Phycocyanin

Phycocyanin is a pigment-protein complex from the light-harvesting phycobiliprotein family, along with allophycocyanin and phycoerythrin. It is an accessory pigment to chlorophyll. All phycobiliproteins are water-soluble, so they cannot exist within the membrane like carotenoids can. Instead, phycobiliproteins aggregate to form clusters that adhere to the membrane called phycobilisomes. Phycocyanin is a characteristic light blue color, absorbing orange and red light, particularly near 620 nm (depending on which specific type it is), and emits fluorescence at about 650 nm (also depending on which type it is). Allophycocyanin absorbs and emits at longer wavelengths than phycocyanin C or phycocyanin R. Phycocyanins are found in cyanobacteria (also called blue-green algae). Phycobiliproteins have fluorescent properties that are used in immunoassay kits. Phycocyanin is from the Greek "phyco" meaning “algae” and "cyanin" is from the English word “cyan", which conventionally means a shade of blue-green (close to "aqua") and is derived from the Greek “kyanos" which means a somewhat different color: "dark blue". The product phycocyanin, produced by "Aphanizomenon flos-aquae" and Spirulina, is for example used in the food and beverage industry as the natural coloring agent 'Lina Blue' or 'EXBERRY Shade Blue' and is found in sweets and ice cream. In addition, fluorescence detection of phycocyanin pigments in water samples is a useful method to monitor cyanobacteria biomass.
The phycobiliproteins are made of two subunits (alpha and beta) having a protein backbone to which 1-2 linear tetrapyrrole chromophores are covalently bound.
C-phycocyanin is often found in cyanobacteria which thrive around hot springs, as it can be stable up to around 70 °C, with identical spectroscopic (light absorbing) behaviours at 20 and 70 °C. Thermophiles contain slightly different amino acid sequences making it stable under these higher conditions. Molecular weight is around 30,000 Da.
Stability of this protein invitro at these temperatures has been shown to be substantially lower. Photo-spectral analysis of the protein after 1 min exposure to 65 °C conditions in a purified state demonstrated a 50% loss of tertiary structure.
Structure.
Phycocyanin shares a common structural theme with all phycobiliproteins. The structure begins with the assembly of phycobiliprotein monomers, which are heterodimers composed of α and β subunits, and their respective chromophores linked via thioether bond.
Each subunit is typically composed of eight α-helices. Monomers spontaneously aggregate to form ring-shaped trimers (αβ)3, which have rotational symmetry and a central channel. Trimers aggregate in pairs to form hexamers (αβ)6, sometimes assisted with additional linker proteins. Each phycobilisome rod generally has two or more phycocyanin hexamers. Despite the overall similarity in structure and assembly of phycobiliproteins, there is a large diversity in hexamer and rod conformations, even when only considering phycocyanins. On a larger scale phycocyanins also vary in crystal structure, although the biological relevance of this is debatable.
As an example, the structure of C-phycocyanin from "Synechococcus vulcanus" has been refined to 1.6 Angstrom resolution. The (αβ) monomer consists of 332 amino acids and 3 thio-linked phycocyanobilin (PCB) cofactor molecules. Both the α- and β-subunits have a PCB at amino acid 84, but the β-subunit has an additional PCB at position 155 as well. This additional PCB faces the exterior of the trimeric ring and is therefore implicated in inter-rod energy transfer in the phycobilisome complex. In addition to cofactors, there are many predictable non-covalent interactions with the surrounding solvent (water) that are hypothesized to contribute to structural stability.
R-phycocyanin II (R-PC II) is found in some "Synechococcus" species. R-PC II is said to be the first PEB containing phycocyanin that originates in cyanobacteria. Its purified protein is composed of alpha and beta subunits in equal quantities. R-PC II has PCB at beta-84 and the phycoerythrobillin (PEB) at alpha-84 and beta-155.
As of March 7, 2018, there are 44 crystal structures of phycocyanin deposited in the Protein Data Bank.
Spectral characteristics.
C-phycocyanin has a single absorption peak at ~621 nm, varying slightly depending on the organism and conditions such as temperature, pH, and protein concentration "in vitro". Its emission maximum is ~642 nm. This means that the pigment absorbs orange light, and emits reddish light. R-phycocyanin has an absorption maxima at 533 and 544 nm. The fluorescence emission maximum of R-phycocyanin is 646 nm.
Ecological relevance.
Phycocyanin is produced by many photoautotrophic cyanobacteria. Even if cyanobacteria have large concentrations of phycocyanin, productivity in the ocean is still limited due to light conditions.
Phycocyanin has ecological significance in indicating cyanobacteria bloom. Normally chlorophyll "a" is used to indicate cyanobacteria numbers, however since it is present in a large number of phytoplankton groups, it is not an ideal measure. For instance a study in the Baltic Sea used phycocyanin as a marker for filamentous cyanobacteria during toxic summer blooms. Some filamentous organisms in the Baltic Sea include "Nodularia spumigena" and "Aphanizomenon flosaquae".
An important cyanobacteria named spirulina ("Arthrospira plantensis") is a micro algae that produces C-PC.
There are many different methods of phycocyanin production including photoautotrophic, mixotrophic and heterotrophic and recombinant production. Photoautotrophic production of phycocyanin is where cultures of cyanobacteria are grown in open ponds in either subtropical or tropical regions. Mixotrophic production of algae is where the algae are grown on cultures that have an organic carbon source like glucose. Using mixotrophic production produces higher growth rates and higher biomass compared to simply using a photoautotrophic culture. In the mixotrophic culture, the sum of heterotrophic and autotrophic growth separately was equal to the mixotrophic growth. Heterotrophic production of phycocyanin is not light limited, as per its definition. "Galdieria sulphuraria" is a unicellular rhodophyte that contains a large amount of C-PC and a small amount of allophycocyanin. "G. sulphuraria" is an example of the heterotrophic production of C-PC because its habitat is hot, acidic springs and uses a number of carbon sources for growth. Recombinant production of C-PC is another heterotrophic method and involves gene engineering.
Lichen-forming fungi and cyanobacteria often have a symbiotic relationship and thus phycocyanin markers can be used to show the ecological distribution of fungi-associated cyanobacteria. As shown in the highly specific association between Lichina species and Rivularia strains, phycocyanin has enough phylogenetic resolution to resolve the evolutionary history of the group across the northwestern Atlantic Ocean coastal margin.
Biosynthesis.
The two genes cpcA and cpcB, located in the cpc operon and translated from the same mRNA transcript, encode for the C-PC α- and β-chains respectively. Additional elements such as linker proteins, and enzymes involved in phycobilin synthesis and the phycobiliproteins are often encoded by genes in adjacent gene clusters, and the cpc operon of Arthrospira platensis also encodes a linker protein assisting in the assembly of C-PC complexes. In red algae, the phycobiliprotein and linker protein genes are located on the plastid genome.
Phycocyanobilin is synthesised from heme and inserted into the C-PC apo-protein by three enzymatic steps. Cyclic heme is oxidised to linear biliverdin IXα by heme oxygenase and further converted to 3Z-phycocyanobilin, the dominant phycocyanobilin isomer, by 3Z-phycocyanobilin:ferredoxin oxidoreductase. Insertion of 3Z-phycocyanobilin into the C-PC apo-protein via thioether bond formation is catalysed by phycocyanobilin lyase.
The promoter for the cpc operon is located within the 427-bp upstream region of the cpcB gene. In "A. platensis", 6 putative promoter sequences have been identified in the region, with four of them showing expression of green fluorescent protein when transformed into "E. coli". The presence of other positive elements such as light-response elements in the same region have also been demonstrated.
The multiple promoter and response element sequences in the cpc operon enable cyanobacteria and red algae to adjust its expression in response to multiple environmental conditions. Expression of the cpcA and cpcB genes is regulated by light. Low light intensities stimulate synthesis of CPC and other pigments, while pigment synthesis is repressed at high light intensities. Temperature has also been shown to affect synthesis, with specific pigment concentrations showing a clear maximum at 36 °C in Arthronema africanum, a cyanobacterium with particular high C-PC and APC contents.
Nitrogen and also iron limitation induce phycobiliprotein degradation. Organic carbon sources stimulate C-PC synthesis in Anabaena spp., but seem to have almost no effector negative effect in A. platensis. In the rhodophytes Cyanidium caldarium and Galdieria sulphuraria, C-PC production is repressed by glucose but stimulated by heme.
Biotechnology.
Pure phycocyanin extractions can be isolated from algae. The basic segregation order is as follows. The rupturing of the cell wall, with mechanical forces (freeze thawing) or chemical agents (enzymes). Then, C-PC is isolated with centrifugation and purified with ammonium sulfate precipitation or chromatography -either ion or gel-filtration. After, the sample gets frozen and dried.<br>.
Applications.
Phycocyanin can be used in many practices, it is particularly used medicine and foods applications. It can also be used in genetics, where it acts a tracer due to its natural fluorescence.
Medicine.
Anti-oxidation and anti-inflammation.
Phycocyanin has both anti-oxidant and anti-inflammation properties. Peroxyl, hydroxyl, and alkoxyl radicals are all oxidants scavenged by C-PC. C-PC, however, has a greater effect on peroxyl radicals. C-PC is a metal binding antioxidant as it prevents lipid peroxidation from occurring. The peroxyl radicals are stabilized by the chromophore (a subunit of C-PC). For hydroxyl radicals to be scavenged, it must be done in low light and with high C-PC levels. Hydroxyl radicals are found at inflamed parts of the body. C-PC, being an anti-oxidant, scavenges these damage-inducing radicals, hence being an anti-inflammation agent.
Neuroprotection.
Excess oxygen in the brain generates Reactive Oxygen Species (ROS). ROS causes damages to brain neurons, leading to strokes. C-phycocyanin scavenges hydrogen peroxide, a type of ROS species, from the inside of astrocyte, reducing oxidative stress. Astrocytes also increase the production of growth factors like BDNF and NDF, therefore, enhance nerve regeneration. C-PC also prevents astrogliosis and glial inflammation.
Hepatoprotection.
C-phycocyanin is found to have hepatotoxicity protection. Vadiraja et al. (1998) found an increase in the serum glutamic pyruvic transaminase (SGPT) when C-PC is treated against heptatoxins such as Carbon tetrachloride (CCl4) or R-(+)-pulegone. C-PC protects the liver by the means of the Cytochrome-P450 system. It can either disturb the production of menthofuran or disturb formation of α, β-unsaturated- γ-ketoaldehyde. Both of which are key components of the cytochrome P-450 system that produced a reactive metabolite that produce toxins when it binds to liver tissues. Another possible protection mechanism by C-PC can be the scavenging of reactive metabolites (or free radicals if the cause is CCl4).
Anti-cancer.
C-phycocyanin (C-PC) has anti-cancer effects. Cancer happens when cells continue to grow uncontrollably. C-PC has been found to prevent cell growth. C-PC stops the formation of tumour before the S phase. DNA synthesis is not performed due to the tumour cell entering G0, resulting in no tumour proliferation. Furthermore, C-PC induces apoptosis. When cells are treated with C-PC, ROS (Radical Oxygen Species) are made. These molecules decrease BCl-2 (regulator of apoptosis) production. Here, BCl-2 inhibits proteins called caspases. Caspases are part of the apoptosis pathway. When BCl-2 decreases, the expression of caspases increases. As a result, apoptosis occurs. C-PC alone is not enough to treat cancer, it needs to work other drugs to overcome the persistence nature of tumour cells.
Food.
C-phycocyanin (C-PC) can be used as a natural blue food colouring. This food colourant can only be used for low temperature prepared goods because of its inability to maintaining its blue colouring in high heats unless there is an addition of preservatives or sugars. The type of sugar is irrelevant, C-PC is stable when there is high sugar content. Knowing so, C-PC can be used for numerous types of foods, one of which being syrups. C-PC can be used for syrups ranging from green to blue colours. It can have different green tints by adding yellow food colourings.
Market.
Phycocyanin market is expected to grow in the period 2019-2028 as a phycocyanin blue pigment got approval from FDA in 2013 which has opened a lot of opportunities for this market and it is anticipated to reach a 1 Billion USD during forecast period 2019-2028.


