A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.     

Intergenerational Metabolomic Analysis of Mothers with a History of Gestational Diabetes Mellitus and Their Offspring

Shared metabolomic patterns at delivery have been suggested to underlie the mother-to-child transmission of adverse metabolic health. This study aimed to investigate whether mothers with gestational diabetes mellitus (GDM) and their offspring show similar metabolomic patterns several years postpartum. Targeted metabolomics (including 137 metabolites) was performed in plasma samples obtained during an oral glucose tolerance test from 48 mothers with GDM and their offspring at a cross-sectional study visit 8 years after delivery. Partial Pearson’s correlations between the area under the curve (AUC) of maternal and offspring metabolites were calculated, yielding so-called Gaussian graphical models. Spearman’s correlations were applied to investigate correlations of body mass index (BMI), Matsuda insulin sensitivity index (ISI-M), dietary intake, and physical activity between generations, and correlations of metabolite AUCs with lifestyle variables. This study revealed that BMI, ISI-M, and the AUC of six metabolites (carnitine, taurine, proline, SM(-OH) C14:1, creatinine, and PC ae C34:3) were significantly correlated between mothers and offspring several years postpartum. Intergenerational metabolite correlations were independent of shared BMI, ISI-M, age, sex, and all other metabolites. Furthermore, creatinine was correlated with physical activity in mothers. This study suggests that there is long-term metabolic programming in the offspring of mothers with GDM and informs us about targets that could be addressed by future intervention studies.     

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeolei VT8

Wax esters (WE) belong to the class of neutral lipids. They are formed by an esterification of a fatty alcohol and an activated fatty acid. Dependent on the chain length and desaturation degree of the fatty acid and the fatty alcohol moiety, WE can have diverse physicochemical properties. WE derived from monounsaturated long-chain acyl moieties are of industrial interest due to their very good lubrication properties. Whereas WE were obtained in the past from spermaceti organs of the sperm whale, industrial WE are nowadays mostly produced chemically from fossil fuels. In order to produce WE more sustainably, attempts to produce industrial WE in transgenic plants are steadily increasing. To achieve this, different combinations of WE producing enzymes are expressed in developing Arabidopsis thaliana or Camelina sativa seeds. Here we report the identification and characterization of a fifth wax synthase from the organism Marinobacter aquaeolei VT8, MaWSD5. It belongs to the class of bifunctional wax synthase/acyl-CoA:diacylglycerol O-acyltransferases (WSD). The protein was purified to homogeneity. In vivo and in vitro substrate analyses revealed that MaWSD5 is able to synthesize WE but no triacylglycerols. The protein produces WE from saturated and monounsaturated mid- and long-chain substrates. Arabidopsis thaliana seeds expressing a fatty acid reductase from Marinobacter aquaeolei VT8 and MaWSD5 produce WE. Main WE synthesized are 20:1/18:1 and 20:1/20:1. This makes MaWSD5 a suitable candidate for industrial WE production in planta.     

Terahertz Heterodyne Array Receivers for Astronomy

Journal of Infrared, Millimeter, and Terahertz Waves - We review the development of multi-pixel heterodyne receivers for astronomical research in the submillimeter and terahertz spectral domains....     

Does thermal control improve visual satisfaction? Interactions between occupants’ self-perceived control,visual, thermal,and overall satisfaction

Occupants’ satisfaction had been researched independently related to thermal and visual stimuli for many decades showing among others the influence of self-perceived control. Few studies revealed interactions between thermal and visual stimuli affecting occupant satisfaction. In addition, studies including interactions between thermal and visual stimuli are lacking different control scenarios. This study focused on the effects of thermal and visual factors, their interaction, seasonal influences, and the degree of self-perceived control on overall, thermal, and visual satisfaction. A repeated-measures laboratory study with 61 participants running over two years and a total of 986 participant sessions was conducted. Mixed model analyses with overall satisfaction as outcome variable revealed that thermal satisfaction and visual satisfaction are the most important predictors for overall satisfaction with the indoor environment. Self-perceived thermal control served as moderator between thermal satisfaction and overall satisfaction. Season had slight influence on overall satisfaction. Random effects explained the highest amount of variance, indicating that intra- and interindividual differences in the ratings of satisfaction are more prevalent than study condition. Future building design and operation plans aiming at a high level of occupant satisfaction should consider personal control opportunities and take into account the moderating effect of control opportunities in multimodal interactions.     

Manganese ferrite-polyaniline hybrid materials: Electrical and magnetic properties

Magnetic MnFe₂O₄ nanopowders were synthesized by an original solvothermal method in the absence and in the presence of tetra-n-butylammonium bromide (TBAB) and Tween 80 (TW) as surfactants. Manganese ferrite/polyaniline (PANI) hybrid materials were synthesized by in situ polymerization of aniline on the surface of MnFe₂O₄ using ammonium persulfate as oxidant. The purpose of the study was to investigate the influence of the two surfactants on the properties of the MnFe₂O₄ powders and of their composites with PANI. The specific surface area, the cumulative surface area of pores and the cumulative volume of pores are influenced by the nature of surfactant in case of MnFe₂O₄ powders and are higher by comparison to those of the MnFe₂O₄/PANI hybrid materials. The values of saturation magnetization in case of MnFe₂O₄ powders are higher than those of the hybrid materials and are not influenced by the surfactant nature. These features revealed that MnFe₂O₄ powders can be efficiently used as adsorbents for the purification of wastewaters. The values of the electrical conductivity of the composites exhibit a significant increase in comparison to the MnFe₂O₄ powders and depend on the surfactant nature. The highest value of electrical conductivity was achieved by the composite obtained using Tween 80 as surfactant (σ_DC = 54.5·10^?5S?m^?1) which was close to that of PANI (σ_DC = 61.2·10^?5 S?m^?1). The fact that the magnetic and electric properties of the synthesized MnFe₂O₄/PANI composites can be changed by design, demonstrate the high potential of these materials to be used in magneto-electric applications.     

