XDNA

xDNA (also known as expanded DNA or benzo-homologated DNA) is a size-expanded nucleotide system synthesized from the fusion of a benzene ring and one of the four natural bases: adenine, guanine, cytosine, and thymine. This size expansion produces an 8 letter alphabet which has a larger information density by a factor of 2n compared to natural DNA's (often referred to as B-DNA in literature) 4 letter alphabet. As with normal base-pairing, A pairs with xT, C pairs with xG, G pairs with xC, and T pairs with xA. The double helix is thus 2.4Å wider than a natural double helix. While similar in structure to B-DNA, xDNA has unique absorption, fluorescence, and stacking properties.
Initially synthesized as an enzyme probe by Nelson J. Leonard's group, benzo-homologated adenine was the first base synthesized. Later, Eric T. Kool's group finished synthesizing the remaining three expanded bases, eventually followed by yDNA ("wide" DNA), another benzo-homologated nucleotide system, and naphtho-homologated xxDNA and yyDNA. xDNA is more stable when compared to regular DNA when subjected to higher temperature, and while entire strands of xDNA, yDNA, xxDNA and yyDNA exist, they are currently difficult to synthesize and maintain. Experiments with xDNA provide new insight into the behavior of natural B-DNA. The extended bases xA, xC, xG, and xT are naturally fluorescent, and single strands composed of only extended bases can recognize and bind to single strands of natural DNA, making them useful tools for studying biological systems. xDNA is most commonly formed with base pairs between a natural and expanded nucleobase, however x-nucleobases can also be paired together. Current research supports xDNA as a viable genetic encoding system in the near future.
Origins.
The first nucleotide to be expanded was the purine adenine. Nelson J. Leonard and colleagues synthesized this original x-nucleotide, which was referred to as "expanded adenine". xA was used as a probe in the investigation of active sites of ATP-dependent enzymes, more specifically what modifications the substrate could take while still being functional. Almost two decades later, the other three bases were successfully expanded and later integrated into a double helix by Eric T. Kool and colleagues. Their goal was to create a synthetic genetic system which mimics and surpasses the functions of the natural genetic system, and to broaden the applications of DNA both in living cells and in experimental biochemistry. Once the expanded base set was created, the goal shifted to identifying or developing faithful replication enzymes and further optimizing the expanded DNA alphabet.
Synthesis.
In benzo-homologated purines (xA and xG), the benzene ring is bound to the nitrogenous base through nitrogen-carbon (N-C) bonds. Benzo-homologated pyrimidines are formed through carbon-carbon (C-C) bonds between the base and the benzene. Thus far, x-nucleobases have been added to strands of DNA using phosphoramidite derivatives, as traditional polymerases have been unsuccessful in synthesizing strands of xDNA. X-nucleotides are poor candidates as substrates for B-DNA polymerases as their size interferes with binding at the catalytic domain. Attempts at using template-independent enzymes have been successful as they have a reduced geometric constraint for substrates. Terminal deoxynucleotidyl transferase (TdT) has been used previously to synthesize strands of bases which have been bound to fluorophores. Using TdT, up to 30 monomers can be combined to form a double-helix of xDNA, however this oligomeric xDNA appears to inhibit its own extension beyond this length due to the overwhelming hydrogen bonding. In order to minimize inhibition, xDNA can be hybridized into a regular helix.
Replication.
For xDNA to be used as a substitute structure for information storage, it requires a reliable replication mechanism. Research into xDNA replication using a Klenow fragment from DNA polymerase I shows that a natural base partner is selectively added in instances of single-nucleotide insertion. However, DNA polymerase IV (Dpo4) has been able to successfully use xDNA for these types of insertions with high fidelity, making it a promising candidate for future research in extending replicates of xDNA. xDNA's mismatch sensitivity is similar to that of B-DNA.
Structure.
Similar to natural bases, x-nucleotides selectively assemble into a duplex-structure resembling B-DNA. xDNA was originally synthesized by incorporating a benzene ring into the nitrogenous base. However, other expanded bases have been able to incorporate thiophene and benzo[b]thiophene as well. xDNA and yDNA use benzene rings to widen the bases and are thus termed "benzo-homologated". Another form of expanded nucleobases known as yyDNA incorporate naphthalene into the base and are "naptho-homologated". xDNA has a rise of 3.2Å and a twist of 32°, significantly smaller than B-DNA, which has a rise of 3.3Å and a twist of 34.2° xDNA nucleotides can occur on both strands—either alone (known as "doubly expanded DNA") or mixed with natural bases—or exclusively on one strand or the other. Similar to B-DNA, xDNA can recognize and bind complementary single-stranded DNA or RNA sequences.
Duplexes formed from xDNA are similar to natural duplexes aside from the distance between the two sugar-phosphate backbones. xDNA helices have a greater number of base pairs per turn of the helix as a result of a reduced distance between neighbour nucleotides. NMR spectra report that xDNA helices are anti-parallel, right-handed and take an "anti" conformation around the glycosidic bond, with a C2'-endo sugar pucker. Helices created from xDNA are more likely to take a B-helix over an A-helix conformation, and have an increased major groove width by 6.5Å (where the backbones are farthest apart) and decreased minor groove width by 5.5Å (where the backbones are closest together) compared to B-DNA. Altering groove width affects the xDNA's ability to associate with DNA-binding proteins, but as long as the expanded nucleotides are exclusive to one strand, recognition sites are sufficiently similar to B-DNA to allow bonding of transcription factors and small polyamide molecules. Mixed helices present the possibility of recognizing the four expanded bases using other DNA-binding molecules.
Properties.
Expanded nucleotides and their oligomeric helices share many properties with their natural B-DNA counterparts, including their pairing preference: A with T, C with G. The various differences in chemical properties between xDNA and B-DNA support the hypothesis that the benzene ring which expands x-nucleobases is not, in fact, chemically inert. xDNA is more hydrophobic than B-DNA, and also has a smaller HOMO-LUMO gap (distance between the highest occupied molecular orbital and lowest unoccupied molecular orbital) as a result of modified saturation. xDNA has higher melting temperatures than B-DNA (a mixed decamer of xA and T has a melting temperature of 55.6 °C, 34.3 °C higher than the same decamer of A and T), and exhibits an "all-or-nothing" melting behaviour.
Conformation.
Under lab conditions, xDNA orients itself in the "syn" conformation. This unfortunately does not expose the binding face of the xDNA nucleotides to face the neighbouring strand for binding, meaning that extra measures must be applied to alter the conformation of xDNA before attempting to form helices. However, the "anti" and "syn" orientations are practically identical energetically in expanded bases. This conformational preference is seen primarily in pyrimidines, and purines display minimal preference for orientation.
Enhanced stacking.
Stacking of the nucleotides in a double helix is a major determinant of the helix's stability. With the added surface area and hydrogen available for bonding, stacking potential for the nucleobases increases with the addition of a benzene spacer. By increasing the separation between the nitrogenous bases and either sugar-phosphate backbone, the helix's stacking energy is less variable and therefore more stable. The energies for natural nucleobase pairs vary from 18 to 52 kJ/mol. This variance is only 14–40 kJ/mol for xDNA.
Due to an increased overlap between and expanded strand of DNA and its neighbouring strand, there are greater interstrand interactions in expanded and mixed helices, resulting in a significant increase in the helix's stability. xDNA has enhanced stacking abilities resultant from changes in inter- and intrastrand hydrogen bonding that arise from the addition of a benzene spacer, but expanding the bases does not alter hydrogen's contribution to the stability of the duplex. These stacking abilities are exploited by helices consisting of both xDNA and B-DNA in order to optimize the strength of the helix. Increased stacking is seen most prominently in strands consisting only of A and xA and T and xT, as T-xA has stronger stacking interactions than T-A.
The energy resultant from pyrimidines ranges from 30–49 kJ/mol. The range for purines is between 40-58kJ/mol. By replacing one nucleotide in a double-helix with an expanded nucleotide, the strength of the stacking interactions increases by 50%. Expanding both nucleotides results in a 90% increase in stacking strength. While xG has an overall negative effect on the binding strength of the helix, the other three expanded bases outweigh this with their positive effects. The change in energy caused by expanding the bases is mostly dependent on the rotation of the bond about the nucleobases' centers of mass, and center of mass stacking interactions improve the stacking potential of the helix. Because the size-expanded bases widen the helix, it is more thermally stable with a higher melting temperature.
Absorption.
The addition of a benzene spacer in x-nucleobases affects the bases' optical absorption spectra. Time-dependent density functional theory (TDDFT) applied to xDNA revealed that the benzene component of the highest occupied molecular orbitals (HOMO) in the x-bases pins the absorption onset at an earlier point than natural bases. Another unusual feature of xDNA absorption spectra is the red-shifted excimers of xA in the low range. In terms of stacking fingerprints, there is a more pronounced hypochromicity seen in consecutive xA-T base pairs.
Implications of xDNA's altered absorption include applications in nanoelectronic technology and nanobiotechnology. The reduced spacing between x-nucleotides makes the helix stiffer, thus it is not as easily affected by substrate, electrode, and functional nanoparticle forces. Other alterations to natural nucleotides resulting in different absorption spectra will broaden these applications in the future.
Fluorescence.
One unique property of xDNA is its inherent fluorescence. Natural bases can be bound directly to fluorophores for use in microarrays, "in situ" hybridization, and polymorphism analysis. However, these fluorescent natural bases often fail as a result of self-quenching, which diminishes their fluorescent intensity and reduces their applicability as visual DNA tags. The pi interactions between the rings in x-nucleobases result in an inherent fluorescence in the violet-blue range, with a Stokes shift between 50–80 nm. They also have a quantum yield in the range of 0.3–0.6. xC has the greatest fluorescent emission.
Other expanded bases.
After the creation of and successful research surrounding xDNA, more forms of expanded nucleotides were investigated. yDNA is a second, similar system of nucleotides which uses a benzene ring to expand the four natural bases. xxDNA and yyDNA use naphthalene, a polycyclic molecule consisting of two hydrocarbon rings. The two rings expand the base even wider, further altering its chemical properties.
yDNA.
The success and implications of xDNA prompted research to examine other factors which could alter B-DNA's chemical properties and create a new system for information storage with broader applications. yDNA also uses a benzene ring, similar to xDNA, with the only difference being the site of addition of the aromatic ring. The location of the benzene ring changes the preferred structure of the expanded helix. The altered conformation makes yDNA more similar to B-DNA in its orientation by changing the interstrand hydrogen bonds. Stability is highly dependent on the bases' rotation about the link between the base and the sugar of the backbone. yDNA's altered preference for this orientation makes it more stable overall than xDNA. The location of the benzene spacer also affects the bases' groove geometry, altering neighbour interactions. The base pairs between y-nucleotides and natural nucleotides is planar, rather than slightly twisted as with xDNA. This decreases the rise of the helix even further than achieved by xDNA.
While xDNA and yDNA are quite similar in most properties, including their increased stacking interactions, yDNA shows superior mismatch recognition. y-pyrimidines display slightly stronger stacking interactions than x-pyrimidines as a result of the distance between the two anomeric carbons, which is slightly larger in yDNA. xDNA still has stronger stacking interactions in model helices, but adding either x- or y-pyrimidines to a natural double helix strengthens the intra- and interstrand interactions, increasing overall helix stability. In the end, which of the two has the strongest overall stacking interactions is dependent on the sequence; xT and yT bind A with similar strength, but the stacking energy of yC bound to G is stronger than xC by 4kJ/mol. yDNA and other expanded bases are part of a very young field which is highly understudied. Research suggest that the ideal conformation still remains to be discovered, but knowing that the benzene location affects the orientation and structure of expanded nucleobases adds information to their future design.
yyDNA and xxDNA.
Doubly-expanded (or "naphtho-homologated") nucleobases incorporate a naphthalene spacer instead of a benzene ring, widening the base twice as much with its two-ringed structure. These structures (known as xxDNA and yyDNA) are 4.8Å wider than natural bases and were once again created as a result of Leonard's research on expanded adenine in ATP-dependent enzymes in 1984. No literature was published on these doubly-expanded bases for nearly three decades until 2013 when the first xxG was produced by Sharma, Lait, and Wetmore and incorporated along with xxA into a natural helix. Although very little research has been performed on xxDNA, xx-purine neighbours have already been shown to increase intrastrand stacking energy by up to 119% (as opposed to 62% in x-purines). xx-purine and pyrimidine interactions show an overall decrease in stacking energies, but the overall stability of duplexes including pyrimidines and xx-purines increases by 22%, more than twofold that of pyrimidines and x-purines.
Uses.
xDNA has many applications in chemical and biological research, including expanding upon applications of natural DNA, such as scaffolding. In order to create self-assembling nanostructures, a scaffold is needed as a sort of trellis to support the growth. DNA has been used as a means to this end in the past, but expanded scaffolds make larger scaffolds for more complex self-assembly an option. xDNA's electrical conduction properties also make it a prime candidate as a molecular wire, as its π-π interactions help it efficiently conduct electricity. Its 8-letter alphabet (A, T, C, G, xA, xT, xC, xG) gives it the potential to store 2n times increase in storage density, where "n" represents the number of letters in a sequence. For example, combining 6 nucleotides of with B-DNA yields 4096 possible sequences, whereas a combination of the same number of nucleotides created with xDNA yields 262,144 possible sequences. Additionally, xDNA can be used as a fluorescent probe at enzyme active sites, as was its original application by Leonard et al.
xDNA has also been applied to the study of protein-DNA interactions. Due to xDNA's natural fluorescing properties, it can easily be visualized in both lab and living conditions. xDNA is becoming more easy to create and oligomerize, and its high-affinity binding to complementary DNA and RNA sequences means that it can not only help locate these sequences floating around in the cell, but also when they are already interacting with other structures within the cell. xDNA also has potential applications in assays that employ TdT as it may improve reporters, and can be used as an affinity tag for interstrand bonding.


