OBJECTIVES/GOALS: Despite highly effective antiretroviral therapy, people living with HIV (PLWH) experience chronic immune activation and inflammation which may influence the progression of infections such as SARS-CoV-2. Here, we explore the immune response and clinical outcomes in HIV(+) and HIV(-) individuals experiencing acute COVID-19 and long COVID (LC). METHODS/STUDY POPULATION: We performed flow cytometric analyses on peripheral blood mononuclear cells from the following: 1) HIV(-) individuals experiencing acute COVID-19, 2) PLWH experiencing acute COVID-19, and 3) pre-COVID-19 pandemic PLWH. Additionally, we will perform similar analyses for the following: 1) PLWH experiencing LC, 2) PLWH previously infected with SARS-CoV-2 who recovered, 3) pre-COVID-19 pandemic PLWH, and 4) HIV(-) individuals experiencing LC. Flow cytometry panels include surface markers for immune cell populations, activation and exhaustion surface markers (with and without SARS-CoV-2-specific antigen stimulation), and intracellular cytokine staining. We will also analyze how chronic HIV infection and other clinical and demographic factors (e.g., age, CD4 %) impact persistent symptomatic burden. RESULTS/ANTICIPATED RESULTS: Acute COVID-19 results–Overall, PLWH had higher baseline expression of activation markers OX40 and CD137 on CD4+ and CD8+ T cells, along with increased levels of TNFa producing CD8+ T cells. Interestingly, PLWH had increased expression of exhaustion markers PD1 and TIGIT but decreased expression of TIM3 on CD4+ and CD8+ T cells. Additionally, PLWH had decreased levels of IL-2 and IFNg producing CD4+ T cells which suggests functional exhaustion. Long COVID-19 expected results–we hypothesize that the activation and inflammation seen in chronic HIV infection will lead to more immune dysregulation and subsequently worsened symptomatic burden. Additionally, we hypothesize that PLWH may have different frequencies of certain LC manifestations, such as increased rates of neurocognitive impairment. DISCUSSION/SIGNIFICANCE: Our findings suggest that chronic HIV infection influences acute immune response during SARS-CoV-2 infection, and that PLWH have variable expression of exhaustion markers which warrants further study. Additionally, our findings in the LC cohort will aid in characterizing clinical manifestations and immunologic mechanisms of LC in PLWH.

OBJECTIVES/GOALS: We have designed an analogue of the Vitamin E tocotrienols called tocoflexol, which improves their pharmacokinetic limitations to make it an effective radiation medical countermeasure. Our goal is to demonstrate that tocoflexol is an effective radiomitigator in vivo when administered after exposure to lethal doses of total body irradiation. METHODS/STUDY POPULATION: Tocoflexol was designed using computational techniques to improve binding to ATTP, the key transporter that reduces the rate of elimination of tocols. In vitro studies compared the antioxidant and cell uptake properties to conventional tocotrienols. Next, we used a mouse model of lethal total body irradiation to evaluate its radioprotection efficacy (treating before radiation). To determine the optimal administration route for radiomitigation (treating after radiation), we will test oral and subcutaneous dosing. Mouse survival will be monitored for 30 days after irradiation. Sample tissues will be taken to evaluate the ability of tocoflexol to protect key organs from acute radiation syndrome. The bioavailability of tocoflexol will be evaluated in a rodent model. RESULTS/ANTICIPATED RESULTS: Known Results: Results show that tocoflexol has potent antioxidant properties and high cell uptake. When tocoflexol was administered 24 hours before exposure to lethal doses of radiation, tocoflexol-treated mice showed 100% survival. Anticipated Results: Because of its improved bioavailability and pharmacokinetic properties, we expect that tocoflexol will show radiomitigation efficacy when administered 24 hours after radiation, improving survival and protecting key organ systems from acute radiation syndrome. DISCUSSION/SIGNIFICANCE: There is an unmet need for safe and effective radiomitigators that can offer multi-organ protection and be rapidly administered in the event of nuclear emergencies. Demonstration of radiomitigation efficacy will position tocoflexol as a prime candidate to be developed into a nuclear medical countermeasure and stockpiled for emergencies.

