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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.Item Essential Oils from Monarda fistulosa: Chemical Composition and Activation of Transient Receptor Potential A1 (TRPA1) Channels(MDPI, 2020-10) Ghosh, Monica; Schepetkin, Igor A.; Ozek, Gulmira; Khlebnikov, Andrei I.; Damron, Derek S.; Quinn, Mark T.Little is known about the pharmacological activity of Monarda fistulosa L. essential oils. To address this issue, we isolated essential oils from the flowers and leaves of M. fistulosa and analyzed their chemical composition. We also analyzed the pharmacological effects of M. fistulosa essential oils on transient receptor potential (TRP) channel activity, as these channels are known targets of various essential oil constituents. Flower (MEOFl) and leaf (MEOLv) essential oils were comprised mainly of monoterpenes (43.1% and 21.1%) and oxygenated monoterpenes (54.8% and 77.7%), respectively, with a high abundance of monoterpene hydrocarbons, including p-cymene, γ-terpinene, α-terpinene, and α-thujene. Major oxygenated monoterpenes of MEOFl and MEOLv included carvacrol and thymol. Both MEOFl and MEOLv stimulated a transient increase in intracellular free Ca2+ concentration ([Ca2+]i) in TRPA1 but not in TRPV1 or TRPV4-transfected cells, with MEOLv being much more effective than MEOFl. Furthermore, the pure monoterpenes carvacrol, thymol, and β-myrcene activated TRPA1 but not the TRPV1 or TRPV4 channels, suggesting that these compounds represented the TRPA1-activating components of M. fistulosa essential oils. The transient increase in [Ca2+]i induced by MEOFl/MEOLv, carvacrol, β-myrcene, and thymol in TRPA1-transfected cells was blocked by a selective TRPA1 antagonist, HC-030031. Although carvacrol and thymol have been reported previously to activate the TRPA1 channels, this is the first report to show that β-myrcene is also a TRPA1 channel agonist. Finally, molecular modeling studies showed a substantial similarity between the docking poses of carvacrol, thymol, and β-myrcene in the binding site of human TRPA1. Thus, our results provide a cellular and molecular basis to explain at least part of the therapeutic properties of these essential oils, laying the foundation for prospective pharmacological studies involving TRP ion channels.Item Activity-based, genome-resolved metagenomics uncovers key populations and pathways involved in subsurface conversions of coal to methane(Springer Science and Business Media LLC, 2021-10) McKay, Luke J.; Smith, Heidi J.; Barnhart, Elliott P.; Schweitzer, Hannah D.; Malmstrom, Rex R.; Goudeau, Danielle; Fields, Matthew W.Microbial metabolisms and interactions that facilitate subsurface conversions of recalcitrant carbon to methane are poorly understood. We deployed an in situ enrichment device in a subsurface coal seam in the Powder River Basin (PRB), USA, and used BONCAT-FACS-Metagenomics to identify translationally active populations involved in methane generation from a variety of coal-derived aromatic hydrocarbons. From the active fraction, high-quality metagenome-assembled genomes (MAGs) were recovered for the acetoclastic methanogen, Methanothrix paradoxum, and a novel member of the Chlorobi with the potential to generate acetate via the Pta-Ack pathway. Members of the Bacteroides and Geobacter also encoded Pta-Ack and together, all four populations had the putative ability to degrade ethylbenzene, phenylphosphate, phenylethanol, toluene, xylene, and phenol. Metabolic reconstructions, gene analyses, and environmental parameters also indicated that redox fluctuations likely promote facultative energy metabolisms in the coal seam. The active "Chlorobi PRB" MAG encoded enzymes for fermentation, nitrate reduction, and multiple oxygenases with varying binding affinities for oxygen. "M. paradoxum PRB" encoded an extradiol dioxygenase for aerobic phenylacetate degradation, which was also present in previously published Methanothrix genomes. These observations outline underlying processes for bio-methane from subbituminous coal by translationally active populations and demonstrate activity-based metagenomics as a powerful strategy in next generation physiology to understand ecologically relevant microbial populations.Item Evaluation of the Antimicrobial Efficacy of N-Acetyl-l-Cysteine, Rhamnolipids, and Usnic Acid—Novel Approaches to Fight Food-Borne Pathogens(MDPI, 2021) Chlumsky, Ondrej; Smith, Heidi J.; Parker, Albert E.; Brileya, Kristen; Wilking, James N.; Purkrtova, Sabina; Michova, Hana; Ulbrich, Pavel; Viktorova, Jitka; Demnerova, KaterinaIn the food industry, the increasing antimicrobial resistance of food-borne pathogens to conventional sanitizers poses the risk of food contamination and a decrease in product quality and safety. Therefore, we explored alternative antimicrobials N-Acetyl-L-cysteine (NAC), rhamnolipids (RLs), and usnic acid (UA) as a novel approach to prevent biofilm formation and reduce existing biofilms formed by important food-borne pathogens (three strains of Salmonella enterica and two strains of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus). Their effectiveness was evaluated by determining minimum inhibitory concentrations needed for inhibition of