Progress 04/01/24 to 03/31/25

Outputs
Target Audience: The project primarily serves undergraduate and graduate students at West Virginia State University, providing them with hands-on training in functional genomics, gene-editing, and neurobiology using Drosophila melanogaster as a model system. Students enrolled in courses such as Genetics, Biotechniques, and Developmental Genetics directly benefit from integrated instruction that incorporates current project data and methodologies. Through lab-based exercises, students gain practical experience with technologies like CRISPR, single-nucleus RNA sequencing, confocal imaging, and behavioral assays, preparing them for advanced research and careers in molecular biology and biotechnology. In addition to students, the project engages faculty and research collaborators at partner institutions, including Ohio University, West Virginia University, and the University of Puerto Rico. These collaborators participate in joint data analysis, training workshops, and protocol development, facilitating a shared learning environment across institutions. The project also extends its reach to pre-college audiences through outreach activities such as Brain Camp and research exposure programs, introducing high school students to molecular biology concepts, laboratory skills, and scientific career pathways. By integrating teaching, research, and outreach, the project builds a robust educational pipeline that supports the development of the next generation of scientists and educators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has created a robust environment for training and professional development by seamlessly integrating teaching and research activities. Undergraduate and graduate students received hands-on experience in cutting-edge molecular biology techniques, including CRISPR gene editing, single-nucleus RNA sequencing (snRNA-seq), confocal imaging, and behavioral phenotyping in Drosophila melanogaster. These research methods were directly incorporated into the curriculum of core courses such as Genetics, Biotechniques, and Developmental Genetics, allowing students to apply what they learned in the classroom to real-world experimental systems. Through independent research projects, students were trained to design experiments, handle and phenotype Drosophila lines, extract RNA, run PCRs, and analyze transcriptomic data. They also conducted behavioral assays, including the proboscis extension reflex (PER), sleep analysis, and lifespan studies, to gain a comprehensive understanding of gene function in the context of dietary interventions. These experiences equipped students with the technical skills and critical thinking needed for careers in biomedical research. In addition to laboratory training, students engaged in scientific writing, data interpretation, and presentation of results. Several students contributed to poster presentations, internal seminars, and the preparation of a peer-reviewed abstract presented at the 66th Annual Drosophila Research Conference. Faculty mentorship and collaborative discussions with partner institutions further enriched their professional development by exposing them to interdisciplinary approaches in functional genomics and neurobiology. Collectively, the project provided a highly integrated training platform that combined formal instruction, experimental research, and professional communication, effectively preparing students for graduate school, research careers, and biotechnology-related professions. How have the results been disseminated to communities of interest? The project's outcomes have been effectively communicated across academic and educational communities through a combination of research presentations, classroom instruction, and institutional engagement. A significant milestone in dissemination was the presentation of a peer-reviewed abstract, "TRPV1-Independent Capsaicin Signatures in the Drosophila Brain Transcriptome," at the 66th Annual Drosophila Research Conference, where the team shared key data and insights with a national and international audience of experts in genetics and neuroscience. Within West Virginia State University, the project's findings were directly integrated into undergraduate and graduate courses, including Genetics, Biotechniques, and Developmental Genetics. Students were exposed to real data sets, experimental workflows, and analytical techniques derived from the project, gaining firsthand experience with transcriptomics, CRISPR applications, and behavioral assays in Drosophila melanogaster. These instructional activities enhanced the learning experience and strengthened the link between classroom theory and practical research.Collectively, these efforts--including conference presentations, educational integration, institutional seminars, and collaboration with partner universities--have ensured the wide dissemination of project findings to key communities engaged in research, teaching, and student training. What do you plan to do during the next reporting period to accomplish the goals?In the upcoming reporting period, the project will build on current progress by expanding both research and educational components. A major research focus will involve continued analysis of the single-nucleus RNA sequencing (snRNA-seq) data to explore differential gene expression across neuronal cell types under various phytochemical treatments. This will include cross-treatment comparisons and pathway enrichment analyses to better understand sex-specific transcriptional responses. In parallel, we will advance our functional validation studies by completing phenotypic screening of newly generated double and triple CRISPR knockout lines, assessing behaviors such as sleep, locomotion, feeding, and lifespan.To further integrate teaching with research, we plan to develop and launch a new upper-level undergraduate course focused on Bioinformatics and Model Organism Research. This course will introduce students to data analysis tools, including transcriptomic and functional genomic datasets generated from this project, while offering foundational instruction in comparative genomics, gene annotation, and pathway analysis. Students will use real experimental data to conduct guided research projects, reinforcing key concepts in genetics, neuroscience, and systems biology.Additional plans includerefining confocal imaging protocols to map neuronal activity and circuit-specific gene expression inDrosophilabrains, as well as preparing manuscriptsfor publication in peer-reviewed journals. Project results will be presented at upcoming scientific conferences, and collaborative efforts with partner institutions will continue to support cross-training and joint data analysis. These activities will ensure steady progress toward the project's research goals while further enriching the educational experiences of participating students.

