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Montana Academy of SciencesAnd

Montana Local Section of theAmerican Chemical Society

2011 Annual Meeting

April 15 & 16, 2011

Abstracts

Index

Plenary Session pp. 1-9Breakout Session I (Jr. Academy) pp. 10-18Breakout Session II (Sr. Academy MAS) pp. 19-28Breakout Session III (ACS) pp. 29-33Breakout Session IV (ACS) pp. 34-37Posters pp. 38-51

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Plenary Session – Sections Combined

Mutant Bacteriophages Capable of Infecting Resistant Hosts

Tyler J. Hilton, Robert J. Lester1, Marisa L. Pedulla; Montana Tech of The University of Montana, 1300 West Park St., Butte, MT, 59701; tjhilton@mtech.edu

(1) East Middle School, 2600 Grand Avenue, Butte, MT, 59701

Introduction: Mycobacteriophage Snicklefritz is a double-stranded DNA virus that infects Mycobacterium smegmatis. A mutant strain of the host bacterium, M. smegmatis, was previously isolated, that resists infection by Snicklefritz. The mutation is presumably in the host gene encoding the presumptive receptor protein and prevents wild-type Snicklefritz from productively infecting the host. A mutant Snicklefritz phage was then isolated that is capable of infecting the mutant bacteria. This project tests the hypothesis that the mutant phage’s gene encoding the tail fiber protein has an alteration in DNA sequence that leads to an altered amino acid sequence in the protein, and thereby to a different phenotype (ability to infect the mutant bacteria) compared to the wild-type mycobacteriophage Snicklefritz.

Methods: This project has tested this hypothesis by: (1) using bioinformatic approaches from preliminary sequence data to identify a fully sequenced related phage (Mycobacteriopahge PG1) and its tail fiber gene region (2) identifying the similar region in Snicklefritz (3) designing primer pairs to amplify three segments of this region using polymerase chain reaction (PCR); (4) amplifying the DNA in the region of the tail fiber locus from both wild type and mutant Snicklefritz phages and (5) comparing the DNA sequences of these segments surrounding minor tail fiber genes of both wild type and mutant mycobacteriophage Snicklefritz.

Results and Conclusions: Three amplicons spanning the tail fiber gene region of each wildtype and mutant Snicklefritz phages were obtained via adjustments of the standard PCR conditions. DNA sequence of these 3 amplicons from wildtype and 2 amplicons from the mutant phage were obtained. Bioinformatic comparisons reveal no changes in the bases sequenced from the gene 30 or gene 33 amplicon. Analysis of the gene 31 amplicon is still underway and updated results will be presented at the conference.

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The Effects of Caffeine on Precision Target Shooters

Mackenzie Enich

Big Sky High School, Missoula, MT. enichm@mac.com

Introduction: The purpose of this project is to determine whether there are any effects of caffeine on marksmanship, when it is not used to compensate for any lack of cognitive or physical performance. Given the calculated dose of caffeine for this study, it is predicted that the positive effects of the caffeine will outweigh the negative such as dilating blood vessels (increase in heart rate). A moderate amount of caffeine, relative to the participant's weight, might improve focus and precision in a competitive precision shooter, because of caffeine’s natural ability to cause bronchial relaxation and stimulate brain activity.Methods: This project was conducted as a randomized double blind experiment, and there was no control group for the experiment. The results of this study are based on individual progress. This project only looks at precision and accuracy to measure the effects of caffeine. Precision is the measure of reproducibility, whereas accuracy is the measure of veracity, which is mainly dependent on the sights. The first analysis is a measure of accuracy which is simply defined as the score of the target, and the second analysis is a measure of precision Results: The results of this project suggest how caffeine can be either beneficial or harmful to the precision of a competitive shooter. The outcome of this experiment is intended to lend knowledge to marksmen about how caffeine effects their hold, score, and overall performance. Although only one type of competitive shooting is tested under the influence of caffeine, the results of this study might be applied to other precision demanding sports and occupations.Conclusions: Only one participant’s results reject the alternate hypothesis and the pooled results do support the null hypothesis, which is caffeine has no effect on the precision of shooters. The applications of these results were originally intended for surgeons and pilots, and other people with precision demanding occupations. The results of this test suggest that generally, at these doses, caffeine has no effect on the precision of the shooter, but it greatly depends on the individual.

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Investigating the use of Ustilago bullata as a Biological Control Agent for Bromus tectorum: Is Genetic Variation Present within Various Ustilago bulllata Populations on MPG Ranch?Stacia Hill1, Beau Larkin2, Sean Gibbons3, Ylva Lekberg4 and Lorinda Hunt5.1slhill@mcps.k12.mt.us2MPG Operations, Missoula, MT. beaularkin@gmil.com.Introduction: The fungi Ustilago bullata has been found to prevent the development of seeds in seed banks of B. tectorum (commonly known as cheatgrass). U. bullata infection naturally occurs to some degree in almost all cheatgrass populations. This makes it a good candidate for use as a biological control agent for this invasive weed. In addition, the fungi’s aptitude to grow in cultures and be used easily in greenhouses has helped to contribute to research concerning potential protocols for the growth and infection of U. bullata within unwanted cheatgrass populations on MPG ranch; a 6,227 acre conservation land located in the northern Sapphire Mountains of southwestern Montana. Methods: Locations where cheatgrass had been found to be infected with the fungi were spaced at least 100 m apart from each other. This distance between the U. bullata populations has led to another question which is one main focus of the research at this time. An idea presented suggests the distance between the 10 spore locations could potentially cause significant genetic variance between separate U. bullata populations. If this distance is large enough to isolate these populations from each other for a significant period of time it’s possible that different evolutionary characteristic in the gnome of each population could be present when analyzed. Results: Some genetic variation has been found to exist within U. bullata populations on the ranch. This variation has not demonstrated a significant effect on infection rates of U. bullata in cheatgrass populations. The U. bullata population on MPG Ranch as a whole appears to be genetically isolated from other U. bullata populations, and capable of infecting and preventing seed development in cheatgrass populations on the ranch. Conclusion: Further investigation will help to understand the conditions under which U. bullata infects B. tectorum, as well as potential risks involving the usage of U. bullata as a biological control agent in the presence of not only cheatgrass on MPG ranch, but also other native plants that coexist in the same area. If Ustilago bullata is able to be used successfully as a biological control for this invasive weed many current issues relating to the control of cheatgrass could be circumvented, and much more economically and environmentally friendly solutions could be available as a solution to this problem.

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“Evaluation of density functionals using valence and core excited spectra of [Ni(SR)4]x”. Matt Queen, Kevin Murray, Harlan Byker, Dr. Robert Szilagyi. Montana State University.

We present a systematic evaluation of the performance of various density functional theories using the novel series of [Ni(SR)4]x complexes. We report the accuracy of the ground state electronic structure from calculations employing gradient corrected (GGA), hybrid GGA, and meta-GGA functionals with systematic variation of exchange and correlation functions. Reference data for these calculations included valence and core excitation spectroscopic results. The core excitation spectra at the S K-edge was analyzed for extracting experimental orbital coefficient information of the lowest lying unoccupied frontier orbitals. Both the core and valence excitation spectra were simulated using ground state and time dependent density functional theory. A new hybrid functional is presented for the most reasonable description of the ground state of nickel(II) complexes.

“An improved synthesis of sterically hindered aromatic Isoxazoles”. Dr. Joseph Mirzaei, Matheaw J. Weaver, Scott Steiger, Lilly Matti, Dr. Nick Natale. The University of Montana.

Base-promoted cyclocondensation of hindered aryl C-chloro oximes or Aromatic nitrile oxides with 1,3-dicarbonyl compounds affords highly functionalized, sterically-encumbered isoxazole products in good yield. The mild reaction conditions tolerate a wide variety of functionality and permit the preparation of precursors to complex polycycles typically inaccessible via direct, intermolecular carbon-carbon bond forming reactions, under convenient reaction conditions. This process enables the synthesis of highly substituted products with notable functional group tolerances. The products obtained are directly converted to a variety of isoxazole containing structures.

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Effect of residual feed intake on temporal patterns of glucose, insulin, and NEFA concentrations after a glucose challenge in Targhee ewes.

J. G. Berardinelli 1 , R.R. Redden2, R.B. McCosh1, R.W. Kott1

1Dept. of Animal and Range Sciences, Montana State University, Bozeman, MT, USA, and2North Dakota State University, Fargo, ND, USAjgb@montana.edu

Introduction: There is little or no knowledge of the biological mechanisms controlling residual feed intake (RFI) which is a measure of feed efficiency. Understanding changes in metabolites and metabolic hormones that regulate RFI could be useful to evaluate the overall metabolic status of sheep that diverge in RFI. Since RFI is independent of the level of production it represents inherent variations in basic metabolic processes related to efficiency. The objective of this study is to evaluate the effect of a glucose challenge on glucose, insulin, and NEFA concentrations in ewes selected for high (inefficient) and low (efficient) RFI scores. The null hypotheses were that temporal concentrations of glucose, insulin, NEFA and glucose to insulin ratios in response to a glucose challenge do not differ between high and low RFI ewes.

Methods: Nine-month-old Targhee ewe lambs were ranked by RFI score after a 49-day feeding trial. The 6 highest and 6 lowest RFI scoring ewes were selected for the glucose challenge. Blood samples were collected at -30, -15, 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min after infusion of glucose (59 mM/kg BW) at time = 0 h (1015h). Glucose (Gluc), insulin (I), and Non-esterified fatty acids (NEFA) were assayed using colorimetric assays, and insulin was assayed by RIA. Temporal patterns of Gluc, I, Gluc:I ratios and NEFA were analyzed by repeated measures of SAS. Gluc and I disappearance from circulation were analyzed by separate one-way ANOVA.

Results: Concentrations of Gluc, I, and NEFA over time did not differ (P > 0.10) between ewes of the RFI classes. However, time to 50% disappearance of Gluc from circulation, estimated from quadratic equation fitting of concentrations after the maximum concentration tended (P = 0.07) to be greater in high RFI ewes than in low RFI ewes (110 and 86 min, respectively), and Gluc to I ratios tended (P = 0.08) to be greater in high RFI ewes than low RFI ewes (273 and 204, respectively).

Conclusions: the response of inefficient or high RFI and efficient or low RFI ewe lambs during the growing phase to a glucose challenge, in terms of temporal concentrations of glucose, insulin and NEFA, indicates that energy homeostasis or glucose utilization is not directly related to RFI and may not be the underlying physiological basis for efficiency as measured by RFI score in sheep. However, there is an indication that low RFI ewe lambs may be more efficient at utilizing glucose than high RFI ewes.

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“Oxygen-atom reactivity with polyimide and POSS-polyimide surfaces”. Brooks Marshall, Sara A Marquez1, Dr. Amy L Brunsvold, Russel Cooper1, Linhan Shen1, Dr. Timothy K Minton1, Sandra J Tomczak2, Michael E Wright4, Andrew J Guenthner4, Brian J. Pettys4, Vandra Vij3, Laura M McGrath3, Joseph M Mabry2. Montana State University; Air Force Research Laboratory, AFRL/RZSM; Air Force Research Laboratory, ERC Incorporated; Chemistry Division NAVAIR-US NAVY, Research and Engineering Sciences Department.

Upon exposure to atomic oxygen in low Earth orbit, Kapton polyimide erodes and becomes rough. An effective approach to prevent this erosion is chemically bonding polyhedral oligomeric silsesquioxane (POSS) into the polyimide matrix by copolymerization of POSS monomers with the polyimide. We have conducted a variety of studies of POSS polyimides by exposing their surfaces to beams of hyperthermal oxygen atoms and measuring mass loss in situ and examining the exposed surfaces. In addition, we have carried out similar studies of POSS polyimides that were exposed to an atomic-oxygen environment on the International Space Station. During POSS polyimide exposure to atomic oxygen, organic material is degraded and a silica passivation layer is formed. Depending on the mole fraction of POSS, the erosion yield of the POSS polyimide may decrease to 0.01 that of the pure polyimide. Additional studies involving O-atom beam-surface scattering reveal the main reaction products to be OH, H2O, CO, and CO2. The kinetics of the decrease in reactivity of the surface with exposure time compare favorably with model predictions.

