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The development of biopharmaceutical downstream processes relies on exhaustive experimental studies. The root cause is the poorly understood relationship between the protein structure of monoclonal antibodies (mAbs) and their macroscopic process behavior. Especially the development of preparative chromatography processes is challenged by the increasing structural complexity of novel antibody formats and accelerated development timelines. This study introduces a multiscale in silico model consisting of homology modeling, quantitative structure-property relationships (QSPR), and mechanistic chromatography modeling leading from the amino acid sequence of a mAb to the digital representation of its cation exchange chromatography (CEX) process. The model leverages the mAbs' structural characteristics and experimental data of a diverse set of 21 therapeutic antibodies to predict elution profiles of two mAbs that were removed from the training data set. QSPR modeling identified mAb-specific protein descriptors relevant for the prediction of the thermodynamic equilibrium and the stoichiometric coefficient of the adsorption reaction. The consideration of two discrete conformational states of IgG4 mAbs enabled prediction of split-peak elution profiles. Starting from the sequence, the presented multiscale model allows in silico development of chromatography processes before protein material is available for experimental studies.

Powder compaction-induced surface chemistry in metal oxide nanocrystal ensembles is important for very diverse fields such as triboelectrics, tribocatalysts, surface abrasion, and cold sintering of ceramics. Using a range of spectroscopic techniques, we show that MgO nanocube powder compaction with uniaxial pressures that can be achieved by gentle manual rubbing or pressing (p ≥ 5 MPa) excites energetic electron-hole pairs and generates oxygen radicals at interfacial defect structures. While the identification of paramagnetic O- radicals and their adsorption complexes with O2 point to the emergence of hole centers, triboemitted electrons become scavenged by molecular oxygen to convert into adsorbed superoxide anions O2 - as measured by electron paramagnetic resonance (EPR). By means of complementary UV-photoexcitation experiments, we found that photon energies in the range between 3 and 6 eV produce essentially the same EPR spectroscopic fingerprints and optical absorption features. To provide insights into this effect, we performed density functional theory calculations to explore the energetics of charge separation involving the ionization of low-coordinated anions and surface-adsorbed O2 - radicals at points of contact. For all selected configurations, charge transfer is not spontaneous but requires an additional driving force. We propose that a plausible mechanism for oxygen radical formation is the generation of significant surface potential differences at points of contact under loading as a result of the highly inhomogeneous elastic deformations coupled with the flexoelectric effect.

Developing simple, inexpensive, and environmentally benign approaches to integrate morphologically well-defined nanoscale building blocks into larger high surface area materials is a key challenge in materials design and processing. In this work, we investigate the fundamental surface phenomena between MgO and water (both adsorption and desorption) with particles prepared via a vapor-phase process (MgO nanocubes) and a modified aerogel process (MgO(111) nanosheets). Through these studies, we unravel a strategy to assemble individual MgO nanoparticles into extended faceted single-crystalline MgO nanosheets and nanorods with well-defined exposed surfaces and edges. This reorganization can be triggered by the presence of H2O vapor or bulk liquid water. Water adsorption and the progressive conversion of vapor-phase grown oxide particles into hydroxides give rise to either one-dimensional or two-dimensional (1D or 2D) structures of high dispersion and surface area. The resulting Mg(OH)2 lamella with a predominant (001) surface termination are well-suited precursor structures for their topotactic conversion into laterally extended and uniform MgO(111) grain surface configurations. To understand the potential of polar (111) surfaces for faceting and surface reconstruction effects associated with water desorption, we investigated the stability of MgO(111) nanosheets during vacuum annealing and electron beam exposure. The significant surface reconstruction of the MgO(111) surfaces observed shows that adsorbate-free (111)-terminated surfaces of unsupported MgO nanostructures reconstruct rather than remain as charged planes of either three-fold coordinated O2– ion or Mg2+ ions. Thus, here we demonstrate the role water can play in surface formation and reconstruction by bridging wet chemical and surface science inspired approaches., The organization of MgO nanocubes as primary building blocks can be achieved by materials contact with water and subsequent thermal treatment in a vacuum.

