Microcavity supported lipid bilayers; biomimetic models of the cell membrane

Biomimetic models of the cell membrane are sought after as they have the potential to provide a realistic representation of an organism’s lipid bilayer. They can be used to understand lipid dynamics, signalling, drug permeability and membrane protein diffusion in an environment that is away from the complexity of the real living cell. This thesis examines the application of a new type of lipid membrane model, the micro-cavity supported lipid bilayer (MSLB), to study drug-membrane interactions and glycolipid containing bilayers using electrochemical impedance spectroscopy (EIS). Chapter 1 outlines the structure and function of the cell membrane and describes current models used to replicate the functions of the cellular bilayer. The limits of these models are also discussed particularly in the context of stability, lipid fluidity and addressability of both sides of the bilayer. The biomimetic MSLB system is then explored as a viable alternative in this thesis and is described in Chapter 2. 2.80 ± 0.04 μm diameter gold arrays were used and their surfaces were chemically modified to render them hydrophilic which aided the assembly of lipid bilayers using Langmuir Blodgett to form the initial monolayer and vesicle disruption to create the final bilayer structure. This model is applied in Chapter 3 as a means of assessing drug plasma membrane interactions of two representative non-steroidal anti-inflammatory drugs; ibuprofen and diclofenac. These drugs were chosen as their log P values are well established and their interactions with membranes have been characterized by other methods. Their impact on the cavity array supported lipid membrane was investigated using EIS. Chapter 4 uses the MSLB model to study the interactions between the ganglioside, GM1, and disease relevant lectins by fabricating asymmetric GM1 containing lipid bilayer membranes. The influence of lipid/sterol composition on GM1-lectin recognition and aggregation was also considered. Overall, this work demonstrates that, using EIS as the interrogation method, it is possible to sensitively explore interactions between external molecules and the lipid bilayer using these MSLBs. The MSLBs are a significant advance on current lipid membrane models as they permit accurate representations of cell membrane in elements of composition, fluidity, asymmetry and deep aqueous well on either side of the membrane

How the citizen-warrior was created in Classical Athens and Sparta

This study explores how Classical Athens and Sparta created the warrior citizen and continued to motivate citizens to fight throughout their lives. It engages with the issues of the 'hoplite ideal', exploring the extent to which it existed in practice and the implications of hoplite ideology for other types of warrior. This study also considers various methods of training and proliferation of state ideology, both formal modes of training such as the Spartan agoge and also informal modes of training such as hunting. Modern sociological and psychological evidence regarding military training and the fostering of aggression are also considered. The important role of religious beliefs and mythology are examined with both the role of gods in battle (for example, in pre-battle sacrifices) and also the depiction of gods and heroes as military figures are considered as potential motivating factors. The potential for inter-state rivalries as played out during Panhellenic festivals and as displayed in Panhellenic sanctuaries is also explored as a motivating factor for individuals and for the state as a whole

Discovery of potent and selective MRCK inhibitors with therapeutic effect on skin cancer

The myotonic dystrophy-related Cdc42-binding kinases MRCKα and MRCKβ contribute to the regulation of actin-myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphological changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent anti-proliferative effects with greatest activity in hematological cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy

Reliability of a smartphone goniometer app compared with traditional goniometer for measuring passive motion at the first metatarsophalangeal joint

BACKGROUND: Adequate sagittal plane motion of the first metatarsalphalangeal joint (1st MTPJ) is important during normal gait and goniometric measurement is commonly used as a diagnostic and outcome assessment tool. We aimed to determine the intra and inter-rater reliability together with the concurrent validity of a universal plastic goniometer (UG) and a smartphone applicationlication (Dr G) for the measurement of dorsiflexion at the 1st MTPJ. METHODS: Measurement of joint position and passive range of motion of the 1st MTPJ dorsiflexion was compared using a UG and DrG goniometer. A double-blind repeated measures design was utilized, with intraclass correlation coefficient (ICC) used to determine levels of reliability. RESULTS: For joint position good intra-rater reliability (ICC >0.861) and good inter-rater reliability (ICC >0.823) was noted. However, the Dr G application consistently measured lower angles (mean 27.8° (SD 8.37)) than the UG (mean 32° (SD 11.7)) and these associations were significant (r = 0.399, p < 0.001). For passive range of motion, the mean total range of dorsiflexion motion (from maximum plantarflexed position to maximum dorsiflexed position) was 82.8° (SD 12.2) for the UG and 82.9° (SD 11.3) for the Dr G application. Both instruments demonstrated high levels of intra-rater reliability (ICC >0.809). Inter-rater reliability was moderate to good for the UG (ICC 0.693 (95 % CI 0.580 to 0.788)) and good for the Dr G application (ICC 0.708 (95 % CI 0.597 to 0.799)). CONCLUSIONS: Moderate to high intra and inter-rater reliability of joint position and passive 1st MTPJ motion can be achieved with traditional and smartphone-based goniometric measurement. The Dr G application may provide a slightly higher reliability, but devices should not be used inter-changeably as significant variation in measurement between devices may occur. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13047-015-0088-3) contains supplementary material, which is available to authorized users

Tumour risks and genotype-phenotype correlations associated with germline variants in succinate dehydrogenase subunit genes SDHB, SDHC and SDHD.