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The Joint Toxicity of Three Pyrethroid Insecticides to Drosophila melanogasterJerome J. Schleier III1, Collin Preftokis1, and Robert K. D. Peterson1 1 Department of Land Resources and Environmental Sciences, Montana State University, 334 Leon Johnson Hall, Bozeman, MT 59717, USA. jeromes@montana.edu

Introduction:Regulatory agencies assess the risk of pesticides based on single toxicant

experiments. The use of pyrethroid insecticides has increased substantially throughout the world over the past few decades as organophosphate, carbamate, and organocholrine insecticides are being removed from the market. Concerns have been expressed that pesticide mixtures, especially pyrethroid insecticides with their widespread use, may have additive or greater than additive toxicity in the environment.

The primary mode-of-action for pyrethroids is binding to the voltage-gated sodium channels (VGSC) causing loss of coordination and paralysis. Multiple lines of evidence show that pyrethroids, as a class, do not act in a similar fashion on the VGSC because of differences in chemical structures. There are three types of pyrethroids which are categorized based on their structure and toxicology: those lacking the -cyano group on the phenoxybenzyl moiety (type I), those with a -cyano group on the phenoxybenzyl moiety (type II), and those that are classified as non-ester pyrethroids (pseudopyrethroids).Methods:

We tested the toxicity of three different pyrethroid types: type I permethrin, type II cypermethrin, and pseudopyrethroid etofenprox alone and in all combinations on Drosophila melanogaster. After adult emergence, D. melanogaster were allowed to mature for two days before exposure. Dose-mortality regressions were estimated by logistic regression analysis. Because overdispersion was identified, a quasibinomial model was used to correct the standard errors. Results and Conclusions:

The order of toxicity was cypermethrin > cypermethrin + etofenprox > permethrin + cypermethrin + etofenprox > permethrin > permethrin + cypermethrin > etofenprox > permethrin + etofenprox. The mode-of-action for permethrin, cypermethrin, and etofenprox are the same, so the mixture should have an additive toxicity. However, we found that the permethrin + etofenprox and permethrin + cypermethrin mixture was 48 and 52 percent lower than would be expected for additive toxicity. The toxicity of permethrin + cypermethrin + etofenprox was similar to what would be expected if the toxicity was additive.

The interactions of pyrethroid insecticide types in the environment may significantly decrease the toxicity of the most toxic pyrethroid. The order of toxicity for the three pyrethroids alone was similar to previous estimates. The toxicity of cypermethrin increased the toxicity of the mixtures because of its greater overall toxicity. The most interesting finding is that the permethrin and etofenprox mixture was significantly less toxic than both permethrin and etofenprox alone. Our results support the hypothesis that when pyrethroids are in mixtures they can decrease the toxicity of the most toxic pyrethroid. The most likely explanation for this phenomenon is due to the

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competitive binding at the VGSC, which is supported by recent physiological and biochemical studies examining the binding of pyrethroids.

“Protein Cage Nanoparticles and Associated Protection Against Respiratory Viruses”. Amy Servid, James Wiley, Ben Johnson, Agnieszka Rynda, Mark Young, Allen Harmsen, Trevor Douglas. Montana State University.

Protein cage nanoparticles (PCNs) are hollow, roughly spherical architectures that self assemble from identical protein subunits. Viral capsids, ferritins, and small heat shock proteins are a few examples of PCNs. Our research focuses on the use and design of PCN architectures in an effort to understand the relationship between the bio-physical properties of PCNs and their in vivo functions.It has been shown that administration of non-infectious PCNs is associated with formation of inducible bronchus-associated lymphatic tissue (iBALT) in murine lungs and offer non-inflammatory protection against two serologically distinct influenza viruses (mouse pneumovirus and SARS virus). Inducible bronchus-associated lymphatic tissue (iBALT) is described as clusters of lymphoid cells in close proximity to blood vessels and the airway epithelium. No obvious deleterious effects, such as respiratory hypersensitivity, are observed as a result of PCN-induced iBALT formation in the animals. Our efforts focus on understanding the bio-physical properties of these PCNs that contribute to the phenotypes of iBALT formation and influenza protection. This work will summarize recent experiments in which we investigated the roles of size, structural stability, particle number of PCNs in relation to iBALT and influenza protection. The engineering of PCN-protein conjugates to incorporate targeting moieties and lower the dosage of PCN required for protection will be discussed. These studies provide a foundation for understanding the role of PCNs in protection from respiratory viruses, and are the first step toward vaccination strategies that could offer broad spectrum, non-inflammatory protection against respiratory viruses.

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Breakout Session I – Junior Academy

Comparison of Heavy Breed Chicken to Cornish Broilers in Meat Quality and Lifespan.

Catherine WittBig Sky High School, Missoula, MT.chwitt@mcps.k12.mt.us .

Introduction: Hybrid chickens, such as Cornish Cross, have been bred to produce the greatest amount of meat in the shortest amount of time. Quick production, though beneficial for the poultry industry, has lead to many health issues in Cornish Cross chickens. Such issues include the following: heart failure, lung failure, fat cysts, pasty butt, hock burns, breast blisters, and breaking down. Other breeds, with slower growth rates, do not suffer such maladies.Methods: Two breeds, Cornish Cross and Delaware roosters, were compared to test weight gain, behavior, feed consumption, and lifespan. Both groups ate a high-protein broiler feed and were monitored daily. Each week, feed consumption and weight gain was measured to show efficiency of each breed. Chickens were also monitored daily for activity and health. From this, conclusions could be made concerning economic and ethical concerns when raising each breed.Results/Conclusion:

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the amount of feed consumed by the Cornish. This shows the Delaware is a more costly choice when raising roosters for meat, but it is a much more ethical choice due to the lower death rates. What effect does invasive species have on a generalist herbivores growth and success compared to a non-invasive species?

Morgan SquiresBig Sky High School, Missoula, MT. mtsquires@gmail.com

Introduction: Invasive species are a major problem in U.S. and across the world. Scientists have not discovered a solution or a distinct explanation to why so many species are introduced into non-native ranges but only some of them are able to become noxious invaders. Centaurea solstitialis, Centaurea calcitrapa, and Centaurea sulhpurea are three species native to Southern Europe that were introduced and established themselves in California circa 200 years ago. Only C. solstitialis became an invasive noxious weed in the non-native range. Thesis: The main goal of this study is to determine why C. solstitialis is so successful in California while the other two species are not. Methods: Herbivores can determine the success of plant populations. Trichoplusia ni is a generalist herbivore caterpillar. We grew plants of each of the three species from seeds that originated from different populations in Spain and California. We used leaves from each plant to feed T. ni individuals.Conclustion: Herbivores survived more when fed with leaves from California, suggesting that Californian plants have less chemical defense than its Spanish counterparts. This suggests that Californian plants could be experiencing less herbivory and therefore could dedicate more resources to growth and reproduction, making them more competitive.Acknowledgements: Thank you to Prof. Ray Callaway (University of Montana) and Dr. Daniel Montesinos (University of Montana) for their critical role in the success of this project. Thanks to the Callaway lab for their help. Special thanks also to Brandon Honzel, and Jim Harkins.

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Improving the Efficiencies of the Linear Accelerator

Dylan Burkhart

Big Sky High School, Missoula, MT.

Introduction: The linear accelerator, a device which projects a mass (in this case any ferromagnetic metal) in a strait path. This is accomplished through a pulse of electric current through a coiled wire to produce the magnetic field necessary for this acceleration. The intention of my research is to augment the efficiency of this device, improving the amount of the stored potential energy that is transferred to the projectile as kinetic energy. This could greatly increasing the possibility of future applications such the ability to launch pay loads into space, public transportation and self defense.

Methods: This project began with the construction of 2 accelerators, qualitative observations of these were made in order to increase my current understanding of the project. Currently I am in the process of constructing the third and final accelerator of which portions of it will be altered and data will be gathered concerning the change in efficiency of each change. These changes will include different triggering methods, coil dimensions and others.

Results: Observations gartered form the construction of the first two accelerators will be implemented in the final, such as the use of capacitors and the ability to pulse current for specific periods of time.

Conclusions: The final accelerator, of which all data will be gathered from, is currently in construction.

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The Use of Duckweed for the Purpose of Bioremediation of BenzeneEmily SterbisBig Sky High School, Missoula, MT.sterbis@montana.comIntroduction:In the BP Deepwater Horizon well explosion, more than 200 million gallons of oil leaked into the Gulf of Mexico. With this oil came benzene, a human carcinogen making up 1 to 20 percent of crude oil. The Environmental Protection Agency has placed a restriction of 5 parts per billion (ppb) of benzene in any drinking water. The Gulf of Mexico had concentrations up to 3000 ppb. This project looks at using duckweed, an aquatic weed, to clean up aquatic spill zones contaminated with benzene.Methods:Duckweed was placed in Teflon capped jars of concentrations ranging from 0-1500 parts per million (ppm) of benzene in intervals of 250 ppm to test the mortality of duckweed in benzene. After three weeks, plant and water samples were taken and tested using a UV spectrometer. The UV spectrometer failed to yield conclusive results, so another experiment was performed leaving duckweed in a concentration of 250 ppm for a week. After one week, plant and water samples were once again removed and tested using gas chromatography coupled with a mass spectrometer (GC/MS) as well as nuclear magnetic resonance (NMR).Results:

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Above and to the left is a mass spectrum obtained using GC/MS from a plant sample in 250 ppm water. This is all that was stored in the plant. When compared with spectrums from the National Institute of Standards and Technology (on the right), it was found to be a match with cyclohexane. This means duckweed is somehow converting benzene to cyclohexane and then storing the cyclohexane.Conclusions:From the successful GC/MS and NMR testing (not shown in results), it can be concluded duckweed is a viable candidate for the bioremediation of benzene. No benzene was found in plant or water samples, only cyclohexane. This means the duckweed somehow (through methods currently unknown) converted benzene to cyclohexane and then stored most of the cyclohexane. Duckweed should be

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considered for removing benzene from contaminated sites, although future efforts will work to figure out how the duckweed is converting benzene to cyclohexane as well as figuring out in what conditions duckweed is most effective.

Analysis of Thermodynamics within Compost and the Utilization of Heat Produced by Decomposition.

Nathaniel GoodburnBig Sky High School, Missoula, MT.ndgoodburn@mcps.k12.mt.us

Introduction: The utilization of decomposition and its byproducts is beneficial in many ways. It produces an inexpensive fertilizer more effective than most synthetic ones. However, there is another byproduct of decomposition which goes widely unused. This is the heat produced by the microbial activity responsible for the process. This heat, sometimes reaching upwards of 70°C, if properly captured could be transformed into usable amounts of energy. This experiment aims to capture and transform this heat into mechanical energy to show the energy's availability and the accessibility of this source.

Methods: The experiment is being carried out on a small scale as a proof of concept. A baseline temperature profile of the activity within compost is being recorded as control data. The experiment then will be largely based around the engineering of several components needed for heat transfer, attempting as much efficiency as possible. Data is still being recorded for the control test. Later testing will include data concerning the effects of heat extraction from the decomposition process on microbial activity. After the control data is collected and used to compare with later data, the experiment will continue in the course of attempting greater efficiency in the usage of the captured energy.

