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Imaging liver-stage malaria parasites Rankin et al, Cellular Microbiology 125, 2010

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Imaging liver-stage malaria parasites Rankin et al, Cellular Microbiology 125, 2010

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    1. Imaging liver-stage malaria parasites Rankin et al, Cellular Microbiology 12(5), 2010 Qasim Rafique Veriah 21.02.2011 Latest paper published in April 2010, discuss many different microscopy techniquesLatest paper published in April 2010, discuss many different microscopy techniques

    2. Main aims of the paper: Investigated: Stages of development of Plamsmodium in liver. Investigated by: Laser scanning Conofocal Microscopy Epifluorescence microscopy Electron microscopy Live cell fluorescence imaging. Mouse Plasmodium infecting type Plamsmodium Bugretii?? Microscopy done after killing the mouse and artificially infecting the cells. Mouse Plasmodium infecting type Plamsmodium Bugretii?? Microscopy done after killing the mouse and artificially infecting the cells.

    3. Sporozoites in salivary gland. gametes unite in stomach. Oocysts in stomach wall of mosquito. Liver phase Release of merozoites from liver Merozoytes infect RBC Life cycle of plasmodium Life cycle of MP, the same as humansLife cycle of MP, the same as humans

    4. Erythrocytes stage of malaria Erythrocytic stage: Long studied. Disease manifestation begin in RBC Current diagnosis in RBC Drug target are in RBC Classically studied since 1937, Classically studied since 1937,

    5. Why liver stage is important? Conserved developmental processes of plasmodium in liver. progression from Sporozoites to merozoites. Potential drug and vaccine target. (Cohen etal., 2010). Liver stage is under studied as compare to blood stage (James and Tate, 1937). Describe the importance as discussed in paper. Mainly, liver stage is very begining of disease so prophylitic therapy can also be used. Describe the importance as discussed in paper. Mainly, liver stage is very begining of disease so prophylitic therapy can also be used.

    6. liver stage parasite developmental findings Hepatic entry of sporozoite and rapid and extensive replication. Electron microscopy used beginning of this century focusing on liver (Sinden, 1978;Meis etal.,1985) Liver stage parasite imaging more complex than blood. In live imaging era, still electron microscopy is important tool in examination of liver stage parasite development e.g. novel parasite protein (Muller et al.,2005; Aly et al., 2008).

    7. liver stage parasite developmental findings Wide field Epifluorescence imaging of live cells and sporozoite movements dont cease. Quantitive analysis of live parasite in hepatoma cells reveal parasite growth rate high close to host nucleus. Manipulation of host cells and prevention of apoptosis. Alteration of host cell cytoskeleton and increase in production of parasite without inducing cell death pathways.

    8. liver stage parasite developmental findings Immunoflourescence study using organelle specific antibody and live microscopy of cultured cells using specific dyes show plasmodium liver stages and reveals host ER gathers around PVM but mitochondria and Golgi bodies do not.

    9. Pre-erythrocytic development of Plasmodium HepG2 cells: Hepatocytes: Transgenic mice expressing Green Fluorescent protein in hepatocytes. Plasmodium berghei: Transgenic expressing mCheery (red coloured fluorescent protein)HepG2 cells: Hepatocytes: Transgenic mice expressing Green Fluorescent protein in hepatocytes. Plasmodium berghei: Transgenic expressing mCheery (red coloured fluorescent protein)

    10. Pre-erythrocytic development of Plasmodium

    11. Late plasmodium exo-erythrocytic stages by different imaging techniques

    12. New range of proteins and techniques for brightness and photo stability The red fluorescent protein mCherry great promise for imaging of plasmodium liver stage parasites (Graewe et al., 2009) GFP photostable used for single labelling experiment. Spinning disc microscopy images of great clarity can be obtained by making large no of slices of each 3D reconstruction.

    13. Drawbacks and limitations Hepatoma cells adherent but still move out of focal plane Photo bleaching Using dyes for labelling fresh dye can be added but not possible in fluorescent proteins GFP fairly photo stable, single labelling experiment

    14. Future directions Genetic manipulation and stage-specific gene expression Host cell inhibition of parasite development Imaging of human malaria parasites in vivo Whole body imaging

    15. CONCLUSION Introduction of new cell dyes to monitor signalling events or viability of parasite and host in real time. New fluorescent proteins in broader array of colours and more photos table (Shaner et al., 2004). In future ability to maintain developing parasite in host cell under ideal conditions Use of double fluorescent P berghei and reduce photo toxicity would increase our understanding of liver stage development of plasmodium

    16. Thanks Any question ?

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