OBJECTIVES/GOALS: SGLT2i therapy is currently a cornerstone in heart failure with preserved ejection fraction (HFpEF) therapy. Similarly, H2S has been shown to be beneficial in preclinical models of heart failure. With this in mind, we sought to investigate the effects of the SGLT2i and H2S donor therapy alone or in combination in a rodent model of cardiometabolic HFpEF. METHODS/STUDY POPULATION: Male C57BL/6N mice (9 weeks of age) were fed a high fat, Western diet (HFD) and received L-NG-Nitro arginine methyl ester (L-NAME) in the drinking water (0.5 g/L) to induce HFpEF. At 5 weeks, animals were randomized to either control, H2S donor (SG-1002, 90 mg/kg/d, P.O), Empagliflozin (155 mg/L, P.O), or the combination of SG-1002 and Empagliflozin for an additional 5 weeks while being maintained on HFD and L-NAME. Echocardiography, left ventricular invasive LV and systemic hemodynamics, and exercise capacity testing were performed to assess cardiovascular disease severity. Fasted glucose, circulating triglyceride and cholesterol content were similarly measured to quantify key clinical metabolic parameters. H2S and its metabolite, sulfane sulfur, were quantified to assure adequate H2S donation. RESULTS/ANTICIPATED RESULTS: Administration of SG-1002 restored H2S and sulfane sulfur to normal circulating levels. All treatment groups exhibited similar improvements in LV diastolic dysfunction as measured by E/E’and LVEDP. Combination therapy significantly improved exercise capacity whereas the monotherapy groups did not. Treatment with SG-1002 decreased fasting glucose and circulating cholesterol while all treatment groups displayed decreased circulating triglycerides and body weight compared to HFpEF control. DISCUSSION/SIGNIFICANCE: These data indicate that restoring H2S or treatment with an SGLT2i in this preclinical HFpEF model attenuates pathology. Combination of both drugs exhibited greater benefit than either monotherapy in important HFpEF parameters such as exercise capacity. Further studies are underway to characterize the benefits observed from combination therapy.

OBJECTIVES/GOALS: To develop an untargeted metabolomics assay that can holistically characterize the small molecule signatures of different inborn errors of metabolism (IEM) for biomarker discovery and identification of novel IEMs, with the goal of implementing the assay into the clinical laboratory to improve testing efficiency. METHODS/STUDY POPULATION: A hydrophilic interaction liquid chromatography (HILIC) column and reverse phase (RP) column were assembled in tandem on a SCIEX X500B quadrupole time-of-flight (QTOF) system to create a dual liquid chromatography (LC), tandem mass spectrometry method. The X500B was operated in data-independent acquisition mode with both positive and negative ionization. A mixture of 165 reference standards from eleven compound classes common to IEMs were used to evaluate the capability of the assay to resolve small molecules. Chromatographic resolution for each standard was determined qualitatively by comparison to a reference spectral database. External validation of the assay will be performed by analyzing a commercial library of reference metabolites. RESULTS/ANTICIPATED RESULTS: A total of 88% (146/165) of the standards were detected by the assay. The RP column alone resolved 71% (117/165) of the standards, the HILIC column resolved 33% (55/165), while 17% (29/165) of the standards were resolved by both columns. The HILIC column resolved standards that were more polar, while the RP column resolved more non-polar compounds. To evaluate matrix effects, the reference standard mixture was spiked into pooled plasma. In the presence of plasma 6/146 (4%) of the standards were suppressed to levels below the limit of detection. We expect external validation with the commercial metabolite library will corroborate these results, and that the high-quality spectral data attained from this reference library can be used to improve identification of unknown metabolites in patient samples. DISCUSSION/SIGNIFICANCE: We have shown our untargeted metabolomics assay can detect known biomarkers for IEMs. Clinical implementation of this method could streamline diagnosis of IEMs while simultaneously improving patient outcomes by leveraging the metabolome for biomarker discovery, and improved understanding of IEM mechanisms to inform novel treatment strategies.

