ABSTRACT IMPACT: Investigating the relationship between ICH volume at presentation and cortical atrophy over time may help to explain the phenomenon of cognitive impairment after ICH. OBJECTIVES/GOALS: Non-traumatic intracerebral hemorrhage (ICH) is the most common type of hemorrhagic stroke. ICH causes both mechanical as well as oxidative injury leading to neurologic deterioration. However, the relationship between ICH volume and brain atrophy is not fully understood. To that end, we aimed to investigate this relationship. METHODS/STUDY POPULATION: A retrospective cohort of adult ICH patients over a 10-year study period were studied. Demographic data, ICH cause, location, laterality, and treatment, as well as other data, were collected. DICOM files of CT or MRI at presentation and all subsequent follow-up imaging up to 5-years post-ICH were obtained. Using a novel semi-automated image segmentation tool developed in MATLAB by our group, ICH volumes were segmented. Using T1w MRI data, ipsi- and contralateral brain volumes were segmented using NeuroQuant, a fully automated software for MRI volumetric processing, for both initial and follow-up MRIs. Ipsilateral and global cerebral volume changes over time were calculated using initial and follow-up MRI images. Spearman rank correlation between volume changes and ICH volumes were calculated for each patient. RESULTS/ANTICIPATED RESULTS: 75 spontaneous ICH patients with adequate imaging follow-up and both early and follow-up MRI comparisons were considered. Of these, 14 patients had MRIs adequate for segmentation by NeuroQuant. There was a positive correlation (R^2 = 0.72, p=0.03) between ABC/2, the traditional ICH volume measuring approach, and our semi-automatic segmentation tool, with ABC/2 tending to overestimate ICH volume. There was an average of -6.51% of total brain volume loss with respect to initial brain volume at follow-up. There was a negative correlation between ICH volume and both global (Spearman rank correlation coefficient (R)=-0.714, p=0.004)) and local atrophy (R=-0.785, p=0.0009), meaning that as ICH volume increases, there is greater brain volume loss. DISCUSSION/SIGNIFICANCE OF FINDINGS: Greater ICH volume is associated with greater brain volume loss both ipsilaterally, reflected as encephalomalacia, and globally. These findings are important as encephalomalacia can result in focal neurologic deficit and other neurological symptoms over time, while global brain atrophy is associated with dementia and cognitive decline.

ABSTRACT IMPACT: This work describes, for the first time, the methylome in patients with T-LGLL, focusing on the IL-15 promoter, and clearly demonstrates that 5-azacytidine decreases IL-15 production leading to T-LGLL cell death. These results form the basis a translational clinical trial in T-LGLL that will begin accrual in 2021 OBJECTIVES/GOALS: T-LGLL is an incurable leukemia with few treatment options driven by overexpression of IL-15. Our objective is to characterize the methylation status of the IL-15 promoter in T-LGLL patients and evaluate the potential use of 5-azacytidine (5-aza) in a translational trial by studying the effect of 5-aza in vitro on IL-15 levels, and the IL-15 promoter. METHODS/STUDY POPULATION: We sorted T-LGLL patient (n=3) and normal donor (ND) samples (n=3) for CD3+/CD8+/CD5-/dim for T-LGLL immunophenotype. We analyzed DNA methylation and gene expression profiling using reduced representation bisulfite and RNA sequencing and determined differential methylation and gene expression using 1-way ANOVA analysis. To determine the functional significance of differential methylation, we evaluated MOTN-1 T-LGLL cell viability in vitro with 5-aza at increasing concentrations. Next, we evaluated IL-15 gene expression in MOTN-1 cells treated with 5-Aza versus MOTN-1 with control using western immunoblot. Finally, we exposed MOTN-1 cells to a novel IL-15 inhibitor, IBI-15, and compared cell viability against MOTN-1 cells exposed to an inactive control. RESULTS/ANTICIPATED RESULTS: There was significant differential methylation (P= 0.0178) and expression (P =0.0059) in T-LGLL patients vs ND. These data revealed significant differential hypermethylation of gene promoters, including an increase in DNA methylation of the IL-15 promoter in T-LGLL cells vs ND. In MOTN-1 cells treated in vitro with 5-Aza at 24 and 48 hours, a dose-dependent decrease in the viability of T-LGLL cells was observed, from 100% to 49.5%, p=0.037. Further, a marked decrease in IL-15 expression was observed at all concentrations of 5-aza compared to control (p=0.0001). Finally, a decrease in cell viability was observed utilizing the IL-15 inhibitor IBI-15 vs control. These results confirm that 5-aza leads to decreased transcription of the IL-15 gene, possibly due to hypomethylation of the IL-15 promoter. DISCUSSION/SIGNIFICANCE OF FINDINGS: Hypermethylation of the IL-15 promoter and subsequent increase in IL-15 is critical to the pathogenesis of T-LGLL. Inhibition of the IL-15 promoter by 5-aza leads to down-regulation of the IL-15 gene transcript, which is sufficient to induce T-LGLL cell death. Based on these results, a phase I trial will be conducted using CC-486 (oral 5-Aza) in T-LGLL.

