Tuberculosis is still one of the leading causes of death worldwide, primarily due to multidrug-resistant tuberculosis and lengthy drug regimens. Efforts are being made to understand the mechanisms of resistance and to develop novel antimycobacterial drugs. Drug resistance has been found in the Esx-3 type VII secretion system of Mycobacterium tuberculosis, which is involved in metal ion homeostasis. Rv0474 is a transcriptional copper-responsive regulator in M. tuberculosis with a copper binding motif. Under toxic copper conditions, Rv0474 binds to the rpoB promoter, which blocks the transcription of the RNA polymerase beta subunit leading to growth arrest. Rv0474 has also been found to be involved in mediating resistance to the aminothiazole (AmT) and 8-hydroxyquinoline (8HQ) drug series. I am studying the Rv0474 homolog (MSMEG_0918) in Mycobacterium smegmatis, a fast-growing species being used as a model organism. MSMEG_0918 lacks the copper binding motif, permitting determination of whether copper binding activity is required to confer resistance. We propose that if copper binding activity is involved in AmT/8HQ resistance, overexpression, or downregulation of MSMEG_0918 would have no effect on compound activity, because it lacks the copper binding motif. To test the effect of changes in MSMEG_0918 expression on AmT and 8HQ activity, I have constructed and sequenced recombinant plasmids containing the CRISPRi backbone and sites essential for target gene knockdown. Using CRISPRi knockdown technique, I have generated strains with reduced expression of MSMEG_0918, along with overexpression strains. Using growth curve measurements and PCR analysis, I have characterized these strains. Knockdown and overexpression strains will enable me to determine growth, AmT/8HQ sensitivity and copper sensitivity. This work will determine whether MSMEG_0918 plays an equivalent role to Rv0474 in conferring resistance to AmT and 8HQ compounds. This research will provide knowledge in understanding the mechanism of resistance to AmT and 8HQ compounds in M. tuberculosis.

The escalating epidemics of obesity and Type 2 Diabetes (T2D) are among the most impactful and costly biomedical challenges confronting modern society. Despite intensive research efforts, few non-surgical treatments are available that have shown sustained efficacy. However, growing evidence points to hypothalamic neurocircuits involved in energy homeostasis as potential therapeutic targets. Previous studies have identified fibroblast growth factor 1 (FGF1) to be an attractive candidate for the treatment of obesity and T2D as exogenous intracerebroventricular administration of FGF1 elicits potent weight-loss and anti-diabetic effects. FGF1 is also endogenously expressed in the hypothalamus, but the potential contribution of endogenous hypothalamic FGF1 signaling in brain control of energy and glucose homeostasis is unknown. We report that in both rats and mice, FGF1 is expressed by tanycytes and neurons in hypothalamic nuclei that are well-recognized to participate in energy and glucose homeostasis including the arcuate nucleus, ventromedial nucleus, dorsomedial hypothalamus, and lateral hypothalamus. However, the extent to which further cell types express FGF1 awaits further investigation. We aim to further these findings by determining endogenous FGF1 expression by hypothalamic nuclei and cell type using double-labeled immunohistochemistry to colocalize FGF1 with cell markers for neurons, astrocytes, and microglia. Next, we found that hypothalamic FGF1 mRNA expression is significantly decreased in fasted mice deprived of food for 24 h. Similarly, hypothalamic FGF1 protein levels were decreased by fasting, but rapidly increase 2 h after refeeding. Surprisingly, we found that in contrast to white adipose tissue in which FGF1 expression increases during high fat diet (HFD) feeding, hypothalamic FGF1 mRNA and protein levels are potently reduced seven days after being switched onto a HFD. These findings demonstrate that endogenous hypothalamic FGF1 mRNA and protein expression are dynamically regulated by acute changes in metabolic state, and that HFD may disrupt endogenous hypothalamic FGF1 signaling. 

