Dronenburg v. Zech: Fundamental Rights and the Military

In Dronenburg v. Zech, the United States Court of Appeals, District of Columbia Circuit, upheld the discharge of a Navy Petty Officer on the grounds that he had engaged in homosexual relations while a member of the service. The initial basis for the discharge was Instruction 1900.9C, promulgated by the Secretary of the Navy, which provided for the separation from the naval service of any member who solicits, attempts, or engages in homosexual acts. In reaching its decision, the court held that the Navy had not violated Dronenburg\u27s constitutional rights to privacy and equal protection of the laws. The court also concluded that the Navy\u27s policy of discharging homosexual members was rationally related to a permissible objective and, in a larger context, that there is no constitutional right to engage in homosexual relations. The broad dicta in Dronenburg and the fact that the court disposed of the appeal on general constitutional grounds applicable in a civil context makes the opinion of the court of more than passing interest to civilians. This Note will discuss some of the implications for both servicepeople and civilians arising out of Dronenburg and its forebears

Dronenburg v. Zech: Fundamental Rights and the Military

In Dronenburg v. Zech, the United States Court of Appeals, District of Columbia Circuit, upheld the discharge of a Navy Petty Officer on the grounds that he had engaged in homosexual relations while a member of the service. The initial basis for the discharge was Instruction 1900.9C, promulgated by the Secretary of the Navy, which provided for the separation from the naval service of any member who solicits, attempts, or engages in homosexual acts. In reaching its decision, the court held that the Navy had not violated Dronenburg\u27s constitutional rights to privacy and equal protection of the laws. The court also concluded that the Navy\u27s policy of discharging homosexual members was rationally related to a permissible objective and, in a larger context, that there is no constitutional right to engage in homosexual relations. The broad dicta in Dronenburg and the fact that the court disposed of the appeal on general constitutional grounds applicable in a civil context makes the opinion of the court of more than passing interest to civilians. This Note will discuss some of the implications for both servicepeople and civilians arising out of Dronenburg and its forebears

Influence of biomaterial nanotopography on the adhesive and elastic properties of Staphylococcus aureus cells

Despite the well-known beneficial effects of biomaterial nanopatterning on host tissue integration, the influence of controlled nanoscale topography on bacterial colonisation and infection remains unknown. Therefore, the aim of the present study was to determine the nanoscale effect of surface nanopatterning on biomaterial colonisation by S. aureus, utilising AFM nanomechanics and single-cell force spectroscopy (SCFS). Nanoindentation of S. aureus bound to planar (PL) and nanopatterned (SQ) polycarbonate (PC) surfaces suggested two distinct areas of mechanical properties, consistent with a central bacterial cell surrounded by a capsullar component. Nevertheless, no differences in elastic moduli were found between bacteria bound to PL and SQ, suggesting a minor role of nanopatterning in bacterial cell elasticity. Furthermore, SCFS demonstrated increased adhesion forces and work between S. aureus and SQ surfaces at 0 s and 1 s contact times. Although WLC modelling showed similarities in contour lengths for attachment to both surfaces, Poisson analysis suggests increased short-range forces for the S. aureus–SQ interactions. In the case of S. aureus–PL, long-range forces were found to not only be dominant but also repulsive in nature, which may help explain the reduced adhesion forces observed during AFM probing. In conclusion, although surface nanopatterning does not significantly influence the elasticity of attached bacterial cells, it was found to promote the early-adhesion of S. aureus cells to the biomaterial surface