ABSTRACT IMPACT: This study characterizes interactions between human limbic circuitry and ventral temporal cortex using single pulse electrical stimulation, which may inform emerging stimulation therapies for epilepsy. OBJECTIVES/GOALS: The goal of electrical brain stimulation treatment is to modulate brain network function. However, stimulation inputs to different brain sites alter the network in a variety of ways. This study examines that variability by characterizing responses in a target region while stimulating multiple other brain sites. METHODS/STUDY POPULATION: We measured voltages in intracranial EEG in 6 patients who had electrodes implanted for epilepsy monitoring. We stimulated pairs of electrodes at multiple sites in the brain with a single pulse every 5 to 7 s and measured the resulting corticocortical evoked potential (CCEP) responses in the ventral temporal cortex (VTC). Using a novel clustering method, we uncovered sets of distinct canonical response shapes from the 20 to 500 ms post-stimulation period. This allowed us to group stimulation sites that evoked similar responses. We then related each group to high frequency, broadband, changes in spectral power as a reflection of local neuronal activity. RESULTS/ANTICIPATED RESULTS: We found that the VTC receives strong inputs specifically from the amygdala and hippocampus, both in terms of amplitude and broadband spectral power change. However, inputs from the hippocampus produced a different canonical shape than those from the amygdala. We also observed that VTC responses to inputs from the insula clustered in shape with those from the amygdala. These clustering patterns were consistent across subjects, although the actual shapes of the clusters showed variability. We further observed that some shapes were more associated with increases in overall neuronal activity than others, as reflected by broadband spectral power change. DISCUSSION/SIGNIFICANCE OF FINDINGS: Stimulation of connected sites may drive excitability at the target region in ways that are described by sets of full-time-course responses. By capturing their shapes, we can begin to decipher canonical input types at the circuit level. This approach might identify how stimulation inputs can be tailored to therapy while mitigating adverse effects.

ABSTRACT IMPACT: A single seismocardiography (SCG) parameter has been shown to accurately classify aortic valve disease (AVD) status in healthy controls and AVD patients. This could support development of SCG as a quick, inexpensive screening tool to better tailor MRI examination to patients’ needs. OBJECTIVES/GOALS: MRI is used commonly for monitoring of aortic valve disease (AVD), but it has high costs. We hypothesize that energy in seismocardiograms (SCG)’‘ signals from chest surface vibrations’‘ is different between healthy controls and AVD patients, and we evaluate potential efficacy of using SCG to recommend MRI only for patients with flow abnormalities. METHODS/STUDY POPULATION: With IRB approval, 45 healthy control subjects (47 ±18years, 18 female) and 9 patients (63 ±16years, 2 female) with aortic valve disease history and known flow abnormalities were recruited. SCG signals were acquired supine, immediately prior to MRI of thoracic aortic blood flow at 1.5T with a time-resolved phase contrast (4D Flow) sequence.

The SCG was processed to calculate late-systole high-frequency (120-240Hz) RMS energy. MR velocity images were analyzed to measure peak velocity and trace pathlines of flow.

Screening efficacy of the SCG energy metric was assessed, with hypothesis testing for differences in energy level distributions between controls and patients, and receiver-operator characteristic (ROC) analysis was used to calculate rates of correct/incorrect classification of disease. RESULTS/ANTICIPATED RESULTS: Healthy subjects had coherent flow pathlines through the aortic arch and mid-ascending aorta peak velocities of 106 ±21cm/s (cohort mean ±standard deviation). All valve disease subjects had flow abnormalities, such as jetting flow near the valve or swirling through the arch, as visualized by pathlines. Patients’ peak mid-ascending aorta velocities were 167 ±69cm/s. The SCG energy for healthy controls was significantly different than that of valve patients (-5.6 ±0.3dBmm/s/s vs. -4.0 ±1.2dBmm/s/s respectively; p<0.001). Thresholding SCG energy to distinguish patients from controls correctly classifies subjects with a high true-positive rate and low false-positive rate. The ROC for this classification has area-under-curve 0.956. DISCUSSION/SIGNIFICANCE OF FINDINGS: A high potential screening efficacy was observed using a single, linear SCG metric to identify AVD patients with flow abnormalities. If used to complement MRI surveillance protocols for AVD, this method has potential to serve as a quick, inexpensive tool for better tailoring MRI exams to patient needs.

ABSTRACT IMPACT: Through conducting this systematic review and meta-analysis, we will elucidate which factors influence thoracic aortic aneurysm growth, which will further help clinicians to properly stratify and manage their patients with TAAs. OBJECTIVES/GOALS: Thoracic aortic aneurysms (TAA) are an indolent but fatal disease, and the patient characteristics that predict both overall growth and growth rate are still not well characterized. Our goal is to conduct a systematic review and meta-analysis in other to better describe different patient characteristics that predict TAA growth. METHODS/STUDY POPULATION: M.M. conducted a search of Ovid MEDLINE, Embase, and Scopus to identify articles. Inclusion criteria were any longitudinal study reporting asymptomatic TAA growth, growth rates, or clinical proxies for growth such as dissection, rupture, emergency surgery, and death. M.H and P.B. independently applied the criteria to the results of the search. Conflicts were resolved by N.B. Data was extracted and risk of bias assessed independently by M.H. and P.B. Summary estimates of the outcome variables are combined across studies using standard meta-analysis methods. Heterogeneity is assessed via forest plots, chi2 test (Q test), and I2 statistic. Sensitivity analysis is conducted to assess robustness of the findings. RESULTS/ANTICIPATED RESULTS: The literature search resulted in 3,419 abstracts, of which 176 were included and thus require a full text review. Cohen’s Kappa coefficient was 0.64, indicating substantial agreement and high inter-rater reliability. We describe four categories of patient characteristics influencing the growth of asymptomatic TAAs: demographics, genetic or inheritable conditions, hemodynamic or biomechanical factors, and serum biomarkers. We describe the measure of effect for all variables. We anticipate there is a significant level of heterogeneity between studies, and potentially moderate risk of bias for many of the included studies as they are retrospective and observational in nature. Furthermore, we anticipate publication bias and evaluate it with funnel plots. DISCUSSION/SIGNIFICANCE OF FINDINGS: Understanding the factors that influence the growth of asymptomatic thoracic aortic aneurysms (TAA) is paramount, as the size and growth rate of TAAs dictates the clinical course. Little is understood about the degree to which different characteristics influence growth. This meta-analysis will help elucidate factors that promote TAA growth.

