Monosodium titanate (MST) is an effective metal ion-exchanging material and has been used for decontaminating nuclear waste solutions. More recently, MST has been considered a candidate to exchange calcium ions in biological applications, especially for therapeutic delivery in dentistry. However, the toxicological properties of MST and MST-Ca(II) are poorly defined. Yet, it is critical for the mechanisms of toxicity to be understood to accurately assess MST as a therapeutic agent. In recent experiments, a decrease in the in vitro growth of MC3T3 osteoblasts exposed to MST or MST-Ca(II) attached to culture wells has been observed, but the cause of the inhibition is unknown. We observed (phase contrast microscopy) a tenacious affinity of the MST for the cell culture plate and the MC3T3 cells. This current study tested the hypothesis that MST limits cell growth in vitro by competing for attachment sites on culture polystyrene using L929 cells. Unlike MC3T3 osteoblasts, the growth of L929 fibroblasts was not inhibited when MST was added to cultures after cells had attached. We added MST or MST-Ca(II) (0 [control]–200 mg/L) to culture wells (n=8) 24 h prior to adding the L929 cells. The cells were then added (5000 cells/cm²) and the culture was incubated for 72 h to allow growth. Cell numbers and metabolism were estimated using a fluorescent-based assay (Cell-Titer Blue®). Statistical differences were detected using ANOVA and Tukey post-hoc tests (α=0.05). We expected that the addition of MST or MST-Ca(II) before cells will impede L929 cell growth; these experiments provided evidence that disruption of cell attachment to substrate may be one factor causing MST to inhibit cell growth in vitro. We will extend our investigation to other cells such as MC3T3 and dental pulp stem cells. 

Calcium is an ubiquitous and critical signaling molecule that mediates a wide range of cellular processes. Cytosolic calcium is maintained at concentrations that are orders of magnitude below both the extracellular space and intracellular organelles. Even small, sustained increases in cytosolic calcium can have harmful effects on the cell. As such, calcium homeostasis mediated in large part by the endoplasmic reticulum (ER) and mitochondria that serve to sequester calcium enable normal functioning of the cell. To examine cellular mechanisms that mediate calcium homeostasis, we are using mechanosensory hair cells of the zebrafish lateral line. Mechanosensory hair cells can be stimulated via a mechanical stimulus or drug-mediated calcium release from intracellular stores. The hair cells' abilities to easily uptake drugs and respond to physical stimuli make them an ideal tool to examine calcium homeostasis. Preliminary studies using transgenic zebrafish in which the calcium indicator GCaMP3 is targeted to the cytosol vs. the mitochondrial matrix demonstrates that stimulation of hair cells causes an immediate increase in cytosolic calcium in contrast to a gradual increase in mitochondria calcium. The question is whether the calcium in the ER is changing in response to the changes in mitochondrial and cytosolic calcium. As a first step to investigating the role of ER in calcium homeostasis, we will use gateway cloning to generate transgenic zebrafish lines in which calcium indicators are targeted to the ER. We predict that stimulation of mechanosensory hair cells will cause changes in ER calcium levels. These studies may provide valuable insight in how cells maintain health by buffering intracellular calcium levels in response to activity. 

O6-methylguanine-DNA methyltransferase (MGMT) is the sole DNA repair activity that removes cytotoxic methyl group adducts from the O6 position of guanine. My overall hypothesis is that MGMT in human glioblastoma (GBM), a malignant brain tumor, limits tumor cell killing by chemotherapeutic methylating agents such as temozolomide. To address this hypothesis I will compare MGMT activity in GBMs with a mutated copy of the IDH1 (isocitrate dehydrogenase) gene with that in wild type tumors. IDH1 mutant GBMs differ from wild type in being much more responsive to methylating agent therapy and having longer survival. Mutant IDH1 converts isocitrate to 2-dihydroglutarate that suppresses the expression of epigenetically regulated genes. As MGMT expression is controlled epigetically by a mechanism sensitive to 2-dihydroglutarate, I hypothesize that the better clinical response of IDH1 mutant GBMs is associated with lower MGMT activity than wild type tumors. To address my secondary hypothesis, I will assay MGMT activity in homogenates of human GBM tissue with known mutation status and a complete history of the course of alkylating agent treatment. Tissue and clinical outcome data will be provided by the Neurosurgery Tumor Bank. When necessary I will determine mutation status of archived GBM tissue by an established PCR assay. Finding significantly lower MGMT activity associated with longer survival in mutant tumors will support my hypotheses. I will also examine the association of MGMT activity with survival following alkylator therapy for wild type and mutant separately to provide additional evidence of the role of MGMT as a mechanism of treatment resistance. An inverse association between tumor MGMT activity and treatment response will suggest (1) that MGMT can be used as a prognostic marker for treatment outcome and (2) that inhibition of MGMT as part of alkylator therapy may improve clinical outcome for GBM patients regardless of tumor IDH1 mutation status.
 

Hair cells are mechanosensory receptors in the inner ear used for hearing and balance. When hair cells are damaged or die, hearing is lost or impaired. Many studies have also shown that genetic mutations affecting mitochondrial function are another cause of hearing loss. However, it is unknown which cells in the auditory system are affected. In this study, we sought to determine whether or not these deafness-causing mutations impact hair cells. To do so, we took advantage of zebrafish as a model system and studied hair cells of the lateral line. To generate mutant fish, we used the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)/Cas system, a genome editing tool that allows for gene-specific changes to be made to DNA. If these mutations do affect hair cells, we predict to see changes in mitochondrial and hair cell function. Characterizing these mutations in zebrafish will inform our understanding of the cellular mechanisms of hearing loss.

Iron is an essential micronutrient, playing key roles in neurodevelopment and erythropoiesis. Maintaining iron sufficiency is essential for brain growth and development, particularly in preterm infants. Current guidelines for iron supplementation of these infants (2-4mg/kg/day) may not be adequate to maintain iron sufficiency. Our objective is to assess concordance of ferritin and zinc protoporphyrin-to-heme ratios (ZnPP/H) in evaluating iron sufficiency in Neonatal Intensive Care Unit patients and clinical conditions in which these values diverge, and to evaluate iron sufficiency in these infants over time. All infants who underwent paired ferritin and ZnPP/H measurements between October 2014 and October 2015 in the University of Washington Medical Center NICU were assessed. Concordance of these measures was evaluated, as were effects of iron supplementation, blood transfusion, and inflammation. 144 patients met inclusion criteria with 352 paired samples collected between day of life 9 to 114. 34% of ferritin values were at concerningly low levels, indicating severe iron deficiency. In infants with low ferritin, ZnPP/H was higher than normal 60% of the time, which shows that ferritin and ZnPP/H were overall concordant. There were 36 abnoramlly high ferritin values in 14 infants; ZnPP/H ranged variably in these infants, with 5 cases of significant discordance. Of the infants with high ferritins, half had proinflammatory states within 2 days of obtaining the ferritin value suggesting it was elevated due to inflammation. Transfusion did not affect either ferritin or ZnPP/H values. Ferritin values decreased with advancing postnatal age. Despite iron supplementation that followed AAP guidelines, infants in this cohort became more iron-deficient with advancing age. This suggests the need for more aggressive iron supplementation among critically ill infants. Either ZnPP/H or ferritin can be used to assess iron status, although elevated ferritin may reflect a proinflammatory state.

