Nucleic acid analogue

Nucleic acid analogues are compounds which are analogous (structurally similar) to naturally occurring RNA and DNA, used in medicine and in molecular biology research.
Nucleic acids are chains of nucleotides, which are composed of three parts: a phosphate backbone, a pentose sugar, either ribose or deoxyribose, and one of four nucleobases.
An analogue may have any of these altered. Typically the analogue nucleobases confer, among other things, different base pairing and base stacking properties. Examples include universal bases, which can pair with all four canonical bases, and phosphate-sugar backbone analogues such as PNA, which affect the properties of the chain (PNA can even form a triple helix).
Nucleic acid analogues are also called Xeno Nucleic Acid and represent one of the main pillars of xenobiology, the design of new-to-nature forms of life based on alternative biochemistries.
Artificial nucleic acids include peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA), threose nucleic acid (TNA) and hexitol nucleic acids (HNA). Each of these is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule.
In May 2014, researchers announced that they had successfully introduced two new artificial nucleotides into bacterial DNA, and by including individual artificial nucleotides in the culture media, were able to passage the bacteria 24 times; they did not create mRNA or proteins able to use the artificial nucleotides. The artificial nucleotides featured 2 fused aromatic rings.
Medicine.
Several nucleoside analogues are used as antiviral or anticancer agents. The viral polymerase incorporates these compounds with non-canonical bases. These compounds are activated in the cells by being converted into nucleotides, they are administered as nucleosides since charged nucleotides cannot easily cross cell membranes.
Molecular biology.
Nucleic acid analogues are used in molecular biology for several purposes:
Investigation of possible scenarios of the origin of life: By testing different analogs, researchers try to answer the question of whether life's use of DNA and RNA was selected over time due to its advantages, or if they were chosen by arbitrary chance;
As a tool to detect particular sequences: XNA can be used to tag and identify a wide range of DNA and RNA components with high specificity and accuracy;
As an enzyme acting on DNA, RNA and XNA substrates - XNA has been shown to have the ability to cleave and ligate DNA, RNA and other XNA molecules similar to the actions of RNA ribozymes;
As a tool with resistance to RNA hydrolysis;
Investigation of the mechanisms used by enzyme;
Investigation of the structural features of nucleic acids.
Backbone analogues.
Hydrolysis resistant RNA-analogues.
To overcome the fact that ribose's 2' hydroxy group that reacts with the phosphate linked 3' hydroxy group (RNA is too unstable to be used or synthesized reliably), a ribose analogue is used. The most common RNA analogues are 2'-O-methyl-substituted RNA, locked nucleic acid (LNA) or bridged nucleic acid (BNA), morpholino, and peptide nucleic acid (PNA). Although these oligonucleotides have a different backbone sugar or, in the case of PNA, an amino acid residue in place of the ribose phosphate, they still bind to RNA or DNA according to Watson and Crick pairing, but are immune to nuclease activity. They cannot be synthesized enzymatically and can only be obtained synthetically using phosphoramidite strategy or, for PNA, methods of peptide synthesis.
Other notable analogues used as tools.
Dideoxynucleotides are used in sequencing . These nucleoside triphosphates possess a non-canonical sugar, dideoxyribose, which lacks the 3' hydroxyl group normally present in DNA and therefore cannot bond with the next base. The lack of the 3' hydroxyl group terminates the chain reaction as the DNA polymerases mistake it for a regular deoxyribonucleotide. Another chain-terminating analogue that lacks a 3' hydroxyl and mimics adenosine is called cordycepin. Cordycepin is an anticancer drug that targets RNA replication. Another analogue in sequencing is a nucleobase analogue, 7-deaza-GTP and is used to sequence CG rich regions, instead 7-deaza-ATP is called tubercidin, an antibiotic.
Precursors to the RNA world.
RNA may be too complex to be the first nucleic acid, so before the RNA world several simpler nucleic acids that differ in the backbone, such as TNA and GNA and PNA, have been offered as candidates for the first nucleic acids.
Base analogues.
Nucleobase structure and nomenclature.
Naturally occurring bases can be divided into two classes according to their structure:
Artificial nucleotides ("Unnatural Base Pairs" (UBPs) named "d5SICS UBP" and "dNaM UBP") have been inserted into bacterial DNA but these genes did not template mRNA or induce protein synthesis. The artificial nucleotides featured two fused aromatic rings which formed a (d5SICS–dNaM) complex mimicking the natural (dG–dC) base pair.
Mutagens.
One of the most common base analogs is 5-bromouracil (5BU), the abnormal base found in the mutagenic nucleotide analog BrdU. When a nucleotide containing 5-bromouracil is incorporated into the DNA, it is most likely to pair with adenine; however, it can spontaneously shift into another isomer which pairs with a different nucleobase, guanine. If this happens during DNA replication, a guanine will be inserted as the opposite base analog, and in the next DNA replication, that guanine will pair with a cytosine. This results in a change in one base pair of DNA, specifically a transition mutation.
Additionally, HNO2, or nitrous acid is a potent mutagen that acts on replicating and non-replicating DNA. It can cause deamination of the amino groups of Adenine, Guanine and Cytosine. Adenine is deaminated to hypoxanthine, which base pairs to cytosine instead of thymine. Cytosine is deaminated to uracil, which base pairs with Adenine instead of Guanine. Deamination of Guanine is not mutagenic. Nitrous acid-induced mutations also are induced to mutate back to wild-type using nitrous acid.
Fluorophores.
Commonly fluorophores (such as rhodamine or fluorescein) are linked to the ring linked to the sugar (in para) via a flexible arm, presumably extruding from the major groove of the helix. Due to low processivity of the nucleotides linked to bulky adducts such as florophores by taq polymerases, the sequence is typically copied using a nucleotide with an arm and later coupled with a reactive fluorophore (indirect labelling):
Fluorophores find a variety of uses in medicine and biochemistry.
Fluorescent base analogues.
The most commonly used and commercially available fluorescent base analogue, 2-aminopurine (2-AP), has a high-fluorescence quantum yield free in solution (0.68) that is considerably reduced (appr. 100 times but highly dependent on base sequence) when incorporated into nucleic acids. The emission sensitivity of 2-AP to immediate surroundings is shared by other promising and useful fluorescent base analogues like 3-MI, 6-MI, 6-MAP, pyrrolo-dC (also commercially available), modified and improved derivatives of pyrrolo-dC, furan-modified bases and many other ones (see recent reviews). This sensitivity to the microenvironment has been utilized in studies of e.g. structure and dynamics within both DNA and RNA, dynamics and kinetics of DNA-protein interaction and electron transfer within DNA. A newly developed and very interesting group of fluorescent base analogues that has a fluorescence quantum yield that is nearly insensitive to their immediate surroundings is the tricyclic cytosine family. 1,3-Diaza-2-oxophenothiazine, tC, has a fluorescence quantum yield of approximately 0.2 both in single- and in double-strands irrespective of surrounding bases. Also the oxo-homologue of tC called tCO (both commercially available), 1,3-diaza-2-oxophenoxazine, has a quantum yield of 0.2 in double-stranded systems. However, it is somewhat sensitive to surrounding bases in single-strands (quantum yields of 0.14–0.41). The high and stable quantum yields of these base analogues make them very bright, and, in combination with their good base analogue properties (leaves DNA structure and stability next to unperturbed), they are especially useful in fluorescence anisotropy and FRET measurements, areas where other fluorescent base analogues are less accurate. Also, in the same family of cytosine analogues, a FRET-acceptor base analogue, tCnitro, has been developed. Together with tCO as a FRET-donor this constitutes the first nucleic acid base analogue FRET-pair ever developed. The tC-family has, for example, been used in studies related to polymerase DNA-binding and DNA-polymerization mechanisms.
Natural non-canonical bases.
In a cell, there are several non-canonical bases present: CpG islands in DNA (are often methylated), all eukaryotic mRNA (capped with a methyl-7-guanosine), and several bases of rRNAs (are methylated). Often, tRNAs are heavily modified postranscriptionally in order to improve their conformation or base pairing, in particular in/near the anticodon: inosine can base pair with C, U, and even with A, whereas thiouridine (with A) is more specific than uracil (with a purine). Other common tRNA base modifications are pseudouridine (which gives its name to the TΨC loop), dihydrouridine (which does not stack as it is not aromatic), queuosine, wyosine, and so forth. Nevertheless, these are all modifications to normal bases and are not placed by a polymerase.
Base-pairing.
Canonical bases may have either a carbonyl or an amine group on the carbons surrounding the nitrogen atom furthest away from the glycosidic bond, which allows them to base pair (Watson-Crick base pairing) via hydrogen bonds (amine with ketone, purine with pyrimidine). Adenine and 2-aminoadenine have one/two amine group(s), whereas thymine has two carbonyl groups, and cytosine and guanine are mixed amine and carbonyl (inverted in respect to each other).
The precise reason why there are only four nucleotides is debated, but there are several unused possibilities.
Furthermore, adenine is not the most stable choice for base pairing: in Cyanophage S-2L diaminopurine (DAP) is used instead of adenine (host evasion). Diaminopurine basepairs perfectly with thymine as it is identical to adenine but has an amine group at position 2 forming 3 intramolecular hydrogen bonds, eliminating the major difference between the two types of basepairs (Weak:A-T and Strong:C-G). This improved stability affects protein-binding interactions that rely on those differences.
Other combination include,
However, correct DNA structure can form even when the bases are not paired via hydrogen bonding; that is, the bases pair thanks to hydrophobicity, as studies have shown using DNA isosteres (analogues with same number of atoms), such as the thymine analogue 2,4-difluorotoluene (F) or the adenine analogue 4-methylbenzimidazole (Z). An alternative hydrophobic pair could be isoquinoline, and the pyrrolo[2,3-b]pyridine
Other noteworthy basepairs:
Metal base-pairs.
In metal base-pairing, the Watson-Crick hydrogen bonds are replaced by the interaction between a metal ion with nucleosides acting as ligands. The possible geometries of the metal that would allow for duplex formation with two bidentate nucleosides around a central metal atom are: tetrahedral, dodecahedral, and square planar. Metal-complexing with DNA can occur by the formation of non-canonical base pairs from natural nucleobases with participation by metal ions and also by the exchanging the hydrogen atoms that are part of the Watson-Crick base pairing by metal ions. Introduction of metal ions into a DNA duplex has shown to have potential magnetic, conducting properties, as well as increased stability.
Metal complexing has been shown to occur between natural nucleobases. A well-documented example is the formation of T-Hg-T, which involves two deprotonated thymine nucleobases that are brought together by Hg2+ and forms a connected metal-base pair. This motif does not accommodate stacked Hg2+ in a duplex due to an intrastrand hairpin formation process that is favored over duplex formation. Two thymines across from each other in a duplex do not form a Watson-Crick base pair in a duplex; this is an example where a Watson-Crick basepair mismatch is stabilized by the formation of the metal-base pair. Another example of a metal complexing to natural nucleobases is the formation of A-Zn-T and G-Zn-C at high pH; Co+2 and Ni+2 also form these complexes. These are Watson-Crick base pairs where the divalent cation in coordinated to the nucleobases. The exact binding is debated.
A large variety of artificial nucleobases have been developed for use as metal base pairs. These modified nucleobases exhibit tunable electronic properties, sizes, and binding affinities that can be optimized for a specific metal. For, example a nucleoside modified with a pyridine-2,6-dicarboxylate has shown to bind tightly to Cu2+, whereas other divalent ions are only loosely bound. The tridentate character contributes to this selectivity. The fourth coordination site on the copper is saturated by an oppositely arranged pyridine nucleobase. The asymmetric metal base pairing system is orthogonal to the Watson-Crick base pairs. Another example of an artificial nucleobase is that with hydroxypyridone nucleobases, which are able to bind Cu2+ inside the DNA duplex. Five consecutive copper-hydroxypyridone base pairs were incorporated into a double strand, which were flanked by only one natural nucleobase on both ends. EPR data showed that the distance between copper centers was estimated to be 3.7 ± 0.1 Å, while a natural B-type DNA duplex is only slightly larger (3.4 Å). The appeal for stacking metal ions inside a DNA duplex is the hope to obtain nanoscopic self-assembling metal wires, though this has not been realized yet.
Unnatural base pair (UBP).
An unnatural base pair (UBP) is a designed subunit (or nucleobase) of DNA which is created in a laboratory and does not occur in nature. In 2012, a group of American scientists led by Floyd Romesberg, a chemical biologist at the Scripps Research Institute in San Diego, California, published that his team designed an unnatural base pair (UBP). The two new artificial nucleotides or "Unnatural Base Pair" (UBP) were named d5SICS and dNaM. More technically, these artificial nucleotides bearing hydrophobic nucleobases, feature two fused aromatic rings that form a (d5SICS–dNaM) complex or base pair in DNA. In 2014 the same team from the Scripps Research Institute reported that they synthesized a stretch of circular DNA known as a plasmid containing natural T-A and C-G base pairs along with the best-performing UBP Romesberg's laboratory had designed, and inserted it into cells of the common bacterium "E. coli" that successfully replicated the unnatural base pairs through multiple generations. This is the first known example of a living organism passing along an expanded genetic code to subsequent generations. This was in part achieved by the addition of a supportive algal gene that expresses a nucleotide triphosphate transporter which efficiently imports the triphosphates of both d5SICSTP and dNaMTP into "E. coli" bacteria. Then, the natural bacterial replication pathways use them to accurately replicate the plasmid containing d5SICS–dNaM.
The successful incorporation of a third base pair is a significant breakthrough toward the goal of greatly expanding the number of amino acids which can be encoded by DNA, from the existing 20 amino acids to a theoretically possible 172, thereby expanding the potential for living organisms to produce novel proteins. Earlier, the artificial strings of DNA did not encode for anything, but scientists speculated they could be designed to manufacture new proteins which could have industrial or pharmaceutical uses. Transcription of DNA containing unnatural base pair and translation of corresponding mRNA were actually achieved recently. On November 2017, the same team at the Scripps Research Institute that first introduced two extra nucleobases into bacterial DNA, reported having constructed a semi-synthetic "E. coli" bacteria able to make proteins using such DNA. Its DNA contained six different nucleobases: four canonical and two artificially added, dNaM and dTPT3 (these two form a pair). Also, this bacteria had two corresponding additional RNA bases included in two new codons, additional tRNAs recognizing these new codons (these tRNAs also contained two new RNA bases within their anticodons) and additional amino acids, making the bacteria able to synthesize "unnatural" proteins.
Another demonstration of UBPs were achieved by Ichiro Hirao's group at RIKEN institute in Japan. In 2002, they developed an unnatural base pair between 2-amino-8-(2-thienyl)purine (s) and pyridine-2-one (y) that functions "in vitro" in transcription and translation, for the site-specific incorporation of non-standard amino acids into proteins. In 2006, they created 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) as a third base pair for replication and transcription. Afterward, Ds and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (Px) was discovered as a high fidelity pair in PCR amplification. In 2013, they applied the Ds-Px pair to DNA aptamer generation by "in vitro" selection (SELEX) and demonstrated the genetic alphabet expansion significantly augment DNA aptamer affinities to target proteins.
Orthogonal system.
The possibility has been proposed and studied, both theoretically and experimentally, of implementing an orthogonal system inside cells independent of the cellular genetic material in order to make a completely safe system, with the possible increase in encoding potentials.
Several groups have focused on different aspects:


