OBJECTIVES/GOALS: A disease-agnostic translational science framework for data mining is proposed for use across disciplines to: Answer clinical questions, justify future clinical research recruitment, and explore under-represented populations. As a case example, male puberty demonstrates utility of the framework. METHODS/STUDY POPULATION: As a case example using the generalizable framework, the following interdisciplinary question was asked: Does early pubertal timing increase the risk of developing type II diabetes (T2d) in boys? A digital phenotype of males < 18 years old was created in the TriNetX Diamond Network utilizing Boolean operator data queries. TriNetX contains patient electronic health record information (ICD-10 diagnoses, anthropometric measures). A case control analysis leveraging patient counts from various digital phenotypes allowed for outcome (T2d) comparison of boys diagnosed with precocious puberty (E30.1, ICD code for early pubertal timing) to those without, while controlling for body mass index (BMI). RESULTS/ANTICIPATED RESULTS: Subjects (N=12,996,132) displayed the following digital phenotype: Male, < 18 years old, without ever having a BMI documented >85th percentile. Boys diagnosed with precocious puberty (E30.1) were 6.89 times more likely to develop T2d when aged 14-18 years old than those without (OR 6.89, 95%CI: 5.17-9.19, p. DISCUSSION/SIGNIFICANCE: Boys are under-represented in the early pubertal timing literature, justifying future human subjects research on male puberty. This case example demonstrates a broader disease-agnostic framework which can be adapted across disciplines. Opportunities may include public health digital phenotyping.

OBJECTIVES/SPECIFIC AIMS: Hepatitis C viral (HCV) infections are rising significantly both in young adults and as newly diagnosed cases in “baby boomers.” New HCV therapeutics cure over 95% of cases, and a call has been made for elimination of the epidemic by 2030; yet major HCV cascade of care (CoC) barriers exist. We secured CTSA pilot funding to obtain preliminary data for an innovative clinical trial utilizing big data modeling toward HCV elimination. METHODS/STUDY POPULATION: Our pilot work has developed a coordinated, real-time clinical data management process across 3 major CTSA affiliated hospital systems (MedStar Health, Emory-Grady, and UT-Southwestern), and additional data will be obtained from a pragmatic clinical trial. Electronic medical records data will be mapped to the OHDSI model, securely transmitted to Oak Ridge National Laboratory, Knoxville, TN and exposed to integrated data, analytics, modeling and simulation (IDAMS). RESULTS/ANTICIPATED RESULTS: Our U01 CTSA application proposes that HCV-IDAMS will model modifications to the established HCV CoC at community and population levels and thus simulate future outcomes. As data volume increases, system knowledge will expand and recursive applications of IDAMS will increase the accuracy of our models. This will reveal real-world reactions contingent upon population dynamics and composition, geographies, and local applications of the HCV CoC. DISCUSSION/SIGNIFICANCE OF IMPACT: Only an innovative, integrated approach harnessing pragmatic clinical data, big data and supercomputing power can create a realistic model toward HCV elimination.

OBJECTIVES/GOALS: Normal fear learning produces avoidance behavior that promotes survival, but excessive and persistent fear after trauma can lead to development of phobias and post-traumatic stress disorder (PTSD). Our goal is to understand the mechanism and identify novel genetic targets underlying fear responses. METHODS/STUDY POPULATION: Involvement of the amygdala in fear acquisition is well established and requires activation of N-methyl-D-aspartic acid receptors (NMDARs). At a cellular level, NMDAR activation leads to production of nitric oxide (NO) by a process mediated by interaction between postsynaptic density protein 95 (PSD95) and neuronal nitric oxide synthase (nNOS). To elucidate mechanisms underlying the role of the PSD95-nNOS-NO pathway in conditioned fear, here we use rodent behavioral paradigms, pharmacological treatment with a small molecular PSD95-nNOS inhibitor, co-immunoprecipitation, Western blotting, and RNA-sequencing. RESULTS/ANTICIPATED RESULTS: We show that fear conditioning enhances the PSD95-nNOS interaction and that the small-molecule ZL006 inhibits this interaction. Treatment with ZL006 also attenuates rodent cued-fear consolidation and prevents fear-mediated shifts in glutamatergic receptor and current densities in the basolateral amygdala (BLA). With RNA-sequencing, expression of 516 genes was altered in the BLA following fear expression; of these genes, 83 were restored by systemic ZL006 treatment. Network data and gene ontology enrichment analysis with Ingenuity Pathway Analysis and DAVID software found that cell-cell interaction, cognition-related pathways, and insulin-like growth factor binding were significantly altered. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results reveal novel genetic targets that underlie plasticity of fear-memory circuitry via their contribution of NMDAR-mediated fear consolidation and can inform future strategies for targeting fear related disorders like PTSD. CONFLICT OF INTEREST DESCRIPTION: Anantha Shekhar and Yvonne Lai are co-founders of Anagin, Inc., which is developing some of the related molecules for the treatment of PTSD.

