The following student awards were presented at the 2023 In Vitro Biology Meeting held at the Hilton Norfolk The Main in Norfolk, Virginia from June 10 – 14, 2023. Information on additional awardees at the 2023 In Vitro Biology Meeting were presented in the last  issue of the In Vitro Report. Information related to the available specific student awards can be found here or by contacting the SIVB Business Office at [email protected].

2023 WILTON R. EARLE AWARD AND 2023 STUDENT TRAVEL AWARD

An Expected Journey: Following the Path of dsRNA-loaded Nanoparticles Through Human Cells

Dominic Dharwadker

Nicholas Jadaa

Immunostimulatory nucleic acids are RNA and DNA molecules that stimulate an innate immune response in a non-sequence-specific manner; one such molecule is long double-stranded (ds)RNA. These molecules are recognized by pattern-recognition receptors (PRRs), found in the cytoplasm, cell surface, and endosome. Due to their structure and sensitivity to nuclease degradation, nucleic acids benefit from the use of a carrier or delivery system such as a nanoparticle. It has not yet been fully elucidated how the addition of a nanocarrier modifies the natural route of dsRNA uptake and movement throughout the cell. Using two ovarian cancer cell lines, SKOV3 and OVCAR3, that show differential responses to dsRNA, we sought to elucidate the receptors and subsequent cellular compartment pathways of dsRNA-carrying nanoparticles. As expected, based on unbound dsRNA molecules, the involvement of class-A scavenger receptors was confirmed using competitive ligands. Furthermore, fluorescent microscopy and lysosome-specific stains demonstrated the particles can be found in lysosomes. The data from this project will contribute to our foundational knowledge surrounding nanocarrier delivery of dsRNA, contributing to future applications for antiviral and anticancer therapies, and vaccine adjuvants.

Nicholas Jadaa, Masters of Integrative Biology, Department of Biology, Wilfrid Laurier University, Waterloo, ON, N2L 3C5, CANADA. Abstract presentation A-2008

2023 GORDON SATO AND WALLY MCKEEHAN AWARD

Encapsulation of Long dsRNA in Extracellular Vesicles, SARS-CoV2 Virus-like Particles, and Liposomes for the Stimulation of Antiviral RNA Interference Against Human Coronaviruses

Dominic Dharwadker

Dominique Daniels

Viruses produce long dsRNA (LdsRNA) during replication, and its presence in the cell triggers the innate antiviral immune responses. In the RNA interference response to LdsRNA (dsRNAi), LdsRNA is used as a template to bind complementary viral mRNA and halt viral protein replication. While in vitro delivery of 700 bp LdsRNA can stimulate the dsRNAi response in human cells against Human coronaviruses, we hypothesize that in vivo antiviral treatments will require carriers for tissue targeting and protection from LdsRNA degradation. We evaluated extracellular vesicles (EVs) and virus-like particles (VLPs) as carriers for 700 bp HCoV-229E N gene LdsRNA in treatments against HCoV-229E. LdsRNA-EVs were produced by treating U937 (human monocytes) with LdsRNA and extraction of EVs with ExoQuickÒ. To encapsulate LdsRNA, SARS-CoV2 VLPs were disassembled in 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1mM EGTA, 20mM DTT, then reassembled with 5mM CaCl2 after LdsRNA addition. A green fluorescent protein (GFP) plasmid was encapsulated in VLPs and delivered to HepG2 (human hepatoblastoma) cells. LdsRNA carriers were treated with RNase III and GFP-VLPs were treated with DNase I. LdsRNA Immunoblot confirmed LdsRNA packaging in EVs and protection against RNase III degradation. HEL-299 were treated with LdsRNA-EVs and dsRNA delivery was confirmed by immunocytochemistry. Agarose gel electrophoresis (AGE) of LdsRNA-VLPs showed successful LdsRNA encapsulation and protection from RNAse III degradation. LdsRNA Immunoblot confirmed LdsRNA packaging in EVs and protection against RNase III degradation. AGE of GFP-VLPs before and after DNase I treatment confirmed plasmid encapsulation and fluorescence microscopy of GFP-VLP treated HepG2 cells showed GFP expression suggesting an intact delivery mechanism. EVs and VLPs can encapsulate and protect LdsRNA from degradation and are suitable LdsRNA carriers for antiviral experiments. This is the first study to explore EVs or VLPs as LdsRNA carriers for antiviral dsRNAi in vertebrates, and if effective this strategy could be applied to other vertebrate viruses.

