Objectives/Goals: Monensin is FDA approved for use in veterinary medicine. Recent studies pointed to its potent anticancer activity. Since de novo drug discovery process typically takes 10 to 15 years and requires an investment of approximately $1.3 to $3 billion, drug repositioning can bypass several steps in this process and increase the potential for success. Methods/Study Population: Cell viability assays were conducted on human MDA-MB-231, MDA-MB-468, and MCF10A breast cancer cell lines and mouse EO771 and 4T1 breast cancer cell lines. MDA-MB-231 cell line was used in all the studies unless specified otherwise. Time course levels of Bcl-2, Bak, p62, and LC3II were assessed via Western blotting with GAPDH as a loading control. Proteomics analysis was conducted by the IDEA National Resource for Quantitative Proteomics. Time course levels of major histocompatibility complex (MHC) I and II and calreticulin were evaluated using flow cytometry. At least three biological replicates have been conducted for each experiment. Results/Anticipated Results: Monensin and several of its novel analogs were potent toward human and mouse breast cancer cell lines. Furthermore, they induced apoptotic cell death as evidenced by Annexin V/PI assay, downregulation of Bcl-2, and upregulation of Bak in MDA-MB-231 cells. Proteomics analysis revealed that several molecular pathways related to MHC class I and II antigen presentation were significantly altered following treatment with these compounds. Additionally, monensin and its analogs significantly increased the expression of MHC class I and II. Our studies also showed that monensin and its analogs increase the surface calreticulin levels. Treatment of MDA-MB-231 cells with these compounds also resulted in an increase in p62 and LC3II expression, suggesting a disruption of the autophagic process. Discussion/Significance of Impact: These results suggest that monensin and its analogs not only exhibit anti-breast cancer cell activity but also modulate immune-related pathways. By disrupting autophagy and enhancing calreticulin levels, these compounds may potentiate antitumor immune responses, providing a promising avenue for drug repositioning in cancer therapy.

Objectives/Goals: Given the challenges that early career research scientists face, especially preparing for promotion and tenure, the decision on whether to join a research team can be fraught. We developed a novel training to support informed decision-making regarding new scientific teaming opportunities. Methods/Study Population: A team science workshop entitled “Should I join this research team” was designed for early career investigators from varied disciplinary backgrounds. Learning objectives for attendees included 1) describing the role of team science in translational research, 2) determining if teaming opportunities are a good fit, and 3) crafting thoughtful responses to requests. The training was initially delivered to 38 attendees (11 K scholars) during a virtual national meeting. We adapted this training for in-person delivery to K and T scholars at our CTSA regional partners. Instructional methods shared across virtual and in-person modalities included self-reflection, think and share activities, and scenario application. In-person delivery also included short video clips and small group discussions. Results/Anticipated Results: Multiple Likert-scale items were completed by workshop participants before and after completing the workshop to evaluate attendees’ confidence in their perceived abilities to explain strengths and limitations of team science, identify characteristics of effective science teams, evaluate a team invitation, assess costs and benefits, negotiate collaborative team invitations, etc. Preliminary data from the virtual workshop suggests that 54.6% of scholars were either not at all or only slightly confident in evaluating a teaming invitation. After the workshop, 45.5% reported being very confident, and 9.1% reported extreme confidence in evaluating a team invitation. Evaluation of the in-person training, along with a comparison of virtual and in-person learning outcomes will also be presented. Discussion/Significance of Impact: Our multimodal training is designed to equip early career investigators with the tools needed to evaluate and respond effectively to research team invitations. We believe this novel training will result in informed teaming decisions for early career research scientists.

As a free-ranging, social species, the housing of horses (Equus caballus) may limit their opportunity to display natural behaviour, compromising well-being. This review records and presents studies that have investigated horse housing design, evaluates the location and number of studies carried out to date, and reports the methods used to assess impact on equine well-being. A Boolean search was conducted in two databases: Web of Science and Scopus, filtered according to Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) protocol, resulting in 60 peer-reviewed papers for evaluation. Key findings are that a significant amount of work to date has been carried out in Europe and the USA, and the frequency of horse housing studies has steadily increased over the last 33 years, with 52% of them occurring in the last eight years. Health and welfare measures indicate benefits of housing horses in more natural management systems, particularly with conspecifics. Generally, the studies reviewed were only conducted in the short term, therefore future research should aim to increase the length of time over which housing is evaluated, particularly to ensure studies continue beyond an adaptation period. The review also highlights a requirement for more standardised methodology in housing welfare evaluation to allow for more meaningful comparisons to be made. Studies seeking to improve horse welfare in existing housing systems, in the face of limited space or other management constraints, are of high value to the end user and are encouraged. The studies reviewed here represent a significant and diverse body of work from which gaps in knowledge and future research directions can be determined.