OBJECTIVES/GOALS: Patient adherence with antidepressant therapy is a critical aspect of clinical management. Drug molecules often interact with other off-target proteins resulting in adverse events. We utilized an interactome model for predicting the likelihood of adverse drug reactions in order to accurately prescribe antidepressant medications to a patient. METHODS/STUDY POPULATION: We utilized an interactome model to study physical interactions between proteins and biological functions disrupted by major depression to understand how changes in genes alter patient-specific drug efficacy and cause adverse drug-reactions. To study how drugs and diseases propagate through the proteins and biological functions, we harnessed diffusion profiles to represent nodes in the graphical model and used a biased random walks model to illustrate how signals propagate in a heterogeneous biological network. The edge weights were defined for the drug, disease, protein, and biological function node types. The interactions studied included drug-protein, disease-protein, protein-protein, protein-biological function, biological function-biological function. RESULTS/ANTICIPATED RESULTS: Based on previous studies, we anticipate that the genetic variants modulating antidepressant response include 5-HTT, STin2, HTR1A, HTR2A, TPH1 and BDNF. Additionally, genetic variants such as SLC6A4 as well as the HPA pathway may play an important role in antidepressant therapeutic response. With regards to medication drug response, the genes, SLC6A4 and HTR2A, have been known for encoding proteins that affect the synaptic cleft of serotonergic neurons. Also, the gene SLC6A4 has been shown to encode for the serotonin reuptake transporter, which is the main pharmacological target of SSRIs. In female patients with major depression, the polymorphism associated with MAOA gene may be involved in the pharmacological response. DISCUSSION/SIGNIFICANCE: The key to successful depression treatment is early adherence but nearly 60% of patients discontinue antidepressants within three months. By developing a strong understanding of the genes that alter treatment efficacy, we can provide patients with more awareness regarding the most effective treatment plan to minimize adverse events.

OBJECTIVES/GOALS: Extracellular vesicles (EVs) in biofluids can reflect pathology in the brain (like alpha-synuclein protein linked to Dementia with Lewy Bodies) making them attractive disease biomarkers to distinguish disease. Here, we will investigate their potential as non-invasive biomarkers to differentiate Dementia with Lewy Bodies & Alzheimer's Disease. METHODS/STUDY POPULATION: We will leverage the collection of antemortem plasma from autopsy confirmed Dementia with Lewy Bodies (DLB), Alzheimer's Disease (AD) & healthy controls (HC) through the Mayo Clinic Brain Bank. We will characterize antemortem brain-derived plasma EVs (bPEVs) & post-mortem brain-derived EVs (bEVs) of the same patient using nanoparticle tracking, electron microscopy, & western blotting for EV-associated proteins. We will then measure levels of total & phosphorylated alpha-synuclein (asyn) using AlphaLisa. We will assess EVs' ability to initiate asyn aggregation using a real-time quaking-induced conversion (RT-QuIC) assay & a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Once characterized, this will allow differentiation of bpEVs in DLB, AD, or healthy controls. RESULTS/ANTICIPATED RESULTS: We hypothesize that asyn levels and seeding capacity will discriminate DLB from AD. With AlphaLisa assay, we expect higher total and phosphorylated asyn levels in DLB bpEVs and their corresponding post-mortem bEVs. Using RT-QuIC and FRET assay, we expect EVs isolated from DLB patient samples to support seeded aggregation, whereas EVs from AD and HC will not. DISCUSSION/SIGNIFICANCE: To date, no diagnostic or less invasive biomarkers can distinguish DLB from AD. The successful completion of the aims outlined in this proposal will identify characteristics of bpEVs that differentiate DLB from AD or HC and support the development of bpEVs as a non-invasive, early biomarker to diagnose patients presenting with dementia from DLB.

OBJECTIVES/GOALS: The goal of this study is to investigate the potential independent relationship between epicardial adipose tissue (EAT) and cardiometabolic health in youth-onset type 2 diabetes (T2D) and explore changes in EAT as a potential mediator of changes in cardiometabolic health in response to vertical sleeve gastrectomy (VSG). METHODS/STUDY POPULATION: We will assess glycemic control, insulin sensitivity and secretion in youth with T2D before and 3 months after VSG. Fasting labs, anthropometrics, and a 4-hour, frequently sampled liquid mixed meal tolerance test (45g carbohydrates, 14g fat, and 14g protein) were performed. Calculations included glucose, insulin, and GLP-1 area under the curve (AUC), Matsuda Index, HOMA-IR, and oral disposition index (DI). These cardiometabolic outcomes will then be assessed for associations between total EAT volume, measured from cardiac MRI. RESULTS/ANTICIPATED RESULTS: Previous studies have shown that individuals with obesity have higher EAT than lean controls, and adults with T2D have even higher EAT than obese controls. Therefore, we anticipate that our participants will have higher volume of EAT than what has been reported in the literature and that they will have worsening cardiometabolic outcomes without MBS. Our anticipated results will include: Weight and BMI, hemoglobin A1c, diabetes medications, Matsuda Index, HOMA-IR, DI, and glucose and insulin AUC during an MMTT. Cardiac MRI's are being analyzed and will give total EAT volume and will be analyzed for correlations with the cardiometabolic outcomes of body composition, aortic stiffness, blood pressure, cardiac structure and function, as well as lipid panel and insulin sensitivity. DISCUSSION/SIGNIFICANCE: This study is the first to specifically assess EAT in adolescents with T2D. The assessment of EAT will be done with gold-standard MRI and correlated with cardiometabolic health assessed by gold-standard methods. Together, the results will give insight into EAT as a potential independent cardiometabolic risk factor in adolescents undergoing VSG.

