OBJECTIVES/GOALS: IgA1 nephropathy, which can lead to kidney failure, is caused by complexes formed between aberrant galactose-deficient IgA1 and antibodies directed against it. Our goals are to characterize shifting glycosylation patterns at the level of single IgA1 molecules and to apply this to patient samples for early detection and understanding of the disease. METHODS/STUDY POPULATION: To characterize glycosylation patterns on single IgA1 molecules, labelled IgA1 in low concentration was physisorbed to borosilicate glass in a fluidic cell and labelled Jacalin was flowed in to bind with Glycans on IgA1. The samples were observed with a Nikon TiE epi-fluorescence microscope. FRET images were created by exciting the Jacalin dye with a blue laser and recording the red emission of the IgA1 dye with an EMCCD camera. FRET emission intensities of individual IgA1 molecules over time were analyzed to determine how frequent and how long Jacalin binds to each of them. The rate of binding is roughly inversely proportional to the amount of abnormal glycans on a given IgA1 molecule. After the method is perfected, we intend to compare the glycosylation patterns of healthy and diseased patient samples. RESULTS/ANTICIPATED RESULTS: Addition of the competitive binder Galactose to the solution led to an increase in the off times of Jacalin and IgA1 and a decrease in the on times in a concentration-dependent manner, yielding an increase in the dissociation constant. Dissociation constants of individual molecules within a single experiment vary by 3 orders of magnitude, which cannot be attributed to stochastic fluctuations but rather reflects differences in the adsorption geometry. Nevertheless, the unaffected dissociation constant can be identified. We expect that when this method is applied to samples from healthy and IgA1 Nephropathy patients, specific IgA1 molecules from patients will have higher dissociation constants for Jacalin compared to those from healthy patients. DISCUSSION/SIGNIFICANCE: The binding rates of Jacalin to single IgA1 vary by 3 orders of magnitude. The observed heterogeneity shows the Jacalin probe can differentiate between different IgA1 populations. An understanding of which IgA1 molecules in patient samples are problematic and what their distribution of Glycans is can lead to discovering biomarkers and treatments.

OBJECTIVES/GOALS: Prior work has established subtypes of OSA and linked them to risks of future adverse events but rarely with the longitudinality and richness of data available in the EMR. Our goal is to leverage EMR data identify clinically meaningful sub-phenotypes of OSA and better study how they affect risks of adverse outcomes. METHODS/STUDY POPULATION: Vanderbilt’s EMR database has over 61,000 adult patients with a literature-validated EMR definition of OSA with a median EMR follow-up period of 4 years after OSA diagnosis. Of these patients, 12,516 have fully recorded sleep study data in addition to EMR variables such as age at study and most recent BMI. We applied several clustering methods including to identify natural sub-phenotypes of OSA and assessed cluster quality. We also applied techniques which allow a single patient to belong to multiple clusters in various degrees. After selecting final clusters, we plan to analyze the associations between OSA sub-phenotypes and risks using statistical tools like logistic regression and Cox proportional hazards regression, with and without adjusting for factors such as age, gender, and certain medications. RESULTS/ANTICIPATED RESULTS: Preliminary clustering with primarily sleep study data has shown overlap with literature-described patient clusters, including a severe, high non-REM stage 1 sleep, high BMI cluster and a high nocturnal limb movement cluster. As we incorporate more EMR variables, we will select a final set of OSA sub-types. We anticipate patients in different clusters to have different risks of various adverse OSA-associated outcomes that are tracked in our EMR data. Notable outcomes with sufficient incidence rates (>3%) after OSA diagnosis include essential hypertension (43.4%), hyperlipidemia (28.8%), type 2 diabetes (21.9%), anxiety disorder (19.2%), coronary atherosclerosis (14.9%), cerebrovascular disease (7.7%), and pulmonary heart disease (5.9%). DISCUSSION/SIGNIFICANCE: If our results match anticipations, we will show how EMR data can be used to define OSA sub-phenotypes and predict patient risks of various OSA-associated outcomes. This analysis enables work in personalized risk and treatment predictions for OSA patients. By better understanding these risks, providers can better tailor treatments to patients.