Vascular malformations (VM) are congenital malformations of veins, arteries, capillaries, and/or lymphatic vessels. They are primarily caused by post-zygotic activating mutations within oncogenes of the PI3K-MTOR-RAS-MAPK pathways. The major types of VM include capillary malformations (CM), lymphatic malformations (LM), venous malformation (VeM), and arteriovenous malformations (AVM). Identifying the molecular etiology of VM is important, as there are increasingly targeted medical therapies. However, current molecular diagnosis requires surgically excised tissues, as the causative mutations are somatic and not present in blood cells. Cell-free DNA (cfDNA) is an emerging diagnostic analyte that is already in wide use in cancer diagnosis and prenatal genetic screening.  Given the role of mutant endothelial cells in the development of VM and their proximity to blood and lymphatic fluid, we hypothesized that mutant cfDNA will be detectable in plasma or cyst fluid from individuals with VMs. This project aims to develop a non-invasive methods to identify genetic causes of VM without surgery to reduce the costs and risks relating to surgical biopsy. Utilizing digital droplet polymerase chain reaction (ddPCR) technology, we are able to amplify cfDNA fragments to detect specific well known “hotspot” DNA mutations in PIK3CA, BRAF, TEK, and others. We continue to determine the sensitivity of allele-specific cfDNA analysis in individuals with known, tissue-based mutations from a retrospective cohort, and test prospective cfDNA from VM patients with unknown mutations using both ddPCR screening and/or a high sensitivity gene sequencing panel. Ultimately, our hope is to bring these techniques into the clinical arena to increase the possibility of medical therapies for individuals with vascular malformations.

Cystic fibrosis (CF) is a genetic disease characterized by polymicrobial lung infections. Staphylococcus aureus, a Gram-positive pathogen, is commonly cultured from the secretions of people with CF (PwCF). Treatment for S. aureus infections requires antibiotics, such as the bactericidal antibiotic trimethoprim-sulfamethoxazole (SXT). SXT prohibits growth by targeting folate biosynthesis, a pathway important for DNA replication and maintenance and production of cell metabolites. In many PwCF, S. aureus generally persists despite antibiotic treatment. Our data shows that S. aureus can survive SXT treatment through the accumulation of adaptive mutations. In this project, we examined these adaptive mutations in S. aureus in vitro to better understand the mechanisms of resistance. We grew several S. aureus isolates with adaptive mutations in Luria Bertani (LB) broth. We sampled the culture tubes at several times in a 24-hour period, measuring viable bacterial counts on chocolate agar. We found that isolates with mutations in the sugar transport gene, ptsI, persisted better under SXT selection, relative to wild-type S. aureus. Other S. aureus isolates with mutations in pathways for aerobic respiration, including menaquinone (menB, thiN) and hemin synthesis (hemB), also better survived SXT compared to wild-type. Many of these mutations were also identified in S. aureus infecting PwCF. These results indicate that adaptive mutations in pathways associated with important metabolic processes may allow survival with folate inhibition. We hypothesize that limiting aerobic respiration may assist in S. aureus surviving SXT. As S. aureus can survive without oxygen, we are currently studying whether limiting oxygen and, consequently, aerobic respiration in wild-type S. aureus will improve survival with SXT. This work will help us understand the mechanisms of SXT action and S. aureus’ response, in an effort to improve treatment for S. aureus infections.

