OBJECTIVES/GOALS: In this study we sought to determine the role of glucagon-like peptide-1 (GLP-1), one of the main gut hormones in regulating glucose metabolism, after protein ingestion in patients with a history of Roux-en-Y gastric bypass (GB) and sleeve gastrectomy (SG). METHODS/STUDY POPULATION: We examined the glucose and islet-cell secretory responses to 50 g protein ingestion with and without a potent GLP-1 receptor antagonist, exendin-(9-39) [Ex-9], in 10 GB-treated subjects, 9 SG-treated, and 7 non-operated controls (CN). The groups were matched for age, BMI, fat-free mass, fasting glucose and insulin, and HbA1c. The surgical groups also were matched for weight loss and time post-surgery. No subjects had diabetes. RESULTS/ANTICIPATED RESULTS: Protein ingestion resulted in an early rise in glycemia (AUCGlucose1hr) in GB and SG, whereas CN had minimal change in glucose (p<0.05). Protein ingestion enhanced C-peptide responses in all groups, but to a larger extent in GB and SG when compared to CN (p<0.05). Early glucagon response to protein ingestion (AUCGlucagon1hr) tended to be larger in GB and SG subjects when compared to CN (p=0.07). Ex-9 increased premeal and prandial glycemia in all groups (p<0.05), but increase in early glycemia (AUCGlucose1hr) was most notable in GB (p=0.1, interaction). This glycemic effect of Ex-9 was associated with a ~25% reduction in prandial C-peptide secretion in GB and SG and ~8% increase in CN (p<0.05, interaction). Early prandial glucagon responses were larger during studies with Ex-9 compared to those without (p<0.05). DISCUSSION/SIGNIFICANCE: Our findings indicate that glucose metabolism after protein ingestion is altered after GB and SG. To our knowledge, this is the first report to demonstrate that endogenous GLP-1 contributes to glucose and islet-cell secretory response to protein ingestion, and that GB and SG exaggerate GLP-1 contribution to insulin secretion after protein ingestion.

OBJECTIVES/GOALS: Post-traumatic stress disorder (PTSD) develops in a subset of individuals (15-25%) exposed to trauma. We report our preliminary findings investigating underlying peripheral immune responses related to risk and resilience to PTSD. METHODS/STUDY POPULATION: Sprague-Dawley rats (half male) were exposed to predator scent stress in the form of the fox pheromone 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) once for 10 minutes. Seven days later, rats were assessed for persistent anxiety-like behavior using the acoustic startle response task (ASR) and elevated plus maze (EPM). Tail blood was collected for later inflammation analysis at three time points: 1) prior to experiment start, 2) after TMT exposure, and 3) after ASR and EPM. Using a with-in subjects design, our experiment elucidates the connection between PTSD-like symptoms and baseline immune function, inflammatory stress responses, and chronic inflammatory state after stress exposure. RESULTS/ANTICIPATED RESULTS: In comparison to healthy controls, humans with PTSD show elevated blood levels of inflammatory markers. Human studies also show a relationship between baseline immune dysfunction and later PTSD development. In agreement with this literature, we anticipate PTSD-like rats will have increased blood levels of inflammation markers at all three time points compared to resilient and control rats. These findings will back-translate human findings in support of the predator scent stress preclinical model of PTSD. DISCUSSION/SIGNIFICANCE: Our findings will elucidate the temporal aspects of proinflammatory markers and PTSD development. This study will indicate if baseline peripheral immune dysfunction, inflammatory response immediately after stress exposure, and/or chronic inflammatory state predicts ptsd-like behavior in rats.