Dominique Daniels, Department of Biology, Faculty of Science, Wilfrid Laurier University, Waterloo, ON, CANADA. Abstract presentation A-1001.

2023 PHILIP R. WHITE AWARD

Understanding the Regulation of Fruit Abscission in Physalis grisea

Dominic Dharwadker

Elise Tomaszewski

Groundcherry (Physalis grisea) is an underutilized species in the Solanaceae family that produces small, sweet, yellow berries enveloped by an inflated calyx (husk). As a semi-domesticated species, groundcherry exhibits undesirable agronomic traits including a sprawling growth habit and extreme fruit abscission. Fruit abscission occurs via detachment of the pedicel along a region of pre-differentiated living cells called the abscission zone (AZ). Compared to other types of plant organ abscission, fruit abscission is not well understood on the molecular level. This project utilizes groundcherry as a model to understand the mechanisms of fruit abscission. It also serves to create improved groundcherry varieties with reduced fruit abscission for large-scale agronomic cultivation. To understand how the abscission zone is regulated, a multifaceted approach is being conducted. Specifically, a reverse genetics approach is being conducted utilizing CRISPR/Cas9 to edit fruit abscission-related genes. This approach will functionally validate abscission development genes for a deeper understanding of the molecular regulation and cellular differentiation behind fruit abscission. We hypothesize that editing specific AZ related genes will disrupt the formation and separation of the AZ, generating mutants with reduced fruit shedding. Lastly, the AZ will be imaged throughout development using paraffin-embedded tissue. This work will characterize and capture the first images of the groundcherry AZ development. It will also aid in illustrating differences in the AZ between abscission mutants and WT. Results from this work will not only lead to an understanding of groundcherry fruit abscission but will also expand the general knowledge of fruit AZ development and detachment.

Elise Tomaszewski, Plant Breeding and Genetics Graduate Program, Cornell University, Ithaca, NY 14853 and Boyce Thompson Institute, 533 Tower Road, Ithaca, NY 14853. Abstract presentation P-2002

2023 CELLULAR TOXICOLOGY AWARD

Improved Potency of Long Double-stranded RNA in Ovarian Cancer Cells Using a Cationic Polystyrene Latex Nanocarrier.

Dominic Dharwadker

Natalie L. Aldor

Ovarian cancer is a deadly cancer, often diagnosed in the late stage with a low survival rate. Immunotherapies are an avenue to produce novel and more effective ovarian cancer therapies. Long double-stranded (ds)RNA is a potent immunostimulatory nucleic acid that induces innate immune responses through type I interferon and proinflammatory cytokines. Nucleic acid therapies require a delivery system, in the present study, we use cationic polystyrene latex nanoparticles (clNPs) as carriers for immunostimulatory dsRNA. The nanocarrier enhanced delivery to the ovarian cancer cell line, SKOV3, demonstrating increased cellular association compared to unbound dsRNA. The clNPs boosted the dsRNA-induced innate immune response in SKOV3 cells as measured by transcript expression of two interferon-stimulated genes, 47- and 108-fold improvement over unbound dsRNA, and the establishment of a protective antiviral state, 3.3-fold improvement. Using clNPs to carry dsRNA potentiated the immunostimulatory power of the nucleic acid, further studies will continue to evaluate the potential of clNPs in novel anticancer therapies.