OBJECTIVES/SPECIFIC AIMS: Objective: To summarize the diagnosis and management of two acute rejection (AR) episodes in the first penis transplant patient in the U.S. Background: Vascularized composite allotransplantation (VCA) has been utilized for state-of-the-art reconstruction of devastating craniofacial defects, limb loss, and recently, severe genitourinary defects. To date, more than 200 VCA’s have been performed, of which four successful penis transplants have been achieved worldwide (Two in the U.S.). However, despite the technical success of VCAs in general, acute rejection episodes remain a significant postoperative management problem, with 80-85% experiencing at least one episode in the first-year post-transplantation. The incorporation of skin in VCAs, which is highly immunogenic, allows early visible recognition of rejection but requires prompt management to prevent allograft failure as well as the progression of chronic rejection, which has been associated with the frequency of acute rejection episodes in preclinical models. We present the first report of acute rejection in a penile allograft. AR episodes in VCA typically manifest with erythema of the allograft skin and/or maculopapular lesions that can be either patchy, focal or diffuse. Histopathologic assessment is essential for diagnosis and management. The Banff classification of histopathologic criteria for degree of AR is most commonly utilized to direct clinical management. METHODS/STUDY POPULATION: We reviewed the clinical course of the first American patient who underwent penis transplantation at Massachusetts General Hospital in 2016. Postoperatively, routine clinical and chemical assessment (immunosuppression levels, routine blood work) was performed, with increased frequency during AR episodes. Skin punch biopsies were obtained during (suspected) AR episodes, analyzed and graded according to the Banff 2007 classification of rejection of skin-containing composite allografts. Histopathologic tissue assessment included CD3, C4d, CD4/8, CD20 FOXP3 and cellular infiltration (hyper keratinization, lymphocytic infiltrate, dermal erosion, macrophage, eosinophilia, T-cell infiltration) and epidermal or perivascular fibrosis. RESULTS/ANTICIPATED RESULTS: The patient is a 65-year-old male with history of penile carcinoma requiring subtotal penectomy in 2012. He is currently 30-months post penile transplantation (as of 11/15/2018). First Rejection Episode: At 28 days post-transplantation, the patient noted induration, swelling and erythema of the allograft, which was diagnosed as AR clinically (Image 1A). Biopsy showed a Banff Grade III AR, with focal keratinocyte apoptosis with lymphocytic infiltration in epidermis and arteriolar endothelialitis with perivascular inflammation. Initially this episode was treated for 2 days with 2 pulse doses of methylprednisolone (500mg/d IV) with clinical improvement. However, recurrent allograft erythema was observed on postoperative day 32 and an acute rejection grade III according the Banff classification was confirmed by a second biopsy that demonstrated epidermal perivascular lymphocytic infiltrates, spongiosis and dyskeratosis, deep dermis focal lymphocytic infiltrates and focal infiltrates in arterioles as well as endothelialitis in venules. Donor specific antibodies and C4d were negative. CD3+ T cells were present in the epidermis and perivascular space. This was treated with anti-thymocyte globulin (thymoglobulin) course for 4 days (1.5mg/kg/day IV) and 3 more pulse doses of methylprednisolone (500mg/d IV.), followed by a prednisone (250mg/d) taper to baseline. This resulted in complete resolution of AR. Second Rejection Episode: At 10.8 months post VCA the patient presented with penile erythema and scrotal swelling suggestive of AR and received three doses of methylprednisolone (day 1: 500mg/d IV, day 2: 1000mg/d IV and day 3: 500mg/d IV respectively) followed by increased baseline prednisone (10mg PO daily; increased dose compared to previous AR episode). A skin biopsy confirmed Banff Grade III AR. Compared to the previous biopsy, this biopsy demonstrated an increased density of lymphocytic inflammation of the dermis with endarteritis. Prominent involvement of epidermis and adnexal structures corresponding to acute T-cell mediated rejection was also observed (Figure 1). Donor specific antibodies and C4d were again negative. Three doses of ATG (1.5mg/kg/day IV) were administered. In addition, tacrolimus was increased and local tacrolimus (1% ointment) treatment was begun. Clinical signs of rejection improved and repeat biopsy showed dramatic histopathological improvement. Current maintenance immunosuppressive regimen consists of tacrolimus, sirolimus, prednisone, mycophenolic mofetil acid (MMF), rapamycin, and tacrolimus ointment, with no new clinical or histopathological signs of rejection (Image 1B). DISCUSSION/SIGNIFICANCE OF IMPACT: We report the first described case of acute T-cell mediated rejection in penile transplantation. These rejection episodes demonstrated that, even on stringent immunosuppressive regimens, severe acute rejection episodes in VCA may still occur. Edema and acute induration preceded the development of erythema in our cases, representing a harbinger for the more severe grade of rejection that eventually developed. Our experience was consistent with other VCAs in that donor specific antibodies did not develop, despite a severe Banff Grade. Consistent use of topical calcineurin inhibitor based immunosuppression on the allograft skin may be helpful in warding off future episodes, as our patient has been rejection free now for 18 months. To date, no histologic signs of chronic rejection were present on 2-year protocol surveillance biopsy. We have added rapamycin to the current drug regimen, with concurrent reduction of tacrolimus dosing for renal protection, which has been demonstrated in cardiac transplantation to deter the intimal hyperplasia/vasculopathy associated with chronic rejection.

