Summer Research Update

August 23, 2022

Summer 2020 summary:

Research update from the May 5th blog post: “Its not just a simple conversation!”

The topic of taking notes while conversing with faculty who have expertise in the possible directions my project may go was explored.

Link to the May 5th 2022 blog post: https://pricketts4582.wixsite.com/prickett/post/it-is-not-just-a-simple-conversation

June 2022

In June, the result from my grant proposal to Sigma Xi was received. Sigma Xi, the Scientific Research Honor Society, is an international honor society for science and engineering that spans all age ranges. The Grants in Aid Research program offers various amounts of aid to undergraduate researchers to help pay for experiment supplies. I met a representative of Sigma Xi at the National McNair Conference in Maryland and decided to submit my proposal for some extra supply funds. My proposal was declined. The best part about this experience is that the results tab in the Sigma Xi portal included feedback so I can improve my proposal and resubmit it for the next round.

Here is a link to Sigma Xi’s Grants in Aid program: https://www.sigmaxi.org/programs/grants-in-aid-of-research

July 2022

July brought the beginning of my experiment. I had some help from the Principal Investigator, Dr. Walsh, in writing up an experimental protocol. Knowing exactly what will be done in an experiment helps to reduce supplies used, confusion on tasks, and makes exploration easier. Sometimes making experimental protocols looks concise on paper but have tasks that take weeks to finish. For example, the shortest, easiest, and most straightforward task in my experiment was growing the MCF-7 cells I am dosing and fixing to slides. The longest part is using the Raman machine to gather spectra.

Waiting does not equal time lost on a project.

There are always going to be factors outside of a researcher’s control that need to be addressed as they occur. For instance, my university only has one Raman machine, and multiple groups use it. The scheduling issues indicate that my project may take longer. Another important research lesson is that waiting is okay. There are always more papers to read, more knowledge to accrue and more experiments to design so waiting does not equal time lost.

Personally, the most vexing thing about my project is the nuances of the Raman machine itself. Starting a block of research in the morning by shutting down and restarting the system only to have the machine act possessed makes the Raman machine very cheeky. The troubleshooting of the Raman machine just makes me more efficient at gathering data for the experience.

Having to troubleshoot is not the end of the day, troubleshooting is valuable experience.

The important thing is to not attach emotions to the glitches of the machinery. It is not the researchers fault it decided to not have its video camera working today. It is not the end of the world, the end of my job, or the ruin of my day. It just needs a stern look from the tech to work properly. Or maybe a few cycles of powering off and on again.

There is always going to be troubleshooting. Being able to troubleshoot is one of the best skills a researcher can have because it shows the knowledge and ability of the researcher. It is also good to have documentation of the errors to show a supervisor if you end up resolving them yourself.

August 2022

All in all, my research project is coming along well. I have conducted a small, preliminary dosage experiment and am now gathering the spectral data for analysis. Another literature review has been suggested so I can learn more about one of the potential outcomes of my experiment: that Monotropa uniflora produces a neurotoxin called grayanotoxin akin to Rhododendrons.

This is a screenshot of multiple spectra before any post-processing to reduce noise. Each peak tells me what is present in the sample. The big peak in the 2900 cm-1 range indicates lipids from the cell membranes. My goal is to discern differences between the experimental groups to show that the extract of M. uniflora effects cells.

It is entirely possible that the concentration of extract and ethanol I chose to use in this experiment is too low. If so, then I will need to troubleshoot and design my next replication with a different concentration. Learning through experimentation takes time. Shortening the time is takes to learn through experiments takes experience.

Oral poster presentation at UW-Superior

In August, I attended and presented at the first Research Summit for the UW system at the University of Wisconsin – Superior to share what I have done so far. I made a poster detailing the literature I have found, my question, and how I plan to answer my research question. The script for the oral poster presentation was written using the feedback from Sigma Xi. The biggest feedback I received was that my proposal was not compelling enough. I outlined the greatest potential my research can have in the script below.

Does Monotropa uniflora extract cause cells to phenotypically exhibit grayanotoxin mechanisms?

“Everyone experiences pain sometime in their lives. If the pain is severe enough, opioid based drug therapy will be prescribed. Unfortunately, opioid drugs are addictive to the point of being an epidemic in this country. Opioids also have other side-effects that hinders people from working and performing daily tasks. Non-opioid drugs like NSAIDs also have negative side-effects which limit the kinds of other medication that can be taken concurrently, such as anti-depressants. Finding a drug to treat pain without addiction, organ damage, and has better compatibility with other medicines is desired.

An alternative medicine that has documentation since the Ancient Greeks is Rhododendron honey. Rhododendrons produce a neurotoxin called grayanotoxin which gets concentrated into the honey. Grayanotoxin binds to sodium voltage channels, depolarizing nerve and muscle cells preventing pain signals from being sent. Grayanotoxin and Rhododendron honey is being researched as a possible therapeutic agent for pain around the Black Sea region.

In North America, a different alternative medicine is used for pain management. Monotropa uniflora is written as a treatment for pain and anxiety. It is unknown what constituent of the plant is the therapeutic compound.

Does Monotropa uniflora produce grayanotoxin like rhododendrons? It is possible since M. uniflora and Rhododendrons are in the same plant family. However, so are blueberries so results could go wither way.

My project is to visually and chemically compare MCF-7 cells that have been untreated, treated with ethanol, and plant extract from M. uniflora. Raman spectroscopy is used to bombard ethanol fixed cells with light in the visible spectrum. The scattered light is detected, producing a line of peaks. The spectral data is run through multidimensional PCA-LDA to determine a difference between groups. This project is still ongoing, and more data needs to be collected at different dosages.

PCA is an “unsupervised” method of detecting differences. The computer looks at every datapoint for differences, regardless of whether the datapoints are relevant. LDA is the “supervised” method, which means the computer knows which datapoint is in each group and only looks at the common peaks causing differences between groups. Once the PCA-LDA test shows the extract being different than the ethanol and untreated groups, more tests will be done to gather phenotype evidence similar to literature reports for grayanotoxin.

Another research update will be posted in the fall.

Thank you for your time.