Trifluoromescaline

Trifluoromescaline (TF-M) is a derivative of the phenethylamine hallucinogen mescaline, which has a trifluoromethoxy group replacing the central methoxy group of mescaline. Synthesis of this compound was first reported by Daniel Trachsel in 2011, alongside many other related compounds. Trifluoromescaline was found to be one of the most potent compounds in the series, with a reported dosage of 15-40mg (and 60mg being described as a "strong overdose"), and a slow onset of action and long duration of effects, lasting 14-24 hours or more.


MMP3

Stromelysin-1 also known as matrix metalloproteinase-3 (MMP-3) is an enzyme that in humans is encoded by the "MMP3" gene. The MMP3 gene is part of a cluster of MMP genes which localize to chromosome 11q22.3. MMP-3 has an estimated molecular weight of 54 kDa.
Function.
Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix proteins and during tissue remodeling in normal physiological processes, such as embryonic development and reproduction, as well as in disease processes, such as arthritis, and tumour metastasis. Most MMPs are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases.
The MMP-3 enzyme degrades collagen types II, III, IV, IX, and X, proteoglycans, fibronectin, laminin, and elastin. In addition, MMP-3 can also activate other MMPs such as MMP-1, MMP-7, and MMP-9, rendering MMP-3 crucial in connective tissue remodeling. The enzyme is also thought to be involved in wound repair, progression of atherosclerosis, and tumor initiation.
In addition to classical roles for MMP3 in extracellular space, MMP3 can enter in cellular nuclei and control transcription.
Gene regulation.
MMP3 itself can enter in nuclei of cells and regulate target gene such as CTGF/CCN2 gene.
Expression of MMP3 is primarily regulated at the level of transcription, where the promoter of the gene responds to various stimuli, including growth factors, cytokines, tumor promoters, and oncogene products. A polymorphism in the promoter of the MMP3 gene was first reported in 1995. The polymorphism is caused by a variation in the number of adenosines located at position -1171 relative to the transcription start site, resulting in one allele having five adenosines (5A) and the other allele having six adenosines (6A). In vitro promoter functional analyses showed that the 5A allele had greater promoter activities as compared with the 6A allele. It has been shown in different studies that individuals carrying the 5A allele have increased susceptibility to diseases attributed to increased MMP expression, such as acute myocardial infarction and abdominal aortic aneurysm.
On the other hand, the 6A allele has been found to be associated with diseases characterized by insufficient MMP-3 expression due to a lower promoter activity of the 6A allele, such as progressive coronary atherosclerosis. The -1171 5A/6A variant has also been associated with congenital anomalies such as cleft lip and palate, where individuals with cleft lip/palate presented significantly more 6A/6A genotypes than controls. Recently, the MMP3 gene was shown to be down-regulated in individuals with cleft lip and palate when compared to controls, reinforcing the nature of cleft lip/palate as a condition resulting from insufficient or defective embryonic tissue remodeling.
Structure.
Most members of the MMP family are organized into three basic, distinctive, and well-conserved domains based on structural considerations: an amino-terminal propeptide; a catalytic domain; and a hemopexin-like domain at the carboxy-terminal. The propeptide consists of approximately 80–90 amino acids containing a cysteine residue, which interacts with the catalytic zinc atom via its side chain thiol group. A highly conserved sequence (. . .PRCGXPD. . .) is present in the propeptide. Removal of the propeptide by proteolysis results in zymogen activation, as all members of the MMP family are produced in a latent form.
The catalytic domain contains two zinc ions and at least one calcium ion coordinated to various residues. One of the two zinc ions is present in the active site and is involved in the catalytic processes of the MMPs. The second zinc ion (also known as structural zinc) and the calcium ion are present in the catalytic domain approximately 12 Å away from the catalytic zinc. The catalytic zinc ion is essential for the proteolytic activity of MMPs; the three histidine residues that coordinate with the catalytic zinc are conserved among all the MMPs. Little is known about the roles of the second zinc ion and the calcium ion within the catalytic domain, but the MMPs are shown to possess high affinities for structural zinc and calcium ions.
The catalytic domain of MMP-3 can be inhibited by tissue inhibitors of metalloproteinases (TIMPs). The n-terminal fragment of the TIMP binds in the active site cleft much like the peptide substrate would bind. The Cys1 residue of the TIMP chelates to the catalytic zinc and forms hydrogen bonds with one of the carboxylate oxygens of the catalytic glutamate residue (Glu202, see mechanism below). These interactions force the zinc-bound water molecule that is essential to the enzyme's function to leave the enzyme. The loss of the water molecule and the blocking of the active site by TIMP disable the enzyme.
The hemopexin-like domain of MMPs is highly conserved and shows sequence similarity to the plasma protein, hemopexin. The hemopexin-like domain has been shown to play a functional role in substrate binding and/or in interactions with the tissue inhibitors of metalloproteinases (TIMPs), a family of specific MMP protein inhibitors.
Mechanism.
The mechanism for MMP-3 is a variation on a larger theme seen in all matrix metalloproteinases. In the active site, a water molecule is coordinated to a glutamate residue (Glu202) and one of the zinc ions present in the catalytic domain. First, the coordinated water molecule performs a nucleophilic attack on the peptide substrate's scissile carbon while the glutamate simultaneously abstracts a proton from the water molecule. The abstracted proton is then removed from the glutamate by the nitrogen of the scissile amide. This forms a tetrahedral gem-diolate intermediate that is coordinated to the zinc atom. In order for the amide product to be released from the active site, the scissile amide must abstract a second proton from the coordinated water molecule. Alternatively, it has been shown for thermolysin (another metalloproteinase) that the amide product can be released in its neutral (R-NH2) form. The carboxylate product is released after a water molecule attacks the zinc ion and displaces the carboxylate product. The release of the carboxylate product is thought to be the rate-limiting step in the reaction.
In addition to the water molecule directly involved in the mechanism, a second water molecule is suggested to be a part of the MMP-3 active site. This auxiliary water molecule is thought to stabilize the gem-diolate intermediate as well as the transition states by lowering the activation energy for their formation. This is demonstrated in the mechanism and reaction coordinate diagram below.
Disease relevance.
MMP-3 has been implicated in exacerbating the effects of traumatic brain injury (TBI) through its disruption of the blood-brain barrier (BBB). Different studies have shown that after the brain undergoes trauma and inflammation has begun, MMP production in the brain is increased. In a study conducted using MMP-3 wild type (WT) and knockout (KO) mice, MMP-3 was shown to increase BBB permeability after traumatic injury. The WT mice were shown to have lower claudin-5 and occludin levels than the KO mice after TBI. Claudin and occludin are proteins that are essential for the formation of the tight junctions between the cells of the blood-brain barrier. Tissue from uninjured WT and KO mice brains was also treated with active MMP-3. Both the WT and KO tissues showed a drop in claudin-5, occludin, and laminin-α1 (a basal lamina protein), suggesting that MMP-3 directly destroys tight junction and basal lamina proteins.
MMP-3 also does damage to the blood-spinal cord barrier (BSCB), the functional equivalent of the blood-brain barrier, after spinal cord injury (SCI). In a similar study conducted using MMP-3 WT and KO mice, MMP-3 was shown to increase BSCB permeability, with the WT mice showing greater BSCB permeability than the KO mice after spinal cord injury. The same study also found decreased BSCB permeability when spinal cord tissues were treated with a MMP-3 inhibitor. These results suggest that the presence of MMP-3 serves to increase BSCB permeability after SCI. The study showed that MMP-3 accomplishes this damage by degrading claudin-5, occludin, and ZO-1 (another tight junction protein), similar to how MMP-3 damages the BBB.
The increase in blood-brain barrier and blood-spinal cord barrier permeability allows for more neutrophils to infiltrate the brain and spinal cord at the site of inflammation. Neutrophils carry MMP-9., which has also been shown to degrade occludin. This leads to further disruption of the BBB and BSCB


Doxazosin

Doxazosin, sold under the brand names Cardura among others, is a medication used to treat symptoms of an enlarged prostate and high blood pressure. For high blood pressure, it is a less preferred option. It is taken by mouth.
Common side effects include dizziness, sleepiness, swelling, nausea, shortness of breath, and abdominal pain. Severe side effects may include low blood pressure with standing, an irregular heart beat, and priapism. Prostate cancer should be ruled out before starting treatment. It is a α1-selective adrenergic blocker in the quinazoline class of compounds.
Doxazosin was patented in 1977 and came into medical use in 1988. It is available as a generic medication. In 2017, it was the 150th most commonly prescribed medication in the United States, with more than four million prescriptions.
Medical uses.
High blood pressure.
Doxazosin is usually added to other antihypertensive therapy such as calcium channel antagonists, diuretics, beta-adrenoreceptor antagonists, angiotensin-converting enzyme inhibitors and angiotensin-2 receptor blockers.
Doxazosin is generally considered to be safe, well tolerated and effective as an add-on antihypertensive drug.
Like other alpha-1 receptor antagonists, it has a role in the peri-operative management of pheochromocytoma.
Benign prostatic hypertrophy.
Doxazosin is considered to be effective in reducing urinary symptom scores and improving peak urinary flow in men with benign prostatic hypertrophy.
History.
The Antihypertensive and Lipid Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) study stopped its arm of the trial looking at alpha blockers, because doxazosin was less effective than a simple diuretic, and because patients on doxazosin had a 25% higher rate of cardiovascular disease and twice the rate of congestive heart failure as patients on diuretics. Pfizer, aware of the results before publication, launched a marketing campaign in early 2000, and sales were largely unaffected, despite the dangers highlighted by the study.