OBJECTIVES/GOALS: To identify an electroencephalographic (EEG) signature of SOR in adults with TS METHODS/STUDY POPULATION: We will recruit 60 adults with CTD and 60 sex- and age-matched healthy controls to complete scales assessing severity of SOR (Sensory Gating Inventory, SGI), tics, and psychiatric symptoms. Subjects will then be monitored on dense-array scalp EEG during sequential auditory and tactile sensory gating paradigms, as such paradigms have been shown to correlate with self-report measures of SOR in other populations. Single-trial EEG data will be segmented into 100-ms epochs and spectrally deconvoluted into standard frequency bands (delta, theta, alpha, beta, gamma) for pre-defined regions of interest. We will conduct between-group contrasts (Wilcoxon rank-sum) of band-specific sensory gating indices and within-group correlations (Spearman rank correlations) between sensory gating indices and SGI scores. RESULTS/ANTICIPATED RESULTS: We hypothesize that, relative to controls, adults with CTD exhibit impaired sensory gating and that extent of impairment correlates with severity of SOR. 14 adults with CTD (9 men, 5 women) and 16 controls (10 men, 6 women) have completed the protocol to date. Within this sample, adults with CTD showed significantly reduced sensory gating compared to controls in frontal (CTD median 0.12 dB (interquartile range -0.15–0.70 dB); control -0.37 dB (-0.80–-0.13 dB); p = 0.01) and parietal (CTD 0.17 dB (-0.08–0.50 dB); control -0.20 dB (-0.43–0.10 dB); p = 0.01) gamma band during the 100-200 ms epoch in the tactile paradigm. No significant between-group differences were evident for the auditory paradigm. Among adults with CTD, multiple sensory gating indices significantly correlated with SGI scores. Enrollment continues. DISCUSSION/SIGNIFICANCE: Results aim to clarify the extent of sensory gating impairment in TS and identify a clinical correlate of neurophysiologic dysfunction in the disorder. Such knowledge has direct implications for identification of candidate neurophysiologic biomarkers, an express goal of the National Institutes of Health.

OBJECTIVES/GOALS: To investigate the relationship between abnormal bacterial colonization of the gastrointestinal (GI) tract and systemic abnormalities in innate immunity as it contributes to the pathogenesis of rosacea. METHODS/STUDY POPULATION: This is a prospective observational study of patients with erythematotelangiectatic or papulopustular rosacea. The study participants will undergo urea breath testing for Helicobacter pylori (Hp) and hydrogen-methane breath testing for small intestinal bacterial overgrowth (SIBO). Colonic microbiome analysis will be performed using 16S rRNA sequencing of fecal samples. Further, key pro-inflammatory cytokines will be quantified from serum samples. Markers for rosacea subjects and subgroups will be compared by standard analysis of variance methods where appropriate, and Tukey studentized range tests will be done for specific comparisons. Chi-square tests will be used to assess group differences in categorical data. At least 42 subjects will be studied to provide 80% power atα-0.05. RESULTS/ANTICIPATED RESULTS: We hypothesize that the results of this study will support an observed relationship between abnormal GI bacterial colonization and systemic innate immunity abnormalities in rosacea as determined by three primary endpoints: a significantly greater prevalence of Hp and SIBO in rosacea participants, presence of pro-inflammatory cytokines linked to rosacea pathogenesis including interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, and Granulocyte-macrophage colony-stimulating factor (GM-CSF), and observation of distinct, metabolically active colonic bacterial communities specific to rosacea participants. DISCUSSION/SIGNIFICANCE: By identifying rosacea as a cutaneous manifestation of a more systemic inflammatory disease, the results of this study will have implications for the development of important pharmacological interventions targeting key inflammatory pathways in rosacea pathogenesis.

OBJECTIVES/GOALS: Strong evidence has implicated oxidative stress (OS) as a disease mechanism in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). The study aim was to assess whether a C>T single nucleotide polymorphism (SNP) (rs1800668), which reduces the activity of glutathione peroxidase 1 (GPX1), is associated with brain OS in patients with ME/CFS. METHODS/STUDY POPULATION: Study population: The study enrolled 20 patients with ME/CFS diagnosed according to Canadian Consensus Criteria, and 11 healthy control (HC) subjects. Genotyping: DNA was extracted from whole blood samples, amplified by PCR, and purified. Sanger sequencing was used for genotyping. 1H MRS: Proton magnetic resonance spectroscopy (1H MRS) was used to measure levels of glutathione (GSH)— a primary tissue antioxidant and OS marker— in a 3x3x2 cm3 occipital cortex (OCC) voxel. GSH spectra were recorded in 15 minutes with the standard J-editing technique. The resulting GSH peak area was normalized to tissue water level in the voxel. Statistical Analysis: T-tests were used to compare OCC GSH levels between ME/CFS and HC groups, and between the study’s genotype groups (group 1: CC, group 2: combined TC and TT). RESULTS/ANTICIPATED RESULTS: Clinical characteristics: ME/CFS and HC groups were comparable on age and BMI but not on sex (p = 0.038). Genotype frequencies: Genotype frequencies in the ME/CFS group were 0.55 (CC), 0.25 (TC) and 0.2 (TT); and 0.636 (CC), 0.364 (TC), and 0 (TT) in the HC group. GSH levels: There was a trend-level lower mean OCC GSH in ME/CFS than in HC (0.0015 vs 0.0017; p = 0.076). GSH levels by genotype group interaction: Within the ME/CFS group but not in the combined ME/CFS and HC group or HC group alone, GSH levels were lower in the TC and TT genotypes than in CC genotypes (0.00143 vs 0.00164; p = 0.018). DISCUSSION/SIGNIFICANCE: This study found that the presence of a C>T SNP in GPX1 is associated with lower mean GSH levels and, hence, brain oxidative stress, in ME/CFS patients. If validated in a larger cohort, this finding may support targeted antioxidant therapy based on their genotype as a potentially effective treatment for patients with ME/CFS.