Atomic dispersion of dopants and control over their defect chemistry are central goals in the development of oxide nanoparticles for functional materials with dedicated electronic, optical or magnetic properties. We produced highly dispersed oxide nanocubes with atomic distribution of cobalt ions in substitutional sites of the MgO host lattice via metal organic chemical vapor synthesis. Vacuum annealing of the nanoparticle powders up to 1173 K has no effect on the shape of the individual particles and only leads to moderate particle coarsening. Such materials processing, however, gives rise to the electronic reduction of particle surfaces, which—upon O2 admission—stabilize anionic oxygen radicals that are accessible to UV/Vis diffuse reflectance and electron paramagnetic resonance (EPR) spectroscopy. Multi‐reference quantum chemical calculations show that the optical bands observed mainly originate from transitions into 4A2g (4F), 4T1g (4P) states with a contribution of transitions into 2T1g, 2T2g (2G) states through spin‐orbit coupling and gain intensity through vibrational motion of the MgO lattice or the asymmetric ion field. Related nanostructures are a promising material system for single atomic site catalysis. At the same time, it represents an extremely valuable model system for the study of interfacial electron transfer processes that are key to nanoparticle chemistry and photochemistry at room temperature, and in heterogeneous catalysis., A MgO host lattice with an atomic distribution of cobalt ions in substitutional sites was achieved via metal organic chemical vapor synthesis, providing a promising material system for single atomic site catalysis and a good model system for the study of interfacial electron transfer processes that are key to nanoparticle chemistry and photochemistry at room temperature, and in heterogeneous catalysis.

Identification and manipulation of transition-metal ion impurities in oxide nanoparticles require an in-depth understanding of their stability, segregation behavior, and, at the same time, knowledge about their surface reactivity. Powders of magnesium oxide nanoparticles with admixtures of iron or cobalt ions, as two next neighbors in the periodic table, were synthesized in the gas phase via injection of metal–organic precursors into the magnesium combustion flame followed by temperature quenching of resulting nanocrystals in argon. In these model systems of cubic nanocrystals, we explored the distinct stability of these impurities in great detail. While Co2+ ions keep their divalent valence state and substitute the host ions in the cationic sublattice, Fe3+ ions emerge due to the energy gain provided by charge compensation and impurity–vacancy complex formation. The very different behavior of Co and Fe ions in the MgO host lattice, their changes in the local environment, and the different trends in segre...

It is an important goal of computational biology to correctly predict the association state of a protein based on its amino acid sequence and the structures of known homologues. We have pursued this goal on the example of anthranilate phosphoribosyltransferase (AnPRT), an enzyme that is involved in the biosynthesis of the amino acid tryptophan. Firstly, known crystal structures of naturally occurring homodimeric AnPRTs were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA) service of the European Bioinformatics Institute (EBI). This led to the identification of two hydrophobic "hot spot" amino acids in the protein-protein interface that were predicted to be essential for self-association. Next, in a comprehensive multiple sequence alignment (MSA), naturally occurring AnPRT variants with hydrophilic or charged amino acids in place of hydrophobic residues in the two hot spot positions were identified. Representative variants were characterized in terms of thermal stability, enzymatic activity, and quaternary structure. We found that AnPRT variants with charged residues in both hot spot positions exist exclusively as monomers in solution. Variants with hydrophilic amino acids in one hot spot position occur in both forms, monomer and dimer. The results of the present study provide a detailed characterization of the determinants of the AnPRT monomer-dimer equilibrium and show that analysis of hot spots in combination with MSAs can be a valuable tool in prediction of protein quaternary structures.

MgO nanocubes are well-suited for the study of defects and interfaces inside metal oxide powders and ceramics. We evaluated the synthesis of MgO nanocubes via flame spray pyrolysis (FSP) and compared the structural and spectroscopic particle properties with materials obtained by chemical vapor synthesis (CVS). Characterization of the as-synthesized and vacuum annealed powder samples revealed slightly larger particle sizes for the FSP material that is absolutely comparable to CVS powders in terms of the particles structural definition, morphology and crystallinity. Moreover, optical absorption and photoluminescence emission studies show fingerprints, which are characteristic of MgO nanocrystals with a high abundance of grain corners, kinks and edges. The opportunity to synthesize MgO nanoparticles of high structural definition via FSP at significantly larger production rates and lower costs compared to CVS carries great potential to upscale powder production to generate MgO based functional oxide nanocomposites for ceramics and catalysis.

