Engineered Bacterial Flavin-Dependent Monooxygenases for the Regiospecific Hydroxylation of Polycyclic Phenols

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H), a flavin-dependent monooxygenase from E. coli that catalyzes the hydroxylation of monophenols to catechols, was modified by rational redesign to convert also more bulky substrates, especially phenolic natural products like phenylpropanoids, flavones or coumarins. Selected amino acid positions in the binding pocket of 4HPA3H were exchanged with residues from the homologous protein from Pseudomonas aeruginosa, yielding variants with improved conversion of spacious substrates such as the flavonoid naringenin or the alkaloid mimetic 2-hydroxycarbazole. Reactions were followed by an adapted Fe(III)-catechol chromogenic assay selective for the products. Especially substitution of the residue Y301 facilitated modulation of substrate specificity: introduction of nonaromatic but hydrophobic (iso)leucine resulted in the preference of the substrate ferulic acid (having a guaiacyl (guajacyl) moiety, part of the vanilloid motif) over unsubstituted monophenols. The in vivo (whole-cell biocatalysts) and in vitro (three-enzyme cascade) transformations of substrates by 4HPA3H and its optimized variants was strictly regiospecific and proceeded without generation of byproducts.     

Nonsingular adsorption/desorption of chlorpyrifos in soils and sediments: experimental results and modeling

At environmentally relevant concentrations in soils and sediments, chlorpyrifos, a hydrophobic organic insecticide, showed strong adsorption that correlated significantly with organic matter content. Chlorpyrifos desorption followed a nonsingular falling desorption isotherm that was estimated using a memory-dependent mathematical model. Desorption of chlorpyrifos was biphasic in nature, with a labile and nonlabile component. The labile component comprised 18-28% of the original solid-phase concentration, and the residue was predicted to slowly partition to the aqueous phase, implying long-term desorption from contaminated soils or sediments. The newly proposed mechanism to explain sorption/desorption hysteresis and biphasic desorption is the unfavorable thermodynamic energy landscape arising from limitation of diffusivity of water molecules through the strongly hydrophobic domain of soils and sediments. Modeling results suggest that contaminated soils and sediments could be secondary long-term sources of pollution. Long-term desorption may explain the detection of chlorpyrifos and other hydrophobic organic compounds in aquatic systems far from application sites, an observation that contradicts conventional transport predictions.     

Simultaneous design and operation optimization of renewable combined cooling heating and power systems

Combined cooling, heating, and power (CCHP) systems are promising solutions for conserving energy and reducing emissions. This article proposes a new mixed-integer linear programming (MILP) model for simultaneous design and operation optimization of a renewable CCHP system, considering component nonlinear operating characteristics and performance degradation with time. A bi-objective MILP problem is solved to achieve a trade-off between total annual cost (TAC) and greenhouse gas emissions (GHGe). A case study of a commercial region is employed to demonstrate our proposed methodology. The results shows, in comparison with conventional cost minimization, our solution features a tardy increase of 12.8% in TAC and a sharp reduction of 75.5% in GHGe. Moreover, we find that ignoring performance degradation leads to an over-estimation of 2.3–13.7% in system economic performance. The proposed methodology provides an effective and flexible framework for optimal design and operational analysis of renewable CCHP systems.     

Identifying the Origin and Contribution of Surface Storage in TiO2(B) Nanotube Electrode by In Situ Dynamic Valence State Monitoring

Fundamental insight into the surface charging mechanism of TiO₂(B) nanomaterials is limited due to the complicated nature of lithiation behavior, as well as the limitations of available characterization tools that can directly probe surface charging process. Here, an in situ approach is reported to monitor the dynamic valence state of TiO₂(B) nanotube electrodes, which utilizes in situ X‐ray absorption spectroscopy (XAS) to identify the origin and contribution of surface storage. A real‐time correlation is elucidated between the rate‐dependent electrode performance and dynamic Ti valence‐state change. A continuous Ti valence state change is directly observed through the whole charging/discharging process regardless of charging rates, which proves that along with the well‐known non‐faradaic reaction, the surface charging process also originates from a faradaic reaction. The quantification of these two surface storage contributions at different charging rates is further realized through in situ dynamic valence state monitoring combined with traditional cyclic voltammetry measurement. The methodology reported here can also be applied to other electrode materials for the real‐time probing of valence state change during electrochemical reactions, the quantification of the faradaic and non‐faradaic reactions, and the eventual elucidation of electrochemical surface charging mechanisms.     