Metabolite

In biochemistry, a metabolite is an intermediate or end product of metabolism.
The term "metabolite" is usually used for small molecules. Metabolites have various functions, including fuel, structure, signaling, stimulatory and inhibitory effects on enzymes, catalytic activity of their own (usually as a cofactor to an enzyme), defense, and interactions with other organisms (e.g. pigments, odorants, and pheromones).
A primary metabolite is directly involved in normal "growth", development, and reproduction. Ethylene exemplifies a primary metabolite produced large-scale by industrial microbiology.
A secondary metabolite is not directly involved in those processes, but usually has an important ecological function. Examples include antibiotics and pigments such as resins and terpenes etc.
Some antibiotics use primary metabolites as precursors, such as actinomycin, which is created from the primary metabolite tryptophan. Some sugars are metabolites, such as fructose or glucose, which are both present in the metabolic pathways.
Examples of primary metabolites produced by industrial microbiology include:
The metabolome forms a large network of metabolic reactions, where outputs from one enzymatic chemical reaction are inputs to other chemical reactions.
Metabolites from chemical compounds, whether inherent or pharmaceutical, form as part of the natural biochemical process of degrading and eliminating the compounds.
The rate of degradation of a compound is an important determinant of the duration and intensity of its action. Understanding how pharmaceutical compounds are metabolized and the potential side effects of their metabolites is an important part of drug discovery.


Looxcie

Looxcie was a mobile-connected, handsfree, streaming video camera created by Looxcie, Inc., a privately owned Sunnyvale, California company. The Looxcie video camera was named a top 50 best invention of 2010 by "Time Magazine" (November 2010), and LooxcieLive, their live streaming video service, was named as a top 100 best innovation of 2011 by "Popular Science" (December 2011).
Mobile-connected streaming video cameras.
Looxcie is a mobile-connected, handsfree, streaming video camera that facilitates live video streaming and capture. The live video streaming is designed for real-time sharing. Video clips can be saved on the camera and shared via a mobile device or uploaded to a computer.
Looxcie video cameras are compatible with Android (2.1+) and iOS (4.2.6+) devices. Looxcie utilizes a dual processor system for simultaneous video capture, wireless streaming and phone calls via Bluetooth. The Looxcie maximizes battery life (up to 4 hours) and optimizes video for viewing (480p) and mobile sharing (320p) in a small wearable, ergonomic form factor.
Looxcie 2.
Looxcie 2 features an adjustable fit system with attachment accessories. Looxcie 2 weighs 22g (0.78 oz) and comes in two configurations: up to 5 hour video storage model and up to 10 hour video storage model.
Looxcie 1.
Looxcie 1 weighs less than 28g (0.99 oz) and provides up to 5 hour video storage.
Mobile apps.
Looxcie companion apps are available for download from LooxcieDesktop, the App Store and Play Store (Android Market).
LooxcieLive.
LooxcieLive lets users stream live video and view other people’s broadcasts in real-time. Push-to-Talk and Text Chat features allow viewers to dialog with the broadcaster while they live stream. Video streaming auto-adapts based on network capabilities – 3G, 4G or Wi-Fi.
LooxcieMoments.
LooxcieMoments allows users to continuously record video into a looping buffer. With the Instant Clip button on Looxcie, users can reach back and save the last 30-seconds of an event, which can then be shared to social networks and email.
LooxcieCam.
LooxcieCam provides users with basic camcorder functionality.


Leucoanthocyanidin reductase

In enzymology, a leucoanthocyanidin reductase () (LAR, aka leucocyanidin reductase or LCR) is an enzyme that catalyzes the chemical reaction
The 3 substrates of this enzyme are (2R,3S)-catechin, NADP+, and H2O, whereas its 3 products are 2,3-trans-3,4-cis-leucocyanidin, NADPH, and H+.
This enzyme belongs to the family of oxidoreductases, specifically those acting on CH or CH2 groups with NAD+ or NADP+ as acceptor. The systematic name of this enzyme class is (2R,3S)-catechin:NADP+ 4-oxidoreductase. This enzyme is also called leucocyanidin reductase. This enzyme participates in flavonoid biosynthesis.
The enzyme can be found in the plant "Hedysarum sulphurescens" and in "Vitis vinifera" (grape).


TRIMU 5

TRIMU 5 is a selective agonist of the mu opioid receptor type 2.


Transformation (genetics)

