WAVLD Symposium Handbook_V4.indd - csiro

More documents

Recommendations

Info

Tues 13 November World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 MULTI-RESOLUTION SPATIO-TEMPORAL ANALYSIS OF MAMMALIAN CELLS RECONSTRUCTED IN 3D BY ELECTRON MICROSCOPE TOMOGRAPHY Brad J Marsh W Group Leader/Senior Research Fellow, Institute for Molecular Bioscience, Centre for Microscopy & Microanalysis and School of Molecular & Microbial Sciences, The University of Queensland, St Lucia, QLD 4072, AUSTRALIA SUMMARY The beta cell - the sole source of the hormone insulin in mammals - resides within the islets of Langerhans in the pancreas. We are focused on understanding the basic mechanisms that underpin normal beta cell function, so that we can elucidate the steps that lead to beta cell/islet dysfunction and ultimately, diabetes. To this end, we combine fast-freezing techniques with electron microscope tomography (ET) to conduct comparative structure-function studies of pancreatic islets isolated from mice and humans. To complement insights from these high-resolution 3D reconstructions (“tomograms”) of parts of cells, and to move toward a more integrated or “holistic” approach to understanding the mammalian cell as a unitary example of an ordered complex system, we have undertaken a multi-scale/multi-resolution approach to reconstructing mammalian (beta) cells in toto in 3D by ET at both high (≤5nm) and intermediate (15-20nm) resolutions. By providing complete sets of 3D spatio-temporal coordinates for cells at a range of resolutions that will uniquely inform advanced in silico studies of 3D cell and molecular organization, we are working to develop the world's first navigable ‘Visible Cell atlas’ within the broader framework of the Visible Cell project (http://www.visiblecell.com/). Such an interactive high-resolution map of the 3D landscape of an entire mammalian cell imaged and reconstructed by ET at the EM level will serve as a unique international resource for protein and organelle annotation, database integration and 3D visualization, and as a framework for 4D animations and computational simulations of cells at pseudo-molecular resolution. KEY REFERENCES (IN CHRONOLOGICAL ORDER) 2001 Marsh BJ, Mastronarde DN, Buttle KF, Howell KE, McIntosh JR. Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography. Proc Natl Acad Sci USA. 98:2399-2406 2001 Marsh BJ, Mastronarde DN, McIntosh JR, Howell KE. Structural evidence for multiple transport mechanisms through the Golgi complex in the pancreatic beta cell line, HIT-T15. Biochem Soc Trans. 29:461-467 2002 Marsh BJ, Howell KE. Timeline: The mammalian Golgi - complex debates. Nat Rev Mol Cell Biol. 3:789-795 2004 Marsh BJ, Volkmann N, McIntosh JR, Howell KE. Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells. Proc Natl Acad Sci USA. 101:5565-5570 2005 Marsh BJ. Lessons from tomographic studies of the mammalian Golgi. Biochimica et Biophysica Acta. 1744:273-29 2006 Marsh BJ. Toward a “Visible Cell”…and beyond. Australian Biochemist. 37:5-10 2007 LR Brunham, JK Kruit, TD Pape, JM Timmins, AQ Reuwer, Z Vasanji, BJ Marsh, B Rodrigues, JD Johnson, JS Parks, CB Verchere and MR Hayden. Beta-cell ABCA1 influences insulin secretion, glucose homeostasis and response to thiazolidinedione treatment. Nature Medicine. 13:340-347 2007 P van der Heide, X Xu, BJ Marsh, D Hanein and N Volkmann. Efficient automatic noise reduction of electron tomographic reconstructions based on iterative median filtering. J Struct Biol. 158:196-204 2007 BJ Marsh, C Soden, C Alarcón, BL Wicksteed, K Yaekura, AJ Costin, GP Morgan and CJ Rhodes. Regulated autophagy controls hormone content in secretory-deficient pancreatic endocrine beta-cells. Mol Endocrinol. 21:2255-2269 2007 BJ Marsh. Reconstructing mammalian membrane architecture by large area cellular tomography. Methods in Cell Biology. 79C:193-220 2007 AB Noske, AJ Costin, GP Morgan and BJ Marsh. Expedited approaches to whole cell electron tomography and organelle mark-up in situ in high-pressure frozen pancreatic islets. J Struct Biol. In press ACKNOWLEDGEMENTS This work has been supported by grants from the Juvenile Diabetes Research Foundation International (2- 2004-275) and the National Institutes of Health (DK-71236) in the USA. BJM is a Senior Research Affiliate of the ARC Special Research Centre for Functional and Applied Genomics, and is a Chief Investigator of the ARC Centre of Excellence in Bioinformatics. World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 DETECTING AND INTERPRETING ARBOVIRAL INFECTIONS IN INSECTS. Dr Simon Carpenter Arbovirology programme, Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey. GU24 0BN. UK. simon.carpenter@bbsrc.ac.uk Vector-borne arboviruses have a significant detrimental impact on both human health and animal health and production world wide. A vital key to understanding the epidemiology of the diseases caused by these viruses lies in the correct identification of the insect vectors responsible for their spread. While this appears at first sight to be a relatively simple task, the complexities of virus-vector interactions commonly lead to results that are difficult to interpret. In this talk I will examine the ways in which vectors have traditionally been implicated, including studies of their ecology, the presence of virus in field collected samples, PCR positives and laboratory-based infection trials. I will begin by distinguishing between different modes of arboviral transmission and their implications for studying the epidemiology of each particular virus. I will then examine the process by which vectors become infected when taking a blood-meal from a viraemic host. This will include a discussion of the various barriers that may inhibit dissemination of the virus through the body of the insect vector, with examples drawn from work carried out upon biting flies. I will then briefly describe the influence of environmental factors upon this process and examine the evidence for likely impacts with regard to climate change. The commonly used techniques of arbovirus detection in insects will then be described in detail and their advantages and disadvantages examined. I will critically explore a case study of ongoing bluetongue virus outbreaks in Europe, (spread via Culicoides midges, which are true biological vectors), to illustrate how these techniques are commonly implemented in practice. This will include a discussion of where vector diagnostics can be integrated into other diagnostic methods to monitor arboviral circulation, and suggestions as to how these can be easily expanded into standard laboratory set-ups. Finally, I will discuss the application of novel and high-throughput diagnostic techniques to virus-vector systems and some of the methodological difficulties that need to be addressed to ensure their correct application. I will discuss the problems intrinsic in attempting to combine entomological and molecular expertise emphasising that input is required from all parties to facilitate the incorporation of new technologies into mainstream diagnostics. Tues 13 November
