OBJECTIVES/GOALS: We will use a novel syngeneic model of prostate cancer to examine impairments and uncover potential changes in inflammatory signaling in the brains of animals with and without tumors. We will then investigate the interaction between peripheral tumor, age, and chemotherapy on cognitive impairments and any accompanying neuroinflammation METHODS/STUDY POPULATION: Male Copenhagen rats (aged 3 or 10 months) were subjected to tumor fragment implantation (Dunning R2237G cells) or sham surgery. Once tumors were palpable, animals received either docetaxel (4.5mg/kg, intraperitoneal) or it’s vehicle once every other day for 5 days (3 injections total) followed by a two-week recovery period. During this time, TNFa and IL-6 was quantified in plasma samples obtained once per week for two weeks. Hippocampal-mediated visuospatial and working memories were assessed using the novel object task and percent alternation in a y-maze, respectively. Afterwards, trunk blood and hippocampal tissue were isolated. TNFa and IL-6 protein was quantified in plasma. Hippocampal tissue was probed for markers of neuroinflammation, including increases in TNFa, IL-6, and reactive microglia RESULTS/ANTICIPATED RESULTS: The presence of a tumor alone produces deficits in hippocampal-mediated visuospatial memory and working memory regardless of treatment and persistent elevations in TNFa and IL-6 in plasma. Docetaxel administration also produces impairments in hippocampal-mediated visuospatial memory, but not in working memory. We anticipate these cognitive impairments will be accompanied by hippocampal neuroinflammation. We expect age and docetaxel chemotherapy to exacerbate working memory deficits and markers in hippocampal neuroinflammation, including increases in TNFa, IL-6 and reactive microglia DISCUSSION/SIGNIFICANCE: This study will provide insight into the interaction between tumor, age, and chemotherapy in impairments in visuospatial memories. This model provides a substrate upon which interventions can be tested to ensure the efficacy of the cancer treatment is maintained when treating these cognitive impairments.

OBJECTIVES/GOALS: The dominant complication of Systemic Sclerosis (SSc) is clinically severe and commonly fatal pulmonary fibrosis (PF). We sought to determine the downstream regulatory role of the basic Helix-Loop-Helix protein 40 (bHLHe40), in response to Insulin-like Growth Factor II (IGF-II) on Pro-Lysyl Oxidase cleavage products. METHODS/STUDY POPULATION: We examined the response of primary pulmonary fibroblasts cultured from the lungs of control donors and SSc lung explants to IGF-II as well as human recombinant Lysyl Oxidase Propeptide (LOX-PP). In addition, we utilized an experimentally-induced model of lung fibrosis with intratracheal bleomycin administration. We used qPCR and immunoblotting to quantify mRNA and protein levels, respectively. We used sequence-specific small-interfering RNA to silence targeted genes. Immunoblots were quantified in ImageJ (NIH) and statistical analyses were performed in GraphPad Prism. RESULTS/ANTICIPATED RESULTS: IGF-II regulates levels of Pro-LOX, active LOX, and LOX-PP, as well as isoforms of proteases Bone Morphogenetic Protein 1 (BMP1) and Tolloid-like 1 (TLL1). The transcription factorbHLHe40 localizes to the nucleus in response to IGF-II. bHLHe40 silencing downregulated TLL1, abrogating the enzymatic cleavage of Pro-LOX. SSc lungs have higher baseline levels of the total (N-glycosylated/unglycosylated) LOX-PP than normal lung tissues, and baseline levels of LOX-PP correlated with TLL1 Isoform 2 in SSc lungs. LOX-PP contributes to the development and progression of SSc-PF by mediating changes consistent with the extracellular matrix deregulation implicated in SSc-PF: elevated levels of Collagen 3A1 (COL3A1), Fibronectin-1 (FN1), and Plasminogen Activator Inhibitor-1 (PAI1). DISCUSSION/SIGNIFICANCE: Our findings indicate that bHLHe40, TLL1, and LOX-PP may serve as targets of therapeutic intervention to stop the progression of SSc-PF. Since activation of common fibrotic pathways are involved in different diseases characterized by lung fibrosis such as IPF, our findings may have wider implications for lung fibrosis associated with other diseases.

