Self-association of calcium-binding protein S100A4 and metastasis

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.

IN VIVO analysis of ocular morphological changes during phakic accommodation

The principal theme of this thesis is the in vivo examination of ocular morphological changes during phakic accommodation, with particular attention paid to the ciliary muscle and crystalline lens. The investigations detailed involved the application of high-resolution imaging techniques to facilitate the acquisition of new data to assist in the clarification of aspects of the accommodative system that were poorly understood. A clinical evaluation of the newly available Grand Seiko Auto Ref/ Keratometer WAM-5500 optometer was undertaken to assess its value in the field of accommodation research. The device was found to be accurate and repeatable compared to subjective refraction, and has the added advantage of allowing dynamic data collection at a frequency of around 5 Hz. All of the subsequent investigations applied the WAM-5500 for determination of refractive error and objective accommodative responses. Anterior segment optical coherence tomography (AS-OCT) based studies examined the morphology and contractile response of youthful and ageing ciliary muscle. Nasal versus temporal asymmetry was identified, with the temporal aspect being both thicker and demonstrating a greater contractile response. The ciliary muscle was longer in terms of both its anterior (r = 0.49, P <0.001) and overall length (r = 0.45, P = 0.02) characteristics, in myopes. The myopic ciliary muscle does not appear to be merely stretched during axial elongation, as no significant relationship between thickness and refractive error was identified. The main contractile responses observed were a thickening of the anterior region and a shortening of the muscle, particularly anteriorly. Similar patterns of response were observed in subjects aged up to 70 years, supporting a lensocentric theory of presbyopia development. Following the discovery of nasal/ temporal asymmetry in ciliary muscle morphology and response, an investigation was conducted to explore whether the regional variations in muscle contractility impacted on lens stability during accommodation. A bespoke programme was developed to analyse AS-OCT images and determine whether lens tilt and decentration varied between the relaxed and accommodated states. No significant accommodative difference in these parameters was identified, implying that any changes in lens stability with accommodation are very slight, as a possible consequence of vitreous support. Novel three-dimensional magnetic resonance imaging (MRI) and analysis techniques were used to investigate changes in lens morphology and ocular conformation during accommodation. An accommodative reduction in lens equatorial diameter provides further evidence to support the Helmholtzian mechanism of accommodation, whilst the observed increase in lens volume challenges the widespread assertion that this structure is incompressible due to its high water content. Wholeeye MRI indicated that the volume of the vitreous chamber remains constant during accommodation. No significant changes in ocular conformation were detected using MRI. The investigations detailed provide further insight into the mechanisms of accommodation and presbyopia, and represent a platform for future work in this field.

Autonomic, biometric and oculomotor correlates of myopia in young adults

The principal work reported in this thesis is the examination of autonomic profile of ciliary muscle innervation as a risk factor in myopia development. Deficiency in sympathetic inhibitory control of accommodation has been proposed as a contributory factor in the development of late onset myopia (LOM). Complementary measurements of ocular biometry, oculomotor function and dynamic accommodation response were carried out on the same subject cohort, thus allowing cross-correlation of these factors with. autonomic profile. Subjects were undergraduate and postgraduate students of Aston University. A 2.5 year longitudinal study of refractive error progression in 40 subjects revealed the onset of LOM in 10, initially emmetropic, young adult subjects (age range 18-24 years) undertaking substantial amounts of near work. A controlled, double blind experimental protocol was conducted concurrently to measure post-task open-loop accommodative regression following distance (0 D) or near (3 D above baseline tonic accommodation) closed-loop tasks of short (10 second) or long (3 minute) duration. Closed-loop tasks consisted of observation of a high contrast Maltese cross target; open-loop conditions were imposed by observation of a 0.2 c/deg Difference of Gaussian target. Accommodation responses were recorded continuously at 42 Hz using a modified Shin-Nippon SRW-5000 open-view infra-red optometer. Blockade of the sympathetic branch of accommodative control was achieved by topical instillation of the non-selective b-adrenoceptor antagonist timolol maleate. Betaxolol hydrochloride (non-selective b1-adrenoceptor antagonist) and normal saline were employed as control agents. Retarded open-loop accommodative regression under b2 blockade following the 3 minute near task indicated the presence of sympathetic facility. Sympathetic inhibitory facility in accommodation control was found in similar proportions between LOM and stable emmetropic subjects. A cross-sectional study (N=60) of autonomic profile showed that sympathetic innervation of ciliary muscle is present in similar proportions between emmetropes, early-, and late-onset myopes. Sympathetic facility was identified in 27% of emmetropes, 21% of EOMs and 29% of LOMs.