Asymbescaline

Asymbescaline (3,4-diethoxy-5-methoxyphenethylamine) is a lesser-known psychedelic drug. It is a homolog of mescaline. Asymbescaline was first synthesized by Alexander Shulgin. In his book "PiHKAL", the dosage range is listed as 200–280 mg, and the duration listed as 10–15 hours. Asymbescaline produces few to no effects. Very little data exists about the pharmacological properties, metabolism, and toxicity of asymbescaline.


Dazopride

Dazopride (AHR-5531) is an antiemetic and gastroprokinetic agent of the benzamide class which was never marketed. It acts as a 5-HT3 receptor antagonist and 5-HT4 receptor agonist. In addition to its gastrointestinal effects, dazopride facilitates learning and memory in mice.


Methylenetetrahydrofolate reductase (ferredoxin)

In enzymology, a methylenetetrahydrofolate reductase (ferredoxin) () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are 5-methyltetrahydrofolate and oxidized ferredoxin, whereas its 3 products are 5,10-methylenetetrahydrofolate, reduced ferredoxin, and H+.
This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-NH group of donors with an iron-sulfur protein as acceptor. The systematic name of this enzyme class is 5-methyltetrahydrofolate:ferredoxin oxidoreductase. This enzyme is also called 5,10-methylenetetrahydrofolate reductase. This enzyme participates in one carbon pool by folate.


Extension Poly(A) Test

The extension Poly(A) Test (ePAT) describes a method to determine the poly(A) tail lengths of mRNA molecules. It was developed and described by A. Jänicke et al. in 2012.
The method consists of three separate steps: in the first step, the poly-adenylated RNA is hybridised to a DNA oligonucleotide featuring a poly-deoxythymidine sequence at its 5’ end. Klenow polymerase then catalyses elongation of the mRNA’s 3’ end, using the DNA oligonucleotide as a template. This reaction takes place at 25 °C. In the second step, reverse transcriptase synthesis extends the DNA oligonucleotides that have annealed to the mRNA’s extended 3’ end. In order to ensure that DNA oligomers hybridised to internal poly(A) sequences do not serve as primers for reverse transcription, the second step is carried out at 55 °C. A third and final step involves amplification of the newly synthesised cDNA via PCR. This PCR requires one gene-specific and one universal primer. Analysis of the amplicons’ lengths allows for estimation of the sequence flanked by the two primers, i.e. the poly(A) tail length of the sample mRNA.
According to the authors, measurement of poly(A) tail lengths and their distribution amongst different transcripts, this method can be used to determine the cell’s translation state instead of the more tedious analysis of protein translation states.


Lewisite 2

Lewisite 2 (L-2) is an organoarsenic chemical weapon like lewisite 1 and lewisite 3 first synthesized in 1904 by Julius Arthur Nieuwland. It is usually found as a mixture of 2-chlorovinylarsonous dichloride (lewisite 1) as well as bis(2-chloroethenyl) arsinous chloride (lewisite 2) and tris(2-chlorovinyl)arsine (lewisite 3). Pure lewisite 1 is an oily, colorless liquid, however, the impure mixture can appear amber to black with an odor distinct to geraniums.
Synthesis.
Lewisite 2 is made as a byproduct along with lewisite 3 in the reaction that makes lewisite 1.
Acetylene reacts with AsCl3 in hydrochloric acid solution, with mercuric chloride as a catalyst, to give lewisite in 80 to 85 percent yield (based on AsCl 3).
Lewisite 2 is formed when there are two additions of acetylene to the arsenic center instead of one like the reaction seen above where only lewisite one is shown.
Exposure.
Adverse health effects caused by lewisite can vary and are dependent on; the amount people are exposed to and, the amount of time someone is exposed to it. Lewisite immediately damages the skin, eyes, and respiratory tract and is a strong irritant and blistering agent. Due to its arsenic center, lewisite may also cause problems similar to arsenic poisonings like stomach ailments and low blood pressure.
Inhalation.
Inhalation, the most common route of exposure, causes a burning pain and irritation throughout the respiratory tract, nosebleed (epistaxis), laryngitis, sneezing, coughing, vomiting, difficult breathing (dyspnea). From one acute exposure, someone who has inhaled lewisite can develop chronic respiratory disease. In severe cases of exposure, can cause fatal pulmonary edema, pneumonitis, or respiratory failure.
Ingestion.
Ingestion results in severe pain, nausea, vomiting, and tissue damage. Those exposed to lewisite can develop refractory hypotension (low blood pressure) known as Lewisite shock, as well as some features of arsenic toxicity. Lewisite causes physical damage to capillaries, which then become leaky, meaning that there is not enough blood volume to maintain blood pressure, a condition called hypovolemia. When the blood pressure is low, the kidneys may not receive enough oxygen and can be damaged.
Eye Exposure.
The results of eye exposure can range from stinging, burning pain and strong irritation to blistering and scarring of the cornea, along with blepharospasm, lacrimation, and edema of the eyelids and periorbital area. Eye exposure to lewisite can cause permanent visual impairment or blindness. The eyes can swell shut, which can keep the eyes safe from further exposure. The most severe consequences of eye exposure to lewisite are globe perforation and blindness. Generalised symptoms also include restlessness, weakness, hypothermia and low blood pressure.
Chronic Exposure.
Chronic exposure to lewisite can cause arsenic poisoning and development of a lewisite allergy. It can also cause various long-term illnesses or permanent damage to organs, depending on where the exposure has occurred, including conjunctivitis, aversion to light (photophobia), visual impairment, double vision (diplopia), tearing (lacrimation), dry mucous membranes, garlic breath, burning pain in the nose and mouth, toxic encephalopathy, peripheral neuropathy, seizures, nausea, vomiting, chronic obstructive pulmonary disease (COPD), bronchitis, dermatitis, skin ulcers, basal cell carcinoma, and squamous cell carcinoma.
Treatment.
Basic first aid for lewisite exposure is decontamination and irrigation of any areas that have been exposed, and when necessary airway management, assisted ventilation, and monitoring of vital signs.
British anti-lewisite, also called dimercaprol, is the antidote for lewisite. It can be injected to prevent systemic toxicity, but will not prevent injury to the skin, eyes, or mucous membranes. Chemically, dimercaprol is a chelating agent that binds to the arsenic in lewisite. It is contraindicated in those with peanut allergies.


Pipoxizine

Pipoxizine () is a first-generation antihistamine as well as serotonin antagonist of the diphenylmethylpiperazine group related to hydroxyzine. It was investigated as a bronchodilator but was never marketed.


JY cell line

The JY cell line is an Epstein–Barr virus (EBV)-immortalised B cell lymphoblastoid line. JY cells express HLA class-I A2 and class-II DR. JY is a suspension cell line, although the cells are known to grow in clumps. The growth medium is RPMI 1640, 10% fetal calf serum and 1% -glutamine. JY cells are positive for murine leukemia virus.


Cyclic di-GMP-I riboswitch

Cyclic di-GMP-I riboswitches are a class of riboswitch that specifically bind cyclic di-GMP, which is a second messenger that is used in a variety of microbial processes including virulence, motility and biofilm formation. Cyclic di-GMP-I riboswitches were originally identified by bioinformatics as a conserved RNA-like structure called the "GEMM motif". These riboswitches are present in a wide variety of bacteria, and are most common in Clostridia and certain varieties of Proteobacteria. The riboswitches are present in pathogens such as "Clostridium difficile", "Vibrio cholerae" (which causes cholera) and "Bacillus anthracis" (which causes anthrax). "Geobacter uraniumreducens" is predicted to have 30 instances of this riboswitch in its genome. A bacteriophage that infects "C. difficile" is predicted to carry a cyclic di-GMP-I riboswitch, which it might use to detect and exploit the physiological state of bacteria that it infects.
The discovery of this riboswitch class answers the question of how genes are regulated in response to cyclic di-GMP levels in many different bacteria. However, some bacteria in which cyclic di-GMP has been studied lack cyclic di-GMP-I riboswitches, e.g. "Pseudomonas aeruginosa". Cyclic di-GMP-I riboswitches are the first kind of riboswitch to be discovered whose role is not primarily in regulating metabolism, but is instead part of signaling. A second class of riboswitch that binds cyclic di-GMP is called the cyclic di-GMP-II riboswitch. The two classes of cyclic di-GMP-binding riboswitches do not share any known sequence or structural features.
High-resolution three-dimensional structures of cyclic di-GMP-I riboswitches have been determined using X-ray crystallography.
Some homologs of the c-di-GMP-I riboswitch structure actually function a riboswitches that recognize another signaling molecule, cyclic AMP-GMP.


