Dehydrogenase

A dehydrogenase is an enzyme belonging to the group of oxidoreductases that oxidizes a substrate by reducing an electron acceptor, usually NAD+/NADP+ or a flavin coenzyme such as FAD or FMN. Like all catalysts, they catalyze reverse as well as forward reactions, and in some cases this has physiological significance: for example, alcohol dehydrogenase catalyzes the oxididation of ethanol to acetaldehyde in animals, but in yeast it catalyzes the production of ethanol from acetaldehyde.
IUBMB Classification.
Oxidoreductases, enzymes that catalyze oxidation-reduction reactions, constitute Class EC 1 of the IUBMB classification of enzyme-catalyzed reactions. Any of these may be called dehydrogenases, especially those in which NAD+ is the electron acceptor (oxidant), but reductase is also used when the physiological emphasis on reduction of the substrate, and oxidase is used "only" when O2 is the electron acceptor. The systematic name of an oxidoreductase is "donor:acceptor oxidoreductase", but when possible it is more conveniently named as "donor dehydrogenase".
Reactions catalyzed.
Dehydrogenases oxidize a substrate by transferring hydrogen to an electron acceptor, common electron acceptors being NAD+ or FAD. This would be considered an oxidation of the substrate, in which the substrate either loses hydrogen atoms or gains an oxygen atom (from water). The name "dehydrogenase" is based on the idea that it facilitates the removal (de-) of hydrogen (-hydrogen-), and is an enzyme (-ase). Dehydrogenase reactions come most commonly in two forms: the transfer of a hydride and release of a proton (often with water as a second reactant), and the transfer of two hydrogens.
Transferring a hydride and releasing a proton.
Sometimes a dehydrogenase catalyzed reaction will look like this: AH + B+ ↔ A+ + BH when a hydride is transferred. A represents the substrate that will be oxidized, while B is the hydride acceptor. Note how when the hydride is transferred from A to B, the A has taken on a positive charge; this is because the enzyme has taken two electrons from the substrate in order to reduce the acceptor to BH.
The result of a dehydrogenase catalyzed reaction is not always the acquisition of a positive charge. Sometimes the substrate loses a proton. This may leave free electrons on the substrate that move into a double bond. This happens frequently when an alcohol is the substrate; when the proton on the oxygen leaves, the free electrons on the oxygen will be used to create a double bond, as seen in the oxidation of ethanol to acetaldehyde carried out by alcohol dehydrogenase in the image on the right.
Another possibility is that a water molecule will enter the reaction, contributing a hydroxide ion to the substrate and a proton to the environment. The net result on the substrate is the addition of one oxygen atom. This is seen for example in the oxidation of acetaldehyde to acetic acid by acetaldehyde dehydrogenase, a step in the metabolism of ethanol and in the production of vinegar.
Transferring two hydrogens.
In the above case, the dehydrogenase has transferred a hydride while releasing a proton, H+, but dehydrogenases can also transfer two hydrogens, using FAD as an electron acceptor. This would be depicted as AH2 + B ↔ A + BH2.
A double bond is normally formed in between the two atoms that the hydrogens were taken from, as in the case of succinate dehydrogenase. The two hydrogens have been transferred to the carrier or the other product, with their electrons.
Identifying a dehydrogenase reaction.
The distinction between the subclasses of oxidoreductases that catalyze oxidation reactions lies in their electron acceptors.
Dehydrogenase and oxidase are easily distinguishable if one considers the electron acceptor. An oxidase will remove electrons from a substrate as well, but only uses oxygen as its electron acceptor. One such reaction is: AH2 + O2 ↔ A + H2O2.
Sometimes an oxidase reaction will look like this: 4A + 4H+ + O2 ↔ 4A+ + 2H2O. In this case, the enzyme is taking electrons from the substrate, and using free protons to reduce the oxygen, leaving the substrate with a positive charge. The product is water, instead of hydrogen peroxide as seen above. An example of an oxidase that functions like this is complex IV in the Electron Transport Chain (ETC).
Note that oxidases typically transfer the equivalent of dihydrogen (H2), and the acceptor is a dioxygen. Similarly, a peroxidase (another subclass of oxidoreductases) will use a peroxide (H2O2) as the electron acceptor, rather than an oxygen.
Electron acceptors.
Dehydrogenase enzymes transfer electrons from the substrate to an electron carrier; what carrier is used depends on the reaction taking place. Common electron acceptors used by this subclass are NAD+, FAD, and NADP+. Electron carriers are reduced in this process and considered oxidizers of the substrate. Electron carriers are coenzymes that are often referred to as "redox cofactors."
NAD+.
NAD+, or nicotinamide adenine dinucleotide, is a dinucleotide, containing two nucleotides. One of the nucleotides it contains is an adenine group, while the other is nicotinamide. In order to reduce this molecule, a hydrogen and two electrons must be added to the 6-carbon ring of nicotinamide; one electron is added to the carbon opposite the positively charged nitrogen, causing a rearrangement of bonds within the ring to give nitrogen more electrons; it will lose its positive charge as a result. The other electron is "stolen" from an additional hydrogen, leaving the hydrogen ion in solution. Reduction of NAD+: NAD+ + 2H+ + 2e− ↔ NADH + H+NAD+ is mostly used in catabolic pathways, such as glycolysis, that break down energy molecules to produce ATP. The ratio of NAD+ to NADH is kept very high in the cell, keeping it readily available to act as an oxidizing agent.
NADP+.
NADP+ differs from NAD+ only in the addition of a phosphate group to the adenosine 5-membered carbon ring. The addition of the phosphate does not alter the electron transport abilities of the carrier. The phosphate group creates enough contrast between the two groups that they bind to the active site of different enzymes, generally catalyzing different types of reactions.
These two electron carriers are easily distinguished by enzymes and participate in very different reactions. NADP+ mainly functions with enzymes that catalyze anabolic, or biosynthetic, pathways. Specifically, NADPH will act as a reducing agent in these reactions, resulting in NADP+. These are pathways that convert substrates to more complicated products, using ATP. The reasoning behind having two separate electron carriers for anabolic and catabolic pathways relates to regulation of metabolism.
The ratio of NADP+ to NADPH in the cell is kept rather low, so that NADPH is readily available as a reducing agent; it is more commonly used as a reducing agent than NADP+ is used as an oxidizing agent.
FAD.
FAD, or flavin adenine dinucleotide, is a prosthetic group (a non-polypeptide unit bound to a protein that is required for function) that consists of an adenine nucleotide and a flavin mononucleotide. FAD is a unique electron acceptor. Its fully reduced form is FADH2 (known as the hydroquinone form), but FAD can also be partially oxidized as FADH by either reducing FAD or oxidizing FADH2. Dehydrogenases typically fully reduce FAD to FADH2. The production of FADH is rare.
The double-bonded nitrogen atoms in FAD make it a good acceptor in taking two hydrogen atoms from a substrate. Because it takes two atoms rather than one, FAD is often involved when a double bond is formed in the newly oxidized substrate. FAD is unique because it is reduced by two electrons "and" two protons, as opposed to both NAD+ and NADP, which only take one proton.
Examples.
Biological implications.
Aldehydes are the natural by-product of many physiological processes, as well as being the consequence of many industrial processes, put out into the environment in the form of smog and motor vehicle exhaust. Build-up of aldehydes in the brain and pericardium can be detrimental to a person's health, as they can form adducts with important molecules and cause their inactivation.
Considering how prevalent aldehydes are, there must be an enzyme to facilitate their oxidation to a less volatile compound. Aldehyde dehydrogenases (ALDH) are NAD+ dependent enzymes that function to remove toxic aldehydes from the body, functioning mostly in the mitochondria of cells. These enzymes are largely responsible for the detoxification of acetylaldehyde, which is an intermediate in the metabolism of ethanol. It has been shown that a mutation in the ALDH2 gene (one of 19 aldehyde dehydrogenase genes) is what leads to the common occurrence in East Asian population of a flushed face after consuming alcohol, due to the build-up of acetaldehyde. This build-up of acetaldehyde also causes headaches and vomiting (hangover symptoms) if not broken down quickly enough, another reason why those with acetaldehyde DH deficiencies have bad reactions to alcohol. Importantly, a lack of this enzyme has been linked to an increase in the risk of myocardial infarction, while activation has shown the enzyme's ability to reduce damage caused by ischaemia.
Deactivation of aldehyde dehydrogenases has been shown to be instrumental in the mechanisms of many cancers. ALDHs function in cell differentiation, proliferation, oxidation, and drug resistance. These enzymes are only one example of the many different types of dehydrogenases in the human body; their wide array of functions, and the impact that their deactivation or mutations has upon crucial cell processes underscores the importance of all dehydrogenases in maintaining body homeostasis.
More examples.
TCA cycle examples:


Limnogale


Methoxypiperamide

Methoxypiperamide (also known as MeOP and MEXP) is a psychoactive drug of the piperazine class that has been sold online as a designer drug. It is the 4-methoxy-α-keto analog of methylbenzylpiperazine.
Very little data exists about the pharmacology and toxicity of methoxypiperamide; however, the US state of Vermont classifies it as a hallucinogen.
Legal status.
Methoxypiperamide is a controlled substance in Vermont as of January 2016, and also in Hungary.


Bunitrolol

Bunitrolol is a beta-adrenergic antagonist.


