Objectives/Goals: The never in mitosis kinase (NEK) family regulates vital processes, namely cell cycle progression, but their potential as therapeutic targets in TNBC has not been fully explored. Our studies aim to develop a toolkit to investigate the functional roles of NEKs in pathologies including carcinogenesis. Methods/Study Population: To assess differential NEK expression in normal and tumor tissues and correlation of gene expression with patient survival, we used Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan–Meier Plotter (KMPlot) pan-cancer analysis, respectively. Basal NEK protein levels were determined by immunoblot across a panel of cell lines, including breast cancer, osteosarcoma, hepatocellular carcinoma, and non-cancerous cells, to identify appropriate systems for evaluation of NEK function. Doxycycline-inducible cell lines were generated by transduction with lentiviral stocks of NEK shRNA and overexpression constructs and antibiotic selection. Expression was analyzed by qPCR and immunoblot. Results/Anticipated Results: Expression of NEK2, 4, 5, 6, 8, and 11 was higher in breast tumors compared to normal tissue by GEPIA analysis. Further examination using KMPlot showed a correlation between elevated NEK6 expression and decreased overall survival in patients with aggressive cancers. As an initial proof-of-concept study, we analyzed NEK6 protein expression in breast cancer cells. Levels of NEK6 were elevated in TNBC cells (MDA-MB-231) compared to hormone receptor positive (HR+) breast cancer cells (MCF7). Using complementary approaches to investigate the functional role of NEK6 in breast cancer, we depleted NEK6 expression using shRNAs in TNBC cells and expressed NEK6 in HR+ cells Discussion/Significance of Impact: Because kinase dysregulation promotes oncogenesis and metastasis, targeting kinases is a key strategy in therapeutic development. A NEK-specific molecular toolkit allows researchers to elucidate NEK functions and contributions to carcinogenesis, promoting advancement of novel therapies.

Defamatory publications may carry any number of related or distinct imputations. Complexities arise where a plaintiff selects one or more imputations for complaint, but ignores other imputations carried by the same publication. In England and Wales, the so-called Polly Peck principle permits defendants to plead and justify an imputation other than one complained of by the plaintiff but bearing a common sting with such an imputation. The Polly Peck principle has not been good law in Australia for more than 20 years. The statutory defence of contextual truth in Australia’s uniform defamation laws, however, permits a defendant to plead and justify imputations that are ‘in addition to’ those complained of by the plaintiff and affords a complete defence where, having regard to the substantial truth of those contextual imputations, the imputations complained of by the plaintiff do not further harm the reputation of the plaintiff. As enacted, the defence was infected with a serious drafting error. This article looks at the implications of the reformulation of the contextual truth defence effected by the recent amendments to Australia’s uniform defamation laws. It posits that those implications are considerably broader than have been recognised to date. It argues that the reformulated defence not only corrects the drafting error in the original defence of contextual truth but also resurrects the Polly Peck principle in Australia and substantially neuters 20 years of confused jurisprudence concerning the extent to which a defendant is or should be constrained by the imputations pleaded by the plaintiff.

Latinos, especially those who recently immigrated, face many obstacles in navigating the political and judicial environment in the United States. While prior scholarship suggests that racial minorities are more likely to be stopped by law enforcement for traffic violations and face harsher penalties for major crimes, little research has explored whether a defendant’s characteristics are influential in routine traffic court cases. Using an original database, this paper examines disparate treatment in speeding ticket reductions. The results indicate that Latino defendants are less likely to receive meaningful reductions to their charges. However, attorney representation greatly lessens the likelihood of disparate treatment for Latino drivers. As traffic court proceedings often represent the only interaction most people have with the judicial system, these findings have significant implications for racial equality, the administration of justice, attorney representation, and public opinion of the judiciary.

Presidents may react to Supreme Court decisions by supporting or opposing them in their public rhetoric and by calling on Congress to take action to alter or implement the Court’s decisions. We investigate this unique form of lawmaking using an original database of presidential calls to Congress and congressional reactions. We find that presidents call for congressional action to pursue their policy goals and enhance the power of the presidency; we also find that Congress reacts when it is asked to do so by both the Court and the president, as well as when presidents support the implementation of the Court’s decisions.