ABSTRACT IMPACT: The impact of this study is to encourage health professionals to screen for violent experiences as potential risk factors for CVD and adapt interventions from the non-abused in children as in adults. OBJECTIVES/GOALS: This study aims to assess the relationship between child sexual abuse and cardiovascular risk factors (CVDRF) that present in children. The objectives will provide the prevalence of CVDRF, their association with types of sexual victimization, and a score to assess the impact. METHODS/STUDY POPULATION: This study is a chart review, cross-sectional study. The Puerto Rico Health Justice Center (PRHJC) is a forensic, transdisciplinary, victim-centered, trauma-informed, and evidence-based service. The demographic variables collected are age, sex; the cardiovascular risk factors variables include a level of physical activity, tobacco exposure/alcohol, vital signs (blood pressure, BMI), lipid profile, and C-reactive protein. Sexual violence variables are the type of victimization (sexual assault, sexual molestation), the number of victimizations, and the relationship with the offender. RESULTS/ANTICIPATED RESULTS: A previous study, which examined types of evidence related to the prosecution of sexual violence cases, found that among female victims, 54% was a victim of sexual assault, and 59% had at least one health concern. The study’s hypothesis includes that older and female victims have a higher prevalence of cardiovascular disease risk factors. Also, children victims of sexual assault will have more cardiovascular risk factors than victims of sexual molestation. The age group, sex, number of victimizations, and relationship with the offender will also impact the relationship between the type of victimization and cardiovascular risk factors. DISCUSSION/SIGNIFICANCE OF FINDINGS: Early identification of child sexual abuse is needed to prevent long-term health impacts. The study’s results will be significant in developing clinical guidelines for health care providers to identify child sexual abuse as a predictor of cardiovascular risk factors and encourage victim advocates to identify cardiovascular risk factors.

ABSTRACT IMPACT: This work will inform the need for more trauma-informed approaches to violence screenings among marginalized populations by health care providers. OBJECTIVES/GOALS: Female sex workers (FSW) experience high rates of intimate partner violence (IPV) which may have negative reproductive health consequences. Routine IPV screening by healthcare providers (HCP) is recommended. This study examines how FSW experience IPV assessments by HCP. METHODS/STUDY POPULATION: This qualitative descriptive study is nested within EMERALD, a longitudinal cohort study of street-based FSW. EMERALD assesses a structural community-level intervention on HIV and STI risks among FSW. Participants were recruited for EMERALD using time-location sampling to identify a sampling frame of venues and times where sex work is likely to occur. Inclusion criteria for this qualitative study include: participating in EMERALD, age 18-49, and willingness to participate in one phone interview. Twenty-two semi-structured individual qualitative interviews were conducted. Two coders, using thematic analysis, applied deductive codes and inductive coding to identify themes. RESULTS/ANTICIPATED RESULTS: Three themes emerged from participant interviews regarding IPV screening. Inconsistent screening practices: Despite frequent reproductive health visits among participants, many women did not recall IPV screening by a HCP. Stigma as a barrier to disclosure: Women described feeling judged by HCP regarding their frequency of visits for reproductive concerns, sex work, and substance use and did not trust disclosing violence to HCP. Transactional health encounters: During visits, HCP were focused on addressing women’s immediate concerns; if the HCP did ask about IPV women felt the questions were asked part of a required protocol and not driven by a concern to deeply understand their lives. DISCUSSION/SIGNIFICANCE OF FINDINGS: FSW described inconsistent IPV screening practices by HPC. Participants expressed feeling stigmatized by HCP and that encounters with HCP did not foster trust for women to disclose IPV experiences. There is a need for HPC training in universal IPV screening focused on relationship and trust building to facilitate disclosure.

OBJECTIVES/GOALS: We conducted interviews with investigators, clinicians, and health system and health agency leaders to assess regional educational needs in implementation and improvement science, including content (knowledge and skill), format, experiential learning, and mentoring, to identify barriers and guide planning. METHODS/STUDY POPULATION: Five CTSAs in the University of California Biomedical Research Acceleration, Integration, & Development consortium (UC BRAID) plus a fifth affiliated CTSA developed a common protocol and interviewed 31 California-based learners (current fellows, early and mid-career investigators, clinicians, and health agency personnel) and system leaders from health care and health agencies. Interviews focused on impact goals, educational needs in dissemination, implementation, and improvement (DII) science, challenges in DII research, preferred learning formats, desired proficiencies and skills, and barriers such as cost, time, awareness, terminology, and suitability and availability of training. A rapid review of literature identified potential domains of knowledge and skills for a proposed curriculum. RESULTS/ANTICIPATED RESULTS: Areas of emphasis varied among interviewees; identified learning needs differed between traditional research perspectives (emphasizing areas such as partner engagement, grant writing, frameworks, study design) and applied perspectives (emphasizing areas such as managing change, complex systems, learning system capacity). Learners had a range of preferences; most interviewees desired formats that are longitudinal, experiential, applied, cooperative, and affordable. Variation in knowledge of, and interpretations of, DII terms and goals limited the ability of some interviewees to specify educational needs. A synthesis reveals areas for potential future co-development and networked approaches to regional training and capacity enhancement. DISCUSSION/SIGNIFICANCE OF IMPACT: In response to a rapidly changing health landscape, our academic health systems are developing capabilities to improve care for their populations. Our work informs the training and education needs that are critical to translation at a system-wide level. Regional convenings can raise awareness while translational programs fill educational gaps.