OBJECTIVES/GOALS: Financial toxicity describes the adverse effects of medical expenses on financial security and health related quality of life. Though dementia caregiving carries serious costs, financial toxicity has not been studied in this context. Here we assess the prevalence of financial toxicity in dementia caregiving and its sociodemographic correlates. METHODS/STUDY POPULATION: We utilized the COmprehensive Score for financial Toxicity (COST)— 12-item questionnaire validated to quantify financial toxicity in patients and their caregivers— to conduct a nationally representative survey of 317 US dementia caregivers, oversampling non-Hispanic Black (n = 75) and Hispanic (n = 61) caregivers. Participants were required to be currently providing unpaid care to someone 50 years or older with dementia. Financial toxicity was defined as COST 0 & RESULTS/ANTICIPATED RESULTS: COST scores ranged between 0 and 44, with a survey-weighted mean of 24.57 and standard deviation of 9.8. Weighted analysis revealed 52.7% of American dementia caregivers experience some degree of financial toxicity. Of those who experience financial toxicity, 73.1% are classified as mild, 25.7% as moderate, and 1.2% as severe. Financial toxicity was identified in 69.5% of non-Hispanic Black, 54.1% of Hispanic, and 42.3% of non-Hispanic White caregivers, with non-Hispanic Black caregivers significantly more likely to experience financial toxicity compared to their non-Hispanic White counterparts (p = 0.017). DISCUSSION/SIGNIFICANCE: Most US dementia caregivers experience financial toxicity, though prevalence varies significantly by caregiver race. Discerning the pervasiveness of financial toxicity in this population and significant correlates will inform the development and expedient delivery of resources for patients and families.

OBJECTIVES/GOALS: Evaluate the impact of COVID-19 on oral clefts services including surgical and dental treatments in Puerto Rico. METHODS/STUDY POPULATION: This Observational retrospective cohort study will consider patients 0-21 y/o with CL/P that visited the UPR school of Dental Medicine, Pediatric University Hospital Dr. Antonio Ortiz and ongoing case-control research project Face-Genes. Records to be used are classified as follow: Pandemic (March 15, 2020 to March 15 2022) Pre-pandemic (March 15, 2015 to March 15, 2017) Power analysis (power=0.80 alpha=0.05) will be calculated. Unavailable and incomplete medical records and those that did not attended study clinic during study period will be excluded. Data extraction instrument will be based on previous published study. Descriptive statistics, Chi-square, Odds Ratios at 95% confidence intervals and multiple logistic regression will be estimated. RESULTS/ANTICIPATED RESULTS: We hypothesize that surgical and dental services in Puerto Rico will be adversely impacted because of COVID-19 pandemic. DISCUSSION/SIGNIFICANCE: CL/P are common congenital diseases that require early interdisciplinary attention. Lack of timely care as well as surgery and treatment delays, could be associated with poorer prognosis, increased morbidity and mortality. If there is high risk of dh services during emergency situations, our findings will help to allocate the available resources