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.
Transformation is one of three processes for horizontal gene transfer, in which exogenous genetic material passes from one bacterium to another, the other two being conjugation (transfer of genetic material between two bacterial cells in direct contact) and transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium). In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium.
As of 2014 about 80 species of bacteria were known to be capable of transformation, about evenly divided between Gram-positive and Gram-negative bacteria; the number might be an overestimate since several of the reports are supported by single papers.
"Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "transformation" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called "transfection".
History.
Transformation in bacteria was first demonstrated in 1928 by the British bacteriologist Frederick Griffith. Griffith was interested in determining whether injections of heat-killed bacteria could be used to vaccinate mice against pneumonia. However, he discovered that a non-virulent strain of "Streptococcus pneumoniae" could be made virulent after being exposed to heat-killed virulent strains. Griffith hypothesized that some "transforming principle" from the heat-killed strain was responsible for making the harmless strain virulent. In 1944 this "transforming principle" was identified as being genetic by Oswald Avery, Colin MacLeod, and Maclyn McCarty. They isolated DNA from a virulent strain of "S. pneumoniae" and using just this DNA were able to make a harmless strain virulent. They called this uptake and incorporation of DNA by bacteria "transformation" (See Avery-MacLeod-McCarty experiment) The results of Avery et al.'s experiments were at first skeptically received by the scientific community and it was not until the development of genetic markers and the discovery of other methods of genetic transfer (conjugation in 1947 and transduction in 1953) by Joshua Lederberg that Avery's experiments were accepted.
It was originally thought that "Escherichia coli", a commonly used laboratory organism, was refractory to transformation. However, in 1970, Morton Mandel and Akiko Higa showed that "E. coli" may be induced to take up DNA from bacteriophage λ without the use of helper phage after treatment with calcium chloride solution. Two years later in 1972, Stanley Norman Cohen, Annie Chang and Leslie Hsu showed that treatment is also effective for transformation of plasmid DNA. The method of transformation by Mandel and Higa was later improved upon by Douglas Hanahan. The discovery of artificially induced competence in "E. coli" created an efficient and convenient procedure for transforming bacteria which allows for simpler molecular cloning methods in biotechnology and research, and it is now a routinely used laboratory procedure.
Transformation using electroporation was developed in the late 1980s, increasing the efficiency of in-vitro transformation and increasing the number of bacterial strains that could be transformed. Transformation of animal and plant cells was also investigated with the first transgenic mouse being created by injecting a gene for a rat growth hormone into a mouse embryo in 1982. In 1907 a bacterium that caused plant tumors, "Agrobacterium tumefaciens", was discovered and in the early 1970s the tumor-inducing agent was found to be a DNA plasmid called the Ti plasmid. By removing the genes in the plasmid that caused the tumor and adding in novel genes, researchers were able to infect plants with "A. tumefaciens" and let the bacteria insert their chosen DNA into the genomes of the plants. Not all plant cells are susceptible to infection by "A. tumefaciens", so other methods were developed, including electroporation and micro-injection. Particle bombardment was made possible with the invention of the Biolistic Particle Delivery System (gene gun) by John Sanford in the 1980s.
Definitions.
Transformation is one of three forms of horizontal gene transfer that occur in nature among bacteria, in which DNA encoding for a trait passes from one bacterium to another and is integrated into the recipient genome by homologous recombination; the other two are transduction, carried out by means of a bacteriophage, and conjugation, in which a gene is passed through direct contact between bacteria. In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium.
Competence refers to a temporary state of being able to take up exogenous DNA from the environment; it may be induced in a laboratory.
It appears to be an ancient process inherited from a common prokaryotic ancestor that is a beneficial adaptation for promoting recombinational repair of DNA damage, especially damage acquired under stressful conditions. Natural genetic transformation appears to be an adaptation for repair of DNA damage that also generates genetic diversity.
Transformation has been studied in medically important Gram-negative bacteria species such as "Helicobacter pylori", "Legionella pneumophila", "Neisseria meningitidis", "Neisseria gonorrhoeae", "Haemophilus influenzae" and "Vibrio cholerae". It has also been studied in Gram-negative species found in soil such as "Pseudomonas stutzeri", "Acinetobacter baylyi", and Gram-negative plant pathogens such as "Ralstonia solanacearum" and "Xylella fastidiosa". Transformation among Gram-positive bacteria has been studied in medically important species such as "Streptococcus pneumoniae", "Streptococcus mutans", "Staphylococcus aureus" and "Streptococcus sanguinis" and in Gram-positive soil bacterium "Bacillus subtilis". It has also been reported in at least 30 species of "Proteobacteria" distributed in the classes alpha, beta, gamma and epsilon. The best studied "Proteobacteria" with respect to transformation are the medically important human pathogens "Neisseria gonorrhoeae" (class beta), "Haemophilus influenzae" (class gamma) and "Helicobacter pylori" (class epsilon)
"Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "transformation" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called "transfection".
Natural competence and transformation.
As of 2014 about 80 species of bacteria were known to be capable of transformation, about evenly divided between Gram-positive and Gram-negative bacteria; the number might be an overestimate since several of the reports are supported by single papers.
Naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s). The transport of the exogenous DNA into the cells may require proteins that are involved in the assembly of type IV pili and type II secretion system, as well as DNA translocase complex at the cytoplasmic membrane.
Due to the differences in structure of the cell envelope between Gram-positive and Gram-negative bacteria, there are some differences in the mechanisms of DNA uptake in these cells, however most of them share common features that involve related proteins. The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through the cytoplasmic membrane via DNA translocase. Only single-stranded DNA may pass through, the other strand being degraded by nucleases in the process. The translocated single-stranded DNA may then be integrated into the bacterial chromosomes by a RecA-dependent process. In Gram-negative cells, due to the presence of an extra membrane, the DNA requires the presence of a channel formed by secretins on the outer membrane. Pilin may be required for competence, but its role is uncertain. The uptake of DNA is generally non-sequence specific, although in some species the presence of specific DNA uptake sequences may facilitate efficient DNA uptake.
Natural transformation.
Natural transformation is a bacterial adaptation for DNA transfer that depends on the expression of numerous bacterial genes whose products appear to be responsible for this process. In general, transformation is a complex, energy-requiring developmental process. In order for a bacterium to bind, take up and recombine exogenous DNA into its chromosome, it must become competent, that is, enter a special physiological state. Competence development in "Bacillus subtilis" requires expression of about 40 genes. The DNA integrated into the host chromosome is usually (but with rare exceptions) derived from another bacterium of the same species, and is thus homologous to the resident chromosome.
In "B. subtilis" the length of the transferred DNA is greater than 1271 kb (more than 1 million bases). The length transferred is likely double stranded DNA and is often more than a third of the total chromosome length of 4215 kb. It appears that about 7-9% of the recipient cells take up an entire chromosome.
The capacity for natural transformation appears to occur in a number of prokaryotes, and thus far 67 prokaryotic species (in seven different phyla) are known to undergo this process.
Competence for transformation is typically induced by high cell density and/or nutritional limitation, conditions associated with the stationary phase of bacterial growth. Transformation in "Haemophilus influenzae" occurs most efficiently at the end of exponential growth as bacterial growth approaches stationary phase. Transformation in "Streptococcus mutans", as well as in many other streptococci, occurs at high cell density and is associated with biofilm formation. Competence in "B. subtilis" is induced toward the end of logarithmic growth, especially under conditions of amino acid limitation. Similarly, in "Micrococcus luteus" (a representative of the less well studied "Actinobacteria" phylum), competence develops during the mid-late exponential growth phase and is also triggered by amino acids starvation.
By releasing intact host and plasmid DNA, certain bacteriophages are thought to contribute to transformation.
Transformation, as an adaptation for DNA repair.
Competence is specifically induced by DNA damaging conditions. For instance, transformation is induced in "Streptococcus pneumoniae" by the DNA damaging agents mitomycin C (a DNA cross-linking agent) and fluoroquinolone (a topoisomerase inhibitor that causes double-strand breaks). In "B. subtilis", transformation is increased by UV light, a DNA damaging agent. In "Helicobacter pylori", ciprofloxacin, which interacts with DNA gyrase and introduces double-strand breaks, induces expression of competence genes, thus enhancing the frequency of transformation Using "Legionella pneumophila", Charpentier et al. tested 64 toxic molecules to determine which of these induce competence. Of these, only six, all DNA damaging agents, caused strong induction. These DNA damaging agents were mitomycin C (which causes DNA inter-strand crosslinks), norfloxacin, ofloxacin and nalidixic acid (inhibitors of DNA gyrase that cause double-strand breaks), bicyclomycin (causes single- and double-strand breaks), and hydroxyurea (induces DNA base oxidation). UV light also induced competence in "L. pneumophila". Charpentier et al. suggested that competence for transformation probably evolved as a DNA damage response.
Logarithmically growing bacteria differ from stationary phase bacteria with respect to the number of genome copies present in the cell, and this has implications for the capability to carry out an important DNA repair process. During logarithmic growth, two or more copies of any particular region of the chromosome may be present in a bacterial cell, as cell division is not precisely matched with chromosome replication. The process of homologous recombinational repair (HRR) is a key DNA repair process that is especially effective for repairing double-strand damages, such as double-strand breaks. This process depends on a second homologous chromosome in addition to the damaged chromosome. During logarithmic growth, a DNA damage in one chromosome may be repaired by HRR using sequence information from the other homologous chromosome. Once cells approach stationary phase, however, they typically have just one copy of the chromosome, and HRR requires input of homologous template from outside the cell by transformation.
To test whether the adaptive function of transformation is repair of DNA damages, a series of experiments were carried out using "B. subtilis" irradiated by UV light as the damaging agent (reviewed by Michod et al. and Bernstein et al.) The results of these experiments indicated that transforming DNA acts to repair potentially lethal DNA damages introduced by UV light in the recipient DNA. The particular process responsible for repair was likely HRR. Transformation in bacteria can be viewed as a primitive sexual process, since it involves interaction of homologous DNA from two individuals to form recombinant DNA that is passed on to succeeding generations. Bacterial transformation in prokaryotes may have been the ancestral process that gave rise to meiotic sexual reproduction in eukaryotes (see Evolution of sexual reproduction; Meiosis.)
Methods and mechanisms of transformation in laboratory.
Bacterial.
Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature. Typically the cells are incubated in a solution containing divalent cations (often calcium chloride) under cold conditions, before being exposed to a heat pulse (heat shock). Calcium chloride partially disrupts the cell membrane, which allows the recombinant DNA to enter the host cell. Cells that are able to take up the DNA are called competent cells.
It has been found that growth of Gram-negative bacteria in 20 mM Mg reduces the number of protein-to-lipopolysaccharide bonds by increasing the ratio of ionic to covalent bonds, which increases membrane fluidity, facilitating transformation. The role of lipopolysaccharides here are verified from the observation that shorter O-side chains are more effectively transformed – perhaps because of improved DNA accessibility.
The surface of bacteria such as "E. coli" is negatively charged due to phospholipids and lipopolysaccharides on its cell surface, and the DNA is also negatively charged. One function of the divalent cation therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface.
DNA entry into "E. coli" cells is through channels known as zones of adhesion or Bayer's junction, with a typical cell carrying as many as 400 such zones. Their role was established when cobalamine (which also uses these channels) was found to competitively inhibit DNA uptake. Another type of channel implicated in DNA uptake consists of poly (HB):poly P:Ca. In this poly (HB) is envisioned to wrap around DNA (itself a polyphosphate), and is carried in a shield formed by Ca ions.
It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure, making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance across the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.
Electroporation is another method of promoting competence. In this method the cells are briefly shocked with an electric field of 10-20 kV/cm, which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock, the holes are rapidly closed by the cell's membrane-repair mechanisms.
Yeast.
Most species of yeast, including "Saccharomyces cerevisiae", may be transformed by exogenous DNA in the environment. Several methods have been developed to facilitate this transformation at high frequency in the lab.
Efficiency – Different yeast genera and species take up foreign DNA with different efficiencies. Also, most transformation protocols have been developed for baker's yeast, "S. cerevisiae", and thus may not be optimal for other species. Even within one species, different strains have different transformation efficiencies, sometimes different by three orders of magnitude. For instance, when S. cerevisiae strains were transformed with 10 ug of plasmid YEp13, the strain DKD-5D-H yielded between 550 and 3115 colonies while strain OS1 yielded fewer than five colonies.
Plants.
A number of methods are available to transfer DNA into plant cells. Some vector-mediated methods are:
Some vector-less methods include:
Fungi.
There are some methods to produce transgenic fungi most of them being analogous to those used for plants. However, fungi have to be treated differently due to some of their microscopic and biochemical traits:
As stated earlier, an array of methods used for plant transformation do also work in fungi:
Animals.
Introduction of DNA into animal cells is usually called transfection, and is discussed in the corresponding article.
Practical aspects of transformation in molecular biology.
The discovery of artificially induced competence in bacteria allow bacteria such as "Escherichia coli" to be used as a convenient host for the manipulation of DNA as well as expressing proteins. Typically plasmids are used for transformation in "E. coli". In order to be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the replication of the cell's own chromosome.
The efficiency with which a competent culture can take up exogenous DNA and express its genes is known as transformation efficiency and is measured in colony forming unit (cfu) per μg DNA used. A transformation efficiency of 1×108 cfu/μg for a small plasmid like pUC19 is roughly equivalent to 1 in 2000 molecules of the plasmid used being transformed.
In calcium chloride transformation, the cells are prepared by chilling cells in the presence of (in solution), making the cell become permeable to plasmid DNA. The cells are incubated on ice with the DNA, and then briefly heat-shocked (e.g., at 42 °C for 30–120 seconds). This method works very well for circular plasmid DNA. Non-commercial preparations should normally give 106 to 107 transformants per microgram of plasmid; a poor preparation will be about 104/μg or less, but a good preparation of competent cells can give up to ~108 colonies per microgram of plasmid. Protocols, however, exist for making supercompetent cells that may yield a transformation efficiency of over 109. The chemical method, however, usually does not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell's native exonuclease enzymes rapidly degrade linear DNA. In contrast, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA.
The transformation efficiency using the method decreases with plasmid size, and electroporation therefore may be a more effective method for the uptake of large plasmid DNA. Cells used in electroporation should be prepared first by washing in cold double-distilled water to remove charged particles that may create sparks during the electroporation process.
Selection and screening in plasmid transformation.
Because transformation usually produces a mixture of relatively few transformed cells and an abundance of non-transformed cells, a method is necessary to select for the cells that have acquired the plasmid. The plasmid therefore requires a selectable marker such that those cells without the plasmid may be killed or have their growth arrested. Antibiotic resistance is the most commonly used marker for prokaryotes. The transforming plasmid contains a gene that confers resistance to an antibiotic that the bacteria are otherwise sensitive to. The mixture of treated cells is cultured on media that contain the antibiotic so that only transformed cells are able to grow. Another method of selection is the use of certain auxotrophic markers that can compensate for an inability to metabolise certain amino acids, nucleotides, or sugars. This method requires the use of suitably mutated strains that are deficient in the synthesis or utility of a particular biomolecule, and the transformed cells are cultured in a medium that allows only cells containing the plasmid to grow.
In a cloning experiment, a gene may be inserted into a plasmid used for transformation. However, in such experiment, not all the plasmids may contain a successfully inserted gene. Additional techniques may therefore be employed further to screen for transformed cells that contain plasmid with the insert. Reporter genes can be used as markers, such as the "lacZ" gene which codes for β-galactosidase used in blue-white screening. This method of screening relies on the principle of α-complementation, where a fragment of the "lacZ" gene ("lacZα") in the plasmid can complement another mutant "lacZ" gene ("lacZΔM15") in the cell. Both genes by themselves produce non-functional peptides, however, when expressed together, as when a plasmid containing "lacZ-α" is transformed into a "lacZΔM15" cells, they form a functional β-galactosidase. The presence of an active β-galactosidase may be detected when cells are grown in plates containing X-gal, forming characteristic blue colonies. However, the multiple cloning site, where a gene of interest may be ligated into the plasmid vector, is located within the "lacZα" gene. Successful ligation therefore disrupts the "lacZα" gene, and no functional β-galactosidase can form, resulting in white colonies. Cells containing successfully ligated insert can then be easily identified by its white coloration from the unsuccessful blue ones.
Other commonly used reporter genes are green fluorescent protein (GFP), which produces cells that glow green under blue light, and the enzyme luciferase, which catalyzes a reaction with luciferin to emit light. The recombinant DNA may also be detected using other methods such as nucleic acid hybridization with radioactive RNA probe, while cells that expressed the desired protein from the plasmid may also be detected using immunological methods.


