Cell culture models of insulin signalling and glucose uptake

Insulin maintains glucose homeostasis through its binding of the insulin receptor and activation of the insulin signalling cascade in insulin sensitive tissues. Skeletal muscle is a major endocrine organ, and is responsible for the majority of post-prandial glucose disposal. The maintenance of glucose homeostasis is a delicate balance and impairments in glucose disposal can have significant physiological effects, resulting in the onset of metabolic diseases such as diabetes mellitus. Insulin stimulated glucose uptake involves a number of signalling proteins to enable uptake to occur. In order to understand the complexities associated with the insulin signalling cascade, cell culture models have provided a controlled and easily manipulated environment in which to investigate insulin stimulated glucose uptake in skeletal muscle. While the majority of these experiments have been conducted in conventional monolayer cultures, the growing field of three-dimensional tissue engineering provides an alternative environment in which skeletal muscle cells can be grown to investigate their physiological function. The purpose of this thesis was to investigate the use of different cell culture models for investigating the effects of acute and chronic insulin exposure on skeletal muscle. Initial investigations aimed to establish glucose uptake in tissue engineering skeletal muscle constructs using tritium labelled (H3) 2-deoxy-d-glucose. Monolayer cultures were used to developed base line conditions. In these cultures, concentrations greater than 0.5 µCi for 15 minutes of insulin stimulation suggested an initial assay window for investigating insulin stimulated glucose uptake. However, the duration of insulin stimulation was not effective in measuring uptake in tissue engineered skeletal muscle constructs based upon western blot experiments of Akt phosphorylation, therefore insulin stimulation in skeletal muscle tissue engineered constructs was increased to 30 minutes. Glucose uptake is mediated via specific glucose transporter protein, GLUT1 and GLUT4. Therefore, the transcriptional profile of these transporters was elucidated in monolayer culture and tissue engineered skeletal muscle constructs. Time course experiments showed an increase in GLUT4 transcription in tissue engineered and monolayer culture systems which is associated with an increase in the transcription of skeletal muscle development and myogenic genes. In two dimensional culture, skeletal muscle cells were exposed to insulin during differentiation and in post-mitotic skeletal muscle myotubes to investigating the potential effects upon metabolic genes and proteins involved in insulin signalling. Chronic exposure to insulin during skeletal muscle differentiation reduced insulin signalling and resulted in an increase in basal glucose uptake and ablated insulin stimulated glucose uptake. In contrast, post-mitotic skeletal muscle myotubes did not shown similar changes and were not as responsive to acute insulin exposure. Therefore future experiments exposed skeletal muscle to insulin during differentiation. Using the previous findings as a basis for experimentation, the effects of chronic and acute insulin exposure upon three dimensional skeletal muscle constructs were investigated. Fibrin and collagen constructs were grown for a total period of 14 days. Constructs were exposed to insulin during differentiation and acutely stimulated for 30 minutes at day 14. Although there was a mean increase in Akt protein phosphorylation in both types of tissue-engineered constructs, these changes were not significant following acute insulin stimulation. In addition, glucose uptake in fibrin skeletal muscle constructs increased as a result of acute insulin stimulation however was not significantly difference to unstimulated constructs. The work presented in this thesis provides initial experimental data of the use of different skeletal muscle cell culture models for investigating insulin signalling and glucose uptake. Further research should further characterise these in vitro models for investigating skeletal muscle metabolism

Scientific research on animal biodiversity is systematically biased towards vertebrates and temperate regions.

Over the last 25 years, research on biodiversity has expanded dramatically, fuelled by increasing threats to the natural world. However, the number of published studies is heavily weighted towards certain taxa, perhaps influencing conservation awareness of and funding for less-popular groups. Few studies have systematically quantified these biases, although information on this topic is important for informing future research and conservation priorities. We investigated: i) which animal taxa are being studied; ii) if any taxonomic biases are the same in temperate and tropical regions; iii) whether the taxon studied is named in the title of papers on biodiversity, perhaps reflecting a perception of what biodiversity is; iv) the geographical distribution of biodiversity research, compared with the distribution of biodiversity and threatened species; and v) the geographical distribution of authors' countries of origin. To do this, we used the search engine Web of Science to systematically sample a subset of the published literature with 'biodiversity' in the title. In total 526 research papers were screened-5% of all papers in Web of Science with biodiversity in the title. For each paper, details on taxonomic group, title phrasing, number of citations, study location, and author locations were recorded. Compared to the proportions of described species, we identified a considerable taxonomic weighting towards vertebrates and an under-representation of invertebrates (particularly arachnids and insects) in the published literature. This discrepancy is more pronounced in highly cited papers, and in tropical regions, with only 43% of biodiversity research in the tropics including invertebrates. Furthermore, while papers on vertebrate taxa typically did not specify the taxonomic group in the title, the converse was true for invertebrate papers. Biodiversity research is also biased geographically: studies are more frequently carried out in developed countries with larger economies, and for a given level of species or threatened species, tropical countries were understudied relative to temperate countries. Finally, biodiversity research is disproportionately authored by researchers from wealthier countries, with studies less likely to be carried out by scientists in lower-GDP nations. Our results highlight the need for a more systematic and directed evaluation of biodiversity studies, perhaps informing more targeted research towards those areas and taxa most depauperate in research. Only by doing so can we ensure that biodiversity research yields results that are relevant and applicable to all regions and that the information necessary for the conservation of threatened species is available to conservation practitioners