OBJECTIVES/GOALS: Retinal inflammation caused by the activation of resident macrophages (microglia) during diabetes exacerbates glial cell dysfunction, resulting in neuronal loss. The goal is to use rAAV gene therapy to deliver neuronal-derived fractalkine (FKN), minimizing inflammation and vascular damage in the diabetic retina. METHODS/STUDY POPULATION: The human microglial receptor (CX3CR1) binds to FKN, a protein that is expressed on neuronal membranes (mFKN), and undergoes constitutive cleavage to release a soluble domain (sFKN). Deficiencies in CX3CR1 or FKN showed increased microglial activation and elevated retinal pathology. To understand the mechanism by which mFKN and sFKN regulate microglia function, recombinant adeno-associated viruses (rAAVs) expressing mFKN or sFKN were delivered to intact retinas during diabetes. Markers of neuronal loss, vascular damage, and inflammation were analyzed. We hypothesize that the administration of rAAV-sFKN but not rAAV-mFKN will prevent vascular and neuronal damage, and improve visual function. RESULTS/ANTICIPATED RESULTS: rAAV-sFKN minimized microglial activation, blood vessel rupture, fibrinogen deposition, and prevented neuronal loss, compared to mice treated with rAAV-mFKN in a mouse model of diabetic retinopathy (DR). rAAV-sFKN treated mice showed improved visual acuity using a two-choice discrimination task through learning-based behavior. rAAV-sFKN treatment correlated with the success rate of the mice finding the reward based on their ability to distinguish visual cues. Future studies will test the effects of rAAV-sFKN and rAAV-mFKN on microglia inflammatory cytokine release, optic nerve damage and synaptic neurotransmission, peripheral immune responses, and transcriptomic changes in microglia during diabetes. DISCUSSION/SIGNIFICANCE: Current therapies for DR are ineffective in restoring vision. rAAVs-sFKN delivery appears to act as a neuroprotective approach in the diabetic retina. sFKN serves as an alternative pathway to implement translational and therapeutic approaches, minimizing pathology and improving visual function.

OBJECTIVES/GOALS: This study investigates whether localization of high mobility group box 1 (HMGB1) controls inflammatory signaling and DNA damage response in human keratinocytes, the cell of origin for squamous cell carcinoma (SCC). SCC is especially metastatic in chronic wounds, burns, and in patients with recessive dystrophic epidermolysis bullosa (RDEB). METHODS/STUDY POPULATION: We used CRISPR/Cas9 gene-editing to knock out HMGB1 in a keratinocyte line, p16INK4a-negative keratinocytes immortalized by ectopic hTERT expression (N/TERT-2G [46,XY]). Following gene editing, clonal keratinocyte populations were screened for knockout by PCR followed by TIDE analysis (Tracking of Indels by Decomposition) to identify indels that would result in a frameshift mutation. Total cell lysates for each clonal population were analyzed by immunoblot and immunofluorescence for HMGB1 protein. These cells will be used to assay for DNA damage sensitivity in the presence of genotoxic agents (etoposide, ultraviolet radiation, γ-irradiation). A lentiviral vector will then be used to express mutant forms of HMGB1 that localize to the nucleus (C23/45S) or cytoplasm (C106S) and DNA damage assays repeated. RESULTS/ANTICIPATED RESULTS: We have confirmed by sequencing, immunoblot, and immunofluorescence that HMGB1 is knocked out in a clonal population of N/TERT-2G human keratinocyte cells. We anticipate that cells with a complete absence of HMGB1 will have high sensitivity to DNA damaging agents, but little change in inflammatory signaling. We also expect that cells expressing mutant HMGB1 that localizes exclusively to the cytoplasm will demonstrate an increased sensitivity to DNA damage relative to wild-type controls, while mutant HMGB1 that localizes exclusively to the nucleus will be protected from DNA damage caused by exposure to genotoxic agents. DISCUSSION/SIGNIFICANCE: HMGB1 is a nuclear protein and damage associated molecular pattern that is elevated systemically in patients with RDEB, many of whom will go on to develop metastatic squamous cell carcinoma. The experiments described here investigate whether HMGB1 plays a mechanistic role in skin carcinogenesis via regulation of the DNA damage response.

