Giardiasis is a disease caused by the protozoan parasite Giardia lamblia which affects approximately 20,000 people per year in the United States alone. To this day, there are few anti-Giardiasis drugs available so new therapeutics to address toxicity and resistance issues are needed. In previous studies, we have examined seven protein kinases in Giardia’s genome and these kinases share an unusual structural feature in their active site, which results from an atypically small gatekeeper residue (amino acid residue). This feature confers sensitivity to a class of compounds called bumped kinase inhibitors (BKIs); BKIs demonstrated minimal toxicity and positive pharmacological properties. We repeated the search for small gatekeeper kinases using an updated multisequence alignment program, which identified four new small gatekeeper kinases. We developed a cloning strategy to 3HA tag each of these kinases. Then, we evaluated the expression and cellular localization of the proteins using Western blot and immunofluorescence (IF) assays. With the tagged kinase lines, we utilized translation blocking morpholinos to test the importance of each kinase using cell growth. This data will be used to prioritize protein purification for screening against a BKI library using thermal shift assays. These results may identify lead compounds toward novel treatment options for giardiasis.

Traditional antibiotics are known to wipe out many species of bacteria, but often leave several resistant microbes to dominate. These resistant bacteria are a huge concern for human health. Medical researchers are on the hunt for new approaches to combat bacterial infections on a large scale. It has recently been discovered that bacteria communicate through Quorum Sensing (QS), which if inhibited, presents a promising strategy to control these pathogenic bacteria. Instead of eliminating the weakest strain of bacteria resulting in a proliferation of antibiotic resistance, inhibition of quorum sensing silences gene expression, virulence and pathogenicity. A variety of marine organisms have demonstrated quorum sensing inhibition (QSI) potential, however there are many marine organisms that have yet to be screened for QSI compounds. In this study, three species of marine algae native to the Pacific Northwest were investigated for quorum sensing inhibition (QSI) capabilities. Ulva Lactuca, Chondracanthus exasperatus, and Iridaea Cordata were subjected to QSI capability testing that involved use of the indicator strain Chromobacterium violaceum Bergonzini (ATC 12472). The indicator bacterium uses the QS signal N-acyl-homoserine lactone (AHL) to produce a purple pigment. Each species of algae was overlaid with agar inoculated with Chromobacterium violaceum and incubated overnight. Absence of pigment in the indicator strain suggested the presence of a QSI compound. Preliminary evidence indicated that two of the three species of algae tested demonstrated QSI activity. The next step of this research involves identification of the specific compounds involved in QSI, thereby contributing to the expanding catalogue of known QSI molecules from marine environments. Ultimately this work aims to make a contribution to the fight against antibiotic resistant pathogens.

Eukaryotic cells are constantly locked in genetic conflict with selfish elements, elements that are detrimental to the host, but rely on the host for replication and propagation to progeny. This puts selective pressure on the host to evolve methods of restricting these elements, and selective pressure on the element to evade those methods. Such pressures could cause rapid evolution of host genes, resulting in an evolutionary arms race. One selfish element is the 2-micron plasmid found in budding yeast; in this case, we are referring to Saccharomyces cerevisiae. The 2-micron plasmid relies on the host to propagate, but also confers a 1-3% growth rate defect to the yeast, making it a selfish element and a possible source of genetic conflict. We hypothesize that the 2-micron plasmid may be undergoing an evolutionary arms race with its host, Saccharomyces cerevisiae. To discover if and how an evolutionary arms race is occurring in yeast, we need to look for positive selection in host genes that the plasmid interacts with. Although brewer’s yeast is a very well studied organism, we still do not fully understand how the yeast and plasmid interact. There exists a yeast deletion collection where there is a collection of strains, each with a single non-essential gene deleted. Using the yeast deletion collection, we have discovered some of the host genes the plasmid requires for propagation. This could illuminate possible host evolutionary pathways for evolving restriction mechanisms against a selfish genetic element as well as additional insights about plasmid propagation.

Gene regulation lessens the cost of cellular operations by altering physiological processes in an as-needed manner depending on resource availability. Previously, it was shown that repeated ecologically-relevant shifts in combination with conditional gene regulation can lead to a population collapse phenomenon. In this prior work, wild-type and regulatory mutant strains of Desulfovibrio vulgaris Hildenborough (DvH) were grown in co-cultures together with wild-type Methanococcus maripaludis in an environment where they were continuously transitioned between sulfate respiration and syntrophy conditions. While the wild-type DvH co-cultures collapsed after few transitions in this environment, the regulatory mutant persisted and never collapsed. This work showed that conditional gene regulation can drive a microbial community to collapse when the environment fluctuates too rapidly. In addition, it provided us with an experimental framework for probing the impact of gene regulation in adaptation of microbial communities to rapidly fluctuating environments. In this work, we further combined the experimental framework of alternating transitions together with transposon mutant libraries to identify active gene regulatory networks. The random barcode transposon-site sequencing (Rb-TnSeq) DvH library was rapidly transitioned between high and low sulfate concentrations and samples were collected for barcode sequencing to determine transcription factors that were enriched over the course of transitions. We will present fitness data and a list of enriched regulatory genes to shed light on how the regulatory mutations impact the organism’s ability for adaptation to fluctuating environments.

