Dual Activity BLEG-1 from Bacillus lehensis G1 Revealed Structural Resemblance to B3 Metallo-β-Lactamase and Glyoxalase II: An Insight into Its Enzyme Promiscuity and Evolutionary Divergence

Metallo-β-lactamases (MBLs) are class B β-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of β-lactam antibiotics. A previous study on BLEG-1 (formerly called Bleg1_2437), a hypothetical protein from Bacillus lehensis G1, revealed sequence similarity and activity to B3 subclass MBLs, despite its evolutionary divergence from these enzymes. Its relatedness to glyoxalase II (GLXII) raises the possibility of its enzymatic promiscuity and unique structural features compared to other MBLs and GLXIIs. This present study highlights that BLEG-1 possessed both MBL and GLXII activities with similar catalytic efficiencies. Its crystal structure revealed highly similar active site configuration to YcbL and GloB GLXIIs from Salmonella enterica, and L1 B3 MBL from Stenotrophomonas maltophilia. However, different from GLXIIs, BLEG-1 has an insertion of an active-site loop, forming a binding cavity similar to B3 MBL at the N-terminal region. We propose that BLEG-1 could possibly have evolved from GLXII and adopted MBL activity through this insertion.     

Dietary fiber from coconut flour: A functional food

To determine the effectiveness of dietary fiber present in coconut flour as a functional food, the following studies were conducted: (a) Dietary Fiber Composition and Fermentability of Coconut Flour; (b) The Effect of Coconut Flour on Mineral Availability from Coconut Flour Supplemented Foods; (c) Glycemic Index of Coconut Flour Supplemented Foods in Normal and Diabetic Subjects; and (d) The Cholesterol Lowering Effect of Coconut Flakes in Moderately Raised Cholesterol Levels of Humans. The dietary fiber content of coconut flour was 60.0 ± 1.0 g/100 g sample, 56% insoluble and 4% soluble. Fermentation of coconut flour produced short chain fatty acids with butyrate (1.73 ± 0.07 mmol/g fiber isolate) > acetate (1.40 ± 0.12; (P < 0.05) > propionate (0.47 ± 0.01; P < 0.05). Iron and zinc availability were highest for carrot cake (Fe, 33.3 ± 0.7%; Zn, 12.6 ± 0.1%) supplemented with 20% coconut flour while multigrain loaf supplemented with 10% and macaroons with 25% coconut flour were highest for calcium availability (63.4 ± 8.0% and 38.7 ± 1.1%, respectively). Increasing concentrations of dietary fiber from coconut flour did not affect mineral availability from all test foods. The significantly low glycemic index foods (< 60 mmol × min/l) investigated were: macaroons (45.7 ± 3.0), carrot cake (51.8 ± 3.3) and brownies (60.1 ± 5.4) with 20–25% coconut flour. The test foods containing 15% coconut flour has a glycemic index ranging from 61 to 77 mmol × min/l. Among the test foods, pan de sal (87.2 ± 5.5) and multigrain loaf (85.2 ± 6.8) gave significantly higher glycemic index with 5% and 10% coconut flour. On the other hand, granola bar and cinnamon which contained 5% and 10% coconut flour, respectively gave a glycemic index ranging from 62 to 76 mmol × min/l and did not differ significantly from the test foods containing 15% coconut flour (P < 0.05). A very strong negative correlation (r = − 85, n = 11, P < 0.005) was observed between the glycemic index and dietary fiber content of the test foods supplemented with coconut. There was a significant reduction (%) in serum total and LDL cholesterol for: oat bran flakes, 8.4 ± 1.4 and 8.8 ± 6.7, respectively; 15% coconut flakes, 6.9 ± 1.1 and 11.0 ± 4.0, respectively; and 25% coconut flakes, 10.8 ± 1.3 and 9.2 ± 5.4, respectively (P < 0.05). Serum triglycerides were significantly reduced for all test foods: corn flakes, 14.5 ± 6.3%; oat bran flakes, 22.7 ± 2.9%; 15% coconut flakes, 19.3 ± 5.7%; and 25% coconut flakes, 21.8 ± 6.0% (P < 0.05). Results from the above study can be a basis in the development of coconut flour as a functional food.