Background: Nursing home (NH) residents and staff were at high risk for COVID-19 early in the pandemic; several studies estimated seroprevalence of infection in NH staff to be 3-fold higher among CNAs and nurses compared to other staff. Risk mitigation added in Fall 2020 included systematic testing of residents and staff (and furlough if positive) to reduce transmission risk. We estimated risks for SARS-CoV-2 infection among NH staff during the first winter surge before widespread vaccination. Methods: Between February and May 2021, voluntary serologic testing was performed on NH staff who were seronegative for SARS-CoV-2 in late Fall 2020 (during a previous serology study at 14 Georgia NHs). An exposure assessment at the second time point covered prior 3 months of job activities, community exposures, and self-reported COVID-19 vaccination, including very recent vaccination (≤4 weeks). Risk factors for seroconversion were estimated by job type using multivariable logistic regression, accounting for interval community-incidence and interval change in resident infections per bed. Results: Among 203 eligible staff, 72 (35.5%) had evidence of interval seroconversion (Fig. 1). Among 80 unvaccinated staff, interval infection was significantly higher among CNAs and nurses (aOR, 4.9; 95% CI, 1.4–20.7) than other staff, after adjusting for race and interval community incidence and facility infections. This risk persisted but was attenuated when utilizing the full study cohort including those with very recent vaccination (aOR, 1.8; 95% CI, 0.9–3.7). Conclusions: Midway through the first year of the pandemic, NH staff with close or common resident contact continued to be at increased risk for infection despite enhanced infection prevention efforts. Mitigation strategies, prior to vaccination, did not eliminate occupational risk for infection. Vaccine utilization is critical to eliminate occupational risk among frontline healthcare providers.

Funding: None

Disclosures: None

OBJECTIVES/GOALS: In triple negative breast cancer (TNBC), obesity is associated with poor outcomes. Adipose stem cells (ASCs) from obese patients (obASCs) secrete higher levels of adipokines compared to ASCs from lean individuals. Leptin, one of these adipokines, has been implicated in many cancers. This study seeks to examine the role of leptin signaling in TNBC. METHODS/STUDY POPULATION: Previous work in conjunction with a collaborating lab has shown that leptin signaling promotes metastasis and increased expression of epithelial-mesenchymal transition (EMT) markers in triple negative breast cancer cell lines. This project expands upon this work through using both patient-derived cell lines and and patient-derived xenografts (PDX), and examines the role of leptin signaling both in vitro and in vivo. To determine the effects of obesity upon a PDX model of TNBC, a high fat diet was used to induce obesity in vivo. A pharmacological inhibitor of the leptin receptor was used to test the requirement for leptin signaling both in vivo and in vitro. RESULTS/ANTICIPATED RESULTS: Exposure to conditioned media harvested from obASCs increased the percentage of TNBC cells that expressed cancer stem cell markers, whereas exposure to an inhibitor of the leptin receptor decreased the percentage of cells with cancer stem cell markers. PDX tumors implanted into mice with diet-induced obesity had an increased volume compared to tumors implanted into lean controls. Further analysis will be conducted on metastasis, circulating tumor cells, and survival in both lean and obese mice. DISCUSSION/SIGNIFICANCE: Understanding the complex signaling events in the obese tumor microenvironment is essential, as these molecular differences may contribute to different outcomes for obese and lean individuals with triple negative breast cancer. Therefore, study of the crosstalk between obASCs and TNBC cells is critical.

OBJECTIVES/GOALS: Current approaches to drug development for the aggressive triple negative breast cancer rely on current 2D and 3D in vitro models which have limited capabilities. We have developed a translational microphysiological system that can maintain the human breast microenvironment to capture the complex interaction with the tumor microenvironment. METHODS/STUDY POPULATION: Three different TNBC cell lines were seeded in BC-MPS: MDA-MB-231 parental cell line, MDA-MB-231wiht the gene, LKB1 overexpressed, which is a tumor suppressor, and MDA-MB-231 with the enzyme, ERK5, an enzyme associated with increased metastasis and drug resistance, knocked out. These three TNBC cell lines were cultured in a standard 2D 96-well plate and in BC-MPS. Time-lapse videos were taken to track cellular mobility. RNA-sequencing was performed to compare different expression levels of various cancer related genes of the cell lines cultured in standard 2D and BC-MPS. RESULTS/ANTICIPATED RESULTS: The LKB1 overexpressed MDA-MB-231 and the ERK5-ko MDA-MB-231 cell lines are expected to have decreased mobility compared to the parental cells. The cell lines are expected to have increased expression of cancer related genes when cultured in BC-MPS than when cultured in standard 2D due to the presence of human breast tissue. DISCUSSION/SIGNIFICANCE: BC-MPS is a promising new translational MPS that facilitates studying long term interactions between real human breast tissue and cancer cells. The BC-MPS systems ability to support the growth of established cell lines has been demonstrated. Future studies will focus on developing the model for personalized medicine.

