2022 SIVB Student Award Winners

2022 GORDON SATO AND WALLY MCKEEHAN AWARD

Assessing the Apoptosis Effect of Prenylated Stilbenoids Combined with Paclitaxel in Triple-negative Breast Cancer Cells

Breast cancer is one of the most prevalent types of cancer in women worldwide. Triple-negative breast cancer (TNBC) is unresponsive to typical hormonal treatments causing it to be one of the deadliest forms of breast cancer. Investigating alternative therapies to increase survival rates for this disease is essential. This study aimed to examine if prenylated stilbenoids from peanut can act as an adjuvant for paclitaxel, a chemotherapeutic drug with severe side effects. The prenylated stilbenoids arachidin-1 (A-1) and arachidin-3 (A-3) are analogs of resveratrol (RES) and were produced in hairy root cultures of peanut. The cytotoxicity activity of A-1, A-3, and RES was studied in TNBC cell lines MDA-MB-231 and MDA-MB-436. Furthermore, the cytotoxicity of A-1, the most potent prenylated stilbenoid, combined with paclitaxel was studied by checkerboard assays in the TNBC cell lines. The apoptotic effects of this combination treatment were studied by western blotting targeting protein expression levels of PARP, caspase-8, caspase-9, and survivin and through the Apo-ONE Homogeneous Caspase-3/7 assay. To further investigate the apoptosis and cell cycle stages, cells treated with prenylated stilbenoids or RES were studied using flow cytometry. After 24 hours of treatment, A-1 exhibited higher cytotoxicity than A-3 and RES with approximately 11-fold and 6-fold lower IC50, respectively, in MDA-MB-231 cells, and 9-fold and 8-fold lower IC50, respectively, in MDA-MB-436 cells. A-1 did not show significant cytotoxicity in the non-cancerous cell line MCF-10A. Cytotoxicity, checkerboard, and flow cytometry assays showed a decrease in paclitaxel concentration when combined with prenylated stilbenoids. This highlights the significance of continuing research with prenylated stilbenoids as an adjuvant in TNBC treatment.

Sepideh Mohammadhosseinpour, Arkansas State University, PO Box 639, State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 58:S19 2022

2022 PHILIP R. WHITE AWARD

Highly Conserved sgRNA Target Sequences Support Cas9-mediated Mutagenesis of LIGULELESS1 in Both Sorghum and Sugarcane

Sugarcane is a prime feedstock for commercial production of biofuel and table sugar. Its highly polyploid genome is the most complex of any domesticated agricultural species (2n = 100–120). This complicates crop improvement by both traditional breeding and genome editing. Sorghum has a diploid genome and its exons have a high level of sequence conservation to those of sugarcane. Establishing protocols to achieve robust, specific, and efficient multiallelic editing remains challenging in most C4 grasses despite the flexibility and efficiency of RNA guided nucleases like CRISPR/Cas9. Therefore, our objectives were to; (1) introduce CRISPR/Cas9-mediated mutations into LIGULELESS1 (LG1) to create a rapidly identifiable phenotype by changing the leaf inclination angle, and (2) evaluate if highly conserved sgRNA target sequences for LG1 support Cas9-mediated mutagenesis in both sorghum and sugarcane. Genome editing reagents were co-delivered into immature embryos of sorghum (var. Tx430; single LG1 copy) and sugarcane calli (var. CP88-1762; 32 LG1 copies) alongside the nptII selectable marker via biolistic gene transfer. The same guide RNAs were used in both crops. Transgenic lines were regenerated following selection, and edits were confirmed via Sanger or NGS sequencing. In sorghum, a single nucleotide, monoallelic insertion at the lg1 target site conferred an upright leaf phenotype in tissue culture that persisted after transfer to soil. T1 progeny of a sorghum event carrying the insertion were analyzed and biallelic lg1 knockouts resulted in a complete lack of ligules and more severe reduction in leaf inclination angle than monoallelic lines. Transgene free, edited events were also recovered following Mendelian segregation. In comparison, sugarcane lines exhibiting between 10-100% lg1 knockout have been obtained and results from phenotypic analysis will be presented. This work highlights lg1 knockout as a suitable strategy for creating a rapidly scorable phenotype and confirms the potential of sorghum as a model species for sugarcane gene editing.

