Spring 2021 Grant Awardees

Cannabinoids Potential as Antibiotics

The CDC lists antibiotic resistance as the number one global health threat in the world1. Certain bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) responsible for various types of tissue infection in humans, Staphylococcus aureus (S. aureus) responsible for skin infections and Escherichia coli (E-coli), a bacteria that commonly causes urinary tract infections are growing increasingly resistant to current antibiotics. The CDC statistics show there are 2.8 million antibiotic resistant infections yearly, resulting in 35,000 deaths1. This is where cannabis cannabinoids could play a vital role as new antibiotics. Cannabinoids are secondary metabolites found in the cannabis sativa plant believed by many to have various medicinal properties. The human body contains an endocannabinoid system that works with cannabinoids to create numerous types of signals and triggers in the immune and nervous system of the body.

The proposed project is to test three cannabinoids, tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabigerol (CBG) against MRSA, Staphylococcus aureus, and E-coli. The project will be performed using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. The MIC value will be a numerical representation of the concentration of cannabinoid required to inhibit the bacteria’s growth. The MBC will be calculated from the MIC achieved and represents the concentration of cannabinoid that causes approximately 99.9% bacterial death. These assays will combine an inoculated bacteria strain at a specific concentration and the cannabinoid at increasing concentrations in 96 well plates. The lower the MIC value the better and the closer the MBC to the MIC the better antimicrobial properties. The need to develop improved antibiotics is long overdue. In this research a goal would be to see all cannabinoids tested effective against both gram-negative (E-coli.) and gram-positive (MRSA, S. aureus) bacterial strains. This would provide evidence to warrant further research into these plants compounds as future antibiotics.

Larval Lake Whitefish/Zooplankton Density at Day and Night

Lake whitefish support some of the most commercially, culturally, and recreationally important fisheries in the Great Lakes. Lake whitefish populations have decreased since the 1990s in the upper Great Lakes, coinciding with the invasion of zebra and quagga (dreissenid) mussels (Pothoven et al. 2001). These invasive mussels have decreased phytoplankton, and subsequently, zooplankton populations (Pothoven and Madenjian 2008). Adequate amounts of zooplankton are critical for the growth and survival of larval fish (Zorn et al. 2020), including lake whitefish, who spend this time in shallow, beach habitats.

One hypothesis for decreased lake whitefish populations in the Great Lakes is a result of decreased zooplankton populations. However, limited data exist that examine zooplankton and larval lake whitefish relations, and existing data are only from daytime beach surveys. Habitat use by zooplankton and fish can vary significantly at day versus night, necessitating day and night comparisons to determine whether daytime sampling is adequate for quantifying lake whitefish-zooplankton relations (Doubek et al. 2020). To address these knowledge gaps, I ask: 1) What is the relationship between larval LAW and zooplankton prey density in Lakes Michigan, Huron, and Superior?, and 2) Does the relationship differ at night? To expand our knowledge on the relations between zooplankton and larval lake whitefish populations, I will work with LSSU and the Sault Tribe of Chippewa Indians to sample zooplankton, larval lake whitefish, and environmental data (water temperature, pH etc.) at 3 locations in each of the following: Lake Michigan, Lake Huron, and Lake Superior. My sampling will occur between April-June in the Eastern UP. In addition to this, I am receiving samples from multiple tribal, state, and federal agencies throughout the Great Lakes region (Green Bay, Muskegon, Duluth etc.). All samples will be processed by myself in the lab at LSSU, and I will run statistical analyses.

The Effects of Diaphragmatic Breathing on Melatonin and Sleep Among Collegiate Student-Athletes

Along with the many responsibilities the average college student experiences, collegiate student-athletes have additional time-consuming responsibilities. These additional responsibilities include practices, competitions, travel, team meetings, and more. The stressors of academics and athletics have proven to have a large impact on the well-being of student-athletes, with almost half of male student-athletes and more than half of female student-athletes reporting that stress from athletics and academics has impacted their mental or emotional health (Humphrey, Yow, and Bowden, 2000). With the high prevalence of stress among student-athletes, it is common to lead to other negative impacts on the body. Chronic stress can lead to a decrease in quantity and quality of sleep (Soffer-Dudek and Shahar, 2011). Insufficient sleep has many consequences to the body, including the weakening of the immune system and a decrease in performance of the hypothalamus, pituitary, and adrenal glands, which leads to further consequences of a decline in physical performance and learning ability (Ghorbani at al., 2019). Melatonin is the main hormone synthesized by the pineal gland that is controlled by the hypothalamus and plays a role in regulating sleep-wake cycles by releasing low levels during the day, and the highest levels at night to prepare the body for sleep (Jennum et al., 2016). Because stress can impact the performance of the hypothalamus, therefore impacting the secretion of melatonin, circadian rhythm can be disrupted, consequently interfering with sufficient sleep in those affected. Due to the damages that stress and lack of sleep can have on an athlete’s performance in the classroom and in competition, it is imperative to give athletes a technique they can utilize on and off the field of play. Diaphragmatic breathing (DB) is a technique used by many yoga and meditation practices that involves breathing deeply into the lungs by expanding the diaphragm instead of the rib cage (Martarelli et al., 2009).