OBJECTIVES/GOALS: COVID-19 disproportionately affects patients with prior health conditions and those living at a lower socioeconomic status. Persons living with HIV (PLWH) are infected with SARS-CoV-2 at a higher rate than seronegative patients. Risk factors and incidence of post-COVID-19 comorbidities in PLWH, specifically, are still unknown METHODS/STUDY POPULATION: We will study PLWH enrolled in the Emory Centers for AIDS Research (CFAR) Registry who receive care at the Grady Ponce de Leon Center in Atlanta, Georgia to 1) investigate the incidence of, and 2) identify risk factors that predispose PLWH to post-COVID-19 comorbidities. All PLWH with documented COVID-19 (by positive SARS-CoV-2 PCR or antigen test) between March 1, 2020, and September 30, 2021, with a clinic visit within 12 months will be included. We will identify comorbidities using problem list diagnoses and ICD9/10 codes. With a predicted sample size of 395, we will use a Cox proportional hazards model for time-to-detection of comorbidity, and bivariate and multivariate logistic regression models to identify predictors of incident comorbidity within 12 months of COVID-19. RESULTS/ANTICIPATED RESULTS: o Previous work demonstrated that in PLWH, age and non-AIDS comorbidities, but not HIV-related factors, were associated with hospitalization for COVID-19 in a dose dependent fashion.18 We anticipate that rate of incident comorbidities will be significantly higher in PLWH after COVID-19 compared to PLWH without a history of COVID-19. We also expect that pre-existing comorbidities including obesity and cardiovascular disease, male sex, Black race, and older age are associated with higher incidence of post-COVID-19 comorbidities in PLWH. When stratifying by organ system, we also anticipate that prior comorbidities of an organ system will predispose patients to later complications of that same system. DISCUSSION/SIGNIFICANCE: By understanding the incidence and risk factors associated with developing post-COVID-19 comorbiditieswe can improve guidelines for treatment of groups experiencing the disproportionate impact of co-infection with HIV and SARS-CoV-2.

OBJECTIVES/GOALS: Breast cancer survivors have a high risk for chronic disease and early mortality, especially if their psychological and physical symptoms persist beyond treatment. We compared survivors’and noncancer patient controls’health trajectories. We also examined how their relationship satisfaction—a key health determinant—impacted their health. METHODS/STUDY POPULATION: In this longitudinal study, participants were women who were married/domestic partners with an initial suggestive test of cancer identified at cancer clinics. After follow-up testing, women received either a malignant diagnosis (cancer survivors; n=139, stages 0–IIIC) or benign diagnosis (noncancer patient controls; n=69). Breast cancer survivors completed a baseline visit prior to beginning cancer treatment and two follow-up visits 6 and 18 months after treatment ended (surgery, radiation, or chemotherapy, whichever came last); noncancer patient controls completed visits within a comparable timeframe. At each visit, all women completed self-report questionnaires assessing their relationship satisfaction, insomnia, and depressive symptoms. RESULTS/ANTICIPATED RESULTS: We used mixed models and adjusted for participant age, comorbidities, cancer treatment and stage, BMI, and menopause status. At the pre-treatment visit, cancer survivors reported greater depressive symptoms than noncancer patient controls. Cancer survivors’depressive symptoms also decreased over time and were higher before treatment than at the 6- and 18-month post-treatment visits. Insomnia in cancer survivors, but not noncancer patient controls, decreased over time: insomnia was higher at the pre-treatment and 6-months post-treatment visits relative to the 18-month post-treatment visit. Survivors, but not noncancer patient controls, had lower depressive symptoms and insomnia at visits when they reported higher satisfaction than at visits when they reported lower satisfaction. DISCUSSION/SIGNIFICANCE: Cancer survivors had poorer psychological health than those without cancer before treatment, but survivors’psychological and physical health improved after finishing treatment. Survivors’satisfying relationships predicted better psychological and physical health, demonstrating the notable health benefits of survivors’relationships.

OBJECTIVES/GOALS: Research ethics services are critical to the clinical, research, and educational missions of an academic health science center. Our ethics program aims to develop a culture where investigators are as intellectually engaged in ethical issues of scientific integrity as they are in study design, data collection, and implementation. METHODS/STUDY POPULATION: This descriptive analysis depicts the historical development, from 2010 to 2022, of our research ethics program as an exemplar of ethics integration into the research enterprise of an academic health science center that engages in translational research. In this culture, clinicians, translational researchers and their scientific peers, research participants, and community members become involved in ethics investigation, deliberation, and innovation. RESULTS/ANTICIPATED RESULTS: There are four pillars to our research ethics program: 1) research ethics consultation service, which fosters the development of ethical best practices and standards for the practice of translational research; 2) education, which provides customized training and educational opportunities in research ethics to diverse stakeholders; 3) leadership, through collaboration and partnerships; 4) scholarly engagement, in the pursuit of innovative ethics research and professional development. Through these initiatives we can engage a broad constituency of stakeholders, become an integral component of research oversight, engage as active participants in the research enterprise, and have a critical role in guiding institutional culture. DISCUSSION/SIGNIFICANCE: The integration of our ethics program mirrors the translational science continuum which promotes the multidirectional flow of ideas among ethics consultants, laboratory/clinical scientists, implementation researchers and the community.