Nanostructured segregates of alkaline earth oxides exhibit bright photoluminescence emission and great potential as components of earth-abundant inorganic phosphors. We evaluated segregation engineering of Ca2+- and Ba2+-admixtures in sintered MgO nanocube-derived compacts. Compaction and sintering transform the nanoparticle agglomerates into ceramics with residual porosities of Φ = 24-28%. Size mismatch drives admixture segregation into the intergranular region, where they form thin metal oxide films and inclusions decorating grain boundaries and pores. An important trend in the median grain size evolution of the sintered bodies with dCa(10 at. %) = 90 nm < dBa(1 at. %) = 160 nm < dMgO = 250 nm ∼ dCa(1 at. %) = 280 nm < dBa(10 at. %) = 870 nm is rationalized by segregation and interface energies, barriers for ion diffusion, admixture concentration, and the increasing surface basicity of the grains during processing. We outline the potential of admixtures on interface engineering in MgO nanocrystal-derived ceramics and demonstrate that in the sintered compacts, the photoluminescence emission originating from the grain surfaces is retained. Interior parts of the ceramic, which are accessible to molecules from the gas phase, contribute with oxygen partial pressure-dependent intensities to light emission.

Adsorption of organic molecules at ZnO nanoparticle surfaces enables the transfer of energy or charge across resulting organic-inorganic interfaces and, consequently, determines the optoelectronic performance of ZnO based hybrids. We investigated on aqueous colloidal ZnO dispersions adsorption-induced changes with photoluminescence (PL) and electron paramagnetic resonance (EPR) spectroscopy. Citrate and acetate ion adsorption increases or decreases radiative exciton annihilation at hν = 3.3 eV and at room temperature, respectively. Searching for a correspondence between PL emission and the yield of trapped charge carriers originating from exciton separation - using photon energies of hν = 4.6 eV and fluxes of = 10(14) cm(-2) s(-1) for excitation - we found that there is a negligible fraction of paramagnetic products that originate from exciton separation. Upon polychromatic excitation with significantly higher photon fluxes (Ṅ(ph)= 10(16) cm(-2)·s(-1)), ZnO specific shallow defects trap unpaired electrons in citrate and acetate functionalized samples. The adsorption dependent PL intensity changes and the excitation parameter dependent yield of separated charges (EPR) in colloidal ZnO nanoparticles underline that the distribution over the different exciton annihilation channels sensitively depends on interface composition and the intensity of the photoexcitation light.

We analyze how a reduction of the tax rate on corporate income from intellectual property (IP) in one country, known as a patent box regime, affects corporate R&D activity in other countries. Combining data on patents and multinational corporation networks, we show that the cross-border effect of tax policy changes depends on whether co-location of the IP and the underlying R&D activity is required. Patent boxes without such a requirement increase patent output in other countries. Patent boxes with such a requirement reduce patent output abroad but only when relocation costs for R&D activity are small.

Existing evidence shows that R&D tax incentives boost countries’ private sector R&D. Given the importance of multinational enterprises (MNEs) for private sector innovation, it is unclear, however, whether firms engage in genuinely new R&D or whether R&D is reallocated across borders. Drawing on data on unconsolidated R&D activity of MNEs in Europe, we provide evidence that responses are dominated by cross-border relocations: More generous tax incentives in one country increase MNEs’ R&D investments in affiliates located there, while lowering R&D investments in affiliates of the same MNE group located in other countries. Globally, firms hardly raise their R&D activities when tax incentives become more generous.

