OBJECTIVES/GOALS: Individuals with a family history of alcoholism (FH+) are more likely to develop an alcohol use disorder than those with no such history. Early life adversity has a high coincidence with FH+ making pathogenic studies difficult in clinical studies. Here, we developed a mouse model to study pathogenic mechanisms underlying these risk factors. METHODS/STUDY POPULATION: Male and female C57BL6/J mice were exposed to increasing concentrations of ethanol (3-21%) or water for 15 days prior to breeding. Ethanol was not present during gestation. Offspring were either removed from the home cage and isolated for 3 hours or left undisturbed from postnatal days 1-21. Beginning on PND 56 offspring mice were assessed for clinically relevant behavioral disruptions in social behavior, cognitive working memory, locomotor activity, anxiety-like phenotypes, ethanol preference and binge drinking behavior. In a separate experiment, brains of Cx3cr2+/GFPxCcr2+/RFP mice from ELA or control conditions were collected every 7 days after birth for assessment of neuroinflammation and central immune cell morphology and density. RESULTS/ANTICIPATED RESULTS: Mice with a family history of ethanol exposure and ELA are predicted to exhibit behavioral changes (impaired working memory, reduced social behavior, increased anxiety-like behaviors, increased ethanol consumption) to a greater extent than mice with a family history of ethanol exposure or ELA alone. We expect markers of neuroinflammation (cytokine expression, immune cell activation) to predict the behavioral changes in these mice. DISCUSSION/SIGNIFICANCE OF IMPACT: Alcohol consumption and stressful life events are known environmental precipitants to neuroinflammation, which in turns may predispose individuals to anti-social and risky behavior. A mouse model of these early postnatal conditions will allow basic scientists to unravel the biological underpinnings of the behaviors driven by these factors.

OBJECTIVES/SPECIFIC AIMS: Hematopoietic stem and progenitor cells (HSPCs) function to maintain steady state production of new blood cells and to rapidly respond to blood cell loss. Little is known regarding how HSPCs develop the ability to sense and respond to blood cell loss during embryogenesis. Gaining insight into the robust ability of HSPCs to regenerate blood during early development may allow us to develop therapies to rejuvenate this capacity at any stage. METHODS/STUDY POPULATION: We generated a new hematopoietic-specific and inducible cell ablation zebrafish model to uncover the origins of regenerative capacity in HSPCs during development. These transgenic zebrafish express a cyan fluorescent protein (CFP)-nitroreductase (NTR) fusion construct under the control of the draculin (drl) promoter (drl:CFP-NTR), which restricts NTR expression to blood cells. Co-expression analyses of drl:CFP-NTR with known markers of other blood types including HSPCs (runx1+23:mCherry), erythroid cells (gata1:dsRed), and lymphoid cells (rag2:RFP), revealed drl:CFP-NTR was restricted to HSPCs and erythrocytes. To delineate the regeneration potential of embryonic HSPCs, we exposed drl:CFP-NTR transgenic zebrafish embryos to Metronidazole (MTZ), which results in selective ablation of only NTR-expressing blood cells. Embryos were treated from 2 to 3 days postfertilization and recovery of drl+ and gata1+ cells was evaluated over a 7-day recovery period. RESULTS/ANTICIPATED RESULTS: Following MTZ exposure, the nadir of drl+ cell ablation occurs at 2 days post MTZ (dpM) treatment. During the renewal phase of blood regeneration, we first observe recovery of drl+ cells by about 4 dpM, while more mature blood cells like gata1+ erythrocytes show a delayed recovery at about 6 dpM. Our results suggest that HSPCs can respond to injury as early as 2 days of life and that the HSPC-driven regeneration of embryonic blood cells occurs in a hierarchical fashion, similar to regeneration of the adult blood system. DISCUSSION/SIGNIFICANCE OF IMPACT: We have established a quantitative method for in vivo real-time monitoring of embryonic and larval blood regeneration. A significant advantage of our system is that we can use these insights to guide an in-vivo drug screen for factors that accelerate HSPC-driven blood regeneration in a complex biological environment.

