A study of low-cost reliable actuators for light aircraft. Part B: Appendices

Computer programs written in FORTRAN are given for time response calculations on pneumatic and linear hydraulic actuators. The programs are self-explanatory with comment statements. Program output is also included

A study of low-cost reliable actuators for light aircraft. Part A: Chapters 1-8

An analysis involving electro-mechanical, electro-pneumatic, and electro-hydraulic actuators was performed to study which are compatible for use in the primary and secondary flight controls of a single engine light aircraft. Actuator characteristics under investigation include cost, reliability, weight, force, volumetric requirements, power requirements, response characteristics and heat accumulation characteristics. The basic types of actuators were compared for performance characteristics in positioning a control surface model and then were mathematically evaluated in an aircraft to get the closed loop dynamic response characteristics. Conclusions were made as to the suitability of each actuator type for use in an aircraft

Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer

Enzymatic oxidation of cell wall polysaccharides by lytic polysaccharide monooxygenases (LPMOs) plays a pivotal role in the degradation of plant biomass. While experiments have shown that LPMOs are copper dependent enzymes requiring an electron donor, the mechanism and origin of the electron supply in biological systems are only partly understood. We show here that insoluble high molecular weight lignin functions as a reservoir of electrons facilitating LPMO activity. The electrons are donated to the enzyme by long-range electron transfer involving soluble low molecular weight lignins present in plant cell walls. Electron transfer was confirmed by electron paramagnetic resonance spectroscopy showing that LPMO activity on cellulose changes the level of unpaired electrons in the lignin. The discovery of a long-range electron transfer mechanism links the biodegradation of cellulose and lignin and sheds new light on how oxidative enzymes present in plant degraders may act in concert.info:eu-repo/semantics/publishe

Quorum sensing-controlled gene expression in lactic acid bacteria

Quorum sensing in lactic acid bacteria (LAB) involves peptides that are directly sensed by membrane-located histidine kinases, after which the signal is transmitted to an intracellular response regulator. This regulator in turn activates transcription of target genes, that commonly include the structural gene for the inducer molecule. The two-component signal-transduction machinery has proven to be indispensable for transcription activation and production of several autoinducers found in LAB, which are predominantly bacteriocins or bacteriocin-like peptides. In the nisin autoregulation process in Lactococcus lactis the NisK protein acts as the sensor for nisin and the NisR protein as the response regulator, activating transcription of target genes. The cis-acting elements for NisR were identified as the nisA and nisF promoter fragments and these were further analysed for inducibility. Based on this knowledge efficient nisin-controlled expression (NICE) systems were developed for several different lactic acid bacteria. A promising application of the NICE system is the development of autolytic starter lactococci, which will lyse in an early stage during cheese ripening thereby facilitating the release of intracellular enzymes which can contribute to flavour formation.

Consensus mutagenesis reveals that non-helical regions influence thermal stability of horseradish peroxidase

The enzyme horseradish peroxidase has many uses in biotechnology but a stabilized derivative would have even wider applicability. To enhance thermal stability, we applied consensus mutagenesis (used successfully with other proteins) to recombinant horseradish peroxidase and generated five single-site mutants. Unexpectedly, these mutations had greater effects on steady-state kinetics than on thermal stability. Only two mutants (T102A, T110V) marginally exceeded the wild type's thermal stability (4% and 10% gain in half-life at 50 °C respectively); the others (Q106R, Q107D, I180F) were less stable than wild type. Stability of a five-fold combination mutant matched that of Q106R, the least-stable single mutant. These results were perplexing: the Class III plant peroxidases display wide differences in thermal stability, yet the consensus mutations failed to reflect these natural variations. We examined the sequence content of Class III peroxidases to determine if there are identifiable molecular reasons for the stability differences observed. Bioinformatic analysis validated our choice of sites and mutations and generated an archetypal peroxidase sequence for comparison with extant sequences. It seems that both genetic variation and differences in protein stability are confined to non-helical regions due to the presence of a highly conserved alpha-helical structural scaffold in these enzymes

Value-based pharmaceutical care at population, hospital and patient level:Integrating value-based health care and cost-effectiveness analysis