Results/Conclusion: The experiment returned results which suggest that, with the current size of compost pile (170lbs livestock manure, 30lbs dead leaves) a temperature differential between the center of the pile and the ambient air was produced which had a magnitude of approximately 10°-15°C. This temperature differential may be large enough to operate a Stirling engine.

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The Investigation of the Biological Properties of the Ayurvedic Plant Bacopa monnieri

Michelle Nemetchek1 and Andrea Stierle2.1mdnemetchek@mcps.k12.mt.us2 Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT.

Introduction: Bacopa monnieri is an Ayurvedic medicinal plant widely used in India for its antiallergic, antistress, and memory-enhancing properties. Such a popular form of medicine in the East has now begun to be tested by Western medicine.

Methods: This project tested the antimicrobial properties of Bacopa against the bacterium Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and the fungi Candida albicans, and Candida tropicales. This plant was also tested for enzyme inhibition of the Caspase-1, Caspase-3, and Matrix Metalloproteinase-3 enzymes.

Results: Each microbe grew normally. Some extracts of B. monnieri inhibited Casp-1 signal transduction up to 98%. Extracts inhibited signal transduction of Casp-3 up to 93%, and signal transduction of MMP-3 as high as 93% as well. Solid-liquid chromatographic extractions of CHCl3-MeOH inhibited Casp-1 signal transduction up to 98%, and MMP-3 up to 77%. Solid-liquid chromatographic extractions of CHCl3 inhibited Casp-1 signal transduction as much as 100%, MMP-3 up to 96%, and Casp-3 up to 75%.

Conclusions: Bacopa has no antimicrobial properties towards any of the five microbes tested, and may be useful for controlling inflammation of the heart, arthritis, and programmed cell death.

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Exploring the Prion DNA Sequence in Montana Elk Genome in Relationship to Susceptibility to Chronic Wasting Disease.Chelsea Anderson

Introduction: Chronic Wasting Disease is a disease occurring in Elk and Deer herds. The causative agent for this disease is the abnormal form of a small protein that occurs in the brains of mammals and birds called the prion protein. At codon 132 in the Elk prion gene, the normally occurring amino acid is Leucine. However, and abnormality in the genetic code sometimes causes Methionine to occur there instead. When Methionine occurs there instead of Leucine the normal form of the prion protein is more likely to fold into the abnormal fold of the protein, causing the animal to be more susceptible to Chronic Wasting Disease.

Methods: Using previously extracted DNA from elk blood samples, perform PCR on the DNA to replicate the 600bp prion gene. To be sure PCR worked correctly run Agarose Gel Electrophoresis. If the bands reach to the 600bp line in the ladder, PCR worked correctly. Before the sample can be sent in to be sequenced, it must be cleaned of the primers, short primer-dimers, and dNTP’s from PCR using a KleanKit. Once the sample has been cleaned the concentrations of the DNA must be changed to 10 ng/uL and then sent into a sequencing lab.

Results and Conclusions: Results from Agarose Gel Electrophoresis show that PCR worked correctly. After having cleaned the PCR sample, the next step is to change the concentration of the DNA to 10ng/uL And then send the samples to the Murdock Lab at the University of Montana in Missoula. Once the samples have been sequenced it will be necessary to see if Methionine or Leucine is at codon 132. By knowing which amino acid occurs at codon 132 it will be evident whether the animal is more or less susceptible to Chronic Wasting Disease.

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Galaxy Composition within Galaxy Clusters Compared to Standalone Galaxies

Haggin Hubert and Kalvin BerdineBig Sky High School, Missoula, MT.Haggin@ymail.com & Mtraptorboy@yahoo.com

Introduction: There are two main types of galaxies the first are the elliptical galaxies which are discs with no defined shape. They normally come in three different forms: spheres, semi-spheres, and cigars. The second type of galaxy is the Spiral Galaxy. This galaxy type has large arms that curl around the core of the galaxies. These two galaxies also have different compositions of materials and can be analyzed using spectroscopy. Spiral galaxies tend to appear to have a higher ultra violet magnitude output while elliptical galaxies usually have a higher infrared light magnitude. Our study was to determine if using spectroscopy to identify galaxy types could be used within galaxy clusters.Methods: our project was conducted using Data from the Sloan Digital Sky survey and placing them within tables based on whether or not they were clustered or isolated from other galaxies. The data we collected included the locational data as well as the galaxies Electro Magnetic Magnitudes. The data from the standalone galaxies was used to create ranges based on standard deviations. We preformed the same test on our clustered galaxies when it became apparent that there was uniform E-M magnitudes between all galaxies within clusters. Results: We have found that galaxies within clusters are very uniform on their E-M Magnitudes. Elliptical galaxies and Spiral galaxies have had magnitudes that overlap very severely this could be also seen with our standalone galaxies. Conclusions: Like humans galaxies are very unique and no two galaxies are the same just because they appear in the same shape. Creating Electro Magnetic ranges doesn’t work because looking at galaxies as two groups is inefficient. To create a more uniform project many more variables must be looked at when creating these standalone galaxy ranges. Such variables include (size and redshift, shape).

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Stress Relevance Affecting Female Productivity Traits in Swine Neela Andres, Big Sky High School, Missoula, MTnandres@mcps.k12.mt.us

Introduction: Stress holds a critical role in the show swine industry. In the 1990’s pigs that were strong competitors in the show ring, meaning they had maximum muscle and minimum fat, were mysteriously dying of stress. These pigs possessed the stress gene or Porcine Stress Syndrome (PSS). In the 90’s pigs that had appeal possess one common trait: a high lean/low fat ratio. This is one benefit of the stress gene. It produces extremely heavily muscled hogs that are practically devoid of fat. It was discovered that pigs that possessed this desired phenotype also acquired at least one copy of the stress gene. The inheritance of the stress gene has many hazardous side affects to the pig industry. Containing the stress gene causes pigs to grow slower. Other symptoms included: excitability, extreme nervousness, excessive tail twitching, foaming at the mouth, and dark pink/ purple colored spots. We can expect this gene to affect sow productivity because of its many negative side effects on swine. Therefore the gene should affect sows’ offspring in a negative and unproductive way. Methods: Blood samples were taken and sent to Biogenetic Services, Inc. for evaluation. Results: This chart includes all the sows and gilts and the factors in calculating Sow Productivity Index or SPI.Name of sow

NBA Standardization Raised

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Sapphire 4 6 4 4 97 110.3 49.5

Diamond*

11 10 4 11 106 109.8 94.15

Jewel*

10 8 5 10 121 148.2 126.05

Pearl

8 9 5 8 112 135.2 100.05

Maggie 7 7 1 8.2 90 126.2 92.35

Emme* 10 9 2 10.9 117 134 117.7

Vanna* 10 9 2 10.9 120 141.5 125.2

Mask 6 6 1 7.2 81 128 86.35

Blueberry 7 7 1 8.2 95 142.2 108.35

Note: * symbolize Stress negative SwineSPI = 100 + [ 6.5 ( L-L ) ] + [ 1.0 ( W-W ) ]

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Conclusion: Sows without Porcine Stress Syndrome were better mothers then stress carrier swine. In comparison stress negative swine are better mothers then stress carrying swine. The piglets gain 0.5 lbs more per day as well as having 3.85 more pigs per litter. Breakout Sessioni II - MAS

Developing Novel Proteomic Approaches to Identify the Targets of Grr1p and Cdc4p in Candida albicans Daniel Willems, Joy Goeffena, David Butler and Kurt Toenjes Montana State University Billings, Billings, MTDanielLWillems@gmail.comIntroduction:The candida species are the fourth most common cause of nosocomial bloodstream infections. C. albicans, an opportunistic pathogen, is also a leading cause of oropharyngeal candidiasis, also known as thrush; a superficial infection may become systemic. Severe systemic infections can lead to death despite available treatment protocols. Candida infection is a significant problem. We have developed a tool to aid in the discovery of new and alternative treatments. Our tool is centered on a method for the purification, separation and analysis of the C. albicans proteome, to identify potential drug targets and their role in morphgenesis. Methods:To analyze the entire C. albicans proteome a novel protein purification protocol was developed to isolate the soluble, cytosolic proteins as well as the membrane-bound proteins. The complete proteome of C. albicans was isolated through a combination of mechanical cold fracturing and chemical detergent digestion. Once the cellular mass was cold fractured the protein fractions were separated by ultra-high force centrifugation. The soluble proteins were separated, then precipitated and washed. The membrane fraction was further extracted, pelleted, washed and re-solubilized with detergents. A 2-dimensional electrophoretic approach was employed to isolate individual proteins. The quantitative differences were evaluated to find possible Grr1p and Cdc4p targets that correspond to an accumulation or depletion of a particular protein. Those proteins were identified and are currently being further studied through reverse genetic approaches.Results and conclusions:We have developed methods to separate soluble proteins from membrane bound proteins for analysis via a 2-dimensional electrophoretic approach. While the soluble fraction of the proteome is readily purified the membrane-bound fraction presents a number of problems due to the strength of the protein-protein interactions and the number of hydrophobic domains in that cellular fraction. First, the membrane-bound fraction of the proteome must be centrifuged out of the soluble fraction; this separation must be carried out at ultra-high force, greater than 100,000 xg. Second, the membrane-bound proteins cannot be precipitated out of solution, so they must first be washed and then re-pelleted while bound to the membrane; the proteins can then be solubilized using zwitterionic detergents. After solubilization, the proteins must first be reduced and alkylated. Next, they were separated based on their isoelectric focusing point. A further alkylation/reduction step is carried out prior to the proteins being separated in a second dimension based on the relative protein size. Finally, the proteins were detected and compared, quantitatively, to each other and to an isogenic wild-type strain. Statistically-significant differences in quantitation will be used to identify potential genes of interest; also the genes and their protein products will be studied further.

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Preparation of an atlas of the ovine hypothalamus for colocalization of key signaling peptides in reproduction.Richard B. McCosh 1 , Carson Butler1, Nicole Cloutier1, Rodney W. Kott1, and James G. Berardinelli11 Montana State University, Bozeman, MT, USA richard.mccosh@msu.montana.edu

Introduction: The biostimulatory effect in sheep is known to cause a relatively rapid increase in Gonadotropin releasing hormone (GnRH) in ewes that accelerates resumption of seasonal ovulatory activity. Furthermore, the 10 amino acid molecule known as kisspeptin has been identified as a key regulator of reproduction. Kisspeptin expression has been found to increase during the breeding season, and increase further during the late follicular phase in ewes. Additionally, kisspeptin has been colocalized with GnRH in the caudal portion of the arcute nucleus in the hypothalamus. The objective of this project was to create an atlas of the ovine hypothalamus so that specific nuclei can be identified and immunohistochemistry can be performed more selectively.

Methods: During the breeding season, one 20-mo-old Targhee ewe was injected with heparin, then euthanized with pentobarbitol; the head was removed and perfused with 4% paraformaldehyde and Na2NO3 in 0.1 M phosphate buffer (pH = 7.4). The brain was removed, the hypothalamic region was dissected out and stored cool (4o C) in 30% sucrose until it was sectioned. Frozen coronal sections (50 µm thick) were cut and stored in a cryoprotectant solution until processed for staining. Sections at 600 µm intervals from the center of the optic chiasm to the center of the mammallary bodies were mounted on gelatinized slides. Sections were washed in water (4x), soaked in 70% ethanol and stained with 0.3% sudan black B, then washed again in 70% ethanol and finally rinsed in water. Coverslips were applied with Tris-buffered glycerol. Digital images of each slide were obtained and annotated by several operators with structures compiled from 1 other sheep brain atlas and 2 human brain atlases. Six additional sections were similarly stained to more closely identify the rostral and caudal aspects of the arcute nucleus.