Industrial relevance

The functionality of coconut flour in terms of prevention for risk of chronic diseases, e.g. diabetes mellitus, cardiovascular diseases (CVD) and colon cancer, revealed increase production of coconut and coconut flour. The production of coconut flour is very economical because it can be produced in a small or large scale. The raw material is obtained from the by-product (waste) of the coconut milk industry and the process and equipment used in its production is simple and cheap. Coconut flour as a good source of dietary fiber can be added to bakery products, recipes and other food products for good health.     

Highly crystalline WO3 thin films with ordered 3D mesoporosity and improved electrochromic performance

WO3 thin layers with nanometer-scale periodicity were prepared by evaporation-induced self-assembly (EISA) using a novel amphiphilic block-copolymer template (poly(ethylene-co-butylene)-block-poly(ethylene oxide)). The evolution of the mesoporous ordered network and the crystallinity of the framework were monitored by 2D-SAXS, WAXS, SEM, XPS, and porosimetry. By annealing the films, the pore-wall crystallinity is adjusted between fully amorphous and highly crystalline without mesostructural degradation. Thus, the crystalline-film framework is composed of phase-pure monoclinic WO3 nanoparticles (12-14 nm in size). Furthermore, heat treatment transforms the originally spherical mesopores into ellipsoids, resulting in a unidirectionally shrunken, but still well-defined and fully accessible bcc mesopore architecture. The influence of mesoporosity and crystallinity on electrochemical/electrochromic characteristics was addressed by monitoring electrochemical features and the absorption changes during Li insertion/extraction (repetitive potentiostatic cycling). Both the amorphous and crystalline mesoporous films possess electrochromic response times on the order of only seconds, which are attributable to the facilitated insertion of guest ions due to shortening of the diffusion path lengths. Also, the insertion/extraction reversibility of crystalline WO3 layers with 3D mesoporosity is improved compared to amorphous ones and reaches values close to 100%.     

Optimization of electron transfer dissociation via informed selection of reagents and operating parameters

Electron transfer dissociation (ETD) has improved the mass spectrometric analysis of proteins and peptides with labile post-translational modifications and larger intact masses. Here, the parameters governing the reaction rate of ETD are examined experimentally. Currently, due to reagent injection and isolation events as well as longer reaction times, ETD spectra require significantly more time to acquire than collision-induced dissociation (CID) spectra (>100 ms), resulting in a trade-off in the dynamic range of tandem MS analyses when ETD-based methods are compared to CID-based methods. Through fine adjustment of reaction parameters and the selection of reagents with optimal characteristics, we demonstrate a drastic reduction in the time taken per ETD event. In fact, ETD can be performed with optimal efficiency in nearly the same time as CID at low precursor charge state (z = +3) and becomes faster at higher charge state (z > +3).     

Surface functionalization of mesoporous antimony doped tin oxide by metalorganic reaction

Electrically conducting mesoporous antimony doped tin oxide was functionalized by the metalorganic reaction with several Grignard reagents. This fast and efficient grafting approach enables a direct connection of the metal atoms with the organic functionalities avoiding the formation of insulating Si–O linkers, which is of special interest for the interfacial charge transfer processes. Using this approach we introduced vinyl, allyl and phenyl groups into the pores of mesoporous ATO, which was confirmed by IR spectroscopy, TGA and nitrogen sorption measurements. We obtained a high loading of organic groups corresponding to about 50–60% of the monolayer surface coverage. The obtained mesoporous inorganic–organic hybrids can serve as a platform for incorporation of electrochemically active species.     

Designing training for process control simulators: a review of empirical findings and current practices

Although simulators have been widely used as training environments in different industries (e.g. oil and nuclear power), there is little rigorous empirical work evaluating the effectiveness of the training methods employed. This article examines the use of simulator training in process control environments. The results of an exploratory field study are reported and the current practices of simulator training are described. The study revealed that simulator training varied considerably across organisations, often with little theoretical or empirical work to guide training design. To evaluate the utility and effectiveness of different methods of simulator training in process control environments, the article also presents a literature review of the research on laboratory- and field-based training. Several training methods are identified as having particular potential for temporal and adaptive transfer and are to be empirically tested in future studies.     