Decreased NADPH oxidase expression and antioxidant activity in cachectic skeletal muscle

Background - Cancer cachexia is the progressive loss of skeletal muscle protein that contributes significantly to cancer morbidity and mortality. Evidence of antioxidant attenuation and the presence of oxidised proteins in patients with cancer cachexia indicate a role for oxidative stress. The level of oxidative stress in tissues is determined by an imbalance between reactive oxygen species production and antioxidant activity. This study aimed to investigate the superoxide generating NADPH oxidase (NOX) enzyme and antioxidant enzyme systems in murine adenocarcinoma tumour-bearing cachectic mice. Methods - Superoxide levels, mRNA levels of NOX enzyme subunits and the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidise (GPx) and catalase was measured in the skeletal muscle of mice with cancer and cancer cachexia. Protein expression levels of NOX enzyme subunits and antioxidant enzyme activity was also measured in the same muscle samples. Results - Superoxide levels increased 1.4-fold in the muscle of mice with cancer cachexia, and this was associated with a decrease in mRNA of NOX enzyme subunits, NOX2, p40phox and p67phox along with the antioxidant enzymes SOD1, SOD2 and GPx. Cancer cachexia was also associated with a 1.3-fold decrease in SOD1 and 2.0-fold decrease in GPx enzyme activity. Conclusion - Despite increased superoxide levels in cachectic skeletal muscle, NOX enzyme subunits, NOX2, p40phox and p67phox, were downregulated along with the expression and activity of the antioxidant enzymes. Therefore, the increased superoxide levels in cachectic skeletal muscle may be attributed to the reduction in the activity of endogenous antioxidant enzymes.

Induction of apoptosis by a cachectic-factor in murine myotubes and inhibition by eicosapentaenoic acid

Treatment of C2C12 myotubes with a tumour-derived proteolysis-inducing factor (PIF) at concentrations between 1 and 10 nM was shown to stimulate the activity of the apoptotic initiator caspases-8 and -9 and the apoptotic effector caspases-2,-3 and -6. This increased caspase activity was attenuated in myotubes pretreated with 50 μM eicosapentaenoic acid (EPA). At least part of the increase in caspase activity may be related to the increased proteasome proteolytic activity, since a caspase-3 inhibitor completely attenuated the PIF-induced increase in 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the proteasome. However, Western blot analysis showed that PIF induced an increase in expression of the active form of caspase-3, which was also attenuated by EPA. Further Western blot analysis showed PIF increased the cytosolic content of cytochrome c, as well as expression of the pro-apoptotic protein bax but not the antiapoptotic protein bcl-2, which were both attenuated by 50 μM EPA. Induction of apoptosis by PIF in murine myotubes was confirmed by an increase in free nucleasomes formation and increased DNA fragmentation evidenced by a nucleasomal ladder typical of apoptotic cells. This process was again inhibited by pre-incubation with EPA. These results suggest that in addition to activating the proteasome, PIF induces apoptosis in C2C12 myotubes, possibly through the common intermediate arachidonic acid. Both of these processes would contribute to the loss of skeletal muscle in cancer cachexia.

Reciprocating power generation in a chemically driven synthetic muscle

A scalable synthetic muscle has been constructed that transducts nanoscale molecular shape changes into macroscopic motion. The working material, which deforms affinely in response to a pH stimulus, is a self-assembled block copolymer comprising nanoscopic hydrophobic domains in a weak polyacid matrix. A device has been assembled where the muscle does work on a cantilever and the force generated has been measured. When coupled to a chemical oscillator this provides a free running chemical motor that generates a peak power of 20 mW kg 1 by the serial addition of 10 nm shape changes that scales over 5 orders of magnitude. It is the nanostructured nature of the gel that gives rise to the affine deformation and results in a robust working material for the construction of scalable muscle devices.

Characterisation of electrospun polystyrene scaffolds for three-dimensional in vitro biological studies

The purpose of this study was to produce a well-characterised electrospun polystyrene scaffold which could be used routinely for three-dimensional (3D) cell culture experimentation. A linear relationship (p<0.01p<0.01) between three principal process variables (applied voltage, working distance and polymer concentration) and fibre diameter was reliably established enabling a mathematical model to be developed to standardise the electrospinning process. Surface chemistry and bulk architecture were manipulated to increase wetting and handling characteristics, respectively. X-ray photoelectron spectroscopy (XPS) confirmed the presence of oxygen-containing groups after argon plasma treatment, resulting in a similar surface chemistry to treated tissue culture plastic. The bulk architecture of the scaffolds was characterised by scanning electron microscopy (SEM) to assess the alignment of both random and aligned electrospun fibres, which were calculated to be 0.15 and 0.66, respectively. This compared to 0.51 for collagen fibres associated with native tissue. Tensile strength and strain of approximately of 0.15 MPa and 2.5%, respectively, allowed the scaffolds to be routinely handled for tissue culture purposes. The efficiency of attachment of smooth muscle cells to electrospun scaffolds was assessed using a modified 3-[4,5-dimethyl(thiazol-2yl)-3,5-diphery] tetrazolium bromide assay and cell morphology was assessed by phalloidin-FITC staining of F-actin. Argon plasma treatment of electrospun polystyrene scaffold resulted in significantly increased cell attachment (p<0.05p<0.05). The alignment factors of the actin filaments were 0.19 and 0.74 for the random and aligned scaffold respectively, compared to 0.51 for the native tissue. The data suggests that electrospinning of polystyrene generates 3D scaffolds which complement polystyrene used in 2D cell culture systems.