MAFA

MAFA (Mast cell function-associated antigen) is a type II membranal glycoprotein, first identified on the surface of rat mucosal-type mast cells of the RBL-2H3 line. More recently, human and mouse homologues of MAFA have been discovered yet also (or only) expressed by NK and T-cells.
The intracellular domain of MAFA contains a single immunoreceptor tyrosine-based inhibitory motif (ITIM), which classifies MAFA as a member of an inhibitory receptor superfamily. The inhibitory capacity of MAFA is best defined in mast cells, where MAFA keeps in check the antigen-induced (i.e. Fc epsilon RI-induced) secretion of inflammatory mediators.


Anaritide

Anaritide (also known as human atrial natriuretic peptide [102-126]) is a synthetic analogue of atrial natriuretic peptide (ANP).
Structure.
Anartidine has the following primary structure:
 RSSCFGGRMDRIGAQSGLGCNSFRY
or
H-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH
This structure is identical to residues 102-126 of human preproANP. In comparison, active human ANP comprises resides 99-126 of human preproANP.
Medical uses.
Anaritide has been investigated as a potential therapy for acute tubular necrosis but was shown not to improve the dialysis-free survivial of these patients. It also appears to exacerbate proteinuria and natriuresis in patients with nephrotic syndrome.


Alloantigen


Molecular cloning

Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word "cloning" refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.
In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of "E. coli" bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. Because they contain foreign DNA fragments, these are transgenic or genetically modified microorganisms (GMO). This process takes advantage of the fact that a single bacterial cell can be induced to take up and replicate a single recombinant DNA molecule. This single cell can then be expanded exponentially to generate a large amount of bacteria, each of which contain copies of the original recombinant molecule. Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as "clones". Strictly speaking, "recombinant DNA" refers to DNA molecules, while "molecular cloning" refers to the experimental methods used to assemble them. The idea arose that different DNA sequences could be inserted into a plasmid and that these foreign sequences would be carried into bacteria and digested as part of the plasmid. That is, these plasmids could serve as cloning vectors to carry genes.
Virtually any DNA sequence can be cloned and amplified, but there are some factors that might limit the success of the process. Examples of the DNA sequences that are difficult to clone are inverted repeats, origins of replication, centromeres and telomeres. Another characteristic that limits chances of success is large size of DNA sequence. Inserts larger than 10kbp have very limited success, but bacteriophages such as bacteriophage λ can be modified to successfully insert a sequence up to 40 kbp.
History.
Prior to the 1970s, the understanding of genetics and molecular biology was severely hampered by an inability to isolate and study individual genes from complex organisms. This changed dramatically with the advent of molecular cloning methods. Microbiologists, seeking to understand the molecular mechanisms through which bacteria restricted the growth of bacteriophage, isolated restriction endonucleases, enzymes that could cleave DNA molecules only when specific DNA sequences were encountered. They showed that restriction enzymes cleaved chromosome-length DNA molecules at specific locations, and that specific sections of the larger molecule could be purified by size fractionation. Using a second enzyme, DNA ligase, fragments generated by restriction enzymes could be joined in new combinations, termed recombinant DNA. By recombining DNA segments of interest with vector DNA, such as bacteriophage or plasmids, which naturally replicate inside bacteria, large quantities of purified recombinant DNA molecules could be produced in bacterial cultures. The first recombinant DNA molecules were generated and studied in 1972.
Overview.
Molecular cloning takes advantage of the fact that the chemical structure of DNA is fundamentally the same in all living organisms. Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for DNA replication, and the resulting recombinant DNA is introduced into the organism from which the replication sequences were obtained, then the foreign DNA will be replicated along with the host cell's DNA in the transgenic organism.
Molecular cloning is similar to polymerase chain reaction (PCR) in that it permits the replication of DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an "in vitro" solution, free of living cells.
Steps.
In standard molecular cloning experiments, the cloning of any DNA fragment essentially involves seven steps: (1) Choice of host organism and cloning vector, (2) Preparation of vector DNA, (3) Preparation of DNA to be cloned, (4) Creation of recombinant DNA, (5) Introduction of recombinant DNA into host organism, (6) Selection of organisms containing recombinant DNA, (7) Screening for clones with desired DNA inserts and biological properties.
Although the detailed planning of the cloning can be done in any text editor, together with online utilities for e.g. PCR primer design, dedicated software exist for the purpose. Software for the purpose include for example ApE (open source), DNAStrider (open source), Serial Cloner (gratis) and Collagene (open source).
Notably, the growing capacity and fidelity of DNA synthesis platforms allows for increasingly intricate designs in molecular engineering. These projects may include very long strands of novel DNA sequence and/or test entire libraries simultaneously, as opposed to of individual sequences. These shifts introduce complexity that require design to move away from the flat nucleotide-based representation and towards a higher level of abstraction. Examples of such tools are GenoCAD, Teselagen (free for academia) or GeneticConstructor (free for academics).
Choice of host organism and cloning vector.
Although a very large number of host organisms and molecular cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium "E. coli" ("Escherichia coli") and a plasmid cloning vector. "E. coli" and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment. If the DNA to be cloned is exceptionally large (hundreds of thousands to millions of base pairs), then a bacterial artificial chromosome or yeast artificial chromosome vector is often chosen.
Specialized applications may call for specialized host-vector systems. For example, if the experimentalists wish to harvest a particular protein from the recombinant organism, then an expression vector is chosen that contains appropriate signals for transcription and translation in the desired host organism. Alternatively, if replication of the DNA in different species is desired (for example, transfer of DNA from bacteria to plants), then a multiple host range vector (also termed shuttle vector) may be selected. In practice, however, specialized molecular cloning experiments usually begin with cloning into a bacterial plasmid, followed by subcloning into a specialized vector.
Whatever combination of host and vector are used, the vector almost always contains four DNA segments that are critically important to its function and experimental utility:
Preparation of vector DNA.
The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted. The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with the ends of the foreign DNA (see DNA end). Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme, for example EcoRI. Most modern vectors contain a variety of convenient cleavage sites that are unique within the vector molecule (so that the vector can only be cleaved at a single site) and are located within a gene (frequently beta-galactosidase) whose inactivation can be used to distinguish recombinant from non-recombinant organisms at a later step in the process. To improve the ratio of recombinant to non-recombinant organisms, the cleaved vector may be treated with an enzyme (alkaline phosphatase) that dephosphorylates the vector ends. Vector molecules with dephosphorylated ends are unable to replicate, and replication can only be restored if foreign DNA is integrated into the cleavage site.
Preparation of DNA to be cloned.
For cloning of genomic DNA, the DNA to be cloned is extracted from the organism of interest. Virtually any tissue source can be used (even tissues from extinct animals), as long as the DNA is not extensively degraded. The DNA is then purified using simple methods to remove contaminating proteins (extraction with phenol), RNA (ribonuclease) and smaller molecules (precipitation and/or chromatography). Polymerase chain reaction (PCR) methods are often used for amplification of specific DNA or RNA (RT-PCR) sequences prior to molecular cloning.
DNA for cloning experiments may also be obtained from RNA using reverse transcriptase (complementary DNA or cDNA cloning), or in the form of synthetic DNA (artificial gene synthesis). cDNA cloning is usually used to obtain clones representative of the mRNA population of the cells of interest, while synthetic DNA is used to obtain any precise sequence defined by the designer. Such a designed sequence may be required when moving genes across genetic codes (for example, from the mitochrondria to the nucleus) or simply for increasing expression via codon optimization.
The purified DNA is then treated with a restriction enzyme to generate fragments with ends capable of being linked to those of the vector. If necessary, short double-stranded segments of DNA ("linkers") containing desired restriction sites may be added to create end structures that are compatible with the vector.
Creation of recombinant DNA with DNA ligase.
The creation of recombinant DNA is in many ways the simplest step of the molecular cloning process. DNA prepared from the vector and foreign source are simply mixed together at appropriate concentrations and exposed to an enzyme (DNA ligase) that covalently links the ends together. This joining reaction is often termed ligation. The resulting DNA mixture containing randomly joined ends is then ready for introduction into the host organism.
DNA ligase only recognizes and acts on the ends of linear DNA molecules, usually resulting in a complex mixture of DNA molecules with randomly joined ends. The desired products (vector DNA covalently linked to foreign DNA) will be present, but other sequences (e.g. foreign DNA linked to itself, vector DNA linked to itself and higher-order combinations of vector and foreign DNA) are also usually present. This complex mixture is sorted out in subsequent steps of the cloning process, after the DNA mixture is introduced into cells.
Introduction of recombinant DNA into host organism.
The DNA mixture, previously manipulated in vitro, is moved back into a living cell, referred to as the host organism. The methods used to get DNA into cells are varied, and the name applied to this step in the molecular cloning process will often depend upon the experimental method that is chosen (e.g. transformation, transduction, transfection, electroporation).
When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation, and cells that are in a physiological state such that they can take up DNA are said to be competent. In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed transfection. Both transformation and transfection usually require preparation of the cells through a special growth regime and chemical treatment process that will vary with the specific species and cell types that are used.
Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present). In contrast, transduction involves the packaging of DNA into virus-derived particles, and using these virus-like particles to introduce the encapsulated DNA into the cell through a process resembling viral infection. Although electroporation and transduction are highly specialized methods, they may be the most efficient methods to move DNA into cells.
Selection of organisms containing vector sequences.
Whichever method is used, the introduction of recombinant DNA into the chosen host organism is usually a low efficiency process; that is, only a small fraction of the cells will actually take up DNA. Experimental scientists deal with this issue through a step of artificial genetic selection, in which cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive.
When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin. Cells harboring the plasmid will survive when exposed to the antibiotic, while those that have failed to take up plasmid sequences will die. When mammalian cells (e.g. human or mouse cells) are used, a similar strategy is used, except that the marker gene (in this case typically encoded as part of the kanMX cassette) confers resistance to the antibiotic Geneticin.
Screening for clones with desired DNA inserts and biological properties.
Modern bacterial cloning vectors (e.g. pUC19 and later derivatives including the pGEM vectors) use the blue-white screening system to distinguish colonies (clones) of transgenic cells from those that contain the parental vector (i.e. vector DNA with no recombinant sequence inserted). In these vectors, foreign DNA is inserted into a sequence that encodes an essential part of beta-galactosidase, an enzyme whose activity results in formation of a blue-colored colony on the culture medium that is used for this work. Insertion of the foreign DNA into the beta-galactosidase coding sequence disables the function of the enzyme so that colonies containing transformed DNA remain colorless (white). Therefore, experimentalists are easily able to identify and conduct further studies on transgenic bacterial clones, while ignoring those that do not contain recombinant DNA.
The total population of individual clones obtained in a molecular cloning experiment is often termed a DNA library. Libraries may be highly complex (as when cloning complete genomic DNA from an organism) or relatively simple (as when moving a previously cloned DNA fragment into a different plasmid), but it is almost always necessary to examine a number of different clones to be sure that the desired DNA construct is obtained. This may be accomplished through a very wide range of experimental methods, including the use of nucleic acid hybridizations, antibody probes, polymerase chain reaction, restriction fragment analysis and/or DNA sequencing.
Applications.
Molecular cloning provides scientists with an essentially unlimited quantity of any individual DNA segments derived from any genome. This material can be used for a wide range of purposes, including those in both basic and applied biological science. A few of the more important applications are summarized here.
Genome organization and gene expression.
Molecular cloning has led directly to the elucidation of the complete DNA sequence of the genomes of a very large number of species and to an exploration of genetic diversity within individual species, work that has been done mostly by determining the DNA sequence of large numbers of randomly cloned fragments of the genome, and assembling the overlapping sequences.
At the level of individual genes, molecular clones are used to generate probes that are used for examining how genes are expressed, and how that expression is related to other processes in biology, including the metabolic environment, extracellular signals, development, learning, senescence and cell death. Cloned genes can also provide tools to examine the biological function and importance of individual genes, by allowing investigators to inactivate the genes, or make more subtle mutations using regional mutagenesis or site-directed mutagenesis. Genes cloned into expression vectors for functional cloning provide a means to screen for genes on the basis of the expressed protein's function.
Production of recombinant proteins.
Obtaining the molecular clone of a gene can lead to the development of organisms that produce the protein product of the cloned genes, termed a recombinant protein. In practice, it is frequently more difficult to develop an organism that produces an active form of the recombinant protein in desirable quantities than it is to clone the gene. This is because the molecular signals for gene expression are complex and variable, and because protein folding, stability and transport can be very challenging.
Many useful proteins are currently available as recombinant products. These include--(1) medically useful proteins whose administration can correct a defective or poorly expressed gene (e.g. recombinant factor VIII, a blood-clotting factor deficient in some forms of hemophilia, and recombinant insulin, used to treat some forms of diabetes), (2) proteins that can be administered to assist in a life-threatening emergency (e.g. tissue plasminogen activator, used to treat strokes), (3) recombinant subunit vaccines, in which a purified protein can be used to immunize patients against infectious diseases, without exposing them to the infectious agent itself (e.g. hepatitis B vaccine), and (4) recombinant proteins as standard material for diagnostic laboratory tests.
Transgenic organisms.
Once characterized and manipulated to provide signals for appropriate expression, cloned genes may be inserted into organisms, generating transgenic organisms, also termed genetically modified organisms (GMOs). Although most GMOs are generated for purposes of basic biological research (see for example, transgenic mouse), a number of GMOs have been developed for commercial use, ranging from animals and plants that produce pharmaceuticals or other compounds (pharming), herbicide-resistant crop plants, and fluorescent tropical fish (GloFish) for home entertainment.
Gene therapy.
Gene therapy involves supplying a functional gene to cells lacking that function, with the aim of correcting a genetic disorder or acquired disease. Gene therapy can be broadly divided into two categories. The first is alteration of germ cells, that is, sperm or eggs, which results in a permanent genetic change for the whole organism and subsequent generations. This “germ line gene therapy” is considered by many to be unethical in human beings. The second type of gene therapy, “somatic cell gene therapy”, is analogous to an organ transplant. In this case, one or more specific tissues are targeted by direct treatment or by removal of the tissue, addition of the therapeutic gene or genes in the laboratory, and return of the treated cells to the patient. Clinical trials of somatic cell gene therapy began in the late 1990s, mostly for the treatment of cancers and blood, liver, and lung disorders.
Despite a great deal of publicity and promises, the history of human gene therapy has been characterized by relatively limited success. The effect of introducing a gene into cells often promotes only partial and/or transient relief from the symptoms of the disease being treated. Some gene therapy trial patients have suffered adverse consequences of the treatment itself, including deaths. In some cases, the adverse effects result from disruption of essential genes within the patient's genome by insertional inactivation. In others, viral vectors used for gene therapy have been contaminated with infectious virus. Nevertheless, gene therapy is still held to be a promising future area of medicine, and is an area where there is a significant level of research and development activity.