ABSTRACT IMPACT: This study will answer key questions that spine surgeons have regarding techniques used in cement augmentation of vertebral compression fractures and will ultimately advance patient care for such injuries. OBJECTIVES/GOALS: The objective of this study is to determine if a difference exists in load-bearing characteristics and load-to-fracture between injecting cement anteriorly prior to screw placement versus cement augmentation via fenestrated pedicle screws. We also expect differences in load-to-failure characteristics between different cement volumes. METHODS/STUDY POPULATION: This study will be performed in a bioengineering laboratory that has access to a Materials Testing System (MTS). Eight cadaveric specimens will be selected from our stock after pre-screening via CT for inclusion and exclusion criteria. The levels T8-L1 will be dissected from the vertebral column along with any soft tissue structures. The vertebral bodies will be potted in an epoxy mold. From each spine, there are 2 groups of three. One vertebral body from each spine will serve as an internal control, one will be augmented with cement via a cannula and then instrumented with a non-fenestrated screw and the third will be instrumented will a fenestrated screw and then augmented with cement. After appropriate curing time, repeat CT imaging will be completed. The specimens will then be loaded to failure and the results analyzed. RESULTS/ANTICIPATED RESULTS: We hypothesize that we will see a better anterior spread with the cannula/non-fenestrated screw method as compared to the fenestrated screw. The reason being is that we would expect the fenestrated screw to experience more cement extruding from the fenestration rather than being directed anteriorly. We believe a better anterior spread of the cement will lead to a greater load-bearing capacity for the vertebral body. We also believe that a difference will exist in load-to-failure testing with the two volumes being tested, though we cannot predict to what a degree this difference will be impactful as there have been few studies prior looking at this. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study is significant because it will aid in determining the optimal technique to implement in the setting of vertebral compression fractures. This will lead to improved patient care as well as a greater understanding of the instrumentation used in such procedures. The results will lay the groundwork for future research on this procedure.

ABSTRACT IMPACT: This project uses rapid prototyping, 3D printing, and cell seeding to solve the most common post-operative complications that arise from the current methods of urethral elongation during phalloplasty. OBJECTIVES/GOALS: Post-op fistula and stricture formation occur in up to 50% of phalloplasty patients. These complications arise from the mismatch between skin and uroepithelium, or from scarring secondary to ischemia. Here we describe the fabrication of a novel vascularized urothelial flap for phalloplasty that contains discrete urothelial and vascular channels. METHODS/STUDY POPULATION: A custom designed 3D negative mold, with a urethral channel and a vascular inlet and outlet channel was prototyped in Adobe Fusion 360 and printed on a Prusa i3 MK3S printer in PLA. A 2mm diameter pluronic sacrificial macrofiber was used to connect the channels to form a vascular loop, and 1% type-I collagen was extruded over the mold. After solidifying, the scaffold was demolded and seeded with grade I urothelial carcinoma (SW780 cells, at 5-10 x 106 cells/mL) in the urethral channel, and adenovirus-infected E4 endothelial cells (at 3x106 cells/mL) in the vascular channel. The scaffolds were cultured up to 14 days and then fixed for histologic analysis. RESULTS/ANTICIPATED RESULTS: Collagen scaffolds were fabricated reliably using the custom 3D negative molds. After both seven and fourteen days of culture, the urothelial channel contained a robust, stable urothelial monolayer lining throughout the channel. By 14 days urothelial multilayer formation was seen, providing definitive evidence of a more mature urothelial layer. Grooves within the collagen allowed for nests of urothelial cells to develop, leading to increased multilayer formation. In addition, the vascular channels supported a healthy endothelial lining at both seven and fourteen days. There were no significant histological differences between constructs seeded with 5x106 urothelial cells/mL and 107 cells/mL. We anticipate that multilayer formation will increase with time, and that constructs will survive beyond 28 days. DISCUSSION/SIGNIFICANCE OF FINDINGS: We have developed a novel strategy to engineer vascularized urethral tissue. These constructs can be kept for at least 14 days and form stable monolayers and multilayers consistent with native urothelial architecture. Using 3D printing and autologous cell seeding promises to create patient-specific vascularized urethral flaps for phalloplasty.