Choi Yang-do

Choi Yang-do () is a South Korean agricultural biotechnologist. His research focuses on the development of crops with stress-tolerant and yield-enhancing traits. One of his academic achievements is the discovery of a new jasmonate which enhances resistance against external stress of the crop. Choi is currently professor at Seoul National University in South Korea.
Biography.
Choi Yang-do was born in Seoul, South Korea. He studied agricultural chemistry at Seoul National University (1972-1976) and graduated with a BS degree. From 1976 to 1978 he received a MS degree in biological science from Korea Advanced Institute of Science and Technology (KAIST) and earned
his Ph.D. in biochemistry, molecular biology and cell biology from 1981 to 1985 at Northwestern University in Evanston.
Researches.
Choi researched jasmonates, which are believed to participate in signal transduction processes between external stresses, such as wounding, pathogenic attack, or cell response by activating the defense genes. He discovered a new enzyme, (EC2.1.1.141) jasmonate carboxyl methyltransferase (JMT), which catalyzes the methylation of jasmonate to MeJA. The overexpression of the JMT gene confers resistance to pathogens. He is currently working to understand the molecular function of MeJA, JMT, and its effector genes. Furthermore, the genetic engineering of crops by application of those genes and knowledge is under investigation.
The major research activity is the discovery of a novel enzyme and its genes encoding jasmonic acid carboxyl methyltransferase, which is involved in stress response and signal transduction in plant. Other achievements are the development of drought tolerant transgenic rice and transferred technology to Mahyco, India and the development of yield-enhanced transgenic rice and transferred technologies to BASF Plant Science in Germany.
Awards.
For his scientific achievements Choi received the New Technology Development Award from the Ministry of Science & Technology (1992), the Special Award from Korean Society for Molecular & Cell Biology from the Korean Society of Molecular and Cellular Biology (1999), the 11th Sangrok Agriculture & Life Science Award from Seoul National University College of Agriculture (2002), the Excellent Monograph Award from the Korean Federation for Science & Technology Societies (2003), the 11th Hwanong Award from Hwanong Academy & Research Foundation (2004), the KSABC Award from the Korean Society for Applied Biology and Chemistry (2006), the 52nd NAS Award from the National Academy of Sciences, and the Most Outstanding Korean Scientist and Engineer Award from the Ministry of Education, Science and Technology & Korean Federation of Science and Technology Societies (2008).


North Negros BioPower

North Negros BioPower is a planned biomass-fired power station in Manapla, Negros Occidental in the Philippines. When commissioned in the 4th quarter 2018 it will be among the biggest biomass power stations in the Philippines and will have a generating capacity of 24.99 megawatts, enough electricity to provide 265,000 people in the region’s urban centres and rural areas on the island of Negros. The power plant is a cooperation between ThomasLloyd CTI Asia Holdings Pte and Bronzeoak Philippines. General Contractor is Wuxi Huaguang Electric Power Engineering.
The plant will be primary fed with cane trash with some grassy and woody energy crop plants. The feedstock utilisation will be 220,000 tonnes per year with a local feedstock availability of 2.1 million tonnes per year within a 50 km-radius catchment area. The plant will be connected to an existing 138 kV substation, 12 km away when operational. The power plant is expected to create 675 new jobs in the plant and 2,500 jobs in feedstock production and collection.
The facility was initially planned to be constructed in 2014 for commissioning in 2017 with the cost of about $114 million. The planned schedule for completion was moved to the third quarter of 2016. The planned commissioned date was moved once again to the fourth quarter of 2018 with construction of the facility set on the second quarter of 2017.


Counsyl


Hydrophobic mismatch

Hydrophobic mismatch is the difference between the thicknesses of hydrophobic regions of a transmembrane protein and of the biological membrane it spans. In order to avoid unfavorable exposure of hydrophobic surfaces to water, the hydrophobic regions of transmembrane proteins are expected to have approximately the same thickness as the hydrophobic (lipid acyl chain) region of the surrounding lipid bilayer. Nevertheless, the same membrane protein can be encountered in bilayers of different thickness. In eukaryotic cells, the plasma membrane is thicker than the membranes of the endoplasmic reticulum. Yet all proteins that are abundant in the plasma membrane are initially integrated into the endoplasmic reticulum upon synthesis on ribosomes. Transmembrane peptides or proteins and surrounding lipids can adapt to the hydrophobic mismatch by different means.
Possible adaptations to mismatch.
In order to avoid unfavorable exposure of hydrophobic surfaces to a hydrophilic environment, biological membrane tends to adapt to such mismatch. For example, an integral membrane protein tends to surround itself by lipids of matching size and shape due to protein and lipid segregation. Since proteins are relatively rigid, whereas lipid hydrocarbon chains are flexible, the condition of hydrophobic matching can be fulfilled by stretching, squashing, and/or tilting of the lipid chains
Protein aggregation.
Since Mouritsen and Bloom proposed the detailed thermodynamic model, which includes adaptation of the lipids and induction of protein segregation at a more extreme mismatch in their “Mattress Model”, more additional insight into mismatch-induced protein aggregation has been obtained. Also some experimental evidence that a hydrophobic mismatch can lead to protein aggregation in fluid bilayer were founded. Electron microscopy studies on bacteriorhodopsin, reconstituted in saturated and unsaturated fluid PC bilayers with varying chain length, showed that protein aggregation occurred only with a rather large mismatch, and that bilayer thicknesses of 4 angstrom thicker and 10 angstrom thinner than the estimated hydrophobic thickness of the protein are allowed without induction of significant aggregation.
Helix tilt.
Tilt is also a possible result if the hydrophobic part of a peptide or protein is too long to span the membrane. A previous study on lactose permease of E. coli showed that upon reconstitution of the protein in PE/PG (3/1) lipid bilayer, an increase in helix tilt occurs at increasing protein content. This tilt was accompanied by a decrease in lipid order, which results in a decrease in bilayer thickness, suggesting that it is a mismatch related response.
In large proteins that span the membrane multiple times, changes in helical tilt may occur with little effect on lipid packing. However, for a single transmembrane helix, it is possible that a tilt would cause a strain on the surrounding lipids to accommodate the helix in the bilayer. Thus, a large degree of tilting can be a less favorable option for single transmembrane proteins.
Surface orientation.
Relatively small hydrophobic peptides may not be able to integrate into the membrane, and in response adopt an orientation at the membrane surface. The experimental evidence was shown by a fluorescence study on an artificial peptide with a 19 amino acid long hydrophobic sequence of mainly leucines and flanked on both sides with lysines as anchoring residues. The results indicated that a conversion from a dominant transmembrane to parallel orientation of the peptide could be induced by modulating bilayer thickness via addition of cholesterol or by increasing lipid chain length.
Backbone conformation change.
To obtain detailed information on the consequences of mismatch for the conformation of peptides and proteins in lipid bilayer, small membrane-spanning peptides are most suitable. Still need some studies.
Theories for the mismatch effects.
Different theoretical approaches have been applied to describe the energy cost and thermodynamic effects of mismatch, including treatment of the membrane as an elastic sheetor a microscopic approach.
Mattress model.
Mattress model was proposed as a phenomenological theory approach in 1984 by Mouritsen and Bloom. It is a two-component real solution theory based on the theory of nonideal solutions and hence allows for phase separation. In their model, they relate the energy stored in the undulations of the membrane surface caused by the mismatch to the elastic properties of the lipids and proteins. They do not include microscopic detail of the lipids, but use as input the known thermodynamic properties of the pure lipid system. They also include indirect lipid-protein interactions induced by the mismatch as well as direct lipid-protein van der Waals-like interactions between the hydrophobic parts of the lipid bilayer and the proteins. The excess "hydrophobic effect" associated with the lipid-protein hydrophobic mismatch, and the elastic deformation free energy of the lipid chains near the protein. The interaction potentials are estimated based on experimental data derived from thermodynamic and mechanical measurements of membrane properties.
Monte Carlo simulation scheme.
The mattress model was later replicated in a Monte Carlo simulation scheme by Sperotto and Mouritsen. They allowed for different microstates of the lipids, classified according to Pink’s 10-state model. hence enabling a pure lipid bilayer phase transition. This version of the model provides a connection between the microscopic characteristics of the system and its thermodynamic behavior.
Molecular theory.
In a molecular theory of the lipid chains of the membrane, peptides, with their hydrophobic length, were treated as providing a boundary condition on the configuration of the lipid chains. A molecular modeling was combined with phenomenological free energy contributions describing lipid head group repulsion and membrane solvent surface tension. Duque et al.
Experimental studies of hydrophobic mismatch and helix tilt.
Knowledge of the response of membrane proteins to mismatch has been obtained from a variety of experimental studies. Different types of experimental approaches provide different kinds of insight into the contributions from the abovementioned hypothetical molecular responses. For example, proteins or peptides outfitted with fluorescent or paramagnetic labeling groups can be employed in fluorescence spectroscopy and electron spin resonance studies. These can reveal the molecular details of both the protein-lipid interactions and protein-protein interactions (characteristic of an aggregation-style response) and how they are affecting by (mis)match conditions. Studies of helix tilting as a function of membrane thickness have also benefited from the use of solid-state NMR techniques, in particular using oriented membranes that provide direct insight into the helix tilt angle. Early studies of model membrane-spanning peptides (such as the WALP peptide) have provided insight into the various factors that influence the response, including membrane composition, peptide sequence and in particular also the presence of interfacial anchoring residues. In recent years, great advances in X-ray crystallography and electron microscopy techniques have yielded new insights of the lipid interactions of larger proteins. This is exemplified by the insights into helix tilting in a crystallized calcium pump protein.
Biological significance of mismatch.
The hydrophobic mismatch is important for the protein sorting and formation of lipid rafts.
Protein sorting.
In eukaryotic cells, the level of cholesterol increases through the secretory pathway, from the endoplasmic reticulum to the Golgi to the plasma membrane, suggesting a concomitant increase in membrane thickness. In line with this, the average length of transmembrane segment of single-span plasma membrane proteins typically is five amino acids longer than the average length of proteins from the Golgi. Experimental evidence was obtained that protein sorting in the Golgi may be based on this length difference: for several proteins that normally reside in the Golgi, it was shown that increasing their hydrophobic length can reroute the proteins to the plasma membrane, or vice versa, that decreasing the hydrophobic length of proteins from the plasma membrane can cause their retention in the Golgi.
Lipid rafts.
Rafts are membrane domains enriched in cholesterol, sphingomyelin (SM), and certain membrane proteins. Rafts have putative roles in many physiological processes, such as signal transduction, endocytosis, apoptosis, protein trafficking, and lipid regulation. Raft lipids typically have saturated hydrocarbon chains. Lipid rafts have a higher hydrophobic thickness than the rest of the lipid bilayer, which may lead to a preferential separation of transmembrane proteins with a higher hydrophobic thickness into the lipid rafts.