Five prime untranslated region

The 5′ untranslated region (5′ UTR) (also known as a leader sequence, transcript leader, or leader RNA) is the region of an mRNA that is directly upstream from the initiation codon. This region is important for the regulation of translation of a transcript by differing mechanisms in viruses, prokaryotes and eukaryotes. While called untranslated, the 5′ UTR or a portion of it is sometimes translated into a protein product. This product can then regulate the translation of the main coding sequence of the mRNA. In many organisms, however, the 5′ UTR is completely untranslated, instead forming complex secondary structure to regulate translation.
The 5′ UTR has been found to interact with proteins relating to metabolism, and proteins translate sequences within the 5′ UTR. In addition, this region has been involved in transcription regulation, such as the sex-lethal gene in "Drosophila". Regulatory elements within 5′ UTRs have also been linked to mRNA export.
General structure.
Length.
The 5′ UTR begins at the transcription start site and ends one nucleotide (nt) before the initiation sequence (usually AUG) of the coding region. In prokaryotes, the length of the 5′ UTR tends to be 3–10 nucleotides long, while in eukaryotes it tends to be anywhere from 100 to several thousand nucleotides long. For example, the "ste11" transcript in "Schizosaccharomyces pombe" has a 2273 nucleotide 5′ UTR while the "lac" operon in "Escherichia coli" only has seven nucleotides in its 5′ UTR. The differing sizes are likely due to the complexity of the eukaryotic regulation which the 5′ UTR holds as well as the larger pre-initiation complex that must form to begin translation.
The 5′ UTR can also be completely missing, in the case of leaderless mRNAs. Ribosomes of all three domains of life accept and translate such mRNAs. Such sequences are naturally found in all three domains of life. Humans have many pressure-related genes under a 2–3 nucleotide leader. Mammals also have other types of ultra-short leaders like the TISU sequence.
Elements.
The elements of a eukaryotic and prokaryotic 5′ UTR differ greatly. The prokaryotic 5′ UTR contains a ribosome binding site (RBS), also known as the Shine–Dalgarno sequence (AGGAGGU), which is usually 3–10 base pairs upstream from the initiation codon. In contrast, the eukaryotic 5′ UTR contains the Kozak consensus sequence (ACCAUGG), which contains the initiation codon. The eukaryotic 5′ UTR also contains "cis"-acting regulatory elements called upstream open reading frames (uORFs) and upstream AUGs (uAUGs) and termination codons, which have a great impact on the regulation of translation (see below). Unlike prokaryotes, 5′ UTRs can harbor introns in eukaryotes. In humans, ~35% of all genes harbor introns within the 5′ UTR.
Secondary structure.
As the 5′ UTR has high GC content, secondary structures often occur within it. Hairpin loops are one such secondary structure that can be located within the 5′ UTR. These secondary structures also impact the regulation of translation.
Role in translational regulation.
Prokaryotes.
In bacteria, the initiation of translation occurs when IF-3, along with the 30S ribosomal subunit, bind to the Shine–Dalgarno (SD) sequence of the 5′ UTR. This then recruits many other proteins, such as the 50S ribosomal subunit, which allows for translation to begin. Each of these steps regulates the initiation of translation.
Initiation in Archaea is less understood. SD sequences are much rarer, and the initiation factors have more in common with eukaryotic ones. There is no homolog of bacterial IF3. Some mRNAs are leaderless.
In both domains, genes without Shine–Dalgarno sequences are also translated in a less understood manner. A requirement seems to be a lack of secondary structure near the initiation codon.
Eukaryotes.
Pre-initiation complex regulation.
The regulation of translation in eukaryotes is more complex than in prokaryotes. Initially, the eIF4F complex is recruited to the 5′ cap, which in turn recruits the ribosomal complex to the 5′ UTR. Both eIF4E and eIF4G bind the 5′ UTR, which limits the rate at which translational initiation can occur. However, this is not the only regulatory step of translation that involves the 5′ UTR.
RNA-binding proteins sometimes serve to prevent the pre-initiation complex from forming. An example is regulation of the "msl2" gene. The protein SXL attaches to an intron segment located within the 5′ UTR segment of the primary transcript, which leads to the inclusion of the intron after processing. This sequence allows the recruitment of proteins that bind simultaneously to both the 5′ and 3′ UTR, not allowing translation proteins to assemble. However, it has also been noted that SXL can also repress translation of RNAs that do not contain a poly(A) tail, or more generally, 3′ UTR.
Closed-loop regulation.
Another important regulator of translation is the interaction between 3′ UTR and the 5′ UTR.
The closed-loop structure inhibits translation. This has been observed in "Xenopus laevis", in which eIF4E bound to the 5′ cap interacts with Maskin bound to CPEB on the 3′ UTR, creating translationally inactive transcripts. This translational inhibition is lifted once CPEB is phosphorylated, displacing the Maskin binding site, allowing for the polymerization of the PolyA tail, which can recruit the translational machinery by means of PABP. However, it is important to note that this mechanism has been under great scrutiny.
Ferritin regulation.
Iron levels in cells are maintained by translation regulation of many proteins involved in iron storage and metabolism. The 5′ UTR has the ability to form a hairpin loop secondary structure (known as the iron response element or IRE) that is recognized by iron-regulatory proteins (IRP1 and IRP2). In low levels of iron, the ORF of the target mRNA is blocked as a result of steric hindrance from the binding of IRP1 and IRP2 to the IRE. When iron is high, then the two iron-regulatory proteins do not bind as strongly and allow proteins to be expressed that have a role in iron concentration control. This function has gained some interest after it was revealed that the translation of amyloid precursor protein may be disrupted due to a single-nucleotide polymorphism to the IRE found in the 5′ UTR of its mRNA, leading to a spontaneous increased risk of Alzheimer's disease.
uORFs and reinitiation.
Another form of translational regulation in eukaryotes comes from unique elements on the 5′ UTR called upstream open reading frames (uORF). These elements are fairly common, occurring in 35–49% of all human genes. A uORF is a coding sequence located in the 5′ UTR located upstream of the coding sequences initiation site. These uORFs contain their own initiation codon, known as an upstream AUG (uAUG). This codon can be scanned for by ribosomes and then translated to create a product, which can regulate the translation of the main protein coding sequence or other uORFs that may exist on the same transcript.
The translation of the protein within the main ORF after a uORF sequence has been translated is known as reinitiation. The process of reinitiation is known to reduce the translation of the ORF protein. Control of protein regulation is determined by the distance between the uORF and the first codon in the main ORF. A uORF has been found to increase reinitiation with the longer distance between its uAUG and the start codon of the main ORF, which indicates that the ribosome needs to reacquire translation factors before it can carry out translation of the main protein. For example, "ATF4" regulation is performed by two uORFs further upstream, named uORF1 and uORF2, which contain three amino acids and fifty-nine amino acids, respectively. The location of uORF2 overlaps with the "ATF4" ORF. During normal conditions, the uORF1 is translated, and then translation of uORF2 occurs only after eIF2-TC has been reacquired. Translation of the uORF2 requires that the ribosomes pass by the "ATF4" ORF, whose start codon is located within uORF2. This leads to its repression. However, during stress conditions, the 40S ribosome will bypass uORF2 because of a decrease in concentration of eIF2-TC, which means the ribosome does not acquire one in time to translate uORF2. Instead, "ATF4" is translated.
Other mechanisms.
In addition to reinitiation, uORFs contribute to translation initiation based on:
Internal ribosome entry sites and viruses.
Viral (as well as some eukaryotic) 5′ UTRs contain internal ribosome entry sites, which is a cap-independent method of translational activation. Instead of building up a complex at the 5′ cap, the IRES allows for direct binding of the ribosomal complexes to the transcript to begin translation. The IRES enables the viral transcript to translate more efficiently due to the lack of needing a preinitation complex, allowing the virus to replicate quickly.
Role in transcriptional regulation.
"msl-2" transcript.
Transcription of the "msl-2" transcript is regulated by multiple binding sites for fly "Sxl" at the 5′ UTR. In particular, these poly-uracil sites are located close to a small intron that is spliced in males, but kept in females through splicing inhibition. This splicing inhibition is maintained by "Sxl". When present, "Sxl" will repress the translation of "msl2" by increasing translation of a start codon located in a uORF in the 5′ UTR (see above for more information on uORFs). Also, "Sxl" outcompetes TIA-1 to a poly(U) region and prevents snRNP (a step in alternative splicing) recruitment to the 5′ splice site.