OBJECTIVES/GOALS: Ebola virus infection causes severe disease and liver injury in humans. Macrophages contribute to inflammatory signaling and are prevalent in the liver. We assessed the activation status, including therapeutic target expression, of hepatic macrophages. METHODS/STUDY POPULATION: We compared formalin-fixed, paraffin-embedded liver tissue from terminal Ebola virus-infected and uninfected control cynomolgus macaques, a gold-standard model for human disease. We characterized region-specific protein and whole transcriptome expression in these tissues using GeoMx Digital Spatial Profiling. Macrophage (CD68+) and leukocyte (CD45+) accumulation in liver tissue was quantified by immunofluorescence image analysis using digital pathology software. RESULTS/ANTICIPATED RESULTS: Macrophage-specific (CD68+) regions in the liver of Ebola virus-infected macaques demonstrated a shift towards an inflammatory gene expression profile, as compared to those from healthy control tissue. These regions showed differential expression of monocyte/macrophage differentiation, antigen presentation, and T cell activation gene sets, which were associated with decreased MHC-II allele expression. Moreover, macrophage-specific regions in the infected macaques showed enriched expression of genes or proteins associated with known immunomodulatory therapeutics, including S100A9, IDO1, and CTLA-4. DISCUSSION/SIGNIFICANCE: These data demonstrate that hepatic macrophages express an inflammatory phenotype, that their ability to present antigens to the adaptive immune system may be impaired, and that they express therapeutically targetable markers for immunomodulation of these cells during Ebola virus infection.

OBJECTIVES/GOALS: 22q.11 deletion syndrome (22q11DS) is a genomic syndrome that elevates risk for psychosis >20-fold. We used a battery of cognitive and psychophysiological psychosis-risk biomarkers in 22q11DS patients and healthy subjects in order to identify biomarkers of psychosis in 22q11DS that could be used as translational targets in intervention studies. METHODS/STUDY POPULATION: We recruited 15 22q11DS individuals (Mean age=30, M/F=9/6) and 19 healthy controls (HCs; Mean age=34, M/F=5/14). Each individual completed the MATRICS Consensus Cognitive Battery (MCCB), the Wechsler Abbreviated Scale of Intelligence, Second edition (WASI-II) Verbal IQ subtests, and the computerized Wisconsin Card Sorting Task (WCST). To examine auditory EEG responses, each participant completed the 'Double-Deviant' target detection paradigm, which presents a pseudorandom sequence of frequent standard tones and infrequent deviant tones. Mismatch negativity (MMN) metrics were generated from this assessment. Welch's t-tests were completed for neurocognitive variables. One-Way ANOVAs were completed to examine EEG results, with sex entered as a separate factor and age entered as a covariate. RESULTS/ANTICIPATED RESULTS: Significant group differences were found in 8 of the 9 neurocognitive measurements (FDR-adjusted p's< 0.02, average Cohen's d=1.62, average observed power= 0.91) indicating widespread cognitive deficits in 22q11DS subjects across multiple domains. The Double-Deviant MMN ERP response was significantly smaller in absolute magnitude in the 22q11DS group (FDR-adjusted p=0.048, Cohen’s d= -0.864, observed power= 0.58). The MMN ERPs for the frequency and duration deviants were not significantly different (FDR-adjusted p's> 0.33). No group by sex interactions were observed in any of the measures. Neurocognitive variables were associated with psychosis positive, negative, general, and disorganized symptom scales. DISCUSSION/SIGNIFICANCE: Our results identify potential psychosis-risk biomarkers in 22q11DS. If replicated, these biomarkers could provide important translational targets for future clinical trials for individuals with 22q11DS and other individuals at-risk for psychosis syndromes.

OBJECTIVES/GOALS: Children with traumatic intracranial hemorrhage are monitored closely for deterioration and need for intervention. Data on risk factors for deterioration in nonsevere head injury are limited. Our objective was to identify children with hemorrhage from non-severe head injury who are at risk for deterioration. METHODS/STUDY POPULATION: We conducted a 10-site cross-sectional study of children 8. Our primary outcome was clinically important hemorrhage after injury and within 96 hours of ED arrival, defined as ED interventions (intubation, hyperosmotic agents, or neurosurgery within 4 hours of arrival) or clinically important deterioration (new or worsening signs/symptoms with an acute change in management). After testing model assumptions, we used logistic regression to identify clinical and neuroradiographic factors associated with clinically important hemorrhage. RESULTS/ANTICIPATED RESULTS: We studied 763 children with intracranial hemorrhage, with a median (IQR) age of 3.0 (0.4, 10.5) years. Initial GCS was mild (14-15) in 89.4% (n=682) and moderate (9-13) in 10.6% (n=81). Clinically important hemorrhage was observed in 19.5% (n=149), and 7.8% (n=59) developed clinically important deterioration. Median (IQR) time to deterioration was 17.6 (4.6, 37.9) hours. In our sample, 16.3% (n=124) underwent critical interventions, 54.9% (n=419) were admitted to an ICU, and 50.1% (n=382) underwent repeat neuroimaging. We found older age (OR 1.6; 95% CI 1.3, 1.9), lower GCS (OR 5.0; 95% CI 2.9, 8.5), and epidural hemorrhage (OR 3.3; 95% CI 2.0, 5.5) was associated with clinically important hemorrhage. DISCUSSION/SIGNIFICANCE: Clinically important hemorrhage occurred in one in five children with non-severe head injury. Clinical and neuroradiographic factors associated with ED interventions and deterioration were identified. Risk stratification algorithms using these data will be developed to assist clinicians caring for children with head injury.