OBJECTIVES/GOALS: The purpose of this study is to identify and quantitatively describe environmental barriers to community engagement and activity participation for adults with stroke and low income. Repeated electronic surveys collected in real time will reduce recall bias and improve characterization of barriers. METHODS/STUDY POPULATION: 20-30 community-dwelling adults with stroke and low income will be recruited for this pilot study. Inclusion criteria: > 1 month post stroke and evidence that they have the vision, literacy, and cognitive capacities to answer survey questions on a smart device. Exclusion criteria: severe aphasia, severe mental illness or substance abuse within 3 months, and ataxia. Participants will complete standardized assessments of daily activities, engagement in and perceptions about community activities, social support, and perceived environmental barriers. Participants then complete four surveys per day for 14 days using an app on an iPod Touch, reporting activities attempted and barriers encountered. RESULTS/ANTICIPATED RESULTS: This is the first study of this kind and is a work in progress. We anticipate that the environmental barriers reported will include physical (e.g. built structures, climate, and natural terrain), social (e.g. support or lack thereof; stigma), political (e.g. access to transportation; healthcare services), and technological barriers (e.g. difficulties with personal equipment and/or technologies such as elevators, ticket kiosks, etc.). DISCUSSION/SIGNIFICANCE OF IMPACT: An increased understanding of the barriers facing community-dwelling adults with stroke and low income will facilitate the development of culturally-appropriate and more accessible self-management programs to help this population re-engage in their communities and return to pre-stroke activities.

OBJECTIVES/GOALS: 1. Identify candidate AN-SAD-causing variants. 2. Estimate the variant effect size of and genotypic relative risk for arrhythmias and AN-SAD. METHODS/STUDY POPULATION: We performed whole genome sequencing (WGS) on 59 Thoroughbred AN-SAD cases and 58 controls. WGS was mapped and variants identified using a modified version of the Genome Analysis Toolkit best practices. Variants will be selected based on case-control analysis using SnpSift and presence in candidate genes. The top 400 candidate AN-SAD-causing variants will be selected based on being common in cases and rare or absent in controls, and uncommon (allele frequency less than 10%) in a catalog of genetic variation for the horse. The 400 variants will be genotyped in our cohort of 1,200 racehorses to determine variant effect size and genotypic relative risk. RESULTS/ANTICIPATED RESULTS: 17,182,003 variants were identified. 230 variants had significantly different allele frequencies (AF) between cases and controls (SnpSift). 723 high and 4,824 moderate impact variants were identified in 1,072 candidate genes. 3,681 variants were present at an AF 10% in the equine variant catalog. Variant effect prediction is ongoing to select the final 400 variants. Cardiac phenotyping (cardiac auscultation and ECG before, during, and after exercise) was performed on 790 racehorses and we will have 1,200 racehorses with cardiac phenotypes and/or AN-SAD. We will genotype the top 400 candidate AN-SAD-causing variants in these 1,200 horses to identify variants of large effect size. DISCUSSION/SIGNIFICANCE: Identification of candidate AN-SAD variants in a spontaneous animal model can facilitate interpretation of candidate variants in humans and horses. This project will provide further support for the racehorse AN-SAD model and will support future work exploring the genetic and environmental risk factors contributing to AN-SAD in this animal model.