As concern over antibiotic resistance continues to grow, there is a critical need to discover and develop new methods of controlling and preventing bacterial infections. Essential oils derived from the peels of oranges are known to possess antimicrobial properties. Previous research has established that limonene, the principle compound present in orange oil extract, is a particularly effective antimicrobial agent that both destroys bacteria and inhibits bacterial growth. To determine whether different varieties of oranges have varying antimicrobial potency, the efficacies of oil distilled from blood, navel, and valencia oranges were compared. Oils extracted through steam distillation were diluted with methanol to concentrations ranging from 1:20 to 1:1. Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli bacteria were exposed to the oils using the disc diffusion method on TSA plates. The minimum inhibitory concentration (MIC) for each oil was determined using the agar diffusion method. Gas Chromatography Mass Spectrometry (GC-MS) was used to compare the relative concentrations of limonene present in each essential oil. A better understanding of the potency of orange oil as an antimicrobial agent could lead to development of novel drugs and products to be used to combat infection and prevent the spread of bacterial pathogens.

Oxygen transportation in red blood cells occurs via a protein called hemoglobin. When hemoglobin is either absent, flawed, or impaired, the transportation of oxygen is hindered and this is known as anemia. Due to the prevalence and dangers of anemia, our goal is to effectively measure the concentration of hemoglobin in small volumes of a patient’s blood in order to diagnose critical levels of anemia. In order to do this, we take advantage of Beer-Lambert’s Law – a physical law that states concentration scales linearly with absorbance (a measure of how much light a molecule can absorb at a given wavelength). With the use of a microfluidic device, whole blood flows through an 8 mm x 250 μm x 50 μm channel and is illuminated by a white LED. A portion of the light is then absorbed by hemoglobin and continues at a reduced intensity to a green filter to focus our measurements to the visible green spectra, because it is known that hemoglobin absorbs the most light at about 540 nm, which is in this region. We demonstrate that our devices can determine hemoglobin concentration effectively with less than 40 μL of blood and at a low cost. The immediate impact of hemoglobin measurements of small volumes is its use towards children. The long-term impact is a diagnostic point of care device to solve global needs.

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. Some children infected with P. falciparum go on to develop CM – a potentially lethal disease complication where 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 parasite ligand, P. falciparum erythrocyte membrane protein 1 (PfEMP1) encoded by a family of 60 var genes. 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. Our study uses computational and molecular biology tools to generate putative gain-of-function CIDRα1.3 mutants. Biophysical properties are assessed and compared across al CIDRa1.x domains to determine features that predict EPCR binders which are subsequently incorporated or removed substituting amino acid residues via cloning of PCR-mutagenized and synthesized gene fragments followed by protein expression in E. coli. Purified CIDRα1.3 mutants will be tested for their ability to bind EPCR using Bio-Layer Interferometry. This study demonstrates the potential for this approach to develop PfEMP1-derived peptides that will mimic the shape and binding potential of diverse CIDRα1 domains for use in vaccines to reduce the incidence of CM.
 

Blood clotting after injury requires processing of Von Willebrand Factor (VWF), a large blood protein that self-associates into long fibers which are then broken down by the enzyme ADAMTS13 into smaller fragments. These cleavage fragments self-associate after injury to stabilize clotting factors and activate platelets for clot formation. When the activity of ADAMTS13 is insufficient or inhibited, overactive clot formation leads to thrombotic thrombocytopenic purpura (TTP). VWF fiber formation can be modeled using a microfluidic device that measures the flow of platelet-free plasma through a microscopic channel. My goal is to investigate the properties of cleaved VWF fragments and how they regulate the capacity of ADAMTS13 to further cleave normal VWF. I hypothesize that the presence of cleaved VWF by ADAMTS13 will inhibit further cleavage and produce longer VWF fibers. To test this hypothesis, I will examine VWF self-association in the microfluidic channel - in the presence and absence of the cleaved VWF fragments. The addition of VWF fragments will be expected to inhibit further cleavage of VWF and increase self-association through the microfluidic channel. Understanding how cleavage products from ADAMTS13 breakdown of VWF can regulate self-association of VWF will provide critical insight into causes of TTP and developing novel treatments for patients with this disease.

Protein glutathionylation is a post-translational modification that can take place under mild oxidative stress. It occurs when the antioxidant glutathione (GSH) forms mixed disulfide bridges with low pKa cysteine residues on proteins. This may play a role in reversibly regulating the activity of proteins that have key cysteine residues or protecting those cysteines from further irreversible oxidation if oxidative stress worsens. GSH itself is synthesized in a two-step process, and the rate limiting step is catalyzed by glutamate-cysteine ligase (GCL), a heterodimer composed of the catalytic subunit (GCLC) and modifier subunit (GCLM). GCLM acts to increase the activity of GCLC. Thus, in the absence of GCLM, GCLC is less efficient in making GSH. We have generated Gclm null mice and these mice have liver GSH levels that are about 10% of normal, resulting in an oxidized thiol redox environment. An important question that has not been answered is whether Gclm null mice have different levels protein glutathionylation than Gclm wild-type mice. The oxidized thiol redox status of Gclm null mice is predicted to increase the level of protein glutathionylation. On the other hand, there may in fact be less protein glutathionylation in Gclm null mice because they have less GSH to begin with and there is a compensatory increase in sulfiredoxin and thioredoxin reductase expression, both of which are known to reverse glutathionylation. Using assays of protein glutathionylation, this project seeks to provide confirmation of the hypothesis that Gclm deficiency results in adaptive regulation of glutathionylation under different degrees of oxidative stress.

The transcription factor hypoxia inducible factor-1 (HIF-1) plays a central role in responding to changes in environmental oxygen levels in both mammals and nematodes. HIF-1 stabilization in Caenorhabditis elegans via knockout of the E3 ubiquitin ligase von Hippel-Lindau 1 gene (vhl-1) improves proteostasis and lifespan. In humans, however, stabilization of HIF-1 via loss of VHL results in von Hippel-Lindau syndrome, a disease characterized by angiomas and renal carcinomas. Because it is a transcription factor, HIF-1 activates multiple genes that are likely responsible for its effects – whether they are improved proteostasis and lifespan in worms or neoplastic disease in humans. Previously, my lab performed a screen to determine which genes downstream of hypoxic signaling are responsible for the beneficial effects of HIF-1 stabilization in worms. This screen yielded a list of genes which were subsequently tested for whether they were necessary and/or sufficient to promote health and longevity, leading to the isolation of flavin-containing monooxygenase-2 (FMO-2), as a protein that increases lifespan and improves multiple measures of healthspan in nematodes. My project involves first characterizing the sensitivity of the yeast FMO1 deletion strain (fmo1Δ) to specific forms of toxic stress. Having established these phenotypes, I am performing a screen to find genes that, when transgenically expressed, rescue the effects of the FMO1 deletion. My strategy is to overexpress a library of other yeast genes as well as orthologous FMO genes to isolate those that rescue the fmo1Δ phenotypes. Additionally, I will overexpress FMO1, and any genes of interest from the screen, for analysis in replicative lifespan assays, testing whether they also play a role in increasing yeast longevity. This screen and the associated replicative lifespan assays will help us characterize the role of yeast FMO1 and the conserved role of FMOs in general, furthering our understanding of the process of aging.

