OBJECTIVES/GOALS: This study examines psychological resilience in adults living with Sickle Cell Disease (SCD) in the U.S. aiming to explore how psychological resilience is defined by this community. METHODS/STUDY POPULATION: Participants were recruited between 2014 – 2018, from across the U.S. as part of an ongoing cross-sectional study: Insights into Microbiome and Environmental Contributions to Sickle Cell Disease and Leg Ulcers Study (INSIGHTS). Inclusion criteria included age of 18 or older, with a clinical history of SCD, and were interviewed if they completed the Brief Resilience Scale (BRS) as part of INSIGHTS. 150 study participants were separated by their BRS scores into “High” and “Low” scoring quartiles. 30 participants were randomly selected, 15 from the lowest quartile and 15 from the highest. All participants completed the Connor Davidson Resilience (CD) measure at the end of their qualitative interview. All identified as Black with an average age of 42.5 (13 F, 17 M). RESULTS/ANTICIPATED RESULTS: Three main concepts emerged within both groups in response to the question “How do you define resilience?” (a) not giving up (b) how one deals with challenges and (c) moving forward. DISCUSSION/SIGNIFICANCE: Our analysis shows that the BRS may not be a precise or accurate indicator of the resilience of adults living with SCD. Therefore, it remains to be seen if these measures are descriptive of these individuals true psychological or physiological state as they have not been used in this community until now.

OBJECTIVES/GOALS: Neonatal hypoxic-ischemic encephalopathy (HIE) is an acute neurologic syndrome where decreased blood flow and oxygen to the brain causes acute and chronic brain dysfunction. The only proven neuroprotective intervention for HIE is hypothermia treatment started within 6 hours of birth and 50% of survivors have long-term deficits. METHODS/STUDY POPULATION: Pre-clinical adult stroke studies demonstrated that vagus nerve stimulation (VNS) has anti-inflammatory effects and attenuates brain damage. Transcutaneous auricular VNS (taVNS) is safe and feasible in infants and may improve the motor skill of bottle feeding. We hypothesize that a combined hypothermia-taVNS treatment shortly after HIE birth will have neuroprotective effects, improve motor function, attenuate infarct volume inflammation compared to hypothermia alone. The HIE model includes ligation of the right common carotid artery in postnatal day 7 (P7) rats followed by 90min hypoxia (8% oxygen) and 2hr hypothermia. taVNS or sham taVNS was administered using a bipolar electrode placed on the auricular concha region for 30min, [30sec trains, 0.5msec duration, 20Hz frequency, followed by 4.5min breaks] RESULTS/ANTICIPATED RESULTS: Experimental groups include +HIE/+taVNS, +HIE/-taVNS, and -HIE/-taVNS. To assess motor function, grasping reflex and forelimb grip strength tasks were assessed prior to surgery through P10. Infarct volume was assessed at 72h after injury by staining coronal sections with cresyl-violet. Thirty-four rat pups underwent surgery with an 8.82% mortality rate. taVNS was well tolerated by the P7 rats when delivered below perceptual threshold (0.4-1.1mA). There was no difference in elementary motor function or infarct volume between any group. DISCUSSION/SIGNIFICANCE: Future studies will include 2.5hr hypoxia for a more severe brain injury and a -HIE/+taVNS control group. These initial pre-clinical studies in neonates are important in determining whether taVNS may translate as a treatment to improve outcomes after neonatal HIE.

OBJECTIVES/GOALS: While anti-seizure medications are effective, nearly one third of patients have seizures that go untreated. Prior studies using evoked seizure models have shown that activation of the Superior Colliculus (SC) display anti-seizure effects. Here we monitored and modulated the DLSC to suppress spontaneous seizures in a genetic model of epilepsy. METHODS/STUDY POPULATION: WAG/Rij rats (4 months old) were employed as study subjects. Animals were surgically prepared for virus injection (ChR2 excitatory opsin, or control vector), fiber optic implantation and cortical EEG for optogenetic studies. For In vivo electrophysiology, animals were implanted with a 16 wire multi-electrode array into the DLSC. In optogenetic experiments, we compared the efficacy of continuous neuromodulation to that of on-demand neuromodulation (real time detection of seizures) paradigms on a within-subject basis. We compared three stimulation frequencies on a within-subject basis (5, 20, 100 Hz). We quantified the number and duration of each spike wave discharge (SWD) during each two-hour-long trial. Electrode array single units were sorted and analyzed for activity before, during and after seizures. RESULTS/ANTICIPATED RESULTS: In vivo electrophysiology found there to be a significant decrease in single unit activity leading up to the start of SWDs. Interestingly, on-demand neuromodulation was effective in both females and males – where the greatest reduction in seizure duration was under 100 Hz light delivery. As expected, male and female animals injected with a control vector did not show a reduction in seizures in response to light delivery. In the open-loop (continuous) stimulation paradigm, optogenetic activation of the DLSC was without effect on the number or duration of SWDs at any of the frequencies examined. DISCUSSION/SIGNIFICANCE: SC activity is significantly decreased prior to the start of seizures. Furthermore, activation of the SC displays anti-seizure effects in a model of spontaneous seizures. A striking difference between open and closed-loop neuromodulation approaches underscores the importance of stimulation paradigm in determining therapeutic effect.