Microstructure, structure, and compositional homogeneity of metal oxide nanoparticles can change dramatically during catalysis. Considering the different stabilities of cobalt and iron ions in the MgO host lattice [M. Niedermaier et al., J. Phys. Chem. C 123, 25991 (2019)], we employed MgO nanocube powders with or without transition metal admixtures for the oxidative coupling of methane (OCM) reaction to analyze characteristic differences in catalytic activity and sintering behavior. Undoped MgO nanocrystals exhibit the highest C2 selectivity and retain the nanocrystallinity of the starting material after 24 h time on stream. For the Co–Mg–O nanoparticle powder, which exhibits the highest activity and COx selectivity and where OCM-induced coarsening is strongest, we found that the Co2+ ions remain homogeneously distributed over the MgO lattice. Trivalent Fe ions migrate to the surface of Fe–Mg–O nanoparticles where they form a magnesioferrite phase (MgFe2O4) with a characteristic impact on catalytic performance: Fe–Mg–O is initially less selective than MgO despite its lower activity. An increase in C2 selectivity and a decrease in the CO2/CO ratio with time on stream are attributed to the increasing fraction of coarsened particles that become depleted in redox active Fe. Surface water is a by-product of the OCM reaction, favors mass transport across the particle surfaces, and serves as a sintering aid during catalysis. The characteristic changes in size and morphology of MgO, Co-doped, and Fe-doped MgO particles can be consistently explained by activity and C2 selectivity trends. The original morphology of the nanocubes as a starting material for the OCM reaction does not impact the catalytic activity.

Metal oxide nanocomposites are non‐equilibrium solids and promising precursors for functional materials. Annealing of such materials can provide control over impurity segregation and, depending on the level of consolidation, represents a versatile approach to engineer free surfaces, particle‐particle interfaces and grain boundaries. Starting with indium‐magnesium‐oxide nanoparticle powders obtained via injection of an indium organic precursor into the magnesium combustion flame and subsequent particle quenching in argon, we investigated the stability of the trivalent In3+ ions in the host lattice of MgO nanoparticles by determining grain growth, morphology evolution and impurity segregation. The latter process is initiated by vacuum annealing at 873 K and can be tracked at 1173 K on a time scale of minutes. In the first instance the surface segregated indium wets the nanoparticle interfaces. After prolonged annealing indium evaporates and leaves the powder via the gas phase. Resulting MgO nanocubes are devoid of residual indium, regain their high morphological definition and show spectroscopic fingerprints (UV Diffuse Reflectance and Photoluminescence emission) that are characteristic of electronically unperturbed MgO cube corner and edge features. The results of this combined XRD, TEM, and spectroscopy study reveal the parameter window within which control over indium segregation is used to introduce a semiconducting metal oxide component into the intergranular region between insulating MgO nanograins.

Existing evidence shows that R&D tax incentives boost countries' private sector R&D. Given the importance of multinational enterprises (MNEs) for private sector innovation, it is unclear, however, whether firms engage in genuinely new R&D or whether R&D is reallocated across borders. Drawing on data on unconsolidated R&D activity of MNEs in Europe, we provide evidence that responses are dominated by cross-border relocations: More generous tax incentives in one country increase MNEs' R&D investments in affiliates located there, while lowering R&D investments in affiliates of the same MNE group located in other countries. Globally, firms hardly raise their R&D activities when tax incentives become more generous.

The selection of optimized enzymes from gene libraries is important, both for basic and applied research. Here, we first describe the generation of plasmid-borne libraries using error-prone PCR and highly competent Escherichia coli cells. We then provide protocols for the use of these libraries for auxotrophic selection assays with E. coli and the extremely thermophilic bacterium Thermus thermophilus as hosts.

The selection of optimized enzymes from gene libraries is important, both for basic and applied research. Here, we first describe the generation of plasmid-borne libraries using error-prone PCR and highly competent Escherichia coli cells. We then provide protocols for the use of these libraries for auxotrophic selection assays with E. coli and the extremely thermophilic bacterium Thermus thermophilus as hosts.