OBJECTIVES/GOALS: Buprenorphine (BUP) is used for opioid use disorder during pregnancy but causes neonatal opioid withdrawal syndrome (NOWS). The goal of this study was to determine the contribution of the active metabolite, norbuprenorphine (NorBUP), to the development of NOWS when the parent drug, BUP, is administered during pregnancy. METHODS/STUDY POPULATION: Subcutaneously implanted osmotic minipumps delivered BUP (0, 0.01, 0.1 or 1 mg/kg/day) ± NorBUP (1 mg/kg/day) to pregnant Long-Evans rats from gestation day 9 until after delivery. NOWS was measured between 3 and 12 hours after delivery. Withdrawal was precipitated by an intraperitoneal injection of a mu opioid receptor antagonist naltrexone (NTX; 0, 1 or 10 mg/kg), and movement duration (MD; a validated proxy for NOWS) was measured using Noldus Ethovision. Concentrations of BUP, NorBUP, and their glucuronide conjugates in the brains of neonatal littermates not undergoing withdrawal testing were determined using LC/MS/MS. Two-way ANOVA and multiple linear regression analyses tested for interactions between BUP and NorBUP on MD and related brain concentrations to MD, respectively. RESULTS/ANTICIPATED RESULTS: There was no interaction effect between BUP and NorBUP on MD for either sex or at any dose of naltrexone. In females, but not males, BUP (1 mg/kg/day) significantly increased NorBUP-induced MD by 58% following an injection with 1 mg/kg NTX. A multiple linear regression model that included BUP and NorBUP brain concentrations as predictors of MD was significant and well-fitting [FEMALES: F (2, 40) = 23.97, P < .0001, adj R2 = 0.52; MALES: F (2, 40) = 5.84, P = .0059, adj R2 = 0.19]. There was a differential contribution of NorBUP brain concentrations to MD based on sex. The partial regression coefficient for NorBUP was 51.34 (p < .0001) for females and 19.21 (p = 0.093) for males. The partial regression coefficient for BUP was similar for females and males (FEMALES:βBUP = 10.62, p = .0017; MALES:βBUP = 11.38, p = .009). DISCUSSION/SIGNIFICANCE: We show for the first time a differential contribution of NorBUP to BUP-associated NOWS in each sex, suggesting sex differences in NorBUP susceptibility and implicating that treatment strategies reducing prenatal NorBUP exposure may be more effective for females than males.

OBJECTIVES/GOALS: Our goal is to explore the extent to which organoids can serve as models for the protective mechanisms of the stomach–the mucus barrier and the pH gradient across it. We aim to first optimize and validate an organoid-based model of the gastric mucus layer, and then define the cellular mechanisms by which the gastric pH gradient is maintained across it. METHODS/STUDY POPULATION: We have developed a method for the in vitro engineering of gastric mucus by growing epithelial cells at the air-liquid interface (ALI). We use microrheology with fluorescent microspheres to define and compare the biophysical and viscoelastic properties of our lab-grown mucus to those of native mucus. We will perform CryoFE-SEM to compare the internal heterogeneity of our lab-grown mucus to fresh mucus obtained from patient tissue. For our mechanistic studies, we will use a pH-sensitive dye (methyl red) to assess the ability of our lab-grown mucus to maintain an artificial pH gradient in a microfluidic device. Next, we will use a pH microelectrode to measure proton flux through our mucus in vitro, investigating the potential for a physiological gradient in both 2D and 3D organoid models. RESULTS/ANTICIPATED RESULTS: Here we show that gastric organoids and their corresponding epithelial monolayers produce a mucus gel that does indeed mimic in vivo functions. Immunohistochemical staining, electron microscopy, microrheology, and particle tracking analyses revealed that our gastric organoid mucus is viscoelastic and structurally heterogeneous–both properties that are crucial to the stomach’s mucosal first line of defense. Mechanically similar mucus was also engineered using two-dimensional air-liquid interface cultures of the same epithelia. Lastly, live confocal imaging revealed that H. pylori motility–an important virulence factor–was drastically hindered by our lab- grown mucus. DISCUSSION/SIGNIFICANCE: We describe a novel method for the in vitro engineering of gastric mucus and highlight biophysical properties that contribute to our stomach’s defense against pathogens. This work will lead to an improved understanding of gastric physiology and may contribute to the development of novel drug delivery systems to tackle diseases of the gastric mucosa.