Preparation of immobilized Trametes pubescens laccase on a cryogel-type polymeric carrier and application of the biocatalyst to apple juice phenolic compounds oxidation

A new biocatalyst was prepared by the immobilization of a Trametes pubescens laccase, into a wide-pore poly(vinyl alcohol) cryogel. The known enzyme was produced and purified by the previously described procedure. The resulted laccase (yield 40%) has an activity of 46.4 U mg⁻¹ and 12.51 mg mL⁻¹ protein content. The enzyme was subsequently immobilized in a functionalized macroporous cryogel beads by a covalent immobilization technique. The time dependence of the immobilization process and the enzyme loading of the carrier material (5.2 mg g⁻¹ cryogel) were determined by measuring the decrease of protein amount in the enzyme solution. In conversion experiments, a higher stability of the immobilized biocatalyst compared to the free enzyme was evidenced. Steady-state kinetic characterization of four phenols (catechol, caffeic and chlorogenic acids, and catechin) has been performed with free and immobilized laccase, the catalytic parameters being determined and compared. The effect of both laccases (free and immobilized) on the phenol content of retailed apple juice samples, having the same initial composition, was also investigated by working in batch conversion. The variation in phenolic compound content has been compared with that of an untreated apple juice sample having initially the same content of phenolic compounds. A number of advantages resulted in using the immobilized laccase for the apple juice treatment (conservation to some extent of enzyme activity, higher content of phenols preserved, easy separation of the enzyme from the apple juice, therefore avoiding the possible unhealthy effects due to the remaining protein, etc.).     

Assessment of tremor activity in the Parkinson's disease using a set of wearable sensors

Tremor is the most common motor disorder of Parkinson's disease (PD) and consequently its detection plays a crucial role in the management and treatment of PD patients. The current diagnosis procedure is based on subject-dependent clinical assessment, which has a difficulty in capturing subtle tremor features. In this paper, an automated method for both resting and action/postural tremor assessment is proposed using a set of accelerometers mounted on different patient's body segments. The estimation of tremor type (resting/action postural) and severity is based on features extracted from the acquired signals and hidden Markov models. The method is evaluated using data collected from 23 subjects (18 PD patients and 5 control subjects). The obtained results verified that the proposed method successfully: 1) quantifies tremor severity with 87 % accuracy, 2) discriminates resting from postural tremor, and 3) discriminates tremor from other Parkinsonian motor symptoms during daily activities.     

Kinetics of L-tryptophan production from indole and L-serine catalyzed by whole cells with tryptophanase activity

The essential amino acid L-tryptophan can be produced by a condensation reaction between indole and L-serine, catalyzed by whole cells of Escherichia coli B1t-7A with tryptophanase activity. The reaction was previously studied using soluble tryptophanase, a kinetic mechanism proposed and the catalytic properties of the enzyme described. It is important, however, to determine the kinetic parameters of the reaction catalyzed by whole cells, if the process is to be designed with the catalyst in this form. The reaction stoichiometry was established, a mole of product being formed from a mole of each reactant, with no indication of side reactions under the conditions used. The two-substrate reaction kinetics were characterized and modelled, assuming an enzyme-substituted mechanism and no product inhibition. Theoretical consumption rates of indole were compared with experimental values obtained in a batch reactor system. The K(m) values of whole cells towards L-serine and indole were 1.79 M and 0.07 M, respectively. These values are, as expected, considerably higher than their counterparts for soluble tryptophanase.     

N‐Methyl‐Substituted Fluorescent DAG–Indololactone Isomers Exhibit Dramatic Differences in Membrane Interactions and Biological Activity

N‐methyl‐substituted diacylglycerol–indololactones (DAG–indololactones) are newly synthesized effectors of protein kinase C (PKC) isoforms and exhibit substantial selectivity between RasGRP3 and PKCα. We present a comprehensive analysis of membrane interactions and biological activities of several DAG–indololactones. Translocation and binding activity assays underline significant variations between the PKC translocation characteristics affected by the ligands as compared to their binding activities. In parallel, the fluorescent properties of the ligands were employed for analysis of their membrane association profiles. Specifically, we found that a slight change in the linkage to the indole ring resulted in significant differences in membrane binding and association of the DAG–indololactones with lipid bilayers. Our analysis shows that seemingly small structural modifications of the hydrophobic regions of these biomimetic PKC effectors contribute to pronounced modulation of membrane interactions of the ligands.