OBJECTIVES/GOALS: To determine the signs, symptoms, and diagnoses that are significantly upregulated in cases of long COVID while identifying risk factors and demographics that increase one’s likelihood of developing long COVID. METHODS/STUDY POPULATION: This is a retrospective, big data science study. Data from Veterans Affairs (VA) medical centers across the United States between the start of 2020 and the end of 2022 were utilized. Our cohort consists of 316,782 individuals with positive COVID-19 tests recorded in the VA EHR with a history of ICD10-CM diagnosis codes in the record for case-control comparison. We looked at all new diagnoses that were not present in the six months before COVID diagnosis but were present in the time period from one month after COVID through seven months after. We determined which were significantly enriched and calculated odds ratios for each, organized by long COVID subtypes by medical specialty / affected organ system. Demographic analyses were also performed for long COVID patients and patients without any new long COVID ICD10-CM codes. RESULTS/ANTICIPATED RESULTS: This profile shows disorders that are highly upregulated in the post-COVID population and provides strong evidence for a broad definition of long COVID. By breaking this into subtypes by medical specialty, we define cardiac long COVID, neurological long COVID, pulmonary long COVID, and eight others. The long COVID cohort was older with more comorbidities than their non-long COVID counterparts. We also noted any differences regarding sex, race, ethnicity, severity of acute COVID-19 symptoms, vaccination status, as well as some analysis regarding medications taken. DISCUSSION/SIGNIFICANCE: This profile can be utilized to decisively define long COVID as a clinical diagnosis and will lead to consistence in future research. Elucidating an actionable model for long COVID will help clinicians identify those in their care that may be experiencing long COVID, allowing them to be admitted into more intensive monitoring and treatment programs.

