in 1784 as "Agaricus radicosus". Adalbert Ricken transferred it to "Hebeloma" in 1915. Historical synonyms have resulted from the transfer of the fungus to the genera "Pholiota" by Paul Kummer in 1871, "Dryophila" by Lucien Quélet in 1886, "Myxocybe" by Victor Fayod in 1889, and "Roumeguerites" by Marcel Locquin in 1979.
Molecular analysis places the species in a basal position of the "Myxocybe" clade. This grouping of phylogenetically related species contains members that form a pseudorrhiza, such as "H. danicum", "H. senescens", "H. calyptrosporum", "H. birrus", "H. pumilium", and "H. cylindrosporum".
The mushroom is commonly known as the "rooting poison pie".
Description.
The fruit bodies have caps in diameter that are initially convex before flattening out in age. The surface of fresh caps is sticky; the color, which ranges from yellowish tan to golden brown to ochre, or pale cinnamon, is lighter towards the margin. The margins of young caps are curled inward, and often have adherent hanging remnants of the partial veil. The gills are notched to nearly free from attachment to the stipe, and have scalloped or fringed edges, especially in maturity. Initially white, the gills change from ochre to reddish brown as the spores mature. The stipe measures long by thick, and is usually swollen in the middle and tapered on each end. The stipe base is covered with pale brownish fibers and cottony scales over a cream ground color. The stipe is solid (i.e., not hollow) and firm, with a ring on the upper portion. The flesh has a mild taste, and an odor of almonds or marzipan. Several aromatic compound are responsible for the mushroom's odor, including benzaldehyde, 2-phenylethanal, 2-phenylethanol, phenylacetic acid, N-formylaniline, and 1-octen-3-ol.
"Hebeloma radicosum" produces a rusty brown to cinnamon-brown spore print. Spores are almond-shaped, covered in small warts, and measure 8–10 by 5–6 μm. The basidia (spore-bearing cells) are four-spored.
The mushroom is poisonous, and can cause gastrointestinal distress in those who consume it.
Similar species.
The Japanese species "Hebeloma radicosoides" resembles "H. radicosum" in appearance and habitat, but can be distinguished by its yellower cap and lack of odor. Additionally, "H. radicosoides" fruits after experimental application of urea to soil, while "H. radicosum" does not. Another lookalike, "Hypholoma radicosum", has a rooting stipe, but it is more slender and smells strongly of the compound iodoform."
Ecology and distribution.
"Hebeloma radicosum" is an ammonia fungus, and associates with the latrines of moles, wood mice, and shrews. The mushroom has been used to study the nesting ecology of moles. Fruit bodies occur on the ground scattered or in groups on soil or grassy areas, and are also associated with bushes and hedges in residential areas. A Japanese field study demonstrated that male flies of the genus "Suillia" rest on the mushrooms and actively defend their territory from others of the same species while waiting to mate with oviparous females. "Hebeloma radicosum" fruit bodies can be grown in pure culture conditions in the laboratory. The fruit bodies are negatively gravitropic and not phototropic. Although they do not require light to form primordia, light is needed for differention and maturation. The sensitivity to gravity and light may be related to the growth habits of the fungus, which colonizes deep in the ground where it forms primordia, and develops mature fruit bodies on the ground.
The species is found in Japan, Europe and North America.


Ying E. Zhang

Ying E. Zhang is a Chinese-American biochemist specialized in TGF-beta signaling and functions of ubiquitin E3 ligase Smurfs to better understand cancer cells and metastasis. She is a senior investigator in the Laboratory of Cellular and Molecular Biology at the National Cancer Institute.
Education.
Zhang received her B.S. degree in chemistry and M.S. degree in biochemistry from Peking University. She obtained her Ph.D. degree from University of Wisconsin–Madison in 1995. Her dissertation was titled, "Insights into mechanisms of F₁F₀ATP synthase by genetic analysis of subunit C from Escherichia coli". Zhang's doctoral advisor was . She completed her postdoctoral training with in University of California, San Francisco.
Career and research.
Zhang joined the National Cancer Institute's (NCI) Laboratory of Cellular and Molecular Biology in 2000 as a tenure-track investigator and became a senior investigator in 2007.
Zhang's research focuses on understanding TGF-beta signaling and functions of ubiquitin E3 ligase Smurfs. She identified and characterized several key molecules in the TGF-beta signaling pathway, including Smads and Smurfs. Discoveries from her group generated mechanistic insight into how TGF-beta controls cellular responses through Smad-dependent and -independent pathways in normal and cancer cells. On the front of Smurf E3 ligases, their findings extended the function of Smurfs beyond TGF-beta pathway to genome stability and metastasis.
Awards and honors.
In 2020, she was elected Fellow of the American Institute for Medical and Biological Engineering.


Bip internal ribosome entry site (IRES)

The BiP internal ribosome entry site (IRES) is an RNA element present in the 5' UTR of the mRNA of BiP protein and allows cap-independent translation. BiP protein expression has been found to be significantly enhanced by the heat shock response due to internal ribosome entry site (IRES)-dependent translation. It is thought that this translational mechanism is essential for the survival of cells under stress.


Beta-Neoendorphin

β-Neoendorphin is an endogenous opioid peptide with a nonapeptide structure and the amino acid sequence Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro.


Adipose-derived hormones

Adipose tissue is an endocrine organ that secretes numerous protein hormones, including leptin, adiponectin, and resistin. These hormones generally influence energy metabolism, which is of great interest to the understanding and treatment of type 2 diabetes and obesity.
Their relative roles in modifying appetite, insulin resistance and atherosclerosis are the subjects of intense research, as they may be modifiable causes of morbidity in people with obesity.
History of adipose-derived hormones.
It had been shown that adipose tissue secreted some unknown factor that influenced appetite. However, the importance of adipose tissue as an endocrine organ was only fully appreciated in 1995 with the discovery of leptin, the protein product of the Ob gene. Leptin is a strong appetite suppressant that, when depleted, causes early onset severe obesity in humans and in animal models. Low levels of leptin in the blood plasma have been heavily associated with people suffering from moderate to severe forms of depression. Leptin is known to influence moods and cognition by inducing some structural and functional changes within the hippocampus and prefrontal cortex. Also, leptin has been shown to activate signal transduction pathways associated with dopamine and mTOR, which can increase synaptogenesis. Leptin's role in neuroplasticity is currently still being elucidated, but it has been proven to be active in regions of the brain closely linked to depression. It was found that leptin has antidepressant-like effects similar to that of selective serotonin reuptake inhibitors (SSRIs).
The discovery of leptin and its effects on appetite led to hopes of a treatment for obesity and type 2 diabetes, a major disease in the developed world. Unfortunately, clinical studies using leptin as a treatment for obesity in humans failed to show improvement, leading some scientists to conclude that the brain can become resistant to leptin, even at supra-physiological levels (the so-called "ceiling effect"), rendering treatment with leptin ineffective. However, although the notion of obesity as a state of 'leptin resistance' has become ingrained in the minds of many researchers, data does not directly support this contention. For example, the work of Rudolph Leibel at Columbia University shows that, in both obese and lean individuals, leptin injections do not reduce body mass. The finding that both lean and obese subjects have a similar lack of response underscores the notion that the brain is not designed to respond to increased leptin by decreasing food intake; rather, lack of leptin acts as a signal to increase food intake. Indeed, Leibel's work has shown that the decreases in serum leptin that occur post-weight-loss constitute a state of leptin deficiency, which drives increased appetite with weight loss. As such, leptin injections in weight-reduced patients can prevent increases in appetite and thereby allow patients to maintain weight loss. These studies, therefore, demonstrate that leptin treatment may be a useful strategy to treat obesity in humans, if not by driving weight loss directly then by allowing weight loss (as a result of diet and exercise) to be more readily maintained.
Moreover, as geneticists learn more from the few cases of leptin gene mutations, the possibility remains that, although leptin was ineffective at treating obesity across the population, some individual obese patients might still benefit from its use as an anti-obesity medication.
Research into the adipose-derived hormones adiponectin and resistin is ongoing. Like leptin, these hormones also affect energy balance and metabolism. Like leptin, observations in both humans and animal models have shown that adiponectin is relevant to insulin sensitivity and energy homeostasis. In contrast, the relationship between resistin and adiposity is not consistent between rodent models and human subjects (See Peter Arner, 2005: "Resistin: yet another adipokine tells us that men are not mice"); hence, the notion of resistin as a genuine adipose-derived hormone remains questionable.


