The study of neonatal neuroanatomy is a complex field because of the rapid changes occurring continuously. The physical development of tissues is critical to understanding the cognitive development of behavior and learning in the neonate. These structures can be visualized and analyzed with Magnetic Resonance Imaging (MRI). The hippocampal structure in particular is vital to memory consolidation and is therefore key to understanding how newborns build upon acquired knowledge and how this affects their behavior as they grow. This strucure has been studied at many different ages with parallel work analyzing how changes in anatomy result in observable cognitive differences and developments. This study in particular has a rare cohort of scans that were taken shortly after birth. Prematurity is an especially important factor to consider here as it affects physical and behavioral growth. Here we looked at 42 premature MRI subjects and used these to build a temporal model of brain development. Our primary aims of the study are to: (1) develop a protocol for labeling the hippocampus in premature neonate scans, (2) create a growth over time model of the structure in order to automatically segment a larger number of scans and (3) observe how volume varies with behavior as projected by Bayley's Scale of Infant and Toddler Development. So far aim 1 has been achieved and current work is increasing the previous atlas size of 32 scans to 42 in order to improve automatic segmentation. The overarching goal of premature neonatal MRI segmentation is to provide further data for early diagnosis of behavior abnormalities that could develop later in the subject. Results here could produce a meaningful indicator of cognitive development through MRI analysis as well as assist in improving the accuracy of MRI automatic segmentation algorithms. 

Premature births, or births before 37 gestational weeks, are associated with structural and functional abnormalities in the brain. Magnetic Resonance Imaging (MRI) is used to examine brain injury in neonates. Previous work demonstrated correlations between neonatal cortical lobes volumes from MRI and scores on the Bayley Scales of Infant and Toddler Development-Third Edition (Bayley-III), which measures cognitive, motor, and language skills of infants. The occipital lobe is important to diagnosis of visual and neurodevelopmental delays. In this work, I examined the relationship between occipital lobe subdivision volumes and Bayley-III scores. In order to do this, I designed a parcellation or labeling protocol that divided the occipital lobe into two regions based on the calcarine sulcus, the cuneus region and the lingual region. I applied the protocol to the 32 MRI training dataset. Using this parcellation protocol, I created a spatio-temporal atlas of brain parcels, which is a time and space model of brain tissue and parcel probability estimates. This atlas was used to validate our protocol using a leave-one-out validation and to automatically parcellate the larger dataset using EM or Expectation-Maximization labeling. The dataset contained 270 T1-weighted MRI scans from premature neonates who took the Bayley-III at 18 months. Once the larger dataset was labeled, I used multi-variate linear regression, normalizing for head size, to examine the relationship between occipital lobe gray matter volumes and scores on the Bayley-III and analyzed the growth pattern of the regions of the occipital lobe. Both regions showed symmetric and exponential growth patterns. Statistically significant correlations between the lingual region of the occipital lobe and Bayley-III scores were found. This suggests that regional occipital lobe volumes are associated with developmental outcomes in premature neonates.

Social media, as a source for social connection and information, has been shown to be a platform by which users display content related to risky health behaviors such as problem drinking. This type of content has both the potential to influence social norms and behaviors especially in populations that are greatly interacting with social media such as young adults. Snapchat, a social media application (app), is used by 77% of college students at least once a day. As an app, Snapchat allows users to display information through pictures and videos, 10 seconds or less in length, that are then erased or self-destroyed after being viewed by a recipient. This unique feature for transient content and popularity among college students positions Snapchat as a potential platform for displaying both intoxication and general alcohol references due to the lack of permanent display. In the current literature there exists a gap in understanding of alcohol displays and college student behavior in regards to Snapchat. The purpose of this study is to investigate self-reported alcohol content and displays on Snapchat among college students. Surveys will include questions related to participants’ own experience with viewing alcohol on Snapchat, their own display or lack of display of alcohol, which social media platforms they believe have the highest amount of alcohol content, and how likely friends are, if intoxicated, to post to Snapchat. Data from this study could indicate the prevalence of alcohol displays on Snapchat among college students, provide evidence for the monitoring of Snapchat use by peers and friends, as well as support a potential area for intervention.