bacterial growth, biofilm formation, metabolic activity, and biofilm reduction. Transmission electron microscopy and confocal scanning laser microscopy followed by image analysis were used to visualize and quantify the impact of tested substances on both planktonic and biofilm-associated cells. The in vitro cytotoxicity of the substances was determined as a half-maximal inhibitory concentration in five different cell lines. The results indicate relatively low cytotoxic effects of NAC in comparison to RLs and UA. In addition, NAC inhibited bacterial growth for all strains, while RLs showed overall lower inhibition and UA inhibited only the growth of Gram-positive bacteria. Even though tested substances did not remove the biofilms, NAC represents a promising tool in biofilm prevention.Item Improving the two-photon absorption properties of fluorescent proteins for neuroscience(Montana State University - Bozeman, College of Letters & Science, 2020) Molina, Rosana Sophia; Chairperson, Graduate Committee: Thomas Hughes; Yong Qian, Jiahui Wu, Yi Shen, Robert E. Campbell, Mikhail Drobizhev and Thomas E. Hughes were co-authors of the article, 'Understanding the fluorescence change in red genetically encoded calcium ion indicators' in the journal 'Biophysical Journal' which is contained within this dissertation.; Tam M. Tran, Robert E. Campbell, Gerard G. Lambert, Anya Salih, Nathan C. Shaner, Thomas E. Hughes and Mikhail Drobizhev were co-authors of the article, 'Blue-shifted green fluorescent protein homologues are brighter than enhanced green fluorescent protein under two-photon excitation' in the journal 'The Journal of physical chemistry letters' which is contained within this dissertation.; Jonathan King, Jacob Franklin, Nathan Clack, Christopher McRaven, Vasily Goncharov, Daniel Flickinger, Karel Svoboda, Mikhail Drobizhev, Thomas E. Hughes were co-authors of the article, 'An instrument to optimize fluorescent proteins for two-photon excitation' which is contained within this dissertation.Untangling the intricacies of the brain requires innovative tools that power basic research. Fluorescent proteins, first discovered in jellyfish, provide a genetically encodable way to light up the brains of animal models such as mice and fruit flies. They have been made into biosensors that change fluorescence in response to markers of neural activity such as calcium ions (Ca 2+). To visualize them, neuroscientists take advantage of two-photon excitation microscopy, a specialized type of imaging that can reveal crisp fluorescence images deep in the brain. Fluorescent proteins behave differently under twophoton excitation compared to one-photon excitation. Their inherent two-photon properties, namely brightness and peak absorption wavelength, limit the scope of possible experiments to investigate the brain. This work aims to understand and improve these properties through three projects: characterizing a set of red fluorescent protein-based Ca 2+ indicators; finding two-photon brighter green fluorescent proteins; and developing an instrument to screen for improved fluorescent proteins for two-photon microscopy. Analyzing nine red Ca 2+ indicators shows that they can be separated into three classes based on how their properties change in a Ca 2+-dependent manner. In one of these classes, the relative changes in one-photon properties are different from the changes in two-photon properties. In addition to characterizing, identifying and directly improving fluorescent proteins for enhanced two-photon properties is important. Presented here is a physical model of the light-absorbing molecule within the green fluorescent protein (the chromophore). The model predicts that green fluorescent proteins absorbing at higher energy wavelengths will be brighter under two-photon excitation. This proves to be the case for 12 blueshifted green fluorescent proteins, which are up to 2.5 times brighter than the commonly used Enhanced Green Fluorescent Protein. A way to directly improve fluorescent proteins is through directed evolution, but screening under two-photon excitation is a challenge. An instrument, called the GIZMO, solves this challenge and can evolve fluorescent proteins expressed in E. coli colonies under two-photon excitation. These results pave the way for better two-photon fluorescent protein-based tools for neuroscience.Item Determining function of the IKAP protein in the peripheral nervous system for targeted therapeutic intervention in familial dysautonomia(Montana State University - Bozeman, College of Letters & Science, 2017) Ohlen, Sarah Beth; Chairperson, Graduate Committee: Frances Lefcort; Magdalena L. Russell, Michael J. Brownstein and Frances Lefcort were co-authors of the article, 'BGP-15 prevents the death of neurons in a mouse model of familial dysautonomia' in the journal 'Proceedings of the National Academy of Sciences' which is contained within this thesis.Familial Dysautonomia (FD) is a recessive genetic disorder that leads to devastation of the peripheral nervous system and is the result of incomplete neurodevelopment and progressive neurodegeneration. The disorder is also marked by a continual loss of retinal ganglion cells that leads to blindness. Even with early identification