ABSTRACT IMPACT: Characterizing the cellular composition of human adipose tissue may contribute to the prevention and/or treatment of obesity-associated metabolic diseases. OBJECTIVES/GOALS: Our aims in this study were to use single-cell techniques 1. to characterize cell types within the stromavascular fraction of human adipose tissue, 2. to identify subsets of cells within each type (sub-clustering), 3. to identify gene sets and pathways that may provide information on the function and significance of each cell cluster. METHODS/STUDY POPULATION: Abdominal subcutaneous adipose tissue samples from n=6 healthy volunteers (1M, 5F, age 28-38 y, BMI 24.5-63.0 kg/m2) were collected by aspiration or during surgery. In 3 subjects, all females, paired femoral samples were also collected. After collagenase digestion approximately n=10,000 cells/sample were used for single-cell RNA sequencing using the 10X Genomics platform. After QC and downstream analysis, data were analyzed in Seurat v.3.1.5. We identified first different cell types and then subclusters in an unbiased fashion. Gene Set Enrichment Analysis (GSEA) was used for pathway analysis. RESULTS/ANTICIPATED RESULTS: Progenitor markers are related to extracellular matrix, eg DCN (logFC progenitors vs other cells types=3.17, expressed in 99.7% (pct.1) of progenitors, 26.1% (pct.2) of others). Endothelial and pericytes shared markers like RBP7 (logFC=1.67, pct.1 0.88, pct.2 0.39); pericytes also showed unique markers, eg RGS5 (logFC=2.29, pct.1 0.89, pct.2 0.17). Progenitors are further divided into 11 sub-clusters, one of which showed enrichment of CD36 (high proliferation potential), FABP4 (differentiation), and of the novel marker PALMD (logFC=7.13, pct.1 0.94, pct.2 0.48). All p<10E-5. GSEA analysis suggests that inflammatory pathways are downregulated in both adipose progenitors and endothelial/pericyte cells in the femoral compared to the abdominal depot. DISCUSSION/SIGNIFICANCE OF FINDINGS: Single cell RNA sequencing provides unique insights into the molecular profile of cell types and the identification of novel subsets of cells within the human adipose tissue. Such cellular heterogeneity may explain differences in adipose function between individuals and eventually in the risk of obesity-associated metabolic diseases.

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.

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.

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.

ABSTRACT IMPACT: Baseline presentation in AT (higher upper trapezius PPT with no difference at calf or tendon) may suggest a mechanism for persistent symptoms: with more advantageous central pain processing and no tradeoff peripherally, they may choose to continue their usual activities without regard for further damage to the affected tendon. OBJECTIVES/GOALS: Exercise-induced hypoalgesia, a reduction in pain with exercise, is often observed in healthy populations but is not well established in Achilles tendinopathy (AT). The aim was to compare pressure-pain threshold (PPT) at baseline and after fatiguing isometric exercise in AT and healthy controls. METHODS/STUDY POPULATION: 21 participants were recruited for the study: 7 AT (26.5 ±8.8 yrs), 14 control (22.1 ±3.2 yrs). After a familiarization session, participants completed an experimental session that involved performance of intermittent maximal voluntary isometric contractions (MVICs) (2s:2s duty cycle) in a Biodex3 dynamometer (Biodex Medical, Shirley, NY) for 4 minutes. PPT was measured at the medial gastrocnemius (calf), Achilles tendon, and upper trapezius at baseline and immediately following the fatiguing isometric task using a Somedic Algometer (Somedic AB, Sweden). Data are expressed as Mean(SD). Change in PPT is expressed as a percentage of baseline PPT. Units for PPT are kPa. A priori alpha was set to 0.05. RESULTS/ANTICIPATED RESULTS: There was no change in tendon or calf PPT following isometric exercise in AT (tendon: p=0.78; calf: p=0.76), while both increased (i.e., exercise-induced hypoalgesia) in controls (tendon: 9.5(17.8), p=0.03; calf: 21.3(22.7), p<0.01). Neither group experienced a post-exercise change in upper trapezius PPT (AT: p=0.35; control: p=0.37). There was no between-group difference in baseline calf (p=0.14) or tendon (p=0.19) PPT. However, baseline and post-exercise upper trapezius PPT were significantly higher in AT (baseline: 335.6(194.8); post-exercise: 321.2(170.1)) than in controls (baseline: 193.7(75.1), p<0.01; post-exercise: 198.1(79.1), p<0.01). DISCUSSION/SIGNIFICANCE OF FINDINGS: These findings suggest: (1) in persons with AT, central pain processing is altered at baseline, but unaffected in response to isometric fatiguing exercise; and (2) in persons with AT, peripheral pain processing is unaffected at baseline, but is altered in response to this mode and dosage of fatiguing isometric exercise.