OBJECTIVES/GOALS: The overall aim of the proposed study is to evaluate the effectiveness and clinical utility of the NMR as a biomarker of response to placebo, transdermal nicotine, and varenicline to be utilized within the clinical practice as a point-of-care predictor to tailor an individual’s smoking cessation treatment. METHODS/STUDY POPULATION: A 12-week phase II, stratified multicenter, randomized, placebo-controlled trial of 3 treatment groups to measure treatment-seeking smokers (n-900:150 slow metabolizers; 150 normal metabolizers), randomized to 12-weeks of nicotine patch (active patch + placebo pill), varenicline (active pill + placebo patch), or placebo (placebo pill + patch). At the end of treatment, patients will be followed for 12 months. Study Population: Adult smokers 18–65 years old reported smoking≥10 cigarettes/day for≥6 months (verified by carbon monoxide (CO) >10 ppm). Drug and route of administration: RESULTS/ANTICIPATED RESULTS: The study would conclude the effectiveness of the interventions well defined. DISCUSSION/SIGNIFICANCE: The report will provide robust evidence to support the effectiveness of NRT intervention on smoking cessation.

OBJECTIVES/GOALS: Approximately 37 million people in the U.S. have chronic kidney disease, which is a major risk factor for cardiovascular and end stage renal diseases. PPAR-αknockout (KO) mice exhibit increased renal inflammation and blood pressure. In this study, we investigated the role of PPAR-αin renal function in a mouse model of hypertension. METHODS/STUDY POPULATION: Male 4-month-old wild type (WT) and PPAR-αKO mice were instrumented with radio transmitters by artery canulation (Data Science Intl). This method minimizes stress and artifacts by avoiding the use of tethering, restraining, or anesthetizing the mice during data sampling. After recovery from surgery, we continuously measured mean arterial pressure (MAP) via radio telemetry in conscious ambulatory mice. After baseline MAP was established, vehicle (Veh; saline) or angiotensin II (Ang II) were infused using an osmotic minipump at a slow pressor dose (400 ng/kg/min) for 12 days. On day 12, we injected an intravenous bolus of fluorescin-sinistrin (3.74µl/g body weight) and collected 8 blood samples (20µl/sample) over 75 minutes to enable calculation of the glomerular filtration rate (GFR) using [GFR = I/(A/α+ B/ß)]. RESULTS/ANTICIPATED RESULTS: Similar to our prior observations, no significant (ns) differences in baseline MAP were observed between WT and PPAR-αKO mice [(mmHg): WT (n=6), 111 ± 20 vs. PPAR-αKO (n=6), 113 ± 10; ns] whereas after 12 days of the slow pressor effect of Ang II, MAP was increased in both strains [(mmHg): WT (n=8), 138 ± 11# vs. PPAR-αKO (n=8) , 156 ± 16#; #p DISCUSSION/SIGNIFICANCE: PPAR-αprotects mice from worsening hypertension and is critical to preserving GFR during normotensive conditions. Ongoing studies are further investigating how PPAR-αregulates renal function. These finding suggest therapeutics designed to increase PPAR-αactivity could have clinical benefit in chronic kidney disease.

OBJECTIVES/GOALS: While mutant TP53 is an attractive therapeutic target in TNBC, attempts to target the mutant p53 protein directly have failed. Thus, we aim to identify pathways critical for the survival of TP53 mutant cells that can be targeted in TNBC. We have identified Kif11 as one such target and aim to further investigate its function in TP53 mutant TNBC. METHODS/STUDY POPULATION: We conducted a dual in silico/in vivo screen that identified Kif11 inhibition as preferentially inhibiting the growth of TP53 mutant TNBC. We obtained data on TP53 mutational status, KIF11 mRNA expression levels, and clinical characteristics from TCGA, METABRIC, and CCLE datasets. We treated breast cancer cell lines with the KIF11 inhibitor SB-743921. Cell counts were obtained through staining with DAPI or Hoechst and imaging on the ImageXPress PICO. We detected cell death by DRAQ7 staining and flow cytometry analysis following Annexin V-PI staining. To investigate mitotic spindle organization, we performed immunofluorescent staining with an anti-tubulin antibody and DAPI co-staining. Cell cycle analysis was performed through flow cytometry. RESULTS/ANTICIPATED RESULTS: KIF11 is highly expressed in TP53 mutant and TNBC clinical samples. High KIF11 expression is associated with poorer clinical outcomes. Kif11 inhibition suppresses growth of both TP53 mutant and wild-type breast cancer cells, but preferentially induces the death of TP53 mutant cells as detected by DRAQ7 and Annexin V/PI staining. Kif11 inhibition induces a G2-M block and growth inhibition in TP53 wild-type cells. On the other hand, following treatment with the Kif11 inhibitor SB-743921, TP53 mutant cells undergo mitotic spindle dysfunction leading to the formation of multinucleated cells and cell death. DISCUSSION/SIGNIFICANCE: These results demonstrate that Kif11 is a promising therapeutic target in aggressive, TP53 mutant TNBCs. Kif11 inhibitors, including SB-743921, have been tested in human trials, and are well tolerated, but it is unclear which patients would most benefit. Our studies show that Kif11 inhibitors may be most useful in patients with TP53 mutant TNBCs.