Metabolic Engineering (journal)

Metabolic Engineering is a peer-reviewed scientific journal covering research on production of fuels and chemicals using microorganisms and other biological systems. The journal specialises in quantitative analyses of metabolic pathways and their relationship to cell physiology in industrial and medical contexts.
Abstracting and indexing.
The journal is abstracted and indexed in Biotechnology Citation Index, Chemical Abstracts Service, Current Contents/Life Sciences, Clinical Medicine, EMBASE, EMBiology, MEDLINE, and Scopus.


Icos

Icos Corporation (trademark ICOS) was an American biotechnology company and the largest biotechnology company in the U.S. state of Washington, before it was sold to Eli Lilly and Company in 2007. It was founded in 1989 by David Blech, Isaac Blech, Robert Nowinski, and George Rathmann, a pioneer in the industry and Chief Executive Officer (CEO) and co-founder of Amgen, Icos focused on the development of drugs to treat inflammatory disorders. During its 17-year history, the company conducted clinical trials of twelve drugs, three of which reached the last phase of clinical trials. Icos also manufactured antibodies for other biotechnology companies.
Icos is famous for tadalafil (Cialis), a drug used to treat erectile dysfunction. This drug was discovered by GlaxoSmithKline, developed by Icos, and manufactured and marketed in partnership with Eli Lilly. Boosted by a unique advertising campaign led by the Grey Worldwide Agency, sales from Cialis allowed Icos to become profitable in 2006. Cialis was the only drug developed by the company to be approved. LeukArrest, a drug to treat shock, and Pafase, developed for sepsis, were both tested in phase III clinical trials, but testing was discontinued after unpromising results during the trials. Eli Lilly acquired Icos in January 2007, and most of Icos's workers were laid off soon after. CMC Biologics, a Danish contract manufacturer, bought the remnants of Icos and retained the remaining employees.
History.
Icos was founded in 1989 by George Rathmann, Robert Nowinski, and Christopher Henney, each of whom had previously started another biotechnology company: Rathmann had created Amgen; Nowinski had launched Genetic Systems, later sold to Bristol-Myers Squibb; and Henney co-founded Immunex, later sold to Amgen. Icos was formed with the goal of developing new drugs to treat the underlying causes of inflammatory diseases and halt the disease process in the early stages. The name "Icos" comes from "icosahedron", a 20-sided polyhedron, which is the shape of many viruses, and was chosen because the founders originally thought retroviruses might be involved in inflammation. The founders raised $33 million in July 1990 from many investors, including Bill Gates – who at the time was the largest shareholder, with 10% of the equity. The company initially had temporary offices in downtown Seattle, but moved to Bothell in September 1990. Icos went public on June 6, 1991, raising $36 million. George Rathmann, seen as a guiding father to Icos, left the company in February 2000, and was replaced as CEO and chairman by Paul Clark, a former executive at Abbott Laboratories. A former Icos manager named short-sighted leadership by Clark as a factor in the failure of the company to develop any other successful drugs apart from Cialis.
Cialis.
Sold as Cialis and initially codenamed IC351, tadalafil is a drug prescribed for erectile dysfunction (ED) and approved for pulmonary arterial hypertension (PAH). It is a phosphodiesterase type 5 (PDE5) inhibitor, similar in function to sildenafil. In addition to ED and PAH, tadalafil has undergone clinical trials for the treatment of benign prostatic hyperplasia and for female sexual dysfunction.
Tadalafil was initially formulated by Glaxo Wellcome (now GlaxoSmithKline) under a new drug development partnership between Glaxo and Icos that began in August 1991. The drug was originally researched as a treatment for cardiovascular diseases such as hypertension and angina, but focus quickly shifted to ED with the success of another PDE5 inhibitor, sildenafil (Viagra), which had been developed by Pfizer. Icos began research on tadalafil in 1993, and clinical trials started two years later. Glaxo let the partnership with Icos lapse in 1996, including the company's 50% share of profits from resulting drugs, because the drugs in development were not in Glaxo's core markets. In 1998, Icos formed a 50/50 joint venture with Indianapolis-based Eli Lilly (Lilly Icos LLC) to develop and commercialize tadalafil as Cialis. The release of Cialis in the United States was delayed in April 2002 when the Food and Drug Administration (FDA) recommended that Icos perform more studies, improve labelling, and address manufacturing issues. Cialis was approved in Europe in November 2002 and in the United States a year later. The drug was approved for once-daily use for ED in Europe in June 2007 and in the United States in January 2008.
In 2006, Cialis generated $971 million in sales, leading Icos to post its first-ever quarterly profit in August. In May 2009, tadalafil, to be sold as Adcirca by United Therapeutics, was approved in the United States for the treatment of pulmonary arterial hypertension based on data from a pivotal study begun before the sale of Icos to Eli Lilly.
Lawsuits with Pfizer.
Pfizer and Lilly Icos have filed many lawsuits against each other in various countries over Cialis and Viagra. Pfizer was given a broad patent on PDE5 inhibitors in Britain in 1993. Lilly Icos filed a complaint in a London court in September 1999, and the patent was overturned in November 2000 on the grounds that Pfizer's patent was based on information already in the public domain when the patent was issued. In the United States, Pfizer filed suit against Lilly Icos soon after receiving a broad US patent for PDE5 inhibitors in October 2002. The United States Patent and Trademark Office ordered a reexamination of the patent, and, as in Britain, the examiner found that PDE5 inhibitors were not a new invention by Pfizer, voiding the patent. In Canada, Pfizer moved to block sales of Cialis five months after it was approved there, arguing that there could be consumer backlash against Pfizer should Cialis be pulled from the market months later as a result of an ongoing patent lawsuit. A federal judge refused, saying he could not "imagine demonstrations in the street or storming of the barricades because one impotence medicine is made unavailable".
Blindness warning on label.
In May 2005, the FDA began investigating reports of sudden blindness in users of sildenafil (Viagra). The FDA said it had received reports of the condition, a permanent blindness in one eye known as non-arteritic anterior ischemic optic neuropathy, in 38 users of sildenafil and 5 users of tadalafil or vardenafil (Levitra). Lilly Icos voluntarily amended the Cialis label to warn of the condition. The FDA was criticized for its handling of the matter, as an FDA safety officer had commented on unusual reports of blindness over 13 months before a journal article was published on the issue. United States Senator Chuck Grassley wrote a letter to the FDA detailing his criticism, saying that the FDA's Office of New Drugs (OND) had taken no action "despite OND's knowledge of the blindness risks since January 2004 and general agreement among FDA staff last spring that the label should be updated". Grassley's letter also suggested that Pfizer resisted adding the blindness warning to Viagra's label. In July 2005, the FDA said that Viagra, Levitra, and Cialis labels would all carry warnings on the risk of sudden blindness, though it was unclear whether the drugs were actually causing the blindness.
Marketing.
Lilly Icos hired the Grey Worldwide Agency in New York, part of the Grey Global Group, to run the Cialis advertising campaign. Cialis advertisements were described as being gentler, warmer and with a more relaxed feel than those of its rivals, to reflect the longer duration of the drug. (Tadalafil has a half-life of 17.5 hours, compared to 3.5 for sildenafil and 4.5 for vardenafil.) Iconic themes in Cialis advertisements include couples in bathtubs and the slogan "When the moment is right, will you be ready?" Cialis advertisements were unique among those of ED drugs in that they went beyond describing ED and mentioning the drug's benefits. As a result, Cialis advertisements were also the first to describe side effects, as the FDA requires advertisements in support of a specific brand name to mention side effects; ads for Levitra and Viagra did not mention the brand name of the drug, therefore circumventing this FDA requirement. One of the first advertisements for Cialis aired during the 2004 Super Bowl; Lilly Icos paid more than $4 million for the one-minute ad. Just weeks before the game, the FDA required more possible side effects, including priapism, to be listed in the advertisement. Although many parents objected to the ad being aired during the Super Bowl, Janet Jackson's halftime "wardrobe malfunction" overshadowed Cialis. In January 2006, a physician was added to the advertisements to describe side effects on-screen, and Icos began running advertisements only where more than 90 percent of the audience was made up of adults, effectively ending Super Bowl advertisements. In 2004, Lilly Icos, Pfizer, and GlaxoSmithKline spent a combined $373.1 million to advertise Cialis, Viagra, and Levitra respectively.
