In this study, we focused on identifying specific genes that make Pseudomonas fluorescens L5.1-96 a potential biocontrol agent to combat wheat take-all disease. This strain is a supercolonizer meaning it suppresses fungal pathogens in the soil and colonizes the rhizosphere of plants. This strain has not yet been fully sequenced; therefore, analyzing genomic data will contribute to its classification. Our first step was increasing the amount of L5.1-96 DNA through culturing E. coli that contained an insert of L5.1-96 DNA. Next we extracted L5.1-96 DNA using the Qiagen miniprep kit, amplified the DNA insert using linear sequencing, and sent our cleaned DNA samples to Bellevue College for Sanger sequencing. We ran bioinformatics software (NCBI BLAST) to compare our DNA insert with previously sequenced genomic data. We found our DNA sample resulted in a 98% match with genes from Pseudomonas brassicacearum strain BS3663; therefore this indicates a phylogenetic relationship between the two Pseudomonas species. While we did not discover any new genes or novel proteins, our sample coded for tRNA preQ1(34)S-adenosylmethionine ribosyltransferase-isomerase QueA which is a nutrient protein found in both eukaryotes and bacteria. We have contributed to the genomic analysis of strain L5.1-96 and with this information it is possible one day all of the data together will lead researchers to fully evaluate strain L5.1-96 and its ability to suppress wheat take-all disease.

Pseudomonas fluorescens is a bacterium recognized for producing an antifungal against the debilitating wheat take-all disease. In this study I am investigating the Pseudomonas fluorescens L5.1-96 strain to identify the genes making it a supercolonizer of take-all disease resistant soil and how it fits phylogenetically with other bacteria. This project will contribute to the understanding of how biological agents may be used to combat wheat take-all disease. To investigate this, a plasmid of DNA from strain L5.1-96 was inserted into Escherichia coli to make a bacterial library. I separated the plasmid from the E. coli and amplified it by linear sequencing then sent the DNA to Bellevue College for Sanger sequencing. I used bioinformatics to analyze my unique insert which I found did not match unknown genes, rather it coded for proteins regarding nitrogen fixation. The sequences matched those of Pseudomonas brassicacearum. This provides evidence to renaming the bacterial strain. A match to an anaerobic enzyme in nitrogen fixation might provide insight to its supercolonizer abilities. As we are sequencing a minor portion of the strain L5.1-96 genome, there is need for further gene sequencing in order to complete its genome.

Few studies have attempted to figure out how humans may alter their behaviors when faced with trade-off decisions surrounding convenience and risk. Walking is still a primary mode of transportation for many humans, and in this study, we aimed to try to understand how pedestrians behave while engaging in risky behavior, specifically jaywalking (here defined as crossing at a crosswalk but against the green light). Subjects were observed at different intersections in Seattle as they crossed the streets while we recorded various characteristics, including speed, group composition, and gender. The video footage was collected using an iPhone; distance was measured using a Kestrel wheel; and the video footage was analyzed using Kinovea software. Statistics were done in SPSS 25. People (N= 652) were more likely to walk faster when they were jaywalking (p=0.006), and were more likely to check the surrounding environment (p<0.001) when crossing against the light. People walking in groups were less likely to walk quickly (p=0.001) (than people walking independently), and less likely to check the surrounding areas when crossing the street (p=0.034); however, groups were also less likely to jaywalk than individuals (p=0.013). Jaywalking and walking-safety data are regularly used by city planning and development committees to determine how to allocate city resources to pedestrian safety measures. Additionally, this study can be used as a foundation for better understanding humans’ walking and movement decisions surrounding risky behaviors, and how walking in groups might increase walking safety and change people’s risk assessment.

Pseudomonas fluorescens strain L5.1-96 has been known to help combat the wheat take-all fungal pathogen, Gaeumannomyces graminis var. tritici (Ggt). Strain L5.1-96 is maintained in the soil longer than others, so it is known as a supercolonizer, which contributes to the phenomenon referred to as Take-All Decline (TAD). The strain L5.1-96 is a non-pathogenic bacterium which grows in the rhizosphere of plants and produces an antifungal agent known as 2.4-diacetylphloroglucinol (DAPG) causing TAD. The DNA fragment we amplified, sequenced, and analyzed was a perfect match to P. brassicacearum strain BS3663. Through bioinformatics, we also found our fragment matched the genetic code for pyridoxal 5'-phosphate (PLP)-dependent aminotransferase, a co-enzyme which catalyzes the transamination of amino acids to alpha-keto acids. We did not identify new proteins that are responsible for the advantages that this bacterium holds against wheat take-all; although, we did find the best phylogenetic fit for this strain. These findings contribute information to the broader study at Washington State University to investigate whether strain L5.1-96 can be used as a biocontrol agent against wheat take-all.​

