‘Aigarchaeota’ is a candidate phylum of Archaea known only by 16S rRNA gene fragments from cultivation-independent microbial surveys and a single composite genome from Candidatus ‘Caldiarchaeum subterraneum’, an inhabitant of a subterranean gold mine in Japan. Gene sequences reported in various publications were found almost exclusively in geothermal settings, but a comprehensive assessment has not yet been performed. The purpose of this study was: (i) to rigorously define the phylum; (ii) to gain insight into the phylogenetic and potential taxonomic structure of the phylum; (iii) to assess the distribution of ‘Aigarchaeota’; and (iv) to design ‘Aigarchaeota’-specific 16S rRNA gene primers. Public databases were mined for 16S rRNA gene sequences related to known ‘Aigarchaeota’ and a combination of approaches were used to rigorously define the phylogenetic boundaries of the phylum and compile a neighbor-joining and maximum likelihood phylogenetic tree. Primer template gene sequences were aligned in ClustalW in order to locate regions that are suitable for targeting genus-level groups. ‘Aigarchaeota’-specific primers for the polymerase chain reaction (PCR) amplification of 16S rRNA genes were designed using sequence alignments and reviewed using the Ribosomal Database Project Probe Match tool. The analyses supported the proposed relationship between ‘Aigarchaeota’, Thaumarchaeota, Crenarchaeota, and Korarchaeota in the so-called ‘TACK’ superphylum, and identified ~300 16S rRNA genes and gene fragments affiliated with ‘Aigarchaeota’, including those recovered from terrestrial geothermal systems on several continents and marine geothermal and subsurface samples. ‘Aigarchaeota’ belonged to at least three family- to order-level groups and at least 13 genus-level groups which are represented in the resulting phylogenetic tree. All genus-level groups were recovered from geographically distant locations, suggesting a global distribution. The primers will be used to determine the presence and abundance of ‘Aigarchaeota’ in a wide variety of samples from terrestrial geothermal systems in the U.S. and Asia using quantitative real-time PCR.

Cochlear implants are electronic devices surgically implanted into the inner ear to restore hearing in those with severe or profound hearing loss. A signal processing strategy in the device’s computer interprets the input sound and sends information to the nerves of the inner ear. Mandarin Chinese, spoken by 900 million people, is a tonal language that consists of four tones, each conveying different meaning. Unfortunately, cochlear implant users who speak Mandarin have difficulty perceiving tonal changes in speech with current signal processing strategies. Improving tone perception in Mandarin-speaking cochlear implant users is an ongoing topic of signal processing research. However, if English speakers’ Mandarin tone identification can be used to evaluate signal processing strategies, a pilot study could begin with English-speaking cochlear implant users, who are easier to recruit in the US. The purpose of this study is to test the validity of using native English speakers to evaluate Mandarin tone perception with novel processing strategies. Using a Mandarin tone perception test, we evaluated the ability of English-speaking subjects to categorize spoken tones into the four Mandarin tones – rising, falling, flat, or falling then rising. Ten normal-hearing English speakers were able to correctly identify tones, on average, 88% of the time and three subjects with cochlear implants achieved 61% correct identification on average. Two processing strategies were evaluated on the normal hearing listeners using acoustic simulations of cochlear implant signal processing: CIS, a common clinically-used program, and HSSE, a novel program developed at UW. HSSE was shown to significantly improve perception of tones in normal hearing subjects (p<0.001). These results suggest the possibility of evaluating novel sound processing strategies for tonal languages using English-speaking cochlear implant listeners as test subjects.