Armillaria gallica

Armillaria gallica (synonymous with "A. bulbosa" and "A. lutea") is a species of honey mushroom in the family Physalacriaceae of the order Agaricales. The species is a common and ecologically important wood-decay fungus that can live as a saprobe, or as an opportunistic parasite in weakened tree hosts to cause root or butt rot. It is found in temperate regions of Asia, North America, and Europe. The species forms fruit bodies singly or in groups in soil or rotting wood. The fungus has been inadvertently introduced to South Africa. "Armillaria gallica" has had a confusing taxonomy, due in part to historical difficulties encountered in distinguishing between similar "Armillaria" species. The fungus received international attention in the early 1990s when an individual colony living in a Michigan forest was reported to cover an area of , weigh at least , and be 1,500 years old. This individual is popularly known as the "humungous fungus", and is a tourist attraction and inspiration for an annual mushroom-themed festival in Crystal Falls. Recent studies have revised the fungus's age to 2,500 years and its size to about , four times the original estimate.
"Armillaria gallica" is a largely subterranean fungus, and it produces fruit bodies that are up to about in diameter, yellow-brown, and covered with small scales. On the underside of the caps are gills that are white to creamy or pale orange. The stem may be up to long, with a white cobwebby ring that divides the color of the stem into pale orange to brown above, and lighter-colored below. The fungus can develop an extensive system of underground root-like structures, called rhizomorphs, that help it to efficiently decompose dead wood in temperate broadleaf and mixed forests. It has been the subject of considerable scientific research due to its importance as a plant pathogen, its ability to bioluminesce, its unusual life cycle, and its ability to form large and long-lived colonies.
Phylogeny, taxonomy and naming.
Confusion has surrounded the nomenclature and taxonomy of the species now known as "Armillaria gallica", paralleling that surrounding the genus "Armillaria". The type species, "Armillaria mellea", was until the 1970s believed to be a pleiomorphic species with a wide distribution, variable pathogenicity, and one of the broadest host ranges known for the fungi. In 1973, Veikko Hintikka reported a technique to distinguish between "Armillaria" species by growing them together as single spore isolates on petri dishes and observing changes in the morphology of the cultures. Using a similar technique, Kari Korhonen showed in 1978 that the European "Armillaria mellea" species complex could be separated into five reproductively isolated species, which he named "European Biological Species" (EBS) A through E. About the same time, the North American "A. mellea" was shown to be ten different species (North American Biological Species, or NABS I through X); NABS VII was demonstrated shortly after to be the same species as EBS E. Because several research groups had worked with this widely distributed species, it was assigned several different names.
The species that Korhonen called EBS B was named "A. bulbosa" by Helga Marxmüller in 1982, as it was thought to be equivalent to "Armillaria mellea" var. "bulbosa", first described by Jean Baptiste Barla (Joseph Barla) in 1887, and later raised to species status by Josef Velenovský in 1927. In 1973, the French mycologist Henri Romagnesi, unaware of Velenovský's publication, published a description of the species he called "Armillariella bulbosa", based on specimens he had found near Compiègne and Saint-Sauveur-le-Vicomte in France. These specimens were later demonstrated to be the same species as the EBS E of Korhonen; EBS B was later determined to be "A. cepistipes". Therefore, the name "A. bulbosa" was a misapplied name for EBS E. In 1987 Romagnesi and Marxmüller renamed EBS E to "Armillaria gallica". Another synonym, "A. lutea", had originally been described by Claude Casimir Gillet in 1874, and proposed as a name for EBS E. Although the name had priority due to its early publication date, it was rejected as a "nomen ambiguum" because of a lack of supporting evidence to identify the fungus, including a specimen, type locality, and incomplete collection notes. "A. inflata" (Velenovský, 1920) may represent another synonym, but the type specimens were not preserved, so it is considered a dubious name ("nomen dubium"). As of 2010, both the Index Fungorum and MycoBank consider "Armillaria gallica" Marxm. & Romagn. to be the current name, with "A. bulbosa" and "A. lutea" as synonyms.
Phylogenetic analysis of North American "Armillaria" species based on analysis of amplified fragment length polymorphism data suggests that "A. gallica" is most closely related to "A. sinapina", "A. cepistipes", and "A. calvescens". These results are similar to those reported in 1992 that compared sequences of nuclear ribosomal DNA.
The specific epithet "gallica" is botanical Latin for "French" (from "Gallia", "Gaul"), and refers to the type locality. The prior name "bulbosa" is Latin for "bulb-bearing, bulbous" (from "bulbus" and the suffix -"osa"). "Armillaria" is derived from the Latin "armilla", or "bracelet".
Description.
The fruit bodies of "Armillaria gallica" have caps that are broad, and depending on their age, may range in shape from conical to convex to flattened. The caps are brownish-yellow to brown when moist, often with a darker-colored center; the color tends to fade upon drying. The cap surface is covered with slender fibers (same color as the cap) that are erect, or sloping upwards.
When the fruit bodies are young, the underside of the caps have a cottony layer of tissue stretching from the edge of the cap to the stem—a partial veil—which serves to protect the developing gills. As the cap grows in size the membrane is eventually pulled away from the cap to expose the gills. The gills have an adnate (squarely attached) to somewhat decurrent (extending down the length of the stem) attachment to the stem. They are initially white, but age to a creamy or pale orange covered with rust-colored spots. The stem is long and thick, and almost club-shaped with the base up to thick. Above the level of the ring, the stem is pale orange to brown, while below it is whitish or pale pink, becoming grayish-brown at the base. The ring is positioned about below the level of the cap, and may be covered with yellowish to pale-brownish woolly cottony mycelia. The base of the stem is attached to rhizomorphs, black root-like structures 1–3 mm in diameter. While the primary function of the below-ground mycelia is to absorb nutrients from the soil, the rhizomorphs serve a more exploratory function, to locate new food bases.
Microscopic features.
When the spores are seen in deposit, such as with a spore print, they appear whitish. They have an ellipsoid or oblong shape, usually contain an oil droplet, and have dimensions of 7–8.5 by 5–6 µm. The spore-bearing cells, the basidia, are club-shaped, four-spored (rarely two-spored), and measure 32–43 by 7–8.7 µm. Other cells present in the fertile hymenium include the cheilocystidia (cystidia present on the edge of a gill), which are club-shaped, roughly cylindrical and 15–25 by 5.0–12 µm. Cystidia are also present on the stem (called caulocystidia), and are broadly club-shaped, measuring 20–55 by 11–23 µm. The cap cuticle is made of hyphae that are irregularly interwoven and project upward to form the scales seen on the surface. The hyphae that make up the surface scales typically measure 26–88 µm long by 11–27 µm thick and can be covered with a crust of pigment. Clamp connections are present in the hyphae of most tissues.
Edibility.
Like all "Armillaria" species, "A. gallica" is considered edible. Thorough cooking is usually recommended, as the raw mushroom tastes acrid when fresh or undercooked. One author advises to consume only a small portion initially, as some people may experience an upset stomach. The taste is described as "mild to bitter", and the odor "sweet", or reminiscent of camembert cheese.
Similar species.
"Armillaria calvescens" is rather similar in appearance, and can only be reliably distinguished from "A. gallica" by observing microscopic characteristics. "A. calvescens" has a more northern distribution, and in North America, is rarely found south of the Great Lakes. "A. mellea" has a thinner stem than "A. gallica", but can be more definitively distinguished by the absence of clamps at the base of the basidia. Similarly, "A. cepistipes" and "A. gallica" are virtually identical in appearance (especially older fruit bodies), and are identified by differences in geographical distribution, host range, and microscopic characteristics. Molecular methods have been developed to discriminate between the two species by comparing DNA sequences in the gene coding translation elongation factor 1-alpha.
Metabolites.
"Armillaria gallica" can produce cyclobutane-containing metabolites such as arnamiol, a natural product that is classified as a sesquiterpenoid aryl ester. Although the specific function of arnamiol is not definitively known, similar chemicals present in other "Armillaria" species are thought to play a role in inhibiting the growth of antagonistic bacteria or fungi, or in killing cells of the host plant prior to infection.
Bioluminescence.
The mycelia (but not the fruit bodies) of "Armillaria gallica" are known to be bioluminescent. Experiments have shown that the intensity of the luminescence is enhanced when the mycelia are disturbed during growth or when they are exposed to fluorescent light. Bioluminescence is caused by the action of luciferases, enzymes that produce light by the oxidation of a luciferin (a pigment). The biological purpose of bioluminescence in fungi is not definitively known, although several hypotheses have been suggested: it may help attract insects to help with spore dispersal, it may be a by-product of other biochemical functions, or it may help deter heterotrophs that might consume the fungus.