ABSTRACT IMPACT: Our improved understanding of the changes in chromatin accessibility that occur in persistent pain states may identify regulatory genomic elements that play essential roles in modulating gene expression in the DRG. OBJECTIVES/GOALS: Efforts to understand genetic variability involved in an individual’s susceptibility to persistent pain support a role for upstream regulation by epigenetic mechanisms. Our objective was to examine the transcriptomic and epigenetic basis of persistent pain following nerve injury. METHODS/STUDY POPULATION: We used a multiomic approach to identify novel molecular pathways associated with nerve injury-induced pain hypersensitivity. Adult Sprague Dawley rats were randomized to Chronic Constriction Injury (CCI) to the sciatic nerve or no treatment (naive). The ipsilateral L4-L6 dorsal root ganglia (DRG)s were removed on Day 14 and used for ChIP-seq for H3K4me1, ATAC-seq, and RNA-seq. We assessed for differential chromatin accessibility, transcription factor motifs, and enrichment for biological processes in chromatin accessible regions associated with cis-regulatory regions identified by ATAC-seq and H3K4me1 enrichment. Luciferase assays determined the functional significance of these sequences. RESULTS/ANTICIPATED RESULTS: We identified 58,446 genomic regions where H3K4me1 enrichment overlapped with chromatin accessibility. Differential analysis identified 2145 of these 58,446 regions that had changes in accessibility after CCI. The majority of these regions were located in introns or intergenic regions. Functional annotation of the differentially accessible regions identified disparate molecular functions enriched following nerve injury which suggests that altered chromatin structure plays a role in the development of mechanical hypersensitivity. Motif analysis identified specific transcription factor families whose binding sequences were enriched in regions of increased or decreased accessibility. Luciferase assays showed significant enhancement or repression of gene transcription. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our data provides a comprehensive map of chromatin accessibility changes in the DRG after CCI and emphasizes the importance of chromatin structure in the development and maintenance of chronic pain.

OBJECTIVES/GOALS: For shoulder osteoarthritis (OA), the understanding of the patient-specific factors that determine success of both non-operative and operative treatment options is limited. This study aims to identify key factors associated with the response and the heterogeneity of outcomes for both types of treatment. METHODS/STUDY POPULATION: Patients diagnosed with shoulder OA and treated with either reverse/anatomic total shoulder arthroplasty (rTSA/TSA) or non-operative management at the University of California, San Francisco were enrolled in this study. They were followed for a year to ascertain phenotypic traits and patient-reported outcomes (PROs). Magnetic Resonance Imaging (MRI) was used to calculate the Shoulder Osteoarthritis Severity (SOAS) score, a semi-quantitative global assessment of shoulder OA, and to measure fat fractions of rotator cuff muscles. A Microsoft Kinect camera was used to determine the Reachable Workspace (RWS). Linear regression models were used to assess the associations between baseline demographic and radiographic factors on outcomes related to shoulder function. RESULTS/ANTICIPATED RESULTS: It is anticipated that the pre-operative MRI-based SOAS score will be inversely correlated with the magnitude of improvement in PROs 1 year after rTSA/TSA and non-operative management of shoulder OA for the surgical replacement and non-operative cohorts, respectively. Additionally, the non-operative patients who convert to rTSA/TSA within 1 year of observation will have higher SOAS scores compared to patients who continue with non-operative management. The surgical replacement patients with an infraspinatus fat fraction of more than 5% will have worse shoulder function, as measured by RWS, compared to patients with an infraspinatus fraction less than 5%. DISCUSSION/SIGNIFICANCE: MRI may be a novel technique to better predict prognosis of shoulder OA management. This will allow for the development of appropriate algorithms in the prescription of treatments and may be used to counsel patients regarding their expected outcomes or to recommend alternative treatments.

ABSTRACT IMPACT: Understanding the biology underpinning head and neck cancer invasion and metastasis could lead to novel targeted therapies that are both effective and tolerable for patients with this debilitating disease. OBJECTIVES/GOALS: The bromodomain and extra-terminal (BET) family of epigenetic regulators has been implicated in the tumorigenesis of various cancers. In head and neck squamous cell carcinoma (HNSCC), the majority of morbidity is due to invasion and metastasis, so there is special interest in understanding the development of these phenotypes in HNSCC cells. METHODS/STUDY POPULATION: Stable SCC9 knockdown cell lines were generated by infecting cells with a lentivirus encoding Cas9-KRAB and a lentivirus encoding gRNA targeting each of the candidate BET proteins: BRD2, BRD3, BRD4, and BRDT. Knockdown was confirmed by qRT-PCR. Next, standard assays for proliferation (CellTiter-glo), invasion (Matrigel), and migration (scratch-wound healing assay) were performed for all candidate knockdowns and compared to a non-target control. RESULTS/ANTICIPATED RESULTS: Proliferation assay results revealed that BRD4 knockdown had a significant negative effect on the proliferative capacity of SCC9 cells in vitro. Similarly, BRD4 knockdown SCC9 cell lines were less invasive and less migratory. Interestingly, knockdown of BRD2, BRD3, and BRDT had no effect on proliferation, invasiveness, or migration. DISCUSSION/SIGNIFICANCE OF FINDINGS: We have identified BRD4 as a key driver of the HNSCC tumorigenic phenotype. In the future, we plan to investigate the role of JQ1, a pan-BET inhibitor, on HNSCC cell phenotypes. Additionally, we will identify the downstream targets of BRD4, which may serve as potential therapeutic targets for both HNSCC as well as other cancers more broadly.

