OBJECTIVES/SPECIFIC AIMS: Earlier pubertal timing has been associated with risk for depression, particularly in girls (e.g., Keenan etal., 2014). Evidence suggests pubertal timing in girls also relates to alterations in the microstructural properties of brain white matter tracts in late adolescence (Chahal etal., 2018), and structural connectivity of cingulate and frontal regions (Chahal etal., in prep), though differences in pubertal development in both boys and girls have not been examined in the context of brain functional connectivity (FC). Individual differences in the course of puberty may have enduring effects on functional coupling among brain regions that may contribute to the risk for psychopathology. To address this question, we explored the relation between pubertal timing and tempo with depression symptoms (age 16). Then, we examined whether brain network FC (age 16) associates with pubertal indices and predicts concurrent and later depressive symptoms (age 18). METHODS/STUDY POPULATION: Sixty-eight adolescents (37 females) completed the Mini-Mood and Anxiety Symptom Questionnaire (MASQ; Clark & Watson, 1995) at ages 14-18. Gompertz growth curve modelling of pubertal development (age 10-15; Waves 1-6) was used to estimate pubertal timing and tempo per individual, separately for males and females (e.g., Chahal etal., 2018). Resting-state MRI data (age 16) were parcellated into 264 cortical and subcortical regions to create region-to-region FC matrices based on correlations of time-series. Individual matrices were fed to the GraphVar program (Kruschwitz etal., 2015) to assess the interaction of pubertal timing and pubertal tempo with functional network connectivity using Network-based statistic (NBS; Zalesky etal., 2010). Subnetworks showing alterations in relation to pubertal timing and tempo were then examined in association with concurrent (age 16) symptoms and used to predict future depressive symptoms (age 18). RESULTS/ANTICIPATED RESULTS: In all youth, earlier pubertal timing was associated with higher depressive symptoms at age 16 (p<.018). This association was stronger in girls with slower pubertal tempo (p<.039). Interregional connectivity analyses revealed that the interaction of earlier pubertal timing and slower tempo was associated with lower FC between the left cingulate gyrus and right precuneus (p<.0001), regions implicated in emotion processing (i.e., Affective Processing Network) and self-referential thinking (i.e., Default Mode Network). FC of the three other emotion- and self-referential processing network regions (i.g., left insula, superior parietal lobule, and precuneus) was lower in youth with greater age 16 depressive symptoms (p<.0001). Finally, lower FC of of the left and right inferior parietal lobule predicted greater depressive symptoms at age 18 (p<.0001). In summary, FC of overlapping affective and default mode network areas was related to earlier pubertal timing and higher concurrent and future depressive symptoms. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings demonstrate individual differences in pubertal maturation are associated with depressive symptoms and differences in brain connectivity in mid-adolescence. Early pubertal development was associated with greater depression symptoms and lower FC of brain regions involved in emotion regulation and self-referential processing. Further, FC between these regions predicted higher depression symptoms two years later. These neurobiological mechanisms may, in part, underlie the link between off-time pubertal development and the risk for depression. These findings also have important implications for precision psychiatry, as we show that a risk-factor of depression (early pubertal timing) may manifest in developing neurobiology in region-specific ways. Previous network models of depression (e.g., Li etal., 2018) implicated affective network connectivity in sustained negative mood and the default mode/ self-referential network in rumination. Other networks implicated in these past models include the reward network, which may be involved in anhedonia and loss of pleasure. Our study only found associations between affective and self-referential regional connectivity, pubertal maturation, and depression, suggesting that pubertal risk factors may relate more closely with emotion-regulation and self-referential processing deficits.

OBJECTIVES/SPECIFIC AIMS: This study’s aims are to optimize the isolation and growth of chondrocytes from pig auricular cartilage; to identify the ideal seeding conditions onto 3D printed auricular bioscaffolds to maximize chondrocyte growth; and to investigate what quantity and types of host tissue can grow on the bioscaffold. Primary outcomes will include comparisons between different seeding conditions in various objective measures of bioscaffold growth and survival as listed in the methods section. Secondary outcomes will include continued optimization of bioscaffolds to minimize extrusion rates and maximize morphologic and histologic similarity to human auricular cartilage. METHODS/STUDY POPULATION: For chondrocyte-seeded scaffolds, cartilage will be collected from freshly harvested porcine auricular tissue and digested in type II collagenase. Chondrocytes derived from the harvest will be seeded into auricular PCL scaffolds using a type I collagen/hyaluronic acid composite gel, which has been previously shown to support chondrogenesis. For scaffolds containing cartilage, punch biopsies will be collected and embedded in specific areas of the scaffold previously shown to experience excessive stress/strain compared to the rest of the construct. From there, five of each chondrocyte-seeded bioscaffolds, chondrocyte-unseeded bioscaffolds, and cartilage-containing bioscaffolds will be implanted into athymic rats. Total follow up will be for six months, with outcomes as measured by clinical assessments, morphologic measurements, radiological imaging, histological analysis, biomechanical evaluation, and photodocumentation. Once these measures are obtained, we will work closely with Dr. Myra Kim, an adjunct professor with the Biostatistics Department, to appropriately analyze differences between the models. RESULTS/ANTICIPATED RESULTS: We believe that while all scaffolds (chondrocyte-seeded, chondrocyte-unseeded, and cartilage-containing) will be structurally sound, the chondrocyte-seeded scaffolds and cartilage-containing scaffolds will exhibit improved soft tissue coverage and have lower exposure and fracture rates. Additionally, between the two, we posit that there will not be appreciable differences histologically, radiologically, or morphologically. DISCUSSION/SIGNIFICANCE OF IMPACT: Auricular reconstruction is a geometrically complex and technically challenging problem. Reconstruction hinges on the physical characteristics of the deformity, patient preferences, and reconstructive materials available. The current gold standard for auricular reconstruction uses autologous rib cartilage as foundational support for overlying soft tissue and these techniques involve freehand carving of the cartilage, requiring high levels of technical skill. Harvesting the materials for this procedure is invasive, and the outcomes of the surgery are largely variable and sometimes undesirable. As alternatives, implantable scaffolds including those made from high density porous polyethylene (commercially referred to as MedPor) have been investigated. However, many of these have proven inadequate due to factors including infection, extrusion, and morphologic and biomechanical dissimilarity from native tissue. 3D printing represents an exciting new avenue through which to address many of these difficulties. Our group has previously demonstrated the successful design, production, and implantation of 3D-printed models: in auricular reconstruction, we have demonstrated the successful creation and implementation of a 3D printed ear scaffold into an athymic rodent model. We now turn our attention to optimization of seeding of our ear scaffold with chondrocytes derived from porcine auricular cartilage or with cartilage punch biopsies, all while maintaining emphasis on regulatory feasibility. With success in this arena, we will be able to provide a much less invasive and technically challenging alternative to the current gold standard, create patient-specific bioscaffolds which are more form fitting and individualized, and provide children with ear malformations better alternatives and treatments for their conditions.

