Membrane curvature

Membrane curvature is the geometrical measure or characterization of the curvature of membranes.
The membranes can be naturally occurring or man-made (synthetic). An example of naturally occurring membrane is the lipid bilayer of cells, also known as cellular membranes. Synthetic membranes can be obtained by preparing aqueous solutions of certain lipids. The lipids will then "aggregate" and form various phases and structures. According to the conditions (concentration, temperature, ionic strength of solution, etc.) and the chemical structures of the lipid, different phases will be observed. For instance, the lipid POPC (palmitoyl oleyl phosphatidyl choline) tends to form lamellar vesicles in solution, whereas smaller lipids (lipids with shorter acyl chains, up to 8 carbons in length), such as detergents, will form micelles if the CMC (critical micelle concentration) is reached.
Basic geometry.
A biological membrane is commonly described as a two-dimensional surface, which spans a three-dimensional space. So, to describe membrane shape, it is not sufficient to determine the membrane curling that is seen in a single cross-section of the object, because in general there are two curvatures that characterize the shape each point in space. Mathematically, these two curvatures are called the principal curvatures, formula_1 and formula_2, and their meaning can be understood by the following thought experiment. If you cross-section the membrane surface at a point under consideration using two planes that are perpendicular to the surface and oriented in two special directions called the principal directions, the principal curvatures are the curvatures of the two lines of intercepts between the planes and the surface which have almost circular shapes in close proximity to the point under consideration. The radii of these two circular fragments, formula_3 and formula_4, are called the principal radii of curvature, and their inverse values are referred to as the two principal curvatures.
formula_5
formula_6
The principal curvatures formula_1 and formula_2 can vary arbitrarily and thereby give origin to different geometrical shapes, such as cylinder, plane, sphere and saddle. Analysis of the principal curvature is important, since a number of biological membranes possess shapes that are analogous to these common geometry staples. For instance, prokaryotic cells such as cocci, rods, and spirochette display the shape of a sphere, and the latter two the shape of a cylinder. Erythrocytes, commonly referred to as red blood cells, have the shape of a saddle, although these cells are capable of some shape deformation. The table below lists common geometric shapes and a qualitative analysis of their two principal curvatures.
Even though often membrane curvature is thought to be a completely spontaneous process, thermodynamically speaking there must be factors actuating as the driving force for curvature to exist. Currently, there are some postulated mechanisms for accepted theories on curvature; nonetheless, undoubtedly two of the major driving forces are lipid composition and proteins embedded and/or bound to membranes.
Lipid spontaneous curvature.
Perhaps the most simple and intuitive driving force in membrane curvature is the natural spontaneous curvature exhibited by some lipids. This is because, depending on their chemical structures, lipids tend to curve with a slight spontaneously negative or positive curvature. Lipids such as DOPC (dioleoyl phosphatidyl choline), diacyl glycerol, dioleyl phosphatidylethanolamine (DOPE) and cholesterol exhibit a negative spontaneous curvature. On the other hand, lipids with smaller acyl chain area to polar head group area ratio tend to curve positively, in other words they exhibit positive spontaneous curvature. The table below lists experimentally determined spontaneous curvatures for different lipids in DOPE (dioleyl phosphatidyl ethanolamine).
The energy requirements to generate a cylinder shaped cell from an originally flat membrane can be expressed as
formula_9
where L is the length of the cylinder, JB is the difference between the spontaneous curvature, Js, for the lipids in the inner and outer leaflet divided by two, and Kb is the bending modulus of the bilayer.
The radii of membrane cylinders that form in intracellular membrane-transport pathways are typically ~25–30 nm. So, the spontaneous curvature necessary to generate such cylinders equals ~(1/50) nm–1. As JB results from a difference in the spontaneous curvatures of the monolayers, an unusual membrane lipid composition would be required to produce such curvature. The lipids cholesterol, dioleoylphosphatidylethanolamine(DOPE) and diacylglycerol are characterized by strongly negative spontaneous curvatures (figure 1) and therefore have the potential to generate a large membrane curvature. However, even for these lipids, the required JB can be reached only if they are extensively concentrated in the internal monolayer.
Proteins can Induce Curvature.
Some biologically occurring lipids do exhibit spontaneous curvature which could explain the shapes of biological membranes. Nevertheless, calculations show that spontaneous lipid curvature alone is either insufficient or would require conditions that are unrealistic to drive the degree of curvature observed in most cells. It is now known that lipid curvature is "aided" by protein structures in order to generate complete cellular curvature.
Currently there are 4 proposed mechanisms to explain protein-mediated membrane bending:
1. Lipid clustering.
Bacterial toxins such as cholera toxin B, shiga toxin B favors binding and thus clustering of certain lipid molecules. The effect of lipid clustering, together with the intrinsic shape of individual lipid molecule, give rise to membrane curvature.
2. Rigid scaffold.
A classical example of membrane bending by rigid protein scaffold is clathrin. Clathrin is involved in cellular endocytosis and is sequestrated by specific signaling molecules. Clathrin can attach to adaptor protein complexes on the cellular membrane, and it polymerizes into lattices to drive greater curvature, resulting in endocytosis of a vesicular unit. Coat protein complex I (COP1) and coat protein complex II (COPII) follow similar mechanism in driving membrane curvature. Figure A shows a protein coating that induces curvature. As mentioned above, proteins such as clathrin are recruited to the membrane through signaling molecules and assemble into larger polymeric structures that form a rigid structure which serves as a frame for the membrane. Clathrin binds to its receptors that are present in the membrane.
Another example of protein interactions that directly affect membrane curvature is that of the BAR (Bin, amphiphysin, Rvs’) domain. The BAR domain is present in a large family of proteins. Relative to the cellular lipid bilayer, this domain is rigid and exhibits a "banana" shape. It has been postulated that the positively charged amino acid residues in the concave region of the BAR domain would come into contact with the negatively charged polar head groups of lipids in the bilayer, thus allows the binding process. Upon binding, the membrane's curvature is increased by the rigid domain. Figure B shows bending of membrane by banana-shape like BAR domain.
3. Insertion of hydrophobic protein motifs.
Hydrophobic portion of protein can act as "wedge" when inserting into lipid bilayer. Epsin is one example that utilizes this mechanism to drive membrane bending. Epsin has several amphipathic alpha helices that allows it to partition between the hydrophobic core of the membrane and surrounding aqueous, hydrophilic environment. Another interesting characteristic of epsin and other proteins that bind to membranes is the fact that it shows high binding affinity for a fairly common membrane lipid, phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2). Unlike other proteins that simply bend the membrane through sheer rigidity, epsin is a globular soluble protein and thus not rigid. The insertion of its helices into the membrane force the neighboring lipids of the leaflet that has been bound to expand laterally. This displacement of lipids on only one of the leaflets increases the bilayer's curvature. Figure C shows membrane bending by insertion of hydrophobic protein parts into lipid bilayer.4.
The figure to the right illustrates the different mechanisms through which proteins can aid and/or induce membrane curvature. In A, an illustration of a BAR domain present in a number of proteins. The curvature is induced by the very shape of this proteic region. This domain attaches to the lipid bilayer through strong coulombic interactions. This idea is supported by the existence of positively charged amino acid residues in the concave region of the BAR domain. These amino acids would come into contact with the negatively charged polar head groups of lipids in the bilayer. This form phenomenon is also referred to as the "scaffold mechanism".
B shows a protein coating that induces curvature. As mentioned above, proteins such as clathrin are recruited to the membrane through signaling molecules and assemble into larger polymeric structures that form a rigid structure which serves as a frame for the membrane. Clathrin binds to its receptors that are present in the membrane.
C illustrates a slightly different mechanism. In this case, the membrane-bending protein does not exhibit intrinsic rigidity. Instead they are often globular and soluble. The protein epsin is an example. Epsin has an ENTH (epsin N-terminal homology) domain which inserts its amphipathic alpha helix into the membrane. Epsin has high binding affinity for the membrane if PI-4,5-P2 is present.
This figure illustrates membrane bending caused by protein crowding. When a high local concentration of proteins (shown in green) are present on membrane surface (shown in black), membrane curvature can be induced. This hypothesis reasoned that the high protein concentration increases the likelihood of repulsions between proteins, therefore generates steric pressure between proteins. To relieve such pressure, lipid membrane has to bend in order to decrease protein repulsions.
4. Protein crowding.
The protein crowding mechanism hypothesizes that proteins can bend membrane without directly perturbing membrane structures like the above mechanisms. When a high enough local concentration of protein is present on membrane surface, repulsion between protein molecules on the membrane surface can induce membrane curvature. Although contribution of this mechanism remains unclear, multiple experimental and computation evidences have shown its potential in bending membrane. A recent study even shows that protein crowding can cause membrane bending and leads to membrane fission. These studies suggest that high local protein concentration can overcome the energy barrier to bend lipid membrane, and thus can contribute to membrane bending.


BioCampus

The Biocampus is an enterprise area in Midlothian, Scotland. It is part of the larger Edinburgh Science Triangle, which includes the Edinburgh BioQuarter and was the first dedicated national bio-manufacturing campus. Development on the site is supported through financial incentives and business rate reduction through the Scottish Government's enterprise area scheme.
The campus was established in 2001 with a site facilitates large-scale biomanufacturing with the initial development costing around £15 million. There is outline plans in place to expand the site with a further for a phase 2 project. The first facility on the campus was opened by Alba Biosceince, a subsidiary of Quotient, as a commercial diagnostics company offering tests related to blood transfusion diagnostics. The firm received a Queen's Award for Enterprise in 2016 for their work in Midlothian both at the BioCampus site and the nearby Pentlands science park.
The campus has close links to a number of University departments including Heriot-Watt, University of Strathclyde and University of Edinburgh.
An evaluation in 2005 estimated that enterprise zones across Scotland had generated 58,000 full time jobs at a total public cost of £17,000 per ten-year job. However, some commentators have criticised the tax breaks and financial support given to private companies through enterprise zones, highlighting evidence that very few new high-quality jobs are created with most of the jobs being transferred from other parts of the country.
See also.
Other Life Sciences Enterprise Areas in Scotland include: Edinburgh BioQuarter; Forres, Moray; Inverness Campus, Highland; Irvine, North Ayrshire.


