The Synthesis of Macrocyclic Polystyrene by Sequential Atom Transfer Radical Reactions

A method for the production of macrocyclic polystyrene via ring closing of a linear !,"-dibrominated polystyrene by an Atom Transfer Radical Coupling (ATRC) reaction is described. The dibrominated polystyrene chain was produced from two simultaneous atom transfer radical polymerizations (ATRPs) originating from a dibrominated benzal bromide initiator. To ensure the retention of the halogen end groups polymerization was allowed to proceed to less than 50% conversion. Using this precursor in an intramolecular ATRC (ring closing) reaction was found to yield in excess of 90% cyclic product based on refractive index-gel permeation chromatography (GPC) analysis. The cyclic architecture of the polymer was verified by GPC, Nuclear Magnetic Resonance (NMR), and mass spectrometry analysis. The utility of this method has been expanded by the addition of 2-methyl-2-nitrosopropane to the coupling reaction, which allows for the coupling to proceed at a faster rate and to yield macrocycles with incorporated alkoxyamine functionality. The alkoxyamine functionality allows for degradation of the cycles at high temperatures (>125° C) and we hypothesize that it may allow the macrocycles to act as a macroinitiator for a ring expansion polymerization in future studies.

Evaluation of colonoscopic allergen provocation as a diagnostic tool in dogs with proven food hypersensitivity reactions

OBJECTIVE: To evaluate the colonoscopic allergen provocation (COLAP) test as a new tool for the diagnosis of IgE-mediated food allergy. METHODS: Oral food challenges as well as COLAP testing were performed in a colony of nine research dogs with proven immediate-type food allergic reactions. In addition, COLAP was performed in five healthy dogs. RESULTS: When compared with the oral challenge test, COLAP accurately determined 18 of 23 (73 per cent) positive oral challenge reactions (73 per cent) in dogs with food allergies and was negative in the healthy dogs. CLINICAL SIGNIFICANCE: The accuracy of this new test may be higher than that for gastric sensitivity testing. Therefore, COLAP holds promise as a new test to confirm the diagnosis of suspect IgE-mediated food allergy in dogs.

Numerical investigation of effects of addition of ethanol to gasoline on Laminar Flame Speed (LFS), autoignition, and wall quenching

Since the advent of automobiles, alcohol has been considered a possible engine fuel1,2. With the recent increased concern about the high price of crude oil due to fluctuating supply and demand and environmental issues, interest in alcohol based fuels has increased2,3. However, using pure alcohols or blends with conventional fuels in high percentages requires changes to the engine and fuel system design2. This leads to the need for a simple and accurate conventional fuels-alcohol blends combustion models that can be used in developing parametric burn rate and knock combustion models for designing more efficient Spark Ignited (SI) engines. To contribute to this understanding, numerical simulations were performed to obtain detailed characteristics of Gasoline-Ethanol blends with respect to Laminar Flame Speed (LFS), autoignition and Flame-Wall interactions. The one-dimensional premixed flame code CHEMKIN® was applied to simulate the burning velocity and autoignition characteristics using the freely propagating model and closed homogeneous reactor model respectively. Computational Fluid Dynamics (CFD) was used to obtain detailed flow, temperature, and species fields for Flame-wall interactions. A semi-detailed validated chemical kinetic model for a gasoline surrogate fuel developed by Andrae and Head4 was used for the study of LFS and Autoignition. For the quenching study, a skeletal chemical kinetic mechanism of gasoline surrogate, having 50 species and 174 reactions was used. The surrogate fuel was defined as a mixture of pure n-heptane, isooctane, and toluene. For LFS study, the ethanol volume fraction was varied from 0 to 85%, initial pressure from 4 to 8 bar, initial temperature from 300 to 900K, and dilution from 0 to 32%. Whereas for Autoignition study, the ethanol volume fraction was varied between 0 to 85%, initial pressure was varied between 20 to 60 bar, initial temperature was varied between 800 to 1200K, and the dilution was varied between 0 to 32% at equivalence ratios of 0.5, 1.0 and 1.5 to represent the in-cylinder conditions of a SI engine. For quenching study three Ethanol blends, namely E0, E25 and E85 are described in detail at an initial pressure of 8 atm and 17 atm. Initial wall temperature was taken to be 400 K. Quenching thicknesses and heat fluxes to the wall were computed. The laminar flame speed was found to increase with ethanol concentration and temperature but decrease with pressure and dilution. The autoignition time was found to increase with ethanol concentration at lower temperatures but was found to decrease marginally at higher temperatures. The autoignition time was also found to decrease with pressure and equivalence ratio but increase with dilution. The average quenching thickness was found to decrease with an increase in Ethanol concentration in the blend. Heat flux to the wall increased with increase in ethanol percentage in the blend and at higher initial pressures. Whereas the wall heat flux decreased with an increase in dilution. Unburned Hydrocarbon (UHC) and CO % was also found to decrease with ethanol concentration in the blend.