OBJECTIVES/GOALS: Current COVID-19 rapid molecular tests require cartridge-reader detection, expensive circuitry, and complex microfluidics making the most accurate tests unavailable to the masses. Here we present a rapid molecular diagnostic leveraging isothermal amplification and paper-based microfluidics for a low-cost ultra-sensitive COVID-19 assay. METHODS/STUDY POPULATION: We designed a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of SARS-CoV-2 and bacteriophage MS2 RNA. RT-RPA is a sequence specific, ultrasensitive, rapid isothermal DNA amplification technique that is well suited to home based testing due to its rapid assay time, robustness, ease of use, and readout options. RT-RPA reagents are added to a tube and incubated at 39°C in a fluorometer. Realtime fluorometer data gives results in under 15 minutes. This assay also provides visual detection via lateral flow readout with results in 23 minutes. RESULTS/ANTICIPATED RESULTS: We have developed a rapid multiplexed nucleic acid amplification assay with an internal process control for SARS-CoV-2 using single-pot RT-RPA. We screened 21 primer combinations to select primers that demonstrated excellent performance and target specificity against common respiratory viruses. We demonstrate the ability to multiplex SARS-CoV-2 and MS2 detection, utilizing MS2 as an internal process control for lysis, reverse transcription, amplification, and readout. We show duplexed detection using both fluorescence readout and visual readout using lateral flow strips. Duplexed fluorescence detection shows a limit of detection of 25 copies per reaction. Duplexed lateral flow readout shows a limit of detection of 50 copies per reaction DISCUSSION/SIGNIFICANCE: We developed a duplexed RT-RPA assay for SARS-CoV-2 with fluorescence or lateral flow readout. Our assay does not re-quire expensive reader, circuity, or fluid handling. The low material cost, temperature, and robustness make it ideal for a more accurate home-based COVID-19 diagnostic.

OBJECTIVES/GOALS: Neuropsychiatric disorders classified as synaptopathies are marked by a glutamate-associated hypofrontality which impacts decision making and impulsivity. We hypothesized that behavioral efficacy of the psychoplastogen ketamine is mediated in part through lasting promotion of markers of synaptic strength in corticoaccumbens circuit. METHODS/STUDY POPULATION: Male, Sprague-Dawley rats received an intraperitoneal (i.p.) injection of saline, single ketamine (10 mg/kg; 1x/day), or repeated ketamine (10 mg/kg; 1x/day for three days). Twenty-four hrs following the dosing regimen, animals were euthanized, and brains dissected to harvest corticoaccumbens structures including the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAc). mRNA was extracted and converted to cDNA. Levels of brain derived neurotrophic factor (BDNF) exon II mRNA were quantified using reverse transcriptase polymerase chain reaction (RT-PCR); cyclophilin A (PPIA) was used as a loading control. Gene expression differences in ketamine-treated rats were identified versus saline-treated rats. BDNF protein levels were quantified using capillary-electrophoresis immunoblotting. RESULTS/ANTICIPATED RESULTS: Repeated, but not single, ketamine administration decreased mPFC, but increased NAc, BDNF exon II mRNA levels versus saline (p<0.05). Single and repeated ketamine administration increased NAC BDNF protein (p<0.05), while both dosing paradigms induced a trend towards an increase in mPFC BDNF levels. DISCUSSION/SIGNIFICANCE: We discovered a dosing regimen-dependent and sustained effects of ketamine administration on BDNF levels in the rodent brain. Taken together, ketamine-mediated BDNF levels may sustain synaptic strengthening mechanisms supporting future investigation into the utility of ketamine for diseases characterized by synaptopathies.

OBJECTIVES/SPECIFIC AIMS: The objective of our collaboration is to develop a strong trans-disciplinary team consisting of microfluidics engineers, cancer biologists, and clinicians, to identify a universal marker to detect circulating osteosarcoma cells (COC) using microfluidic devices. Our goals are 3 fold: 1) Identify cell surface markers unique to osteosarcoma (OS) for COC isolation, 2) Develop a Geometrically Enhanced Mixing (GEM) device to isolate COCs, and 3) Evaluate the efficacy of GEM device to detect COCs in patients with OS. The long term goal of this collaboration is to utilize this cell detection approach to evaluate treatment efficacy and correlate the presence of circulating osteosarcoma cells with metastatic incidence. METHODS/STUDY POPULATION: In this phase of our study, we have identified an abundant and conserved cell surface marker across a panel of OS cell lines. Flow cytometry was used to evaluate the relative expression of Epithelial Cell Adhesion Molecule (EpCAM), and Ganglioside 2 or/and 3 (GD2/3) on a panel of OS cell lines. An antibody coated GEM microfluidic device is used to affirm the efficacy of GD2/3 to capture COCs. Further capture studies will be conducted using OS cell spiked blood samples. Analysis of variance (ANOVA) will be used to determine any significant difference in capture efficiency between EpCAM, GD2/3 cell surface markers. RESULTS/ANTICIPATED RESULTS: Our results demonstrate that EpCAM is not a suitable marker for COC detection. Results from our flow cytometry studies demonstrate that GD2/3 expression is significantly higher than EpCAM expression, across all OS cell lines within our panel. The cell capture efficiency strongly correlates with the cell surface expression data obtained from flow cytometry analysis. DISCUSSION/SIGNIFICANCE OF IMPACT: OS is the most common primary bone tumor and the third leading cause of pediatric cancer deaths. At diagnosis, 80% of patients will present with metastasis, however only 20% of these cases are clinically detectable. Innovative strategies to identify patients at risk of metastasis would allow for stratification of intervention therapies. Liquid biopsies are a novel alternative to current diagnostic imaging systems to monitor metastatic incidence and treatment efficacy. The detection of circulating tumor cells (CTCs) through routine blood sampling has the potential to be used clinically for earlier detection, monitoring the treatment of metastatic cancers and surveying the effect of therapeutic interventions on metastasis. To date, the majority of the studies on CTCs have evaluated their presence in carcinomas. Although sarcomas are rare, they generally have a poorer prognosis. This study will address one of the unmet medical needs in the field of CTC detection; the identification of cell surface OS makers to improve binding specificity, increase purity, and maintain a high capture efficiency.

