2024 SIVB Student Award Winners

2024 WILTON R. EARLE AWARD AND 2024 STUDENT TRAVEL AWARD

Fibrotic Responses Differ after Treatment with Artemisia sp., Artemisinin, and Its Derivatives

Fibrosis is a pathology linked to most diseases and prior studies indicated that various artemisinin (ART) derivatives (artesunate (AS), artemether (AM), dihydroartemisinin (DHA)) reduced fibrosis in vitro and in vivo. Artemisia annua L., is the natural source of ART and globally used as a tea infusion especially in underdeveloped countries to treat many diseases including malaria. A. afra contains no ART but is also antimalarial. Using human dermal fibroblasts (CRL-2097), we compared A. annua and A. afra tea infusions, ART, AS, AM, DHA, and a liver metabolite of ART, deoxyART (dART) for their effects on fibroblast growth and expression of key fibrotic marker genes and proteins after 1 and 4 d treatment. A resazurin assay showed that AS, DHA, and both Artemisia teas significantly reduced fibroblast proliferation after 1 d post-treatment, whereas ART and AM did not. After 4 d, all treatments showed less proliferation except AM and dART. Unlike AS and DHA, ART had no significant effect on fibrotic gene expression. Although A. annua contains ART, it had a significantly greater anti-fibrotic effect than ART. Using mouse anti-SMA and anti-mouse Alexa488 conjugated secondary antibodies an immunofluorescent stain for smooth muscle actin (α-SMA) correlated with PCR results of drug-treated fibroblasts. Together, the PCR and immunocytochemistry results showed that ART, AS, DHA, and the two Artemisia teas have different anti-fibrotic responses at both the level of transcription and translation of α-SMA. The fibroblast fibrotic phenotype is inducible by TGF-β. A resazurin assay showed that CRL-2097 cell proliferation decreased by 16% and 22%, 2 and 4 d, respectively, post TGF-β treatment suggesting faster induction to myofibroblasts. The effects of the two Artemisia teas, ART, and its derivatives are being studied further to determine their relative anti-fibrotic effects on the same target genes post TGF-β treatment. These results provide a foundation for studying possibly new artemisinin-based therapeutics

Trevor Bush, Worchester Polytechnic Institute, 100 Institute Rd., Worcester, MA 01609. Abstract presentation: A-2012

2024 GORDON SATO AND WALLY MCKEEHAN AWARD

Enhancing dsRNA Delivery and Uptake in Salmonids Using Liposomes and Nanophytoglycogen

Therapeutics are most effective when they are delivered to their target intact and concentrated enough to perform their function with minimal toxicity to the host. One class of prophylactic molecules of interest is long dsRNA, which is produced by all viruses at some point during their replication and serves as a robust stimulant of the innate immune system against all viruses. Another is CpG-ODNs, which are short, pro-inflammatory DNA sequences. The use of a carrier molecule serves to mitigate many of the challenges posed to drug delivery by the host immune system, such as enzymatic degradation. In this study, simple cationic liposomes demonstrated significant toxicity in rainbow trout (Oncorhynchus mykiss) gill (RTgill-W1) and gonadal (RTG-2) cell lines, rendering them an impractical carrier choice for stimulation of the innate immune response. Antiviral trials proved the need for an alternative carrier to prevent infection by Chum Salmon Reovirus (CSV). Contrastingly, initial studies in RTG-2 cells revealed that cationic nanophytoglycogen NanodendrixTM (NDx) was considerably less toxic than the liposomes when conjugated with either dsRNA or CpG-ODNs and could therefore be applied in much higher concentrations. This nanoparticle, derived from sweet corn, is a potential candidate to reduce CSV infection in rainbow trout cell lines. Chinook salmon embryonic cells (CHSE-214) that had their dsRNA sensor, melanoma differentiation-associated protein 5 (MDA5), gene knocked out by CRISPR modification naturally lack the membrane scavenger receptors (SR-As) required to internalize extracellular nucleotides. They are being employed to examine the ability of NDx to translocate long dsRNA into the cytosol without the assistance of a dsRNA receptor. Further, these modified cells will help determine if NDx can enhance the innate immune response in cells lacking key cytosolic dsRNA sensors and act as an adjuvant for peritoneal injection vaccines in rainbow trout. Developing prophylactic drug delivery systems for antiviral therapeutics can help protect Ontario’s thriving aquaculture industry, situating Canada as a global competitor as both the incidence of aquaculture disease and food demand continue to rise.