A more complete understanding of protective HIV-specific immune responses is critical to designing effective vaccine strategies. A recently concluded Phase III HIV vaccine trial identified antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) as a correlate of protection. While researchers have characterized hundreds of neutralizing antibodies (Abs), much less is known about Abs that mediate ADCC. ADCC can be measured through a distinct assay known as the rapid and fluorometric ADCC assay (RF-ADCC). This assay quantifies HIV-coated cell lysis mediated by antibodies and primary effector cells. Here we describe seven ADCC-mediating Abs isolated from an individual who developed a potent HIV-specific response. This project aims to examine the biological development of these seven ADCC Abs. To accomplish this, we deep sequenced longitudinal blood samples to reconstruct phylogenetic trees that illustrate the mutative developmental pathway of this patient’s Abs. We selected and cloned Ab sequences that represent intermediate states between germline and mature ADCC-capable Abs. These intermediaries are currently undergoing HIV binding studies. Next, we will test these Abs in the RF-ADCC assay to assess when the Ab lineages acquired ADCC activity. Finally, epitope mapping studies will be conducted on Ab intermediates of interest. This project will offer understanding on the developmental pathways of ADCC-mediating Abs and inform us on how we can exploit these antibodies in vaccine development.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS), a cancer of endothelial cell origin that is the most common malignancy among AIDS patients worldwide. Previous research in our lab has established that the number of peroxisomes is increased during latent KSHV infection. Peroxisomes are multifunctional cellular organelles involved in a variety of metabolic pathways important to KSHV pathogenesis. My project is to evaluate the cellular mechanism by which KSHV induces peroxisome biogenesis, thereby elucidating one of the key pathways involved in KSHV latency. I hypothesize that KSHV increases the transcription of specific regulatory genes responsible for peroxisome biogenesis. I evaluated gene expression of a known transcription factor, peroxisome proliferator-activated receptor alpha (PPARA), that has been implicated in peroxisome biogenesis during mock and KSHV latent infection of endothelial cells. I used real-time PCR to quantify gene expression of PPARA, in addition to other genes involved in peroxisome formation and function. My data demonstrates that KSHV infection upregulates PPARA and peroxisome-associated genes, suggesting that PPARA may be a key regulator of the expression of peroxisome biogenesis. To further establish the role of PPARA in peroxisome biogenesis, I am currently working on silencing PPARA expression using small interfering RNA (siRNA). I will determine if the knockdown of PPARA prevents KSHV upregulation of peroxisome-associated genes. In the absence of PPARA, I expect that expression of peroxisome-associated genes will be downregulated, suggesting that PPARA regulates them at the transcriptional level. These results will establish a key mechanism in KSHV pathogenesis, and potentially contribute to the development of novel therapeutic avenues for KS treatment.

Pathogenic microorganisms cause a large burden of disease globally. Rapid and accurate detection of these pathogens is essential for treatment. The goal of our project is to evaluate the analytical and clinical sensitivity of Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) for detecting such microorganisms compared to conventional qPCR. qPCR targets and amplifies specific DNA sequences and is widely used in laboratories to detect these organisms. However, when there is only a single organism in a diagnostic sample, the DNA amplified from such a limited number of template copies may not be detectable by qPCR. RT-qPCR overcomes this by targeting ribosomal RNA (rRNA), which is in greater abundance compared to the rRNA-encoding DNA. rRNA-targeted assays can provide more sensitive detection because the target is more abundant. In addition, rRNA is relatively resistant to degradation, unlike more labile mRNA targets. To evaluate RT-qPCR, we selected important human pathogens, designed reagents for DNA dye based (LCGreen) or probe-based amplification/detection assays, and isolated total nucleic acids from samples by using a silica-based extraction instrument (bioMérieux). Nucleic acids from extracted samples were tested on a real-time PCR machine using with and without RT (+RT/-RT). We evaluated the relative increase in rRNA compared to coding DNA by comparing the +RT and -RT standard curves’ cycle thresholds (Ct). In all experiments, RT-qPCR detected the target organism earlier than qPCR although the relative enrichment of rRNA varied by organism. For instance, in Toxoplasma gondii, 18S rRNA was up to 500 times more abundant than the coding DNA. In contrast, in Cryptosporidium parvum rRNA was only modestly enriched at 50-fold increase compared to coding DNA. Our work will provide a survey of relative enrichment of rRNA and will evaluate whether this target should be widely pursued as an improved approach for pathogen detection.