Comparison of the anticatabolic effects of leucine and Ca-β-hydroxy-β-methylbutyrate in experimental models of cancer cachexia

Objective: Loss of skeletal muscle is the most debilitating feature of cancer cachexia, and there are few treatments available. The aim of this study was to compare the anticatabolic efficacy of L-leucine and the leucine metabolite β-hydroxy-β-methylbutyrate (Ca-HMB) on muscle protein metabolism, both invitro and invivo. Methods: Studies were conducted in mice bearing the cachexia-inducing murine adenocarcinoma 16 tumor, and in murine C2 C12 myotubes exposed to proteolysis-inducing factor, lipopolysaccharide, and angiotensin II. Results: Both leucine and HMB were found to attenuate the increase in protein degradation and the decrease in protein synthesis in murine myotubes induced by proteolysis-inducing factor, lipopolysaccharide, and angiotensin II. However, HMB was more potent than leucine, because HMB at 50 μM produced essentially the same effect as leucine at 1 mM. Both leucine and HMB reduced the activity of the ubiquitin-proteasome pathway as measured by the functional (chymotrypsin-like) enzyme activity of the proteasome in muscle lysates, as well as Western blot quantitation of protein levels of the structural/enzymatic proteasome subunits (20 S and 19 S) and the ubiquitin ligases (MuRF1 and MAFbx). Invivo studies in mice bearing the murine adenocarcinoma 16 tumor showed a low dose of Ca-HMB (0.25 g/kg) tobe 60% more effective than leucine (1 g/kg) in attenuating loss of body weight over a 4-d period. Conclusion: These results favor the clinical feasibility of using Ca-HMB over high doses of leucine for the treatment of cancer cachexia. © 2014 Elsevier Inc.

Ciliary muscle morphology in emmetropia and ocular biometric correlates

Purpose: Recent studies have documented a link between axial myopia and ciliary muscle morphology; yet, the variation in biometric characteristics of the emmetropic ciliary muscle are not fully known. Ciliary muscle morphology, including symmetry, was investigated between both eyes of emmetropic participants and correlated to ocular biometric parameters. Methods: Anterior segment optical coherence tomography (Zeiss, Visante) was utilised to image both eyes of 49 emmetropic participants (mean spherical equivalent refractive error (MSE) ≥ -0.55; < +0.75 D), aged 19 to 26 years. High resolution images were obtained of nasal and temporal aspects of the ciliary muscle in the relaxed state. MSE of both eyes was recorded using the Grand Seiko WAM 5500; axial length (AXL), anterior chamber depth (ACD) and lens thickness (LT) of the right eye were obtained using the Haag-streit Lenstar LS 900 biometer. A bespoke semi-objective analysis programme was used to measure a range of ciliary muscle parameters. Results: Temporal ciliary muscle overall length (CML) was greater than nasal CML, in both eyes (right: 3.58 ± 0.40 mm and 3.85 ± 0.39 mm for nasal and temporal aspects, respectively, P < 0.001; left: 3.65 ± 0.35 mm and 3.88 ± 0.41 mm for nasal and temporal aspects, respectively, P < 0.001). Temporal ciliary muscle thickness (CMT) was greater than nasal CMT at 2 mm and 3 mm from the scleral spur (CM2 and CM3, respectively) in each eye (right CM2: 0.29 ± 0.05 mm and 0.32 ± 0.05 mm for nasal and temporal aspects, respectively, P < 0.001; left CM2: 0.30 ± 0.05 mm and 0.32 ± 0.05 mm for nasal and temporal aspects, respectively, P < 0.001; right CM3: 0.13 ± 0.05 mm and 0.16 ± 0.04 mm for nasal and temporal aspects, respectively, P < 0.001; left CM3: 0.14 ± 0.04 mm and 0.17 ± 0.05 mm for nasal and temporal aspects, respectively, P < 0.001). AXL was positively correlated with ciliary muscle anterior length (AL) (e.g. P < 0.001, r2 = 0.262 for left temporal aspect), CML (P = 0.003, r2 = 0.175 for right nasal aspect) and ACD (P = 0.01, r2 = 0.181). Conclusions: Morphological characteristics of the ciliary muscle in emmetropic eyes display high levels of symmetry between the eyes. Greater CML and AL are linked to greater AXL and ACD, indicating ciliary muscle growth with normal ocular development.