In their books Pihkal and Tihkal, Dr. Alexander and Ann Shulgin describe their experiences ingesting 234 psychoactive compounds, most of them newly synthesized by Dr. Shulgin. The goal of this project is to use natural language processing techniques to map the semantic space of the Shulgins’ qualitative comments and determine what commonalities, if any, exist between compounds of similar molecular structures. I first created a TF-IDF matrix to determine the importance of each word for each compound. I then applied three clustering techniques (k-means, DBSCAN, and affinity propagation) to group compounds based on their meaning and used UMAP after each technique to graph the clusters in two dimensions. Unfortunately, despite attempting different combinations of pre/post processing and hyperparameter tuning, each method resulted in only weakly associated clusters. My next analytical method is using Sentence-BERT modeling to compare the semantic meanings at the sentence level. Since sentences hold more meaning than single words, I anticipate that this technique will differentiate the compounds to a greater extent, therefore leading to more visibly divided clusters. I also have the compounds clustered by similarity in molecular structure and determined the most common words associated within each group. By quantifying the subjective experiences of these psychoactive compounds and mapping them to molecular structures, this knowledge could allow us to synthesize molecules to obtain a desired effect on a patient’s consciousness. This could in turn aid in synthesizing new medications to treat mental health disorders.

Hemophilia A (HemA)—a severe genetic bleeding disorder affecting 1 in 10,000 people—is caused by mutations in the F8 gene. These mutations cause an inability to produce the coagulation factor eight protein (FVIII) necessary to stop bleeding after a wound. Current treatment- repeated FVIII replacement, is costly and frequently ineffective, as around 30% of patients develop inhibitor antibodies causing the immune system to reject the foreign protein. Alternatively, our lab hopes to utilize gene therapy to restore the functional gene and allow the body to continue producing the essential FVIII protein itself. 45% of human HemA cases are caused by a mutation of the human F8 gene where a large portion called Intron 22 (In22) is inverted. The In22 inversion halts translation of the rest of the gene, and the resulting FVIII protein is truncated and non-functional. To address the mutation, our lab aims to use a CRISPR-based knock-in approach to the DNA following In22, upstream of the mutation site. We expect that this strategy can restore endogenous production of missing FVIII and potentially provide curative treatment for affected patients. To test this treatment’s efficacy, this project utilizes a HemA mouse model (E16) in which a neo cassette insertion at the 3’ end of exon 16 disrupts FVIII expression. We propose using the same strategy to integrate the missing DNA upstream of the mutation and restore FVIII function in HemA mice. I use molecular cloning to construct and evaluate different versions of the CRISPR-Cas9 plasmid containing sgRNA, and a plasmid containing the donor DNA. This research allows us to determine the safety and efficacy of our gene therapy strategy, and evaluate how to maximize recovery of FVIII production. This project aims to eventually contribute to treatment of human HemA patients, without the expensive and unreliable replacement of the protein.

Protein kinases have been found to regulate cellular processes such as growth and stress response. Thus, they act as excellent targets for drug treatment. The Mycobacterium tuberculosis (Mtb) genome encodes 11 serine/threonine protein kinases. Our lab has previously found that two of these kinases, PknF and PknL, show a large survival deficit when induced. They phosphorylate similarly throughout central carbon metabolism (CCM), a process known to be involved in cellular survival. To test kinase regulation of different pathways in CCM, I tested the growth of avirulent Mycobacterium tuberculosis (aMtb) strains expressing PknF or PknL using two different carbon sources: propionate and succinate. Propionate is broken down into propionyl-CoA, a toxic co-intermediate, which passes through the methylmalonyl or methylcitrate pathway to enter the citric acid cycle at succinate. The methylmalonyl pathway requires vitamin B12 to proceed and prevent toxic propionyl-CoA build up. Thus, propionate + B12 was tested to further elucidate regulation of these pathways. I measured colony-forming units (CFU) to quantify aMtb survival in these growth conditions. I compared survival measurements of the PknF and PknL induced strains relative to an empty vector control strain. I found that PknF induced grown with propionate showed a greater survival deficit by day 7 compared to the strain grown in succinate. Interestingly, the addition of B12 did not rescue growth as it did in the empty vector control. PknL induced grown with propionate shows a greater survival deficit compared to succinate; however, the addition of B12 decreased the survival deficit experienced in propionate. Due to this difference between B12 phenotypes, we hypothesize that PknF induction is regulating the methylmalonyl pathway, resulting in no rescue of the survival deficit. These findings can be used to inform future studies on PknF and PknL as potential targets for tuberculosis treatment during infection.

Neonatal tracheal intubation (TI) is a high-risk procedure requiring careful coordination to minimize complications and improve outcomes. The Personalized Intubation Neonate Safety (PINS) Bundle was developed to enhance team communication and optimize intubation practices for neonates at risk. The bundle incorporates five key domains: patient risk assessment, treatment threshold for intubation, premedication plan, equipment specification, and provider selection with escalation strategies. At the University of Washington Neonatal Intensive Care Unit, eligibility criteria for the PINS bundle include neonates requiring non-invasive respiratory support with >30% FiO₂, those meeting surfactant administration criteria (≥30% FiO₂ and PEEP 6 on NCPAP), already intubated and mechanically ventilated infants, those with difficult airway diagnoses (e.g., craniofacial anomalies, large tongue, micro/retrognathia), and extremely preterm infants (<1000g or <4 weeks old). We will evaluate the impact of PINS implementation by comparing intubation attempts and adverse events before (January – June 2024) and after (July – December 2024) the bundle’s introduction. Data was adjusted to exclude ineligible patients, ensuring comparable cohorts. Primary outcomes include the number of intubation attempts before success and the incidence of tracheal intubation adverse events (TIAEs). We hypothesize that PINS implementation will be associated with a reduction in intubation attempts and TIAEs, reflecting improved preparation and procedural success by the healthcare team. By standardizing an individualized pre-intubation plan, the PINS bundle aims to enhance neonatal safety and streamline team response during intubation. Findings from this study will inform future clinical protocols and may support the broader adoption of personalized intubation strategies in the NICU. Further assessment of long-term outcomes for infants will strengthen the bundle’s clinical utility.

The first-in-class capsid (CA) binding antiretroviral, Lenacapavir (Len), inhibits viral spread at multiple steps in the viral life cycle. Structural studies show that Len interacts with an FG-binding pocket between the N-terminal and C-terminal domains of adjacent CA monomers resulting in destabilization of the CA core lattice. Three key binding functional groups within Len that interact with CA were identified. Subsequently, six Len analogues (Lenalogs) were designed and synthesized. These Lenalogs vary by the removal or replacement of one of the identified functional groups. My work investigates the impact of Len and Lenalog binding on CA assembly rate, as well as, the structure of the assembled protein. Using an IPTG  E. coli expression system and ion exchange chromatography, I have expressed and purified CA protein. I induced in vitro assembly of the purified CA protein by the addition of inositol hexakisphosphate (IP6) in both the presence and absence of Len or the Lenalogs. Relative to Len, LL-10.4 and LL-15 promoted assembly, LL-14 was similar, while LL-11, LL-19 and LL-20 promoted assembly to a lesser extent. Samples with LL-10.4 and LL-15 were chosen for cryo-EM analysis as these promoted assembly to a greatest extent. CA was assembled on lipid vesicles (templated CA-like particles or CLPs) by the Dick lab (Emory University, Atlanta, GA), and these were subjected to cryo-EM data collection and analysis. Both LL-10.4 and LL-15 bound to the FG-binding pocket like Len. Negative stain transmission electron microscopy and light scattering will be used to further assess the effect of Len and Lenalogs on assembly kinetics. My work will be used to inform the design of next generation CA-targeting antiretrovirals.