Humongous fungus.
Researchers reported finding "Armillaria gallica" in the Upper Peninsula of Michigan in the early 1990s, during an unrelated research project to study the possible biological effects of extremely low frequency radio stations, which were being investigated as a means to communicate with submerged submarines. In one particular forest stand, "Armillaria"-infected oak trees had been harvested, and their stumps were left to rot in the field. Later, when red pines were planted in the same location, the seedlings were killed by the fungus, identified as "A. gallica" (then known as "A. bulbosa"). Using molecular genetics, they determined that the underground mycelia of one individual fungal colony covered , weighing over , with an estimated age of 1,500 years. The analysis used restriction fragment length polymorphism (RFLP) and random amplification of polymorphic DNA (RAPD) to examine isolates collected from fruit bodies and rhizomorphs (underground aggregations of fungal cells that resemble plant roots) along transects in the forest. The 15-hectare area yielded isolates that had identical mating type alleles and mitochondrial DNA restriction fragment patterns; this degree of genetic similarity indicated that the samples were all derived from a single genetic individual, or clone, that had reached its size through vegetative growth. In their conclusion, the authors noted: "This is the first report estimating the minimum size, mass, and age of an unambiguously defined fungal individual. Although the number of observations for plants and animals is much greater, members of the fungal kingdom should now be recognized as among the oldest and largest organisms on earth." After the "Nature" paper was published, major media outlets from around the world visited the site where the specimens were found; as a result of this publicity, the individual acquired the common name "humongous fungus". There was afterward some scholarly debate as to whether the fungus qualified to be considered in the same category as other large organisms such as the blue whale or the giant redwood.
The fungus has since become a popular tourist attraction in Michigan, and has inspired a "Humongous Fungus Fest" held annually in August in Crystal Falls. The organism was the subject of a Late Show Top Ten List on Late Night with David Letterman, and an advertising campaign by the rental company U-Haul.
Life cycle and growth.
The life cycle of "A. gallica" includes two diploidization–haploidization events. The first of these is the usual process of cell fusion (forming a diploid) followed by meiosis during the formation of haploid basidiospores. The second event is more cryptic and occurs before fruit body formation. In most basidiomycetous fungi, the hyphae of compatible mating types will fuse to form a two-nucleate, or dikaryotic stage; this stage is not observed in "Armillaria" species, which have cells that are mostly monokaryotic and diploid. Genetic analyses suggest that the dikaryotic mycelia undergo an extra haploidization event prior to fruit body formation to create a genetic mosaic. These regular and repeating haploidization events result in increased genetic diversity, which helps the fungus to adapt to unfavorable changes in environmental conditions, such as drought.
The growth rate of "A. gallica" rhizomorphs is between per year. Population genetic studies of the fungus conducted in the 1990s demonstrated that genetic individuals grow mitotically from a single point of origin to eventually occupy territories that may include many adjacent root systems over large areas (several hectares) of forest floor. Based on the low mutation rates observed in large, long-lived individuals, "A. gallica" appears to have an especially stable genome. It has also been hypothesized that genetic stability may result from self-renewing mycelial repositories of nuclei with stem cell-like properties.
Habitat and distribution.
"Armillaria gallica" can normally be found on the ground, but sometimes on stumps and logs. Mushrooms that appear to be terrestrial are attached to plant roots underneath the surface. It is widely distributed and has been collected in North America, Europe, and Asia (China, Iran, and Japan). The species has also been found in the Western Cape Province of South Africa, where it is thought to have been introduced from potted plants imported from Europe during the early colonization of Cape Town. In Scandinavia, it is absent in areas with very cold climates, like Finland or Norway, but it is found in southern Sweden. It is thought to be the most prevalent low altitude species of "Armillaria" in Great Britain and France. The upper limits of its altitude vary by region. In the French Massif Central, it is found up to , while in Bavaria, which has a more continental climate, the upper limit of distribution reaches . In Serbian forests, it is the most common "Armillaria" between elevations of . Field studies suggest that "A. gallica" prefers sites that are low in organic matter and have high soil pHs.
In North America, it is common east of the Rocky Mountains, but rare in the Pacific Northwest. In California, where it is widely distributed, the fungus is found in a variety of plant communities, including aspen, coastal oak woodland, Douglas Fir, Klamath mixed conifer, montane hardwood, montane hardwood-conifer, montane riparian, Redwood, Sierran mixed conifer, valley oak woodland, valley-foothill riparian, and White Fir. It was found to be the most common "Armillaria" species in hardwood and mixed oak forests in western Massachusetts.
A Chinese study published in 2001 used the molecular biological technique restriction fragment length polymorphism to analyze the differences in DNA sequence between 23 "A. gallica" specimens collected from the Northern Hemisphere. The results suggest that based on the restriction fragment length polymorphism patterns observed, there are four global "A. gallica" subpopulations: the Chinese, European, North American–Chinese, and North American–European geographical lineages. A 2007 study on the northeastern and southwestern Chinese distribution of "Armillaria", using fruit body and pure culture morphology, concluded that there are several unnamed species (Chinese biological species C, F, H, J and L) that are similar to the common "A. gallica".
Ecology.
"Armillaria gallica" is a weaker pathogen than the related "A. mellea" or "A. solidipes", and is considered a secondary parasite—typically initiating infection only after the host's defenses have been weakened by insect defoliation, drought, or infection by another fungus. Fungal infection can lead to root rot or butt rot. As the diseased trees die, the wood dries, increasing the chance of catching fire after being struck by lightning. The resulting forest fire may, in turn, kill the species that killed the trees. Plants that are under water stress caused by dry soils or waterlogging are more susceptible to infection by "A. gallica". It has been shown to be one of several "Armillaria" species responsible for widespread mortality of oak trees in the Arkansas Ozarks. The fungus has also been shown to infect Daylily in South Carolina, Northern highbush blueberry ("Vaccinium corymbosum") in Italy and vineyards ("Vitis" species) of Rías Baixas in northwestern Spain. The latter infestation "may be related to the fact that the vineyards from which they were isolated were located on cleared forestry sites". When "A. solidipes" and "A. gallica" co-occur in the same forest, infection of root systems by "A. gallica" may reduce damage or prevent infection from "A. solidipes".
"Armillaria gallica" can develop an extensive subterranean system of rhizomorphs, which helps it to compete with other fungi for resources or to attack trees weakened by other fungi. A field study in an ancient broadleaved woodland in England showed that of five "Armillaria" species present in the woods, "A. gallica" was consistently the first to colonize tree stumps that had been coppiced the previous year. Fractal geometry has been used to model the branching patterns of the hyphae of various "Armillaria" species. Compared to a strongly pathogenic species like "A. solidipes", "A. gallica" has a relatively sparse branching pattern that is thought to be "consistent with a foraging strategy in which acceptable food bases may be encountered at any distance, and which favours broad and divisive distribution of potential inoculum." Because the rhizomorphs form regular networks, mathematical concepts of graph theory have been employed to describe fungal growth and interpret ecological strategies, suggesting that the specific patterns of network attachments allow the fungus "to respond opportunistically to spatially and temporally changing environments".
"Armillaria gallica" may itself be parasitized by other soil flora. Several species of the fungus "Trichoderma", including "Trichoderma polysporum", "T. harzianum" and "T. viride", are able to attack and penetrate the outer tissue of "A. gallica" rhizomorphs and parasitize the internal hyphae. The infected rhizomorphs become devoid of living hyphae about one week after the initial infection. "Entoloma abortivum" is another fungus that can live parasitically upon "A. gallica". The whitish-gray malformed fruit bodies that may result are due to the "E. abortivum" hyphae penetrating the mushroom and disrupting its normal development.