High-risk human papillomavirus (HR HPV) can establish a persistent infection in epithelial cells through its ability to evade immune detection. HR HPV type E6 is an oncogenic protein that is known to downregulate the innate immune response in human keratinocytes. Previous studies have shown that NFX1-123, an endogenously expressed epithelial protein, acts as a novel binding a partner for HR HPV type 16 E6 (16E6). We examined whether or not NFX1-123 also plays a role in the repression of the innate immune response in human foreskin keratinocytes (HFKs) transduced with HR HPV type 16E6. By stimulating the immune response with Poly(I:C) or double stranded DNA in HFKs, cells with overexpressed levels of NFX1-123 (FWT), and cells with endogenous levels of NFX1-123 (LXSN), we demonstrated that stimulation occurs both at the RNA and protein levels. We found, however, that the immune response of 16E6 HFKs with overexpressed levels of NFX1-123 were more downregulated than those with endogenous levels, confirming the protein’s association with 16E6 in the repression of the innate immune response. By understanding the roles of 16E6 and NFX1-123, we will further understand how together they cause a persistent infection, which can ultimately lead to cancer.

Eating disorders (ED) such as anorexia and bulimia nervosa currently affect more than 30 million individuals in the United States. Eating disorders are linked to adverse health consequences including osteoporosis, multi-organ damage, and even death. Many studies have found a clear association between social media exposure and social comparison with idealized body figures leading to disordered eating. It remains unknown the impact that Instagram, a visually driven social media platform has on individuals already experiencing the symptoms of disordered eating. The purpose of this study is to compare the attitudes and motivations displayed on pro-ED and ED-recovery Instagram profiles, in order to understand how social media platforms can be utilized as a tool for encouraging and sustaining ED recovery. In this retrospective content analysis study, 30 public pro-ED and 30 public ED-recovery profiles were selected based on the profiles use of specific ED related hashtags. Posts within this three month period on these profiles were thematically categorized and coded based upon themes in the grounded theory tradition. Data collected from both types of profiles were comparatively analyzed. Descriptive data included the quantity of followers, comments, and likes received per post displayed on the profile. With the pro-ED and ED-recovery data, categorical and quantitative comparisons were conducted using Chi-squared tests and t-tests. Findings from this study could potentially inform ED related agencies and clinicians on how social media can be used as a tool for effective prevention, screening, and early treatment for eating related disorders.

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

Epileptic encephalopathies (EEs) are devastating childhood epilepsies characterized by multiple seizure types that are usually refractory to medication. Next-generation sequencing has revealed the prominent role of de novo mutations in causing EEs with clinical diagnostic labs reporting a molecular diagnosis in as many as 24-40% of cases. Despite this significant progress, the majority of cases are unexplained. Exome sequencing, a next-generation sequencing technique, allows one to look at all the protein coding regions of an individual’s genome, offering an affordable and unbiased approach to gene discovery. Using exome sequence data for 50 trios (proband and unaffected parents), in which the child had seizure onset in the first 2 years of life, we identified 76 candidate genes, of which 37 are confirmed de novo variants through Sanger sequencing. Notably, 2 affected individuals were identified to have a de novo variant in the guanine nucleotide-binding protein (G-protein) beta1 subunit, GNB1. This was a statistically significant finding (p=0.005), and in collaboration with colleagues we have now identified 13 patients with neurodevelopmental delays with and without seizures. This previously unrecognized genetic condition is characterized by global developmental delays (13/13), hypotonia (11/13), seizures (10/13), ocular manifestations (8/13) and growth delay (6/13). These genetic findings will have immediate impact to clinicians and families when discussing diagnosis and prognosis, and hopefully guide future studies of finding effective therapies for these patients. Future work will screen for genetic variants in the candidate genes identified by exome sequencing using a targeted sequencing approach in a cohort of 600 patients with EE of unknown etiology.