Results: Our results show the anatomical arrangement of nuclei and tracts of the hypothalamus within the ovine brain. The arcute nucleus originates 0.21 cm posterior to the center of the optic chiasm. This section is also the first in which the optic tracts separate from the hypothalamus. The most caudal portion of the arcute nucleus is located 0.69 cm posterior of the center of the optic chiasm. This section can be identified as the last section that contains any evidence of the inferior branch of the 3rd ventricle.

Conclusion: The creation of this atlas of the sheep hypothalamus is a critical first step in performing immunohistochemistry on hypothalamic sections. The hypothalamus is a highly structured region of the brain, and as such, spatial location and proximity are important features. This atlas will support future studies by enabling others to identify specific centers in sections of interest, thereby reducing the total amount of antibodies and reagents needed to examine the immunocytochemical expression of specific neuropeptides of these centers. The ability to identify key structures such as the arcute nucleus based on histostructure shows the utility of this atlas.

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Commercially made fish feeds could potentially be exposing VHSV to P.Herring and causing different growth in the pacific

herring.

Josh Beaulaurier 1 , Paul Hershberger2, Nate Bickford1, Jake Gregg2, Courtney Grady2, and Rachael Collin2

1University of Great Falls, Great Falls, Montana2United States Geological Survey: Marrowstone Marine Field Station, Nordland Washington

Introduction: Viral Hemorrhaging Septicemia Virus (VHSV) is a serious problem with marine and freshwater fishes. In order to study these pathogens in the laboratory setting, a pathogen free environment is required for experimental purposes with fish and pathogens before the study starts. This study was developed to identify any possible pathogen particles of VHSV that were in commercially made fish feeds such as Bio-diet, Bio-Vita, and Washington Fish and wildlife feeds over two years. This study was also initiated to find out which fish feed increases growth in herring the greatest.

Methods: P. Herring eggs were collected on macrophytes by the Washington Department of Fish and Wildlife. Herring were raised to metamorphosis stage then switched to the different feed types. The fish were fed to cessation daily until there was a visible difference in the size of the fish. The fish were then exposed to VHSV in a controlled environment and all results were recorded.

Results: All but FWP feeds could be exposing the fish to VHSV or causing an immune response towards the virus. A long with that the growth difference between the feeds fed to the Herring had a difference in weight and length.

Conclusions: Bio-diet, and bio-vita could be potential exposing the fish to the pathogen and/or is increasing the immune system of the fish. This is inhibiting the mortality rate that is supposed to be seen in the first exposure of the virus to the fish. However, the commercially made feeds are producing larger fish then the FWP feeds.

Bird Diversity Among Land Use

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Jenni S. Hill1, Nate Bickford2 and Jason Gibbons3.1Wildlife Ecology, The University of Great Falls, Great Falls, MT.jhill02@ugf.edu2Professor of Biology, The University of Great Falls, Great Falls, MT. 3Cheif of Conservation Program, Malmstrom Air Force Base, Great Falls, MT

Introduction:Malmstrom Air Force Base is located northeast of Great Falls, Montana

with the Missouri River located 1 mile north of the base. Vegetation on base includes lawn grasses and native prairie grasses. A small pond covers 1.7 acres on base and is located south east of the airfield. The pond and prairie attracts many seed-eating and migratory birds. A survey was started in June of 2010 to determine current bird activity, species diversity among land use, and provide statistical data for bird awareness around base. Methods:

Starting in June of 2010, a survey was conducted to gather information on current bird species and activity on base. Ten point locations were determined to gather information from around base. Each location was picked to obtain information on species around the airfield, developed areas, wetland habitat, pond habitat, rural, and urban areas. Each location was observed for 5 minute intervals to gather the necessary information. By using binoculars, spotting scope, and reference books, species were identified and recorded on a data sheet. From each location, information on temperature, weather condition, cloud cover, time, wind speed, species, number, activity, cover type and any other comments were recorded. The same points were used throughout the entire survey.Results:

Conclusions:From the data gathered so far, we are able to determine species diversity

decreases in developed areas (sites 1-4).

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Identification of Potential Targets of the Grr1 Ubiquitin Ligase in Candida albicansCameron Fox 1 , Jon Laurent2, Ron Hull1, Ryan Merril-Johnson1, Kim Eiselein1, Kurt Toenjes1, and David Butler1

1Montana State University-Billings, Billings, MT 59101. cameron.fox@student.msubillings.edu2University of Texas, Austin, TX

Introduction: The opportunistic human pathogen Candida albicans is aleading cause of fungal disease. C. albicans can grow in different cell morphologies, including yeast-like cells and a variety of filamentous forms that are likely essential for virulence. Previously, we have shown that deletion of the GRR1 gene of C. albicans results in the constitutive formation of filamentous growth forms. The Grr1 protein is a ligase component of the ubiquitin-proteasome system that selectively targets proteins for degradation. Presumably, the loss of Grr1-mediated proteolysis leads to the stabilization, and inappropriate activity, of proteins that positively regulate filamentous growth. The spectrum of proteins targeted for degradation by Grr1 is not known in C. albicans.

Methods: We have used the yeast two-hybrid system to screen a variety of proteins involved in the regulation of filamentous growth in C. albicans for interaction with Grr1.

Results: We have identified binding interactions between Grr1 and the proteins Cdc24 and Ssy1. This suggests that both proteins are targets of Grr1 and are subject to the ubiquitin-proteasome pathway.

Discussion and Conclusions: Cdc24 is a guanine nucleotide exchange factor for the highly conserved cell polarity protein Cdc42. This result connects Grr1 and the ubiquitin-proteasome system with a key regulatory pathway of cell polarity in C. albicans. Ssy1 is a component of the SPS plasma membrane amino acid sensory system which senses external concentrations of amino acids and produces internal signals that regulate amino acid permease gene expression. This interaction links external signals to the ubiquitin-proteasome system in C. albicans. External signals appear to be an important determinate in hyphal growth.

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What Makes a Plant Invasive? It Might be a Matter of MicrobesMarnie E. Rout 1,2 , Ragan M. Callaway1, and William E. Holben1,2 1The University of Montana, Division of Biological Sciences, 2 Montana- Ecology of Infectious Diseases. 32 Campus Drive, Missoula, MT 59812 marnie.rout@mso.umt.edu

Introduction: Invasive plants profoundly alter ecosystem processes, and tremendous economic costs are associated with these disturbances. Attributes like higher growth rates, increased biomass, and enhanced chemical defenses have been documented in many invasive plants, often allowing them to outcompete in native communities. Current theories suggest these characteristics are plant-regulated; however, this work shows that bacterial symbionts can regulate these traits. Methods: Using culture and molecular approaches, we found that a highly invasive grass (Sorghum halepense) harbors several bacterial organisms inside below-ground tissues (rhizomes). Using a novel antibiotic approach, we manipulated the bacterial symbiont loads in plant tissues and measured effects on plant growth, competition, and defense. Results: We confirmed numerous physiological functions of these bacteria: nitrogen fixation, iron chelation, phosphate solubilization, and plant-growth hormone production. Alterations to several soil biogeochemical cycles were recorded from field studies; invaded soils had increased plant-available nitrogen, phosphorus, potassium, and several trace metals. Manipulating bacterial loads with antibiotics showed that the bacteria significantly increased plant biomass, and altered resource allocation enhancing rhizome growth. Plants with symbionts significantly inhibited the growth of a native prairie grass in competition experiments. Restricting bacterial growth completely removed these competitive effects. Plants with bacterial symbionts also had increased production of the herbivore-defense compound, dhurrin, contained in leaves. When leaves from plants with symbionts were fed to a generalist insect herbivore, the insect could not grow and experienced significant mortality. Restricting bacterial growth resulted in <6-fold decrease in dhurrin, and significant increases in insect growth and survival. Conclusions: These results suggest bacterial symbionts significantly contribute to S. halepense invasions by enhancing the plant traits of biomass, growth rate, competitive effects, and herbivore-defense. A new plant-invasion strategy, Microbially Enhanced Competitive Ability (MECA), is hypothesized based on these findings, where microbial partnerships with plants enhance various invasive plant traits.

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Ecosystem effects and suitability of microbes as sentinels for elevated CO2.

Sergio E. Morales and William E. Holben. University of Montana, Missoula, MT. sergio.morales@mso.umt.edu

Introduction: Carbon dioxide (CO2) represents one of the major greenhouse gases emitted by soils, with nitrous oxide (N2O) and methane (CH4) being two additional greenhouse gases produced in terrestrial ecosystems. One proposed approach to attenuating elevated atmospheric CO2 is long-term storage in geologic formations. However, the potential effects on the environment due to failure in containment and subsequent release of billions of tons of CO2 are unknown. Previous work in soils demonstrated that emissions of all three gases exhibit a similar response under field conditions and are directly linked to differences in microbial populations. Thus changes in microbial metabolism or community structure in response to elevated CO2 could affect cycling of the other two gases. This poses a new concern regarding the influence of elevated CO2 on the emission of other greenhouse gases, and on biogeochemical cycles in general.

Methods: We sampled soils from a field site undergoing simulated CO2 seepage

from underground geologic storage. Total nucleic acids (RNA/DNA) were extracted in order to track temporal changes in microbial communities in response to elevated CO2. Quantitative PCR analysis targeting both DNA and mRNA was conducted on nitrogen and carbon cycling genes and transcripts related to the consumption and production of CO2, N2O and CH4. This method allowed quantification of genes and transcripts representing specific enzymes involved in carbon and nitrogen cycling.

Results: No significant effects were detected for most of the targets tested at the DNA level. Nitrogenase gene (nitrogen fixation) abundances were negatively impacted by elevated CO2.

Conclusions: As temperature and CO2 concomitantly go up, as a result of a leak from storage or global climate change, less N is going to be fixed making N even more limiting in future scenarios.

This work was funded by U.S. Department of Energy Grant No. DE-FG02-08ER46527.

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Breakout Session III - ACS

“Using Sol-Gel Chemistry for New Hybrid Materials”. Erik Johnston, Dr. Ed Rosenberg. The University of Montana.

Recently sol-gel chemistry and the use of organic-inorganic hybrid materials have attracted considerable attention for use as remediation materials for aqueous streams contaminated with heavy metals. Heavy metal ions from manufacturing sources, such as mine waste, can contaminate local water sources and have very negative effects on various biological processes as well as human health, either through drinking water or through the food chain. Remediation of these aqueous streams is necessary to ensure the health of the ecosystem. The metal remediation can be done using many different technologies including ion exchange polystyrene resins and also membrane technologies. The problem with these remediation technologies is that they are either too expensive (membrane technologies) or lack specificity and have low selectivity for toxic metal ions over other ions in solution (ion exchange). The use of sol-gel chemistry to make hybrid materials for the removal of these metal ions from the contaminated streams has been sought after recently because of the high selectivity for specific heavy metal ions previously encountered with hybrids made from amorphous silica gels. The sol-gels are synthesized from a mixture of tetramethoxysilane (TMOS), chloropropyltrimethoxysilane (CP) and methyltrimethoxysilane (Me) in which the reaction first occurs over an acid catalyzed step and then gelates through a base catalyzed step. The synthesized sol-gel is then converted to the organic-inorganic hybrid material using polyallylamine (PAA). These hybrid materials, known as silica polyamine composites (SPC), have a high affinity towards heavy metal ions and with the modification of these SPCs with organic functional groups can be used to selectively remove metal ions in up to 98% purity. Recent work has been done to optimize the use of TMOS in the synthesis of the sol-gels. Silica leaching and copper capacity studies as well as surface characterization using scanning electron microscope (SEM), mercury porosimetry and elemental analysis have all been used to characterize the sol-gels and corresponding composites.

“Probing the Mo site of FeMo-co by Synthesis, Spectroscopy, and Theory”. Bradley Towey, Dr. Robert Szilagyi. Montana State University.