Store-and-Forward Teledermatology is used as a system for increasing access to dermatology care for patients in the Veterans Health Administration (VA). Where dermatologists are not available, primary care clinics partner with teledermatology to help patients access high-quality, timely healthcare. Rather than being seen face-to-face by a dermatologist, patients’ skin lesions are imaged and sent asynchronously to a teledermatology reader (a dermatologist) in another location. Many types of skin conditions can be diagnosed through this system, including melanoma, a dangerous form of skin cancer. While this health system has demonstrated good diagnostic sensitivity for melanoma, little is known about its positive predictive value (PPV), also called precision. PPV is the proportion of melanoma diagnoses that are confirmed by pathology to be truly melanoma. Pathology diagnosis is considered the ‘gold standard’ for diagnosing dermatological conditions. Because the threat of melanoma is taken very seriously, low PPV leads to more unnecessary biopsy procedures and pathology consultations- actions that strain VA health system resources. This quality improvement study investigated the PPV of all teledermatology readers in the Seattle VA program from February 2015 – January 2016. Chart reviews (n=552) were completed and data were analyzed to determine an overall PPV rating, to find individual provider PPV ratings, and to identify factors correlated with high PPV. We hope to use this analysis for two primary quality improvement actions. First, we aim to develop PPV standards for teledermatology providers to meet. Secondly, we aim to identify what steps could be taken to maximize teledermatology reader PPV. Policy changes could be applied to help minimize waste in our teledermatology program.

In the past few years, cholera has caused many outbreaks of disease. Many studies have been done on its transmission pathways and its effect on non-pregnant patients. This literature review focused on the application of research to discover the preferred recommendations for pregnant women in an outbreak of cholera. This project explored research in academic journals and other professional publications with keywords such as “cholera and pregnancy” and “cholera transmission.” We explored existing transmission pathways of cholera, as well as current recommendations for these specific cases. Such recommendations would be to prevent maternal illness and death, as well as diminishing the risk of fetal demise. It was found that cholera is present in biofilms and endemic in certain regions. Its contamination can be prevented through sewage, chlorination of water, and proper food preparation. This review used the disciplinary lenses of microbiology, clinical medicine, epidemiology, and public health. The information concerning cholera in pregnancy is conflicting regarding fetal demise pertaining to first or third trimester outcomes. However, there is overwhelming evidence pointing to the recommendation to vaccinate in the circumstances of an epidemic. Literature suggests that women who are pregnant are not at a higher risk of getting infected with cholera, however given a change in immune response, the fetus is at a higher mortality risk due to the mother’s emesis and dehydration. This review will suggest implementation in regards to vaccination and treatment options.

Cytosolic pH is sustained by the movement of protons out of the cytosol. The vacuolar ATPase (V-ATPase) is a conserved protein complex that localizes to the membrane of organelles and transfers protons from the cytosol into the organelle lumen. The V-ATPase couples the energy of ATP hydrolysis to transport protons. Proper pH homeostasis is important for many organelles such as endosomes, lysosomes, and secretory vesicles. In Saccharomyces cerevisiae, the Pma1 protein localizes to the plasma membrane and pumps protons out of the cell, helping to maintain cellular pH homeostasis. In Saccharomyces cerevisiae, we have found that loss of activity of the V-ATPase results in a growth arrest at low temperature. We generated 66 yeast V-ATPase mutant strains that are able to suppress the low temperature induced growth arrest. In complementation tests of these mutants, our results suggest that all of the mutants belong in the same complementation group. We have performed whole genome sequencing on one strain and analysis of the whole genome sequencing data has identified a single putative mutation. We are validating that the identified mutation is causative for the phenotypic suppression, and performing targeted sequencing of the candidate gene in the other suppressor strains.

Atypical sensory behaviors have only recently been included as an explicit symptom of DSM-5 Autism Spectrum Disorder (ASD), despite a considerable history of anecdotal evidence. As a result, sensory behaviors are not well characterized in ASD research. In the current study, we aim to fill a gap in the literature by specifically investigating auditory processing in 143 children with ASD and/or a likely gene disrupting mutation (LGDM) associated with ASD. Participants’ parents completed the Sensory Profile Caregiver Questionnaire about their child and psychiatric symptoms were assessed via a comprehensive evaluation with a licensed clinical psychologist. We hypothesized that responses on the Sensory Profile would reflect either hypo- or hyper-sensitivity to auditory stimuli. We predicted that extreme hypo- or hyper-auditory responses would reflect separate deficits, rather than a continuum of auditory sensitivity. We also predicted that we would see unique auditory response profiles associated with LGDMs in particular gene groups. Our results will be discussed with regard to current theories and information about sensory abnormalities in children with ASD. The results and findings from this study could be used to help lay a foundation regarding sensory markers and auditory processing deficiencies in children with ASD, a topic that is fairly new and has not been thoroughly investigated.