OBJECTIVES/GOALS: To describe and compare clinical data and outcomes for patients with CNS tumors and tumor mimics in Puerto Rico who are undergoing surgical and nonsurgical management. Thus, increasing data from an underrepresented group which can serve as a foundation for investigating determinants of outcomes. METHODS/STUDY POPULATION: This proposal will examine patient charts, radiology and pathology reports, financial data, and treatment details from the electronic medical record of patients receiving surgical and nonsurgical treatment for CNS tumors and mimics in the University of Puerto Rico Medical Sciences Campus and all associated institutions. Data will be analyzed retrospectively between January 1, 2014 and June 30, 2022, and prospectively for ten years until December 31, 2032. Patients with primary and metastatic CNS tumors and tumor mimics in the brain, meninges, ventricles, spinal cord, cranial nerves, orbit, facial sinuses, bony skull, vasculature will be included. The registry will include patients from birth onward. RESULTS/ANTICIPATED RESULTS: We plan to compare different surgical and non-surgical techniques and devices in terms of technical and clinical outcomes after surgical interventions for CNS tumors. We are collaborating with the CNS Tumor Outcome Registry at Emory (CTORE) and plan to continue collaboration with other institutions. Combining our data, we aim to develop predictive models of patient outcomes after surgical and nonsurgical intervention for CNS pathologies using supervised and unsupervised machine learning strategies. DISCUSSION/SIGNIFICANCE: There is a significant lack of literature on CNS intervention outcomes in Puerto Rico. This registry will provide the platform for cost-analysis studies for techniques and clinical protocols applicable to pre-operative, intra-operative, post-operative, and conservative management of patients, in Puerto Rico and beyond.

OBJECTIVES/SPECIFIC AIMS: Drug repositioning has the potential to accelerate translation of novel cancer chemotherapeutics from bench to bedside. The goal of this study was to determine the effects of ciclopirox olamine (CPX) on esophageal tumor cells. METHODS/STUDY POPULATION: We tested the effect of CPX on four esophageal cancer cell lines, assessing cell proliferation and viability by hexosaminidase and clonogenicity assay, respectively. We analyzed the effects of CPX on three-dimensional (3D) esophageal tumor cell spheroids. We also analyzed effects on cell cycle by flow cytometry. For mechanism, we performed western blots for proteins involved in cell cycle regulation, apoptosis and the Wnt/β-catenin pathway. For in vivo effects, we performed a murine xenograft model with intraperitoneal administration of CPX (100 mg/Kg body weight daily). RESULTS/ANTICIPATED RESULTS: CPX inhibited growth of all cell lines in a time and concentration-dependent manner. CPX also inhibited growth of esophageal spheroids. Cell cycle analysis demonstrated G0/G1 arrest in cells treated with CPX. Western blot analyses demonstrated decreased expression of cyclinD1, CDK4, CDK6, and transcriptionally active β-catenin, supporting the role of CPX in cell cycle inhibition and decreased β-catenin activity. Finally, treatment of nude mice with CPX significantly decreased tumor xenograft volume. DISCUSSION/SIGNIFICANCE OF IMPACT: CPX demonstrates anti-tumor properties in esophageal cancer cell lines. The current results justify further research into the mechanism of this inhibition. Additionally, given its established safety in humans, CPX is a potential candidate for repositioning as an adjunct treatment for esophageal cancer.

OBJECTIVES/GOALS: The primary objective is to measure the independent association of hospital-level and sociodemographic variables on the rate of hospital-onset bacteremia among infants admitted to the neonatal intensive care unit in a United States of America retrospective cohort. The secondary outcome will be relative blood culture collection rate. METHODS/STUDY POPULATION: The study is an analysis of a retrospective cohort comprised of infants admitted to 322 neonatal intensive care units (NICUs) in the United States of America between 2016-2021. The primary outcome will be hospital-onset bacteremia (HOB), defined as a positive blood culture with a bacteria or fungi after day 3 of admission. Independent risk factors will include birthweight, postnatal age, central venous catheter presence, sociodemographic variables (race, ethnicity, insurance status and ZIP code-level demographic data from the US Census American Community Survey (ACS), and hospital-level variables. Infants will be stratified by sociodemographic groups and a Poisson model will be utilized to measure the adjusted association between risk of HOB and clinical and hospital-level variables. RESULTS/ANTICIPATED RESULTS: I anticipate that infants in sociodemographic groups with a history of socioeconomic marginalization will have a higher unadjusted rate of HOB; however, sociodemographic variables will not be independently associated with HOB risk after adjusting for markers of hospital quality and acuity, such as quartiles of the following: mean admissions per year, percentage of infants born <1500g, annual blood culture contamination rate, and percentage infants born at another facility. DISCUSSION/SIGNIFICANCE: Neonatal bacteremia has high morbidity and mortality; however, its contribution to known infant mortality inequities is unknown. This study will estimate the burden of infant HOB stratified by sociodemographic groups and measure the independent association of sociodemographic and hospital-level variables on the adjusted rate of HOB.