OBJECTIVES/GOALS: We have shown that glioblastoma upregulates blood-brain barrier drug efflux transporters via a mechanism that likely involves TNFα and PI3K/Akt. Our goal is to repurpose FDA-approved PI3K/Akt inhibitors to increase anticancer drug brain concentrations, which holds the potential for translation into the neuro-oncology clinic. METHODS/STUDY POPULATION: GL261 Red-FLuc and MBR525-1 Red-FLuc cells (2μl; 2.5K cells/μl; 1μl/min) were injected into the right hemisphere of 8-week old female J:NU mice (coordinates relative to bregma: AP -2 mm, ML -2 mm, DV -3 mm). Tumor burden was assessed weekly with IVIS® Spectrum in vivo imaging; tumor volume and invasiveness were measured by MRI and histopathology, respectively. On day 14 post-injection, mice received 5-ALA (200 mg/kg ip), and tumors were resected with a 2 mm punch biopsy tool and surgical fluorescence microscope (ex/em: 405/635nm). Drug efflux transporter expression and activity in isolated brain capillaries were determined by Western blot and substrate fluorescence assays, respectively. Cytotoxicity was assessed after 48-hour drug incubation using CyQuant MTT Cell Proliferation Assay kits. RESULTS/ANTICIPATED RESULTS: IC50 values of temozolomide, lapatinib, alpelisib, and miltefosine were N/A, 32, 20, and 190 μM for GL261 Red-FLuc cells and N/A, 49, 36, and 148 μM for MBR525-1 Red-FLuc cells, respectively. Median survival of GL261 Red-FLuc mice was 26.5d and significantly increased to 34d with resection (p=0.116). In GBM mice, drug efflux transporter expression and activity levels in brain capillaries isolated from the contralateral hemisphere were significantly upregulated compared to sham controls. Furthermore, treatment with FDA-approved PI3K/Akt inhibitors, alpelisib and miltefosine, significantly reduced drug efflux transporter expression and activity to control levels. In PK and survival studies, we expect that PI3K/Akt inhibition will increase brain uptake of anticancer drugs and prolong GBM mouse survival. DISCUSSION/SIGNIFICANCE: We have previously shown that PI3K/Akt inhibition reduces P-gp/BCRP levels in brain capillaries. Here, we vertically extend this strategy by repurposing the FDA-approved PI3K/Akt inhibitors alpelisib/miltefosine to improve brain uptake of anticancer drugs in GBM resection models.

OBJECTIVES/GOALS: Studies have shown that SARS-CoV-2 specific memory B cells can be maintained at least a year after exposure. However, reports show an altered B cell response during infection in severe COVID-19 cases. This study aims to describe the B cell response during COVID-19 convalescence with a focus on signatures that contribute to durable and robust immunity. METHODS/STUDY POPULATION: Our study cohort consisted of individuals who had recovered from non-severe (hospitalized) or severe (hospitalized and requiring invasive mechanical ventilation) COVID-19. In our comparative analysis, samples from both groups were carefully matched to fall within 4-5 weeks post-symptom onset. We also performed a longitudinal analysis of non-severe patients with sampling ending 5 months post-symptom onset. Using high parameter flow cytometry, we characterized the phenotype of memory B cells using 19 distinct cell markers and fluorescently labeled probes to identify B cells reactive with SARS-CoV-2 spike and receptor-binding domain protein. Additionally, serum collected from individuals was used to quantify antibody titers. RESULTS/ANTICIPATED RESULTS: The frequency of spike-specific B cells and serum antibody titers were similar between severe and non-severe groups. However, we observed that individuals recovered from severe COVID-19 have a significantly reduced frequency of spike specific IgG+ memory B cells expressing Tbet and FcRL5 (markers associated with long lived immunity). In the non-severe patients, we observed IgG+Tbet+ B cells targeting the spike protein peak at 2-3 weeks post-symptom onset, decrease by almost fifty percent 4-5 weeks post-symptom onset, and return to baseline 5 months post-symptom onset. Our study also validated previous findings of a short-lived primary response of IgM+ B cells targeting the spike protein. DISCUSSION/SIGNIFICANCE: Our findings highlight potential implications for long-term immunity against re-infection or severity of the resulting disease in patients with severe COVID-19. Further investigation will be necessary to determine whether the maintenance of immunological protection is hindered in patients who overcame severe COVID-19.