Clinical research studies run the risk of being in a deficit leading to premature study termination or a desperate struggle to find new funding to continue the research. It is important for institutions, small or large, to have financial oversight during the research process. We created a financial audit process for a core clinical research department at a pediatric hospital. Understanding where to find your costs, what costs are important, and other elements of the audit process are essential. Knowing how to replicate a financial audit process can help you eliminate the risk of a financial deficit.

OBJECTIVES/GOALS: The effect of immunosuppressive metabolites on anti-tumor immunity in human papillomavirus (HPV)-associated vs carcinogen-driven head and neck cancer is unknown. The objective of this study is to define the extent to which metabolites impair this response and identify novel metabolic targets for enhancing anti-tumor immunity. METHODS/STUDY POPULATION: HPV-associated and carcinogen-driven head and neck squamous cell carcinoma specimens were frozen following surgical excision, and tumor sections were cut onto glass slides. Slides were coated in alpha-cyano-4-hydroxy-cinnamic acid (CHCA) matrix and subjected to mass spectrometry imaging using matrix-assisted laser desorption ionization (MALDI) on a Bruker SolariX XR 12T Hybrid QqFT-ICR mass spectrometer run in positive mode. Slides were then stained for immunohistochemistry (IHC) using markers of CD8 T cells, macrophages (CD163), B cells (CD20), and tumor cells (panCK). Mass spectrometry imaging and IHC spatially resolved data will be co-registered and metabolite intensity in regions of interest (cell types) quantified. RESULTS/ANTICIPATED RESULTS: A total of seven HPV-associated (three metastatic lymph nodes and four primary tumors) and six carcinogen-driven (primary tumors) HNSC specimens were subjected to MALDI and IHC. Metabolites significantly enriched in HPV-associated HNSC relative to carcinogen-driven HNSC include 2,3-diphosphoglyceric acid, xanthine, 2,3,5-Trichloromaleylacetate, and indole-3-carboxyaldehyde. Metabolites significantly enriched in carcinogen-driven HNSC relative to HPV-associated HNSC include hesperetin 3'-O-sulfate, hypoxanthine, phosphorylcholine, and L-homocysteine sulfonic acid. In ongoing analyses, we anticipate identifying a relationship between CD8+ T cell enriched vs depleted regions and immunosuppressive metabolites (e.g., kynurenine, adenosine monophosphate). DISCUSSION/SIGNIFICANCE: Defining the extent to which CD8+ T cells interact with the metabolic milieu of the microenvironment will provide a foundation for metabolic Precision Medicine. Strategically targeting metabolic pathways to enhance the anti-tumor immune response will be leveraged for the design and implementation of immune modulatory metabolic therapy.

The obesity epidemic has continued to rise at an alarming rate and has increased health complications in children, adolescents, and adults. Resistance training, aerobic training, and a combination of the two have been shown to be effective at reducing excess adiposity and improving outcomes of obesity. The continued development of programs, community centers, or medical exercise facilities prescribing these treatments is nearing an absolute necessity to slow the advancing nature of obesity and related metabolic diseases. In this brief review, we summarize the effectiveness of these three training paradigms from a population-centric perspective.

OBJECTIVES/GOALS: The primary objective of this study was to define the transcriptomes and transcriptional regulatory network required for the development and function of adaptive Natural Killer (NK) cells in donors with latent human cytomegalovirus (HCMV) infection. METHODS/STUDY POPULATION: Eight healthy adult human spleens were obtained from four HCMV seropositive and four HCMV seronegative donors. Spleens were provided by the Versiti Organ Donor Center of Wisconsin and were processed to a single cell suspension. CD7+ CD3E- CD14- CD19- CD20- NK cells were isolated, using the BD FACSAria sorter. Following cell sorting, single-cell RNA sequencing (scRNA-seq) was performed, and cDNA libraries were constructed and sequenced via NextSeq 550. Cell Ranger was then used to algin the cDNA reads and the Seurat R package was used to analyze the transcriptional data. Cells were filtered and clustered based on the number of uniquely expressed genes. The monocle software was used for single cell trajectory analysis and the SCENIC software was used to decipher gene regulatory networks. RESULTS/ANTICIPATED RESULTS: Eight healthy spleens from four HCMV seropositive and four HCMV seronegative donors were obtained and their NK cells were sorted and captured for scRNA-seq. Donor median age was 59 [IQR 48.5-56.5], 50% (n=4) were female and all donors were not experiencing any acute or chronic symptoms. Using scRNA-seq, we observed elevated numbers of NKG2C+ adaptive NK cells in HCMV seropositive individuals when compared to HCMV seronegative individuals. In addition, we identify a set of transcription markers and regulators that are responsible for the development and function of adaptive NKG2C+ NK cells. Finally, our trajectory analysis of adaptive NKG2C+ NK cells revealed a unique developmental pathway. DISCUSSION/SIGNIFICANCE: Here, we demonstrate that HCMV infection can induce the formation of adaptive NKG2C+ NK cells that display a unique transcriptional and developmental profile. These findings have the potential to influence the future application of adaptive NK cells in cellular immunotherapies.