The Mountain West Clinical Translational Research – Infrastructure Network (MW CTR-IN), established in 2013, is a research network of 13 university partners located among seven Institutional Development Award (IDeA) states targeting health disparities. This is an enormous undertaking because of the size of the infrastructure network (encompassing a third of the US landmass and spanning four time zones in predominantly rural and underserved areas, with populations that have major health disparities issues). In this paper, we apply the barriers, strategies, and metrics to an adapted educational conceptual model by Fink (2013). Applying this model, we used four tailored approaches across this regional infrastructure network to: (1) assess individual faculty specific needs, (2) reach out and engage with faculty, (3) provide customized services to meet the situational needs of faculty, and (4) utilize a “closed communication feedback loop” between Professional Development (PD) core and MW CTR-IN faculty within the context of their home institutional environment. Summary statement results from participating faculty show that these approaches were positive. Grounded in best educational practice approaches, we have an opportunity to refine and build from this sound foundation with implications for future use in other CTR-IN networks and institutions in the IDeA states.

OBJECTIVES/GOALS: Commercial health insurance payers invest in disease management programs (DM) to coordinate care for complex patients. To overcome gaps in connecting patients hospitalized with heart failure to DM, we implemented a novel warm handoff referral between hospital providers and payer DM using the Implementation Research Logic Model (IRLM). METHODS/STUDY POPULATION: A research and quality improvement team collaborated with champions from one hospital and three payers to build and pilot an inpatient-based referral for hospitalized patients with heart failure who were beneficiaries of one of three payers. The standard process of payers initiating contact with patients by phone was restructured to enable inpatient teams to initiate referrals by screening eligible patients prior to discharge. Between August 2020 and October 2021, 285 patients were hospitalized and eligible for screening. Patient registries were built to track patient referral, eligibility, and enrollment status. Monthly stakeholder meetings were used to collect referral rates and review barriers and facilitators related to implementation. RESULTS/ANTICIPATED RESULTS: Of the 63.6% (N=168) patients screened, 31.4% (N=83) were referred, 17.4% (N=46) declined referral, and 14.8% (N=39) were deemed ineligible by payers. Inpatient screenings were challenged by variability across five units with incomplete/missed referrals, primarily attributed to COVID-19-related staff shortages. Payers were challenged by delayed/incomplete referrals and varying access to the hospitals EHRs. Building patient registries helped inpatient champions track eligibility and referral status, and centralizing screening to one champion improved screening rates and reduced incomplete referrals. Additional challenges being addressed include clarifying each payers unique eligibility requirements, refining payers review of referral emails, and creating descriptions of DM for patients. DISCUSSION/SIGNIFICANCE: Implementing inpatient-based DM referrals requires patient and staff engagement, real-time data sharing, and iterative process improvement. Referrals using robust health IT systems could improve patient engagement by connecting payers, providers, and patients; and improve evaluation efforts with real-time process and outcome data.

OBJECTIVES/GOALS: Same-day discharge (SDS) after mastectomy without reconstruction (MwoR) is cost-effective with equivalent complications rates when compared to patients discharged the day after surgery. Despite this, facilities SDS rates after MwoR vary from <10%-80% (20% overall). We aimed to explore barriers to and strategies for implementing SDS after MwoR. METHODS/STUDY POPULATION: We conducted semi-structured interviews with surgeons currently performing MwoR and practicing in the state of Michigan. Recruitment was done through purposeful and snowball sampling methods. Interviews ranged from 40-60 minutes in length. The interview guide was based on the Tailored Implementation of Chronic Disease framework. Interviews were transcribed then analyzed by 3 independent reviewers. A framework matrix was created to identify common themes. RESULTS/ANTICIPATED RESULTS: Participants (n=15) included general surgeons, breast surgeons, surgical oncologists, and both university and non-university affiliated practices were represented. Surgeons either discharged patients the same-day or the day after MwoR. All surgeons who didnt routinely discharge patients the day of surgery (n=9) believed their facility had the resources to implement this practice and it would be safe for most patients. Identified barriers to same-day discharges included provider preoperative expectation setting, patient anxiety about managing their wound drain, and practice standards at their facility. Potential strategies for implementation included incorporating drain teachings with patients before surgery and explaining the benefits of same-day discharge with other providers at your facility. DISCUSSION/SIGNIFICANCE: We identified novel barriers to same-day discharge after MwoR. These barriers are potentially targetable with interventions addressing patient anxiety surrounding drain management, and education of providers across the care continuum.