Adoptive cell immunotherapy (ACT) with engineered T cells has shown impressive efficacy against some cancers, particularly CD19+ leukemias. However, ACT efficacy in solid tumors can be limited by restrictive tumor microenvironments (TMEs), with increased inhibitory signals, reduced T cell infiltration/accumulation, and inadequate metabolic substrates. We engineer T cells with novel switch receptors that combine an inhibitory ectodomain with a costimulatory signaling endodomain, to “replace a break with an accelerator”. FasL is a death receptor ligand that is overexpressed in the majority of human TMEs and can protect tumor cells from immunity by giving T cells inhibitory signals upon binding. We engineered a Fas-4-1BB switch receptor to convert the Fas death signal to a costimulatory 4-1BB signal and demonstrated enhanced Fas switch-receptor-T cell in vivo persistence and therapeutic efficacy in a murine pancreatic cancer model (KPC). We developed new switch receptor candidates by combining CD200R, an inhibitory signaling receptor, ectodomain with various co-stimulatory endodomains. Our aim is to screen for CD200R switch receptors that exhibit the best expression on T cells, best ability in tumor-lysis and proliferation with the least exhaustion in TMEs, and test the best candidates in KPC models. We are able to transduce mouse T cells to express our CD200R constructs, which is verified by flow cytometry, and ready to start in vitro killing assay to assess their ability in tumor-lysis. This T cell engineering strategy may help overcome the restrictive TMEs, catalyze an endogenous immune response, and greatly improve T cell anticancer efficacy.

In neonates, hypoxic-ischemic encephalopathy (HIE) increases the risk of impaired neurodevelopmental outcomes and death. In high resource settings, HIE occurs in 1-4 per 1,000 live births, and in low resource settings, 12 per 1,000 live births. Where available, therapeutic hypothermia (TH) is the standard of care for neuroprotection in HIE. However, the protection provided by TH is incomplete, motivating the search for adjunctive therapies. To assist in testing new therapies, researchers have attempted to predict injury severity in animals before ex vivo quantification, but the association between early injury and long-term outcomes has not been studied in the Vannucci model of hypoxic-ischemic brain injury - the most widely used HIE model in rats. This study sought to determine the association of early injury identified by in vivo magnetic resonance imaging (MRI) with weight gain and behavioral testing data. Using the Vannucci model, postnatal day (P) 10 rats underwent ligation of the left carotid artery followed by 3 hours of hypoxia and then 5 hours of normothermia in room air. Animals were then randomized, with half receiving an additional 3 hours of TH while the other half were returned to their cage. On P12, MRIs were performed, and injury quantification was determined using imaging software FSLeyes. Animals were weighed daily for one week following the HIE injury and then every 2-3 days until P43. Behavioral testing was conducted on P42 and P43 and included open field, novel object recognition, and CatWalk. We hypothesize that greater injury on the early MRI will be associated with reduced weight gain after injury and worse performance on behavioral testing.

Inherited retinal diseases (IRDs) are a diverse family of disorders which cause progressive vision loss and retinal degeneration. IRDs impact over two million people globally, with an estimated prevalence of 1 in 2000 individuals. With more than 280 genes currently implicated in IRD phenotypes, a retinal gene therapy approach offers hope for resolving the underlying genetic causes of IRDs. In 2017, the first FDA-approved retinal gene therapy for Leber congenital amaurosis, an IRD causing severe vision loss at birth, served as an encouraging landmark for their efficacy in therapeutic application. The therapy employs adeno-associated virus (AAV) vectors to replace mutant RPE65 genes. However, AAVs only have a carrying capacity of approximately 4.7kb, significantly limiting their application to other target genes. We hypothesize that lipid nanoparticle (LNP)-mediated delivery of messenger RNA (mRNA) may be a viable alternative to existing retinal gene therapy methods. We characterized cell type specificity of enhanced Green Fluorescent Protein (EGFP) expression in retinas after intravitreal injection of mRNA LNPs to anesthetized male, CD-1 mice. Retinas were subsequently prepared for cross section and flat mount confocal imaging 48 hours after injections. Immunohistochemical staining with SOX9, GFAP, and RPE65 indicated EGFP expression in retinal pigment epithelial cells (RPE) and Müller Glia (MG). These neuronal cell types are impacted in various IRDs and other degenerative disorders, making them promising targets for retinal gene therapy. In future work, we will quantify the intensity and duration of EGFP expression in mouse retinas after intravitreal injection with EGFP mRNA using flow cytometry analysis at timepoints 6 hours to 14 days post-injection. Additionally, we aim to deliver therapeutic replacement mRNAs to improve IRD phenotypes. The results of our investigation may demonstrate the therapeutic prospects of LNP-mediated gene therapy in mouse retinas.