Shayne Oberhoffner, Department of Biology, Wilfrid Laurier University, Waterloo, ON N2L 3C5, CANADA. Abstract Presentation A-1013

2024 HOPE E HOPPS AWARD AND STUDENT TRAVEL AWARD

Integrating Histology and Phytohormone/Metabolite Profiling to Understanding of Yellow Camellia and American Chestnut Cutting Rooting

Adventitious roots (ARs) are plant roots that form from tissues other than roots via an organogenic process. For numerous plants, AR formation is crucial for successful vegetative propagation. However, many species are recalcitrant to adventitious root formation, imposing a major limitation in the clonal propagation of elite germplasms. Due to their relevance to basic plant biology and applications for commercial plants, the mechanisms underlying AR formation and the cause for variation among plant species and genotypes in AR production are of great interest. We integrated histology, endogenous hormone analysis, and metabolite profiling to study AR induction in two recalcitrant species, American chestnut (Castanea dentata) (AC) and yellow camellia (Camellia nitidissima) (YC), and conducted comparisons with easy-to-root willow and poplar. We found that auxin treatment is required for rooting AC and YC cuttings, and AC is more recalcitrant to AR induction than YC (rooting rate of YC 86% vs AC 50%). Preexisting primordia are present in willow cuttings, while AC cuttings exhibit a sclerenchyma ring, which may hinder AR formation and emerging. ARs are initiated from cambial derivatives and develop a vascular system connected with that of the stem. AR-promoting endogenous hormones, such as IAA, are at a lower level in YC and AC, while AR-inhibiting hormones, such as cytokinin and salicylic acid, are at a higher level in AC and YC cuttings than in poplar. Hormone distribution between leaves and stems also differs between the recalcitrant species and poplar. Profiling of primary and secondary metabolites indicates that AC and YC contain fewer AR-promoting compounds and more AR-inhibiting ones. We conclude that both AC and YC have unfavorite endogenous hormone and metabolite profiles that may contribute to their recalcitrance to rooting. Our study lays the foundation for future investigation of genes and gene networks that regulate AR induction in recalcitrant species.

Xinya Lu, Department of Biochemistry and Genetics, Clemson University, Clemson, SC 29631. Abstract presentation: P-2035

2024 HONOR B. FELL AWARD AND 2024 STUDENT TRAVEL AWARD

EGR1, ZNF24 and VEGF Gene Expression are Altered in Response to NSC348884 Treatment in a NLF Neuroblastoma Cell Line