Recommendations for HLA-B*15:02 and HLA-A*31:01 genetic testing to reduce the risk of carbamazepine-induced hypersensitivity reactions.

OBJECTIVE To systematically review evidence on genetic risk factors for carbamazepine (CBZ)-induced hypersensitivity reactions (HSRs) and provide practice recommendations addressing the key questions: (1) Should genetic testing for HLA-B*15:02 and HLA-A*31:01 be performed in patients with an indication for CBZ therapy to reduce the occurrence of CBZ-induced HSRs? (2) Are there subgroups of patients who may benefit more from genetic testing for HLA-B*15:02 or HLA-A*31:01 compared to others? (3) How should patients with an indication for CBZ therapy be managed based on their genetic test results? METHODS A systematic literature search was performed for HLA-B*15:02 and HLA-A*31:01 and their association with CBZ-induced HSRs. Evidence was critically appraised and clinical practice recommendations were developed based on expert group consensus. RESULTS Patients carrying HLA-B*15:02 are at strongly increased risk for CBZ-induced Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) in populations where HLA-B*15:02 is common, but not CBZ-induced hypersensitivity syndrome (HSS) or maculopapular exanthema (MPE). HLA-B*15:02-positive patients with CBZ-SJS/TEN have been reported from Asian countries only, including China, Thailand, Malaysia, and India. HLA-B*15:02 is rare among Caucasians or Japanese; no HLA-B*15:02-positive patients with CBZ-SJS/TEN have been reported so far in these groups. HLA-A*31:01-positive patients are at increased risk for CBZ-induced HSS and MPE, and possibly SJS/TEN and acute generalized exanthematous pustulosis (AGEP). This association has been shown in Caucasian, Japanese, Korean, Chinese, and patients of mixed origin; however, HLA-A*31:01 is common in most ethnic groups. Not all patients carrying either risk variant develop an HSR, resulting in a relatively low positive predictive value of the genetic tests. SIGNIFICANCE This review provides the latest update on genetic markers for CBZ HSRs, clinical practice recommendations as a basis for informed decision making regarding the use of HLA-B*15:02 and HLA-A*31:01 genetic testing in patients with an indication for CBZ therapy, and identifies knowledge gaps to guide future research. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.

TrypanoCyc: a community-led biochemical pathways database for Trypanosoma brucei.

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.

MONITORING ENZYME REACTIONS BY FAST ATOM BOMBARDMENT MASS SPECTROMETRY

The potential for the direct analysis of enzyme reactions by fast atom bombardment (FAB) mass spectrometry has been investigated. Conditions are presented for the maintenance of enzymatic activity under FAB conditions along with FAB mass spectrometric data showing that these conditions can be applied to solutions of enzyme and substrate to follow enzymatic reactions inside the mass spectrometer in real-time. In addition, enzyme kinetic behavior under FAB mass spectrometric conditions is characterized using trypsin and its assay substrate, TAME, as an enzyme-substrate reaction model. These results show that two monitoring methods can be utilized to follow reactions by FAB mass spectrometry. The advantages of each method are discussed and illustrated by obtaining kinetic parameters from the direct analysis of enzyme reactions with assay or peptide substrates. ^

Effect of increased pCO2 on bacterial assemblage shifts in response to glucose addition in Fram Strait seawater mesocosms