ABSTRACT IMPACT: To track recovery and mitigate additional spinal cord injury (avoiding further paralysis), we are assessing the applicability of an implantable ultrasound device that can monitor the tissue health postoperatively. OBJECTIVES/GOALS: To date, no method has been developed that monitors spinal cord perfusion rate (mL/min/g) or pressure (mmHg) successfully after the surgery. Our goal is to design, construct, and validate (in animal models) a novel sensor that quantifies postoperative tissue perfusion in patients with SCI at the site of and downstream from the injury. METHODS/STUDY POPULATION: A sample size of 10 animals will allow us to test our hypothesis to track tissue perfusion before and after the SCI using ultrasound. After prepping and scrubbing the animal, the skin will be incised with a blade, bony structures will be removed and the spinal cord will be revealed. A 25-g weight will then be dropped from a height of 15 cm, and the animal will be observed for contraction of the lower extremities, a sign that the cord was damaged. Using Doppler ultrasound settings available on commercial transducers, we will investigate the acceptable frequency, as well as proper Doppler mode with and without contrast agents, and with and without elastography (stiffness mapping of the tissue). A range of frequencies will be tested (5 -25 MHz). RESULTS/ANTICIPATED RESULTS: It is expected that at frequencies 12 MHz and above, our radiologist collaborators would be able to easily detect the blood flow. It is also expected that the injury will have a noticeable effect on the changes of this detected blood flow. We aim to present figures demonstrating ultrasound image qualities obtained at various frequencies. We expect three such figures: one for gray scale ultrasound imaging, one for color Doppler and finally, one for spectral Doppler, which is the one mostly used to quantify blood flow. DISCUSSION/SIGNIFICANCE OF FINDINGS: To monitor recovery and mitigate secondary injury in patients with traumatic SCI, there is a need to monitor tissue perfusion intra-operatively. To address this need, we will design, construct, and validate a novel sensor that will postoperatively quantify tissue perfusion for the SCI patients at the site of the injury.

ABSTRACT IMPACT: The Bench Tutorials Program is an independent study course in biomedical research in which high school students are paired with graduate and post-doctoral students during the academic year. The purpose is to enhance the rigor of high school science education and build the pipeline of tomorrow’s researchers. OBJECTIVES/GOALS: The Bench Tutorials Program: ο Proficiency in research design, implementation, and presentation; ο Acquisition of hands-on laboratory skills; ο Increase in scientific literacy; ο Increase in analytical skills and critical thinking; ο Career in science; ο Build the pipeline of tomorrow’s biomedical researchers METHODS/STUDY POPULATION: High School seniors are paired with graduate and postdoc mentors through a matching process. Students spend approximately four hours/week in supervised instruction and research from a participating laboratory in addition to classroom experience at their High School. Mentors design research projects relating to the larger research framework of their laboratories. In light of COVID-19, approaches have been adjusted to maintain the program safely through a hybrid method of using the high school lab for hands-on learning and through the use of Go-Pros ’s to enable our mentors to video and narrate as they conduct experiments in their own labs to teach their mentees scientific methods and processes. RESULTS/ANTICIPATED RESULTS: Since inception, more than 400 students and mentors have participated in the Bench Tutorial’s program. This year we found a way to continue the program under COVID-19 restraints without putting anyone in harms way. Go-Pros have been essential for our program to maintain continuity for high school students who receive academic credit for this course. This program is also one of few in which our graduate students have the opportunity to serve as mentors in the scientific setting. Using Go-Pro’s will also enable us to provide teaching videos online for other academic institutions, so even in the absence of COVID-19 in the future, the continued use of these devices will still be of great value. DISCUSSION/SIGNIFICANCE OF FINDINGS: High school students are afforded the ability to work on cutting edge research projects alongside graduate students and postdocs, who are afforded the chance to mentor and teach. Due to the COVID-19 pandemic, we have successfully adjusted our methods for teaching through the use of Go-Pro technology.