Body image dissatisfaction is a leading contributor to the development of eating disorders. Eating disorders have the highest mortality of all mental illnesses, are most common among females, and most often develop between ages 16 and 20. Research shows that Facebook use is associated with increased risk of eating disorders among female college students, and that appearance comparisons mediate this relationship. However, it remains unclear whether Instagram increases the risk of eating disorders among this population. Instagram is an image-based social networking platform that is more commonly used among women compared to men ages 18-29. The purpose of this pilot study is to examine the influence of Instagram use on body image perceptions among college females. Higher Instagram use is expected to be associated with an increased risk of body dissatisfaction. Female undergraduate public health majors were recruited through an email list-serve to complete an online survey. The survey asked about frequency and patterns of Instagram use. Additional questions assessed appearance comparison to peers, family members, celebrities, and models on Instagram. The survey also assessed prevalence of and appearance comparison to fitness motivation accounts on feeds. Finally, body dissatisfaction is assessed with the Body Dysmorphic Disorder (BDD) scale. Analyses include descriptive statistics for quantitative data and thematic analysis for open-ended responses. Predictive models will be created to predict BDD from Instagram use per week and per day. Results include descriptive statistics of demographic information, Instagram use, and body dysmorphic scores. Assessment of social group appearance comparison on Instagram will also be included. Additionally, open-ended responses may reveal themes in fitness motivation accounts or general Instagram use. Findings will inform relationship patterns between Instagram use and body image dissatisfaction. Future studies could determine whether Instagram should be targeted for eating disorder interventions.

Epileptic encephalopathy (EE) is an umbrella-term for a class of severe epilepsies characterized by debilitating seizures. Epileptic activity often begins in early adolescence, contributing to developmental, cognitive, and behavioral abnormalities. Identification of genetic mutations in EE-patients has implicated >50 genes, with clinical diagnostic labs reporting a molecular diagnosis in 24-40% of cases. Current EE gene discovery focuses primarily on identifying de novo mutations in genomic coding regions, which alter the amino acid sequence of the resulting protein, disrupting the protein’s function and leading to neural abnormalities. A de novo mutation is not inherited from either unaffected parent; it first occurs in an affected individual, suggesting a genetic cause for the phenotype. My research focuses on identifying novel noncoding mutations in a cohort of 167 patients with EE of unknown cause. The proportion of rare diseases caused by noncoding mutations is unknown and relatively few examples of functional noncoding causative mutations exist. My goal is to identify de novo variants in intronic and untranslated genomic regions, and also to identify synonymous variants in which the resulting amino acid is unaltered. Initially, the 167-patient cohort was screened for variants in 44 established EE genes using targeted next-generation DNA sequencing. I have filtered this data for high-quality noncoding variants, which are unreported in a healthy population. I am currently screening patients’ parents for these noncoding variants to determine the mode of inheritance. Confirmed de novo variants will be followed-up with functional studies to test their molecular consequences (via mRNA, splicing, gene expression). Determining genetic causes of EE unveils molecular mechanisms behind phenotypes, allowing for precise diagnoses, and leading to treatment of severe epilepsies based on their causes. I hope to identify causative noncoding variants in epilepsy genes to expand epigenetic analysis of EE, and contribute to a better understanding of EE genotype-phenotype relationship.

The Mefford lab is identifying the genetic mutations that cause early-onset epileptic encephalopathies. This is a rare class of epilepsy that is characterized by refractory seizures that often begin in infancy and carry a poor prognosis for the patient. We accomplish this by identifying de novo mutations in affected children using Sanger sequencing to compare the children’s DNA to that of their unaffected parents. This approach, however, lacks the sensitivity to detect genetic mosaicism. A mosaic mutation occurs post-fertilization and causes one’s body to contain cells with different genotypes from one another. These mutations can be difficult to identify with Sanger sequencing because they are often present at very low levels. However, recent advances in gene-sequencing technology has enabled us to detect mosaicism in patients at increasingly lower levels via deep sequencing using smMIPs (single-molecule molecular inversion probes), a technique that allows us to quantify the number of alleles assayed. Recent studies have reported that 1.7% - 10% of alleged de novo mutations were actually inherited from a mosaic parent, inspiring us to find the rate of falsely identified de novo mutations in our own research. We re-sequenced the parents in our cohort of 50 solved de novo cases using smMIPs to investigate how often the mutation had actually come from a mosaic parent. This revealed 10 parents as candidate mosaic carriers. We are still performing further testing on those parents and are adding more families to the study. Knowing the origin of a mutation is crucial for genetic counselors, and knowing whether a child’s epilepsy came from a de novo or a mosaic mutation in the parent can have huge effects on the recurrence risk of the disease. The data from this study will also help other researchers assess the accuracy of their research on disease inheritance as well.