Miscellaneous drugs.
Icos developed several drugs whose purpose was to disrupt the process of inflammation in the body. The research program focused on the underlying causes of inflammation rather than specific disorders. The compounds developed by Icos were tested in clinical trials in the areas of sepsis, multiple sclerosis, ischemic stroke, heart attack, pancreatitis, pulmonary arterial hypertension, chronic obstructive pulmonary disease, interstitial cystitis, psoriasis, hemorrhagic shock, sexual dysfunction, benign prostatic hyperplasia, rheumatoid arthritis, emphysema, chronic bronchitis, and acute respiratory distress syndrome.
LeukArrest.
Rovelizumab, trade-named LeukArrest and also known as Hu23F2G, was developed to treat patients suffering from hemorrhagic shock, which is caused by massive blood loss. The drug is a monoclonal antibody that inhibits the recruitment of white blood cells to the site of inflammation. During testing, few patients were given the drug, because LeukArrest had to be administered within four hours of the injury and informed consent was required; patients were often unconscious, and relatives had to be reached to give consent. In June 1998, Icos and many medical centers asked the FDA to waive consent requirements in situations where the patient was at high risk of dying and relatives could not be reached. While some medical ethicists opposed waiving consent, the FDA approved the proposal in August 1998 for five medical centers. Development of LeukArrest was halted in April 2000 when interim data from phase III clinical trials did not meet Icos's goals of significantly reducing the chance of multiple organ failure and reducing the death rate from shock at 28 days. LeukArrest was also tested unsuccessfully for treatment of heart attack, multiple sclerosis, and stroke.
Pafase.
Pafase, also known as rPAF-AH, was developed to treat severe sepsis. Pafase is the recombinant form of "platelet-activating factor acetylhydrolase" (PAF-AH, also known as lipoprotein-associated phospholipase A2), an enzyme made naturally by macrophages and found in human blood. PAF-AH inactivates platelet-activating factor, a phospholipid that plays a role in the inflammation seen in sepsis. The enzyme was discovered in the mid-1980s by graduate student Diana Stafforini and researchers Steve Prescott, Guy Zimmerman, and Tom McIntyre at the University of Utah. The gene that codes for Pafase was discovered by Icos. Early trials for sepsis showed that the drug reduced the death rate after 28 days and patients were less likely to develop severe respiratory problems. Icos also tested Pafase for acute respiratory distress syndrome (ARDS). In phase II trials for ARDS, Pafase reduced the death rate after 28 days and reduced the chance that the lungs of the patient would fail. However, Icos halted development in December 2002 when interim data from phase III trials for sepsis showed that the drug did not help patients survive. Scientists at Northwestern University later studied Pafase for necrotizing enterocolitis, and there is ongoing research on the enzyme for atherosclerosis at the University of Utah.
Sitaxentan sodium and TBC3711.
In June 2000, Icos and Texas Biotechnology formed a 50/50 partnership to research endothelin receptor antagonists for use in the areas of pulmonary hypertension and chronic heart failure. Two drugs, sitaxentan sodium (also spelled "sitaxsentan") and TBC3711, were tested in clinical trials under the partnership. Sitaxentan was designed to treat pulmonary arterial hypertension, and TBC3711 was designed to treat cardiovascular diseases such as hypertension. In April 2003, Icos sold its part of the 50/50 partnership, including any share of sitaxentan and TBC3711, to Texas Biotechnology for $4 million at closing and another $6 million within 18 months. Sitaxentan sodium was later approved in Europe, Canada, and Australia, and was marketed under the brand name Thelin. In 2010, Thelin was voluntarily withdrawn from the market worldwide due to concerns about irreversible liver damage.
Other drugs tested in clinical trials.
Icos tested many other drugs that were not approved. They are:
Manufacturing.
Icos manufactured many antibodies for various companies. In August 2001, the company partnered with Seattle Genetics to manufacture a component of their top experimental antibody drug SGN-15. In November 2001, Icos signed a production agreement with GPC Biotech to manufacture a class of GPC's antibodies that targeted B-cell lymphomas. In January 2002, Icos signed an agreement with Eos Biotechnology, under which Icos would produce Eos's most promising monoclonal antibody candidate, and Eos would have non-exclusive rights to Icos's CHEF1 enhanced mammalian protein production technology. Eos's antibody inhibited angiogenesis (the formation of new blood vessels) and was being researched as a treatment for solid tumors. In October 2003, Icos partnered with Protein Design Labs to manufacture their M200 antibody.
Acquisition by Eli Lilly.
After Icos's experimental drugs failed in clinical trials, Eli Lilly was in a prime position to purchase the company. In October 2006, Eli Lilly announced that it had reached terms to acquire Icos for $2.1 billion, or $32 a share. After receiving pressure from large institutional shareholders as well as proxy advisory firm Institutional Shareholder Services (ISS) suggesting the deal should be rejected, Lilly increased its offer to $2.3 billion or $34 per share, a 6% increase. Again, resistance was voiced by some large shareholders, and ISS advised shareholders against accepting the new offer, which it still deemed insufficient. On January 25, 2007, at a special meeting, 77% of the shareholders voted in support of the acquisition. Eli Lilly closed the transaction to acquire Icos for $2.3 billion on January 29, 2007.
As a result of the acquisition, Eli Lilly gained complete ownership of Cialis, and promptly shut down Icos operations and laid off Icos personnel, except for 127 employees working at the biologics facility. Icos was the largest biotechnology company in the state of Washington at the time of the acquisition, and employed around 700 people. In December 2007, CMC Biopharmaceuticals A/S, a Copenhagen-based provider of contract biomanufacturing services, bought the Bothell biologics facility and retained the existing 127 employees.
Controversy.
In addition to the layoff of Icos employees, other aspects of the acquisition were equally controversial, such as assertions that Icos was being sold too cheaply and that conflicts of interest existed. The latter related to Icos senior executives, who – despite poor stock performance, in part from failed clinical development programs and an inability to successfully license drugs over the preceding years – were to be massively compensated upon a successful acquisition.
Senior executives at Icos received cash payments worth a combined $67.8 million for selling the company to Eli Lilly. Icos chairman, chief executive, and president Paul Clark received "a 'golden parachute' worth $23.2 million in severance pay, cashed-out stock options, restricted stock awards and other bonuses for retention and closing the deal." Nine senior Icos executives received similar packages, each worth more than $1 million.


Pregnanediol glucuronide

Pregnanediol glucuronide, or 5β-pregnane-3α,20α-diol 3α-glucuronide, is the major metabolite of progesterone and the C3α glucuronide conjugate of pregnanediol (5β-pregnane-3α,20α-diol). Approximately 15 to 30% of a parenteral dose of progesterone is metabolized into pregnanediol glucuronide. While this specific isomer is referred to as "pregnanediol glucuronide" and is the most major form, there are actually many possible isomers of the metabolite.


