Rational design of serine protease inhibitors

Proteases regulate a spectrum of diverse physiological processes, and dysregulation of proteolytic activity drives a plethora of pathological conditions. Understanding protease function is essential to appreciating many aspects of normal physiology and progression of disease. Consequently, development of potent and specific inhibitors of proteolytic enzymes is vital to provide tools for the dissection of protease function in biological systems and for the treatment of diseases linked to aberrant proteolytic activity. The studies in this thesis describe the rational design of potent inhibitors of three proteases that are implicated in disease development. Additionally, key features of the interaction of proteases and their cognate inhibitors or substrates are analysed and a series of rational inhibitor design principles are expounded and tested. Rational design of protease inhibitors relies on a comprehensive understanding of protease structure and biochemistry. Analysis of known protease cleavage sites in proteins and peptides is a commonly used source of such information. However, model peptide substrate and protein sequences have widely differing levels of backbone constraint and hence can adopt highly divergent structures when binding to a protease’s active site. This may result in identical sequences in peptides and proteins having different conformations and diverse spatial distribution of amino acid functionalities. Regardless of this, protein and peptide cleavage sites are often regarded as being equivalent. One of the key findings in the following studies is a definitive demonstration of the lack of equivalence between these two classes of substrate and invalidation of the common practice of using the sequences of model peptide substrates to predict cleavage of proteins in vivo. Another important feature for protease substrate recognition is subsite cooperativity. This type of cooperativity is commonly referred to as protease or substrate binding subsite cooperativity and is distinct from allosteric cooperativity, where binding of a molecule distant from the protease active site affects the binding affinity of a substrate. Subsite cooperativity may be intramolecular where neighbouring residues in substrates are interacting, affecting the scissile bond’s susceptibility to protease cleavage. Subsite cooperativity can also be intermolecular where a particular residue’s contribution to binding affinity changes depending on the identity of neighbouring amino acids. Although numerous studies have identified subsite cooperativity effects, these findings are frequently ignored in investigations probing subsite selectivity by screening against diverse combinatorial libraries of peptides (positional scanning synthetic combinatorial library; PS-SCL). This strategy for determining cleavage specificity relies on the averaged rates of hydrolysis for an uncharacterised ensemble of peptide sequences, as opposed to the defined rate of hydrolysis of a known specific substrate. Further, since PS-SCL screens probe the preference of the various protease subsites independently, this method is inherently unable to detect subsite cooperativity. However, mean hydrolysis rates from PS-SCL screens are often interpreted as being comparable to those produced by single peptide cleavages. Before this study no large systematic evaluation had been made to determine the level of correlation between protease selectivity as predicted by screening against a library of combinatorial peptides and cleavage of individual peptides. This subject is specifically explored in the studies described here. In order to establish whether PS-SCL screens could accurately determine the substrate preferences of proteases, a systematic comparison of data from PS-SCLs with libraries containing individually synthesised peptides (sparse matrix library; SML) was carried out. These SML libraries were designed to include all possible sequence combinations of the residues that were suggested to be preferred by a protease using the PS-SCL method. SML screening against the three serine proteases kallikrein 4 (KLK4), kallikrein 14 (KLK14) and plasmin revealed highly preferred peptide substrates that could not have been deduced by PS-SCL screening alone. Comparing protease subsite preference profiles from screens of the two types of peptide libraries showed that the most preferred substrates were not detected by PS SCL screening as a consequence of intermolecular cooperativity being negated by the very nature of PS SCL screening. Sequences that are highly favoured as result of intermolecular cooperativity achieve optimal protease subsite occupancy, and thereby interact with very specific determinants of the protease. Identifying these substrate sequences is important since they may be used to produce potent and selective inhibitors of protolytic enzymes. This study found that highly favoured substrate sequences that relied on intermolecular cooperativity allowed for the production of potent inhibitors of KLK4, KLK14 and plasmin. Peptide aldehydes based on preferred plasmin sequences produced high affinity transition state analogue inhibitors for this protease. The most potent of these maintained specificity over plasma kallikrein (known to have a very similar substrate preference to plasmin). Furthermore, the efficiency of this inhibitor in blocking fibrinolysis in vitro was comparable to aprotinin, which previously saw clinical use to reduce perioperative bleeding. One substrate sequence particularly favoured by KLK4 was substituted into the 14 amino acid, circular sunflower trypsin inhibitor (SFTI). This resulted in a highly potent and selective inhibitor (SFTI-FCQR) which attenuated protease activated receptor signalling by KLK4 in vitro. Moreover, SFTI-FCQR and paclitaxel synergistically reduced growth of ovarian cancer cells in vitro, making this inhibitor a lead compound for further therapeutic development. Similar incorporation of a preferred KLK14 amino acid sequence into the SFTI scaffold produced a potent inhibitor for this protease. However, the conformationally constrained SFTI backbone enforced a different intramolecular cooperativity, which masked a KLK14 specific determinant. As a consequence, the level of selectivity achievable was lower than that found for the KLK4 inhibitor. Standard mechanism inhibitors such as SFTI rely on a stable acyl-enzyme intermediate for high affinity binding. This is achieved by a conformationally constrained canonical binding loop that allows for reformation of the scissile peptide bond after cleavage. Amino acid substitutions within the inhibitor to target a particular protease may compromise structural determinants that support the rigidity of the binding loop and thereby prevent the engineered inhibitor reaching its full potential. An in silico analysis was carried out to examine the potential for further improvements to the potency and selectivity of the SFTI-based KLK4 and KLK14 inhibitors. Molecular dynamics simulations suggested that the substitutions within SFTI required to target KLK4 and KLK14 had compromised the intramolecular hydrogen bond network of the inhibitor and caused a concomitant loss of binding loop stability. Furthermore in silico amino acid substitution revealed a consistent correlation between a higher frequency of formation and the number of internal hydrogen bonds of SFTI-variants and lower inhibition constants. These predictions allowed for the production of second generation inhibitors with enhanced binding affinity toward both targets and highlight the importance of considering intramolecular cooperativity effects when engineering proteins or circular peptides to target proteases. The findings from this study show that although PS-SCLs are a useful tool for high throughput screening of approximate protease preference, later refinement by SML screening is needed to reveal optimal subsite occupancy due to cooperativity in substrate recognition. This investigation has also demonstrated the importance of maintaining structural determinants of backbone constraint and conformation when engineering standard mechanism inhibitors for new targets. Combined these results show that backbone conformation and amino acid cooperativity have more prominent roles than previously appreciated in determining substrate/inhibitor specificity and binding affinity. The three key inhibitors designed during this investigation are now being developed as lead compounds for cancer chemotherapy, control of fibrinolysis and cosmeceutical applications. These compounds form the basis of a portfolio of intellectual property which will be further developed in the coming years.