Lash (comics)

Lash is a fictional supervillain appearing in American comic books published by Marvel Comics. The character is an Inhuman. Created by writer Charles Soule and artist Joe Madureira, he first appeared in "Inhuman" #1 (June 2014), and plays a key role after the release of the Terrigen Mists around the globe at the conclusion of the "Infinity" storyline.
Lash appeared in the third season of the Marvel Cinematic Universe series "Agents of S.H.I.E.L.D.", portrayed by Matt Willig as Lash and Blair Underwood as his human alter-ego Andrew Garner.
Publication history.
Lash was created by writer Charles Soule and artist Joe Madureira, and first appeared in "Inhuman" #1 (June 2014).
According to Charles Soule, the character comes from Orollan, another Inhuman city:
Soule has said that "He's trying to do what he can to both collect strong Inhumans to help his own society, but also cleanse the planet of Inhumans who should never have received Terrigenesis in the first place", because "the idea is that not everyone on Earth sees all these new Inhumans popping up as a good thing."
He also appears in All-New Invaders Issues # 14 to recruit more Inhumans.
Fictional character biography.
Born in the hidden Inhuman city of Orollan (which is somewhere in Greenland), Lash was among the few Inhumans of his generation chosen to undergo Terrigenesis. When the Inhuman King Black Bolt activated a Terrigen Bomb above New York flooding the world with Terrigen Mist and awakening the powers of Inhuman descendants living among humanity (as seen at the end of the "Infinity" storyline and the start of the "Inhumanity" storyline), Lash embarked on a mission to find all the individuals affected and judge for himself whether they were worthy to live with their new abilities. Upon arriving in Illinois, Lash encountered a newly created Inhuman named Dante Pertuz. He attempted to persuade Dante to join him at Orollan until he was stopped by the Inhuman Queen Medusa.
Recruiting a NuHuman named Jason in Minnesota, Lash teleports them both to Orollan. There, he explains that Jason and his family were descendants of the Inhumans and while his family may have died during Terrigenesis, Jason has survived and will now have a home among his fellow survivors. With their gifts, they can rebuild the city and perform wonders. Lash shows Jason the most sacred space in Orollan—the Terrigenesis chamber. Here, they hoarded the crystals that bestow Terrigenesis that will show the worthy their true form. Lash says Jason must call himself Korvostax now and introduces him to other NuHumans who explain that humans have formed mobs and attacked them in their communities. The other NuHumans in Lash's group blame Medusa. Lash's new recruits are rebuilding a wall despite a near-total lack of prior experience. Jason asks why Lash isn't showing them how to do this, and the others tell him it isn't wise to speak like that—there used to be two more NuHumans in the group. The New Attilan group teleports nearby, looking to capture Lash at the advice of Lineage. Lash tells the others that Queen Medusa means to kill them, and urges them to fight. As the battle develops between the New Attilan group and Lash's group, Jason taps into his powers for the first time and begins throwing rocks at Gorgon. Lash absorbs the energy of a volcanic spring to power energy blasts that he fires randomly into the scrum. Jason is saved from death by Gorgon who then stuns Lash with a small earth tremor. Taking hold of Lash, Medusa pins him, then invites Lineage to step forward and tell Lash what he told her. Lineage explains that Black Bolt originally released the Terrigen Cloud because something is coming for the Inhumans and they need everyone to hold it back with as many hidden bloodlines from every nation on Earth united. If they cannot prepare, their species (and maybe humans as well) will become extinct. Medusa gathers the NuHumans and tells Lash that he can either help her, stay away, or face defeat. It is also mentioned that Lash can tell Ennilux and the others from her what has transpired.
During the "Civil War II" storyline, Lash is told about Iron Man abducting Ulysses Cain from New Attilan by Maximus. He considered what Iron Man did as an act of war against all Inhumans. This led to Lash's group destroying the Stark Manufacturing Facility SZ-4 near Zurich, Switzerland so that they can force Medusa's hand for an attack on Iron Man. This led to Medusa and the Ultimates fighting Lash's group until Medusa teleports them to the Triskelion where Lash and his followers are taken into custody.
After escaping from the Triskelion, Lash and his followers learn that Black Bolt is back on Earth and that he is no longer powerful than before. They take advantage of this and attack Black Bolt. After Lash abducted Blinky and took him to Orollan, Black Bolt surrendered to him as Lash states that he will use Black Bolt's blood to make his own Terrigen bomb to make new Inhumans. Before Lash can go ahead with this plan, he and Jack Chain were killed by Blinky who was possessed by the Jailer.
Powers and abilities.
Lash has various energy conversion abilities:
According to Soule "Let's say someone's running at him — that's kinetic energy. Lash can change all of that into heat — and whoosh! — the other guy goes up in flames."
In other media.
Television.
Lash appears in the third season of "Agents of S.H.I.E.L.D." portrayed by Matt Willig. Executive producer Jed Whedon explains that the show's adaptation of the character is different from the comics, but some elements are inspired from the source material. This version of Lash is depicted to having black skin. Lash first appeared in the episode "Laws of Nature" and the episode "Devils You Know" revealed that he can transform into a human form. The episode "Among Us Hide..." revealed that Andrew Garner (portrayed by Blair Underwood), a psychologist at Culver University working for S.H.I.E.L.D. and ex-husband of Agent Melinda May (portrayed by Ming-Na Wen), is Lash's human form who went through Terrigenesis while investigating an Inhuman genealogy ledger that was kept by Jiaying (portrayed by Dichen Lachman). The show explains Lash's ability to return to his human form as his transition phase before he fully becomes an Inhuman. Similar to his comics' version, Lash searches for and kills every unworthy Inhuman without prejudice. Lash even enlisted an Inhuman named Dwight Frye who can react to any nearby Inhumans to help find any worthy and unworthy ones. While he has attempted to kill Lincoln Campbell, Lash's victim included Alisha Whitely's clone, levitating Inhuman Shane Henson, pyrokinetic Inhuman Lori Henson, Dwight Frye after he served his purpose, and an assortment of unnamed Inhumans and Hydra Agents. Garner later surrenders himself to S.H.I.E.L.D. before the transformation becomes permanent and manages to say goodbye to May before completely becoming Lash. After saving Daisy Johnson (portrayed by Chloe Bennet) from Hive in Grant Ward's body (portrayed by Brett Dalton) and his "Inhuman Primitives" in the episode "Emancipation," he purged her of Hive's control and killed at least three Inhuman primitives before dying upon being impaled by Hellfire's chain. May later mourned his death. In the episode "The Real Deal," a fear manifestation of Lash is among the fear manifestations released when the Kree Beacon exploded near the three monoliths. It was destroyed by Phil Coulson and Deathlok.
Video games.
Lash is a playable character in "".