ABSTRACT IMPACT: This research will promote understanding the role of the Type-I Interferon signaling pathway during embryo implantation, potentially leading to a new diagnostic or treatment target in early pregnancy failure. OBJECTIVES/GOALS: Studies suggest interferon signaling regulation is tightly balanced between physiologic and pathophysiologic growth in early pregnancy. We propose to determine the impact of interferon-mediated inflammation on embryo implantation and early pregnancy failure in normal conditions and chronic inflammatory diseases in a novel mixed-mouse model. METHODS/STUDY POPULATION: To probe the role of type-I interferons (IFNs) in implantation, we will utilize a mouse model and non-surgically transfer both Ifnar1-/- and Ifnar1-/+ embryos into an immune-competent pseudopregnant wild-type female recipient. This will allow analysis of a litter with distinct genotypes within the same, immune-competent, uterine environment. Type-I IFN stimulation will be systemically induced with Poly-(I:C) at various time points around implantation. A similar approach will be used in mouse models of chronic inflammatory disease states associated with early pregnancy loss (e.g. systemic lupous erythematous). With this model, we will be able to control for deficiencies in maternal immune response to specifically determine the embryonic response to inflammation during implantation and development. RESULTS/ANTICIPATED RESULTS: We anticipate the Ifnar1-/+ embryos - those able to respond to Type-I IFN - and their surrounding implantation sites will exhibit more maternal-fetal barrier dysfunction in the form of impaired trophoblast fusion, improper formation of the microvascular architecture, and increased permeability of the maternal-fetal barrier, compared to embryos unable to respond to IFN. We will also conduct similar analyses in mouse models of chronic inflammatory diseases. We hypothesize these mice to have baseline endometrial inflammation that stimulated the IFN-pathway in IFN-capable embryos, producing breakdown of the maternal-fetal barrier. In these mice, we predict Ifnar1-/- embryos will show improved molecular outcomes when compared to Ifnar1-/+ embryos, and thus improved associated pregnancy outcomes. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work can insight into the immunological mechanisms that govern embryo implantation and early placentation. This could provide more pointed means for management and intervention of early pregnancy failure and/or disease states that are commonly associated with poor reproductive outcomes.

ABSTRACT IMPACT: Screening the effect of thousands of non-coding genetic variants will help identify variants important in the etiology of diseases OBJECTIVES/GOALS: Massively parallel reporter assays (MPRAs) can experimentally evaluate the impact of genetic variants on gene expression. In this study, our objective was to systematically evaluate the functional activity of 3’-UTR SNPs associated with neurological disorders and use those results to help understand their contributions to disease etiology. METHODS/STUDY POPULATION: To choose variants to evaluate with the MPRA, we first gathered SNPs from the GWAS Catalog that were associated with any neurological disorder trait with p-value < 10-5. For each SNP, we identified the region that was in linkage disequilibrium (r2 > 0.8) and retrieved all the common 3’-UTR SNPs (allele-frequency > 0.05) within that region. We used an MPRA to measure the impact of these 3’-UTR variants in SH-SY5Y neuroblastoma cells and a microglial cell line. These results were then used to train a deep-learning model to predict the impact of variants and identify features that contribute to the predictions. RESULTS/ANTICIPATED RESULTS: Of the 13,515 3’-UTR SNPs tested, 400 and 657 significantly impacted gene expression in SH-SY5Y and microglia, respectively. Of the 84 SNPs significantly impacted in both cells, the direction of impact was the same in 81. The direction of eQTL in GTEx tissues agreed with the assay SNP effect in SH-SY5Y cells but not microglial cells. The deep-learning model predicted sequence activity level correlated with the experimental activity level (Spearman’s corr = 0.45). The deep-learning model identified several predictive motifs similar to motifs of RNA-binding proteins. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study demonstrates that MPRAs can be used to evaluate the effect of non-coding variants, and the results can be used to train a machine learning model and interpret its predictions. Together, these can help identify causal variants and further understand the etiology of diseases.

ABSTRACT IMPACT: Characterizing specialized pro-resolving lipid mediators (SPMs) of inflammation in a cohort of adult patients with traumatic brain injury (TBI) will potentially identify novel biomarkers of disease and would generate hypotheses regarding the role of SPMs in the pathophysiology and recovery after brain injury OBJECTIVES/GOALS: Primary aim: evaluate the association between plasma and cerebrospinal fluid (CSF) SPM levels with inflammatory markers known to mediate blood brain barrier (BBB) disruption. Secondary aim: evaluate the association between SPM levels and disease severity, 14-day Glasgow Coma Scale score (GCS) and survival. METHODS/STUDY POPULATION: This is a single-center, prospective, observational cohort study (target N=50). Adult participants with moderate-to-severe non-penetrating TBI will have blood and CSF sampled serially for laboratory measures of SPMs (resolvins, protectins, lipoxins, maresins) and inflammatory peptides (TNF-α, IL-1β, IL-6, MMP-9, TIMP-1) by liquid chromatography’‘ mass spectrometry and RT-PCR & ELISA, respectively. Baseline patient characteristics, admission GCS, and 14-day GCS and mortality will be prospectively collected. To evaluate the association between SPMs and other continuous variables, Pearson’s and Spearman’s correlation will be used. Cox regression will be used to evaluate the association between SPM lipidomes and 14-day survival. RESULTS/ANTICIPATED RESULTS: As the primary outcome measure, the association between SPM levels with assayed inflammatory markers will be determined at 24 and 72 hours post-TBI. We hypothesize that SPMs will be negatively correlated with TNF-α, IL-1β, IL-6, MMP-9 and positively correlated with TIMP-1. As secondary outcome measures, the association between SPM levels with disease severity by admission GCS will be determined at 24 hours post-TBI, and the association between SPM levels and GCS and mortality will be determined at 14-days. We hypothesize that SPM levels will be positively associated with admission and 14-day GCS and will be independently associated with 14-day survival. DISCUSSION/SIGNIFICANCE OF FINDINGS: Using the SPM lipidome as a biomarker of disease is a novel tool for future translational research. It will inform a foundational mechanistic framework for TBI pathophysiology and attenuation of neuroinflammation post-TBI, providing rationale for pre-clinical and clinical research focused on novel therapeutics