Defining standards for modelling the biomechanics of the foot and ankle : a systematic review

The complex interaction of the bones of the foot has been explored in detail in recent years, which has led to the acknowledgement in the biomechanics community that the foot can no longer be considered as a single rigid segment. With the advance of motion analysis technology it has become possible to quantify the biomechanics of simplified units or segments that make up the foot. Advances in technology coupled with reducing hardware prices has resulted in the uptake of more advanced tools available for clinical gait analysis. The increased use of these techniques in clinical practice requires defined standards for modelling and reporting of foot and ankle kinematics. This systematic review aims to provide a critical appraisal of commonly used foot and ankle marker sets designed to assess kinematics and thus provide a theoretical background for the development of modelling standards.

A Systematic Review of kinematic models used in foot & ankle biomechanics

Over the past decade our understanding of foot function has increased significantly[1,2]. Our understanding of foot and ankle biomechanics appears to be directly correlated to advances in models used to assess and quantify kinematic parameters in gait. These advances in models in turn lead to greater detail in the data. However, we must consider that the level of complexity is determined by the question or task being analysed. This systematic review aims to provide a critical appraisal of commonly used marker sets and foot models to assess foot and ankle kinematics in a wide variety of clinical and research purposes.

Defining standards for modelling the biomechanics of the foot and ankle: A systematic review

The complex interaction of the bones of the foot has been explored in detail in recent years, which has led to the acknowledgement in the biomechanics community that the foot can no longer be considered as a single rigid segment. With the advance of motion analysis technology it has become possible to quantify the biomechanics of simplified units or segments that make up the foot. Advances in technology coupled with reducing hardware prices has resulted in the uptake of more advanced tools available for clinical gait analysis. The increased use of these techniques in clinical practice requires defined standards for modelling and reporting of foot and ankle kinematics. This systematic review aims to provide a critical appraisal of commonly used foot and ankle marker sets designed to assess kinematics and thus provide a theoretical background for the development of modelling standards.