Traumatic brain injury (TBI) results from a blow to the skull that causes shearing forces in the brain. Elevating intracranial pressure (ICP) at the moment of impact may protect the brain from TBI by stiffening the brain tissue and decreasing shearing. When they expect an impact, humans naturally brace and perform a Valsalva maneuver (exhaling against a closed airway), which momentarily elevates ICP. In a ferret TBI model, we conducted abdominal compression using a blood pressure cuff to induce a Valsalva-like response (VLR) and determine whether VLR resulted in neuroprotection. The ferret model was chosen for its gyrified brain structure and white to grey matter ratio that closely resembles the human. TBI was induced using a CHIMERA (Closed-Head Impact Model of Engineered Rotational Acceleration) device, which is designed to deliver high-energy, controlled skull impacts. Initial work showed that the abdominal compression procedures increased ICP. The TBI study involved a total of 36 adult ferrets of both sexes randomized into three groups: (1) a sham control group exposed to isoflurane with a cuff but no compression, (2) a TBI group with a cuff but no compression, and (3) a TBI group with a cuff and abdominal compression. Baseline behavioral assessments (CatWalk, Novel Object Recognition, Swim Test, and Open Field) were conducted one week prior to injury. Post-injury behavioral testing, using the same assessments, was performed at 24–48 hours and 8 days post-TBI to evaluate functional outcomes. On day 8, ferrets were euthanized, and their brain tissue was collected and assessed for neuropathological outcomes. We hypothesize that abdominal compression will mitigate deleterious TBI outcomes. If these findings are supported, this intervention could improve the lives of those at risk of TBI and contribute to ongoing research in the field.

During neonatal transport, specialized pediatric transport teams closely monitor the status of critically ill newborns. Hyperspectral imaging, a method of manipulating light for medical imaging, can be used for remote monitoring using video of the patient’s physical appearance and to measure vital signs. Appropriate light intensity is critical for clear visibility of the newborn and hyperspectral imaging accuracy, but this must be balanced with safety for sensitive eyes. My previous studies have determined the minimum range of light needed to accurately view the neonate in a transport incubator. My current research is focused on developing a novel method for vital sign analysis by using hyperspectral imaging. A smartphone camera will be used to take RGB photos of a calibration chart and a short video of the wrist of study participants. After taking the RGB photographs, custom MATLAB code will be used to extract physiological data such as hemoglobin and bilirubin content from the skin. Data analysis will compare the vital sign data collected using hyperspectral imaging and using a pulse oximeter to understand the feasibility of hyperspectral imaging for vital sign extraction. The expected result of this study is that the heart rate and blood oxygen levels measured using light and a pulse oximeter will be highly correlated. In conclusion, this research will demonstrate the potential application of hyperspectral imaging to pediatric transport.

Malaria, caused by Plasmodium parasites and transmitted via mosquito bite, caused over 600,000 deaths in 2022, making the disease a pertinent public health problem. After injection into mammalian hosts through mosquito bite, Plasmodium parasites travel into the liver and develop in hepatocytes, where they undergo massive replication but cause no symptomatic disease. The parasites then egress into the bloodstream, where they infect red blood cells and cause the clinical symptoms and mortality associated with malaria, along with transmission to mosquitos to continue the cycle of infection. Although the liver-stage of the parasite is clinically silent, parasite infection of the liver results in incompletely understood hepatic immune responses that impact the development of immune memory, which is necessary for protection from future infections. Innate-like αβ and γδ T cells make up a significant proportion of intrahepatic lymphocytes, leading us to become interested in how these immune cells respond to a primary Plasmodium parasite infection of the liver. To address the role of these T cells in combating a primary liver-stage infection, we infected wildtype mice, mice that lack αβ T cells, and mice that lack γδ T cells with Plasmodium parasites and examined parasite density, size, and hepatic localization using immunofluorescence microscopy. Preliminary results demonstrate no significant differences in malaria parasite susceptibility between wildtype mice, mice without αβ T cells, and mice without γδ T cells, indicating that these cell types alone may not mount a significant anti-Plasmodium response upon primary infection. Future work will involve examining T cell localization within infected tissues to determine how T cell localization is impacted by primary infection and characterizing subsets of T cells that are present in infected livers. We hope these results add to a greater understanding of the entire hepatic immune response to primary Plasmodium parasite infection of the liver.

Neonatal hypoxic-ischemic encephalopathy (HIE) occurs when the brain receives insufficient oxygen and blood supply before or during childbirth. HIE is a leading cause of neonatal mortality and morbidity that may also affect later brain development, specifically gyrification - folding of the cerebral cortex creating gyri and sulci. The nonhuman primate (NHP) brain is gyrified, similar to humans, making NHPs a highly translatable model to examine brain development after injury, which has not been well-studied in HIE. In our nonhuman primate (NHP) model of neonatal HIE, we induced injury through in utero umbilical cord occlusion (UCO) for 20 minutes, mimicking the cause of HIE in humans. Twenty-two term-equivalent pigtailed macaques (Macaca nemestrina) underwent UCO and were randomized to no treatment (n = 11) or treatment with therapeutic hypothermia and erythropoietin (TH + Epo [5x1000 U/kg]; n = 11), while non-UCO animals served as controls (n = 7). All animals were delivered via cesarian section. Injury severity was determined by physiological parameters (Apgar score), lactate, and pH levels after resuscitation. To evaluate the impact of injury on gyrification, we will utilize magnetic resonance imaging (MRI) taken 6-months post-injury to measure the gyrification index (GI). GI will be calculated by taking brain’s inner-to-outer hemispheric ratio; the inner trace following the contours of the gyri and sulci, and the outer trace following the circumference of the cerebral cortex. We hypothesize that global and regional GI will be altered in animals exposed to UCO, corresponding with decreased brain volume and greater injury. We also hypothesize that treatment will mitigate some of these changes, leading to a GI closer to control. These results will help determine whether hypoxia-ischemia alters the trajectory of cortical development, as well as the association between injury severity, brain volume, and gyrification.