Neuroblastoma is a solid tumor in children targeting the primary neural cells of the sympathetic nervous system. The aggressive form of neuroblastoma, high-risk neuroblastoma, has a low 5-year survival and few targeted treatments exist. The small molecule, NSC348884, has been shown to apoptosis in many solid tumors. Previously, we showed that NSC348884 induces apoptosis in neuroblastoma cell lines. To better understand what effects NSC348884 has on neuroblastoma cells, we used RNASeq and qRT-PCR to identify genes altered after NSC348884 treatment. NLF neuroblastoma cells were treated with 6.5 µM of NSC348884 or DMSO vehicle for 6 hours. RNA was isolated from cells and RNASeq or RT-qPCR performed. For RNASeq, samples were analyzed by Poly(A) RNA sequencing usingTruSeq mRNA sample preparation and Illumina NovaSeq 6000 sequencing. For RT-qPCR, RNA was converted to cDNA and relative expression levels of EGR1, ZNF24 and VEGF were compared to the 18S control gene using Taqman assay probes. RNASeq analysis revealed 99 genes altered after NSC348884 treatment in NLF neuroblastoma cells. The differentially expressed mRNAs were selected with log2 fold change above 1 and with statistical significance (p value < 0.05) by R package edgeR. Two of the altered genes were Early Growth Response 1 (EGR1) and Zinc Finger Protein 24 (ZNF24) and they both decreased in abundance after NSC348884 treatment. We validated expression levels of these genes using RT-qPCR. In concordance with RNASeq results, EGR1 and ZNF24 levels decreased in response to 6-hour treatment of 6.5µM NSC348884 in comparison to the 18S control gene. GraphPad Prism software was used for T-test and significance denoted at p < 0.05. VEGF levels were also measured, and it was found to have increased. EGR1 and ZNF24 are both transcription factors with many roles varying from cell proliferation to metastasis and angiogenesis. VEGF is involved in angiogenesis and ZNF24 has previously been described as a repressor of VEGF. Further studies are needed to elucidate the specific roles each of these genes play and how the inhibitor induces apoptosis.

Caitlin Nicolai, Midwestern University, 555 31st St., Downers Grove, IL 60515. Abstract presentation A-2002

2024 CELLULAR TOXICOLOGY AWARD

Antioxidant, Anticancer, and Anti-inflammatory Activities of Isoflavone-Rich Extracts from Elicited Hairy Root Cultures of Pigeon Pea

Plant-based natural products have attracted attention due to their diverse biological activities, encompassing antioxidant, anticancer, and anti-inflammatory properties. Among these, the pigeon pea plant stands out for its production of isoflavones such as genistein, known for various health benefits. However, the extraction and purification of these isoflavones from natural sources pose challenges and consume time. This project aimed to utilize a pigeon pea hairy root system to generate an isoflavone-rich extract and evaluate its potential health benefits. To achieve this, hairy root cultures of pigeon pea were established and co-elicited with methyl jasmonate, methyl-β-cyclodextrin, hydrogen peroxide, and magnesium chloride. Two isoflavones, genistein and its prenylated analog, isowighteone, were purified and identified in the elicited culture medium extract using different analytical chemistry techniques. The antioxidant activity of these isoflavones and extracts enriched in these compounds was determined through the DPPH antioxidant assay. The isoflavone-rich extract exhibited higher antioxidant activity compared to individual compounds at equimolar concentration. Moreover, the study examined the effects of genistein and isowighteone in MDA-MB-231 triple-negative breast cancer cells and RAW 264.7 macrophages, each at a concentration of 25 µM. Notably, isowighteone exhibited greater cytotoxicity than genistein in MDA-MB-231 cells over a 72-hour period and demonstrated higher anti-inflammatory activity at 24 hours compared to genistein. In conclusion, the combination of pigeon pea hairy roots and elicitation provides a sustainable platform for producing bioactive isoflavones.

Gaurav Gajurel, Arkansas State University, 2105 Aggie Rd., Jonesboro, AR 72401. Abstract presentation: A-1010