OBJECTIVES/SPECIFIC AIMS: This study aims to estimate patient attitudes and receptiveness towards stem cell-based cell replacement devices to management Type 1 Diabetes. The primary outcomes of this study are mean response values to questions interrogating patient attitudes, knowledge, and receptiveness. METHODS/STUDY POPULATION: A RedCap survey was generated for this study. 100 participants will be drawn from Mayo Clinic Rochester, MN patients living with Type 1 Diabetes. RESULTS/ANTICIPATED RESULTS: Response values will be used to estimate broader patient attitudes. DISCUSSION/SIGNIFICANCE OF IMPACT: The response values of this survey will help inform future directions of cell replacement device development. Additionally, understanding patient attitudes may be useful in crafting more effective education strategies.

OBJECTIVES/SPECIFIC AIMS: Cardiovascular diseases (CVD) is the leading cause of death worldwide in both men and women due to lack of cardiac regeneration after disease or damaged is caused. There are many challenges to studying CVD since native cardiomyocytes cannot be cultured in vitro. With the advancements in biomaterial and pluripotent stem cells research, scientists are now able to produce engineered cardiac tissue models in vitro that mimic the native myocardium. This study shows our methods for producing engineered cardiac tissue with potential applications in cardiac regeneration, disease modeling, and scalable production. METHODS/STUDY POPULATION: In this study, human induced pluripotent stem cells (hiPSCs) were combined with two different photocrosslinkable hybrid biomaterials, poly (ethylene)- glycol fibrinogen (PF) and gelatin methacrylate (GelMa), in various tissue geometries to form 3D human engineered cardiac tissues (3D-hECTs). To study tissue growth and contraction, image and video analysis was performed at specific timepoints. To analyze differentiation efficiency and cell population, flow cytometry was performed using cardiac markers. To evaluate gene expression, qPCR was performed using pluripotency and cardiac specific primers. RESULTS/ANTICIPATED RESULTS: Direct cardiac differentiation of encapsulated hiPSCs resulted in synchronously contracting 3D-hECTs in both biomaterials and all tissue geometries. Spontaneous contractions started on Day 7 and increased in velocity, frequency, and synchronicity over time. 3D-hECTs had high cell viability with > 70% of cells positive for cardiac markers. Engineered tissues showed appropriate temporal changes in gene expression over time with pluripotency gene expression decreasing and cardiac gene expression increasing. DISCUSSION/SIGNIFICANCE OF IMPACT: This study shows the potential for direct differentiation of encapsulated hiPSCs to produce physiologically relevant engineered cardiac tissues. Resulting 3D-hECTS showed features of mature myocardium with appropriate cardiomyocyte populations, mechanical motion, and gene expression. Using this platform, we are able to produce engineered cardiac tissue in a variety of biomaterials and tissue geometries to study new therapeutics, mechanism of disease, and scalable tissue culture.

OBJECTIVES/SPECIFIC AIMS: These studies seek to evaluate hormonal regulation of luminal breast cancer lipid metabolism and to identify targetable progesterone-mediated changes in lipid biology that contribute to therapeutic resistance in breast cancer. METHODS/STUDY POPULATION: Established and patient-derived luminal breast cancer cell lines, which express ER and PR, were used for this study. RNA transcript and protein expression levels were evaluated by qRT-PCR and immunoblot, respectively. Broad scale lipidomics of progesterone-treated cells was conducted via ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) through the UCD Skaggs School of Pharmacy Mass Spectrometry Core. RESULTS/ANTICIPATED RESULTS: Data mining of previously published microarray data of CK5+ and CK5− syngeneic cancer sublines revealed that CK5+ cells have increased expression of lipid processing genes, including LPL and PPARG. As progestin treatment induces a subpopulation of cells to turn on CK5 expression in luminal breast cancers, UHPLC-MS-based lipidomics analysis will expose whether modulation of the lipid landscape occurs in all cells with progesterone treatment, or whether this phenomenon is heightened specifically in CK5+ cells. I also expect that ER+ breast cancers with progestin induced-altered lipid content, such as lipid droplet formation, will evade therapy-induced death. DISCUSSION/SIGNIFICANCE OF IMPACT: There are numerous approved and developmental therapeutics targeting lipid biology. By determining if progestins alter lipid metabolic genes specifically in CK5+ CSCs, which are endocrine resistant, strategies may be devised to target these resistant cells using combination therapy in conjunction with existing therapies to prevent tumor recurrence.

OBJECTIVES/SPECIFIC AIMS: We have reported that radiofrequency renal denervation (RF-RDN) in SHR at 20-weeks of age, decreased blood pressure (BP) and fibrosis in kidney cortex and medulla when rats were sacrificed at 6 months. However, whether RF-RDN can have similar benefits in older rats remains unknown. This study examined whether performing RF-RDN in older rats also has a beneficial effect on BP and renal fibrosis. METHODS/STUDY POPULATION: Baseline systolic and diastolic BP (SBP/DPB) was measured (telemetry) in nine-month-old SHR and Wistar Kyoto rats (WKY). Groups of rats then received bilateral RF-RDN or Sham-RDN (SHR-RDN, n=9; SHR-Sham, n=10; WKY-RDN, n=5; WKY-Sham, n=8). Rats were then sacrificed at 12-months of age. Kidneys were harvested, sectioned, and assessed for fibrosis by Masson’s trichrome stain. A pathologist, who was blinded to treatment groups, evaluated each kidney section for fibrosis. RESULTS/ANTICIPATED RESULTS: Compared to SHR with Sham-RDN, RF-RDN prevented a further increase in systolic and diastolic BP from baseline (9-month) in SHR as they aged to 12-months (SHR-Sham mmHg: 9-month 193±4/127±4; 12-month 207±3/142±5; SHR-RDN mmHg: 9-month 197±3/132±2; 12-month 197±4/132±3). RF-RDN did not alter SBP or DBP in aged WKY. One-year-old SHR with prior Sham-RDN showed extensive renal fibrosis in kidney cortex and medulla. In contrast, RF-RDN significantly decreased renal fibrosis in the medulla, but not cortex. There was no fibrosis in kidneys of age matched WKY. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings suggest that RF-RDN may be a potential therapy for halting progression of hypertension and decreasing medullary fibrosis in the aged population.

