Scholarship & Research
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Item Antibiotic Potential of Flathead Fungi and Flora(Montana State Univeristy, 2017-04) Powell, RaserWidespread overuse of antibiotics in medical and agricultural industries has resulted in extensive antibiotic resistance at the global level, which poses an immediate threat to human health. The most commonly used antibiotics are currently synthesized from fungi & bacteria, yet other organisms such as lichens, bryophytes and pteridophytes have sparked scientific interest as potential sources of antimicrobial compounds, but only a small fraction of species have been tested. The overarching goal of our research is to determine whether locally occurring fungi, lichens, bryophytes, and pteridophytes have antibiotic potential against several pathogenic bacteria. One-hundred-and-ten plant and fungal specimens were collected, identified, dried and deposited in the FVCC herbarium. Samples were later prepared for antibiotic analyses using ethanol extractions and tested using the Kirby-Bauer disk diffusion method. Extractions from 9 different lichen, 3 bryophytes, and 1 pteridophyte species inhibited growth of Staphylococcus epidermidis, but none inhibited that of E. coli. Our continued research in this area will involve testing extractions from additional fungal, plant and lichen species against these bacteria, and combining various extracts to determine whether we can produce more synergistically effective antibiotics.Item Targeted Deletion of IFNγ- and GM-CSF-Activated STAT Proteins(Montana State Univeristy, 2017-04) Dupuis, Lydia; Domanico, LukeVarious CRISPR-Cas systems act as adaptive immune system in the archaeal and bacterial domains. These systems utilize captured fragments of foreign genetic sequences to enable the prokaryote to defend against specific threats such as viral genomes. The CRISPR associated proteins (Cas), when expressed along with short segment of guide RNA (gRNA), are able to be used as tools for editing genomes with exquisite precision across all domains of life. Here, we created tools designed to employ CRISPR-Cas technology to target genes that code for STAT1 and STAT5A/B proteins and hypothesize that the resulting STAT knockout cells will be unable to adequately respond to transgenic leishmanial parasites expressing recombinant human IFNγ and GM-CSF, respectively. STAT1 and STAT5A oligonucleotide duplexes were successfully cloned into the pSpCas9(BB)-2A-EGFP plasmid at the tandem BbsI restriction sites. HEK293 cells were successfully transfected with the pSpCas9(STAT1)-2A-EGFP and pSpCas9(STAT5A)-2A-EGFP plasmids as demonstrated by EGFP expression in these cells. Monoclonal strains of HEK293 cells are being screened for unresponsiveness to STAT pathway-activating stimuli. Upon confirmation of successful gRNA-directed Cas9 mutations in STAT genes, lentiviral vectors containing these gRNA-encoding sequences will be used to similarly mutate human monocytic cell lines as an important tool for characterizing human IFNγ- and GM-CSF-expressing leishmanial parasite-mediated monocytic cell activation.Item Generation of Human Monocytic-Activating Leishmanial Parasites(Montana State Univeristy, 2017-04) Kempainnen, Winter; Taylor, TealMacrophages and other monocyte-derived cells are used by the immune system in response to environmental stimuli and the polarized effect of the response can have beneficial or detrimental outcomes in different settings. This project evaluates the efficiency of the specific transgenic Leishmania tarentolae to activate human macrophages and, if successful, the parasites could be used for macrophage-activating therapeutics for infected hosts. We hypothesize that nonpathogenic L. tarentolae expressing human cytokines from transgenes, will activate human macrophages in a consistent and controllable manner. DNA recombination methods were used to clone hIFNG and hGM-CSF into plasmid vectors capable of recombining with the highly repetitive ribosomal RNA locus of leishmanial parasites. In addition, fluorescent protein-coding genes were cloned into the plasmid vectors as a marker of successful genomic integration. Current efforts are focused on optimizing the efficiency of generating transgenic L. tarentolae. In addition to the potential of these transgenic parasites for use as macrophage-activating therapeutics, it is possible through cross protection for these parasites to be used as vaccines against the pathogenic forms of this neglected tropical disease.Item Analysis of Correlations Between Low Resting Heart Rate, Personality Tendencies, and Decision Making(Montana