Joseph Wang

Joseph Wang is an American researcher and inventor. He is Distinguished Professor, SAIC Endowed Chair, and former Chair of the Department of Nanoengineering at the University of California, San Diego specializing in nanomachines, biosensors, nanobioelectronics, wearable devices, and electrochemistry. He also serves as the Director of the Center for Wearable Sensors at the University of California San Diego Jacobs School of Engineering.
Biography.
Joseph Wang studied chemistry at the Technion and was awarded the BSc degree in 1972 and an MSc degree in 1974. After completing his D.Sc. at the Technion in 1978, he served as a postdoctoral research associate at the University of Wisconsin, Madison. In 1980, he joined the Department of Chemistry and Biochemistry at New Mexico State University, where he became a Regents Professor and holder of the Manasse Chair.
Wang founded the journal "Electroanalysis" (Wiley-VCH) in 1988 and was Editor-in-Chief for three decades until 2018. From 2004 to 2008, he served as the Director of the Center for Bioelectronics and Biosensors at the Biodesign Institute and as a Professor of Chemical Engineering and Chemistry at Arizona State University (ASU). Since 2008, Wang has served as Distinguished Professor of Nanoengineering at the University of California, San Diego, and as Department Chair between 2014 and 2019.
Fields of research.
Wang's earlier research focused on electrochemical biosensors and detectors for clinical diagnostics and environmental monitoring, with particular emphasis on blood glucose monitoring for diabetes management. His current research interests include the development of nanomotors and nanomachines, wearable non-invasive sensors, electrochemical biosensors, bioelectronics, microfluidic (“Lab-on-a-Chip”) devices, and remote sensors for environmental and security monitoring. His contributions in these directions have been of major impact in the development of electrochemical sensing techniques and man-made nanomachines His books include "Stripping Analysis" (VCH-1985), "Analytical Electrochemistry" (Wiley 2006) and "Nanomachines" (Wiley-VCH 2013).
Wang led a team that successfully merged efforts in the fields of biosensors, bioelectronics and nanotechnology to fashion nanocrystals that can act as amplifying tags for DNA or protein biosensors. His work in the field of nanomachines, involving novel motor designs and applications, has led to the world's fastest nanomotor, first demonstration of nanomotor operation in living organism, a novel motion-based DNA biosensing, and nanomachine-enabled isolation of biological targets, such as cancer cell identification, and advanced motion control in the nanoscale.
Wang has also pioneered the use of body-worn printed flexible electrochemical sensors and biofuel cells (including textile and epidermal-tattoo devices), microneedle-based electrochemical biosensors for real-time, pain-free quantification of circulating metabolites and electrolytes, 'green' bismuth electrodes for sensing toxic metals and remote submersible devices for continuous environmental monitoring. The advances made by Wang's team have been described in over 1200 research papers and reviews, that were cited over 130,000 times, leading to a H Index of 177.
Published books.
Wang has also been the Editor-in-Chief of the journal "Electroanalysis."