Influenza is a prolific and hazardous virus, affecting even those in the population who have been previously infected or vaccinated. The innate immune system serves as a key first line of defense against this pathogen, with the signaling components, called interferons, driving the production of a potent cellular antiviral response. Studies have indicated that viral populations replete in defective virus particles, virions with a deletion in a portion of their genome, are less efficient at blocking the antiviral response, as shown by increased interferon in the host. Our project seeks to explore this phenomenon of RNA deletions leading to increased interferon expression in host cells by testing the hypothesis that deletions in the three polymerase genes of influenza alone are sufficient to cause an increase in the interferon response. To begin answering this question, an interferon reporter system was used to analyze the viral genome of interferon positive cells, and deletions within various lengths of the PB1, PB2, and PA polymerase genes were found to be enriched. I then created pure populations of influenza with these empirically derived gene deletions in PB1 while my mentor, Dr. Alistair Russell, created pure PB2 deletion populations, which were grown on cell lines expressing functional PB1 and PB2 proteins respectively. When used to infect unmodified cell lines, it was found that pure populations of both PB1 and PB2 defective influenza were sufficient to induce the host interferon response. In the future, I will create PA expressing cell lines and influenza with deletions in PA to analyze the effect of PA deletions within the influenza genome on the host interferon response, and it is hoped that these results can be used to explore a mechanism for how influenza occasionally fails to escape the innate immune response.

Synaptic plasticity influences many functions in the body, some of which include the mechanisms of learning and memory. However, isolating these cells for further study is inefficient as grinding up a portion of the brain yields a mixture that includes synaptosomes, neuronal and glial contaminants, and free myelin as well as mitochondria. One way to sort synapses from the mixture is to use FACS (fluorescence-activated cell sorting). However, this method is limited as it is very time consuming. Instead, our lab is developing methods to sort specific synapses based on immunology cell-sorting techniques that can isolate activity-dependent synapse species from the assortment of synapse types in the brain. We have created TAG proteins that targets glutamate synapses, therefore potentially enabling us to utilize the more efficient MACS (Magnetic Cell Sorting) method that is proven to isolate cell types at higher rate. My project is to develop a method to ensure that the TAG RNA is translated only in specific neurons. I utilized Cre-lox recombination methods so that the TAG RNA is translated only when a CRE protein removes the lox-stop-lox gene. To do so, I used a plasmid with an AS-LSL-TAG insert previously made by the lab. I then used HEK 293 cells, each with a different condition to evaluate how well the CRE protein will be able to remove the LSL portion of the tag construct: one with untransfected cells (control), another with the CRE-RFP plasmids only, a third with only AS-LSL-TAG plasmids, and the last with both CRE-RFP and AS-LSL-TAG plasmids. If I achieve HEK 293 cells with the AS-LSL-TAG and the CRE-RFP that express the both the green fluorescence from the AS-LSL-TAG and the red fluorescence from CRE-RFP, I will then insert the LSL TAG construct into neurons and analyze the results by western blots and microscopy.

Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is the etiological agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor predominantly made up of cells of endothelial origin. KSHV establishes a predominantly latent infection in endothelial cells in culture and in the KS tumor. KSHV latent infection alters cellular metabolism to improve the survival of the infected cells. These metabolic changes resemble a common alteration in cancer cells, termed the Warburg effect. This refers to an increase in glycolysis in the presence of oxygen and a decrease in oxidative phosphorylation. The mechanism of KSHV induction of the Warburg effect is currently unknown. Hypoxia-induced factors (HIFs) are likely candidates for KSHV induction of the Warburg effect as both HIF1α and 2α are induced by KSHV latent infection. To determine if HIF1α or 2α is required for the survival of KSHV infected endothelial cells, I constructed HIF1α or 2α knockouts using the CRISPR/Cas9 gene editing technique in a lentivirus vector. I expressed the endonuclease Cas9 and guide RNAs that lead Cas9 to cut at the HIF1α or 2α genomic sequences leading to genomic mutations in HIF1α and 2α respectively. To determine if HIF1α or 2α is required for the survival of latently infected endothelial cells, I infected the HIF1α and 2α knockout cells with KSHV and looked for cell death at 48 hours after infection. To evaluate if HIFs are required for KSHV induction of the Warburg effect, I will determine if one or both of the knockout cells produce less lactic acid and increase oxygen consumption at 48 hours post KSHV infection as compared to infection of wild type cells indicating the Warburg effect is not being induced by KSHV in the knockout cells. These results will aid in the future efforts to develop antiviral drugs by inhibiting viral latency.