OBJECTIVES/GOALS: 30,000,000 people in the U.S. have hearing loss, negatively impacting quality of life and work. Understanding auditory axon guidance for spiral ganglia neurons (SGNs) will aid development of new therapies. I study role of Eph/Ephrin signaling in mediating type II SGN turning events, and how planar cell polarity (PCP) signaling modulates this process. METHODS/STUDY POPULATION: This quantitative study was conducted on Efna3 and Vangl2 null mice possessing Neurog1CreERT2 and R26RtdTomato mutations. Spontaneous Cre activity within the Neurogenin 1 CreERT2 line causes recombination and expression of fluorescent Rosa26 Reporter (R26R)tdTomato in a restricted number of SGNs, including type IIs. Together these lines permit SGN sparse labeling. Bulk-labeling was used for Efna3;Vangl2 double knockout (DKO) mutants. Immunostaining and confocal imaging was used to analyze dsRed in Efna3; Vangl2 and NF-200 in DKOs to quantify type II SGN turning. In combination, 3D rendering in Imaris software was used to quantify type II SGN turning, branching and other growth and navigation characteristics. 5-6 cochleae per genotype were analyzed and compared by t-test to wildtype controls. RESULTS/ANTICIPATED RESULTS: EPHRIN-A3 is expressed on the membranes of outer pillar and Deiters’cells of the cochlear epithelium. Efna3 nulls showed a small rise in type II SGNs incorrectly turning toward the apex at an error frequency of 16.9% compared to controls (n=6; p=0.05). Efna3 nulls had reduced branch number/fiber compared to controls, 4.14 and 7.22, respectively (n=129; p DISCUSSION/SIGNIFICANCE: Our results suggest that Eph/Ephrin signaling acts parallel of PCP signaling to mediate type II SGN guidance during development. The clinical implications of these findings are that therapeutics targeting EPHRIN-A3 and/or VANGL2 in this pathway could stimulate new outer hair cell innervation by type II SGNs following auditory damage.

OBJECTIVES/GOALS: The goals of this project are to: i) investigate the cargo such as immune mediators (cytokines) and small non coding RNAs (sncRNAs) of EVs derived from nasopharyngeal secretions (NPS) of children with episodes of viral infections and exposed to SHTS; ii) test the biological activity of EVs released from upper airway mucosa on target/recipient lung cells METHODS/STUDY POPULATION: EVs were isolated from ten NPS samples of children with episodes of acute respiratory infections. EVs were characterized by particle sizing (size and concentration), EV markers, and protein arrays for interferons, cytokines, and other immune mediators content. RNA was extracted from ten samples of NPS-derived EVs by column for next generation high throughput sequencing (NGS) to identify sncRNAs in EVs. In studies currently in progress, EVs will be isolated from RSV-infected human nose organoids (HNOs) cells in air liquid interface (ALI) culture, with or without pre-exposure to tobacco smoke. EVs will be then tested for antiviral activity on recipient RSV-infected lower airway cells. Viral titers will be measured in recipient infected lung cells. RESULTS/ANTICIPATED RESULTS: We isolated EVs from NPS samples and confirmed by immunoblot EV markers CD63 and Alix. The average size of NPS-derived EVs of virus positive and negative patients was 170 nm and 145 nm, respectively. We determined the particles number of EVs, concentrations of IFN-βand IFN-λin NPS and NPS-derived EVs of these children. While IFN-βlevels were below the limit of detection in both NPS and NPS-derived EVs of all children, IFN-λwas detected in NPS and NPS-derived EVs from infected patients, except for the two patients with no viral infections. We extracted RNA from control-, virus infected- and/or SHTS- EVs and found that piR-36511, piR-40926, piR-49645, piR-32679 and piR- 53263 were all significantly enriched in EVs derived from NPS of children exposed to TS compared to those not exposed. DISCUSSION/SIGNIFICANCE: RSV leads to approximately 22,000 hospitalizations of children due to second-hand smoke. A vaccine is not currently available for RSV infection. EVs represent a novel translational approach to target undruggable. Airway mucosa EVs in viral respiratory infection function as antiviral messengers and tobacco smoke impairs the EV antiviral activity.