OBJECTIVES/GOALS: The goal of this study is to determine the role of the tRNA modifications in the translation of Hfq. Hfq is an RNA chaperone that acts as a co-factor for the action of the largest class of small RNAs in E. coli. RNA modifications have been known to play critical roles in the translational fidelity of many cellular proteins in bacteria. METHODS/STUDY POPULATION: In this study, we used an hfq-lacZ translation fusion to screen several RNA modification mutant genes to uncover additional RNA modifications that may play a role in Hfq translation. We measured hfq-lacZ activity in genetic backgrounds mutated for several additional RNA modification enzymes previously untested for Hfq effects. RESULTS/ANTICIPATED RESULTS: We identified 5 RNA modification genes that were defective for hfq-lacZ fusion activity, and we subsequently performed western blot analysis on the Hfq protein in the absence of these modification mutant genes to determine the effect of these mutants more directly on Hfq protein levels. We identified 2 out of these 5 RNA modification mutants that also affect Hfq protein levels. DISCUSSION/SIGNIFICANCE: Since Hfq is critically important for small RNA function is a wide range of bacteria, it is possible tRNA modifications regulate Hfq expression in other bacteria. These processes, when further investigated, could provide us with the basic information to develop new antibiotics needed to address emerging antibiotic resistance.

OBJECTIVES/GOALS: Inflammatory Breast Cancer (IBC) is the most aggressive form of breast cancer and does not have targeted therapy. GPR30, a 7-transmembrane estrogen receptor, may play a role in regulating cell growth and proliferation of cancerous cells. Here, we evaluated changes in gene expression while inhibiting GPR30 to determine putative targets to treat IBC. METHODS/STUDY POPULATION: IBC cell lines (SUM149PT) were cultured in medium with serum stripped from growth factor and hormones for 48 hours. Cells were then exposed to either G15 (GPR30 inhibitor) at a concentration of 1µM or ETOH (vehicle negative control) 3 hours in triplicates. After exposure, total RNA was extracted using the Qiagen RNAeasy Mini kit and RNA was sequenced using the Illumina NextSeq (2 X 75bp). The higher-quality reads were aligned, annotated, and quantified to the human genome (HG38) using STAR and RSEM softwares. Gene expression analysis was performed in R statistical software (packages tximport and DESeq2). Functional and enrichment analyses were performed using Metascape and STRING database, respectively. RESULTS/ANTICIPATED RESULTS: There were 656 significantly expressed genes (p < 0.05) between groups (G15 vs. ETOH). The top 5 significant genes include: SMIM7, FANCG, ARID1A, MAML2, and ATF3. Significantly impacted biological processes and pathways include: electron transport chain, mitotic cell cycle process, microtubule cytoskeleton organization, cellular component morphogenesis and DNA-dependent DNA replication (adj p < 0.05). Additionally, physical and functional interaction networks showed 3 major clusters (≥ 12 genes), which contained several gene hubs including BRCA1, BRCA2, FOS (proto-oncogene), PLK1 and PAK1 (both serine/threonine-protein kinases), among others. Interestingly, the network analysis showed the previously known interaction between FANCG and BRCA2, which were both dysregulated by GPR30 inhibition. DISCUSSION/SIGNIFICANCE: Through gene expression, functional and enrichment analyses we found several targets genes that could be associated with the pathogenesis of IBC. Validation of candidates genes (qRT-PCR and Western blot), and functional assays (cell proliferation, motility, and invasion) will be performed to understand the potential of these genes in treating IBC.

OBJECTIVES/GOALS: This research project aims to isolate and identify bioprospects capable of metabolizing estrogen using culture-dependent, culture independent methods and the identification of the gene/genes responsible for the metabolization of estrogen by the bioprospects. METHODS/STUDY POPULATION: For the culture dependent technique, samples were collected from the water treatment plant in Mayagüez, cultivated on TSA medium and selected specific and diverse colonies were patched on M-9NC (no carbon sources), M-9-glucose (M9G) and M-9-hormone mixture (M9H: 17-beta-Estradiol and 17-alpha-Ethynylestradiol). After the 48hrs incubation at 25 and 37 Celsius, growth was scored on the different media, to choose those potential bioprospects that use the hormones as the sole carbon source. For the culture independent approach, metagenomic clones from libraries generated from the Guajatacas water reservoir and Cabo Rojos Saltern microbial mats were cultivated on the appropriate M9NC, M-9G and M-9H, supplemented with Leucine and Thiamine for 48 hours at 37 Celsius. RESULTS/ANTICIPATED RESULTS: The culture-dependent approach showed a total of 6 potential bioprospects capable of growing on hormones as a sole carbon source on M9H at 25 and 37 Celsius and no-growth on the M-9 media used. Currently, we are in the process of determining the identities of the cultivable isolates genetically, any preference on hormone metabolization, monitoring a larger number of metagenomic clones and optimizing the selection conditions. DISCUSSION/SIGNIFICANCE: The identification of bioprospects capable of metabolizing hormones could potentially lead to the generation of new water treatments that could help remove unwanted chemicals, such as high concentrations of estrogens.

