Montana State University Billings
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Item Optimization of DNA extraction and size selection for NGS sequencing across plant families found in dover memorial park(Montana State University Billings, 2023) Schwartz, Olivia ; Comer (Faculty Mentor), Jason; Jason ComerBiodiversity can be explored in a variety of ways, from species richness to phylogenetic diversity. This project aims to investigate the plant biodiversity of Montana through analyses of species diversity (floristic collections) and phylogenetic diversity (next-generation sequencing [NGS]). Plants are well known for their secondary metabolites that interfere with downstream applications, such as DNA extraction and sequencing. To investigate phylogenetic diversity, optimized protocols for DNA extraction, fragmentation, and size selection need to be developed first. By optimizing extraction protocols, unique plant family characteristics will minimally affect yields and save time spent troubleshooting downstream applications. Plant specimens collected from Dover Memorial Park over the 2022 growing season were used to optimize an NGS workflow. This study found additional fragmentation of genomic DNA was unnecessary and automated size selection was sufficient to select the optimum fragment size range.Item Characterizing the growth patterns of novel S. aureus mutants; both in vitro and ex vivo(Montana State University Billings, 2022) Estes, Dominic ; Wynter, Doyle ; Byrn, Lien ; Collins (Faculty Mentor), Madison; Madison, CollinsStaphylococcus aureus (S. aureus) is a ubiquitous commensal of the human anterior nares that is estimated to permanently colonize ~30% of the population. S. aureus is also a predominant infectious pathogen that causes significant morbidity and mortality and bears a considerable burden on the healthcare industry. Options for treating this “superbug” are dwindling at an alarming rate. Although initially being considered a hospital-acquired pathogen, community-associated strains have emerged. These strains have the ability to avoid normal immune cell killing and cause disease in healthy individuals. Mechanisms for how S. aureus can escape the defenses of the body are incompletely defined. Previously published work has demonstrated a role for the two-component gene regulatory system, SaeR/S, in S. aureus and that the SaeR/S system influences the ability for the immune system to perform effectively1–3. Although initially considered a two-component system, SaeR/S is actually composed of four genes: saeP, saeQ, saeR, and saeS and the roles of saeP and saeQ are yet to be fully discovered. It is speculated that SaeR/S inhibits the proper function of attacking innate immune cells that circulate in the blood, although the role of the accessory proteins on the blood are completely unknown. We have begun to characterize the role of these accessory genes by using a clinically relevant strain of S. aureus USA300 and isogenic deletion mutants (deficient in either saeP and saeQ; USA300ΔsaeP and USA300ΔsaeQ, respectively). Experiments first began by quantifying the growth patterns of these mutants during in vitro broth culture, as well as, ex vivo during growth in heparinized human whole blood. These studies will help to fill clinically relevant gaps in our understanding of how S. aureus escapes the host immune system to advance disease during septicemic infection. Defining how this pathogen can survive immune defenses in our circulatory system can help identify new potential targets for the design of therapeutics.Item An Elongator Knock Out Mouse Model for ALS(Montana State University Billings, 2022) Snow, Magge ; Snyder, Sara ; Trudell, Rachel ; Pond, Renzie; Cameron, BreAnna ; George (Faculty Mentor), Lynn; Lynn GeorgeAmyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that results in the death of motor neurons. As a consequence of motor neuron death, the muscles they innervate atrophy, causing patients to lose their ability to walk, talk, eat, and eventually breath, such that patients typically die within 4 years of diagnosis. Worldwide, ALS is the most common motor neuron disease. Fifteen people are diagnosed with ALS every day and importantly, the number of cases is projected to increase 69% by the year 2040. The George Lab studies a molecular complex called Elongator, and specific mutations in genes encoding Elongator subunits are associated with ALS. To determine whether motor neurons express Elongator, we used a genetically engineered reporter mouse that “reports” the expression of Elp1, encoding the scaffolding subunit for Elongator. Our results indicate that Elp1 is in fact expressed by alpha motor neurons, a subpopulation of motor neurons in the spinal cord that is most impacted in ALS. To investigate Elongator’s specific function in this cell type, we then generated a conditional knockout (CKO) mouse, where Elp1 is selectively ablated in motor neurons. These mice exhibit reduced motor function, as evidenced by PaGE testing, motor fasciculations, diminished muscle mass and overall body weight (~ ½ the weight of their littermate controls), and a shortened life span (averaging only 3 months). All of these symptoms are hallmark features of ALS. We hypothesized that the phenotype of our CKO mice is due to the death of motor neurons. To investigate this question, the number of alpha motor neurons in the lumbar enlargement was quantified in control and CKO mice using immunohistochemistry and Image J software. Alpha motor neuron numbers were found to be significantly decreased in the CKO. In conclusion, these data demonstrate that Elongator function is essential for the function and survival of motor neurons. Additionally, our Chat-Cre; Elp1LoxP/LoxP mice represent a new Elongator mouse model for studying the cellular and molecular mechanisms that contribute to ALS.