OBJECTIVES/SPECIFIC AIMS: The objective of this study was to determine if dopamine signaling is altered in a mouse model displaying excessive self-grooming and further elucidate the potential utility of compounds targeting the striatal DA system in modulating repetitive behaviors. METHODS/STUDY POPULATION: Here, we report studies using fast-scan cyclic voltammetry (FSCV) in mice lacking the postsynaptic protein SAP90/PSD95-associated protein (SAPAP3 KO mice) as well as control littermates. Rodent self-grooming provides a behavioral output with which one can monitor repetitive, self-directed, patterned behavior that has great translational value to OCD-like disorders. Total time spent grooming was monitored in SAPAP3KO mice and control littermates. To further examine the role of DA in regulating repetitive grooming behaviors the magnitude and kinetics of DA transients were assessed using FSCV in ex vivo slice preparations as well as in anesthetized mice in vivo. DA transients were elicited in the dorsolateral striatum (DLS), dorsomedial striatum (DMS); and nucelus accumbens core (NAcc). In some experiments mice were crossed with DAT-Cre animals and channelrhodopsin 2 (ChR2) was virally expressed in DA neurons to allow optical stimulation of DA transients. RESULTS/ANTICIPATED RESULTS: As previously reported, SAPAP3 KO mice showed excessive grooming compared to control littermates at the age assessed (4-5 months). DA transients evoked by a single electrical pulse in slices from SAPAP3 KO mice were not significantly different from those observed in slices from control littermates in any of the regions tested including the DLS, DMS and NAcc. However, when four electrical pulses were applied at a frequency of 10Hz to mimic DA neuron bursting, the magnitude of DA transients observed in the DMS and NAcc of SAPAP3 mice were greater than those evoked in control littermates.Interestingly, phasic stimulation produced similar DA transients in the DLS of both genotypes suggesting that phasic DA signaling was not globally altered. To confirm this finding we crossed SAPAP3 KO mice with DAT-Cre mice and injected ChR2 containing virus into the midbrain to selectively express ChR2 in DA neurons. Transients were then optically evoked resulting in selective activation of DA neurons. Optical stimulation produced a pronounced enhancement of DA release in SAPAP3 KO mice specifically in the DMS and only following phasic-like stimulation. DISCUSSION/SIGNIFICANCE OF IMPACT: These exciting findings suggest that DA signaling in SAPAP3KO mice is dysregulated in a very precise manner that is sub-region specific as well as dependent on the pattern of stimulation. These results suggest that targeted therapies that can modulate these specific modes of dopaminergic signaling in these distinct striatal subregions could provide improved efficacy in OCD patients that are resistant to SSRI treatment.

OBJECTIVES/SPECIFIC AIMS: This study aims to determine if a bronchiolitis specific microbiome exists and how it evolves through disease course. Objective 1. Determine the microbiome profile of the airway in virus induced bronchiolitis-associated respiratory failure. Objective 2. Identify changes in the airway microbiome profile through the course of virus induced bronchiolitis associated respiratory failure, and the relationship between microbiome composition and clinical respiratory status. Objective 3. Determine the impact of rhinovirus infection on the lung and stool microbiome in a murine asthma model. METHODS/STUDY POPULATION: Objectives 1 &2: We are conducting a single-center prospective case-control study of patients admitted to the Komansky - Weill Cornell Pediatric Critical Care Unit. Infants less than two years of age with a diagnosis of bronchiolitis requiring intubation and mechanical ventilation are enrolled as subjects. Infants less than two years of age intubated and requiring mechanical ventilation without primary lung pathology are enrolled as controls. To evaluate our primary objective, tracheal aspirates will be collected from both subjects and controls on the day of intubation. We will perform 16s RNA sequencing on the tracheal aspirate samples and compare the resulting microbiomes. To evaluate secondary objective, we will collect tracheal aspirates of our study population on a daily basis and map the microbiome in parallel with objective measures of respiratory status including oxygen index and successful extubation. Both subjects and controls are being enrolled as a convenience sample. Objective 3: Mice, heterozygous for the sptlc2 gene (Spltc2 +/−) demonstrate reduced de-novo sphingolipids and increased airway hyperresponsiveness with methacholine challenge. Airway hyper-responsiveness is a cardinal feature of asthma. This airway hyperresponsiveness is exacerbated in the setting of rhinovirus (Figure 1). Using 16s sequencing, we will examine the lung microbiome of Sptlc2 +/− ad Sptlc2 +/+ at 1- and 7-days following rhinovirus infection. RESULTS/ANTICIPATED RESULTS: This clinical study is currently IRB approved and enrollment is ongoing. We have enrolled 12 subjects and 5 controls. Sample analysis will begin following the 2018-2019 respiratory season, with an anticipated cohort of 20 subjects and 20 controls. With regards to the murine studies, we have demonstrated that the lung microbiome of Sptlc2 +/− and Sptlc2 +/+ mice is similar at baseline (Figure 2) and remains similar following 1-day infection with rhinovirus. We do however, see a distinct change in the microbiome profile of the stool of Sptlc2 +/− mice following rhinovirus infection (Figure 3). Lung analysis at day 7 post infection is pending. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will lay the groundwork for detailing the functional role of the airway microbiome in bronchiolitis, with the objective of developing new modalities for disease treatment and prevention. In addition our murine studies allow us to view the microbiome in the context of sphingolipid deficiency, providing a potential mechanistic link to rhinovirus and ORMDL3 associated asthma.