Thuc-Quyen Nguyen

Thuc-Quyen Nguyen is director and Professor at the Center for Polymers and Organic Solids (CPOS), and a professor of the Chemistry & Biochemistry department at the University of California Santa Barbara. Her research focuses on organic electronic devices, using optical, electrical, and structural techniques to understand materials and devices such as photovoltaics, LEDs, and field-effect transistors.
Early life and education.
Professor Nguyen was born in Ban Me thuot Vietnam in 1970. She was curious from an early age, always trying to understand how things work. She also says that she was inspired from an early age to become a teacher. There are four generations of teachers in her family, and as a young child, she went along to her mother's classes as there was no daycare to attend. These experiences sparked interest in being an effective teacher
In 1991, when she was 21, she moved with her family to the United States, arriving with very little knowledge of English. To try to improve her language skills and progress through school, she attended three schools at once, going to morning, afternoon, and evening classes. Her first term at Santa Monica College, she took four ESL courses at the same time, and after a year, was able to begin normal coursework.
After graduating from Santa Monica College in 1995 with an A.S., she began working toward a bachelor's degree at UCLA, while also work in the library in evenings to help pay for University. She also began working in a biology lab, beginning by washing glassware, before becoming more involved in experiments that further increased her interest in scientific research.
Research and career.
Professor Nguyen completed her masters in 1998, and PhD in 2001, both from UCLA.  In her PhD, she processed and studied conducting polymers using ultrafast spectroscopy under the supervision of Professor Benjamin Schwartz.
After her PhD, Professor Nguyen worked as a research associate at Columbia University, with Professor Louis Brus. She also worked for some time after her PhD at the IBM Thomas J Watson Research Center. In 2004, Nguyen joined UCSB Chemistry and Biochemistry department as an assistant professor, and received appointment to full professor in 2011.
Professor Nguyen's current research focuses on organic electronic devices. She studies how chemical structure influence performance and function of organic devices like PVs, OLEDs, OFETs. She is interested in improving organic solar cells as well as developing flexible electronics.


Panus

Panus is a genus of fungi in the family Polyporaceae.


Elaine Mardis

Elaine R. Mardis (born September 28, 1962) is the co-executive director of the Institute for Genomic Medicine at Nationwide Children's Hospital, where she also serves as the Nationwide Foundation Endowed Chair in Genomic Medicine. She also is professor of pediatrics at the Ohio State University College of Medicine. Mardis’s research focuses on the genomic characterization of cancer and its implications for cancer medicine. She was part of the team that reported the first next-generation-based sequencing of a whole cancer genome, and participated extensively in The Cancer Genome Atlas (TCGA) and the Pediatric Cancer Genome Project (PCGP).
Biography.
Mardis was born in North Platte, Nebraska. She gained a passion for science at an early age, and credits her father, a chemistry professor for more than 30 years, for nurturing this passion.
She received her undergraduate degree in Zoology from the University of Oklahoma in 1984. During her senior year she took a course in biochemistry that was taught by Bruce Roe, PhD, which says opened her eyes to the world of molecular biology. She stayed at the University of Oklahoma for her doctoral studies under the supervision of Dr. Roe, who was one of the first academic scientists to have a fluorescent DNA sequencer in the laboratory. As a result, during her doctoral work Mardis learned the art of DNA sequencing, at a time when few others were doing this.
After obtaining her PhD in chemistry and biochemistry in 1989, Mardis did postgraduate work in industry at Bio-Rad Laboratories in Hercules, California.
In 1993, Mardis joined the faculty of Washington University School of Medicine. Over the next 23 years, she held several and academic and leadership roles at the University, including serving as co-director of the McDonnell Genome Institute. In that position, she contributed substantially to the sequencing and analysis of the human genome, and was instrumental in establishing the utility of massively parallel sequencing technologies for understanding cancer biology. Her work in cancer genetics and genomics has provided insights into the genetic drivers of many types of cancer, including acute myeloid leukemia, breast cancer, glioblastoma, and lung adenocarcinoma. By better defining the landscape of germline and somatic alterations, this research helps drive new strategies for treating cancer, and is central to the concept of precision medicine.
Since joining Nationwide Children's Hospital in 2016, Dr. Mardis has turned the focus of her research to the incorporation of next-generation sequencing assays and established knowledge about cancer genomics into clinical and therapeutic decision-making, and into the design of new approaches to cancer immunotherapy.
In 2015, Mardis helped launch an open access, precision medicine journal, "Molecular Case Studies", in conjunction with Cold Spring Harbor Laboratory Press. She currently serves as Editor-in-Chief.
Mardis was elected as president of the American Association for Cancer Research for 2019–2020.


Rich Ingram, 5th Viscount of Irvine

Colonel Rich Ingram, 5th Viscount of Irvine (6 January 1688 – 10 April 1721), was an English peer and politician.
Early life.
Ingram was born on 6 January 1688. He was the second of nine sons born to Isabella Machell (1670–1764) and Arthur Ingram, 3rd Viscount of Irvine, the Vice-Admiral of Yorkshire and Member of Parliament for Yorkshire and Scarborough. His brothers were Edward (who died of smallpox after his Grand Tour), Arthur (MP for Horsham), Henry (also MP for Horsham), John, George (the Chaplain to the House of Commons), Charles (an Army Officer and also MP for Horsham), Thomas, and William Ingram. Of the nine brothers, three died in infancy, five acceded to the Viscountcy, and only one, Charles (father of the 9th and final Viscount of Irvine), had children.
His paternal grandparents were Henry Ingram, 1st Viscount of Irvine and the former Lady Essex Montagu (a daughter of Edward Montagu, 2nd Earl of Manchester). His maternal grandparents were the former Helena Warmestry and John Machell, MP for Horsham, of Hills, Sussex. His grandmother Helena was a daughter of Gervase Warmestry, and step-daughter of Sir John Covert, 1st Baronet,
He schooled at Eton College and was admitted Fellow-Commoner at Christ's College, Cambridge, aged 14 in 1703, where his elder brother the 4th Viscount had studied with the Master, John Covel. He was admitted a student at the University of Leyden in September 1704.
Career.
Under the terms of his grandfather Machell's will, Rich inherited the property of Hills, with 190 acres, at Horsham, together with a burgage at Horsham, several properties in London and in other parts of England, at his age of 21 years: the will having been proved in 1704 by the executors, Rich was sworn to probate, aged 22, as Rich Ingram alias Machell on 10 March 1711/12.
After his elder brother Edward died from smallpox, he succeeded in the viscountcy in 1714. This was a Scottish peerage and did not entitle him to a seat in the House of Lords. He was appointed Lord-Lieutenant of the East Riding of Yorkshire the same year, a post he held until 1721. He was also Governor of Kingston-upon-Hull between 1715 and 1721 and a colonel of the 16th Regiment of Foot between 1715 and 1717 and of the 1st Regiment of Dragoon Guards between 1717 and 1721. In 1720 he was appointed Governor of Barbados but died of smallpox before taking up the post.
Personal life.
In 1717, Lord Irvine married Lady Anne Howard (–1764), daughter of Charles Howard, 3rd Earl of Carlisle and Lady Anne Capel, daughter of the Earl of Essex and Lady Elizabeth Percy. Their portrait was painted by Jonathan Richardson (the elder, 1667-1745). There were no children from the marriage.
He died from smallpox in April 1721, aged 33. He is buried in Westminster Abbey. Lord Irvine was succeeded by his younger brother, Arthur, to whom the Horsham estate also passed by entail in default of male issue from Rich. The Viscountess of Irvine was a Lady of the Bedchamber to the Princess of Wales (mother of George III) in 1736 and married, as her second husband, Brig-Gen. William Douglas (MP for Kinross-shire) in 1737. She died in December 1764.
References.


Neamphamide A

Neamphamide A is an HIV-inhibitory isolate of the sea sponge "Neamphius huxleyi".


L-ascorbate oxidase

In enzymology, a L-ascorbate oxidase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are L-ascorbate and O2, whereas its two products are dehydroascorbate and H2O.
Function.
This enzyme belongs to the family of oxidoreductases, specifically those acting on diphenols and related substances as donor with oxygen as acceptor. This enzyme participates in ascorbate metabolism. It employs one cofactor, copper.
Nomenclature.
The systematic name of this enzyme class is L-ascorbate:oxygen oxidoreductase. Other names in common use include ascorbase, ascorbic acid oxidase, ascorbate oxidase, ascorbic oxidase, ascorbate dehydrogenase, L-ascorbic acid oxidase, AAO, L-ascorbate:O2 oxidoreductase, and AA oxidase.


Birbeck granules

Birbeck granules, also known as Birbeck bodies, are rod shaped or "tennis-racket" cytoplasmic organelles with a central linear density and a striated appearance. First described in 1961 (where they were simply termed "characteristic granules"), they are solely found in Langerhans cells. Although part of normal Langerhans cell histology, they also provide a mechanism to differentiate Langerhans cell histiocytoses (which are a group of rare conditions collectively known as histiocytoses) from proliferative disorders caused by other cell lines.
Formation is induced by langerin.
Function.
The function of Birbeck granules is debated, but one theory is that they migrate to the periphery of the Langerhans cells and release their contents into the extracellular matrix. Another theory is that the Birbeck granule functions in receptor-mediated endocytosis, similar to clathrin-coated pits.
History.
Birbeck granules were discovered by Michael Stanley Clive Birbeck (1925–2005), a British scientist and electron microscopist who worked at the Chester Beatty Cancer Research Institute or Institute of Cancer Research, London from 1950 until 1981.


Cell and Tissue Research

Cell and Tissue Research presents regular articles and reviews in the areas of molecular, cell, stem cell biology and tissue engineering. In particular, the journal provides a forum for publishing data that analyze the supracellular, integrative actions of gene products and their impact on the formation of tissue structure and function. Articles emphasize structure–function relationships as revealed by recombinant molecular technologies. The coordinating editor of the journal is Klaus Unsicker.
Subjects covered in journal.
Areas of research frequently published in "Cell and Tissue Research" include: neurobiology, neuroendocrinology, endocrinology, reproductive biology, skeletal and immune systems, and development.
Editors.
The coordinating editor of the journal is Klaus Unsicker, of the University of Heidelberg. Section editors are K. Unsicker, neurobiology/sense organs/endocrinology; M. Furutani-Seiki, Development/growth/regeneration; W.W. Franke, molecular/cell biology; Andreas Oksche and Horst-Werner Korf, neuroendocrinology; T. Pihlajaniemi, extracellular Extracellular matrix; D. Furst, muscle; Joseph Bonventre, kidney and related subjects; P. Sutovsky, reproductive biology; B. Singh, immunology/hematology; and V. Hartenstein, invertebrates.