Lonza Group

Lonza Group is a Swiss multinational chemicals and biotechnology company, headquartered in Basel, with major facilities in Europe, North America and South Asia. Lonza was established under that name in the late 19th-century in Switzerland. The company provides product development services to the pharmaceutical and biologic industries, including organic, fine and performance chemicals, custom manufacturing of biopharmaceuticals, chemical synthesis capabilities, detection systems and services for the bioscience sector.
History.
Lonza was founded in 1897 in the small Swiss town of Gampel, situated in the canton of Valais, taking its name from the nearby river. In the course of the 20th century Lonza evolved from hydroelectricity and C2 chemistry, through nitrogen chemistry to petrochemistry before moving into fine chemistry and biochemistry.
Initially the company produced electricity. The following year, calcium carbide manufacture began using the electricity to heat a furnace to the 2000 °C required for quicklime to react with coal. Lonza moved to neighbouring Visp (where it retains a production site today) in 1909. Competition from electric lighting reduced demand for calcium carbide and in 1915 Lonza started industrial production of calcium cyanamide which became popular as a cheap nitrogenous fertilisers, only to be supplanted by urea. Several new processes were launched in the 1920s. In addition to an electrolysis plant capable of making 5800 m3 hydrogen per hour, came metaldehyde, ammonia and ketene. In 1923, Lonza started to convert calcium carbide to metaldehyde (sold as a solid fuel under the brand 'Meta' and then slug repellant) via acetylene and acetaldehyde. In 1925, ammonia production was started, with a process licensed from Casale. Ammonia was also converted to nitric acid. With calcium cyanamide and calcium nitrate (made from nitric acid), Lonza commercialised formulated fertilisers starting in the 1930s. At the start of WWII, Lonza was contracted by the Swiss government to produce synthetic fuel, which it did by converting acetaldehyde to paraldehyde, used as an additive in transport fuel. In 1928, ketene production started using acetic acid made by oxidation of acetaldehyde. Ketene was initially used to make cellulose acetate for rayon manufacture, but after 1947 Lonza converted most of its ketene to diketene, which was then converted to a range of chemical precursors. Diketene capacity rose to 18'000t per year in 1993. In 1956, the production of niacin (an important vitamin) via 5-ethyl-2-methyl-pyridine. In the 1940s, Lonza used its C2 basis to enter the vinyl chloride business. The production was relocated to Sins and finally discontinued.
At the 1959 AGM, it was decided to back integrate into petrochemistry, and Montecatini was contracted to build the world's smallest cracker in Visp. The initial failure of this plant to meet its specifications triggered a CHF12mio penalty which Lonza used to build a research center.
the new headquarters building in Basel.
Lonza is involved in the manufacturing of biologics with several pharmaceutical companies. Lonza entered into a partnership with Teva in 2009 to develop and manufacture biosimilars. In 2010, they made a deal with GlaxoSmithKline to manufacture therapeutic monoclonal antibodies. In 2014, Lonza entered into an agreement with Bristol-Myers Squibb to manufacture two biologic drugs. Lonza also manufactures Imbruvica for Pharmacyclics and Mydicar for Celladon. In 2015, Lonza contracted with Alexion to construct a new facility dedicated to Alexion manufacturing. In 2017, Lonza and Sanofi partnered to construct a new facility for production of biologics.
In May 2020, Moderna struck a manufacturing deal with Lonza to produce its (mRNA-1273) COVID-19 vaccine active ingredient while expanding sterile production and fill-finish activities to 500 million doses starting in 2021. Production of the mRNA bulk vaccine started in January 2021, soon after an official visit by Swiss Federal Councilor Alain Berset. Lonza Group is responsible for the global production of two-thirds of the raw materials for the Moderna vaccine at combined U.S. and Swiss facilities. The company also started to collaborate with Altimmune Inc. for the development of a nasal COVID-19 vaccine.
Corporate structure.
As of 2020, Lonza's business areas include:
In November 2019, Chairman Albert Baehny became both chairman and interim CEO of Lonza after chief executive officer (CEO) Marc Funk left after 9 months.
Business operations.
, Lonza had twenty-four self-described major sites located in the United States, India, the Czech Republic, Belgium, Spain, China, France, South Africa, Switzerland, Singapore, Mexico, Japan, Brazil and the United Kingdom. This site accounting shows significant changes from a 2016 self-description, which listed sixteen major sites. The presence in India was established in Genome Valley, where ground was broken in 2011.
In 2018, Lonza opened a facility in the United States in Pearland, Texas (a suburb of Houston). This is one of four sites which are focused on cell and gene therapy, the other three being in Portsmouth, New Hampshire; Geleen, Netherlands; and Singapore.
, the company has employed over 15,000 people, across about 35 countries. employment was noted as 14,500 across 120 sites.


Molecular genetics

Molecular genetics is a sub-field of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens.  The field of study is based on the merging of several sub-fields in biology: classical Mendelian inheritance, cellular biology, molecular biology, biochemistry, and biotechnology. Researchers search for mutations in a gene or induce mutations in a gene to link a gene sequence to a specific phenotype. Molecular genetics is a powerful methodology for linking mutations to genetic conditions that may aid the search for treatments/cures for various genetics diseases.
History.
For molecular genetics to develop as a discipline, several scientific discoveries were necessary.  The discovery of DNA as a means to transfer the genetic code of life from one cell to another and between generations was essential for identifying the molecule responsible for heredity.  (in conjunction with Franklin and Wilkins) figured out the structure of DNA, a cornerstone for molecular genetics.  The isolation of a restriction endonuclease in "E. coli" by Arber and Linn in 1969 opened the field of genetic engineering.  Restriction enzymes were used to linearize DNA for separation by electrophoresis and Southern blotting allowed for the identification of specific DNA segments via hybridization probes. In 1971, Berg utilized restriction enzymes to create the first recombinant DNA molecule and first recombinant DNA plasmid.  In 1972, Cohen and Boyer created the first recombinant DNA organism by inserting recombinant DNA plasmids into "E. coli", now known as bacterial transformation, and paved the way for molecular cloning.  The development of DNA sequencing techniques in the late 1970s, first by Maxam and Gilbert, and then by Frederick Sanger, was pivotal to molecular genetic research and enabled scientists to begin conducting genetic screens to relate genotypic sequences to phenotypes. Polymerase chain reaction (PCR) using Taq polymerase, invented by Mullis in 1985, enabled scientists to create millions of copies of a specific DNA sequence that could be used for transformation or manipulated using agarose gel separation. A decade later, the first whole genome was sequenced ("Haemophilus influenzae"), followed by the eventual sequencing of the human genome via the Human Genome Project in 2001. The culmination of all of those discoveries was a new field called genomics that links the molecular structure of a gene to the protein or RNA encoded by that segment of DNA and the functional expression of that protein within an organism. Today, through the application of molecular genetic techniques, genomics is being studied in many model organisms and data is being collected in computer databases like NCBI and Ensembl. The computer analysis and comparison of genes within and between different species is called bioinformatics, and links genetic mutations on an evolutionary scale.
The Central Dogma.
This image shows an example of the central dogma using a DNA strand being transcribed then translated and showing important enzymes used in the processes
The Central Dogma is the basis of all genetics and plays a key role in the study of molecular genetics. The Central Dogma states that DNA replicates itself, DNA is transcribed into RNA, and RNA is translated into proteins. Along with the Central Dogma, the genetic code is used in understanding how RNA is translated into proteins. Replication of DNA and transcription from DNA to mRNA occurs in the mitochondria while translation from RNA to proteins occurs in the ribosome. The genetic code is made of four base pairs: adenine, cytosine, uracil, and guanine and is redundant meaning multiple combinations of these base pairs (which are read in triplicate) produce the same amino acid. Proteomics and genomics are fields in biology that come out of the study of molecular genetics and the Central Dogma.
Techniques.
Forward genetics.
Forward genetics is a molecular genetics technique used to identify genes or genetic mutations that produce a certain phenotype. In a genetic screen, random mutations are generated with mutagens (chemicals or radiation) or transposons and individuals are screened for the specific phenotype. Often, a secondary assay in the form of a selection may follow mutagenesis where the desired phenotype is difficult to observe, for example in bacteria or cell cultures. The cells may be transformed using a gene for antibiotic resistance or a fluorescent reporter so that the mutants with the desired phenotype are selected from the non-mutants.
Mutants exhibiting the phenotype of interest are isolated and a complementation test may be performed to determine if the phenotype results from more than one gene. The mutant genes are then characterized as dominant (resulting in a gain of function), recessive (showing a loss of function), or epistatic (the mutant gene masks the phenotype of another gene). Finally, the location and specific nature of the mutation is mapped via sequencing. Forward genetics is an unbiased approach and often leads to many unanticipated discoveries, but may be costly and time consuming. Model organisms like the nematode worm "Caenorhabditis elegans", the fruit fly "Drosophila melanogaster", and the zebrafish "Danio rerio" have been used successfully to study phenotypes resulting from gene mutations.
Reverse genetics.
Reverse genetics is the term for molecular genetics techniques used to determine the phenotype resulting from an intentional mutation in a gene of interest. The phenotype is used to deduce the function of the un-mutated version of the gene. Mutations may be random or intentional changes to the gene of interest. Mutations may be a mis-sense mutation caused by nucleotide substitution, a nucleotide addition or deletion to induce a frameshift mutation, or a complete addition/deletion of a gene or gene segment. The deletion of a particular gene creates a gene knockout where the gene is not expressed and a loss of function results (e.g. knockout mice). Mis-sense mutations may cause total loss of function or result in partial loss of function, known as a knockdown. Knockdown may also be achieved by RNA interference (RNAi). Alternatively, genes may be substituted into an organism's genome (also known as a transgene) to create a gene knock-in and result in a gain of function by the host. Although these techniques have some inherent bias regarding the decision to link a phenotype to a particular function, it is much faster in terms of production than forward genetics because the gene of interest is already known.