OBJECTIVES/SPECIFIC AIMS: Scavenger receptor (SR) surface proteins are highly conserved motifs and are implicated in the uptake of nanotherapies. Gold nanoparticles functionalized with DNAzymes (DzNP) represent a promising novel nanotherapy for lung diseases such as asthma, particularly because they can be delivered directly to the lung. Our lab has been studying the therapeutic potential of a DzNP targeting GATA-3, a master transcription factor regulating Th2 inflammation. Although nanoparticle uptake through scavenger receptors has been described in macrophages in other models, the role of SRs in DzNP uptake in the lung is poorly understood. We hypothesize that scavenger receptors mediate DzNP uptake in alveolar macrophages. To begin examining this hypothesis, we examined whether DzNP exposure and uptake regulates gene expression of MARCO and MSR1, two class A scavenger receptors. METHODS/STUDY POPULATION: Using a silver stain, we measured dose dependent DzNP uptake in murine alveolar macrophages (MH-S). Using qRT-PCR, we measured gene expression of scavenger receptors MSR1 and MARCO in murine alveolar macrophages (MH-S) and after 24 hour exposure to 2251 DzNP, a DzNP targeting GATA-3, and dextran sulfate sodium (DSS), a known SR-A blocker. RESULTS/ANTICIPATED RESULTS: 2251 DzNP uptake in alveolar macrophages is dose dependent. MARCO gene expression levels significantly increase in murine alveolar macrophages when cultured with increasing concentrations of 2251 DzNP (10 pM-2 nM) or DSS 25-200 ug/ml) for 24 hours. However, MSR1 gene expression levels have minimal change when exposed to low concentrations of 2251 DzNP and DSS. At higher concentrations of 2251 DzNP and DSS, MSR1 expression levels are decreased. DISCUSSION/SIGNIFICANCE OF IMPACT: Alveolar macrophages exhibit a dose dependent increase in MARCO gene expression levels with increasing concentrations of 2251 DzNP and DSS, but MSR1 gene expression is not affected in a similar fashion. 2251 DzNP-induced increases in MARCO gene expression suggests that 2251 DzNP may facilitate its own uptake through MARCO. 2251 DzNP exposure negatively regulates MSR1 expression at higher doses and suggests that 2251 DzNP may inhibit its own uptake thought MSR1.

OBJECTIVES/SPECIFIC AIMS: The goal of this study was to determine if there are any sex differences in the pathophysiological effects of sFR. METHODS/STUDY POPULATION: Male Fischer rats (4-month-old) were maintained on a control (CT) (ad libitum regular chow; n=8) or sFR (60% reduction of daily food intake, n=8) diet for 2 weeks. On days 1, 2, 3 and 14, the rats were placed in metabolic cages for food and water intake and 24-hour urine collection. Body weight (BW) is measured daily. After 2 weeks, the animals are given free access to normal chow for 3 months. Short-term and long-term effects of sFR on blood pressure and heart rate will be measured. RESULTS/ANTICIPATED RESULTS: After 2 weeks, the male CT group gained 7% BW (p <0.05), while BW in the sFR males was reduced by 12% (p<0.05 vs. CT). In contrast, female controls did not gain BW while the sFR females lost 18% of their BW. Water intake was reduced by 35%, which was similar to the reduction in females (p=0.18). The hematocrit of sFR male rats was higher (51.1%) than the CT group (45.2%, p<0.05), which was most likely due to the 6% reduction in plasma volume. A similar effect on hematocrit was observed in sRF females. Similarly, also to female rats, sFR had no effect on Na+ and K+ plasma or urine concentrations by day 14 in the male rats. DISCUSSION/SIGNIFICANCE OF IMPACT: sFR has similar effects on electrolyte balance in males and females. Ongoing studies will determine if there is any sex difference in the effects of sFR on blood pressure, heart rate and susceptibility to hypertension and cardiac injury.

OBJECTIVES/SPECIFIC AIMS: Overall Research Aim: To develop an iNKT-cell engaging reagent (“CAb”)to induce neuroblastoma-directed cytotoxicity in vitro and in a mouse model of neuroblastoma. Objective 1: Explore the contribution of different GD2 affinities to the cytotoxicity against neuroblastoma cells in vitro. Objective 2: Deteremine whether use of different stimulatory glycolipids (alpha-GalCer vs. C34) alter the activation and cytotoxicity of iNKT cells against neuroblastoma in vitro. Objective 3: To analyze survival of an immunocompetent mouse model of neuroblastoma treated with C34-loaded vs alpha-GalCer-loaded CAb molecule, and to analyze the tumor microenvironment in each treatment condition. METHODS/STUDY POPULATION: CAb molecule will be generated by fusing a CD1d protein to an scFv domain for GD2 using cloning techniques. Previous work by our group has used a streptavidin-biotin system to link CD1d to an antibody against GD2, which is large and immunogenic. Protein expression of this novel fusion protein will occur in HEK293 cells. This new CAb molecule will then be loaded with alpha-GalCer or C34 for use in cytotoxicity and in vivo experiments. Cytotoxicity Assessment: Chromium assays will be used to assess the specific cytotoxicity generated by iNKT cells against neuroblastoma cells in vitro. iNKT cells will be activated by “CAb’s” with relatively high and low affinity for GD2, and also with Alpha-GalCer and C34 glycolipid antigen. flow cytometry will be used to assess for CD107a and Interferon Gamma. Mouse Model of Neuroblastoma: TH-MYCN +/+ mice will be used as an immunocompetent model of neuroblastoma. These mice have the MYCN gene under the control of a tyrosine hydroxylase promoter, and spontaneously develop neuroblastomas by 2 weeks of life which are uniformly fatal by 8 weeks of life. In vivo survival studies will be conducted by injecting CAb of relatively high and low affinity, loaded with glycolipid antigen intraperitonealy into TH-MYCN+/+ mice starting at 2 weeks of age, twice weekly. There will also be a matched negative control. Treatment groups are listed below: 1. alpha-GalCer loaded high-affinity Cab 2. alpha-GalCer loaded low-affinity Cab 3. C34-loaded high-affinity Cab 4. C34-loaded low-affinity Cab 5. Unloaded high-affinity Cab 6. Unloaded low-affinity Cab Enrollment will be 6 mice per group for the survival curves. Tumor Microenvironment analysis: 2 additional mice will be included in each group listed above to be sacrificed 2 weeks into treatment for tumor assessment with flow cytomtetry for iNKT cell, NK cell, T-Lymphocyte frequencies as well as interferon-Gamma expression. RESULTS/ANTICIPATED RESULTS: Objective 1: We expect to find that the highest affinity scFv domains for GD2 result in the greatest amount of cytotoxicity against neuroblastoma cells via iNKT cells. Objective 2: We expect that the C34 molecule will induce the greatest amounts of iNKT cell activation against neuroblastoma cells and higher cytotoxicity against neuroblastoma, which has not been shown previously. Objective 3: We expect to see prolonged survival of mice treated with the high affinity GD2 CAb loaded with C34 or alpha GalCer compared with the low affinity CAb loaded with C34 or alpha GalCer. We also expect that the C34 loaded CAb in both groups will have prolonged survival when compared with the alpha-GalCer loaded CAbs of either affinity. DISCUSSION/SIGNIFICANCE OF IMPACT: iNKT cells have been shown previously to confer an improved prognosis in neuroblastoma and other malignancies. Furthermore, high risk neuroblastomas tend to downregulate expression of a chemokine that attracts iNKT’s to the site of the neuroblastoma. Directing iNKT to the site of neuroblastoma holds promise as an effective immunotherapy option. Our preliminary data demonstrate that CAbs directed against GD2 are capable of exerting cytotoxicity of neuroblastoma in vitro. Furthermore a trend towards prolonged survival has been shown in TH-MYCN mice in early experiments. The development of a novel antibody that has reduced immunogenicity, incorporates a glycolipid antigen that does not induce iNKT cell anergy, and is specific for the GD2 tumor specific antigen has potential to result in increased iNKT-mediate neuroblastoma cytotoxicity and prolonged survival in TH-MYCN+/+ mice.