This breathing technique utilizes techniques that strengthen the diaphragm, decrease the work of breathing by slowing respiration rate, and decrease oxygen demand (Cleveland Clinic, 2018). Diaphragmatic breathing exercises have proven to be a non-invasive treatment that positively impacts insomnia, cardiac autonomic function, depression, and anxiety, all of which could improve the mental and physical health of an athlete (Ghorbani et al., 2019). Not only does diaphragmatic breathing improve these important aspects of athlete health, but it also has been proven to reduce testing stress and increase testing performance of the general college student population (Paul, Elam, and Verhulst, 2007). The purpose of this experiment is to investigate the impact of diaphragmatic breathing exercises on sleep in the collegiate student-athlete population by testing and observing melatonin levels. Participants will be given a five-minute diaphragmatic breathing exercise and asked to perform it every day for a period of two weeks. Melatonin levels will be collected once before they begin the diaphragmatic breathing exercises, and again after the two weeks of completing the exercises. It is hypothesized that sleep quantity and quality will improve among the student-athletes performing the breathing exercises and will be reflected in the increase of melatonin levels. Methods: A group of twenty-four student-athletes at Lake Superior State University will be asked to volunteer in this study. They will be given instructions on the diaphragmatic breathing exercises they will be asked to perform, and written consent will be obtained. There will be a control group of twelve students-athletes that will not perform diaphragmatic breathing exercises, and an experimental group of twelve student-athletes that will perform the breathing exercises. Data will be collected in two ways, which will consist of a survey and measurements of melatonin levels that will be completed before and after the study.

Each participant will be assigned a numerical code to maintain confidentiality of the participants. The experimental group will be shown a five-minute diaphragmatic breathing exercise they can find in any mobile App Store called, “Strategic Breathing,” that was created by Max Strom, an author and teacher with many accolades regarding his work with diaphragmatic breathing (Strom, 2021). The exercise within the app includes visual and audio instructions. The experimental group will be asked to perform the breathing exercise 30 minutes before bed every night for the two-week period in their own residency (Chander, 2020). The control group will not be asked to perform the breathing exercises and to continue on with their daily habits. The survey that will be conducted will ask a series of questions that will be beneficial to determine the effectiveness of the breathing exercises from a subjective point of view within the participants. Adapted from Assessment of Recovery after Scheduled Time off to Hospital Employees Working Night Shift, questions will be asked about sleep quality, sleep quantity, athletic performance, academic performance, and levels of stress participants experience. (Blyly & Ranson Olson, 2018). These surveys will be given before and after the study to each participant in both the control and experimental group. To measure melatonin levels, a melatonin saliva ELISA Assay kit will be used. Saliva will be collected by unstimulated passive drool. Participants will be asked to not eat 60 minutes before sample collection, as well as to avoid consuming alcohol 12 hours prior to sample collection. To analyze the data, a t-test will be used to observe the changes in melatonin levels within each individual from the beginning of the two-week experiment compared to the end. Another t-test will be conducted to test the average effect between the experimental and control groups. An ANOVA will be performed to determine any significant differences. If there is a significant difference that supports the hypothesis that diaphragmatic breathing increases melatonin levels, therefore improving sleep, the results will be presented to coaching staffs throughout the athletic department at Lake Superior State University. If implemented, diaphragmatic breathing could be a beneficial tool for student-athletes to improve their physical and mental health. Literature Cited Blyly, McKenna, B. Ranson Olson. 2018. Assessment of Recovery after Scheduled Time off to Hospital Employees Working Night Shift. Chander, T. , Lakeview Internal Medicine, personal communication. Cleveland Clinic. 2021. Diaphragmatic breathing exercises & techniques.