The molybdenum site of the iron-molybdenum cofactor (FeMo-co) of nitrogenase has been proposed as the catalytic center where biological nitrogen fixation and reduction to ammonia occurs. Toward the creation of functionally active biomimetic compounds that catalyze ammonia production for fertilizer, a one pot-synthesis of a single cubane MoFe3S4(S2CNEt2)5 cluster was conducted. To understand the electronic structure of this Mo-3Fe-4S cluster, the contributions of Mo-S and Mo-S2CNEt2 fragments are currently being determined using multi-edge X-ray absorption spectroscopy (XAS) and density functional theory (DFT). The XAS data yields, experimentally, information about the donations of the sulfur ligands to the Mo atom, and when coupled with DFT can determine an accurate electronic structure of the MoFe3S4(S2CNEt2)5

cluster. This cluster will then serve as a template for obtaining other Mo-3Fe-4S clusters, which will also be studied with XAS and DFT. The electronic structures of Mo-3Fe-4S clusters are important for being able to understand the molecular basis of dinitrogen binding and reduction.

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“CVD Diamond Oxidation by Hyperthermal Atomic Oxygen”. Linhan Shen, T. K. Minton1, Z. Shpilman2, 3, I. Gouzman2, E. Grossman2, R. Akhvledianai4, A. Hoffman4, J. T. Paci5. Montana State University; Space Environment Section , Israel; Department of Physics, Technion, Israel; Schulich Faculty of Chemistry, Technion, Israel; University of Victoria, British Columbia.

Behaviors of chemical vapor deposited (CVD) diamond films under exposure to hyperthermal (5 eV and 7.5 eV) atomic oxygen (AO) were studied by using high resolution electron energy loss spectroscopy (HREELS) and atomic force microscopy (AFM). The chemical bonding and morphology studies suggest a selective etching on the diamond surfaces: (111) oriented surfaces are severely etched, while (100) oriented surfaces have high resistance. Theoretical calculations of reactions on these surfaces were studied using the self-consistent charge density functional tight binding (SCC-DFTB) based and pseudopotentials (PSPs) implemented in direct dynamics simulations. The calculations suggest that although the unfuctionalized, H-atom terminated diamond (111) surface is more stable than the (100) surface, fuctionalization produces more stable bonds and therefore less reactive species on (100) surface. In the case of diamond (100), the atop oxygen atom has a ketone-type carbonyl arrangement with the carbon bonded to the oxygen atom bound to two neighboring carbon atoms. This arrangement is more stable than the atop oxygen atom on a (111) surface, where the oxygen atom is single-bonded to the carbon surface because the carbon atom to which it is attached is also bonded to three other carbon atoms. Therefore, the atop oxygen atom on a (111) surface must remain as a radical. As a result, the atop oxygen arrangement on the (111) surface is more reactive than the ketone arrangement on the (100) surface. Thus the (111) surface is more easily eroded under the attack of hyperthermal oxygen atoms.

“Attachment of Ruthenium Organometallic Probes to a Solid Silica Polyamine Composite(SPC) Surface”. Geoffrey Abbott, Dr. Ed Rosenberg, Dr. Alexander Ross, Dr. Ayesha Sharmin. The University of Montana.

Ruthenium probes have long been used for investigating biomacromolecular dynamics due in part to their long lifetimes and visible emissions. Recently, we have synthesized a series of mono-diimine ruthenium complexes that have photophysical properties that are not only for use in biomacromolecular studies but also for studies of Resonance Energy Transfer, between a solid state matrices and the probe; as well as for use as photosensitizers. The goal of the current project is to study the changes that occur when these probes are anchored to a the hybrid organic-inorganic composite materials, silica polyamine composites(SPC), through various assemblies and anchor points. The project will reveal whether or not the anchoring of these probes to the SPC has any major affect on their photophysical properties compared to the probes in solution.

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“Data analysis for ESI-MS of peptides prepared by Labeling Electrospray Deposition (LESD): a New Process for studying protein interactions”. Douglas Steiner, Jonathan Burden, Patrick Ingham, Nicole Therrien, Christopher Holmes, Daniel Therrien, Dr. Paul Martino. Flathead Valley Community College.

Labeling Electrospray Deposition (LESD) is a new process for performing protein or peptide structural studies. The process involves covalent attachment of methylene labels along the polypeptide chain during electrospray ionization. ESI-MS analysis reveals data shifted by various mass units depending on location and number of methylene labels. Protein structural information produced by this process, including solvent accessible and inaccessible regions, is confirmed by X-ray crystallography. Labeled proteins are run through an MSMS process on a Thermo-Fisher LCQ. Data is then exported into an Excel spreadsheet, and fed into the UCSF Protein Prospector MS-Product Utility Program. MS-Product identifies labeled and unlabeled peaks, which are then exported to an Excel file for side by side comparison of ion intensities. The intensity of labeled peaks for each peptide fragment ion is divided by itself plus the unlabeled peak, yielding a numerical ratio, which increases towards 1 as the full peptide chain is analyzed. The data is further processed into a format compatible with the web utility JMolScripter, which provides a visual representation showing the extent of labeling at each residue. Developed and evaluated is a new semi-automated method for analysis of LESD data- a mass spectrometry-based process for structural studies.

“Molecular Basis of Uranium Toxicity”. Katie Burbank, Dr. Robert Szilagyi. Montana State University.

Uranium(VI) is a common environmental contaminant, which occurs as dioxo-uranium cation, UO2

2+ or uranyl in aqueous media. A combination of in vivo, in vitro, and in silico studies recently demonstrated that UO2

2+ nearly completely inhibited microbial activity at very low concentrations by strongly binding to pyrroloquinoline quinine (PQQ) cofactor of bacterial dehydrogenases.   Using the PQQ/uranyl interaction as a model system, this project aims to gain computational insight for uranium toxicity on a molecular basis.

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“Electroless palladium film deposition for thin membrane fuel cell applications”. Matt Berlin, William G. Pinson, Dr. Ed Rosenberg, Dr. William Gleason, Jay McCloskey. The University of Montana & Montana Tech.

The goal for the project was to develop new methods to control electroless deposition of palladium membranes on different substrates. The use of palladium membranes to purify hydrogen has been explored extensively. Purifying hydrogen is a must for fuel cell applications. Palladium membranes allow the diffusion of hydrogen through the membrane, leaving all of the other gasses and impurities behind. The removal of sulfur containing compounds and trace amounts of carbon monoxide are critical to lifetime of the fuel cell catalyst.Electroplating palladium has been shown to be inefficient when compared to electroless plating. Electroless plating also allows the ability to vary the conditions favorable to the formation of membrane thickness and type of depositional morphology. The development of these techniques can allow controllable membrane deposition on different substrates. Differing membrane substrates can significantly affect gas selectivity, flow characteristics, cost, and working lifetime.

“Evaluation of the Performance of Solvation Models in Ni(SR)4 Density Functional Theory Calculations”. Kevin A. Murray, Matt Queen, Dr. Robert Szilagyi. Montana State University.

We present an evaluation of the performance of simulated solvation environments to better model the diverse electronic structures of a novel series of Ni(SR)4 complexes. Reference data from this series were collected using X-ray Absorption Spectroscopy (XAS) at the sulfur K-edge energy range. These core excitation spectra were analyzed to extract orbital coefficient information of the lowest lying frontier orbitals. These spectra where simulated using both ground state Density Functional Theory (DFT) as well as excited state Time Dependent Density Functional Theory (TDDFT). We show systematic variation of the solvation environment. We use a gas phase, a Polarized Continuum Model (PCM), and a Conductor-like Screening Model (CPCM) environment, as well as a full cell crystal structure encircled by a CPCM. We report spin densities, and MO energies from these ground state DFT calculations, as well as the excitation energies from TDDFT calculations. These are compared to the experimental XAS data to assess the accuracy of each simulated environment.

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“Multifunctional nanoapatite particles as potential materials for nano-enhanced applications”. Collette Chorney, Dr. Michael Klem, Margaret Davis, Dr. Rajendra Kasinath. Montana Tech.

The synthesis of functional nano-materials for potential biomedical applications, such as drug delivery systems, non-invasive cancer imaging, hyperthermia, and MRI has produced remarkable and consistent breakthroughs over the last decade. Individual particles (polymeric, inorganic, and/or hybrids) have become smaller, more defined, and have shown potential for well defined functionality. Low toxicity, unwanted agglomeration in relevant physiological conditions, longer blood half-lives, and tissue and cell specificity have been some of the areas that have received particular attention. However, there are still unresolved problems such as complicated chemistry involving non-aqueous media, environmentally undesirable reactants/side-products, harsh synthetic conditions, finer control over size and morphology, colloidal stability, and process scale-up to meet commercial demands.

This works makes use of nanoscale hydroxyapatite particles as a platform for divalent and trivalent transition-metal doping. These doped nanoapatites (nHA) were synthesized using an aqueous green chemical approach under mild conditions of temperature and pH. The resulting nHAs exhibited novel properties as a function of both the dopant atom (Fe, Gd, Nd, and Ti) and concentration. The physical properties of these doped apatites were characterized by a variety of techniques including TEM, dynamic light scattering, magnetometry, and fluorescence-based measurements. Further synthetic work has been carried out using multiple dopants to create multifunctional nanoapatites (e.g. particles with both fluorescence and enhanced MRI contrast).

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Beakout Session IV - ACS

“Enhanced relaxivity and in vivo imaging of macrophage-rich carotid lesions with protein cage based T1-contrast agents”. Shefah Qazi, L. O. Liepold1, M. Uchida1, M. J. Abedin1, H. Kosuge2, T. Kitagawa2, P. Prevelige3, M. V. McConnell2, T. Douglas*1. Montana State University1, Stanford University School of Medicine2, University of Alabama.

The objective of this work was to develop a highly sensitive T1 contrast agent (CA) by chemically attaching a multitude of Gd-DTPA chelates onto a branched polymer (BP) constrained inside a protein cage nanoparticle (PCN). CA enhances r1 relaxivity, consequently lowering Gd dosage administered and providing greater sensitivity in MR imaging. When a Gd-DTPA chelate is tethered to a PCN, the rotational correlation time of the lanthanide ion is reduced, improving r1 relaxivity. Small heat shock protein cage (Hsp), with a 12 nm diameter, was utilized to attach Gd-DTPA chelates to BP synthesized on the interior of Hsp via an iterative azide-alkyne click reaction. Quantitative analysis of Gd and protein concentration showed there were approximately 160 Gd-DTPAs per cage, a high Gd-payload per PCN. At a clinically relevant field strength of 0.73T, Hsp-BP-DTPA-Gd exhibited high r1 values, with ionic (per Gd) and particle r1 values equal to 25mM-1sec-1 and 4,200mM-1sec-1, respectively. At 3T, the ionic r1 value was 9.7 mM -1sec-1 (3x greater than Magnevist) and the particle r1 value was 1630 mM-1sec-1. Macrophage-rich lesions were imaged with low Gd dosage of Hsp-BP-DTPA-Gd at 1/5th the clinical dosage, making Hsp-BP-DTPA-Gd a promising material for detection of atherosclerosis at an early stage. This synthetic strategy was successfully extended to a 55nm PCN, the P22 bacteriophage capsid. P22 has 230x greater interior volume than Hsp, allowing for an even higher Gd-payload per PCN. At generation 3.5, P22-BP-DTPA-Gd accumulated 4,200 Gd ions, maintaining high ionic and particle r1 values of 23mM-1sec-1 and 96,600mM-1sec-1, respectively.

“Genetically Programmed in vivo Packaging and Controlled Release of Protein Cargo from Bacteriophage P22”. Alison O’Neil, Dr. Trevor Douglas, Courtney Reichhardt, Benjamin Johnson, Peter E. Prevelige Jr. Montana State University.