(R)-3-Nitrobiphenyline

("R")-3-Nitrobiphenyline is a drug which acts as an α-adrenergic agonist, selective for the α subtype, as well as being a weak antagonist at the α and α subtypes.


Intrahealth Systems Limited

Intrahealth Systems Limited is a privately held company that develops, licences, supports and sells electronic health record software, medical practice management software, and related services. Founded in Auckland, New Zealand by Dr. Mark Matthews and Dr. Andrew Hall in 1997, they have since moved their headquarters to the City of North Vancouver, British Columbia, Canada in 2005. Apart from the head office, Intrahealth maintains offices in Auckland, New Zealand; Sydney; and Toronto, Canada.
History.
Intrahealth was founded in 1997 and spent its early years developing Profile and moving towards becoming one of New Zealand's largest vendors of Practice Management Systems (PMS).
The ACT (Australian Capital Territory) Division of General Practice entered into an alliance with Intrahealth to supply clinical and patient management software for general practitioners in Canberra and surrounding areas in 2003.
In 2005, Intrahealth expanded into British Columbia, Canada after winning a significant contract with Fraser Health Authority. The expansion saw additional contracts awarded from Vancouver Coastal, Interior, and Intertribal Health Authorities between that year and 2006. Also, in its home country, Intrahealth won a contract with the New Zealand Defense Force which would see its software used for the medical records of the defence force's 10,000 employees.
Profile for iOS was announced as a portable version of its practice management system in 2012, combining both a live online mode with syncing for offline use.
In 2013, Intrahealth was selected as the sole approved EHR provider for New Brunswick's provincial program. The selection followed a rigorous procurement process conducted by Velante, a subsidiary of the New Brunswick Medical Society, in which 16 other vendors submitted proposals. Additionally, Profile was granted dual National Class certification as both an Electronic Medical Record (EMR) and Ambulatory EMR by Canada Health Infoway, the first company to do so.


South Negros BioPower

South Negros BioPower is a biomass-fired power station in La Carlota, Negros Occidental in the Philippines. When commissioned in the 4th quarter 2017 it will be among the biggest biomass power stations in the Philippines and will have a generating capacity of 24.99 megawatts, enough electricity to provide 265,000 people in the region’s urban centres and rural areas on the island of Negros. The power plant is a cooperation between ThomasLloyd CTI Asia Holdings Pte and Bronzeoak Philippines. General Contractor is Wuxi Huaguang Electric Power Engineering.
The plant will be primary feed with cane trash with some grassy and woody energy crop plants. The feedstock utilisation will be 220,000 tonnes per year with a local feedstock availability of 1.4 million tonnes per year within a 50 km-radius catchment area. The plant will be connected to an existing 69 kV substation in San Enrique, 8 km away when operational. The power plant is expected to create 675 new jobs in the plant and 2,500 jobs in feedstock production and collection.
The plant has been chosen by the Philippine government as an "Awarded Biomass Project" in January 2015.


Antizyme RNA frameshifting stimulation element

Antizyme RNA frameshifting stimulation element is a structural element which is found in antizyme mRNA and is known to promote frameshifting. Antizyme genes have two partially overlapping open reading frames, the second, which encodes the functional (antizyme) protein requires +1 translational frameshifting. This frameshift is stimulated by a pseudoknot present 3' of the frameshift site in the antizyme mRNA. The frameshifting efficiency is dependent on the concentration of polyamines in the cell, when the polyamine concentration is high frameshifting is more likely to occur which leads to an increase in the quantity of functional antizyme produced. The functional antizyme acts to reduce ornithine decarboxylase (ODC) activity which leads to a drop in polyamines present in the cell. Therefore, this family can be thought of as a biosensor for intracellular free polyamines that functions via a negative feedback loop.


Thiol oxidoreductase

Thiol oxidoreductases are proteins that redox control by utilizing catalytic cysteine (Cys) residues for oxidation or reduction of their substrates. Examples of such proteins include thioredoxin, thioredoxin reductase, glutathione reductase, glutaredoxin, glutathione peroxidase, and peroxiredoxin.
They are involved in various processes, such as sulfur metabolism, DNA synthesis and repair, signaling, protein degradation, oxidative folding, protein modification, regulation of gene expression.
Some form functional complexes/modules, where one thiol oxidoreductase acts on another. For example, thioredoxin reductase provides reducing equivalents to thioredoxin, which in turn reduces peroxiredoxin.


Stan Rose

Stan Rose (born February 2, 1956) is an American biologist, biotechnology executive, and serial entrepreneur. Born in the Bronx, New York, and raised in Greenburgh, New York, Rose earned his B.A. from Cornell University in 1978 and his Ph.D. from the Massachusetts Institute of Technology in 1985.
In his professional career, Rose has spent over three decades working with leading scientists and clinicians to commercialize inventions related to genome analysis. He has raised more than $100 million in funding for early stage companies that have gone on to create more than $1 billion in shareholder value. Since 2008, he has served as President of Rose Ventures, an advisory and investment firm that works with early stage companies developing innovative, high-impact life science products and services.
Through April 2021 Dr. Rose served as Executive Chairman of Eurofins-TGI following the 2019 acquisition of Transplant Genomics Inc. (TGI) by Eurofins Scientific SE (ERF.PA). Rose was co-founder, Director and CEO of TGI, a molecular diagnostics company commercializing tests to help physicians better assess adequacy of immunosuppression for organ transplant recipients, and thereby improve outcomes.
Rose is a Director of Invenra, a biotechnology company focused on the discovery and development of multi-specific antibodies for immuno-oncology.
Rose is a Senior Fellow with the Council on Strategic Risks, participating in The Alliance to End Biological Threats.
Rose was a Director of Cellular Dynamics International, a leader in pluripotent stem cell technology for drug discovery and cell therapy acquired by FujiFilm in 2015, and Chair of the Market Advisory Board of Cyvek, a protein immunoassay and immunodiagnostics company acquired by Bio-Techne in 2014.
Highlights from earlier in his career include serving as CEO of NimbleGen Systems, Inc., a genomics tool company, from 2003 through its acquisition by Roche in 2007; co-founding and growing Genetic MicroSystems, a microarray instrumentation company that was acquired by Affymetrix in 2000; and directing the PCR business for Perkin-Elmer and Applied Biosystems during its most dynamic growth phase in the 1990s.


Anisodine

Anisodine, also known as daturamine and α-hydroxyscopolamine, is an antispasmodic and anticholinergic drug used in the treatment of acute circulatory shock in China. It is a tropane alkaloid and is found naturally in plants of the family Solanaceae like Scopolia tanguticus. Anisodine acts as a muscarinic acetylcholine receptor antagonist and α1-adrenergic receptor antagonist.
Synthesis.
(-)-Anisodine can be efficiently prepared using 6-beta-acetyltropine as the starting material via a key step of the Sharpless asymmetric dihydroxylation (AD).


Bacillus subtilis BSR sRNAs

In a screen of the "Bacillus subtilis" genome for genes encoding ncRNAs, Saito et al. focused on 123 intergenic regions (IGRs) over 500 base pairs in length, the authors analyzed expression from these regions. Seven IGRs termed bsrC, bsrD, bsrE, bsrF, bsrG, bsrH and bsrI expressed RNAs smaller than 380 nt. All the small RNAs except BsrD RNA were expressed in transformed "Escherichia coli" cells harboring a plasmid with PCR-amplified IGRs of "B. subtilis", indicating that their own promoters independently express small RNAs. Under non-stressed condition, depletion of the genes for the small RNAs did not affect growth. Although their functions are unknown, gene expression profiles at several time points showed that most of the genes except for bsrD were expressed during the vegetative phase (4–6 h), but undetectable during the stationary phase (8 h). Mapping the 5' ends of the 6 small RNAs revealed that the genes for BsrE, BsrF, BsrG, BsrH, and BsrI RNAs are preceded by a recognition site for RNA polymerase sigma factor σA.
Type I Toxin/Antitoxin system.
It was shown that "bsrE", "bsrG" and "bsrH" pair through intermolecular interactions with newly identified antisense sRNAs. It was suggested that they form type I toxin/antitoxin system that includes an mRNA encoding for a short, toxic peptide ("bsrE", "bsrG" and "bsrH" ) and an antitoxin that consists of an antisense RNA.
Further studies established that the 294-nucleotide "bsrG" encodes a 39-amino-acid toxin, and the 180 nucleotide antisense sRNA called SR4 acts as the antitoxin (they overlap by 123 nucleotides). SR4 interaction with the 3'UTR of "bsrG" RNA promotes "bsrG" degradation and inhibits its translation. BsrG interferes with cell envelope biosynthesis, causes membrane invaginations and delocalisation of the cell wall synthesis and initiates autolysis.
The 256 nucleotide "bsrE" RNA encodes 30 amino-acid toxin peptide. Its antitoxin gene, SR5 overlaps by 112 nucleotides at the 3' end of bsrE. The antitoxin SR5 promotes bsrE degradation but unlike SR4 it does not directly inhibits toxin mRNA translation.


Phenylalanine racemase (ATP-hydrolysing)

The enzyme phenylalanine racemase (, "phenylalanine racemase", "phenylalanine racemase (adenosine triphosphate-hydrolysing)", "gramicidin S synthetase I") is the enzyme that acts on amino acids and derivatives. It activates both the L & D stereo isomers of phenylalanine to form L-phenylalanyl adenylate and D-phenylalanyl adenylate, which are bound to the enzyme. These bound compounds are then transferred to the thiol group of the enzyme followed by conversion of its configuration, the D-isomer being the more favorable configuration of the two, with a 7 to 3 ratio between the two isomers. The racemisation reaction of phenylalanine is coupled with the highly favorable hydrolysis of adenosine triphosphate (ATP) to adenosine monophosphate (AMP) and pyrophosphate (PP), thermodynamically allowing it to proceed. This reaction is then drawn forward by further hydrolyzing PP to inorganic phosphate (Pi), via Le Chatelier's principle.
Links to disease.
Problems in the digestion of phenylalanine (phe) to tyrosine (tyr) lead to the buildup of both phe and phenylpyruvate, in a disease called Phenylketonuria (PKU). These two compounds build up in the blood stream and cerebral spinal fluid, which can lead to mental retardation if left untreated. Treatment consists of a restricted diet of foods that contain phe or compounds that can breakdown into phe. Children in the US are routinely tested for this at birth. For more information see the Phenylketonuria page or the link below.
The reaction.