Methylglutamate dehydrogenase

In enzymology, a methylglutamate dehydrogenase () is an enzyme that catalyzes the chemical reaction
The 3 substrates of this enzyme are N-methyl-L-glutamate, acceptor, and H2O, whereas its 3 products are L-glutamate, formaldehyde, and reduced acceptor.
This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-NH group of donors with other acceptors. The systematic name of this enzyme class is N-methyl-L-glutamate:acceptor oxidoreductase (demethylating). Other names in common use include N-methylglutamate dehydrogenase, and N-methyl-L-glutamate:(acceptor) oxidoreductase (demethylating). This enzyme participates in methane metabolism.


Nucleotidyltransferase

Nucleotidyltransferases are transferase enzymes of phosphorus-containing groups, e.g., substituents of nucleotidylic acids or simply nucleoside monophosphates. The general reaction of transferring a nucleoside monophosphate moiety from A to B, can be written as:
For example, in the case of polymerases, A is pyrophosphate and B is the nascent polynucleotide.
They are classified under [[Enzyme Commw9iudn owiddmlsi;ja \l- groups
Role in metabolism.
Many metabolic enzymes are modified by nucleotidyltransferases. The attachment of an [[Adenosine monophosphate|AMP]] (adenylylation) or [[Uridine monophosphate|UMP]] (uridylylation) can activate or inactivate an enzyme or change its specificity (see figure). These modifications can lead to intricate regulatory networks that can finely tune [[Enzyme|enzymatic]] activities so that only the needed compounds are made (here: glutamine).
Role in DNA repair mechanisms.
Nucleotidyl transferase is a component of the repair pathway for single nucleotide [[base excision repair]]. This repair mechanism begins when a single nucleotide is recognized by DNA [[glycosylase]] as incorrectly matched or has been mutated in some way (UV light, chemical mutagen, etc.), and is removed. Later, a nucleotidyl transferase is used to fill in the gap with the correct base, using the [[template strand]] as the reference.
External links.
[[Category:Transferases]]
[[Category:EC 2.7.7|*]]


Mycena fera

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


L-methionine (S)-S-oxide reductase

In enzymology, a L-methionine (S)-S-oxide reductase () is an enzyme that catalyzes the chemical reaction
The 3 substrates of this enzyme are L-methionine, thioredoxin disulfide, and H2O, whereas its two products are L-methionine (S)-S-oxide and thioredoxin.
This enzyme belongs to the family of oxidoreductases, specifically those acting on a sulfur group of donors with a disulfide as acceptor. The systematic name of this enzyme class is L-methionine:thioredoxin-disulfide S-oxidoreductase. Other names in common use include fSMsr, methyl sulfoxide reductase I and II, acetylmethionine sulfoxide reductase, methionine sulfoxide reductase, L-methionine:oxidized-thioredoxin S-oxidoreductase, methionine-S-oxide reductase, and free-methionine (S)-S-oxide reductase. This enzyme participates in methionine metabolism.


Polymerase

A polymerase is an enzyme (EC 2.7.7.6/7/19/48/49) that synthesizes long chains of polymers or nucleic acids. DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules, respectively, by copying a DNA template strand using base-pairing interactions or RNA by half ladder replication.
A DNA polymerase from the thermophilic bacterium, "Thermus aquaticus" ("Taq") (PDB 1BGX, EC 2.7.7.7) is used in the polymerase chain reaction, an important technique of molecular biology.
A polymerase may be template dependent or template independent. Poly-A-polymerase is an example of template independent polymerase. Terminal deoxynucleotidyl transferase also known to have template independent and template dependent activities.
Types.
By structure.
Polymerases are generally split into two superfamilies, the "right hand" fold () and the "double psi beta barrel" (often simply "double-barrel") fold. The former is seen in almost all DNA polymerases and almost all viral single-subunit polymerases; they are marked by a conserved "palm" domain. The latter is seen in all multi-subunit RNA polymerases, in cRdRP, and in "family D" DNA polymerases found in archaea. The "X" family represented by DNA polymerase beta has only a vague "palm" shape, and is sometimes considered a different superfamily ().
Primases generally don't fall into either category. Bacterial primases usually have the Toprim domain, and are related to topoisomerases and mitochrondrial helicase twinkle. Archae and eukaryotic primases form an unrelated AEP family, possibly related to the polymerase palm. Both families nevertheless associate to the same set of helicases.


Neutrophil swarming

Neutrophil swarming is a type of coordinated neutrophil movement that acts in response to acute tissue inflammation or infection. The term comes from the swarming characteristics of insects that are similar to the behavior of neutrophils in response to an infection. These processes have mostly been studied in tissues of mice and studies of mouse ear tissue has proved to be very effective at observing neutrophil movement. Neutrophil swarming typically aggregates at surface layers of tissue so the thin nature of the mouse ear tissue makes for a good model to study this process. Additionally, zebrafish larvae have been used for the study of neutrophil movement mainly because of their translucence during the first few days of their development. With transgenic lines that fluorescently label zebrafish neutrophils, the cells can be tracked by epifluorescence or confocal microscopy during the course of an inflammatory response. Through this method, specific subpopulations of neutrophils can be tracked and their origin and fate during the induction and resolution of inflammation is observed. Another advantage for using zebrafish to study neutrophil swarming is that adaptive immunity for this organism does not develop until around 4 weeks of age. This allows for the study of neutrophil movement and other host immune responses independent of adaptive immune responses.
Variations.
A study of the lymph nodes of mice that were infected by injection of parasites into their earflaps revealed two types of neutrophil swarming: transient and persistent swarms. Transient swarms are characterized by groups of 10-150 neutrophils forming multiple small cell clusters within 10-40 minutes that quickly dispersed. Persistent swarms showed clusters of more than 300 neutrophils and recruitment lasted for more than 40 minutes. For both the transient and persistent swarms, the formed neutrophil clusters appeared to be competing with each other with the larger clusters attracting neutrophils from the smaller clusters. The study also revealed two distinct phases in swarm formation. The first phase occurs when a small number of “pioneer” neutrophils respond to an initial signal and form small clusters and this is followed by the second phase where there are a large scale migration of neutrophils leading to the growth of multiple cell clusters.
The exact size or duration of swarms depends on the specific inflammatory conditions as well as the tissue type of the infection location. Several factors that influence the swarm phenotype are: the size of the initial tissue damage, the presence of pathogens, the induction of secondary cell death, and the number of recruited neutrophils. A study that compared large scale tissue damage of sterile mouse tissue by a needle prick with small injuries by a laser beam showed that the needle prick provoked a larger and longer swarm response. After the needle injury, hundreds to thousands of neutrophils were recruited that formed stable cell clusters that sometimes were prolonged for days. In comparison, the neutrophil swarms resulting from the laser induced injury only recruited around 50-330 neutrophils which persisted for a few hours. The presence of pathogens can also increase the size of neutrophil swarms, not necessarily because of their presence as a foreign body, but because of the additional cell death that they can cause in infection sites. When cells are lysed in an infection site, they release an assortment of signaling factors that augment the recruitment of neutrophils to the site. Additionally, neutrophil death during a swarm releases more signaling factors to recruit more neutrophils so the initial amount of neutrophils recruited plays a role in how large the propagation effect is during swarming.
Stages.
Stages 1-3.
The neutrophil swarming process is categorized into 5 phases: swarm initiation, swarm amplification, additional swarm amplification through intercellular signaling, swarm aggregation and tissue remodeling, and recruitment of myeloid cells and swarm resolution. The first stage of neutrophil swarming details the “pioneer” neutrophils responding to an infection or inflammation site. The neutrophils close to the injury will switch from random motility to chemotactic movement within a period of 5-15 minutes and swarm towards the infection site. In the second stage, the pioneer neutrophils attract a second wave of neutrophils that come from more distant regions of the tissue. The methods of movement to the region of injury depends on the tissue environment the neutrophils are moving towards. Neutrophil swarming in extravascular spaces such as the connective tissue in the skin involves movement without the assistance of integrin proteins and neutrophil attraction by a gradient of chemoattractants. Neutrophils will be guided by the forces generated by the actomyosin cytoskeleton through the path of least resistance to the site of infection. However, for intravascular tissue environments, neutrophil movement is dependent on integrins and chemoattractant signals on the luminal surface of endothelial cells. In this process, distant neutrophils will be recruited by an inflammatory signal and perform integrin-mediated crawling along the vascular walls to reach the neutrophil swarming sites.
In the third stage, swarming neutrophils can amplify their recruitment in a feed forward manner through intercellular communication by leukotriene B4 (LTB4). The propagation of neutrophil recruitment leads to multiple, dense neutrophil cell clusters at the site of inflammation. A 2013 study showed that neutrophils lacking the high affinity receptor for LTB4 (LTB4R1) decreased the recruitment of neutrophils at later stages of swarming. In addition, proximal cells to the inflammation site showed chemotaxis similar to the control cells while distant cells were poorly attracted. This finding suggests that the proximal neutrophils that are recruited early on are not affected by the lack of LTB4R1, but distant neutrophils that are required for the propagation of neutrophil swarming are not able to be recruited to the swarming site. These results present LTB4 as a key signaling molecule for a prolonged neutrophil swarm response and recruitment of neutrophils from distant areas of the tissue.
Stages 4-5.
After stages 1-3, neutrophils slow down in the cell clusters and begin to form aggregates. In this fourth stage, the neutrophil aggregates will aid in rearranging the surrounding extracellular tissue area and create a collagen-free zone at the inflammation center eventually resulting in a wound seal which isolates the site from the rest of the tissue. The exact mechanisms of this are unknown but it is believed that neutrophil proteases from the cell clusters play a role in clearing out the surrounding tissue environment. These neutrophil aggregates become stable as opposed to the constant movement in stages 1-3 by development of high chemoattractant concentrations within the clusters that promote local neutrophil interactions within the cluster. Additionally, neutrophils are switched to an adhesive mode of migration within clusters which further stabilize the aggregates and can prevent neutrophils from leaving the cluster. This switch is believed to be caused by additional secretions of LTB4 and other chemoattractants within the neutrophil aggregates.
In stage 5, the swarming response terminates and the clusters dissolve with the resolution of inflammation. Little is known about the mechanisms of this stage but the process may be regulated by neutrophils or external factors from the tissue environment. In a laser-induced skin injury model, neutrophil aggregation typically stopped after 40-60 minutes which occurs at the same time as the appearance of secondary myeloid cell swarms. Knock-in mice studies have shown that the myeloid cells move slower than neutrophils and assemble around the neutrophil aggregates during this stage. These myeloid cells may disrupt the propagation signals of neutrophil chemoattractants or to create competing attractants in the tissue space so that the neutrophil aggregation is less strong.