OBJECTIVES/GOALS: Adolescents with congenital heart defects (CHD) have an elevated risk for future cardiovascular events, but information about their risk for e-cigarette use (“vaping”) is unknown. This study aims to present preliminary findings on gender differences in the association of impulsive behavior and vaping susceptibility from an ongoing investigation. METHODS/STUDY POPULATION: Adolescents with CHD (12-18 years; N=63) reported their vaping susceptibility and completed subjective (UPPS-P)/objective (Iowa Gambling Task; IGT) assessments of impulsive behavior previously associated with tobacco use. The UPPS-P includes 5 facets: 1) negative urgency (impulsivity under negative emotions), 2) positive urgency (impulsivity under positive emotions), 3) lack of premeditation (acting without thinking), 4) lack of perseverance (inability to focus), and 5) sensation seeking (seeking thrilling experiences). The IGT is a computerized task that creates conflict between immediate reward and delayed punishment via selections from advantageous/disadvantageous card decks. Linear regressions stratified by gender determined associations between vaping susceptibility and impulsivity. RESULTS/ANTICIPATED RESULTS: Nearly 30% (29.7%) of adolescents with CHD were susceptible to vaping. Negative urgency was associated with vaping susceptibility among females (β = 0.44, p = .035) but not males (β = 0.25, p = .128). Positive urgency was associated with vaping susceptibility among males (β = 0.37, p = .021) and trended toward significance among females (β = 0.40, p = .058). Lack of premeditation was associated with vaping susceptibility among males (β = 0.36, p = .025) but not females (β = 0.15, p = .490). The association between lack of perseverance and vaping susceptibility trended toward significance among males (β = 0.30, p = .064) but not females (β = -0.18, p = .413). IGT performance was not associated with susceptibility to vaping among either gender. UPPS-P facets and IGT performance were not significantly correlated. DISCUSSION/SIGNIFICANCE: The association of impulsivity and vaping susceptibility appears to be characterized by emotion-based rash action (positive/negative urgency) for females and by decreased conscientiousness (lack of premeditation/perseverance) for males. If replicated, the findings have implications for assessment of vaping risk and tailored intervention.

OBJECTIVES/GOALS: Developmental dyslexia is a common reading disability (RD) which negatively impacts academic success. To address the role of early language experience on RD, we tested if the reported differences in gray matter volume (GMV) in RD also manifests in poor readers with a bilingual language background. METHODS/STUDY POPULATION: We studied 54 Spanish-English Bilingual Typical Readers with Oral Reading Recognition Test (ORRT) scores above 100 (avg. =113 ± 10), 51 Spanish-English Bilinguals with RD with ORRT scores below 92 (avg. =84 ± 7), 54 English Monolingual Typical Readers with ORRT scores above 100 (avg. =113 ± 10) and 51 English Monolinguals with RD with ORRT scores below 92 (avg. =84 ± 7) from the Adolescent Brain & Cognitive Development Study. All groups had an average age of 12 ± 0.7 years and were matched for sex and self-ratings of English ability. Structural magnetic resonance images were analyzed using Voxel-Based Morphometry and the bilingual and monolingual groups were separately compared in two-sample t-tests (p < 0.05). RESULTS/ANTICIPATED RESULTS: Monolinguals with RD had less GMV than the Monolingual Typical Readers in the right supramarginal gyrus (Brodmanns Area (BA) 40; MNI Coordinates: 69, -27, 39) (p=0.011) similar to the right superior temporal finding reported in the existing literature. However, a comparison of Bilinguals with RD and Bilingual Typical Readers did not show any GMV differences in superior temporal regions. Instead, our Bilinguals with RD had less GMV compared to the Spanish-English Bilingual Typical Readers in the right superior frontal gyrus (BA 11; MNI Coordinates: 21, 44, -24) extending to the middle frontal gyrus (BA 10) (p = 0.014). DISCUSSION/SIGNIFICANCE: Our findings suggest that the neuroanatomical bases of RD in Spanish-English Bilingual children are not the same as those observed for monolinguals, and biological models developed in monolinguals cannot be generalized. This has implications for diagnoses and treatment of RD in bilinguals.

