2022 Student Awards

2022 WILTON R. EARLE AWARD AND 2022 STUDENT TRAVEL AWARD

Targeted Mutagenesis of Vacuolar H+ Translocating Pyrophosphatase (V-PPase) Promoter Limits Sucrose Formation and Disturbs Cytosolic pH During Germination in Rice

Germination and seedling growth in rice relies on the breakdown of starch to generate sucrose in the endosperm. This process is inhibited by inorganic pyrophosphate (PPi). An enzyme, Vacuolar H+ translocating pyrophosphatase (V-PPase), hydrolyzes PPi into inorganic phosphate (Pi) as it translocates H+ from the cytoplasm into the vacuole. Although the enzymatic activity of V-PPase is known, its function in germination and seedling growth is poorly understood. To explore the role of V-PPase in these stages, it was downregulated by heat and mutagenesis using CRISPR/Cas9. Downregulation by heat provides understanding on its mechanism in maintaining cytoplasmic pH while mutagenesis elucidates its contribution in germination and growth. In mutagenesis by CRISPR/Cas9, the resulting lines harbored a mutation in one of the four predicted promoter elements, an ATC deletion in GATA. Interestingly, when six selected lines with homozygous ATC deletions were subjected to gene expression analysis, V-PPase was found to be downregulated. Additionally, these lines showed lower germination rates and slower seedling growth. Understanding that V-PPase downregulation can inhibit sucrose formation due to the potential build-up of PPi, the lines were tested for sucrose content to explain their poor germination and growth. All lines had a lower sucrose content, but external supply reverted their growth to normal. In downregulation by heat, calli expressing the pH sensitive reporter, Green Fluorescent Protein (GFP), demonstrated a decrease in cytoplasmic pH. Altogether, V-PPase impacts cytoplasmic pH, a regulator of enzyme activities and the breakdown of starch to form sucrose, an important substrate for seedling growth.

Dominic Dharwadker, University of Arkansas Fayetteville, 495 N. Campus Drive, Plant Sciences 125, Fayetteville, AR. In Vitro Cellular and Developmental Biology, 58:S26 2022

2022 JOHN S. SONG AWARD

Comparative Assessment of Antioxidant Activity of Prenylated Stilbenoid-Rich Extracts from Elicited Hairy Root Cultures of Three Different Cultivar of Peanut

Over-production of reactive oxygen species (ROS) induces oxidative stress by damaging lipids, membranes, proteins, and DNA at the cellular level. This phenomenon results in several pathogeneses like cardiovascular diseases, cancer, neurodegenerative disease, and aging. Stilbenoids and their derivatives possess antioxidant activity and act to counteract these ROS, thereby alleviating oxidative stress. However, the antioxidant capacity of these stilbenoids is yet to be explored in depth. This project aimed to assess the antioxidant capacity of stilbenoid-rich extracts from hairy root cultures of peanut cultivars Hull, Tifrunner, and Georgia Green in vitro using the DPPH (2,2-diphenyl-1picrylhydrazyl) assay. To produce the stilbenoid-rich extracts, hairy root cultures of the three cultivars of peanut were co-treated with methyl jasmonate, methyl-β-cyclodextrin, hydrogen peroxide, and magnesium chloride for 168 h. Different levels of resveratrol and prenylated stilbenoids were detected in the ethyl acetate extract of the culture medium. The stilbenoid-rich extracts from the Tifrunner hairy root culture showed a higher amount of arachidin-1 and arachidin-6. Upon reaction with DPPH, the Tifrunner stilbenoid-rich extracts had significantly higher antioxidant activity at the lower concentration of 6.25 µg/mL and 3.125 µg/mL when compared to extracts from cultivars Hull and Georgia Green. The stilbenoid-rich extracts from peanut hairy root cultures may provide a formulation for nutraceuticals to promote human health.

Gaurav Gajurel, Arkansas State University, 105 N Caraway #443, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 58:S38 2022

Lipid nanoparticles (LNPs) encapsulating nucleic acids such as mRNA and siRNA have been used in many types of research and clinical settings as they allow for non-viral gene delivery. For example, siRNA has been used in eukaryotic cells to induce RNAi and cause subsequent degradation of target mRNA. Although there has been plenty of research done on the therapeutic use of LNP systems in humans, there has been little use of LNPs or other nanoparticles to induce gene expression or gene silencing in aquaculture. Fish mortality due to infectious diseases is a major concern in the aquaculture industry, but the reliance on antibiotics can lead to complications such as antibiotic resistance in both fish and humans. LNPs encapsulating nucleic acids such as siRNA (LNP-siRNAs) can potentially be utilized as replacements for these antibiotics. The cell lines used in this research include three established rainbow trout cell lines: a gill epithelial cell line (RTgill-W1), a gut epithelial cell line (RTgutGC), and a macrophage cell line that stably expresses Turbo green fluorescent protein (RTS11-GFP). The present research aims to examine cellular uptake, gene expression, gene silencing, and cytotoxicity through the use of fluorescently Dil-labeled LNP-siRNA formulations. Preliminary results indicate that Dil-LNP-siRNAs are successfully taken up by the tested rainbow trout cell lines and that there are no significant cytotoxic effects. GFP-DsiRNA was also transfected into the RTS11-GFP cell line and RT-PCR was used to verify gene expression and gene silencing prior to LNP encapsulation.