Dorothy Jordan Lloyd

Dorothy Jordan Lloyd (1 May 1889 – 21 November 1946 ) was an early protein scientist who studied the interactions of water with proteins, particularly gelatin. She was also Director of the British Leather Manufacturers' Research Association. She was the first to propose that the structure of globular proteins was maintained by hydrogen bonds, an idea championed later by Linus Pauling and others.
Early life.
She was born in Birmingham, the daughter of George Jordan Lloyd, surgeon and later professor of surgery at the University of Birmingham, and his wife, Marian Hampson Simpson. One of four children, she was educated at the King Edward VI High School for Girls, Birmingham, and entered Newnham College, Cambridge, in 1908.
At the age of 12 when she decided she wanted to become a scientist. She was placed in the first class in part one of the natural sciences tripos in 1910 and in part two (zoology) in 1912, was a Bathurst student, and became the third Newnham fellow (1914–21).
She worked for a time at Cambridge on problems of regeneration and osmotic phenomena in muscle, and this led her to a study of osmotic phenomena in simpler non-living colloidal systems. Her researches were interrupted by the First World War when she investigated – for the Medical Research Committee – substitute culture media for bacteriology, and the causes and prevention of ropiness in bread.
Research and later life.
She accepted an invitation from R. H. Pickard in 1921 to join the newly formed British Leather Manufacturers' Research Association. While maintaining her interest in fundamental research (particularly into the behaviour of protein fibres in aqueous systems), she rapidly acquired an insight into the art of leather manufacture, and introduced many methods of control which have since become normal tannery practice.
In 1927, she succeeded Pickard as director, and was, until her death, the only woman leading such an association for industrial research. In spite of many set-backs, including the destruction of new laboratories by German bombing in 1940, support for the association increased under her directorship, and it was recognised as an integral part of the industry. She served on the councils and committees of many societies, including the executive committee of the International Society of Leather Trades' Chemists.
In 1939, she was awarded the Fraser Muir Moffat medal by the Tanners' Council of America for her contributions to leather chemistry. She was also vice-president of the Royal Institute of Chemistry (1943–46) and a member of the Chemical Council.As Dorothy got old she started to enjoy mountain climbing.
Besides many contributions to scientific journals, Dorothy Jordan Lloyd was the author of "The Chemistry of the Proteins" (1926; 2nd edn, with Agnes Shore, 1938), and planned and contributed to "Progress in Leather Science, 1920–45" (3 vols., 1946–48), which became one of the world's foremost textbooks on leather technology.
Death.
A keen mountaineer, in 1928 Dorothy Jordan Lloyd achieved the distinction of making the first ascent and descent in one day of the Mittellegi Ridge of the Eiger. She died at Kenilworth Lodge, Great Bookham, Surrey, on 21 November 1946, aged 57.


Atlantic Storm

Atlantic Storm was a ministerial exercise simulating the top-level response to a bioterror incident. The simulation operated on January 14, 2005 in Washington, D.C. It was created in part to reveal the current international state of preparedness and possible political and public health issues that might evolve from such a crisis.
The project was sponsored by the Alfred P. Sloan Foundation, the German Marshall Fund of the United States, and the Nuclear Threat Initiative. Organization efforts were provided by the Center for Biosecurity of UPMC, the Center for Transatlantic Relations of Johns Hopkins University, and the Transatlantic Biosecurity Network.
Scenario.
In the scenario, smallpox was released in the following major cities in a covert attack: Istanbul, Rotterdam, Warsaw, Frankfurt, New York City, and Los Angeles. Cases were initially reported in Germany, Turkey, Sweden, and the Netherlands, but the pathogen was exported to other nations within hours. Specifically, the 51 cases confirmed in these four nations grew to 3,320 cases with transatlantic spread after no more than 4.5 hours. This rapid spread of disease forced the attending representatives to grapple with a quickly escalating crisis and revealed difficult tensions between domestic politics and international relations, revealing the need for improving response systems for such a crisis. The scenario was propelled by continual briefings by “Summit Staff”, breaking news segments from the “Global News Network”, and private updates for representatives from their “national advisors”. The scenario assumes that the viral ingredients were obtained from a bioweapons facility in Russia. The terrorist group responsible used publicly available knowledge as well as training in US and Indian universities in order to create the strain of smallpox. To spread the virus, members walked around public areas with canisters releasing the virus in high traffic areas.
Considerations.
The initial problem facing the participants was whether to use a ring vaccination strategy over a mass vaccination one in order to deal with the small number of those thought to be infected with the smallpox virus. While ring vaccination is recommended for initial control over an outbreak, states may quickly choose to switch to mass vaccination if it is unsuccessful. In addition, the participants for countries with no infected persons faced pressures to share available vaccine resources with countries currently experiencing outbreaks. As more countries began to experience outbreaks, domestic pressures forced participants to withhold the sharing of vaccines in order to preserve their supply for their own citizens. Other strategies, such as vaccine dilution, became necessary as the amount of those suspected to be infected grew. Participants also considered the viability of closing borders to prevent the further spread of the outbreak to their own countries. Certain dire measures, such as the use of military quarantines, were considered as participants also had the obligation to ensure public safety in civilian populations.
Findings.
Conclusions from the exercise's published documents:
At the end of the exercise, participants were given an opportunity to share insights gained during the scenario. The lack of current international strategy and planning represented a shortfall in bioterrorism protection. In addition, members with defense backgrounds warned against the widespread unawareness that bioterrorism presented to safety at home and abroad. They believed that states without adequate protection would pose a dangerous threat to neighboring states with sufficient resources and infrastructure.
Recommendations.
Atlantic Storm highlights the importance of international communication and coordination in responding to a bioterrorist attack. Participants of the exercise indicated that the WHO would be the ideal entity to coordinate an international response. In addition, Atlantic Storm proved that most countries lack the structure and resources to handle such an outbreak. Therefore, the establishment of more resources and infrastructure is a necessity for the success of future response. Next, entities like the EU and NATO should further develop plans of action for bioterror incidents. Strong international coalitions would ensure that response time in an epidemic is not slowed by complications between neighboring countries. Additionally, many politicians are unaware of the threat of bioterrorism and require more information regarding safety and defense implications. Inclusion of knowledgeable defense authorities and scientists would give politicians the capability to form a response. Finally, the international community should do more, as a whole, to bolster developing countries' resources for defending against epidemics.
Other Bioterrorism Response Exercises.
Below are further exercises by other organizations with similar scenarios:
Criticisms & Weaknesses.
Some observers raised concerns about the exercise how it was conducted. At the time, the World Health Organization, whom participants indicated to be the ideal response coordinator, lacked the resources and staffing to handle the effort. Finally, leaders in today's world need to possess experience in homeland security and bioterrorist threats in order to handle a situation similar to the scenario proposed in Atlantic Storm. The credentials of some participating members were questioned due to a lack of experience in these fields.
The rate of infection transmission used by the Atlantic storm exercise was higher than historical records of smallpox transmission. As a result, most otherwise appropriate measures would still fail in the hypothetical scenario. This raises some concerns with conclusions of Atlantic Storm and its application to a real world scenario. In addition, some defense research questions the ability of terrorists to create and distribute such a virus.


Dimemebfe

Dimemebfe (5-MeO-BFE) is a recreational drug and research chemical. It acts as an agonist for the 5-HT1A and 5-HT2 family of serotonin receptors. It is related in structure to the psychedelic tryptamine derivative 5-MeO-DMT, but with the indole nitrogen replaced by oxygen, making dimemebfe a benzofuran derivative. It is several times less potent as a serotonin agonist than 5-MeO-DMT and with relatively more activity at 5-HT1A, but still shows strongest effects at the 5-HT2 family of receptors.
Legal status.
Dimemebfe is a Schedule I controlled substance in the US state of Alabama.


ORG-37684

ORG-37684 is a drug developed by Organon, which acts as a potent and selective agonist for the 5-HT2 receptor family, with highest affinity at 5-HT2C and lowest at 5-HT2B subtypes. It has anorectic effects in animal studies and has been researched as a potential weight loss drug for use in humans.


Alpha operon ribosome binding site

The alpha operon ribosome binding site in bacteria is surrounded by this complex pseudoknotted RNA structure. Translation of the mRNA produces 4 ribosomal protein products, one of which (S4) acts as a translational repressor by binding to the nested pseudoknot region. The mechanism of repression is thought to involve a conformational switch in the pseudoknot region and ribosome entrapment.


2-OH-NPA

2-OH-NPA is a drug used in scientific research which acts as a potent and selective agonist for the dopamine D2 receptor.