ABSTRACT IMPACT: Gaining a better understanding on the role of opioids in opioid use disorder (OUD) can help us find better diagnostics, treatments, and procedures to treat the disorder. OBJECTIVES/GOALS: While we are familiar with brain areas and pathways that are implicated in opioid use disorder (OUD), we do not have a full understanding of the neural circuits activated upon drug exposure. METHODS/STUDY POPULATION: In order to identify areas of the brain most activated by opioids, we ran a pilot study using transgenic cFos-GFP mice that were injected with saline or heroin and examined the brain-wide activity patterns using a quantitative high-resolution mapping method. We observed many brain regions highly activated upon drug exposure. To examine cFos based brain activation in rats, we also ran a pilot study using a tissue clearing and 3D immunolabeling method combined with light sheet microscopy. RESULTS/ANTICIPATED RESULTS: We would expect to see higher cFos activation for brain areas in the reward pathway [including the Nucleus Accumbens (NAc), Ventral Tegmental Area (VTA), Prefrontal Cortex (PFC)] in heroin animals compared to saline animals. We can also expect higher activation in more novel areas like the lateral hypothalamus. DISCUSSION/SIGNIFICANCE OF FINDINGS: If we are able to track OUD effects through imaging in mice and rats, this can help us find better diagnostics, therapeutics, and procedures to treat the disorder. We can also eventually have a human brain atlas that outlines these affected areas as well in order to gain a better understanding on OUD particularly in the human population.

OBJECTIVES/GOALS: A major barrier to older adult (65+) over-the-counter (OTC) safety is the lack of actionable research on factors affecting older adult decisions during OTC selection. This goal of this study is to develop and validate a survey instrument based on the Protection Motivation Theory to measure older adults perceived risk of OTCs. METHODS/STUDY POPULATION: The 24-item OTC Protection Motivation survey underwent revision using a consult from the UW Survey Center as well as rigorous cognitive interviewing and pilot testing with 8 older adults. It was administered to 103 community-dwelling older adults. OTC misuse data was gathered from 15 of the participants. OTC misuse evaluated by three pharmacy experts on the basis of OTC appropriateness given participants age, existing conditions, concurrent medication use, and intention to adhere to label guidelines. Multivariate linear regression was used to examine the relationship between OTC misuse and protection motivation. Exploratory factor analysis (EFA) was used to determine a model of best fit to describe the factor structure of the OTC Protection Motivation survey. RESULTS/ANTICIPATED RESULTS: The EFA yielded a 5-factor model of protection motivation, which included the components of deliberative risk perception, a combination of experiential and affective risk perception, threat severity, protective behavior intent, and perceived efficacy in protective behavior engagement. The EFA-based item reduction resulted in a final 18-item OTC Protection Motivation survey. DISCUSSION/SIGNIFICANCE: Older adults, who have the lowest health literacy levels, are responsible for 30% of OTC medication use and 61.5% of emergency department visits due to adverse drug events. This study is the first to characterize the relationship of OTC protection motivation and OTC misuse to inform patient-centered interventions for older adult OTC safety.