Elazer R. Edelman

Elazer R. Edelman is an American engineer, scientist and cardiologist. He is the Edward J. Poitras Professor in Medical Engineering and Science at the Massachusetts Institute of Technology (MIT), Professor of Medicine at Harvard Medical School and at Brigham and Women's Hospital (BWH), and a practicing cardiologist at BWH. He is the director of MIT's Institute for Medical Engineering and Science (IMES), the Harvard-MIT Biomedical Engineering Center, and the MIT Clinical Research Center. He is also the Program Director of the MIT Graduate Education in Medical Sciences program within the Harvard-MIT Division of Health Sciences and Technology. At BWH, he serves as a senior attending physician in the coronary care unit. He is currently the Chief Scientific Advisor for the journal Science Translational Medicine. Edelman has been elected to the American Academy of Arts and Sciences, National Academy of Inventors, National Academy of Engineering and the National Academy of Medicine.
Background and education.
Edelman was raised in the greater Boston area. He attended the Massachusetts Institute of Technology, where he received bachelor's degrees in Electrical Engineering and Computer Science (EECS) and in Applied Biology, and a master's degree in EECS. He earned his M.D. degree with distinction from Harvard Medical School and Ph.D. in Medical Engineering and Medical Physics within the Harvard-MIT Division of Health Sciences and Technology. He conducted his Ph.D. thesis work under the direction of Robert Langer to define the mathematics of regulated and controlled drug delivery. Edelman completed his medical training at Brigham and Women's Hospital and is board certified in internal medicine and cardiology. Following this, he spent six years as a research fellow under the tutelage of Prof. Morris J. Karnovsky to work on the biology of vascular repair. Edelman and his wife, Cheryl, have 3 children: Alex, A.J., and Austin.
Research.
Edelman is a major advocate of multidisciplinary research. Through his research centers, he combines teams of clinicians, engineers, and scientists from both academia and industry to create highly effective and clinically relevant solutions to medical problems. Through this approach, Edelman and his students have been credited as some of the key contributors and pioneers of the coronary stent. They critically aided in the development, characterization, and optimization of the first bare-metal stents and subsequent iterations including drug-eluting stents. Edelman's research programs fall in the following general categories:
Awards and honors.
Edelman has authored or co-authored more than 680 original scientific publications, holds some 80 patents, and has trained more than 300 students and post-doctoral fellows. He has served on several advisory boards including the Science Board to the Food and Drug Administration.
Edelman has been elected a fellow of the Association of American Physicians, American College of Cardiology, American Heart Association, American Institute for Medical and Biological Engineering, American Society of Clinical Investigators, American Society of Mechanical Engineers, Association of University Cardiologists, American Academy of Arts and Sciences, National Academy of Engineering, National Academy of Medicine, and National Academy of Inventors. He received the Officer's Cross of the Spanish Order of Civil Merit in 2010.
Selected awards received by Edelman include:


Hepatitis C virus cis-acting replication element

The Hepatitis C virus (HCV) cis-acting replication element (CRE) is an RNA element which is found in the coding region of the RNA-dependent RNA polymerase NS5B. Mutations in this family have been found to cause a blockage in RNA replication and it is thought that both the primary sequence and the structure of this element are crucial for HCV RNA replication.


Cruzipain

Cruzipain is a cysteine protease expressed by "Trypanosoma cruzi".
It is classified under .
Cruzipain is expressed by all strains and developmental forms of "Trypanosoma cruzi". It is secreted and can be found in the membrane of the parasite.
The study of "Trypanosoma cruzi" virulence is difficult due to the complexity of the parasite’s biology. "Trypanosoma cruzi" has two different infective forms which are both biochemically and antigenically distinct. The two forms, MTs and blood trypomastigotes use distinct sets of surface molecules to interact with their respective hosts. "Trypanosoma cruzi" also uses unique processes for gene expression, such as RNA editing, trans-splicing, and constitutive polycistronic transcription of protein-coding genes. In the absence of mechanisms controlling initiation of transcription, extracellular signals will trigger subsets of "Trypanosoma cruzi" genes to be post-transcriptionally co-regulated.
Cruzipain is part of clan CA, a group of papain-like enzymes. Clan CA is the most studied class of cysteine proteinases in parasitic protozoa. The localization of cruzipain within the cell differs based on which stage of the biological cycle the parasite is in. Cruzipain is involved in aiding the parasite in penetrating and evading the immune response of the host. The cysteine peptidases within parasitic protozoa are pivotal for several biological processes. Metamorphosis, immune evasion, and adaptation to certain hosts are some of the processes that cruzipain can exert influence over.
Cruzipain is a sulfated glycoprotein which plays a role in the parasitic disease known as Chagas disease. It is found to aid the parasite in entering the host cell and in evading an immune response.
Cruzipain can help parasites escape the response from the adaptive immune system by interfering with the functions of immunoglobulins from the immunoglobin G subclasses. These immunoglobulins are bound to receptors and cruzipain interacts with these immunoglobulins by cleaving their hinges.
During smooth muscle cell invasion, cruzipain may mobilize vasoconstricting endothelin receptors, which may interfere with the vasoconstrictor's ability to cause the blood vessels to become narrower.
Cruzipain aids in the process of breaking down host tissue and is prepared to signal the escape mechanism if it detects any response from the host's immune system.
Cruzipain as a DNA-Based Therapeutic Vaccine.
It has been reported that cruzipain is an efficient prophylactic vaccine, combined with several adjuvants, and administered through different routes. Results have been found using a cruzipain DNA-based vaccine that has demonstrated a decrease in parasitemia, inflammatory cell infiltrate, and tissue damage in models of infection caused by "Trypanosoma cruzi". In the experiment, chagasin, a natural protein of "Trypanosoma cruzi", and a tight binding inhibitor of papain-like cysteine proteases is used. Chagasin regulates the activity of cruzipain by controlling the functions that are necessary for parasitic invasion into mammalian cells. By combining the DNA of cruzipain and chagasin to synthesize a vaccine, a balanced immune response that decreases blood and tissue parasites, as well as the tissue damage caused by "Trypanosoma cruzi" infection can be induced.
Chemotherapy of Chronic Chagas Disease.
There are currently only two drugs in use for the treatment of Chagas disease, specifically nifurtimox and benznidazole. Both of these drugs require long use, cause adverse side effects, and have controversial efficacy in adults infected with "Trypanosoma cruzi". Recently, through drug repurposing, a strategy which utilizes existing drugs for new therapeutic purposes, the trypanocidal effects of benidipine and clofazimine have been discovered. These drugs showed the ability to inhibit cruzipain, by diminishing the burden of the parasite in skeletal and cardiac muscles, as well as by reducing the inflammatory response in the tissues of "Trypanosoma cruzi" infected mice.


Cyclohexylamine oxidase

In enzymology, a cyclohexylamine oxidase () is an enzyme that catalyzes the chemical reaction
The 3 substrates of this enzyme are cyclohexylamine, O2, and H2O, whereas its 3 products are cyclohexanone, NH3, and H2O2.
This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-NH2 group of donors with oxygen as acceptor. The systematic name of this enzyme class is cyclohexylamine:oxygen oxidoreductase (deaminating). This enzyme participates in caprolactam degradation. It employs one cofactor, FAD.


List of bioterrorist incidents

This is a list of bioterrorist incidents.
Guidelines.
The definitions of bioterrorism for the purpose of this article are:
The following criteria of violence or threat of violence fall outside of the definition of this article:


Kraig Biocraft Laboratories

Kraig Biocraft Laboratories, Inc. is an American biotechnology company headquartered in Ann Arbor, Michigan. It develops and manufactures recombinant spider silks and other high-performance polymers using spider silk gene sequences. Kraig Labs holds intellectual property on various spider silk technologies, including exclusive global license agreements with US universities for patented core technologies and has a proprietary production process for their recombinant spider silks.
Its flagship fiber is Dragon Silk which has been demonstrated to be tougher than many fibers used in bullet proof vests. In the fall of 2019, the company’s wholly owned subsidiary Prodigy Textiles LLC established a 45,000 sq ft production facility in Vietnam for the production of spider silk.
History.
Kim Kraig Thompson, a retired lawyer,
 invented the protein expression platform in 2002, which would become the basis for Kraig Lab’s work with spider silk., Inc. was He founded Kraig Biocraft Laboratories in April 2006 to develop and commercialize spider silks and other high performance polymers gene and sequences using platform technology in combination with genetic engineering concepts d.. Kraig Labs has successfully created numerous (approximately 20 by 2010) transgenic silkworm variants expressing different properties derived from the spider silk proteins.
The original scientific work to reduce Thompson’s invention to practice was performed in the biological laboratories of the University of Notre Dame. The University of Notre Dame was chosen in large part because the co-inventor of the PiggyBac transposon system, Dr. Malcom Fraser was in residence there. This transposon was utilized by Kraig Labs and the University of Notre Dame to create the world’s first transgenic silkworm producing recombinant spider silk. This work was subsequently the subject of a peer reviewed article in the Publication of the National Academy of Sciences (PNAS).
The Science.
The Company's production platform is based upon genetic modification of the domesticated silk worm (Bombyx mori). In interviews, the founder has stated that this decision was based largely on economics and the practicality of production. By utilizing the silkworm as the basis of production, it is able to produce the desired spider silk protein and have the silkworm spin it into a continuous fiber. Each individual transgenic silkworm produces one continuous fiber approximately one Kilometer in length.
In 2020, The firm successfully developed a significantly more advanced technology platform. This utilized a non-CRISPR gene editing, large plasmid knock-in knock-out technology. The new platform allows for the creation of essentially pure spider silk. Other than the silkworm’s remaining specifically desired native silk protein elements, Kraig Labs is now able to produce nearly pure spider silk. This is also an eco-friendlier and cost-effective silkworm production system, which is significantly more advanced than any of the competing methods. The knock-in knock-out technology allows Kraig Labs to work with very complex protein sequences in the silkworms, which are about four times more complex than published technologies. The Company’s Generation III Spider Silk Technology is purposed for specific customization.
Kraig Labs originally used the PiggyBac Transposon plasmid vector that was developed in collaboration with the University of Notre Dame.
In all methods, specific sequences of spider DNA are inserted into the genetic makeup of the silkworm to create a silkworm that produces spider silk. That transgenic silkworm is then used as the basis for establishing a genetic line silkworms that also produce spider silk.
The firm is able to customize the sequences that it inserts into the silkworm, thus giving them the ability to customize the resulting silk thread’s strength, flexibility and possibly other properties.
Spider Silk Applications.
Silk with strength equaling native spider silk has potential applications such as: car airbags, bulletproof vests, seat belts, parachutes, nets, sporting goods. sporting apparel. and medical applications includingy are bandages that have the ability to reduce scarring versus using traditional bandages and as a scaffolding material for artificial tendon and ligament repair.