Cancer immunotherapy, specifically Adoptive Cell Therapy (ACT), has revolutionized treatment approaches using genetically modified T cells to recognize and eliminate cancer cells. However, tumors combat this by creating an immunosuppressive tumor microenvironment (TME) blocking effective antitumor immune responses.  Dendritic cells (DCs) are innate immune cells that act as messengers between the innate and adaptive systems.   In the Oda lab we have designed Dual Costimulatory Receptor (DCRs) that combine a FLT3L or CD40L ectodomain with different costimulatory endodomains (e.g. CD40, 4-1BB, OX40), to provide both T cell-extrinsic and -intrinsic costimulatory signals.  These DCRs are expressed on the surface of antigen specific T cells, and the combination of these signals allows for enhanced tumor antigen presentation and dendritic cell activation, leading to an increase of the immune response to target and destroy cancer tumors. I will investigate how incorporating DCRs on T cells will enhance ACT effectiveness. I hypothesize these DCR signals on T cells will enhance dendritic cell function in the TME, allowing for increased T cell activation and antitumor immune responses. To test this, I will conduct in vitro coculture experiments to determine how DCR-T cells, dendritic cells, and pancreatic cancer cells interact together. I will study the interactions of these immune cells using live cell imaging technology such as the Incucyte. Additionally, I will analyze the phenotypes of our distinct cell populations via flow cytometry.  This research aims to enhance the development of immunotherapy for Pancreatic and all solid cancers by improving the recognition of cancer cells from the immune system. These results could help pave the way for improving solid tumor cancer treatment.

Background: Premature infants or those with hypoxic-ischemic encephalopathy are at high-risk for cerebral palsy (CP). Early detection of CP can significantly improve outcomes, however, inconsistent attendance at developmental follow-up after UW NICU discharge puts infants at risk of missed diagnoses. Objective: Implementing a process for early detection of CP in the NICU to increase high-risk infant follow-up (HRIF) rates at 3–4 months from 51% to 80% within 10 months. Design/methods: A single-center Quality Improvement initiative. Baseline data included patient characteristics, comorbidities, and CP risk factors. Process mapping and stakeholder meetings began June 2024, informing changes implemented beginning in August 2024. Process measures included % of eligible infants receiving a General Movements Assessment (GMA) and % of infants with a 3-4-month follow-up scheduled before NICU discharge. Outcome measures include % of infants seen at HRIF by 3-4 months corrected gestational age (cGA) and number diagnosed with High Risk for CP or CP before 12 months. Balancing measures addressed % of clinic No-Shows or appointment cancellations. Results: At baseline, 67 infants, discharged from UW NICU between Dec 2024 and July 2024 and met criteria for HRIF; 66 (99%) were very premature, GA ≤ 32 weeks and/or birthweight ≤1500g, 3(5%) were extremely premature, GA 22 to 24 weeks 6 days. 65(97%) infants had a GMA before discharge. No infants had cramped synchronized movements. 97% received referral for HRIF and none were scheduled for a 3–4-month appointment before NICU discharge. Monthly follow-up rate (number of infants seen at 3-4 months cGA / number of eligible infants discharged per month) was 51%. By January 2025, 57/62 (92%) eligible infants were scheduled for HRIF before NICU discharge. Process changes are ongoing. Conclusion: A standardized pathway for high-risk infants in the NICU can improve local follow-up rates, enabling earlier CP detection and intervention.

During embryonic development, gene expression is temporally and spatially coordinated to control organogenesis and fetal growth. We previously identified a subset of 140 genes that conferred lethal and sub-viable phenotypes in mice and are likely to be haploinsufficient in humans. These genes presumptively play essential roles in fetal development, but their function is unknown. I aim to uncover the role of these genes in mouse embryonic development using Weighted Gene Co-Expression Analysis (WGCNA). Co-expression analysis will be conducted on mouse embryonic stem cell RNA sequencing data obtained at three different stages of in vitro differentiation and across two different genetic backgrounds, creating a subset of nine samples encompassing 12555 genes. Choosing three different time points allows us to see how expression of our genes of interest changes over time, and choosing two different genotypes (wild type and knock-in) allows us to investigate if expression changes due to a single point mutation. We performed dynamic clustering on this RNA sequencing data to identify co-expressed gene clusters. I will map these gene clusters to biological pathways to make inferences about which cellular processes, metabolic functions, or structural components the genes of interest are involved in. This may indicate the role of these genes in fetal development and help reveal why fetal viability is compromised. In future studies, the functional characterization of these genes will generate new ideas and hypotheses about the basis of genetic disease.

Currently, nearly 10% of Americans have type 2 diabetes (T2D), placing it among the most common chronic diseases in the United States.  Growing evidence points to brain neurocircuits that regulate glucose homeostasis as potential targets for developing novel therapeutics to treat T2D. Recent studies have revealed that the brain can induce sustained remission of hyperglycemia in rodent models following intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1). FGF1 stimulates the MAPK/ERK signal transduction system, which translates signals from the activation of cell surface receptors into gene expression changes within the cell nucleus. Following FGF1 injection, tanycytes and astrocytes are sites of transcriptional changes related to the ERK pathway. Inhibition of MAPK/ERK signaling blocks FGF1-induced diabetes remission. Like other growth factors, FGF1 requires integrin signaling to elicit the full spectrum of its cellular responses.  The integrin receptor αvβ3, which is implicated in the chronic cellular response to FGF1 is expressed in hypothalamic neurons and tanycytes and is required for FGF1 to induce sustained activation of MAPK/ERK signaling. To determine if integrin signaling in tanycytes is required for FGF1 to induce diabetes remission, we utilized adult, male, diabetic mice expressing either floxed Integrin β3 (ITβ3) or floxed Integrin αV (ITαV). Mice received a single 500µl injection of TAT-cre or heat-inactivated TAT-cre into the 3rd ventricle. TAT-cre administration inactivates the expression of genes encoding either ITβ3 or ITαV. Next, each cohort received a single icv injection of either FGF1 (2 µg) or saline vehicle. Food intake, body weight, and blood glucose measurements were taken daily throughout the investigation.  Our data demonstrates that the ability of icv FGF1 to induce lowering of blood glucose levels is not blocked by knockout of either ITβ3 or ItαV in tanycytes suggesting that integrin signaling isn't required for FGF1 to induce diabetes remission.

Skin-to-skin (STS) care, in which a baby is held directly against a caregiver’s bare chest, has health benefits. However, preterm newborns born before 32 weeks of gestation commonly do not receive STS in the first two weeks after birth in the neonatal intensive care unit (NICU). Research suggests that early STS reduces adverse outcomes such as life-threatening sepsis and mortality within low- and middle-income countries worldwide. Yet, evidence demonstrating the importance of early STS within high-resourced NICUs is limited. To develop evidence-based guidelines for NICU care, it is necessary to examine the relationship between time until first STS and outcomes such as sepsis and mortality. We hypothesize that earlier STS is associated with lower rates of sepsis and mortality. To investigate this, we conducted a retrospective study of very preterm neonates admitted to a level III NICU in Washington state. Newborns were categorized into three groups based on time until first STS: (1) STS within 72 hours of birth, (2) STS between 72 hours and 7 days, and (3) no STS within the first week. We statistically compared the rates of culture-positive sepsis and mortality rates between these groups while also assessing associations with potential confounding variables such as gestational age, birth weight, and fluid intake. Preliminary findings suggest associations between timing of first STS and outcomes of sepsis and mortality, although confounding factors may bias these results and, thus, require future multivariate models to account for confounding variables and their impact on outcomes. Thus, we are conducting an ongoing study to expand the sample size to overcome these limitations. We plan to evaluate the impact of STS on newborn outcomes among other sites to expand generalizability of findings in the future. Ultimately, research on STS care can help improve hospital documentation policies, neonatal care guidelines, and neonatal health outcomes.

