Wednesday, 3 July 2013

Cell Biology of Infectious Pathogens - Ghana 2013

For the last four years there has been a cell biology workshop in West Africa, organised by Dick McIntosh, an intense two week course aiming to help young African scientists around the master's degree stage of their careers. This course ran again this year, and was the first organised by Kirk Deitsch (malaria expert and a regular from the previous courses) and I was fortunate enough to be invited to teach the trypanosome half of the course. For its fifth incarnation the course returned to a location where it has previously been held, the Department of Biochemistry, Cell and Molecular Biology in the University of Ghana, and was organised with Gordon Awandare.


The focus this year was teaching basic cell biology and the associated lab techniques, emphasising how this helps understand and fight some of the major parasitic diseases in Africa: African trypanosomiasis (sleeping sickness), leishmaniasis and malaria. All three of these diseases impact Ghana and the surrounding countries and these diseases are of enormous interest to students embarking on a scientific career in Africa.


Of the three diseases we were teaching about malaria is by far the most well known, both locally and internationally. It is caused by Plasmodium parasites (which are single cell organisms) which force themselves inside the red cells in the blood to hide from the host immune system. Malaria is often viewed as the iconic neglected tropical disease, however in the last 10 years or so the understanding of the disease and efforts to find a vaccine and new drugs has improved vastly. Unfortunately it is still very common (we had one case in the participants on the course in the two weeks), drug resistance is rising, and it places a huge cost and health burden on the affected countries. It also impacts a huge area; almost all of sub-Saharan Africa is at risk.

Looking at Leishmania. One of the lab practicals was making light microscopy samples from non-human infective Leishmania using Giemsa stain.

Leishmaniasis and trypanosomiasis are caused by two related groups of parasites, Leishmania and trypanosomes (also single cell organisms), and if malaria is a neglected tropical disease the these are severely neglected tropical diseases. The two parasites live in different areas in the host, trypanosomes swim in the blood while Leishmania live inside macrophages, a type of white blood cell that should normally eat and kill parasites. In comparison to malaria fewer drugs are available, the drugs are less effective and several have severe side effects. Even diagnosis is thought to often be inaccurate. The impact of these diseases is less than malaria; human trypanosomiasis is thought to be relatively rate and leishmaniasis is confined to a semi-desert band just to the south of the Sahara. Trypanosomiasis does have a huge economic impact though, as it infects cattle and prevents milk and meat production, and cases of leishmaniasis are probably under-reported.

Staining trypanosomes. One practical was making immunofluorescence samples. In this sample the flagellum of trypanosomes was stained fluorescent green using the antibody L8C4.

So what did we teach?

The teaching was a mixture of lectures, small group discussions, lab practicals and lab demonstrations and we taught for 14 hours a day for 11 days; we could cover a lot of material! All the teaching was focused on linking basic cell biology to parasites and to practical lab techniques. Topics taught included how parasites avoid the host immune system, molecular tools to determine parasite species, light microscopy techniques, using yeast as tool to analyse cell biology of proteins from other species, host cell interaction of parasites, and many more.

Detecting human-infective trypanosomes. This gel of PCR products shows whether the template DNA was from a human-infective or non-human infective subspecies of T. brucei. If there was a DNA product of the correct size (glowing green) then the sample was human-infective.

A great example of how all the teaching tied together was polymerase chain reaction (PCR) to determine species. Human-infective trypanosomes have a single extra gene which lets them resist an innate immune factor in human blood which would otherwise kill them, and I taught about why this is important for understanding the disease and how it was discovered. This gene can be detected by PCR, and this technique is used to tell if a particular trypanosome sample could infect people. We ran a practical actually doing this in the lab.

PCR is a simple, adaptable and easy technique for checking any parasite for a particular species-defining or drug resistance gene, and we also taught how to use online genome sequence data to design PCR assays. We even worked through PCR assay design for many individual participant's personal research projects, really transferring the skills we were teaching to their current research. Finally we looked at papers using PCR techniques to critically analyse the experiment and assay design to help people avoid pitfalls in their own work.

This was a great demonstration of how basic cell biology and lab techniques can have real practical application with medical samples and help with surveillance of a disease. We designed all of the teaching to have this kind of practical application.


7x speed timelapse video of fish melanophores responding to adrenaline.

One practical with massive visual impact was the response of fish melanophores to adrenaline/epinephrine. Fish normally use these cells to change colour in response to stimuli and melanin particles (melanosomes) inside specialised cells (melanophores) run along the microtubules which make up a large portion of the cell cytoskeleton. We used it to demonstrate signalling; adrenaline can be used to stimulate movement of the melanosomes towards the centrosome.

This is really flexible experimental system for demonstrating the functions of the cytoskeleton, motor proteins and signalling pathways because the output (movement of the pigment particles) is so easy to observe with a cheap microscope or even a magnifying glass. This experiment was particularly chosen as it is a useful and accessible teaching tool for cell and molecular biology, and many of the course participants had teaching obligation in addition to their research.


Western blots in Western Africa. 100x timelapse of loading and running a SDS-PAGE gel.

We aimed to cover all the major molecular and cell biology techniques and had practicals doing microscopy, immunofluorescence, growing a microorganism (in this case yeast), PCR, agarose gel electrophoresis SDS-PAGE and Western blotting. The yeast practicals were particularly cool; using genetically modified cell lines the students analysed the function of p53, a transcription factor with a major role in recognising genetic damage and avoiding cancer, and how well it promotes transcription from different promoter sequences. These practicals taught growing yeast, temperature sensitive mutants, several types of reporter proteins in yeast and Western blotting, all concerning a transcription factor with huge clinical relevance in cancer!

Exploring DNA and protein structures through PyMol in a bioinformatics session.

Practicals weren't just limited to lab practicals though. We also ran interactive bioinformatics sessions looking at the kinds of data which are freely available in genome and protein structure databases online. These were also very popular, especially as so much data is available for free online.

All in all the course was a great success. The participants were all extremely enthusiastic, hard working and scarily smart! Feedback so far has also been very positive. I feel that courses like this can have a huge impact on the careers of young African scientists, and I sincerely hope that funding can be secured to continue running this type of course in the future.

You can also read more about this course at the ASCB website.

Software used:
ImageJ: Image processing and timelapse video creation.
Tasker for Android: Timelapse video capture.
Pymol: Protein structure analysis

7 comments:

  1. Thanks Richard, the bioinformatics had a great impact on me i will be seeking to advance my knowledge in the area and make it more useful in my career if you hear any course on advanced bioinformatics that has sponsorship please do let me know i will be happy to attend
    Thanks

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    1. Thank you for your kind words. I will let you know if I see anything!

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  2. Woooooowww, I got all these information in two weeks...!!! I am sooo glad I got the opportunity as a participant! This time around I had a practical feel of all the techniques I had to imagine like Western blotting, SDS-PAGE, among others. I also got enlightened on Malaria, Burulli Ulcer, Leishmaniasis and Trypanosomiasis and how these techniques could be used in curbing such diseases! Thanks Richard for a great summary, keep up with the good work... Kudos to all the organizers of ASCB workshop 2013!

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    Replies
    1. It is great to get feedback like that, thanks for the message :)

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