G-less cassette

The G-less cassette transcription assay is a method used in molecular biology to determine promoter strength "in vitro". The technique involves quantification of an mRNA product with the use of a plasmid. The G-less cassette is part of a pre-constructed vector, usually containing a multiple cloning site (MCS) upstream of the cassette. For this reason, promoters of interest can be inserted directly into the MCS to ultimately measure the accuracy and efficiency of a promoter in recruiting transcription machinery.
Method.
The G-less cassette is a reporter gene that encodes a transcript lacking guanine nucleotides in the sense strand of the DNA (hence "G-less"). A plasmid containing such a gene is located downstream of a MCS. After the promoter is inserted into the MCS, transcription proceeds with the addition of radiolabeled UTP, CTP, and ATP (as well as non-radiolabeled/cold nucleotides) and continues until the end of the G-less cassette is reached and guanine residues are once again apparent in the sense strand of the DNA. The absence of GTP "in vitro" results in transcription being prematurely terminated at the first guanine residue in the sense strand following the cassette. Gel electrophoresis is performed on the transcription products and the amount of radioactivity is quantified by autoradiography or phosphorimaging to determine the strength of the promoter of interest.
Application.
The G-less cassette technique is used to determine promoter strength beyond basal levels of transcription (i.e. in the presence of transcription activators or transcription factors). For example, to measure the effects of a TATA box consensus sequence modification in "Saccharomyces cerevisiae" in the presence of TFIID, G-less cassettes were implemented to measure the relative strength of each promoter.
Advantages.
The G-less assay can be performed on a circular plasmid to measure levels of transcription. A circular plasmid provides a more efficient template in many systems when compared to other assays such as runoff transcription, in which a cleaved end is required. This method generates radiolabeled transcripts very efficiently because it bypasses the unnecessary process of performing other indirect mRNA product measurements. The promoter is inserted into a circular plasmid containing the G-less cassette, which will generate a transcript of a certain length that omits random and nonspecific transcription throughout the plasmid. Most crude systems, such as HeLa nuclear extracts, are used because they contain low amounts of contaminating GTP that lead to background transcription and may occasionally cause random transcription to read through the G-less cassette.


4,5-MDO-DiPT

4,5-MDO-DiPT, or 4,5-methylenedioxy-N,N-diisopropyltryptamine, is a lesser-known psychedelic drug. It is the 4,5-methylenedioxy analog of DiPT. 4,5-MDO-DiPT was first synthesized by Alexander Shulgin. In his book "TiHKAL" ("Tryptamines I Have Known and Loved"), 4,5-MDO-DiPT produces slight LSD-like effects after several hours. Very little data exists about the pharmacological properties, metabolism, and toxicity of 4,5-MDO-DiPT.


Mir-30 microRNA precursor

miR-30 microRNA precursor is a small non-coding RNA that regulates gene expression. Animal microRNAs are transcribed as pri-miRNA (primary miRNA) of varying length which in turns are processed in the nucleus by Drosha into ~70 nucleotide stem-loop precursor called pre-miRNA (precursor miRNA) and subsequently processed by the Dicer enzyme to give a mature ~22 nucleotide product. In this case the mature sequence comes from both the 3' (miR-30) and 5' (mir-97-6) arms of the precursor. The products are thought to have regulatory roles through complementarity to mRNA.
A screen of 17 miRNAs that have been predicted to regulate a number of breast cancer associated genes found variations in the microRNAs
miR-17 and miR-30c-1, these patients were noncarriers of BRCA1 or BRCA2 mutations, lending the possibility that familial breast cancer may be caused by variation in these miRNAs.
Members of the miR-30 family have been found to be highly expressed in heart cells.
Targets of miR-30.
It has been shown that the integrin ITGB3 and the ubiquitin conjugating E2 enzyme UBC9 are downregulated by miR-30. It has also been suggested that the TP53 protein may be a target of miR-30c and miR-30e. An immunoblot analysis revealed that p53 expression levels were elevated upon knockdown of miR-30c and miR-30e.


Arylalkanolamine

Arylalkanolamines (ArROHNR2) are a class of medicinal molecules that are structurally related to one another in certain respects.
Detailed understanding of their structure-activity relationship is expected to aid in drug discovery.


Nested gene

A nested gene is a gene whose entire coding sequence lies within the bounds (between the start codon and the stop codon) of a larger external gene. The coding sequence for a nested gene differs greatly from the coding sequence for its external host gene. Typically, nested genes and their host genes encode functionally unrelated proteins, and have different expression patterns in an organism.
There are two categories of nested genes:
Nested intronic genes.
A nested intronic gene lies within the non-coding intronic region of a larger gene, and occurs relatively frequently, especially in the introns of metazoans and higher eukaryotes. Because only eukaryotic DNA contains intronic regions, this type of gene does not occur in bacteria or archaea.
The human genome contains a relatively high proportion of nested intronic genes. It is predicted to contain at least 158 functional intronic nested genes, with an additional 212 pseudogenes and three snoRNA genes nested in intronic regions. These genes seem to be distributed randomly across all chromosomes, and the majority code for proteins that are functionally unrelated to their host genes.
Genes nested opposite coding sequences.
Genes nested opposite the coding sequences of their host genes are very rare, and have been observed in prokaryotes, and more recently, in yeast ("S. cerevisiae") and in "Tetrahymena thermophila". These non-intronic nested genes remain to be identified in metazoan genomes. As with intronic nested genes, nonintronic nested genes typically do not share functions or expression patterns with their host genes.


LPAR6

Lysophosphatidic acid receptor 6, also known as LPA6, P2RY5 and GPR87, is a protein that in humans is encoded by the "LPAR6" gene. LPA6 is a G protein-coupled receptor that binds the lipid signaling molecule lysophosphatidic acid (LPA).
The protein encoded by this gene belongs to the family of G-protein coupled receptors, that are preferentially activated by adenosine and uridine nucleotides. This gene aligns with an internal intron of the retinoblastoma susceptibility gene in the reverse orientation.
Role in hair growth/loss.
In February 2008, researchers at the University of Bonn announced they have found the genetic basis of two distinct forms of inherited hair loss, opening a broad path to treatments for baldness. They found that mutations in the gene P2RY5 causes a rare, inherited form of hair loss called hypotrichosis simplex. It is the first receptor in humans known to play a role in hair growth. The fact that any receptor plays a specific role in hair growth was previously unknown to scientists, and with this new knowledge a focus on finding more of these genes may be able to lead to therapies for many different types of hair loss.
In 2013, it was found that mutations in "LPAR6" give rise to the Cornish Rex cat breed, which has a form of ectodermal dysplasia characterised by short woolly hair which is susceptible to loss.