OBJECTIVES/GOALS: Pancreatic ductal adenocarcinoma (PDAC) is a relatively radioresistant disease, and inhibition of DNA homologous recombination (HR) repair in combination with radiation therapy (RT) is a potentially attractive strategy to overcome radioresistance. We have found that the expression of the HR protein RAD18 is upregulated in PDAC cells. METHODS/STUDY POPULATION: Standard clonogenic assays, γH2aX foci staining, HR-GFP reporter assay, and western blot analysis of DNA damage response proteins were performed in MIA-PaCa2 (MP2) and PANC-1 cells following knockdown of RAD18 in cells via short hairpin RNA (shRNA). Drug targeting of RAD18 was achieved through the use of a USP-7 inhibitor, P5091. Cells with or without stable knockdown of RAD18 were implanted orthotopically in the pancreas of athymic nude mice and treated with sham radiation or radiation to a dose of 20 Gy in 5 daily fractions once tumors reached 100-150 mm3. RESULTS/ANTICIPATED RESULTS: Stable knockdown of RAD18 in MP2 and PANC-1 resulted in decreased radiation clonogenic survival in vitro (dose enhancement factor (DEF)=1.52 and 1.51, respectively), decreased DNA repair after radiation as measured by the increased number of γH2aX nuclear foci assay at 6, 12, and 24 hours (all p<0.05), decreased HR activation following DNA damage via an HR-GFP reporter assay (p=0.039), and increased tumor growth delay following radiation in vivo (p<0.001). P5091 treatment of both MP2 and PANC-1 resulted in efficient knockdown of RAD18, which was confirmed through western blotting, qRT-PCR, and luciferase reporter assays. P5091 increased radiosensitization, yH2aX nuclear foci remained elevated at 12 and 24 hours (p<0.05), and HR repair was also reduced (p=0.014). DISCUSSION/SIGNIFICANCE: Herein, we show the HR repair protein RAD18, and that modulation of RAD18 expression correlates with in vitro and in vivo radiosensitization through altered HR-mediated DNA repair. USP7 inhibition successfully reduced RAD18 expression and resulted in enhanced radiosensitization.

Justice-involved youth (JIY) have high rates of behavioral health disorders, but few can access, much less complete, treatment in the community. Behavioral health treatment completion among JIY is poorly understood, even within treatment studies. Measurement, reporting, and rates of treatment completion vary across studies. This systematic review and meta-analysis synthesizes the literature on rates of treatment completion among JIY enrolled in research studies and identifies potential moderators. After systematically searching 6 electronic databases, data from 13 studies of 20 individual treatment groups were abstracted and coded. A meta-analysis examined individual prevalence estimates of treatment completion in research studies as well as moderator analyses. Prevalence effect sizes revealed high rates of treatment completion (pr = 82.6). However, analysis suggests a high likelihood that publication bias affected the results. Treatment groups that utilized family- or group-based treatment (pr = 87.8) were associated with higher rates of treatment completion compared to treatment groups utilizing individual treatment (pr = 61.1). Findings suggest that it is possible to achieve high rates of treatment completion for JIY, particularly within the context of family- and group-based interventions. However, these findings are limited by concerns about reporting of treatment completion and publication bias.