Hypoxia ischemia (HI) is one of the leading injuries in term neonates and can lead to death or long-term disability. The current standard of care is therapeutic hypothermia (TH). Our lab has developed a model for HI injury in rats which is used to test the efficacy of potential treatments. At postnatal day (P) 10, which is the equivalent of a full-term human neonate, rats undergo unilateral carotid artery ligation followed by hypoxia. The animals are given a 30-minute rest period before rectal temperatures are gauged to determine the post-surgical (time 0) temperatures. Animals are then placed in warm water baths for a period of normothermia (NT). Every 15-30 minutes rectal temperatures are recorded, and water baths are adjusted in order for the rats to maintain the goal temperature of 36.5°C (± 0.5°C). After 6 hours of NT, a randomized subset of animals undergo 3 hours of TH to reach 32°C (± 0.5°C). On P43 the rats are euthanized and perfused transcardially for brain removal and formalin fixation. The brains are assigned an injury severity score between 0 and 4, and % injury by volume is detected by MRI. The correlation between time 0 temperature and injury score will be calculated, as well as the correlation between the coefficient of variation of the temperatures for each animal throughout NT and the injury score. Further statistical analysis will be conducted throughout this experiment as the project is currently ongoing, including analysis by treatment group (NT vs TH). This project aims to determine if time 0 temperatures and variation of temperature are indicative of injury severity. Based on a similar study observing the relationship between the time 0 temperatures of ferrets and HI injury severity score, we expect to see that rats with lower time 0 temperature and greater temperature variation will have more severe brain injuries.

 For 9 months, a human fetus is fully dependent on the placenta which provides oxygen and nutrients, regulates waste transport, and acts as an immunological barrier. These 9 months are critical as the placenta has been linked to various health outcomes later in life. Glial Cells Missing Transcription Factor 1 (GCM1) is a transcription factor (TF) that plays a crucial role in placental development. We hypothesize that reducing the expression of GCM1 will affect placenta-specific genes such as the placenta growth factor gene (PlGF), and pathways that control embryonic development such as the sonic hedgehog. Studying how GCM1 alters gene expression in the placenta will reveal specific biological pathways that are affected by GCM1. Primary villous trophoblast cells were collected from 4 male and 5 female placentas following routine c-sections and a siRNA knockdown was conducted to decrease GCM1 expression. High-quality RNA has been isolated and sequenced from these samples. I am using the RNA sequencing data to compare gene expression between two groups: normal placental cells and cells where GCM1 has been knocked down. Using standardized differential gene expression approaches in R, I have identified genes with statistically significant differences between the two groups. Preliminary results indicate that 15 genes are decreased and 2 are increased. A sex-stratified analysis has been performed to determine if fetal sex plays a role in the different placental expression levels between the two conditions. I also performed a pathway enrichment analysis to determine if certain biological pathways are enriched for genes whose expression was associated with a reduction in GCM1. These results will help us better understand the molecular changes that occur due to a reduction of GCM1 and potentially how that contributes to maternal health conditions such as preeclampsia which harms both the mother and fetus.