OBJECTIVES/GOALS: Pain Catastrophizing is a negative coping mechanism involving rumination, magnification, and helplessness and is associated with worse chronic pain. The neurobiological mechanisms underlying this relationship are poorly understood. We aim to examine the intrinsic activity of a functional pathway in patients with chronic orofacial pain. METHODS/STUDY POPULATION: This is the second phase of a parent study examining genetics, placebos, and the brain in temporomandibular disorders (TMD). We intend to recruit 120 of the original 398 TMD patients for this phase. Participants completed the Graded Chronic Pain Scale to assess TMD pain intensity and disability and the Pain Catastrophizing Scale. Behaviorally, pain catastrophizing scores and pain intensity and disability will be analyzed using structural equation modeling. Resting-state functional magnetic resonance imaging will be used to record intrinsic brain activity. The functional connectivity between the posterior cingulate, anterior insula, and periaqueductal grey will be assessed as a causal pathway relating pain catastrophizing to pain intensity and disability. Mediation analyses will be used to test causality. RESULTS/ANTICIPATED RESULTS: We anticipate that greater engagement in catastrophic thinking about pain increases the functional connectivity strength between the posterior cingulate, anterior insula, and periaqueductal grey, which ultimately leads to heightened perception of pain intensity and disability. Therefore, we expect to see increased functional connectivity in those with high pain catastrophizing levels as compared to those with low pain catastrophizing levels, and that this pathway will mediate the relationship between pain catastrophizing and pain intensity and disability. Further, we predict that helplessness will most strongly correlate with the change in functional connectivity as compared to rumination and magnification. Results will be presented in full at the conference. DISCUSSION/SIGNIFICANCE: Understanding how pain catastrophizing can influence chronic pain pathways will not only promote a more integrative approach to chronic pain management but will also help identify the mechanisms by which pain itself develops and persists in the particularly vulnerable pain population of TMD.

OBJECTIVES/GOALS: Insulin dosing is crucial for regulating blood glucose in cardiac surgery patients, yet it requires the use of cumbersome insulin dosing charts. To streamline this process, an electronic insulin calculator (IC) is trialed in the cardiac operating rooms and intensive care unit. This study compares glycemic control prior to and after institution of IC. METHODS/STUDY POPULATION: Using the EHR, a retrospective population of 3,164 cardiac surgery patient charts from 12/19/19 - 11/06/20 were obtained. Baseline, intra-operative, and ICU blood glucose values were obtained from each patients admission. Using this data, a baseline level of glycemic control throughout a cardiac surgery patients stay was established. A preliminary cohort of 244 patients were then chosen to be managed with the new IC. Baseline, intra-operative, and ICU blood glucose values were obtained in this group for comparison. Paired t tests were used to compare the IC group of patients to the retrospective group of patients. Additional subgroup analysis was performed to assess IC efficacy for on pump vs off pump cardiac surgeries. RESULTS/ANTICIPATED RESULTS: The 244 patients managed with the IC showed significantly reduced average blood glucose values during their time in the ICU compared to those previously not managed with the IC (185 mg% vs 153 mg%, p= 0.02). Additionally, a trend towards a reduction in last operating room blood glucose level was also noted. Lastly, average blood glucose levels were significantly reduced for patients undergoing on pump cardiac surgeries compared to off pump surgeries (157 mg% vs 149 mg%, p = 0.03). DISCUSSION/SIGNIFICANCE: Preliminary results suggest IIC to be associated with better intra and postoperative blood glucose control. More data is being collected to test its association with outcomes.