Robert Plonsey

Robert Plonsey (July 17, 1924 – March 14, 2015) was the Pfizer-Pratt University Professor Emeritus of Biomedical Engineering at Duke University. He is noted for his work on bioelectricity.
Education.
Plonsey was born in New York City in 1924. He received the B.E.E. degree in electrical engineering from the Cooper Union School of Engineering in New York in 1943, and the M.E.E degree from New York University in 1948.
He obtained his PhD from the University of California, Berkeley in 1957. In addition, he completed the first year and a half of the MD curriculum and the Case Western Reserve University School of Medicine (1969–1972).
Career.
Plonsey was a professor at Case Western Reserve University from 1968–1983, including a term as chair of the Department of Biomedical Engineering (1976–1980). In 1983, he moved to Duke University. He was a fellow of the American Association for the Advancement of Science and was elected as a member of the National Academy of Engineering in 1986 for "the application of electromagnetic field theory to biology, and for distinguished leadership in the emerging profession of biomedical engineering." He retired from Duke in 1996 as the Pfizer Inc./Edmund T. Pratt Jr. University Professor Emeritus of Biomedical Engineering.
Research.
Plonsey's research centered on bioelectric phenomena, including the electrical activity of nerves and muscle. With his student John Clark, he derived a mathematical relationship between the transmembrane potential and the extracellular potential produced by a propagating action potential in a nerve axon.
Some of Plonsey's most influential work addressed the electrical properties of the heart, often in collaboration with Roger Barr. They played a role in the development of the bidomain model, a mathematical model of the anisotropic electrical properties of cardiac muscle,
and developed a hypothesis of the mechanism for defibrillation based on the idea that individual cardiac cells are depolarized on one end and hyperpolarized on the other during the shock, sometimes known as the saw-tooth model.
Plonsey also collaborated with Yorum Rudy to calculate the relationship between body surface and epicardial electrical potentials,
and with Frank Witkowski to analyze action potential wave fronts recorded during defibrillation shocks.
Books.
Plonsey was the author of several books, including:


Discontinuous electrophoresis

Discontinuous electrophoresis (colloquially disc electrophoresis) is a type of polyacrylamide gel electrophoresis. It was developed by Ornstein and Davis. This method produces high resolution and good band definition. It is widely used technique for separating proteins according to size and charge.
Method.
In this method, the gel is divided into two discontinuous parts, resolving and stacking gel, both have different concentrations of polyacrylamide. The one with lower concentration is stacked on top of the one with higher concentration. Discontinuity is based on four parameters: gel structure, pH value of the buffer, ionic strength of the buffer, and the nature of the ions in the gel and electrode buffer. The electrode buffer contains glycine. Glycine has very low net charge at pH 6.8 of stacking gel, so it has low mobility. The proteins are separated according to the principle of isotachophoresis and form stacks in the order of mobility (stacking effect). Mobility depends on net charge, not on the size of the molecule. Proteins move towards anode slowly at constant speed till they reach limit of separation gel. Suddenly, frictional resistance increases but glycine is not affected and it passes the proteins and becomes highly charged in resolving zone. Proteins present in homogeneous buffer start to separate based on principles of zone electrophoresis. Now their mobility depends on size as well as charge. pH value rises to 9.5 and net charge increases.


Piribedil

Piribedil (trade names Pronoran, Trivastal Retard, Trastal, Trivastan, Clarium and others) is an antiparkinsonian agent and piperazine derivative which acts as a D2 and D3 receptor agonist. It also has α2-adrenergic antagonist properties.
Medical uses.
Other uses.
The drug has been shown to enhance working memory capacities in normal aging adults.
In age-related memory impairment, it has a positive effect on psychophysiological state of elderly people, improving memory and attention and increasing the velocity of psychomotor reactions and lability of nervous processes.
It enhances cognitive skill learning in healthy older adults.
It showed a positive effect in restless legs syndrome.
Dosage.
Parkinson's disease.
Administration of piribedil should be initiated with one sustained-release tablet (50 mg) daily during the first week. Dosage should then be gradually increased every week until achieving the optimal therapeutic dose:
Other indications.
One tablet daily at the end of the main meal. In severe cases: two tablets daily in two doses.
Side effects.
As with other dopamine agonists (like pramipexole and ropinirole), compulsive behavior like pathological gambling, overeating, excessive shopping, increased libido, sexual and/or other intense urges, may develop.
Another rare side effect of piribedil is excessive daytime sleepiness and unintended sleep episodes.
Overdose.
At very high doses, piribedil has an emetic action on the chemoreceptor trigger zone (CTZ). Tablets will thus be rapidly rejected, which explains why no data are currently available concerning the risk of overdosage.
Interactions.
Dopamine antagonists reduce the effect of piribedil.


ACell

ACell, Incorporated is a Columbia, Maryland-based biotechnology company. The company works in regenerative medicine, in which it owns several extracellular matrix patents. ACell develops, manufactures and markets products for medical and veterinary applications. The company is founded by Alan R. Spievack, a former associate professor at Harvard Medical School and currently run by Patrick A. McBrayer.
ACell’s ECM is derived from the lamina propria and basement membrane of a porcine Urinary Bladder Matrix (UBM). The lamina propria acts as a scaffold for cell infiltration while the basement membrane encourages the growth of site-specific tissue as the UBM resorbs. The UBM is offered in both sheet and powder form. The powder form has been informally referenced as “pixie dust” but is sold under the brand name of MicroMatrix. The Company offers products for wound management (Cytal and MicroMatrix), hernia repair (Gentrix) and Pelvic Organ Prolapse repair (Pelvic Floor Matrix).
The clinical data related to the use of ACell’s MatriStem UBM continues to grow with over 100 published papers.
ACell's use of porcine cellular structure, called MatriStem, as a scaffold for human tissue regeneration was named the "medical breakthrough of the year" by "Esquire". The use of pig bladder ground up into "magical pixie dust" to regrow Spievak's brother's finger received considerable mainstream coverage. Ken Muneoka of Tulane University, who works with ACell's scientific advisors on US-government funded investigations into regenerative medicine, said that the news should be viewed with caution because it was not a controlled study.
On January 5, 2020, Integra LifeSciences acquired the company for $400 million.


Crustacean cardioactive peptide

Crustacean cardioactive peptide (CCAP) is a highly conserved, amidated cyclic nonapeptide with the primary structure PFCNAFTGC-NH2 (ProPheCysAsnAlaPheTyrGlyCys-NH2) and a disulfide bridge between Cys3 and Cys9. It is found in crustaceans and insects where it behaves as a cardioaccelerator, neuropeptide transmitter for other areas of the nervous system and a hormone. CCAP was first isolated from the pericardial organs of the shore crab "Carcinus maenas", where it has a role in regulating heartbeat. It was assumed that this was the peptide's main function and its name reflects this.
Function.
As well as "C. maenas", CCAP has cardioacceleratory properties in "Manduca sexta" and "Drosophila melanogaster"; it is a highly conserved protein found in species as diverse as moths and fruit flies in addition to the crustacean it was originally identified in. Under specific conditions, CCAP has also been shown to induce cardiac reversal in "M. sexta" and "D. melanogaster".
Since CCAP was first identified and its possible role suggested, it has been proven it serves many other functions. It regulates release of adipokinetic hormone during sustained flight in "L. migratoria". It is also important in metabolism and digestion in other species; CCAP is involved as a hormone in the cockroach "Periplaneta americana" by upregulating digestive hormones after the insect has fed on nutritional matter. In the crab "Cancer borealis" CCAP controls passage of food through the foregut by modulating the stomatogastric ganglion's pyloric rhythm. This suggests CCAP is involved primarily in movement of food along the gut, facilitating digestion by secretion of digestive hormones and increasing flow of blood.
The effects of CCAP on the reproductive organs of females of the species "L. migratoria" have also been studied. The peptide stimulates the contraction of the oviducts and spermatheca. There is no CCAP or CCAP-like immunoreactivity shown in the oviduct or the nerves innervating the oviduct so it is probable that CCAP acts as a neurohormone to perform these functions.
CCAP is one of several peptides and hormones to be involved in the moulting process of crustaceans and insects.
Effects of lack of CCAP.
There have been no mutant individuals of any species found to possess CCAP who have a genetic defect meaning they are not able to produce the peptide. It is likely any such mutation would be lethal. In "Drosophila" the destruction of CCAP-containing neurons during development causes major changes in and disruptions to timing and behaviour of moulting.


Iprocrolol

Iprocrolol is a beta blocker that was never marketed.