Homeoviscous adaptation

Homeoviscous adaptation is the adaptation of the cell membrane lipid composition to keep the adequate membrane fluidity.
The maintenance of proper cell membrane fluidity is of critical importance for the function and integrity of the cell, essential for the mobility and function of embedded proteins and lipids, diffusion of proteins and other molecules laterally across the membrane for signaling reactions, and proper separation of membranes during cell division.
A fundamental biophysical determinant of membrane fluidity is the balance between saturated and unsaturated fatty acids. Regulating membrane fluidity is especially important in poikilothermic organisms such as bacteria, fungi, protists, plants, fish and other ectothermic animals. The general trend is an increase in unsaturated fatty acids at lower growth temperatures and an increase in saturated fatty acids at higher temperatures.
A recent work§ has explored the importance of the homeoviscous adaptation of the cell membrane for a psychrotolerant bacteria living in the cold biosphere of earth.


Lactate 2-monooxygenase

In enzymology, a lactate 2-monooxygenase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are (S)-lactate and O2, whereas its 3 products are acetate, CO2, and H2O.
This enzyme belongs to the family of oxidoreductases, specifically those acting on single donors with O2 as oxidant and incorporation of two atoms of oxygen into the substrate (oxygenases). The oxygen incorporated need not be derived from O with incorporation of one atom of oxygen (internal monooxygenases o internal mixed-function oxidases). The systematic name of this enzyme class is (S)-lactate:oxygen 2-oxidoreductase (decarboxylating). Other names in common use include lactate oxidative decarboxylase, lactate oxidase, lactic oxygenase, lactate oxygenase, lactic oxidase, L-lactate monooxygenase, lactate monooxygenase, and L-lactate-2-monooxygenase. This enzyme participates in pyruvate metabolism. It employs one cofactor, FMN.
Structural studies.
As of late 2007, only one structure has been solved for this class of enzymes, with the PDB accession code .


TBST

In molecular biology, TBST (or TTBS) is a mixture of tris-buffered saline (TBS) and Polysorbate 20 (also known as Tween 20). It is a buffer used for washing nitrocellulose membrane in western blotting and microtiter plate wells in ELISA assays. Tris is a pH buffer that maintains a pH that usually lies around pH=8. Buffered Saline is a salt that provides an isotone solution. Tween is a soap that removes fat droplets from the blot or well.
Contents of TBS-Tween.
The following is a sample recipe for TBST:
Adjust pH with HCl to pH 7.4–7.6
The simplest way to prepare a TBS-Tween solution is to use TBS-T tablets. They are formulated to give a ready to use TBST solution upon dissolution in 500 ml of deionized water.
References.
https://www.gbiosciences.com/Buffers-Reagents-Chemicals/Electrophoresis-Related-Buffers-Chemicals/TBST-10X


Chiral link


Anthony Cary, 5th Viscount Falkland

Anthony Cary, 5th Viscount Falkland PC (16 February 1656 – 24 May 1694; the surname is spelt Carey in some sources) was an English born, Scottish nobleman and English politician.
He was born at Farley Castle, Somerset, the son of Henry Cary, 4th Viscount Falkland, to whose peerage he succeeded as a child in 1663.
He married Rebecca Lytton and had one daughter:
As a Scottish peer he was entitled to be a Member of the Parliament of England. He thus served as Tory MP for Oxfordshire for 1685–1689, Great Marlow from 1689 to 1690, and Great Bedwyn from 1690 until his death.
He was sworn of the Privy Council of England in 1692 and served as First Lord of the Admiralty from 1693 to 1694. He had previously held office with the latter department as Treasurer of the Navy from 1681 to 1689, under Charles II and James II, and as Commissioner of the Admiralty from 1690 to 1693. Samuel Pepys had a rather low opinion of his abilities, while admitting that he suffered from chronic ill-health.
In March 1694 he was committed to the Tower of London on charges of peculation and died of smallpox in May, aged 38, without male issue. He was buried in Westminster Abbey.
The Falkland Islands are named in his honour. The Viscounts Falkland in turn take their title from the Scottish monarchs' residence Falkland Palace, in Falkland, Fife, Scotland.


Robert J. Cotter

Robert J. Cotter (July 15, 1943 – November 12, 2012 ) was an American chemist and mass spectrometrist. His research contributed to many early advances in the field of time-of-flight mass spectrometry. From 1998 to 2000 he was president of the American Society for Mass Spectrometry. Cotter was also a co-investigator on the Mars Organic Molecule Analyzer (MOMA) project, developing a miniaturized, low power consumption ion trap/time-of-flight mass spectrometer that was to be deployed with the ExoMars rover.
Early life.
Cotter was raised in Abington, Massachusetts and was the oldest of seven children. After graduating from Boston College High School in 1961, he attended the College of the Holy Cross in Worcester, Massachusetts. After receiving a B.S. degree in 1965, he studied under W.S. Koski at Johns Hopkins University. He received his PhD in 1972 and joined the faculty of Towson University and Gettysburg College.
Academic research career.
From 1978 until his death in 2012, Robert Cotter was a member of the faculty at Johns Hopkins University in the departments of Pharmacology and Molecular Sciences and Biophysics and Biophysical Chemistry.
Curved-field reflectron.
To increase the mass resolution in time-of-flight mass spectrometry, a reflectron is often employed. Traditional, single-stage or linear reflectrons suffered from a lack of sensitivity and resolving power when ion velocities (and thus kinetic energies were not equal. In MALDI mass spectrometry, metastable ions generated spontaneously after ionization (post-source decay) exhibit a wide spectrum of kinetic energies. This is also known to occur during collision-induced dissociation. In 1993, Cotter's research group discovered that, using a non-linear electric field, the spread of ion kinetic energies could be compensated, increasing the resolution of time-of-flight mass spectrometers and forming the basis for many modern TOF/TOF mass spectrometers.
Ionization and biological mass spectrometry.
While he is primarily known for his contributions to the field of time-of-flight mass spectrometry, his research in the development and application of new ionization techniques for complex biomolecules. These include thermal desorption, laser desorption, fast atom bombardment, thermospray and plasma desorption.
His research into ionization sources also enabled many other discoveries in biomedical sciences. In 1993, he was part of the research team responsible for implicating the 42 amino acid form of the protein Aβ in the pathology of Alzheimer's disease.
Mars organic molecule analyzer.
The Mars Organic Molecule Analyzer (MOMA) project is part of a collaboration to search for potential signatures of Martian life. Cotter was a deputy principal investigator and responsible for the design and development of a low-power, ion trap-time-of-flight mass spectrometer to be deployed with the ESA ExoMars rover. However, in February 2012, NASA cancelled its participation in the rover and defunded projects related to it. By November 2012 NASA reestablished the funding for the US part of MOMA.


Protofection

Protofection is a protein-mediated transfection of foreign mitochondrial DNA (mtDNA) into the mitochondria of cells in a tissue to supplement or replace the native mitochondrial DNA already present. The complete mtDNA genome or just fragments of PCR-generated mtDNA can be transferred into the target mitochondria through the technique of protofection.
Scientists have hypothesized for the last couple of decades that protofection can be beneficial for patients with mitochondrial diseases. This technique is a recent development and is continuously being improved.As mitochondrial DNA becomes progressively more damaged with age, this may provide a method of at least partially rejuvenating mitochondria in old tissue, restoring them to their original, youthful function.
Method.
Protofection is a developing technique and is continuously being improved. A specific protein transduction system has been created that is complexed with mtDNA, which enables the mtDNA to move across the targeted cell's membrane and specifically target mitochondria. The transduction system used consists of a protein transduction domain(PTD), mitochondrial localization sequences(MLS), and mitochondrial transcription factor A(TFAM). Each of these play a specific role in protofection:
This process can lead to an increase in the amount of mtDNA present in the mitochondria of the target cells.
The transduction system has been tweaked and modified, since the first use of protofection. To shorten the name of the complex, which was previously called PTD-MLS-TFAM complex, it is now named MTD-TFAM. MTD stands for mitochondrial transduction domain and it includes the PTD and the MLS.
Possible Therapeutic Uses.
One hypothesis for mitochondrial diseases is that mitochondrial damage and dysfunction play an important role in aging. Protofection is being researched as a possibly viable laboratory technique for constructing gene therapies for inherited mitochondrial diseases, such as Leber's hereditary optic neuropathy. Studies have shown that protofection can lead to improved mitochondrial function in targeted cells.
Protofection could be applied to modified or artificial mitochondria. Mitochondria could be modified to produce few or no free radicals without compromising energy production. Recent studies have demonstrated that mitochondrial transplants may be useful to rejuvenate dead or dying tissue, such as in heart attacks, for which the mitochondria is the first part of the cell that dies.