OBJECTIVES/SPECIFIC AIMS: Our main objectives are to study sensory encoding in the adult cortex and quantify rodents’ ability to use intracortical microstimulation to guide behavior. METHODS/STUDY POPULATION: Three rats were implanted with unilateral bipolar stimulating electrodes. The electrodes were connected to a wireless neural stimulator housed in the rat’s backpack. The rat’s swim path was tracked by a video camera above the circular pool, and stimulation parameters were updated in real-time based on distance from the platform. Stimulation was delivered as the distance from the platform increased. Stimulation amplitude was determined through behavioral threshold testing, and parameters ranged from 15–75 μA with 100-Hz pulse trains and 0.2-ms pulses. Rats were first challenged with the 4-platform task in which the submerged platform was randomized across 4 possible locations. This dissociated visual cues from the platform location, as rats had knowledge of the 4 possible locations, but had to use stimulation to guide them efficiently. Next, rats were tasked with the more challenging random-platform task. Visual cues were completely dissociated from the platform location by randomizing the platform location across the entire pool. Performance using the neuroprosthetic device was assessed by comparing trials when the device was on (stimulation trial) Versus off (no-stim trial) for the 2 tasks. RESULTS/ANTICIPATED RESULTS: 4-platform task: Rats visited less potential platform locations when the neuroprosthetic was on Versus off. Rats were also more likely to visit the correct platform location on their first swim trajectory when brain stimulation was delivered. When artificial cues were not available, rats had a greater chance of visiting the platform location from the previous trial. This indicated that rats relied on visuospatial memory without the neuroprosthetic. Random platform task: Performance was measured by taking the ratio of the rat’s actual path length to the optimal path length. When the neuroprosthetic was on, rats demonstrated superior performance through a smaller path to length ratio compared with when the device was off. The platform locations of catch trials were matched to a random subset of stimulation trials, permitting a paired sample t-test. Both rats had significantly shorter path lengths when the device was on. DISCUSSION/SIGNIFICANCE OF IMPACT: Rodents have excellent navigation skills that have been well studied. They have been shown to rely on multimodal sensory information from visual, olfactory, auditory, and idiothetic cues to navigate through their environment. The importance of these cues depends on both their environmental presence and task relevance. In the original Morris water maze experiment, rats use vision to form a visuospatial map of the platform location for allocentric navigation. Here, we have shown that sensory augmented rats can pick up on novel sensory information delivered through ICMS to efficiently find a hidden platform when visual cues are made irrelevant.Our results have implications for the design of the bi-directional sensorimotor neuroprosthetic. We have demonstrated that mammals can interpret artificial sensory information to guide behavior. Future directions include investigating sensory encoding in other primary sensory areas and downstream targets along the somatosensory neuraxis.

OBJECTIVES/GOALS: The goal was to create and deploy an intuitive, easy-to-use tool that clinical investigators can apply to their data to identify erroneous or inconsistent data entries. Investigators can then correct any errors prior to sharing the data with their statistician for analysis. METHODS/STUDY POPULATION: We developed an interactive shiny app, the Data Loofah, using R Studio that researchers or data analysts can use to examine data. After an investigator uploads data, the app reports which variables are numeric or categorical. Means, standard deviation, median, 25th and 75th quantiles, range and number of missing values are reported for numeric variables. Counts and percentages of categorical variables are summarized. Graphical displays further enhance identification of errors. Access to the Data Loofah is through a secure, university-maintained website with access restricted to university personnel. Supporting materials consisting of instructional step-by-step handouts and videos were developed to assist investigators in the use of the app. RESULTS/ANTICIPATED RESULTS: We will integrate use of the Data Loofah into our Clinical and Translational Science Program’s biostatistics consultative practice. Investigators will use the Data Loofah to pre-screen their data prior to sending it to a statistician, identify errors and inconsistencies, and facilitate making necessary corrections. Statisticians will also use the Data Loofah to review data with investigators prior to starting analyses. Through use of this app, investigators are expected to develop a better understanding of their data specifically and more generally about requirements for preparing data for statistical analysis. Most significantly, regular use of the Data Loofah is expected to result in higher quality data and more efficient use of statistician resources due to reduced effort for data cleaning. DISCUSSION/SIGNIFICANCE: Data cleaning is a time-consuming task and finding data errors can be difficult for data analysts not familiar with clinical variables under study. Further, failure to identify data errors can lead to erroneous results. By facilitating identification of data errors by clinical investigators, the Data Loofah will improve and enhance research output.

