A Genome-Edited ERα-HiBiT Fusion Reporter Cell Line for the Identification of ERα Modulators Via High-Throughput Screening and CETSA.

The estrogen receptor α (ERα) is a target of intense pharmacological intervention and toxicological biomonitoring. Current methods to directly quantify cellular levels of ERα involve antibody-based assays, which are labor-intensive and of limited throughput. In this study, we generated a post-translational reporter cell line, referred to as MCF7-ERα-HiBiT, by fusing a small pro-luminescent nanoluciferase (NLuc) tag (HiBiT) to the C-terminus of endogenous ERα in MCF7 cells. The tag allows the luminescent detection and quantification of endogenous ERα protein by addition of the complementary NLuc enzyme fragment. This MCF7-ERα-HiBiT cell line was optimized for quantitative high-throughput screening (qHTS) to identify compounds that reduce ERα levels. In addition, the same cell line was optimized for a qHTS cellular thermal shift assay to identify compounds that bind and thermally stabilize ERα. Here, we interrogated the MCF7-ERα-HiBiT assay against the NCATS Pharmacological Collection (NPC) of 2,678 approved drugs and identified compounds that potently reduce and thermally stabilize ERα. Our novel post-translational reporter cell line provides a unique opportunity for profiling large pharmacological and toxicological compound libraries for their effect on ERα levels as well as for assessing direct compound binding to the receptor, thus facilitating mechanistic studies by which compounds exert their biological effects on ERα.

Genomics of Environmental Salmonella: Engaging Students in the Microbiology and Bioinformatics of Foodborne Pathogens.

We have developed and implemented an undergraduate microbiology course in which students isolate, characterize, and perform whole genome assembly and analysis of Salmonella enterica from stream sediments and poultry litter. In the development of the course and over three semesters, successive teams of undergraduate students collected field samples and performed enrichment and isolation techniques specific for the detection of S. enterica. Eighty-eight strains were confirmed using standard microbiological methods and PCR of the invA gene. The isolates' genomes were Illumina-sequenced by the Center for Food Safety and Applied Nutrition at the FDA and the Virginia state Division of Consolidated Laboratory Services as part of the GenomeTrakr program. Students used GalaxyTrakr and other web- and non-web-based platforms and tools to perform quality control on raw and assembled sequence data, assemble, and annotate genomes, identify antimicrobial resistance and virulence genes, putative plasmids, and other mobile genetic elements. Strains with putative plasmid-borne antimicrobial resistance genes were further sequenced by students in our research lab using the Oxford Nanopore MinIONTM platform. Strains of Salmonella that were isolated include human infectious serotypes such as Typhimurium and Infantis. Over 31 of the isolates possessed antibiotic resistance genes, some of which were located on large, multidrug resistance plasmids. Plasmid pHJ-38, identified in a Typhimurium isolate, is an apparently self-transmissible 183 kb IncA/C2 plasmid that possesses multiple antimicrobial resistance and heavy-metal resistance genes. Plasmid pFHS-02, identified in an Infantis isolate, is an apparently self-transmissible 303 kb IncF1B plasmid that also possesses numerous heavy-metal and antimicrobial resistance genes. Using direct and indirect measures to assess student outcomes, results indicate that course participation contributed to cognitive gains in relevant content knowledge and research skills such as field sampling, molecular techniques, and computational analysis. Furthermore, participants self-reported a deeper interest in scientific research and careers as well as psychosocial outcomes (e.g., sense of belonging and self-efficacy) commonly associated with student success and persistence in STEM. Overall, this course provided a powerful combination of field, wet lab, and computational biology experiences for students, while also providing data potentially useful in pathogen surveillance, epidemiological tracking, and for the further study of environmental reservoirs of S. enterica.