Next Generation Lab turns large and hitherto unstudied urban assemblages of archaeological leather and bone into a laboratory learning experience for high school students. The students, in turn, provide species identifications and thus increase knowledge on medieval and Renaissance livestock exploitation and material selection by craftsmen.

Field trials were conducted to determine the effects of glyphosate and/or dicamba simulated drift rates on chipping potatoes (Solanum tuberosum L.) ‘Atlantic’ and ‘Dakota Pearl.’ Sublethal herbicide rates were applied at the tuber initiation stage and consisted of dicamba at 99 g ae ha−1 or glyphosate at 197 g ae ha−1 applied alone or the combinations of dicamba at 20 or 99 g ae ha−1 and glyphosate at 40 or 197 g ae ha−1, respectively. At 7 days after treatment (DAT), the high spray combination of glyphosate plus dicamba resulted in the greatest plant damage (28%). Plant injury from plants treated with the low combination of glyphosate plus dicamba did not differ from the nontreated control. At 21 DAT, visible injury increased to 40% for plants treated with the high combination of glyphosate plus dicamba. Total yield suggested that dicamba and glyphosate caused similar yield reductions as plants that received glyphosate at 197 g ha−1 or dicamba at 99 g ha−1 had lower total yields compared to the nontreated and plants that received the combination of glyphosate (197 g ha−1) and dicamba (99 g ha−1) had lower total yields compared to plants that received either herbicide alone. However, ‘Dakota Pearl’ plants were more sensitive to glyphosate at 197 g ha−1 than were ‘Atlantic’ plants, causing the interaction for most tuber grades. Tuber specific gravity was lower for plants that received glyphosate at 197 g ha−1, dicamba at 99 g ha−1, or this combination, but this reduction would not prevent chip processing. Results reinforce the need for diligence when applying these herbicides in proximity to a susceptible crop, such as chipping potatoes, and the need to thoroughly clean sprayers before application to a sensitive crop.

The Subglacial Antarctic Lakes Scientific Access (SALSA) Project accessed Mercer Subglacial Lake using environmentally clean hot-water drilling to examine interactions among ice, water, sediment, rock, microbes and carbon reservoirs within the lake water column and underlying sediments. A ~0.4 m diameter borehole was melted through 1087 m of ice and maintained over ~10 days, allowing observation of ice properties and collection of water and sediment with various tools. Over this period, SALSA collected: 60 L of lake water and 10 L of deep borehole water; microbes >0.2 μm in diameter from in situ filtration of ~100 L of lake water; 10 multicores 0.32–0.49 m long; 1.0 and 1.76 m long gravity cores; three conductivity–temperature–depth profiles of borehole and lake water; five discrete depth current meter measurements in the lake and images of ice, the lake water–ice interface and lake sediments. Temperature and conductivity data showed the hydrodynamic character of water mixing between the borehole and lake after entry. Models simulating melting of the ~6 m thick basal accreted ice layer imply that debris fall-out through the ~15 m water column to the lake sediments from borehole melting had little effect on the stratigraphy of surficial sediment cores.