Nicotinate-nucleotide adenylyltransferase

In enzymology, a nicotinate-nucleotide adenylyltransferase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are ATP and nicotinate ribonucleotide, whereas its two products are diphosphate and deamido-NAD+.
This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing nucleotide groups (nucleotidyltransferases). The systematic name of this enzyme class is ATP:nicotinate-ribonucleotide adenylyltransferase. Other names in common use include deamido-NAD+ pyrophosphorylase, nicotinate mononucleotide adenylyltransferase, deamidonicotinamide adenine dinucleotide pyrophosphorylase, NaMN-ATase, and nicotinic acid mononucleotide adenylyltransferase. This enzyme participates in nicotinate and nicotinamide metabolism.
Structural studies.
As of late 2007, 9 structures have been solved for this class of enzymes, with PDB accession codes , , , , , , , , and .


George Bugliarello

George Bugliarello, (May 20, 1927—February 18, 2011) was an Italian President Emeritus, Institute Professor and former chancellor of the Polytechnic Institute of NYU (formerly Polytechnic University).
Early life.
George Bugliarello was born as Georgio Bugliarello-Wondrich to Spera Bugliarello-Wondrich and Colonel Federico Bugliarello Magnano di San Lio in Trieste, Italy. He studied engineering at the University of Padua from which he graduated in 1951 with summa cum laude. He then completed his Master's degree at the University of Minnesota in civil engineering in 1954 and five years later he obtained Ph.D. from the Massachusetts Institute of Technology in both hydrodynamics and civil engineering. He also have various honorary degrees from the University of Trieste, the Milwaukee School of Engineering, the Illinois Institute of Technology, Pace University, Trinity College and Rensselaer Polytechnic Institute.
Career.
George had joined the faculty at the Carnegie Mellon University in 1959 as an assistant professor of biotechnology and civil engineering and worked there along with Tin-Kan Hung who was a professor of biomedical engineering until 1969. In 1964 he became the founding director of the bioengineering program at CMU and five years later became Dean of engineering at the University of Illinois at Chicago.
From 1969 to 1972 he was a Director of the Biomedical Engineering Society.
On October 15, 1973, he was inaugurated as the 13th President of the Polytechnic Institute of New York and served as such till 1994.
Since 1987 he was a member of the National Academy of Engineering and in 2003 he was elected as a Foreign Secretary of the NAE.
In 1994 George had launched the Center for Technology and Financial Services at Polytechnic and from 1994 to 1997 he served as chairman on the Board on Infrastructure and the Constructed Environment of the National Research Council (BOSTID). From 1997 to 2011 he was an interim editor of "The Bridge" (the quarterly publication of the National Academy of Engineering), and was co-founder and co-editor of "Technology in Society" - An International Journal. George also served on several editorial advisory boards, had authored over 300 professional papers, and was the author, co-author or editor of numerous books.
He was a lifetime member of the National Associate of The National Academies and served as chair of the National Academy of Engineering Council’s International Affairs Committee.
George also was a chairman of the National Medal of Technology Nomination Evaluation Committee, the Advisory Panel for Technology Transfer to the Middle East of the Office of Technology Assessment, and the Committee on Science, Engineering and Public Policy (COSEPP) of the American Association for the Advancement of Science. He served as chair of the Board on Infrastructure and the Constructed Environment of the National Research Council, and of the National Academies Megacities project for the Habitat II conference. He also served as chair of the National Academies steering committee on the Megacities. Other committee service included the U.S. National Academies-Russian Academy of Sciences Committee on Terrorism Confronting the U.S. and Russia and co-chairmanship of a joint Russian-American Task Force on Urban Security.
Dr. Bugliarello’s international experience included consultantships abroad for OECD as reviewer of the science policy of several countries, and for UNESCO, assignments as specialist for the U.S. Department of State in Venezuela and Central Africa. He also held a NATO Senior Faculty Fellowship at the Technical University of Berlin, was a member on the U.S.-Egypt Joint Consultative Committee of the National Academy of Sciences, and member on the Scientific Committee of the Summer School on Environmental Dynamics in Venice. He had been the U.S. member of the Science for Stability Steering Committee, and of the Science for Peace Steering Committee, of the Scientific Affairs Division of NATO.
George Bugliarello had died of pneumonia on February 18, 2011 in Roslyn, New York.
Fellowships and honors.
He also was the founding member of the Marconi Society, a member of the Council on Foreign Relations and a Fellow of the New York Academy of Sciences, and the Biomedical Engineering Society.
Personal life.
George Bugliarello was married to Virginia Upton Harding. Together they had two sons; David Wondrich and Nicholas Bugliarello as well as three grandchildren. Besides engineering, his other job was education in which he had broad background ranging from fluid mechanics to computer languages, the environment, biomedical engineering and science policy.


List of chemical warfare agents

A "chemical weapon agent (CWA)" is a chemical substance whose toxic properties are used to kill, injure or incapacitate human beings. About 70 different chemicals have been used or stockpiled as chemical weapon agents during the 20th century. These agents may be in liquid, gas or solid form.
In general, chemical weapon agents are organized into several categories according to the physiological manner in which they affect the human body. Division may also be by tactical purpose or chemical structure. The names and number of categories varies slightly from source to source, but in general, types of chemical warfare agents are as follows:
Harassing agents.
These are substances that are not intended to kill or injure. They are often referred to as Riot Control Agents (RCAs) and may be used by civilian police forces against criminals and rioters, or in the military for training purposes. These agents also have tactical utility to force combatants out of concealed or covered positions for conventional engagement, and preventing combatants from occupying contaminated terrain or operating weapons. In general, harassing agents are sensory irritants that have fleeting concentration dependent effects that resolve within minutes after removal. Casualty effects are not anticipated to exceed 24-hours nor require medical attention.
Tear agents.
These sensory irritants produce immediate pain to the eyes and irritate mucous membranes (aka "lachrymatory agent" or "lachrymator").
Vomiting agents.
These sensory irritants are also termed sternators or nose irritants. They irritate the mucous membranes to produce congestion, coughing, sneezing, and eventually nausea.
Malodorants.
These are compounds with a very strong and unpleasant smell, which produce powerfully aversive effects without the toxic effects of tear agents or vomiting agents.
Incapacitating agents.
These are substances that produce debilitating effects with limited probability of permanent injury or loss of life. The casualty effects typically last over 24 hours, and though medical evacuation and isolation is recommended, it is not required for complete recovery. These, together with harassing agents, are sometimes called nonlethal agents. There may be as high as 5% fatalities with the use of these agents.
Psychological agents.
These are substances that produce casualty effects through mental disturbances such as delirium or hallucination.
Other incapacitating agents.
These substances have also been investigated as incapacitants, though they operate more through interactions outside the central nervous system.
Lethal agents.
These substances are for producing chemical casualties without regard to long-term consequences or loss of life. They cause injuries that require medical treatment.
Blister agents.
A "blister agent" is a chemical compound that irritates and causes injury to the skin. These substances also attack the eyes, or any other tissue they contact.
Vesicants.
The vesicants are substances that produce large fluid-filled blisters on the skin.
Urticants.
The urticants are substances that produce a painful weal on the skin. These are sometimes termed skin necrotizers and are known as the most painful substances produced.
Blood agents.
These substances are metabolic poisons that interfere with the life-sustaining processes of the blood.
Choking agents.
These substances are sometime referred to as "pulmonary agent" or "lung irritants" and cause injury to the lung-blood barrier resulting in Asphyxia.
Nerve agents.
Nerve agents are substances that disrupt the chemical communications through the nervous system. One mechanism of disruption, utilized by the G, GV, and V series of chemicals is caused by blocking the acetylcholinesterase, an enzyme that normally destroys and stops the activity of acetylcholine, a neurotransmitter. Poisoning by these nerve agents leads to an accumulation of acetylcholine at the nerve axon, producing a perpetual excited state in the nerve (e.g. constant muscle contraction). The eventual exhaustion of muscles leads to respiratory failure and death. A separate class of nerve agents are related to Tetrodotoxin, frequently abbreviated as TTX, is a potent neurotoxin with no known antidote. Tetrodotoxin blocks action potentials in nerves by binding to the voltage-gated, fast sodium channels in nerve cell membranes, essentially preventing any affected nerve cells from firing by blocking the channels used in the process.
G series.
These are high volatility nerve agents that are typically used for a nonpersistent to semipersistent effect.
GV series.
These agents have a volatility between the V and G agents and are typically used for a semi-persistent to persistent effect.
V series.
These agents have low volatility and are typically used for a persistent effect or liquid contact hazard.
T series.
These agents are related to the puffer fish


Tert-Amyl alcohol

"tert"-Amyl alcohol (TAA) or 2-methylbutan-2-ol (2M2B), is a branched pentanol.
Historically TAA has been used an anesthetic and more recently used as a recreational drug. TAA is mostly a positive allosteric modulator for GABAA receptors in the same way as ethanol. The effects of TAA and ethanol are similar.
TAA is a colorless liquid with a burning flavor and an unpleasant odor similar to paraldehyde with a hint of camphor. TAA remains as a liquid at room temperature making it a useful alternative solvent to "tert"-butyl alcohol.
Production.
TAA is primarily made by the hydration of 2-methyl-2-butene in the presence of an acidic catalyst.
Natural occurrence.
Fusel alcohols like TAA are grain fermentation byproducts and therefore trace amounts of TAA are present in many alcoholic beverages. Traces of TAA have been detected in other foods, like fried bacon, cassava and rooibos tea.
History.
From about 1880s to 1950s, TAA was used as an anesthetic with the contemporary name of "amylene hydrate", but was rarely used solely because of the existence of more efficient drugs. In the 1930s, TAA was mainly used as a solvent for the primary anesthetic tribromoethanol (TBE). Like chloroform, TBE is toxic for the liver, so the use of such solutions declined in the 1940s in humans. TBE-TAA-solutions remained in use as short-acting anesthetics for laboratory mice and rats. Such solutions are sometimes called "Avertin", which was a brand name for the now discontinued TAA and TBE solution with a volume ratio of 0.5:1 made by the Winthrop Laboratories. Nowadays TAA has found use as a recreational drug.
Use and effects.
Ingestion or inhalation of TAA causes euphoria, sedative, hypnotic, and anticonvulsant effects similar to ethanol. When ingested, the effects of TAA may begin in about 30 minutes and can last up to 1–2 days. 2–4 grams of TAA causes unconsciousness. About 100 g of ethanol induces a similar level of unconsciousness.
Overdose and toxicity.
The smallest known dose of TAA that has killed a person is 30 ml.
An overdose produces symptoms similar to alcohol poisoning and is a medical emergency due to the sedative/depressant properties which manifest in overdose as potentially lethal respiratory depression. Sudden loss of consciousness, simultaneous respiratory and metabolic acidosis, fast heartbeat, increased blood pressure, pupil constriction, coma, respiratory depression and death may follow from an overdose.The oral in rats is 1 g/kg. The subcutaneous LD50 in mice is 2.1 g/kg.
Metabolism.
In rats, TAA is primarily metabolized via glucuronidation, as well as by oxidation to 2-methyl-2,3-butanediol. It is likely that the same path is followed in humans, though older sources suggest TAA is excreted unchanged.
The use of TAA cannot be detected with general ethanol tests or other ordinary drug tests. Its use can be detected from a blood or a urine sample by using gas chromatography–mass spectrometry for up to 48 hours after consumption.


