Solid‐State NMR Spectroscopy Reveals that E. coli Inclusion Bodies of HET‐s(218–289) are Amyloids

Protein deposition frequently occurs as inclusion bodies (IBs) during heterologous protein expression in E. coli. The structure of these E. coli IBs of the prion‐forming domain from the fungal prion HET‐s is the same as that previously determined for fibrils assembled in vitro, and show prion infectivity. These results demonstrate that the IBs of HET‐s(218–289) are amyloids.

     

Mechanism of membrane interaction and disruption by α-synuclein

Parkinson's disease is a common progressive neurodegenerative condition, characterized by the deposition of amyloid fibrils as Lewy bodies in the substantia nigra of affected individuals. These insoluble aggregates predominantly consist of the protein α-synuclein. There is increasing evidence suggesting that the aggregation of α-synuclein is influenced by lipid membranes and, vice versa, the membrane integrity is severely affected by the presence of bound aggregates. Here, using the surface-sensitive imaging technique supercritical angle fluorescence microscopy and F?rster resonance energy transfer, we report the direct observation of α-synuclein aggregation on supported lipid bilayers. Both the wild-type and the two mutant forms of α-synuclein studied, namely, the familiar variant A53T and the designed highly toxic variant E57K, were found to follow the same mechanism of polymerization and membrane damage. This mechanism involved the extraction of lipids from the bilayer and their clustering around growing α-synuclein aggregates. Despite all three isoforms following the same pathway, the extent of aggregation and their effect on the bilayers was seen to be variant and concentration dependent. Both A53T and E57K formed cross-β-sheet aggregates and damaged the membrane at submicromolar concentrations. The wild-type also formed aggregates in this range; however, the extent of membrane disruption was greatly reduced. The process of membrane damage could resemble part of the yet poorly understood cellular toxicity phenomenon in vivo.     

Product line optimization in the presence of preferences for compromise alternatives

Recent advances in customer choice analysis demonstrated the strong impact of compromise alternatives on the behaviour of decision-makers in a wide range of decision situations. Compromise alternatives are characterized by an intermediate performance on some of the relevant attributes. For instance, price compromises are well known in the sense that customers tend to buy neither the cheapest, nor the most expensive alternative, but the mid-priced one. However, thus far, the literature on product line optimization has not considered such context effects.In this paper, we propose a model-based approach for optimal product line selection which incorporates customers’ preferences for compromise alternatives. We consider customer choice in a realistic, sophisticated fashion by applying an established utility model that integrates compromise variables into a multinomial logit model. We formulate the resulting optimization problem as a mixed-integer linear program. The challenging feature for modelling – making the formulation substantially more complicated than existing ones without compromises – are the endogenous effects of selected products on other alternatives’ utilities that need to be adequately captured via compromise variables. Based on data we collected by a stated choice experiment in a retail setting, we perform a computational study and demonstrate the superiority of our product line selection approach compared to a reference model that does not take compromises into account. Even under uncertainty of the estimated utility parameters, profit gains of, on average, 23% can be achieved in our experimental setting.     

Integrated revenue management approaches for capacity control with planned upgrades

In many service industries, firms offer a portfolio of similar products based on different types of resources. Mismatches between demand and capacity can therefore often be managed by using product upgrades. Clearly, it is desirable to consider this possibility in the revenue management systems that are used to decide on the acceptance of requests. To incorporate upgrades, we build upon different dynamic programming formulations from the literature and gain several new structural insights that facilitate the control process under certain conditions. We then propose two dynamic programming decomposition approaches that extend the traditional decomposition for capacity control by simultaneously considering upgrades as well as capacity control decisions. While the first approach is specifically suited for the multi-day capacity control problem faced, for example, by hotels and car rental companies, the second one is more general and can be applied in arbitrary network revenue management settings that allow upgrading. Both approaches are formally derived and analytically related to each other. It is shown that they give tighter upper bounds on the optimal solution of the original dynamic program than the well-known deterministic linear program. Using data from a major car rental company, we perform computational experiments that show that the proposed approaches are tractable for real-world problem sizes and outperform those disaggregated, successive planning approaches that are used in revenue management practice today.     