OBJECTIVES/SPECIFIC AIMS: This study will assess the effect of essential amino acid (EAA) supplementation on plasma triglyceride (TG) in elderly adults. We will also explore the mechanisms mediating EAA mediated changes in fat metabolism and to suggest promising routes to refine therapy of hypertriglyceridemia. METHODS/STUDY POPULATION: In total, 7 nondiabetic male and female subjects ages 50–75 years with elevated plasma TG levels (130–500 mg/dL) were recruited to participate in an acute (5 h) and long-term (8 wk) EAA supplementation study. We measured changes in regional and whole body fat metabolism, including changes in body composition, plasma TG levels, whole body fat metabolic rates, tissue mitochondrial respiratory capacity, and metabolomic profiles before and after supplementation. RESULTS/ANTICIPATED RESULTS: Long-term EAA supplementation decreased fasted plasma TG levels by 19% (p<0.01). Metabolomics of skeletal muscle found acute EAA supplementation resulted in increased EAA metabolic products while long-term supplementation resulted in increased anaplerosis [flux into the tricarboxylic acid cycle (TCA) intermediate pool] and anaplerotic substrates [propionyl (p<0.01) and succinyl (p<0.01) carnitine] and intermediates of long-chain fatty acid metabolism [stearoyl (p<0.01) and myristoyl (p<0.05) carnitine]. However, tissue level respiratory capacity appeared to be unaffected by EAA supplementation. DISCUSSION/SIGNIFICANCE OF IMPACT: EAA supplementation has potential to improve lipid metabolism and plasma TG levels in non-diabetic older adults. Mitochondrial metabolomics suggest that insufficient TCA pool size may limit tissue fatty acid oxidation and may provide an additional route for nutritional therapy.

OBJECTIVES/GOALS: To characterize the oncogenic potential of HNSCC cell lines harboring 17 non-canonical PIK3CA mutations. METHODS/STUDY POPULATION: Non-canonical PIK3CA mutant constructs generated via site-directed mutagenesis are subcloned into doxycycline-inducible vector pLVX-Puro. Serum-dependent HNSCC cell line (PCI-52-SD1) is then stably transfected with vectors and undergo doxycycline-induction. Cell survival is determined by depriving cells of fetal bovine serum for 72 hours and quantifying remaining cells with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell proliferation and migration is evaluated with colony formation assays and transwell assays respectively. RESULTS/ANTICIPATED RESULTS: To date, the survival behavior of eight non-canonical mutants was assessed. Three mutants – Q75E, V71I, and E970K – exhibited 18.7-26.7% greater survival rate relative to cells transfected with wild-type. Five mutants – R519G, Y606C, W328S, C905S, and M1040I – demonstrated survival rates that differed only by −4.3% to +6.6% relative to wild-type. We hypothesize the three activating mutants that exhibited increased survival will also demonstrate increased cell proliferation and migratory behavior whereas the three neutral mutants will not differ from control. DISCUSSION/SIGNIFICANCE OF IMPACT: Ongoing HNSCC PI3K inhibitor trials could be more effective if all PIK3CA hyperactivation mutations are known. Identifying non-canonical mutation effects could result in greater efficacy if drugs are restricted only to those with activating mutations. CONFLICT OF INTEREST DESCRIPTION: JRG and DEJ are co-inventors of cyclic STAT3 decoy and have financial interests in STAT3 Therapeutics, Inc. STAT3 Therapeutics, Inc. holds an interest in a cyclic STAT3 decoy oligonucleotide. The remaining authors declare no conflicts.

OBJECTIVES/SPECIFIC AIMS: The objectives of this study were to compare different methods for determining the disciplines involved in a research article. We sought to address the following questions: To what extent does the number of disciplines reported by an article’s corresponding author agree with their description of the article as unidisciplinary or interdisciplinary? (Q1) and To what extent does the corresponding author’s description of the research as unidisciplinary or interdisciplinary agree with its classification as unidisciplinary or interdisciplinary based on the affiliation of its co-authors? (Q2). METHODS/STUDY POPULATION: Using Scopus, we randomly selected 100 articles from 2010 and 2015 from science teams that had at least 1 author affiliated with Johns Hopkins. Author affiliations were grouped into common academic disciplines: Basic Science, Medicine (and all clinical specialties), Public Health, Engineering, Social Science, Computer Science, Pharmacy, Nursing, and Other. Articles with more than 1 discipline were considered, interdisciplinary. We then sent an online Qualtrics survey to the corresponding author of each article and asked them to indicate (1) all of the disciplines that contributed to the research article at hand, and (2) to indicate whether they considered the research to be “unidisciplinary” or “interdisciplinary” based on definitions that we provided. RESULTS/ANTICIPATED RESULTS: For Q1, we asked corresponding authors to indicate the number of disciplines involved in their research and then to choose the definition that best described their research. Among 76 respondents, 42 indicated that their research consisted of 1 discipline, and 34 indicated that their research consisted of more than 1 discipline. Of the 42 respondents who indicated that their research consisted of one discipline, 21 (50%) respondents described their research as “unidisciplinary” and 21 (50%) described their research as “interdisciplinary.” However, of the 34 respondents who indicated that their research consisted of more than 1 discipline, all but 1 (97%) described their research as “interdisciplinary.” For Q2, we assigned a discipline to each co-author based on his/her affiliation and counted the number of disciplines involved. Among 76 respondents, of the 22 who described their research as “unidisciplinary,” 16 (73%) were categorized as “unidisciplinary” and 6 (27%) were categorized as “interdisciplinary,” using this method. Of the 54 respondents who described their research as “interdisciplinary,” 30 (56%) were categorized as “interdisciplinary” and 24 (44%) as “unidisciplinary.” DISCUSSION/SIGNIFICANCE OF IMPACT: Our results highlight that different methods for determining whether a given research article is interdisciplinary are likely to yield different results. Even when researchers indicate that their research is based within one major discipline, they may still consider it interdisciplinary. Likewise, classifying an article as either unidisciplinary or interdisciplinary based on the affiliations of its co-authors, may not be consistent with the way it is viewed by its authors. It is important to acknowledge that assessing the interdisciplinarity of research is complex and that objective and subjective views may differ.