 North American river otter (Lontra canadensis) populations are difficult to assess by means such as live-capture; however, non-invasive fecal sampling has become a viable way to assess many aspects of river otter ecology including diet and genetics. L. canadensis uses specific locations called latrines to regularly defecate and scent mark. Fecal samples collected from latrine sites can be used for DNA analysis to distinguish unique individuals and evaluate genetic diversity in a population. Within Possession Sound, minimal research on the abundance and diversity of L. canadensis has been conducted. In fall of 2012, students at the Ocean Research College Academy (ORCA) became the first to study the species in this area. As of fall 2013, two latrine sites of L. canadensis in Possession Sound have been monitored using motion-sensing camera traps. Weekly checks for fresh scat and anal jellies are performed, with 153 samples collected so far. Diet is assessed by identifying prey remains present in feces. Currently, 27 scat samples have gone through DNA extraction, and Polymerase Chain Reaction (PCR) will be performed in the near future using the primers RIO-05, RIO-10, LUT-701, and LUT-801. DNA analysis results will provide information about population size, which is difficult to estimate using camera footage. It is expected that DNA analysis will identify a relatively small number of individuals, as the maximum number of otters simultaneously captured on camera footage is seven. With a small population size, we also expect some amount of genetic interrelatedness in the individuals identified through DNA analysis.

While language delay and speech impairment in individuals with Autism Spectrum Disorders (ASD) have been widely investigated, few studies have analyzed the role that non-verbal forms of communication, such as gestures, may play in augmenting impaired speech in individuals with ASD. One system that has been heavily studied, which has the potential to enhance or inhibit communication, is the Mirror Neuron System (MNS). While the MNS could serve to augment speech, the dysfunction of this system could prove detrimental to those who already possess impaired language skills. If the MNS were to be equally affected by ASD, individuals with this disorder would be unable to compensate for impairments in verbal communication with non-verbal forms of interaction. Although reduced activity in MNS areas in ASD individuals has been identified, the implications of this dysfunction remain unclear. In order to identify if the dysfunction of the MNS impairs non-verbal forms of communication, we recorded 16 typically developing (TD) adolescents and 13 ASD adolescents as they played a game of charades. We utilized charades that were not associated with speech centers, but instead have been shown to rely heavily on an intact MNS and motor cortex to mimic the action. This allowed us to identify whether reduced activation of mirror neurons impacts neural centers that serve to augment language through nonverbal means. Using video footage, two blind coders scored the quality of gestures based upon a 0-8 point scale that was unique to each gesture. Our preliminary results suggest that adolescents with ASD do not have significantly lower scores of functional gesture quality compared to TD individuals. One possible implication of these data is that other regions of the brain may compensate for MNS dysfunction and, importantly, may serve as potential therapeutic targets for strategic interventions to minimize the language impairments associated ASD.

Possession Sound is located in the northeast arm of Whidbey Basin of Puget Sound. Students at the Ocean Research College Academy, a Running Start program at Everett Community College, conduct monthly research cruises to collect oceanographic metrics in Possession Sound. Initial questions about seasonal changes, demonstrated by water temperature, influencing photosynthesis led to questions about correlations with dissolved oxygen, chlorophyll and silicate levels. We hypothesize that dissolved oxygen (D.O.) and chlorophyll concentrations will increase during the spring and summer due to increased photosynthesis with rising temperatures as a result of increased direct sunlight. Additionally, diatomaceous phytoplankton make silica shells out of the silicate nutrients absorbed from the water. Therefore, the silicate levels will decrease during the spring and summer because of an increase in the abundance of diatoms. Water chemistry data were compared seasonally at Mukilteo (MUK) during 2012-2014. The YSI 650 was deployed in meter increments, from the surface to a maximum of 20 meters, to collect temperature, D.O., and chlorophyll data. The Niskin was deployed at the surface and cline, and was used to collect silicate nutrient concentration trends with depth. For quantitative analysis of nutrient data - done by the University of Washington Marine Chemistry Laboratory - the silicate levels correspond to the depth the halocline, which varied for each deployment. Our data show that dissolved oxygen (D.O.) and chlorophyll both increased during the spring and summer, which supports our original hypothesis. Phytoplankton data samples were collected at the halocline with a 20 µm net for a three minute horizontal tow. Further studies on identifying diatom bloom events through plankton species abundance will further support seasonal trends and identify other factors that affect primary production in the local estuary system.

