Week Two

Hello again, and hope everyone has had a great second week with their SRPs. Mine has been very busy. I spent a lot of time collecting virgin D42 females and TDP-43 males, which I talked about in my first post, but I also got to learn and practice some new techniques this week. One of these was larval turning. This is a way for us to check to see if the food we are feeding the fruit flies has any affect on their motor functions. I collected larvae from their containers and put them on an agar plate that contained grape juice. I then would, while looking at the larvae under a microscope, flip them onto their backs. After flipping them, I would record the time it took for them to flip back over and begin moving again. In larvae that contain both the TDP-43 and the D42 genes and therefore the characteristics for ALS, the time it takes to flip back over is significantly longer. When larval turning, two times have to be recorded, the 'freeze time' and the 'turning time'. Often, when a larvae is turned on to its back, it initial reaction is to stop moving and try to assess its situation before flipping over. The time we care more about is the flipping time, so was have to record both the time it take to actually flip and the time it spends frozen on its back. A larvae isn't considered fully flipped until you can see the entire strip on its back and it begins to move in a forward motion. Sometimes larvae will partially flip over and wiggle on their sides to move instead of completely flipping over. I was larval turning with white eyed flies. There are the flies we use as controls to show that they do not have TDP-43 genes or D42 genes. Flies with either of those genes will have red eyes instead of white eyes.

Wednesday was lab meeting for my group, the drug screen team. They went over what we are doing right now, results of past experiments, and what we plan to do in the future. It was cool how the entire lab pitched ideas for reasons behind results, thing that should be standardized or re-done, and things that could be tested in the future. My team also discussed two previous experiments they had been working on that I had been briefly involved with the previous week. They had been using enoxacin (an antibacterial) and different peptides to see if there were increased rescues in the fruit fly population. Both had no statistically significance, and large amounts of error due to inconsistency in the number if rescues in the vials and the variability of the number of flies both produced and rescued. They also commented on how drugs in the past had work only in specific tissues, and that they would often rescue glia and motor phenotypes but not muscle phenotypes. To finish the lab meeting, they outlined their plans for future experiments, which right now include testing fruit flies who ate food with high concentrations of sugar. To set up this experiment, we had to make our own fruit fly food.

The making of the food was a new experience for everyone in my team, not just me. The food is prepared using agar, yeast, several other components, and then the component we are testing, the drug, or in this case, the sucrose, is mixed in. It was quite an adventure making the food, resulting in several overcooked batches and a lab that smelled vaguely of burned marshmallows. We made a batch with the normal concentration of sugar, and ones with two, four and eight times that concentration. We are testing to see if high concentrations of sugar have an effect on the condition of the fruit flies due to past experiments that showed very low larval turning times, meaning that the larvae had greater control over their motor functions and muscles. Additionally, the larvae were all very robust and juicy across multiple phenotypes.

After we made the food, I set up crosses of fruit flies with the flies I had been collecting all week. I put six virgin D42 females and three TDP-43 males in each vial. I set up crosses with three phenotypes for the TDP-43 flies: human wildtype, white (control), and G298S. There were four different concentrations of food, and there were three vials for each phenotype for each concentration, resulting in a total of 36 vials, over 200 virgins female fruit flies, and over 100 male fruit flies.

So that was my week, hope everyone else is having a great time with their SRPs too!