Chronic Recurrent Multifocal Osteomyelitis (CRMO), is an autoinflammatory bone disorder that is notable by the recurrent bone lesions with potential long-term complications that include growth impairment in pediatric patients. Growth impairment can be illustrated through z-scores for weight and height. Z-scores represent how far a patient’s weight and height measurements deviate from the average for their age and sex. Z-scores below -1 and -2 could indicate the negative disturbance by CRMO or inadequate treatment. Standard second-line treatments for CRMO include bisphosphonates, disease-modifying antirheumatic drugs (DMARDs), and tumor necrosis factor inhibitors (TNFi). Bisphosphonates are frequently prescribed pediatric medications for their ability to improve bone health and reduce inflammation. However, the impact of these medications – alone or in combination with DMARDs and TNFis – on growth patterns are understudied. Medication practices may demonstrate varying results on height and weight outcomes in CRMO patients. CHOIR included prospective longitudinal data from >500 patients from multiple sites across continents, which allows us to compare the effects of these treatment regimens on the change of Z-score. We expect that the proportion of patients with low Z-scores is similar across all groups before the treatments. By gathering patients based on treatment regimen, this review will compare the prevalence of lower z-scores. Understanding these correlations is vital for identifying whether certain treatments contribute to growth improvement, offering insights into optimizing care for CRMO patients.

Cardiopulmonary bypass (CPB) is essential for most cardiac surgeries but often leads to systemic inflammation and multiorgan dysfunction in neonatal and pediatric patients. These adverse inflammatory responses are driven by severe shear stress on the blood, contact with plastic tubing, and rapid cooling/rewarming. However, the molecular mechanisms underlying these complications are poorly understood, creating a significant barrier in improving clinical outcomes. The Nigam Lab has identified Interleukin 8 (IL-8) and Tumor Necrosis Factor alpha (TNF-α) as inflammatory cytokines upregulated in blood cells in response to CPB-associated shear stress. We hypothesize that Lamins (LMNA) play a key role in driving these transcriptional responses, as these structural proteins form the nuclear lamina and can sense mechanical forces acting on the cell. To investigate this, we performed in-vitro experiments using THP-1 human monocytic cells to simulate bypass conditions, applying shear stress and collecting samples at various time points to study the cells’ response and recovery from CPB. Using mass spectrometry-based proteomics (MS), we have identified changes in LMNA phosphorylation between sheared and static cells, providing insight into the mechanisms driving LMNA modifications under CPB conditions. We are also employing techniques such as proximity-dependent biotin identification (BioID) to explore kinase interactions with LMNA. Furthermore, to understand how LMNA influences chromatin organization, transcription factor binding, and regulation of inflammatory genes, we will perform greenCUT&RUN to map LMNA localization on chromatin in both sheared and static THP-1 cells. We aim to uncover the specific molecular mechanisms by which LMNA is altered under shear stress and how it influences chromatin dynamics and transcription of inflammatory genes during CPB. Ultimately, this research will help us understand the underlying causes of systemic inflammation post-CPB and inform novel drug targets and therapeutics to enhance the quality of life for pediatric patients undergoing cardiac surgery.

Preterm birth is a leading cause of under-5 morbidity and mortality. No treatments exist to address the neurological complications of premature birth, which include loss of oligodendrocytes and activation of microglia, leading to white matter injury and inflammation, respectively. Our study explored repurposing azithromycin, an FDA-approved antibiotic with anti-inflammatory properties, to mitigate preterm brain injury caused by hypoxia-ischemia. We used a postnatal day (P)14 neonatal ferret model, equivalent to extremely preterm infants. We induced brain injury through a combination of inflammatory stimulus, bilateral carotid artery ligation, and oxygen fluctuations (hypoxia/hyperoxia). Ferrets were randomized into control, vehicle (saline)-treated, and azithromycin-treated groups. Littermate controls were not exposed to injury. Body weights and ex-vivo brain measurements (sulci and gyri widths) were recorded at P21, seven days after injury. Quantitative immunohistochemistry (qIHC) was performed to analyze microglia (Iba-1) and oligodendrocyte (Olig-2) density, and data were analyzed using Kruskal-Wallis tests. In our preliminary findings, post-surgical weights from the azithromycin-treated ferrets were similar to those of vehicle-treated animals. Azithromycin-treated ferrets also showed similar global microglia and oligodendrocyte staining compared to the vehicle group. The vehicle group had lower summed gyri measurements than controls (p=0.04), while azithromycin-treated ferrets had more similar gyri widths to controls (p=0.21). We will continue investigating microglial and oligodendrocyte density using qIHC across additional brain regions using pathology software (VisioPharm), including subregions of each gyrus (cortex, subcortical white matter, and coronal radiata), corpus callosum, hippocampus, and upper and lower thalamus. This will allow us to identify the brain regions most impacted by the injury and investigate if there are regional neuroprotective responses to azithromycin. By deepening our understanding of preterm brain injury and azithromycin-mediated neuroprotection, these findings could lay the groundwork for advancing azithromycin toward clinical trials, offering new hope for saving the lives of the tiniest neonates.

Traumatic brain injury (TBI), characterized by a physical impact to the skull, is a significant health concern among veterans, athletes, and the elderly, with over 200,000 TBI-related hospitalizations in 2020. TBI causes shearing forces and physical damage to the brain, resulting in increased risk of neurodegeneration and mental health problems. When they expect an impact, humans brace, exhaling against a closed airway in what is known as a Valsalva maneuver. This prevents venous return from the head, pressurizes the vascular network in the brain, and increases intracranial pressure (ICP) in a way that may protect the brain from TBI. We aim to mimic a Valsalva-like response (VLR) through external abdominal stimulation and measure corresponding ICP changes. First, we performed a 3mm-wide craniotomy in anesthetized ferrets and implanted a pressure transducer inside the brain to collect baseline pressure readings. After skull closure, VLR was performed both supine and upright (body at 45°), either physically (pVLR, 80-120mmHg by abdominal compression using a blood pressure cuff, n=4) or electrically (eVLR, bilateral 25-30mA stimulus of the rectus muscles, n=4). pVLR resulted in a 2-4mmHg increase in ICP over 2-5 sec. By comparison eVLR resulted in a larger and faster ICP increase - 3-7mmHg with an onset of 250-750ms. Consequently, we will utilize eVLR to modulate ICP in a TBI model to determine whether it is neuroprotective. Ferrets will be assigned to control or randomized to receive a TBI impact with either sham eVLR or eVLR. Animals will be subjected to baseline (pre-TBI), acute, and long-term behavioral testing. Additionally, we will perform brain cell specific histological staining. Results from behavioral testing and histology will inform us of the potential neuroprotective effects of eVLR against TBI and provide future direction towards translating the findings into a wearable device for at-risk individuals.