OBJECTIVES/SPECIFIC AIMS: Despite significant advances in screening and treatment, colorectal cancer is the second leading cancer killer in the United States today. Some of the most promising recent developments in cancer therapy have come from immune-based therapy. Immune-based therapy, however, has shown limited utility in patients with colorectal cancer. Studies have previously shown that certain chemotherapy regimens may be more effective in combination with immune-based therapy due to induction of inflammation in the tumor microenvironment. In this study, we sought to determine how standard chemotherapy (FOLFOX) affects the generation of antigen-specific anti-tumor immunity in colorectal cancer. METHODS/STUDY POPULATION: To determine the how antigen-specific immunity and T cell responses are affected by FOLFOX, we utilized a model antigen expressing murine colon cancer cell line syngeneic to C57BL/6 (MC38-CEA). Treatment was initiated when tumor size reached 50 mm2. Mice were treated with either vehicle (PBS), 5-Fluorouracil (5-FU), Oxaliplatin, or combination (FOLFOX). Antigen-specific cytotoxic T cell (tet+Tc) were detected using Db-CEA-tetramer obtained from the NIH-tetramer core facility. Flow cytometry was performed for phenotypic analysis and tetramer positivity. Tumor growth was measured using standard caliper measurements. Statistical analysis was performed using t-test for continuous variables and ANOVA was used when comparing multiple groups. Statistical analysis was performed using SPSS. All arms were completed with n=3–7. RESULTS/ANTICIPATED RESULTS: To determine how systemic treatment with chemotherapy affects cytotoxic T cell development (Tc), we established that we could detect antigen-specific Tc (tet+Tc) in the spleen, tumor, and draining lymph nodes of tumor-bearing mice. After establishing that the system worked appropriately, tumor-bearing mice were treated with different chemotherapy regimens and tumor growth was monitored. As expected, the combination of FOLFOX was significantly better than either drug individually (2-way ANOVA, p<0.01). FOLFOX therapy also showed a significant (p<0.05) increase in the number of tumor-associated tet+Tc, and tet+Tc expressing phenotypic markers of effector (Te) and resident memory (Trm) subsets. Tumor-associated tet+Tc highly expressed PD-1 (>50%); however, this was not significantly different between treatment or vehicle arms. Since 5-FU, one component of FOLFOX has previously shown a selective reduction of myeloid-derived suppressor cells, we also investigated the myeloid compartment. There were no significant differences in conventional or plasmacytoid dendritic cells, myeloid-derived suppressor cells, or tumor-associated macrophages. DISCUSSION/SIGNIFICANCE OF IMPACT: The future of cancer care involves multi-modality care tailored to patients. To more effectively combine therapy it is critical that we understand how currently utilized therapy works. In this study, we show that the primary chemotherapy regimen utilized in colorectal cancer increases tumor-associated antigen-specific cytotoxic T cells and the majority of these cells are PD-1 positive. This suggests that FOLFOX may work in concert with immune-based therapy when selected appropriately. Further study is warranted to determine optimal combination therapy and ways to maximize anti-tumor immunity in order to improve the treatment of patients with this deadly disease.

The consent process for research studies can be burdensome for potential participants due to complex information and lengthy consent forms. This pragmatic study aimed to improve the consent experience and evaluate its impact on participant decision making, study knowledge, and satisfaction with the In Our DNA SC program, a population-based genomic screening initiative. We compared two consent procedures: standard consent (SC) involving a PDF document and enhanced consent (EC) incorporating a pictograph and true or false questions. Decision-making control, study knowledge, satisfaction, and time to consent were assessed. We analyzed data for 109 individuals who completed the SC and 96 who completed the EC. Results indicated strong decision-making control and high levels of knowledge and satisfaction in both groups. While no significant differences were found between the two groups, the EC experience took longer for participants to complete. Future modifications include incorporating video modules and launching a Spanish version of the consent experience. Overall, this study contributes to the growing literature on consent improvements and highlights the need to assess salient components and explore participant preferences for receiving consent information.

OBJECTIVES/SPECIFIC AIMS: Microarray technology has produced large volumes of gene expression data profiling differences in gene expression in a vast array of conditions, much of which is publicly available. Methods to query these data for similarities in patterns of gene regulation are limited to comparisons between preannotated groups. In response, we developed openSESAME to find experiments where a set of genes is similarly coregulated without regard to experimental design. An important application of openSESAME is drug repositioning: if a pattern associated with disease is reversed by a given drug, the drug might target disease-related processes. METHODS/STUDY POPULATION: Experiments from the Gene Expression Omnibus (GEO) were normalized, signature-association (SA) scores computed for each sample, experiments assigned enrichment scores, and ANOVAs used to assign significance to experimental variables automatically extracted from GEO. SA scores were also generated for hundreds of publicly available signatures, and pairwise correlations used to create a relevance network. RESULTS/ANTICIPATED RESULTS: Using signatures of estrogen and p63, we recovered relevant experimental variables, and with the network approach, we recovered previously reported associations between disease states and/or drug treatments. DISCUSSION/SIGNIFICANCE OF IMPACT: openSESAME has the potential to illuminate “dark data” and discover novel relationships between drugs and diseases on the basis of common patterns of differential gene expression.