Aldehyde tag

The aldehyde tag is a short peptide tag which can be introduced into fusion proteins and by subsequent treatment with the formylglycine-generating enzyme (FGE) a reactive aldehyde group is generated for further coupling. Since there already is an array of aldehyde-specific reagents commercially available (such as aminooxy or hydrazide reagents), possible applications are diverse and include the conjugation of fluorophores, glycans, PEG (polyethylene glycol) chains or reactive groups for further synthesis (see applications).
Development.
The aldehyde tag is an artificial peptide tag recognized by the formylglycine-generating enzyme (FGE). Formylglycine is a glycine with a formyl group (-CHO, an aldehyde) at the α-carbon. The sulfatase motif is the basis for the sequence of the peptide which results in the site-specific conversion of a cysteine to a formylglycine residue. The peptide tag was engineered after studies on FGE recognizable sequences in sulfatases from different organisms. Carrico et al. discovered a high homology in the sulfatase motif in bacteria, archaea as well as eukaryotes.
Aldehydes and ketones find use as chemical reporters due to their strong electrophilic properties. This enables a reaction under mild conditions when using a strong nucleophilic coupling partner. Typically, hydrazides and aminooxy probes are used in bioconjugation. They form stabilized addition products with carbonyl groups that are favoured under the physiological reaction conditions. At neutral pH, the equilibrium of Schiff base formation, is lying far to the reactant's side. To make more product named compounds are used to form stable hydrazones and oximes. Since the pH-optimum of 4 to 6 cannot be achieved by adding a catalyst due to associated toxicity, the reaction is slow in live cells. A typical reaction constant is 10−4 to 10−3 M−1 s−1.
A carbonyl group is introduced into proteins as a chemical reporter using different techniques, including modern methods like stop codon suppression and the herein discussed aldehyde tag. Limiting the use of aldehydes and ketones is their restricted bioorthogonality in certain cellular environments.
Limitations of aldehydes and ketones as chemical reporters are due to
Aldehydes and ketones are therefore best used in compartments where such unwanted side reactions are decreased. For experiments with live cells, cell surfaces and extracellular space are typical fielding areas. Nevertheless, a feature of carbonyl groups is the vast number of organic reactions that involve them as electrophiles. Some of these reactions are readily convertible to ligations for probing aldehydes. A rather exotic reaction recently employed for bioconjugation by Agarwal et al. is the adaptation of the Pictet-Spengler-reaction as a ligation. The reaction is known from natural product biosynthetic pathways and has the major advantage that a new carbon-carbon bond is formed. This guarantees long-term stability compared to carbon-heteroatom bonds at same reaction kinetics.
The modification of cysteine or, more rarely, serine by FGE is a rather unusual posttranslational modification and was discovered already in the late 1990s. The deficiency of FGE leads to an overall deficiency of functional sulfatases due to a lack of α-formylglycine formation vital for the sulfatases to perform their function. FGE is essential for protein modification and need of high specificity and conversion rate is given in the native setting, which makes this reaction applicable in chemical and synthetic biology.
Carrico et al. pioneered the insertion of the modified sulfatase motif peptide into proteins of interest in 2007. Such use of aldehydes and ketones as a chemical reporter in bioorthogonal applications has been applied in self-assembly of cell-lysing drugs, the targeting of proteins, as well as glycans and the preparation of heterobifunctional fusion proteins since then.
Genetically encoding the aldehyde tag.
The formylglycine tag or aldehyde tag is a convenient 6- or 13-amino acids long tag fused to a protein of interest. The 6-mer tag represents the small core consensus sequence and the 13-mer tag the longer full motif.
The experiments on the genetically encoded aldehyde tag by Carrico et al. clearly showed the high conversion efficiency with only the core consensus sequence present. Four proteins were produced recombinantly in E.coli with an 86% efficiency of for the full-length motif and >90 % efficiency for the 6-mer determined by mass spectrometry.
The size of the sequence is analogous to the commonly used 6x His-Tag and has the advantage that it can also be genetically encoded. The sequence is recognized in the ER solely depending on primary sequence and subsequently targeted by FGE. Notably, in the setup of recombinant expression proteins in "E. coli" a coexpression of exogenous FGE aids full conversion, although "E. coli" has endogenous FGE-activity.
The introduction of an aldehyde tag as proposed by Carrico et al. has a workflow that consists of three segments: A the expression of the fusion protein, that carries the peptide tag derived from the sulfatase motif, B the enzymatic conversion of Cys to f(Gly) and C the bioorthogonal probing with hydrazides or alkoxy amines (Fig. 1).
As seen in Fig. 1, the engineered aldehyde tag consists of six amino acids. A set of organisms from all domains of life was chosen and the sequence homology of the sulfatase motif was determined. The sequence used is the best consensus for sequences found in bacteria, archaea, worms and higher vertebrates.
FGE-mechanism of cysteine-formylglycine conversion.
The catalytic mechanism of FGE is well studied. A multistep redox reaction with a covalent enzyme:substrate intermediate is proposed. The role of the cysteine residue for the occurring conversion was studied by mutating the cysteine to alanine. No conversion was found using mass spectrometry when the mutated peptide tag was used. The mechanism shows the important role of the redox active thiol group of cysteine in the formation of f(Gly), as seen in Fig. 2.
The key step of the catalytic cycle is the monooxidation of the cysteine residue of the enzyme, forming a reactive sulfenic acid intermediate. Subsequently, the hydroxyl group is transferred to the cysteine of the substrate and after hetero-analogous β-elimination of H2O, a thioaldehyde is formed. This compound is very reactive and easily hydrolyzed, releasing the aldehyde and a molecule of H2S,
Applications.
The aldehyde tag is a technique which recently found increased application because of the introduction of bioorthogonal chemical reporters. Bioorthogonal agents contain functional groups such as azides or cyclooctynes for coupling which are not naturally found in the cell. Due to their foreignness, they seem inert and do not disrupt the native metabolism,
Fig. 3 gives an overview of possible labelling methods for formylglycine. For example, it can be coupled to probes such as biotin or a protein tag like Flag that are useful for purification and detection. Furthermore, fluorophores can be directly conjugated for live cell imaging. The conjugation of polyethylene glycol (PEG) chains to potential drug candidates extends the stability against proteases in body fluids and at the same time reduces renal clearance and immunogenicity. The first application described here, deals with the formation of protein-protein conjugates through bioorthogonal probes. Since, the aldehyde tag is strictly speaking not a true bioorthogonal agent as it can be found in various metabolites, it can cause cross reactions during protein labelling. However, coupling bioorthogonal probes such as azides or cyclooctynes can be applied to overcome this obstacle. As a second application, the coupling of glycan moieties to proteins is presented here. It can be utilised in the strategy of chemically introduced glycosylation patterns.
Forming protein-protein conjugates via Cu-free click chemistry.
Hudak et al. explored the strategy of producing protein-protein conjugates with the help of the aldehyde tag. Their aim was to connect full length human IgG (hIgG) to the human growth hormone (hGH). These protein-protein conjugates can be superior to monomeric proteins in terms of serum half life in protein therapeutics and, additionally, have appealing dual binding properties.
In order to achieve protein fusion, the five-residue aldehyde tag (CxPxR) was incooperated into hIgG and hGH. In hIgG, the aldehyde tag was introduced at the C termini of the two heavy chains, resulting in two possible conjugation sites. FGE then oxidizes the cysteine residue to formylglycine (fGly) during protein expression. For the subsequent conjugation steps, the strategy of the copper-free click chemistry was selected. A strain-promoted 1,3-dipolar cycloaddition of a cyclooctynes and an azide was carried out forming a covalent linkage (also termed the Cu-free azide-alkyne cycloaddition). Thus, the aldehyde bearing proteins react under oxime formation with different heterobifunctional linkers which carry an aminooxy residue on one end and either an azide or cyclooctynes on the other. This results in the attachment of hIgG to a linker containing a cyclooctyne (here dibenzoazacyclooctyne (DIBAC)) and hGH to a linker holding an azide function (Fig.: 2A and B). The proteins hGH and hIgG were also treated with DIBAC-488, azide Alexa Fluor 647 and analysed by SDS-PAGE and Western blot to validate oxime formation. Next, the DIBAC-hIgG and azide-hGH derivatives are joined by Cu-free click chemistry (Fig.: 2C). The resulting fusion proteins were purified and analyzed by immunoblot (see Hudak et al. 2012).
The Western blots were first stained with Ponceau and then incubated with IgG antibodies against hGH and subsequently treated with α-mIgG HRP and α-hIgG 647 for visualisation. In the hIgG-hGH conjugate Western blot (nonreducing conditions), two separate bands with different molecular weights are visible after immunodetection. These can be contributed to the formation of mono- and bi-conjugated hGH to hIgG.
Chemical glycosylation of the IgG Fc fragment.
Nature has perfected glycosylation of proteins through a complex interaction of enzymes and carbohydrates over thousands of years. However, chemical glycosylation is still an obstacle due to the difficult synthesis of glycan in general. The synthesis of carbohydrate derivatives can be slow and tedious. Nonetheless, the interest in technologies to structurally mimic protein glycosylation is an appealing application as some protein functions solely depend on the pattern of the attached glycan. The Fc fragment of the IgG antibody, for example, is a homodimer with a highly conserved N-glycosylation site. The attached sugar moieties modulate the binding to specific immunoreceptors, thereby modifying the whole antibody function.
Smith et al. demonstrate the application of the aldehyde tag as a chemical conjugation site for glycans. The aldehyde tag sequence was incooperated into the Fc construct and introduced into CHO (Chinese hamster ovary) cells. As controls, gene constructs were used in which the cysteine residue was mutated to an alanine. After expression, the Fc proteins were purified using a protein A/G agarose column. The conversion in CHO cells of cystein to formylglycine was examined using aminooxy AlexaFluor 488 and subsequent SDS-PAGE. However, fluorescence scanning displayed no fluorescence labelling, i.e. no formylglycine formation by endogenous FGE in CHO cells. The unaltered proteins were then treated with recombinant FGE from Mycobacterium tuberculosis in vitro in which the aldehyde group was successfully installed at the glycosylation site of Fc (Fig. 3A).
Next, the introduction of N-acetylglucoseamine (GlcNAc) to the aldehyde tagged proteins via oxime formation was carried out through the treatment with aminooxy GlcNAc (AO-GlcNAc) (Fig. 3B). The conjugation was confirmed by liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) and lectin blot with the GlcNAc-binding wheat germ agglutinin attached to AlexaFluor 647. Having successfully introduced GlcNAc, the monomer was extended with a glycan structure containing GlcNAc, mannose (Man) and galactose (Gal) (Fig. 3C). A mutant endoglycosidase EndoS (EndoS-D233Q) was utilised as it is highly specific for IgG Fc N-linked GlcNAc residues and does not elongate Asn-GlcNAc sites on other proteins or on denatured IgGs. Product formation was again monitored by LC-ESI-MS and lectin blot probing with the sialic acid-binding sambucus nigra agglutinin attached to fluorescein isothiocyanate.
A successful chemical glycosylation of the Fc IgG fragment was achieved which resembles the natural occurring glycosylation pattern. The study discussed above focused on the IgG antibody, however, the application of the aldehyde tag for glycan conjugation could potentially be extended to other proteins.