Advances in molecular techniques have allowed for genome-wide studies to examine which genes are associated with many different human diseases, including those of the skeletal system. In one such study, mutations in the human SOST gene were linked to the skeletal disease Sclerosteosis 1, which presents as hyperostosis in the skull and long bones of the axial skeleton. Functional SOST genes influence the production of sclerostin, a protein which inhibits osteoblastic bone formation. The homologous gene in zebrafish is hypothesized to have a similar function based on ongoing work examining the axial skeleton in mutant zebrafish. Our goal for the current study is to test how the zebrafish cranial skeleton is affected by the SOST gene. I used the open-source 3DSlicer software to place landmarks on micro-CT scans of 27 zebrafish (9 SOST mutants, 9 SOST heterozygotes, and 9 wildtype fish) from the same clutch. I also used 3DSlicer to generate digital models of the cranial skeleton as well as to place 308 pseudolandmark points on the models. From here, I used geometric morphometric methods implemented in R to analyze the complex shape differences between the three groups. Preliminary results suggest that SOST mutants have narrower posterior cranial skeletons than heterozygous or wildtype fish, and that groups may vary in their degree of cranial asymmetry. In addition to quantifying the effect of SOST on zebrafish cranial morphology, this study is part of a larger project to establish a baseline craniofacial analysis method, and create a screening tool for examining genotype-phenotype relationships in genes associated with human skeletal diseases within zebrafish models.

Genome Wide Association Studies enable researchers to identify which genes are associated with human diseases. Once genes are identified, gene editing of zebrafish models allows researchers to further examine the link between genotype and phenotype, with the long-term goal of treatment development. One such gene is MEOX1, which encodes for transcription factor Mesenchyme Homeobox 1. MEOX1 mutations in humans have been associated with Klippel-Feil syndrome, a congenital disorder with the most frequent symptoms being a shortened neck caused by the fusion of cervical vertebrae, leading to a decreased range of motion. Previous studies have established that zebrafish with loss-of-function meox1 mutations present with similar phenotypes in the axial skeleton. However, fusion of the vertebral column could also impact other aspects of fish morphology. The goal of this study is to test if a semi-automated screening tool can be used to quantify craniofacial variations in meox1 crispant zebrafish. Here, I utilized micro-CT scans of CRISPR-modified zebrafish with meox1 mutations (n=12) and compared them to wild type zebrafish (N=12) from the same clutch. I used 3D Slicer to manually landmark 23 major anatomical points on each individual wildtype and crispant zebrafish. I then used a semi-automated process to distribute pseudolandmarks on the surface of each zebrafish. Anatomical differences between the groups were quantified using a geometric morphometrics approach. Preliminary results show that meox1 mutations are associated with a wider posterior section of the skull and a shorter skull length. There were also differences found in the degree of asymmetry between groups. This last result in particular aligns with previous human studies of Klippel-Feil syndrome. The findings from this investigation are important for the understanding of how diseases from meox1 mutations present clinically as well as the testing of a semiautomated pipeline that will be used as a screening tool for crispant zebrafish.

Congenital Heart Defects (CHDs) are the most common birth defect within humans and are characterized by having multiple structural problems with the heart, often leading to problems with blood circulation. Our lab has identified a set of genes potentially involved in birth defects that are also expressed during heart development, thus identifying candidate genes for congenital heart defects. Amongst the gene candidates is a gene encoding proteasome maturation protein (POMP). Proteasomes are the main system for protein degradation, but the role of proteasome factors in CHD development is not known. To establish the importance of POMP and the proteasome in proper heart development, I examined zebrafish embryos that are mutant for the POMP gene and the effects of chemical proteasome inhibitors on zebrafish heart development. Zebrafish (Danio rerio) are commonly used to study human development and birth defects because their genes and organs strongly resemble those of humans, and the zebrafish heart develops in only 3-4 days. Using fish that have been tagged with green fluorescent proteins to visualize heart development, I tracked the developing hearts of POMP mutants and proteasome for signs of heart malformation. I expect to find structural similarities between the developing hearts of the POMP mutants and those with inhibited proteasome function, thus indicating the proteasome system as having a prominent role in CHDs. Gaining insight into the molecular mechanisms and genes behind CHDs aids in the development of human patient diagnoses, targeted therapeutic treatments, and an increased understanding of complex diseases.