ABSTRACT IMPACT: This work represents a novel way in which genetic information can be used to improve clinical decision making as it pertains to both treatment and management of congenital heart disease. OBJECTIVES/GOALS: Our lab found that MYH6 variants are both enriched in hypoplastic left heart syndrome (HLHS) and associated with decreased cardiac transplant-free survival. To elucidate the mechanisms of MYH6 variant pathogenicity, we are assessing their impact on atrial function during HLHS development and progression. METHODS/STUDY POPULATION: We are using 2D speckle-based tracking to retrospectively evaluate echocardiograms (echos) from 51 HLHS patients, 17 with MYH6 variants and 34 matched controls. Atrial function will be assessed by myocardial strain and strain rate at seven time points, beginning at the time of the patients’ earliest prenatal echo, and ending with their last available echo before death or cardiac transplant. Early left atrial function will examine the role of MYH6 variants in the development of HLHS in vivo, while longitudinal right atrial function will be assessed in order to look for differences that could be contributing to the decreased transplant-free survival seen in MYH6 variant carriers. RESULTS/ANTICIPATED RESULTS: We hypothesize that MYH6 variants cause HLHS by impairing early left atrial (LA) contractility, resulting in altered left ventricular hemodynamics and consequent hypoplasia. We therefore expect to find diminished prenatal LA function in HLHS patients with MYH6 variants. We also hypothesize that MYH6 variants continue to impair right atrial (RA) function in surgically-reconstructed HLHS hearts, necessitating earlier transplantation. Accordingly, we expect variant carriers to exhibit lower RA function at birth versus controls. We expect differences between groups to persist over time, and possibly increase in magnitude. In HLHS patients with MYH6 variants, we anticipate declining RA function will precede right ventricular function and therefore be an early indicator of transplant need. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study represents a novel way in which genetic information can inform clinical decision-making. Identifying MYH6 variants as an early cause of HLHS offers chances for intervention. Understanding long-term effects of MYH6 on right atrial function in HLHS may aid in cardiac transplant risk stratification, thus improving patient outcomes.

ABSTRACT IMPACT: Plasma and CSF are not reliable estimates of drug exposure in tissue compartments relevant for treatment and prevention of infectious diseases. OBJECTIVES/GOALS: Globally, high dose fluconazole is widely used in the management of cryptococcal meningitis. While it is known to readily penetrate into cerebrospinal (CSF), less is known about drug concentrations in brain parenchymal tissues. Similarly, distribution of fluconazole into gynecological tissues has not been robustly characterized. METHODS/STUDY POPULATION: With informed consent from next-of-kin, we conducted autopsies within 24h of death for hospitalized Ugandans receiving fluconazole for treatment or secondary prophylaxis of cryptococcal meningitis. Dosing history was abstracted from medical chart and caregiver interviews. Fluconazole concentrations were determined using high-performance liquid chromatography- tandem mass spectrometry (LC-MS/MS) in plasma, CSF, 10 brain compartments (frontal, parietal, and occipital lobes, corpus callosum, globus pallidus, hippocampus, midbrain, medulla oblongata, spinal cord, and choroid plexus) and 4 female genital compartments (cervix, vagina, ovary, and uterus), depending on tissue availability. Descriptive statistics of tissue to plasma ratios were used to describe concentrations relative to plasma. RESULTS/ANTICIPATED RESULTS: Fluconazole concentrations were measured in available tissues of 21 individuals with detectable fluconazole in plasma. Daily doses of fluconazole were 200 mg (n=4), 400 mg (n=1), 800 mg (n=4), 1200 mg (n=9) or unknown (n=3). CSF concentrations (n=10) ranged from 93-1380% (median 100%) of plasma while brain concentrations (n=3) across all 10 compartments ranged from 45% to 89% (median 69%) of plasma. In the female genital tract, cervical concentrations (n=10) were 9-78% (median 65%) of plasma and in the 2 individuals with available tissue, concentrations in vaginal, ovarian, and uterine tissues were similar to cervix, ranging from 63-105% of plasma. DISCUSSION/SIGNIFICANCE OF FINDINGS: Measuring drug concentrations directly in tissues, the presumed site of action, improves estimates of drug efficacy. While fluconazole concentrations in CSF were similar to plasma, actual brain tissues were consistently lower. Concentrations were similar between upper and lower female genital tracts, but were consistently lower than plasma.

ABSTRACT IMPACT: As the number of poorly water-soluble drugs in development increases, our research will expand on the science behind improving drug solubility and absorption and ensuring that promising poorly-water solubility drugs do not fail drug development. OBJECTIVES/GOALS: Spray-dried dispersion (SDD) tablet formulation is an approach to increase oral drug solubility and absorption. Methods to predict SDD performance in humans are poorly developed. We aim to develop an in vivo in vitro correlation (IVIVC) between in vitro dissolution and in vivo absorption of itraconazole SDD tablets. METHODS/STUDY POPULATION: This research project involves tablet manufacturing, in vitro dissolution experiments, and a clinical study. We manufactured fast-, medium-, and slow-release SDD tablets containing amorphous solid dispersion of itraconazole (100 mg) and different grades of the polymer hypromellose acetate succinate (HPMC-AS). Tablets differed in slug pressure, tablet compression force, and formulation composition. Dissolution studies were performed using the United States Pharmacopeia (USP) type II apparatus. The clinical study is an ongoing randomized, cross-over, open-label, fasted, single-dose trial in healthy participants (n=12). An IVIVC will be created by comparing the rank order of drug in vitro dissolution with in vivo absorption. RESULTS/ANTICIPATED RESULTS: Tablet manufacturing was successful, and the tablets displayed the same dissolution rate ranking order as anticipated. Fast-release tablets showed the highest percentage of drug dissolved by 10 min (74%) compared to medium- (62%) and slow-release (1.2%) tablets. Percentage drug dissolved differs by at least 10% at all time points among the different release-rate tablets. The clinical study is currently ongoing, and we expect that the pharmacokinetic (PK) profiles differ among the different tablets. We predict that the rank order of tablet absorption in humans will agree with the order of drug dissolved observed in the dissolution experiments. DISCUSSION/SIGNIFICANCE OF FINDINGS: Spray-dried dispersions are a formulation method to try to improve drug solubility and oral drug absorption. This research will elucidate manufacturing parameters that can impact tablet performance and expand on the ability of in vitro dissolution to predict human PK and streamline drug development of poorly soluble drug candidates.