F-22 (psychedelic)

F-22 is a lesser-known psychedelic drug of the substituted amphetamine class. F-22 was first synthesized by Alexander Shulgin. In his book "PiHKAL", the minimum dosage is listed as 15 mg, and the duration unknown. F-22 produces few to no effects. Very little data exists about the pharmacological properties, metabolism, and toxicity of F-22.
Legality.
United Kingdom.
This substance is a Class A drug in the Drugs controlled by the UK Misuse of Drugs Act.


Dictyostelium class I RNA

The class I RNA is a non-coding RNA. This family was identified in Shotgun sequencing approach of full-length cDNA libraries from small RNAs from "Dictyostelium discoideum". The function of this RNA is unknown, but it bears some resemblance to riboswitches found primarily in bacteria. These RNAs are 42–65 nucleotides (nt) long and they share 5' and 3' sequence elements of 16 and 8 nt respectively. These elements can partially base-pair forming a short hairpin.
There are multiple copies of class I RNA in the "D. discoideum" genome and the RNA is highly expressed i.e. 14 unique sequences were identified in the small RNA library and it comprised ~12% of all RNA in the small RNA library. Homologs to class I RNA have only been located in "D. discoideum" to date. Some of the members of this family were also identified in a bioinformatic screen. This family has also been the subject of reviews.


GLD-2

GLD-2 (which stands for Germ Line Development 2) is a cytoplasmic poly(A) polymerase (cytoPAPs) which adds successive AMP monomers to the 3’ end of specific RNAs, forming a poly(A) tail, which is a process known as polyadenylation.
For RNA specificity, GLD-2 associates with an RNA-binding protein, typically a GLD-3, to form a heterodimer that acts as a cytoplasmic PAP. This protein has an enzymatic function and belongs to a family (DNA polymerase type-B-like family) which includes several similar enzymes such as GLD-1, GLD-3 and GLD-4.
This family of cytoplasmic PAPs has been described in several different species including "Homo sapiens", "Caenorhabditis elegans", "Xenopus", "Mus musculus" and "Drosophila".
Moreover, as it is a cytoplasmtaic PAP it differs from nuclear PAPs in some aspects. While nuclear PAPs contain a catalytic domain and an RNA-binding domain, GLD-2 family members have only a catalytic domain.
Localization.
GLD-2 is a common and abundant, but yet quite unknown protein that has already been found in each of the five kingdoms. In the animal kingdom, it has been specially detected in "Homo sapiens, Drosophila, Xenopus" and "Mus musculus". However, there has also been noticed the presence of GLD-2 in "Arabidopsis thaliana" which belongs in the plants kingdom; "Escherichia Coli" in monera and "Candida albicans" in fungi.
In human beings it is mostly expressed in the brain and within it, in the cerebellum, hippocampus and medulla. We can also find them in some other source tissues are the fibroblast, HeLa cell, MCF-7 cell, melanoma cell line and thymus. Inside those cells, it can be located in the nucleus and mitochondrion since its main function is related with DNA polyadenilation and these cell organelles are the only ones were DNA can be found. However, there are also GLD-2 in a soluble way in the cytosol, although the reason why they are there is still unsure.
In "Escherichia Coli", this enzymatic protein can be found in the cell membrane and in the cytosol, whereas in "Drosophila melanogaster", it predominates in the brain's nucleus and cytoplasm, oocyte, ovary and testis’ cells. Finally, in the "Arabidopsis thaliana", it is located in the flower's nucleus, root, stem and leaf cells.
Related functions.
GLD-2 primarily stabilizes mRNAs that are translationally repressed as well as it strongly promotes bulk polyadenylation. Surprisingly, those functions seem to have little impact on dynamizing efficient target mRNA translation, as it is an efficient Poly(A) Polymerase which helps developing polyadenylation activity. This activity is stimulated by its interaction with a putative RNA-binding protein: GLD-3. It is proposed by some studies that GLD-3 stimulates GLD-2 by recruiting it to the RNA. If so, then bringing GLD-2 to the RNA by other means also should stimulate its activity.
Molecular function.
ATP binding.
GLD-2, as a poly(A) polymerase (PAP) acts incorporating ATP at the 3' end of mRNAs in a template-independent manner.
Enzymatic activity: Polynucleotide adenylyltransferase activity.
It has been discovered that this protein has a catalytic activity, in other words, it has the ability to increase the speed of chemical reactions which would not occur so fast. It is known to catalysis the following reaction (which requires the following cofactor: Mg(2+)):
 ATP + RNA(n) ⇄ diphosphate + RNA(n+1)
Depending on the surroundings the optimal pH varies from 8 in the cytoplasm to 8.3 in the nucleus.
Biological process.
Hematopoietic progenitor cell differentiation.
The GLD-2 protein together with 136 proteins more, is involved in the molecular process of hematopoietic progenitor cell differentiation, in the human proteome. This is the process in which precursor cell type acquires the specialized features of a hematopoietic progenitor cell, a kind of cell types including myeloid progenitor cells and lymphoid progenitor cells.
mRNA processing by RNA polyadenylation.
The polyadenylation activity of GLD-2, as we previously mentioned, is stimulated by physical interaction with an RNA binding protein, GLD-3. To test whether GLD-3 might stimulate GLD-2 by recruiting it to RNA, some studies tethered C. elegans GLD-2 to mRNAs in "Xenopus" oocytes by using MS2 coat protein. Tethered GLD-2 adds poly(A) and stimulates translation of the mRNA, demonstrating that recruitment is sufficient to stimulate polyadenylation activity. PAP heterodimer in which GLD-2 contains the active site and GLD-3 provides RNA-binding specificity. MS2 coat protein was joined to GLD-2 to recruit it to an RNA.
Furthermore, GLD-2 activity is also important to maintain or up-regulate the abundance of many mRNAs, as the cytoplasmic polyadenylation has an essential role in activating maternal mRNA translation during early development. In vertebrates, the reaction requires CPEB, an RNA-binding protein and the poly(A) polymerase GLD-2.
The "Xenopus" enzyme, which exists in two closely related forms, polyadenylates RNAs to which it is tethered and enhances their translation. Likewise, it interacts with cytoplasmic polyadenylation factors, including Cleavage and polyadenylation specificity factor and CPEB, and with target mRNAs. These findings confirm and extend a recent report that a GLD-2 enzyme is the long-sought PAP responsible for cytoplasmic polyadenylation in oocytes.
In addition, the formation of long-term memory is believed to lack translational control of localized mRNAs. In mammals, dendrite mRNAs are kept in a repressed state and are activated upon repetitive stimulation. Several regulatory proteins required for translational control in early development are thought to be needed for memory formation, suggesting similar molecular mechanisms. In an experiment using "Drosophila", it has been detected the enzyme responsible for poly(A) elongation in the brain and it has been demonstrated too that its activity is required specifically for long-term memory. These findings provide strong evidence that cytoplasmic polyadenylation is critical for memory formation, and that GLD2 is the responsible enzyme.
Medical implications.
It has also been discovered that GLD2 has medical uses.
For example, such enzyme is overexpressed in patients who suffer from cancer; that's why it can be used as a prognostic factor for early appearance in breast cancer patients. Moreover, PAP activity is used to measure the effect of anticancer drugs as etoposide and cordycepin in two carcinoma cell lines: HeLa, which is the human epithelioid cervix carcinoma, and MCF-7 (human breast cancer).
However, in spite its utilities it can also be involved in the expression of several common diseases such as: leukemia, liver cirrhosis, brain injuries, hepatitis and in some cases infertility in male patients.


Jared Roach

Jared C. Roach is an American biologist who introduced the pairwise end sequencing strategy while at the University of Washington.
Education and early career.
Roach attended Cornell University, where he received his Bachelor of Science in Biology in 1990. He then attended the University of Washington, where he received his PhD in Immunology in 1998, and his MD in 1999. He trained in Internal Medicine at the University of Utah through 2001.
Career.
Starting as graduate student in the 1990s, Roach worked on the Human Genome Project from its early days through the end of the project in 2003. He contributed strategic and algorithmic designs to the Human Genome Project, including the pairwise end-sequencing strategy.
Roach was a Senior Fellow at the Department of Molecular Biotechnology at the University of Washington from 1999-2000. In 2001, he became a Research Scientist at the Institute for Systems Biology.
In 2009, he was a leader of a team that sequenced the whole genomes of a human family of four. This effort identified the cause of Miller syndrome, a simple recessive Mendelian disorder. It also produced the first complete whole-chromosomal haplotypes in humans. Haplotyping is the process of assigning all the variants in the genome to paternal and maternal chromosomes. The team applied these techniques to identify the causes of several Mendelian diseases, including genes for Adams-Oliver syndrome, alternating hemiplegia of childhood, epilepsy, palmoplantar keratoderma, and Fanconi anemia.
From 2007 to 2009, he was Scientific Director of the High-throughput Data Analysis Laboratory at Seattle Children’s Research Institute. Since then, he has been a Senior Research Scientist at the Institute for Systems Biology. Roach's group currently applies systems biology and genomics to complex diseases, focussing on the systems biology architecture of Alzheimer’s disease.
In 2020, Roach was a spokesperson for the molecular phylogenetics of SARS-CoV-2.
Selected publications.
Roach has over 80 publications with over 8000 citations. He has also translated a book on the Asian game of Go. His most-cited articles are:


Squelching

Squelching is a biological phenomenon in which a strong transcriptional activator acts to inhibit the expression of another gene. Squelching has been mostly studied in yeast, and most of the ideas regarding its mechanisms have come from research into modes of transcriptional control in yeast. One important study of this topic was conducted using the Gal4-VP16 artificial transcription factor system, where it was shown that the activating complex formed by VP-16 was sequestering adapters required for transcription of other targets.
The primary cause of squelching is believed to be the interaction of activator molecules disrupting the biochemical pathways associated with related processes due to structural similarity between the activators and important substrates along that pathway. In particular, the activator binds to transcription factors along alternative biochemical pathways, inhibiting the ability of these transcription factors to bind to their true targets. As in the example above, sequestration of an intermediate in a metabolic pathway is a confounding variable in genetic studies because knowledge of the expected binding targets of the primary molecules involved does not help predict why unexpected behavior results.