MicF RNA

The micF RNA is a non-coding RNA stress response gene found in "Escherichia coli" and related bacteria that post-transcriptionally controls expression of the outer membrane porin gene ompF. The micF gene encodes a non-translated 93 nucleotide antisense RNA that binds its target ompF mRNA and regulates ompF expression by inhibiting translation and inducing degradation of the message. In addition, other factors, such as the RNA chaperone protein StpA also play a role in this regulatory system. Expression of micF is controlled by both environmental and internal stress factors. Four transcriptional regulators are known to bind the micF promoter region and activate micF expression.
Species distribution.
Homologues of MicF RNA have been characterised by Southern blotting in a variety of bacteria including "Salmonella typhimurium", "Klebsiella pneumoniae", and "Pseudomonas aeruginosa". MicF has also been identified computationally in "Yersinia pestis" and "Yersinia enterocolitica".


Alpha blocker

Alpha-blockers, also known as α-blockers or α-adrenoreceptor antagonists, are a class of pharmacological agents that act as antagonists on α-adrenergic receptors (α-adrenoceptors).
Historically, alpha-blockers were used as a tool for pharmacologic research to develop a greater understanding of the autonomic nervous system. Using alpha blockers, scientists began characterizing arterial blood pressure and central vasomotor control in the autonomic nervous system. Today, they can be used as clinical treatments for a limited number of diseases.
Alpha blockers can treat a small range of diseases such as hypertension, Raynaud's disease, benign prostatic hyperplasia (BPH) and erectile dysfunction. Generally speaking, these treatments function by binding an α-blocker to α receptors in the arteries and smooth muscle. Ultimately, depending on the type of alpha receptor, this relaxes the smooth muscle or blood vessels, which increases fluid flow in these entities.
Classification.
When the term "alpha blocker" is used without further qualification, it can refer to an α1 blocker, an α2 blocker, a nonselective blocker (both α1 and α2 activity), or an α blocker with some β activity. However, the most common type of alpha blocker is usually an α1 blocker.
Non-selective α-adrenergic receptor antagonists include:
Selective α1-adrenergic receptor antagonists include:
Selective α2-adrenergic receptor antagonists include:
Finally, the agents carvedilol and labetalol are both α and β-blockers.
Below are some of the most common drugs used in the clinic.
Medical uses.
While there are limited clinical α-blocker uses, in which most α-blockers are used for hypertension or benign prostatic hyperplasia , α-blockers can be used to treat a few other diseases, such as Raynaud's disease, congestive heart failure (CHF), pheochromocytoma, and erectile dysfunction.
Furthermore, α-blockers can occasionally be used to treat anxiety and panic disorders, such as posttraumatic stress disorder (PTSD) induced nightmares. Studies have also had great medical interest in testing alpha blockers, specifically α2 blockers, to treat Type II diabetes and psychiatric depression.
Hypertension.
Hypertension is due to an increase in vascular resistance and vasoconstriction. Using α1 selective antagonists, such as prazosin, has been efficacious in treating mild to moderate hypertension. This is because they can decrease vascular resistance and decrease pressure. However, while these drugs are generally well tolerated, they have the potential to produce side effects such as orthostatic hypotension and dizziness.
Another treatment for hypertension is using drugs that have both α1 blocking activity, as well as nonselective β activity, such as Labetalol or carvedilol. In low doses, labetalol and carvedilol can decrease the peripheral resistance and block the effects of isoprenaline to reduce hypertensive symptoms.
Pheochromocytoma.
Pheochromocytoma is a disease in which a catecholamine secreting tumor develops. Specifically, norepinephrine and epinephrine are secreted by these tumors, either continuously or intermittently. The excess release of these catecholamines increases central nervous system stimulation, thus causing blood vessels to increase in vascular resistance, and ultimately giving rise to hypertension. In addition, patients with these rare tumors are often subject to headaches, heart palpitations, and increased sweating.
Phenoxybenzamine, a nonselective α1 and α2 blocker, has been used to treat pheochromocytoma. This drug blocks the activity of epinephrine and norepinephrine by antagonizing the alpha receptors, thus decreasing vascular resistance, increasing vasodilation, and decreasing blood pressure overall.
Congestive heart failure.
Blockers that have both the ability to block both α and β receptors, such as carvedilol, bucindolol, and labetalol, have the ability to mitigate the symptoms in congestive heart failure. By binding to both the α and β receptors, these drugs can decrease the cardiac output and stimulate the dilation of blood vessels to promote a reduction in blood pressure.
Erectile dysfunction.
Yohimbine, an α2 blocker derived from the bark of the "Pausinystalia johimbe" tree, has been tested to increase libido and treat erectile dysfunction. The proposed mechanism for yohimbine is blockade of the adrenergic receptors that are associated with neurotransmitters inhibition, including dopamine and nitric oxide, and thus aiding with penile erection and libido. By doing so, they can alter the blood flow in the penis to aid in achieving an erection. However, some side effects can occur, such as palpitation, tremor, elevated blood pressure, and anxiety.
Yohimbe bark contains both alpha-1 and alpha-2 adrenergic receptors blocking alkaloids.
Phentolamine, a non-selective alpha blocker, has also been tested to treat erectile dysfunction. By reducing vasoconstriction in the penis, there appears to be increased blood flow that aids in penile erection. Side effects associated with phentolamine include headache, flushing, and nasal congestion.
Benign prostatic hyperplasia.
In benign prostatic hyperplasia (BPH), men experience urinary obstruction and are unable to urinate, thus leading to urinary retention. α1 specific blockers have been used to relax the smooth muscle in the bladder and enlarged prostate. Prazosin, doxazosin, and terazosin have been particularly useful for patients with BPH, especially in patients with hypertension. In such patients, these drugs can treat both conditions at the same time. In patients without hypertension, tamsulosin can be used, as it has the ability to relax the bladder and prostate smooth muscle without causing major changes in blood pressure.
Raynaud's disease.
Both α1 blockers and α2 blockers have been examined to treat Raynaud's disease. Although α1 blockers, such as prazosin, have appeared to give slight improvement for the sclerotic symptoms of Raynaud's disease, there are many side effects that occur while taking this drug. Conversely, α2 blockers, such as Yohimbine, appear to provide significant improvement of the sclerotic symptoms in Raynaud's Disease without excessive side effects.
Post traumatic stress disorder.
Patients with posttraumatic stress disorder (PTSD) have often continued to be symptomatic despite being treated with PTSD-specific drugs. In addition, PTSD patients often have debilitating nightmares that continue, despite their treatments. High doses of the α1 blocker, prazosin, have been efficacious in treating patients with PTSD induced nightmares due to its ability to block the effects of norepinephrine.
Adverse effects of prazosin to treat PTSD nightmares include dizziness, First Dose Effect (a sudden loss of consciousness), weakness, nausea, and fatigue.
Adverse effects.
Although alpha blockers have the ability to reduce some disease pathology, there are some side effects that come with these alpha blockers. However, because there are several structural compositions that make each alpha blocker different, the side effects are different for each drug. Side effects that arise when taking alpha blockers can include the first dose effect, cardiovascular side effects, genitourinary side effects, as well as other side effects.
First dose effect.
One of the most common side effects with alpha blockers is the first dose effect. This is a phenomenon in which patients with hypertension take an alpha blocker for the first time, and suddenly experience an intense decrease in blood pressure. Ultimately, this gives rise to orthostatic hypotension, dizziness, and a sudden loss of consciousness due to the drastic drop in blood pressure.
Alpha blockers that possess these side effects include prazosin, doxazosin, and terazosin.
Cardiovascular side effects.
There are some alpha blockers that can give rise to changes in the cardiovascular system, such as the induction of reflex tachycardia, orthostatic hypotension, or heart palpitations via alterations of the QT interval.
Alpha blockers that may have these side effects include yohimbine, phenoxybenzamine, and phentolamine.
Genitourinary side effects.
When alpha blockers are used to treat BPH, it causes vasodilation of blood vessels on the bladder and the prostate, thus increasing urination in general. However, these alpha blockers can produce the exact opposite side effect, in which edema, or abnormal fluid retention, occurs.
In addition, due to the relaxation of the prostate smooth muscle, another side effect that arises in men being treated for BPH is impotence, as well as the inability to ejaculate. However, if any ejaculation activity does occur, oftentimes, it results in a phenomenon called retrograde ejaculation, in which semen flows into the urinary bladder instead of exiting through the urethra.
Drugs that may produce such side effects include prazosin, terazosin, tamsulosin, and doxazosin.
Other side effects.
Finally, there are other general side effects that can be caused by most alpha blockers (however, more frequently in alpha-1 blockers). Such side effects include dizziness, drowsiness, weakness, fatigue, psychiatric depression, and dry mouth.
Priapism, an unwanted, painful long term erection not brought on by sexual arousal and lasting several hours has been associated with alpha blocker use. While this is extremely rare, particularly with tamsulosin, it can cause permanent impotence if not treated in a hospital setting. Male patients should be made aware of this as it can result from a single dose or develop over time.
Contraindications.
There is only one compelling indication for alpha blockers, which is for benign prostatic hyperplasia. Patients who need alpha blockers for BPH, but have a history of hypotension or postural heart failure, should use these drugs with caution, as it may result in an even greater decrease in blood pressure or make heart failure even worse. The most compelling contraindication is urinary incontinence and overall fluid retention. To combat such fluid retention, patients can take a diuretic in combination with the alpha-blocker.
In the absence of compelling indications or contraindications, patients should take alpha blockers as a step 4 therapy to reduce blood pressure, but only if the use of ACE inhibitors, angiotensin-II receptor blockers, calcium channel blockers, or thazide diuretics (in full dose or in combinations) have not been efficacious.
Drug interactions.
As with any drug, there are drug interactions that can occur with alpha blockers. For instance, alpha blockers that are used for the reduction of blood pressure, such as phenoxybenzamine or phentolamine can have synergy with other drugs that affect smooth muscle, blood vessels, or drugs used for erectile dysfunction (i.e. sildenafil, tamsulosin, etc.). This stimulates exaggerated hypotension.
Alternative alpha blockers, such as prazosin, tamsulosin, doxazosin, or terazosin can have adverse interactions with beta blockers, erectile dysfunction drugs, anxiolytics, and antihistamines. Again, these interactions can cause dangerous hypotension. Furthermore, in rare cases, drug interactions can cause irregular, rapid heartbeats or an increase blood pressure.
Yohimbine can interact with stimulants, hypertension drugs, naloxone, and clonidine. Interactions with such drugs can cause either an unintended increase in blood pressure or potentiate an increase in blood pressure.
Finally, in drugs with both alpha and beta blocking properties, such as carvedilol and labetalol, interactions with other alpha or beta blockers can exaggerate a decrease in blood pressure. Conversely, there are also drug interactions with carvedilol or labetalol in which blood pressure is increased unintentionally (such as with cough and cold medications). Finally, there may also be some alpha/beta blocker drug interactions that can worsen previous heart failure.
Mechanism of action.
Alpha blockers work by blocking the effect of nerves in the sympathetic nervous system. This is done by binding to the alpha receptors in smooth muscle or blood vessels. α-blockers can bind both reversibly and irreversibly.
There are several α receptors throughout the body where these drugs can bind. Specifically, α1 receptors can be found in most vascular smooth muscle, the pupillary dilator muscle, the heart, the prostate, and pilomotor smooth muscle. On the other hand, α2 receptors can be found in platelets, cholinergic nerve terminals, some vascular smooth muscle, postsynaptic CNS neurons, and fat cells.
The structure of α receptors is a classic G protein–coupled receptors (GPCRs) consisting of 7 transmembrane domains, which form three intracellular loops and three extracellular loops. These receptors couple to heterotrimeric G proteins composed of α, β, and γ subunits. Although both of the α receptors are GPCRs, there are large differences in their mechanism of action. Specifically, α1 receptors are characterized as Gq GPCRs, signaling through Phospholipase C to increase IP3 and DAG, thus increasing the release of calcium. Meanwhile, α2 receptors are labeled as Gi GPCRs, which signal through adenylyl cyclase to decrease cAMP.
Because the α1 and α2 receptors have different mechanisms of action, their antagonists also have different effects. α1 blockers can inhibit the release of IP3 and DAG to decrease calcium release, thus, decreasing overall signaling. On the other hand, α2 blockers prevent the reduction of cAMP, thus leading to an increase in overall signaling.