ABSTRACT IMPACT: Specific recommendations will be suggested in this presentation as to how a health equity lens can be applied to implementation science frameworks. OBJECTIVES/GOALS: This systematic review consolidated literature on how implementation science (IS) frameworks (e.g., RE-AIM) have been used in cancer prevention services (e.g., screening, tobacco cessation programs) to reduce health disparities. METHODS/STUDY POPULATION: The systematic review was conducted in accordance with PRISMA and registered with PROSPERO. Searches were conducted in Ovid MEDLINE, PubMed, PsycINFO, CINAHL, and EMBASE between January-May 2020. Search strategies used the combinations of terms related to implementation science frameworks, cancer prevention and/or intervention, and all search algorithms were validated by a public health librarian. RESULTS/ANTICIPATED RESULTS: A total of 1,025 articles were screened and 84 were deemed eligible for full-text screening. After full-text screening, n=27 articles were included for data abstraction and synthesis. Of the 27 studies that used an implementation science framework, only one-third of studies (N=9, 33.3%) used an IS framework to address cancer-related health disparities. Of those nine studies, six of them used the Consolidated Framework for Implementation Research (CFIR) to guide, inform, and/or adapt the implementation of a cancer prevention intervention to target health disparities. However, the variability in how this framework was applied remains a challenge. DISCUSSION/SIGNIFICANCE OF FINDINGS: Recommendations for how various IS frameworks can be used to address cancer prevention disparities will be presented, such as, guiding principles on how to intentionally select domains within the CFIR that will capture input from key stakeholders in health disparities populations.

OBJECTIVES/GOALS: We will use control- and DMD-engineered heart tissues to better model and investigate DMD cardiomyopathy. We will primarily assess cardiac calcium handling, mitochondrial function, and mitochondrial calcium handling, as calcium regulation and mitochondrial function are known to be affected in DMD. METHODS/STUDY POPULATION: We will use patient-derived stem cells, differentiated into cardiomyocytes in bioprinted 3D heart tissue muscle chambers to better model DMD cardiomyopathy. We will look at calcium handling and general mitochondrial function, as well as mitochondrial calcium handling, using a novel multifunctional genetic probe I previously developed allowing for simultaneous observation of cytosolic and mitochondrial calcium in real time. Optical mapping will also be used for tissue-level analysis. We will establish the functional differences at baseline, and then progress heart failure in the tissues to see how the abnormalities seen in the DMD tissues may get worse. Finally, we will investigate the effects of early restoration of dystrophin function on the effects of DMD cardiomyopathy development. RESULTS/ANTICIPATED RESULTS: We anticipate that DMD tissues will show more irregular/abnormal calcium handling, as seen in 2D hiPSC-CMs, as well as disruptions to mitochondrial function and ultrastructural development, as well as a decreased synchronization between cytosolic and mitochondrial calcium dynamics. We anticipate that these abnormalities will be exacerbated as the disease state progresses, but at least partially ameliorated with the restoration of dystrophin function. DISCUSSION/SIGNIFICANCE: DMD is a fatal disease with no known cure. Patients develop heart failure in their teens and die in their 20s, so any new insight that may prolong life and improve quality of life for patients is drastically needed. This would be the most accurate preclinical model of DMD cardiomyopathy to date and would investigate yet-untapped aspects of the disease state.