In 2015/16, the photomultiplier cameras of the H.E.S.S. Cherenkov telescopes CT1-4 have undergone a major upgrade. The entire electronics has been replaced, using NECTAr chips for the front-end readout. A new ventilation system has been installed and several auxiliary components have been replaced. Besides this, the internal control and readout software was rewritten from scratch in a modern and modular way. Ethernet technology was used wherever possible to ensure both flexibility, stability and high bandwidth. An overview of the installed components will be given. In 2015/16, the photomultiplier cameras of the H.E.S.S. Cherenkov telescopes CT1-4 have undergone a major upgrade. The entire electronics has been replaced, using NECTAr chips for the front-end readout. A new ventilation system has been installed and several auxiliary components have been replaced. Besides this, the internal control and readout software was rewritten from scratch in a modern and modular way. Ethernet technology was used wherever possible to ensure both flexibility, stability and high bandwidth. An overview of the installed components will be given.

For production of different monoclonal antibodies (mAbs), biopharmaceutical companies often use related upstream and downstream manufacturing processes. Such platforms are typically characterized regarding influence of upstream and downstream process (DSP) parameters on critical quality attributes (CQAs). CQAs must be monitored strictly by an adequate control strategy. One such process-related CQA is the content of host cell protein (HCP) which is typically analyzed by immunoassay methods (e.g., HCP-ELISA). The capacity of the immunoassay to detect a broad range of HCPs, relevant for the individual mAb-production process should be proven by orthogonal proteomic methods such as 2D gel electrophoresis or mass spectrometry (MS). In particular MS has become a valuable tool to identify and quantify HCP in complex mixtures. We evaluate up- and DSP parameters of four different biopharmaceutical products, two different process variants, and one mock fermentation on the HCP pattern by shotgun MS analysis and ELISA. We obtained a similar HCP pattern in different cell culture fluid harvests compared to the starting material from the downstream process. During the downstream purification process of the mAbs, the HCP level and the number of HCP species significantly decreased, accompanied by an increase in diversity of the residual HCP pattern. Based on this knowledge, we suggest a control strategy that combines multi product ELISA for in-process control and release analytics, and MS testing for orthogonal HCP characterization, to attain knowledge on the HCP level, clusters and species. This combination supports a control strategy for HCPs addressing safety and efficacy of biopharmaceutical products.

International audience; The High Energy Stereoscopic System (H.E.S.S.) is an array of imaging atmospheric Cherenkov telescopes (IACTs) located in Namibia. It was built to detect Very High Energy (VHE, >100 GeV) cosmic gamma rays, and consists of four 12 m diameter Cherenkov telescopes (CT1-4), built in 2003, and a larger 28 m telescope (CT5), built in 2012. The larger mirror surface of CT5 permits to lower the energy threshold of the array down to 30 GeV. The cameras of CT1-4 are currently undergoing an extensive upgrade, with the goals of reducing their failure rate, reducing their readout dead time and improving the overall performance of the array. The entire camera electronics has been renewed from ground-up, as well as the power, ventilation and pneumatics systems, and the control and data acquisition software. Technical solutions forseen for the next-generation Cherenkov Telescope Array (CTA) observatory have been introduced, most notably the readout is based on the NECTAr analog memory chip. The camera control subsystems and the control software framework also pursue an innovative design, increasing the camera performance, robustness and flexibility. The CT1 camera has been upgraded in July 2015 and is currently taking data; CT2-4 will upgraded in Fall 2016. Together they will assure continuous operation of H.E.S.S at its full sensitivity until and possibly beyond the advent of CTA. This contribution describes the design, the testing and the in-lab and on-site performance of all components of the newly upgraded H.E.S.S. camera.

International audience; The 14 years old cameras of the H.E.S.S. 12-m telescopes have been upgraded in 2015/2016, with the goals of reducing the system failure rate, reducing the dead time and improving the overall performance of the array. This conference contribution describes the various tests that were carried out on the cameras and their sub-components both in the lab and on site. It also gives an overview of the commissioning and calibration procedures adopted during and after the installation, including e.g. flat-fielding and trigger threshold scans. Finally, it reports in detail about the overall performance of the four new H.E.S.S-I cameras, using very recent data.