Antitermination

Antitermination is the prokaryotic cell's aid to fix premature termination of RNA synthesis during the transcription of RNA. It occurs when the RNA polymerase ignores the termination signal and continues elongating its transcript until a second signal is reached. Antitermination provides a mechanism whereby one or more genes at the end of an operon can be switched either on or off, depending on the polymerase either recognizing or not recognizing the termination signal.
Antitermination is used by some phages to regulate progression from one stage of gene expression to the next. The lambda gene N, codes for an antitermination protein (pN) that is necessary to allow RNA polymerase to read through the terminators located at the ends of the immediate early genes. Another antitermination protein, pQ, is required later in phage infection. pN and pQ act on RNA polymerase as it passes specific sites. These sites are located at different relative positions in their respective transcription units.
Antitermination may be a regulated event.
Antitermination was discovered in bacteriophage infections. A common feature in the control of phage infection is that very few of the phage genes can be transcribed by the bacterial host RNA polymerase. Among these genes, however, are regulators whose products allow the next set of phage genes to be expressed. One of these types of regulator is an antitermination protein. In the absence of the antitermination protein, RNA polymerase terminates at the terminator. When the antitermination protein is present, it continues past the terminator.
The best characterized example of antitermination is provided by lambda phage, in which the phenomenon was discovered. It is used at two stages of phage expression. The antitermination protein produced at each stage is specific for the particular transcription units that are expressed at that stage.
The host RNA polymerase initially transcribes two genes, which are called the immediate early genes (N and cro). The transition to the next stage of expression is controlled by preventing termination at the ends of the immediate early genes, with the result that the delayed early genes are expressed. The antitermination protein pN acts specifically on the immediate early transcription units. Later during infection, another antitermination protein pQ acts specifically on the late transcription unit, to allow its transcription to continue past a termination sequence.
The different specificities on pN and pQ establish an important general principle: RNA polymerase interacts with transcription units in such a way that an ancillary factor can sponsor antitermination specifically for some transcripts. Termination can be controlled with the same sort of precision as initiation.
The antitermination activity of pN is highly specific, but the antitermination event is not determined by the terminators tL1 and tR1; the recognition site needed for antitermination lies upstream in the transcription unit, that is, at a different place from the terminator site at which the action eventually is accomplished.
The recognition sites required for pN action are called "nut" (for N utilization). The sites responsible for determining leftward and rightward antitermination are described as "nut"L and "nut"G, respectively.
When pN recognizes the "nut" site, it forms a persistent antitermination complex in cooperation with a number of E. coli host proteins. These include three host Nus proteins, NusA, B, and C. NusA is an interesting protein. By itself in E. coli, it is part of the transcription termination system. However, when co-opted by N, it participates in antitermination. The complex must act on RNA polymerase to ensure that the enzyme can no longer respond to the terminator. The variable locations of the "nut" sites indicate that this event is linked neither to initiation nor to termination, but can occur to RNA polymerase as it elongates the RNA chain past the nut site. Phages that are related to lambda have different N genes and different antitermination specificities. The region on the phage genome in which the "nut" sites lie has a different sequence in each of these phages, and each phage must therefore have characteristic "nut" sites recognized specifically by its own pN. Each of these pN products must have the same general ability to interact with the transcription apparatus in an antitermination capacity, but each product also has a different specificity for the sequence of DNA that activates the mechanism.
Processive antitermination.
Antitermination in lambda is induced by two quite distinct mechanisms. The first is the result of interaction between lambda N protein and its targets in the early phage transcripts, and the second is the result of an interaction between the lambda Q protein and its target in the late phage promoter. We describe the N mechanism first. Lambda N, a small basic protein of the arginine-rich motif (ARM) family of RNA binding proteins, binds to a 15-nucleotide (nt) stem-loop called BOXB. (We will capitalize the names of sites in RNA and italicize the names of the corresponding DNA sequences; e.g., BOXB and "boxB".) "boxB" is found twice in the lambda chromosome, once in each of the two early operons. It is close to the start point of the PL operon transcript and just downstream of the first translated gene of the PR operon. Neither the distance between the transcription start site and "boxB", nor the nature of the promoter (at least in the case of sigma-70-dependent promoters), nor the nature of the terminator is relevant to N action. Although the "boxB" sequence is not well conserved in other bacteriophages of the lambda family, most of these phages encode proteins that are analogous to lambda N and have sequences capable of forming BOXB-like structures in their PL and PR operons. In some cases, it has been shown that these structures are recognized by the cognate N analogs. It is believed that this accounts for the phage specificity of N-mediated antitermination.
Processive antitermination requires the complete antitermination complex. The assembly of NusB, S10, and NusG onto the core complex involves nt 2 to 7 of lambda BOXA (CGCUCUUACACA), as well as the carboxyl-terminal region of N, which interacts with RNAP. The role of NusG in the N antitermination reaction is not clear. NusG binds to termination factor Rho and to RNAP. It stimulates the rate of transcription elongation and is required for the activity of certain Rho-dependent terminators. NusG is a component of the complete antitermination complex and enhances N antitermination in vitro. However, alteration of lambda BOXA to a variant called BOXA consensus (CGCUCUUUAACA) allows NusB and S10 to assemble in the absence of NusG. Furthermore, depletion of NusG has no effect on lambda N antitermination in vivo, and unlike nusA, nusB, and nusE, no point mutations in nusG that block N activity have been isolated. A NusG homolog, RfaH, enhances elongation of several transcripts in E. coli and S. typhimurium. The possibility that RfaH and NusG are redundant for N antitermination has not yet been tested, although for several other functions, the two proteins are not interchangeable.
Processive antitermination can be mediated by RNA as well as proteins. Coliphage HK022, alone among the known lambdoid phages, does not encode an analog to lambda N. Instead, it promotes antitermination of early phage transcription through the direct action of transcribed sequences called "put" (for polymerase utilization) sites. There are two closely related "put" sites, one located in the PL operon and the other located in the PR operon, roughly corresponding to the positions of the "nut" sequences in lambda and in other lambda relatives. "put" sites act in cis to promote readthrough of downstream terminators in the absence of all HK022 proteins. The "put" transcripts are predicted to form two stem-loops separated by a single unpaired nucleotide. This prediction is supported by mutational studies and the pattern of sensitivity of the two RNAs to cleavage with single- and double-strand-specific endoribonucleases. RNA structure is critical to antitermination because mutations that prevent the formation of base pairs in the stems reduce function, and these mutations can be suppressed by additional mutations that restore base pairing. Like lambda N and Q, the "PUT" sequences suppress polymerase pausing and promote processive antitermination in a purified in vitro transcription system. In contrast to lambda N, no phage or auxiliary bacterial factors are required. The only mutations known to block "PUT"-mediated antitermination change highly conserved amino acids located in a cysteine-rich amino-proximal domain of the RNAP beta' subunit. Strains carrying these mutations are unable to support lytic growth of HK022 but are normal in all other respects tested, including lytic growth of lambda and other lambda relatives. The phage-restricted phenotypes conferred by these mutations suggest that they alter a domain of RNAP-beta’ that interacts specifically with nascent "PUT" RNA in the transcription elongation complex, but this idea has not been directly tested. The stability of the putative "PUT"-RNAP interaction and the nature of the "PUT"-induced modification to the elongation complex are unknown.
Processive antitermination was first discovered in a bacteriophage, but examples have since been found in bacterial operons. The E. coli rrn operons are regulated by an antitermination mechanism that is dependent on sites that are closely related to lambda "boxA" and located promoter proximal to the 16S and 23S structural genes in each operon. The sequences of the rrn BOXA sites are more similar to the bacteriophage consensus than is that of lambda, and they bind NusB-S10 more efficiently. Although stem-loop structures analogous to BOXB are found promoter proximal to the BOXA sites, they are not essential for antitermination. An rrn BOXA sequence confers full antitermination activity against Rho-dependent but not against intrinsic terminators. BOXA also increases the rate of transcription elongation by RNAP. Point mutations in BOXA induce premature transcription termination. rrn antitermination requires NusB in vivo, as shown by a NusB depletion experiment. NusA stimulates the elongation rate of rrn RNA chains carrying BOXA. A role for NusA is further suggested by the observation that the nusA10 (Cs) mutation inhibits both antitermination and the rate of transcription elongation in an rrn operon. The role of other Nus factors in rrn regulation in vivo is not clear. In vitro, an antitermination complex that includes NusA, NusB, S10, and NusG forms at the BOXA sequence of rrnG, but these components are not sufficient for antitermination by themselves. An additional factor or factors that can be supplied by a cellular extract are required, but their identities are unknown.