5,6-MeO-MiPT

5,6-MeO-MiPT, or 5,6-dimethoxy-N-methyl-N-isopropyltryptamine, is a lesser-known psychedelic drug. It is the 5,6-dimethoxy analog of MiPT. 5,6-MeO-MiPT was first synthesized by Alexander Shulgin. In his book "TiHKAL" ("Tryptamines I Have Known and Loved"), 5,6-MeO-MiPT produces no noticeable psychoactive effects. Very little data exists about the pharmacological properties, metabolism, and toxicity of 5,6-MeO-MiPT.


Latrophilin 2

Latrophilin 2 is a protein that in humans is encoded by the "ADGRL2" gene.
This gene encodes a member of the latrophilin subfamily of G-protein coupled receptors (GPCR). Latrophilins may function in both cell adhesion and signal transduction. In experiments with non-human species, endogenous proteolytic cleavage within a cysteine-rich GPS (G-protein-coupled-receptor proteolysis site) domain resulted in two subunits (a large extracellular N-terminal cell adhesion subunit and a subunit with substantial similarity to the secretin/calcitonin family of GPCRs) being non-covalently bound at the cell membrane. While several transcript variants have been described, the biological validity of only one has been determined.


Enhancer-FACS-seq

Enhancer-FACS-seq (eFS), developed by the Bulyk lab at Brigham and Women’s Hospital and Harvard Medical School, is a highly parallel enhancer assay that aims for the identification of active, tissue-specific transcriptional enhancers, in the context of whole "Drosophila melanogaster" embryos. This technology replaces the use of microscopy to screen for tissue-specific enhancers with fluorescence activated cell sorting (FACS) of dissociated cells from whole embryos, combined with identification by high-throughput Illumina sequencing.
Introduction.
Transcriptional regulation.
In metazoans, in order to respond to environmental stress, differentiate properly, and progress normally through the cell cycle, a eukaryotic cell needs a specific and coordinated gene expression program, which involves the highly regulated transcription of thousands of genes. This gene regulation is in large part controlled, in a tissue-specific manner, by the binding of transcription factors to noncoding genomic regions referred to as cis-regulatory modules (CRMs), activating or repressing gene expression by modulating the structure of the chromatin and therefore having a positive or negative effect on transcription regulation. CRMs activating gene expression are often referred to as transcriptional enhancers, whereas those repressing gene expression are referred to as transcriptional silencers.
Enhancer detection in Drosophila melanogaster.
Despite being a powerful model organism for biology and the study of transcriptional enhancers, the tissue specific activity of less than 5% of the estimated 50,000 transcriptional enhancers in "Drosophila melanogaster" have been discovered. Over the past decade, the main method for detection of tissue- or cell-type specific activities of enhancers in "Drosophila melanogaster" was to test candidate enhancers by traditional reporter assays, which are low-throughput and costly. Over the past few years, even though enhancer discovery has been improved and other parallel reporter assays have been developed, none so far allowed the direct identification of enhancer activity in a genomic context in cell types of interest in a whole embryo.
Methodology.
Each candidate CRM (cCRM) is cloned upstream of a reporter gene. Compared to traditional reporter assays, the main innovation is the use of fluorescence activated cell sorting (FACS) of dissociated cells, instead of microscopy, to screen for tissue-specific enhancers. This approach utilizes a two-marker system: in each embryo, one marker (here, the rat CD2 cell surface protein) is used to label cells of a specific tissue for being sorted by FACS, and the other marker (here, green fluorescent protein GFP) is used as a reporter of CRM activity.
Cells are sorted according to their tissue type and then by GFP fluorescence, and the cCRMs are recovered by PCR from double-positive sorted cells, and from total input cells. High-throughput sequencing of both populations then allows measuring the relative abundance of each cCRM in input and sorted populations; one can then assess the enrichment or depletion of each cCRM in double-positive cells versus input as a measure of activity in the CD2-positive cell type being tested.
Significant results.
In the initial report on this method, a library of ~500 cCRMs was drawn from a variety of genomic data sources (e.g., TF-bound regions, coactivator-bound regions, DNase I hypersensitive sites, and predictions from the Bulyk lab’s PhylCRM algorithm ) by PCR from genomic DNA, and then screened for activity in embryonic mesoderm and in specific mesodermal cell types. The results were validated by traditional reporter gene assay in "Drosophila melanogaster" embryos for 68 cCRMs tested by eFS. The specificity of eFS was excellent among significantly enriched cCRMs, while sensitivity was good where the majority of the CD2-positive cells express GFP. It was found that the known enhancer-associated chromatin marks H3K27ac, H3K4me1, and Pol II are significantly enriched among the enhancers found to be active in mesoderm.
Advantages and future applications.
Future applications.
The eFS assay could be used to analyze other cell or tissue types. By assessing enrichment in GFP-expressing CD2-negative as well as CD2-positive cells, and by crossing a common pool of reporter transformant male flies to females expressing CD2 in different cell types, it is possible to assay specificity as well as activity. Accelerating the annotation of the regulatory genome in Drosophila should in principle generate the kind of large-scale regulatory interaction data that would allow exploring the network properties of transcriptional regulation.


Flusoxolol

Flusoxolol is a selective beta-1 receptor blocker.


Iron-starvation-induced protein A

Iron-starvation-induced protein A, also known as isiA, is a photosynthesis-related chlorophyll-containing protein found in cyanobacteria. It belongs to the chlorophyll-a/b-binding family of proteins, and has been shown to have a photoprotection role in preventing oxidative damage via energy dissipation. It was originally identified under Fe starvation, and thus received the name iron-starvation-induced protein A. However, the protein has more recently been found to respond to a variety of stress conditions such as high irradiance. It can aggregate with carotenoids and form rings around the PSI reaction center complexes to aid in photoprotective energy dissipation.
Antenna function.
IsiA functions as an antenna for photosystem I (PSI) under iron-limiting conditions, when phycobilisomes disappear. In the (PSI)3(Isi3)18 complex most of the harvested energy is probably used by PSI; in other PSI-containing supercomplexes a large part of the energy will probably not be used for light harvesting, but rather is dissipated to protect the organism from light damage.
Under iron-starvation, it forms a complex with PSI trimers, where the trimer is surrounded by a ring composed of 18 isiA subunits. When the PSI subunits PsaF and PsaJ are missing the ring is composed of 17 isiA subunits, indicating that each isiA subunit has a different interaction with the trimer. This suggests that the size of the PSI complex determines the number of isiA units in the surrounding ring. In the absence of PsaL, it has a tendency to form incomplete rings with PSI monomers, suggesting PsaL helps form the rings. Also it can form other aggregates of varying sizes depending on the level of iron deprivation.
Photoprotective function.
IsiA aggregates forming empty multimeric rings (without PSI) also accumulate and are very abundant in long-term iron-depleted cells. When isolated, these aggregates are in a strongly quenched state, suggesting they are responsible for thermal dissipation of absorbed energy. IsiA is also synthesized in cells grown under high light irradiances, protecting from photodamage
External links.
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Ketosis