Christine S. Chow

Christine "Christy" Chow is a Professor of Chemistry (Biochemistry Division) and former Associate Dean of the College of Liberal Arts and Sciences at Wayne State University. She works on modified RNAs, RNA-ligand interactions and RNA therapeutics. She is a Fellow of the American Chemical Society (ACS).
Early life and education.
Chow was born New Jersey. She studied environmental science at Bowdoin College and graduated in 1987. She was a graduate student at Columbia University, earning a master's degree in 1988. Chow earned her doctorate in inorganic chemistry with Jacqueline Barton at the California Institute of Technology in 1992.
Research and career.
Chow was a postdoctoral research fellow at Massachusetts Institute of Technology, where she worked with Stephen J. Lippard. In 1994 Chow joined Wayne State University. Her research has been supported by the National Institutes of Health since she joined Wayne State University. Chow studies the structural and functional roles of modified nucleosides in RNA. So far, several hundred modifications have been found in RNA and Transfer RNA, but their contributions to structure and function have yet to be fully established. Chow develops the methodologies to incorporate modified nucleosides at specific points in RNA; in particular helix 69 of 23S ribosomal RNA. The pseudouridine modification helps to maintain fidelity during protein synthesis, but its exact role in regulating the function of a ribosome is unknown. They use inorganic complexes (such as platinum(II)) to examine the structure of nucleic acid.
Chow has used fluorescence spectroscopy and mass spectrometry to study drug-RNA interactions in an effort to inform the design of new antibiotics. Improved RNA binding ligands indicate that drugs have potential, and should be developed further. She developed assays to investigate aminoglycoside analogues. She is working on new anti-infectives that combat antimicrobial resistance.
Academic service and advocacy.
Chow is an advocate for undergraduate research opportunities and diversity in the scientific community. In 2006 one of Chow's students, Uzoma Azuh, was diagnosed with acute myeloid leukemia. When he died in 2007, Chow created the Uzoma Azuh Endowed Memorial Research Scholarship, a fellowship for undergraduate students in chemistry and biochemistry, in his honour. She is a faculty mentor for an National Institutes of Health grant, the Wayne State University "Initiative for Maximizing Student Development", which supports students from underrepresented minorities in their scientific careers. In 2016, Wayne State University and Chow were awarded a multi-million dollar BEST (Broadening Experiences in Scientific Training) grant from National Institutes of Health to develop a doctoral training program for graduate students. She is a member of the leadership team of the American Chemical Society Women's Chemistry Committee and the Division of Biological Chemistry.


Tamara Minko

Tamara Minko is a distinguished professor and chair of the department of pharmaceutics at Rutgers University, Ernest Mario School of Pharmacy. Her work has been included in over 400 publications.
Career.
Minko's scientific career began in Ukraine, but in 1997 she moved to the United States to work with Professor J. Kopecek at the University of Utah. Minko is the chair of the pharmaceutics at Rutgers University. Her research focuses on nanoparticles used to deliver medicine more efficiently to the body, specifically to fight cancer. In 2009, she became the CRS Bioactive Materials Track Program Chair. She was also a member of the board of Scientific Advisors and director-at-large of the Controlled Release Society, Academy of Applied Pharmaceutical Sciences Fellow Research, the Rutgers Cancer Institute of New Jersey, and Environmental and Occupational Health Science Institute.
Minko is a Fellow of American Institute for Medical and Biological Engineering (AIMBE), Controlled Release Society (CRS), and American Association of Pharmaceutical Scientists (AAPS). She is a member of the Study Sections of the Center for Scientific Review at the National Institutes of Health, the Department of Defense, and the American Heart Association. Her research is funded by grants from the National Institutes of Health, the American Lung Association, National Science Foundation, and the Department of Defense among other sources.
Publications and speaking events.
Minko is executive editor of Advanced Drug Delivery Reviews and editor of Pharmaceutical Research. She has been author or co-author for more than 400 publications including PNAS, Nature Nanotechnology, Cancer Research, Advanced Drug Delivery Reviews, Journal of Controlled Release, Small, and ACS Nano. Minko is on the editorial board of nine scientific journals.
Minko has spoken at symposiums and seminars including the Nanomedicine and Drug Delivery Symposium 2008 at Northeastern University, and the Chemistry in Cancer Research from the American Association for Cancer Research and the American Chemical Society in 2009. In 2012 she spoke at the 9th International Conference and Workshop on Biological Barriers 2012. Minko spoke at the Center for Targeted Therapeutics and Translation Nanomedicine Symposium, and the Capital Region Cancer Research New Frontiers Symposium in 2014. In March 2015, she spoke at the Spring Seminar Series at University of Pittsburgh. Minko has also spoken at the New Strategies and Technologies in Cancer Research for Bioconnex and Capital Region Cancer Research Group.


Naphazoline

Naphazoline is a medicine used as a decongestant. It is a sympathomimetic agent with marked alpha adrenergic activity. It is a vasoconstrictor with a rapid action in reducing swelling when applied to mucous membrane. It acts on alpha-receptors in the arterioles of the conjunctiva to produce constriction, resulting in decreased congestion.
It was patented in 1934 and came into medical use in 1942.
Chemistry.
The non-hydrochloride form of Naphazoline has the molecular formula C14H14N2 and a molar mass of 210.28 g/mol. The HCl salt form has a molar mass of 246.73 g/mol.
Side effects.
A few warnings and contraindications that apply to all naphazoline-containing substances intended for medicinal use are:
A possible association with stroke has been suggested.
Brand names.
It is an active ingredient in several over-the-counter formulations including Rohto, Eucool, Clear Eyes and Naphcon eye drops.


Non-stop decay

Non-stop decay (NSD) is a cellular mechanism of mRNA surveillance to detect mRNA molecules lacking a stop codon and prevent these mRNAs from translation. The non-stop decay pathway releases ribosomes that have reached the far 3' end of an mRNA and guides the mRNA to the exosome complex, or to RNase R in bacteria for selective degradation. In contrast to Nonsense-mediated decay (NMD), polypeptides do not release from the ribosome, and thus, NSD seems to involve mRNA decay factors distinct from NMD.
Non-Stop Decay (NSD).
Non-stop decay (NSD) is a cellular pathway that identifies and degrades aberrant mRNA transcripts that do not contain a proper stop codon. Stop codons are signals in messenger RNA that signal for synthesis of proteins to end. Aberrant transcripts are identified during translation when the ribosome translates into the poly A tail at the 3' end of mRNA. A non-stop transcript can occur when point mutations damage the normal stop codon. Moreover, some transcriptional events are more likely to preserve gene expression on a lower scale in particular states.
The NSD pathway discharges ribosomes that have stalled at the 3' end of mRNA and directs the mRNA to the exosome complex in eukaryotes or RNase R in bacteria.  Once directed to their appropriate sites, the transcripts are then degraded. The NSD mechanism requires the interaction of RNA exosome with the Ski complex, a multi-protein structure that includes the Ski2p helicase and (notably) Ski7p.  The combination of these proteins and subsequent complex formation activates the degradation of aberrant mRNAs. Ski7p is thought to bind the ribosome stalled at the 3’ end of the mRNA poly(A) tail and recruit the exosome to degrade the aberrant mRNA. However in mammalian cells, Ski7p is not found, and even the presence of the NSD mechanism itself has remained relatively unclear. The short splicing isoform of HBS1L (HBS1LV3) was found to be the long-sought after human homologue of Ski7p, linking the exosome and SKI complexes. Recently, it has been reported that NSD also occurs in mammalian cells, albeit through a slightly different system.  In mammals, due to the absence of Ski7, the GTPase Hbs1, as well as its binding partner Dom34, were identified as potential regulators of decay.  Together, Hbs1/Dom34 are capable of binding to the 3’ end of a mis-regulated mRNA, facilitating the dissociation of malfunctioning or inactive ribosomes in order to restart the process of translation.  In addition, once the Hbs1/Dom34 complex has dissociated and recycled a ribosome, it has also been shown to recruit the exosome/Ski complex.
Liberation of the Ribosome.
In bacteria, trans-translation, a highly conserved mechanism, acts as a direct counter to the accumulation of non-stop RNA, inducing decay and liberating the misregulated ribosome.  Originally discovered in "Escherichia coli", the process of trans-translation is made possible by the interactions between transfer-messenger RNA (tmRNA) and the cofactor protein SmpB, which allows for the stable binding of the tmRNA to the stalled ribosome.  The current tmRNA model states that tmRNA and SmpB interact together in order to mimic tRNA. The SmpB protein recognizes the point of stalling, and directs the tmRNA to bind to the ribosomal A site. Once bound, SmpB engages in a transpeptidation reaction with the improperly functioning polypeptide chain through the donation of charged alanine.  Through this process, the stalled and defective mRNA sequence is replaced with the SmpB RNA sequence, which encodes for the addition of an 11 amino acid tag on the C-terminus of the mRNA, which promotes degradation. The modified portion of RNA, along with the amino acid tag, are translated, and demonstrate incomplete characteristics, alerting and allowing for intracellular proteases to remove these harmful protein fragments, causing stalled ribosomes on damaged mRNA to resume function.
mRNA Degradation.
Many enzymes and proteins play role in degrading mRNA. For example, in "Escherichia coli" there are three enzymes: RNase II, PNPase, and RNase R. RNase R is a 3’-5’ exoribonuclease that is recruited to degrade a defective mRNA. RNase R has two structural domains, an N-terminal putative helix-turn-helix (HTH) and a C-terminal lysine(K-rich) domain. These two domains are unique to RNase R, and are attributed as being the determining factors for the selectivity and specificity of the protein. Evidence has been shown that the K-rich domain is involved in the degradation of non-stop mRNA. These domains are not present in other RNases. Both RNase II and RNase R are members of RNR family, and they share a noteworthy similarity in primary sequence and domain architecture. However, RNase R has the ability to efficiently degrade mRNA, while RNase II has less efficiency in the degrading process. Nevertheless, the specific mechanics of degrading mRNA via RNase R has remained a mystery.