OBJECTIVES/GOALS: The goal of this study is to decipher the mechanisms by which breast cancers evade the immune system to facilitate metastasis through the lymphatic vasculature. We believe this is caused, in part, by semaphorin 7A (SEMA7A) as its expression is associated with increased lymphatic vessels, tumor-associated macrophages, and metastasis in breast cancer. METHODS/STUDY POPULATION: We have observed that breast cancer cells, lymphatics, and macrophages exhibit SEMA7A-dependent expression of PD-L1 in vitro, which may make SEMA7A+ tumors more sensitive to anti-PD-1/PD-L1 immunotherapies. Therefore, we utilized multiple orthotopic, immunocompetent mouse models to determine if SEMA7A expression makes tumors more susceptible to immune checkpoint blockade. E0771 and 66cl4 mouse mammary carcinoma cells were injected orthotopically into the #4 mammary fat pads. Once tumors reached 50-100 mm^3 mice were injected intraperitoneally with 250ug of anti-PD-1, anti-PD-L1, or IgG control every third day. Tumors were measured with calipers every other day and harvested for flow cytometry to determine the effect of immune checkpoint blockade on the TME in SEMA7A+ tumors. RESULTS/ANTICIPATED RESULTS: We reveal that growth of SEMA7A overexpressing (OE) tumors, but not controls, was significantly slowed with both anti-PD-1 and anti-PD-L1 treatments. Flow cytometric analysis of cells from the TME revealed increased LECs, TAMs, and PoEMs in SEMA7A+ tumors, compared to controls – all populations had higher PD-L1 expression, which was decreased with both anti-PD-1 or anti-PD-L1. We also observed a decrease in PD-L1 expression on the tumor cells with treatment. Furthermore, LEC, TAM, and PoEM presence was decreased within the TME alongside increased presence of activated CD4 and CD8 T cells. Collectively, our results suggest that SEMA7A expression in breast cancers activates a major pathway associated with immune evasion and helps to establish a pro-tumor microenvironment. DISCUSSION/SIGNIFICANCE: SEMA7A expression establishes a pro-tumor microenvironment, which can be targeted with readily available FDA approved drugs such as immune checkpoint-based therapies. Since SEMA7A+ breast cancers have high rates of metastasis, more specific treatments for these patient populations should be explored.

OBJECTIVES/GOALS: This project aimed to investigate differences between isometric and isotonic systems for low and high frequency contractions. Intracellular changes in pH caused by the rate of exercise differ in their effect on PCr. The project assessed the reproducibility between these exercise methods. METHODS/STUDY POPULATION: Four subjects were studied (2F/2M, avg. age 27 years). 31P-MRS spectra were measured in the vastus lateralis (VL) using a 3T MRI scanner using a 31P/1H surface coil. In the isometric studies, the (supine) subjects legs were supported by a wedge pad and restrained by straps over the hips and knee with a resistance strap over one ankle. In the isometric studies, the (prone) subjects legs were fit into machine designed to induce isometric exercise. There were 4 exercise bouts that differed by exercise type and frequency and, consequently, duration. 31P-MR spectra were acquired from a 25mm slab of leg positioned parasagittal along the VL. Analyses were done using jMRUI and R. RESULTS/ANTICIPATED RESULTS: With an increase in sample size, we expect differences between recovery time constants, tau, of PCr between the different exercise frequency groups alluded to by other studies investigating recovery rates in bicep skeletal muscle. From the preliminary data, we obtained large limits of agreement of tau = 0.053 s-1 & tau =-0.074 s-1 from using Bland-Altman analysis. As we increase our sample size, we anticipate that we will see differences of PCr recovery rates between the exercise types as well. DISCUSSION/SIGNIFICANCE: Current studies that investigate the differences in PCr recovery rates between different metabolic states differ in the exercise methods employed on subjects. This study aims to standardize PCr recovery assessment in the VL for more interpretable and applicable assessments of metabolic function.

OBJECTIVES/GOALS: Parent stress and coping impacts reward and motivation circuits during child development which influence self-regulation. One well known maladaptive coping response is alcohol or drinking-to-cope (DTC). This study assessed differences in stress and child behaviors among DTC parents as compared to non-DTC parents. METHODS/STUDY POPULATION: Baseline data was used from parents of a 2-5-year-old who were screened for a larger study assessing a mindfulness-based parent stress reduction intervention to improve healthy choices for themselves and their families. The sample included 172 parent-child dyads, mean parent age was 34.4 (6.1) years old, 56.3% white, mean child age was 3.6 (1.2) years old, 52.3% male. Subjective stress was assessed using the Perceived Stress Scale (PSS), parent-specific stress was assessing using the Parenting Stress Index (PSI), DTC was assessed using the COPE inventory, and child behaviors were assessed using the Devereux Early Childhood Assessment. To investigate the differences in stress and child behaviors between DTC parents and non-DTC parents independent samples t-tests were conducted. RESULTS/ANTICIPATED RESULTS: DTC was significantly correlated with PSS (r= 0.23, p<.01), PSI (r= 0.26, p<.01), child self-control (r= -0.16, p= .03), child attention problems (r= 0.22, p<.01), and total behavioral concerns (r= 0.16, p=.04). After excluding those who do not drink alcohol at all, we found significantly higher perceived stress among DTC parents (M= 27.83, SD= 9.79) compared to non-DTC parents (M= 23.79, SD= 8.40), t(80)= 2.02, p= .02. For children, we found significantly higher aggression scores for children of DTC parents (M= 47.16, SD= 31.69) compared to children of non-DTC parents (M=35.83, SD=25.72), t(84)= 1.83, p= .04. And greater attention problems among children of DTC parents (M= 73.97, SD= 26.77) compared to children of non-DTC parents (M= 56.71, SD= 34.09), t(84)=2.63, p=.01. DISCUSSION/SIGNIFICANCE: Stress and DTC may contribute to negative behaviors in children. An intervention designed to decrease stress and increase adaptive coping mechanism in parents could benefit health child socioemotional and behavioral development. Future analyses will examine third variable effects in the relationship between stress, coping, and child behaviors.