Lipid bilayer mechanics

Lipid bilayer mechanics is the study of the physical material properties of lipid bilayers, classifying bilayer behavior with stress and strain rather than biochemical interactions. Local point deformations such as membrane protein interactions are typically modelled with the complex theory of biological liquid crystals but the mechanical properties of a homogeneous bilayer are often characterized in terms of only three mechanical elastic moduli: the area expansion modulus Ka, a bending modulus Kb and an edge energy formula_1. For fluid bilayers the shear modulus is by definition zero, as the free rearrangement of molecules within plane means that the structure will not support shear stresses. These mechanical properties affect several membrane-mediated biological processes. In particular, the values of Ka and Kb affect the ability of proteins and small molecules to insert into the bilayer. Bilayer mechanical properties have also been shown to alter the function of mechanically activated ion channels.
Area expansion modulus.
Since lipid bilayers are essentially a two dimensional structure, Ka is typically defined only within the plane. Intuitively, one might expect that this modulus would vary linearly with bilayer thickness as it would for a thin plate of isotropic material. In fact this is not the case and Ka is only weakly dependent on bilayer thickness. The reason for this is that the lipids in a fluid bilayer rearrange easily so, unlike a bulk material where the resistance to expansion comes from intermolecular bonds, the resistance to expansion in a bilayer is a result of the extra hydrophobic area exposed to water upon pulling the lipids apart.
Based on this understanding, a good first approximation of Ka for a monolayer is 2γ, where gamma is the surface tension of the water-lipid interface. Typically gamma is in the range of 20-50mJ/m2. To calculate Ka for a bilayer it is necessary to multiply the monolayer value by two, since a bilayer is composed of two monolayer leaflets. Based on this calculation, the estimate of Ka for a lipid bilayer should be 80-200 mN/m (note: N/m is equivalent to J/m2). It is not surprising given this understanding of the forces involved that studies have shown that Ka varies strongly with solution conditions but only weakly with tail length and unsaturation.
The compression modulus is difficult to measure experimentally because of the thin, fragile nature of bilayers and the consequently low forces involved. One method utilized has been to study how vesicles swell in response to osmotic stress. This method is, however, indirect and measurements can be perturbed by polydispersity in vesicle size. A more direct method of measuring Ka is the pipette aspiration method, in which a single giant unilamellar vesicle (GUV) is held and stretched with a micropipette. More recently, atomic force microscopy (AFM) has been used to probe the mechanical properties of suspended bilayer membranes, but this method is still under development.
One concern with all of these methods is that, since the bilayer is such a flexible structure, there exist considerable thermal fluctuations in the membrane at many length scales down to sub-microscopic. Thus, forces initially applied to an unstressed membrane are not actually changing the lipid packing but are rather “smoothing out” these undulations, resulting in erroneous values for mechanical properties. This can be a significant source of error. Without the thermal correction typical values for Ka are 100-150 mN/m and with the thermal correction this would change to 220-270 mN/m.
Bending modulus.
Bending modulus is defined as the energy required to deform a membrane from its natural curvature to some other curvature. For an ideal bilayer the intrinsic curvature is zero, so this expression is somewhat simplified. The bending modulus, compression modulus and bilayer thickness are related by formula_2 such that if two of these parameters are known the other can be calculated. This relationship derives from the fact that to bend the inner face must be compressed and the outer face must be stretched. The thicker the membrane, the more each face must deform to accommodate a given curvature (see bending moment). Many of the values for Ka in literature have actually been calculated from experimentally measured values of Kb and t. This relation holds only for small deformations, but this is generally a good approximation as most lipid bilayers can support only a few percent strain before rupturing.
Curvature.
Only certain classes of lipids can form bilayers. Two factors primarily govern whether a lipid will form a bilayer or not: solubility and shape. For a self assembled structure such as a bilayer to form, the lipid should have a low solubility in water, which can also be described as a low critical micelle concentration (CMC). Above the CMC, molecules will aggregate and form larger structures such as bilayers, micelles or inverted micelles.
The primary factor governing which structure a given lipid forms is its shape (i.e.- its intrinsic curvature). Intrinsic curvature is defined by the ratio of the diameter of the head group to that of the tail group. For two-tailed PC lipids, this ratio is nearly one so the intrinsic curvature is nearly zero. Other headgroups such as PS and PE are smaller and the resulting diacyl (two-tailed) lipids thus have a negative intrinsic curvature. Lysolipids tend to have positive spontaneous curvature because they have one rather than two alkyl chains in the tail region. If a particular lipid has too large a deviation from zero intrinsic curvature it will not form a bilayer.
Edge energy.
Edge energy is the energy per unit length of a free edge contacting water. This can be thought of as the work needed to create a hole in the bilayer of unit length L. The origin of this energy is the fact that creating such an interface exposes some of the lipid tails to water, which is unfavorable. formula_1 is also an important parameter in biological phenomena as it regulates the self-healing properties of the bilayer following electroporation or mechanical perforation of the cell membrane. Unfortunately, this property is both difficult to measure experimentally and to calculate. One of the major difficulties in calculation is that the structural properties of this edge are not known. The simplest model would be no change in bilayer orientation, such that the full length of the tail is exposed. This is a high energy conformation and, to stabilize this edge, it is likely that some of the lipids rearrange their head groups to point out in a curved boundary. The extent to which this occurs is currently unknown and there is some evidence that both hydrophobic (tails straight) and hydrophilic (heads curved around) pores can coexist.
Finite element modeling.
FE Modeling is a powerful tool for testing the mechanical deformation and equilibrium configuration of lipid membranes. In this context membranes are treated under the thin-shell theory where the bending behavior of the membrane is described by the Helfrich bending model which considers the bilayer as being a very thin object and interprets it as a two-dimensional surface. This consideration imply that Kirchhoff-Love plate theory can be applied to lipid bilayers to determine their stress-deformation behavior. Furthermore, in the FE approach a bilayer surface is subdivided into discrete elements, each described by the above 2D mechanics.
Under these considerations the weak form virtual work for the entire system is described as the sum of the contribution of all the discrete elements work components.
formula_4
For each discrete element the virtual work is determined by the force vector formula_5and the displacement vector formula_6 each for an applied stress formula_7 and a bending momentum formula_8
formula_9
The FE force vectors due to the applied bilayer stress formula_10 are given as
formula_11
Here formula_12 is the displacement state function at point formula_13, and formula_14 the tangent vector to the bilayer surface at point formula_15
The above individual element vectors for the internal force formula_16 and the internal work formula_17can be expressed in a global assembly to obtain a discretized weak form as follows:
formula_18
In the above equation formula_6 is the deformation in each discrete element while formula_20 is the Lagrange multiplier associated with area-incompressibility. The discretized weak form is satisfied when formula_21 and formula_22. The resulting nonlinear equations are solved using Newton ‘s method. This allows the predictions of equilibrium shapes that lipid membranes adopt under different stimuli.
Most analysis are done for lipid membranes with uniform properties (isotropic), while this is partly true for simple membranes containing a single or few lipid species, this description can only approximate the mechanical response of more complex lipid bilayers which can contain several domains of segregated lipids having distinct material properties or intermembrane proteins as in the case of cellular membranes. Other complex cases requiring surface flow analysis, pH, and temperature dependency would require a more discrete model of a bilayer such as MD simulations. FE methods can predict the equilibrium conformations of a lipid bilayer in response to external forces as shown in the following cases.
Tethering.
In this scenario a point in the bilayer surface is pulled with a force normal to the surface plane, this leads to the elongation of a thin tether of bilayer material. The rest of the bilayer surface is subject to a tension S reflecting the pulling force of the continuous bilayer. In such this case a finer mesh is applied near the pulling force area to have a more accurate prediction of the bilayer deformation. Tethering is an important mechanical event for cellular lipid bilayers, by this action membranes are able to mediate docking into substrates or components of the cytoskeleton.
Budding.
Lipid bilayer budding is a commonplace phenomenon in living cells and relates to the transport of metabolites in the form of vesicles. During this process, a lipid bilayer is subject to internal hydrostatic stresses, in combination with strain restrictions along a bilayer surface, this can lead to elongation of areas of the lipid bilayer by elastic shear or viscous shear. This eventually leads to a deformation of a typical spherical bilayer into different budding shapes. Such shapes are not restricted to be symmetrical along their axes, but they can have different degrees of asymmetry. In FE analysis this results in budding equilibrium shapes like, elongate plates, tubular buds and symmetric budding.