OBJECTIVES/GOALS: Intraocular pressure is the most significant risk factor for glaucoma. Mechanosensitive proteins may have a critical role in transducing mechanical stimuli that ultimately lead to death of retinal ganglion cells. Our goal is to use genetic and functional approaches to discover mechanosensitive ion channels that mediate the progression of glaucoma. METHODS/STUDY POPULATION: Association data, obtained using a logistic regression model that included age, gender and population substructure as co-variates, for 2,576 SNPs located in the PIEZO1 and PIEZO2 genomic regions were extracted from the NEIGHBORHOOD genome-wide association study results for primary open angle glaucoma (POAG) (3,853 cases and 33,480 controls) and the subset of cases with intraocular pressure (IOP) measurements > 21mmHg (high-tension, HTG) (1868 cases and 33,480 controls). Rare coding PIEZO1 and PIEZO2 variants were evaluated using logistic regression and SNP data from the Human Exome array in 2606 POAG cases and 2606 controls and the subset of 1868 HTG cases and 2606 controls. Immunohistochemistry was used to characterize the expression of Piezo1 and Piezo2 in mouse eye sections. RESULTS/ANTICIPATED RESULTS: Exome data analysis identified two protein-altering variants associated with lower glaucoma risk (P<0.05): a PIEZO1 missense allele (Arg1527His; OR= 0.18, P= 0.001) and a variant disrupting a splice donor site (c.1107+1G>C; OR=0.38, P= 0.02), that prematurely truncates the protein. Investigation of the NEIGHBORHOOD GWAS dataset identified nominal association with common PIEZO2 variants (minor allele frequency > 0.3) in POAG overall (top SNP rs264179, P= 0.008) and in the HTG subgroup (top SNP rs264160, P= 0.001). The associated PIEZO2 SNPs are significantly associated with gene expression in lymphocytes (P< 1x10-8) with the risk allele correlated with decreased gene expression. Piezo1 and Piezo2 are expressed in many ocular tissues in the mouse, including cornea, ciliary body and retina. DISCUSSION/SIGNIFICANCE: We identify rare, protein-altering PIEZO1 variants associated with lower glaucoma risk and show that Piezo1 and Piezo2 are broadly expressed in the eye. Common variants influencing PIEZO2 expression also show nominal association with POAG risk. Inhibition of Piezo1 or augmentation of Piezo2 could be novel therapeutic strategies for glaucoma.

OBJECTIVES/GOALS: This study aimed to investigate the mechanistic effects of fish oil (âμ-3 fatty acids) or evening primrose oil (âμ-6 fatty acids) supplementation on platelet reactivity in postmenopausal women. METHODS/STUDY POPULATION: Postmenopausal women were recruited from the Ann Arbor community and the University of Michigan Medicine Center. All subjects were recruited under study protocols approved by the University of Michigan IRB between November 2015 and March 2017. We conducted a randomized, double-blind, two-period crossover trial, consisting of a 60-day supplementation period followed by a 14-day washout period in between and at the end of the study. Subjects were treated daily in random order with 2g of fish oil supplement and 2g of evening primrose oil. Blood was drawn at baseline, post-supplementation, and after washout. The effects of fatty acid supplementation on platelet aggregation, dense granule secretion and activation of basal integrin âºIIbÎ23 were assessed following supplementation and washout period. RESULTS/ANTICIPATED RESULTS: The study started with 90 postmenopausal women. A total of 78 subjects completed the study, with 12 subjects dropping out due to non-compliance and medical reasons. Supplementation with fish oil attenuated the thrombin receptor PAR4-induced platelet aggregation, whereas primrose oil supplementation attenuated aggregation mediated by PAR4 or collagen. Supplementation with âμ-3 or âμ-6 fatty acids decreased platelet dense granule secretion and attenuated basal levels of integrin âºIIbÎ23 activation. Post-washout following supplementation with primrose oil, the thrombin receptor PAR1-induced platelet aggregation was similarly attenuated. For either treatment, the observed effects post supplementation on dense granule secretion and basal integrin activation were sustained after the washout. DISCUSSION/SIGNIFICANCE: Postmenopausal women are at increased risk for a cardiovascular event due to platelet hyperactivity. This study indicates that supplementation with âμ-3 and âμ-6 fatty acids may offer significant protection for postmenopausal women against cardiovascular diseases and occlusive thrombotic events by reducing platelet reactivity.

