Mechanistic Analysis of an Engineered Enzyme that Catalyzes the Formose Reaction

An enzyme that catalyzes the formose reaction, termed “formolase”, was recently engineered through a combination of computational protein design and directed evolution. We have investigated the kinetic role of the computationally designed residues and further characterized the enzyme's product profile. Kinetic studies illustrated that the computationally designed mutations were synergistic in their contributions towards enhancing activity. Mass spectrometry revealed that the engineered enzyme produces two products of the formose reaction—dihydroxyacetone and glycolaldehyde—with the product profile dependent on the formaldehyde concentration. We further explored the effects of this product profile on the thermodynamics and yield of the overall carbon assimilation from the formolase pathway to help guide future efforts to engineer this pathway.     

Exploratory analysis of excitation-emission matrix fluorescence spectra with self-organizing maps—A tutorial

Large datasets are common in chemical and environmental engineering applications and tools for their analysis are in great demand. Here, the outputs of a series of fluorescence spectroscopy analyses are utilised to demonstrate the application of the self-organising map (SOM) technique for data analysis. Fluorescence spectroscopy is a well-established technique of organic matter fingerprinting in water. The technique can provide detailed information on the physico-chemical properties of water. However, analysis of fluorescence spectra requires the application of robust statistical and computational data pre-processing and analysis tools.This paper presents a tutorial for training engineering postgraduate researchers in the use of SOM techniques using MATLAB^®. Via a tutorial, the application of SOM to fluorescence spectra and, in particular, the characterisation of organic matter removal in water treatment, is presented. The tutorial presents a step-by-step example of the application of SOM to fluorescence data analysis and includes the source code for MATLAB^®, together with presentation and discussion of the results. With this tutorial we hope to popularise this robust pattern recognition technique for fluorescence data analysis and large data sets in general, and also to provide educational practitioners with a novel tool with which to train engineering students in SOM.     

Mechanical and thermal properties of poly(ethylene terephthalate) fibers crosslinked with disulfonyl azides

A novel method for the crosslinking of poly(ethylene terephthalate) fibers is described using 1,6‐hexanedisulfonyl azide, 1,3‐benzenedisulfonyl azide, and 2,6‐naphthalenedisulfonyl azide. The azides are diffused into poly(ethylene terephthalate) fibers (Dacron) from perchloroethylene solution, and the fibers are heat treated to bring about decomposition of the sulfonyl azide and give rise to crosslinking. A study is made of the mechanical and thermal properties of the resultant fibers, which are changed considerably in comparison to the untreated fiber. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 1517–1527, 2002     

Behavioral and electrophysiological activity of (Z,E)-7,9,11-dodecatrienyl formate,a mimic of the major sex pheromone component of carob moth,Ectomyelois ceratoniae

The behavioral and electrophysiological activity of a mimic [(Z,E)7,9,11-dodecatrienyl formate] of the major sex pheromone component [(Z,E) 9,11,13-tetradecatrienal] of carob moth was assessed. Wind-tunnel bioassays demonstrated that the formate was as effective as natural gland extracts, and significantly more effective than the trienal alone or than the trienal blended with two minor pheromone components, in evoking source contact. Dispensers containing the formate were as effective as trienal-containing blend lures in attracting males when placed at the same dosage in traps in date gardens. Single-cell recordings showed that at least two olfactory neurons, differentiated by spike amplitude, are located in the long trichoid hairs on male carob moth antennae. Dose-response relationships indicated that puffs from cartridges loaded with at least 0.1 g of the formate or of the trienal were necessary to elicit spiking by either the small or the large-spiking cell within a sensillum. Cross-adaptation studies demonstrated that both compounds stimulated the same large-spiking cell. The frequencies of spikes evoked from the large cell when stimulated by emissions from 0.1-g, 1-g, or 10-g cartridges of either the formate or the trienal were not significantly different, suggesting that the formate is an effective mimic of the trienal at the antennal receptor cell level.     