Dandy-Walker Malformation (DWM) is the most common cerebellar malformation in humans affecting one in every 10,000 live births. DWM is characterized by underdevelopment of the cerebellar vermis, as well as an enlarged fourth ventricle and posterior fossa. Symptoms range from intellectual disability to autism, although some patients are minimally affected. Recent research performed in our laboratory implicated deletion of a transcription factor called FOXC1 in DWM. Surprisingly, Foxc1 expression is absent in the developing cerebellum of mice. Instead, it is abundantly expressed in the meninges overlying the growing cerebellum. Therefore, we hypothesized that Foxc1-mediated meningeal signaling plays a critical role in cerebellar development. In Foxc1-/- mice, we used a combination of immunohistochemical and in vitro assays to demonstrate devastating decreases in neuronal proliferation in the cerebellar ventricular zone, concomitant with increased neuronal differentiation during crucial embryonic stages of cerebellar development. These contributed to a severe reduction of the neural progenitor pool and significantly reduced cerebellar size. Aberrant cell migration and dysmorphic radial glial cells were also detected in mutant mice. Sfd1α is a Foxc1-dependant secreted factor in the meninges that signals to Cxcr4 in the cerebellum. We demonstrated that disruption of Sdf1α-Cxcr4 signaling in Cxcr4 mutant mice phenocopied the Foxc1-/- mutant cerebellum. We conclude that interactions between head mesenchyme and the early embryonic cerebellum are crucial to the development of the cerebellum. Most recently we have extended these studies to show that the developing cerebellum in FOXC1 +/- human DWM patients largely resembles the Foxc1 and conditional Cxcr4 mouse mutant phenotypes. This suggests that key mechanisms controlling early cerebellar development are conserved between mouse and human. This also strongly argues that we have identified the key developmental pathological disruptions causing human DWM.

The prevalence of non-medical prescription drug use has recently increased dramatically. It is estimated that 52 million Americans over the age of 12 have used prescription drugs for non-medical purposes in their lifetime. Prolonged use of benzodiazepines, and Alprazolam (Xanax) in particular, has detrimental effects ranging from physical and psychological dependence to increased risk of Alzheimer’s disease. Xanax displays are present on social media such as Twitter, a popular social networking site with approximately 320 million users. These social media displays provide novel opportunities to capture epidemiologic trends in displays about Xanax. While Xanax abuse is more common among females, it’s unclear whether and how Xanax social media displays are present across genders. The purpose of this study is to examine the different types of Xanax displays on Twitter and compare them between males and females. To identify profiles, we identified common Internet nicknames for Xanax including “Xanax”, “xan”, “zanland”, “zannies”, and “zans” and will use these as search terms on Twitter. Inclusion criteria include profiles being publicly available and the gender of the profile owner clearly being determined by the profile picture and/or pronouns used in their tweets. Profiles were excluded if the age was clearly stated under 18 years old. One hundred participants’ Twitter profiles will be analyzed. We will conduct content analysis on the profiles’ tweets for a 3 month span. The tweets will be coded into 1 of 3 categories based on content: anti-Xanax sentiment, Xanax use in reference to “blacking out”, and describing Xanax co-use with another substance. Analyses will include evaluating the proportion of Xanax-related tweets among total amount of tweets in the coding period and will be compared between males and females. Results from this study could inform gender based interventions for prescription drug use.

Cleft lip and palate (CLP) is one of the most common birth defects and has been found to be associated with multiple dentofacial anomalies, including hypoplastic teeth, and defects of the orbicularis oris muscle. In view of this associated spectrum of anomalies, numerous studies have investigated the risk of caries development in the CLP population, with the vast majority reporting an increase in caries incidence and severity. However, the cause of the observed increase in caries is largely unknown, making it difficult to manage and significantly prevent. In the Cox lab, we have established conditionally targeted mice for the cleft gene, Irf6 (Irf6 null), by deleting the gene only in the oral epithelium after the development of the lip has been completed, thus permitting investigation of the gene’s other roles in the oral phenotypes. Therefore, these mice provide an ideal system with which to investigate the susceptibility and risk factors for caries progression in CLP population. Initial observations revealed these mice to have an array of tooth anomalies and salivary gland hypofunction. Using null and control mice, we employed an established caries induction protocol that involved infection of the oral cavity with S. mutans, a cariogenic bacterium, followed by feeding with a high-sucrose diet for eight weeks. After eight weeks, bacteria were recovered from the animals by sonication of the lower jaw in normal saline and bacterial numbers counted and the existence and severity of caries scored. The data reveal a massive increase in carious lesions in the CLP mice but not controls and this was associated with a significant increase in both total cultivable flora and the S. mutans population (5.2x105 in Irf6 null vs. 2.6 x105 wildtype mice; p value<0.001). These results may ultimately translate into more effective caries preventive approaches in patients with cleft lip and palate.