Towards a comprehensive proteome of normal and malignant human colon tissue by 2-D-LC-ESI-MS and 2-DE proteomics and identification of S100A12 as potential cancer biomarker

The aim of this study was to characterize the proteome of normal and malignant colonic tissue. We previously studied the colon proteome using 2‐DE and MALDI‐MS and identified 734 proteins (Roeßler, M., Rollinger, W., Palme S., Hagmann, M.‐L., et al.., Clin. Cancer Res. 2005, 11, 6550–6557). Here we report the identification of additional colon proteins from the same set of tissue samples using a complementary nano‐flow 2‐D‐LC‐ESI‐MS. In total, 484 proteins were identified in colon. Of these, 252 had also been identified by the 2‐DE/MALDI‐MS approach, whereas 232 proteins were unique to the 2‐D‐LC‐ESI‐MS analysis. Comparing protein expression in neoplastic and normal colon tissue indicated elevated expression of several proteins in colorectal cancer, among them the well established tumor marker carcinoembryonic antigen, as well as calnexin, 40S ribosomal protein S15a, serpin H1, and S100A12. Overexpression of these proteins was confirmed by immunoblotting. Serum levels of S100A12 were determined by ELISA and were found to be strongly elevated in colorectal cancer patients compared to healthy individuals. We conclude, that 2‐D‐LC‐ESI‐MS is a powerful approach to identify and compare protein profiles of tissue samples, that it is complementary to 2‐DE/MALDI‐MS approaches and has the potential to identify novel biomarkers.     

Experimental Estimation of Model Error Bounds Based on Modified Stochastic Approximation

A modification of the estimation algorithm stochastic approximation is presented. With assumptions to the statistical distribution of the training data it becomes possible, to estimate not only the mean value but also well directed deviating values of the data distribution. Thus, detailed error models can be identified by means of parameter-linear formulation of the new algorithm. By definition of suitable probabilities, these parametric error models are estimating soft error bounds. That way, an experimental identification method is provided that is able to support a robust controller design. The method was applied at an industrial robot, which is controlled by feedback linearisation. Based on a dynamic model realised by a neural network, the presented approach is utilised for the robust design of the stabilising decentral controllers.     

Trends of energy efficiency in Finnish road freight transport 1995–2009 and forecast to 2016

A framework for modeling and analyzing the energy efficiency of road freight transport is presented in this paper. This framework is tested by using the data from the Finnish Goods Transport by Road statistics. The data was enhanced by calculating the fuel consumption for each trip in the data. To calculate this, weight-fuel consumption functions were estimated for each Euro-class vehicles and road type. This is a new method for analyzing the energy efficiency of road freight transport and it could be applied also in other countries gathering freight transport data with continuous company surveys. The analysis show that the energy efficiency of road freight transport in Finland improved during 1995–2002, but has declined since. The major drivers in the development have been the changes in the level of empty running and vehicle fuel efficiency. Extrapolating current statistical trends of factors that influence the energy efficiency show that the target set by the Finnish government for improving energy efficiency by 9% until 2016 will not be achieved. However, the target is possible to be achieved by a combination of small changes to some determinants.     

High-sensitivity MALDI-MRM-MS imaging of moxifloxacin distribution in tuberculosis-infected rabbit lungs and granulomatous lesions

MALDI-MSI is a powerful technology for localizing drug and metabolite distributions in biological tissues. To enhance our understanding of tuberculosis (TB) drug efficacy and how efficiently certain drugs reach their site of action, MALDI-MSI was applied to image the distribution of the second-line TB drug moxifloxacin at a range of time points after dosing. The ability to perform multiple monitoring of selected ion transitions in the same experiment enabled extremely sensitive imaging of moxifloxacin within tuberculosis-infected rabbit lung biopsies in less than 15 min per tissue section. Homogeneous application of a reference standard during the matrix spraying process enabled the ion-suppressing effects of the inhomogeneous lung tissue to be normalized. The drug was observed to accumulate in granulomatous lesions at levels higher than that in the surrounding lung tissue from 1.5 h postdose until the final time point. MALDI-MSI moxifloxacin distribution data were validated by quantitative LC/MS/MS analysis of lung and granuloma extracts from adjacent biopsies taken from the same animals. Drug distribution within the granulomas was observed to be inhomogeneous, and very low levels were observed in the caseum in comparison to the cellular granuloma regions. In this experiment the MALDI-MRM-MSI method was shown to be a rapid and sensitive method for analyzing the distribution of anti-TB compounds and will be applied to distribution studies of additional drugs in the future.     

Resistance of pure and mixed rare earth silicates against calcium-magnesium-aluminosilicate (CMAS): A comparative study

Rare earth silicate environmental barrier coatings (EBCs) are state of the art for protecting SiC ceramic matrix composites (CMCs) against corrosive media. The interaction of four pure rare earth silicate EBC materials Yb₂SiO₅, Yb₂Si₂O₇, Y₂SiO₅, Y₂Si₂O₇ and three ytterbium silicate mixtures with molten calcium-magnesium-aluminosilicate (CMAS) were studied at high temperature (1400°C). The samples were characterized by SEM and XRD in order to evaluate the recession of the different materials after a reaction time of 8 hours. Additionally, the coefficient of thermal expansion (CTE) was determined to evaluate the suitability of Yb silicate mixtures as EBC materials for SiC CMCs. Results show that monosilicates exhibit a lower recession in contact with CMAS than their disilicate counterparts. The recession of the ytterbium silicates is far lower than the recession of the yttrium silicates under CMAS attack. Investigation of the ytterbium silicate mixtures exposes their superior resistance to CMAS, which is even higher than the resistance of the pure monosilicate. Also their decreased CTE suggests they will display better performance than the pure monosilicate.