The ACT Network was funded by NIH to provide investigators from across the Clinical and Translational Science Award (CTSA) Consortium the ability to directly query national federated electronic health record (EHR) data for cohort discovery and feasibility assessment of multi-site studies. NIH refunded the program for expanded research application to become “Evolve to Next-Gen ACT” (ENACT). In parallel, the US Food and Drug Administration has been evaluating the use of real-world data (RWD), including EHR data, as sources of real-world evidence (RWE) for its regulatory decisions involving drug and biological products. Using insights from implementation science, six lessons learned from ACT for developing and sustaining RWD/RWE infrastructures and networks across the CTSA Consortium are presented in order to inform ENACT’s development from the outset. Lessons include intentional institutional relationship management, end-user engagement, beta-testing, and customer-driven adaptation. The ENACT team is also conducting customer discovery interviews with CTSA hub and investigators using Innovation-Corps@NCATS (I-Corps™) methodology for biomedical entrepreneurs to uncover unmet RWD needs. Possible ENACT value proposition hypotheses are presented by stage of research. Developing evidence about methods for sustaining academically derived data infrastructures and support can advance the science of translation and support our nation’s RWD/RWE research capacity.

OBJECTIVES/GOALS: Single-cell and spatial transcriptomics have revealed high heterogeneity in the tumor and microenvironment. Identifying populations of cells that impact a patient’s prognosis is an important research goal, so researchers can generate hypotheses and clinicians can provide targeted treatment. METHODS/STUDY POPULATION: DEGAS uses deep-transfer-learning to identify patterns between patient tumor RNA-seq and clinical outcomes and map these associations on to higher-resolution data like spatial and single-cell transcriptomics. We apply DEGAS to prostate and pancreatic cancer spatial transcriptomics samples, as well as one normal sample of prostate tissue. We used the TCGA prostate cancer cohort to with the accompanying survival information and publicly accessible prostate cancer ST data from 10X Genomics to predict survival associations in the ST slides derived from the TCGA patients. Based on these survival associations, we identify higher risk subsections of ST slides which can be further studied. RESULTS/ANTICIPATED RESULTS: We were able to validate our method by comparing it to Scissor and were able to show that the number of high-risk regions in prostate cancer slides increased with the stage of disease. Furthermore, we identify transcriptomic signatures enriched for ontology terms associated with growth regulation and apoptosis, inflammation, immune signaling, and autophagy in histologically normal prostate tissues and adjacent normal pancreatic cancer tissues that were identified as high-risk by DEGAS. The regions highlighted by DEGAS could reflect transcriptional precursors to intraepithelial neoplasia–a well-recognized premalignant morphological change in glandular epithelium. DISCUSSION/SIGNIFICANCE: Identifying biomarkers of tissue stress that precede morphologic diagnosis of high-grade pre-malignant lesions by a pathologist may help triage patients at high risk for future development of cancer, or aid in better understanding whether histologically normal pre-malignant tissues at tumor margins contribute to recurrence.

