CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering**

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.     

Aging behavior of a hot‐cured epoxy system

The thermal and hydro‐thermal aging of a hot‐cured epoxy system (diglycidylether of bisphenol A (DGEBA) + dicyandiamide (DDA)) in the glassy state is revisited using DSC and IR attenuated total reflection spectroscopy. Because of the diffusion of DDA from the solid particles into the liquid DGEBA matrix, curing produces a highly crosslinked amorphous matrix that contains low crosslinked amorphous regions. After full curing, the network possesses a relatively low molecular mobility and no residual reactive groups. Thermal and hydro‐thermal loading is performed at 60°C, well below the principal glass transition temperature (T_g1 = 171°C). Both aging regimes cause significant chemical and structural changes to the glassy epoxy. It undergoes a phase separation of relatively mobile segments inside the low mobile matrix, providing a second glass transition that shifts from T_g2 = 86–114°C within 108 days of aging. This phase separation is reversible on heating into the viscoelastic state. Hydro‐thermal aging leads to a reversible and a nonreversible plasticizing effect as well. On thermal aging, no chemical changes are observed but hydro‐thermal aging causes significant chemical modifications in the epoxy system. These modifications are identified as a partial degradation of crosslinks produced by the cyano groups of the DDA and correspond to the nonreversible plasticitation. These changes in the cured epoxy should exert an influence on the mechanical properties of an adhesive bond. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci, 2006     

Impacts of new technologies on media usage and social behaviour in domestic environments

Technological infrastructure at home is changing continuously and is becoming increasingly interconnected. Media devices, including the TV set, provide access to the Internet and offer manifold opportunities to consume media on demand. Additionally, personal devices, such as smartphones, also enable flexible consumption and sharing of media. Questions about how these technologies change the user's media usage and how these changes affect the social structure of a household, however, remain largely unanswered. In order to gain insight into the adoption of new technologies into daily routines, we explored these changes in respect of people's media usage in a qualitative long-term Living Lab study. We will present findings regarding personal routines, flexible integration of new devices into existing practices, influences on households as social systems and related issues in device access and collective use. We will highlight potentials and conflicts regarding device shifts and roles; restrictions in device access; social influences in the living room; and individual changes in media consumption.     

GRADIENT OF THE MECHANICAL MODULUS IN GLASS-EPOXY-METAL JOINTS AS MEASURED BY BRILLOUIN MICROSCOPY

The newly developed Brillouin microscopy is used for the first time to measure in situ the longitudinal elastic stiffness coefficient in the GHz-range inside of glass-epoxy-metal joints as a function of distance from the substrates. Interphases with a local variation of mechanical properties are quantitatively characterized. These interphases possess unexpected widths of tens to hundreds of microns. Inside the interphases, the spatial variation of the longitudinal stiffness coefficient depends on the type of substrate, on the curing conditions for the epoxy and probably on the distribution of internal stresses. The obtained spatial mechanical profiles provide valuable insight into the morphology-driven mechanics of the interphase, but additional information is needed for a full understanding of their physical and chemical origin. The presented results prove the sensitivity of the Brillouin microscopy; the elastic stiffness coefficients are detected with an accuracy in the subpercentage range. The spatial resolution is better than 10 µm.     

Data collection in global software engineering research: learning from past experience

Global Software Engineering has become a standard in today’s software industry. Research in distributed software development poses severe challenges that are due to the spatial and temporal distribution of the actors, as well as to language, intercultural and organizational aspects. These challenges occur in addition to “traditional” challenges of the domain itself in large-scale software projects, like coordination and communication issues, requirements volatily, lack of domain knowledge, among others. While several authors have reported empirical studies of global software development projects, the methodological difficulties and challenges of this type of studies have not been sufficiently discussed. In this paper, we share our experiences of collecting and analysing qualitative data in the context of Global Software Engineering projects. We discuss strategies for gaining access to field sites, building trust and documenting distributed and complex work practices in the context of several research projects we have conducted in the past 9 years. The experiences described in this paper illustrate the need to deal with fundamental problems, such as understanding local languages and different cultures, observing synchronous interaction, or dealing with barriers imposed by political conflicts between the sites. Based on our findings, we discuss some practical implications and strategies that can be used by other researchers and provide some recommendations for future research in methodological aspects of Global Software Engineering.     

Locating computer clubs in multicultural neighborhoods: How collaborative project work fosters integration processes

Located in socially and culturally diverse neighborhoods, we have built a network of intercultural computer clubs, called come_IN. These clubs offer a place to share practices among children and adults of diverse ethnical backgrounds. We show how this initiative ties into the striving for the integration of migrant communities and host society in Germany. In this paper, we analyze how collaborative project work and the use of mobile media and technologies contribute to integration processes in multicultural neighborhoods. Qualitative data gathered from interviews with club participants, participative observation in the computer clubs, as well as the analysis of artifacts created during project work provides the background needed to match local needs and peculiarities with (mobile) technologies. Based on these findings we present two approaches to add to the technological infrastructure: (1) a mesh-network extending the clubs into the neighborhood and (2) a project management tool, which supports projects and stimulates the sharing of ideas among projects.     

Structures of the N-Terminal Domain of PqsA in Complex with Anthraniloyl- and 6-Fluoroanthraniloyl-AMP: Substrate Activation in Pseudomonas Quinolone Signal (PQS) Biosynthesis

Pseudomonas aeruginosa, a prevalent pathogen in nosocomial infections and a major burden in cystic fibrosis, uses three interconnected quorum-sensing systems to coordinate virulence processes. At variance with other Gram-negative bacteria, one of these systems relies on 2-alkyl-4(1H)-quinolones (Pseudomonas quinolone signal, PQS) and might hence be an attractive target for new anti-infective agents. Here we report crystal structures of the N-terminal domain of anthranilate-CoA ligase PqsA, the first enzyme of PQS biosynthesis, in complex with anthraniloyl-AMP and with 6-fluoroanthraniloyl-AMP (6FABA-AMP) at 1.4 and 1.7 Å resolution. We find that PqsA belongs to an unrecognized subfamily of anthranilate-CoA ligases that recognize the amino group of anthranilate through a water-mediated hydrogen bond. The complex with 6FABA-AMP explains why 6FABA, an inhibitor of PQS biosynthesis, is a good substrate of PqsA. Together, our data might pave a way to new pathoblockers in P. aeruginosa infections.