Anorexia nervosa (AN) is characterized by severe dietary restriction that results in significant malnourishment and weight loss. Regions in the brain related to appetitive drives and interoceptive awareness could be key biological factors that contribute to this severe dietary restriction. The insular cortex may be a particularly important region, as it is thought to be involved in a neural network that processes gustation and the integration of emotional valence with visceral input. A systematic review was conducted to explore links between AN and the insula using the following inclusion criteria: peer-reviewed fMRI studies that incorporated only adult samples of the AN population, were published in the last ten years, and measured activation in response to food-related stimuli. Eleven fMRI studies that addressed patients with current or past anorexia nervosa and the insula met inclusion criteria and were reviewed for qualitative analysis. Studies used a range of different methods, including different task paradigms, making it difficult to make between-study comparisons. Thus, although the insular cortex has been linked in the past to AN, if or how the insular cortex is implicated in restrictive eating in AN remains unclear. Additional studies are needed to clarify links between the insular cortex and AN to distinguish between disturbances that are present during the ill-state versus those that may occur prior to illness onset or following recovery. Studies that use larger sample sizes from both ill and recovered groups with AN will be necessary to further elucidate the potential insula dysregulation in AN and its role in food motivation.

Viral infection is associated with asthma exacerbations. Normally, viral presence is detected by Toll-like receptor 7 (TLR7)—an innate immune receptor in airway epithelium that responds to single-stranded RNA viruses (like influenza) and triggers an immune response to infection. TLR7 is also expressed on airway sensory nerves, but its specific role is unknown. Here, I tested the role of TLR7 on sensory nerves and identified which sensory nerve populations express TLR7 using nerves isolated from dorsal root ganglia from female Hartley guinea pigs (~400 g). Ganglia were isolated, plated onto matrigel, and treated with a TLR7 agonist R837 (0.1-100 microM) for 16 hours. Neurite number per cell was not changed by stimulating TLR7, but neurite length, and the number of branch points were significantly increased. Separate experiments measured which types of sensory nerves expressed TLR7. Vagal and dorsal root ganglia were isolated and fixed in zinc formalin. Nerves were immunolabeled with antibodies against TLR7 and either neurofilament-1 (NF-H) to identify A𝛿 fiber sensory nerves or transient receptor potential V1 (TRPV1) to identify C-fiber sensory nerves. I found that airway sensory nerves originating in the vagal and dorsal root ganglia expressed TLR7. However, TLR7 was expressed predominantly on small TRPV1-expressing C-fiber neurons, but not on large NF-H-positive A𝛿 neurons. In conclusion, TLR7 is highly expressed by airway sensory C-fibers nerves, and its activation stimulates neurite growth. These findings suggest TLR7 may increase airway C-fibers supplying the lungs which may enhance airway reactivity and be a mechanism for virus induced exacerbations of asthma.

Standard medical practice does not consider a lateral leaning uterus to be medically relevant. However, many indigenous cultures globally have used uterine abdominal massage to correct a “displaced” uterus to enhance fertility. A leaning uterus could theoretically interfere with ovarian function. Additionally, Research indicates that the circulatory functions, the autonomic innervations of the uterus, and pelvic vein incompetence may negatively affect ovarian function, and fertilization. This study examines a population of 30 U.S. fertility patients undergoing ovarian stimulation for an In Vitro Fertilization (IVF) procedure (an assisted reproductive medical technique used to assist with the conception of a child). We will identify differences in the quantity, and size of follicles that are produced by the ovaries of patients with a leaning uterus. At the beginning of a patient’s IVF cycle, researchers will palpate the lower abdomen to determine the position of the uterus. After the patient completes their series of gonadotropins, prescribed hormones used to stimulate follicular growth in the ovaries, physicians will record the number, size, and location of each follicle observed. We predict that a laterally leaning uterus reduces follicle number and size. If our results identify a correlation between uterine displacement and follicle development, then additional research should explore potential causes. A positive correlation would also suggest that complementary treatment options for a laterally leaning uterus should be investigated.