OBJECTIVES/GOALS: Leigh Syndrome, French Canadian-Type (LSFC) is a neurometabolic disorder caused by mutation of mitochondria-related gene, LRPPRC. White matter lesions and demyelination in central nervous system are common in LSFC. LRPPRC is enriched in myelinating glial cells, yet its role is not known. Our goal is to elucidate its mechanistic role in myelination. METHODS/STUDY POPULATION: We crossed C57BL/6N mice bearing a LRPPRC-loxP allele with mice bearing a Plp-CreERT2 allele. Mice with the Plp-CreERT2 allele expresses a tamoxifen-inducible Cre under the control of the Plp promoter, which drives expression in oligodendrocytes. Using these strains, we can target the deletion of LRPPRC, via tamoxifen injection, in both newly formed myelin and mature myelin. Plp-CreERT2; LRPPRCL/L (LRPPRC-KO) or control littermate mice will be injected for LRPPRC deletion at developmental and maturation stages of myelin. Immunofluorescence and electron microscopy of isolated brain tissues will be used for myelin integrity analysis. Cognitive functions of the mice will be measured via behavioral tests. Lastly, we will submit tissues for lipidomic analyses to observe any lipid metabolite variation. RESULTS/ANTICIPATED RESULTS: Behavioral and motor defects would be expected in LRPPRC-KO mice performing in cognitive function tasks across myelin maturation stages. Electron microscopy-based structure analysis of optic nerve, corpus callosum, and spinal cord should reveal thin or loss of myelin on the axons of LRPPRC-KO compared to control. Immunofluorescence staining of major myelin structural proteins, including myelin proteolipid protein (PLP), myelin basic protein (MBP), and myelin-associated glycoprotein (MAG) would be expected have lower levels in LRPPRC deficient tissues. Since myelin is a lipid-rich species, we would also expect lipid concentrations to be affected. LRPPRC-KO lipidomic analyses of myelin-related lipids should depict lower levels in comparison to control, which would imply dysfunctional lipid metabolism. DISCUSSION/SIGNIFICANCE: There are limited studies in ameliorating neural deficits caused by LS and LSFC. Successful completion of this project would help elucidate the functions of LRPPRC in myelination and lipid metabolism and potentially provide insights for developing novel therapeutic strategies for alleviating the demyelination and neural deficits in LSFC.

OBJECTIVES/GOALS: Interleukin (IL)-6 is produced in excess in Systemic Sclerosis (SSc). Likewise, microarray analysis of Insulin-like Growth Factor (IGF)-II-treated NL fibroblasts revealed increased expression of the basic helix-loop-helix transcription factor, BHLHB2. Our goal is to delineate the role of BHLHB2 in the fibrotic response to IGF-II and IL-6. METHODS/STUDY POPULATION: Primary lung fibroblasts were cultured from human lung tissues at 37oC and 5% CO2. Cell cultures were stimulated with IL-6. Gene expression was measured using quantitative PCR (qPCR). IGF-II mRNA expression levels after IL-6 stimulation were compared with those of the housekeeping gene PPIB (Peptidylprolyl Isomerase B). Western blot was performed on nuclear and chromatin-bound subcellular fractions from treated lung fibroblasts. BHLHB2 protein levels were assayed in response to IGF-II in comparison to PBS as vehicle control. RESULTS/ANTICIPATED RESULTS: Results: Our results show that IL-6 increases IGF-II levels in fibroblasts. In turn, IGF-II increases BHLHB2 nuclear localization. We further show that IL-6 increases BHLHB2 levels and its nuclear localization in lung fibroblasts. Our findings are novel since the role of the transcription factor BHLHB2 in the IL-6 induced/ IGF-II-mediated fibrotic response in SSc lung disease remains unexplored. DISCUSSION/SIGNIFICANCE: Our findings may provide a rationale for combination therapy to block IL-6 and IGF-II function concomitantly and thus halt the progression of SSc pulmonary fibrosis (PF). Our findings may have wide implications for lung fibrosis associated with various diseases, since SSc-PF, is characterized by the activation of common fibrotic pathways.