Cell migration on material-driven fibronectin microenvironments

Cell migration is a fundamental process involved in a wide range of biological phenomena. However, how the underlying mechanisms that control migration are orchestrated is not fully understood. In this work, we explore the migratory characteristics of human fibroblasts using different organisations of fibronectin (FN) triggered by two chemically similar surfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA); cell migration is mediated via an intermediate layer of fibronectin (FN). FN is organised into nanonetworks upon simple adsorption on PEA whereas a globular conformation is observed on PMA. We studied cell speed over the course of 24 h and the morphology of focal adhesions in terms of area and length. Additionally, we analysed the amount of cell-secreted FN as well as FN remodelling. Velocity of human fibroblasts was found to exhibit a biphasic behaviour on PEA, whereas it remained fairly constant on PMA. FA analysis revealed more mature focal adhesions on PEA over time contrary to smaller FAs found on PMA. Finally, human fibroblasts seemed to remodel adsorbed FN more on PMA than on PEA. Overall, these results indicate that the cell–protein–material interface affects cell migratory behaviour. Analysis of FAs together with FN secretion and remodelling were associated with differences in cell velocity providing insights into the factors that can modulate cell motility

Two strategies to engineer flexible loops for improved enzyme thermostability

The stereospecifically controlled carbon-carbon bond forming ability of Escherichia coli transketolase (TK) makes it very promising as a biocatalyst in industry. However, as a mesophilic enzyme the enzyme suffers the limitation of low stability to elevated temperatures and extremes of pH, limiting its current use in industrial processes. Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. In this study, two parallel strategies were applied to identify mutation candidates within the flexible loops of TK. The first was a “back to consensus mutations” approach, and the second was computational design based on ΔΔG calculations in Rosetta. Forty-nine single variants were generated and characterised experimentally. From these, three single-variants I189H, A282P, D143K were found to be more thermostable than wild-type TK. The combination of A282P with H192P, a variant constructed previously, resulted in the best all-round variant with a 3-fold improved half-life at 60 °C, 5-fold increased specific activity at 65 °C, 1.3-fold improved kcat and a Tm increased by 5 °C above that of wild type. Based on a statistical analysis of the stability changes for all variants, the qualitative prediction accuracy of the Rosetta program reached 65.3%. Furthermore, molecular dynamics (MD) simulations of variants confirmed a good inverse correlation between protein stability and local flexibility which was determined by the magnitude of fluctuations with respect to the average conformations. Both of the two strategies investigated were useful in guiding mutation candidates to flexible loops, and had the potential to be used for other enzymes

Engineer flexible loops for improved enzyme thermostability

Enzyme thermostability is a critical factor for its wide applications in industrial fields. Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. The stereospecifically controlled carbon-carbon bond forming ability of Escherichia coli transketolase (TK) makes it very promising as a biocatalyst in industry. However, as a mesophilic enzyme, it suffers the limitation of low stability to elevated temperatures and extremes of pH, limiting its current use in industrial processes. In order to improve thermostability of TK, we have applied two parallel strategies to identify mutation candidates within the flexible loops. The first was a “back to consensus mutations” approach, and the second was computational design based on ΔΔG calculations in Rosetta. Forty-nine single variants were generated and characterized experimentally. From these, three single-variants I189H, A282P, D143K were found to be more thermostable than wild-type TK. The combination of A282P with H192P, a variant constructed previously, resulted in the best all-round variant with a 3-fold improved half-life at 60 °C, 5-fold increased specific activity at 65 °C, 1.3-fold improved kcat and a Tm increased by 5 °C above that of wild type. Based on a statistical analysis of the stability changes for all variants, the qualitative prediction accuracy of the Rosetta program reached 65.3%. Furthermore, molecular dynamics (MD) simulations of variants confirmed a good inverse correlation between protein stability and local flexibility which was determined by the magnitude of fluctuations with respect to the average conformations. Both of the two strategies investigated were useful in guiding mutation candidates to flexible loops, and had the potential to be used for other enzyme

Stability Convergence in Antibody Coformulations

Combined administration of antibody therapeutics has proven to be beneficial for patients with cancer or infectious diseases. As a result, there is a growing trend toward multiple antibodies premixed into a single product form and delivered to patients as a fixed-dose coformulation. However, combining antibodies into a single coformulation could be challenging as proteins have the potential to interact and alter their stability and degradation profiles in the mixture, compared to that in isolation. We show that in two specific antibody-antibody coformulations, the more stable antibody component increased the stability of the less stable component, which in return destabilized the more stable component, hence exhibiting an overall convergence of stability in the coformulation