Insulator (genetics)

An insulator is a type of cis-regulatory element known as a long-range regulatory element. Found in multicellular eukaryotes and working over distances from the promoter element of the target gene, an insulator is typically 300 bp to 2000 bp in length. Insulators contain clustered binding sites for sequence specific DNA-binding proteins and mediate intra- and inter-chromosomal interactions.
Insulators function either as an enhancer-blocker or a barrier, or both. The mechanisms by which an insulator performs these two functions include loop formation and nucleosome modifications. There are many examples of insulators, including the CTCF insulator, the "gypsy" insulator, and the β-globin locus. The CTCF insulator is especially important in vertebrates, while the "gypsy" insulator is implicated in "Drosophila." The β-globin locus was first studied in chicken and then in humans for its insulator activity, both of which utilize CTCF.
The genetic implications of insulators lie in their involvement in a mechanism of imprinting and their ability to regulate transcription. Mutations to insulators are linked to cancer as a result of cell cycle disregulation, tumourigenesis, and silencing of growth suppressors.
Function.
Insulators have two main functions:
While enhancer-blocking is classified as an inter-chromosomal interaction, acting as a barrier is classified as an intra-chromosomal interaction. The need for insulators arises where two adjacent genes on a chromosome have very different transcription patterns; it is critical that the inducing or repressing mechanisms of one do not interfere with the neighbouring gene. Insulators have also been found to cluster at the boundaries of topologically associating domains (TADs) and may have a role in partitioning the genome into "chromosome neighborhoods" - genomic regions within which regulation occurs.
Some insulators can act as both enhancer blocker and barriers, and some just have one of the two functions. Some examples of different insulators are:
Mechanism of action.
Enhancer-blocking insulators.
Similar mechanism of action for enhancer-blocking insulators; chromatin loop domains are formed in the nucleus that separates the enhancer and the promoter of a target gene. Loop domains are formed through the interaction between enhancer-blocking elements interacting with each other or securing chromatin fibre to structural elements within the nucleus. The action of these insulators is dependent on being positioned between the promoter of the target gene and the upstream or down stream enhancer. The specific way in which insulators block enhancers is dependent on the enhancers mode of action. Enhancers can directly interact with their target promoters through looping (direct-contact model), in which case an insulator prevents this interaction through the formation of a loop domain that separates the enhancer and promoter sites and prevents the promoter-enhancer loop from forming. An enhancer can also act on a promoter through a signal (tracking model of enhancer action). This signal may be blocked by an insulator through the targeting of a nucleoprotein complex at the base of the loop formation.
Barrier insulators.
Barrier activity has been linked to the disruption of specific processes in the heterochromatin formation pathway. These types of insulators modify the nucleosomal substrate in the reaction cycle that is central to heterochromatin formation. Modifications are achieved through various mechanisms including nucleosome removal, in which nucleosome-excluding elements disrupt heterochromatin from spreading and silencing (chromatin-mediated silencing). Modification can also be done through recruitment of histone acetyltransferase(s) and ATP-dependent nucleosome remodelling complexes.
CTCF insulator.
The CTCF insulator appears to have enhancer blocking activity via its 3D structure and have no direct connection with barrier activity. Vertebrates in particular appear to rely heavily on the CTCF insulator, however there are many different insulator sequences identified. Insulated neighborhoods formed by physical interaction between two CTCF-bound DNA loci contain the interactions between enhancers and their target genes.
Regulation.
One mechanism of regulating CTCF is via methylation of its DNA sequence. CTCF protein is known to favourably bind to unmethylated sites, so it follows that methylation of CpG islands is a point of epigenetic regulation. An example of this is seen in the "Igf2-H19" imprinted locus where methylation of the paternal imprinted control region (ICR) prevents CTCF from binding. A second mechanism of regulation is through regulating proteins that are required for fully functioning CTCF insulators. These proteins include, but are not limited to cohesin, RNA polymerase, and CP190.
"gypsy" insulator.
The insulator element that is found in the "gypsy" retrotransposon of "Drosophila" is one of several sequences that have been studied in detail. The "gypsy" insulator can be found in the 5' untranslated region (UTR) of the retrotransposon element. "Gypsy" affects the expression of adjacent genes pending insertion into a new genomic location, causing mutant phenotypes that are both tissue specific and present at certain developmental stages. The insulator likely has an inhibitory effect on enhancers that control the spatial and temporal expression of the affected gene.
β-globin locus.
The first examples of insulators in vertebrates was seen in the chicken β-globin locus, "cHS4". "cHS4" marks the border between the active euchromatin in the β-globin locus and the upstream heterochromatin region that is highly condensed and inactive. The "cHS4" insulator acts as both a barrier to chromatin-mediated silencing via heterochromatin spreading, and blocks interactions between enhancers and promoters. A distinguishing characteristic of "cHS4" is that it has a repetitive heterochromatic region on its 5' end.
The human β-globin locus homologue of "cHS4" is "HS5". Different from the chicken β-globin locus, the human β-globin locus has an open chromatin structure and is not flanked by a 5' heterochromatic region. HS5 is thought to be a genetic insulator "in vivo" as it has both enhancer-blocking activity and transgene barrier activities.
CTCF was first characterized for its role in regulating β-globin gene expression. At this locus, CTCF functions as an insulator-binding protein forming a chromosomal boundary. CTCF is present in both the chicken β-globin locus and human β-globin locus. Within cHS4 of the chicken β-globin locus, CTCF binds to a region (FII) that is responsible for enhancer blocking activity.
Genetic implications.
Imprinting.
The ability of enhancers to activate imprinted genes is dependent on the presence of an insulator on the unmethylated allele between the two genes. An example of this is the "Igf2-H19" imprinted locus. In this locus the CTCF protein regulates imprinted expression by binding to the unmethylated maternal imprinted control region (ICR) but not on the paternal ICR. When bound to the unmethylated maternal sequence, CTCF effectively blocks downstream enhancer elements from interacting with the "Igf2" gene promoter, leaving only the "H19" gene to be expressed.
Transcription.
When insulator sequences are located in close proximity to the promoter of a gene, it has been suggested that they might serve to stabilize enhancer-promoter interactions. When they are located farther away from the promoter, insulator elements would compete with the enhancer and interfere with activation of transcription. Loop formation is common in eukaryotes to bring distal elements (enhancers, promoters, locus control regions) into closer proximity for interaction during transcription. The mechanism of enhancer-blocking insulators then, if in the correct position, could play a role in regulating transcription activation.
Mutations and cancer.
CTCF insulators affect the expression of genes implicated in cell cycle regulation processes that are important for cell growth, cell differentiation, and programmed cell death (apoptosis). Two of these cell cycle regulation genes that are known to interact with CTCF are "hTERT" and "C-MYC." In these cases, a loss of function mutation to the CTCF insulator gene changes the expression patterns and may affect the interplay between cell growth, differentiation and apoptosis and lead to tumourigenesis or other problems.
CTCF is also required for the expression of tumour repressor retinoblastoma (Rb) gene and mutations and deletions of this gene are associated with inherited malignancies. When the CTCF binding site is removed expression of Rb is decreased and tumours are able to thrive.
Other genes that encode cell cycle regulators include BRCA1, and p53, which are growth suppressors that are silenced in many cancer types, and whose expression is controlled by CTCF. Loss of function of CTCF in these genes leads to the silencing of the growth suppressor and contributes to the formation of cancer.