OBJECTIVES/SPECIFIC AIMS: We are exploring the structure of the interaction between an immunogenic neoantigen and a T cell receptor (TCR) that recognizes the neoantigen while tolerating the counterpart self antigen. No structural example exists to date of how a TCR can discriminate between a neoantigen and the self antigen. We aim to determine the structural and biophysical features that underlie the immunogenicity for this neoantigen, and the features we determine are likely to be present in other immunogenic neoantigens. Algorithms to predict the immunogenicity of neoantigens are available, but do not incorporate structural or biophysical factors. We aim to improve these methods for immunogenic neoantigen prediction by determining structural and biophysical factors that result in recognition by the immune system. METHODS/STUDY POPULATION: Recombinant protein expression, production, and purification. Protein x-ray crystallography. Biophysical protein-protein binding experiments RESULTS/ANTICIPATED RESULTS: The T cell receptor (TCR) bound to the neoantigen with an affinity 15-fold higher than the self antigen. The leucine to phenylalanine mutation occurs at position 8 of a 9-amino acid long peptide antigen. This position is typically in the interface bound by the T cell receptor. The structures of the unbound neoantigen and self antigen showed that the mutated residue was in the TCR interface. Additionally we noted a change in the side chain position of a proximal tryptophan, potentially due to clashes with the larger phenylalanine residue. The structure of the TCR bound to the neoantigen showed that the TCR interacted with the tryptophan in the mutation-induced conformation and with the phenylalanine residue. Thus the mutation may be altering TCR binding affinity by interactions of the residue itself with the TCR, and by locking the proximal tryptophan residue in an optimal position to interact with the TCR. We are testing the contributions of each of these factors to the overall affinity change. Hydrophobicity has been linked to immunogenicity, so mutations that increase hydrophobicity compared to the self antigen are likely to be immunogenic. However, leucine and phenylalanine are similar on hydrophobicity scales. On the other hand, a side chain rotation is unlikely to represent a large energy barrier. Therefore, we hypothesize that another property of the phenylalanine, such as size or aromaticity, is driving the affinity difference. DISCUSSION/SIGNIFICANCE OF IMPACT: Traditional forms of cancer therapy do not specifically target cancer cells, and their toxicity to healthy cells limits their effectiveness. Immunotherapy, which involves orchestrating a specific anti-cancer immune response, is now an established cancer therapy. Several forms of immunotherapy target “neoantigens,” which are derived from mutated proteins in cancer, and are therefore are cancer-specific. Neoantigens represent a foothold that can allow the immune system to distinguish between cancer cells and healthy cells, and thus specifically target cancer cells for destruction while imparting no activity toward healthy cells that lack the neoantigen. Most cancer mutations that result in neoantigens arise from random passenger mutations in cancer and will be different among patients. Neoantigen-based cancer therapies are thus a precision medicine technique. The quality of neoantigens to induce an immune response (immunogenicity), which relates to how likely they are to be presented to the immune system and recognized as foreign, has been shown to be a critical factor in predicting the outcome of immunotherapy treatment. We are investigating, on a structural and biophysical level, features that may increase the likelihood of a neoantigen being recognized as foreign by the immune system. The structural insight we gain can be incorporated into algorithms that predict neoantigens from cancer exome sequencing for patient-specific identification of immunogenic neoantigens for immunotherapeutic intervention.

OBJECTIVES/SPECIFIC AIMS: This study aims to first describe the unique cytokine profile and TGFbeta levels of young children with CF, then understand the pathologic effects of TGFbeta on lung function in a CF animal model. These powerful translational studies linking observations in clinical disease with a transgenic mouse model allow us a unique opportunity to investigate the role of TGFbeta in early CF lung disease. METHODS/STUDY POPULATION: Cytokine levels (TGFbeta, TNFalpha, IL-8, IL-6, HNE, and IL-1beta) in bronchoalveolar lavage fluid (BALF) from CF patients (n = 15) and non-CF control patients (n = 21) under 6 years old were determined by ELISA and Luminex assay. Tracheotomized patients without significant underlying lung disease were chosen as non-CF inflamed control patients, as they had similar levels of neutrophilic inflammation and infection as CF patients. The percentage of BAL neutrophils (% PMNs) in each sample was assessed. The relationships between cytokines were analyzed using linear regression and principal components analysis. In animal studies, CF and non-CF mice (n = 4-5 per group) were treated with intratracheal adenoviral TGFbeta1 vector, an empty vector control, or PBS. After one week, animals were collected; lung function, response to the bronchoconstrictor methacholine, and rescue with albuterol were measured utilizing a FlexiVent machine. Lungs were collected for histology. Immunohistochemistry for alpha-SMA was performed and pictures of all cross-sectional airways were obtained. Burden of ASM was assessed by dividing the square root of alpha-SMA stained airway smooth muscle by the basement membrane perimeter length of each airway. RESULTS/ANTICIPATED RESULTS: Patient characteristics of CF and non-CF inflamed control patients were similar in terms of age (3.6 yrs vs 3.3 yrs respectively, p = 0.49), positive BAL culture (13% vs 14%, p = 0.94), and % PMNs (65% vs 56%, p = 0.64). Despite these similarities, TGFbeta levels were 2-fold higher in CF versus non-CF BAL (p = 0.034). Analysis of BAL cytokines from both patient groups showed that three principal components describe 86% of total variance across the cytokine variables. These components represent different contributions from the cytokines, with TGFbeta, IL6, and % PMNs comprising one component of similarly regulated inflammatory markers. These components can distinguish CF versus non-CF patients with 77% accuracy (area under the curve: 0.77). TGFbeta concentrations were uniquely associated with increased IL-6 in CF samples (r = 0.74; p = 0.0015) but did not demonstrate association with other cytokines. After TGFbeta exposure, CF mice demonstrated greater abnormalities in airway resistance than non-CF mice, with heightened response to methacholine. Importantly, this increase in airway obstruction in CF mice was reversible with albuterol treatment, indicating airway smooth muscle dysfunction as a principal driver of lung function abnormalities. Furthermore, TGFbeta induced an increased ASM burden on lung histology in both CF and non-CF mice (p<0.05). IL-6 levels in the BAL of CF mice showed greater increases after TGFbeta treatment compared to non-CF mice (p<0.05). Empty vector control treatment did not cause lung pathology. DISCUSSION/SIGNIFICANCE OF IMPACT: Young children with CF have a unique pattern of pulmonary inflammation compared to inflamed non-CF control patients. In CF, TGFbeta pulmonary levels are uniquely associated with IL-6, a driver of ASM dysfunction in other pulmonary diseases. We followed up this clinical observation study by investigating the effect of TGFbeta on pulmonary disease in a mouse model. CF mice demonstrate increased pulmonary IL-6, airway obstruction, and ASM dysfunction after TGFbeta exposure. This study provides evidence that TGFbeta is associated with a distinct cytokine pattern that may promote ASM dysfunction in early CF lung disease. Understanding the mechanism of early CF pathophysiology will be critical in developing targeted therapeutics that can prevent early lung damage.