Thomas Young’s investigations in gradient-index optics

Purpose: James Clerk Maxwell is usually recognized as being the first, in 1854, to consider using inhomogeneous media in optical systems. However, some fifty years earlier Thomas Young, stimulated by his interest in the optics of the eye and accommodation, had already modeled some applications of gradient-index optics. These applications included using an axial gradient to provide spherical aberration-free optics and a spherical gradient to describe the optics of the atmosphere and the eye lens. We evaluated Young’s contributions. Method: We attempted to derive Young’s equations for axial and spherical refractive index gradients. Raytracing was used to confirm accuracy of formula. Results: We did not confirm Young’s equation for the axial gradient to provide aberration-free optics, but derived a slightly different equation. We confirmed the correctness of his equations for deviation of rays in a spherical gradient index and for the focal length of a lens with a nucleus of fixed index surrounded by a cortex of reducing index towards the edge. Young claimed that the equation for focal length applied to a lens with part of the constant index nucleus of the sphere removed, such that the loss of focal length was a quarter of the thickness removed, but this is not strictly correct. Conclusion: Young’s theoretical work in gradient-index optics received no acknowledgement from either his contemporaries or later authors. While his model of the eye lens is not an accurate physiological description of the human lens, with the index reducing least quickly at the edge, it represented a bold attempt to approximate the characteristics of the lens. Thomas Young’s work deserves wider recognition.

Hot temperatures and morbidity : a systematic review and meta–analysis

Extreme temperatures have been shown to have a detrimental effect on health. Hot temperatures can increase the risk of mortality, particularly in people suffering from cardiorespiratory diseases. Given the onset of climate change, it is critical that the impact of temperature on health is understood, so that effective public health strategies can correctly identify vulnerable groups within the population. However, while effects on mortality have been extensively studied, temperature–related morbidity has received less attention. This study applied a systematic review and meta–analysis to examine the current literature relating to hot temperatures and morbidity.

A systematic review : the effects of orientation programs for cancer patients and their family/carers

Objectives To assess the effects of information interventions which orient patients and their carers/family to a cancer care facility and the services available within the facility. Design Systematic review of randomised controlled trials (RCTs), cluster RCTs and quasi-RCTs. Data sources MEDLINE, CINAHL, PsycINFO, EMBASE and the Cochrane Central Register of Controlled Trials. Methods We included studies evaluating the effect of an orientation intervention, compared with a control group which received usual care, or with trials comparing one orientation intervention with another orientation intervention. Results Four RCTs of 610 participants met the criteria for inclusion. Findings from two RCTs demonstrated significant benefits of the orientation intervention in relation to reduced levels of distress (mean difference (MD): −8.96, 95% confidence interval (95%CI): −11.79 to −6.13), but non-significant benefits in relation to the levels state anxiety levels (MD −9.77) (95%CI: −24.96 to 5.41). There are insufficient data on the other outcomes of interest. Conclusions This review has demonstrated the feasibility and some potential benefits of orientation interventions. There was a low level of evidence to suggest that orientation interventions can reduce distress in patients. However, other outcomes, including patient knowledge recall/satisfaction, remain inconclusive. The majority of trials were subjected to high risk of bias and were likely to be insufficiently powered. Further well conducted and powered RCTs are required to provide evidence for determining the most appropriate intensity, nature, mode and resources for such interventions. Patient and carer-focused outcomes should be included.

MD investigations for mechanical properties of copper nanowires with and without surface defects

Based on the molecular dynamics (MD) method, the single-crystalline copper nanowire with different surface defects is investigated through tension simulation. For comparison, the MD tension simulations of perfect nanowire are firstly carried out under different temperatures, strain rates, and sizes. It has concluded that the surface-volume ratio significantly affects the mechanical properties of nanowire. The surface defects on nanowires are then systematically studied in considering different defect orientation and distribution. It is found that the Young’s modulus is insensitive of surface defects. However, the yield strength and yield point show a significant decrease due to the different defects. Different defects are observed to serve as a dislocation source.