Impulsivity is defined as behavior characterized by little to no forethought of the consequences of an action, particularly one involving risk taking. Impulsivity is a multifaceted personality trait that can take on both pathological and non-pathological, but still problematic, forms. A period of time characterized in part by heightened impulsivity is adolescence, defined as ages 10-25 years. Currently, 81% of adolescents use some kind of social media; however, the manner in which impulsivity exists in this setting is largely unknown. Because social media has been demonstrated to affect older adolescents’ relationship with substance use, understanding their impulsivity may inform interventions to prevent future problems with substance use. Therefore, this pilot study aims to understand the relationship between older adolescents’ impulsivity, social media use, and substance use. Participants, ages 18-22, were recruited through a snowball sampling method on a college campus. Participants were invited and completed (1) a validated impulsivity questionnaire, (2) a social media profile evaluation of their Facebook account for displayed references to substance use on their Wall and Photos within the past 6-months, and (3) a semi-structured phone interview intended to provide self-reported relationships of impulsivity, social media use, and substance use. Analysis included examining correlations between participants’ impulsivity scores provided by the questionnaire, displayed social media use and substance use, and content from the interview. Expected results include finding a positive correlation between participants’ impulsivity, social media use, and substance use. Implications of this research will guide future studies examining impulsivity, social media use, and substance use, and potentially provide evidence for future interventions for adolescents.

Alcohol use is associated with high amounts of significant injury and mortality among college students. Experimental research has revealed gender differences in how males and females communicate about alcohol use, with females favoring more moderate terms such as “tipsy." However, it is unclear whether these gender differences are present on social media. The purpose of this study was to explore the gender differences in alcohol and intoxication terms in status updates (SU) posted among college students. Incoming freshmen from two large state universities were identified through registrar lists and recruited to a longitudinal study. Participants accepted a Facebook friend request from the study team. During freshman and sophomore year, participants’ Facebook posts were evaluated for references to alcohol using a validated codebook. SU with alcohol references underwent further analysis. Content analysis included reference type (i.e., alcohol state, type of drink, etc.) and whether alcohol use was problematic based on a validated problem drinking criteria. Inter-rater agreement was 83.3%. A total of 166 alcohol references were eligible for further analysis. Of ‘alcohol state’ posts (i.e., drunk, tipsy, etc.), females used the term ‘drunk’ 88% of the time with no mention of tipsy or buzzed. No gender difference between displays of problematic alcohol use was observed. Overall, the gender differences frequently present in studies of non-digital settings were often not observed on Facebook. This data could prove useful in gender-specific prevention and intervention efforts, and understanding the differences in interactions observed on social media compared to real life interactions.

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

Congenital heart disease is a problem with the structure of the heart that is present at birth. In the United States, about 40,000 babies are affected by this disease annually. In our lab, zebrafish are used to investigate the genetic causes of this disease. Previous research investigations from our lab have shown that two important genes that are responsible for the regulation of heart muscle are Pbx4 and Pbx2, which are homeodomain transcription factors. Pbx2 and Pbx4 are genes of interest because their roles in heart development are not completely understood. CRISPR/Cas9 method is a method that can be used to target specific DNA sequences and create modifications in the genome. We used this method to engineer different strains of zebrafish with a variety of Pbx gene alleles to determine the role of the Pbx factors in heart development. In order to figure out how the different alleles affect the Pbx genes, each of the strains were observed for phenotype. The null alleles of Pbx2 and Pbx4 were used to demonstrate that embryos lacking both of these factors have severe defects in heart muscle differentiation and outflow tract formation. Then, in-frame deletions were made in the Pbx4 gene demonstrating the importance of an alanine tract for critical functions in heart development. Next, a strain was engineered which contains a Pbx gene variant associated with congenital heart disease in humans. Crosses were set up between strains to compare the effect of each mutation. Thus, the use of genome engineering allowed us to conduct analyses of vital transcription factors involved in heart development.