OBJECTIVES/GOALS: Illuminate processes and findings of the Environmental Scan of Adaptive Capacity and Preparedness of CTSAs done by the CTSA Working Group. Share challenges, strategies, and lessons learned for CTSAs to build Capacity to address clinical and translational barriers while responding to emergencies. METHODS/STUDY POPULATION: An Environmental Scan approach for searching, collecting, analyzing and using information from diverse sources regarding CTSA hubs experiences during emergency as related to research implementation, translation, support, adaptation, and preparedness: - Triangulating multiple data sources and mixed methods (e.g., literature review, document/RPPR analysis, and expert review); - Secondary analysis of the JCTS COVID-19 Survey of the CTSAs: challenges, lessons learned, and practices that work in various program components/areas; - Using feedback of CTSA professionals from multiple disciplines to enhance our knowledge of emergency preparedness and efficient adaptation to public health and other crises. RESULTS/ANTICIPATED RESULTS: The E-scan approach helped identify challenges, successful practices, and evidence-based strategies for building adaptive capacity and preparedness of CTSAs across various scientific sectors of the translational science spectrum. Some of the findings include: - Roadmaps for the creation of new collaborative research resources (biobanks; data repositories, etc.); - Rapid clinical and research decision making during public health crises; - New community-based research strategies to facilitate communication, research dissemination, and participant recruitment based on existing trust-based networks; - Innovative resource allocation to guarantee continuity of training and research opportunities for trainees. DISCUSSION/SIGNIFICANCE: The Environmental Scan of the Adaptive Capacity of CTSA hubs provides useful knowledge and tools to diverse clinical research stakeholders for mitigating the impact of a disaster via adjusting programs, practices, and processes, and building capacity for effective, emergency-ready and responsive research, training, and community engagement.

OBJECTIVES/GOALS: The host immune response during sepsis is now recognized to have anti-inflammatory pathophysiology. We aim to determine whether mitochondrial dysfunction of leukocytes predicts which critically ill septic patients develop immune paralysis and to identify differences in cellular metabolites between patients with and without immune paralysis. METHODS/STUDY POPULATION: Critically ill septic and control adult patients were recruited from one of 6 ICUs in a single-center tertiary care academic hospital. After enrollment, peripheral blood mononuclear cells (PBMCs) were isolated from a tube of whole blood on day 0-1 after ICU admission. Flow cytometry to quantify monocyte HLA-DR was performed to determine whether patients were immune paralyzed or not. Mitochondrial functional assays of PBMCs were performed with inhibitors of the electron transport chain to assess for differences in oxidative phosphorylation and glycolysis utilization. Metabolic profiling of cell pellets was performed to evaluate for specific metabolites and pathways associated with immune paralyzed patients. RESULTS/ANTICIPATED RESULTS: A total of 101 patients were recruited, including 62 control and 39 septic patients. 81 patients had immune paralysis status available for analysis. 52% of all recruited subjects were immune paralyzed. Of these, 58% were controls and 75% were septic. Immune paralyzed septic and control patients showed features of reduced utilization of oxidative phosphorylation (ox phos) including reduced basal respiration, ATP production and maximal respiration compared with non-immune paralyzed septic and control patients. Immune paralyzed septic patients showed diminished glycolysis utilization compared with septic non-immune paralyzed patients. Finally, cellular kynurenine and quinolinate levels were low in both immune paralyzed control and septic patients compared with non-immune paralyzed patients. DISCUSSION/SIGNIFICANCE: The PBMCs of immune paralyzed septic patients show evidence of mitochondrial dysfunction, with reduced ox phos and glycolysis utilization. Low levels of kynurenine and quinolinate, metabolite precursors to NAD+, in immune paralyzed patients may signal key deficiencies and targetable therapeutic avenues for reversal of an immune paralyzed state.