The Thermal imaging-based Temperature After Within-limb Calibration (TAWiC) algorithm has been used and validated by our team in detection of arthritis in the knees of children. It has previously performed well through using a smart-phone FLIR thermal camera attachment. We are now hoping to explore and expand its potential by developing TAWic thresholds to find other inflammatory arthritis in the knee, ankle/subtalar, elbow and wrist joints. Patients 4 years or older with suspected active inflammatory arthritis in at least one knee, ankle/subtalar, elbow or wrist were enrolled at Harborview Medical Center or Seattle Children’s Hospital after consent was obtained. Joint exams were conducted and infrared thermal imaging was obtained through the use of a FLIR One Pro camera by doctors. I analyzed images in MATLAB by manually selecting elbow, wrists, knees, and ankle joints to generate reports and scores for virtual doctor evaluation. With 89 adults and 85 children enrolled, we found the most commonly affected joints in children to be knees while in adults, it was the wrists. Further validation of applying the TAWiC threshold to detect arthritis was conducted, and the sensitivity and specificity of this algorithm for adults with active inflammatory arthritis in the knees were 50% and 83%, respectively. To our knowledge, this is the first reported validation of the TAWiC algorithm for knee inflammatory arthritis in adults. Ongoing and future studies will seek to validate use of the TAWiC algorithm for assessing arthritis in other joints. We hope that in the future, the technology can be used remotely by patients in telehealth efforts to send imaging to minimize costs, increase efficiency, and save time in caregiving efforts.

Bacteria are constantly under dynamic environmental pressures and must promptly respond to survive. Bacterial general stress responses (GSRs) allow adaptation to perceived environmental changes via two-component and phosphorelay systems. The pathogenic alphaproteobacteria Bartonella quintana uses the body louse as a vector for infecting its target host, humans. It must adapt to two disparate environments, the human bloodstream and the gut of the body louse. Upon niche transfer, B. quintana is able to activate its GSR via a partner-switching mechanism involving an elegant molecular dance between alternative sigma factor RpoE, anti-sigma factor NepR, and anti-anti-sigma factor PhyR. The switching transfers NepR away from RpoE to PhyR, which activates gene transcription. Published works have revealed a molecular mechanism for sequestration via formation of a 1:1 dimer triggered by post-translational modification (PTM). However, a protein data bank (PDB) crystal structure (4QIC) shows a 2:2 tetramer, although it has not been observed in solution. We utilized size exclusion chromatography, multi-angle light scattering (MALS), small-angle X-ray scattering (SAXS), and protein modeling under various buffer conditions to identify conditions favorable for tetramer formation. MALS was chosen to determine the precise molecular weight of our chromatogram peaks, while SAXS was chosen to compare specific chromatogram peak scattering curves to PDB crystal structures and provide an overall shape for relevant peaks. Strikingly, our results revealed the tetramer forms in the absence of phosphorylation in solution, and the dimer is the dominant species under PTM favorable conditions. These results are loosely consistent with the literature but indicate the complexity of the alphaproteobacteria GSR is not fully understood. A possible explanation for the tetramer is that it maintains stress-related transcription despite the absence of a PTM.

Stress-related factors can have a direct impact on cancer biology and patient outcomes. Exposure to a stressor leads to the sympathetic nervous system (SNS) activating downstream signaling pathways that impact cancer-related processes; SNS activity can be measured with heart rate variability (HRV). Low HRV indicates less ‘autonomic flexibility’ and has been associated with poor health outcomes, while high HRV has been associated with better health outcomes. Psychosocial factors such as resilience, stress, and social support are important for adolescents and young adults (AYAs), but the relationship between psychosocial factors and HRV is unknown. The goal of this study is to examine changes in HRV among AYAs with cancer during a qualitative interview about psychosocial factors. Eligible participants were 12-24 years old within six months of initial cancer diagnosis and undergoing treatment at Seattle Children’s Hospital. Once enrolled, participants wore an HRV sensor while participating in a 1:1 semi-structured interview querying topics including stress, resilience, and social support. I used a commonly reported HRV metric, the standard deviation of normal-to-normal intervals (SDNN) to quantify HRV. I defined baseline HRV as the first 5 minutes of the interview, reactive HRV as 5 minutes at the midpoint of the interview and recovered HRV as the last 5 minutes of the interview. I compared baseline HRV to reactive HRV and recovered HRV. I expect to find that both reactive HRV and recovered HRV are lower (‘worse’) than baseline HRV. Results from this study can give insight on the impact psychosocial factors have on the biomarkers of stress in AYAs with cancer.

B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematologic malignancy characterized by the overproduction of malignant B-lymphoblasts in the bone marrow. It is the most common pediatric cancer and the second leading cause of cancer death among children. Standard therapies, including drugs such as dexamethasone and vincristine, achieve remission in approximately 90% of cases, but 10% of patients exhibit resistance. Furthermore, standard therapies result in significant short- and long-term toxicities. Thus, alternative treatment strategies are needed. FHD-286, a BRG1/BRM ATPase inhibitor currently in clinical trials for acute myeloid leukemia (AML), is a potential candidate for improving B-ALL therapy by targeting chromatin remodeling dependencies and reducing reliance on less tolerable chemotherapies. Our study evaluates the efficacy of FHD-286 in combination with dexamethasone and vincristine, hypothesizing that the combination may overcome treatment resistance in B-ALL. We tested these combinations across genetically diverse B-ALL cell lines. We treated the cell lines with varying doses of each drug alone and in combination and, after 3 to 5 days, assessed cell counts and apoptosis using Annexin V staining. Compared to vincristine or dexamethasone alone, when combined with FHD-286, we observed an increase in apoptosis. After three days of treatment, we detected a significant decrease in cell count, while after five days, cell viability dropped, suggesting that the drug combination may induce both cell cycle arrest and followed by apoptosis over time. Notably, FHD-286 demonstrated effectiveness in KMT2A-rearranged B-ALL, a high-risk subtype prone to relapse, while also demonstrating potent effects in non-KMT2A-rearranged B-ALL. Our findings suggest that FHD-286 enhances therapeutic efficacy in B-ALL when combined with current standard treatments, offering a potential strategy to overcome resistance and reduce chemotherapy toxicity across multiple leukemia subtypes.

Hypoxic-ischemic encephalopathy (HIE) is a leading cause of neonatal morbidity and mortality worldwide. The ferret provides a highly translational model to investigate HIE; the gyrified ferret brain has a similar grey-to-white matter ratio to humans, allowing for better assessment of white matter injury and impairment of cortical development compared to rodents. Our previous work has suggested that ferret brains also show greater resilience to hypoxia-ischemia (HI) than rats. Ferrets tolerate exposure to much longer and more significant HI, and 100-fold larger doses of inflammatory stimuli, than rats do. We seek to identify signatures of the ferret's protective mechanisms by comparing differentially regulated genetic pathways in the ferret versus the rat when exposed to identical insults. Whole-hemisphere organotypic brain slices were obtained from term-equivalent ferrets and rats and cultured for 72 hours. Slices were randomly assigned to control or oxygen-glucose deprivation (OGD), an in-vitro model of HIE. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release, while global transcriptomics were analyzed via a 770-gene digital transcriptomics panel. Preliminary results show significantly lower LDH release in ferrets compared to rats, reaffirming the ferrets' resilience to OGD. We identified 90 differentially expressed genes in ferrets following OGD, and 11 genes in the rat. Ferrets upregulated CCL2 and LGALS, genes associated with inflammatory responses, and downregulated ADRB1 and NOS2, suggesting reduced oxidative stress. Rats downregulated KIR3DL1/2 and TGM1, which suppress natural killer cells and form the cell envelope, respectively. The experiment will be repeated with double the sample size and region-specific analysis of gene regulation. We hypothesize the ferret will display lower injury markers globally, which will be associated with regional differences in gene expression compared to the rat. We hope this will enable us to identify potential treatment targets for infants with HIE that can increase resilience and repair after injury. 