Exposure to 17a ethinyl-estradiol (EE2), an endocrine-disrupting compound found commonly in birth control and the environment, impacts both development and reproduction in fish. Although it has been established to also cause aneuploidy in those exposed, the effect of EE2 on disease resistance is not well understood. This study uses the zebrafish model to address the effect of EE2 on fish health. Embryos will have been exposed to three concentrations of EE2 (1, 10, 50 ng/L) and a vehicle control from 10-72 hrs post fertilization. At 72 hrs, embryos will have been infected with Edwardsiella ictaluri (gram negative pathogen of fish) and monitored for differential mortality. In addition, the host immune response (via induction of immune genes) is assessed on day two and three post infection using quantitative RT-PCR. Quantitative RT-PCR is a method where RNA is transcribed to cDNA using reverse transcriptase and then is then used as a template for the PCR reaction. Specifically, we will have assessed the regulation of serum amyloid A1 (SAA1-acute phase response) and the proinflammatory gene interleukin 1 b (IL1B). From previous experiments and test runs, these two genes are expected to be upregulated after the exposure to EE2 and the subsequent infection. Overall, this study addresses the hypothesis that exposure to endocrine disrupting compounds can lead to increased disease susceptibility in fish at the individual and population level.

Cholangiocarcinoma, better known as bile duct cancer, is a rare and aggressive form of cancer in the biliary system. Bile duct cancer has the third lowest 5-year survival rate among all forms of cancer in the US. Due to its location, access to the biliary system for detection or diagnosis is difficult without invasive methods. Early detection of bile duct cancer is crucial and could be achieved by using the multimodal scanning fiber endoscope (SFE) developed in our lab. The SFE is able to noninvasively enter the biliary tree through the digestive system and present realistic information about the potential malignancies or disease state. Typically, a phantom is used to mimic the in-vivo presentation of disease state in order to validate the use of a new medical device. To prepare the SFE for this kind of cancer detection, a bile duct phantom with three dimensional (3D) cell culture was developed to test the sensitivity and specificity of the real-time in-vivo imaging. A biocompatible and optically clear polydimethylsiloxane (PDMS) device was first made for the cast of the hydrogel phantom. The device was utilized for structural stability of the phantom like a cast. The phantom was constructed by folding a flat sheet of hydrogel into a cylindrical tube with a lumen that reflects the true diameter of bile duct. The cancer cells were suspended at specific locations in order for spatial validation of SFE. The end result was the development of a 3D cell culture phantom with fluorescent probes targeted to cancer biomarkers. The phantom was imaged with SFE to evaluate its ability to accurately locate malignancies, distinguishing cancer cells from normal cells. Future work will extend to validation of fluorescent probes and ultimately lead to development of a new cancer imaging method with SFE to reduce late diagnosis.

Methylomicrobium buryatense 5GB1c is a strain of methanotrophic bacteria that serves as an attractive model for production of valuable compounds from methane that can be used in industrial applications. Using this bacteria to transform cheap methane into a commercial commodity is not only a profitable venture, but an effective use of this potent greenhouse gas. This project attempted to genetically manipulate 5GB1 to increase its concentration of cellular lipids, which can be used as precursors to diesel, and introduce new metabolic pathways to produce three stereoisomers of 2,3-butanediol, a compound that can be used in the synthesis of materials such as rubber, plastics, and several others. 5GB1 has a complex membrane structure to support methane oxidation, from which the desired lipids can be utilized in fuel production. By overexpressing five genes previously determined to be involved in high-lipid generating conditions, we expected a greater yield of the product. To accomplish this, direct chromosomal insertion of a genetic construct with a highly active promoter increased transcription of the target genes. After this successful transformation of the cells through homologous recombination, sample cultures were analyzed to determine if the level of lipid production increased. A knockout of the cell-shape determining gene was also made in an attempt to increase lipid concentration; however it was lethal. For 2,3-butanediol biosynthesis, cells were transformed with plasmid constructs containing genes encoding enzymes that are downstream of existing pathways within 5GB1. Three different sets of plasmids were made for the three stereoisomers of the compound, resulting in the production of mostly pure diols. After transformation, 5GB1 was expected to be able to convert methane to the desired products. This process can be scaled up at the industrial level for manufacture and profit.

With 9.5 million new cases of active disease annually and 30% of the world latently infected, tuberculosis (TB) disease is a public health crisis on a massive scale. Despite decades of research, treatment options are still suboptimal, with standard drug regimens ranging from 6-12 months for drug-susceptible cases and up to 24 months in instances of drug resistance. Detection of drug resistance is essential to getting patients the appropriate treatment regimen, but the diagnosis of resistance can take 4-6 weeks using standard culturing approaches. Current TB research is often done with “batch culture” techniques, which are tests done on millions to billions of bacteria and the average response is used to make inferences about individual bacteria. While useful, batch culturing contributes to the lengthy diagnosis of drug resistance and is at odds with the clinical course of TB infections, where such numbers of bacteria are seen in only the most extreme cases of disease. To address this limitation we developed a platform to watch and track single cells responding to a variety of environments. Our platform features a semi-solid growth substrate, brightfield and fluorescent microscopy, and the ability to faithfully track  single cells for more than 5 days. Using our platform we tested TB susceptibility to isoniazid, a common first line TB drug. Benchmarking against batch culture, we found the killing dose of isoniazid to be comparable with our platform; however, unexpectedly, we found that sublethal doses of drug have the opposite effect: they increase bacterial growth rates. In addition to impacting knowledge about the basic biology of TB, our platform has the potential to evolve into a diagnostic test for drug resistance, with the ability to identify drug resistant strains of TB in <4 days.

Freshwater lakes are an important source of the greenhouse gas methane contributing around 6-16% of the total atmospheric methane budget. Methane oxidation in freshwater lakes is the result of different functional guilds of bacteria working together as opposed to just methanotrophs themselves. Microbial communities in methane-fed lake sediment microcosms have not only displayed a clear response to methane by methanotrophs, but also by non-methanotrophic methylotrophs. To obtain a better understanding of the interaction between methanotrophs and methylotrophs, we investigated the role of methanol as a key methane-derived carbon compound enabling stable co-culture formations between a non-methanotrophic methylotroph (Methylotenera mobilis JLW8) and a methanotroph (Methylobacter tundripaludum 31/32). Along with JLW8 wild type, we tested different mutants of Methylotenera with deletions in the methanol oxidation pathway; XoxF mutants 1170, 2048 and 48/70-9. By monitoring the populations of Methylobacter and Methylotenera in the co-cultures with the use of flow cytometry, we find evidence that methane-derived methanol is likely the growth substrate used by Methylotenera in the artificially created methane-fed microcosms. All co-cultures formed stable communities with the exception of the double mutant, which is unable to grow on methanol, indicating that methanol is the key methane-derived component in community establishment. The wild type and both single mutants established co-cultures in similar patterns resulting in somewhat equal distributions of Methylobacter and Methylotenera by the 4th week. Both single mutants, however, showed consistently higher abundances of Methylotenera in comparison to the wild type co-cultures. This data also suggests that both single mutants may be better at oxidizing methanol than the wild type although the mechanisms for this are still unclear. These results provide insight into the growing significance of methanol in facilitating the complex communities invovled in methane oxidation.