OBJECTIVES/GOALS: A negative circumferential resection margin (CRM) after surgical resection of rectal cancer decreases local recurrence and increases overall survival. While MRI is used to predict this risk, there is no predictive model that incorporates clinical factors to predict the risk of CRM positivity. METHODS/STUDY POPULATION: Utilizing the National Cancer Database from 2010-2014, we performed a retrospective study evaluating factors predictive for positive CRM after surgical resection of rectal cancer. The primary outcome was positive CRM (tumor≤1 mm from the surgical margin). Our population included patients with clinical stage I-III rectal cancer who underwent total mesorectal excision. For the primary outcome, multivariable logistic models were used to estimate the probability of a positive CRM. Model performance was evaluated by using the area under the receiver operating characteristic curve (AUC). Model calibration was assessed by examining the calibration plot. Bootstrapping method (300-iteration) was used to internally validate and estimate optimism-adjusted measures of discrimination and overall model fit. RESULTS/ANTICIPATED RESULTS: There were 28,790 patients included. 2,245 (7.8%) had positive CRM. Older age, race, larger tumor size, higher tumor grade, mucinous and signet tumor histology, APR, open operative approach, facility location, higher T stage, lymphovascular invasion, lack of neoadjuvant chemotherapy/radiation, and perineural invasion were all significantly associated with positive CRM (p DISCUSSION/SIGNIFICANCE: An objective model that predicts positive CRM and associated poor clinical outcomes is possible to be used in conjunction with MRI. Positive CRM is associated with specific patient demographics, tumor characteristics, and operative approach. These factors can be used to predict CRM positivity in the preoperative period and plan accordingly.

OBJECTIVES/GOALS: Identify and stratify clinical presentations of thrombotic pathology in PCOS patients. This will be accomplished by 1) evaluating clinical assays for the detection of hypofibrinolysis, and 2) analyzing clinical symptomology of thrombosis in PCOS via Symptom-Disease Pair Analysis of Diagnostic Error (SPADE). METHODS/STUDY POPULATION: Preliminary study populations include n=3 for each of the following groups PCOS with thrombotic complications, PCOS without thrombotic complications, healthy controls, and healthy control samples treated ex vivo with PAI-1. Coagulation assays include ACL coagulation panel, antiphospholipid antibody assays, viscoelastic testing with TEG and Quantra devices, and global fibrinolytic capacity. Coagulation assays will be performed on three samples taken at 4-week intervals. The SPADE techniques will be used to evaluate symptomology of thrombosis in the study period and patient electronic medical history. Molecular testing will be performed for pro-thrombotic polymorphisms (PAI-1 and Apo E) and mutations (Factor V Leiden and Prothrombin). RESULTS/ANTICIPATED RESULTS: We anticipate PCOS and ex vivo PAI-1 samples to show signs of hypofibrinolysis on TEG and Quantra devices outside of reference ranges and with statistical significance. We also anticipate seeing a statistical significance with ACL coagulation panels, however, these results are expected to still be within the reference ranges, as seen in previous studies. We believe the SPADE method will identify clinical presentations of hypofibrinolysis in PCOS patients coinciding with patterns in laboratory tests that were misdiagnosed due to being within reference ranges. We hope to stratify clinical presentations predictive of thrombosis in PCOS patients with standard clinical assays and with increased precision using viscoelastic assays. DISCUSSION/SIGNIFICANCE: The incidence rate of thrombosis is 40x higher in PCOS compared to healthy populations. However, the mechanisms of thrombosis in PCOS are unknown and are undetectable with current clinical assays. We hypothesize that chronic cellular stress in PCOS disrupts the regulation of interconnected immune pathways, causing hypofibrinolysis.

Well-being is a multifaceted construct that is used across disciplines to portray a state of wellness, health, and happiness. While aspects of well-being seem universal, how it is depicted in the literature has substantial variation. The aim of this scoping review was to identify conceptual and operational definitions of well-being within the field of occupational health. Broad search terms were used related to well-being and scale/assessment. Inclusion criteria were (1) peer-reviewed articles, (2) published in English, (3) included a measure of well-being in the methods and results section of the article, and (4) empirical paper. The searches resulted in 4394 articles, 3733 articles were excluded by reading the abstract, 661 articles received a full review, and 273 articles were excluded after a full review, leaving 388 articles that met our inclusion criteria and were used to extract well-being assessment information. Many studies did not define well-being or link their conceptual definition to the operational assessment tool being used. There were 158 assessments of well-being represented across studies. Results highlight the lack of a consistent definitions of well-being and standardized measurements.