The natural viral system of specific nucleic acid encapsulation, cell targeting, and controlled release has inspired many to mimic this scheme for nano-material synthesis. Salmonella typhimuriam bacteriophage P22 assembles into a T = 7 capsid with the assistance of a scaffold protein (SP) resulting in a procapsid packaged with SP. By exploiting this naturally occurring encapsulation, we have engineered a molecular system through which the C-terminal 162 amino acids of the SP (the C terminus being essential for assembly) are genetically fused to a fluorescent protein cargo. The SP acts as an encapsulation signal and templates accurate spontaneous assembly in vivo. Here, we demonstrate the in vivo encapsulation of the easily detectable green fluorescent protein (GFP) and red fluorescent protein, mCherry, inside of P22. These cargo filled capsids are able to undergo the interesting morphological changes available to P22: an expanded icosahedral form and the ‘wiffle ball’ form which is an icosahedral capsid transected by 12 10nm pores. The fluorescent protein cargo appears to act as an anchor in the expanded form, holding the SP, which is usually absent in this form, inside the capsid. When the capsid is forced to wiffle ball form, the fluorescent cargo diffuses out of the capsid (whether

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attached to the SP or not). The designed molecular system will allow for easy genetic manipulation to encapsulate any protein of interest and control its release from the capsid.

“Anthracenyl isoxazole amides (AIMs) as potential anticancer therapeutics”. Nathan S. Duncan, Alison K. Kearns, Howard D. Beall, Dr. Nick Natale. The University of Montana.

Several well-known theories recognize the G-quadruplex (G4) conformation of DNA as a potential molecular target. A novel series of anthracenyl isoxazole amides (AIMs) have been synthesized and will be described. The compounds consist of an isoxazole that pre-organizes a planar aromatic moiety thereby potentially increasing anticancer efficacy. The new conjugate molecules were prepared via doubly activated amidation modification of Weinreb’s amide formation technique, using SmCl3 as an activating agent which produces improved yields for sterically hindered 3-aryl-4-isoxazolecarboxylic esters. The results of the National Cancer Institute’s (NCI) 60 cell line screening assay show a distinct structure activity relationship (SAR), wherein a trend of the highest activity for molecules with one N-methylpyrrole peptide. Evidence consistent with a mechanism of action via the interaction of these compounds with G-quadruplex (G4) DNA and a structural based rational for the observed selectivity of the AIMs for G4 is presented.

“Lateral metalation studies of isoxazolel[3,4-d]pyridazinones”. Chris Koerner, Dr. Joseph Mirzaei, Dr. Nick Natale. The University of Montana.

Isoxazoles are of interest both for their intrinsic biological activity as well as their synthetic utility. Our research group has, for a number of years, been intrigued by the lateral metalation and electrophilic quenching of isoxazoles, which in several examples has provided a mild and selective entry into increasing the functional complexity of the isoxazole. One great remaining problem, however, has been the difficulty of accomplishing asymmetric synthesis in a catalytic fashion during the lateral metalation process. After reviewing our past attempts, we attribute the lack of chiral induction to the conformational flexibility of the isoxazole systems we have studied in the past. The dal Piaz group has reported many uses of isoxazolo[3,4-d]pyridazinones in medicinal chemistry, and we recently observed formation of such rings in another context, which at that time we considered an undesired nuisance. While only a few literature precedents are available, we postulate that the conformational rigidity of the isoxazolo[3,4-d]pyridazinones may allow us to finally make progress towards this last quest in isoxazole chemistry. Our studies are underway, and we will discuss our progress.

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“Synthesis of divalent isoxazole-dihydropyridine for use as a potential drug to combat multiple drug resistance”. Scott Steiger, Dr. Nick Natale. The University of Montana.

The development of multidrug resistance in tumor cells has been recognized as one of the major obstacles to successful cancer treatments. Tumor cells in vitro and in vivo can develop multidrug resistance (MDR) to the lethal effects of a variety of cytotoxic drugs. The over expression of multiple drug resistance gene 1 has been correlated with the expression of P-glycoprotein (P-gp). Consequently, the overproduction of P-gp is seen in cancer cells. The overall goal of this research is to halt the normal operation of the P-gp, in the hope that it will prevent the outward transport of chemotherapeutic agents. Other studies have shown that bivalent tethered dimers act as efficient P-gp inhibitors. Our working hypothesis is then to synthesis novel small molecule bivalent inhibitors that will bind P-gp and halt it’s normal operation in cancer cells. Currently, synthesis of a divalent dihydropyridine (DHP) has been achieved. With the Hantzsch pyridine syntheses being utilized to produce DHPs, and click chemistry was used to attach both DHP to the tether. The characterization of the molecules was done via NMR and Mass Spectrometry. Computer modeling has also been done to evaluate potential P-gp binding sites. The computer modeling produced a working hypothesis of a binding box where we believe the newly synthesized inhibitors can bind. If successful this research would give an option in the treatment of cancer that would normally not be available via pharmaceutical means. The progress on the synthesis of the new ligands will be discussed.

“Microwave Accelerated Synthesis of isoxazole inhibitors of the System Xc-

transporter”. Lilly Matti, Dr. Nick Nitale. The University of Montana.

The purpose of this study is the synthesis of selective ligands, which are important to understand the classes of Glu binding proteins and to define their mechanism of action. Increased selectivity at the various Glu binding proteins should result in better therapeutic outcomes to a host of neurological disorders of the CNS. In previous work from our group, it was found that synthesis of hydrazone isoxazole analogs in the ACPA class had promising Xc- activity, however, the synthesis was plagued by the observation of ring closure which produced isoxazolo[3,4-d]pyridazinones that had lowered biological activity. This study discusses a comparison between the Microwave Accelerated Reaction System (MARS) and the conventional methods. MARS proved to produce isoxazoles with the open hydrazone form, constituting a complete SAR series, and exhibit robust active in biological assays.

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“Polymers inside the P22 protein architecture”. Janice Lucon, Dr. Trevor Douglas, Ben Johnson, Masaki Uchida. Montana State University.

In our previous work, we have modified a 12 nm diameter cage (small heat shock protein) with a ‘click’ chemistry based organic polymer allowing us to build in primary amine sites than can be modified later with a fluorescent dye, drugs, or a MRI contrast agent. While effective, this method becomes less desirable as the diameter of the protein cage increases due to the number of reaction cycles required and therefore time and material to reach the final filled product. To accomplish polymer cross-linking and introduction of addressable sites in fewer steps, we have turned to atom transfer radical polymerization (ATRP) which allows us to create a polymer in a single less time intensive reaction.For several years, ATRP has been successfully applied to biological materials, including virus–like particles, generally with the intent of changing the protein’s solubility or altering its interaction with other materials. Because the polymer grows from a specific initiation site, the polymer growth can be directed to the interior or exterior of a cage-like protein through the attachment of an initiator to specific sites on the wall of the capsid. We have applied ATRP to the 60 nm diameter P22 virus capsid using the protein itself as a barrier to further polymer growth, limiting the polymer to the confines of the protein cage. This is the first report to our knowledge of ATRP being applied to the formation of linear polymers synthesized within the confines of a protein cage architecture.

“Will Cation Binding in the G-quadruplex Cavity Lead to Enhanced Efficacy?”. Matthew J. Weaver”. Dr. Joseph Mirzaei, Dr. Nick Natale. The University of Montana.

The focus of this project is to synthesize specialized molecules that bind to the cation of G-quadruplex (G4) DNA and repress the replication of the oncogenes in cancerous tumors. G4 is a DNA structure with unique folding and is found in the human telomere and promoter regions of many oncogenes. Cations such as K+ and Na+ are found in the cavity of G4 and might provide a binding site for repressors. Anthracenyl isoxazolyl amides (AIMs) represent a new class of antitumor agents developed in our laboratories and show potential to bind G4. Stabilization of G4 limits its structural flexibility and inhibits replication of oncogenes. AIMs contain an isoxazole moiety that anchors the anthracene in a fixed position, allowing π-π stacking between the G4 cavity and anthracene. We propose that an electronegative atom such as nitrogen or oxygen along with an aryl group will increase affinity for G4 via cation binding and increased pi-interactions. We will attach a heteroaryl group to C-10 and predict that the lone pair of electrons will increase the affinity of the novel AIM to bind the cation in the cavity of G4 through dipole and van der Waals interactions. To synthesize this molecule, we will apply the Buchwald-Hartwig palladium-catalyzed cross-coupling reaction. This approach will allow us to work from a common AIM precursor to which we can add substituents. Current work is focused on perfecting the cross-couplings and will be described.

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“Validation of labeling electrospray deposition (LESD) by the examination of the melittin footprint”. Daniel J. Therrien (author), Douglas A. Steiner, Jonathan E. Burden, Jacob L. Harris, Patrick M. Ingham, Nicole Therrien, Christopher A. Holmes, Clayton J. Hamilton, and Paul A. Martino (PI), Flathead Valley Community College.

Current methods utilized to study protein interactions are unable to contend with the speed of protein transitions. We report an approach to covalently label proteins within the electrospray stream. LESD offers distinct advantages to current footprinting techniques, as reactive neutral carbene quickly and nonspecifically reacts with gas phase protein to form covalent methylene labels. Solvent is effectively eliminated by electrospraying which significantly speeds the labeling process and avoids formation of chaotropic solvent-derived side- products.Differential labeling studies of two samples were prepared by LESD- melittin tetramer and monomer. After separate labeling experiments, the samples were analyzed by electrospray ion-trap mass spectrometry. MSMS spectral data of mono-labeled monomer and tetramer were used in combination with Microsoft Excel, and web utilities to create on protein databank renderings a visual representation of the level of labeling on each residue.Independently, data for the tetramer shows heavier labeling on the exposed regions of the alpha helix, specifically at the outermost regions, and lower levels of labeling on the inner regions, specifically at the sections where the tetramer overlaps. This labeling footprint is consistent with published x-ray crystallography data for the tetramer. Data for the monomer presents significant variation from our tetramer data. The monomer shows a relatively even level of labeling on residues 1-13, and heavier levels of labeling towards the N-terminus. Residues 1-13 yield expected results, as one assumes the monomer should present even labeling throughout the peptide. The uneven labeling on residues 14-26 suggests the possibility the intermediate dimer formation.

Poster Session Abstracts (Friday)

Geometric Realization of (13,3) and (13,4) Round Robin Tournaments.Corey Manack1, Alex Mackin1

1The University of Montana Western, Dillon, MT. c_manack@umwestern.edu, a_mackin@umwestern.eduIntroduction: The (n,m) tournament scheduling problem (TSP) can be stated as follows: how can one group n players into a collection of m-player games, so that every pair of players play exactly once? Solutions to the (n,m) TSP are a special class of Balanced Incomplete Block Designs. Aside from tournament scheduling, the applications of block designs are numerous, including error-correcting codes and experimental design. However, many block designs are notoriously difficult to construct. Motivated by their utility and inspired by their symmetry,

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mathematicians have developed a battery of tools to better understand the structure of block designs. Here, we attack the (n,m) TSP from the geometric side, constructing visual models for solutions to the (13,3) TSP and (13,4) TSP. While the solutions of (13,3) TSP and (13,4) TSP are well-known, the corresponding geometric models could not be found in the literature.Methods: Using a known model for the (7,3) tournament, we extended the construction by using combinatorial geometry, pipe cleaners, and trial and error. Constructing verifiable models required a bit of linear algebra, geometry, and the use of Mathematica.

Geometric Realization of a (13,4) round robin tournament.Results and Conclusions: We were able to successfully construct and verify geometric models of (13,3) and (13,4) round robin tournaments. The techniques used in the (13,4) case can be generalized to model solutions of (m^2-m+1,m) tournaments. We suspect the (13,3) model can be generalized as well.