ABSTRACT IMPACT: This project aims to better understand mechanisms of sensory and motor deficits in individuals with ASD with the goal of informing diagnosis and treatment development. OBJECTIVES/GOALS: Over-reliance on both visual and proprioceptive feedback have both been observed during motor behavior in persons with Autism Spectrum Disorders (ASD), suggesting that separate sensory feedback processes may be selectively altered during different behaviors. The objective of this study is to clarify sensory mechanisms of fine motor control in ASD. METHODS/STUDY POPULATION: Participants with ASD (N=43) and controls (N=23) matched on age (10-20 yrs) and non-verbal IQ completed tests of precision gripping. Participants were instructed to press on force sensors with their index finger and thumb so that a moving bar corresponding to their force output reached and stayed as stable as possible at the level of a stationary target bar. Visual feedback was manipulated by changing the visual gain of the force bar (low, medium and high). The force bar moved more per change in force output at higher gains. Proprioceptive feedback was manipulated by applying 80 Hz tendon vibration at the wrist to induce an illusion of muscle contraction. This was compared to a condition with the tendon vibrator turned off. Force variability (standard deviation) and regularity (sample entropy) were examined. RESULTS/ANTICIPATED RESULTS: Controls showed increased force variability with the tendon vibration on compared to off (t = -3.372, p < 0.001); however, the ASD group showed no difference in force variability between the tendon vibration conditions (t = -0.960, p = 0.338). Individuals with ASD had stronger age-associated reductions in force variability relative to controls across tendon vibrator and gain conditions (Group x Age: t = -4.05, p < .001). The ASD group also had greater age-associated increases in force regularity relative to controls, especially at higher gain levels (Group x Gain Level x Age: t = -3.22, p = 0.001). Unlike the ASD group for whom regularity increased with age in both tendon vibration conditions, controls only showed these age-related gains when the tendon vibrator was off (Group x Vibration Frequency x Age: t = 2.46, p = .014). DISCUSSION/SIGNIFICANCE OF FINDINGS: Our findings indicate that while controls integrate proprioceptive and visual feedback online to accurately adjust fine motor behavior, persons with ASD rely mostly on visual feedback. Our results suggest delayed development of sensory integration and reduced reliance on multisensory feedback during online fine motor control in persons with ASD.

ABSTRACT IMPACT: Knowledge of which aspects of social connectedness most strongly associate with caregiver health and health behaviors can inform intervention targets to improve caregiver health OBJECTIVES/GOALS: Stressed dementia caregivers are at risk of poor health. Social connectedness may reduce adverse health effects, yet it is unknown about which aspects relate most strongly to health. This is a barrier to intervention development. Our study identifies aspects of social connectedness most strongly associate with caregivers’ daily health behaviors. METHODS/STUDY POPULATION: Data. Enrolled spousal caregivers completed 14 consecutive days of online surveys. Measures. We examined multiple health behaviors each day, which included: 1) number of occurrences of 3 potential binge-eating behaviors (range 0 to 30), 2) whether participants engaged in at least 30 minutes of physical activity, and 3) perceived sleep quality, rated 1 (very bad) to 5 (very good). We also examined a count of health symptoms caregivers experienced (e.g., backache; range: 0 to 7). Measures of social connectedness included: spousal emotional support, perceived spousal appreciation, emotional support from any source, and loneliness. Analysis. We applied bivariate multi-level mixed effects models to examine the association between each aspect of social connectedness and health behaviors day-to-day. RESULTS/ANTICIPATED RESULTS: Since November 2020, 5 of N=40 participants were enrolled, of whom 3 had completed all diary surveys. Participants were women ages 59 to 73, and included 4 non-Hispanic white and 1 Hispanic caregivers. Data included 51 days of surveys (93% adherence). No differences in eating behaviors nor physical activity according to social connectedness were found. Emotional support from any source was positively associated with sleep quality (B= 0.20; SE=0.08; p-value 0.015). On days when caregivers indicated their spouse appreciated them ‘A lot’ versus ‘Not at all,’ sleep quality was marginally better (B=0.73, SE=0.42; p-value-0.08). Finally, days when caregivers felt lonely at least ‘Some of the time’ versus ‘Not at all’ were associated with experiencing more adverse health symptoms (B=1.54; SE=0.58; p-value<0.001). DISCUSSION/SIGNIFICANCE OF FINDINGS: Improved emotional support from any source may support better sleep quality among caregiving spouses, while loneliness appears to contribute to more adverse health symptoms. Findings, if confirmed, can be translated to develop intervention programs that target loneliness and perceived emotional support among caregivers.