OBJECTIVES/SPECIFIC AIMS: Accumulation of cholesterol-laden macrophages in arterial walls leads to atherosclerosis. LXRs induce expression of genes that are atheroprotective in macrophages including CCR7, a chemokine receptor that promotes their emigration from the plaque. CCR7 expression has been shown to be negatively regulated by phosphorylation of LXRα at S198 and is reduced in diabetic mice that show impaired plaque regression. I hypothesized that LXRα phosphorylation at S198 diminishes macrophage emigration from atherosclerotic plaque and contributes to impaired regression in diabetes. METHODS/STUDY POPULATION: Inducible LXRα S198A phosphorylation deficient knock in mouse were used as donors for bone marrow transplantation into mice prone to develop atherosclerosis. Plaques were developed by placing mice on western diet; and regression was induced by lowering their lipid levels. Aortic plaques were then analyzed by using morphometric, histological, and molecular analyses in control and diabetic mice expressing either LXRα WT or LXRα S198A during regression. RESULTS/ANTICIPATED RESULTS: Surprisingly, lack of phosphorylation increased plaque macrophage content and impaired regression under normoglycemic condition; however, it did not exacerbate diabetic regression. Plaques in diabetic mice were associated with increased LXRα S198 phosphorylation. Consistent with this, LXRα phosphorylation is enhanced in macrophages cultured under hyperglycemic conditions indicating glucose-dependent regulation of LXRα phosphorylation. Monocyte trafficking studies reveal that lack of phosphorylation and diabetes independently increase recruitment of monocytes in the plaque that might contribute to increased macrophage content. Importantly, I found that diabetes also increases macrophage retention in the plaque, which is reversed in the absence of phosphorylation. We predict that this increased retention results from inhibition of emigration of plaque macrophages through enhanced phosphorylation in diabetes. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings suggest that inhibiting LXRα phosphorylation could be beneficial in diabetic atherosclerosis to reverse the accumulation of macrophages in the plaque. This study imparts insight on regulation of plaque macrophage trafficking through LXRα S198 phosphorylation.

OBJECTIVES/GOALS: Breast cancer metastases are stochastic and difficult to detect. Therapy is often ineffective due to phenotypic changes of tumor cells at these sites. We engineered a synthetic metastatic niche to study the role of phenotypic transitions in the microenvironment on tumor cell phenotype. METHODS/STUDY POPULATION: The engineered metastatic niche is composed of a porous polycaprolactone scaffold implanted subcutaneously in Balb/c mice. The mice received an orthotopic inoculation of 4T1 cells (murine triple negative breast cancer) in the fourth right mammary fat pad and the disease was allowed to progress for 7-21 days (pre-metastatic to overt metastatic disease). The scaffolds and lungs (native metastatic site) were explanted and analyzed by single cell RNA-seq via Drop-seq. Cell phenotypes were identified and tracked over time with the Seurat and Monocle3 pipelines. Assessment of the impact of these cell populations on tumor cell phenotype was conducted through Transwell co-cultures. RESULTS/ANTICIPATED RESULTS: Healthy scaffolds are primarily composed of macrophages, dendritic cells, and fibroblasts – consistent with a foreign body response. Despite differences in the lung and scaffold prior to tumor inoculation, both tissues were marked by >5-fold increase in neutrophils/MDSCs. Additionally, 79% of genes at the scaffold that significantly changed over time were also identified in the lung, indicating key similarities in niche maturation. However, many immune cells at the scaffold had distinct phenotypes, with pro-inflammatory/cytotoxic characteristics. These changes clearly impacted tumor cell phenotype, as cells from the scaffold increased tumor cell migration and apoptosis in vitro. DISCUSSION/SIGNIFICANCE OF IMPACT: Early phenotypic changes at the engineered metastatic niche can identify signs of metastasis prior to colonization of tumor cells. Furthermore, dynamics of immune and stromal cells change throughout niche maturation, influencing tumor cell phenotype and may suggest targeted therapies. CONFLICT OF INTEREST DESCRIPTION: Lonnie Shea, Jacqueline Jeruss, and Grace Bushnell are named inventors on patents or patent applications.