Greater hedgehog tenrec

The greater hedgehog tenrec ("Setifer setosus"), also known as the large Madagascar hedgehog or sokina, is a species of mammal in the family Tenrecidae. It is endemic to Madagascar. Its natural habitats are subtropical or tropical dry forest, subtropical or tropical moist lowland forest, subtropical or tropical moist montane forest, dry savanna, moist savanna, subtropical or tropical dry shrubland, subtropical or tropical moist shrubland, subtropical or tropical high-altitude shrubland, subtropical or tropical dry lowland grassland, subtropical or tropical high-altitude grassland, rural gardens, urban areas and zoos.
It is the only species in the genus Setifer. Despite the close resemblance, it is not closely related to hedgehogs.
Distribution and Habitat.
Greater hedgehog tenrecs are found throughout the island of Madagascar and are endemic to this island.
The island of Madagascar ranges from sea level to 2,250 meters above sea level, and Setifer setosus is found throughout the island, with the exception of wetlands and marshes. Greater hedgehog tenrecs are seen in urban areas and even in areas with extensive human disturbance. The prime habitat for greater hedgehog tenrecs are the eastern forests and lower elevations because of their eating habits, but they are found in wet and dry areas.


ChromaDex

ChromaDex is a dietary supplement and food ingredient company based in Los Angeles, California founded in 1999 that is publicly traded on the NASDAQ.
In 2011, ChromaDex licensed patents from Dartmouth College, Cornell University, and Washington University regarding nicotinamide riboside, which it markets and sells as an ingredient under the brand name Niagen and sells directly to consumers as Tru Niagen. ChromaDex licensed patents from the University of Mississippi and the USDA to commercially develop pterostilbene and sell the compound under the brand name pTeroPure. ChromaDex no longer sells pTeroPure.
Elysium Health bought the ingredients in its "Basis" product from ChromaDex. The two firms had an agreement under which Elysium didn't have to acknowledge ChromaDex as the source of the ingredients, but then after Elysium recruited the VP of business development from ChromaDex and stopped paying ChromaDex, ChromaDex sued Elysium and the information became public.


Ribosomal pause

Ribosomal pause refers to the queueing or stacking of ribosomes during translation of the nucleotide sequence of mRNA transcripts. These transcripts are decoded and converted into an amino acid sequence during protein synthesis by ribosomes. Due to the pause sites of some mRNA’s, there is a disturbance caused in translation. Ribosomal pausing occurs in both eukaryotes and prokaryotes.
It's been known since the 1980s that different mRNAs are translated at different rates. The main reason for these differences was thought to be the concentration of varieties of rare tRNAs limiting the rate at which some transcripts could be decoded. However, with research techniques such as ribosome profiling, it was found that at certain sites there were higher concentrations of ribosomes than average, and these pause sites were tested with specific codons. No link was found between the occupancy of specific codons and amount of their tRNAs. Thus, the early findings about rare tRNAs causing pause sites doesn't seem plausible.
Two techniques can localize the ribosomal pause site in "vivo"";" a micrococcal nuclease protection assay and isolation of polysomal transcript. Isolation of polysomal transcripts occurs by centrifuging tissue extracts through a sucrose cushion with translation elongation inhibitors, for example cycloheximide.
Ribosome pausing can be detected during preprolactin synthesis on free polysomes, when the ribosome is paused the other ribosomes are tightly stacked together. When the ribosome pauses, during translation, the fragments that started to translate before the pause took place are overrepresented. However, along with the mRNA if the ribosome pauses then specific bands will be improved in the trailing edge of the ribosome.
Some of the elongation inhibitors, such as: cycloheximide (in eukaryotes) or chloramphenicol, cause the ribosomes to pause and to accumulate in the start codons. Elongation Factor P regulates the ribosomal pause at polyproline in bacteria, and when there is no EFP the density of ribosomes decreases from the polyproline motifs. If there are multiple ribosome pauses, then the EFP won't resolve it.
Effects on gene expression.
During protein synthesis, rapidly changing conditions in the cell can cause ribosomal pausing. In bacteria, this can affect growth rate and trigger translational abandonment. This releases the ribosome from the mRNA and the incomplete polypeptide is targeted for destruction.
In eukaryotes, ribosomal pausing can initiate an analogous process which triggers endonucleolytic attack of the mRNA, a process termed mRNA no-go decay. Ribosomal pausing also aids co-translational folding of the nascent polypeptide on the ribosome, and delays protein translation while its encoding mRNA; this can trigger ribosomal frameshifting.
Protein synthesis must occur in a specific way for ribosomal pausing to impact or change the outcome of this process. The products that are made because of the ribosomal pausing can be broken down by Ribosome Quality Control (RQC). RQC can happen after the ribosomal pausing. Even though RQC works to undo the effects of the ribosomal pause, there are specific situations relating to proteins when the ribosomal pause is needed. Ribosomal pausing does have an impact on the rate of protein synthesis and it may decrease the rate that it occurs.
Advantage of the ribosomal pause.
When the ribosome movement on the mRNA is not linear, the ribosome gets paused at different regions without a precise reason. The ribosome pause position will help to identify the mRNA sequence features, structure, and the transacting factor that modulates this process. The advantage of ribosomal pause sites that are located at protein domain boundaries are aiding the folding of a protein. There are times when the ribosomal pause does not cause an advantage and it needs to be restricted. In translation, elF5A inhibits ribosomal pausing for translation to function better. Ribosomal pausing can cause more non-canonical start codons without elF5A in eukaryotic cells. When there is a lack of elF5A in the eukaryotic cell, it can cause an increase in ribosomal pausing. The ribosomal pausing process can also be used by amino acids to control translation.
The location of the ribosome pause event in "vitro".
It is known that ribosomes pause at distinct sites, but the reasons for these pauses are mostly unknown. Also, the ribosome pauses if the pseudoknot is disrupted. 10% of the ribosome pauses at the pseudoknot and 4% of the ribosomes are terminated. Before the ribosome is obstructed it passes the pseudoknot. An assay was put together by a group from the University of California in an effort to show a model of mRNA. The translation was monitored in two in vitro systems. It was found that translating ribosomes aren't uniformly distributed along an mRNA. Protein folding "in vivo" is also important and is related to protein synthesis. For finding the location of the ribosomal pause "in vivo", the methods that have been used to find the ribosomal pause "in vitro" can be changed to find these specific locations "in vivo."
Ribosome profiling.
Ribosome profiling is a method that can reveal pausing sites through sequencing the ribosome protected fragments (RPFs or footprints) to map ribosome occupancy on the mRNA. Ribosome profiling has the ability to reveal the ribosome pause sites in the whole transcriptome. When the kinetics layer is added, it discloses the time of the pause, and the translation takes place. Ribosome profiling is however still in early stages and has biases that need to be explored further. Ribosome profiling allows for translation to be measured more accurately and precisely. During this process, translation needs to be stopped in order for ribosome profiling to be performed. This may cause a problem with ribosome profiling because the methods that are used to stop translation in an experiment can impact the outcome, which causes incorrect results. Ribosome profiling is useful for getting specific information on translation and the process of protein synthesis.


Catechol dioxygenase

Catechol dioxygenases are metalloprotein enzymes that carry out the oxidative cleavage of catechols. This class of enzymes incorporate dioxygen into the substrate (biochemistry). Catechol dioxygenases belong to the class of oxidoreductases and have several different substrate specificities, including catechol 1,2-dioxygenase (), catechol 2,3-dioxygenase (), and protocatechuate 3,4-dioxygenase (). The active site of catechol dioxygenases most frequently contains iron, but manganese-containing forms are also known.
The "Pseudomonas putida" xylE gene, which encodes catechol 2,3-dioxygenase, is often used as a reporter to quantitate gene expression.
An example of the reaction carried out by catechol 1,2-dioxygenase is the formation of "cis,cis"-muconic acid from catechol, shown below.