Ketosis is a metabolic state characterized by elevated levels of ketone bodies in the blood or urine. Physiologic ketosis is a normal response to low glucose availability, such as low-carbohydrate diets or fasting, that provides an additional energy source for the brain in the form of ketones. In physiologic ketosis, ketones in the blood are elevated above baseline levels, but the body's acid–base homeostasis is maintained. This contrasts with ketoacidosis, an uncontrolled production of ketones that occurs in pathologic states and causes a metabolic acidosis, which is a medical emergency. Ketoacidosis is most commonly the result of complete insulin deficiency in type 1 diabetes or late-stage type 2 diabetes. Ketone levels can be measured in blood, urine or breath and are generally between 0.5 and 3.0 millimolar (mM) in physiologic ketosis, while ketoacidosis may cause blood concentrations greater than 10 mM.
Trace levels of ketones are always present in the blood and increase when blood glucose reserves are low and the liver shifts from primarily metabolizing carbohydrates to metabolizing fatty acids. This occurs during states of increased fatty acid oxidation such as fasting, starvation, carbohydrate restriction, or prolonged exercise. When the liver rapidly metabolizes fatty acids into acetyl-CoA, some acetyl-CoA molecules can then be converted into ketone bodies: acetoacetate, beta-hydroxybutyrate, and acetone. These ketone bodies can function as an energy source as well as signalling molecules. The liver itself cannot utilize these molecules for energy, so the ketone bodies are released into the blood for use by peripheral tissues including the brain.
When ketosis is induced by carbohydrate restriction, it is sometimes referred to as nutritional ketosis. A low-carbohydrate, moderate protein diet that can lead to ketosis is called a ketogenic diet. Ketosis is well-established as a treatment for epilepsy and is also effective in treating type 2 diabetes. The possible effect on a range of neurological diseases, metabolic syndrome, cancer, and other conditions is currently under investigation.
Definition.
Physiologic ketosis.
Physiologic ketosis is a physiologic state characterized by elevated serum ketones and normal blood glucose and blood pH. Increasing production of ketone bodies is a response to low glucose availability that creates an alternate energy source for the brain. Physiologic ketosis can result from any state that increases fatty acid oxidation including fasting, prolonged exercise, or very low-carbohydrate diets such as the ketogenic diet. When physiologic ketosis is induced by carbohydrate restriction, it is sometimes referred to as nutritional ketosis. Ketone levels generally remain below 3 mM.
Ketoacidosis.
Ketoacidosis is a pathological state of uncontrolled production of ketones that results in a metabolic acidosis. Ketoacidosis is most commonly caused by a deficiency of insulin in type 1 diabetes or late stage type 2 diabetes but can also be the result of chronic heavy alcohol use, salicylate poisoning, or isopropyl alcohol ingestion. Ketoacidosis causes significant metabolic derangements and is a life-threatening medical emergency. Ketoacidosis is distinct from physiologic ketosis as it requires failure of the normal regulation of ketone body production.
Causes.
Elevated blood ketone levels are most often caused by accelerated ketone production but may also be caused by consumption of exogenous ketones or precursors.
When glycogen and blood glucose reserves are low, a metabolic shift occurs in order to save glucose for the brain which is unable to use fatty acids for energy. This shift involves increasing fatty acid oxidation and production of ketones in the liver as an alternate energy source for the brain as well as the skeletal muscles, heart, and kidney. Low levels of ketones are always present in the blood and increase under circumstances of low glucose availability. For example, after an overnight fast, 2-6% of energy comes from ketones and this increases to 30-40% after a 3-day fast.
The amount of carbohydrate restriction required to induce a state of ketosis is variable and depends on activity level, insulin sensitivity, genetics, age and other factors, but ketosis will usually occur when consuming less than 50 grams of carbohydrates per day for at least three days.
Neonates, pregnant women and lactating women are populations that develop physiologic ketosis especially rapidly in response to energetic challenges such as fasting or illness. This can progress to ketoacidosis in the setting of illness, although it occurs rarely. Propensity for ketone production in neonates is caused by their high-fat breast milk diet, disproportionately large central nervous system and limited liver glycogen.
Biochemistry.
The precursors of ketone bodies include fatty acids from adipose tissue or the diet and ketogenic amino acids. The formation of ketone bodies occurs via ketogenesis in the mitochondrial matrix of liver cells.
Fatty acids can be released from adipose tissue by adipokine signaling of high glucagon and epinephrine levels and low insulin levels. High glucagon and low insulin correspond to times of low glucose availability such as fasting. Fatty acids bound to coenzyme A allow penetration into mitochondria. Once inside the mitochondrion, the bound fatty acids are used as fuel in cells predominantly through beta oxidation, which cleaves two carbons from the acyl-CoA molecule in every cycle to form acetyl-CoA. Acetyl-CoA enters the citric acid cycle, where it undergoes an aldol condensation with oxaloacetate to form citric acid; citric acid then enters the tricarboxylic acid cycle (TCA), which harvests a very high energy yield per carbon in the original fatty acid.
Acetyl-CoA can be metabolized through the TCA cycle in any cell, but it can also undergo ketogenesis in the mitochondria of liver cells. When glucose availability is low, oxaloacetate is diverted away from the TCA cycle and is instead used to produce glucose via gluconeogenesis. This utilization of oxaloacetate in gluconeogenesis can make it unavailable to condense with acetyl-CoA, preventing entrance into the TCA cycle. In this scenario, energy can be harvested from acetyl-CoA through ketone production.
In ketogenesis, two acetyl-CoA molecules condense to form acetoacetyl-CoA via thiolase. Acetoacetyl-CoA briefly combines with another acetyl-CoA via HMG-CoA synthase to form hydroxy-β-methylglutaryl-CoA. Hydroxy-β-methylglutaryl-CoA form the ketone body acetoacetate via HMG-CoA lyase. Acetoacetate can then reversibly convert to another ketone body—D-β-hydroxybutyrate—via D-β-hydroxybutyrate dehydrogenase. Alternatively, acetoacetate can spontaneously degrade to a third ketone body (acetone) and carbon dioxide, which generates much greater concentrations of acetoacetate and D-β-hydroxybutyrate. The resulting ketone bodies cannot be used for energy by the liver so are exported from the liver to supply energy to the brain and peripheral tissues.
In addition to fatty acids, deaminated ketogenic amino acids can also be converted into intermediates in the citric acid cycle and produce ketone bodies.
Measurement.
Ketone levels can be measured by testing urine, blood or breath. There are limitations in directly comparing these methods as they measure different ketone bodies.
Urine testing.
Urine testing is the most common method of testing for ketones. Urine test strips utilize a nitroprusside reaction with acetoacetate to give a semi-quantitative measure based on color change of the strip. Although beta-hydroxybutyrate is the predominant circulating ketone, urine test strips only measure acetoacetate. Urinary ketones often correlate poorly with serum levels because of variability in excretion of ketones by the kidney, influence of hydration status, and renal function.
Serum testing.
Finger-stick ketone meters allow instant testing of beta-hydroxybutyrate levels in the blood, similar to glucometers. Beta-hydroxybutrate levels in blood can also be measured in a laboratory.
Medical uses.
Epilepsy.
Ketosis induced by a ketogenic diet is a long-accepted treatment for refractory epilepsy. It was first used in the 1920s and is now widely implemented for pediatric and adult patients.
Type 2 diabetes.
Ketosis induced by a very low-carbohydrate diet is supported as an effective treatment for type 2 diabetes and can improve blood glucose metrics even with reduction or discontinuation of antidiabetic medications. Ketosis can be an effective treatment for this population by reducing dietary glucose load, increasing insulin sensitivity as well as reducing hepatic glucose output. This results in reduction in fasting glucose and insulin and hemoglobin A1c as well as reduced need for exogenous insulin.
Obesity and metabolic syndrome.
Ketosis can improve markers of metabolic syndrome through reduction in serum triglycerides, elevation in high-density lipoprotein (HDL) as well as increased size and volume of low-density lipoprotein (LDL) particles. These changes are consistent with an improved lipid profile despite potential increases in total cholesterol level.
Research.
Ketosis is being investigated for a growing number of conditions, however clinical recommendations for these conditions cannot yet be made based on the current level of evidence.
Neurological disorders: In addition to its use for epilepsy, ketosis is being investigated in other neurological disorders because of its proposed neuroprotective effects including Alzheimer's disease, amyotrophic lateral sclerosis (ALS), autism, migraine headache, neurotrauma, pain, Parkinson's disease, and sleep disorders.
Cancer: Preclinical studies have indicated ketosis may have anti-tumor effects, although clinical trials have been limited by small sample sizes and have not shown conclusive benefit.
Glycogenosis Ketosis has been reported to alleviate symptoms related to glycogenosis as in some glycogen storage diseases. GSDs which are characterised by the inability of tissues to utilise glycogen stores, such as McArdle disease, may be managed with a ketogenic diet by ensuring that tissues are using ketone bodies for energy and not the impaired pathways of glycogen utilisation for glycolysis.
Other conditions: There is emerging evidence for the use of ketosis in other conditions such as type 1 diabetes, non-alcoholic fatty liver disease, acne, polycystic kidney disease and polycystic ovary syndrome, although evidence quality is limited by small sample sizes.
Safety.
The safety of ketosis from low-carbohydrate diets is often called into question by clinicians, researchers and the media. A common safety concern stems from the misunderstanding of the difference between physiologic ketosis and pathologic ketoacidosis. There is also continued debate whether chronic ketosis is a healthy state or a stressor to be avoided. Some argue that humans evolved to avoid ketosis and should not be in ketosis long-term. The counter-argument is that there is no physiologic requirement for dietary carbohydrate as adequate energy can be made via gluconeogenesis and ketogenesis indefinitely. Alternatively, the switching between a ketotic and fed state has been proposed to have beneficial effects on metabolic and neurologic health. The effects of sustaining ketosis for up to two years are known from studies of people following a strict ketogenic diet for epilepsy or type 2 diabetes, and these including short-term adverse effects leading to potential long-term ones. However, literature on longer term effects or intermittent ketosis is lacking.
Medication considerations.
Some medications require attention when in a state of ketosis, especially several classes of diabetes medication. SGLT2 inhibitor medications have been associated with cases of euglycemic ketoacidosis- a rare state of high ketones causing a metabolic acidosis with normal blood glucose levels. This usually occurs with missed insulin doses, illness, dehydration or adherence to a low-carbohydrate diet while taking the medication. Additionally, medications used to directly lower blood glucose including insulin and sulfonylureas may cause hypoglycemia if they are not titrated prior to starting a diet that results in ketosis.
Adverse effects.
The most common side effects of ketosis include headache, fatigue, dizziness, insomnia, difficulty in exercise tolerance, constipation, and nausea, especially in the first days and weeks after starting a ketogenic diet. This is commonly referred to as the "keto flu", and can be remedied by sufficient electrolyte intake. Breath may develop a sweet, fruity flavor via production of acetone that is exhaled because of its high volatility.
Most adverse effects of long-term ketosis reported are in children because of its longstanding acceptance as a treatment for pediatric epilepsy. These include compromised bone health, stunted growth, hyperlipidemia, and kidney stones.
Contraindications.
Ketosis induced by a ketogenic diet should not be pursued by people with pancreatitis because of the high dietary fat content. Ketosis is also contraindicated in pyruvate carboxylase deficiency, porphyria, and other rare genetic disorders of fat metabolism.
Veterinary medicine.
In dairy cattle, ketosis commonly occurs during the first weeks after giving birth to a calf and is sometimes referred to as "acetonemia". This is the result of an energy deficit when intake is inadequate to compensate for the increased metabolic demand of lactating. The elevated β-hydroxybutyrate concentrations can depress gluconeogenesis, feed intake and the immune system, as well as have an impact on milk composition. Point of care diagnostic tests can be useful to screen for ketosis in cattle.
In sheep, ketosis, evidenced by hyperketonemia with beta-hydroxybutyrate in blood over 0.7 mmol/L, is referred to as "pregnancy toxemia". This may develop in late pregnancy in ewes bearing multiple fetuses and is associated with the considerable metabolic demands of the pregnancy. In ruminants, because most glucose in the digestive tract is metabolized by rumen organisms, glucose must be supplied by gluconeogenesis. Pregnancy toxemia is most likely to occur in late pregnancy due to metabolic demand from rapid fetal growth and may be triggered by insufficient feed energy intake due to weather conditions, stress or other causes. Prompt recovery may occur with natural parturition, Caesarean section or induced abortion. Prevention through appropriate feeding and other management is more effective than treatment of advanced stages of pregnancy toxemia.