OBJECTIVES/SPECIFIC AIMS: Preliminary animal studies showed that low-intensity ultrasound (US) coincident with intravascularly administered microbubbles locally disrupts tumor vasculature. This study translates the novel therapy of antivascular ultrasound (AVUS) into an autochthonous model of hepatocellular carcinoma (HCC). The differential effects produced by AVUS at low and high doses are evaluated. METHODS/STUDY POPULATION: HCC was induced in 12 Wistar rats by ingestion of 0.01% diethylnitrosamine in drinking water for 12 weeks. Rats received AVUS treatment at low and high doses. Low dose group (n=6) received 1 W/cm2 US for 1 minute with 0.2 mL microbubbles injected IV. High dose group (n=6) received 2 W/cm2 for 2 minute with 0.7 mL microbubbles IV. Perfusion was measured before and after AVUS with contrast-enhanced ultrasound (CE-US) and power Doppler (PD-US). Peak enhancement (PE) and perfusion index (PI) were measured from each US mode. Histology after sacrifice or natural death was compared to pre/post US. Analysis of H&E and trichrome sections was evaluated for percent area of hemorrhage and findings of tissue injury and repair including inflammation, necrosis, and fibrosis. RESULTS/ANTICIPATED RESULTS: After high dose AVUS, PE, and PI of CE-US decreased from baseline by an average of 33.3% and 29.7%, respectively. Histology showed extensive tissue injury (hemorrhage, necrosis, fibrosis) in 58% of tumor cross-sectional area. Conversely, low dose AVUS increased PE and PI of CE-US by an average of 39.3% and 67.8%, respectively. Histology showed smaller areas of microhemorrhage Versus large pools of hemorrhage (only 17% area). PD-US changes were similar to CE-US. DISCUSSION/SIGNIFICANCE OF IMPACT: In summary, the opposing effects of AVUS observed at 2 doses allows for multiple roles in tumor therapy. Enhanced perfusion at a low dose may improve drug delivery or radiation therapy. Whereas, vascular disruption at high doses of AVUS may allow noninvasive ischemic therapy. Furthermore, AVUS is ripe for translation given the use its component parts clinically: low-intensity long-tone burst for physiotherapy and microbubbles as an US contrast agent. Thus, AVUS should be evaluated for translation of its differential effects into noninvasive therapies for HCC and other tumors.

ABSTRACT IMPACT: Our study will integrate state-of-the-art methods in pathogen genomics, epidemiology, and geospatial analysis to identify both host- and pathogen-factors driving the MDR-TB transmission and the study outcome can inform the design of targeted interventions OBJECTIVES/GOALS: The emergence of multidrug-resistant tuberculosis (MDR-TB) poses serious challenges for the global eradication of tuberculosis. Recent research has shown that transmission is now the dominant driver of MDR-TB. However, our limited understanding of where and among whom MDR-TB is transmitted hampers efforts to control person-to-person spread. METHODS/STUDY POPULATION: We used several analytic approaches to characterize the dynamics of MDR-TB transmission in Shanghai, China. We identified all culture-confirmed MDR cases between 2009-2016 in the city and 1) estimated individual-level risk factors for MDR disease; 2) mapped the TB cases by their home addresses and used a Bayesian spatial disease mapping method to identify regions with an elevated risk of MDR-TB; and 3) we sequenced all MDR isolates to understand whether transmission explained variance in risk that was not attributable to the distribution of individual or location-specific risk variates. RESULTS/ANTICIPATED RESULTS: There were 1034 MDR-TB cases among 16,315 culture-confirmed TB cases during the study period. Bayesian disease mapping identified spatial heterogeneity of MDR-TB and determined four hotspots with an elevated risk of MDR-TB, none of which were fully explained by individual or regional-covariates (Figure 1). Sequencing revealed that more than 40% of the MDR-TB strains were in genomic clusters, indicating recent MDR-TB transmission. Most importantly, MDR-TB cases in three of the four large clades (>8 isolates) were spatially concentrated in three strain-specific hotspots (Figure 2). DISCUSSION/SIGNIFICANCE OF FINDINGS: With the combination of traditional epidemiological tools, geographical, and genomic methods, this study revealed multiple loci of transmission of specific MDR-TB clades within a single city. Identification of where and among whom MDR-TB is transmitted can inform the design of targeted interventions.