Biofilms are surface-associated aggregates of bacteria encased in a protective, extracellular matrix. Biofilm bacteria resist antibiotic treatment and killing by the host immune response, leading to persistence in a variety of chronic infections. Pseudomonas aeruginosa is an opportunistically pathogenic bacterium and a model organism for studying biofilm formation. One major factor that drives biofilm formation in P. aeruginosa and other bacteria is the intracellular second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP). Elevated c-di-GMP levels promote cell surface adhesiveness by up-regulating production of biofilm extracellular matrix components and down-regulating motility genes. In contrast, planktonic P. aeruginosa cells are known to have comparatively lower c-di-GMP levels than their biofilm counterparts. While factors contributing to the formation of mature biofilms have been well-characterized, early biofilm formation, when a bacterium first senses a surface and transitions from a planktonic state to a surface-attached state, remains largely understudied. Using a fluorescent, transcriptional reporter of intracellular c-di-GMP and confocal microscopy, we have monitored the dynamics of c-di-GMP production at the single cell level. Our major finding is that c-di-GMP levels are elevated in only a subpopulation of P. aeruginosa cells (30-69%) during this time. We have used flow-assisted cell sorting (FACS) to separate surface-attached cells with high and low c-di-GMP, to examine ways in which these populations are physiologically distinct. We have found by lectin staining and by qRT-PCR that surface-associated cells with high intracellular c-di-GMP produce more polysaccharide than their surface-associated, but low c-di-GMP counterparts. We hypothesize that the heterogeneity in c-di-GMP observed during P. aeruginosa surface sensing represents a specialization among the genetically homogenous population into subpopulations of non-motile, early polysaccharide producers and motile, surface-exploratory cells, both of which contribute to downstream biofilm maturation.

Nitric oxide (NO) is a reactive radical produced by cells of the innate immune system. NO reversibly binds to protein cysteine residues through a process called S-nitrosylation, often resulting in reduced protein function. This antimicrobial molecule serves as an important defense mechanism used by the immune system to fight off infections by many different pathogens, including the Gram positive bacterium Staphylococcus aureus. S. aureus is a common opportunistic pathogen and a major cause of wound infections, food poisoning, pneumonia, and invasive disease. Its ability to cause acute and chronic infections throughout the body is the result of numerous virulence factors. While many of these virulence factors have been extensively studied, little is known about how S. aureus pathogenesis is affected by its interaction with NO. We show that NO inhibits the expression of protein A, an extracellular IgG binding protein that is a key immune evasion factor in Staphylococcal disease. Reverse transcriptase quantitative PCR (RT-qPCR) shows that S. aureus cultures treated with NO exhibit dose-dependent inhibition of protein A transcription with increasing concentrations of NO. Western blots further confirm that transcription inhibition results in reduced protein A levels. Using allelic exchange mutagenesis, we determined that inhibition is likely due to NO targeting XdrA, a transcription activator of the spa gene encoding protein A. Using the Biotin-switch method, we have shown that XdrA is directly modified by NO and structural analysis of the protein suggests that NO modifications could detrimentally affect its ability to bind the spa promoter as it does when exogenous NO is not present. These findings introduce a novel mechanism by which the innate immune system could compromise the ability of S. aureus to evade immune detection during infection through NO inhibition of protein A.

Cerebral malaria (CM) is a multifactorial disease with lethal neurological complications plus increased risks of cognitive and neurological impairments making CM a leading cause of childhood neuro-disability. The malaria parasite, Plasmodium falciparum, accounts for most mortality from malaria, and primarily kills children under five in sub-Saharan Africa. The deadly CM complication develops when red blood cells infected with parasites (iRBCs) bind to the endothelial cell wall in brain microvasculature. This process of parasite sequestration via cytoadhesion is mediated by the parasite ligand, P. falciparum erythrocyte membrane protein 1 (PfEMP1) encoded by a family of 60 var genes. An individual parasite, however, strategically undergoes allelic exclusion to express only a single distinct PfEMP1 variant. PfEMP1 proteins possess the ability to bind relatively few host receptors despite seemingly limitless sequence diversity. Requirements for maintaining binding surfaces conserved in structure and binding potential despite significant sequence diversity remain a mystery. Understanding these requirements will be critical for developing therapeutics to mitigate disease severity. A subset of PfEMP1 proteins associated with severe/cerebral malaria bind highly conserved regions of the host receptor, endothelial protein C receptor (EPCR), and inhibit binding of its natural ligand, activated protein C (APC). There are six PfEMP1 subdomains that bind EPCR (CIDRα1.1 and CIDRα1.4–1.8 domains), which share limited sequence identity among PfEMP1 variants. The data so far suggests the presence or otherwise absence of a coiled coil domain within CIDRα1 determines binding to EPCR. Our study uses computational and molecular biology tools to investigate the molecular and structural determinants for malaria parasite adhesion to EPCR. 
 