We present results for the fluctuations of the displacement of a tracer particle on a planar lattice pulled by a step force in the presence of impenetrable, immobile obstacles. The fluctuations perpendicular to the applied force are evaluated exactly in first order of the obstacle density for arbitrarily strong pulling and all times. The complex time-dependent behavior is analyzed in terms of the diffusion coefficient, local exponent, and the non-Skellam parameter, which quantifies deviations from the dynamics on the lattice in the absence of obstacles. The non-Skellam parameter along the force is analyzed in terms of an asymptotic model and reveals a power-law growth for intermediate times., Comment: Physical Review E; 12 pages, 5 figures

We analyze the cross-border effect of tax cuts on R&D activity in the context of profit shifting. A tax cut in one location of a multinational enterprise reduces the user cost of capital for the whole group if profit shifting is possible and exerts a positive cross-border effect on R&D output. Using micro-level data, we find an increase of patent output of 15% upon the implementation of a foreign tax cut for firms with cross-border links. In addition, we find that foreign tax cuts prohibiting profit shifting generate a negative cross-border effect on average patent quality., Series: WU International Taxation Research Paper Series

International audience; The High Energy Stereoscopic System (HESS) is an array of imaging atmospheric Cherenkov telescopes (IACTs) located in the Khomas highland in Namibia. It was built to detect Very High Energy (VHE >100GeV ) cosmic gamma rays. Since 2003, HESS has discovered the majority of the known astrophysical VHE gamma-ray sources, opening a new observational window on the extreme non-thermal processes at work in our universe. HESS consists of four 12-m diameter Cherenkov telescopes (CT1–4), which started data taking in 2002, and a larger 28-m telescope (CT5), built in 2012, which lowers the energy threshold of the array to 30GeV . The cameras of CT1–4 are currently undergoing an extensive upgrade, with the goals of reducing their failure rate, reducing their readout dead time and improving the overall performance of the array. The entire camera electronics has been renewed from ground-up, as well as the power, ventilation and pneumatics systems, and the control and data acquisition software. Only the PMTs and their HV supplies have been kept from the original cameras. Novel technical solutions have been introduced, which will find their way into some of the Cherenkov cameras foreseen for the next-generation Cherenkov Telescope Array (CTA) observatory. In particular, the camera readout system is the first large-scale system based on the analog memory chip NECTAr, which was designed for CTA cameras. The camera control subsystems and the control software framework also pursue an innovative design, exploiting cutting-edge hardware and software solutions which excel in performance, robustness and flexibility. The CT1 camera has been upgraded in July 2015 and is currently taking data; CT2-4 have been upgraded in fall 2016. Together they will assure continuous operation of HESS at its full sensitivity until and possibly beyond the advent of CTA. This contribution describes the design, the testing and the in-lab and on-site performance of all components of the newly upgraded HESS camera.

The recombinant expression and biocatalytic characterization of two thermostable pyrimidine nucleoside phosphorylases (PyNP), isolated from Geobacillus thermoglucosidasius (Gt) and Thermus thermophilus (Tt) is described. Both enzymes are highly thermostable (half life of GtPyNP is 1.6 h at 70 °C, half life of TtPyNP is >24 h at 80 °C). Kinetic parameters for the phosphorolysis of natural substrates were determined for GtPyNP at 60 °C ( K m for uridine 2.3 mM, K m for thymidine 1.3 mM) and TtPyNP at 80 °C ( K m for uridine 0.15 mM, K m for thymidine 0.43 mM). The k cat values for uridine are almost identical for both enzymes (ca. 277 s −1 ), while the k cat value for thymidine is about 8 times higher for TtPyNP than for GtPyNP (679 s −1 vs. 83 s −1 ). Both enzymes were tested towards the ability to catalyze the phosphorolytic cleavage of 2′-fluorosubstituted pyrimidine nucleosides – a prerequisite for the efficient synthesis of a number of relevant purine nucleoside analogues. GtPyNP showed poor activity towards 2′-deoxy-2′-fluorouridine (dUrd 2′F ; 0.4% substrate conversion after 30 min), and the phosphorolysis of the epimeric counterpart 1-(2-deoxy-2-fluoro-β- d -arabinofuranosyl)uracil (dUrd 2′F ) could not be detected at all. By contrast, TtPyNP showed dramatically higher conversion rates (15.6% and 1.6% conversion in 30 min of both substrates, respectively). The amount of converted pyrimidine nucleosides increased significantly with time. After 17 h 65% of dUrd 2′F and 46% of dUrd 2′F was phosphorolytically cleaved. Our results demonstrate the potential of TtPyNP as a biocatalyst in transglycosylation reactions aiming at the production of 2′-fluorosubstituted purine nucleosides that are highly bioactive but hardly accessible by chemical methods.