OBJECTIVES/GOALS: Ocular graft versus host disease (oGVHD) affects ~50% of individuals after an allogeneic hematopoietic stem cell transplant for treating blood cancers. OGVHD results in severe dry eye disease and decreased vision. Quantifying specific cytokine changes in tears may reveal biomarkers and future treatment targets for patients with oGVHD. METHODS/STUDY POPULATION: The goal of this study is to determine if cytokines can be measured in tears with the Isoplexis platform. This pilot validation study evaluates the tears of a patient with oGVHD utilizing the Isoplexis platform. The Isoplexis has specific advantages for tear samples including high-throughput analysis, and small sample requirements, but has yet to be validated in tears. A sample from normal and oGVHD patient tears were collected for comparison. Samples were analyzed on two separate backgrounds–standard Bovine Serum Albumin (BSA) background and artificial tears (ATs). The negative control was ATs and positive control was a concentrated cytokine solution. Analysis of 22 cytokines was performed. RESULTS/ANTICIPATED RESULTS: Analysis of 22 cytokines was performed. As expected, the cytokine levels of the ATs alone were below the limit of detection (LOD). The oGVHD patient tears showed elevated TNF-alpha, TNF-beta, perforin, MIP-1a, MIP-1β, MCP-1, IL2, IL4, IL5, IL-7A, IL9, IL-13, IL-15, IFN-γ, granzyme B, and GM-CSF with ATS background, but no cytokines above the LOD in the BSA background plate. The control tears had elevated IP-10. The elevated cytokines for the oGVHD patient corresponded to symptom severity and clinical findings. DISCUSSION/SIGNIFICANCE: These results suggest that using ATs as the background with the Isoplexis platform improves the sensitivity to detect tear cytokines. Findings of elevated IL-7A and GM-CSF in tears parallels literature findings for oGVHD. Further evaluation of samples will continue to validate the Isoplexis multiplex assay for tear cytokine analyses.

OBJECTIVES/GOALS: Although gut fungi have been implicated in the immunopathogenesis of inflammatory bowel disease, the fungal microbiome has not been deeply explored across endo-histologic activity and treatment-exposure in ulcerative colitis. METHODS/STUDY POPULATION: Our retrospective cohort was derived from the Study of a Prospective Adult Research Cohort with Inflammatory Bowel Disease. We evaluated the fungal composition of fecal samples from 98 ulcerative colitis patients across endoscopic activity (n=43), endo-histologic activity (n=41), and biologic-exposure (n=98). Across all subgroups, we assessed fungal diversity and differential abundance of specific taxonomic groups. RESULTS/ANTICIPATED RESULTS: We identified 504 unique fungal amplicon sequence variants across the cohort of 98 patients, dominated by phylum Ascomycota. Compared to endoscopic remission, patients with endoscopic activity had an increased global fungus load (p DISCUSSION/SIGNIFICANCE: Endoscopic inflammation in ulcerative colitis is associated with altered fungal diversity driven by expansion of Saccharomyces and Candida compared to remission. The role of these fungal taxa as potential biomarkers and targets for personalized approaches to therapeutics in ulcerative colitis should be evaluated.

OBJECTIVES/GOALS: There is increasing evidence that cannabidiol (CBD) has promising potential to treat PTSD. However, more research is warranted to fully understand the benefits of CBD for PTSD. This poster will describe the design and methodology of one of the first ever pilot RCTs examining CBD (vs. placebo) combined with prolonged exposure therapy for PTSD. METHODS/STUDY POPULATION: This study is an early Phase II double-blind, pilot RCT. Participants are 24 individuals 18-65 years old who meet DSM-5 criteria for PTSD on the CAPS-5 and were recruited from local hospitals and the community. Individuals complete a standardized baseline assessment with an independent evaluator to assess study eligibility. Participants who meet study inclusion are randomized to 18 days of CBD 250mg (BID) or placebo delivered in combination with 10-sessions Prolonged Exposure (PE) psychotherapy over 2 weeks. Individuals begin medication 3 days prior to beginning PE to ensure steady state. Participants complete self-report and biomarker outcomes at select timepoints during study participation, and are also asked to complete a 1-month follow-up assessment following treatment. RESULTS/ANTICIPATED RESULTS: This aims of this study are to: 1) examine the safety, feasibility, and PTSD symptom reductions associated with the combined intervention; 2) evaluate biomarkers associated with the endocannabinoid system and stress response; 3) determine the association between changes in biomarkers and PTSD symptoms following treatment. It is expected that CBD+PE will be safe and feasible, and that there will be a detectable signal of CBD vs. placebo in the reduction of PTSD symptoms. It is also anticipated that CBD will have higher levels of endocannabinoids and lower stress response levels compared to placebo. Lastly, we expect that greater changes in biomarkers will be associated with lower levels of PTSD severity following treatment. DISCUSSION/SIGNIFICANCE: Although there is growing interest in cannabinoids for psychiatric conditions, such as PTSD, controlled trials are limited and have yet to examine the proposed intervention for PTSD. If successful, this study will enhance the feasibility of a larger, adequately powered RCT to address immediate and long-term improvements for PTSD treatments.

