2023 JOHN S. SONG AWARD
Production of Isowighteone in Hairy Root Cultures of Pigeon Pea (Cajanus cajan) using an Optimized Elicitation Procedure.
Gaurav Gajurel
Isowighteone, a prenylated flavonoid derivative, exhibits anti-inflammatory, antibacterial, and pro-apoptotic properties. The extraction and purification of isowighteone from natural sources can be challenging and time-consuming. This study aimed to establish a hairy root culture system for pigeon pea via Agrobacterium rhizogenes-mediated transformation as a sustainable production platform for isowighteone. The hairy root cultures were co-treated with methyl jasmonate, cyclodextrin, H2O2, and MgCl2 for different periods to study their effect on the accumulation of isowighteone in the culture medium and hairy root tissue. Most of the isowighteone accumulated in the culture medium. Isowighteone was identified from extracts of culture medium and root tissues using tandem mass spectroscopy and semi-preparative HPLC was used for its purification. The combined yield of isowighteone in the medium and root tissue of the 12-day-old culture elicited for 144 h was 8058.62 ± 445.78 μg/g DW. The total yield of isowighteone in the elicited hairy root culture was approximately 277-fold higher than under non-elicited conditions. The morphological differences between the normal and elicited hairy roots were studied using scanning electron microscopy. The cells in the non-elicited hairy root tips were tube-like and smooth. Interestingly, the cells in elicited hairy root tips seemed to have a non-uniform shape along with some ruptures on the surface. Pigeon pea hairy roots provide a sustainable platform for producing isowighteone.
Gaurav Gajurel, Arkansas State University, 105 N Caraway #443, Jonesboro, AR. Abstract presentation P-1006
2023 GORDON SATO AND WALLY MCKEEHAN AWARD
Histamine 2 Receptor Influence on Chemokine Production in Macrophages is STAT6-independent
The prevalence of asthma has grown to afflict over 25 million patients in recent decades, showing the importance of studying allergic disease. Macrophages are elevated in allergic asthma and contribute to disease. Histamine is a mediator used in an immune response to allergens and its receptors are expressed on macrophages. Previous work has found that mice deficient in histamine 2 receptor (H2R) are protected from allergic asthma. We have found that H2R KO macrophages have impaired gene expression of CCL11, an important eosinophil recruiting chemokine. Our goal is to determine if H2R expression on macrophages is important for this protection. Bone marrow-derived macrophages were collected and cultured from wild-type (WT) and H2R KO mice. The macrophages were treated with the Th2 cytokines interleukin-4 (IL-4) or interleukin-13 (IL-13), to promote alternative activation of macrophages. Through flow cytometry, we studied the expression of the Type I and Type II IL-4 receptor subunits expression on macrophages. We also studied the phosphorylation of STAT6 in response to stimulation by the Th2 cytokines in WT and H2R KO macrophages using western blot. Our results suggest H2R does not function through the Type I or Type II receptor subunits. This is seen through no significant difference between the WT and H2R KO when looking at the Type I and Type II receptor subunits. Additionally, we found through western blot, that H2R KO macrophages do not have reduced phosphorylation of STAT6, a transcription factor important for CCL11 production. These findings have gained us insight in the fact that while H2R is involved in protection from allergic disease, it is not due to changes in macrophage expression of the Type I receptor, Type II receptor, or the phosphorylation of STAT6. Future work is planned to further study the function of macrophages in the absence of H2R, as well as alternative signaling pathways important for CCL11 production.
Peter Alfano, College of Graduate Studies, Midwestern University, Downers Grove, IL 60515. Abstract presentation A-2006
2023 JOSEPH F. MORGAN AWARD AND 2023 STUDENT TRAVEL AWARD
Ease of Development of Cell Cultures from Fish Olfactory Rosettes and Initial Characterization of a Continuous Cell Line From Rainbow Trout Olfactory Tissue
Olfactory rosettes (ORs) are the primary organs of olfaction in fish. Most teleost ORs are defined structures found within a nasal cavity anterior to the eye, composed of folded sheets of olfactory mucosa (lamellae) held by a central support (raphe) that connects to the fish brain. The olfactory mucosa is composed of pseudostratified epithelium and an underlying lamina propria. Olfactory sensory neurons, which function in odour recognition, help compose the epithelial layers along with supportive sustentacular cells and regenerating basal stem cells. Mucus producing goblet cells, dendritic cells, macrophages and melanocytes are also abundant within the ORs. Over the past 5 years, attempts to culture ORs from various species led to successful primary cultures from all attempted nasal explants. These included ORs from Rainbow Trout, Atlantic Salmon, Chinook Salmon, Coho Salmon, Sea Snapper, Bluefin Tuna. Despite the ease of establishing primary cultures, continuous maintenance was hindered in several of these cultures by microbial contamination. In the present study we focus on a novel Rainbow Trout olfactory cell line (RTolf-UFV1) that has been passaged for over 20 generations and has been successfully cryopreserved and thawed. L15 media with added fetal bovine serum (FBS) in plastic tissue culture vessels allowed OR cells to adhere and proliferate. Cultures are polymorphic with a mixture of epithelial, fibroblastic, dendritic and macrophage-like cells. Characterization of the cultures is currently underway, including confirmation of species of origin by DNA barcoding, establishing growth characteristics at different temperatures and concentrations of FBS, immunohistochemical assays for structural markers and cell specific functional assays including phagocytosis, cell migration, cell depolarization.
Ryan Goldbach, Faculty of Science, University of the Fraser Valley, Abbotsford, BC V2S 7M8, CANADA. Abstract presentation A-2027.