OBJECTIVES/GOALS: Recent in vitro evidence suggests that diverse parasite protein families called RIFINs and STEVORs are displayed on the surface of infected red blood cells and may have a role in severe malaria, but they remain sparsely studied in natural infections. We measured the RNA expression of these antigens in Malian children with severe or mild malaria illness. METHODS/STUDY POPULATION: We collected blood samples from Malian children aged six months to five years, including 14 with cerebral malaria, 10 with severe malarial anemia, and demographic-matched controls with mild, uncomplicated malaria. We extracted total RNA from each patient and used a custom capture array to selectively enrich Plasmodium falciparum parasite RNA. We then performed Illumina next-generation RNA sequencing and reconstructed parasite transcriptomes using reference-free de novo assembly. We identified RNA encoding RIFINs and STEVORs using an in-house classifier, then measured the diversity and abundance of gene expression for each infection. Expression diversity was defined as the number of unique variants transcribed. Expression abundance was calculated as transcripts per million (TPM). RESULTS/ANTICIPATED RESULTS: Cerebral malaria cases, but not severe malarial anemia cases, had higher diversity and abundance of RIFIN expression compared to mild infections. Type A RIFINs predominated over Type B RIFINs, and the same two RIFINs were predominantly expressed in all disease phenotypes. We anticipate that predominantly expressed RIFINs share high sequence homology with variants previously shown to bind blood antigens or immune inhibitory receptors. STEVOR expression was also higher in cerebral malaria compared to mild malaria, but STEVOR transcripts were sparse overall. DISCUSSION/SIGNIFICANCE: Elevated RIFIN expression in cerebral malaria over mild malaria supports a role for these antigens in pathogenesis. Severe malarial anemia may progress through a different pathogenic mechanism. Predominantly expressed RIFIN variants may be promising targets for vaccines and therapeutics to protect children against cerebral malaria.

OBJECTIVES/GOALS: Dystonia is a brain disorder which causes excessive muscle activation, manifesting as abnormal movements. Neuroimaging studies of dystonia have revealed changes in a network involving the cerebellum. We sought out to determine whether subjects with dystonia in the UK Biobank exhibit MRI-based cerebellar pathology. METHODS/STUDY POPULATION: This nested case-control study drew from the UK Biobank, a cohort of >500,000 subjects in the United Kingdom, aged 40-69, enrolled 2006-2010. Eligible subjects must have undergone diffusion-weighted brain MRI. Dystonia cases were ascertained using ICD10 codes. We selected controls without neurologic diagnoses, matched (1:3) on age, sex, imaging site, and medical comorbidities. Mean diffusivity, fractional anisotropy, intracellular and isotropic volume fractions, and orientation dispersion were extracted from four white-matter tracts (inferior, middle, superior cerebellar peduncles; superior thalamic radiations) along with cerebellum, basal ganglia, and whole-brain grey and white-matter volumes. Means were compared using two-tailed t-tests and the Benjamini-Hochberg procedure (FDR = 0.05). RESULTS/ANTICIPATED RESULTS: 23 cases of dystonia and 69 control subjects were selected and ascertained. After correcting for multiple-comparisons (40 volumetric and 35 diffusion-related), intracellular-volume-fraction (ICVF) of the middle- and superior-cerebellar peduncles was significantly lower in subjects with dystonia, suggesting reduced axon density. Volumetric analysis showed significantly reduced volumes of the motor cerebellum (lobules VI and VIII). There were no differences in basal ganglia, cortical, or whole-brain volumes. DISCUSSION/SIGNIFICANCE: These findings support the hypothesis that abnormalities of cerebellar networks contribute to dystonia. In future we will use similar techniques to assess these tracts in subjects with dystonia who are undergoing Deep Brain Stimulation, with the goal of guiding stimulation targeting, and predicting therapeutic outcomes.

Ongoing professional development is important for collaborative biostatisticians, as it enables them to remain current with the latest advances in statistical methodology and software, refine their analytical skills, and expand their domain knowledge, thereby facilitating their ability to contribute effectively to biomedical research. Although external opportunities for professional development, such as attending conferences and workshops, are widely recognized and valued in the field of biostatistics, there has been comparatively little attention given to internal opportunities for enhancing the skills and knowledge of biostatisticians which can be implemented with lower financial and time investment than external offerings. The purpose of this paper is to offer guidance for ongoing internal professional development activities that can be employed by collaborative biostatistics units in universities and academic medical centers to complement structured curricula and initial training. Specific examples of activities are provided so that collaborative biostatisticians and/or managers of biostatistical units can flexibly combine components to create an appropriately scaled, customized program that meets the needs of themselves or of the unit.