The brain's ability to learn and adapt in response to its environment lies in the plasticity of its synaptic connections. Studying these neuroadaptations is complicated by the tremendous heterogeneity of the brain. Simply isolating a portion of the brain and homogenizing it results in a preparation that includes irrelevant cell types like mitochondria and glia, as well as the specific synaptic targets under consideration. Thus, neurobiologists use synaptosome preparations, which are enriched for synaptic particles, to study synaptic proteins. These preparations are the foundation for the study of synaptic neurobiology. While these synaptosomes preps are of great use, they also have limitations in regards to specificity and purity, with non-synaptic neuronal contaminants occupying a large part of the preparation. Beyond the creation of “cleaner” preparations, it would also be desirable to isolate specific cell types or projections (eg. Thalamo-cortical vs. cortico-cortical synapses). To tackle this issue, we created the Fingr-Sare “TAG” construct, which will allow us to physically isolate synapses that express the construct with high specificity. The FINGR insert is a fibronectin intrabody that binds endogenous PSD-95 and localizes the construct to the synapse, while the “TAG” serves as a target for antibody-based magnetic sorting of glutamatergic synapses. Following transfection of neurons in culture, the construct shows correct dendritic spine localization, with the CD4 portion appearing extracellular of the synaptic membrane. We have been able to sort cells using the TAG construct, and future research will focus on isolating pure populations of “tagged” synaptosomes. Overall, the versatility of this construct could provide researchers with the opportunity to customize and pinpoint specific types synapses from the brain in the hopes of advancing future neurobiological research.
 

 Our lab is investigating the process of horizontal transfer of mobile DNA molecules in microbes, focusing on plasmids of bacteria. A plasmid is a circular piece of DNA that replicates separately from the host chromosome and can be passed from one bacterial cell to another via conjugation, allowing advantageous traits to spread among diverse species. My work has focused on the pQil plasmid, which was originally found in pathogen Klebsiella pneumonia, but has been observed to transfer to other bacterial species in human hosts. The pQil plasmid encodes resistance to the carbapenem antibiotics, a class of antibiotics used to combat multi-drug resistant bacterial infections and important in the treatment of bacterial pneumonia. We are characterizing the mobility of the pQil plasmid to create a laboratory model of a multi-plasmid system. We paired pQil with the F plasmid in E. coli to measure the transfer of these plasmids in the laboratory. We mated a plasmid-free recipient with the E. coli donor strain containing pQil, F, or both. The recipient cells were then characterized for the presence of the plasmids. We determined that the frequency of transfer for pQil is increased in the presence of F’42 and that the transfer of F’42 is slightly inhibited by the presence of pQil. Continuing studies will analyze the mechanisms through which bacterial conjugation occurs. Ultimately our work will inform us on how complex microbial systems evolve in natural environments, such as humans infected with pathogenic bacteria. With this information we can begin to study ways to halt plasmid transfer to slow the global spread of antimicrobial resistance.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS), a cancer of endothelial cell origin that commonly presents in immunocompromised individuals. While relatively rare in the United States, KS remains endemic to certain parts of Africa, where it is the most common cancer. Previous research has established that the number of peroxisomes is altered during latent KSHV infection. Peroxisomes are multifunctional cellular organelles involved in a variety of metabolic pathways important to KSHV pathogenesis. We propose to evaluate the cellular mechanism by which KSHV induces peroxisome biogenesis. I hypothesized that KSHV increases the transcription of specific regulatory genes responsible for the increase of peroxisome biogenesis. After mock and KSHV infecting cells, I evaluated gene expression of a known transcription factor, peroxisome proliferator-activated receptor alpha (PPARA), that has been shown to be responsible for peroxisome biogenesis. I used real time PCR to evaluate gene expression of the transcription factor PPARA. Our preliminary data shows that PPARA is increased, suggesting that PPARA regulates the expression of peroxisome associated genes and peroxisome biogenesis. Currently, I am evaluating if other peroxisome genes are increased, including those involved in peroxisome formation and enzyme activity. In addition, I am using small interfering RNA (siRNA) to transiently knock down PPARA. Silencing the expression of PPARA will determine its specific role in peroxisome biogenesis. I will mock and KSHV infect the cells in which PPARA is knocked down and use real time PCR to evaluate peroxisome biogenesis. In the absence of PPARA, I expect that PEX gene expression will be downregulated, which would suggest that PPARA regulates PEX genes at the gene transcription level. These results will help elucidate one of the key pathways involved in KSHV latency, and potentially contribute to the development of novel therapeutic avenues for KS treatment. 


Staphylococcus aureus is a global human pathogen and a major cause of wound infections, food poisoning, and invasive disease. Its success as a pathogen requires virulence factors used to evade the human immune system and subvert host defenses. Protein A is a multifunctional immune evasion factor that inhibits bacterial uptake by immune cells, modulates inflammatory responses, and disrupts development of adaptive immunity. We found that protein A expression decreased when cultures of S. aureus were exposed to the antimicrobial molecule nitric oxide (NO). NO is a reactive radical produced at high levels by macrophages and other host cells during infection. Reverse transcriptase quantitative PCR (RT-qPCR) showed that transcription of the spa gene, encoding protein A, was significantly decreased in the presence of NO. Dose response curves were generated comparing the sensitivity of spa and other genes to NO as well as to hydrogen peroxide (H₂O₂), another host-produced antimicrobial compound. Western blots confirmed that NO inhibition of spa transcription resulted in reduced protein A levels. Through allelic exchange mutagenesis, we mutated multiple spa transcription factors of S. aureus and identified the regulator protein XdrA as a likely NO target. Given that NO modifies cysteine thiols of proteins, we hypothesized that XdrA is S-nitrosylated by NO, leading to inhibition of spa transcription and concurring decrease in protein A. Single cysteine mutants of XdrA (C54S, C73S) have shown partial resistance to NO inhibition. A double cysteine XdrA mutant will be tested to assess the cumulative effect that multiple NO modifications have on spa transcription. This will allow us to understand the molecular mechanism of NO dependent protein A inhibition. Additional experiments demonstrating that NO produced by macrophages inhibits production of an important S. aureus virulence factor would characterize one mechanism by which innate immunity controls bacterial infections.
 