OBJECTIVES/GOALS: 5-nitroimidazoles are the only FDA-approved medications for T. vaginalis treatment. Resistance has been observed in 5-10% of cases, but may be rising. We aimed to delineate mechanisms of resistance in isolates of T. vaginalis using transcriptome profiling of resistant and sensitive T. vaginalis isolates. METHODS/STUDY POPULATION: T. vaginalis isolates (4 metronidazole (MTZ)-resistant were grown in triplicate in Diamond’s Trypticase-Yeast-Maltose medium. MTZ susceptibility testing confirmed MTZ MLCs of T. vaginalis isolates. Total RNA extraction was done using Trizol reagent (Invitrogen; Carlsbad; CA); according to the manufacturer’s instructions. RNA sequencing (RNAseq) and bioinformatics analyses were performed to identify significantly differentially expressed genes (DEGs) in MTZ-resistant vs. sensitive isolates. Subsequent qPCR was performed to confirm and extend RNAseq data and gene targets related to 5-nitroimidazole resistance. RESULTS/ANTICIPATED RESULTS: RNAseq identified key DEGs in MTZ-resistant vs. sensitive isolates. DEGs from MTZ-resistant isolates included those involved in metabolic pathways relevant to 5-nitroimidazole resistance such as energy production (glycolytic enzymes) and oxygen-scavenging (thioredoxin). Other DEGs included those encoding transcription factors (MYB DNA-binding protein), ribosomal proteins (30S, 40S, 50S, 60S), protein kinases (CAMK, ser/thr, CMGC), Ankyrin repeat proteins, surface proteins (Surface antigen BspA-like) and various uncharacterized hypothetical proteins. RT-qPCR experiments confirmed reduced expression of genes encoding ferredoxin (drug activation) and flavin reductase 1 (oxygen scavenging) in MTZ-resistant T. vaginalis isolates as compared to MTZ-sensitive isolates. DISCUSSION/SIGNIFICANCE: In this study, we identified several DEGs in resistant T. vaginalis isolates. Further studies with large number of isolates representing a broad range of MTZ-susceptibility patterns are needed to identify genes that may represent new targets for future drug development.

The COVID-19 pandemic has exacerbated health disparities and rendered them acutely more visible. Special and underrepresented populations need to be fully integrated into the translational research process from the very beginning and all the way through. This article presents findings and rapid analysis mini-case studies from the Environmental Scan (E-Scan) of adaptive capacity and preparedness of Clinical and Translational Science Award hubs, specific to the goal of integrating special and vulnerable populations in different institutional research settings. In our discussion of the findings and case studies, we flexibly apply local adaptive capacity framework concepts and characteristics, and, whenever possible, we present ideas on how to enhance capacity in those areas, based on the challenges and practices identified through the E-Scan. Although the past year has recorded incredible achievements in vaccine development, clinical trials, diagnostics, and overall biomedical research, these successes continue to be hampered by our inability to turn them into achievements equally available and accessible to all populations.

OBJECTIVES/GOALS: Evaluate the migration and immune suppressive functions of CCR2+/CX3CR1+ myeloid-derived suppressor cells (MDSCs). Integrate experimental data and biologically relevant mathematical models of infiltrating MDSCs in the context of glioblastoma (GBM). METHODS/STUDY POPULATION: CCR2+/CX3CR1+ cells were enriched from bone marrow obtained from CCR2(+/RFP)/CX3CR1(+/GFP) glioma-bearing mice to evaluate their immune-suppressive phenotype and ability to migrate to CCL2 and CCL7. Fluorescent imaging and quantification were performed on a range of tumor sizes to acquire vasculature, tumor, T cell, and MDSC densities. A system of ordinary differential equations was constructed to represent the temporal dynamics of glioma cells, T cells, and MDSCs within the tumor microenvironment. The Approximate Bayesian Computation method was used to determine probability distributions of important parameters, such as the suppression rate of T cells by MDSCs. RESULTS/ANTICIPATED RESULTS: CCR2+/CX3CR1+ M-MDSCs isolated from the bone marrow of tumor-bearing mice suppress CD8+ T cell proliferation and IFNγ production. CCR2+/CX3CR1+ cells migrate to recombinant and KR158B glioma sourced CCL2 and CCL7. Parameter values determined by the Approximate Bayesian Computation method agreed with parameter values from experimental data. This result further validated the structure and results of the mathematical model when performing computer simulations; thus, we can predict CCR2+/CX3CR1+ M-MDSC infiltration over time. DISCUSSION/SIGNIFICANCE: The immune-suppressive microenvironment in GBM contributes to poor outcomes despite standard of care. This study integrates biological and mathematical models to better understand infiltrating immune-suppressive cells, namely CCR2+/CX3CR1+ M-MDSCs. Future directions include modeling immunotherapies.