At both the European and national levels, the introduction of numerous novel and innovative therapies is gaining momentum, often designed for and applied to large patient populations. From a population-level perspective, it is critical to ensure that these therapies are deployed efficiently, targeting the appropriate patient groups or subgroups. Efficiency in this context encompasses the avoidance of unnecessary treatments and the prevention of systematic overdosing, which could lead to suboptimal patient outcomes and increased healthcare costs. From a pharmaceutical point, it is imperative to systematically assess the long-term implications of these therapies for patient populations and their incorporation into clinical guidelines within hospital settings. The rapid evolution of hospital pharmacy practices, including advancements in pharmaceutical preparation, the expansion of specialized advisory roles, and the implementation of pricing models that align with real-world effectiveness, further underscores the need for a comprehensive evaluation. Moreover, it is essential to integrate the concept of value-based healthcare (VBHC), which prioritizes patient-centered care by focusing on patient-reported outcomes and linking these outcomes to the effectiveness of expensive drugs. The complementary nature of VBHC and cost-effectiveness analysis (CEA) offers the potential to develop new and more sustainable healthcare payment in the future perspective, where scarcity of resources and employees has an impact on the sustainability of pharmaceutical care in the Netherlands.The main aim of this thesis is the integration of the concepts value-based healthcare (VBHC) and cost-effectiveness analysis (CEA) in pharmaceutical care on population, hospital and patient levels; mostly in the context of drugs often labeled as ‘expensive’

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease

Cavities in the hydrophobic core of the neutral protease of Bacillus stearothermophilus were analyzed using a three-dimensional model that was inferred from the crystal structure of thermolysin, the highly homologous neutral protease of B.thermoproteolyticus (85% sequence identity). Site-directed mutagenesis was used to fill some of these cavities, thereby improving hydrophobic packing in the protein interior. The mutations had small effects on the thermostability, even after drastic changes, such as Leu284 --> Trp and Met168 --> Trp. The effects on T50, the temperature at which 50% of the enzyme is irreversibly inactivated in 30 min, ranged from 0.0 to +0.4-degrees-C. These results can be explained by assuming that the mutations have positive and negative structural effects of approximately the same magnitude. Alternatively, it could be envisaged that the local unfolding steps, which render the enzyme susceptible towards autolysis and which are rate limiting in the process of thermal inactivation, are only slightly affected by alterations in the hydrophobic core

In the light of directed evolution: Pathways of adaptive protein evolution

Directed evolution is a widely-used engineering strategy for improving the stabilities or biochemical functions of proteins by repeated rounds of mutation and selection. These experiments offer empirical lessons about how proteins evolve in the face of clearly-defined laboratory selection pressures. Directed evolution has revealed that single amino acid mutations can enhance properties such as catalytic activity or stability and that adaptation can often occur through pathways consisting of sequential beneficial mutations. When there are no single mutations that improve a particular protein property experiments always find a wealth of mutations that are neutral with respect to the laboratory-defined measure of fitness. These neutral mutations can open new adaptive pathways by at least 2 different mechanisms. Functionally-neutral mutations can enhance a protein's stability, thereby increasing its tolerance for subsequent functionally beneficial but destabilizing mutations. They can also lead to changes in “promiscuous” functions that are not currently under selective pressure, but can subsequently become the starting points for the adaptive evolution of new functions. These lessons about the coupling between adaptive and neutral protein evolution in the laboratory offer insight into the evolution of proteins in nature

Heterologous expression of a recombinant lactobacillal -galactosidase in Lactobacillus plantarum: effect of different parameters on the sakacin P-based expression system

Background: Two overlapping genes lacL and lacM (lacLM) encoding for heterodimeric -galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system. In this study, we analyzed different factors that affect the production of recombinant L. reuteri -galactosidase. Results: Various factors related to the cultivation, i.e. culture pH, growth temperature, glucose concentration, as well as the induction conditions, including cell concentration at induction point and inducer concentration, were tested. Under optimal fermentation conditions, the maximum -galactosidase levels obtained were 130 U/mg protein and 3540 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium. As calculated from the specific activity of the purified enzyme (190 U/mg), -galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism. It was observed that pH and substrate (glucose) concentration are the most prominent factors affecting the production of recombinant -galactosidase. Conclusions: The over-expression of recombinant L. reuteri -galactosidase in a food-grade host strain was optimized, which is of interest for applications of this enzyme in the food industry. The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.(VLID)90704