Carbohydrate catabolism

Digestion is the breakdown of carbohydrates to yield an energy rich compound called ATP. The production of ATP is achieved through the oxidation of glucose molecules. In oxidation, the electrons are stripped from a glucose molecule to reduce NAD+ and FAD. NAD+ and FAD possess a high energy potential to drive the production of ATP in the electron transport chain. ATP production occurs in the mitochondria of the cell. There are two methods of producing ATP: aerobic and anaerobic.
In aerobic respiration, oxygen is required. Oxygen as a high-energy molecule increases ATP production from 4 ATP molecules to about 30 ATP molecules.
In anaerobic respiration, oxygen is not required. When oxygen is absent, the generation of ATP continues through fermentation. There are two types of fermentation: alcohol fermentation and lactic acid fermentation.
There are several different types of carbohydrates: polysaccharides (e.g., starch, amylopectin, glycogen, cellulose), monosaccharides (e.g., glucose, galactose, fructose, ribose) and the disaccharides (e.g., sucrose, maltose, lactose).
Glucose reacts with oxygen in the following reaction, C6H12O6 + 6O2 → 6CO2 + 6H2O. Carbon dioxide and water are waste products, and the overall reaction is exothermic because of the relative weak, high-energy double bond of O2.
The reaction of glucose with oxygen releasing energy in the form of molecules of ATP is therefore one of the most important biochemical pathways found in living organisms.
Glycolysis.
Glycolysis, which means “sugar splitting,” is the initial process in the cellular respiration pathway. Glycolysis can be either an aerobic or anaerobic process. When oxygen is present, glycolysis continues along the aerobic respiration pathway. If oxygen is not present, then ATP production is restricted to anaerobic respiration. The location where glycolysis, aerobic or anaerobic, occurs is in the cytosol of the cell. In glycolysis, a six-carbon glucose molecule is split into two three-carbon molecules called pyruvate. These carbon molecules are oxidized into NADH and ATP. For the glucose molecule to oxidize into pyruvate, an input of ATP molecules is required. This is known as the investment phase, in which a total of two ATP molecules are consumed. At the end of glycolysis, the total yield of ATP is four molecules, but the net gain is two ATP molecules. Even though ATP is synthesized, the two ATP molecules produced are few compared to the second and third pathways, Krebs cycle and oxidative phosphorylation.
Fermentation.
Even if there is no oxygen present, glycolysis can continue to generate ATP. However, for glycolysis to continue to produce ATP, there must be NAD+ present, which is responsible for oxidizing glucose. This is achieved by recycling NADH back to NAD+. When NAD+ is reduced to NADH, the electrons from NADH are eventually transferred to a separate organic molecule, transforming NADH back to NAD+. This process of renewing the supply of NAD+ is called fermentation, which falls into two categories.
Alcohol Fermentation.
In alcohol fermentation, when a glucose molecule is oxidized, ethanol (ethyl alcohol) and carbon dioxide are byproducts. The organic molecule that is responsible for renewing the NAD+ supply in this type of fermentation is the pyruvate from glycolysis. Each pyruvate releases a carbon dioxide molecule, turning into acetaldehyde. The acetaldehyde is then reduced by the NADH produced from glycolysis, forming the alcohol waste product, ethanol, and forming NAD+, thereby replenishing its supply for glycolysis to continue producing ATP.
Lactic Acid Fermentation.
In lactic acid fermentation, each pyruvate molecule is directly reduced by NADH. The only byproduct from this type of fermentation is lactate. Lactic acid fermentation is used by human muscle cells as a means of generating ATP during strenuous exercise where oxygen consumption is higher than the supplied oxygen. As this process progresses, the surplus of lactate is brought to the liver, which converts it back to pyruvate.
Respiration.
The Citric acid cycle (also known as the Krebs cycle).
If oxygen is present, then following glycolysis, the two pyruvate molecules are brought into the mitochondrion itself to go through the Krebs cycle. In this cycle, the pyruvate molecules from glycolysis are further broken down to harness the remaining energy. Each pyruvate goes through a series of reactions that converts it to acetyl coenzyme A. From here, only the acetyl group participates in the Krebs cycle—in which it goes through a series of redox reactions, catalyzed by enzymes, to further harness the energy from the acetyl group. The energy from the acetyl group, in the form of electrons, is used to reduce NAD+ and FAD to NADH and FADH2, respectively. NADH and FADH2 contain the stored energy harnessed from the initial glucose molecule and is used in the electron transport chain where the bulk of the ATP is produced.
Oxidative phosphorylation.
The last process in aerobic respiration is oxidative phosphorylation, also known as the electron transport chain. Here NADH and FADH2 deliver their electrons to oxygen and protons at the inner membranes of the mitochondrion, facilitating the production of ATP. Oxidative phosphorylation contributes the majority of the ATP produced, compared to glycolysis and the Krebs cycle. While the ATP count is glycolysis and the Krebs cycle is two ATP molecules, the electron transport chain contributes, at most, twenty-eight ATP molecules. A contributing factor is due to the energy potentials of NADH and FADH2. As they are brought from the initial process, glycolysis, to the electron transport chain, they unlock the energy stored in the relatively weak double bonds of O2. A second contributing factor is that cristae, the inner membranes of mitochondria, increase the surface area and therefore the amount of proteins in the membrane that assist in the synthesis of ATP. Along the electron transport chain, there are separate compartments, each with their own concentration gradient of H + ions, which are the power source of ATP synthesis. To convert ADP to ATP, energy must be provided. That energy is provided by the H+ gradient. On one side of the membrane compartment, there is a high concentration of H+ ions compared to the other. The shuttling of H+ to one side of the membrane is driven by the exergonic flow of electrons throughout the membrane. These electrons are supplied by NADH and FADH2 as they transfer their potential energy. Once the H+ concentration gradient is established, a proton-motive force is established, which provides the energy to convert ADP to ATP. The H+ ions that were initially forced to one side of the mitochondrion membrane now naturally flow through a membrane protein called ATP synthase, a protein that converts ADP to ATP with the help of H+ ions.


Dieticyclidine

Dieticyclidine (PCDE), or diethylphenylcyclohexylamine, is a psychoactive drug and research chemical of the arylcyclohexylamine chemical class related to phencyclidine (PCP) and eticyclidine (PCE). It acts as an NMDA receptor antagonist but has low potency and acts mainly as a prodrug for eticyclidine.


Methamnetamine

Methamnetamine (also known as methylnaphetamine, MNA, MNT and PAL-1046) is a triple monoamine releasing agent and "N"-methyl analog of the non-neurotoxic experimental drug naphthylaminopropane and the naphthalene analog of methamphetamine. It has been sold online as a designer drug.
It acts as a releasing agent of serotonin, norepinephrine, and dopamine, with EC50 values of 13 nM, 34 nM, and 10 nM, respectively.
Legal status.
Methamnetamine is illegal in Japan.


Trizol

TRIzol is a chemical solution used in the extraction of DNA, RNA, and proteins from cells. The solution was initially used and published by Piotr Chomczyński and Sacchi, N. in 1987.
TRIzol is the brand name of guanidinium thiocyanate from the Ambion part of Life Technologies, and Tri-Reagent is the brand name from MRC, which was founded by Chomczynski.
Uses in extraction.
The correct name of the method is guanidinium thiocyanate-phenol-chloroform extraction. The use of TRIzol can result in DNA and RNA yields comparable to other extraction methods. An alternative method for RNA extraction is phenol extraction and TCA/acetone precipitation. Chloroform should be exchanged with 1-bromo-3-chloropropane when using the new generation TRI Reagent.
DNA and RNA from TRIzol and TRI reagent can also be extracted using the Direct-zol Miniprep kit by Zymo Research. This method eliminates the use of Chloroform and 1-bromo-3-chloropropane completely, bypassing phase-separation and precipitation steps.
TRIzol is light-sensitive and is often stored in a dark-colored, glass container covered in foil. It is stored at room temperature.
When used, it resembles cough syrup, bright pink. The smell of the phenol is extremely strong. TRIzol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components.
Hazards.
Caution should be taken while using TRIzol (due to the phenol and chloroform).
Exposure to TRIzol can be a serious health hazard. Exposure can lead to serious chemical burns and permanent scarring. A lab coat, gloves and a plastic apron are recommended.


6S / SsrS RNA

In the field of molecular biology the 6S RNA is a noncoding RNA that was one of the first to be identified and sequenced. What it does in the bacterial cell was unknown until recently. In the early 2000s scientists found out the function of 6S RNA to as a regulator of sigma 70-dependent gene transcription. All bacterial RNA polymerases have a subunit called a sigma factor. The sigma factors are important because they control how DNA promoter binding and RNA transcription start sites. Sigma 70 was the first one to be discovered in "Escherichia coli."
Structure.
The structure of 6S RNA was defined in 1971. It is a small RNA strand consisting of 184 nucleotides. 6S RNA is a long double-stranded structure and has a single strand loop. Its structure is similar to an open promoter complex of DNA structure. Varies analyses discovered that 6S RNAs are capable of forming a secondary structure. The secondary structure consists of two irregular helical stem regions, making a large core loop which is called a central knot.
Function and Regulation.
The function of 6S RNA is to regulate transcription for "E. Coli" cell survival because it is essential in the process. 6S RNA specifically associates with RNA polymerase holoenzyme containing the sigma70 specificity factor. This interaction represses expression from sigma70-dependent promoters during stationary phase. Which will lead to activate the transcription from sigma 70 dependent promoters. Therefore, during the change in "E. coli" from logarithmic growth to stationary phase, the 6S RNA performs as a regulator of transcription. 6S RNA homologs have recently been identified in most bacterial genomes. polymerase holoenzyme, which contains the housekeeping sigma factor and be expressed during different stages of growth. In many proteobacteria, 6S RNA may be processed from a transcript encoding homologs of the "E. coli" YgfA protein, which is a putative methenyltetrahydrofolate synthetase. Diverged 6S RNAs have been identified in additional bacterial lineages. The purD RNA motif has been experimentally shown to overlap with 6S RNA. One way to examine if the activity of 6S RNA by doing a knockout of 6S RNA. Strains with mutations in 6S RNA have a reduction of lifespan in contrast to the wild-type cells after more than 20 days of nonstop culture. When mutant 6S cells cultured with wild-type cells, it will be at a modest disadvantage in the following days of growth.


GosNIIOKhT

The State Research Institute of Organic Chemistry and Technology (GosNIIOKhT) is a Russian research institute engaged in the development of chemical technologies for use in the national economy and the production of relevant goods and products.
History.
GosNIIOKhT was founded in 1924, during the time of the Soviet Union, to conduct research work in organic synthesis. In the early 1930s, the research institute was engaged in the development of chemical weapons.
Currently, its activities include the destruction of chemical weapons and other hazardous materials. Other areas of work include the development and production of drugs, toxicological research, preclinical testing, chemical technology, and environmental safety.
On October 15, 2020, European Union sanctions were imposed on the institute in connection with the poisoning of politician Alexei Navalny.


Clostridiales-3 RNA motif

The Clostridiales-3 RNA motif is a conserved RNA structure that was discovered by bioinformatics.
Clostridiales-3 motifs are found in Clostridiales.
Clostridiales-3 RNAs likely function in "trans" as sRNAs and, structurally, largely consist of several hairpins.