State Univeristy, 2017-04) Pancost, AngieLow resting heart rate has been found as a prevalent biological marker for personality tendencies along the antisocial spectrum. Additional characteristics that emerge along the antisocial spectrum include superficial charm, grandiose sense of self-worth, stimulation seeking, and a lack of empathy for others. Studies suggest that this lack of empathy could play a role in jury decisions. This research examines possible correlations between people’s resting heart rates, personality tendencies, and decisions made on court cases. We hypothesize that low resting heart rate will correlate to high prevalence of the three researched personality traits as well as less empathetic decision making in the court case analysis. In order to test the hypothesis, we ran a social survey that collected data on low-resting heart rates, personality traits, and evaluations of court cases. We focused on three main personality tendencies seen in the antisocial personality disorder spectrum: callousness, deceitfulness, and thrill seeking. The court cases selected were particularly difficult scenarios that hinged on perspective. Our goal was to examine the degree to which low resting heart rates, personality traits, and extreme sentencing showed a statistical relationship. Understanding the ways in which biomarkers affect decision making could benefit the legal system tremendously.Item Medium pH and nitrate concentration effects on accumulation of triacylglycerol in two members of the chlorophyta(2010-12) Gardner, Robert D.; Peters, P.; Peyton, Brent M.; Cooksey, Keith E.Algal-derived biodiesel is of particular interest because of several factors including: the potential for a near-carbon-neutral life cycle, the prospective ability for algae to capture carbon dioxide generated from coal, and algae’s high per acre yield potential. Our group and others have shown that in nitrogen limitation, and for a single species of Chlorella, a rise in culture medium pH yields triacylglycerol (TAG) accumulation. To solidify and expand on these triggers, the influence and interaction of pH and nitrogen concentration on lipid production was further investigated on Chlorophyceae Scenedesmus sp. and Coelastrella sp. Growth was monitored optically and TAG accumulation was monitored by Nile red fluorescence and confirmed by gas chromatography. Both organisms grew in all treatments and TAG accumulation was observed by two distinct conditions: high pH and nitrogen limitation. The Scenedesmus sp. was shown to grow and produce lipids to a larger degree in alkaliphilic conditions (pH >9) and was used to further investigate the interplay between TAG accumulation from high pH and/or nitrate depletion. Results given here indicate that TAG accumulation per cell, monitored by Nile red fluorescence, correlates with pH at the time of nitrate depletion.Item Microbial and algal alginate gelation characterized by magnetic resonance(2012-10) Fabich, H. T.; Vogt, Sarah J.; Sherick, Matthew L.; Seymour, Joseph D.; Brown, Jennifer R.; Franklin, Michael J.; Codd, Sarah L.Advanced magnetic resonance (MR) relaxation and diffusion correlation measurements and imaging provide a means to non-invasively monitor gelation for biotechnology applications. In this study, MR is used to characterize physical gelation of three alginates with distinct chemical structures; an algal alginate, which is not O-acetylated but contains poly guluronate (G) blocks, bacterial alginate from Pseudomonas aeruginosa, which does not have poly-G blocks, but is O-acetylated at the C2 and/or C3 of the mannuronate residues, and alginate from a P. aeruginosa mutant that lacks O-acetyl groups. The MR data indicate that diffusion-reaction front gelation with Ca2+ ions generates gels of different bulk homogeneities dependent on the alginate structure. Shorter spin–spin T2 magnetic relaxation times in the alginate gels that lack O-acetyl groups indicate stronger molecular interaction between the water and biopolymer. The data characterize gel differences over a hierarchy of scales from molecular to system size.Item Surprises and insights from long-term aquatic datasets and experiments(2012-08) Dodds, W. K.; Robinson, C. T.; Gaiser, E. E.; Hansen, G. J. A.; Powell, H.; Smith, J. M.; Morse, N. B.; Gregory, S. V.; Bell, Tisza A. S.; Kratz, T. K.; McDowell, W. H.Long-term research on freshwater ecosystems provides insights that can be difficult to obtain from other approaches. Widespread monitoring of ecologically relevant water-quality parameters spanning decades can facilitate important tests of ecological principles. Unique long-term data