The biological basis behind aging in eukaryotic species is defined by a wide range of factors, from diet to environmental conditions and metabolic predisposition. The genetic factors associated with aging are of particular interest because they are highly conserved among all eukaryotes. Determining longevity genes that are broadly conserved among eukaryotic species is a critical step in understanding aging, and is one of the primary goals of this project. The Kaeberlein lab has analyzed the replicative lifespan (RLS) of more than 4700 single-gene deletion mutants of Saccharomyces cerevisiae, a eukaryotic model organism. RLS experiments measure the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Mother and daughter cells are visibly differentiated under a light microscope and physically separated using a fiber-optic needle. The number of daughter cells dissected are quantitated and recorded. The total number of cell divisions are then summed for statistical analysis. The result of the investigation yielded 238 gene deletion mutants that exhibited a statistically significant lifespan extension. Longevity genes were found to be clustered within specific parts of an organism's biology. Ribosomal genes and genes related to metabolic responses to nutrients were particularly significant. One particularly significant gene is the LOS1 gene, which functions as a tRNA exporter from the nucleus. Deletion of the LOS1 gene resulted in a dramatic enhancement of yeast RLS, opening the possibilities of lifespan research regarding the subcellular localization of tRNA. Also significant was the discovery that a majority of the longevity genes were concentrated among highly evolutionarily conserved parts of the genome. Ultimately, the success of the yeast RLS project in identifying novel longevity genes and establishing a database of conserved longevity genes in eukaryotes points to the efficacy of such methodology.

The process of aging brings about susceptibility to disease and death, but its mechanisms are still poorly understood. Dietary restriction is a potent approach to slow down the aging process, which has shown efficacy in multiple species, including the roundworm, Caenorhabditis elegans, a popular model organism for aging studies. Specifically, pre-reproductive adult C. elegans subjected to starvation enter a state of adult reproductive diapause (ARD), which preserves their lifespan and reproductive potential. Intriguingly, diapaused animals show signs of age-related deterioration upon prolonged starvation, but exhibit dramatic morphological improvements and have normal lifespans following return to feeding. However, the mechanisms regulating this lifespan preservation and apparent rejuvenation are not known. In this project, we aim to identify the genes responsible for the recovery during diapause exit. For that purpose, we used an RNA interference (RNAi)-based screen to inactivate individual genes during exit from diapause and analyzed the resulting phenotypes. Our current results reveal many regulators of metabolism and cellular homeostasis as crucial mediators of post-diapause recovery. Further studies based on these findings will discover the critical steps required for post-diapause tissue rejuvenation and preservation of longevity.

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a method of organism identification that is currently used widely in clinical microbiology. Compared to traditional biochemical testing, it offers a faster turnaround time and provides a more cost-efficient way to identify clinical isolates. Currently at the University of Washington clinical microbiology laboratory, if a yeast grows from any media other than inhibitory mold agar (IMA) media, it is subcultured onto IMA and incubated to test by MALDI-TOF the following day. This validation study will allow medical laboratory scientists (MLS) to bypass the subculturing step, which will decrease the turnaround time by approximately 24 hours. We evaluated five different media types and incubation conditions, with a sample size of 51 organisms. Through this validation study, it was concluded that the various media and incubation conditions had little to no effect on yeast identification by MALDI-TOF. Because the different conditions tested had no effect on the MALDI-TOF MS score values, this successful validation will allow for shorter turn-around time for yeast identification, which in turn will allow for faster and more accurate patient care.

NASA confronts a forward contamination problem with the launch of spacecraft and probes into extraterrestrial environments with potential for detection of life. Microbial control is required in order to avoid cross contamination that would compromise data collected. Currently approved decontamination methods are costly and involve high heat and chemical treatments which risk damaging thermally sensitive spacecraft materials and components. Alternative methods, including atmospheric pressure plasma jets (APPJs), show promise as effective technologies for microbial control. Researchers at Edmonds Community College partnered with Eagle Harbor Technologies (EHT) of Seattle, WA to test the capabilities of an APPJ developed by EHT, performing characterization of killing efficacy and demonstrating proof of principle for large-area spacecraft sterilization. Research demonstrated killing capabilities of APPJ when applied to various conditions including different surface materials, meshes, tapered and elevated holes. The plume of the APPJ traveled through tapered and elevated holes yielding greater than 99% killing of endospores. Application of APPJ as a “brush” demonstrated greater than a 6 log reduction of endospores over 56.75 cm²/10 minute exposure. Findings suggest a brush composed of multiple jets could be effective for spacecraft sterilization over large surface areas of differing materials with capability to penetrate unusual shapes and surfaces. Anticipated improvements in killing efficacy resulting from better characterization of increased voltage, frequency, humidity, and the inclusion of low O₂ concentrations should lead to the development of an APPJ brush effective at spacecraft sterilization.