Polymerase chain reaction optimization

The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization which have been developed by molecular biologists to improve PCR performance and minimize failure.
Contamination and PCR.
The PCR method is extremely sensitive, requiring only a few DNA molecules in a single reaction for amplification across several orders of magnitude. Therefore, adequate measures to avoid contamination from any DNA present in the lab environment (bacteria, viruses, or human sources) are required. Because products from previous PCR amplifications are a common source of contamination, many molecular biology labs have implemented procedures that involve dividing the lab into separate areas. One lab area is dedicated to preparation and handling of pre-PCR reagents and the setup of the PCR reaction, and another area to post-PCR processing, such as gel electrophoresis or PCR product purification. For the setup of PCR reactions, many standard operating procedures involve using pipettes with filter tips and wearing fresh laboratory gloves, and in some cases a laminar flow cabinet with UV lamp as a work station (to destroy any extraneomultimer formation). PCR is routinely assessed against a negative control reaction that is set up identically to the experimental PCR, but without template DNA, and performed alongside the experimental PCR.
Hairpins.
Secondary structures in the DNA can result in folding or knotting of DNA template or primers, leading to decreased product yield or failure of the reaction. Hairpins, which consist of internal folds caused by base-pairing between nucleotides is inverted repeats within single-stranded DNA, are common secondary structures and may result in failed PCRs.
Typically, primer design that includes a check for potential secondary structures in the primers, or addition of DMSO or glycerol to the PCR to minimize secondary structures in the DNA template, are used in the optimization of PCRs that have a history of failure due to suspected DNA hairpins.
Polymerase errors.
Taq polymerase lacks a 3′ to 5′ exonuclease activity. Thus, Taq has no error-proof-reading activity, which consists of excision of any newly misincorporated nucleotide base from the nascent (i.e., extending) DNA strand that does not match with its opposite base in the complementary DNA strand. The lack in 3′ to 5′ proofreading of the Taq enzyme results in a high error rate (mutations per nucleotide per cycle) of approximately 1 in bases, which affects the fidelity of the PCR, especially if errors occur early in the PCR with low amounts of starting material, causing accumulation of a large proportion of amplified DNA with incorrect sequence in the final product.
Several "high-fidelity" DNA polymerases, having engineered 3′ to 5′ exonuclease activity, have become available that permit more accurate amplification for use in PCRs for sequencing or cloning of products. Examples of polymerases with 3′ to 5′ exonuclease activity include: KOD DNA polymerase, a recombinant form of "Thermococcus kodakaraensis" KOD1; Vent, which is extracted from "Thermococcus litoralis"; Pfu DNA polymerase, which is extracted from "Pyrococcus furiosus"; and Pwo, which is extracted from "Pyrococcus woesii".
Magnesium concentration.
Magnesium is required as a co-factor for thermostable DNA polymerase. Taq polymerase is a magnesium-dependent enzyme and determining the optimum concentration to use is critical to the success of the PCR reaction. Some of the components of the reaction mixture such as template concentration, dNTPs and the presence of chelating agents (EDTA) or proteins can reduce the amount of free magnesium present thus reducing the activity of the enzyme. Primers which bind to incorrect template sites are stabilized in the presence of excessive magnesium concentrations and so results in decreased specificity of the reaction. Excessive magnesium concentrations also stabilize double stranded DNA and prevent complete denaturation of the DNA during PCR reducing the product yield. Inadequate thawing of MgCl2 may result in the formation of concentration gradients within the magnesium chloride solution supplied with the DNA polymerase and also contributes to many failed experiments .
Size and other limitations.
PCR works readily with a DNA template of up to two to three thousand base pairs in length. However, above this size, product yields often decrease, as with increasing length stochastic effects such as premature termination by the polymerase begin to affect the efficiency of the PCR. It is possible to amplify larger pieces of up to 50,000 base pairs with a slower heating cycle and special polymerases. These are polymerases fused to a processivity-enhancing DNA-binding protein, enhancing adherence of the polymerase to the DNA.
Other valuable properties of the chimeric polymerases TopoTaq and PfuC2 include enhanced thermostability, specificity and resistance to contaminants and inhibitors. They were engineered using the unique helix-hairpin-helix (HhH) DNA binding domains of topoisomerase V from hyperthermophile "Methanopyrus kandleri". Chimeric polymerases overcome many limitations of native enzymes and are used in direct PCR amplification from cell cultures and even food samples, thus by-passing laborious DNA isolation steps. A robust strand-displacement activity of the hybrid TopoTaq polymerase helps solve PCR problems that can be caused by hairpins and G-loaded double helices. Helices with a high G-C content possess a higher melting temperature, often impairing PCR, depending on the conditions.
Non-specific priming.
Non-specific binding of primers frequently occurs and may occur for several reasons. These include repeat sequences in the DNA template, non-specific binding between primer and template, high or low G-C content in the template, or incomplete primer binding, leaving the 5' end of the primer unattached to the template. Non-specific binding of degenerate primers is also common. Manipulation of annealing temperature and magnesium ion concentration may be used to increase specificity. For example, lower concentrations of magnesium or other cations may prevent non-specific primer interactions, thus enabling successful PCR. A "hot-start" polymerase enzyme whose activity is blocked unless it is heated to high temperature (e.g., 90–98˚C) during the denaturation step of the first cycle, is commonly used to prevent non-specific priming during reaction preparation at lower temperatures. Chemically mediated hot-start PCRs require higher temperatures and longer incubation times for polymerase activation, compared with antibody or aptamer-based hot-start PCRs.
Other methods to increase specificity include Nested PCR and Touchdown PCR.
Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design.
Touchdown polymerase chain reaction or touchdown style polymerase chain reaction is a method of polymerase chain reaction by which primers will avoid amplifying nonspecific sequences. The annealing temperature during a polymerase chain reaction determines the specificity of primer annealing. The melting point of the primer sets the upper limit on annealing temperature. At temperatures just below this point, only very specific base pairing between the primer and the template will occur. At lower temperatures, the primers bind less specifically. Nonspecific primer binding obscures polymerase chain reaction results, as the nonspecific sequences to which primers anneal in early steps of amplification will "swamp out" any specific sequences because of the exponential nature of polymerase amplification.
The earliest steps of a touchdown polymerase chain reaction cycle have high annealing temperatures. The annealing temperature is decreased in increments for every subsequent set of cycles (the number of individual cycles and increments of temperature decrease is chosen by the experimenter). The primer will anneal at the highest temperature which is least-permissive of nonspecific binding that it is able to tolerate. Thus, the first sequence amplified is the one between the regions of greatest primer specificity; it is most likely that this is the sequence of interest. These fragments will be further amplified during subsequent rounds at lower temperatures, and will out compete the nonspecific sequences to which the primers may bind at those lower temperatures. If the primer initially (during the higher-temperature phases) binds to the sequence of interest, subsequent rounds of polymerase chain reaction can be performed upon the product to further amplify those fragments.
Primer dimers.
Annealing of the 3' end of one primer to itself or the second primer may cause primer extension, resulting in the formation of so-called primer dimers, visible as low-molecular-weight bands on PCR gels. Primer dimer formation often competes with formation of the DNA fragment of interest, and may be avoided using primers that are designed such that they lack complementarity—especially at the 3' ends—to itself or the other primer used in the reaction. If primer design is constrained by other factors and if primer-dimers do occur, methods to limit their formation may include optimisation of the MgCl2 concentration or increasing the annealing temperature in the PCR.
Deoxynucleotides.
Deoxynucleotides (dNTPs) may bind Mg2+ ions and thus affect the concentration of free magnesium ions in the reaction. In addition, excessive amounts of dNTPs can increase the error rate of DNA polymerase and even inhibit the reaction. An imbalance in the proportion of the four dNTPs can result in misincorporation into the newly formed DNA strand and contribute to a decrease in the fidelity of DNA polymerase.


Prince Gennaro of Naples and Sicily

Prince Gennaro of Naples and Sicily (Gennaro Carlo Francesco; 12 April 1780 – 1 January 1789) was a Prince of Naples and Sicily. He died of smallpox at age 8. He and his mother are the central figures of a portrait by Angelica Kauffman in 1783.
Biography.
Born at the Royal Palace of Naples in the Kingdom of Naples, he was his parents' third son and eighth child. He was named "Gennaro" in honour of Saint Januarius, the patron saint of Naples and a martyr saint of both the Roman Catholic and the Eastern Orthodox Churches.
A member of the Bourbons of Naples, he was a Prince of Naples and Sicily by birth.
His sisters included the future Holy Roman Empress, Grand Duchess of Tuscany. His younger sister's Princess Maria Cristina, was the wife of the future Charles Felix of Sardinia and Queen of Sardinia; Maria Cristina's twin Princess Maria Cristina Amelia died in 1783 of smallpox. Another sister was the Queen of the French and the youngest was the future Princess of Asturias.
His cousins included a Duke of Parma, Grand Duke of Tuscany, Holy Roman Emperor, Queen of Portugal, King of Spain, a Duchess of Calabria, the first wife of his older brother Ferdinand.
He was the second in line to the throne, second only to his oldest surviving brother Prince Francis, "then" Duke of Calabria. At the age of three, he survived a smallpox epidemic which killed his older sister Maria Cristina Amelia and younger brother Giuseppe, the two dying within a week of each other in February 1783. Later that year, his mother gave birth to a stillborn daughter named Maria Cristina in honour of the deceased Maria Cristina Amelia.
In August 1788, his mother gave birth to another son named Carlo. In December 1788, Gennaro caught smallpox and died on 2 January 1789 at the Caserta Palace. Gennaro passed the illness onto his infant brother who died a month after him. Both are both buried at the Basilica of Santa Chiara, Naples.


Adenylosuccinate

Adenylosuccinate is an intermediate in the interconversion of purine nucleotides inosine monophosphate (IMP) and adenosine monophosphate (AMP). The enzyme adenylosuccinate synthase carries out the reaction by the addition of aspartate to IMP and requires the input of energy from a phosphoanhydride bond in the form of guanosine triphosphate (GTP). GTP is used instead of adenosine triphosphate (ATP), so the reaction is not dependent on its products.


CRISPR Therapeutics

CRISPR Therapeutics AG is a Swiss–American biotechnology company headquartered in Zug. In fiscal year 2019, the company had revenues of $289.59 million, with net income of $66.86 million. In the same year, the number of employees stood at 304. As of early 2021, the company had a market capitalization of over $13 billion. CRISPR Therapeutics' investors include German chemical company Bayer. The company operates R&D in Cambridge, Massachusetts.
History.
CRISPR Therapeutics was founded in 2012. One of the co-founders Emmanuelle Charpentier later shared the Nobel Prize in Chemistry in 2020 with Jennifer Doudna. As part of a working group, she provided the first scientific documentation on the development and use of CRISPR gene editing. This allows DNA to be specifically modified and exchanged, which can for example prevent diseases. The company CRISPR Therapeutics is to apply this new technology commercially and advance research.
In 2016, the company went public on NASDAQ. In August 2016 the company started to operate Casebia Therapeutics, as a joint venture with Bayer. In 2019, Casebia Therapeutics came directly under the control of CRISPR Therapeutics.
Products.
The company has several drugs in development. These include the drug CTX001 for the treatment of the rare blood disorders Beta thalassemia and sickle cell disease, which is being developed jointly with Vertex Pharmaceuticals. In May 2020, CTX001 received Orphan drug Designation from the U.S. Food and Drug Administration for transfusion-dependent beta thalassemia and from the European Medicines Agency for sickle cell disease and transfusion-dependent beta thalassemia. Early clinical trial results support the safety and efficacy of this treatment.