Impacts
What was accomplished under these goals? During the reporting period, the project successfully integrated teaching and research to advance its primary objectives--exploring the health effects of dietary phytochemicals such as capsaicin, curcumin, resveratrol, and bitter melon using Drosophila melanogaster as a model organism. Significant accomplishments include the completion of both bulk and single-nucleus RNA sequencing (snRNA-seq) of Drosophila brains exposed to capsaicin, which revealed 44 distinct neural cell clusters and highlighted key pathways involved in metabolism, neural signaling, and stress response (e.g., mTOR, AMPK, and TGF-β). These data form the foundation for downstream functional validation and behavioral studies. The project made substantial progress in functional genomics by generating and phenotyping CRISPR-based single and double knockout mutant lines for candidate genes associated with capsaicin response. These mutants were analyzed in sleep and locomotion assays, confirming that capsaicin and curcumin increase sleep fragmentation, while resveratrol improves sleep quality and promotes activity, particularly in female flies. Lifespan studies also demonstrated that capsaicin and its non-pungent analog capsiate promote longevity, underscoring their potential anti-aging properties. Teaching and research were strategically integrated into the curriculum of Genetics, Biotechniques, and Developmental Genetics courses. Students were introduced to real-world applications of CRISPR, transcriptomics, and phenotyping through laboratory modules that used data and techniques developed in this project. Lab sessions involved RNA isolation, PCR, fly handling, behavioral scoring, and data interpretation, directly linking course content to active research objectives. This integration enhanced student engagement and fostered a deeper understanding of experimental design and functional genomics. Additionally, students participated in independent research projects aligned with the project's goals, contributing to ongoing experiments and co-authoring abstracts and manuscripts. One major dissemination effort was the presentation of a peer-reviewed abstract at the 66th Annual Drosophila Research Conference, showcasing the transcriptomic and behavioral effects of capsaicin in Drosophila. These efforts not only advanced the research aims of the project but also provided valuable professional development opportunities for students through mentorship, data analysis training, and scientific communication.Overall, the project successfully synthesized research advancements and student education, establishing a dynamic framework for experiential learning while making meaningful contributions to the fields of functional genomics, behavioral biology, and nutraceutical science.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Corral-Guerrero IA, Mart�nez-Medina AE, Alvarado-Mata LY, Ch�vez ACF, Mu�oz-Garc�a R, Lu�vanos-Escare�o MP, Sosa-Mart�nez JD, Castro-Alonso MJ, Nimmakayala P, Reddy UK. Capsaicin as a Microbiome Modulator: Metabolic Interactions and Implications for Host Health. Metabolites. 2025; 15(6):372.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Belcher, S., Flores-Iga, G., Natarajan, P., Crummett, G., Talavera-Caro, A., Gracia-Rodriguez, C., ... & Reddy, U. K. (2024). Dietary Curcumin Intake and Its Effects on the Transcriptome and Metabolome of Drosophila melanogaster. International Journal of Molecular Sciences, 25(12).
• Type: Conference Papers and Presentations Status: Other Year Published: 2025 Citation: Flores-Iga, G., Amirthalingam, M., Kayastha, P., Lopez-Ortiz, C., Nimmakayala, P., & Reddy, U. (2025). TRPV1-independent capsaicin signatures in the Drosophila brain transcriptome. Paper presented at the 66th Annual Drosophila Research Conference. https://submissions.mirasmart.com/Drosophila2025/Itinerary/PresentationDetail.aspx?evdid=1401