Systemic lupus erythematosus (SLE) is a life-threatening disease that affects over 500,000 individuals in the United States. One in five patients is diagnosed as a child and they are at higher risk for kidney disease, early mortality and infection due to current immunosuppressive medications that serve as the only treatments. The voltage-gated potassium channel Kv1.3 sustains intracellular calcium levels needed for the activation of effector memory T-lymphocytes (TEMs), a cell associated with serious complications of SLE including lupus nephritis (LN). Because Kv1.3 is highly expressed on TEMs, we hypothesize that it could serve as a viable drug target for SLE. Preliminary data suggests that Kv1.3 expression is high on peripheral blood CD4+ (helper) and CD8+ (cytotoxic) TEMs from SLE patients compared to controls, but the inflammatory cells of interest are found within the tissues. In order to test for the expression of Kv1.3 on immune cells active in LN, we are characterizing Kv1.3 expression on cells in urine from pediatric SLE LN patients. Urinary leukocytes were isolated from 16 patients and applied to slides for analysis via multicolor immunofluorescence for the co-expression of Kv1.3 and CD3 (as a marker for T lymphocytes). Kv1.3 expression was observed on CD4+ TEMs verified by flow cytometry analysis. Further evaluation of patient lymphocytes will be performed by immunofluorescence in conjunction with flow cytometry assays showing the dual expression of Kv1.3 and CD3 on TEMs, and other cell types with potential pathogenic roles in LN, including B lymphocytes (CD19), monocytes (CD14) and epithelial cells (cytokeratin). Positive results will indicate the viability of Kv1.3 as a drug target for SLE treatment. This research has great implications for patients with SLE and those with a wide range of autoimmune disorders including rheumatoid arthritis/juvenile idiopathic arthritis, uveitis, psoriasis, and multiple sclerosis.

As our planet warms, organisms that cannot migrate to more suitable habitats will be forced to adapt to the increasing temperatures or face extinction. An important question to address is how the rate of environmental change affects an organism’s chance to adapt. If an environment changes suddenly, then organisms must also adapt rapidly, whereas slower rates of change allow the organism more time to adapt. Microorganisms, such as the virus phi-6, can be utilized to answer this question because they evolve rapidly, allowing us to watch evolution in real time. We evolved phi-6 over 11 weeks to survive high temperatures. In different treatments, the viruses experienced sudden, moderate, or gradual changes in temperature. Sequencing the evolved lineages at two genes revealed multiple mutations. We added the mutations one by one to the ancestor to determine how each mutation affects thermotolerance. We find that each mutation can increase or decrease thermotolerance by a different amount. Interestingly enough, mutations that give the greatest increase in thermotolerance appeared in viruses that experienced slower temperature change. There is also a trend where viruses that experienced slower temperature change had more mutations per lineage than viruses that experienced sudden change in temperature. My results demonstrate that rate is an important parameter when taking adaptation into consideration. If the temperature increases too rapidly, there may be fewer ways for organisms to adapt, and they are unlikely to survive climate change.

A more complete understanding of a protective HIV-specific immune response is critical to designing effective vaccine strategies. Researchers have characterized hundreds of antibodies (Abs) that elicit broad and potent neutralizing activity but much less is known about Abs that mediate FcR-dependent responses, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular virus inhibition (ADCVI) activity. ADCC involves antibody-dependent recruitment of natural killer cells through Fc/FcR-dependent interactions and subsequent lysis of infected cells whereas ADCVI activity includes ADCC but also involves chemokine release and FcR-mediated phagocytosis. Intriguing evidence from the recent RV144 vaccine trial demonstrated a modest 31.2% protection that was subsequently correlated with higher levels of ADCC activity, rendering previously uncharacterized ADCC- and ADCVI-mediating Abs of increased interest. Here we describe 13 HIV-specific Abs identified from an individual who developed a uniquely broad and potent HIV-specific response early following infection. All 13 Abs were tested for neutralizing and ADCC activity and a small subset were tested for ADCVI activity. While only two Abs demonstrated modest neutralizing activity, two Abs demonstrated broad, cross-clade ADCC activity as well as ADCVI activity. Our project aims to firstly evaluate all 13 HIV-specific Abs for ADCVI activity and secondly, to map the binding properties of the mAbs identified to mediate ADCVI. These latter studies will be completed first using a high-throughput binding antibody multiplex assay (BAMA), and secondly through a more refined series of competition-based enzyme-linked immunosorbent assays (ELISAs). Preliminary analysis has indicated that at least one of these 13 Abs mediates ADCVI but neither neutralizing nor ADCC activity and mapping studies suggest this Ab may bind to the gp41 portion of the HIV envelope. Further understanding of the epitope specificities of Abs that mediate ADCVI activity will help us characterize how this individual mounted a broad and potent response early following infection.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi’s sarcoma (KS)—the most common malignancy of AIDS patients worldwide. Because KS is an endothelial cell based cancer, it relies on mechanisms unique to endothelial cells to proliferate. One such way is through expression of vascular endothelial growth factor receptors 1 and 3 (R1 and R3). The goal of my project is to determine the viral mechanisms by which KSHV upregulates these receptors. Because the majority of KSHV-infected cells are in latent phase, I hypothesized that latent genes are responsible for R1 and R3 upregulation. By infecting cells with an adenovirus modified to express only genes within KSHV latency-associated region (KLAR), I was able to confirm that KLAR genes are sufficient to upregulate R1, but not R3. To determine which KLAR genes are required for R1 upregulation, I created lentiviruses to express one viral gene at a time. By infecting cells with these lentiviruses and monitoring R1 mRNA transcript production by PCR, I was able to rule out three of the four genes in KLAR. Using a cell line modified to constituently express the remaining KLAR gene, latency-associated nuclear antigen (LANA), I found mRNA transcript levels for R1 were significantly higher in LANA-expressing cells than in control cells. This suggests LANA is responsible for R1 upregulation. I am currently conducting a luciferase assay to determine if LANA interacts with the R1 promoter to modify R1 expression. To narrow the search for genes involved in R3 upregulation, I will continue to make lentiviruses to test the involvement of viral genes not located in KLAR. I will present current work examining if conditioned media induces R3 to determine if virus induced secreted factors could be responsible for R3 upregulation. My research will contribute to the ongoing search for treatment strategies for KSHV and KS.

Chronic inflammation in human immunodeficiency virus (HIV) infected individuals is associated with increased mortality, even in patients who are virally suppressed by antiretroviral therapy.The goal of this research is to understand how the gastrointestinal (GI) tract is damaged during early HIV/SIV infection. In many infections, neutrophils play an important part during early response to infection because of their microbial killing properties. The role of neutrophils in the establishment of HIV infection and subsequent chronic inflammation has not been examined. Our purpose was to study neutrophil supporting cytokines before and during acute simian immunodeficiency virus (SIV) infection and their potential role in the kinetics of the neutrophil response. We used SIV in the Rhesus macaque model instead of HIV so that we could obtain longitudinal samples very early after infection to observe the neutrophil kinetics. The neutrophil supporting cytokines we looked at were IL-8, IL-17, and G-CSF. We hypothesized neutrophils would decrease during acute SIV infection in peripheral blood in response to the decreasing neutrophil-supporting cytokines. We used Luminex to measure cytokine levels in the blood at each time point, and used Complete Blood Count to get blood neutrophil concentrations. After infection, neutrophils initially increased, but then 14 days post-infection there was a decrease in most of the animals. Prior to the post-infection decrease of neutrophils, we saw a decline of IL-8 levels, which may contribute to neutrophil decline. In contrast to other types of infection, we saw no increase in G-CSF or IL-17. However, we did observe increases in pro-inflammatory cytokines 14 days post-infection, and the pro-inflammatory cytokine IL-15 correlated with neutrophil decrease. These data suggest that reduced neutrophil supporting cytokines and increased pro-inflammatory cytokines may be contributing to the neutrophil decline observed in acute HIV and SIV infection.