Ewing sarcoma (ES) is a bone and soft tissue tumor that primarily occurs in children and young adults. The tumor is driven by an oncogenic fusion gene that fuses the EWSR1 gene located on chromosome 22 to FLI1, an ETS family transcription factor located on chromosome 11. EWS-FLI1 promotes tumorigenesis through transcriptional and epigenetic dysregulation. Despite maximally intensive cytotoxic therapy, the outcome for patients with metastatic ES remains poor, thus the need to identify new therapeutic agents. Given ES’s epigenetic dependencies, there is strong rationale to investigate epigenetic modifying drugs. Bromodomain and extra terminal domain (BET) proteins function as epigenetic readers that facilitate transcription and are upregulated in many tumors. I have shown that BET inhibition slows the growth of ES cells in vitro and in mice. However, BET inhibitors (BETi) will not be successful as a single agent. I hypothesize that combining BETi with other biologically targeted agents will lead to synergistic effects and tumor regression. Based on established literature, RNA-seq data of BETi-treated ES cells, and in silico drug screen, we selected Copanlisib (PI3K inhibitor), GSK-126 (EZH2 inhibitor), PP121 (Src, mTOR, VEGFR2, PI3K inhibitor), Danusertib (Aurora Kinase inhibitor) and Infigratinib (FGFR inhibitor) to test in combination with BETi (BMS-986158). Using standard in vitro cytotoxic assays and calculating synergy using the Chou-Talalay method, my preliminary results showed strong synergy between Copanlisib and BMS-986158. I detected no synergy between BMS-986158 and GSK-126. Our initial results from our in silico drug screen predict that Danusertib, Infigratinib and PP121 may be synergistic with BETi. Ongoing in vitro studies are testing this. Promising combinations will be tested in vivo xenograft models. It is our goal to identify drug combinations from the drug screen that will enhance the cytotoxic effects of BETi in vitro and lead to tumor regression in vivo.

Juvenile Idiopathic Arthritis (JIA) is the most common rheumatic disease in children. It causes joint swelling and stiffness and can last for months to years. Current methods to screen for JIA include Magnetic Resonance Imaging (MRI) and Musculoskeletal Ultrasound, both of which are often time-consuming and expensive. Using our developed thermal imaging algorithm, the ability to screen for JIA would become more accessible, affordable, and less time-consuming to kids and their families. The goal of our study was to determine if using a smartphone-attached thermal camera was reliable for image detection of arthritis in children. We also wanted to test the effect that physical activities, such as walking, would have on lower extremity temperature data within our imaging algorithm. Using an industrial-grade thermal imaging camera, we took thermal images of the lower extremities from the anterior, posterior, medial, and lateral views. We also repeated this using the smartphone-based thermal imaging camera. The temperature data was extracted from the thermal images and analyzed for temperature fluctuations in the regions of interest. Even though the smartphone-attached camera had lower resolution and less precision than the industrial-grade camera, both performed well in their sensitivity and specificity to detect the inflamed joints compared to the common standard of doing a physical joint exam. In addition, the cohort which performed physical activity demonstrated significant temperature changes which were consistent over time and did not return to pre-activity levels. The results of this study show potential for faster and more accessible JIA imaging platforms in the future.

Chronic non-bacterial osteomyelitis (CNO), also known in its severist form as Chronic Recurrent Multifocal Osteomyelitis (CRMO), is a rare, auto-inflammatory disease with no present cure. The disease involves the chronic inflammation of normal, healthy bone without the presence of infection. Currently, no medications have been approved by the US Food and Drug Administration specifically for CNO. Consequently, many different types of medications, including disease modifying anti-rheumatic drugs (DMARDs) and tumor necrosis factor (TNF) inhibitors, and Bisphosphonates are being prescribed off label. However, patients can have significant side effects after taking these medications and consistent reports on the prevalence of these serious adverse events (SAE) among CNO patients are lacking. We will be examining instances of COVID-19 infection, hospitalizations, and psoriasis while taking CNO medication. We aim to investigate the association between taking various medications with the prevalence of SAEs among patients under 21 years old. For our research, we are drawing information from one of the largest CNO clinical research databases, Seattle Children’s Hospital’s database from January 2014 - present day. We hypothesize that all medications will be well tolerated by CNO patients under 21 years old. Through self-reported patient data and physician examination, information on SAE prevalence was collected. The patient population includes 351 patients treated with DMARDs, 294 treated with TNFs, and 89 treated with Bisphosphonates. General statistical methods were used to summarize the data and determine correlation; descriptive statistics was used to report the incidence rate per 100 patient years.As there is minimal knowledge about effective treatments for CNO, we expect that the results of this study will shed light on the reliability of various medications, improving patient disease management and possibly lead to a cure.