Levobunolol

Levobunolol (trade names AKBeta, Betagan, Vistagan, among others) is a non-selective beta blocker. It is used topically in the form of eye drops to manage ocular hypertension (high pressure in the eye) and open-angle glaucoma.
Contraindications.
Like other beta blockers, levobunolol is contraindicated in patients with airway diseases such as asthma and severe chronic obstructive pulmonary disease (COPD), as well as heart problems such as sinus bradycardia, second- or third-degree atrioventricular block, sick sinus syndrome, and cardiogenic shock. Combination with MAO-A inhibitors is also contraindicated because it could cause a dangerous rise in blood pressure.
Levobunolol is not useful for the treatment of closed-angle glaucoma.
Side effects.
The most common side effect is eye irritation felt as stinging or burning, which occurs in up to a third of patients. Blepharoconjunctivitis occurs in up to 5% of patients. Rarer adverse effects include keratitis, edema and increased lacrimation. Allergies are rare, but seem to be more common than under the related drug timolol.
If the substance reaches the nasal mucosa via the tear duct, it can be absorbed into the bloodstream and cause systemic side effects. These include orthostatic hypotension (low blood pressure) and other effects on the heart and circulatory system, breathing problems in people with asthma, and skin symptoms such as itching and aggravation of psoriasis.
Interactions.
Even in the form of eye drops, levebunolol may cause hypotension when combined with alpha blockers, calcium channel blockers, tricyclic antidepressants, and other drugs that lower blood pressure. It can also cause severe hypertension ("high" blood pressure) when combined with sympathomimetic drugs or MAO-A inhibitors, bradycardia (low heart rate) when combined with antiarrhythmics or mefloquine, and hypoglycemia (low blood sugar) when combined with antidiabetic drugs such as insulin.
Pharmacology.
Mechanism of action.
Levobunolol is a non-cardioselective beta blocker, that is, it blocks beta-1 receptors as well as beta-2 receptors. The latter type dominates in the ciliary body, where it controls aqueous humour production. Blocking this type of receptor reduces aqueous humour production, lowering intraocular pressure. The substance has no relevant membrane stabilizing effect or intrinsic sympathomimetic activity. Like other beta blockers, and unlike the anti-glaucoma medication pilocarpine, levobunolol has no effect on accommodation and pupil size.
Pharmacokinetics.
The substance quickly penetrates the cornea and reaches the aqueous humour. It is reduced to dihydrolevobunolol, which is equally active, in the eye's tissues. The drug starts to lower intraocular pressure within an hour, reaches its maximum effect after two to six hours, and remains effective for up to 16 hours. It has an elimination half-life of six hours and is mainly excreted via the kidney.
Chemistry.
Levobunolol is the pure L-enantiomer of bunolol and has more than 60 times the pharmacological activity of D-bunolol. It is used as the hydrochloride, which melts at and is soluble in water and methanol and slightly soluble in ethanol.


Dendroaspis natriuretic peptide

Dendroaspis natriuretic peptide (DNP) is a 38-residue peptide and a member of natriuretic peptide family. It is structurally similar to the atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) and possesses biologic properties similar to these natriuretic peptides.
DNP was originally isolated from the venom of the green mamba snake ("Dendroaspis angusticeps"), from which its name is derived.


Chloroplast membrane

Chloroplasts contain several important membranes, vital for their function. Like mitochondria, chloroplasts have a double-membrane envelope, called the chloroplast envelope, but unlike mitochondria, chloroplasts also have internal membrane structures called thylakoids. Furthermore, one or two additional membranes may enclose chloroplasts in organisms that underwent secondary endosymbiosis, such as the euglenids and chlorarachniophytes.
The chloroplasts come via endosymbiosis by engulfment of a photosynthetic cyanobacterium by the eukaryotic, already mitochondriate cell. Over millions of years the endosymbiotic cyanobacterium evolved structurally and functionally, retaining its own DNA and the ability to divide by binary fission (not mitotically) but giving up its autonomy by the transfer of some of its genes to the nuclear genome.
Envelope membranes.
Each of the envelope membranes is a lipid bilayer that is between 6 and 8 nm thick. The lipid composition of the outer membrane has been found to be 48% phospholipids, 46% galactolipids and 7% sulfolipids, while the inner membrane has been found to contain 16% phospholipids, 79% galactolipids and 5% sulfolipids in spinach chloroplasts.
The outer membrane is permeable to most ions and metabolites, but the inner membrane of the chloroplast is highly specialised with transport proteins. For example, carbohydrates are transported across the inner envelope membrane by a triose phosphate translocator. The two envelope membranes are separated by a gap of 10–20 nm, called the intermembrane space.
Thylakoid membrane.
Within the envelope membranes, in the region called the stroma, there is a system of interconnecting flattened membrane compartments, called the thylakoids. The thylakoid membrane is quite similar in lipid composition to the inner envelope membrane, containing 78% galactolipids, 15.5% phospholipids and 6.5% sulfolipids in spinach chloroplasts. The thylakoid membrane encloses a single, continuous aqueous compartment called the thylakoid lumen.
These are the sites of light absorption and ATP synthesis, and contain many proteins, including those involved in the electron transport chain. Photosynthetic pigments such as chlorophylls a,b,c and some others, e.g., xanthophylls, carotenoids, phycobilins are also embedded within the granum membrane. With exception of chlorophyll a, all the other associated pigments are "accessory" and transfer energy to the reaction centers, Photosytems I and II.
The membranes of the thylakoid contain photosystems I and II which harvest solar energy to excite electrons which travel down the electron transport chain. This exergonic fall in potential energy along the way is used to draw (not pump!) H+ ions from the lumen of the thylakoid into the cytosol of a cyanobacterium or the stroma of a chloroplast. A steep H+ gradient is formed, which allows chemiosmosis to occur, where the thylakoid, transmenbrane ATP-synthase serves a dual function as a "gate" or channel for H+ ions and a catalytic site for the formation of ATP from ADP + a PO43− ion.
Experiments have shown that the pH within the stroma is about 7.8, while that of the lumen of the thylakoid is 5. This corresponds to a six-hundredfold difference in concentration of H+ ions. The H+ ions pass down through the ATP-synthase catalytic gate. This chemiosmotic phenomenon also occurs in mitochondria.


Alison Todd

Professor Alison Todd is holder of 18 patents (as at July 2019), and a co-founder and chief scientific officer of SpeeDx. The company manufactures and sells tests for detecting infectious pathogens and identifying antibiotic resistance. The biomedical company, co-founded by Todd, develops diagnostic tools. Todd mentors younger scientists and entrepreneurs, as well as advocating for greater gender diversity in leaders in STEM. ‘Nearly 60 per cent of medical science and health graduates are women, but we hold only 20 per cent of senior leadership positions in the field’.
Career.
Todd is the Chief Scientific Officer of SpeeDx, which is a molecular diagnostics company which she and Elisa Mokany started. Todd and Mokany have 18 patent families between them. They have brought 11 medical diagnostic tests for the management of clinical disease.
Todd developed several novel molecular analytical technologies which have been used for basic research, preclinical/clinical drug development and in vitro diagnostics. Her expertise include nucleic acid chemistry, particularly target amplification and catalytic DNA technologies, and the biology of cancer and viral diseases. Prior to founding SpeeDx, Todd was a Senior Research Director at Johnson and Johnson Research Pty Limited, Sydney.
Todd describes her work, "We are proud of what we have created because we believe we are making a positive contribution to the welfare of patients around the world. As the old-timer of the pair, I will take the liberty of having the last word and sum up our relationship as a clear case of ‘two heads are better than one’.
Research.
Todd describes her "Eureka moment", "It all began when Elisa joined my group at Johnson & Johnson Research (JJR), we were already exploring ways to exploit DNAzymes (deoxyribozymes) for diagnostic applications. These fascinating molecules are simple, short, synthetic DNA sequences (oligonucleotides) that can catalyse reactions in a manner analogous to protein enzymes. Although catalytic RNA (ribozymes) had been found in nature, catalytic DNA had not, and it had been assumed DNA would not have similar properties. However, a few years earlier, undeterred by dogma, Jerry Joyce and co-workers at Scripps had conducted ‘evolution in a test tube’".
Select publications.
Todd's publications can be found at (Google Scholar).
Media.
2017 — Todd's work was referenced in "The Australian Financial Review", where she was described as a 'frontline fighter in the war on superbugs'.
2017 — An article from "Australian Biochemist" describes in technical detail, the complexity, yet elegant simplicity that Todd's company uses technology to test infectious diseases.
2017 — "The Sydney Morning Herald" described the formation of Todd's company, as well as their concern that women were under-represented in STEMM, and the hiring and mentoring practices reflected in their company.