Understanding the clinical risk factors for COVID-19 disease severity and outcomes requires a combination of data from electronic health records and patient reports. To facilitate the collection of patient-reported data, as well as accelerate and standardize the collection of data about host factors, we have constructed a COVID-19 survey. This survey is freely available to the scientific community to send electronically for patients to complete online. This patient survey is designed to be comprehensive, yet not overly burdensome, to gather data useful for a range of clinical investigations, and to accommodate a wide variety of implementation settings including at a COVID-19 testing site, at home during infection or after recovery, and/or for individuals while they are hospitalized. A widely adopted standardized survey that can be implemented online with minimal resources can serve as a critical tool for combining and comparing data across studies to improve our understanding of COVID-19 disease.

ABSTRACT IMPACT: This work provides supporting evidence for the development of a novel immunosuppression therapy for transplant patients. OBJECTIVES/GOALS: Our laboratory reported that inhibition of the kinase DNA-PK(cs) in mice delays allogeneic graft rejection in part by mitigating the induction of certain cytokines. We hypothesized that this was due to an inhibition of intracellular signaling programs in T cells and designed studies to identify the mechanism(s) by which this occurs. METHODS/STUDY POPULATION: The immortalized Jurkat T cell line was used to evaluate the effect of the DNA-PK(cs) inhibitor NU7441 on T cell activation by PMA/Ionomycin or PMA/PHA. Mouse primary splenocytes also were used to demonstrate the universality and reproducibility of our observations. Initially, protein mass spectrometry of lysates from untreated and NU7441-treated Jurkat cells identified proteins of interest regulated by DNA-PK(cs) that play a role in T cell activation and cytokine production. CRISPR genome editing was used to validate a potential downstream target of DNA-PK(cs). Western blot, ELISA, and flow cytometry were used to document changes in protein levels with respect to treatments. RESULTS/ANTICIPATED RESULTS: We observed that expression of the transcription factor Egr1 was highly induced after activation but attenuated after treatment with NU7441 in both Jurkat T cells and mouse splenocytes. Phosphorylated serine 301 of Egr1 was identified by mass spectrometry in stimulated cells and fits the kinase consensus sequence for DNA-PK(cs). Both an endogenous CRISPR-generated serine 301 to alanine mutant and expression of a plasmid-based S301A mutant resulted in an unstable form of Egr1 that was barely detectable. In contrast, expression of either a S301 to D or E phospho-mimetic mutant resulted in a stable form of the protein detectable by Western blot. Further evaluation of these mutants and Egr1 phosphorylation is underway to determine the mechanism by which DNA-PK(cs) kinase regulates protein stability. DISCUSSION/SIGNIFICANCE OF FINDINGS: We previously reported a role for DNA-PK(cs) in immunomodulation. We now have evidence that this occurs in part through stabilization of Egr1. We believe this novel finding will lead to uncovering a broader role for DNA-PK(cs) as a mediator of protein stability in T cells and provide support for targeting DNA-PK(cs) in immunosuppression therapy.