Hypoxic Ischemic Encephalopathy (HIE) is a brain injury caused by a lack of oxygen and blood flow in the peripartum period. Cardiac dysfunction occurs in up to 80% of infants with HIE and is associated with worse neurodevelopmental outcomes. The current standard of care for HIE is whole body therapeutic hypothermia (TH). The expected physiologic response to TH is a decrease in cardiac output by 10%, and heartrate (HR) by 10bpm, per 1-degree Celsius decrease in body temperature. However, neonates with cardiac dysfunction tend to have normal or elevated HR to compensate for decreased cardiac output. Therefore, normal or elevated HR during TH may indicate compromised cardiac function. We hypothesize that in neonates with HIE, HR trends during TH reflect cardiac function, and a sustained HR above 100bpm is indicative of cardiac dysfunction. Using echocardiograms performed within the first 2 days after birth in babies with HIE treated with TH at the Seattle Children's neonatal intensive care unit (NICU; n=19), we categorized neonates by cardiac function: normal, right ventricular (RV) dysfunction, and RV plus left ventricular (LV) dysfunction. We then extracted continuous HR data and compared median HR during TH across groups using linear regression during specific periods: 12-24h, 24-36h, and 36-48h after birth. Results showed that infants with RV+LV dysfunction had a higher HR than those with RV dysfunction only or normal function. Across all time periods, infants with any kind of cardiac dysfunction had an average HR above 100bpm, while those without dysfunction had average HRs less than 100bpm. Therefore, it appears that HR can be utilized as a proxy for cardiac dysfunction in neonates with HIE. Utilizing HR as screening biomarker for cardiac dysfunction may allow improve optimal resource utilization of echocardiograms as well as real-time, cost-effective monitoring and targeted treatment initiation. 

Pediatric sleep disorders, such as obstructive sleep apnea (OSA), impact 5-10% of children. Children are diagnosed with sleep-disordered breathing through polysomnography (PSG), which requires hours of sleep and physiology data including EEG tracings, cardiograms, pulse-oximetry, and airflow monitoring. PSG data can be used to create individualized therapy and advance the care of children with sleep disorders. To facilitate novel diagnostic and validation studies using data collected on PSG and patient questionnaires, we created a data bank of PSG and patient data. Through creating a patient database in R, we can analyze sleep behaviors and medical diagnosis of pediatric patients and support future investigations. We aimed to create a pediatric sleep disorder research database to analyze sleep behaviors of pediatric patients, hypothesizing that chronotype classification would differ with age. We created a custom R script to analyze the raw data bank for medical diagnosis, age, sex, PSG diagnosis, chronotype, patient symptom scales and reported summary statistics including count, range, and standard deviation. Dplyr and tidyverse packages were used to create data summaries and ggplot2 for graphical presentation. An initial cohort of 111 participants were analyzed for correlation of chronotype and age range (>11, <=11). Initial analysis revealed a cohort of 111 participants with an age range of 6 months to 18 years (median: 7), medical history of 15 prematurity, 16 allergic rhinitis, and 3 congenital heart disease patients, PSG diagnosis of 44 normal and 8 severe, chronotype scoring of 8 evening to 34 morning patients, OSA-18 scores ranging from 34 to 102 (median: 61). Correlation analysis revealed that chronotype distribution is statistically different between age groups. We have created a custom analysis tool to create a summary report of a new sleep data bank repository. Future studies will use the tool to inform preliminary summaries of available demographic and data. 

Neuroscientists have conventionally used enriched preparations of synaptosomes, or isolated nerve terminals containing particles such as synaptic vesicles and synaptic mRNAs, to study biochemistry in the brain and the physiological features of the synapses. However, the molecular diversity of the brain limits the ability to study specific types of synapses with conventional preparations. Here we present a synthetic protein “TAG”, comprised of PSD95Δ1.2, part of the postsynaptic scaffolding protein, guides the TAG construct to be recruited at the targeted synapse; an extracellular binding site that binds to a CD4 antibody to allow us to sort synaptosomes that express TAG, and intracellular mVenus green fluorescent protein for easy visualization. TAGed synapses provide a tool for scientists to more robustly investigate the biochemical properties of synapses by increasing signal over noise. My work was done to optimize a novel preparation method by isolating rare TAGed synaptosomes in the TAGxCAGGCre-ER mouse model. I induced conditional TAG gene expression by injecting tamoxifen into a TAG-crossed transgenic double-floxed inverted open-reading frame Cre mouse. I also optimized a new preparation method using a high-salt buffer, filtration system, bead conjugation, and magnetic separation to isolate TAGed synaptosomes. The product was the input of a series of Western Blots to assess the enrichment of TAG in sorted and pre-sorted samples. From my latest results, the CD4-sorted lysate showed significant enrichment in the GFP band, meaning the TAGed synaptosome has been purified. By replacing the CAG promoter in the TAGxCAGGCre-ER model, scientists can also use this method to target region-specific neuron subtypes and isolate rare synaptosomes. The precision and flexibility of the TAG construct allow scientists to observe subcellular connections with more specificity and allow for the discovery of biological mechanisms underlying neuronal diseases.

Malaria, caused by Plasmodium parasites, infects millions of people across the globe and leads to over half a million deaths annually. Infection begins when a mosquito takes an infectious blood meal, resulting in the deposition of infectious parasites known as sporozoites into the skin. Sporozoites traffic from the skin into the liver and undergo clinically silent development in hepatocytes.  This liver stage development is required for the transition into blood stage development where all the clinical symptoms of malaria and transmission back into mosquito vectors occur. No highly efficacious malaria vaccines exist, but one promising vaccination strategy is immunization with sporozoites that are impaired in their ability to complete liver stage development.  These attenuated whole parasite vaccines provide robust immune protection in malaria-naive individuals, but further refinement of this approach is required before this strategy can be deployed globally in endemic regions.  We have shown that the type 1 interferon (IFN-1) signaling regulates the immune response induced by whole parasite vaccines. My project aims to spatially characterize how IFN-1 influences parasite development within the liver using immunofluorescence. Interferon-alpha/beta receptor knockout (Ifnar-) mice (which are impaired for IFN-1 signaling) and wildtype C57Bl/6 mice were infected with Plasmodium yoelli. We then harvested livers from infected mice at various time points during liver stage development. I observed that IFN-1 restricts parasite development beginning at 24 hours post-infection but does not impact parasite size in hepatocytes. Future studies will selectively eliminate IFNAR on hepatocytes or on distinct immune cells to identify if IFN-1 mediated parasite restriction is hepatocyte intrinsic or is immune cell mediated.

