Alteration of the Route to Menaquinone towards Isochorismate-Derived Metabolites

Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double-variant Asn117Arg–Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHD), the hydrolysis product of isochorismate, with a >70-fold higher ratio than that for the wild type. The single-variant Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHA) as substrate with >6-fold conversion compared to wild-type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L⁻¹). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild-type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.     

Fouling Detection in an Optically Accessible Microstructured Heat Exchanger

The application of highly effective microstructured devices in continuous production and industrial environments is frequently prone to fouling. A new method is presented to characterize fouling in these microstructures. Thermal fouling of aqueous solutions containing whey protein were used as a test system. Different fouling effects could be observed and distinguished. Integral fouling indicators, such as thermal fouling resistance and pressure drop, as conventional criteria for the occurrence of fouling were compared with direct local optical observation. Low thermal fouling resistances could be detected.     

Modeling and in-depth analysis of the start-up of dividing-wall columns

Saving potentials of up to 30% in capital and operating costs are the driving forces behind the increase in the application of dividing-wall columns in industry. However, a lack of knowledge still exists when dealing with the start-up of dividing-wall columns, which is inherently a strongly nonlinear process. Here, for the first time the start-up of dividing-wall columns is explored, where the starting point is an empty column at ambient conditions. A model is presented which is capable of predicting the dynamic discrete-continuous changes which are characteristic of dividing-wall columns. The proposed process model takes into account the heat transfer across the dividing wall as well as the vapor distribution below the dividing wall. The degree of accuracy of the model is clearly determined by comparing different simplifications, e.g. a constant vapor distribution ratio equal to the steady-state value. The detailed studies were carried out with strict product specifications so that the influence of process parameters could be quantified. The rigorous process model and the obtained simulation results presented in this study provide a promising basis for developing and applying optimal start-up policies for dividing-wall columns.     

An Experimental Workflow for Studying Barrier Integrity,Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell–cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.     

Killing Me Softly—Future Challenges in Apoptosis Research

The induction of apoptosis, a highly regulated and clearly defined mode of cell dying, is a vital tenet of modern cancer therapy. In this review we focus on three aspects of apoptosis research which we believe are the most crucial and most exciting areas currently investigated and that will need to be better understood in order to enhance the efficacy of therapeutic measures. First, we discuss which target to select for cancer therapy and argue that not the cancer cell as such, but its interaction with the microenvironment is a more promising and genetically stable site of attack. Second, the complexity of combination therapy is elucidated using the PI3-K-mediated signaling network as a specific example. Here we show that the current clinical approach to sensitize malignancies to apoptosis by maximal, prolonged inhibition of so-called survival pathways can actually be counter productive. Third, we propose that under certain conditions which will need to be clearly defined in future, chronification of a tumor might be preferable to the attempt at a cure. Finally, we discuss further problems with utilizing apoptosis induction in cancer therapy and propose a novel potential therapeutic approach that combines the previously discussed features.     

Structure‐Based Mechanism of Oleate Hydratase from Elizabethkingia meningoseptica

Hydratases provide access to secondary and tertiary alcohols by regio‐ and/or stereospecifically adding water to carbon‐carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure‐based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two‐electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio‐ and stereoselective hydration.     

Synthesis,morphology and properties of segmented poly(ether ester amide)s comprising uniform glycine or β-alanine extended bisoxalamide hard segments

Segmented poly(ether ester amide)s comprising glycine or β-alanine extended bisoxalamide hard segments are highly phase separated thermoplastic elastomers with a broad temperature independent rubber plateau. These materials with molecular weights, M_n, exceeding 30 × 10³ g mol^?1 are conveniently prepared by polycondensation of preformed bisester–bisoxalamides and commercially available PTHF diols. FT-IR revealed strongly hydrogen bonded and highly ordered bisoxalamide hard segments with degrees of ordering between 73 and 99%. The morphology consists of fiber-like nano-crystals randomly dispersed in the soft polymer matrix. The micro-structural parameters of the copolymers were addressed by simultaneous small- and wide-angle X-ray scattering. It is shown that the crystals have strictly identical thickness, which is close to the contour length of the hard segment. The long dimension of the crystals is identified with the direction of the hydrogen bonds. The melting transitions of the hard segments are sharp, with temperatures up to 170 °C. The studied polymers have an elastic modulus in the range of 139–170 MPa, a stress at break in the range of 19–31 MPa combined with strains at break of higher than 800%. The segmented copolymer comprising the β-alanine based bisoxalamide hard segment with a spacer of 6 methylene groups has a melting transition of 141 °C which is higher than the melting transition of its glycine analogue of 119 °C. Likewise, the fracture stress increased from 22 to 31 MPa when the glycine ester group in the hard segment was replaced with β-alanine. The improved thermal and mechanical properties of the latter polymers is related to the crystal packing of the β-alanine based hard segments in the copolymer compared to the packing of the hard segments comprising glycine ester groups.     

Chemical-Shift Perturbations Reflect Bile Acid Binding to Norovirus Coat Protein: Recognition Comes in Different Flavors

Bile acids have been reported as important cofactors promoting human and murine norovirus (NoV) infections in cell culture. The underlying mechanisms are not resolved. Through the use of chemical shift perturbation (CSP) NMR experiments, we identified a low-affinity bile acid binding site of a human GII.4 NoV strain. Long-timescale MD simulations reveal the formation of a ligand-accessible binding pocket of flexible shape, allowing the formation of stable viral coat protein–bile acid complexes in agreement with experimental CSP data. CSP NMR experiments also show that this mode of bile acid binding has a minor influence on the binding of histo-blood group antigens and vice versa. STD NMR experiments probing the binding of bile acids to virus-like particles of seven different strains suggest that low-affinity bile acid binding is a common feature of human NoV and should therefore be important for understanding the role of bile acids as cofactors in NoV infection.