OBJECTIVES/SPECIFIC AIMS: To better understand host, viral and bacterial responses underlying disparate outcome. METHODS/STUDY POPULATION: We utilized metagenomic analysis of 559 genes, the NanoString nCounter Immunology Panel-Plus kit and FFPE mouse lungs from a published BSI time course. RESULTS/ANTICIPATED RESULTS: Results show an overall increased level of gene expression during BSIs associated with survival when compared to gene expression during peak viral titers. Early viral clearance and the presence of S. pyogenes in the lungs of TX98 infected lungs 24 hours after BSI was confirmed. Host responses tied to differences in early viral detection and clearance consisted of RIG-I, OAS, TLR3, TLR8 and TLR9. Key changes were noted in the expression of type I and II interferons, complement proteins, cytokines, chemokines, Fc receptors, scavenger receptors, the immunoproteasome and genes associated with T, B, Treg and NK cell activation. Interestingly, there was no significant increase in host antimicrobial peptides during BSIs associated with survival while CAMP and Nos2 were significantly increased 24hrs prior to death in lethal BSIs. DISCUSSION/SIGNIFICANCE OF IMPACT: To our knowledge this is the first side by side metagenomics study of influenza BSIs associated with death and survival. Results offer mechanistic insight into clinical outcomes.

OBJECTIVES/GOALS: Guidelines suggest people with HIV (PWH) receive routine HIV viral load (VL) testing at least yearly and upon diagnosis with multidrug-resistant tuberculosis (MDR-TB). Many PWH and MDR-TB in South Africa seem to be missing VL results. This study’s goal was to find results which may be available from the National Health Laboratory Service (NHLS). METHODS/STUDY POPULATION: We abstracted HIV laboratory results, specifically baseline VL and CD4 count and VL at MDR-TB cure, from PWH enrolled in a cluster-randomized clinical trial of nurse case management for PWH and MDR-TB in South Africa who were cured of MDR-TB. For any participant missing one or more of these results, we thoroughly searched the electronic NHLS database using multiple separate searches varying terms including patient name, surname, date of birth, medical record number where available, and South African identification number. Returned results were compared to results abstracted from the parent study and any additional results were entered into the parent study data. RESULTS/ANTICIPATED RESULTS: Of 929 PWH cured of MDR-TB, 879 (94.6%) were missing at least one expected VL or CD4 result in the parent study database. Though our search strategy was successful in identifying participants and returning CD4 and VL results, we rarely found additional results that were not already in the parent study database. Following the search and entry of the few additional results retrieved, 116 (12.4%) participants were missing a baseline CD4, 309 (33.3%) missing baseline VL, and 385 (41.4%) missing VL at MDR-TB cure, representing 572 individuals or 61.6% of participants with at least one unavailable result. This high level of unavailability of key laboratory results used to guide MDR-TB and HIV treatment suggests that these tests were either not ordered, not collected, or not completed due to electronic gatekeeping at NHLS. DISCUSSION/SIGNIFICANCE: Unknown CD4 count or VL leaves PWH open to including MDR-TB treatment failure and death. Our search strategy found additional results but was time-consuming and cumbersome. Limitations included lack of information on why laboratory results were missing, which limits our ability to make recommendations for better collection and reporting.