Directed evolution strategies for improved enzymatic performance

The engineering of enzymes with altered activity, specificity and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. However, the general acceptance of these methods as a route to new biocatalysts for organic synthesis requires further improvement of the methods for both ease-of-use and also for obtaining more significant changes in enzyme properties than is currently possible. Recent advances in library design, and methods of random mutagenesis, combined with new screening and selection tools, continue to push forward the potential of directed evolution. For example, protein engineers are now beginning to apply the vast body of knowledge and understanding of protein structure and function, to the design of focussed directed evolution libraries, with striking results compared to the previously favoured random mutagenesis and recombination of entire genes. Significant progress in computational design techniques which mimic the experimental process of library screening is also now enabling searches of much greater regions of sequence-space for those catalytic reactions that are broadly understood and, therefore, possible to model. Biocatalysis for organic synthesis frequently makes use of whole-cells, in addition to isolated enzymes, either for a single reaction or for transformations via entire metabolic pathways. As many new whole-cell biocatalysts are being developed by metabolic engineering, the potential of directed evolution to improve these initial designs is also beginning to be realised

Two-substrate enzyme engineering using small libraries that combine the substrate preferences from two different variant lineages

Improving the range of substrates accepted by enzymes with high catalytic activity remains an important goal for the industrialisation of biocatalysis. Many enzymes catalyse two-substrate reactions which increases the complexity in engineering them for the synthesis of alternative products. Often mutations are found independently that can improve the acceptance of alternatives to each of the two substrates. Ideally, we would be able to combine mutations identified for each of the two alternative substrates, and so reprogramme new enzyme variants that synthesise specific products from their respective two-substrate combinations. However, as we have previously observed for E. coli transketolase, the mutations that improved activity towards aromatic acceptor aldehydes, did not successfully recombine with mutations that switched the donor substrate to pyruvate. This likely results from several active site residues having multiple roles that can affect both of the substrates, as well as structural interactions between the mutations themselves. Here, we have designed small libraries, including both natural and non-natural amino acids, based on the previous mutational sites that impact on acceptance of the two substrates, to achieve up to 630× increases in kcat for the reaction with 3-formylbenzoic acid (3-FBA) and pyruvate. Computational docking was able to determine how the mutations shaped the active site to improve the proximity of the 3-FBA substrate relative to the enamine-TPP intermediate, formed after the initial reaction with pyruvate. This work opens the way for small libraries to rapidly reprogramme enzyme active sites in a plug and play approach to catalyse new combinations of two-substrate reactions

Influence of biomaterial nanotopography on the adhesive and elastic properties of Staphylococcus aureus cells

Despite the well-known beneficial effects of biomaterial nanopatterning on host tissue integration, the influence of controlled nanoscale topography on bacterial colonisation and infection remains unknown. Therefore, the aim of the present study was to determine the nanoscale effect of surface nanopatterning on biomaterial colonisation by S. aureus, utilising AFM nanomechanics and single-cell force spectroscopy (SCFS). Nanoindentation of S. aureus bound to planar (PL) and nanopatterned (SQ) polycarbonate (PC) surfaces suggested two distinct areas of mechanical properties, consistent with a central bacterial cell surrounded by a capsullar component. Nevertheless, no differences in elastic moduli were found between bacteria bound to PL and SQ, suggesting a minor role of nanopatterning in bacterial cell elasticity. Furthermore, SCFS demonstrated increased adhesion forces and work between S. aureus and SQ surfaces at 0 s and 1 s contact times. Although WLC modelling showed similarities in contour lengths for attachment to both surfaces, Poisson analysis suggests increased short-range forces for the S. aureus–SQ interactions. In the case of S. aureus–PL, long-range forces were found to not only be dominant but also repulsive in nature, which may help explain the reduced adhesion forces observed during AFM probing. In conclusion, although surface nanopatterning does not significantly influence the elasticity of attached bacterial cells, it was found to promote the early-adhesion of S. aureus cells to the biomaterial surface