AIMing towards an alternate working hypothesis for anticancer activity.Nathan S. Duncan1, Alison K. Kearns1, Howard D. Beall1, Mariusz P. Gajewski1, Philip Reigan2, Nicholas R. Natale1*. 1Department of Biomedical and Pharmaceutical Sciences, The University of Montana, 32 Campus Drive, Missoula, MT 59812.2 School of Pharmacy, University of Colorado Denver, 12700 East 19th Avenue, Room P15-4002 Aurora, CO 80045.nicholas.natale@mso.umt.edu

Introduction: Surgical resection of many cancers, especially brain tumors or gliomas, have poor success rate because of the difficulty of judging the border between cancerous and healthy tissue. Recently, promising improvements in visualizing tumors during surgery have emerged. Recently reported anthracenyl isoxazole amides (AIMs) have shown promising activity in the National Cancer Institute’s 60 cell line screening protocol (NCI 60). One potential useful aspect of

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AIMs is their fluorescence. Despite their encouraging anticancer activity, a persistent critique of this class of compounds, as well as other planar aromatic moieties, arises from their lipophilicity. Indeed, it is often stated that as many as half of all investigational new drugs fail because of poor pharmacokinetic properties. Methods: From previous studies on structure activity relationship (SAR) of AIMs, it became apparent that in each example examined, the presence of two dimethylaminopropyl groups, or “double tails,” led to increased efficacy. The hypothesis was that enhanced activity was attributed to increased bioavailability arising from the lowered lipophilicity, and another point to note out is that C(10) groups bearing lone pairs, i.e. chloride, appeared to be superior as well. Although, these two factors combined had not been examined, until the present work.Results: The use of SNB-19 as a pre-screen is a good indicator of anti-tumor activity in the NCI 60, and represents the most active anti-tumor compound in this series to date. The overall mid-graph mean point (which approximates the average - log GI50) of the “double tail” compound is 5.71, which compares favorably to several agents currently used in general medical practice, such as fluorouracil (3.5), bleomycin (5.2) and rubidazone (5.6). A new working hypothesis has been developed using the most active of the AIMs docked with the most recent G4 crystal structure, pdb accession 3QCR, and will be described.Conclusions: This study illustrates the use of SAR to guide the design of pharmacokinetic properties relatively early in the discovery process. Clearly, the next hurdle this class of anticancer agents must pass is on the basis of its structural similarity to some polynuclear aromatic hydrocarbons, and thus potentially metabolic toxicity, to which our counter gambit is the proposition that the isoxazole moiety presents a metabolic "sollbruchstellen" site, that is built to metabolize on demand. We will address this issue in forthcoming synthetic methodology and metabolism manuscripts and will report on our progress in due course.

Effects of Road Removal to Total Suspended Solids in Headwater StreamsErin Fenger1, Nate Bickford1, and Stan VanSickle2

1. Biology Department, University of Great Falls, Great Falls Montana, 59405 USA

2. United States Forest Service, Lewis and Clark National Forest, Great Falls Montana, 59405

Introduction:Roads have a big impact on streams. Gravel roads produce large amounts of sediment loads that runoff into streams and rivers. This study identifies the effect of total suspended solids and macro invertebrate densities after road and culvert have been removed from a stream. This study will also identify the effectiveness of using sedimats for sediment removal in streams. Sedimats are made up of three layers, the first layer is made up of jute mesh and then there’s a layer of excelsior, the last layer is made of burlap, which helps catch the sediment.

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Methods:There were a total of three sedimats put in the stream. One sedimat was placed above the culvert as a control, while the other two were placed below the culvert about 100 yards down stream of each other to catch the sediment from the removal of the culvert. The sedimats were brought back and allowed to dry and the dry weight of the sedimats was taken.

Water samples and macro invertebrates were taken before the culvert was pulled both above and below the culvert. Water samples were then taken during the pulling of the culvert and one month after removal. Water samples were taken back to the lab and set up a vacuum apparatus to separate the sediment and water. After the water was filtered and the filters dried, dry weight of the filters was used to determine how much total suspended solids were present.

Results:The mass of the total suspended solids present in the water samples before the removal of the culvert showed that upstream of the culvert had the highest amount of total suspended solids present with a weight of 0.042g. The mass of the total suspended solids present in the water samples that were taken during the removal of the culvert showed that the water sample above the first “sedimat” had more total suspended solids present with a mass of 17.748g. Downstream of the second “sedimat” during the removal process, the water sample had fewer total suspended solids with a mass of 7.379g.

The samples collected was dominated by Trichoptera with 58.29%, the second dominate taxa was Plecoptera with 17.34%.

Conclusion:Find the exciting conclusion at a poster near you on April 15th and 16th.

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Lateral metalation studies of isoxazolel[3,4-d]pyridazinones

Chris Koerner, Joseph Mirzaei1, Nicholas R. Natale1*. 1Department of Biomedical and Pharmaceutical Sciences, The University of Montana, 32 Campus Drive, Missoula, MT 59812.

nicholas.natale@mso.umt.edu

Introduction: Isoxazoles are of interest both for their intrinsic biological activity as well as their synthetic utility. Our research group has, for a number of years, been intrigued by the lateral metalation and electrophilic quenching (LM & EQ) of isoxazoles, which in several examples has provided a mild and selective entry into increasing the functional complexity of the isoxazole. One great remaining problem, however, has been the difficulty of accomplishing asymmetric synthesis in a catalytic fashion during the lateral metalation process. After reviewing our past attempts, we attribute the lack of chiral induction to the conformational flexibility of the isoxazole systems we have studied in the past. The dal Piaz group has reported many uses of isoxazolo[3,4-d]pyridazinones in medicinal chemistry, and we recently observed formation of such rings in another context, which at that time we considered an undesired nuisance. While only a few literature precedents are available, we postulate that the conformational rigidity of the isoxazolo[3,4-d]pyridazinones may allow for defined approach vectors in the LM & EQ process. Our studies are underway, and we will discuss our progress.Methods: The dal Piaz group has reported many uses of isoxazolo[3,4-d]pyridazinones in medicinal chemistry, and we recently observed formation of such rings in another context, which at that time we considered an undesired nuisance. While only a few literature precedents are available, we postulate that the conformational rigidity of the isoxazolo[3,4-d]pyridazinones may allow us to finally make progress towards this important problem in isoxazole chemistry.

Results: The 4-acetyl isoxazole -3- carboxylate has been prepared, and when reaction is

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conducted with hydrazines in many cases undergoes ring closure to the isoxazolo[3,4-d] pyridazinone. Reaction of the [3,4-d] with lithium amide base at low temperature, followed by electrophilic quenching, indeed provided the adduct as evidenced by LC-MS.Conclusions: We believe this to be the first example of LM & EQ of the [3,4-d] system with lithium amide bases, and this reaction holds much promise. Our continuing studies are underway, and we will discuss our progress.

Isolation and Characterization of a Warm Springs Extremophile

Katherine A. McElroy and Marisa L. Pedulla; Montana Tech of The University of Montana, 1300 West Park St., Butte, MT, 59701; kamcelroy@mtech.edu

Introduction: With increasing DNA sequences and bioinformatic characterization of organisms, the diversity and abundance of microbial life has become apparent. In a hunt for possible extremophilic inhabitants, water samples were collected from a nearby hot springs in Warm Springs, MT. Extremophiles are organisms that inhabit niches once thought to be too harsh for cellular life to thrive, including hot springs. Never-before-sequenced thermo-tolerant bacteria were isolated from the hot spring water sample.

Methods: Microbiological and molecular techniques were used to isolate purified cultures and to determine the cells’ morphological nature, optimal growth conditions, heat tolerance, and antibiotic resistance profiles. Polymerase chain reaction (PCR) was used with primers designed from various species of known DNA sequence, including other thermophilic bacteria.

Results and Conclusions: Although various species were isolated, one bacterium in particular, “L2,” became the focus of DNA sequencing. Initial PCR reactions followed by DNA sequencing and bioinformatic comparisons placed L2 in the Bacillus genus; further rounds focused on isolating and amplifying the gene encoding the L2 DNA polymerase. The purified L2 DNA polymerase gene was cloned into a pMOS blue vector and used to transform Escherichia coli. Steps to induce protein expression, purify this protein, and to perform a biochemical analysis of a novel and potentially useful thermostable polymerase are underway.

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Laboratory evolution of mutant bacteria and viruses

Robert J. Lester1,Tyler J. Hilton, Rachel L. Conrad , Marisa L. Pedulla; Montana Tech of The University of Montana, 1300 West Park St., Butte, MT, 59701; mpedulla@mtech.edu

(2) East Middle School, 2600 Grand Avenue, Butte, MT, 59701

Introduction: There are three aims to the project: one is to grow and characterize (study) the mutant Mycobacterium smegmatis strain that is resistant to Mycobacteriophage Snicklefritz. Two is to determine to which other phages the M. smegmatis mutant is resistant. The third is to isolate and characterize a mutant Snicklefritz bacteriophage that is capable of infecting and killing the mutant M. smegmatis bacteria.

Methods: This project has achieved these aims by: (1) growing the bacteria on liquid and solid media from frozen stocks (2) testing 32 mycobacteriophages and bacterial supernatant for the ability to infect the mutant bacteria vs. wildtype M.smegmatis.; (3) purifying of a mutant Snicklefritz bacteriophage from a plaque resulting from infection of the mutant bacteria and comparing its ability to infect both wildtype and resistant mutant M. smegmatis bacteria.

Results and Conclusions: The mutant looks similar in colony morphology, color, and size to wildtype

when grown on solid media and has a similar odor. As previously observed by Rachel Conrad, the mutant bacteria grow slower than wildtype in liquid culture.

Resistance profiles of mutant and wildtype bacteria to different phages in lab showed that the mutant bacteria is resistant to Snicklefritz, Mida, and Pineapple.

The supernatant of the mutant did not kill the wildtype strain, indicating it is NOT a lysogen strain.

A mutant Snicklefritz phage was isolated by its ability to kill the mutant bacteria.

Wildtype Snicklefritz phage could not efficiently kill the mutant bacteria, but the mutant phage could kill mutant and wildtype bacteria in equal numbers; however, plaque size of the mutant phage on the mutant

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bacteria was smaller than plaques formed by the mutant phage on wildtype bacteria.

Studies in collaboration with Tyler Hilton are underway to determine whether the genetic basis of the phenotypic alteration in the mutant phage resides in the tail fiber gene.

Generating a 297-AH130 host strain for the lac-based Borrelia burgdorferi inducible promoter system.Patricia T. Lacey and Michael A. Gilbert Department of Biology, The University of Montana Western, Dillon, MT 59725 m_gilbert@umwestern.edu

Introduction:Borrelia burgdorferi, the causative agent of Lyme disease, has an enzootic life cycle alternating between Ixodes ticks and mammalian hosts, typically small rodents. Although humans are only inadvertently infected, Lyme disease remains the most common arthropod borne disease in the United States and combating this disease requires a thorough understanding of the molecular mechanisms of pathogenesis used by this organism. To understand this mechanism it must be possible to regulate expression of genes to determine if they are important in the tick host or in the mammal host. An inducible promoter system which provides tight regulation of gene expression has been previously developed for B. burgdorferi strain B31–A3, but not for strain. 297-AH130. Before this promoter system can be put to use, a host strain in 297-AH130 must developed that contains all the necessary virulence plasmids as well as produces the lacI protein. This required us to make two constructs. The first was designed to insert the lacI gene as well as an associated streptomycin antibiotic resistance cassette into the “Junky Region” (JR) of lp28-1. The second was designed to insert a gentamicin antibiotic resistance cassette into an intergenic region (IR) of lp25. The development of this strain will allow this strain to be better understood. Methods: We amplified the JR of lp28-1 from 297-AH130 genomic DNA using PCR. This piece was TOPO cloned into pCR4. We also amplified using PCR the lacIgene with associated strep cassette from an existing plasmid using primers which introduced HindIII sites at each end of this piece. This piece was also TOPO cloned into pCR4. We then excised the lacI and strep cassette from pCR4 by digesting with HindIII. The lacI and strep cassette was then inserted into the unique HindIII site of JR and the two pieces were ligated together. We amplified the IR of lp25 from 297-AH130 genomic DNA using PCR. This piece was TOPO cloned into pCR4. We also used PCR to amplify the gent cassette from an existing plasmid using primers which introduced NsiI sites at each end of the piece. This was also TOPO cloned into pCR4. We digested the gent cassette from pCR4 using NsiI and inserted this piece into the unique NsiI site of IR. These two pieces were then ligated together. Results: After the digestion of the pCR4-JR::lacI-strepR, gel electrophoresis confirmed the orientation of the construct. Also, the correct orientation of pCR4-IR::gentR digestion was confirmed by gel electrophoresis. Conclusion:The development of both constructs was successful. The next step will be to electroporate B. burgdorferi 297-AH130 cells with the pCR4-JR::lacI-StrepR construct, select for transformants in

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streptomycin, and screen them for LacI production and the retention of lp 28-1. A LacI producing clone containing lp 28-1 will then be electroporated with the pCR4-IR::gentR construct and selected for transformants in both streptomycin and gentamycin. We hypothesize that generating a derivative of this strain that constitutively expresses LacI and carries selectable markers will provide researchers with another infective host strain that is suitable for the introduction of flacp fusions.