Tues 13 November World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 MOLECULAR METHODS FOR SPECIATION Dr Hez Hird, Senior Molecular Biologist Biotechnology and Molecular Genetics Central Science Laboratory Sand Hutton York YO41 1LZ United Kingdom E-mail h.hird@csl.gov.uk The advent of the polymerase chain reaction (PCR) has revolutionised the diagnosis of diseases over the last 2 decades. The challenge though has been to develop practical routine methods, which could be used in diagnostic laboratories, where reliable and simple amplicon detection would allow the objective analysis of results. The main breakthrough in achieving this aim came at the end of the 1990s with the development of real-time PCR using TaqMan technology. In TaqMan PCR, the exponential amplification of target specific DNA is measured by the use of an oligonucleotide probe complementary to sequence internal to the PCR primers and labelled at each end with fluorescence reporter and quencher molecules. When the probe is intact, the reporter dye fluorescence is absorbed by the quencher molecule by fluorescence resonance energy transfer (FRET). During amplification, the 5’ nuclease activity of Taq polymerase causes the probe to be cleaved and a signal is generated from the free fluorescence reporter dye. The increase in fluorescence is related to the amount of template amplified and can be measured using a fluorimeter. TaqMan PCR offers a number of advantages over conventional PCR, including closed tube assay format with no need for post- PCR manipulations, increased sensitivity, capacity for high throughput and the objective analysis of results. This presentation will focus on the application of real-time PCR based methods for the detection and identification of zoonotic diseases and will be illustrated using Mycobacterium bovis and Bacillus anthracis. M. bovis is a problem in the UK where eradication of the disease from the national cattle heard has been unsuccessful. This is probably due to the presence of M. bovis in wildlife reservoirs, primarily in the badger, which is highly susceptible to the disease. One strategy to eradicate the disease is the vaccination of both badgers and cattle using M. bovis BCG, however differentiation between M. bovis shed by infected animals and M. bovis BCG shed by vaccinated animals is impossible using current PCR based assays. Ongoing work at the Central Science Laboratory, to develop a rapid, robust real-time PCR based assay, to differentiate M. bovis and M. bovis BCG, will be discussed. Bacillus anthracis is an example of a zoonotic disease which causes the rapid death of humans after inhalation exposure and as such, is a possible biological warfare agent. This powerful driver has led to the development of real-time PCR assays for the rapid identification of B. anthracis in the field, and which has necessitated the development of robust portable real-time PCR equipment. There are a number of portable real-time PCR machines available, however the Cephaid Smart cycler appears to be currently the most popular for non-military uses. The Smart cycler has sixteen cycling modules which can be independently programmed, via a controlling computer, to perform four colour, real-time fluorometric detection. The system software enables one or more operators to define and simultaneously carry out separate experiments, each with a unique set of cycling protocols, threshold criteria and data analysis. The data can be viewed in realtime and when used with assays optimised to return results in 30-40 minutes, the time to positive identification for a sample can be kept to a minimum. An additional advantage of the Smart cycler over the laboratory based real-time machines is the much reduced price: making real-time PCR more accessible. The application of field based portable real-time PCR methods for disease diagnosis will also be discussed. In summary, this presentation will demonstrate the use of real-time PCR for the identification of zoonotic diseases, which for the first time offers the opportunity for field-based PCR testing. 13th International World Association of Veterinary Laboratory Diagnosticians Syposium 11 - 14 November 2007 Wednesday 14 November
Page 1 and 2: Symposium Proceedings HOSTS GOLD SP
Page 3 and 4: SPONSORSHIP SPONSORS WAVLD 2007 ext
Page 5 and 6: INVITED SPEAKERS CONTINUED Professo
Page 7 and 8: Wednesday 14 November (continued) 1
Page 9 and 10: WAVLD POSTER PRESENTATIONS (continu
Page 11 and 12: DINING IN MELBOURNE Melbourne’s m
Page 13 and 14: World Association of Veterinary Lab
Page 35 and 36: Tues 13 November World Association
Page 37 and 38: Tues 13 November World Association
Page 39: Tues 13 November World Association
Page 57 and 58: Wed 14 November World Association o
Page 63 and 64: Poster Presentations World Associat
Page 79 and 80: Poster Presentations Objectives Wor
Page 91 and 92:
Poster Presentations World Associat
Page 93 and 94:
Page 95 and 96:
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Page 103 and 104:
Page 105 and 106:
Page 107 and 108:
Page 109 and 110:
Poster Presentations Aims World Ass
Page 111 and 112:
Page 113 and 114:
Page 115 and 116:
Page 117 and 118:
Page 119 and 120:
show all

WAVLD Symposium Handbook_V4.indd - csiro

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?