Evolution of elephants


Glyceroneogenesis

Glyceroneogenesis is a metabolic pathway which synthesizes glycerol 3-phosphate or triglyceride from precursors other than glucose. Usually glycerol 3-phosphate is generated from glucose by glycolysis, but when glucose concentration drops in the cytosol, it is generated by another pathway called glyceroneogenesis. Glyceroneogenesis uses pyruvate, alanine, glutamine or any substances from the TCA cycle as precursors for glycerol 3-phosphate. Phosphoenolpyruvate carboxykinase (PEPC-K), which is an enzyme that catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate is the main regulator for this pathway. Glyceroneogenesis can be observed in adipose tissue and also liver. It is a significant biochemical pathway which regulates cytosolic lipid levels. Intense suppression of glyceroneogenesis may lead to metabolic disorder such as type 2 diabetes.
Summary.
In mammals, triglycerol or its backbone, glycerol 3- phosphate, is usually synthesized from glucose through glycolysis. Glucose will be degraded though glycolysis until fructose 1,6-bisphosphate is broken down to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Dihydroxyacetone phosphate is significant in synthesizing triglycerides because it can be used to generate glycerol 3-phosphate. However, glycerol 3-phosphate is generated through a different pathway when an organism is deficient of carbohydrates such as glucose. During fasting or low carbohydrate diet, glycerol 3-phosphate is generated by another metabolic pathway called glyceroneogenesis which uses precursors other than glucose. Glyceroneogenesis is considerably important because it is the dominant pathway to generate lipids during fasting or starvation. Not only does it produce lipids for the organism but it also regulates lipid levels in the cell. Glyceroneogenesis involves re-esterification of fatty acids to generate triglycerides. In other words, it can regulate fatty acid concentration in the cytosol. Strong activity in glyceroneogenesis will induce the re-esterification of fatty acid which will result in decrease of fatty acid concentration in the cytosol. Therefore, glyceroneogenesis is significantly related to lipid control of mammals.
Metabolic pathway.
The main precursors of glyceroneogenesis are pyruvate, lactate, glutamine, and alanine. Glyceroneogenesis is also known as branched pathway of gluconeogenesis because the first few steps in glyceroneogenesis are exactly the same as gluconeogenesis (Figure 3).
When pyruvate or lactate is used as the precursor for glycerol 3-phosphate, glyceroneogenesis follows exactly the same pathway as gluconeogenesis until it generates dihydroxyacetone phosphate. Lactate catalysed by lactate dehydrogenase will form pyruvate with expense of NAD+. Furthermore, by using 1 ATP and bicarbonate, pyruvate will be converted to oxaloacetate. which is catalysed by pyruvate carboxylase. Oxaloacetate will be catalysed by PEPC-K to generate phosphoenolpyruvate. This phosphorylation and decarboxylation of oxaloacetate is the significant step in glyceroneogenesis because the entire pathway is regulated by this reaction. After the production of phosphoenolpyruvate, gluconeogenesis will continue until dihydroxyacetone phosphate is generated, which produces 2-phosphoglycerate, 3-phosphoglycerate, 1,3-bisphosphoglycerate and glyceraldehyde 3-phosphate as intermediates. When dihydroxyacetone phosphate is produced, glyceroneogenesis will branch off from gluconeogenesis. With expense of NADH, dihydroxyacetone phosphate will convert to glycerol 3- phosphate (Figure 4), which is the final product of glyceroneogenesis. In addition, triglyceride can be generated by re-esterification of 3 fatty acid chains on glycerol 3-phosphate. Therefore, glyceroneogenesis is a metabolic pathway starting from lactate or pyruvate, and it is similar to gluconeogenesis but the pathway will branch out when dihydroxyacetone phosphate is generated. Instead of producing fructose 1,6- bisphosphate as gluconeogenesis does, glyceroneogenesis converts dihydroxyacetone phosphate to glycerol 3-phosphate.
Alanine can also be used as a precursor of glyceroneogenesis because alanine can be degraded to pyruvate. Alanine will degrade to pyruvate by transferring its amino group to 2-oxoglutarate with an enzyme called alanine aminotransferase. Alanine aminotransferase will cleave off the amino group from alanine and binds it to 2-oxoglutarate which generates pyruvate from alanine and glutamate from 2-oxoglutarate. Pyruvate generated from alanine will enter glyceroneogenesis and generate glycerol 3-phosphate.
Glutamate is also a known metabolite substance which can enter glyceroneogenesis. Since the key reaction of glyceroneogenesis is the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate, in theory, any biochemical pathway which generates oxaloacetate is related to glyceroneogenesis. For example, glutamate can generate oxaloacetate in 2 steps. First of all, glutamate can be converted to 2-oxoglutarate with expense of NAD+ and H2O with help of glutamate dehydrogenase. Secondly, 2-oxoglutarate can enter tricarboxyl acid cycle in order to generate oxaloacetate. Therefore, theoretically any metabolites in the TCA cycle or any metabolites generating the metabolites of TCA cycle can be used as a precursor of glyceroneogenesis, but glutamate is the only precursor confirmed,
Regulation.
Phosphoenolpyruvate carboxykinase (PEPC-K).
Glyceroneogenesis can be regulated at two reaction pathways. First of all, it can be regulated at the decarboxylation of oxaloacetate to phosphoenolpyruvate. Secondly, TCA cycle can affect glyceroneogenesis when the glutamate or substrates in TCA cycle are being used as a precursor.
Decarboxylation of oxaloacetate to phosphoenolpyruvate is catalysed by enzyme PEPC-K. PEPC-K is known as the essential enzyme which regulates glyceroneogenesis. Increasing the amount of PEPC-K or over expressing the gene for PEPC-K will increase the activity of glyceroneogenesis. More oxaloacetate can be decarboxylated to phosphoenolpyruvate when there is more PEPC-K that can catalyse the reaction. Furthermore, gene expression of PEPC-K can be suppressed by hormones named norepinephrine, glucocorticoid, and insulin. Norepinephrine is a neurotransmitter hormone which decreases the activity of PEPC-K when the cell is oriented in a cold environment. As a result, glyceroneogenesis is more likely to decrease in activity in cold environment. Glucocorticoid is a steroid hormone which is involved in the reciprocal regulation of glyceroneogenesis in liver and adipose tissues. Unfortunately, the actual mechanism of the reciprocal regulation is not well understood, but glucocorticoids induce transcription of PEPC-K in liver while decreasing the transcription in adipose tissues. Insulin is a peptide hormone which induces cells to intake glucose. In glyceroneogenesis, insulin down regulates the expression of PEPC-K in both liver and adipose tissues.
TCA cycle.
When metabolites from TCA cycle or glutamate are used as a precursor for glyceroneogenesis, the regulator in the TCA cycle can also cause flux to products formed by glyceroneogenesis. Regulation of TCA cycle is mostly determined by product inhibition and substrate availability. TCA cycle will slow down when excess product is present in the environment or deficient of substrate such as ADP and NAD+
Location.
Since glyceroneogenesis is related to lipid regulation, it can be found in adipose tissue and liver. In adipose tissue, glyceroneogenesis restrains the release of free fatty acids by re-esterifying them and in liver, triglycerides are being synthesized for lipid distribution.
White adipose tissue.
White adipose tissue, also known as white fat, is one of the 2 types of adipose tissue in mammals. White adipose tissue stores energy in form of triglycerides that can be broken down to free fatty acids. Its normal function is to store free fatty acids as triglycerides within the tissue. However, when the glucose level of in the cell drops in situations like fasting, white adipose tissue generates glycerol 3-phosphate. The presence of glyceroneogenesis in white adipose tissues is proven by an experiment with mouse. Since glycerol 3-phosphate is usually generated from glucose through glycolysis, level of triglyceride content was compared with normal mouse and mouse which cannot intake glucose in to their cells. Glucose transporter 4, also known as GLUT 4 (Figure 6) is the transporter protein of glucose which intakes extracellular glucose to intracellular environment. In order to examine the presence of glyceroneogenesis in mouse, genes expressing GLUT4 were deleted and triglyceride content of adipose tissue was compared with normal mouse. Since glucose cannot enter the cell, synthesis of glycerol 3-phosphate was expected to decrease. However, the result showed no change in triglyceride concentration. This experiment proved the presence of an alternate metabolic pathway to synthesize triglyceride in adipose tissues of mouse. Furthermore, an additional experiment was performed in order to examine the relation of the alternate glycerol 3-phosphate synthesizing pathway and PEPC-K. Triglyceride content in white adipose tissue of mouse with mutated gene which expresses PEPC-K was observed. Sine PEPC-K is the essential regulatory enzyme for glyceroneogenesis, mutation in PEPC-K genes would lower the activity of glyceroneogenesis. The result showed no production of triglyceride in white adipose tissues as expected. Hence, glyceroneogenesis was present in white adipose tissues because it was able to generate triglycerides without glucose and it was unable to synthesize when PEPC-K was mutated. Therefore, during fasting or low carbohydrate diet, white adipose tissues generate glycerol 3-phosphate using glyceroneogenesis.
Brown adipose tissue.
Brown adipose tissue is another type of adipose tissue which stores free fatty acids. Brown adipose tissue is especially abundant in new born mammals and hibernating mammals. The differences between brown and white adipose tissue are brown adipose tissues have higher activity in glyceroneogenesis than white adipose tissues and glyceroneogenesis in brown adipose tissue is related to thermogenesis. The activity of glyceroneogenesis in brown adipose tissue is greater than that of white adipose tissue because it contains more enzymes involved in the glyceroneogenesis. Compared to white adipose tissue, brown adipose tissue has considerably higher activity of PEPC-K and glycerol kinase. The activity of PEPC-K in brown adipose tissue is almost 10 times the activity of that in white adipose tissue. PEPC-K which is involved in the conversion of oxaloacetate to phosphoenolpyruvate is the key enzyme that regulates glyceroneogenesis. Increase in activity of the enzyme will increase the activity of the pathway. Furthermore, not only PEPC-K but brown adipose tissue is also rich in activity of glycerol kinase. Glycerol kinase is the enzyme which phosphorylates glycerol in order to generate the backbone of triglycerides, glycerol 3-phosphate. Increase in the activity of glycerol kinase will result in an increase in production of glycerol 3- phosphate. As a result, brown adipose tissue will have greater activity in glyceroneogenesis, because it contains more enzymes involved in the pathway.
In addition, glyceroneogenesis in brown adipose tissue is related to thermogenesis in the organism. In mammals, heat is generated by delivering free fatty acids to the mitochondria. When glyceroneogenesis is proceeding regularly, the concentration of free fatty acid is low in intercellular environment because glyceroneogenesis re-esterifies fatty acids to triglycerides. In other words, thermogenesis by free fatty acids is less likely to occur when glyceroneogenesis is proceeding. However, when exposed to cold, a neurotransmitter hormone called norepinephrine will suppress the activity of PEPC-K. When the activity of PEPC-K is suppressed, glyceroneogenesis will be unable to re-esterify the free fatty acids. Eventually, the free fatty acid concentration within the cell will increase leading to excessive free fatty acids in cytosol, which will consequently be delivered to the mitochondria for thermogenesis. Therefore, when a mammal is exposed to cold, heat is generated in the brown adipose tissue by decreasing the activity of glyceroneogenesis.
Liver.
Although glyceroneogenesis was first found in adipose tissues, it was not recognized in the liver until 1998 (Source?). Glyceroneogenesis was unexpected in the liver for 2 reasons; triglyceride synthesis in liver was thought to be not natural because gluconeogenesis is taking huge part in the liver, and liver was believed to have sufficient glycerol 3-phosphate collected from the bloodstream. However, several experiments, which used stable isotopes to track the glycerol in liver and bloodstream, showed that 65% of the glycerol backbone of triglyceride flowing through the bloodstream is actually synthesised in the liver. Therefore, glycerol 3-phosphate synthesis in liver was discovered. In fact, liver synthesizes more than half of the glycerol which mammals need to regulate lipid in their body.
Glyceroneogenesis in liver and adipose tissues regulate lipid metabolism in opposite ways. On one hand, lipids in the form of triglycerides are released from the liver. However, on the other hand, glyceroneogenesis restrains the fatty acid release from adipose tissues by re-esterifying them. In other words, glyceroneogenesis in liver and adipose tissues are alternately regulated. When the lipid concentration in the blood is relatively high, glyceroneogenesis in liver will negatively regulated to stop the synthesis of triglyceride, but glyceroneogenesis in adipose tissues will be induced in order to restrain the release of free fatty acid to the bloodstream. Conversely, glyceroneogenesis in liver will be induced and suppressed in adipose tissues when lipid level of the blood is low. Even though the reciprocal regulation of glyceroneogenesis is not well understood, a hormone called glucocorticoid is a best example of the regulation. Glucocorticoids induce gene transcription of PEPC-K in liver but repress the transcription in adipose tissues.
Disease.
Type 2 Diabetes.
Failure in regulating glyceroneogenesis may lead to Type 2 diabetes. Type 2 diabetes is a metabolic disorder that results in high levels of blood glucose and blood lipid. Type2 diabetes is associated with over production of triglycerides in liver due to excessively active glyceroneogenesis and excess release of fatty acids from adipose tissues. Since the activity of glyceroneogenesis is mostly dependent on PEPC-K, fluctuating the expressions for PEPC-K will dramatically influence the activity of glyceroneogenesis. Over expressing PEPC-K in liver will eventually result in over production of triglycerides which can elevate the lipid level in the bloodstream. Conversely, in adipose tissue, down regulated glyceroneogenesis may decrease de novo lipogenesis. Suppressed glyceroneogenesis will result in increase of free fatty acids in the adipose tissues and its subsequent export to the bloodstream, because re-esterification of free fatty acids into triglycerides will decrease due to the decreased availability of the glycerol backbone. Therefore, glyceroneogenesis overly induced in liver and reduced in adipose tissues can lead, respectively, to hepatic steatosis and lipodystrophy, both highly associated to Type 2 Diabetes.
Treatment.
Regulation of Glyceroneogenesis is a therapeutic target of Type II diabetes. The release of triglycerides in the liver should be inhibited as well as release of free fatty acid in adipose tissues. Insulin is used as a down regulator in liver of glyceroneogenesis. Suppression in glyceroneogenesis will decrease the triglyceride being released in to the bloodstream from liver. However, the problem with insulin is that it also suppresses glyceroneogenesis in adipose tissue. In order to restrict the release of free fatty acid from adipose tissues, fatty acids must be re-esterified by glyceroneogenesis. Thiazolidinedione (Figure 8) is a substance which only affects glyceroneogenesis in adipose tissue. Thiazolidinedione will increase the transcription of PEPC-K and eventually induce the activity of glyceroneogenesis. As a result, re-esterification of fatty acids take place in the cell and prevents the release of fatty acids to the bloodstream.