Premature neonates are particularly vulnerable to electrolyte and fluid imbalances due to their increased insensible fluid losses and immature kidney function. Clinicians carefully monitor and document electrolyte and fluid intake. However, research suggests that saline flushes, small volumes of sodium chloride solution administered to clear intravenous lines after medication delivery, are a source of electrolytes and fluid in the NICU that are often unaccounted for. In the first week after birth, frequent medication administration leads to multiple flushes, and the relative contribution of flushes to total sodium and chloride intake may be substantial for the smallest newborns. Particularly, sodium imbalances contribute to pathologies and have been associated with adverse health outcomes, including intraventricular hemorrhage (IVH) and all-cause mortality. Understanding the impact of saline flush administration on fluid and electrolyte balance is essential for developing evidence-based neonatal care guidelines. We hypothesize that saline flushes in the first have greater relative contributions of sodium, chloride, and total fluid intake for smaller and more premature newborns. To investigate, we are conducting a retrospective study of very preterm newborns admitted to a level III NICU in Washington state. We will statistically compare sodium, chloride, and total fluid intake before and after accounting for saline flushes, and we will descriptively analyze the amount of each in relation to gestational age and birthweight. We will additionally evaluate via univariate models how sodium intake with and without inclusion in total fluid intake correlates with serum sodium daily values. This ongoing study aims to expand the sample size to increase our ability to perform multivariate regression models to account for confounders which may bias our findings. Ultimately, results from this research can improve neonatal care guidelines, helping clinicians optimize sodium, chloride, and fluid intake. 

Cerebellar development relies on the coordinated proliferation and differentiation of progenitors from the ventricular zone (VZ) and rhombic lip (RL). To systematically map their spatiotemporal dynamics, we performed EdU pulse labeling by injecting pregnant mice with EdU and collecting embryonic cerebella at daily intervals over five consecutive days as well as an acute half-an-hour post EdU injection. EdU labeling identifies actively dividing progenitor cells at the time of injection. As development progresses, EdU+ cells can be tracked to study their differentiation and migration, revealing the temporal dynamics of VZ and RL progenitor-derived neurons in the cerebellum. Using multiplex immunohistochemistry with VZ- and RL-derived cell-type specific markers, we tracked the spatial distribution and differentiation of EdU-labeled cells, distinguishing VZ- and RL-derived progenitor lineages. Additionally, we outline a strategy to isolate EdU+ cells for single-cell RNA sequencing (scRNA-seq) and ATAC sequencing (ATAC-seq), enabling a comprehensive molecular characterization of progenitor fate transitions. This approach provides a high-resolution developmental trajectory of cerebellar progenitors, offering new insights into the regulatory mechanisms driving cerebellar neurogenesis and their disruptions in neurodevelopmental disorders.

Acomys Cahirinus (spiny mice) are remarkable creatures that exhibit key differences in inflammatory response, regeneration, and aging compared to mice. Adult neurogenesis - the production of new neurons- in the hippocampal niche declines with age in most mammals, yet Acomys exhibits sustained neurogenic potential, presenting a unique model for regenerative neuroscience. This study leverages advanced image analysis software (Imaris) to develop robust pipelines for quantifying neural stem cell (NSC) and intermediate progenitor (IP) proliferation and fate determination in Acomys versus standard laboratory mice (Mus musculus). Using EdU incorporation to track S-phase entry and a 4D pulse-labeling approach, we assess neurogenic niche activity across species. Additionally, we extend this analysis to aging Acomys, utilizing consistent sectioning, staining, and imaging parameters to confirm continuous progenitor proliferation in young and old cohorts. Our findings provide critical insights into the cellular and molecular mechanisms underlying sustained neurogenesis in Acomys, offering prospective therapeutic targets for age-related neurodegenerative conditions.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental condition characterized by differences in attention, focus, and emotional regulation. ADHD has high heritability rates, meaning children commonly inherit ADHD from their parents. Despite this, there is little research on parental ADHD symptoms and how they affect parenting. We hope to bridge this knowledge gap by investigating the relationship between temperament and emotional socialization in ADHD parent-child dyads (parents and their children with ADHD). Temperament refers to innate behavioral traits shaping one's personality. Individuals with ADHD are known to experience higher rates of negative affect, a temperamental factor including significant aversion from feelings like sadness. Emotional socialization is the process through which individuals recognize, understand, and manage their emotions in a social context. This process is especially complex with ADHD parenting, as parents with ADHD symptoms may have differences in emotional regulation and temperament that could influence parenting behaviors and emotional socialization in their children. We hypothesize that negative affect in parents is positively correlated with (1) negative talk towards children and (2) perceived anxiety and lack of anger control in their children. To test these hypotheses, parents complete the self-report Adult Temperament Questionnaire (ATQ), which assesses negative affect, and the Behavior Assessment System for Children (BASC), which assesses their child's perceived anxiety and anger control. We evaluate negative talk via behavioral coding of video-recorded standardized parent-child interactions. Using these laboratory-based interactions, we use the Dyadic Parent-Child Interaction Coding System (DPICS) to analyze the frequency of parents' verbal disapproval of child behavior and/or attributes. I then use bivariate correlation analysis to determine the relationship between the proposed variables of interest. Through our anticipated findings, we hope to better inform care for children with ADHD by identifying individualized support strategies to use in parental interventions to better facilitate emotional socialization in ADHD families. 

Obstructive sleep apnea (OSA) in children is linked to early life viral infections and increased severity of viral respiratory illnesses. As respiratory viral infections occur in airway epithelial cells, we investigated differences in viral responses using an organotypic epithelial cell model in children with OSA as compared to children without. We hypothesized that gene expression in response to rhinovirus (RV16) infection would differ between healthy children and children with OSA. Primary airway epithelial cells (AECS), from both healthy pediatric donors and children diagnosed with OSA by polysomnography, cultured at an air-liquid interface, were infected with RV16 on the apical surface at a multiplicity of infection of 0.5. RNA-sequencing quantified gene expression at baseline and after RV16 infection. Limma was used to identify genes with differential expression post-infection in healthy AECs as compared to AECs from donors with OSA. Weighted gene co-expression network analysis (WGCNA) was able to organize the identified genes into groups of interest. Using Enrichr, the primary biological functions of the gene groupings were analyzed. Following infection, 122 genes were found to have differing gene expression responses to RV16 in OSA when compared to healthy cell lines. WGCNA identified two modules of gene expression with opposite expression patterns following infection in OSA compared to healthy. One module consists of 43 genes enriched for glycogen metabolism which are downregulated in healthy but upregulated in OSA following infection. A second module consists of 23 genes enriched for DNA repair and replication which are upregulated in healthy but downregulated in OSA after infection. Epithelial cell gene expression differs in response to RV16 in healthy AECs as compared to AECs from children with OSA. Given the small sample size, further studies are needed to investigate the relationship of OSA severity and clinical phenotypes of OSA with epithelial responses to viral infection. 

Understanding the dynamic behaviors of cells in the developing human brain is essential for elucidating the mechanisms that drive both normal and abnormal neurodevelopment. Using lentiviruses encoding fluorescent proteins, we infected cells in slices from different regions of the developing human cerebellum to track their movements over several hours. We then captured timelapse images of these fluorescent slices under a microscope, allowing us to visualize their dynamic behavior. Using live imaging analysis software, hundreds of individual cells were then tracked and characterized. Our analysis found several key processes, including novel modes of cell division and differentiation, neuronal migration, and intercellular communication. This approach allowed us to map a timeline of critical events that shape cerebellar architecture. This research aims to help us gain insight into neurodevelopmental disorders, where disturbances in fundamental biological processes underlie disease progression.

