Student Poster Competition

In Vitro Animal Cell Sciences

1st Place

Developing a Cell-linked Sandwich Immunoassay to Visualize the Spatial Distribution of Cytokines in Ex Vivo Lymph Node Slices

The lymph node (LN) is a spatially organized organ where lymphocytes communicate by secreting cytokines in response to stimuli. Since most cytokines are believed to act locally, detection at the secretion site is needed to understand cell-cell communication in this complex tissue. However, no current technologies detect cytokine spatial distribution in live tissue except for genetically modified mice; an expensive option with a limited cytokine pool. Other methods rely on supernatant sampling and immunostaining fixed tissue which lose dynamic or spatial information. To address this gap, we are designing a tissue-anchored sandwich immunoassay using a cytokine-binder with low molecular weight, such as a peptide or nanobody, to capture cytokines at their secretion site in 300-µm thick murine LN tissue slices. We plan to covalently anchor cytokine-binding peptides in tissue by using click chemistry. We optimized the incubation time of the covalent anchoring reagents to achieved 50 µm penetration, and confirmed the click reaction occurs successfully in tissue. To identify cytokine-binding peptides, multiple phage libraries were screened against cytokine targets and the most promising peptide candidates were synthesized for testing by biolayer interferometry (BLI). To complete the sandwich assay, we are currently expressing and purifying his-tagged anti-cytokine nanobodies. Next steps will focus on testing the cytokine-binding peptide candidates and nanobodies by BLI to determine binding affinity, and optimize further if needed. Once a candidate is found with good affinity (nM range) and high selectivity, it will be synthesized and tested in tissue alongside the detection nanobody. Once completed, we envision that this assay will enable researchers to study the complex cytokine signaling pathways within both healthy and diseased tissue, allowing us to detect changes in cell communication.

Sahana Sankaran, University of Virginia, Charlottesville, VA 22903.  Abstract Presentation: A-2002.

2nd Place

Investigating CD40 and CD40L Expression and Neuronal Synaptogenesis Following Glioblastoma Invasion in Culture

Glioblastoma (GBM) is an aggressive and fatal brain cancer, with seizure-like activity presenting in ~50% of patients. These seizures suggest disruptions in neuronal signaling and immune pathways, but the effect of GBM on surrounding neurons remains unclear. Our lab previously confirmed that U-87 GBM cells express CD40 and CD40L, implicating these immune co-stimulatory molecules in tumor progression and neuron-tumor interactions. This study investigates how GBM invasion affects CD40/CD40L expression and neuronal synaptogenesis to better understand links between tumor growth, neuroinflammation, and epilepsy. To measure CD40/CD40L in mouse hippocampal and rat cortical neurons following GBM co-culture and monitor neuronal electrical activity. The study will evaluate other biomarkers, such as pAkt, generated during neuron-tumor interaction, and will assess Pi3k and activated Mapk signaling pathways. Neuron-tumor interactions are modeled by co-culturing rodent neurons or neurons derived from wt or Pten-deleted neural stem precursor cells (NSPCs) with GL261 (murine) or U-87 (human) GBM cells expressing EYFP. CD40/CD40L expression is assessed by immunocytochemistry (ICC) and fluorescence microscopy. Neuronal activity is recorded using microelectrode arrays (MEAs) and analyzed with a 3Brain system. Initial ICC and fluorescence microscopy confirmed CD40/CD40L expression in U-87 cells, supporting the hypothesis that this pathway contributes to tumor-induced neuronal changes. Data collection from co-culture experiments is ongoing, with current analysis focused on quantifying CD40/CD40L expression in neurons and assessing alterations in excitability following GBM invasion. This study aims to clarify molecular and functional mechanisms underlying GBM-associated seizures and may identify targets for early detection, prevention, and treatment in GBM-related epilepsy. By investigating both immune signaling and neuronal activity, this study contributes to a broader understanding of epilepsy and the neuroinflammatory processes involved in its onset.

Jack Campbell, Department of Biomedical and Translational Science and Department of Neurology Center for Integrative Neuroscience and Inflammatory Diseases, Macon & Joan Brock Virginia Health Sciences, Eastern Virginia Medical School, Old Dominion University, Norfolk, VA. Abstract Presentation: A-2010.

Plant Biotechnology

1st Place

Off-target Effects of the Site-specific Recombinases in Perennial Grasses

Recent advances in biotechnology have enabled efficient plant genetic transformation and expanded the application of genome editing for crop improvement. However, transgenes and other transformation components often require removal due to potential side effects and biosafety concerns, which are central to regulatory approval. Site-specific recombination systems offer a compelling approach for transgene excision, particularly in clonally propagated, self-incompatible, or polyploid crops (e.g., perennial grasses) where sexual segregation is impractical. Although commonly used for DNA excision and transgene containment, these systems can induce unexpected phenotypic changes through off-target genetic and epigenetic modifications. To investigate these effects, we constitutively expressed Cre and PhiC31 recombinases in creeping bentgrass (Agrostis stolonifera). Preliminary data show that PhiC31 significantly enhances plant growth and abiotic stress resistance, whereas Cre modestly improves development but impairs salt stress tolerance. RNA-seq analysis on PhiC31 transgenic plants suggests that an elevated anti-recombinase response may underline increased growth and stress resilience. We also identified numerous putative recombinase recognition sites and are developing a reporter system to confirm their activity. This study will provide insights into the safe and effective use of site-specific recombination systems for producing environmentally responsible transgenic crops.

Xiaotong Chen, Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634. Abstract Presentation: P-2045.

2nd Place

An RNA/DNA Helicase May Be Involved in Double-strand Break Repair (DSBR) In Plant Mitochondria.

Plant mitochondrial DNA (mtDNA) is known to contribute to agriculturally relevant traits including fertility, yet the control of these traits is not well understood. MtDNA is subject to damage from respiration byproducts, which needs to be repaired. Plant mitochondrial genomes are large and variable in size (180-12,000kb), often expand, rearrange frequently, yet have a very low mutation rate in genes. Plant mitochondria seem to use DSBR to repair most DNA damage, which explains both accurate repair in genes, and abundant rearrangements in junk DNA. To study the mechanism of DSBR repair in plant mitochondria, we used CRISPR to generate knockout mutants in nuclear-encoded mtDNA repair proteins to study their function in DSBR in Arabidopsis thaliana. Candidate genes were identified via a forward genetics approach. Briefly, seeds were mutagenized with EMS and plated onto ciprofloxacin. Ciprofloxacin is an antibiotic that causes DSBs in the mitochondrial genome. Plants susceptible to ciprofloxacin are assumed to have mutations in mtDNA repair genes. In this case, a point mutation was found in the mt-targeted RNA/DNA helicase, SUV3. To study the function of SUV3 on mtDNA repair, we designed two stacked gRNAs to target SUV3. Plants were transformed with the vector containing Cas9 and the gRNAs via floral dip with Agrobacterium. Transformed plants were identified with BASTA selection and through seed coat GFP. The resulting deletion is 417bp and was confirmed by sequencing. Plating the CRISPR suv3^– mutant on ciprofloxacin confirmed that these mutants are sensitive to DSBs. The suv3^– mutant was found to be sensitive to salt and heat stress, and to have a lower germination rate. Root assays, which can be an indicator for mtDNA stress, showed no difference to WT. Illumina sequencing showed little structural variation in the mitochondrial genome, although we suspect the short reads may miss low heteroplasmic rearrangements, so we await results with nanopore sequencing. These results highlight the complexity of plant mtDNA, and the need to better understand the proteins involved in DSBR.

Moira Rodriguez, Department of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588. Abstract Presentation: P-2051.