Eleanor Jane Brant, University of Florida, 3054 McCarty Hall, Gainesville, FL. In Vitro Cellular and Developmental Biology, 58:S49-50 2022

2022 JOSEPH F. MORGAN AWARD AND STUDENT TRAVEL AWARD

Investigation of Endocytic Mechanisms of Lipid Nanoparticles (LNP-siRNA Systems) into Rainbow Trout Fish Cells In Vitro

Lipid nanoparticles (LNPs) are small particles (~50-60 nm in diameter) used to encapsulate drugs or nucleic acids (such as siRNA) for delivery to cells or tissues. Endocytosis is a regulatory process through which cells internalize particles in membrane vesicles. RTgill-W1 and RTgutGC are well-characterized, metabolically active rainbow trout (Oncorhynchus mykiss) epithelial cell lines used for in vitro studies. The aim of this research was to establish the minimum time required for endocytosis of LNP-siRNAs occur in RTgill-W1 and RTgutGC cells, and to determine which endocytic mechanism fish cells use to take-up LNPs. DiI-fluorescent labeled LNP-siRNA particles were used to assess cellular uptake in RT cells using 2, 4, 6, 8, 12, 16, and 24 hour time points. RTgutGC began to show uptake by 2 hours; RTgill-W1 began to show uptake by 4 hours, and both cell lines had significant uptake by 8 hours. A variety of inhibitors were used to study the mechanism of endocytosis including chlorpromazine (inhibits clathrin-mediated endocytosis), nystatin (inhibits caveolae-dependent endocytosis), genistein (inhibits caveolae-mediated endocytosis) and amiloride (inhibits micropinocytosis). To determine the mechanism(s) of endocytosis, cells were pre-treated with inhibitors and DiI-fluorescent LNP-siRNAs. Results forthcoming on LNP-siRNA cellular uptake following endocytic inhibition.

Katriana Van Woudenberg, Department of Biology, University of the Fraser Valley, Abbotsford, BC, CANADA. In Vitro Cellular and Developmental Biology, 58:S32-33 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

Knock-down of Vital Gene(s) of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) Using in Planta RNAi

RNA interference is a highly sequence-specific silencing process and can inhibit specific gene functions in targeted insect pests by utilizing dsRNA. To combat crop losses from damaging insect pests, dsRNA-based technologies proved to be an efficient and promising insect pest management strategy. Tomato is the seventh most important crop species after maize, rice, wheat, potatoes, soybeans, and cassava. Turkey is the fourth-largest producer of tomatoes and currently facing serious production losses due to pest infestation especially American tomato pinworm, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), which is a devastating insect pest of tomato. In this study, we reported a dsRNA-based approach against American Tomato Pinworm vital genes which comprises of development of transgenic tomato lines expressing Sec23dsRNA and Ache1dsRNA, which is reported for the first time and developed tomato cv. Rio Grande transgenic lines showed a significant level of resistance against Tuta absoluta (Meyrick). The gene Acetylcholine esterase1 (AChE1) in insects particularly functions in neurotransmission, and Sec23 protein present in insects is one of the components of the coat protein complex II (COPII) that promotes the formation of transport vesicles from the endoplasmic reticulum (ER). Insects fed on transgenic lines expressing dsRNA against AChE1 and Sec23 mRNA resulted in the mortality of larvae, delayed pupation, abnormal growth causing the suppression of Tuta absoluta population. Hence, the reported dsRNA-based insect pest management strategy will serve as a breakthrough to be the cost-effective and eco-friendly approach.

Muneeb Hassan Hashmi, University of Siegen and Nigde Omer Halisdemir University, 7076 Siegen, Germany. In Vitro Cellular and Developmental Biology, 58:S24-25 2022

2022 STUDENT TRAVEL AWARD

Efficient, Multi-allelic Editing for the Genetic Improvement of Bahiagrass (Paspalum notatum Flüggé)

Polyploidy, apomixis and self-incompatibility complicate the genetic improvement of bahiagrass (Paspalum notatum Flüggé), an important forage and turf grass in the Southeast United States. Gene editing techniques such as CRISPR/Cas9 would bypass chromosome doubling and repeated crosses necessary for conventional breeding of this species and could result in production of improved cultivars in a single generation. A pair of guide RNAs targeting magnesium-protoporphyrin IX chelatase (MgCh) along with Cas9 and the NPTII selectable marker were delivered to embryogenic callus cultures of the apomictic, autotetraploid bahiagrass cultivar ‘Argentine’ using particle bombardment. Chlorophyll-depleted lines, which were confirmed for multi-allelic edits with Sanger and next generation sequencing, were obtained across two independent experiments at efficiencies of 23 and 50% of total transgenic events. This approach has supported further endeavors to genetically improve turf and forage quality in bahiagrass including confirmed targeted mutagenesis of genes involved in functional stay-green, lipid accumulation and lignin composition. In addition, the transmission of edits to apomictic progeny was analyzed, and the feasibility of production of transgene-free edited apomictic lines via heat-inducible Cre/lox site-specific recombination was explored. Overall, these efforts have resulted in an efficient, reproducible CRISPR/Cas9 gene editing protocol for Paspalum notatum, a development with beneficial implications for future breeding efforts in this species.

David May, University of Florida, Agronomy Department, 3062 McCarty Hall D, Gainesville, FL. In Vitro Cellular and Developmental Biology, 58:S25-26 2022