The International Study of Comparative Health Effectiveness with Medical and Invasive Approaches (ISCHEMIA) found that there was no statistical difference in cardiovascular events with an initial invasive strategy as compared with an initial conservative strategy of guideline-directed medical therapy for patients with moderate to severe ischemia on noninvasive testing. In this study, we describe the reasons that potentially eligible patients who were screened for participation in the ISCHEMIA trial did not advance to enrollment, the step prior to randomization. Of those who preliminarily met clinical inclusion criteria on screening logs submitted during the enrollment period, over half did not participate due to physician or patient refusal, a potentially modifiable barrier. This analysis highlights the importance of physician equipoise when advising patients about participation in randomized controlled trials.

OBJECTIVES/GOALS: Murine and clinical data suggest that antibody-dependent cellular cytotoxicity (ADCC) is associated with greater protection against disseminated neonatal HSV disease. To quantify the relative transfer of Abs with different functions and targets, we conducted a prospective study of mother-infant term and preterm dyads pre and during COVID-19 METHODS/STUDY POPULATION: Total and HSV lysate, glycoprotein D (gD) and glycoprotein B (gB)-specific IgG, IgG1 and IgG3 as well as HSV neutralizing Abs (nAbs) and ADCC were quantified in paired 3rd-trimester pregnant women and their newborns (cord) blood. Transfer ratios (TR) were defined as cord:maternal Ab levels. IgG1 and IgG3 subclass and gD or gB-specific Abs were isolated by column purification and glycan profiles were assessed by mass spectrometry. The study population included 21 term and 15 preterm dyads who were HSV-1 (+/- HSV-2) seropositive enrolled between 2018-2019 (pre-COVID) and 25 additional HSV-1 (+/- HSV-2) seropositive term dyads whose mothers were SARS-CoV-2 PCR and COVID Ab+ at delivery; 14 were asymptomatic and 11 had mild-moderate COVID disease. None of the mothers had active genital HSV lesions during delivery RESULTS/ANTICIPATED RESULTS: Anti-HSV IgG, IgG1 and IgG3 TR were higher in term vs. preterm dyads (p<0.05). The nAb TR was 2.4 in term vs. 0.8 in preterm (p<0.001) but the ADCC TR was < 1.0 for both. To determine if the latter reflected antigenic target, subclass or glycans, we enriched for gD and gB specific and IgG1 and IgG3 Abs. The gD Abs were IgG1 and had only neutralizing activity. In contrast, gB Abs were polyfunctional and included IgG1 and IgG3 but only the IgG1 Abs had ADCC activity. The gD Abs were enriched for glycans associated with an affinity for the neonatal Fc receptor (FcRn); gB Abs expressed glycans associated with both FcRn and FcγRIIIa binding. There was no significant difference in total HSV-specific IgG TR in pre-COVID vs post-COVID dyads but the nAb TR was lower (p=0.018) and ADCC TR higher (p<0.001) in the COVID compared to pre-COVID cohort DISCUSSION/SIGNIFICANCE: HSV ADCC Abs, which may provide greater protection than nAbs against neonatal disease, transfer poorly particularly to preterm newborns. However, in the setting of SARS-CoV-2, the TR of HSV ADCC is significantly higher. This may reflect alterations in the placental architecture and/or glycan composition which is currently being investigated.