Flavin containing monooxygenases (FMO) are a highly conserved and ancient protein family. They have been suggested to have evolved pre-Last Universal Common Ancestor (LUCA) and to be present in all kingdoms of life. FMOs are known as catalysts for detoxification of xenobiotic compounds, and their activity in the nematode Caenorhabditis elegans, animal model par excellence for aging research, has recently been found to modulate stress resistance and aging. However, the mechanisms by which FMOs affect these processes remain vastly undercharacterized. Our project seeks to define the subcellular localization and endogenous substrates of the C. elegans FMO, FMO-2, as well as investigate its role in redox balance and iron homeostasis. To compare fmo-2 knockout worms, FMO-2 overexpressor worms, and wild-type worms, we crossed the three strains into various established reporters, biochemically stained worms for changes in iron and fat, and performed stress epistasis experiments feeding them RNAi. We also assessed subcellular localization via several fluorescent translational fusion reporters including crossing them into organelle-marked worms. With FMOs’ newly uncovered role as longevity- and health-promoting enzymes, characterizing them more fully may lead in time to human therapies making use of these enzymes.

Chlamydia trachomatis is an obligate, intracellular, Gram-negative bacterium that infects approximately 106 million people annually, making it the most common cause of bacterial sexually transmitted infection worldwide. These staggering statistics reflect the substantial lack of scientific understanding regarding the pathogen’s developmental cycle, creating a huge obstacle for effective vaccine development. So far, it is known that after binding and host cell entry, Chlamydia replicates within a vacuole called an inclusion. Once replication is complete, exit occurs by two different pathways, one of which is the lysis pathway. This pathway specifically involves the release of free, infectious bacteria by inclusion and host cell rupture. Results from previous experiments strongly indicate that cysteine proteases serve a crucial role in inclusion membrane degradation central to the lysis pathway late in the developmental cycle (about 48 hours post-infection). My primary research goal is to apply a novel labeling approach called activity-based protein profiling (ABPP) to determine the identity of the specific chlamydial cysteine proteases involved as a critical first step towards determining the molecular mechanisms of the chlamydial lysis exit strategy. ABPP utilizes a specific probe consisting of a reactive group, which covalently binds to the active site of the protease, and a tag, which is usually a fluorophore or affinity label. After extensive optimization of probe concentrations, I exploited this beneficial specificity to isolate successfully labeled cysteine proteases from Chlamydia-infected cells and assess proper probe binding via SDS-PAGE gels. Following isolation and purification, samples will be sent to collaborator Dr. Aaron Wright at Pacific Northwest National Laboratory for mass spectrometry analysis. Successful identification of these proteases will confirm which specific proteases are essential to lysis, enhancing our understanding the Chlamydia lysis pathway and allowing for evaluation of future vaccine targets to reduce the global burden of Chlamydia transmission.
 

The budding yeast, Saccharomyces cerevisiae, has been an invaluable model organism in the study of aging. Yeast cells divide asymmetrically. During division, the “mother” cell retains any accumulated cellular damage and produces a rejuvenated “daughter” cell. Thus, each yeast cell has a finite replicative lifespan defined by the number of daughter cells it produces before senescence. The historical belief in the field of aging has been that the asymmetrical inheritance of damage is absolute; until the last few divisions of a mother cell’s lifespan, daughters are born with a full lifespan potential, completely independent of the mother’s remaining lifespan. In this project we measure the replicative lifespans of daughter cells that budded off from mothers at different ages. We found a loss of daughter lifespan potential and an increase in mother-daughter remaining lifespan correlation in middle-aged mother cells. By tracking the granddaughter lifespans of aged mother cells, we find that this effect is compounded across generations. The implications of this research are to improve our understanding of yeast replication so as to better interpret the data gained from experiments that, as an example, use yeast aging as a model for human cell aging.

 Cyclic diadenosine monophosphate (c-di-AMP) is an essential nucleotide for bacterial virulence. C-di-AMP has been extensively studied in Listeria monocytogenes (Lm), but is also produced by other pathogens including E. coli, C. trachomatis, and M. tuberculosis. Although c-di-AMP is a signaling molecule in bacteria, host cells detect it as a pathogen-associated molecular pattern. During infection, the host receptor STING senses c-di-AMP and stimulates expression of interferon-beta, an anti-viral molecule. To date, STING is the only known sensor of c-di-AMP, and its promotion of an anti-viral versus anti-bacterial response has been somewhat perplexing. However, our lab has recently discovered another host c-di-AMP sensor, the murine aldo-keto reductase1C13 (AKR1C13) or “RECON”, that induces an alternative, anti-bacterial cellular response to infection. Based on the importance of bacterial clearance and the ubiquity of AKRs across species, we began to investigate whether other murine and human AKRs respond similarly to c-di-AMP. Sequence alignment of AKR genes and data on tissue-specific expression of these genes returned six candidates: the murine AKR1C12 as a potential paralogue, and human AKRs 1C1, 1C2, 1C3, and 1C4 as potential homologues. Cloning techniques allowed me to insert these genes into an E. coli expression vector that I used to purify recombinant protein via affinity chromatography. The binding affinity with c-di-AMP of each protein was tested with radio-ligand binding assays to determine dissociation constants (K_d) and we additionally used enzyme activity assays to quantify inhibition of protein activity. AKRs that bind c-di-AMP will be further characterized in vivo in mammalian cell lines in which expression has been knocked down via lentiviral transduction. Further work in cell lines may elucidate novel pathways that are activated downstream of c-di-AMP detection. Understanding the pathways that naturally clear infections is important to potential medical advances in bacterial infection management and prevention.

Coordination of the initiation of flowering with the optimum season is important for plants in both natural and agricultural systems. In the model plant, Arabidopsis thaliana, timing of flowering is strongly regulated by photoperiod. The main factors important for flowering are CONSTANS (CO) protein, which is an activator of flowering in response to photoperiod and FLOWERING LOCUS T (FT), the direct target of CO. FT protein is a mobile factor which can initiate flowering. Additionally there is a family of repressor transcription factors, the CYCLING DOF FACTOR (CDF) transcription factors that are able to limit the timing of both CO and FT expression so that flowering is repressed during short days. Functionally, CDF expression is controlled by the circadian clock so that their effect is constrained to the morning period of the day. In the afternoon of long days, CDF protein is degraded, enabling CO and FT to promote flowering. Recently, our laboratory has determined that CDFs may require a co-repressor protein, TOPLESS (TPL) to form a repressor complex to regulate flowering genes. To explore the importance of this repressor complex on flowering time, I observed how FT, CO, and other flowering genes expression levels change over the course of development in several genetic backgrounds. To remove TPL function where flowering time genes are expressed, we created a transgenic plant that expresses a dominant negative mutant of TPL in phloem companion cells. We hypothesized that this mutant would resemble a CDF loss of function, so I compared these lines and wild type plants over developmental time. Additionally, I am investigating the genetic interactions and epistasis of TPL with other flowering time pathway components by checking the flowering time and gene expression of transgenic plants which express mutated TPL and harbor mutations in other flowering time genes.