Circadian rhythms are daily biological processes regulated by circadian clocks, endogenous 24-hr biological clocks. Our master circadian clock, located within the suprachiasmatic nucleus (SCN) in the brain, modulates the activity of circadian oscillators in peripheral organs, including the adrenal gland. While the circadian rhythm in glucocorticoid hormone production correlates with adrenal clock gene expression, the molecular mechanisms of adrenal circadian clock regulation require further investigation. Since the transcription factor phosphorylated cAMP response element binding (pCREB) protein is involved in the regulation of the clock gene PER1 in the SCN, we hypothesize that similar pathways to those found in the SCN control clock gene expression and circadian hormone production in the adrenal. Previous results from our laboratory display that the phase of PER1 circadian expression, in cultured adrenal tissue, can be shifted by drug treatments that increase cyclic adenosine monophosphate (cAMP) levels. Since increases in cAMP lead to CREB phosphorylation, we propose that pCREB ultimately regulates adrenal expression of PER1 in vivo. We measured adrenal pCREB levels in rats housed under constant laboratory conditions and sacrificed 11 hours after starting their subjective day (circadian time 11, CT11) or 11 hours after starting their subjective night (CT23), equivalent time points of peak and trough cAMP levels in the SCN respectively. Immunohistochemical detection of pCREB showed significantly higher number of cells expressing pCREB in the adrenal medulla at CT11 than at CT23. Elevated expression of pCREB before the onset of nocturnal activity in rats is consistent with the predicted peak of glucocorticoid levels. Currently we are investigating the presence of pCREB in the adrenal cortex. Exploring the relationship between pCREB and PER1 expression in the adrenal, and the role of pCREB in phase shifting the adrenal clock, may help elucidating how the master circadian clock relays circadian time to the adrenal gland.

Puget Sound consists of many smaller bodies of water including Possession Sound, located between the cities of Everett and Mukilteo, Washington. The Asarco Smelter, located in North Everett was active around 100 years ago, resulting in heavy pollutants such as lead into the Snohomish River leading to Possession Sound. The Ocean Research College Academy (ORCA) students sample Possession Sound for sediments monthly. It can be hypothesized that the lead concentration will be higher at the location Buoy, because it is closer to the fresh water outputs such as the Snohomish River. Consequently, the location Mukilteo/Mount Baker Terminal (MUK/MBT), that is further south in Possession Sound, will have a lower lead concentration since it is further away from the Snohomish River in comparison to Buoy. According to the data collected, from 2009 to 2013, Buoy had a higher amount of lead concentration in comparison to MBT/MUK. However, factors to be considered are other anthropogenic influences, river discharge, tidal stages, time such as time of day, what season and the time of year the data was collected, as well as water currents that could impact the lead concentration in Possession Sound. Future studies will consist of scrutinizing organic matter in sediments in correlation with lead concentration to analyze if biological organisms are impacted by the amount of lead present in sediment.

The Possession Sound, located within the Puget Sound, is a diverse estuarine environment where freshwater from the Snohomish River mixes with the ocean water from the Strait of Juan de Fuca. A key variable that influences fluctuations in an estuary system is tidal stage changes that result in mixing and stratification that influence water chemistry. The working hypothesis is that increased high salinity, cold water during high tide will result in decreased pH and increased dissolved oxygen (D.O.) Data from 2010 to 2013 were collected monthly at the Mukilteo and Mount Baker Terminal stations. YSI 85 and 650 data for salinity, temperature, pH, and D.O. were organized based on tidal stages. The data did not support our hypothesis; trend lines indicate that as tidal stages increase pH increases from 7.5 to 7.6. On average, D.O. decreased as tidal stages increased, from 9.1mg/L to 8.4 mg/L. Chemical levels in the water column are also influenced by phytoplankton through photosynthesis and respiration. Future research will be conducted to determine the correlation between the amount of plankton in the water and the levels of pH and D.O. in the water column.