Implementing a systematic process for rapidly embedding sustainability within chemical engineering education: a case study of James Cook University, Australia

Sustainability has emerged as a primary context for engineering education in the 21st Century, particularly the sub-discipline of chemical engineering. However, there is confusion over how to go about integrating sustainability knowledge and skills systemically within bachelor degrees. This paper addresses this challenge, using a case study of an Australian chemical engineering degree to highlight important practical considerations for embedding sustainability at the core of the curriculum. The paper begins with context for considering a systematic process for rapid curriculum renewal. The authors then summarise a 2-year federally funded project, which comprised piloting a model for rapid curriculum renewal led by the chemical engineering staff. Model elements contributing to the renewal of this engineering degree and described in this paper include: industry outreach; staff professional development; attribute identification and alignment; program mapping; and curriculum and teaching resource development. Personal reflections on the progress and process of rapid curriculum renewal in sustainability by the authors and participating engineering staff will be presented as a means to discuss and identify methodological improvements, as well as highlight barriers to project implementation. It is hoped that this paper will provide an example of a formalised methodology on which program reform and curriculum renewal for sustainability can be built upon in other higher education institutions.

Should athletes return to sport after applying Ice? A systematic review of the effect of local 3 cooling on functional performance

Applying ice or other forms of topical cooling is a popular method of treating sports injuries. It is commonplace for athletes to return to competitive activity, shortly or immediately after the application of a cold treatment. In this article, we examine the effect of local tissue cooling on outcomes relating to functional performance and to discuss their relevance to the sporting environment. A computerized literature search, citation tracking and hand search was performed up to April, 2011. Eligible studies were trials involving healthy human participants, describing the effects of cooling on outcomes relating to functional performance. Two reviewers independently assessed the validity of included trials and calculated effect sizes. Thirty five trials met the inclusion criteria; all had a high risk of bias. The mean sample size was 19. Meta-analyses were not undertaken due to clinical heterogeneity. The majority of studies used cooling durations >20 minutes. Strength (peak torque/force) was reported by 25 studies with approximately 75% recording a decrease in strength immediately following cooling. There was evidence from six studies that cooling adversely affected speed, power and agility-based running tasks; two studies found this was negated with a short rewarming period. There was conflicting evidence on the effect of cooling on isolated muscular endurance. A small number of studies found that cooling decreased upper limb dexterity and accuracy. The current evidence base suggests that athletes will probably be at a performance disadvantage if they return to activity immediately after cooling. This is based on cooling for longer than 20 minutes, which may exceed the durations employed in some sporting environments. In addition, some of the reported changes were clinically small and may only be relevant in elite sport. Until better evidence is available, practitioners should use short cooling applications and/or undertake a progressive warm up prior to returning to play.

Cryotherapy and joint position sense in healthy participants: a systematic review

Objective: To (1) search the English-language literature for original research addressing the effect of cryotherapy on joint position sense (JPS) and (2) make recommendations regarding how soon healthy athletes can safely return to participation after cryotherapy. Data Sources: We performed an exhaustive search for original research using the AMED, CINAHL, MEDLINE, and SportDiscus databases from 1973 to 2009 to gather information on cryotherapy and JPS. Key words used were cryotherapy and proprioception, cryotherapy and joint position sense, cryotherapy, and proprioception. Study Selection: The inclusion criteria were (1) the literature was written in English, (2) participants were human, (3) an outcome measure included JPS, (4) participants were healthy, and (5) participants were tested immediately after a cryotherapy application to a joint. Data Extraction: The means and SDs of the JPS outcome measures were extracted and used to estimate the effect size (Cohen d) and associated 95% confidence intervals for comparisons of JPS before and after a cryotherapy treatment. The numbers, ages, and sexes of participants in all 7 selected studies were also extracted. Data Synthesis: The JPS was assessed in 3 joints: ankle (n 5 2), knee (n 5 3), and shoulder (n 5 2). The average effect size for the 7 included studies was modest, with effect sizes ranging from 20.08 to 1.17, with a positive number representing an increase in JPS error. The average methodologic score of the included studies was 5.4/10 (range, 5–6) on the Physiotherapy Evidence Database scale. Conclusions: Limited and equivocal evidence is available to address the effect of cryotherapy on proprioception in the form of JPS. Until further evidence is provided, clinicians should be cautious when returning individuals to tasks requiring components of proprioceptive input immediately after a cryotherapy treatment.