ABSTRACT IMPACT: Circadian disruption is known to cause significant human pathology but has not been evaluated in pancreas cancer carcinogenesis; through understanding how disruption of circadian rhythms can lead to pancreas cancer development and spread, preventive and therapeutic strategies can be devised. OBJECTIVES/GOALS: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer due to early spread and poor response to therapy. Identifying factors driving PDAC growth could lead to new therapeutic strategies. Thus, we evaluated the extent to which circadian rhythm disruption, a factor strongly associated with cancer formation, contributes to PDAC pathogenesis. METHODS/STUDY POPULATION: To achieve the objective, we evaluated mice with pancreas lineage Kras-mutation (KC mice), which are predisposed to develop the full spectrum of pancreas cancer precursor lesions (pancreatic intra-epithelial neoplasia or PANIN-1, 2, 3) and PDAC. We subjected KC mice to a light-dark phase shift protocol known to induce circadian disruption (KCCD, n = 18), and another group to standard lighting conditions (KCNC, n = 31), with equal numbers of males and females in each group. The mice were allowed access to food and water ad libitum until sacrifice at age 9 months. Histopathologic evaluation of the pancreas was then performed to assess for pancreatic inflammation, pancreatic precursor lesions (PANIN) and PDAC. Fisher’s Exact Test was used to evaluate differences in incidence. RESULTS/ANTICIPATED RESULTS: As expected, both groups of mice demonstrated 100% incidence of chronic pancreatitis and PANIN-1 (low-grade precursor lesion) at age 9 months. This is consistent with the KC phenotype. However, the KCCD mice demonstrated a significant increase in acute pancreatic inflammation (61.1% vs 19.4%, p = 0.005) compared to KCNC mice. Furthermore, intermediate grade precursor lesions (PANIN-2) were also significantly increase in the KCCD mice (38.9% vs 6.5%, p = 0.006). Incidence of high-grade precursor lesions (PANIN-3, or carcinoma in situ: 22.2% vs 9.7%) and PDAC (27% vs 19%) were also increased, but these were not statistically significant. These results are notable given the established progression from higher grade premalignant PANIN lesions (PANIN-2, PANIN-3) to PDAC. DISCUSSION/SIGNIFICANCE OF FINDINGS: Insight into how circadian disruption leads to increased PANIN-2 formation and increase in acute inflammation may be advantageous for understanding circadian disruption in PDAC carcinogenesis. The circadian clock is present in immune cells and disruption can induce immune dysregulation. This mechanism will be evaluated in follow up studies.

The pervasive problem of irreproducibility of preclinical research represents a substantial threat to the translation of CTSA-generated health interventions. Key stakeholders in the research process have proposed solutions to this challenge to encourage research practices that improve reproducibility. However, these proposals have had minimal impact, because they either 1. take place too late in the research process, 2. focus exclusively on the products of research instead of the processes of research, and/or 3. fail to take into account the driving incentives in the research enterprise. Because so much clinical and translational science is team-based, CTSA hubs have a unique opportunity to leverage Science of Team Science research to implement and support innovative, evidence-based, team-focused, reproducibility-enhancing activities at a project’s start, and across its evolution. Here, we describe the impact of irreproducibility on clinical and translational science, review its origins, and then describe stakeholders’ efforts to impact reproducibility, and why those efforts may not have the desired effect. Based on team-science best practices and principles of scientific integrity, we then propose ways for Translational Teams to build reproducible behaviors. We end with suggestions for how CTSAs can leverage team-based best practices and identify observable behaviors that indicate a culture of reproducible research.

ABSTRACT IMPACT: Pharmacological activation of KATP channels may provide analgesia and attenuate opioid tolerance and withdrawal OBJECTIVES/GOALS: Our long term goal is to develop therapeutics for the treatment of the overuse of opioids. The objective of this application is to test novel KATP channel-targeting prodrugs in rodent models of neuropathic and inflammatory pain in addition to opioid tolerance after chronic morphine administration. METHODS/STUDY POPULATION: In one study, two different measures for chronic pain were implemented in mice. Male and female mice (n=10) were subjected to spinal nerve ligation (SNL) or intraplantar injection of Complete Freund’s Adjuvant (CFA) to induce neuropathic and inflammatory pain, respectively. Administration of KATP channel prodrugs (60ug, it) attenuated mechanical hypersensitivity after SNL or CFA compared to vehicle (saline). In a separate study, changes in mechanical hypersensitivity were tested while mice undergo chronic morphine treatment (15mg/kg, 2x, 5 days) with administration of the prodrugs. Tolerance was measured as the loss of antinociception, and withdrawal is measured ˜24 hours after the final morphine injection. RESULTS/ANTICIPATED RESULTS: Intrathecal administration of either KATP channel prodrugs significantly attenuated mechanical hypersensitivity after SNL and significantly attenuated mechanical hypersensitivity after CFA in mice. We predict that intrathecal administration of these prodrugs will also attenuate morphine tolerance and withdrawal in mice. This hypothesis is based off our previous data indicating non-water soluble KATP channel agonists produce analgesia and attenuate morphine tolerance in mice. DISCUSSION/SIGNIFICANCE OF FINDINGS: Pharmaceutical strategies to utilize KATP channels for therapeutics have been hindered due to the low solubility and low ability to cross the neurovascular unit. Newly developed, water-soluble KATP channel openers could be useful pharmaceutical strategy to reduce chronic pain, opioid tolerance, and withdrawal in human populations.