Natalie L. Aldor, Department of Health Sciences, Wilfrid Laurier University, 75 University Avenue W, Waterloo, ON N2L 3C5, CANADA. Abstract presentation A-2009.

2023 HOPE E HOPPS AWARD AND STUDENT TRAVEL AWARD

Mapping Interneuron Migration During Late Neurodevelopment in the Piglet Brain

Dominic Dharwadker

Mst. Sayaduhhhara

As photosynthetic prokaryotes, pigments such as chlorophyll, carotenoids and phycobiliproteins in cyanobacteria are exploited for their high commercial value in cosmetics and dyes. In the present study, we evaluated the impact of 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 mg/L nano-titanium dioxide (n-TiO2) nanoparticles on the growth, chlorophyll a fluorescence, and phycocyanin accumulation in Fremyella diplosiphon strains, B481-WT and B481-SD. In addition, oxidative stress in response to n-TiO2 was quantified using the 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) probe and ATP synthase activity detected using densitometric analysis. We observed significantly higher growth of B481-SD and B481-SD at 2 mg/L n-TiO2 on day 12. In addition, we observed higher phycocyanin and chlorophyll content at 2 mg/L on day 9 in B481-SD strain while there was no significant enhancement of growth in any of the concentrations and intervals tested in B481-WT. Comparison of oxidative stress using the DCFH-DA probe indicated significantly different ROS in both strains treated with 2 and 16 mg/L n-TiO2. Densitometric analysis of ATP synthase in B481-SD revealed significantly higher activity at 0.5, 2, and 128 mg/L n-TiO2 than the untreated control; however significantly higher levels of activity was observed only at 2, and 128 mg/L in B481-WT. Results of the present study indicated 2 mg/L n-TiO2 to be optimal in enhancing growth, pigment accumulation and ATP synthase activity. Future efforts will be aimed at studying transduction mechanisms that cause metabolic signaling as well as photosynthesis in response to n-TiO2 in F. diplosiphon. This research was supported by the National Science Foundation’s Nanoscale Interactions Program grant under award number 1900966 and co-supported by the Excellence in Research. 

Mst. Sayaduhhhara, Morgan State University, 1700 E. Cold Spring Lane, Baltimore, MD 21251. Abstract presentation A-2024.

2023 STUDENT TRAVEL AWARD

Pyramiding MicroRNAs for Transgene Containment and Broad Plant Abiotic Stress Resistance

Dominic Dharwadker

Zhaohui Chen

Abiotic stresses such as salinity, heat and drought seriously impair plant growth and development, causing significant losses in crop yield and ornamental value. Plants have evolved different defense mechanisms responding to the varying environmental adversities. Biotechnology approaches manipulating specific genes prove to be an effective strategy to engineer traits that are difficult to obtain with traditional breeding. In perennial grasses, however, the risk of transgene escape and the unforeseen environmental consequences by the use of transgenic technology require development of strategies for engineering male or total sterility for transgene containment. Here, we have developed a novel approach to produce self-contained superior transgenic turfgrass using the dual roles of microRNAs in plant reproductive development and beneficial agronomic trait improvement. Specifically, a microRNA gene miR396 that regulates plant sterility and abiotic stress responses was introduced into turfgrass along with three other miRNA genes miR319, miR528, and miR393, all of which are positive regulators of plant abiotic stress responses. Transgenic lines with stacked miRNA genes are being evaluated for the efficacy of miRNA396-mediated sterility induction and transgene containment, as well as the synergistical effect of all miRNA genes on multiple beneficial agronomic traits. The feasibility of controlling multiple miRNA genes by a single promoter to facilitate biotechnology application will also be evaluated. The results of this study will allow the establishment of a new genetic containment system in grasses and provide new insights into the streamlined strategy for effective miRNA gene stacking for plant genetic engineering.

Zhaohui Chen, Masters of Biomedical Science, Department of Genetics and Biochemistry, Clemson University, Clemson, SC 296345. Abstract presentation P-2000.

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