Kula Diamond

 is a fictional character from SNK's fighting game franchise "The King of Fighters". She first appears as the sub-boss and playable character in "The King of Fighters 2000"; she enters as a normal participant in her other appearances. Originally a member of the organization NESTS, Kula is the result of an experiment to kill the rebellious K', earning the title of . However, during NESTS' destruction, Kula forms a bond with K' and his allies to the point of joining his team. In following titles she lives with K' under the care of the cyborg Maxima. She is also present in a number of spin-off games in the franchise, most notably " as well as Akihiko Ureshino's novelizations of the games and comic books from China.
Kula was created to add more characters to the NESTS syndicate and to add more depth to the series' second story arc. Designed carefully to appeal to female players, SNK was satisfied with her physical appearance. Yumi Kakazu has voiced Kula in all of her appearances, while Kat Cassteneda provides her English voice in the spin-off game ".
At first the character got a mixed reception as comparisons were made between her and earlier SNK fighters. Subsequently, journalists and video game reviewers have been largely positive about Kula liking her fighting style, mostly for her use of ice, as well as her contrast with K'. However, the character's attractiveness also earned a mixed reaction. Critics were divided over her beauty and her skimpy designs in "SNK Heroines: Tag Team Frenzy".
Creation and design.
SNK created Kula Diamond to bring more complexity to the NESTS' story arc in the series. Her character added another dimension to the villainous NESTS organization in the "NESTS Chronicles". She was to add excitement to the story as an enemy and a foil of K'. Newly hired female staff designed her to depict a "14-year-old girl" with "appropriately girlish" gestures. The unnamed character designer was pleased with Kula's design saying: "Kula had an easy delivery. She didn't give me any pain." Despite being K"s foil, Kula's was designed to closely resemble her rival as both fighters wear similar leather outfits with contrasting colors. According to artist Hiroaki, Kula does not look like a 14 year old girl in the illustrations. Nevertheless, he claims he grew attached to drawing her alongside K'.
Yumi Kakazu has voiced Kula in all of her appearances. In "", Falcoon gave her a color palette that makes her look like fighter Ash Crimson as well as the robot Candy. Her alternative costume is that of a coquettish ice skater. Falcoon also once envisioned an adult version of Kula for the game "Kimi wa Hero".
For "The King of Fighters XIII", the staff decided to make Kula similar to her original incarnation in "The King of Fighters 2000". The team had no problems in designing her gameplay and added taunts where the character becomes boring. Similar to K's moves, the developers wanted Kula's trademarks, most notably her ice moves, to be appealing to see. A move that exemplifies this was the "Crow Bites". Kula's "Diamond Breath" move was recommended for new players, while the "Reispin" was designed to make the gameplay more complex and challenging as Kula becomes stronger when executing it. Kula's strongest technique in the game involves the appearance of Foxy and Diana who attack the enemy alongside her. This technique went through numerous trials before it was added to the game.
When developing "The King of Fighters XIV" with the Unreal Engine, the original game had more realistic visuals but they clashed with character designs of Kula and Athena Asamiya. The designs looked like those in an anime series so the designers opted for the current look. A programmer with the alias of "Ando" designed the blades on Kula's boots during special attacks. Kula was the first character in "KOF XIV" to be given this type of design, requiring multiple attempts to work with her 3D character model. Ando further believed Kula could wield other type of weapons based on the variety used by the cast but in the end felt using blades could prove to be more challenging. An extra outfit for Kula was released for "The King of Fighters XIV" as downloadable content.
In "", Kula appears wearing Angel's skimpy clothing. Director Kaito Soranaka said during an interview that Kula was his favorite character in the game. Ogura said that having Kula wear Angel's outfit brought up the fact that Kula hates Angel, so the game's villain, Kukri, gave her that appearance to humiliate her.
Appearances.
Kula Diamond is a teenage girl who was experimented on by NESTS in order to exterminate the cartel's traitor, K'. Despite the fact their subject was a mindless puppet with no sign of emotions, the NESTS cartel created an android called Candy Diamond to monitor Kula's behavior and to ensure that she would accomplish her missions. During "KOF 2000", Kula can be fought as a mid-boss during her debut. Shortly afterwards, Kula destroys the main Zero Cannon from its titular creator who is betraying NESTS. Candy shields Kula's descent from space by sacrificing her body in the process, as it is badly burned by Earth's atmosphere. While recovering from the trauma of Candy's destruction, in the following game, "The King of Fighters 2001", Kula enters into the fighting tournament with fellow NESTS' agents Foxy, K9999 and Angel to capture K'. During the story, Kula and Foxy are betrayed by K9999 and Angel. However, in K's Team ending among others, she is seen as being safe and forms a friendship with her original target and allies. In the story-less game, "KOF 2002", Kula appears as member of a team with Angel and K9999.
In "KOF 2003", she appears in the ending of K' Team where the group go to recruit her following an apparent threat. She participates in "KOF XI" and "KOF XIII" with K' and Maxima as her allies, having also joined forces with the Ikari Team. They are assigned to investigate a group known as "Those From The Past". In "KOF XIV", she once again participates in the tournament following the Ikari's orders to investigate NESTS' agents. During these games, the K' Team become allied with the Ikari Team to help them on missions though they tend to avoid them.
Kula is also present in the spin-off games, "Neowave" which does not feature a story and ' where looks for a doctor to repair Maxima's cybernetic body. She also appears in "The King of Fighters '98: Unlimited Match Online" and "The King of Fighters Online". She is also present "KOF: Sky Stage", ' and "". In "SNK Heroines" the female fighters must fight their way home but are confronted by Kukri and defeat him before he can initiate his grand plan on the kidnapped female fighters. She also makes a cameo appearance alongside Maxima in the ending of K' from "NeoGeo Battle Coliseum". She is playable in the mobile phone game "Kimi wa Hero" (as an adult) and "Brave Frontier", while also making a cameo in the dating sim "Days of Memories" should the player interact with K'. She has since made guest appearances in more games, including the role-playing game "Valkyrie Connect". She also appears as a downloadable character in Koei Tecmo's fighting game "Dead or Alive 6". In her story chapter, NiCO pulls her into the "Dead or Alive" dimension while experimenting with subspace portals. Kula and NiCO briefly spar, and NiCO later thanks her for providing "interesting data" while swearing her to secrecy.
The printed adaptations of "The King of Fighters" retell Kula's role in the NESTS' story arc where she constantly clashes with K', having a more antagonistic characterization until the events of "2001" and "2002". She also appears in the CD dramas from "KOF 2000", in which she attacks K' before the events of her the story. However, this ends when K' is knocked out by Maxima for fighting with his wounded arm and the fugitive escapes with the unconscious K' from Kula. A cyborg named Rugal Bernstein kidnaps Kula during the "2002" storyline to absorb her powers. Using this, Rugal manages to defeat the fighters Kyo Kusanagi and Iori Yagami, but in the end he is stopped by K' and Maxima who rescue their former enemy. In the comic of "The King of Fighters XII", Kula engages the mysterious Magaki alongside K' and former NESTS' agent Nameless.
Reception.
Kula's character has been mostly well received. Den of Geek listed her as the 35th best character of the franchise, citing her appealing upbeat characterization while liking the idea of her not only being a foil for K', but how she "simply centers him by being his loving opposite". In an article on the history of SNK, GameSpot described Kula as the yin to the yang due of K' to how different both characters are in terms of the elements they control while fighting. Falcoon noted the character had become popular with fans of the series as after a poll on his Twitter account asking what character he should draw. GameSpot, however, criticized Kula and Hinako as the least interesting additions to the cast of "The King of Fighters 2000."
IGN found Kula to be one of the most appealing characters in "The King of Fighters 2000" though the reviewer felt newcomer Lin might be superior. In another article, the same site liked the fact Kula was as a regular playable character unlike the original version, but felt she was still overshadowed by Lin. In a later review, IGN lamented the lack of Geese Howard but believed both Kula and K' could fill the gap. IGN felt Kula's inclusion in "The King of Fighters 2002" alongside K', rather than other famous characters in the series, works with those that are included. Eurogamer and other publications enjoyed return of the originally "inexplicably" absent from "The King of Fighters XII" in following titles of "familiar faces" alongside Maxima and K'. 4thLetter enjoyed the contrast between K's characterization from that of his teammates, Maxima and Kula, noting that the team's ending from "XIII" had him enjoying his vacation by beating up unknown enemies. PlayStation Universe opined fans should look forward to Kula and K' in "The King of Fighters XIV" as their game styles would appeal to gamers. In a review for "KOF: Maximum Impact 2", GameSpot felt Kula and Billy Kane were two of the best new characters. In a museum of videogames sponsored by the municipality of Rome, a special illustration lists Kula as the mascot of the new millennium era of Neo Geo.
GameZone enjoyed her fighting mechanics in "The King of Fighters XI", comparing her style to "athletes participating in the acclaimed Olympic event" enjoying SNK's execution of the character despite the reviewer's dislike of the concept. 3DJuegos felt that Kula's moves in "The King of Fighters XI" were overpowered; she was far more balanced in "XIV". Siliconera found her fighting style in "SNK Heroines: Tag Team Frenzy" to be ridiculous due to the use of "cups of what appears to be sorbet".
Other reviewers focused on the character's sex appeal. Cheat Code Central referred to Kula as one of the most sexually appealing female characters in gaming thanks to her design, abilities, and traits, comparing her looks to those of Hatsune Miku. Universo Nintendo also enjoyed the alternative outfits for Kula, especially her dancer costume. Shoryuken liked Kula's alternative outfits in "SNK Heroines", noting how they give her distinct looks. However, the site noted the fact that Kula can wear skimpy dresses might divide players and make them uncomfortable due to her young age. "Atomix" was more critical of the release of "SNK Heroines" for having their female characters fight in skimpy dresses. A similar response was given when Kula appeared as a guest character in "Dead or Alive 6" for her young age. Nevertheless, Destructoid found the character's moves appealing.


Yeast display

Yeast display (or yeast surface display) is a protein engineering technique that uses the expression of recombinant proteins incorporated into the cell wall of yeast for isolating and engineering antibodies.
Development.
The yeast display technique was first published by the laboratory of Professor K. Dane Wittrup. The technology was sold to Abbott Laboratories in 2001.
How it works.
A protein of interest is displayed as a fusion to the Aga2p protein on the surface of yeast. The Aga2p protein is naturally used by yeast to mediate cell–cell contacts during yeast cell mating. As such, display of a protein via Aga2p projects the protein away from the cell surface, minimizing potential interactions with other molecules on the yeast cell wall. The use of magnetic separation and flow cytometry in conjunction with a yeast display library is a highly effective method to isolate high affinity protein ligands against nearly any receptor through directed evolution.
Advantages and disadvantages.
Advantages of yeast display over other "in vitro" evolution methods include eukaryotic expression and processing, quality control mechanisms of the eukaryotic secretory pathway, minimal avidity effects, and quantitative library screening through fluorescent-activated cell sorting (FACS). Yeast are eukaryotic organisms that allow for complex post-translational modifications to proteins that no other display libraries are able to provide.
Disadvantages include smaller mutant library sizes compared to alternative methods and differential glycosylation in yeast compared to mammalian cells. These disadvantages have not limited the success of yeast display for a number of applications, including engineering the highest monovalent ligand-binding affinity reported to date for an engineered protein (Boder, E.T. 2000). Currently the yeast display library created by Boder is no longer available, as the INVSc1 cell line from Invitrogen is no longer available due to IP issues.
Alternative methods for protein evolution "in vitro" are mammalian display, phage display, ribosome display, bacterial display, and mRNA display.