Towelie

"Towelie" is the eighth episode of the fifth season of the American animated sitcom "South Park", and the 73rd episode of the series overall. It originally aired on Comedy Central in the United States on August 8, 2001. In the episode, the boys attempt to recover their stolen video game console from the middle of a feud between a paramilitary group and extraterrestrials.
The episode was written and directed by series co-creator Trey Parker and is rated TV-MA in the United States, except on syndicated broadcasts, where the episode is instead rated TV-14. It features the first appearance of recurring character Towelie, created to satirize the degree to which "South Park" was being merchandised.
Plot.
While at Stan Marsh's house, Eric Cartman discovers a used tampon in the garbage which he mistakes for an aborted fetus. In an effort to get the kids never to mention the tampon again, Stan's mother, Sharon buys the kids a video game system, the Okama Gamesphere. The boys are completely fascinated by the Okama Gamesphere and intend to play with it for the entire weekend, without sleeping. During their playing, a talking towel called "Towelie" comes in whenever they mention any subject involving water and advises them to keep a towel handy, before asking if they want to get high; however, the boys largely ignore him and tell him to go away.
Returning to school on Monday, the boys wait the bus stop, where a man asks them if they have seen Towelie. When they indicate that they have, the man yells into a walkie-talkie and drives off. Returning to Stan's house after school, the boys find the Okama Gamesphere is gone. In its place is a ransom note, saying that if the boys want the console, they will have to take Towelie to a secluded gas station during the night. The boys locate Towelie and take him to the gas station. An elderly man that works for the company that made Towelie is there, and he thanks the boys for bringing Towelie to him. When the boys ask for their Okama Gamesphere, the old man realizes that it is a trap, and the United States Military ambushes them. During the fight, Towelie and the boys escape.
For the rest of the episode, the boys and Towelie go back and forth between a military base and the company that made Towelie (Tynacorp) in an effort to get their Okama Gamesphere. The plot gets increasingly thick, involving aliens trying to take over Earth using genetically modified towels. Throughout the story, the boys show no interest in these revelations, as they single-mindedly want nothing more than to retrieve their Okama Gamesphere; in spite of their utter indifference, the increasingly complicated plot continues to surround them and the two sides attempt to play them against the other. In the midst of a confrontation between the military and Tynacorp, the boys find their Okama Gamesphere, but before they can play for long the building is blown up in order to kill the aliens that want to rule the world. Kenny McCormick falls in a pool of lava, but the other boys and Towelie escape with the Okama Gamesphere. The boys and Towelie then go home and play the Okama Gamesphere. As they share a laugh, Cartman comments to Towelie, "You're the worst character ever, Towelie", to which the towel complacently replies, "I know."
Production.
Towelie's catchphrase, "don't forget to bring a towel," originated on a boat trip the writers took, during which people were constantly telling each other "don't forget to bring a towel," with the phrase having eventually morphed into "Towelie says to bring a towel." Parker and Stone have explained that they were becoming increasingly aware as to how heavily merchandised and exploited their creations were becoming, and created Towelie to poke fun at this. They designed Towelie as a shallow, two-dimensional (both literally and figuratively) character who has no real purpose except to "spout catch phrases and merchandise the hell out of." This is the reason for the fake commercial during the show, and why Cartman calls Towelie the "worst character ever" at the end of the episode.
Shortly after "Towelie" was originally broadcast, South Park Studios, the official "South Park" website, featured T-shirts and hooded sweatshirts based on the episode. The shirt design featured a waving Towelie saying, "Don't forget to bring a towel."
The console in "Towelie" was originally a PlayStation 2, whose name was used during the production of the episode, in its storyboards and scripts. The fictional console ultimately used in the episode, the Okama Gamesphere, was seen as a reference to the Nintendo Game Cube.
Before the episode cuts to a commercial break, what appears to be a fake commercial is shown advertising Towelie merchandise; however, when this episode first aired, the T-shirts shown were available over a Comedy Central 1-800 line.
Home media.
"Towelie," along with the thirteen other episodes from "South Park: the Complete Fifth Season", were released on a three-disc DVD set in the United States on February 22, 2005. The set includes brief audio commentaries by Parker and Stone for each episode.


Janssen Biotech

Janssen Biotech, Inc., formerly Centocor Biotech, Inc., is a biotechnology company that was founded in Philadelphia in 1979 with an initial goal of developing new diagnostic assays using monoclonal antibody technology.
History.
In 1982, Centocor transitioned into a publicly traded company. In the early 1980s, the company moved to Malvern, Pennsylvania. In 1984, Centocor opened an overseas plant in Leiden, the Netherlands.
In 1997, eighteen years after its foundation, Centocor achieved its first year of operating profitability. In 1998, Centocor sold its diagnostic division to Fujirebio, Inc.
In 1999, Centocor became a wholly owned subsidiary of Johnson & Johnson.
In 2004, Centocor purchased a new manufacturing plant in St. Louis, Missouri, and is currently opening a new manufacturing facility in County Cork, Ireland. The Dutch plant has been expanded substantially with a $250 million investment in additional production facilities, which were opened in 2006.
In 2007, Centocor broke new ground in advertising by releasing Innerstate, believed to be the first theatrically released documentary film both created and entirely funded by a drug company, to promote Remicade (Infliximab).
In 2008, Centocor, Inc. and Ortho Biotech Inc. merged to form Centocor Ortho Biotech Inc.
In June 2010, Centocor Ortho Biotech acquired RespiVert, a privately held drug discovery company focused on developing small-molecule, inhaled therapies for the treatment of pulmonary diseases.
In June 2011, Centocor Ortho Biotech changed its name to Janssen Biotech, Inc. as part of a global effort to unite the Janssen Pharmaceutical Companies around the world under a common identity.
In December 2014, the company announced it would co-develop MacroGenics cancer drug candidate (MGD011) which targets both CD19 and CD3 proteins in treating B-cell malignant tumours. This could net MacroGenics up to $700 million.
In January 2015, the company announced it would utilise Ionis Pharmaceuticals' (formerly Isis Pharmaceuticals) Rna-targeting technology to discover and develop antisense drugs targeting autoimmune disorders of the gastrointestinal tract.
In December 2019, XBiotech Inc. announced it would sell its novel antibody treatment (bermekimab) that neutralizes interleukin-1 alpha (IL-1⍺) to Janssen Biotech, Inc.
Products.
In 1984, Centocor developed their first product approved by the U.S. Food and Drug Administration (FDA) — a diagnostic test used to detect the rabies virus.
In 1998, the company launched its top-selling monoclonal antibody Remicade (infliximab) for its first FDA approved indication in Crohn's disease. Subsequently, Remicade's market has expanded with approvals for rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, ulcerative colitis, and pediatric Crohn's disease. Remicade was approved for plaque psoriasis in September 2006.
Centocor also markets ReoPro (abciximab), a biologic agent indicated as an adjunct to coronary angioplasty (PTCA).
In 2009, the U.S. FDA approved Simponi, a human monoclonal antibody for treatment for arthritis, which was co-developed with Medarex, Inc.


Brefonalol

Brefonalol is a beta-adrenergic antagonist.


Morphiceptin

Morphiceptin is a tetrapeptide (Tyr-Pro-Phe-Pro-NH2) that is a selective μ-opioid receptor agonist. It is derived from β-casomorphin and has over 1,000 times selectivity for μ- over δ-opioid receptors. When injected intracerebroventricularly (into the ventricular system of the brain), morphiceptin had an analgesic ED50 of 1.7 nmol per animal. The analgesic effects of morphiceptin were reversed by naloxone, meaning that the analgesic effect is mediated by the μ-opioid receptor.
Morphiceptin is the (1S,2S,3S,4S)-form whereas deproceptin is the (1S,2S,3S,4R)-form [84799-23-5].


Arthrobacter woluwensis

Arthrobacter woluwensis is a species of Gram-positive bacteria.


Alfuzosin

Alfuzosin, sold under the brand name Uroxatral among others, is a medication of the α1 blocker class. It is used to treat benign prostatic hyperplasia (BPH).
As an antagonist of the α1 adrenergic receptor, it works by relaxing the muscles in the prostate and bladder neck, making it easier to urinate.
Alfuzosin was patented in 1978 and approved for medical use in 1988. It was approved in the US for BPH in 2003. In 2017, it was the 266th most commonly prescribed medication in the United States, with more than one million prescriptions.
Side effects.
The most common side effects are dizziness (due to postural hypotension), upper respiratory tract infection, headache, fatigue, and abdominal disturbances. Side effects include stomach pain, heartburn, and congested nose. Adverse effects of alfuzosin are similar to that of tamsulosin with the exception of retrograde ejaculation.
Contraindications.
Alfuzosin should be used with caution in patients with severe chronic kidney disease, and should not be prescribed to patients with a known history of QT prolongation or those who are taking medications known to prolong the QT interval.
Chemistry.
Alfuzosin contains a stereocenter and is therefore chiral. There are two enantiomeric forms, ("R")-alfuzosin and ("S")-alfuzosin. The drug is used as a racemate, ("RS")-alfuzosin, a 1: 1 mixture of the ("R")- and ("S")-forms.
It is provided as the hydrochloride salt.
Brand names.
It is marketed under the brand name Uroxatral and elsewhere under the tradenames Xat, Xatral, Prostetrol and Alfural.