 The Ocean Research College Academy (ORCA) is a Running Start program through Everett Community College. Monthly State of Possession Sound (SOPS) cruises are conducted that analyze the overall health of the Possession Sound ecosystem. By monitoring physical, chemical, biological, and geological parameters through these cruises, conclusions can be drawn from the data collected. Sediment samples were gathered from different locations throughout Possession Sound--a smaller portion of the Puget Sound located between Whidbey Island and Snohomish County in Washington where the Snohomish River converges with Puget Sound . These samples were analyzed for the relative concentrations of arsenic, copper, and lead by the Everett Environmental Laboratory and distribution and relative concentrations at each site were further examined. The effects of human interaction on the environment--farming, industry, transportation, and recreation--as well as natural fluctuations in tides, currents, and precipitation influence heavy metal deposition in certain locations. The impact of heavy metals spreads to the overall marine health, affecting the food chain through bioaccumulation of these metals in plankton at the basis of the food chain and concentrating further through consumption by other marine life and animals that eat marine life. From the heavy metal information produced by our study, we plan to continue mirroring similar bay studies conducted by the Department of Ecology (DOE), where we can compare heavy metal data collected from this study with water chemistry data and the overall marine health of the Possession Sound ecosystem.

Currently there is extensive interest in the health of honeybee colonies, both in the United States, and worldwide. A healthy and functioning agricultural system requires honeybees, and bees in turn, require nutritious, diverse forage that includes both nectar and pollen. This study involved analyzing the pollen collected by honeybee colonies maintained on the campus of Edmonds Community College, and matching that with pollen collected directly from local plants. We isolated pollen from honey collected and stored by the bees and also collected pollen directly from cells in the hive. In order to identify the different pollen species, the pollen was chemically extracted and acetolyzed to remove sugars and waxes. The treated pollen was then stained, slide-mounted and viewed by light microscopy. These pollen samples were compared to samples of pollen that were collected directly from local plants as well as to existing pollen databases. Preliminary analysis revealed the presence of pollen from brassica, salvia, and trifolium. We were unable to identify some of the extracted pollen species. During the coming foraging season we will expand our database of locally-collected pollen and will generate a local pollen identification key. This will allow us to conclusively identify all of the pollen found in the hives. Results of this study will help us track what the bees are eating and will inform decisions about what best to plant to insure healthy colonies.

Forensic entomology uses the life cycles of insects to determine the time of death (post mortem interval, PMI) of victims of homicide and abuse. Currently, PMI is not used on bodies that have been burned with the use of an accelerant because little is known about how the accelerants affect the life cycles of insects at the scene. The purpose of this study was to investigate whether flesh flies (Sarcophaga bullata) will colonize and complete a full life cycle on charred carrion that has been burned with an accelerant. Laboratory reared adult flesh flies were provided with various substrates; one not burned, one organically burned and one burned with the accelerant, gasoline. Early results of this experiment have shown normal progression of the fly life cycle with larvae deposited on the control carrion. No progression of the life cycle of the flies has been observed on either of the charred substrates. The results of these experiments can show how carrion burned with accelerants affects the colonization and life cycle of an important forensic indicator species, the flesh fly. This pilot study can lead to further studies regarding using PMI for charred remains. The broad scope of this study is to gain insight into the use of insect life cycles to determine the time of death of victims burned with accelerants.

Understanding the energy expenditure required for the activities of daily living is a critical step in assessing the caloric needs both of those individuals whose body mass exceeds recommended standards and those who are chronically under/malnourished. Knowing how much energy people require to accomplish their activities of daily living, which typically includes walking with burdens (carrying excess weight) and on sloped surfaces, is a critical component in developing guidelines for appropriate caloric intake. Activity energy expenditure is the second largest component of daily energy use, after the energy required to maintain bodily function (usually called resting metabolic rate (RMR)). This study allows us to understand the relationship between burden, gradient and walking which is necessary to accurately account for the daily caloric needs of humans. To evaluate this relationship, data is collected from a large number of female participants. Participants walk at three self-selected velocities (slow, normal, and fast), on five gradients (flat, slight uphill and downhill at a gradient of (6.7%), and steep uphill and downhill at a gradient of (12.6%)) and two burdens (no burden and a burden of a 10 kg backpack).“Self-selected” velocity means that the subjects are able to choose their own pace, be it slow, normal or fast. This approach allows flexibility for the subjects to choose speeds that reflect their normal routine. The energy consumption is measured by the volumetric rate of oxygen consumption (VO2, inspiration) and carbon dioxide (VCO2, expiration) with a portable Cosmed K4b2 device. Unlike previous work that has used treadmills, this study is completed outside in an urban setting. Preliminary results indicate that walking outside requires different energy expenditure than walking on a treadmill. This study provides data collected from real-world experience, thereby providing more accurate understanding of the caloric needs to achieve more day-to-day activities.