Non-vascular epiphytes have been determined to grow in a non-random distribution in the canopies of Acer macrophyllum in the Hoh Rainforest, Washington. Nitrogen (N) source has been suggested as a potention driver of the distribution, and stable isotopic analyses were used to consider the influence of this factor. Three epiphyte species, the lycophyte Selaginella oregana, and bryophytes Rhytidiadelphus loreus, and Neckera douglasii, and four trunk dwelling species, Selaginella oregana, and the bryophytes Hypnum subimponens, Leucolepis acanthoneuron, and Metaneckera menziesii, were selected for analyses on the basis of their distribution on five host trees. Samples of plant tissues, as well as canopy and trunk soil were analyzed using isotope ratio mass spectrometry to identify the N source of each species. N source varied significantly by species and growing location. δ¹⁵N values of all bryophyte species confirmed an atmospheric N source. Results for the lycophyte S. oregana varied significantly in response to its relationship with R. loreus, which grows above it in mats in the inner canopy. Selaginella oregana appears to use canopy soil N in the presence of R. loreus, and atmospheric N in its absence on the trunk. These findings suggest the role of niche partitioning in the coexistence of S. oregana and R. loreus within the canopy, and illustrate the likely ability of S. oregana to vary its N source based on competition for the resource. No study of this kind has been conducted in the Hoh Rainforest to date, and this work highlights the intersection of abiotic and biotic factors in determining epiphyte species distributional patterns in the temperate forest ecosystem. 

Probiotics are an area of significant clinical research, as they have been shown to aid digestion, fight infections, and even mitigate irritable bowel syndrome; however, probiotic colonies must grow and thrive in the extreme pH environment present in the human digestive tract. Previous experiments focused on the production of the surface protein Elongation Factor Thermally Unstable (EF-Tu), which attaches to glycoproteins lining the intestines and has been used as a biomarker for probiotic health. The growth of several strains of probiotics (Lactobacillus bulgaricus, Lactobacillus acidophilus, and Bifidobacterium longum) in both lysogeny broth (LB) and De Man, Rogosa, and Sharpe broth (MRS) was evaluated. The fractions of secreted, cytosolic, and membrane proteins were quantified using a Bradford assay. Finally, the relative amount of EF-Tu was determined by tryptic digestion and liquid chromatography-mass spectrometry (LC-MS). LB was found to be a more effective growth medium for the range of bacteria tested, with 24 hours at 37°C the ideal incubation period. To isolate the secreted proteins, the supernatant was drawn off the broth culture after centrifugation. Freeze-thaw lysis was used to extract cytosolic proteins, with three cycles determined as optimal for protein recover. Sodium deoxycholate was used to separate proteins from the membrane. All three protein samples were separated and run through a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the EF-Tu bands (roughly 43 kDa) were excised and sent for analysis by LC-MS. Using the information obtained from this research study, pharmaceutical companies can create more suitably tailored probiotic products, and make them more accessible and understandable to the general population. 

Oil-spills are a widespread hazardous environmental issue, often caused by mistakes when drilling or transporting crude oil. The effects are devastating to the ecosystem. There are very few ways to quickly clean up an oil spill efficiently, at low cost and without additional environmental damage, as often they are cleaned by using expensive chemical dispersants. We conducted research on an environmentally friendly and inexpensive solution to this problem by using pearl oyster mushroom mycelium. The pearl oyster mushroom has a lignin decomposing basidiomycotina that produces a set of extracellular ligninolytic enzymes that have bioremediation properties. Our hypothesis is that, oyster mushrooms can be used to clean oil spills. We cultivated the mushroom mycelium in straw on trays with different amounts of oil on them. We grew this in a greenhouse with controlled temperature and allowed 4-6 weeks for the mycelium to eat the oil. We are currently observing the results. After 6 weeks, we will filter out the remaining hydrocarbons with a solvent and determine if the mycelium reduced the amount of hydrocarbons or consumed them entirely. Our anticipated results are that the mushroom will clean up the oil in six weeks. For future work, we plan on conducting this same experiment but in a salt water environment to explore applications for mycoremediation in a simulated ocean environment.