We aim to find diversity in the HHV-6B genome via sequencing of the U90 gene. We have previously sequenced 120 whole genomes of HHV-6B from New York, Japan, and Fred Hutch chromosomally integrated (Ci-HHV6B) strains and found the U90 gene to encode the most diversity. Our hypothesis was that candidate U90 sequencing of clinical samples for which whole genome sequence could not be accomplished due to low viral concentrations would give the most amount of phylogenetic information of candidate loci available. Eleven excess serum samples from Seattle Cancer Care Alliance and Fred Hutch for which genome sequencing failed were amplified for the U90 locus using nested PCR, pooled, and Nextera/Illumina sequenced. Of those nine samples three had fewer than five changes across the 3kb locus, and one sample accounted for 95% of the diversity in the gene among these eleven samples. We found sequence clusters outside of sequences published to genbank, as well as clusters outside of the 120 whole genome sequences. Nine of the eleven samples clustered with the New York isolates. One shows significant genetic distance from the Japanese group, while another isolate shows similarity to the Z29 reference strain. This demonstrates an unexplored diversity in HHV6B genomes with much more diversity to be expected. Further U90 sequencing studies would expose more diversity in HHV-6B in a low cost, but effective, manner. 

The ion exchanger monosodium titanate (MST) has been proposed to therapeutically deliver calcium ions in dental situations that require calcium. However, previous results suggest that MST limits cell growth in vitro by mechanisms that remain obscure. It is critical to understand these mechanisms for any successful therapeutic use of MST. The current study tested the hypothesis that MST limits cell growth in vitro by binding to culture polystyrene, reducing attachments sites available to cells, thereby limiting cell culture growth. MST was added to culture wells (8 concentrations between 0 and 200 mg/L) either 24 h after addition of L929 fibroblasts/OSC2 osteoblasts or prior to cell addition. Cell cultures (n = 8/condition) were left in contact with the MST for 72 h, after which cell growth was assessed using Cell TiterBlue®. Control groups received no MST; statistical differences were detected using ANOVA with Tukey post-hoc tests (α = 0.05). When MST was added after plating of the cells, cell growth was inhibited in a dose-dependent fashion to a maximum of 40% (p < 0.05 compared to no-MST controls). Yet when MST was added prior to the cells, the maximal inhibition of growth was 80% vs. controls. (p < 0.05). The addition of MST before cells significantly decreased growth in the cultures (relative to conditions where cells were established first). Microscopic inspection of the culture wells revealed more attachment of the MST to the culture polystyrene than when cells were added first. These results and observations are consistent with our hypothesis that the MST attaches the polystyrene and reduces the ability of cells to attach and proliferate. Further testing with other cell types and additional cell densities is in progress.

Iron sufficiency is essential for optimal brain growth and development, particularly among preterm infants. Current guidelines for iron supplementation of these infants (2-4 mg/kg/day) may be inadequate to maintain iron sufficiency. Our objective was to evaluate zinc protoporphyrin-to-heme (ZnPP/H) ratio and ferritin as measures of iron status in neonates. Iron sufficiency over time was also assessed. All UWMC NICU inpatients who received paired ferritin and ZnPP/H measurements between October 2014 and May 2016 were evaluated retrospectively. Concordance of iron measures were evaluated, as were effects of sepsis, transfusion and iron supplementation over time. There were in total of 601 paired samples from 228 patients and the paired values were assessed during culture-positive sepsis, defined as positive blood culture within 2 days before or 1 day after ZnPP/H or ferritin measurement. Mean ZnPP/H in infants with and without sepsis were not significantly different whereas ferritin values were significantly higher during sepsis. Ferritin values were also more affected by red blood cell transfusion given within seven days prior to ferritin and ZnPP/H measurements. A trend toward worsened iron sufficiency in infants born to diabetic mothers was noted. Small for gestational age infants also showed a trend towards poorer iron status. The mean iron supplementation at 30, 60 and 90 days was 5.4 mg/kg/day, 6.9 mg/kg/day and 7.4 mg/kg/day. Despite this, ferritin values decreased with advancing postnatal age, with 66% of ferritin values <76 ng/dL. In conclusion, ferritin is more significantly affected by sepsis and transfusion, and therefore ZnPP/H may be a more reliable marker of iron status during proinflammatory states. Infants showed worsening iron sufficiency over time despite supplementation above AAP guidelines suggesting a potential need for more aggressive iron supplementation.