ABSTRACT IMPACT: Identifying an important pathway in treatment resistant TNBC will allow for the future development of clinical therapeutics specific for this disease. OBJECTIVES/GOALS: Triple Negative Breast Cancer (TNBC) is a subtype of breast cancer characterized by negative expression of estrogen receptor, progesterone receptor, and HER2/neu amplification. It resists therapies and has a high recurrence rate after resection. The goal of my research is to identify & characterize a TNBC pathway for future development of therapies. METHODS/STUDY POPULATION: The project uses a combination of cell lines, patient derived xenograft (PDX) models, as well as patient databases. Standard cellular and molecular biology techniques will be used including: Cell culture, qPCR, western blotting, and flow cytometry. RESULTS/ANTICIPATED RESULTS: LKB1 is a master kinase that activates 14 possible downstream kinases. The signaling pathway has been demonstrated to play a role in energy homeostasis and metabolism. Mutation of LKB1 signaling results in Peutz-Jeghers Syndrome and is associated with neoplasias of the lung, pancreas, and breast. Based on preliminary analysis, overexpression of LKB1 by shRNA in TNBC cell lines results in suppression of EMT and reduction of the cancer stem cell population. Additional studies show that LKB1 overexpression has no effect on growth rate in 2D culture while significant reduction in 3D mammosphere formations can be seen. Downstream studies using commercially available SIK1 inhibitor HG-9-91-01 is able to induce a larger fraction of CSC from reduced LKB1 overexpression as well as from baseline levels. DISCUSSION/SIGNIFICANCE OF FINDINGS: Overall, our results suggest that LKB1 acts through SIK1 to suppress EMT and the generation of cancer stem cells. This results in reduced cancer functionality, as evidenced by inhibition of mammosphere formation. These results establishes a foundation for future mechanistic studies on the LKB1 axis and its mechanisms in TNBC.

Among 353 healthcare personnel in a longitudinal cohort in 4 hospitals in Atlanta, Georgia (May–June 2020), 23 (6.5%) had severe acute respiratory coronavirus virus 2 (SARS-CoV-2) antibodies. Spending >50% of a typical shift at the bedside (OR, 3.4; 95% CI, 1.2–10.5) and black race (OR, 8.4; 95% CI, 2.7–27.4) were associated with SARS-CoV-2 seropositivity.

The ratification of the African Continental Free Trade Agreement (AfCFTA) marked a landmark event in the quest to achieve intra-African free trade. AfCFTA is poised to represent the largest free trade area outside the World Trade Organization. Although AfCFTA aspires to liberalize intra-African trade in goods and services to foster socio-economic development, there are concerns that capacity constraints may stultify the underlying goals. AfCFTA is expected to build on the considerable successes already achieved by Africa's regional economic communities. However, it fails to clarify how the overlapping regimes will be reconciled and harmonized. Nevertheless, the agreement is laudable for its quest to facilitate intra-African trade, foster regional value chains that can facilitate integration into the global economy, and energize industrialization, competitiveness and innovation. This article examines the celebrated AfCFTA to understand its potential amid local realities and the possible implications for the multilateral trading system.

The principal aim of this study was to optimize the diagnosis of canine neuroangiostrongyliasis (NA). In total, 92 cases were seen between 2010 and 2020. Dogs were aged from 7 weeks to 14 years (median 5 months), with 73/90 (81%) less than 6 months and 1.7 times as many males as females. The disease became more common over the study period. Most cases (86%) were seen between March and July. Cerebrospinal fluid (CSF) was obtained from the cisterna magna in 77 dogs, the lumbar cistern in f5, and both sites in 3. Nucleated cell counts for 84 specimens ranged from 1 to 146 150 cells μL−1 (median 4500). Percentage eosinophils varied from 0 to 98% (median 83%). When both cisternal and lumbar CSF were collected, inflammation was more severe caudally. Seventy-three CSF specimens were subjected to enzyme-linked immunosorbent assay (ELISA) testing for antibodies against A. cantonensis; 61 (84%) tested positive, titres ranging from <100 to ⩾12 800 (median 1600). Sixty-one CSF specimens were subjected to real-time quantitative polymerase chain reaction (qPCR) testing using a new protocol targeting a bioinformatically-informed repetitive genetic target; 53/61 samples (87%) tested positive, CT values ranging from 23.4 to 39.5 (median 30.0). For 57 dogs, it was possible to compare CSF ELISA serology and qPCR. ELISA and qPCR were both positive in 40 dogs, in 5 dogs the ELISA was positive while the qPCR was negative, in 9 dogs the qPCR was positive but the ELISA was negative, while in 3 dogs both the ELISA and qPCR were negative. NA is an emerging infectious disease of dogs in Sydney, Australia.