BACKGROUND: Germline pathogenic variants in SDHB/SDHC/SDHD are the most frequent causes of inherited phaeochromocytomas/paragangliomas. Insufficient information regarding penetrance and phenotypic variability hinders optimum management of mutation carriers. We estimate penetrance for symptomatic tumours and elucidate genotype-phenotype correlations in a large cohort of SDHB/SDHC/SDHD mutation carriers. METHODS: A retrospective survey of 1832 individuals referred for genetic testing due to a personal or family history of phaeochromocytoma/paraganglioma. 876 patients (401 previously reported) had a germline mutation in SDHB/SDHC/SDHD (n=673/43/160). Tumour risks were correlated with in silico structural prediction analyses. RESULTS: Tumour risks analysis provided novel penetrance estimates and genotype-phenotype correlations. In addition to tumour type susceptibility differences for individual genes, we confirmed that the SDHD:p.Pro81Leu mutation has a distinct phenotype and identified increased age-related tumour risks with highly destabilising SDHB missense mutations. By Kaplan-Meier analysis, the penetrance (cumulative risk of clinically apparent tumours) in SDHB and (paternally inherited) SDHD mutation-positive non-probands (n=371/67 with detailed clinical information) by age 60 years was 21.8% (95% CI 15.2% to 27.9%) and 43.2% (95% CI 25.4% to 56.7%), respectively. Risk of malignant disease at age 60 years in non-proband SDHB mutation carriers was 4.2%(95% CI 1.1% to 7.2%). With retrospective cohort analysis to adjust for ascertainment, cumulative tumour risks for SDHB mutation carriers at ages 60 years and 80 years were 23.9% (95% CI 20.9% to 27.4%) and 30.6% (95% CI 26.8% to 34.7%). CONCLUSIONS: Overall risks of clinically apparent tumours for SDHB mutation carriers are substantially lower than initially estimated and will improve counselling of affected families. Specific genotype-tumour risk associations provides a basis for novel investigative strategies into succinate dehydrogenase-related mechanisms of tumourigenesis and the development of personalised management for SDHB/SDHC/SDHD mutation carriers

Clinical and genetic aspects of KBG syndrome.

KBG syndrome is characterized by short stature, distinctive facial features, and developmental/cognitive delay and is caused by mutations in ANKRD11, one of the ankyrin repeat-containing cofactors. We describe 32 KBG patients aged 2-47 years from 27 families ascertained via two pathways: targeted ANKRD11 sequencing (TS) in a group who had a clinical diagnosis of KBG and whole exome sequencing (ES) in a second group in whom the diagnosis was unknown. Speech delay and learning difficulties were almost universal and variable behavioral problems frequent. Macrodontia of permanent upper central incisors was seen in 85%. Other clinical features included short stature, conductive hearing loss, recurrent middle ear infection, palatal abnormalities, and feeding difficulties. We recognized a new feature of a wide anterior fontanelle with delayed closure in 22%. The subtle facial features of KBG syndrome were recognizable in half the patients. We identified 20 ANKRD11 mutations (18 novel: all truncating) confirmed by Sanger sequencing in 32 patients. Comparison of the two ascertainment groups demonstrated that facial/other typical features were more subtle in the ES group. There were no conclusive phenotype-genotype correlations. Our findings suggest that mutation of ANKRD11 is a common Mendelian cause of developmental delay. Affected patients may not show the characteristic KBG phenotype and the diagnosis is therefore easily missed. We propose updated diagnostic criteria/clinical recommendations for KBG syndrome and suggest that inclusion of ANKRD11 will increase the utility of gene panels designed to investigate developmental delay. © 2016 The Authors. American Journal of Medical Genetics Part A Published by Wiley Periodicals, Inc.This article is freely available via Open Access. Click on the Additional Link above to access the full-text via the publisher's site

Prevalence and architecture of de novo mutations in developmental disorders.

The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year

All-sky Medium Energy Gamma-ray Observatory: Exploring the Extreme Multimessenger Universe

The All-sky Medium Energy Gamma-ray Observatory (AMEGO) is a probe class mission concept that will provide essential contributions to multimessenger astrophysics in the late 2020s and beyond. AMEGO combines high sensitivity in the 200 keV to 10 GeV energy range with a wide field of view, good spectral resolution, and polarization sensitivity. Therefore, AMEGO is key in the study of multimessenger astrophysical objects that have unique signatures in the gamma-ray regime, such as neutron star mergers, supernovae, and flaring active galactic nuclei. The order-of-magnitude improvement compared to previous MeV missions also enables discoveries of a wide range of phenomena whose energy output peaks in the relatively unexplored medium-energy gamma-ray band