Peptide library

A peptide library is a tool for protein-related study. A peptide library contains a great number of peptides that have a systematic combination of amino acids. Usually, peptide library is synthesized on solid phase, mostly on resin, which can be made as flat surface or beads. The peptide library provides a powerful tool for drug design, protein–protein interactions, and other biochemical as well as pharmaceutical applications.
You can create a mix of desired amino acids at each point in a sequence. In this way, you could create a library of 20 different polypeptides with only one amino acid residue at random and the rest being the same.
The usefulness of this form of peptide synthesis is limited as you can't go beyond 70 acids in length. This would give you 2070 possible combinations and that's only if you don't include the plethora of available amino acids with pre-installed post-translational modifications. Most drug development does not involve such a random assortment of proteins as you wouldn't learn very much.
Important example. Say you wanted a peptide chain 10 residues in length to use in native chemical ligation with a larger recombinantly expressed protein.
residue 1: Alanine only
residue 2: glutamine, glycine, arginine, glutamic acid, serine, or methionine
residue 3: any one of all 20 amino acids
residue 4: acetyl-lysine only
residue 5: alanine
residue 6: isoleucine
residue 7: aspartic acid
residue 8: phenylalanine
residue 9: acetyl-lysine
residue 10: argineine with the carboxy terminal thioester.
there would be 20 different proteins at residue 3. each one of those would have 7 different proteins at residue 2. the end result would be 20*7 or 140 different polypeptides.
this peptide library would be useful for analyzing the effect of the post-translational modification acetylation on lysine which neutralizes the positive charge. having the library of different peptides at residue 2 and 3 would let you see if some change in chemical properties in the N-terminal tail of the ligated protein make the protein more useful or useful in a different way.
As a general formula, if you made a completely random peptide library with n amino acids for each link in the chain with a length of x, the total number of possible sequences is n^x.


TB9Cs4H2 snoRNA

TB9Cs4H2 is a member of the H/ACA-like class of non-coding RNA (ncRNA) molecule that guide the sites of modification of uridines to pseudouridines of substrate RNAs. It is known as a small nucleolar RNA (snoRNA) thus named because of its cellular localization in the nucleolus of the eukaryotic cell. TB9Cs4H2 is predicted to guide the pseudouridylation of LSU3 ribosomal RNA (rRNA) at residue Ψ1336.


Itai Yanai

Itai Yanai (born 6 February 1975) is an American-Israeli biomedical scientist and Founding Director of the Institute for Computational Medicine at the NYU Grossman School of Medicine. He is also a Professor in the Department of Biochemistry and Molecular Pharmacology at NYU.
Early life and education.
Yanai was born in Haifa, Israel, and moved with his family to Boston in his early teens, when his father Moshe Yanai was appointed Chief Engineer at EMC. He graduated from Boston University in 1998 with degrees in computer engineering and philosophy, both summa cum laude. In 2002 he became the first person in the nation to receive a Ph.D. in, what was at the time, the fledgling field of Bioinformatics. After several years at the Weizmann Institute and Harvard University, he returned to Israel in 2008 as a faculty member at the Technion-Israel Institute of Technology. He moved to the United States in 2016, when he assumed his current position.
Research.
Yanai has published extensively in bioinformatics and theory. He has also done seminal experimental research in cell biology.
In 2016 Yanai co-authored a popular book for the general public.


C0343 RNA

The C0343 RNA is a bacterial non-coding RNA of 74 nucleotides in length that is found between the "ydaN" and "dbpA" genes in the genomes of "Escherichia coli" and "Shigella flexneri", "Salmonella enterica" and "Salmonella typhimurium". This ncRNA was originally identified in E.coli using high-density oligonucleotide probe arrays (microarray). The function of this ncRNA is unknown.
FnrS RNA was later found to be transcribed from the same intergenic region as C0343 RNA.


TB3Cs2H2 snoRNA

TB3Cs2H2 is a member of the H/ACA-like class of non-coding RNA (ncRNA) molecule that guide the sites of modification of uridines to pseudouridines of substrate RNAs. It is known as a small nucleolar RNA (snoRNA) thus named because of its cellular localization in the nucleolus of the eukaryotic cell. TB3Cs2H2 is predicted to guide the pseudouridylation of LSU5 ribosomal RNA (rRNA) at residue Ψ1658.


CFU-GEMM

CFU-GEMM is a colony forming unit that generates myeloid cells. CFU-GEMM cells are the oligopotential progenitor cells for myeloid cells; they are thus also called common myeloid progenitor cells or myeloid stem cells. "GEMM" stands for granulocyte, erythrocyte, monocyte, megakaryocyte.
The common myeloid progenitor (CMP) and the common lymphoid progenitor (CLP) are the first branch of cell differentiation in hematopoiesis after the hemocytoblast (hematopoietic stem cell).
Structure.
In current terminology, CFU-S refers to the pluripotent stem cells that can differentiate into all types of blood cells. CFU-S divides into two lineages: the lymphoid precursor (CFU-LSC) and the myeloid precursor (CFU-GEMM). The CFU-GEMM cell is capable of differentiating into white blood cells, red blood cells, and platelets, all of which are normally found in circulating blood.
It has been suggested that eosinophils do not derive from the common myeloid progenitor in humans.
In the adjacent image, CFU-GEMM is the scientific name for the "common myeloid progenitor" that is responsible for forming all the cells of the myeloid lineages. As observed in the image, CFU-GEMM is capable of producing a diverse set of cells. It matures into the megakaryocyte, erythrocyte, mast cell or myeloblast based on the presence of specific factors that encourage the cell to choose a lineage to follow.
Surface markers.
The cells are characterized by expressing the cell surface markers CD33, CD34 and HLA-DR. These surface markers are proteins on the surface that are unique to specific cells and certain maturation periods, allowing researchers to differentiate between two different cells as well as what stage the cell is found in its developmental progression.
Development.
Growth factors.
The differentiation and proliferation of CFU-GEMM are promoted by growth factors, such as interleukins and cytokines. IL-3 and GM-CSF as single factors are equally active in stimulating CFU-GEMM, but the combination of both factors produces additive stimulatory effects upon CFU-GEMM. The growth of CFU-GEMM is stimulated by the stem cell factor, or SCF. SCF has been found also to synergize with GM-CSF, IL-6, IL-3, IL-11 or erythropoietin to increase the numbers of CFU-GEMM.
CFU-GEMM gives rise to CFU-GM (leading to monoblasts and myeloblasts), CFU-Meg (leading to megakaryoblasts), and CFU-E (leading to proerythroblasts). The stem cell will follow a specific lineage depending on the presence of certain growth factors and cytokines. The GM-CSF and IL-3 both work together to stimulate production of all lines. When erythropoietin (EPO) is present, red blood cell production from the CFU-GEMM will be activated. G-CSF, M-CSF, IL-5, IL-4, and IL-3 stimulate the production of neutrophils, monocytes, eosinophils, basophils, and platelets, respectively.
Research studies.
Since the CFU-GEMM cell is a very early ancestor of the mature cells of the blood, it is not normally found in the blood. While present in bone marrow, the place where CFU-GEMM is most common is in the umbilical cord between a mother and baby. It has been discovered that these cells have a high replating efficiency, meaning that when taken from the umbilical cord and grown in culture, a high percentage of these cells are able to produce colonies. The results of studies conducted by Carow, Hangoc, and Broxmeyer in 1993 reveal that the CFU-GEMM can be classified as a stem cell due to its high replating efficiency in the presence of certain growth factors and cytokines.
The growth and production of CFU-GEMM and BFU-E depend on stimulatory factors from a source of burst-promoting activity (BPA) such as the release of interleukin-1 (IL-1) by monocytes, a has been studied in 1987. It has also been shown that fibroblasts are capable of secreting these BPAs, however only respond to regulatory molecule such as interleukin-1. The results showed that IL-1 increases the stimulatory effects of CFU-GEMM in a dose-dependent fashion with a maximum efficacy around 140 ng/mL. This study revealed that IL-1 plays an important role in the regulation of the production of stimulatory factors that influence the progenitor cells of hematopoiesis.
In another study in 2014, researchers were in search of molecules to stimulate the proliferation of long-term hematopoietic stem cells (LT-HSC). They tested a library of more than 5000 small molecules, with all except one (UM729) suppressing growth. A more potent analog was generated and named UM171. When compared to other similar chemicals, UM171 allowed for more HSC proliferation and lower apoptotic cell number compared to controls, along with a higher number in multipotential progenitors like CFU-GEMM. Furthermore, UM171 did not affect division rate. When used in conjunction with SR1, a known transcription factor, UM171 allowed for suppression of differentiation and led to increased CFU-GEMM growth. These results suggest that UM171+SR1 together enhance proliferation of progenitor cells and suppress differentiation.


Katrina Miranda

Katrina Miranda is an Associate Professor of Biochemistry at the University of Arizona. She works on nitric oxide and their role in diseases like breast cancer, stroke and chronic pain.
Early life and education.
Miranda studied chemistry at Northern Arizona University. She moved to the University of California, Santa Barbara, and earned her PhD in 1996. Miranda was a postdoctoral fellow at the University of New Mexico School of Medicine.
Research.
Miranda studies the chemical and biological basis of redox signalling agents. She was a National Institutes of Health postdoctoral fellow at the National Cancer Institute from 1998 to 2002, before joining the University of Arizona. She looks to identify the biomarkers of disease development. They do this by investigating the molecular redox chemistry new donor systems and designing ways to detect biological signals. Nitric oxide is synthesised in the body when L-Arginine enzymatically oxidises. Miranda looked at what happens when nitric oxides accumulate, including their impact on chronic pain, strokes and breast cancer. She demonstrated that nitric oxide can modify how enzymes bind to metal centres. She studied the reactivity of azanone to clarify the activity of biomolecules. She published the textbook "Chemical Biology of Nitric Oxide" in 2008. Her research group attach chemical moieties that can improve drug delivery. They are using gene expression, genomic and proteomic techniques to analyse the cellular effects of redox active signalling.
Miranda runs outreach activities for faculty at the Arizona Community College to take part in research in her faculty. In 2013 she developed a massive open online course with Google that would explain abstract chemistry concepts to students around the world. In 2018 she filed a class action lawsuit against the University of Arizona for gender discrimination against women professors. She is not the first to sue the University of Arizona; emeritus dean Patricia MacCorquodale and former dean Janice Cervelli took them to court on behalf of women deans in early 2018.