2023 HONOR B. FELL AWARD AND 2023 STUDENT TRAVEL AWARD
Identification of Stratifin as a Potential Protein Biomarker Indicative of Oral Squamous Cell Carcinoma Progression
It has been shown that the expression of a squamous epithelium keratinocyte protein, Stratifin, is downregulated in cancerous tissues. This study aims to examine Stratifin expression at the cellular level in four cell lines representative of the successive progression steps of oral squamous cell carcinoma (OSCC), a major type of head and neck cancer. Thus, examining the utility of Stratifin as a diagnostic and prognostic indicator for the disease.This study evaluated the differential expression of Stratifin across four cell lines representing successive progression steps of OSCC. Western blotting was completed to quantify the expression of Stratifin in primary gingival keratinocytes, dysplastic oral keratinocytes (DOK), squamous cell carcinoma 25 (SCC25) cells, and Detroit 562 cells that represent normal oral keratinocytes, oral premalignant lesions, locally invasive OSCC cells, and metastatic OSCC cells, respectively. Stratifin expression was found to be downregulated alongside the progression from normal oral keratinocytes to premalignant, and from premalignant to metastatic cancer cells. Quantitative analysis revealed that SFN was downregulated significantly in the metastatic cells compared to the normal and dysplastic cells (p< 0.05). There was also a significant downregulation in the expression of SFN in the dysplastic cells compared to the normal (p< 0.05). However, there was no significant difference in SFN expression between the dysplastic and the locally invasive cancer cells. Stratifin proved to be differentially expressed at the cellular level in cell lines. Particularly, we documented a significant decrease of Stratifin expression with the progression from normal to premalignant to metastatic cells. Ultimately, the results showed that the expression of SFN was downregulated as OSCC progressed in a step-wise manner. Thus, Stratifin can potentially serve as a diagnostic and prognostic biomarker for patients suffering from oral cancer.
Shuaa Rizvi, Masters of Biomedical Science, College of Graduate Studies, Midwestern University, Downers Grove, IL 60515. Abstract presentation A-1002.
2023 STUDENT TRAVEL AWARD
The Effects of Thermal Stress on the Cellular Responses of Common and Phylogenetically Diverse Florida Reef Sponges
It has been shown that the expression of a squamous epithelium keratinocyte protein, Stratifin, is downregulated in cancerous tissues. This study aims to examine Stratifin expression at the cellular level in four cell lines representative of the successive progression steps of oral squamous cell carcinoma (OSCC), a major type of head and neck cancer. Thus, examining the utility of Stratifin as a diagnostic and prognostic indicator for the disease.This study evaluated the differential expression of Stratifin across four cell lines representing successive progression steps of OSCC. Western blotting was completed to quantify the expression of Stratifin in primary gingival keratinocytes, dysplastic oral keratinocytes (DOK), squamous cell carcinoma 25 (SCC25) cells, and Detroit 562 cells that represent normal oral keratinocytes, oral premalignant lesions, locally invasive OSCC cells, and metastatic OSCC cells, respectively. Stratifin expression was found to be downregulated alongside the progression from normal oral keratinocytes to premalignant, and from premalignant to metastatic cancer cells. Quantitative analysis revealed that SFN was downregulated significantly in the metastatic cells compared to the normal and dysplastic cells (p< 0.05). There was also a significant downregulation in the expression of SFN in the dysplastic cells compared to the normal (p< 0.05). However, there was no significant difference in SFN expression between the dysplastic and the locally invasive cancer cells. Stratifin proved to be differentially expressed at the cellular level in cell lines. Particularly, we documented a significant decrease of Stratifin expression with the progression from normal to premalignant to metastatic cells. Ultimately, the results showed that the expression of SFN was downregulated as OSCC progressed in a step-wise manner. Thus, Stratifin can potentially serve as a diagnostic and prognostic biomarker for patients suffering from oral cancer.
Megan Conkling, Harbor Branch Oceanographic Institute, Florida Atlantic University, 5600 US 1 North, Fort Pierce, FL. In Vitro Cellular and Developmental Biology, 58:S19 2022
2023 STUDENT TRAVEL AWARD
MiR169-NF-Y Module Associates with Creeping Bentgrass Biomass Production and Stress Response
Abiotic stresses, such as salinity, drought and heat, are important limiting factors for plant growth and development, significantly impacting crop production and agriculture economy. Plants have evolved various protection mechanisms coping with different environmental adversities. Manipulation of genes involved in plant stress regulation to genetically engineer enhanced performance in transgenics plays an increasingly important role in sustainable modern agriculture. MicroRNAs (miRNAs) are endogenous small non-coding RNAs identified in plants that engage in post-transcriptional target gene regulation, crucial for plant development and environmental adaptation. Here, we investigated the role of miR169g, a conserved plant miRNA that targets NUCLEAR FACTOR Y (NF-Y) transcription factors in regulating plant development and stress response and the underlying physiological and molecular mechanisms using transgenic approach to overexpress and knockdown miR169 through target mimicry in an important perennial grass species, creeping bentgrass (Agrostis Stononifera). Our data indicate that miR169 regulates expression of the specific NF-Y transcription factor genes leading to significantly altered plant biomass yield and responses to drought and salt stresses that are associated with modified plant development and physiological and molecular characteristics. The on-going RNA-seq analysis would provide additional information for a better understanding of the miR169-mediated plant development and stress response. The results obtained so far have demonstrated the importance of miR169 as a key coordinator in plant development and stress responses, providing information for the development of novel biotechnology approaches to genetically engineer crops for enhanced agricultural production.
Xiaotong Chen, Masters of Biomedical Science, Department of Genetics and Biochemistry, Clemson University, Clemson, SC 296345. Abstract presentation P-1005.