Curing studies of a polyimide precursor. II. Polyamic acid

The cure of pyromellitic dianhydride–oxydianiline polyamic acid is compared with that of a model compound, diphenylcarbamido–dicarboxy–benzene, both having been synthesized in 1-methyl-2-pyrrolidinone (NMP). Laser interferometry and thermogravimetric analysis showed that, analog to the diamic acid model compound, the polyamic acid forms complexes with NMP (repeat unit/NMP: 1/4 and 1/2). Thermal stability of these complexes was found to be similar to the ones of the diamic acid model compound as revealed by comparison of decomplexation temperature–heating rate curves. Activation energies of the decomplexation processes for the diamic acid are given. Anhydride formation, a reaction found in the cure of the model compound to compete with imidization, takes place also during the polyamic acid cure at fairly low temperatures. The molecular weight drop due to chain scission resulting from anhydride formation was followed by viscometry.     

Quantitative determination of cationic modified polysaccharides on hair using LC–MS and LC–MS–MS

Cationic polysaccharides containing N-hydroxypropyl-N,N,N-trimethylammonium substituents are widely used as conditioning agents for hair-care products. A sensitive method has been developed for the quantitation of these polymers. After acidic extraction from hair the polysaccharides are hydrolyzed using trifluoroacetic acid. The cationic monoglycosides are determined using liquid chromatography–tandem mass spectrometry (LC–MS–MS). The developed method is independent of hair treatment. Even hair cut from test persons after customary hair wash can be analyzed. After treatment of natural and bleached hair tresses using a real-life treatment procedure 180 g and 300 g of polymer per gram hair were quantified, respectively. Additionally the fragmentation mechanism of the cationic N-hydroxypropyl-N,N,N-trimethylammonium group during electrospray ionization was investigated. A mass loss of 60 Da in combination with loss of a single charge is observed and associated with cleavage of trimethylamine and a proton. It is assumed that this process is promoted by the anionic counter-ion which might be hydroxide in an aqueous environment.     

(13)C,(13)C]- and [(13)C,(1)H]-TROSY in a triple resonance experiment for ribose-base and intrabase correlations in nucleic acids.

A novel TROSY (transverse relaxation-optimized spectroscopy) element is introduced that exploits cross-correlation effects between (13)C-(13)C dipole-dipole (DD) coupling and (13)C chemical shift anisotropy (CSA) of aromatic ring carbons. Although these (13)C-(13)C effects are smaller than the previously described [(13)C,(1)H]-TROSY effects for aromatic (13)C-(1)H moieties, their constructive use resulted in further transverse relaxation-optimization by up to 15% for the resonances in a 17 kDa protein-DNA complex. As a practical application, two- and three-dimensional versions of the HCN triple resonance experiment for obtaining ribose-base and intrabase correlations in the nucleotides of DNA and RNA (Sklenar, V.; Peterson, R. D.; Rejante, M. R.; Feigon, J. J. Biomol. NMR 1993, 3, 721-727) have been implemented with [(13)C,(1)H]- and [(13)C,(13)C]-TROSY elements to reduce the rate of transverse relaxation during the polarization transfers between ribose (13)C1' and base (15)N1/9 spins, and between (13)C6/8 and N1/9 within the bases. The resulting TROSY-HCN experiment is user-friendly, with a straightforward, robust experimental setup. Compared to the best previous implementations of the HCN experiment, 2-fold and 5-fold sensitivity enhancements have been achieved for ribose-base and intrabase connectivities, respectively, for (13)C,(15)N-labeled nucleotides in structures with molecular weights of 10 and 17 kDa. TROSY-HCN experiments should be applicable also with significantly larger molecular weights. By using modified TROSY-HCN schemes, the origins of the sensitivity gains have been analyzed.     

Characterization of hydrogen bond lengths in Watson-Crick base pairs by cross-correlated relaxation

Hydrogen bond lengths in Watson-Crick base pairs can be characterized by cross-correlated relaxation between 1H chemical shift anisotropy and dipole-dipole coupling of 1H and its hydrogen bond acceptor 15N. As a reference, the cross-correlated relaxation between 1H chemical shift anisotropy and dipole-dipole coupling of 1H and its hydrogen bond donor 15N is used. With the two measured cross-correlated relaxation rates, an apparent hydrogen bond length can be determined, which is composed by the hydrogen bond length multiplied by a term representing the amplitude of inter-base motions. Data are presented for the 15N3-1H3...15N1 hydrogen bonds in A=T base pairs of the Antennapedia homeodomain-DNA complex with a correlation time of global rotational diffusion of 20 ns.