Acute military psychiatric casualties from the war in Iraq

Background: The view that most military personnel evacuated from war zones are suffering from combat stress reactions, or are otherwise traumatised by the horrors of war, has an impact on all aspects of military psychiatry. Aims: To delineate the reasons for psychiatric aeromedical evacuation from Iraq from the start of build-up of UK forces in January 2003 until the end of October that year, 6 months after the end of formal hostilities. Method: A retrospective study was conducted of field and in-patient psychiatric assessments of 116 military personnel evacuated to the UK military psychiatric in-patient facility in Catterick Garrison. Results: Evacuees were mainly non-combatants (69%). A significant proportion were in reserve service (21%) and had a history of contact with mental health services (37%). Only 3% had a combat stress reaction. In over 85% of cases evacuation was for low mood attributed to separation from friends or family, or difficulties adjusting to the environment. Conclusions: These findings have implications especially for screening for suitability for deployment, and for understanding any longer-term mental health problems arising in veterans from Iraq

Does fatigue influence joint-specific work and ground force production during the first steps of maximal acceleration?

During initial acceleration, the first steps of a maximal-effort (sprint) run often determine success or failure in the capture and evasion of an opponent, and is therefore a vital factor of success in many modern sports. However, accelerative events are commonly performed after having already run considerable distances, and the associated fatigue should impair muscle force production and thus reduce acceleration. Despite this, the effects of running-induced fatigue on our ability to accelerate as well as the running technique used to achieve it have received little attention. We recorded 3-D kinematics and ground reaction forces during the first three steps of the acceleration phase from a standing start before and after performing a high-speed, multi-directional, fatiguing run-walk protocol in well-trained running athletes who were habituated to accelerative sprinting. We found that the athletes were able to maintain their acceleration despite changing running technique, which was associated with use of a more upright posture, longer ground contact time, increased vertical ground reaction impulse, decreased hip flexion and extension velocities, and a shift in peak joint moments, power, and positive work from the hip to the knee joint; no changes were detected in ankle joint function. Thus, a compensatory increase in knee joint function alleviated the reduction in hip flexor-extensor capacity. These acute adaptations may indicate that the hip extensors (gluteal and hamstring muscle groups) were more susceptible to fatigue than the ankle and knee musculature, and may thus be a primary target for interventions promoting fatigue resistance

A Discontinuous Galerkin Chimera scheme

The Chimera overset method is a powerful technique for modeling fluid flow associated with complex engineering problems using structured meshes. The use of structured meshes has enabled engineers to employ a number of high-order schemes, such as the WENO and compact differencing schemes. However, the large stencil associated with these schemes can significantly complicate the inter-grid communication scheme and hole cutting procedures. This paper demonstrates a methodology for using the Discontinuous Galerkin (DG) scheme with Chimera overset meshes. The small stencil of the DG scheme makes it particularly suitable for Chimera meshes as it simplifies the inter-grid communication scheme as well as hole cutting procedures. The DG-Chimera scheme does not require a donor interpolation method with a large stencil because the DG scheme represents the solution as cell local polynomials. The DG-Chimera method also does not require the use of fringe points to maintain the interior stencil across inter-grid boundaries. Thus, inter-grid communication can be established as long as the receiving boundary is enclosed by or abuts the donor mesh. This makes the inter-grid communication procedure applicable to both Chimera and zonal meshes. Details of the DG-Chimera scheme are presented, and the method is demonstrated on a set of two-dimensional inviscid flow problems

Draft genome comparison of representatives of the three dominant genotype groups of dairy Bacillus licheniformis Strains

The spore-forming bacterium Bacillus licheniformis is a common contaminant of milk and milk products. Strains of this species isolated from dairy products can be differentiated into three major groups, namely, G, F1, and F2, using random amplification of polymorphic DNA (RAPD) analysis; however, little is known about the genomic differences between these groups and the identity of the fragments that make up their RAPD profiles. In this work we obtained high-quality draft genomes of representative strains from each of the three RAPD groups (designated strain G-1, strain F1-1, and strain F2-1) and compared them to each other and to B. licheniformis ATCC 14580 and Bacillus subtilis 168. Whole-genome comparison and multilocus sequence typing revealed that strain G-1 contains significant sequence variability and belongs to a lineage distinct from the group F strains. Strain G-1 was found to contain genes coding for a type I restriction modification system, urease production, and bacitracin synthesis, as well as the 8-kbp plasmid pFL7, and these genes were not present in strains F1-1 and F2-1. In agreement with this, all isolates of group G, but no group F isolates, were found to possess urease activity and antimicrobial activity against Micrococcus. Identification of RAPD band sequences revealed that differences in the RAPD profiles were due to differences in gene lengths, 3' ends of predicted primer binding sites, or gene presence or absence. This work provides a greater understanding of the phylogenetic and phenotypic differences observed within the B. licheniformis species