OBJECTIVES/GOALS: To demonstrate safety and limit damage to off-target organs, we will be determining dosimetry parameters through experimentation and Monte Carlo simulations with our brachy therapy applicator designed to improve upon current designs to treat a 3-dimensional volume. METHODS/STUDY POPULATION: Low-cost materials were used to manufacture our High Dose Rate (HDR) applicator and a readily available after loading system was used to load our configuration with a radioactive source. The dosimetry of our device was analyzed using commercially available software and external beam therapy films to generate depth dose profiles and superficial dose distribution. Additionally, we attempt to confirm Task Group No. 43 (TG-43) dosimetry parameters using Monte Carlo simulations for our device. These data were compared with currently available applicators used for intraoperative radiotherapy. RESULTS/ANTICIPATED RESULTS: We anticipate that we will be able to validate dosimetry parameters for our device in preparation for clinical use. We aim to show our dose distributions align well with proposed target volumes while considering the composition and shape of our applicator. We hope to demonstrate that, unlike current applicators, our design is more effective at treating a 3-dimensional target volume. DISCUSSION/SIGNIFICANCE: By 2040, pancreatic cancer will be the second-largest cause of cancer-related deaths. Even with current brachytherapy applicators, 30-40% of pancreatic cancer seems to recur near the surgical site after surgery. By preventing local recurrence, we hope to improve patient outcomes.

OBJECTIVES/GOALS: To develop a two-staged convolutional neural network to identify the ovary and antral follicles within ovarian ultrasound images and determine its reliability and feasibility compared to conventional techniques in 2D and 3D ultrasonography image analysis. METHODS/STUDY POPULATION: De-identified and archived ultrasonographic images of women across the reproductive spectrum (N=500) will be used in the study. These ultrasound images will be labeled by experienced raters to train a two-staged convolutional neural network (CU-Net). CU-Net will first separate the entire ovary from the background and subsequently identify all antral follicles within the ovary. Following training, the CU-Net will evaluate a second set of independent images (N=100) to determine performance accuracy. Three specialized raters will establish the reliability and feasibility of CU-Net compared to conventional 2D and 3D ovarian ultrasound image analysis methods. RESULTS/ANTICIPATED RESULTS: The labeled training dataset of ovarian ultrasound images is expected to successfully train the CU-Net and allow for accurate identification of the ovary and the total number of antral follicles in the second testing set of ultrasound images. When compared to conventional 2D and 3D ultrasound image analysis methods, CU-Net is expected to have similar accuracy when compared to the gold-standard method (2D-Offline with Grid) and outperform other approaches, such as 2D-Real Time and 3D volume software (VOCAL and Sono-AVC). However, CU-Net is anticipated to be the fastest and most reliable method across users, supporting its clinical feasibility. DISCUSSION/SIGNIFICANCE: This study will immediately translate to providing a standardized platform that can improve the accuracy, reliability, and time demand required for the evaluation of ovarian ultrasounds across users and clinical and research settings.

OBJECTIVES/GOALS: MiaA is a highly conserved prenyl transferase that catalyzes synthesis of the i6A37 tRNA modification in E. coli. While transcriptional regulation of MiaA is well characterized, there is no information on the MiaA post-transcriptional regulation. The aim of this study is to characterize the post-transcriptional regulation of the MiaA gene in E. coli. METHODS/STUDY POPULATION: To characterize the post-transcriptional regulation of miaA, we executed a targeted genetic screen of an E. coli small RNA library on a miaA-lacZ translational reporter fusion strain to identify small RNAs (sRNAs) that modulate MiaA translation or transcription termination. We also measured MiaA mRNA levels and miaA-lacZ activity in the absence or over-expression of candidate sRNA regulators of MiaA. We also measured MiaA mRNA levels in the absence of RNaseE and PNPase, two enzymes involved in mRNA turnover. Finally, we measured the ability of purified recombinant CsrA to bind to the MiaA mRNA transcript in vitro. RESULTS/ANTICIPATED RESULTS: We identified the carbon sensing sRNA CsrB and its cognate protein interaction partner CsrA, as potential post-transcriptional regulators of MiaA. Over-expression of CsrB fully repressed miaA-lacZ activity and MiaA mRNA levels. The absence of CsrA resulted in a defective miaA-lacZ activity and a 10-fold decrease in MiaA mRNA levels. We also identified an increase in the MiaA mRNA half-life particularly in the absence of RNaseE. Our results demonstrate an additional layer of regulation for the miaA operon by the CsrA/CsrB protein-sRNA system. DISCUSSION/SIGNIFICANCE: MiaA is a highly conserved bacterial protein. Our data may represent phenomena in an array of bacteria that could be targeted by novel antibiotics. The human MiaA homologue, TRIT1, plays a role in mitochondrial disorders. We anticipate that information garnered from MiaA studies will elucidate TRIT1 function and its role in mitochondrial disorders.