Pilin

Pilin refers to a class of fibrous proteins that are found in pilus structures in bacteria. These structures can be used for the exchange of genetic material, or as a cell adhesion mechanism. Although not all bacteria have pili or fimbriae, bacterial pathogens often use their fimbriae to attach to host cells. In Gram-negative bacteria, where pili are more common, individual pilin molecules are linked by noncovalent protein-protein interactions, while Gram-positive bacteria often have polymerized LPXTG pilin.
Type IV pilin.
Type IV pilin proteins are α+β proteins characterized by a very long N-terminal alpha helix. The assembly of these pili relies on interactions between the N-terminal helices of the individual monomers. The pilus structure sequesters the helices in the center of the fiber lining a central pore, while antiparallel beta sheets occupy the exterior of the fiber.
Role of ComP pilin in bacterial transformation.
Genetic transformation is the process by which a recipient bacterial cell takes up DNA from a neighboring cell and integrates this DNA into the recipient’s genome by homologous recombination. In "Neisseria meningitidis", DNA transformation requires the presence of short DNA uptake sequences (DUSs) which are 9-10mers residing in coding regions of the donor DNA. Specific recognition of DUSs is mediated by a type IV pilin, ComP. Menningococcal type IV pili bind DNA through the minor pilin ComP via an electropositive stripe that is predicted to be exposed on the filament's surface. ComP displays an exquisite binding preference for selective DUSs. The distribution of DUSs within the "N. meningitidis" genome favors certain genes, suggesting that there is a bias for genes involved in genomic maintenance and repair.
Chaperone-usher pilin.
The Cup family is known for its use of a chaperone and at least an usher. They exhibit an Ig fold.
Saf, N-terminal extension.
The Saf pilin N-terminal extension protein domain helps the pili to form, via a complex mechanism named the chaperone/usher pathway. It is found in all c-u pilins.
This protein domain is very important for such bacteria, as without pili formation, they could not infect the host. Saf is a "Salmonella" operon containing a c-u pilus system.
Function.
This protein domain, has an important function in forming pili. These are virulence factors crucial for cell adhesion to the host and biofilm formation with successful infection.
Structure.
This protein domain consists of the adjacent Saf-Nte and Saf-pilin chains of the pilus-forming complex. They are Chaperone/usher (CU) pili, and have an N-terminal extension (Nte) of around 10-20 amino acids. Salmonella Saf pili, which are assembled by FGl chaperones. The structure has been well conserved, as they contain a set of alternating hydrophobic residues that form an essential part of the subunit–subunit interaction.
Mechanism.
The mechanism for the assembly reaction is termed donor strand exchange DSE which
Pilus assembly in Gram-negative bacteria involves a Donor-strand exchange mechanism between the C- and the N-termini of this domain. The C-terminal subunit forms an incomplete Ig-fold which is then complemented by the 10-18 residue N terminus of another.
The N terminus sequences contain a motif of alternating hydrophobic residues that occupy the P2 to P5 binding pockets in the groove of the first pilus subunit.
LPXTG pilin.
LPXTG pilin is common in gram-positive cocci. They are named for a C-terminal motif used by the sortase. There is also a LPXTGase.
Development of molecular tools.
LPXTG Pili in Gram-positive bacteria contain spontaneously formed isopeptide bonds. These bonds provide enhanced mechanical and proteolytic stability to the pilin protein. Recently, the pilin protein from "Streptococcus pyogenes" has been split into two fragments to develop a new molecular tool called the isopeptag. The isopeptag is a short peptide that can be attached to a protein of interest and can bind its binding partner through a spontaneously formed isopeptide bond. This new peptide tag can allow scientists to target and isolate their proteins of interest through a permanent covalent bond.


Psychrobacter immobilis

Psychrobacter immobilis is a Gram-negative, oxidase- and catalase-positive, psychrotrophic, nonmotile bacterium of the genus "Psychrobacter" which was isolated from cheese, fish, and processed meat and poultry products.
Further reading.


Gerald Penn

Gerald Penn is a clinical immunologist, and a pioneer in the field of clinical immunoelectrophoresis. He was a student of Henry Kunkel at Rockefeller University from 1968 to 1970, and holds both an M.D. and Ph.D. from the Ohio State University. His research also includes one of the earliest studies on the negative effects of naturally occurring stressors on the response of the human immune system. He is a clinical associate professor of pathology at the Ohio State University.


Small temporal RNA

Small temporal RNA (abbreviated stRNA) regulates gene expression during roundworm development by preventing the mRNAs they bind from being translated. In contrast to siRNA, stRNAs downregulate expression of target RNAs after translation initiation without affecting mRNA stability. Nowadays, stRNAs are better known as miRNAs.
stRNAs exert negative post-transcriptional regulation by binding to complementary sequences in the 3' untranslated regions of their target genes. stRNAs are transcribed as longer precursor RNAs that are processed by the RNase Dicer/DCR-1 and members of the RDE-1/AGO1 family of proteins, which are better known for their roles in RNA interference (RNAi). stRNAs may function to control temporal identity during development in "C. elegans" and other organisms.


Louise de Maisonblanche

Louise Marie Antoinette Josèphe Jeanne de Maisonblanche (17 June 1676 – 12 September 1718), was an illegitimate daughter of Louis XIV of France and Claude de Vin des Œillets, the called "Mademoiselle des Œillets", who was the lady-in-waiting to Madame de Montespan, Louis' long term mistress.
Life.
Born in Paris in 1676, she was one of many illegitimate children that Louis XIV had. She was officially declared "daughter of Philippe de Maisonblanche, old Captain of the Guards, and of Lady Gabrielle de La Tour, his spouse." Brought up in Paris by her mother, she did not receive the same attention that the daughters of Madame de Montespan and Louise de La Vallière did. She lived for a while at the château de Suisnes where her mother died in 1687 aged 50. Louise was then put in the care of François Le Signerre and her sister Catherine Le Signerre in Mulcent.
Louise remained confined at Mulcent with the Le Signerre sisters till the age of 20 in 1696. On 17 April that year, she married Bernard de Prez, Baron of La Queue. He was the lieutenant to the regiment of Burgundy. The Abbé de Brisacier undertook the service which was overseen by Alexandre Bontemps, the first gentleman of the chamber of Louise's father, Louis XIV. It was Bontemps who secured the small dowry of 40,000 Livres along with silver and jewels for the bride. This dowry was nothing compared to the dowries of her other half sisters; the Dowager Princess of Conti (1666–1739) who married Louis Armand de Bourbon in 1680 was given 1 Million Livres as did the Duchess of Bourbon who married in 1685 to Louis de Bourbon. In 1692 another sister the Françoise Marie de Bourbon married Philippe d'Orléans and had been given 2 Million Livres as well as the Palais-Royal in Paris.
Her husband was later named the Kings gardes du corps and this allowed Louise to frequent the court at Versailles with her husband.
Issue.
From her marriage, she was named "dame Louise de Maisonblanche, fille naturelle du Roi". That "style" was shown on the birth certificates of her children. The couple had 11 children, 5 of which died in infancy:
Two of her daughters, Charlotte Angélique and Louise Catherine were raised at Saint-Cyr-l'École, the home of Madame de Maintenon's famous school for young ladies of the nobility. They were both called a "petit-fille du Roi" in their dossiers at Saint-Cyr.
Louise died of smallpox on 12 September 1718 at La Queue-les-Yvelines, during the Regency of her brother in law, Philippe d'Orléans.
Because Louise's mother was known to had several lovers, the king always doubt about his paternity, and for this he never recognised Louise and kept her away from court, treating her poorly at the time of her marriage. Although eyewitnesses say she strongly resembled to him, the king never acknowledged her as his daughter.


Tenrecinae

Tenrecinae is a tenrec subfamily endemic to the island of Madagascar. It contains the largest species in the family, "Tenrec ecaudatus". All members of the genus possess spines, analogous to those of hedgehogs, for defense against predators.
Tenrecinae is thought to have split from the lineages of all other extant tenrecs about 36 million years (Ma) ago. The deepest phylogenetic split within the subfamily, that between two clades composed of "Echinops" plus "Setifer" and "Hemicentetes" plus "Tenrec", is thought to have occurred about 26 Ma ago. In turn, "Hemicentetes" is thought to have diverged from "Tenrec" about 16 Ma ago, and "Echinops" from "Setifer" about 10 Ma ago.
Extant species.
Subfamily Tenrecinae