sets and analytical tools are increasingly available, allowing for powerful and synthetic analyses across sites. Long-term measurements or experiments in aquatic systems can catch rare events, changes in highly variable systems, time-lagged responses, cumulative effects of stressors, and biotic responses that encompass multiple generations. Data are available from formal networks, local to international agencies, private organizations, various institutions, and paleontological and historic records; brief literature surveys suggest much existing data are not synthesized. Ecological sciences will benefit from careful maintenance and anlayses of existing long-term programs, and subsequent insights can aid in the design of effective future long-term experimental and observational efforts. Long-term research on freshwaters is particularly important because of their value to humanity.Item Abandoned well CO2 leakage mitigation using biologically induced mineralization: Current progress and future directions(2013-02) Cunningham, Alfred B.Methods of mitigating leakage or re-plugging abandoned wells before exposure to CO2are of high potential interest to prevent leakage of CO2 injected for geologic carbon sequestration in depleted oil and gas reservoirs where large numbers of abandoned wells are often present. While CO2resistant cements and ultrafine cements are being developed, technologies that can be delivered via low viscosity fluids could have significant advantages including the ability to plug small aperture leaks such as fractures or delamination interfaces. Additionally there is the potential to plug rock formation pore space around the wellbore in particularly problematic situations. We are carrying out research on the use of microbial biofilms capable of inducing the precipitation of crystalline calcium carbonate using the process of ureolysis. This method has the potential to reduce well bore permeability, coat cement to reduce CO2–related corrosion, and lower the risk of unwanted upward CO2 migration. In this spotlight, we highlight research currently underway at the Center for Biofilm Engineering (CBE) at Montana State University (MSU) in the area of ureolytic biomineralization sealing for reducing CO2 leakage risk. This research program combines two novel core testing systems and a 3-dimensional simulation model to investigate biomineralization under both radial and axial flow conditions and at temperatures and pressures which permit CO2 to exist in the supercritical state.This combination of modeling and experimentation is ultimately aimed at developing and verifying biomineralization sealing technologies and strategies which can successfully be applied at the field scale for carbon capture and geological storage (CCGS) projects.Item Nutrient resupplementation arrests bio-oil accumulation in Phaeodactylum tricornutum(2013-08) Valenzuela, Jacob J.; Carlson, Ross P.; Gerlach, Robin; Cooksey, Keith E.; Peyton, Brent M.; Bothner, Brian; Fields, Matthew W.Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.Item Investigation of coal-associated bacterial and archaeal populations from a diffusive microbial sampler (DMS)(2013-08) Barnhart, Elliott P.; Bowen De León, Kara; Ramsay, Bradley D.; Cunningham, Alfred B.; Fields, Matthew W.The Powder River Basin (PRB) in southeastern Montana and northeastern Wyoming contains massive coal deposits with biologically generated coal bed methane (CBM). The microbial ecology of an area within a coal bed influenced by recent groundwater recharge was sampled with a diffusive microbial sampler (DMS). The DMS contained native coal material and was incubated in situ (57 m depth) to allow colonization of the coal particles. Pyrotag sequence analyses of SSU rRNA gene sequences from the coal contained within the post-incubation DMS detected methylotrophic and hydrogenotrophic methanogenic archaea along with diverse bacterial communities. Microbial enrichments (coal or acetate/H2) were established from the DMS, and the enriched bacterial and archaeal communities were characterized via clone library analysis. The in situ bacterial communities were more diverse than the archaeal communities, and the archaeal populations differed between coal incubated in situ and in laboratory enrichments. In addition, bacterial diversity was higher for laboratory enrichments with coal compared to enrichments without coal. The elucidation of relationships between microorganisms involved in coal degradation and metabolite (acetate, H2) utilization within coal-dependent microbial communities is crucial to understanding and improving in situ coal bed methane production.