EFSH

eFSH is a follicle-stimulating hormone obtained from equine species, used to stimulate fertility.
Clinical Studies.
The effects of eFSH on mares is observed by creating a treatment group that received 12.5 milligrams of eFSA injections twice a day when the follicle of the mare was 25 mm in diameter then received human chorionic gonadotropin(hCG) at 35 mm follicle diameter while a control group only receives hCG at 35mm follicle diameter.
Embryo Transfer.
The application of utilizing eFSH to increase the efficiency of equine embryo transfer was tested at the University of Saskatchewan. Embryos from 12 donor mares at 8 days of ovulation were recovered, scored, then transferred to 37 recipients mares who were tested for pregnancy after 7-10 days. 5 pregnancies resulted from the 15 embryos were recovered from the eFSA treatment group and 4 pregnancies occurred from the 6 embryos collected from the control. The usage of eFSA in the study had a correlation with a lower score.
Equine Chorionic Gonadotropin (eCG) Production.
The study conducted by the Department of Veterinary Sciences at the University of Kentucky has found that eFSH injections increase the probability superovulation occurring in mares and that carried twin result in a greater eCG concentration than one. Weekly blood samples were examined for eCG levels and the mares were compared gestation, twin pregnancy, and test grouping. 9 of the 10 mares in the eFSH group carried twin pregnancies and 1 of the 9 mares in the control groups carried twins. The mares carrying twins averaged around twice the eCG concentration of a regular pregnancy.


Genta (company)

Genta Incorporated was a biopharmaceutical company started in La Jolla, California, which discovered and developed innovative drugs for the treatment of patients with cancer. Founded in 1989 by a highly skilled entrepreneur, the company focused on a novel technology known as antisense, which targets gene products that are associated with the onset and progression of serious diseases. At that time, only Ionis Pharmaceuticals, Inc. () (formerly Isis Pharmaceuticals until December 2015) was conducting significant research with this technology. Antisense is a short span of oligonucleotides – modified DNA structures ranging from about 12-24 bases that selectively bind to specific RNA. The intent is to block expression of an aberrant protein that contributes to the disease of interest. Genta in-licensed three different antisense molecules that blocked Bcl-2, a fibroblast growth factor (FGF), and the gene c-myb, respectively.
Overview.
Products.
Genasense (oblimersen sodium).
A potent Bcl-2 antisense molecule was discovered and patented by Dr. John Reed, one of the world's highly ranked scientists. Working with Dr. Carlo Croce, Reed was a key figure in understanding the central role of apoptosis in cancer cell death. Reed and others showed that cell-death in multiple types of cancer cells was blocked by expression of Bcl-2 protein. As a corollary, they showed that inhibitors of Bcl-2 function – or reduced expression of Bcl-2 protein – greatly enhanced cancer-killing activity when combined with conventional anticancer therapy.
Through the mid 1990s, drug development of oligonucleotides was slowed by the enormous cost of synthesizing custom DNA molecules. Moreover, animal studies showed the drug had to be injected in order to be effective, whereas oral administration led to rapid breakdown.
In 1995, Genta developed a clinical protocol for the first in-human study with a Bcl-2 inhibitor. While experts were highly enthusiastic about the approach, there was concern that very low drug supplies (due to very high cost) would constrain observations of clinical benefit. Nonetheless, based on pre-clinical work showing chemotherapy sensitization of melanoma cells, a small Phase 1 study restricted to 14 patients with advance malignant melanoma was initiated in 1997 that reported acceptable safety and evidence of clinical activity.
By then, a publicly traded company with financial constraints, external investors assumed control of Genta and initiated reductions in activity and expenses. The company was eventually able to restore market confidence and secured sufficient financing to expand clinical trials using “Genasense®”, the new name for its Bcl-2 inhibitor.
In clinical trials, the underlying scientific premise was that blocking Bcl-2 expression would enhance the cancer-killing effects of standard anticancer therapy. As a corollary, however, this concept implied that convincing evidence of activity would require a direct comparison with standard treatment. Between 2001 and 2004, Genta initiated and completed three, global, randomized studies in melanoma, myeloma, and chronic lymphocytic leukemia.
Given exceptionally poor patient outcomes in melanoma, a randomized double-blind trial was initiated using a standard drug (dacarbazine) with or without Genasense. The initial size of this trial was expanded to enable a direct comparison of safety and efficacy, which involved 139 research sites in 9 countries. Results showed statistically significant benefits (p < 0.05) in progression-free survival (PFS), overall response rate, and complete response rate. Overall survival also increased (9.0 vs.7.8 mos.; p = 0.077) but did not achieve statistical significance.
Genasense had already been granted “Fast Track” status by the U.S. Food and Drug Administration (FDA). External reviews by melanoma experts suggested these data could be sufficient for conditional regulatory approval by FDA, subject to conduct of a follow-on confirmatory study. External reviewers also noted: (1) no drug had ever shown a survival improvement in melanoma; (2) no drug had ever been required to show increased survival by any regulatory authority; (3) the melanoma drug most recently considered for approval by FDA in melanoma (temozolomide; Temodar®; Schering Plough) had been reviewed on the basis of whether there was statistically significant improvement in progression-free survival, which had not been achieved.
Together, these considerations strongly suggested that the significant increase in PFS, together with increased response rates and the trend in overall survival, would be favorably considered. At a FDA Advisory Committee on May 3, 2004, the Genasense data were felt to be insufficient, and the drug was not approved. A followup study by Genta, which was intended to show a survival benefit in a subset of patients with advanced melanoma, was unsuccessful.
Genasense faced a similar regulatory outcome in the review of its randomized trial of standard chemotherapy with or without Genasense in patients with chronic lymphocytic leukemia (CLL). In this trial, the primary endpoint was response rate – a primary approval endpoint for other CLL drugs, but more rigorously defined in this study as limited to complete plus nodular partial response (CR/nPR). In a study of 241 patients, the CR/nPR rate was significantly increased to 17% in patients who received Genasense plus chemotherapy vs. 7% who received chemotherapy alone (p = 0.025). Patents who achieved CR/nPR on the Genasense arm also demonstrated statistically superior increases in both overall survival as well as progression-free survival compared with patients who achieved CR/nPR using chemotherapy alone. Despite achieving the primary endpoint, on September 6, 2006 at an FDA Advisory Committee meeting, the overall patient benefit was considered modest and insufficient for approval.
Followup of the Genasense Development Program.
Throughout the period from 2004-2015, progression-free survival (PFS) was widely accepted as a primary approval endpoint for patients with solid tumors. Temozolomide (Temodar®) had been approved in brain cancer, and the drug continued to be widely used off-label in patients with melanoma.
In 2016, a different Bcl-2 inhibitor (venetoclax: Venclexta®) (Genentech/AbbVie) was approved by FDA after a single non-randomized trial of relapsed CLL patients with 17p deletion, and on the basis of overall response rate (i.e., all types, complete plus partial), with a safety profile similar to Genasense.
Genta halted the development of Genasense in 2008. The company also elected not to pursue clinical development of antisense drugs targeting FGF or c-myb. Genasense was the first systemic antisense drug to undergo global regulatory review. Since then, more than 10 different antisense drugs have been approved for patients with a wide range of serious clinical illnesses.
Tesetaxel.
Tesetaxel is a novel taxane that is being developed as cancer therapy. Unlike standard taxanes (paclitaxel Taxol®; docetaxel (Taxotere®) that require slow intravenous administration and are mainstays of cancer treatment, tesetaxel is taken by mouth. The drug also appeared to cause fewer side-effects than IV taxanes. Preclinically, tesetaxel demonstrated substantially higher activity against cancer cell lines that were resistant to paclitaxel and docetaxel, since acquired resistance to this drug class is not mediated by the multidrug-resistant p-glycoprotein.
Discovered by Daiichi Sankyo Pharmaceutical Company, Tokyo, Japan, tesetaxel was tested by Daiichi in early Phase 1 and Phase 2 studies. However, the drug was placed on “clinical hold” by the U.S Food and Drug Administration due to deaths of several patients with severe blood count depression, and Daiichi's efforts to remove the regulatory hold were unsuccessful.
On May 7, 2008, Genta entered into a licensing agreement for global rights to tesetaxel with Daiichi Sankyo. Shortly thereafter, Genta secured FDA's agreement to remove the clinical hold with a brief submission that ensured patient safety.
Between 2008-2012, Genta conducted an array of Phase 1 and Phase 2 studies, and subsequently the company initiated two global Phase 3 trials of tesetaxel in combination with capecitabine in breast cancer and gastric cancer, respectively. With positive Phase 3 data, Genta projected potential FDA approval and commercial U.S. launch of tesetaxel for treatment of metastatic breast cancer in 2014. However, financial constraints forced termination of the Phase 3 programs in 2012.
Global rights to tesetaxel were acquired from the Genta estate in 2013. Clinical development of tesetaxel was resumed by Odanate Therapeutics (ODT; Nasdaq), which is conducting Phase 3 studies for its approval in breast cancer.
Gallium Products.
With the emergence of platinum-based drugs as cancer treatment, the U.S. National Cancer Institute initiated a screening program for other metals as anticancer drugs in the 1970s. 67Ga was already being used in radiology scans to image potential areas of inflammation and some types of cancer, and gallium nitrate emerged as a potentially effective anticancer agent. Early clinical studies unexpectedly showed that gallium nitrate markedly reduced serum calcium, which when elevated is a potentially life-threatening complication in cancer patients.
With this discovery, a team at Memorial Sloan-Kettering in New York initiated a clinical development program that showed the drug was highly effective. Follow-on Phase 3 randomized trials showed the drug was superior to other agents used at that time, including injectable calcitonin and intravenous etidronate. Concurrent studies also suggested clinical activity in patients with severe hypercalcemia due to parathyroid carcinoma as well as patients with bone diseases, including Paget's disease, multiple myeloma, and bone metastases. In 2003, the drug was approved as Ganite® (gallium nitrate solution) for the treatment of hypercalcemia resistant to hydration, and the drug was marketed by Fujisawa Inc.
Work at the Hospital for Special Surgery in New York and elsewhere suggested that gallium reduced bone resorption and might also stimulate bone formation. Collectively, this information suggested the drug might be useful for treatment of diseases associated with accelerated bone loss, included bone metastases, Paget's disease, and osteoporosis. Genta Inc. acquired global rights to the marketed drug in 2001, and filed an IND and NDA (both approved by FDA) in 2002 and 2003, respectively. However, the regulatory failure of Genasense in melanoma caused termination of the marketing program for Ganite. Nonetheless, research with gallium-containing compounds was pursued in women with breast cancer and metastatic bone disease.
To improve patient convenience, Genta developed and patented an oral formulation of gallium known as G4544, and the company filed an IND and conducted a preliminary clinical study of G544 that showed superior oral absorption.
Based on discovery work at the University of Washington, in 2010 Genta tested an infusion of Ganite in the first patient with "cystic fibrosis", demonstrating safety and potential therapeutic concentrations of gallium for reducing "Pseudomonas aeruginosa" bacterial infections. Genta also supported the clinical follow-on study at the University of Washington with regulatory cross-reference of both the Ganite IND and drug supply.
Despite promising results, all work on therapeutic gallium compounds by Genta ceased in 2012 due to financial conditions. The cystic fibrosis work has continued to be supported by the Cystic Fibrosis Foundation. Gallium nitrate is currently being administered to patients via inhalation.
Other Products.
The Genasense setbacks prompted termination of two other Genta projects, both in preclinical stages. Galaterone, a drug in-licensed from the University of Maryland, was a small molecule intended for treatment of prostate cancer. As a compound acting as both an androgen receptor antagonist and an inhibitor of CYP17, the drug appeared to have substantial promise. Upon Genta's termination of its license, Gelaterone was re-licensed to Tokai Pharmaeuticals, Inc. In order to expand its nucleotide franchise, Genta had purchased Salus Therapeutics and acquired a portfolio of small interfering ribonucleic acid technologies (siRNA), which also could not be developed due to financial constraints.
Company Operations.
With funding constraints and serial layoffs in the mid-to-late 1990s, Genta had been reduced to six employees. In 1997, the La Jolla facility was closed and the company was moved to Lexington, MA. A new CEO was recruited in late 1999 who opened a temporary office in New York. New Jersey was then a home for global pharmaceutical companies and experienced employees. Genta moved from Lexington, MA into temporary space in Short Hills, NJ, and then into permanent space in Berkeley Heights, NJ, where it remained for the next 12 years.
Financial History.
New management initiated a vigorous business development program to secure co-development funding for the costly Genasense programs. In April 2002, having initiated Phase 3 trials and solicited interest from a number of multinational companies, Genta signed a global development and licensing deal with Aventis Incorporated. At that time, the nominal $480 million deal was the second largest ever concluded for a single-product oncology compound (just behind Erbitux®; Imclone Systems to Bristol Myers Squibb). However, the Genta deal also included funding for U.S. clinical development expenses estimated at an additional $400 million, U.S. co-marketing rights with 50% coverage of Genta's marketing expenses, 100% coverage of all ex-U.S. clinical development expenses, escalating double-digit royalties on U.S. and global sales, and a $75 million investment via a convertible loan.
Despite disappointing survival results from the Phase 3 melanoma trial, both companies favored submission of a New Drug Application, which was not approved by FDA in 2004. Aventis and Genta terminated their agreement in May 2005 with provisions for continued funding of the Phase 3 CLL program. Over the 3-year duration, Aventis had contributed $270 million to Genta.
Following termination, fund-raising to support ongoing programs was considerably constrained, which prompted serial workforce reductions and cost-saving initiatives. Despite positive Phase 3 data, in 2006 the New Drug Application in CLL was not approved by FDA. Having previously secured FDA's Special Protocol Assessment requiring a post-approval confirmatory study, FDA's decision not to consider the initial study as supportive was appealed by Genta and it was reversed by FDA in 2009. FDA reaffirmed the requirement for a confirmatory trial, which Genta had not contested. However, the extended appeal and continued funding challenges precluded further study of Genta's Bcl-2 inhibitor in CLL.
By 2008, company funds were insufficient to proceed, and Genta issued a series of convertible notes to supportive investors, which provided credence to its subsequent development programs. Over the subsequent four years, however, Genta stock came under intensive pressure as it became a “penny stock” that required several “reverse stock splits”. The company stock was also temporarily delisted from the Nasdaq stock exchange.
By mid June 2012, the company believed it had secured additional financing to both remain on Nasdaq and to continue its Phase 3 studies with tesetaxel, which ultimately failed to close. Lacking financial alternatives, Genta filed for Chapter 7 bankruptcy in August 2012 and ceased operations.