Brain computer interfaces (BCIs) hold an amazing potential to increase our knowledge of the brain and to help improve the lives of countless people. However, almost all work currently being done with BCIs is performed in a strictly controlled laboratory setting. In order to create BCIs that can function robustly in the real world we must examine brain signals during naturalistic tasks like speech. Through our partnership with Harborview Neurology we are able to get week long continuous audio/visual/and electrocorigraph (ECoG) data from long term monitoring of epilepsy patients. A significant problem with this data set is that there is so much data that manual annotation of the subjects' speech would be incredibly time consuming. Our work focuses on using high throughput analysis techniques to automatically annotate our data set in order to efficiently study the brain. We present a pipeline to perform automatic speaker tracking of the implanted patients in our clinical recordings. Speaker tracking is the task of figuring out all the times in which a particular speaker spoke during a recording or across several recordings. We have developed an automatic pipeline that extracts all human speech from a recording and then uses a support vector machine (a machine learning algorithm) to differentiate the subject's speech from anyone else that might speak inside the hospital room. Accuracy for every step is above 80%. The methods developed in the study will allow us to automatically generate timestamps of subject speech across our entire data set. This will allow us to perform useful analysis on the ECoG signal in order to better understand the underlying neural basis of natural speech and develop more robust BCIs to improve the lives of countless people.

Listeria monocytogenes is the causative agent of listeriosis, an infection which causes flu-like symptoms in most individuals after ingesting contaminated food, but can severely harm immunocompromised individuals, newborn children, and the fetus of an infected pregnant woman. L. monocytogenes is a facultative intracellular pathogen which escapes a phagocytic vacuole early in its life cycle. Once in the cytosol of the enterocyte, the master virulence regulator PrfA activates virulence factor production. However, what initiates PrfA and allows L. monocytogenes to sense that it is in the cytosolic environment is still not understood. Recent discoveries have indicated that the translation of at least one of the nine virulence genes, actA, requires several genes involved in bacterial redox regulation, including the protein YjbH. In the Bacillus subtilis model, a homologous YjbH interacts with the protein Spx, an important regulator of thiol-specific oxidative stress. Under normal conditions, YjbH binds to Spx and facilitates its degradation via the ClpXP protease. During disulfide stress, YjbH aggregates, freeing Spx to regulate oxidative stress response genes. Although the B. subtilis and L. monocytogenes YjbH are homologs, the function of the L. monocytogenes YjbH is uncharacterized. My research project seeks to extract and purify the protein. I first generated two strains of E. coli that express a tagged version of either the wild type YjbH or a mutant YjbH which lacks two cysteines that may be involved in its interaction with Spx. The second half of my project will focus on limiting the aggregation of recombinant YjbH. After successful purification of the proteins, these two strains will be a useful tool in characterizing the function of L. monocytogenes YjbH, and its role in the virulence pathway. Understanding the role of YjbH in the virulence pathway could ultimately lead to novel ways to inhibit the pathogenicity of L. monocytogenes.

 Many children have lasting pain problems after surgery that can greatly impact their recovery and health-related quality of life. Emerging research has identified psychosocial factors as important predictors of children’s postoperative outcomes, suggesting that an intervention addressing these factors could positively impact both short-term and long-term recovery from pain and quality of life. However, there are few data from children, families, or healthcare providers assessing psychosocial needs and receptiveness to a behavioral pain management intervention in the perioperative period. Involving healthcare provider stakeholders in the development of an intervention is essential to ensuring feasibility, acceptability, and successful implementation of the intervention. Seventeen semi-structured interviews were conducted with pain clinicians, nurses, surgeons, physical therapists, and child life specialists who provide perioperative care at a children’s hospital. Interviews probed the following areas: 1) current experience with perioperative preparation and care, 2) recommendations on content and format of an ideal psychosocial intervention, and 3) potential barriers to implementation and participation in a perioperative program. After transcription, we coded and analyzed interviews using inductive thematic analysis to identify consistent and relevant themes across provider groups. Independent members of the research team reviewed codes at each stage for credibility and representativeness. Providers said that a pain management program would address current gaps in surgery preparation, and that children and their parents need to learn effective coping strategies to positively impact their recovery. Providers also suggested necessary content, delivery methods, and ways to enhance program effectiveness. Provider themes, as well as themes from patient and family interviews, are being used to develop a family-based psychosocial intervention targeting children at risk for poor postsurgical outcomes. Next steps for this research include design, implementation, and evaluation of the perioperative intervention aimed at reducing the likelihood that children will develop lasting pain problems after surgery.