Wild-type Escherichia coli lacZ was subjected to error-prone PCR to generate two plasmid-encoded gene libraries containing approximately 2.6 (SD 1.9) nucleotide exchanges resulting in 1.8 (SD 1.4) amino-acid substitutions. The libraries were used, along with a plasmid containing wild-type lacZ, to transform E. coli lacking genomic lacZ. Cells expressing functional β-galactosidase were identified by blue/white screening. Cell lysates containing the populations of heterogeneously mutagenized β-galactosidase were subjected to single molecule assays using a capillary electrophoresis laser-induced fluorescence-based protocol. There was no significant difference in the average catalytic rate between the random mutagenized and wild-type enzyme populations. Furthermore, there was no clear pattern between error rates and the variances in the population catalytic rates. This suggests that random sequence errors are not a substantial source of the catalytic heterogeneity of this enzyme.

Anthranilate phosphoribosyltransferase from the hyperthermophilic archaeon Sulfolobus solfataricus (ssAnPRT) is encoded by the sstrpD gene and catalyzes the reaction of anthranilate (AA) with a complex of Mg(2+) and 5'-phosphoribosyl-alpha1-pyrophosphate (Mg.PRPP) to N-(5'-phosphoribosyl)-anthranilate (PRA) and pyrophosphate (PP(i)) within tryptophan biosynthesis. The ssAnPRT enzyme is highly thermostable (half-life at 85 degrees C = 35 min) but only marginally active at ambient temperatures (turnover number at 37 degrees C = 0.33 s(-1)). To understand the reason for the poor catalytic proficiency of ssAnPRT, we have isolated from an sstrpD library the activated ssAnPRT-D83G + F149S double mutant by metabolic complementation of an auxotrophic Escherichia coli strain. Whereas the activity of purified wild-type ssAnPRT is strongly reduced in the presence of high concentrations of Mg(2+) ions, this inhibition is no longer observed in the double mutant and the ssAnPRT-D83G single mutant. The comparison of the crystal structures of activated and wild-type ssAnPRT shows that the D83G mutation alters the binding mode of the substrate Mg.PRPP. Analysis of PRPP and Mg(2+)-dependent enzymatic activity indicates that this leads to a decreased affinity for a second Mg(2+) ion and thus reduces the concentration of enzymes with the inhibitory Mg(2).PRPP complex bound to the active site. Moreover, the turnover number of the double mutant ssAnPRT-D83G + F149S is elevated 40-fold compared to the wild-type enzyme, which can be attributed to an accelerated release of the product PRA. This effect appears to be mainly caused by an increased conformational flexibility induced by the F149S mutation, a hypothesis which is supported by the reduced thermal stability of the ssAnPRT-F149S single mutant.

The anthranilate phosphoribosyltransferase from Sulfolobus solfataricus (ssAnPRT) forms a homodimer with a hydrophobic subunit interface. To elucidate the role of oligomerisation for catalytic activity and thermal stability of the enzyme, we loosened the dimer by replacing two apolar interface residues with negatively charged residues (mutations I36E and M47D). The purified double mutant I36E+M47D formed a monomer with wild-type catalytic activity but reduced thermal stability. The single mutants I36E and M47D were present in a monomer-dimer equilibrium with dissociation constants of about 1 microM and 20 microM, respectively, which were calculated from the concentration-dependence of their heat inactivation kinetics. The monomeric form of M47D, which is populated at low subunit concentrations, was as thermolabile as monomeric I36E+M47D. Likewise, the dimeric form of I36E, which was populated at high subunit concentrations, was as thermostable as dimeric wild-type ssAnPRT. These findings show that the increased stability of wild-type ssAnPRT compared to the I36E+M47D double mutant is not caused by the amino acid exchanges per se but by the higher intrinsic stability of the dimer compared to the monomer. In accordance with the negligible effect of the mutations on catalytic activity and stability, the X-ray structure of M47D contains only minor local perturbations at the dimer interface. We conclude that the monomeric double mutant resembles the individual wild-type subunits, and that ssAnPRT is a dimer for stability but not for activity reasons.