Plasma, the fourth state of matter, is created by introducing a gas - in this case, helium - to an electric field. This field ionizes the helium atoms, which in turn ionize other molecules, creating a wide variety of highly reactive species. Previous work at Edmonds Community College demonstrated the efficacy of an atmospheric pressure plasma jet (APPJ) in killing endospores of Bacillus atrophaeus for the purpose of spacecraft sterilization for NASA (Bernard et al., 2017). With increasing rates of antibiotic resistance, there is interest in using an APPJ in healthcare settings to eliminate harmful pathogens and therefore promote wound healing. However, given its destruction of endospores, it is unclear if the APPJ would damage living tissue. The purpose of this research was to analyze the effect of an APPJ on multicellular eukaryotic organisms. The impact of different exposure times of the APPJ upon both germinated and ungerminated Zinnia elegans seeds, and the effects on plant growth, was studied. The optimal exposure time for germinated seeds was between one and two minutes, while treatments greater than 2 minutes may increase germination rate.

The purpose of this study was to explore the interrelationship between two major DNA repair systems--photoreactivation, with blue light as an energy source, and excision repair, which uses ATP as an energy source--of Chlamydomonas reinhardtii, a single-celled algae, by studying the in vivo efficiency of photoreactivation when the excision repair system was suppressed. Working with a mutant which was completely lacking in excision repair, we found that this strain was also deficient in photoreactivation, as compared to wild-type, at low levels of post-UV visible light treatment, measured by killing on plates. We hypothesized that the level of light intensity might have an impact on the efficiency of the photoreactivating enzyme. Using alkaline agarose gel electrophoresis analysis, we quantitatively evaluated the extent of repair of DNA damage over 24 hours under two different light intensities. We found less damage removal in the mutant under low light intensity, suggesting that a positive correlation does indeed exist between the flux of visible light used in our experiments and the amount of photoreactivation for the mutant. This correlation was not observed in the wildtype. Our work is interesting in the context of human-caused changes to Earth’s atmosphere; both photoreactivation and excision repair of DNA damage are present in virtually all organisms studied, including bacteria, fungi, plants and most animals (although not in placental mammals). With the weakening of the stratospheric ozone layer, there is the possibility of increasing solar UV flux to the surface of Earth, with possible negative downstream effects on biological systems. Understanding the functional relationship between these two DNA repair systems could provide information of fundamental importance to ecological and agricultural problems arising from increased solar UV flux to Earth.

In the US more than 100 million people are living with diabetes or pre-diabetes. The economic burden caused by these conditions, including medical costs, is approximately $322 billion annually as of 2013. Conventionally, transgenic Escherichia coli has been the primary source of commercial insulin production, a process that requires extensive purification to ensure shelf stability and complete removal of contaminants. This study seeks to establish an alternative mode of insulin production using polyethelyne glycol (PEG) to transform the oyster mushroom, Pleurotus ostreatus, with the human insulin gene. P. ostreatus is a valuable target for genetic transformation due to its lack of endotoxins and fully sequenced genome. P. ostreatus was transformed using PEG with a plasmid containing the human insulin gene and a carboxin resistance gene. Transformed cells were selected using carboxin, extracted, and regenerated on plates composed of yeast extract, malt extract, and glucose (YMG). Integration of the human insulin gene in to the mushroom genome was confirmed through PCR analysis of the transformants. Successful PEG transformation of P.ostreatus offers a new avenue for insulin production, potentially diversifying the market and treatment options for diabetics.

The overuse of antibiotics by the health and agriculture industries has resulted in an increase of antibiotic resistance in bacteria, leading to a wide range of untreatable illnesses. The U.S. Department of Health and Human Services Centers for Disease Control and Prevention estimates that at least two million illnesses and 23,000 deaths each year have been caused by antibiotic resistance. Understanding the mechanisms and potential reduction of fitness associated with antibiotic resistance is therefore essential for addressing the ongoing battle with disease-causing bacteria. This study investigates streptomycin resistance in E. coli and explores how resistance may affect the bacteria’s fitness under two stresses, temperature and pH. Mutant strains of E. coli were isolated and exposed to a range of temperatures (30-42°C) and pH levels (4.0-5.0). The results suggest that while antibiotic resistance is a beneficial mutation in the presence of streptomycin, it may impact fitness under environmental stress. Understanding the potential costs of antibiotic resistance could ultimately help us create better models to predict and combat the rapid evolution of drug resistant bacteria.