OBJECTIVES/SPECIFIC AIMS: The primary objective of this research is to determine whether being hand-reared, and deprived of early maternal interaction, will affect telomere length in orphaned kittens. The secondary goal is to examine how early maternal separation impacts the health, growth and behavior of orphaned kittens. METHODS/STUDY POPULATION: Kittens were fostered through local rescue groups and shelters. We collected blood samples from 42 orphaned kittens during the first week of their lives. Due to high mortality of this population, we obtained a second blood sample at eight weeks of age from only 30 of these kittens. We collected blood samples from 12 control kittens raised with mothers at during the first and eighth weeks of life. Blood samples are currently being processed with real time quantitative PCR (qPCR) by the Real-time PCR Research and Diagnostics Core Facility at the UC Davis School of Veterinary Medicine (SVM). This includes RNA extraction, cDNA synthesis, Reference Gene Validation, and qPCR analysis. Relative telomere length (RTL) will be calculated by comparing the average telomere abundance across three samples cells with that of a reference gene (single copy number) for each sample. The resulting T/S ratio (telomere to single copy) is proportional to the average telomere length. If T/S = 1, then telomere length in the sample and the reference are the same. RESULTS/ANTICIPATED RESULTS: Because telomeres show the fastest rate of shortening early in life, we predict that maternal separation will increase the rate of telomere shortening in kittens. We also predict that the telomeres of orphaned kittens will be shorter at both one week and eight weeks of age, compared to controls. DISCUSSION/SIGNIFICANCE OF IMPACT: This study will increase our understanding of early life adversity, a finding that can translate to other mammals. It will inform the practice of fostering neonatal kittens, and illuminate whether these kittens might be at higher risk than mother-reared kittens for health problems (which could be investigated in future studies). If significant telomere shortening occurs between collection periods, then future studies can take more frequent blood samples to determine what stages of early development are potentially most sensitive. If differences between groups are found, this will establish a protocol for several future research projects, such as testing whether these detrimental effects can be mitigated by environmental enrichment via activation of telomerase. Telomerase is an enzyme that appears to counteract some shortening of telomeres, and is activated by several external factors, including exercise. Thus, a logical follow up study would be developing and testing age-specific and appropriate enrichments that may activate telomerase and reduce telomere loss. Physical contact, whether human, mother, or siblings, is another possible source of telomerase activation in young kittens. Future studies also could quantify the effects of different sources of physical contact on telomere shortening. Finally, a positive finding would establish a need for longitudinal studies of the effects of early weaning on feline health and behavior and whether differences in early-life telomere lengths predict health and longevity of cats.

OBJECTIVES/SPECIFIC AIMS: 1. To identify novel genetic modifiers that result in a mild bleeding phenotype in patients with FVIII <1%. 2. To examine the feasibility of a practice model that incorporates the principles and methods for both obtaining consent for NGS and returning individual research results from the sources described above. METHODS/STUDY POPULATION: 1. We plan a 3-step approach for identifying novel genetic modifiers of hemophilia: a. Obtain samples from individuals with an extremely mild bleeding phenotype: The study will be narrowed to patients with confirmed FVIII <1%, a null mutation in the gene for FVIII, and a mild bleeding phenotype according to a detailed bleeding history. b. Identify variants that modify phenotype: Whole exome sequencing will be performed, followed by a focused analysis of genes known or suspected to be involved in thrombosis and hemostasis and prediction of variant impact using algorithms that account for conservation and deleteriousness of all variants. c. Verify the impact of novel variants in independent samples: In silico (analyze genetic databases for suspected variants), in vivo (assess bleeding in animal models of hemophilia after introducing presumed modifier variants). 2. We will employ a model for obtaining informed consent and communicating individual genetic research results and results with potential clinical impact to research participants: a. The informed consent process will be performed after potential participants read a pamphlet entitled “Genetic Research at The Rockefeller University Hospital and Center for Clinical and Translational Science.” The pamphlet includes 16 questions that the potential participants are urged to ask the investigator, including, “What will you look for in my genetic information?”, “Will I receive results from this study?”. Potential participants will also be informed of the meaning of clinically actionable variants, either pathogenic variants related to phenotype or secondary (“incidental”) findings (i.e. variants unrelated to phenotype, the knowledge of which could lead to actions that may improve health). Participants who do not want to receive information about potentially actionable variants will be excluded from the study to avoid a situation where the investigator has clinically important information that cannot be shared with the participant. b. Genetic testing will be performed in a CLIA-certified lab to allow investigators to share the results with the study participants. c. Results will be reported to study participants according to a standard operating procedure (SOP) that classifies the report of variants according to the relation to phenotype and the pathogenic potential. d. Participant satisfaction with the informed consent process and the return of results will be assessed by a questionnaire for obtaining participants’ perceptions of their research experience, based on a standard set of validated research participation experiences measures (Kost RG et al, J Clin Transl Sci. 2018;2:31). RESULTS/ANTICIPATED RESULTS: Samples from individuals with severe null mutation hemophilia and a mild bleeding phenotype will be enriched in genetic modifier variants. After completing participation, participants will express satisfaction with the informed consent process and the results of the return of genetic information. DISCUSSION/SIGNIFICANCE OF IMPACT: Genetic risk assessment to predict bleeding risk has the potential to provide hemophilia patients with tailored therapy, allowing for very early initiation of treatment (prophylactic thrice weekly IV administration of FVIII) in patients with a high bleeding risk and deferring this costly and burdensome treatment in patients who are expected to be mild bleeders. Genetic modifier variants of hemophilia may be found to predict thrombosis in non-hemophiliac patients and profoundly impact the treatment of venous thrombosis. A structured process for obtaining consent for NGS and return of genetic results to study participants can protect them from uncertain genetic information. Moreover, this process will prevent a situation in which investigators have knowledge about clinically actionable variants but they are not allowed to report them to the participants or do not have a process for doing so. Sharing individual research results and results with clinical significance with participants of studies that involve whole exome sequencing can promote transparency and engagement of participants throughout the research enterprise.