RNA polymerase

In molecular biology, RNA polymerase (abbreviated RNAP or RNApol, and officially DNA-directed (dependent) RNA polymerase), is an enzyme that synthesizes RNA from a DNA template.
Using the enzyme helicase, RNAP locally opens the double-stranded DNA so that one strand of the exposed nucleotides can be used as a template for the synthesis of RNA, a process called transcription. A transcription factor and its associated transcription mediator complex must be attached to a DNA binding site called a promoter region before RNAP can initiate the DNA unwinding at that position. RNAP not only initiates RNA transcription, it also guides the nucleotides into position, facilitates attachment and elongation, has intrinsic proofreading and replacement capabilities, and termination recognition capability. In eukaryotes, RNAP can build chains as long as 2.4 million nucleotides.
RNAP produces RNA that, functionally, is either for protein coding, i.e. messenger RNA (mRNA); or non-coding (so-called "RNA genes"). At least four functional types of RNA genes exist:
RNA polymerase is essential to life, and is found in all living organisms and many viruses. Depending on the organism, a RNA polymerase can be a protein complex (multi-subunit RNAP) or only consist of one subunit (single-subunit RNAP, ssRNAP), each representing an independent lineage. The former is found in bacteria, archaea, and eukaryotes alike, sharing a similar core structure and mechanism. The latter is found in phages as well as eukaryotic chloroplasts and mitochondria, and is related to modern DNA polymerases. Eukaryotic and archaeal RNAPs have more subunits than bacterial ones do, and are controlled differently.
Bacteria and archaea only have one RNA polymerase. Eukaryotes have multiple types of nuclear RNAP, each responsible for synthesis of a distinct subset of RNA:
Structure.
The 2006 Nobel Prize in Chemistry was awarded to Roger D. Kornberg for creating detailed molecular images of RNA polymerase during various stages of the transcription process.
In most prokaryotes, a single RNA polymerase species transcribes all types of RNA. RNA polymerase "core" from E. coli consists of five subunits: two alpha (α) subunits of 36 kDa, a beta (β) subunit of 150 kDa, a beta prime subunit (β′) of 155 kDa, and a small omega (ω) subunit. A sigma (σ) factor binds to the core, forming the holoenzyme. After transcription starts, the factor can unbind and let the core enzyme proceed with its work. The core RNA polymerase complex forms a "crab claw" or "clamp-jaw" structure with an internal channel running along the full length. Eukaryotic and archaeal RNA polymerases have a similar core structure and work in a similar manner, although they have many extra subunits.
All RNAPs contain metal cofactors, in particular zinc and magnesium cations which aid in the transcription process.
Function.
Control of the process of gene transcription affects patterns of gene expression and, thereby, allows a cell to adapt to a changing environment, perform specialized roles within an organism, and maintain basic metabolic processes necessary for survival. Therefore, it is hardly surprising that the activity of RNAP is long, complex, and highly regulated. In "Escherichia coli" bacteria, more than 100 transcription factors have been identified, which modify the activity of RNAP.
RNAP can initiate transcription at specific DNA sequences known as promoters. It then produces an RNA chain, which is complementary to the template DNA strand. The process of adding nucleotides to the RNA strand is known as elongation; in eukaryotes, RNAP can build chains as long as 2.4 million nucleotides (the full length of the dystrophin gene). RNAP will preferentially release its RNA transcript at specific DNA sequences encoded at the end of genes, which are known as terminators.
Products of RNAP include:
RNAP accomplishes "de novo" synthesis. It is able to do this because specific interactions with the initiating nucleotide hold RNAP rigidly in place, facilitating chemical attack on the incoming nucleotide. Such specific interactions explain why RNAP prefers to start transcripts with ATP (followed by GTP, UTP, and then CTP). In contrast to DNA polymerase, RNAP includes helicase activity, therefore no separate enzyme is needed to unwind DNA.
Action.
Initiation.
RNA polymerase binding in bacteria involves the sigma factor recognizing the core promoter region containing the −35 and −10 elements (located "before" the beginning of sequence to be transcribed) and also, at some promoters, the α subunit C-terminal domain recognizing promoter upstream elements. There are multiple interchangeable sigma factors, each of which recognizes a distinct set of promoters. For example, in "E. coli", σ70 is expressed under normal conditions and recognizes promoters for genes required under normal conditions ("housekeeping genes"), while σ32 recognizes promoters for genes required at high temperatures ("heat-shock genes"). In archaea and eukaryotes, the functions of the bacterial general transcription factor sigma are performed by multiple general transcription factors that work together. The RNA polymerase-promoter closed complex is usually referred to as the "transcription preinitiation complex."
After binding to the DNA, the RNA polymerase switches from a closed complex to an open complex. This change involves the separation of the DNA strands to form an unwound section of DNA of approximately 13 bp, referred to as the "transcription bubble". Supercoiling plays an important part in polymerase activity because of the unwinding and rewinding of DNA. Because regions of DNA in front of RNAP are unwound, there are compensatory positive supercoils. Regions behind RNAP are rewound and negative supercoils are present.
Promoter escape.
RNA polymerase then starts to synthesize the initial DNA-RNA heteroduplex, with ribonucleotides base-paired to the template DNA strand according to Watson-Crick base-pairing interactions. As noted above, RNA polymerase makes contacts with the promoter region. However these stabilizing contacts inhibit the enzyme's ability to access DNA further downstream and thus the synthesis of the full-length product. In order to continue RNA synthesis, RNA polymerase must escape the promoter. It must maintain promoter contacts while unwinding more downstream DNA for synthesis, "scrunching" more downstream DNA into the initiation complex. During the promoter escape transition, RNA polymerase is considered a "stressed intermediate." Thermodynamically the stress accumulates from the DNA-unwinding and DNA-compaction activities. Once the DNA-RNA heteroduplex is long enough (~10 bp), RNA polymerase releases its upstream contacts and effectively achieves the promoter escape transition into the elongation phase. The heteroduplex at the active center stabilizes the elongation complex.
However, promoter escape is not the only outcome. RNA polymerase can also relieve the stress by releasing its downstream contacts, arresting transcription. The paused transcribing complex has two options: (1) release the nascent transcript and begin anew at the promoter or (2) reestablish a new 3'OH on the nascent transcript at the active site via RNA polymerase's catalytic activity and recommence DNA scrunching to achieve promoter escape. Abortive initiation, the unproductive cycling of RNA polymerase before the promoter escape transition, results in short RNA fragments of around 9 bp in a process known as abortive transcription. The extent of abortive initiation depends on the presence of transcription factors and the strength of the promoter contacts.
Elongation.
The 17-bp transcriptional complex has an 8-bp DNA-RNA hybrid, that is, 8 base-pairs involve the RNA transcript bound to the DNA template strand. As transcription progresses, ribonucleotides are added to the 3' end of the RNA transcript and the RNAP complex moves along the DNA. The characteristic elongation rates in prokaryotes and eukaryotes are about 10–100 nts/sec.
Aspartyl (asp) residues in the RNAP will hold on to Mg2+ ions, which will, in turn, coordinate the phosphates of the ribonucleotides. The first Mg2+ will hold on to the α-phosphate of the NTP to be added. This allows the nucleophilic attack of the 3'OH from the RNA transcript, adding another NTP to the chain. The second Mg2+ will hold on to the pyrophosphate of the NTP. The overall reaction equation is:
Fidelity.
Unlike the proofreading mechanisms of DNA polymerase those of RNAP have only recently been investigated. Proofreading begins with separation of the mis-incorporated nucleotide from the DNA template. This pauses transcription. The polymerase then backtracks by one position and cleaves the dinucleotide that contains the mismatched nucleotide. In the RNA polymerase this occurs at the same active site used for polymerization and is therefore markedly different from the DNA polymerase where proofreading occurs at a distinct nuclease active site.
The overall error rate is around 10−4 to 10−6.
Termination.
In bacteria, termination of RNA transcription can be rho-dependent or rho-independent. The former relies on the rho factor, which destablizes the DNA-RNA heteroduplex and causes RNA release. The latter, also known as intrinsic termination, relies on a palindromic region of DNA. Transcribing the region causes the formation of a "hairpin" structure from the RNA transcription looping and binding upon itself. This hairpin structure is often rich in G-C base-pairs, making it more stable than the DNA-RNA hybrid itself. As a result, the 8 bp DNA-RNA hybrid in the transcription complex shifts to a 4 bp hybrid. These last 4 base pairs are weak A-U base pairs, and the entire RNA transcript will fall off the DNA.
Transcription termination in eukaryotes is less well understood than in bacteria, but involves cleavage of the new transcript followed by template-independent addition of adenines at its new 3' end, in a process called polyadenylation.
Other organisms.
Given that DNA and RNA polymerases both carry out template-dependent nucleotide polymerization, it might be expected that the two types of enzymes would be structurally related. However, x-ray crystallographic studies of both types of enzymes reveal that, other than containing a critical Mg2+ ion at the catalytic site, they are virtually unrelated to each other; indeed template-dependent nucleotide polymerizing enzymes seem to have arisen independently twice during the early evolution of cells. One lineage led to the modern DNA polymerases and reverse transcriptases, as well as to a few single-subunit RNA polymerases (ssRNAP) from phages and organelles. The other multi-subunit RNAP lineage formed all of the modern cellular RNA polymerases.
Bacteria.
In bacteria, the same enzyme catalyzes the synthesis of mRNA and non-coding RNA (ncRNA).
RNAP is a large molecule. The core enzyme has five subunits (~400 kDa):
In order to bind promoters, RNAP core associates with the transcription initiation factor sigma (σ) to form RNA polymerase holoenzyme. Sigma reduces the affinity of RNAP for nonspecific DNA while increasing specificity for promoters, allowing transcription to initiate at correct sites. The complete holoenzyme therefore has 6 subunits: β'βαI and αIIωσ (~450 kDa).
Eukaryotes.
Eukaryotes have multiple types of nuclear RNAP, each responsible for synthesis of a distinct subset of RNA. All are structurally and mechanistically related to each other and to bacterial RNAP:
Eukaryotic chloroplasts contain an RNAP very highly similar to bacterial RNAP ("plastid-encoded polymerase, PEP"). They use sigma factors encoded in the nuclear genome.
Chloroplast also contain a second, structurally and mechanistically unrelated, single-subunit RNAP ("nucleus-encoded polymerase, NEP"). Eukaryotic mitochondria use POLRMT (human), a nucleus-encoded single-subunit RNAP. Such phage-like polymerases are referred to as RpoT in plants.
Archaea.
Archaea have a single type of RNAP, responsible for the synthesis of all RNA. Archaeal RNAP is structurally and mechanistically similar to bacterial RNAP and eukaryotic nuclear RNAP I-V, and is especially closely structurally and mechanistically related to eukaryotic nuclear RNAP II.
The history of the discovery of the archaeal RNA polymerase is quite recent. The first analysis of the RNAP of an archaeon was performed in 1971, when the RNAP from the extreme halophile "Halobacterium cutirubrum" was isolated and purified. Crystal structures of RNAPs from "Sulfolobus solfataricus" and "Sulfolobus shibatae" set the total number of identified archaeal subunits at thirteen.
Archaea has the subunit corresponding to Eukaryotic Rpb1 split into two. There is no homolog to eukaryotic Rpb9 (POLR2I) in the "S. shibatae" complex, although TFS (TFIIS homolog) has been proposed as one based on similarity. There is an additional subunit dubbed Rpo13; together with Rpo5 it occupies a space filled by an insertion found in bacterial β' subunits (1,377–1,420 in "Taq"). An earlier, lower-resolution study on "S. solfataricus" structure did not find Rpo13 and only assigned the space to Rpo5/Rpb5. Rpo3 is notable in that it's an iron–sulfur protein. RNAP I/III subunit AC40 found in some eukaryotes share similar sequences, but does not bind iron. This domain, in either case, serves a structural function.
Archaeal RNAP subunit previously used an "RpoX" nomenclature where each subunit is assigned a letter in a way unrelated to any other systems. In 2009, a new nomenclature based on Eukaryotic Pol II subunit "Rpb" numbering was proposed.
Viruses.
Orthopoxviruses and some other nucleocytoplasmic large DNA viruses synthesize RNA using a virally encoded multi-subunit RNAP. They are most similar to eukaryotic RNAPs, with some subunits minified or removed. Exactly which RNAP they are most similar to is a topic of debate. Most other viruses that synthesize RNA use unrelated mechanics.
Many viruses use a single-subunit DNA-dependent RNAP (ssRNAP) that is structurally and mechanistically related to the single-subunit RNAP of eukaryotic chloroplasts (RpoT) and mitochondria (POLRMT) and, more distantly, to DNA polymerases and reverse transcriptases. Perhaps the most widely studied such single-subunit RNAP is bacteriophage T7 RNA polymerase. ssRNAPs cannot proofread.
"B. subtilis" prophage SPβ uses "YonO", a homolog of the β+β' subunits of msRNAPs to form a monomeric (both barrels on the same chain) RNAP distinct from the usual "right hand" ssRNAP. It probably diverged very long ago from the canonical five-unit msRNAP, before the time of the last universal common ancestor.
Other viruses use a RNA-dependent RNAP (an RNAP that employs RNA as a template instead of DNA). This occurs in negative strand RNA viruses and dsRNA viruses, both of which exist for a portion of their life cycle as double-stranded RNA. However, some positive strand RNA viruses, such as poliovirus, also contain RNA-dependent RNAP.
History.
RNAP was discovered independently by Charles Loe, Audrey Stevens, and Jerard Hurwitz in 1960. By this time, one half of the 1959 Nobel Prize in Medicine had been awarded to Severo Ochoa for the discovery of what was believed to be RNAP, but instead turned out to be polynucleotide phosphorylase.
Purification.
RNA polymerase can be isolated in the following ways:
And also combinations of the above techniques.