“Microwave Accelerated Synthesis of isoxazole inhibitors of the System Xc- transporter” Lilly Matti, Jayme Newell, Richard J. Bridges, Sarjubhai A. Patel, Michael Braden, Joseph Mirzaei, and Nicholas R. Natale.

The University of Montana, Biomedical & Pharmaceutical Sciences, Missoula, MT 59812. Email:am138832@umconnect.umt.edu

Introduction:The purpose of this study was the synthesis of selective ligands which are important to understand the classes of Glutamate (Glu) binding proteins, especially as regards Glu receptors verses transporters, and also to define their mechanism of action. In previous work from our group (Patel, 2010), it was found that synthesis of hydrazone isoxazole analogs in the ACPA class had promising system Xc

_ activity, however, the synthesis was plagued by the observation of ring closure to the isoxazolo[3.4-d]pyridazinones which had lowered biological activity. Methods: This study discusses a comparison between the Microwave Accelerated Reaction System (MARS) and conventional reflux methods. Glutamate up-take inhibition was studied as previously described (Patel, 2010), the homology model was threaded through the related ApcT x-ray recently reported by Gouaux (pdb accession: 3GIA).Results: MARS proved to produce isoxazoles with the open hydrazone form predominating, constituting a complete SAR series, and exhibited robust activity in biological assays (Table).

Conclusions: A homology

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model of the best ligand (space filling) binding with xCT (ribbon) has been developed (Figure), and will be described.References: Isoxazole analogues bind the System xc

- Transporter: Structure-activity Relationship and Pharmacophore Model, Sarjubhai A. Patel, Trideep Rajale, Erin O’Brien, David J. Burkhart, Jared K. Nelson, Brendan Twamley, Alex Blumenfeld, Monika I. Szabon-Watola, John M. Gerdes, Richard J. Bridges, and Nicholas R. Natale, Bioorg. Med. Chem., 2010, 18, 202-213.

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Synthesis of Highly Functionalized Aromatic Isoxazoles

Joseph Mirzaei, Mathew J. Weaver, Scott Steiger, Lilly Matti, and N.R. Natale

Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula MT 59812 joseph.mirzaei@mso.umt.edu

Introduction: Our approach relies on base-promoted cyclocondensations of 1,3-dicarbonyl compounds and nitrile oxides (Scheme 1), affording highly functionalized isoxazoles.1 Methods: A series of hindered aromatic nitrile oxides2 or aryl C-chloro oximes2 was prepared. Base-promoted cyclocondensation3 of these nitrile oxides with β-keto amides, both aryl and alkyl β -diketones, and β -ketoesters afforded twelve (12) new highly functionalized, sterically-encumbered isoxazole products in good yield. The mild reaction conditions tolerate a wide variety of functionality and permit the preparation of precursors to complex polycycles typically inaccessible via direct, intermolecular carbon-carbon bond forming reactions, under convenient reaction conditions. Results: Twelve (12) new highly functionalized, sterically-encumbered isoxazole products are synthesized in good yield. This process enables the synthesis of highly substituted products with notable functional group tolerancesConclusions: Base-promoted cyclocondensations of 1,3-dicarbonyl compounds and nitrile oxides, afforded highly functionalized isoxazoles.References:(1) For a review of isoxazole chemistry, see: Wakefield, B. J.; Wright, D. J. Adv. Heterocycl. Chem. 1979, 25, 147-204.(2) Grundman, C.; Grünanger, P. “The Nitrile Oxides”, Springer-Verlag, New York, 1971.(3) a) Bode, J. W.; Hachisu, Y.; Matsuura, T.; Suzuki, K. Org. Lett. 2003, 5, 391-394. b) Bode, J. W.; Hachisu, Y.; Matsuura, T.; Suzuki, K. Tetrahedron Lett. 2003, 44, 355-3558.

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Synthesis of a divalent isoxazole-dihydropyridine for use as a potential drug to combat multiple drug resistance

Scott Steiger 1 , Dr. N.R. Natale2 1Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula,

MT. 59812Introduction: The development of multidrug resistance in tumor cells has been recognized as one of the major obstacles to successful cancer treatments. Tumor cells in vitro and in vivo can develop multidrug resistance (MDR) to the lethal effects of a variety of cytotoxic drugs. The over expression of multiple drug resistance gene 1 has been correlated with the expression of P-glycoprotein (P-gp). Consequently, the overproduction of P-gp is observed in cancer cells. The overall goal of this research is to halt the normal operation of the P-gp, in the hope that it will prevent the outward transport of chemotherapeutic agents. Other studies have shown that bivalent tethered dimers act as efficient P-gp inhibitors. Our working hypothesis is then to synthesis novel small molecule bivalent inhibitors that will bind P-gp and halt it’s normal operation in cancer cells.Methods: Currently, synthesis of a divalent dihydropyridine (DHP) has been achieved. With the Hantzsch pyridine synthesis being utilized to produce DHP’s and click chemistry was used to attach both DHPs to the tether. The characterization of the molecules was accomplished via NMR and Mass Spectrometry. Computer modeling has also been performed to evaluate potential P-gp binding sites. The computer modeling produced a working hypothesis of a binding box where it is plausible that the newly synthesized inhibitors will bind.Results: Currently the first synthesis of a divalent isoxazole-DHP has been accomplished. Evidence of divalent isoxazole-dihydropyridine via NMR and Mass Spectrometry is in hand. Efforts are being made to synthesis additional derivatives of isoxazole-dihydropyridines to further explore the structure activity relationship. The biological testing of the compounds is being hindered by the difficulty of the culturing P-gp expressing cells, but continuing efforts are being made to produce testable cells.Conclusions: If successful this research would give an option in the treatment of cancer that would normally not be available via pharmaceutical means. The progress on the synthesis of the new ligands will be discussed.

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Monitoring and evaluation of benthic macroinvertebrates in the Big Hole River and tributaries, Montana.W. Kyle Tate1, Michelle L. Anderson2 and Michael A. Bias3.1 University of Montana Western, Dillon, MT.2 Western Department of Biology, University of Montana Western, Dillon, MT.m_anderson@umwestern.edu3 Big Hole River Foundation, Butte, MT.mikebias@3rivers.net

Introduction: This study was put together to evaluate benthic macroinvertebrate assemblages in the Big Hole River and important tributaries. Macroinvertebrate-based surveys are used as a way to assess a stream’s biological heath. Through identifying and comparing the different aquatic macroinvertebrate assemblages located at specific target sights within a water way, overall water quality can be established. Due to this conservation measures and efforts can be made to improve overall stream health.

Methods: The targeted sights for 2002 included 7 total sites. These 7 initial sites were expanded to 20 total from 2008-2010. Samples from these locations were taken twice a year, once during the summer, and an additional sample during the fall. The techniques used during this study involved two traveling kick samples, conducted at specific riffles located at each site. The D-net used was (50 cm wide by 25 cm tall; 1 mm mesh) and held immediately downstream from the area being disturbed. Once samples were in the net they were placed in collection jars in the field and preserved with ethanol. The concentrations of the ethanol varied from 30-80%. Once back at the lab the concentrations were brought back to 80% for further preservation. The macroinvertebrates in the sample jars were later picked and sorted by eye, and the use of a microscope until 300 individuals were reached or the entire sample had been gone through. The 5 initial categories used for this were: Ephemeroptera, Plecoptera, Trichoptera, Diptera, and other. The 2002 samples were sent were sent to D. McGuire for taxonomic identification to the species level where possible. The 2008 samples were sent to Dr. Robert Newell a taxonomic specialist in order to further identify those samples to the same levels. The results were put together in tables and assessed allowing for multiple metrics to be calculated, these included: total organisms, taxa richness, EPT richness, and % dominant taxon.

Results and Conclusion: In the 2002 data the total number of organisms and taxa richness varied throughout the 7 sights. Diversity within each of the samples was observed showing both taxa richness, and EPT richness to be the highest at location 7 within the study area. 2008 data shows diversity varying in both total abundance and taxa richness across sites. Current calculation for determining overall water quality and comparative analysis within the data are being put together, and the remaining 2008 data is being assessed for further results. This is part of an ongoing project with further picking and identification of 2009-2010 samples to follow.

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Will Cation Binding in the G-quadruplex Cavity Lead to Enhanced Efficacy?Matthew J. Weaver, Joseph Mirzaei, and Nicholas R. Natale Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT. matt.weaver@umontana.edu

Introduction:The focus of this project is to synthesize specialized molecules that bind to the cation of G-quadruplex (G4) DNA and repress the replication of the oncogenes in cancerous tumors. G4 is a DNA structure with unique folding and is found in the human telomere and promoter regions of many oncogenes.

Methods:Cations such as K+ and Na+ are found in the cavity of G4 and might provide a binding site for repressors. Anthracenyl isoxazolyl amides (AIMs) represent a new class of antitumor agents developed in our laboratories and show potential to bind G4. Stabilization of G4 limits its structural flexibility and inhibits replication of oncogenes. AIMs are a good candidate for stabilization of G4 because they contain an isoxazole moiety that anchors the anthracene in a fixed position, allowing π-π stacking between the G4 cavity and anthracene. We propose that an electronegative atom such as nitrogen or oxygen along with an aryl group will increase affinity for G4 via cation binding and increased pi-interactions. We will attach N or O, with an aryl group, to the C-10 of a 3-(9’-anthracenyl)-5-methyl-4-isoxazolecarboxylate and predict that the lone pair of electrons will increase the affinity of the novel AIM to bind the cation in the cavity of G4 through dipole and van der Waals interactions. To synthesize this molecule, we will apply the Buchwald-Hartwig palladium-catalyzed cross-coupling reaction. This approach will allow us to work from a common AIM precursor to which we can add substituents. We will then identify the AIM variants that bind G4 and test their ability to repress replication.

Results:Our first task was to increase the yields of the reactions leading up to the AIM precursor. Along with increasing the yield of these reactions, we also streamlined the synthesis, saving us time and resources. For one step in particular the yield was increased from >30% to <80%, more than doubling our productivity. The next step is to efficiently cross-couple substituents to the AIM precursor molecule.

Conclusions:Now that the process for synthesizing the AIM precursor has been established, we must now work towards the addition of subunits to the scaffold. Current work is focused on perfecting the cross-couplings with multiple examples. After obtaining multiple examples, we will then identify the AIM variants that bind G4 and test their ability to repress replication.