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

During pregnancy, women and the developing fetus face the risk of invasive bacterial infection, which can lead to adverse birth outcomes such as preterm birth or fetal death. Inflammatory cytokines play a key role during infection-related preterm birth. Previous studies have shown that lipopolysaccharide (LPS), a gram-negative cell wall component, causes increases in inflammatory cytokines and preterm birth in mice. However, limitations exist when comparing the pregnant mouse model to humans. Recently, we conducted a non-human primate (NHP) experiment to identify putative targets of inflammation caused by LPS. However, contrary to expectation, no signs of preterm labor were observed. To determine if the LPS batch used in the NHP model induced inflammation in immune cells, I used an in vitro human macrophage cell culture model.  A human immortalized monocyte cell line, THP-1 cells, was differentiated to macrophages using phorbol myristate acetate (PMA) and treated with a range of concentrations of LPS, similar to those used for the NHP experiment. Cytokines in the medium were quantified by the Enzyme-Linked Immunosorbent Assay (ELISA). LPS increased IL-1B in a dose-dependent manner. Additional inflammatory cytokines associated with preterm birth such as, IL-6, IL-8, and TNF-a, will also be measured. The results will be compared to the ELISA results of the same cytokines released in the NHP samples. This work will guide insight into addressing the inflammatory response associated with preterm birth.

Cysteine knot peptides (knottins) are small proteins found in a wide variety of organisms. Due to their durable cysteine knotted core and small size, knottins are a promising new drug scaffold. Optimized knottins (optides) bind with high specificity to certain cancer cell types indicating that optides have the potential to deliver drug payloads or be utilized as tumor imaging tools. In order to determine if optides are localizing to certain organs or tissues of interest they must be radiolabeled and injected in vivo. The goal of my project was to develop a method to recover radiolabeled optides from mouse plasma or tissue samples and confirm via liquid chromatography that the optides are intact and reaching their target. I developed a column chromatography extraction technique that filtered out plasma proteins in order to recover optides. Analysis of the optides using high performance liquid chromatography showed high recovery rates and preserved structural integrity. The second phase of the project was to do a plasma stability assay. I incubated samples in mouse plasma over a time course and used the extraction method to determine if samples were degraded by plasma proteases and found that there was no visible degradation after incubation. This project set the groundwork for a more detailed examination of optide modifications that could lead to longer optide half-life in plasma and greater drug delivery. This recovery technique is a critical tool for analyzing the pharmacokinetics and biodistribution of optide drug candidates. 

Epileptic encephalopathies (EE) are severe forms of childhood epilepsies. Recent studies have shown that some cases of pediatric EE are caused by copy number variants (CNV), which are chromosomal deletions, duplications, or insertions of DNA. CNVs may contain genes that are important for neural health, causing disease when there is a copy number change. I am investigating rare CNVs in EE patients where the genetic cause is not known. I will screen EE patients for CNVs using a technique called array comparative genomic hybridization (aCGH) to identify rare CNVs that may be disease-causing. If a CNV is found, I will conduct aCGH on parents to determine if the CNV is de novo (arising for the first time in that individual and not inherited from either parent). We also expect that in cases with multiple affected family members, patients may inherit rare mutations from an affected parent. Preliminary results show that out of 69 patients screened thus far, 15 rare CNVs that range in size and number of genes affected have been identified in 13 patients. Of these, 3 de novo CNVs were identified that include a single exon deletion of the gene SLC2A1, a partial deletion of the gene NRXN1, and a 2.4 mb deletion in chromosomal region 7q11.23, affecting 40 genes. In addition, 2 patients each have a 1.4-1.5 mb deletion encompassing the region 16p13.11, which has been reported to be associated with epilepsy and developmental delay. Parental and segregation testing is ongoing for many patients. Rare CNVs can highlight genes implicated for proper neural function and development. This will help advance our understanding of the pathology and genetic basis of epilepsy. Furthermore, identification of particular pathogenic genes will be useful in the clinical setting for genetic counseling of affected patients and families.