GDP-L-galactose phosphorylase

GDP-L-galactose phosphorylase (, "VTC2", "VTC5") is an enzyme with systematic name "GDP:alpha-L-galactose 1-phosphate guanylyltransferase". This enzyme catalyses the following chemical reaction
The enzyme catalyses a reaction of the Smirnoff-Wheeler pathway.


Metabolic intermediate

Metabolic intermediates are molecules which are the precursors or metabolites of biologically significant molecules.
Although these intermediates are of relatively minor direct importance to cellular function, they can play important roles in the allosteric regulation of enzymes.
Clinical significance.
Some can be useful in measuring rates of metabolic processes (for example, 3,4-dihydroxyphenylacetic acid or 3-aminoisobutyrate).
Because they can represent unnatural points of entry into natural metabolic pathways, some (such as AICA ribonucleotide) are of interest to researchers in developing new therapies.


BOHD (psychedelic)

BOHD (4-methyl-2,5-dimethoxy-beta-hydroxyphenethylamine) is a lesser-known psychedelic drug. It is the beta-hydroxy derivative of 2C-D. BOHD was first synthesized by Alexander Shulgin. In his book "PiHKAL", the minimum dosage is listed as 50 mg, and the duration unknown. BOHD produces a marked drop in blood pressure. Very little data exists about the pharmacological properties, metabolism, and toxicity of BOHD.
Legality.
United Kingdom.
This substance is a Class A drug in the Drugs controlled by the UK Misuse of Drugs Act.


Non-canonical base pairing

Non-canonical base pairing occurs when nucleobases hydrogen bond, or base pair, to one another in schemes other than the standard Watson-Crick base pairs (which are adenine (A) -- thymine (T) in DNA, adenine (A) -- uracil (U) in RNA, and guanine (G) -- cytosine (C) in both DNA and RNA). There are three main types of non-canonical base pairs: those stabilized by polar hydrogen bonds, those having interactions among C−H and O/N groups, and those that have hydrogen bonds between the bases themselves. The first discovered non-canonical base pairs are Hoogsteen base pairs, which were first described by American biochemist Karst Hoogsteen.
Non-canonical base pairings commonly occur in the secondary structure of RNA (e.g. pairing of G with U), and in tRNA recognition. They are typically less stable than standard base pairings. The presence of non-canonical base pairs in double stranded DNA results in a disrupted double helix.
History.
James Watson and Francis Crick published the double helical structure of DNA and proposed the canonical Watson-Crick base pairs in 1953. Ten years later, in 1963, Karst Hoogsteen reported that he had used single crystal X-ray diffraction to investigate alternative base pair structures, and he found an alternative structure for the nucelobase pair adenine-thymine in which the purine (A) takes on an alternative conformation with respect to the pyrimidine (T). Five years after Hoogsteen proposed the A-T Hoogsteen base pair, optical rotary dispersion spectra which provided evidence for a G-C Hoogsteen base pair were reported. The G-C Hoogsteen base pair was first observed via X-ray crystallography years later, in 1986, by co-crystallizing DNA with triostin A (an antibiotic). Ultimately, after years of studying both Watson-Crick and Hoogsteen base pairs, it has been determined that both occur naturally in DNA, and that they exist in equilibrium with one another; the conditions in which the DNA exists ultimately determine which form will be favored. Since the structures of the canonical Watson-Crick and non-canonical Hoogsteen base pairs were determined, many other types of non-canonical base pairs have been presented and described.
Structure.
Base pairing.
An estimated 60% of bases in structured RNA participate in canonical Watson-Crick base pairs. Base pairing occurs when two bases form hydrogen bonds with each other. These hydrogen bonds can be either polar or non-polar interactions. The polar hydrogen bonds are formed by N-H...O/N and/or O-H...O/N interactions. Non-polar hydrogen bonds are formed between C-H...O/N.
Edge interactions.
Each base has three potential edges where it can interact with another base. The Purine bases have 3 edges which are able to hydrogen bond. Those are known as the Watson-Crick edge(WC), the Hoogsteen edge(H), and the Sugar edge(S). Pyrimidine bases also have three hydrogen-bonding edges. Like the puring, there is the Watson-Crick edge(WC) and the Sugar edge(S) but the third edge is referred to as the "C-H" edge(H) on the pyrimidine bases. This C-H edge is sometimes also referred to as the Hoogsteen edge for simplicity. The various edges for the purine and pyrimidine bases are shown in Figure 2.
Besides the three edges of interaction, base pairs can also vary in their cis/trans forms. The cis and trans structures depend on the orientation of the ribose sugar as opposed to the hydrogen bond interaction. These various orientations are shown in Figure 3. Therefore, with the cis/trans forms and the 3 hydrogen bond edges, there are 12 basic types of base pairing geometries which can be found in RNA structures. Those 12 types are WC:WC (cis/trans), W:HC (cis/trans), WC:S (cis/trans), H:S (cis/trans), H:H (cis/trans), and S:S (cis/trans).
Classification.
These 12 types can be further divided into more subgroups which are dependent on the directionality of the glycosidic bonds and steric extensions. With all of the various base pair combinations there are 169 theoretically possible base pair combinations. The actual number of base-pair combinations is lower because some combinations result in non-favorable interactions. This number of possible non-canonical base pairs is still being determined as it depends strongly on base pairing criteria . Understanding base pair configuration is similarly difficult since the pairing is depends on the bases surroundings. These surroundings can consist of adjacent base pairs, adjacent loops, or third interactions (such as a base triple).
The bonds between various bases are well defined because of their rigid and planar shape. The spatial interactions between the two bases can be classified in 6 rigid-body parameters or intra-base pair parameters (3 translational, 3 rotational) as shown in Figure 4. These parameters describe the base pairs' three dimensional conformation.
The three translational arrangements are known as shear, stretch, and stagger. These three parameters are directly related to the proximity and direction of the hydrogen bonds. The rotational arrangements are buckle, propeller, and opening. Rotational arrangements relate to the non-planar confirmation (as compared to the ideal coplanar geometry). Intra-base pair parameters are used to determine the structure and stabilities of non-canonical base pairs and were originally created for the base pairings in DNA, but were found to also fit the non-canonical base models.
Types.
The most common non-canonical base pairs are trans A:G Hoogsteen/sugar edge, A:U Hoogsteen/WC, and G:U Wobble pairs.
Hoogsteen base pairs.
Hoogsteen base pairs occur between adenine (A) and thymine (T); and guanine (G) and cytosine(C); similarly to Watson-Crick base pairs. However, the purine (A and G) takes on an alternative conformation with respect to the pyrimidine. In the A-U Hoogsteen base pair, the adenine is rotated 180° about the glycosidic bond, resulting in an alternative hydrogen bonding scheme which has one hydrogen bond in common with the Watson-Crick base pair (adenine N6 and thymine N4), while the other hydrogen bond, instead of occurring between adenine N1 and thymine N3 as in the Watson-Crick base pair, occurs between adenine N7 and thymine N3. The A-U base pair is shown in Figure 5. In the G-C Watson-Crick base pair, like the A-T Hoogsteen base pair, the purine (guanine) is rotated 180° about the glycosidic bond while the pyrimidine (cytosine) remains in place. One hydrogen bond from the Watson-Crick base pair is maintained (guanine O6 and cytosine N4) and the other occurs between guanine N7 and a protonated cytosine N3 (note that the Hoogsteen G-C base pair has two hydrogen bonds, while the Watson-Crick G-C base pair has three).
Wobble base pairs.
Wobble base pairing occur between two nucleotides that are not Watson-Crick base pairs and was proposed by Watson in 1966. The 4 main examples are guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C). These wobble base pairs are very important in tRNA. Most organisms have less than 45 tRNA molecules even though 61 tRNA molecules would technically be necessary to canonically pair to the codon. Wobble base pairing allows for the 5' anticodon to bond to a non-standard base pair. Examples of wobble base pairs are given in Figure 6.
3-D Structure.
The secondary and three-dimensional structures of RNA are formed and stabilized through non-canonical base pairs. Base pairs make up many secondary structural blocks which aid the folding of RNA complexes and three dimensional structures. The overall folded RNA is stabilized by the tertiary and secondary structures canonically base pairing together. Due to the many possible non-canonical base pairs, there are an unlimited amount of structures, which allows for the diverse functions of RNA. The arrangement of the non-canonical bases also allow long-range RNA interactions, recognition of proteins and other molecules, and structural stabilizing elements. Many of the common non-canonical base pairs can be added to a stacked RNA stem without disturbing its helical character.
Secondary.
Basic secondary structural elements of RNA include bulges, double helices, hairpin loops, and internal loops. An example of a hairpin loop of RNA is given in Figure 7. As shown in the figure, hairpin loops and internal loops require a sudden change in backbone direction. Non-canonical base pairing allows for the increased flexibility at junctions or turns required in the secondary structure.
Tertiary.
Three-dimensional structures are formed through the long-range intra-molecular interactions between the secondary structures. This leads to the formation of pseudoknots, ribose zippers, kissing hairpin loops, or co-axial pseudocontinuous helices. The three-dimensional structures of RNA are primarily determined through molecular simulations or computationally guided measurements. An example of a Pseudoknot is given in Figure 8.
Experimental Methods.
Watson-Crick canonical base pairing is not the only edge-to-edge conformation possible for the nucleotide since non-canonical pairing can take place as well. Sugar-phosphate backbone has an ionic character, which makes the bases sensitive to their environment, leading to conformational changes, such as non-canonical pairing. There are various methods of prediction for these conformations, such as NMR structure determination and X-ray crystallography.
Applications.
RNA has a multitude of purposes throughout the cell including regulating many important steps in gene expression. Various conformations of the non-Watson-Crick base pairs allow for a multitude of biological functions such as mRNA splicing, siRNA, transport, protein recognition, protein binding, and translation.
One common example of a biological application of non-canonical base pairs is the kink turn. A kink-turn is found throughout many functional RNA species. It consists of a three-nucleotide bulge due to three Hoogsteen base pairs. This kink-turn acts as a marker where various proteins such as the human 15-5k protein or proteins in the L7Ae family can bind. A similar scenario is described in the binding of the HIV-1 Rev-response element (RRE) RNA. RRE RNA has an extra wide deep groove that is caused by cis Watson-Crick G:A pair followed by a trans Watson-Crick G:G. The HIV-1 Rev-response element is then able to bind due to the deepened groove.