Ocean acidification may stimulate primary production through increased availability of inorganic carbon in the photic zone, which may in turn change the biogenic flux of dissolved organic carbon (DOC) and the growth potential of heterotrophic bacteria. To investigate the effects of ocean acidification on marine bacterial assemblages, a two-by-three factorial mescosom experiment was conducted using surface sea water from the East Greenland Current in Fram Strait. Pyrosequencing of the V1-V2 region of bacterial 16S ribosomal RNA genes was used to investigate differences in the endpoint (Day 9) composition of bacterial assemblages in mineral nutrient-replete mesocosms amended with glucose (0 µm, 5.3 µm and 15.9 µm) under ambient (250 µatm) or acidified (400 µatm) partial pressures of CO2 (pCO2). All mesocosms showed low richness and diversity by Chao1 estimator and Shannon index, respectively, with general dominance by Gammaproteobacteria and Flavobacteria. Nonmetric multidimensional scaling analysis and two-way analysis of variance of the Jaccard dissimilarity matrix (97% similarity cut-off) demonstrated that the significant community shift between 0 µm and 15.9 µm glucose addition at 250 µatm pCO2 was eliminated at 400 µatm pCO2. These results suggest that the response potential of marine bacteria to DOC input may be altered under acidified conditions.

Thermal behaviour of bitumen modified by sulphur addition

The most frequent use of bitumen is as binder for pavement applications. The effect of sulphur addition on the properties of the bitumen has been extensively studied several decades ago. Recently, there is a renewed interest in researching the behaviour of sulphur-bitumen combination, because off 1.The future availability of bitumen may be limited and 2. The beneficial consumption of great amounts of sulphur compounds from petroleum refining is advisable. The addition of sulphur to bitumen provokes the beginning of chemical reactions depending on the sulphur content and heating temperature. At heating temperatures T< 140 ºC liquid sulphur reacts with naphthenic-aromatic fraction forming polysulphides. At temperatures above 150 ºC dehydrogenization reactions with emission of hydrogen sulfide take place and naphthenic-aromatic molecules are transformed into asphaltenes. Therefore, the addition of sulphur to bitumen provokes changes in the chemical structure of the bitumen. The objective of this work is to analyze, the thermal behaviour of sulphur-bitumen mixtures of different composition (0-35 %wt sulphur content) prepared at 130 and 140 ºC, by means of differential scanning calorimetry (DSC). Besides, the volatile emissions of the mixtures at high temperature have been estimated from loss weight measurements as a function of stored time

Versatile 5′ phosphoryl coupling of small and large molecules to an RNA

A Ca2+-requiring catalytic RNA is shown to create 5′ phosphate–phosphate linkages with all nucleotides and coenzymes including CoA, nicotinamide adenine dinucleotide phosphate, thiamine phosphate, thiamine pyrophosphate, and flavin mononucleotide. In addition to these small molecules, macromolecules such as RNAs with 5′-diphosphates, and nonnucleotide molecules like Nɛ-phosphate arginine and 6-phosphate gluconic acid also react. That is, the self-capping RNA isolate 6 is an apparently universal 5′ phosphate-linker, reacting with any nucleophile containing an unblocked phosphate. These RNA reactions demonstrate a unique RNA catalytic capability and imply versatile and specific posttranscriptional RNA modification by RNA catalysis.

Pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon, Pyrococcus furiosus, functions as a CoA-dependent pyruvate decarboxylase

Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from the hyperthermophilic archaeon, Pyrococcus furiosus, an organism that grows optimally at 100°C by fermenting carbohydrates and peptides. The enzyme contains thiamine pyrophosphate and catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 and reduces P. furiosus ferredoxin. Here we show that this enzyme also catalyzes the formation of acetaldehyde from pyruvate in a CoA-dependent reaction. Desulfocoenzyme A substituted for CoA showing that the cofactor plays a structural rather than a catalytic role. Ferredoxin was not necessary for the pyruvate decarboxylase activity of POR, nor did it inhibit acetaldehyde production. The apparent Km values for CoA and pyruvate were 0.11 mM and 1.1 mM, respectively, and the optimal temperature for acetaldehyde formation was above 90°C. These data are comparable to those previously determined for the pyruvate oxidation reaction of POR. At 80°C (pH 8.0), the apparent Vm value for pyruvate decarboxylation was about 40% of the apparent Vm value for pyruvate oxidation rate (using P. furiosus ferredoxin as the electron acceptor). Tentative catalytic mechanisms for these two reactions are presented. In addition to POR, three other 2-keto acid ferredoxin oxidoreductases are involved in peptide fermentation by hyperthermophilic archaea. It is proposed that the various aldehydes produced by these oxidoreductases in vivo are used by two aldehyde-utilizing enzymes, alcohol dehydrogenase and aldehyde ferredoxin oxidoreductase, the physiological roles of which were previously unknown.