OBJECTIVES/GOALS: A barrier to the proliferation of team science is that academicians are often trained in disciplinary silos where “independent” research contributions are lauded. To tackle some of the most pressing scientific challenges, dismantling silos and increasing team science training efforts that focus on early career investigators is a must. METHODS/STUDY POPULATION: A team science training workshop for early career investigators from varied disciplinary backgrounds was informed by a 20-item needs assessment that addressed essential team science competencies and was completed by early career investigators participating in federally funded professional development programs on our campus. During the workshop, the benefits of cross-disciplinary teaming was discussed. Strategies including team formation, team effectiveness and/or dysfunction, diagnosing team strengths and weaknesses, and teaming in community settings were discussed. Instructional methods included short presentations, video clips, case studies, group discussions, pair and share activities, and panel discussions with expert role models encouraged active learning. RESULTS/ANTICIPATED RESULTS: The impact and value of the workshop series to participant’s professional development and knowledge of team science concepts will be evaluated before and after the workshop. Multiple Likert-scale items focused on team science competencies (e.g., confidence in your ability to carry out responsibilities specific to your role on a team, recognize when the team is not functioning well; engage team science practices in on-going research), and open-ended questions (e.g., importance of engaging community partners in academic research teams, vision of what factors contribute to an effective team science collaboration) will be completed by program participants before and after completing the workshop. DISCUSSION/SIGNIFICANCE: Effective collaboration among scientists with expertise in different disciplines is needed to address and solve complex scientific problems. We believe our interactive approach to team competency training sessions would work in a variety of settings and improve team skills.

OBJECTIVES/GOALS: Although the approval of immune checkpoint inhibitors (ICIs) revolutionized the treatment of metastatic breast cancer (BC), yet about 30% remain unresponsive. Since the potency of ICIs depends on the efficient presentation of tumor-specific antigens by cancer cells, compounds which increase such presentation could increase efficacy of ICIs. METHODS/STUDY POPULATION: A library of the ester and urethane derivatives of polyether ionophore antibiotic, monensin (MON) has been synthesized. MTT cell viability assays were performed on the panel of human and mouse BC cell lines, and non-cancerous breast epithelial cells to determine IC50 values of MON and its derivatives. Selectivity Indexes were calculated to identify the most selective compounds towards cancer versus non-cancer cells. Major Histocompatibility Complex (MHC) class I and II presentation and Programmed death-ligand 1 (PD-L1) expression have been determined using flow cytometry. Proteins involved in apoptosis, autophagy and immunogenic cell death were identified through immunoblotting. At least three biological replicates have been performed for each experiment. RESULTS/ANTICIPATED RESULTS: MON and several of its derivatives shown activity in nanomolar range against MDA-MB-231 human BC cell line. MON and its most potent derivatives significantly increased MHC class I and II presentation and downregulated the expression of PD-L1 in BC cell lines. DISCUSSION/SIGNIFICANCE: Present findings will lead to the development of new therapeutic approaches that can serve as single agents or be used in combination with existing ICIs for the treatment of metastatic BC. By pushing the boundaries of our understanding and developing new therapies, this research can make an impact in improving outcomes for patients with metastatic BC.

Little research has explored relationships between prenatal substance use policies and rates of maternal mortality across all 50 states, despite evidence that prenatal substance use elevates risk of maternal death. This study, utilizing publicly available data, revealed that state-level mandated testing laws predicted maternal mortality after controlling for population characteristics.

Empowering the Participant Voice (EPV) is an NCATS-funded six-CTSA collaboration to develop, demonstrate, and disseminate a low-cost infrastructure for collecting timely feedback from research participants, fostering trust, and providing data for improving clinical translational research. EPV leverages the validated Research Participant Perception Survey (RPPS) and the popular REDCap electronic data-capture platform. This report describes the development of infrastructure designed to overcome identified institutional barriers to routinely collecting participant feedback using RPPS and demonstration use cases. Sites engaged local stakeholders iteratively, incorporating feedback about anticipated value and potential concerns into project design. The team defined common standards and operations, developed software, and produced a detailed planning and implementation Guide. By May 2023, 2,575 participants diverse in age, race, ethnicity, and sex had responded to approximately 13,850 survey invitations (18.6%); 29% of responses included free-text comments. EPV infrastructure enabled sites to routinely access local and multi-site research participant experience data on an interactive analytics dashboard. The EPV learning collaborative continues to test initiatives to improve survey reach and optimize infrastructure and process. Broad uptake of EPV will expand the evidence base, enable hypothesis generation, and drive research-on-research locally and nationally to enhance the clinical research enterprise.