OBJECTIVES/SPECIFIC AIMS: To study the rate of recruitment to the Pulmonary Research Registry (PRR) at the University of Chicago using HealtheRx recruitment Versus usual practice. METHODS/STUDY POPULATION: CommunityRx is a health information technology, integrated with electronic medical record (EMR) platforms, that generates personalized referrals (“HealtheRxs”) for community-based programs and services that address basic and other health-related self-care needs. The target population included people ages 18 and older, English speaking, living in 1 of 16 ZIP codes on Chicago’s south and west sides (106 mi2) who received care at ≥1 of 28 CommunityRx partner sites and had a diagnosis of asthma or COPD. Between December 2015 and December 2016, information about pulmonary research participation opportunities was included on the HealtheRxs of eligible patients contemporaneously with usual registry recruitment methods. Usual methods, used since 2010 by the PRR group, included public advertisements requiring the patient to call the research team for more information and education of eligible patients identified during routine clinical care with a Pulmonary/Critical Care clinician or when enrolling in a pulmonary clinical trial. We hypothesized that, compared with usual recruitment practices, the HealtheRx recruitment strategy would increase the rate and decrease the per subject cost of patient recruitment to a prospective registry. Total annual recruitment costs for each method were calculated and divided by the number of consented PRR enrollees per method. RESULTS/ANTICIPATED RESULTS: Between December 22, 2015 and December 15, 2016 13,437 HealtheRxs (8762 for asthma, 3842 for COPD, and 833 for both asthma and COPD) were generated with the recruitment advertisement. In total, 41 patients responded to the ad and participated in the phone survey. In which 15 (36.5%) participants self-reported a diagnosis of asthma only (65% of all HealtheRxs with advertisement were for asthma only), 9 (22%) reported a diagnosis of COPD only (28.5% of all HealtheRxs with advertisement were for COPD only), and 17 (41.5%) reported diagnoses of both asthma and COPD (6.2% of all HealtheRxs with advertisement were for asthma and COPD). Most participants were female (n=28), non-Hispanic black (n=37), and not employed (n=39). The median age was 57. The majority (n=31) had never participated in health or medical research and was not aware of current opportunities to participate in research (n=25). All 41 participants expressed interest in joining PRR and were mailed a blank PRR consent form and a prepaid return envelope with their incentive check for the telephone survey. To date, 5 participants returned a signed consent form via mail and were enrolled in PRR. During the same period, 4 patients enrolled in PRR via usual recruitment methods. The cost per subject to enroll in PRR was $364.40 using the HealtheRx recruitment and $4590 using usual practice. DISCUSSION/SIGNIFICANCE OF IMPACT: NIH has called for innovation in research recruitment methodologies to increase enrollment especially of people who are under-represented in clinical trials research. This study demonstrates the feasibility and efficiency of using an EMR-integrated recruitment method to enroll people of under-represented minority groups to a research registry.

OBJECTIVES/GOALS: Endovascular peripheral vascular interventions (PVIs) are increasingly utilized for the treatment of peripheral artery disease (PAD). We aimed to assess characteristics of patients receiving PVI at ambulatory surgical centers and office-based labs (ASC/OBL) versus the outpatient hospital (hospital) site of service. METHODS/STUDY POPULATION: We performed a retrospective analysis using 100% Medicare fee-for-service claims data between January 1, 2017 and December 31, 2022. We used Current Procedural Terminology (CPT) codes to identify patients undergoing angioplasty, stenting, or atherectomy. Patient demographics were collected from the Medicare Master Beneficiary Summary File and associated comorbidities and PVI indications were identified using International Classification of Disease (ICD)-10 codes. We used patient ZIP codes to determine patients’ residence densities and regions. We used site of service codes to determine whether PVI were performed in the ASC/OBL versus hospital. Results were analyzed with descriptive statistics. RESULTS/ANTICIPATED RESULTS: Of 817,241 patients undergoing PVI for PAD, 461,068 (56.4%) were treated in an ASC/OBL. Compared to patients treated in the hospital, patients receiving PVI at ASC/OBLs were more likely to be older, female, non-white race, with fewer comorbidities (end stage renal disease, diabetes, hypertension, and any history of tobacco use) (all, P<0.001). Patients treated in ASC/OBLs more frequently resided in urban (vs. rural) locations, and in the South and West (both, P<0.001). Indication for PVI was predominately chronic limb-threatening ischemia, and clinically similar between groups (77.1% vs. 76.2%). There was a significant change in site of service over time: a minority (47.6%) of PVIs were performed in the ASC/OBL in 2017, whereas the majority (64.7%) of PVIs were performed in the ASC/OBL in 2022 (P<0.001). DISCUSSION/SIGNIFICANCE: Patients treated in ASC/OBLs were less medically complex compared to those treated in the outpatient hospital setting. Further study is needed to examine whether differences in patient characteristics versus other factors (e.g. reimbursement) are driving the increase in PVIs performed in the ASC/OBL over time.

A foundational principle and practice for translational research is active participation of a range of disciplines, referred to as “team science.” It is increasingly apparent that to be relevant and impactful, these teams must also include stakeholders outside the usual academic research community, such as patients, communities, and not-for- and for-profit organizations. To emphasize the need to link the practices of team science and of community-engaged research, we propose a framework that has community members and stakeholders as integral members of the research team, which we term, “broadly engaged team science.” Such transdisciplinary and multi-stakeholder teams will be best suited to pose translational research questions, conduct the research, and interpret and disseminate the results. We think this will generate important and impactful science, and will support the public’s regard for, and participation in, research.