A yeast sterol auxotroph (erg25) is rescued by addition of azole antifungals and reduced levels of heme

Genetic disruption of the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene leads to sterol auxotrophy. We have characterized a suppression system that requires two mutations to restore viability to this disrupted strain. One suppressor mutation is erg11, which is blocked in 14α-demethylation of lanosterol and is itself an auxotroph. The second suppressor mutation required is either slu1 or slu2 (suppressor of lanosterol utilization). These mutations are leaky versions of HEM2 and HEM4, respectively; addition of exogenous hemin reverses the suppressing effects of slu1 and slu2. Suppression of erg25 by erg11 slu1 (or erg11 slu2) results in a slow-growing strain in which lanosterol, the first sterol in the pathway, accumulates. This result indicates that endogenously synthesized lanosterol can substitute for ergosterol and support growth. In the triple mutants, all but 1 (ERG6) of the 13 subsequent reactions of the ergosterol pathway are inactive. Azole antibiotics (clotrimazole, ketoconazole, and itraconazole) widely used to combat fungal infections are known to do so by inhibiting the ERG11 gene product, the 14α-demethylase. In this investigation, we demonstrate that treatment of the sterol auxotrophs erg25 slu1 or erg25 slu2 with azole antibiotics paradoxically restores viability to these strains in the absence of sterol supplementation via the suppression system we have described.

Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups

Induction of phase 2 enzymes and elevations of glutathione are major and sufficient strategies for protecting mammals and their cells against the toxic and carcinogenic effects of electrophiles and reactive forms of oxygen. Inducers belong to nine chemical classes and have few common properties except for their ability to modify sulfhydryl groups by oxidation, reduction, or alkylation. Much evidence suggests that the cellular “sensor” molecule that recognizes the inducers and signals the enhanced transcription of phase 2 genes does so by virtue of unique and highly reactive sulfhydryl functions that recognize and covalently react with the inducers. Benzylidene-alkanones and -cycloalkanones are Michael reaction acceptors whose inducer potency is profoundly increased by the presence of ortho- (but not other) hydroxyl substituent(s) on the aromatic ring(s). This enhancement correlates with more rapid reactivity of the ortho-hydroxylated derivatives with model sulfhydryl compounds. Proton NMR spectroscopy provides no evidence for increased electrophilicity of the β-vinyl carbons (the presumed site of nucleophilic attack) on the hydroxylated inducers. Surprisingly, these ortho-hydroxyl groups display a propensity for extensive intermolecular hydrogen bond formation, which may raise the reactivity and facilitate addition of mercaptans, thereby raising inducer potencies.

Requirement of the Lec35 Gene for All Known Classes of Monosaccharide-P-Dolichol-dependent Glycosyltransferase Reactions in Mammals

The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-β-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.

Solvent-Free Enantioselective Organocatalyzed Aldol Reactions

The use of proline as catalyst for the aldol process has given a boost to the development of organocatalysis as a research area. Since then, a plethora of organocatalysts of diverse structures have been developed for this and other organic transformations under different reaction conditions. The use of an organic molecule as catalyst to promote a reaction meets several principles of Green Chemistry. The implementation of solvent-free methodologies to carry out the aldol reaction was soon envisaged. These solvent-free processes can be performed using conventional magnetic stirring or applying ball milling techniques and are even compatible with the use of supported organocatalysts as promoters, which allows the recovery and reuse of the organocatalysts. In addition, other advantages such as the reduction of the required amount of nucleophile and the acceleration of the reaction are accomplished by using solvent-free conditions leading to a “greener” and more sustainable process.