Juvenile idiopathic arthritis (JIA) is the most common rheumatic disease in children and frequently presents in knees, followed by ankles, wrists and elbows. JIA is typically evaluated by pediatric rheumatologists using joint exams. However, musculoskeletal ultrasound or MRI with contrast can be used for greater sensitivity but are more expensive and require extensive operator training. In contrast, infrared thermal imaging is a noninvasive tool that is quick, economical, and precise in detecting temperatures of different body parts. Thermal cameras are becoming increasingly accessible and can be used with smartphones, like the FLIR ONE Pro camera by Teledyne FLIR. Recent efforts with temperature after within limb calibration (TAWiC) algorithms have made use of thermal imaging and demonstrated promising results in detecting arthritis in knees and ankles. However, the current TAWiC algorithm implementation has limited scalability due to its dependency on trained technicians to identify key anatomical points. We leveraged existing computer vision libraries like OpenCV and human pose estimation models like OpenPose to automate the TAWiC algorithm. We designed a pipeline that first entails extracting thermal and visible images from a radiometric JPEG generated by a FLIR ONE Pro. The extracted images are subsequently co-registered. Key anatomical points are then labeled in the visible image using OpenPose and contours drawn around the regions of interest. The contours coupled with the labeled key points are used to segment and retrieve the temperatures of specific anatomical regions. We collected preliminary data that demonstrated the feasibility of this workflow. We anticipate that similar TAWiC measurements will be generated when using the automated approach to re-analyze participant thermal images. Automating this algorithm will increase the scalability of this approach and allows for extending this algorithm to other joints.

Fewer than 20% of adolescents and young adults (AYA) with diabetes achieve optimal glycemic control. The use of diabetes technology, including insulin pumps and continuous glucose monitors (CGM), are associated with improved outcomes; however, there are inequities in technology use. The University of Washington (UW) AYA Diabetes Program aims to address disparities in AYA with diabetes by providing tailored education and access to multidisciplinary specialists, including a social worker and psychologist. We examined the impact of the UW AYA Diabetes Program on change in diabetes technology use among AYA and assess whether this varied by sex, race/ethnicity, and health insurance. The study included participants seen in the UW AYA Diabetes Program between 2017-2021. For each binary outcome of insulin pump use and CGM use, we calculated proportions and 95% CIs (confidence intervals) using generalized linear models. We assessed for possible effect modification by sex, race/ethnicity, and health insurance. In this cohort of 453 patients (51% male, 87% non-Hispanic White, 27% public insurance, 55% CGM and pump use) there was evidence of effect modification by health insurance type for both pump and CGM use (p=0.01 and 0.03 respectively) but not by sex or race/ethnicity. Baseline CGM use was significantly lower among public vs private insurance participants (38%, 95% CI: 29-46%; vs 61%, 95% CI: 56-66%) but not after 6-months of program participation (58%, 95% CI: 45-72% vs. 74%, 95% CI: 68-80%). Pump use was also lower at baseline among publicly-insured AYA (29%, 95% CI: 20-37%; vs. 67%, 95% CI: 62-72% for privately-insured). However, by 12-months, pump use for publicly-insured AYA had increased and was no longer statistically significant (44%, 95% CI: 30-59% vs. 64%, 95% CI: 56-72% for privately-insured). We found that the UW AYA Diabetes Program proved to be successful in mitigating baseline disparities and increasing diabetes technology use in AYA with diabetes.