OBJECTIVES/GOALS: The COVID-19 pandemic has impacted nursing frontline professionals. The aims of this study were to explore experiences of nursing professionals in Puerto Rico during the pandemic, examine the impact on their health and provide research development opportunities enhance research capacity. METHODS/STUDY POPULATION: This interpretative phenomenological study recruited graduate nurses who participated in one in-depth semi-structured virtual interviews. Interviews were audio recorded and transcribed. The data analysis process was guided using the following steps: 1. Reading and re-reading, 2. Initial noting, 3. Developing emergent themes, 4. Searching for connections across emergent themes, 5. Moving to the next case, 6. Looking for patterns across cases, and 7. Writing up. In addition, Van Manen's thematic structure of four foundations was used as a complement to guide reflection and interpretation. Faculty and students participated throughout the process. RESULTS/ANTICIPATED RESULTS: Seven nursing professionals’lived experiences caring for Covid-19 patients were gathered. Their ages ranged from 31 to 45 and had worked between 2 and 14 years providing direct care. Themes that emerged from narrations include compassion fatigue, teamwork, working beyond clinical role, and gratification. Nurses expressed dealing with a very difficult situation, fear of being infected , or infecting my family , and working together to get through it and better help patients . Nurses also expressed feelings of anxiety and lack of institutional support. Additionally, the impact of working with patients, feeling good for being there, good or bad and support from families. DISCUSSION/SIGNIFICANCE: Nurses’narrations point to the complexities of their experiences working during the pandemic. They had to transcend usual demands even though they often lacked needed support. We must recognize the value of nursing and reflect upon changes in healthcare that are essential to move nursing forward.

OBJECTIVES/GOALS: The need for mechanisms of ethical discourse and guidance has increased as translational research collaborations become more complex. The goal of this project is to analyze the stakeholder engagement and ethical issues our research ethics consultation service (RECS) conducted over a two year period. METHODS/STUDY POPULATION: We conducted a retrospective review of our RECS database from 2020 to 2022. We examined the nature of the research and ethical issues of concern from consult requestors, including whether or not consults were preventative. In addition, we assessed the educational outreach conducted during that timeframe as a measure of service awareness. RESULTS/ANTICIPATED RESULTS: There was a total of 42 consults conducted over the previous year. There were a wide variety of issues related to informed IRB-related processes (31%), consent (24%), QA/QI determination (12%), authorship (10%), confidentiality (7%), diversity/inclusion (7%), grant preparation (7%). Many of the consults (n=28, 67%) included secondary issues. A few consults (n=4, 10%) were preventative, meaning that the consult was requested in anticipation or consideration of a potential ethical issue. Outreach efforts extended to a diverse array of institutional stakeholders and trainees. DISCUSSION/SIGNIFICANCE: The RECS serves numerous constituencies throughout our institution on ethical issues spanning nearly all aspects of research design, conduct, and analysis. These data highlight initiatives to increase study efficiency (in collaboration with institutional research oversight) and helps to direct educational efforts and outreach.

OBJECTIVES/GOALS: Phagocytes, diverse cells that ingest material, are the primary cell type infected by Mycobacterium tuberculosis (Mtb) and the executors of protective mechanisms. T cells play a critical role by helping phagocytes control the infection. Understanding the precise T cell-dependent mechanisms by which phagocytic cell types contain Mtb is critical. METHODS/STUDY POPULATION: To determine the impact T cells have on different phagocyte cell populations’ host defense mechanisms, groups of wild–type and T cell deficient TCRa-/- mice were infected with an Mtb strain expressing fluorescent mScarlet protein. At four weeks post-infection, a time when T cell help contributes to control of Mtb, lungs were homogenized and cells sorted based on detection of mScarlet, indicating Mtb-infected cells. Cell suspensions from each mouse background were underwent single-cell RNA sequencing analysis to reveal the heterogenous cellular transcriptional response of different phagocyte populations. RESULTS/ANTICIPATED RESULTS: We found that Mtb-infected phagocytes from wild-type and TCRa-/- mouse lungs contain the same dominant cell phenotypic clusters, but these have different patterns of gene expression. Without T cells, phagocytes are prone to a more inflammatory phenotype. DISCUSSION/SIGNIFICANCE: This will translate fundamental biological data to test the hypothesis that Mtb encounters different environmental stresses exerted by different phagocytic cell types. This work could reveal host intracellular niches that enable bacterial persistence and elucidate new pathways that could be targeted for traditional antibiotic therapies for TB.