The High Energy Stereoscopic System (H.E.S.S.) is an array of imaging atmospheric Cherenkov telescopes (IACTs) located in the Khomas Highland in Namibia. It consists of four 12-m telescopes (CT1-4), which started operations in 2003, and a 28-m diameter one (CT5), which was brought online in 2012. It is the only IACT system featuring telescopes of different sizes, which provides sensitivity for gamma rays across a very wide energy range, from ~30 GeV up to ~100 TeV. Since the camera electronics of CT1-4 are much older than the one of CT5, an upgrade is being carried out; first deployment was in 2015, full operation is planned for 2016. The goals of this upgrade are threefold: reducing the dead time of the cameras, improving the overall performance of the array and reducing the system failure rate related to aging. Upon completion, the upgrade will assure the continuous operation of H.E.S.S. at its full sensitivity until and possibly beyond the advent of CTA. In the design of the new components, several CTA concepts and technologies were used and are thus being evaluated in the field: The upgraded read-out electronics is based on the NECTAR readout chips; the new camera front- and back-end control subsystems are based on an FPGA and an embedded ARM computer; the communication between subsystems is based on standard Ethernet technologies. These hardware solutions offer good performance, robustness and flexibility. The design of the new cameras is reported here., Comment: Proceedings of the 34th International Cosmic Ray Conference, 30 July- 6 August, 2015, The Hague, The Netherlands

Objectives: To evaluate the effects of MK-0677, an orally active growth hormone (GH) secretagogue, on functional recovery from hip fracture in previously mobile older individuals. Design: Placebo-controlled, randomized, double-blind trial. Setting: Thirteen medical centers in England, Sweden, Denmark, Belgium, Switzerland, Canada, and the United States. Patients were recruited between 3 and 14 days postoperatively, or no more than 18 days postfracture, at acute care hospitals and rehabilitation centers. Participants: One hundred sixty-one hip-fracture patients were enrolled. Entry criteria included consenting hip-fracture patients who were aged 65 and older and who were ambulatory before their fracture, medically stable postoperatively, and mentally competent. Patients were excluded if they had multiple fractures or severe trauma, diabetes mellitus, cancer, uncontrolled hypertension, congestive heart failure, or total hip replacement in the involved extremity. Intervention: Random assignment to 6 months of daily treatment with MK-0677 or placebo. Patients were followed for an additional 6 months after completion of therapy. Measurements: Change from Week 6 to Week 26 in a panel of functional performance measures. Additional outcome measures included change in the Sickness Impact Profile for Nursing Homes (SIP-NH), the ability to live independently, and insulin-like growth factor I (IGF-I) levels. Results: MK-0677 treatment increased serum IGF-I levels by 84% (95% confidence interval (CI)=63-107), compared with an increase of 17% (95% CI=8-28) on placebo. There were no significant differences between MK-0677 and placebo in improvement in functional performance measures or in the overall SIP-NH score. Although MK-0677 patients showed greater improvement relative to placebo in three of four lower extremity functional performance measures, in the physical domain of the SIP, and in the ability to live independently, these differences were not statistically significant. Conclusion: Although MK-0677 treatment increased serum IGF-I, it is uncertain whether clinically significant effects on physical function were achieved. Measuring function in clinical trials in hip-fracture patients is difficult because of the lack of validated outcome measures, high variability, and the lack of a baseline assessment. Present functional performance measures may not be sufficiently responsive for use as the primary endpoint of small intervention studies; alternatively, stimulation of GH may not result in significant functional improvement.