Heather Kelly, Department of Biology, University of the Fraser Valley, Abbotsford BC, CANADA. In Vitro Cellular and Developmental Biology, 58:S32 2022

2022 HONOR B. FELL AWARD AND STUDENT TRAVEL AWARD

Novel Mammalian Fibroblast Cell Culture Media Technique for Ultraviolet Cell Reduction

Carcass bruising from horns costs the Canadian cattle industry $10 million a year (Anderson, 2012). It is vital to develop safe and cost-effective advancements in dehorning procedures. Removal of horns raises risk of discomfort and pain to the animal; utilizing ultraviolet (UV) light as treatment can be effective when compared to current methods. The objective was to develop a novel media and technique to effectively culture horn producing cells for in vitro UV experimentation. Three media variations failed due to lack of cell reproduction and presence of fungal or bacterial colonies. Horn producing cells demonstrated an average increase of 31.39% in a novel media mixture of 20 ml Ham’s F12, 2% Amphotericin B from Streptomyces sp., and 1% penicillin. A UV prototype, developed in previous research was improved to guarantee isolated UV treatment of horn producing cells. Varying times were adapted for ultraviolet treatment. Horn producing cells were cultured in this media and evaluated by cell count using ImageJ analysis of digital images. Statistical analysis found significant relationships between cell death or reduction and run time of the UV lamp. As light run time increased, mitosis decreased. Ultraviolet light substantially reduced mitosis as compared to controls without UV exposure. Prototype design and implementation were effective for isolated treatment of cells in novel media.

Jasmyn M. Hoeger, University of Dubuque and Tri-Vet Associates, 32078 Floyd Road, Dyersville, IA. In Vitro Cellular and Developmental Biology, 57:S32 2022

2022 CELLULAR TOXICOLOGY AWARD

Antimicrobial Studies of 1,3-Diphenylpyrazole-derived Anilines Against Methicillin-resistant Staphylococcus aureus

Several bacteria (the “ESKAPE” pathogens) are associated with nosocomial infections and antimicrobial resistance, including Staphylococcus aureus, is considered a high threat to human health. The development of new antibiotics is one of the strategies recommended by the CDC to combat the increased prevalence of antibiotic-resistant bacterial infections. Following compound synthesis, compounds were screened for antimicrobial activity using minimum inhibitory concentration testing (MIC) against various strains of S. aureus including methicillin-resistant S. aureus (MRSA). Compounds with low MICs were further investigated to determine their Minimum Bactericidal Concentration (MBC),ability to inhibit biofilm formation and destruction of preformed biofilms in vitro, time-kill assay to determine their bacteriostatic or bactericidal properties, persister kill assay to determine their ability to eliminate MRSA persisters, and multistep resistance to determine resistance accumulation. MIC values as low as 0.78 µg/mL were observed among the compounds tested. For additional testing, compounds with MICs ranging from 6.25 µg/mL to 0.78 µg/mL were selected. MBC test eliminated bacteria at as low as 3.125 μg/mL concentration. At 2×MIC concentrations, biofilm inhibition was observed almost 100% and preformed biofilm removal was around 90%. Three of the compounds were found to be bactericidal and one bacteriostatic against MRSA in a time-kill assay. MRSA persisters were significantly reduced over the four-hour treatment period at 8×MIC in comparison to the untreated and positive controls. The preliminary result of multistep resistant testing showed a four-fold increase in initial MIC value in the course of 14 passages. In this presentation, we will discuss the above antimicrobial properties of the 1,3-diphenylpyrazole derivatives along with on-going in vivo antimicrobial studies to clear bacterial infection in Galleria mellonella.

Hansa Raj KC, Arkansas State University, 2009 Cedar Heights Dr. Apt B1, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 58:S20-21 2022

2022 STUDENT TRAVEL AWARD

Mapping Interneuron Migration During Late Neurodevelopment in the Piglet Brain

Inhibitory neurons (interneurons) are a neuronal subpopulation that has been strongly implicated as causal to neurodevelopmental disorders, conditions such as autism spectrum disorder and epilepsy. Recent evidence has shown robust interneuron migration into the frontal cortex of the human brain during the first months of life from a region known as the ganglionic eminence. This suggests that the human cortex, the brain region associated with higher cognitive functions, remains dynamic even after birth. The migration patterns of these late-migrating interneurons are not well understood or how they might contribute to the final stages of cortical development. Our gaps in knowledge arise from the rarity of samples from this late neurodevelopmental stage and limitations in current animal models, such as the rodent brain. My study will implement a novel animal model, the domestic pig brain, to study the molecular diversity and organization of these cells and generate a map of interneuron migration in the early piglet cortex. Understanding this normal late cortical development is critical to identifying disruptive processes that could lead to neurodevelopmental diseases.

Keira Nakamura, University of California, San Francisco, 1651 4th St, San Francisco, CA 94158. In Vitro Cellular and Developmental Biology, 58:S33 2022