OBJECTIVES/SPECIFIC AIMS: The study aims to further investigate how cyclophilin D (CypD), the key mPTP opening regulator, affects BMSCs fate and to determine potential regulatory mechanisms involved in CypD regulation during osteogenesis. METHODS/STUDY POPULATION: We evaluated CypD mRNA expression in mouse BMSCs and in osteogenic-like (OL) cells during the course of OB differentiation. CypD protein level was also probed. Moreover, BMSCs had their mPTP activity recorded during osteoinduction. We further analyzed the effect of CypD genetic deletion on osteogenesis in vitro and in vivo. For our in vivo model, we performed the ectopic bone formation assay to asses differences in ossicle formation when CypD KO BMSCs were transplanted compared to wild type littermate BMSCs. In our in vitro model, we transfected OL cells with either CypD gain of function or CypD loss of function vector and measured their osteogenic differentiation potential. Additionally, we treated BMSCs with CypD inhibitor and compare to non-treated BMSCs for mineralization level. To determine potential regulatory mechanisms involved in CypD regulation, we analyzed the CypD gene (Ppif) promoter for potential transcription factor (TF) binding sites and found multiple Smad-binding elements within this promoter. Smads (Smad1, 5, 8) are TFs downstream from Bone Morphogenic Protein (BMP) signaling pathway that transmit cell differentiation signaling, and exert either activating or inhibitory effects on a variety of genes. We also transfect OL cells with Smad1 vector and analyzed for CypD mRNA levels. RESULTS/ANTICIPATED RESULTS: - Our data showed that CypD mRNA levels decreased in both primary cells and OL cells at day 7 and day 14 in osteogenic media. - Osteogenic induction also decreased mPTP activity. - In vivo ectopic bone formation assay showed increased ossicle fo DISCUSSION/SIGNIFICANCE OF IMPACT: Our data suggest that downregulation of CypD increases OB differentiation due to improved OxPhos activity led by mPTP closure. Our results corroborate reports of CypD downregulation and mPTP closure during neuronal differentiation in developing rat brains as well as in cardiomyocyte differentiation in developing mouse hearts. Our studies also suggest a yet unknown mechanism linking differentiation signaling with mitochondrial function – BMP/Smad mediated downregulation of CypD transcription. As initially mentioned, in a previous study, our lab showed that CypD KO mice present higher mitochondrial function and osteogenicity in aged BMSCs and less osteoporosis burden. Taken together, these results suggest that CypD can be a potential target to prevent bone loss in aging.

OBJECTIVES/SPECIFIC AIMS: Our primary objective is to determine the mechanism of action of myostatin on osteoblasts by measuring markers of osteoblast differentiation. With these experiments we will evaluate the effects of myostatin on an osteoblastic cell line (MC3T3 cells) and primary murine osteoblasts during baseline and hyperglycemic conditions and assess whether these effects are altered in the presence of a hyperglycemic environment. METHODS/STUDY POPULATION: Primary osteoblasts from calvaria of WT mice will be isolated and cultured per previously published protocol. MC3T3 cells (murine pre-osteoblast cell line) and primary osteoblasts will be plated in 6-well plates until they reach confluency. They will subsequently be stimulated with or without myostatin at various concentrations under control and hyperglycemic conditions. Additional experiments will assess myostatin stimulation during cell differentiation/maturation in the presence of osteogenic induction medium. Subsequently, cells will be lysed and processed for gene analysis with qPCR. Genes of interest (e.g., myostatin, RUNX2, osteocalcin etc.) will be assessed. Additionally, cells will be collected and processed for protein quantification with western blot to assess myostatin-related pathways, such as Smad2/3 and MAPK signaling. RESULTS/ANTICIPATED RESULTS: We have demonstrated that the receptor for myostatin (Activin receptor 2b, AcvR2b) is present in MC3T3 cells and we have evidence of Smad2 phosphorylation in MC3T3 cells as a result of myostatin stimulation, confirming that myostatin can exert intracellular signaling events in bone cells (Fig 1). We anticipate to observe negative effects of myostatin on differentiation of primary osteoblasts and MC3T3 cells. Specifically, we anticipate suppression of Runt-related transcription factor 2 (RUNX-2), a transcription factor known as the “ master regulator” of osteogenic gene expression and programming, as a result of signaling downstream of Smad 2/3. Additionally we anticipate downregulation of osterix and osteocalcin, two essential genes for osteoblast differentiation and activity. We anticipate that hyperglycemia will potentiate the negative effects of myostatin on osteoblastogenesis. DISCUSSION/SIGNIFICANCE OF IMPACT: We have demonstrated that myostatin can directly act on osteoblastic cells. As myostatin is a negative regulator or bone mass, its direct effects on bone cells can be detrimental to the bone health of patients with elevated myostatin levels and/or activity. There is evidence suggesting that myostatin is elevated in Type 1 diabetes, and its effects might be potentiated in a hyperglycemic environments. Future experiments will be evaluating the role of myostatin on a diabetic animal model and in humans. Our experiments provide an additional mechanism by which muscle-bone interactions could be contributing to the development of diabetic bone disease.