Melanin-concentrating hormone receptor 2

Melanin-concentrating hormone receptor 2 (MCH2) also known as G-protein coupled receptor 145 (GPR145) is a protein that in humans is encoded by the "MCHR2" gene.
MCH2 is also found in dogs, ferrets, and some other primates and carnivores, but is not found in mice or rats. This has delayed research into the receptor as a therapeutic target, due to most early pharmaceutical research usually being conducted in small mammals such as mice, rats or rabbits which lack the MCH2 gene and its receptor product.
Clinical significance.
Treatment of human cells expressing MCHR2 with MCH resulted in upregulation of IDH3A, PCK1 and PFKFB4 and the downregulation of INSIG2 and ACOT8.


Muscarinic acetylcholine receptor M3

The muscarinic acetylcholine receptor, also known as cholinergic/acetylcholine receptor M3, or the muscarinic 3, is a muscarinic acetylcholine receptor encoded by the human gene CHRM3.
The M3 muscarinic receptors are located at many places in the body, e.g., smooth muscles, the endocrine glands, the exocrine glands, lungs, pancreas and the brain. In the CNS, they induce emesis. Muscarinic M3 receptors are expressed in regions of the brain that regulate insulin homeostasis, such as the hypothalamus and dorsal vagal complex of the brainstem. These receptors are highly expressed on pancreatic beta cells and are critical regulators of glucose homoestasis by modulating insulin secretion. In general, they cause smooth muscle contraction and increased glandular secretions.
They are unresponsive to PTX and CTX.
Mechanism.
Like the M1 muscarinic receptor, M3 receptors are coupled to G proteins of class Gq, which upregulate phospholipase C and, therefore, inositol trisphosphate and intracellular calcium as a signalling pathway. The calcium function in vertebrates also involves activation of protein kinase C and its effects.
Effects.
Smooth muscle.
Because the M3 receptor is Gq-coupled and mediates an increase in intracellular calcium, it typically causes constriction of smooth muscle, such as that observed during bronchoconstriction. However, with respect to vasculature, activation of M3 on vascular endothelial cells causes increased synthesis of nitric oxide, which diffuses to adjacent vascular smooth muscle cells and causes their relaxation and vasodilation, thereby explaining the paradoxical effect of parasympathomimetics on vascular tone and bronchiolar tone. Indeed, direct stimulation of vascular smooth muscle M3 mediates vasoconstriction in pathologies wherein the vascular endothelium is disrupted.
Diabetes.
The muscarinic M3 receptor regulates insulin secretion from the pancreas and are an important target for understanding the mechanisms of type 2 diabetes mellitus.
Some antipsychotic drugs that are prescribed to treat schizophrenia and bipolar disorder (such as olanzapine and clozapine) have a high risk of diabetes side-effects. These drugs potently bind to and block the muscarinic M3 receptor, which causes insulin dysregulation that may precede diabetes.
Other.
The M3 receptors are also located in many glands, both endocrine and exocrine glands, and help to stimulate secretion in salivary glands and other glands of the body.
Other effects are:
Agonists.
No highly selective M3 agonists are yet available as of 2018, but a number of non-selective muscarinic agonists are active at M3.
Interactions.
Muscarinic acetylcholine receptor M3 has been shown to pre-couple with Gq proteins. The polybasic c-tail of the receptor is necessary for the pre-coupling. It has also been shown to interact with Arf6 and ARF1.