ABSTRACT IMPACT: Reversing tumor microenvironment (TME) immunosuppression will help to increase the overall efficacy of treatment of chemo-resistant triple negative breast cancer (TNBC) and mitigate racial disparities in treatment response. OBJECTIVES/GOALS: We have developed an ex-vivo whole tissue culture model to test the feasibility of reversing local immunosuppression in TME by chemokine modulatory (CKM) regimen. Our current objective is to analyze the molecular changes in CKM-treated chemoresistant TNBC from White and Black women and identify factors determining response to CKM. METHODS/STUDY POPULATION: Freshly resected residual TNBC from 20 White and 20 Black women ≥18 yrs old treated with neoadjuvant chemotherapy (NAC) will be procured. Tumor explants will be prepared & cultured in the absence and presence of CKM (Interferon-ð 〉1/4, TLR3 agonist rintatolimod and COX-2 inhibitor celecoxib). Chemokines implicated in cytotoxic T-lymphocyte (CTL)- & MDSC/ Treg attraction will be analyzed using Taqman & ELISA. We will have 80% power to detect a 0.7 standard deviation difference in chemokines between untreated & treated samples within and between cohorts using ANCOVA. Bulk RNA sequencing will be performed on both untreated & treated samples from CKM responding (highest aggregate increase in CTL- and highest decrease in Treg/MDSC-favoring chemokines in the top quartile) and non-responding (bottom quartile) tissues. RESULTS/ANTICIPATED RESULTS: Our preliminary data show that Black patients (pts) with breast cancer (BC) have an immunosuppressive TME associated with poor outcomes. This is similar to other existing literature showing that Black pts with BC have less favorable and more unfavorable chemokines in the TME. We anticipate the chemokine changes with CKM treatment will be larger in the Black cohort given their ability to elicit a robust inflammatory response. Therefore, we expect that CKM treatment will result in favorable TME in both groups and improve outcomes in TNBC, which has the worst prognosis of all subtypes, eliminating a key area of disparity in BC. The proposed transcriptome analysis will help identify key gene networks involved in response to CKM treatment and guide modulating the targets for non-responsiveness to improve efficacy of CKM. DISCUSSION/SIGNIFICANCE OF FINDINGS: Pts with residual disease (RD) after NAC have a 3-yr overall survival 68% vs. 94% for pts with complete response. Blacks have a higher incidence of TNBC with more likelihood of RD & mortality. We anticipate that the existing TME differences can be abrogated by our current CKM regimen or via developing an alternative CKM regimen optimized for Black pts.

ABSTRACT IMPACT: Triple negative breast cancer (TNBC) affects 10-20% of women with breast cancer and is biologically more aggressive than other subtypes. The novel compound we have developed, DL7076, would give clinicians a vital strategy to improve the commonly used cyclophosphamide (CPA) and doxorubicin (DOX) regimen in the treatment of TNBC. OBJECTIVES/GOALS: The objective of this research project is to develop a novel compound which can activate both 1) the constitutive androstane receptor (CAR) and subsequently enhance the CYP2B6-mediated activation of CPA, and 2) the nuclear factor erythroid- related factor-2 (Nrf2) leading to the cardiomyocyte protection from DOX-associated cardiotoxicity. METHODS/STUDY POPULATION: Following the identification of the compound candidate, DL7076 was evaluated for tissue specific induction of CAR and Nrf2 using qPCR, western blot analysis, and luciferase reporter assays.

Further, we have developed a multicellular coculture model incorporating human primary hepatocytes for metabolism, TNBC spheroids as the target, and cardiomyocytes as a side target of DOX. We have investigated the anticancer effects of CPA/DOX on TNBC cells and the toxic effects on cardiomyocytes with/without a CAR-Nrf2 activator, in a multicellular environment where hepatic metabolism is well-retained. RESULTS/ANTICIPATED RESULTS: We found that our dual activator of CAR and Nrf2, DL7076, exhibits tissue specific induction of CAR and Nrf2. Inclusion of DL7076 in combination with the CPA/DOX regimen improves anticancer efficacy, through the subsequent increase in the formation of the active CPA metabolite. With the addition of DL7076, DOX-mediated off-target cardiotoxicity was markedly reduced.

Lastly, utilizing the novel coculture system with human primary hepatocytes, TNBC spheroids, and cardiomyocytes, the inclusion of DL7076 to the CPA/DOX regimen shows decreased spheroid viability and improved cardiomyocyte viability and function. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our findings suggest that DL7076 can facilitate DOX/CPA containing regimens by increasing CAR-mediated metabolism and subsequent CPA bioactivation while selectively protecting cardiomyocytes from DOX-induced toxicity. This research is expected to translate our basic scientific findings into therapeutic interventions for women with TNBC.