Ferdinando Hastings, 6th Earl of Huntingdon

Ferdinando Hastings, 6th Earl of Huntingdon (18 January 1609 – 13 February 1656), was the son of Henry Hastings, 5th Earl of Huntingdon, and Lady Elizabeth Stanley, the daughter of Ferdinando Stanley, 5th Earl of Derby, and Alice Spencer. He married Lucy, daughter of Sir John Davies, on 7 August 1623.
He was MP for Leicestershire in 1625 and again in 1628–29.
Ferdinando's family seat, Ashby de la Zouch Castle, was destroyed by Oliver Cromwell's troops in the English Civil War in 1646. He died of smallpox at age 47 and was succeeded by his son, Theophilus Hastings, 7th Earl of Huntingdon.
There is a monument to him in St Helen's Church, Ashby-de-la-Zouch.


Aerobic anoxygenic phototrophic bacteria

Aerobic anoxygenic phototrophic bacteria (AAPBs) are alphaproteobacteria and gammaproteobacteria that are obligate aerobes that capture energy from light by anoxygenic photosynthesis. Anoxygenic photosynthesis is the phototrophic process where light energy is captured and stored as ATP. The production of oxygen is non-existent and, therefore, water is not used as an electron donor. They are widely distributed marine plankton that may constitute over 10% of the open ocean microbial community. They can be particularly abundant in oligotrophic conditions where they were found to be 24% of the community. Aerobic anoxygenic phototrophic bacteria are photoheterotrophic (phototroph)microbes that exist in a variety of aquatic environments. Photoheterotrophs (Gk: photo = light, hetero = (an)other, troph = nourishment), are heterotrophic organisms that use light to produce energy, but are unable to utilize carbon dioxide as their primary carbon source. Most are obligately aerobic, meaning they require oxygen to grow. One remarkable aspect of these novel bacteria is that they, unlike other similar bacteria, are unable to utilize BChl (bacteriochlorophyll) for anaerobic growth. The only photosynthetic pigment that exists in AAPB is BChl a. Anaerobic phototrophic bacteria, on the contrary, can contain numerous species of photosynthetic pigments like bacteriochlorophyll a, b, c, d, e, f, etc. There is still a large void in the areas regarding the abundance and genetic diversity of the AAPB, as well as the environmental variables that regulate these properties.
Cellular structure.
Research suggests that all currently known AAPB contain Gram-negative cell walls. The majority, have shapes that resemble cylinders, as well as flagella and cilia. AAPB cell dimensions are normally, 1.2 μm long, 0.7 μm in diameter, and a cell volume of 0.5 μm3. Their dry weight is 0.05 pg and wet weight is 0.5 pg. 3 types of cell division are known to exist within AAPB, 2 daughter-cell division, 4 daughter-cell division, and the non-typical 3 daughter-cell division, commonly referred to as Y-cell division. AAPB are usually pink or orange in color when isolated from water. Current data suggests that marine bacteria have generation times of several days, whereas new evidence exists that shows AAPB to have a much shorter generation time. All species of AAPB produce large amounts of carotenoid pigments. The color of each species is due to the presence of carotenoids, giving peaks in the blue and green absorption spectra.
Taxonomy.
Aerobic anoxygenic phototrophic bacteria are classified in two marine ("Erythrobacter" and "Roseobacter") and six freshwater ("Acidiphilium", "Erythromicrobium", "Blastomonas" (synonym: "Erythromonas"), "Porphyrobacter", "Roseococcus", and "Sandaracinobacter") genera, which phylogenetically belong to the -1, -3, and -4 subclasses of the class Proteobacteria. Phylogenetically, they are not classified into single group. Species so far described are distributed rather widely within the α-subclass of Proteobacteria in which most of the purple nonsulfur bacteria as well as many non-photosynthetic bacteria are included. Apparently, these aerobic BChl-containing bacteria represent an evolutionary transient phase from anaerobic phototrophs to aerobic non-phototrophs. However, some characteristic features distinct from anaerobic phototrophs suggest that most of them are in an evolutionary stable state. Phototrophy is a noticeable and significant marker that should always play a primary role in bacterial classification.
Carbon cycling.
AAPBs play a key role in carbon cycling but to what extent is still questionable. The key to determining their role in marine ecosystems is the AAPB in total bacteria (AAPB%). Knowing that the BCHl a linked phototrophic function is an added feature to their standard heterotrophic diet of dissolved organic carbon, it is thought that AAPBs would fare better when a lack organic carbon electron donors existed in respiration. They also exhibit a selective advantage in oligotrophic environments.
Distribution.
They are widely distributed in coastal and oceanic environments. One study revealed that the surface water of the Indian Ocean ranked the highest of the oceans in AAPB% at 3.79. The Atlantic Ocean surface waters followed with 1.57 AAPB%. Last, the Pacific Ocean followed closely at 1.08 AAPB%. There was a positive correlation with oceans that held higher values of AAPB% and those with higher levels of chlorophyll a. More specific, the coastal/shelf waters of these oceans had greater amounts of AAPBs, some as high as 13.51% AAPB%. Phytoplankton also affect AAPB%, but little research has been performed in this area. They can also be abundant in various oligotrophic conditions, including the most oligotrophic regime of the world ocean. They are globally distributed in the euphotic zone and represent a hitherto unrecognized component of the marine microbial community that appears to be critical to the cycling of both organic and inorganic carbon in the ocean.