OBJECTIVES/SPECIFIC AIMS: Pancreatic ductal adenocarcinoma (PDA) has a dismal 5-year survival rate of 9%, making this disease one of the deadliest human malignancies (https://seer.cancer.gov/). Primary barriers to the treatment of pancreatic cancer include extensive stromal interactions and sustained immune suppression. Aberrant Hedgehog (HH) pathway activity is a hallmark of pancreatic tumorigenesis. Tumor-derived HH ligands signal in a paracrine fashion to the surrounding stroma to influence tumor growth. Expression of HH ligands increases during PDA progression, and previous work has shown that genetic deletion of Sonic HH (Shh) from the epithelium of mice with pancreatic tumors results in increased Indian HH (Ihh) expression. This research aims to investigate the translational impact of changes in immune infiltration following deletion of IHH in a preclinical mouse model of pancreatic cancer. METHODS/STUDY POPULATION: Ihh was deleted in tumor cells lines (IhhKO) derived from a genetically engineered mouse model of pancreatic cancer (LSL-KrasG12D/+;LSL-TrpR270H;P48-Cre), using CRISPR/Cas-9 gene editing to assess the role of Ihh in the tumor microenvironment. The level of HH signaling was determined using tumor cell co-cultures with Gli1lacZ fibroblasts (derived from mice with a lacZ reporter allele knocked into the Gli1 locus), in which Beta Galactosidase activity serves as a readout for HH signaling. WT and IhhKO tumor cells were orthotopically transplanted into the pancreas of syngeneic C57BL/6 mice. Human pancreas samples were obtained from surgical resection of pancreatic adenocarcinoma, or fine needle biopsy procedure (FNB). Immune profiling of mouse and human pancreatic tumors was performed using Cytometry Time-of-Flight analysis (CyTOF), and tumor composition was analyzed by single-cell RNA sequencing (scRNA seq). In vitro cultures with pancreatic fibroblasts treated with either WT or IhhKO tumor cell conditioned media (CM) were cultured with bone-marrow derived macrophages to assess tumor crosstalk. RESULTS/ANTICIPATED RESULTS: Tumor cells lacking Ihh were generated through CRISPR/Cas-9 deletion, and this was confirmed by qRT-PCR. Co-culture of IhhKO tumor cells with Gli1lacZ fibroblasts results in decreased Gli1 expression both in vitro and in vivo. Immune profiling revealed that tumors lacking Ihh have significantly fewer tumor associated macrophages (CD11b+/F4/80+/CD206+), resulting in decreased presence of immunosuppressive factors such as arginase 1 and PDL1. Immune phenotyping of human pancreatic tissues revealed similar populations of immunosuppressive myeloid cells present in tumors. In vitro co-cultures demonstrated that, in the presence of bone-marrow derived macrophages, immunosuppressive IL-6 production was reduced in pancreatic fibroblasts cultured with IhhKO-CM, as compared to fibroblasts cultured with WT-CM, providing mechanistic insight into the in vivo phenotype observed. Further, scRNA seq analysis suggests that modulation of HH signaling in the tumor microenvironment alters chemokine and immunomodulatory signaling pathways driven by fibroblasts in the pancreatic tumor microenvironment. DISCUSSION/SIGNIFICANCE OF IMPACT: HH signaling in pancreatic fibroblasts contributes to the establishment of an immune suppressive environment in pancreatic cancer. Combining methods to target HH signaling and immune checkpoint therapy has translational potential in treating pancreatic cancer patients.

OBJECTIVES/GOALS: This project adopts a multifaceted approach to improving aquaculture management practices in Kenya’s Lake Victoria region by identifying fish pathogens, measuring algal toxin levels in commonly consumed fish, surveying fish farming practices, and educating the public. METHODS/STUDY POPULATION: Limited existing data on the state of floating cage culture in Kenya influenced our decision to begin this portion of the project with a brief literature review of potential Nile tilapia pathogens. Databases were screened for mention of disease in either wild or caged Nile tilapia, with emphasis given to those in Lake Victoria. Results were compiled into a spreadsheet and analyzed for frequently occurring pathogens. The next portion involved creating an interview style survey to assess current cage culture management practices in the region. Editing was done to ensure questions remained unbiased, non-leading, culturally sensitive, multilingual and relevant to the situation. Data went through a quality control screening and analysis was conducted through the R programming language. RESULTS/ANTICIPATED RESULTS: Beginning with mortality, of the 93 farms surveyed, data analysis revealed that there is a higher probability that farms will have a mortality of approximately 20%, over the course of a production cycle. For biosecurity and fish health practices, data shows that 97% of farms do not disinfect scooping nets or other fish handling materials when moving from one cage to another. During the 2022-2023 production cycle, 44% of farms experienced fish kills of over 50 fish. 73% of the 93 farms do not contact any organization when a fish kill occurs. In a qualitative answer, it also appears that many farm workers dispose of their dead fish within the lake, feed it to livestock or dogs, or eat it. Algae blooms have been experienced at 80% of the farms surveyed and 43% of farms say they have seen fish gasping at the surface for air. DISCUSSION/SIGNIFICANCE: While farms are implementing good management practices in the areas of cage design, stocking, and feeding practices, there is room for improvement in fish health, biosecurity, and managing algal blooms. The findings provide insight into the areas that should be considered when taking action to improve the welfare of the region.