Creativity in policing: building the necessary skills to solve complex and protracted investigations

Despite an increased focus on proactive policing in recent years, criminal investigation is still perhaps the most important task of any law enforcement agency. As a result, the skills required to carry out a successful investigation or to be an ‘effective detective’ have been subjected to much attention and debate (Smith and Flanagan, 2000; Dean, 2000; Fahsing and Gottschalk, 2008:652). Stelfox (2008:303) states that “The service’s capacity to carry out investigations comprises almost entirely the expertise of investigators.” In this respect, Dean (2000) highlighted the need to profile criminal investigators in order to promote further understanding of the cognitive approaches they take to the process of criminal investigation. As a result of his research, Dean (2000) produced a theoretical framework of criminal investigation, which included four disparate cognitive or ‘thinking styles’. These styles were the ‘Method’, ‘Challenge’, ‘Skill’ and ‘Risk’. While the Method and Challenge styles deal with adherence to Standard Operating Procedures (SOPs) and the internal ‘drive’ that keeps an investigator going, the Skill and Risk styles both tap on the concept of creativity in policing. It is these two latter styles that provide the focus for this paper. This paper presents a brief discussion on Dean’s (2000) Skill and Risk styles before giving an overview of the broader literature on creativity in policing. The potential benefits of a creative approach as well as some hurdles which need to be overcome when proposing the integration of creativity within the policing sector are then discussed. Finally, the paper concludes by proposing further research into Dean’s (2000) skill and risk styles and also by stressing the need for significant changes to the structure and approach of the traditional policing organisation before creativity in policing is given the status it deserves.

Time dependency of molecular rate estimates and systematic overestimation of recent divergence times

Studies of molecular evolutionary rates have yielded a wide range of rate estimates for various genes and taxa. Recent studies based on population-level and pedigree data have produced remarkably high estimates of mutation rate, which strongly contrast with substitution rates inferred in phylogenetic (species-level) studies. Using Bayesian analysis with a relaxed-clock model, we estimated rates for three groups of mitochondrial data: avian protein-coding genes, primate protein-coding genes, and primate d-loop sequences. In all three cases, we found a measurable transition between the high, short-term (<1–2 Myr) mutation rate and the low, long-term substitution rate. The relationship between the age of the calibration and the rate of change can be described by a vertically translated exponential decay curve, which may be used for correcting molecular date estimates. The phylogenetic substitution rates in mitochondria are approximately 0.5% per million years for avian protein-coding sequences and 1.5% per million years for primate protein-coding and d-loop sequences. Further analyses showed that purifying selection offers the most convincing explanation for the observed relationship between the estimated rate and the depth of the calibration. We rule out the possibility that it is a spurious result arising from sequence errors, and find it unlikely that the apparent decline in rates over time is caused by mutational saturation. Using a rate curve estimated from the d-loop data, several dates for last common ancestors were calculated: modern humans and Neandertals (354 ka; 222–705 ka), Neandertals (108 ka; 70–156 ka), and modern humans (76 ka; 47–110 ka). If the rate curve for a particular taxonomic group can be accurately estimated, it can be a useful tool for correcting divergence date estimates by taking the rate decay into account. Our results show that it is invalid to extrapolate molecular rates of change across different evolutionary timescales, which has important consequences for studies of populations, domestication, conservation genetics, and human evolution.

Ambient temperature and cardiorespiratory morbidity : a systematic review and meta-analysis

BACKGROUND: The effect of extreme temperature has become an increasing public health concern. Evaluating the impact of ambient temperature on morbidity has received less attention than its impact on mortality. METHODS: We performed a systematic literature review and extracted quantitative estimates of the effects of hot temperatures on cardiorespiratory morbidity. There were too few studies on effects of cold temperatures to warrant a summary. Pooled estimates of effects of heat were calculated using a Bayesian hierarchical approach that allowed multiple results to be included from the same study, particularly results at different latitudes and with varying lagged effects. RESULTS: Twenty-one studies were included in the final meta-analysis. The pooled results suggest an increase of 3.2% (95% posterior interval = -3.2% to 10.1%) in respiratory morbidity with 1°C increase on hot days. No apparent association was observed for cardiovascular morbidity (-0.5% [-3.0% to 2.1%]). The length of lags had inconsistent effects on the risk of respiratory and cardiovascular morbidity, whereas latitude had little effect on either. CONCLUSIONS: The effects of temperature on cardiorespiratory morbidity seemed to be smaller and more variable than previous findings related to mortality.