ABSTRACT IMPACT: Indiana CTSI Team Science to maximize rapid collection, analyses and dissemination of biosamples collected from patients with Covid-19 to provide preliminary data for grant applications on the pathogenesis and outcomes of patients with Covid-19. OBJECTIVES/GOALS: When Covid-19 hit Indiana in April, there was an immediate need to respond rapidly to coordinate research across our healthcare systems. The CTSI became a point of contact for coordinating research endeavors including activation of clinical trials and use of precious samples from patients with Covid-19 to maximize preliminary data for grants. METHODS/STUDY POPULATION: The Indiana CTSI coordinated collection of biospecimens at multiple hospitals using in person and remote consenting via telephone or on a smartphone utilizing a QR code. We also retrieved existing samples from the Indiana Biobank previously collected for future research and from subject positive for Covid-19 by search of the linked electronic health record (EHR). A total of 224 subject samples (7 children, 36 previously collected, and 6 with both acute and recovered specimens) were obtained over a four month period. Our CTSI cores ran varied analyses collated to a single database, linked to the EMR for use as preliminary data for grant applications to avoid redundancy of measures on limited samples. RESULTS/ANTICIPATED RESULTS: The 224 subject samples were used for whole exome DNA sequencing, RNA seq, analyses of 48 plasma cytokine/chemokines by multiplex analyses, and PBMC isolated for culture and assessment of secreted cytokines. The clinical data were linked and included demographics, hospitalization length of stay and need for mechanical ventilation, max and min oxygen levels, liver function tests, IL-6, D-dimer, CRP, LDH, and ferritin, need for dialysis, and echocardiography. Additional clinical data were available upon request. A survey was sent to our CTSI email to query for potential interest in the data with 87 inquiries, and to date 46 investigators have requested data and/or additional samples. DISCUSSION/SIGNIFICANCE OF FINDINGS: During the first surge of Covid-19, the CTSI coordinated analyses for the dissemination of results for use by CTSI investigators to minimize duplication of assays and increase availability. The collaboration of research coordinators, biobank, research cores, and informatics demonstrates the power and agility of team science in the Indiana CTSI.

ABSTRACT IMPACT: This study highlights the importance of creatine in developmental myelination and remyelination to investigate whether creatine provides a therapeutic value during a central nervous system (CNS) demyelinating insult with a potential value in patients with Multiple Sclerosis. OBJECTIVES/GOALS: Creatine is vital for ATP buffering in the brain. Interestingly, the cells that generate myelin express the main enzyme for creatine synthesis, Gamt. Patients with Gamt mutations display intellectual delays and impaired myelination. Therefore, we hypothesize that creatine is essential for developmental myelination and improves remyelination. METHODS/STUDY POPULATION: To investigate these hypotheses, we developed a new transgenic mouse model with LoxP sites flanking exons 2-6 of the guanidinoacetate methyltransferase (Gamt) gene where excision leads to expression of a green fluorescent tag allowing us to track the cells normally expressing Gamt. We used immunohistochemistry techniques to look at the corpus callosum, the main white matter tract in the brain, and evaluate the number of oligodendrocytes (OL), glial cells responsible for generating myelin. We also used the cuprizone model of toxic demyelination to investigate whether dietary creatine and cyclocreatine, a planar analog of creatine that more efficiently crosses the blood-brain barrier, can enhance remyelination. RESULTS/ANTICIPATED RESULTS: In this mouse model, we show a 95% (+/-0.47%, n=3) co-localization of Gamt within mature OL during postnatal (P) day P14 with no co-localization in neurons or other glial cells. This suggests that mature OL are the main cells making creatine in the CNS. Next, we show that knocking out Gamt leads to a significant reduction in OL in the developing corpus callosum, at P14 and P21 (P14: 0.007, n=3; P21: 0.04, n=3). We also show that creatine supplementation causes a trending increase in mature OL density in the corpus callosum following cuprizone demyelination (2% creatine; p=0.052; n=4). Interestingly, cyclocreatine supplementation significantly increased mature OL density in the corpus callosum following cuprizone demyelination (0.1% cyclocreatine; p=0.034; n=4). DISCUSSION/SIGNIFICANCE OF FINDINGS: These studies highlight the important role creatine plays in developmental myelination and remyelination to investigate whether creatine and cyclocreatine provide a therapeutic value during a CNS demyelinating insult. This work investigates a potential therapeutic value of creatine to patients with Multiple Sclerosis.

ABSTRACT IMPACT: Lipidomics is emerging as a powerful strategy to identify biomarkers for Major Depressive Disorder, as well as therapeutic targets in lipid metabolic pathways. OBJECTIVES/GOALS: Lipidomics is increasingly recognized in precision psychiatry for global lipid perturbations in patients suffering from Major Depressive Disorder (MDD). We will test the hypothesis that lipid metabolism dysregulation is associated with familial risk of depression. METHODS/STUDY POPULATION: Patients with MDD (G1), children (G2), and grandchildren (G3) have been part of a longitudinal study since 1982. If a parent G2 and grandparent G1 have MDD, G3 is considered a high risk of depression. Biospecimens (saliva and serum) were collected for full exome sequencing and RNA analysis. Samples will also be extracted for lipid content and lipids will be identified by mass spectrometry. A panel of nearly 600 lipid species can reliably be identified and quantified using liquid chromatography paired with tandem mass spectrometry (LC-MS/MS). Dysregulated lipids will be correlated with familial risk of depression in samples of G3. RESULTS/ANTICIPATED RESULTS: We hypothesize that dysregulation of lipids and lipid metabolism will be apparent in biospecimens from the high risk compared to the low risk of depression. Also, alterations in RNA transcriptomics of genes involved in lipid metabolic networks are associated with familial risk of depression. Several differential lipid species were previously identified to be associated with MDD. Reduced phosphatidylcholine(PC), phosphatidylethanolamine(PE), phosphatidylinositol(PI), and increased LysoPC, LysoPE, ceramide, triacylglycerol, and diacylglycerol levels have been correlated to MDD. However, these results need to be replicated in independent studies using lipidomics analysis. DISCUSSION/SIGNIFICANCE OF FINDINGS: It is highly likely that completely novel cellular targets will emerge from these studies by uncovering the convergence of lipidomics and genetic variance of lipid metabolic enzymes as biomarkers for predisposition to MDD as well as potential targets for therapeutic development for MDD.