In the past few years waterborne pathogens have caused many outbreaks of disease after natural disasters. Water flux is influenced by climate change, so it will also have an effect on the epidemiology of these diseases. This means that diseases that are endemic in certain regions might migrate to other regions as the climate changes. This literature review focused on research pertinent to constructing a sanitation system that would, after a natural disaster, be feasibly built by the affected population. We explored existing sanitation system designs which can be easily built by the lay population in a limited time frame, without the need for outside help. Such a sanitation system would prevent waterborne diseases that are known to cause disease outbreaks, and be sturdy enough to withstand other catastrophes. We also presented other generalized models for sanitation systems which may be transposed in any other region with similar topography and climate. It was found that climate change will become increasingly devastating to populations around the world. Sanitation systems, while a better investment, would require more infrastructures which are not always possible in developing countries. It was determined that if an affected population is to avoid outbreaks, arborloos and urine diverting toilets were the simplest solutions, both economically and with regard to ease of construction, especially in developing countries. This literature review used several disciplinary lenses including economics, engineering, microbiology, health, geography, climate change, and finally the epidemiological lens. This project explored research in academic journals and other professional publications with keywords such as “sanitation system” and “waterborne infection.”

In 2012, the World Health Organization (WHO) estimated that in low resource countries 4 in 10 deaths among children under the age of 15 were associated with infectious disease. Point-of-care diagnostic devices that can quickly and effectively identify the source of the infection will drastically improve patient treatment times increasing rates of survival. We therefore investigated whether traditional RNA handling techniques used in a laboratory setting are necessary for these point-of-care devices, by assessing the stability of RNA under various conditions. Using respiratory syncytial virus (RSV) as an example, we discovered that RSV can be thermally lysed without significantly degrading the integrity of the RNA. In addition, the use of RNase inhibitors or tRNA does not have a statistically significant effect on sample integrity. A comparison between RSV RNA derived directly from patient nasal swabs, with swabs containing purified RSV RNA, shows that RSV virions protect their RNA prior to lysis. Amplification by qRT-PCR, indicates that RSV RNA is directly amplifiable suggesting that RNases are inactivated at 95°C, and do not regain activity prior to amplification. Our data provides the necessary framework for performing point-of-care testing without the need for traditional RNA handling procedures. The results have important implications since we expect that the research concerning RNA handling and integrity will translate to other RNA based viruses which include Ebola haemorrhagic fever, influenza, hepatitis C, West Nile fever, polio, and measles. With recognition of these capabilities, we can implement minimal RNA sample processing into point-of-care devices.

The study of crop foods, such as maize and corn, offer us important insights on how to create healthier and more abundant crop yields. The initiation and maintenance of callus tissue cultures is a process essential for researching transgenic plants and is constantly plagued by contamination. The media components can influence the formation of callus, shoot, or root tissue as well as culture longevity and general health. The goal of this project is to find an optimal medium for callus formation that contains anti-microbials in order to avoid contamination and promote callus, rather than shoot or root, production. The monocots used are rice and maize. The base medium used consists of Chu N6 Basal salts, 0.7% phytablend agar, and 3% various sugars. I grew tissues on the base media with varying amounts of hormones, additives, anti-microbials, and sugar blends and assessed callus, root, and shoot development as well as the presence or absence of tissue necrosis and microbial contamination. Various tissue cleaning procedures were also tested. I hypothesize that rinsing tissues in antibiotics before plating them would decrease initial contamination and that antibiotic plates could be used to eliminate contamination from already growing cultures. The presence of various hormones and chemicals, such as 2,4-D and silver nitrate, influenced tissue differentiation when applied at certain times. The use of anti-microbials in the washing process as well as in the initial cultures has eliminated most of the contamination issues. It appears that the use of an initial growth medium that promotes callus development and a long-term growth medium to keep cultures healthy will be the key to achieving long-lived callus cultures. The ability to create and maintain these cultures allows the study of transgenic plants and their DNA.

Aging is characterized by time-dependent deterioration of cellular and physiological functions. Due to the numerous challenges and limitations of studying aging in humans, model organisms are used to further extend our understanding of basic mechanisms of aging. The Kaeberlein laboratory uses the single celled budding yeast Saccharomyces cerevisiae as one such model. Advantages of this model organism include the ability to measure the lifespan of great numbers of organisms under various environmental conditions. One method of measuring a yeast cell’s longevity is by assaying its replicative lifespan (RLS). The RLS model refers to the number of daughter cells that can be produced by a mother cell before senescence. The number of budded daughters can be determined by micro-dissection with fiber optic needles from mother cells after each round of incubation. This process is repeated until mother cells no longer generate daughters. Approximately 600 mother cells are assessed weekly and incubation cycles last one month. Yeast lifespan can be manipulated through genetic changes, altering media content, addition of pharmacological agents, and change of temperature. Data collected is compared to control groups and to past and expected results. These results clarify how senescence is regulated. RLS is thought to be a better representation of senescence in mitotically active cells, like certain tissues in multicellular organisms. By analyzing thousands of cells, a variety of genes and environmental conditions that impact aging have been identified. These genes and interventions can be further studied and applied to other organisms. Studying budding yeast enhances our understanding of the molecular basis of aging.​

Escherichia coli is a major causative agent of extra-intestinal infections and highly clonal. Our goal was to use the newly developed septatyping method to determine the clonal structure of E. coli population in routinely collected urine samples from a clinical microbiology lab, evaluate the patterns of antibiotic resistance of individual clones, their temporal stability and prevalence in samples with clinically significant load. Isolated E. coli were assigned a clonotype using septatyping, which is a quantitative real-time PCR test consisting of seven reactions that identify the absence or presence of clone-specific SNP’s in two loci – fumC and fimH. The eighth reaction is a reference control, detecting E. coli specific loci, uidA. We identified 40 (out of 128 possible) clonotypes from 647 E. coli isolates. The most prevalent was the 561 clonotype, aka H30 clone (114 isolates, 17.6%), followed by clones 571, 760, 620 and 271 (7-8% of isolates per clone). Clonal structure of E. coli remained relatively stable over the 3-year period (less than 2% difference). Furthermore, clones varied significantly in their antibiotic susceptibility profiles. For example, 561 (H30) is a highly multi-drug resistant clone, whereas 760 and 530 are highly susceptible to all tested antibiotics. Such differential susceptibility can be extremely useful for choosing the proper course of treatment for the patient. When we compared the clonal structure of E. coli isolated from samples with low bacterial load (<10⁴ cfu/ml, usually discarded as contamination) and high load (≥10⁴, clinically significant), most of the clonotypes were evenly split between the two groups. The fact that the same clones can grow both to low and to high numbers in patients’ urine indicates that the cutoff of 10⁴ cfu/ml may need revision as a clinically significant level.