Prenatal Cocaine Exposure (PCE) can cause movement disorders and developmental problems in affected children. Statistic from Substance Abuse and Mental Health Services Administration stated that over 4% of pregnant women in the United States use illicit drugs, and up to 1% of pregnant women use cocaine. Investigations in the Bamford Lab have shown that PCE causes long-lasting abnormalities in corticostriatal activity. By using slice preparation techniques and Marianas imaging system, we quantify GABA interneuron density within the striatum in 30 and 60-day-old mice that are exposed to cocaine in utero. Lhx6-GFP BAC transgenic mice are used as they express enhanced green fluorescent protein (GFP) in the GABA interneurons which makes imaging analysis more precise and accurate. From our findings, we have found that there is a decreased population of GABA neurons in the striatum than wild type mice. PCE inhibits the tangential migration of GABA interneurons, which results in a reduction of GABAergic neurons within the striatum. The decreased GABA neurons may contribute to the structural and functional deficits in the exposed offsprings. This research will also help future studies to investigate changes in GABA interneuron complementing in the prenatal period, apoptosis, and changes in dendritic spine density in hopes for better understanding of the mechanisms and effects of prenatal drugs use.

Simian immunodeficiency viruses (SIVs) naturally infect over 40 species of African primates. From these SIV strains, only two have crossed to humans giving rise to HIV-1 and HIV-2 which have now infected over 40 million people worldwide leading to several million AIDS-related deaths per year. The goal of this project is to understand what determines the ability of the SIVs to cross between hosts and to evaluate the risk of future SIV cross-species transmissions to humans. To efficiently transmit between species, an SIV needs to be able to overcome the multiple immunological barriers imposed by a new host or rapidly adapt to gain this ability. Several of these barriers are encoded by the antiviral proteins that limit virus replication and thus constitute an innate immunological response against lentiviral infection. In this study, we studied the evolution and adaptation of SIVs during cross-species transmission by identifying the amino acid mutations that the viral protein, Vif, needs to undergo to antagonize the human antiviral protein APOBEC3G (A3G). Since, A3G has been described as one of the major barriers for cross-species transmission its degradation by the virus might facilitate SIV transmission to humans. In order to identify the adaptive mutations required to gain anti-human A3G activity, a virus encoding for an HIV-1 backbone and a SIV Vif protein was serially passaged in a human CD4+T cell line expressing human A3G. Preliminary data suggests that in short-term infection the virus is not able to overcome human A3G. We will continue to passage the virus to determine whether or not viral replication is rescued which would indicate that Vif acquired resistance to A3G. The acquired resistance then will be mapped to specific amino acid mutations through sequencing. To test the anti-human A3G activity of these mutations, molecular clones of the virus containing the mutations will be generated using site-directed mutagenesis and tested in specific A3G degradation assays.

Glucose and glutamine are primary carbon sources for cells to produce biomolecules and energy. Increased aerobic glycolysis, de novo fatty acid synthesis (FAS), and glutaminolysis are hallmarks of cancer cell metabolism and many tumors are dependent upon these for their survival. Likewise, virally infected cells also have considerably altered metabolic requirements. Our lab studies Kaposi’s Sarcoma Herpes Virus (KSHV), the etiologic agent of Kaposi’s Sarcoma (KS). KS is the most common tumor found in AIDS patients. Previous work from our lab showed that latent KSHV infected endothelial cells also require increased aerobic glycolysis and FAS. Our lab also published a global metabolic analysis of latent KSHV infection, revealing that latent KSHV infection also induces increased glutamine levels. Exogenous glutamine is metabolized through Glutaminolysis; glutamine is converted to glutamate and alpha-ketoglurarate (αKG) through two key enzymes, glutaminase (GLS) and glutamate dehydrogenase (GDH). Further, αKG can enter the tricarboxylic acid cycle (TCA) and support ATP production. Cancer cells utilize glutamine to replenish the TCA cycle when glucose is primarily used to produce lactate and fatty acids. Preliminary data shows that exogenous glutamine is required for latent KSHV infection through starving infected endothelial cells of glutamine. While glutamine starvation does not reduce viability of mock-infected endothelial cells, the absence of glutamine leads to significant cell death in KSHV infected cells. I hypothesize that glutamine is required during latent KSHV infection to replenish the TCA cycle. To study this, I inhibited glutaminolysis using a compound called BPTES, an inhibitor of GLS. Inhibiting glutaminolysis also leads to significant cell death of KSHV infected cells. This data indicates that glutamine is required during latent KSHV infection to replenish the TCA cycle. In the future I will add back TCA cycle intermediates to rescue cell death. Through our metabolomics studies of latent KSHV infection, we are identifying potential targets for KS therapy.