and treatment, the disorder is ultimately fatal. FD is caused by mutation in the IKBKAP gene that leads to cell-type specific loss of the IKAP protein, also known as ELP1. IKAP functions as a part of the six-unit Elongator complex. The role of Elongator is unresolved, although data has accumulated that support Elongator function in tRNA modification and efficient translation of proteins and that its absence leads to cell stress and neurological impairment. We have a mouse model of FD in which mouse Ikbkap is deleted from the peripheral nervous system, and it recapitulates the death of autonomic and TrkA+ sensory neurons observed in FD patients. As we can culture TrkA+ neurons in vitro, while also studying this neuronal population in vivo, we have a system to investigate our goals of (1) determining cellular processes that go awry in absence of Ikap and (2) targeting these cell types and events to prevent their progressive death. We have determined that mitochondrial and cytoskeletal function are disrupted in Ikbkap -/-, TrkA+ neurons and show activation of stress signaling. Interestingly, disrupted mitochondrial function is an emerging hallmark common to most neurodegenerative diseases. We have identified that the compound, BGP-15, is able to restore aspects of mitochondrial function and stress signaling in vitro and can restore neuronal survival of TrkA+ neurons lacking Ikap in vitro and in vivo. BGP-15 also improves actin cytoskeletal function and target innervation. Additionally, we have determined that introduction of the C-terminal half of human IKAP is sufficient to increase neuronal survival in vitro. This smaller protein fragment is compatible with viral delivery to retinal ganglion cells and could be utilized for gene therapy, potentially preventing this neuronal death that lead to blindness. Our goals now are to further explore stress pathways common to many neurodegenerative disorders and optimize rescue strategies in vivo.Item Curating viscoelastic properties of icosahedral viruses, virus-based nanomaterials, and protein cages(2018-06) Kant, Ravi; Rayaprolu, Vamseedhar; McDonald, Kaitlyn; Bothner, BrianThe beauty, symmetry, and functionality of icosahedral virus capsids has attracted the attention of biologists, physicists, and mathematicians ever since they were first observed. Viruses and protein cages assemble into functional architectures in a range of sizes, shapes, and symmetries. To fulfill their biological roles, these structures must self-assemble, resist stress, and are often dynamic. The increasing use of icosahedral capsids and cages in materials science has driven the need to quantify them in terms of structural properties such as rigidity, stiffness, and viscoelasticity. In this study, we employed Quartz Crystal Microbalance with Dissipation technology (QCM-D) to characterize and compare the mechanical rigidity of different protein cages and viruses. We attempted to unveil the relationships between rigidity, radius, shell thickness, and triangulation number. We show that the rigidity and triangulation numbers are inversely related to each other and the comparison of rigidity and radius also follows the same trend. Our results suggest that subunit orientation, protein–protein interactions, and protein–nucleic acid interactions are important for the resistance to deformation of these complexes, however, the relationships are complex and need to be explored further. The QCM-D based viscoelastic measurements presented here help us elucidate these relationships and show the future prospect of this technique in the field of physical virology and nano-biotechnology.Item Sulfide product inhibition of desulfovibrio desulfuricans in batch and continuous cultures(1995-02) Okabe, Satoshi; Nielsen, P. H.; Jones, Warren L.; Characklis, William G.Sulfide product inhibition kinetics for growth and activity of Desulfovibrio desulfuricans was investigated in batch and continuous cultures at pH = 7.0. A non-competitive inhibition model adequately described sulfide product inhibition kinetics. Inhibition coefficient (Ki) for maximum specific growth rate (μinhmax) was 251 mg l−1 S in a batch experiment. Cell yield determined in a chemostat was reduced in half by a sulfide concentration of about 250 mg l−1 S, which was very close to the Ki value for the batch growth. Maximum specific growth rate (μinhmax) and cell yield (YcLac) were strongly inhibited by high levels of sulfide concentrations, whereas specific lactate utilization rate increased with increasing sulfide concentrations. The results indicated an increase in the relative energy needed for maintenance to overcome sulfide inhibition and uncoupling growth from energy production. However, D. desulfuricans to some extent could recover from the shock of high sulfide concentrations. Stoichiometry for catabolic reactions (energy producing) did not change at high sulfide concentrations, while anabolic reactions (cellular synthesis) were strongly inhibited by high sulfide concentrations. These results suggested that separation of sulfide product inhibition into growth (cell yield) and activity (substrate utilization rate) was important to incorporate the sulfide product inhibition kinetics in a variety of applications.