A neuropeptide called corticotropin-releasing factor (CRF) is shown to be necessary in fear behavior, but it is currently unknown whether upregulated CRF expression in the central amygdala is sufficient for enhancing fear behavior. An increase in CRF expression in a controlled manner will specifically test CRF's ability to enhance fear learning. To achieve this, a Cas9-CIBN complex will be directed towards CRF promoter by small-guiding RNAs where it will recruit the Cry-Vp64 complex. When these two complexes come together, they form a light-inducible transcriptional effector (LITE) that can recruit the transcriptional machinery to the CRF promoter and up-regulate CRF expression in the presence of light. LITE machinery will also provide a method of improved spatial and temporal control to the upregulation compared to currently used methods of upregulation. However, in order to accurately analyze this uniquely controlled method, certain comparable controls are required. This project will serve as the comparison to temporally controlled CRF expression by creating a constitutive CRF overexpression vector. This will upregulate CRF without temporal control and cannot be switched “off”. This continuous effect may elicit compensatory changes to the system over time and reveal important differences between constitutive and temporally controlled CRF expression. The three objectives of this project are to 1) clone the CRF gene into a DNA vector called adeno-associated virus vector (AAV vector), 2) package the AAV-CRF vector into virus, and 3) inject the packaged virus into central amygdala for behavioral analysis. The first objective has been achieved using a standard cloning protocol. The second and third objectives are still in the preliminary phase.

Escherichia coli (E. coli) is the major causative agent of urinary tract infections (UTI) and is highly clonal. One of the major clones is a multidrug-resistant H30 clone, which has spread worldwide and is almost exclusively resistant to fluoroquinolones, accounting for 50% of all ciprofloxacin-resistant uropathogenic E. coli. The goal of this study was to determine if H30 clone is prevalent among fecal E. coli, which might explain its high occurrence in extraintestinal infections. We obtained 555 fecal samples from female volunteers and screened them for the H30 clone using a multiplex quantitative PCR test, ST200. This test contains primers for uidA (E. coli specific loci), and primer-probe combinations for ST131, ST69, and fimH30. The combination of positive ST131 and fimH30 probes identifies the H30 clone. The results of fecal screenings were compared to the data on H30 found among urine isolates from women with UTIs. Out of 555 fecal samples, 531 (96%) had detectable presence of E. coli ranging from 10⁴ to 10⁷ cfu/ml. H30 clone was found only in 25/555 samples (4.5%), while it was significantly more prevalent in urine (7.7%, P=0.008). The prevalence of H30 clone in younger (<50 years old) versus older (>50 years old) women didn’t differ significantly for fecal samples (6.1% vs 8.1%, P=0.24). However H30 was significantly more prevalent in older women’s urine samples (5.9% vs 8.6 %, P=0.014). Overall, 44/555 (7.9 %) fecal samples contained ciprofloxacin-resistant isolates. Similarly to urine, H30 clone accounted for almost half of isolates (21/44, 47.7 %),  although resistance of fecal H30 isolates to fluorquinolones was slightly decreased in comparison to urine H30 isolates (84% vs 95%). In summary, the H30 clone preferentially causes UTIs in elderly women and is the major reservoir of ciprofloxacin-resistant E. coli in gut microflora, potentially leading to higher infection rates due to mismatched antibiotic therapy.

The goal of this project is to better understand the functions of symbiotic bacteria that live in earthworm kidneys. This project will be focusing on the composting earthworm, Eisenia fetida. Earthworms play an important role in soil ecosystems, breaking down organic matter, aerating the soil, and enriching soil with nutrients beneficial for plants. Many earthworms harbor bacterial symbionts in their osmoregulatory excretory organs, the nephridia, which function like kidneys to manage salt balance and process waste. Two nephridia occur in each segment of the worm (~ 200 nephridia total) with each containing symbiotic bacteria. Eisenia fetida has three species of bacteria that live within their nephridia: Verminephrobacter eiseniae, a Cytophagales member, and an Actinobacteria. These bacteria are passed into egg capsules from the mother to the next generation. The function of these bacteria is unknown. This project is testing the hypothesis that these bacteria are important for supplementing the diet of the worm. To test this, worms are being cured of their symbionts by treating them with antibiotics during early development to prevent colonization. A colony of cured worms has been established and control and erythromycin-treated stocks will be fed various diets to test their ability to handle a lack of nutrients. Their mortality and fertility will be observed to determine the contribution of the nephridial bacteria to the success of E. fetida. We expect that treated worms will have higher mortality than the control group. 

Eukaryotic cells are constantly locked in genetic conflict with selfish elements. These elements, such as transposons, are detrimental to the host, but rely on the host for their own replication and passage to host progeny. This results in selective pressure on the host to evolve mechanisms to restrict these genetic parasites, and selective pressure on the parasite to evade those mechanisms to survive. I hypothesize that these elements could be a driving factor in the evolution of the host and parasite. One example of the host and selfish element relationship is in yeast and its 2 micron plasmid. The 2-micron plasmid confers a 1-3% growth rate defect to the yeast while utilizing host machinery, making it a selfish element and a source of genetic conflict. The plasmid is naturally occurring in most yeast strains, making this model a currently occurring and natural model of study, as well as having a wide array of genetic tools already developed for this organism. In order to understand how the plasmid is shaping evolution in different yeast strains, we need to first fully understand how it is utilizing host machinery. This allows us to identify genetic changes due to pressure from the plasmid versus other factors. We have screened a yeast deletion collection where non-essential yeast genes were knocked out one by one. DNA was extracted from each strain and checked strains for decrease in plasmid copy number using southern dot blots and/or PCR. In future work, we will then check each candidate gene using bioinformatics tools to look for evidence of positive selection as evidence of an evolutionary arms race between the yeast host and plasmid. This will allow us to form secondary hypotheses about the effects of the plasmid on yeast genome evolution.

While HIV treatment is improving, there are still problems associated with it. Antiretroviral therapy (ART) has unfavorable side effects, is expensive, and requires a lifelong daily pill intake. However, it has been proposed that a therapeutic vaccine could provide durable control of viremia in the absence of ART. This is a difficult approach due to the high mutation rate of the virus. Research has shown that there are conserved elements (CE) within the virus that do not change because they are important for the viral fitness. Accordingly a therapeutic DNA vaccine is being investigated that targets the CE epitopes. To address this, rhesus macaques were infected with SIV (Simian Immunodeficiency Virus), put on ART, vaccinated with four doses of DNA vaccine expressing CE, removed from ART, and their viral loads monitored. A successful vaccine is expected to reduce the viral loads to negligible amounts post ART cessation. My research project investigates the role of CE responses developed during acute infection. I hypothesize that macaques that naturally develop higher CD4+ and CD8+ T cells (types of white blood cells) with CE specificity in response to the acute SIV infection will respond better to ART and CE vaccinations. To investigate this I collected plasma and lymphocytes from the blood during acute infection and analyzed the CD4+ and CD8+ T cells using enzyme linked immunospot (ELISpot) and intracellular cytokine stain (ICS) assays. The viral loads were monitored by measuring the concentration of viral RNA in the plasma. T cell responses measured at each time point was then correlated to viral loads post ART-pre vaccinations or to CE responses measured after the final DNA vaccination by Spearmann Rank correlative test. My findings will help us better understand the significance of conserved elements in controlling the HIV and thus design better human therapeutic vaccines.