OBJECTIVES/GOALS: Our long-term goal is to understand how both genetic and environmental (GxE) factors contribute to neurodevelopmental disorders (NDDs) so that we may potentially intervene in disease pathogenesis and design therapies to address functional deficiencies. METHODS/STUDY POPULATION: Our studies use a novel GxE model to determine how cephalosporin antibiotic exposure alters the gut microbiome, hippocampal neurogenesis, and behavior in the genetically vulnerable 16p11.2 microdeletion (16pDel) mouse. This mouse models one of the most frequently observed genetic risk variants implicated in NDDs, including ~1% of autism diagnoses. Wildtype and 16pDel littermates were exposed to saline or the cephalosporin, cefdinir, from postnatal days 5-9. We quantified changes in gut microbiota composition using 16S rRNA gene sequencing and utilized immunoblotting, immunohistochemistry, and bulk RNA gene sequencing to assess changes in hippocampal neurogenesis. An additional cohort of saline or cefdinir-exposed mice were subjected to a behavioral battery to assess changes in sociability and anxiety. RESULTS/ANTICIPATED RESULTS: We leveraged the next-generation microbiome bioinformatics platform, Quantitative Insights Into Microbial Ecology 2 (QIIME2) to analyze 16S rRNA gene sequencing datasets of P13 cecal samples from saline- and cefdinir-exposed mice. We found successful perturbations to the gut microbiome following early life cefdinir exposure. Further, we found a robust 50% reduction in hippocampal cyclin E protein in cefdinir-exposed 16pDel male mice, which was replicated in a second independent experiment. This reduction extended to the S-phase cell entry and general stem cell population, quantified by EdU+ and Ki67+ cell numbers, respectively. Lastly, in our first cohort of mice for behavioral studies, we found reduced sociability and increased anxiety-like behaviors in cefdinir-exposed mice. DISCUSSION/SIGNIFICANCE: The findings from this GxE model will provide mechanistic insights into the causes of NDDs; they may inform practice guidelines so as to reduce this environmental exposure; and may suggest interventions like probiotics for those at risk in order to overcome altered gut microbiome composition and restore hippocampal neurogenesis defects.

OBJECTIVES/GOALS: Persons with Parkinson’s disease (PwPD) have set-shifting impairments that may limit motor learning of protective responses to prevent falls. This study compares the effect of blocked versus random practice schedules on acquisition and retention of protective stepping stability in PwPD & age-matched controls. METHODS/STUDY POPULATION: Twenty PwPD & 20 age-matched controls will be randomly assigned to random or blocked practice groups to experience slip and trip-like perturbations which induce stepping responses. Blocked practice is repeating a task (e.g., slip) several times before the next task, while random practice is randomly practicing two or more tasks (e.g., slips & trips). Each subject will wear a safety harness to prevent falls, each fitted with a load cell that indicates when a fall would have occurred. We will assess the anterior-posterior and mediolateral margin of stability at 1st protective step, step length, and in-task fall incidence before, 10 minutes, and two days after practice. We will also compare outcomes between participants in the blocked vs. random practice schedule using two ANOVA. RESULTS/ANTICIPATED RESULTS: To date, we have recruited three control subjects. We anticipate that blocked practice will be superior to random practice for protective stepping acquisition and retention PwPD. Existing literature on motor learning of voluntary tasks indicates that PwPD have difficulty acquiring and retaining motor skills via random practice due to the higher cognitive processing required than blocked practice. Furthermore, since the basal ganglia’s set-shifting role is crucial for random practice, it is expected that random practice will limit protective steps acquisition and retention in PwPD. In healthy control, while blocked practice is better for acquisition, random practice will be superior for retention. DISCUSSION/SIGNIFICANCE: The study’s findings will provide knowledge that may foster the development of robust balance rehabilitation protocols to improve postural responses in PwPD and reduce delayed and ineffective stepping responses that results in falls among PwPD.