FMN riboswitch

The FMN riboswitch (also known as RFN element) is a highly conserved RNA element that is found frequently in the 5'-untranslated regions of prokaryotic mRNAs that encode for flavin mononucleotide (FMN) biosynthesis and transport proteins. This element is a metabolite-dependent riboswitch that directly binds FMN in the absence of proteins. In "Bacillus subtilis", the riboswitch controls gene expression by causing premature transcription termination within the 5' untranslated region of the ribDEAHT operon and precluding access to the ribosome-binding site of ypaA mRNA.


Cicloprolol

Cicloprolol is a beta-adrenoceptor antagonist.


CD Genomics

CD Genomics, a genomics services company, provides sequencing services, as well as genotyping and library construction to develop integrated systems of genomic products and services. The company sells products in the United States, Europe, Asia. Scientists from CD Genomics have participated in many genome projects, such as Illumina, the genome sequence, DNA analyzer, Bacillus thuringiensis, Thermoanaerobacter tengcongensis, Streptococcus suis, and quite a few virus genomes.
Founding.
CD Genomics, is a next-generation sequencing provider that was established in 2004. Equipped with both traditional and modern genotyping platforms, the company is headquartered in Shirley, New York. It also maintains operations in North Carolina, Europe and Asia. In 2007, CD Genomics expanded its services to molecular biology research.


James Gegan Miller

James (Jim) Gegan Miller is an American physicist, engineer, and inventor whose primary interests center around biomedical physics. He is currently a Professor of Physics, Medicine, and Biomedical Engineering, emeritus, at Washington University in St. Louis, where he holds the Albert Gordon Hill Endowed Chair in the Faculty of Arts and Sciences. He is notable for his interdisciplinary contributions to biomedical physics, echocardiography, and ultrasonics.
Biography.
Miller grew up in St. Louis, Missouri, and received his bachelor's degree in physics, summa cum laude, from Saint Louis University in 1964. He went on to receive master's and doctoral degrees from Washington University in St. Louis in 1966 and 1969, respectively. His doctoral advisor was Daniel Isadore Bolef.
Thereafter he was hired as assistant professor of physics and earned tenure 2 years later, in 1970. He is now Albert Gordon Hill Chair of the Faculty of Arts and Sciences and Director of the Laboratory of Ultrasonics.
Teaching.
For about four decades, Miller taught a course titled "Physics of the Heart" at Washington University. He won the college's Faculty Teaching Award in 1989 and the Emerson Teaching Award in 2004. Over the course of his career, he mentored 35 graduate students and numerous undergraduates, including Nobel Laureate in Chemistry William E. Moerner.
Research.
Miller has published more than 165 refereed journal articles and 110 conference proceedings and book chapters. His work has contributed greatly to ultrasonics, myocardial tissue characterization and has been incorporated into echocardiography devices in use throughout the world.


Sphingomonas alpina

Sphingomonas alpina is a Gram-negative, aerobic, psychrophilic and motile bacteria from the genus of Sphingomonas which has been isolated from alpine soil from the High Tauern in Austria.


Psychrobacter pacificensis

Psychrobacter pacificensis is a Gram-negative, aerobic, non-spore-forming, catalase- and oxidase-positive, psychrophilic, nonmotile bacterium of the genus "Psychrobacter", which was isolated from 6000-m-deep seawater of the Japan Trench on the Hachijo Island in Japan.


William DeGrado

William F. "Bill" DeGrado, Ph.D., is the Professor of Pharmaceutical Chemistry at the University of California, San Francisco (UCSF) and a member of the National Academy of Sciences.
He received a B.S. (chemistry) from Kalamazoo College and a Ph.D. (Chemistry) from the University of Chicago in 1977 working with Emil T. Kaiser and F. Kezdy. His graduate work focused on the design of the oxime resin for solid-phase synthesis, which was used for synthesis of protected peptides and is still in use for various types of combinatorial chemistry today. He also used peptide design to demonstrate that melittin adopts an amphiphilic helical structure, which is responsible for its membrane-disrupting activity.
He first held an industrial position at DuPont Central Research & Development (later DuPont Merck Pharmaceutical Company). He transitioned to academia in 1996, joining the University of Pennsylvania as the George W. Raiziss professor of biochemistry and biophysics and then moved to UCSF in 2011.
His published research includes contributions to the fields of protein design, synthesis of peptidomimetics, and characterization of membrane-active peptides and proteins, most notably the M2 protein.
"The M2 proton channel from Influenza A virus. "DeGrado’s early work with the groups of Robert Lamb and Larry Pinto established the overall structure and mechanism of the M2 proton channel, which is the target of the anti-influenza drugs, amantadine and rimantadine. A decade later their crystallographic, and NMR structures defined the fine details of the binding site for these drugs and explained the mechanism of the growing problem of amantadine-resistance. With Michael Klein, Robert Lamb and Larry Pinto, DeGrado extensively characterized the physiological properties of many drug-resistant mutants of the channel, identified those most likely to lead to resistance, and designed new drugs to address the problem of drug-resistant forms of influenza A virus.


Collagen hybridizing peptide

A collagen hybridizing peptide (CHP) is a synthetic peptide sequence with typically 6 to 10 repeating units of the Gly-Xaa-Yaa amino acid triplet, which mimics the hallmark sequence of natural collagens. A CHP peptide usually possesses a high content of Proline and Hydroxyproline in the Xaa and Yaa positions, which confers it a strong propensity to form the collagen’s unique triple helix conformation. In the single-stranded (monomeric) status, the peptide can recognize denatured collagen strands in tissues by forming a hybridized triple helix with the collagen strands. This occurs via the triple helical chain assembly and inter-chain hydrogen bonding, in a manner similar to primers binding to melted DNA strands during PCR. The binding does not depend on a specific sequence or epitope on collagen, enabling CHPs to target denatured collagen chains of different types.
Collagen, CHP, CMP, and CLP.
Collagen is the main component of the extracellular matrix (ECM). The collagen superfamily consists of 28 different types of collagen. Although the function and hierarchical structure of these collagens may vary, they all shall the defining structural feature known as the triple helix, where three left handed polyproline II-type (PPII) helices assemble to form a right-handed supercoiled helical motif. Short synthetic peptides known as collagen mimetic peptides (CMPs) or collagen-like peptides (CLPs) have played a major role in elucidating the 3D structure of the collagen triple helix, its folding kinetics, and thermal stability as small triple helical models. CMPs, CLPs, and CHPs are all very similar in terms of their amino acid sequences but only when CMPs or CLPs are heated above their melting temperatures, do they exist in the dissociated, single-stranded state and can be considered as CHPs.
Binding mechanism.
Single-stranded CHPs bind to denatured collagen chains and gelatin in a manner that is unique from other targeting mechanisms, in that they specifically recognize a unique structural motif (collagen triple helix) for folding and chain assembly, as opposed to specific epitopes binding that is seen for monoclonal antibodies (mAbs), for example. Due to their unique targeting mechanism, CHPs have a high binding specificity towards denatured collagen chains but have almost no affinity for intact (triple helical) collagen. CHPs can broadly target collagen chains that have been denatured by thermal, chemical, mechanical, or enzymatic processes, as well as multiple collagen types (e.g., Col I, II, IV). Studies also showed CHPs and their fluorophore conjugates have superior stability in contact with serum.
Denatured collagen as a biomarker for tissue remodelling and damage.
Controlled collagen turnover is crucial for embryonic development, organ morphogenesis, as well as tissue maintenance and repair. However, changes of collagen homeostasis are associated with numerous diseases and pathological conditions. Excessive collagen degradation may be associated with cancer metastasis, skin ageing, arthritis, and osteoporosis. CHPs can target tissues undergoing remodelling based on their ability to bind to degraded and unfolded collagen strands through triple helix formation. As a targeting moiety, CHPs offer great potential in histopathology, diagnostics, and drug delivery for a wide range of diseases.
Most methods for the evaluation of collagen denaturation in disease states are indirect, such as detecting matrix metalloproteinase (MMP) activity or quantifying collagen peptide fragments in urine, serum, or synovial fluid. Using conventional methods for directly targeting collagen, researchers have to relied on collagen binding peptides selected by phage display, derived from collagen binding proteins, or antibodies raised against collagens. Unfortunately, these compounds cannot target denatured collagens which are unstructured and do not present a defined 3D epitope. In addition, antibodies that were reported to distinguish specific degraded collagen fragments can only recognize one or few collagen types. In contrast, CHPs, in principle, can bind to all types of denatured collagens.
Applications.
Tissue staining.
Fluorophore- or biotin-labeled CHPs are used as a staining agent for detecting collagen degradation and denaturation via immunofluorescence and immunohistochemistry applications. CHPs can stain frozen tissue sections, formalin-fixed paraffin embedded (FFPE) sections, as well as fresh tissues. CHP is applicable to tissue specimens from multiple species and a range of diseases, such as myocardial infarction, arthritis, nephritis, and fibrosis.
In vivo imaging.
CHPs can also be labelled with near-infrared fluorophores for "in vivo" fluorescent imaging.
Collagen identification.
CHPs can be used for visualizing many different types of collagen bands in SDS-PAGE gels. Collagen is denatured by heating in the presence of SDS prior to loading the gel. The collagen bands are visualized through CHP-collagen hybridization when the gels are stained by fluorescently-labeled CHPs.
Detecting mechanical damage to connective tissue.
Collagen offers mechanical strength in load bearing tissues in the body such as tendons, ligaments, and bone. As forces are applied to these tissues, the collagen triple helix can be damaged and unwind, and CHPs allow for molecular level detection of mechanical damage in such connective tissues.