Identification of odors from overripe mango that attract vinegar flies,Drosophila melanogaster

Bioassays with a variety of overripe fruits, including mango, plum, pear, and grape, and their extracts showed that odors from overripe mango were most attractive to adult vinegar flies, Drosophila melanogaster. Combined gas chromatography–electroantennographic detection (GC-EAD) analyses of solid-phase microextraction (SPME) and Tenax extracts of overripe mango odors showed that several volatile compounds, including ethanol, acetic acid, amyl acetate, 2-phenylethanol, and phenylethyl acetate elicited significant EAG responses from antennae of female flies. Most of the volatile compounds in the extracts were identified by mass spectral and retention index comparisons with synthetic standards. In cage bioassays, lures with a blend of ethanol, acetic acid, and 2-phenylethanol in a ratio of 1:22:5 attracted six times more flies than any single EAG-active compound. This blend also attracted four times more flies than traps baited with overripe mango or unripe mango. However, in field trials, the blend was not as attractive as suggested by the laboratory bioassay.     

A study of the large strain deformation and failure behaviour of mixed biopolymer gels via in situ ESEM

A mixed biopolymer gel, consisting of a protein (gelatin) and polysaccharide (maltodextrin) mixture has been investigated. By controlling the composition it was possible to construct an ‘emulsion-like’ structure, with included spherical particles of one phase (maltodextrin) within a continuous matrix of the second (gelatin). Large strain deformation and failure behaviour of this system has been examined via in situ environmental scanning electron microscopy (ESEM). ESEM has been employed to explore the changes in the structure of the material, whilst allowing the sample to stay hydrated as it was subjected to tensile strain, thereby allowing simultaneous imaging and determination of stress-strain data of the native sample. Ductile behaviour was observed, which has been attributed to the stretching, tearing and fracture of gelatin ligaments and debonding at the interface between the maltodextrin particles and continuous gelatin matrix. Deformation and fracture of the maltodextrin particles during tensile testing was also observed. The interfacial fracture energy of the composite has been calculated following an elastomer composite-debonding model, although there are several limitations to this approach for the mixed gel. It was found in samples tested after different ageing times that the debonding stress and strain was decreasing with ageing leading to a lower interfacial fracture energy. Samples were also tested after successive loading cycles, which resulted in a mechanical strength decrease after each cycle.     

Lycopene from two food sources does not affect antioxidant or cholesterol status of middle-aged adults

Background

Epidemiological studies have reported associations between reduced cardiovascular disease and diets rich in tomato and/or lycopene. Intervention studies have shown that lycopene-containing foods may reduce cholesterol levels and lipid peroxidation, factors implicated in the initiation of cardiovascular disease. The objective of this study was to determine whether consumption of lycopene rich foods conferred cardiovascular protection to middle-aged adults as indicated by plasma lipid concentrations and measures of ex vivo antioxidants.

Methods

Ten healthy men and women consumed a low lycopene diet with no added lycopene (control treatment) or supplemented with watermelon or tomato juice each containing 20 mg lycopene. Subjects consumed each treatment for three weeks in a crossover design. Plasma, collected weekly was analyzed for total cholesterol, high density lipoprotein cholesterol (HDL-C) and triglyceride concentrations and for the antioxidant biomarkers of malondialdehyde formation products (MDA), plasma glutathione peroxidase (GPX) and ferric reducing ability of plasma (FRAP). Data were analyzed using Proc Mixed Procedure and associations between antioxidant and lipid measures were identified by Pearson's product moment correlation analysis.

Results

Compared to the control diet, the lycopene-containing foods did not affect plasma lipid concentrations or antioxidant biomarkers. Women had higher total cholesterol, HDL-C and triglyceride concentrations than did the men. Total cholesterol was positively correlated to MDA and FRAP while HDL-C was positively correlated to MDA and GPX. GPX was negatively correlated to triglyceride concentration.

Conclusions

The inclusion of watermelon or tomato juice containing 20 mg lycopene did not affect plasma lipid concentrations or antioxidant status of healthy subjects. However, plasma cholesterol levels impacted the results of MDA and FRAP antioxidant tests.     