Guanylyltransferase

Guanylyl transferases are enzymes that transfer a guanosine mono phosphate group, usually from GTP to another molecule, releasing pyrophosphate. Many eukaryotic guanylyl transferases are capping enzymes that catalyze the formation of the 5' cap in the co-transcriptional modification of messenger RNA. Because the 5' end of the RNA molecule ends in a phosphate group, the bond formed between the RNA and the GTP molecule is an unusual 5'-5' triphosphate linkage, instead of the 3'-5' linkages between the other nucleotides that form an RNA strand. In capping enzymes, a highly conserved lysine residue serves as the catalytic residue that forms a covalent enzyme-GMP complex.
The transfer RNA (tRNA) for histidine is unique among eukaryotic tRNAs in requiring the addition of a guanine nucleotide before being aminoacylated by the histidine tRNA synthetase. The yeast guanylyl transferase specific to tRNAHis is unique in being the only known non-tRNA synthetase enzyme that specifically recognizes the tRNA anticodon.
Guanylyl transferases also exist for transferring guanosine nucleotides to sugar molecules, such as mannose and fucose.


Gerhard Braunitzer

Gerhard Braunitzer (24 September 1921 – 27 May 1989) was a German biochemist who was a pioneer in protein sequencing. He refined a method of C-terminal analysis that was used in early sequencing work, and he generated the first full sequence of a hemoglobin chain (β-hemoglobin). He worked at the Max Planck Institute of Biochemistry. Later in his career he was a significant researcher in the field of molecular evolution.


Tuscaridium

Tuscaridium is a genus of phaeodarian, (formerly thought to be radiolarians). The genus contains bioluminescent species. It one of two known bioluminescent phaeodarean genera, the other being "Aulosphaera".
Species.
The following species are known (incomplete list):


Alexander Kittson

Alexander Kittson (February 26, 1853 – April 27, 1883) was a political figure in Manitoba. He represented Ste. Agathe from 1879 to 1883 in the Legislative Assembly of Manitoba as a Liberal-Conservative.
He was born in North Pembina, the son of Norman Kittson and Elise Marion, and was educated at St. Boniface College. In 1875, he married Elise Gingras. Kittson died in office of smallpox at St. Boniface.
His daughter Annie Jane married Horace Chevrier.
His former home in St. Boniface, known as the Maison Kittson, is recognized as a heritage landmark by the city of Winnipeg.


Genetex

GeneTex is a life science, biotechnology company that develops, licenses, and sells a product catalog of research reagents related to the study of the biomedical sciences. The company provides antibodies and proteins for use by academic and industry investigators involved in basic and translational research in biology and medicine.
The 5 Pillars of Antibody Validation.
The validation practices used in antibody research have become an area of growing concern among various research groups. Consequently, several antibody research companies convened an ad hoc group to help standardize the best practices in antibody validation. In 2016, Genetex, Novus Biologicals, R&D Systems, and Atlas Antibodies joined the International Working Group on Antibody Validation (IWGAV).
Recombinant Monoclonal Antibody Technology.
GeneTex has incorporated recombinant monoclonal antibody technology into its product development platform. This technology has numerous well-documented advantages, with consistency of performance being of paramount significance. The protocol employed is a multi-parameter FACS-based approach to isolate antigen-specific memory B cells from an immunized animal, with subsequent cloning of the antibody variable-region genes into an IgG backbone and expression in mammalian cells. The entire development procedure can be completed in weeks.


Choline-phosphate cytidylyltransferase

Choline-phosphate cytidylyltransferase () is an enzyme that catalyzes the chemical reaction
where the two substrates of this enzyme are CTP and choline phosphate, and the two products are diphosphate and CDP-choline. It is responsible for regulating phosphatidylcholine content in membranes.
This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing nucleotide groups (nucleotidyltransferases). The systematic name of this enzyme class is CTP:choline-phosphate cytidylyltransferase. Other names in common use include phosphorylcholine transferase, CDP-choline pyrophosphorylase, CDP-choline synthetase, choline phosphate cytidylyltransferase, CTP-phosphocholine cytidylyltransferase, CTP:phosphorylcholine cytidylyltransferase, cytidine diphosphocholine pyrophosphorylase, phosphocholine cytidylyltransferase, phosphorylcholine cytidylyltransferase, and phosphorylcholine:CTP cytidylyltransferase. This enzyme participates in aminophosphonate metabolism and glycerophospholipid metabolism.
Structural studies.
As of late 2007, two structures have been solved for this class of enzymes, with PDB accession codes and .


Toliprolol

Toliprolol is a beta adrenergic receptor antagonist.


Shankar Balasubramanian

Sir Shankar Balasubramanian (born 30 September 1966) is an Indian-born British chemist and Herchel Smith Professor of Medicinal Chemistry in the Department of Chemistry at the University of Cambridge, Senior Group Leader at the Cancer Research UK Cambridge Institute and Fellow of Trinity College, Cambridge. He is recognised for his contributions in the field of nucleic acids. He is scientific founder of Solexa and Cambridge Epigenetix.
Education.
Born in Madras (now Chennai) India in 1966, Shankar Balasubramanian moved to the UK with his parents in 1967. He grew up in a rural area just outside Runcorn in Cheshire and attended Daresbury Primary School, then Appleton Hall High School (which has since amalgamated to form Bridgewater High School). He then went on to study the Natural Sciences Tripos at Fitzwilliam College, Cambridge, where he did his undergraduate degree from 1985–88 and continued with a PhD for research on the reaction mechanism of the enzyme chorismate synthase supervised by Chris Abell (1988-1991).
Career and research.
Following his PhD, Balasubramanian travelled to the USA as a SERC/NATO Research Fellow and worked in the group of Stephen J. Benkovic at Pennsylvania State University (1991-1993). He started his independent academic career in 1994 in the University of Cambridge and has remained there ever since, first as College Lecturer, then University Lecturer (1998), University Reader in Chemical Biology (2003) and Professor of Chemical Biology (2007). He was most recently appointed Herchel Smith Professor of Medicinal Chemistry in 2008. He currently directs research laboratories in the Department of Chemistry and also the Cancer Research UK (CRUK) Cambridge Institute at the Cambridge Biomedical Campus. His former doctoral students include Julian Huppert.
Balasubramanian works in the field of nucleic acids. His citation on election to the Royal Society reads:
More recently Balasubramanian has been inventing and applying new chemical methods to study epigenetic changes to DNA bases including single base resolution sequencing of 5-formylcytosine, 5-hydroxymethylcytosine and 5-methylcytosine.
Honours and awards.
Honours and awards include:


Squamosa promoter binding protein

The SQUAMOSA promoter binding protein-like (SBP or SPL) family of transcription factors are defined by a plant-specific DNA-binding domain. The founding member of the family was identified based on its specific "in vitro" binding to the promoter of the snapdragon SQUAMOSA gene. SBP proteins are thought to be transcriptional activators.
Function.
SPB proteins have roles in leaf development, vegetative phase change, flower and fruit development, plant architecture, sporogenesis, Gibberelic acid signaling and toxin response.
Structure.
The domain contains 10 conserved cysteine and histidine residues that probably are zinc ligands.
The SBP domain is a highly conserved DNA-binding domain. It is approximately 80 amino acids in length and contains a zinc finger motif
with two zinc-binding sites: Cys-Cys-His-Cys and Cys-Cys-Cys-His. It has a three-stranded antiparallel beta-sheet.