We tested 85 isolates of β-hemolytic Streptococcus spp. against trimethoprim/sulfamethoxazole (TMP/SMX), clindamycin, and doxycycline by broth microdilution (BMD) and BD Phoenix. Susceptibility rates via BMD for TMP/SMX, clindamycin, and doxycycline were 100%, 85.5%, and 56.6%, respectively. TMP/SMX is a potential monotherapy agent for β-hemolytic Streptococcus skin and soft tissue infections.

A cluster of Rhizobium radiobacter isolates isolated from six unique surgical tissue cultures prompted an investigation ultimately identifying a pseudo-outbreak linked to errant laboratory tissue processing with contaminated, nonsterile saline. Timely response and multidisciplinary collaboration led to tangible system-level interventions and avoidance of unnecessary antibiotic exposures.

Background: Antibiotics alone are often insufficient to treat recurrent C. difficile infection (rCDI) because they have no activity against C. difficile spores that germinate within a disrupted microbiome. SER-109, an investigational, oral, microbiome therapeutic comprised of purified Firmicutes spores, was designed to reduce rCDI through microbiome repair. We report an integrated efficacy analysis through week 24 for SER-109 from phase 3 studies, ECOSPOR III and ECOSPOR IV. Methods: ECOSPOR III was a randomized, placebo-controlled phase 3 trial conducted at 56 US or Canadian sites that included 182 participants with ≥2 CDI recurrences, confirmed via toxin EIA testing. Participants were stratified by age (<65 years or ≥65 years) and antibiotic regimen (vancomycin, fidaxomicin) and were randomized 1:1 to placebo or SER-109 groups. ECOSPOR IV was an open-label, single-arm study conducted at 72 US or Canadian sites including 263 participants with rCDI enrolled in 2 cohorts: (1) rollover participants from ECOSPOR III who experienced on-study recurrence diagnosed by toxin EIA (n = 29) and (2) participants with ≥1 CDI recurrence (diagnosed by PCR or toxin EIA), inclusive of the current episode (n = 234). In both studies, the investigational product was administered orally as 4 capsules over 3 consecutive days following symptom resolution after standard-of-care antibiotics. The primary efficacy end point was rCDI (recurrent toxin-positive diarrhea requiring treatment) through week 8. Other end points included CDI recurrence rates and safety through 24 weeks. Results: These 349 participants received at least 1 dose of SER-109 in ECOSPOR III or ECOSPOR IV (mean age 64.2; 68.8% female). Overall, 77 participants (22.1%) enrolled with their first CDI recurrence. Four participants received blinded SER-109 in ECOSPOR III followed by a second dose of open-label SER-109 in ECOSPOR IV. Overall, the proportion of participants who received any dose of SER-109 with rCDI at week 8 was 9.5% (33 of 349; 95% CI, 6.6 %–13.0%), and the CDI recurrence rate remained low through 24 weeks (15.2%, 53 of 349; 95% CI, 11.6%–19.4%), corresponding to sustained clinical response rates of 90.5% (95% CI, 87.0%–93.4%) and 84.8% (95% CI, 80.6%–88.4%), respectively (Fig. 1). Most rollover participants (25 of 29, 86.2%) were from the placebo arm; 13.8% had rCDI by week 8. Conclusions: In this integrated analysis, the rates of rCDI were low and durable in participants who received the investigational microbiome therapeutic SER-109, with sustained clinical response rates of 90.5% and 84.8% at weeks 8 and 24, respectively. These data further support the potential benefit of microbiome repair with SER-109 following antibiotics for rCDI to prevent recurrence in high-risk patients.

Financial support: This study was funded by Seres Therapeutics.

Disclosure: None

In 2017, ancient DNA analysis of the Harper Road burial from Southwark (London) found that the individual had male chromosomes. Now analysis has discovered that the individual had female chromosomes, data which match the osteological estimation of sex and the interpretation of the grave-goods.