2024 STUDENT TRAVEL AWARD

Off-target Effects of the Site-specific Recombinases in Plants

Recent research advances in biology have facilitated efficient plant genetic transformation allowing extended application of genome editing for crop genetic improvement. However, the components employed for enhanced genetic transformation and genomic editing usually need to be removed, driven by concerns about the potential “side effects” of transgenes and biosafety issues, which are crucial for regulatory approval. Site-specific recombination system is an attractive platform for use in transgene excision and is particularly common in clonally propagated, self-incompatible, or polyploid crops (perennial grass, for example), where sexual segregation is challenging or impossible. Although widely used in plants for DNA excision and transgene containment studies, site-specific recombination systems implemented in plants can cause unexpected phenotypic changes, likely due to off-target genetic and epigenetic modifications in the host genomes. In this study, we constitutively expressed Cre or PhiC31, two representative recombinase genes in creeping bentgrass (Agrostis stolonifera), an important perennial grass species, to investigate the potentially unintended morphological and genetic impacts of recombinase activity. Preliminary data show that PhiC31 significantly enhanced plant growth and drought resistance, whereas Cre severely impaired plant growth. Ongoing RNA-seq analysis will provide further insights into which genes are most affected, allowing us to trace back to the genomic data to explore the mechanisms of off-target genetic and epigenetic modifications, providing information for appropriate use of site-specific recombination systems in developing environmentally safe transgenic products.

Xiaotong Chen, Clemson University, Clemson, SC 29634. Abstract presentation P-2034.

2024 STUDENT TRAVEL AWARD

Understanding Innate Immune Mechanisms in Non-immune Cells Using Nanoparticles

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 and their activation leads to the production of mediators of innate antiviral immune pathways, including type I interferon (IFN), and proinflammatory cytokines. A subset of PRRs are found on all cells in the human body, however, their role in the immune response is often overlooked. A key hurdle to exploring these pathways in non-immune cells is the low level of responsiveness. A tool that can enhance these nucleic acid-mediated responses are nanocarriers. The exact mechanism remains unknown, but evidence suggests nanocarriers create more efficient cellular delivery, provide protection from nuclease, and concentrate nucleic acids, enabling multivalent cellular presentation, resulting in a more potent response. Cationic nanoparticles can be used to ionically interact with anionic nucleic acids and used as a delivery system. Phytoglycogen (PhG) nanoparticles have been demonstrated to be effective carrier particles for dsRNA delivery to human cells. This research proposal will explore the mechanisms of dsRNA-loaded nanoparticles as immune stimulants for a multitude of cell lines. This study will investigate the following: (1) what receptors are involved in the uptake of nanoparticle-bound dsRNA, (2) how do nanocarriers modify dsRNA’s natural route of cellular entry and localization, and (3) what cellular mechanisms are responsible for the production of a heightened immune response. The data from this project will contribute to the knowledge base surrounding nanoparticle-mediated PAMP recognition and the innate immune pathway in non-immune cells, ultimately leading to a better understanding of the fundamental mechanisms of innate immunity in vertebrates and contributing to antiviral and anticancer therapeutics.

Nicholas Jadaa, Wilfrid Laurier University, 75 University Ave. W, Waterloo, ON N2L 3C5, Canada. Abstract presentation A-2011.

2024 STUDENT TRAVEL AWARD

Enhancing Solubility of Cytotoxic Peptides Affects Their Efficacy Toward Breast Cancer Cells

Breast cancer is the most common cancer among women and the most prevalent cancer overall. Producing novel and effective therapies is paramount for reducing mortality rates and enhancing the quality of life of individuals affected by this disease. Peptides and their derivatives comprise a substantial sector of drug development initiatives. However, reduced peptide solubility significantly impairs their biological availability and prevents further testing in vivo. Here, we report a physicochemical analysis of cytotoxic peptides that inhibit the CMG helicase and are prone to aggregation in vitro. Our previous research identified a short amino acid motif of X-Leu-Met-Leu-X responsible for these peptide aggregation. To enhance the solubility of our peptides, we generated their derivatives with Leu-Ala and Leu-Ser substitutions. We observed a gradual enhancement in peptide solubility by utilizing light microscopy and fluorescence-based peptide aggregation assay. Further, we observed a pronounced effect on BT-549 breast cancer cells using a colorimetric cell viability assay.

Greg Luis Connelly, Saint Leo University, Dept. of Natural Sciences, 33701 State Road 52, Saint Leo, FL 33574-6665. Abstract presentation A-2000.