OBJECTIVES/SPECIFIC AIMS: Selective loss of long-projecting neural circuitry is a common feature of many neurodegenerative diseases, such as the vulnerable nigrostriatal pathway in Parkinson’s disease (PD). Current in vitro approaches for studying disease development generally do not mimic complex anatomical features of the afflicted substrates such as long axonal pathways between stereotypical neural populations. Such exquisite features are not only crucial for neural systems function but may also contribute to the preferential vulnerability and pathophysiological progression of these structures in neurodegenerative disease. We have previously developed micro-tissue engineered neural networks to recapitulate the anatomy of long-projecting cortical axonal tracts encased in a tubular hydrogel.1 Recently, we have extended this work to include the first tissue-engineered nigrostriatal pathway that was anatomically-inspired to replicate the structure and function of the native pathway.2 Notably, this tissue-engineered brain pathway possesses three-dimensional (3D) structure, multicellular composition, and architecture of long axonal tracts between distinct neuronal populations. Therefore, in the current study we apply this system as a biofidelic test-bed for evaluating axonal pathway development, maturation, and pathophysiology. METHODS/STUDY POPULATION: Dopaminergic neurons from the ventral mesencephalon and medium spiny neurons (MSNs) from the striatum were separately isolated from rat embryos. Tissue-engineered nigrostriatal pathways were formed by initially seeding dopaminergic neuron aggregates at one end of hollow hydrogel micro-columns with a central extracellular matrix, collectively spanning up to several centimeters in length. Several days later, tissue-engineered MSN aggregate was seeded on the other end and was allowed to integrate. Immunocytochemistry (ICC) and confocal microscopy were used to assess health, cytoarchitecture, synaptic integration, and mitochondrial dynamics with stains that label cell nuclei (Hoechst) and mitochondria (MitoTracker Red) and antibodies that recognize axons (anti-β-tubulinIII), neurons/dendrites (anti-MAP2), dopaminergic neurons/axons (anti-tyrosine hydroxylase; TH), and MSNs (anti-DARPP-32). RESULTS/ANTICIPATED RESULTS: Seeding tubular micro-columns with dopaminergic neuronal aggregates resulted in unidirectional axonal extension, ultimately spanning >5mm by 14 days in vitro. For constructs also seeded with Tissue-engineered, ICC confirmed the presence of the appropriate neuronal sub-types in the two aggregate populations, specifically TH+ dopaminergic neurons and DARPP-32+ MSNs. Moreover, confocal microscopy revealed extensive axonal-dendritic integration and synapse formation involving the dopaminergic axons and MSN somata/dendrites. Collectively, these constructs mimicked the general cytoarchitecture of the in vivo nigrostriatal pathway: a discrete population of dopaminergic neurons with long-projecting 3D axonal tracts that were synaptically integrated with a population of MSNs. Mitochondria structure along axonal tracts was also observed using MitoTracker staining, revealing dynamic intra-axonal mitochondrial motility in this system. Ongoing studies are evaluating real-time mitochondrial dynamics and axonal physiology in this tissue-engineered nigrostriatal pathway in vitro, under both baseline conditions as well as following the addition of exogenous α-Synuclein fibrils to model synucleinopathy in PD. DISCUSSION/SIGNIFICANCE OF IMPACT: This tissue-engineered nigrostriatal pathway provides an anatomically-inspired platform with neuronal-axonal architecture that structurally and functionally emulates the nigrostriatal pathway in vivo. We are applying this paradigm as a powerful in vitro test-bed for understanding mitochondrial activity and inter-axonal energetics pathways under homeostatic as well as PD pathological conditions. Successful demonstration will serve as proof-of-concept that this technique can be used to study mitochondria pathology in personalized constructs built using cells derived from PD patients in order to evaluate pharmacological therapies targeted at improving mitochondrial resiliency and fitness so as to delay and/or prevent dopaminergic axonal/neuronal degeneration in tailored to specific PD patients.

OBJECTIVES/SPECIFIC AIMS: Triple-negative breast cancer (TNBC) accounts for one-fifth of the breast cancer patient population. The heterogeneous nature of TNBC and lack of options for targeted therapy make its treatment a constant challenge. The co-deficiency of tumor suppressors p53 and ARF is a significant genetic signature enriched in TNBC, but it is not yet clear how TNBC is regulated by this genetic alteration. METHODS/STUDY POPULATION: To answer this question, we established p53/ARF-defective murine embryonic fibroblast (MEF) to study the molecular and phenotypic consequences in vitro. Moreover, transgenic mice were generated to investigate the effect of p53/ARF deficiency on mammary tumor development in vivo. RESULTS/ANTICIPATED RESULTS: Increased transformation capability was observed in p53/ARF-defective cells, and formation of aggressive mammary tumors was also seen in p53-/-ARF-/- mice. RNA-editing enzyme ADAR1 was identified as a potential mediator for the elevated oncogenic potential. Interestingly, we found that the overexpression of ADAR1 is also prevalent in human TNBC cell lines and patient specimen. Using short hairpin RNA (shRNA) to reduce ADAR1 expression abrogated the oncogenic potential of human TNBC cell lines, while non-TNBC cells are less susceptible. Different levels of RNA editing of known ADAR1 targets were detected in shRNA-treated human TNBC cell lines, suggesting that ADAR1-mediated RNA editing contributes to TNBC pathogenesis. DISCUSSION/SIGNIFICANCE OF IMPACT: These results indicate critical roles played by the tumor suppressors p53 and ARF in the pathogenesis of TNBC, partially through affecting ADAR1-mediated RNA editing. Further understanding of this pathway could shed light on potential vulnerabilities of TNBC and inform the development of personalized therapies based on patients’ genetic signiatures.

OBJECTIVES/SPECIFIC AIMS: To study the biology of Phosholamban (PLN) in a human relevant model. METHODS/STUDY POPULATION: State of the art stem-cell technologies using iPSC-CMs derived from carriers of a lethal PLN mutation. RESULTS/ANTICIPATED RESULTS: Our preliminary data demonstrate that this particular PLN mutation (L39) results in reduced expression and mis-localization of PLN as well as increased incidence of early after depolarization in isolated iPSC-CMs. DISCUSSION/SIGNIFICANCE OF IMPACT: Phospholamban (PLN) is a critical regulator of Ca++ homeostasis yet many uncertainties still remain regarding its role in humans. Our study will provide unique insights into the pathophysiology of this protein in HF.