Affinity capture

Affinity capture is a technique in molecular biology used to isolate desired compounds based on their chemical properties and a solid substrate. Commonly, plates out of solid materials such as glass are coated with various reagents to allow for covalent bonding of a capturing molecule such as an antibody. Afterwards, a solvent containing a desired compound for isolation is poured onto the plate, and the compound binds to the receptors on the plate (hence the capturing of the compound). Washing the plate and removing the desired compound completes the purification process.
Applications.
Affinity capture has been used to isolate proteins by means of binding a peptide sequence to the solid substrate, thus allowing for protein capture. The process has also been examined for potential automation, but the unique circumstances for any given experiment may impede reproducibility.


Terminal deoxynucleotidyl transferase

Terminal deoxynucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells. TdT adds N-nucleotides to the V, D, and J exons of the TCR and BCR genes during antibody gene recombination, enabling the phenomenon of junctional diversity. In humans, terminal transferase is encoded by the "DNTT" gene. As a member of the X family of DNA polymerase enzymes, it works in conjunction with polymerase λ and polymerase μ, both of which belong to the same X family of polymerase enzymes. The diversity introduced by TdT has played an important role in the evolution of the vertebrate immune system, significantly increasing the variety of antigen receptors that a cell is equipped with to fight pathogens. Studies using TdT knockout mice have found drastic reductions (10-fold) in T-cell receptor (TCR) diversity compared with that of normal, or wild-type, systems. The greater diversity of TCRs that an organism is equipped with leads to greater resistance to infection. Although TdT was one of the first DNA polymerases identified in mammals in 1960, it remains one of the least understood of all DNA polymerases. In 2016–18, TdT was discovered to demonstrate "in trans" template dependant behaviour in addition to its more broadly known template independent behaviour
TdT is absent in fetal liver HSCs, significantly impairing junctional diversity in B-cells during the fetal period.
Function and regulation.
Generally, TdT catalyses the addition of nucleotides to the 3' terminus of a DNA molecule. Unlike most DNA polymerases, it does not require a template. The preferred substrate of this enzyme is a 3'-overhang, but it can also add nucleotides to blunt or recessed 3' ends. Further, TdT is the only polymerase that is known to catalyze the synthesis of 2-15nt DNA polymers from free nucleotides in solution "in vivo". "In vitro", this behaviour catalyzes the general formation of DNA polymers without specific length. The 2-15nt DNA fragments produced "in vivo" are hypothesized to act in signaling pathways related to DNA repair and/or recombination machinery. Like many polymerases, TdT requires a divalent cation cofactor, however, TdT is unique in its ability to use a broader range of cations such as Mg2+, Mn2+, Zn2+ and Co2+. The rate of enzymatic activity depends on the available divalent cations and the nucleotide being added.
TdT is expressed mostly in the primary lymphoid organs, like the thymus and bone marrow. Regulation of its expression occurs via multiple pathways. These include protein-protein interactions, like those with TdIF1. TdIF1 is another protein that interacts with TdT to inhibit its function by masking the DNA binding region of the TdT polymerase. The regulation of TdT expression also exists at the transcriptional level, with regulation influenced by stage-specific factors, and occurs in a developmentally restrictive manner. Although expression is typically found to be in the primary lymphoid organs, recent work has suggested that stimulation via antigen can result in secondary TdT expression along with other enzymes needed for gene rearrangement outside of the thymus for T-cells. Patients with acute lymphoblastic leukemia greatly over-produce TdT. Cell lines derived from these patients served as one of the first sources of pure TdT and lead to the discovery that differences in activity exist between human and bovine isoforms.
Mechanism.
Similar to many polymerases, the catalytic site of TdT has two divalent cations in its palm domain that assist in nucleotide binding, help lower the pKa of the 3'-OH group and ultimately facilitate the departure of the resultant pyrophosphate by-product.
Isoform Variation.
Several isoforms of TdT have been observed in mice, bovines, and humans. To date, two variants have been identified in mice while three have been identified in humans.
The two splice variants identified in mice are named according to their respective lengths: TdTS consists of 509 amino acids while TdTL, the longer variant, consists of 529 amino acids. The differences between TdTS and TdTL occur outside regions that bind DNA and nucleotides. That the 20 amino acid difference affects enzymatic activity is controversial, with some arguing that TdTL's modifications bestow exonuclease activity while others argue that TdTL and TdTS have nearly identical "in vitro" activity. Additionally, TdTL reportedly can modulate the catalytic activity of TdTS "in vivo" through an unknown mechanism. It is suggested that this aids in the regulation of TdT's role in V(D)J recombination.
Human TdT isoforms have three variants TdTL1, TdTL2, and TdTS. TdTL1 is broadly expressed in lymphoid cell lines while TdTL2 is predominantly expressed in normal small lymphocytes. Both localize in the nucleus when expressed and both possess 3'->5' exonuclease activity. In contrast, TdTS isoforms do not possess exonuclease activity and perform the necessary elongation during V(D)J recombination. Since a similar exonuclease activity hypothesized in murine TdTL is found in human and bovine TdTL, some postulate that bovine and human TdTL isoforms regulate TdTS isoforms in a similar manner as proposed in mice. Further, some hypothesize that TdTL1 may be involved in the regulation of TdTL2 and/or TdTS activity.
Role in V(D)J Recombination.
Upon the action of the RAG 1/2 enzymes, the cleaved double-stranded DNA is left with hairpin structures at the end of each DNA segment created by the cleavage event. The hairpins are both opened by the Artemis complex, which has endonuclease activity when phosphorylated, providing the free 3' OH ends for TdT to act upon. Once the Artemis complex has done its job and added palindromic nucleotides (P-nucleotides) to the newly opened DNA hairpins, the stage is set for TdT to do its job. TdT is now able to come in and add N-nucleotides to the existing P-nucleotides in a 5' to 3' direction that polymerases are known to function. On average 2-5 random base pairs are added to each 3' end generated after the action of the Artemis complex. The number of bases added is enough for the two newly synthesized ssDNA segments to undergo microhomology alignment during non-homologous end joining according to the normal Watson-Crick base pairing patterns (A-T, C-G). From there unpaired nucleotides are excised by an exonuclease, like the Artemis Complex (which has exonuclease activity in addition to endonuclease activity), and then template-dependent polymerases can fill the gaps, finally creating the new coding joint with the action of ligase to combine the segments. Although TdT does not discriminate among the four base pairs when adding them to the N-nucleotide segments, it has shown a bias for guanine and cytosine base pairs.
Template Dependent Activity.
In a template-dependant manner, TdT can incorporate nucleotides across strand breaks in double-stranded DNA in a manner referred to as "in trans" in contrast to the "in cis" mechanism found in most polymerases. This occurs optimally with a one base-pair break between strands and less so with an increasing gap. This is facilitated by a subsection of TdT called Loop1 which selectively probes for short breaks in double-stranded DNA. Further, the discovery of this template dependant activity has led to more convincing mechanistic hypotheses as to how the distribution of lengths of the additions of the N regions arise in V(D)J recombination.
Polymerase μ and polymerase λ exhibit similar "in trans" templated dependant synthetic activity to TdT, but without similar dependence on downstream double-stranded DNA. Further, Polymerase λ has also been found to exhibit similar template-independent synthetic activity. Along with activity as a terminal transferase, it is known to also work in a more general template-dependent fashion. The similatrities between TdT and polymerase μ suggest they are closely evolutionarily related.
Uses.
Terminal transferase has applications in molecular biology. It can be used in RACE to add nucleotides that can then be used as a template for a primer in subsequent PCR. It can also be used to add nucleotides labeled with radioactive isotopes, for example in the TUNEL assay (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) for the demonstration of apoptosis (which is marked, in part, by fragmented DNA). It is also used in the immunofluorescence assay for the diagnosis of acute lymphoblastic leukemia.
In immunohistochemistry and flow cytometry, antibodies to TdT can be used to demonstrate the presence of immature T and B cells and pluripotent hematopoietic stem cells, which possess the antigen, while mature lymphoid cells are always TdT-negative. While TdT-positive cells are found in small numbers in healthy lymph nodes and tonsils, the malignant cells of acute lymphoblastic leukemia are also TdT-positive, and the antibody can, therefore, be used as part of a panel to diagnose this disease and to distinguish it from, for example, small cell tumors of childhood.
TdT has also seen recent application in the De Novo synthesis of oligonucleotides, with TdT-dNTP tethered analogs capable of primer extension by 1 nt at a time. In other words, the enzyme TdT has demonstrated the capability of making synthetic DNA by adding one letter at a time to a primer sequence.


MicC RNA

The MicC non-coding RNA (previously known as IS063 ) is located between the "ompN" and "ydbK" genes in "E. coli". This Hfq-associated RNA is thought to be a regulator of the expression level of the OmpC porin protein, with a 5′ region of 22 nucleotides potentially forming an antisense interaction with the "ompC" mRNA. Along with MicF RNA this family may act in conjunction with EnvZ-OmpR two-component system to control the OmpF/OmpC protein ratio in response to a variety of environmental stimuli. The expression of "micC" was shown to be increased in the presence of beta-lactam antibiotics.