OBJECTIVES/GOALS: Aiming to better understand the molecular pathogenesis of familial partial lipodystrophy (PL), we initiated whole-exome sequencing for our patients with PL syndromes. A novel variant of early B cell factor 2 (EBF2) was identified. Here we report the biological impact of a novel truncating EBF2 variant. METHODS/STUDY POPULATION: Using 3T3-L1 and human primary subcutaneous preadipocytes, we performed loss-of-function and gene rescue experiments. All cells were cultured in DMEM with 10% bovine calf serum (Invitrogen) at 5% CO2. After lentivirus transfection, cells were grown to confluence and then exposed to adipogenesis induction media containing dexamethasone (0.25µM), insulin (1µg/ml) and isobutyl methylxanthine (0.5 mM). Total RNA was extracted using RNeasy Mini Kit (Qiagen) and cDNA was synthesized using IScript (Bio-Rad). Real-time qPCR was performed using TaqMan probes for Pparg and Fabp4, two key adipogenesis markers. RESULTS/ANTICIPATED RESULTS: Patient was found to carry a heterozygous nonsense mutation in exon 6 of EBF2, causing the premature termination of the protein at amino acid position 165. Adipogenesis was significantly suppressed in 3T3L1 cells when endogenous Ebf2 was suppressed with siRNA and lentiviral shRNA. Adipocytes with suppressed Ebf2 expression showed marked reduction of intracellular lipid content and Pparg and Fabp4 expression (>80% reduction). With lentiviral gene transfer, EBF2 fully rescued adipogenic potential, whereas the truncated variant EBF2 did not. Of note, 3T3-L1 cells transfected with the EBF2 variant displayed impaired adipogenesis, suggesting a dominant-negative effect of the EBF2 variant on adipogenesis. We confirmed the dominant effect of the EBF2 variant in human adipocyte differentiation. DISCUSSION/SIGNIFICANCE: Our data suggest that EBF2 is indispensable for adipogenesis. The loss of function and dominant-negative effect of the truncating variant of EBF2 likely plays a pathogenic role in PL. Whole exome sequencing of PL patients and ex-vivo functional analysis help identify novel gene variants and better understand the molecular pathogenesis of PL.

OBJECTIVES/GOALS: Accurate classification of disorders of consciousness (DoC) is key in developing rehabilitation plans following brain injury. The Coma Recovery Scale-Revised (CRS-R) is a sensitive measure of consciousness. We explore feasibility, safety and impact of CRS-R guided rehab in hemorrhagic stroke patients with DoC and evaluate predictors of recovery. METHODS/STUDY POPULATION: Consecutive patients with non-traumatic hemorrhagic stroke, defined as subarachnoid hemorrhage (SAH) or intracerebral hemorrhage (ICH), receiving serial CRS-R assessments during their ICU stay at University of Maryland Medical Center from 2017-2021 were retrospectively identified. Outcomes of interest included the association with CRS-R and discharge disposition, therapy-based function and mobility and occurrence of safety events during CRS assessment. We also examined the association between CRS-R and physiological and anatomical injury pattern on electroencephalography (EEG) and magnetic resonance imaging (MRI), respectively. CRS-R RESULTS/ANTICIPATED RESULTS: 76 patients with≥2 CRS-R assessments were identified (22 SAH, 54 ICH, median age = 59, 50% female). Median CRS-R completed was 3 with no SAEs identified during sessions. We identified 4 patterns: persistent VS/UWS (49%), persistent MCS or better (13%), emergence from VS/UWS to MCS or better (27%) and regression from MCS or better to VS/UWS (11%). Persistent low CRS-R correlated with older age in SAH (p=0.01), female gender in ICH (p=0.04), and history of diabetes (p=0.01). 2% of patients with final CRS-R DISCUSSION/SIGNIFICANCE: Early neurorehabilitation guided by CRS-R appears to be feasible and safe acutely following hemorrhagic stroke complicated by prolonged DoC and may enhance access to inpatient rehabilitation with a lasting benefit on recovery. Further characterization of DoC patterns and their correlation to clinical markers, including EEG and MRI is needed.