Heuristic Methods for Minimum-Cost Pipeline Network Design – a Node Valency Transfer Metaheuristic

Designing low-cost network layouts is an essential step in planning linked infrastructure. For the case of capacitated trees, such as oil or gas pipeline networks, the cost is usually a function of both pipeline diameter (i.e. ability to carry flow or transferred capacity) and pipeline length. Even for the case of incompressible, steady flow, minimizing cost becomes particularly difficult as network topology itself dictates local flow material balances, rendering the optimization space non-linear. The combinatorial nature of potential trees requires the use of graph optimization heuristics to achieve good solutions in reasonable time. In this work we perform a comparison of known literature network optimization heuristics and metaheuristics for finding minimum-cost capacitated trees without Steiner nodes, and propose novel algorithms, including a metaheuristic based on transferring edges of high valency nodes. Our metaheuristic achieves performance above similar algorithms studied, especially for larger graphs, usually producing a significantly higher proportion of optimal solutions, while remaining in line with time-complexity of algorithms found in the literature. Data points for graph node positions and capacities are first randomly generated, and secondly obtained from the German emissions trading CO₂ source registry. As political will for applications and storage for hard-to-abate industry CO₂ emissions is growing, efficient network design methods become relevant for new large-scale CO₂ pipeline networks.

     

Synthesis,structural and morphological characteristics,magnetic and optical properties of Co doped ZnO nanoparticles

Zn_1−xCo_xO (x==0.05, 0.10, 0.15) nanoparticles have been synthesized by an alternative wet-chemical synthesis route using the SimAdd technique. The as-obtained powders were investigated by FT-IR spectroscopy, X-ray diffraction and thermal analysis correlated with evolved gas analysis (TG–DTA–FT-IR) in order to determine their chemical nature, crystalline structure and to establish the decomposition sequences. The precipitates are generally amorphous, but low-intensity reflection peaks assigned both to the zinc oxalate dihydrate, and zinc hydroxide can be observed in the recorded patterns, indicating that hydroxy-oxalate precipitates were obtained. The structure, morphology and magnetic properties of the thermally treated samples have been investigated by X-ray diffraction, FT-IR, HRTEM, SAED, UV–vis and EPR. XRD studies reveal a hexagonal wurtzite-type structure for all Zn_1−xCo_xO samples. TEM investigations show particle size between 28 and 37 nm, with spherical and polyhedral shapes and with tendency to form aggregates. The presence of a Co₃O₄ secondary phase was evidenced by XRD, UV–vis and EPR for the Zn_0.85Co_0.15O sample. The ferromagnetic behavior of the samples was revealed. The paper highlights that by varying the cobalt concentration it is possible to modulate the structural, morphological, optical and magnetic properties.     

Degradation of poly(vinyl alcohol)‐graft‐lactic acid copolymers by Trichotecium roseum fungus

The biodegradation of poly(vinyl alcohol) and poly(vinyl alcohol)‐graft‐lactic acid copolymers was analyzed, using Trichotecium roseum fungus. The samples were examined during biodegradation at different periods of exposure. Structural modifications of the biodegraded samples were investigated by Fourier transform infrared‐attenuated total reflectance spectroscopy, and the surface morphology was investigated by scanning electron microscopy. The static light scattering results concluded that the weight average molecular mass (M_w) of the copolymers increased after biodegradation, because the fractions with low molecular weight of the copolymers were destroyed. The thermal behavior and stability of the samples before and after the biodegradation period were investigated by differential scanning calorimetry (DSC) and thermogravimetric analyses. The thermogravimetric analyses and their derivatives (TG‐DTG) showed that the thermal stability of the biodegraded samples was more raised comparatively to the unbiodegraded ones. The DSC results demonstrated that biodegradation took place in the amorphous domains of the investigated polymer samples and the crystallinity degree increased after 24 biodegradation days. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41777.     

Functionalization of hybrid poly(n‐isopropylacrylamide) hydrogels for Escherichia coli cell capture via adsorbed intermediate dye molecule

Poly(n‐isopropylacrylamide) Laponite (PNIPAM‐Lap) hybrid hydrogels, which use the synthetic clay Laponite as a crosslinker, permanently adsorb cationic laser dyes out of solution. This proof‐of‐concept expounds on this capability by adsorbing an intermediate dye molecule and using it as the foundation for successfully conjugating microbial antibodies to the surface of a PNIPAM hydrogel. The study involves using acriflavinium chloride molecules, adsorbed by a PNIPAM‐Lap hydrogel from an acriflavine laser dye solution, as an intermediate molecule to attach antibodies raised against E. coli to the hydrogel and demonstrate cell capture. Furthermore, this system exemplifies a novel biotechnological platform for greatly expanding PNIPAM hydrogels' capabilities and applicability through conjugation chemistry to surface‐bound molecules. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41557.