OBJECTIVES/GOALS: Evidence suggests that individuals with generalized joint hypermobility (GJH), or excessive joint range of motion, are at higher risk of developing chronic neck pain. The objective of this study is to determine the prevalence and clinical presentation of chronic neck pain in GJH and investigate its associations with other measures of spine health. METHODS/STUDY POPULATION: Data was collected at the Driven to Discover Research Facility at the 2022 Minnesota State Fair. Individuals 18 years and older were invited to participate. All enrolled participants completed Phase 1, which included: the Beighton Score (measure of GJH), the 5-Point-Questionnaire (self-report survey for current or historical GJH), and a custom self-report survey for demographics and musculoskeletal pain. A subset of participants was also asked to complete Phase 2 of the study. Phase 2 consisted of additional self-report surveys (Neck Disability Index (NDI) and PROMIS-10 Global) and the following physical measures: neck range of motion in all planes, neck strength in flexion-extension and lateral bending, and grip strength. RESULTS/ANTICIPATED RESULTS: A total of 559 participants were enrolled in the study. All participants completed Phase 1, and 285 of those individuals completed Phase 2. Those with a Beighton Score≥4 were categorized as having GJH. The overall prevalence of GJH was 23.8% for females and 9.1% for males. Consistent with previous studies, multiple linear regression analysis (R2=0.20, F(2,552) = 69.37, p DISCUSSION/SIGNIFICANCE: This is one of the largest studies investigating GJH, pain, and physical measures of neck function in the general population. The results highlight the higher prevalence of chronic neck pain in those with GJH and will form the basis for a subsequent study to identify mechanisms and potential therapeutic targets for individuals with GJH and chronic pain.

OBJECTIVES/GOALS: The objective of this study was to determine whether residential radon and proximity to horizontal oil and drilling (fracking) are risk factors for the development of multicentric lymphoma in pet dogs, a spontaneous, immunocompetent model for non-occupational risk for NHL in humans. METHODS/STUDY POPULATION: Two case-control populations of dogs with multicentric lymphoma were utilized, with a focus on two dog breeds at high risk for lymphoma. Control dogs were matched for age, breed, and sex. Home addresses were collected for 54 Golden retrievers with lymphoma and 108 Golden retriever controls, and for 56 boxer dogs with lymphoma and 84 unaffected boxer controls. Counties of residence were matched to radon zones and percentage of home radon tests that exceeded the actionable level of 4 pCi/L, available by county through the EPA and the CDC National Environmental Public Health Tracking Network from 2008 to 2017. Locations of horizontal oil and gas wells were obtained from the Enverus Database, and distances from dog homes to the closest well, and well density by county, were calculated for each case and control. RESULTS/ANTICIPATED RESULTS: We found no significant differences in radon zones, county level radon measurements, or residential proximity to active fracking wells between dogs with lymphoma and unaffected controls in either the Golden Retriever or boxer populations. DISCUSSION/SIGNIFICANCE: Canine multicentric lymphoma resembles human NHL and is a valuable model of non-occupational environmental risk for NHL in people. Although we did not find geographic associations between radon and fracking wells, follow-up studies will measure household radon, as well as household air, water, and dog urine for potentially genotoxic chemicals.

OBJECTIVES/GOALS: Our goal is to determine the structure of the CACHE domain of the Vibrio cholerae quorum sensing receptor CqsR as well as its autoinducer (AI). We are performing X-ray crystallography on the protein in its apo form, with the fractions containing the AI, and with known ligand ethanolamine (ETA). METHODS/STUDY POPULATION: We have transformed BL21(DE3) E. coli cells with a pTB146 vector to contain the gene for the CqsR CACHE domain. We grow these cells to high optical density and induce protein expression, at which point we harvest them and purify the protein. This entails lysing the cells, separating the protein with Ni-NTA resin, cleaving our protein tag, and column chromatography. With purified protein, high-throughput screens are set up to find crystallization conditions of apo CqsR, CqsR-ETA, and CqsR-AI. We then determine conditions that best lead to crystal formation and optimize them. Crystals are then diffracted with X-rays, process the data, and determine the structure of protein and AI. RESULTS/ANTICIPATED RESULTS: We anticipate finding the structure of the CqsR CACHE domain to a high resolution in addition to the identity of its autoinducer. Previous results found that the structure is homologous to another V. cholerae chemoreceptor, Mlp37, and we expect the results from this project to confirm this. In addition, we know that the autoinducer weighs approximately 62 daltons, the same as the known ligand, ethanolamine. Given that CACHE domains bind specifically to their ligands, we anticipate that the autoinducer will be structurally similar to ethanolamine. DISCUSSION/SIGNIFICANCE: The results will reveal the structure of the CqsR CACHE domain and its autoinducer. This knowledge will better allow researchers to treat cholera, as both autoinducer identity and receptor conformational changes will be uncovered, allowing for drug development to inhibit cell growth.