Validation of a Method for Quantitation of Soybean Lectin in Commercial Varieties

Soybean agglutinin (SBA) protein, also known as soybean lectin, is regarded as an anti‐nutrient due to its negative effect on the ability of monogastric animals to gain weight following consumption of raw soybean seed. Historically, SBA has been measured using a time‐consuming and cumbersome hemagglutination procedure. The objective of our research was to obtain a validated methodology that is precise and accurate in the measurement of SBA while allowing minimally equipped laboratories to effectively conduct the analysis, thus our focus was on evaluating an existing commercially available ELISA, an enzyme‐linked‐lectin‐assay (ELLA), and a hybrid ELISA/ELLA. A new ELLA technique that can detect and quantify lectins was chosen and modified specifically for the quantitation of SBA in soybean seed. The proposed ELLA methodology is similar to a standard sandwich ELISA, and uses polyacrylamide‐linked N‐acetylgalactosamine (Gal–NAc–PAA) for a capture phase and the biotinylated version (Gal–NAc–PAA–Biotin) for detection. Based upon the validation data, the ELLA method can precisely and accurately determine soybean lectin levels in soybean seed. The validated ELLA method was used to quantify SBA in nine commercial soybean varieties introduced between 1972 and 2008 and demonstrated that the natural variability of SBA is subject to the effects of genotype and environment.     

Mass Spectrometry-Based Proteomics for Pre-Eclampsia and Preterm Birth

Pregnancy-related complications such as pre-eclampsia and preterm birth now represent a notable burden of adverse health. Pre-eclampsia is a hypertensive disorder unique to pregnancy. It is an important cause of maternal death worldwide and a leading cause of fetal growth restriction and iatrogenic prematurity. Fifteen million infants are born preterm each year globally, but more than one million of those do not survive their first month of life. Currently there are no predictive tests available for diagnosis of these pregnancy-related complications and the biological mechanisms of the diseases have not been fully elucidated. Mass spectrometry-based proteomics have all the necessary attributes to provide the needed breakthrough in understanding the pathophysiology of complex human diseases thorough the discovery of biomarkers. The mass spectrometry methodologies employed in the studies for pregnancy-related complications are evaluated in this article. Top-down proteomic and peptidomic profiling by laser mass spectrometry, liquid chromatography or capillary electrophoresis coupled to mass spectrometry, and bottom-up quantitative proteomics and targeted proteomics by liquid chromatography mass spectrometry have been applied to elucidate protein biomarkers and biological mechanism of pregnancy-related complications. The proteomes of serum, urine, amniotic fluid, cervical-vaginal fluid, placental tissue, and cytotrophoblastic cells have all been investigated. Numerous biomarkers or biomarker candidates that could distinguish complicated pregnancies from healthy controls have been proposed. Nevertheless, questions as to the clinically utility and the capacity to elucidate the pathogenesis of the pre-eclampsia and preterm birth remain to be answered.     

Characterization of particle emissions from consumer fused deposition modeling 3D printers

Particle emissions from multiple fused deposition modeling consumer 3D printers were systematically quantified utilizing an established emission testing protocol (Blue Angel) to allow quantitative exposure assessments for printers operating in different environments. The data are consistent with particle generation from volatilization of the polymer filament as it is heated by the extruder. Typically, as printing begins, a burst of new particle formation leads to the smallest sizes and maximum number concentrations produced throughout the print job. For acrylonitrile butadiene styrene (ABS) filaments, instantaneous concentrations were up to 10⁶ #/cm³ with mean particle sizes of 20 to 40 nm when measured in a well mixed 1 m³ chamber with 1 air change per hour. Particles are continuously formed during printing and the size distribution evolves consistent with vapor condensation and particle coagulation. Particles emitted per mass of filament consumed (particle yield) varied widely due to factors including printer brand, and type and brand of filament. Higher extruder temperatures result in larger emissions. For filament materials tested, average particle number yields ranged from 7.3 × 10⁸ to 5.2 × 10¹⁰ g^?1 (approximately 0.65 to 24 ppm), with trace additives apparently driving the large variations. Nanoparticles (diameters less than 100 nm) dominate number distributions, whereas diameters in the range of 200 to 500 nm contribute most to estimated mass. Because 3D printers are often used in public spaces and personal residences, the general public and particularly susceptible populations, such as children, can be exposed to high concentrations of non-engineered nanoparticles of potential toxicity.

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