OBJECTIVES/SPECIFIC AIMS: The knee injury osteoarthritis and outcomes survey (KOOS) is a commonly used instrument to measure patient-reported quality of life (QOL) post-ACLR. The purpose is to evaluate the psychometric properties of the QOL subscale of the KOOS. METHODS/STUDY POPULATION: Rasch analysis of KOOS QOL subscale from 39 individuals 1–2 years post ACLR was conducted. Measurement properties and model fit of the rating scale, items, and persons were evaluated. Relationship of item difficulties and person measures was evaluated using probability curves and item maps. Reliability indicators were also examined. RESULTS/ANTICIPATED RESULTS: All items demonstrated infit and outfit mean squares and standard z-scores. The majority of persons (n=38, 97.4%) demonstrated fit to the Rasch model. However, ceiling effects were noted (n=4, 10.26%), indicating some participants report higher QOL than is measurable. The mean person measure was 1.73 logits higher than the mean item measure: this sample is skewed toward higher QOL. Person reliability was adequate (0.67) and person separation was 1.42. Calculation of person strata revealed that the KOOS QOL separated participants into 2 strata. DISCUSSION/SIGNIFICANCE OF IMPACT: Although all items of the KOOS QOL fit the model, not all categories of the rating scale were used. Overall, this sample reported high QOL, which is to be expected given the time since ACLR. If participants with a broader range of time since ACLR were included, that the KOOS QOL could identify additional person strata.

OBJECTIVES/SPECIFIC AIMS: Long interspersed element-1s (L1s) are autonomous, mobile elements that are able to copy and insert themselves throughout the genome with their own reverse transcriptase and endonuclease. These elements make up 17% of the human genome with over 500,000 copies, though the vast majority of L1s are defective with only a few dozen potentially responsible for L1 activity. Full-length L1s have the potential to contribute to mutagenesis through random insertion and increased genetic instability. Here we set out to study L1 expression at the specific loci level in bone marrow-derived stem cells (bmSCs) and adipose-derived stem cells (ASCs) and compare the levels of expression from ASCs from donor patients who are young and lean, obese, and old. Our hypothesis is that L1-related damage may contribute to mutation and inflammation that alters the function of these stem cells throughout the life of an individual. METHODS/STUDY POPULATION: ASCs and bmSCs were isolated from patient donors. The following samples were collected: ASCs from 3 young (under the age of 59) and lean (BMI<30) patients, ASCs from 3 older patients (over the age of 59), ASCs from 3 patients with BMI>30, and bmSCs from 4 young and lean patients. Cytoplasmic RNA from the cell populations was isolated and sequenced by RNA-Seq from the cell populations. Using our recently developed bioinformatics pipeline, we set out to quantify L1 expression and identify the few culprit L1s at specific loci that are actively transcribing to RNA in the ASC and bmSC samples. RESULTS/ANTICIPATED RESULTS: Here we provide proof of concept with the application of this novel method in characterizing full-length expressed L1s at the specific loci level in ASCs and bmSCs. We identified L1 loci that are commonly expressed in these cell types and observed an increase in L1 expression in the obese and old ASC cells compared with the young, lean ASCs and bmSCs. DISCUSSION/SIGNIFICANCE OF IMPACT: ASCs hold the promise of broad application in the biomedical field including regenerative treatment. There are reports that ASCs cultivated from older and obese donors are less effective in regenerative treatments. By demonstrating an increased expression of the mutagenic L1 element in ASCs from obese and old donors, this study provides further evidence suggesting the preferable use of ASCs from young and lean donors for regenerative therapies. These studies will also help us to understand the potential contribution of L1 expression to loss of stem cell function during the aging process.