D-aspartate oxidase

In enzymology, a D-aspartate oxidase () is an enzyme that catalyzes the chemical reaction
The 3 substrates of this enzyme are D-aspartate, H2O, and O2, whereas its 3 products are oxaloacetate, NH3, and H2O2.
This enzyme belongs to the FAD dependent oxidoreductase family, specifically those acting on the CH-NH2 group of donors with oxygen as acceptor. The systematic name of this enzyme class is D-aspartate:oxygen oxidoreductase (deaminating). Other names in common use include aspartic oxidase, and D-aspartic oxidase. This enzyme participates in alanine and aspartate metabolism. It employs one cofactor, FAD.
The enzyme is encoded by DDO gene.


Lindsay Rosenwald

Lindsay A. Rosenwald is an American doctor and finance expert in asset management, investment banking, venture capital and direct investing within the biotechnology and life-sciences industry, and one of two co-founders and partners of Opus Point Partners, an asset management company that invests in the healthcare space, primarily in biotechnology. He has been a prolific founder of development stage biotech companies.
Rosenwald has started many biotechnology companies who have over 100 licensed clinical-stage medicines. Many of those drugs were approved by the United States Food and Drug Administration as well as in foreign countries. One medicine was approved for Acute promyelocytic leukemia, a routinely fatal disease, that has cured many thousands of incurable patients over the last decade and continues that achievement today, marking a change to how this disease is treated. Another cancer drug developed by one of his start-up companies is today a near billion dollar-selling drug for prostate cancer. Additionally, that company was the first company to be acquired for approximately $1 billion after only phase 2 studies. Other drugs approved by the United States and many other countries include drugs for schizophrenia, fibromyalgia, obesity, influenza, and infant respiratory distress syndrome.
Early life.
Rosenwald graduated from Abington Senior High School, located in southeast Pennsylvania, in 1973, and went on to graduate from Pennsylvania State University with a major in Finance and Economy in 1977, graduating Beta Gamma Sigma. Then he worked as an independent management consultant for health care companies from 1977 to 1979 before entering Temple University at their School of Medicine. After graduating from Temple in 1983, Rosenwald took an internship at Abington Medical Hospital and remained in private medical practice until 1986. At that time, he moved to Wall Street to serve as a physician/financial analyst, a specialized combination of fields which very few boasted at that time.
Career.
In 1988 Lindsay A. Rosenwald became a managing director of corporate finance for D.H. Blair & Co., a privately owned investment firm headed by J. Morton ("Morty") Davis. D.H. Blair underwrote hundreds of companies, many in the biotechnology industry. Several D.H. Blair brokers were indicted for fraud and the firm was shut down. Rosenwald headed a team of three physician-financiers who searched in the medical community for the latest developments that could be marketed with the help of private investment. They got in touch with hospitals, medical schools, pharmaceutical companies, universities, scientific firms and research groups.
In 1991, Lindsay Rosenwald founded Paramount BioCapital, where he dealt with fields of bioresearch and biotechnology. Since 1992, he became a NASD-member dealer. In 1995 Paramount Capital Investments LLC, a merchant and investment bank, was founded. Paramount Capital Asset Management, Inc., owned by Rosenwald, managed the investments of several funds specializing in the technology and biotechnology sectors, since 1994. Rosenwald also serves as a member of the Columbia-Presbyterian Health Sciences Advisory Counsel.
Lindsay A. Rosenwald was number one in the 2002 edition of Genetic Engineering News' 100 Molecular Millionaires. Since 2002, Rosenwald was in the board of directors for Keryx Biopharmaceuticals, Inc., which he resigned in 2006 to devote more time to the biotechnology market.
Cougar Biotechnology, another company founded by Rosenwald, was purchased by Johnson & Johnson in 2009 based solely on abiraterone acetate. This drug was not yet in phase III clinical trials for its application in prostate cancer treatment and therapy, but was so promising that Johnson & Johnson bid & purchased the company in a "short-term merger" for approximately $1 billion.
In addition to his activities which have provided capital and funding to many portfolio companies, Rosenwald also started the Rosenwald Foundation, a nonprofit organization which has provided millions of dollars to support various scientific and medical education institutions.
In 2009, Lindsay A. Rosenwald and Michael S. Weiss founded a new company called OpusPoint Partners. This company specializes in healthcare and life sciences investments and consulting. In October 2010, he invested in securities in National Holdings Corporation purchasing approximately 23.6% of NHC.


Degarelix

Degarelix, sold under the brand name Firmagon among others, is a hormonal therapy used in the treatment of prostate cancer.
Testosterone is a male hormone that promotes growth of many prostate tumours and therefore reducing circulating testosterone to very low (castration) levels is often the treatment goal in the management of advanced prostate cancer. Degarelix has an immediate onset of action, binding to gonadotropin-releasing hormone (GnRH) receptors in the pituitary gland and blocking their interaction with GnRH. This induces a fast and profound reduction in luteinizing hormone (LH), follicle-stimulating hormone (FSH) and in turn, testosterone suppression.
Medical uses.
The GnRH antagonist degarelix, through its ability to reduce serum testosterone, is used to treat hormone-sensitive prostate cancer.
Side effects.
As with all hormonal therapies, degarelix is commonly associated with hormonal side effects such as hot flashes and weight gain. Due to its mode of administration (subcutaneous injection), degarelix is also associated with injection-site reactions such as injection-site pain, erythema or swelling. Injection-site reactions are usually mild or moderate in intensity and occur predominantly after the first dose, decreasing in frequency thereafter. Less common: Anemia. Diarrhea, nausea. Hyperhidrosis including night sweats, rash. Gynecomastia, testicular atrophy, erectile dysfunction. Increased transaminases. Musculoskeletal pain and discomfort. Dizziness, headache. Insomnia. Weight gain. Chills, fever, fatigue, flu-like illness.
Pharmacology.
GnRH antagonists (receptor blockers) such as degarelix are synthetic peptide derivatives of the natural GnRH decapeptide – a hormone that is made by neurons in the hypothalamus. GnRH antagonists compete with natural GnRH for binding to GnRH receptors in the pituitary gland. This reversible binding blocks the release of LH and FSH from the pituitary. The reduction in LH subsequently leads to a rapid and sustained suppression of testosterone release from the testes and subsequently reduces the size and growth of the prostate cancer. This in turn results in a reduction in prostate-specific antigen (PSA) levels in the patient's blood. Measuring PSA levels helps to monitor how patients with prostate cancer are responding to treatment.
Unlike GnRH agonists, which cause an initial stimulation of the hypothalamic-pituitary-gonadal axis (HPGA), leading to a surge in testosterone levels, and under certain circumstances, a flare-up of the tumour, GnRH antagonists do not cause a surge in testosterone or clinical flare. Clinical flare is a phenomenon that occurs in patients with advanced disease, which can precipitate a range of clinical symptoms such as bone pain, urethral obstruction, and spinal cord compression. Drug agencies have issued boxed warnings regarding this phenomenon in the prescribing information for GnRH agonists. As testosterone surge does not occur with GnRH antagonists, there is no need for patients to receive an antiandrogen as flare protection during prostate cancer treatment. GnRH agonists also induce an increase in testosterone levels after each reinjection of the drug – a phenomenon that does not occur with GnRH antagonists such as degarelix.
GnRH antagonists have an immediate onset of action leading to a fast and profound suppression of testosterone and are therefore especially valuable in the treatment of patients with prostate cancer where fast control of disease is needed.
History.
In December 2008, the US Food and Drug Administration (FDA) approved degarelix for the treatment of patients with advanced prostate cancer in the United States. It was subsequently approved by the European Commission at the recommendation of the European Medicines Agency (EMA) in February 2009, for use in adult males with advanced, hormone-dependent prostate cancer. Ferring Pharmaceuticals markets the drug under the name "Firmagon".
Research.
Degarelix is studied for use as a chemical castration agent on sex offenders in Sweden. A study published on April 29, 2020 in "JAMA Psychiatry" demonstrated a reduced the risk score for committing child sexual abuse in men with pedophilic disorder 2 weeks after initial injection.


Sanjib Senapati

Sanjib Senapati is an Indian biophysicist, biochemist, biotechnologist and a professor at the Bhupat and Jyoti Mehta School of Biosciences, Department of Biotechnology of the Indian Institute of Technology Madras. He is known for his studies in the fields of protein dynamics, green chemistry and nanoclusters and is a Fulbright scholar. The Department of Biotechnology of the Government of India awarded him the National Bioscience Award for Career Development, one of the highest Indian science awards, for his contributions to biosciences, in 2015.
Biography.
Born in the Indian state of West Bengal, Sanjib Senapati secured his master's degree from the University of Calcutta and did his doctoral studies at the Indian Institute of Technology Kanpur which earned him a PhD. Subsequently, he joined the Indian Institute of Technology Madras (IITM) as a faculty member of the department of biotechnology and holds the position of a professor at the Bhupat and Jyoti Mehta School of Biosciences at IITM. The team led by him focuses their research in the fields of protein dynamics, green chemistry and nanoclusters. His studies have been documented by way of a number of articles and Google Scholar, an online repository of scientific articles has listed 110 of them.
The Department of Biotechnology (DBT) of the Government of India awarded Senapati the National Bioscience Award for Career Development, one of the highest Indian science awards in 2015. In 2017, he was selected for the Fulbright Scholarship program by the United States Department of State Bureau of Educational and Cultural Affairs for working on a project at the laboratory of Andrej Šali of the University of California, San Francisco, during 2017–18.


ZinT protein domain

In molecular biology, ZinT (formerly known as YodA) is a family of protein domains found in prokaryotes. The domain contains a single binding site that can accommodate a divalent cation, with a geometry suggestive of zinc binding. This family was first thought to be part of the bacterial response to a toxic heavy metal cadmium by binding to the metal to ensure its elimination; however, more recent work has suggested a role in zinc homeostasis.
Function.
There is strong evidence for involvement of the ZinT domain in zinc homeostasis and management of zinc in the periplasm. It may also facilitate zinc uptake from the environment through interactions with the znuABC zinc transporter. It is regulated by the metalloregulator gene Zur ("z"inc "u"ptake "r"egulator).
The domain was originally discovered in the bacterial stress response to cadmium. Further studies have found that it binds to cadmium, zinc, nickel, and mercury, but not other common metals such as cobalt, copper, iron, and manganese. It may have a secondary function in managing heavy-metal toxicity.


Reductase

A reductase is an enzyme that catalyzes a reduction reaction.


Survival activating factor enhancement

Survivor Activating Factor Enhancement (SAFE) is a metabolic pathway. It is an intrinsic protective signaling programme to limit cell death activated by the heart. This pathway allows ischaemic postconditioning that helps protect against reperfusion injury. This path involves the activation of a transcription factor called "signal transducer and activator of transcription 3" (STAT3).
Activation.
A 2011 study concludes, “Our data demonstrate that both melatonin and resveratrol, as found in red wine, protect the heart in an experimental model of myocardial infarction via the SAFE pathway.”