Balance control, or stability, is the ability to maintain the body’s center of mass over a base of support. Measurements of stability have a wide range of applications in both clinical and research settings. Many methods have been developed for measuring stability, including functional balance tests, force plate analysis, and video analysis. The preferred method to evaluate standing stability employs the use of a triaxial accelerometer (TA) due to ease of use, portability, cost, and reliability. The aim of this study was to develop an accelerometry-based system for measuring inverted stance (handstand) stability. Using a TA, we collected accelerometry data from a group of acrobats and gymnasts who performed handstands over a period of 30 seconds in four different test conditions: eyes open/firm surface, eyes closed/firm surface, eyes open/compliant surface, and eyes closed/compliant surface. We calculated a set of performance parameters, including medial/lateral displacement, anterior/posterior displacement, and mean speed for each condition from the TA data. Our statistical analysis showed that the TA reliably distinguishes between test conditions for handstands. This suggests that accelerometry can be expanded from regular stance analysis to the evaluation of inverted stance stability. Future applications of accelerometry may include evaluating an acrobat’s handstand stability, comparing the relative stability of differing handstand techniques, or as an additional tool for those investigating the balance control mechanisms of the inverted stance.

Giardia lamblia is a protozoan parasite common in mammalian hosts and belongs to one of the earliest diverging eukaryotic groups. Subsequently, Giardia has a simplified and highly divergent genome that lacks classical actin associated proteins, yet has retained a conserved microtubule (MT) cytoskeleton. This unique cell biology could provide a window into ancient cellular processes, particularly cytoskeletal function and regulation. Giardia has evolved novel and elaborate MT-arrays that mediate important stages in a complex life cycle and assemble into structures critical to its virulence, motility, and cellular organization. Like most eukaryotes, Giardia possesses a superfamily of five tubulin paralogs (gene duplicates) that constitute the MT cytoskeleton (alpha, beta, delta, epsilon, and gamma-tubulin). Interestingly, the Giardia genome contains multiple paralogs of the alpha- (TUBA1, TUBA2) and beta- (TUBB1, TUBB2, and TUBB3) types. It remains unresolved why multiple, highly conserved TUB paralogs are maintained in the Giardia genome. To address this question, we localized tubulin proteins by expressing GFP-tagged tubulin under its native promoter in a pGFPa.pac expression vector. These constructs were transfected into cells and proteins were localized using 3D fluorescence microscopy. Microscopy of fixed cells revealed that TUBA and TUBB proteins localize similarly in interphase cells and are largely interchangeable (ventral disk, median body, flagella), yet display differential levels of expression. Namely, GFP::TUBB3 and GFP::TUBBA1 signal is much brighter than that of GFP::TUBB1, GFP::TUBB2, or GFP::TUBA2. Microscopy also demonstrated delta-tubulin is diffuse throughout the cytoplasm, epsilon-tubulin localizes faintly to the ventral disk, and gamma-tubulin appears to track the internal axonemes of the flagella and concentrate at the flagellar tips. This data could suggest Giardia utilizes alpha- and beta-tubulin paralogs in different stages of the life or cell cycle, which has not yet been demonstrated. This data will also inform the development of a robust live cell marker for Giardia’s MT-arrays.