Joubert syndrome (JS) is a neurodevelopmental disorder diagnosed by the appearance of a “molar tooth sign” on an axial brain magnetic resonance imaging (MRI). JS patients present with hypotonia (low muscle tone), abnormal eye movements, and ataxia (uncoordinated movements). Subsets of patients develop progressive medical complications. Autosomal recessive, X-linked and rare dominant causes of JS in >40 different genes can be identified by DNA sequencing in ~75% of families. Cryptic DNA variants and alternative inheritance mechanisms are thought to account for the other 25% of families. The goal of this project is to evaluate the role of non-canonical splice variants in the pathogenesis of JS. We identified candidate splice variants in MKS1 from whole genome and targeted sequencing data and prioritized using SpliceAI annotation. A synonymous variant in patient 1 and a 30 base pair intronic deletion in patient 2 were identified. For each variant, we designed two sets of primers to flank the affected splice junction. Next, we extracted RNA from patient fibroblasts. We converted RNA into complementary DNA (cDNA) and amplified using polymerase chain reaction (PCR). Using gel electrophoresis and Sanger sequencing, we compared PCR products from patients versus controls.  We identified differences in DNA band sizes between unaffected control and patient samples. Based on Sanger sequencing, we determined that exon 4 is skipped in patient 1, but we were unable to determine the specific aberrant splicing event in patient 2. We confirmed the pathogenicity of candidate splice variants, identifying the precise genetic cause. An accurate genetic diagnosis informs prognosis, avoids unnecessary work-up, and guides monitoring for associated complications. Defining all genetic causes of JS expands our knowledge of the genetic mechanisms underlying recessive Mendelian conditions, confirming a substantial role for non-canonical splice variants.

Adolescents with type 1 diabetes (T1D) are at risk for poor physical and psychosocial outcomes. Diabetes-related family conflict has previously been associated with youths' glycemic control (HbA1c). However, less is known about how family conflict associates with other health outcomes. This project aimed to explore correlations between adolescent and parent reported family conflict with diabetes-distress, depressive symptoms, resilience, and health-related quality of life (HRQOL) for both adolescent and parent. Eligible patients were enrolled in a two-site randomized controlled psychosocial intervention study. Participants were ages 13-18 with T1D for over a year and elevated levels of diabetes distress. At baseline, patients and their parent completed measures of: diabetes-specific family conflict (DFCS), HRQOL (T1DAL), diabetes distress (PAID-T), depressive symptoms (PHQ-8), and resilience (CD-RISC). HbA1c was pulled from participants medical charts. Descriptive statistics were used to summarize demographic variables, and bivariate correlation analyses were used to investigate the association between DFCS and the other psychosocial variables. Adolescents (N= 131; 53.4% female, 6.1% identified as another gender, 78.6% White, 9.9% Black, 2.3% Asian, 3.1% American Indian/Alaskan Native, and 80.9% Non-Hispanic, average age 15.38  1.5) DFCS scores correlated with more diabetes-distress (r=0.386, p<0.001), depressive symptoms (r=0.334, p<0.001), and less HQOL (r= -0.303, p<0.001). Parents’ (N=131; 79.4% White, 9.2% Latino/Mexican 6.1% Black, 2.3% Asian, 0.8% other, 79.4% private insurance) DFCS scores correlated with higher youth A1C (r=0.280, p<0.001), higher parent diabetes distress (r= 0.479, p <0.001), and lower parent resilience (r= - 0.200, p = 0.022) and HQOL (r = -0.369, p < 0.001). Both parent and patient reports appear to be an important area of further investigation for determining correlates of poor physical and psychosocial wellbeing in this high-risk group. While further investigation is needed, screening for family conflict may be important in clinical procedure, as it may become a future target for intervention.