Cardiopulmonary bypass (CPB) is a conventional way to treat the majority of cardiac surgeries. CPB is used during heart surgeries to circulate blood out of the patient's body in order for surgeons to operate on the heart. However, CPB has led to inflammation and multiorgan dysfunction especially leading to post CPB complications in neonates. Lack of questioning and understanding behind the complications of the technique have posed issues for improvements to clinical outcomes. Specifically, lack of understanding of molecular mechanisms and CPB-associated post surgery inflammation have posed obstacles to improvement of methods in recent years. To better understand these mechanisms, we performed mRNA and ATAC sequencing on circulating leukocytes from neonatal CPB patients. Notably, IL-8 and TNF-α were strongly upregulated in leukocytes. To explore these findings, I performed in-vitro experiments of running THP-1 human monocytic cells to CPB-like conditions, including high shear stress and cooling/rewarming. These experiments were collected and studied at times pre and post shear, and recovery post shear. Experiments regarding blood plasma changes were proposed and this plasma was similarly collected during varying conditions pre and post bypass. ELISA kits were run on antigens AREG and EREG to determine how antigen binding changes with shearing. Sheared then rested samples were found to show a significant increase in antigen binding in both kits AREG and EREG. Sheared and processed samples also showed an increase in binding when compared to the static samples. I have shifted my focus from plasma experiments to investigating the effects of commonly used plasticizers on blood composition. Specifically, I am analyzing how these plasticizers influence changes in blood and plasma using a PIPSeq kit.

Antibiotic treatment is commonly used to manage bacterial infections in very preterm infants (defined as born before 32 weeks of gestation) admitted to the neonatal intensive care unit (NICU). Because immediate treatment is crucial to treat life-threatening sepsis, antibiotics are often administered empirically before microbiology test results confirm infection. As a result, it is common practice that some infants without confirmed infection receive multiple days of antibiotics, which can disrupt the newborn’s developing microbiota. Research suggests that empiric antibiotic therapy is associated with adverse long-term outcomes, including retinopathy of prematurity (ROP), a disease of the eyes, and bronchopulmonary dysplasia (BPD), a chronic disease of the lungs. Understanding the implications of empiric antibiotic use is essential for developing evidence-based guidelines for preterm infection management. We hypothesized that empiric antibiotic exposure is associated with higher rates of ROP,  BPD, and mortality after adjusting for confounding variables. To investigate this, we are conducting a retrospective study of very preterm newborns admitted to a level III NICU in Washington state (N = 55). We statistically modeled the association between the number of days exposed to antibiotics within the first 14 days after birth and the incidence of ROP, BPD, and all-cause mortality after 2 weeks from birth. Preliminary findings indicate a non-significant trend toward higher rates of ROP, BPD, and all-cause mortality (after 2 weeks) with longer duration of empiric antibiotic therapy within the first two weeks. We are conducting an ongoing study to expand the sample size and refine statistical models to account for additional confounding variables. Research on the effects of empiric antibiotic use can improve clinical practice guidelines for treating unconfirmed infection and reduce potential harms associated with early antibiotic exposure.

Childhood liver transplants have several risks, including rejection, homeostatic complications, and lifelong immunosuppression. Precisely controlling cell identity would enable the generation of transplantable tissue from a patient's cells through cell reprogramming, minimizing these risks and expanding transplant access. Cell identity is partly maintained by heterochromatin states that block transcription factor binding and restrict gene activation. Work from the McCarthy Lab has shown that diverse heterochromatin-associated proteins repress lineage-specific genes, and depleting these proteins can de-repress heterochromatin domains, enabling transcription factor binding, gene activation, and cell reprogramming. However, which proteins regulate distinct heterochromatin domains is poorly understood. My goal is to understand the connection between chromatin state and gene activation permissibility and investigate the roles of specific proteins in maintaining specific chromatin states. We hypothesize that we could utilize an enzymatically dead Cas9 (dCas9) fused with a transcriptional activation domain (VP64) as a programmable transcription factor proxy to investigate specific heterochromatin domains and the function of proteins that maintain them. I identified target genes in H3K27me3, H3K9me3, and unmarked heterochromatin domains in human fibroblasts, focusing on genes only expressed or elevated in the liver. I designed guide RNAs to target dCas9-VP64 to sites 75 to 150 base pairs upstream of gene transcription start sites. I transfected guide RNA plasmids for 14 genes into dCas9-VP64 expressing human fibroblasts and assayed gene activation and transfection efficiency by RT-qPCR. Like transcription factors, dCas9-VP64 could activate unmarked genes and weakly activate genes in H3K27me3 but failed to activate genes in H3K9me3. Knocking down heterochromatin protein ERH using siRNA enabled dCas9-VP64 to activate H3K9me3-marked genes. Future work will investigate connections between additional heterochromatin domains and regulatory proteins. Understanding distinct protein roles in maintaining heterochromatin and repressing genes will improve our ability to control cell identity to reprogram patient cells.

Mast cells are key contributors to allergic disease including asthma, food allergies, rhinitis and atopic dermatitis. Therefore, understanding mast cell biology more deeply is critical for the discovery of new targets to modulate mast cell function in health and disease. The research question being addressed in the Piliponsky Lab is what proteins play a role in mast cell activation and release of mediators that contribute to allergic disease. DOCK8 deficiency is a rare, combined immunodeficiency (CID) associated with allergic diseases which led our lab to investigate the impact of DOCK8 on mast cell function. We took microscopic images of mast cells and enumerated mast cell numbers in mucosal and connective tissues using mice with mast cells deficient in DOCK8, DOCK8 mutant mice, and littermate controls. We used western blots to confirm the absence of DOCK8 protein in the mutant mice and genotyped mice with DOCK8 deficiencies. Our findings suggest that mast cell intrinsic DOCK8 deficiency can cause increased mast cell degranulation in skin and mast cell mediator release at baseline. Learning more about mast cells can help increase understanding of the mechanisms of allergic disease and inflammation, leading to more treatment options. 

The cerebellar ventricular zone (VZ) is the primary source of progenitor cells that give rise to all cerebellar GABAergic neurons, including Purkinje cells (PCs) and interneurons (INs). While the VZ has been well studied in mice, much less is known about its role in human brain development. In this study, we investigated how progenitors and neurons form in the human cerebellar VZ, using in situ hybridization, immunohistochemistry, and single-cell RNAseq analysis. Our findings reveal several key differences from the mouse model. We found that Purkinje cells are generated during a brief two-week period, even before the cerebral cortex begins to develop. Interneurons, on the other hand, start differentiating a few weeks later and mature on a timescale of months to years. A unique feature of human cerebellar development is the presence of specialized inner and outer subventricular zones (SVZ), which are absent in mice. Most differentiation occurs in these regions, with the first wave taking place in the outer SVZ. Additionally, we observed variations in Purkinje cell arrangement and number, including a subset of Purkinje cells that continue expressing cell cycle genes, suggesting a more complex and prolonged developmental profile compared to mice. By characterizing these developmental processes, our study provides new insights into human cerebellar development, highlighting important structural and temporal differences from animal models. These findings may have implications for understanding neurodevelopmental disorders.