Undergraduate Research Day 2024
Permanent URI for this collectionhttp://hdl.handle.net/1903/31825
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Item Defining how two avian double-stranded RNA viruses affect lipid droplet (LD) formation and lipid metabolism in vitro(2024) Liu, Ying-Rong (Megan); Kehlbeck, Declan; Egana-Labrin, Sofia; Brodrick, Andrew; Sunny, Nishanth; Broadbent, Andrew; Broadbent, AndrewSome mammalian and fish double-stranded (ds)RNA viruses hijack lipid droplets (LDs) during their replication cycle, however, our understanding of how avian dsRNA viruses interact with LDs is incomplete. Here, we report data from avian reovirus (ARV) and infectious bursal disease virus (IBDV)- infected cells. Following ARV infection, there was a significant increase in the number (p < 0.0001) and size (p < 0.0001) of LDs compared to mock-infected controls, 6-24 hours post infection (hpi), in both immortalized LMH cells and primary gut cultures. Treatment with the ACCA inhibitor, TOFA, reduced ARV-mediated LD induction, indicating de novo lipogenesis was partially involved in their formation. Moreover, ARV infection decreased the expression of PGC-1α (p < 0.05) and ELOVL2 (p < 0.001). Previous reports demonstrated that inhibition of PGC-1α and ELOVL expression in mice led to cellular lipid accumulation and hepatic steatosis, suggesting ARV could upregulate LD synthesis by decreasing expression of these genes. However, it is unclear whether LD induction was pro- or anti-viral, since both TOFA treatment to inhibit LD synthesis, and oleic acid/palmitic acid treatment to overexpress LDs did not significantly alter ARV replication. In contrast, infection with IBDV did not substantially increase the size or number of LDs, or significantly reduce the expression of PGC-1α and ELOVL2. In contrast to some other dsRNA viruses like rotavirus and mammalian orthoreovirus, neither ARV nor IBDV cytoplasmic puncta colocalized with LDs. Taken together, our findings demonstrate that the two dsRNA viruses had distinct effects on cellular lipid metabolism.Item Role of Mesenchymal Stem Cells in Intestinal Epithelial Cell Growth(2024) Desai, Om; Vaughn, Sarah; Garg, Vanshika; Jin, Younggeon; Ayansola, HammedMucosal healing derived from intestinal epithelial regeneration is crucial in the complete remission of patients with inflammatory bowel diseases (IBDs). Human Bone Marrow Mesenchymal (HBMMSCs) stem cells have been researched as a therapeutic approach to promote mucosal healing through immunomodulation, angiogenesis, and tissue repair. Most preclinical animal studies focus on immune-modulatory functions, but the mechanisms related to epithelial regeneration are poorly understood. We hypothesized that colonoids co-cultured with HBMMSC would increase cell growth and stemness, which are principal for epithelial regeneration. Two different co-culture methods were used: direct and indirect. In the direct method, we analyzed the impact of HBMMSC on colonoid growth and differentiation when the cells were embedded together in Matrigel. In the indirect method, the transwell mimicked the intestine's structure, with the colonoids layered over the HBMMSCs, and isolated each cell type separately to evaluate the gene expression using real-time PCR. Colonoids with HBMMSCs exhibited enhanced stemness and growth, indicated by increased spheroidal surface area and decreased budding counts compared to those without HBMMSCs. Intestinal stem cell genes, like LGR5, ASCL2, and OLFM4, showed a significant increase in the co-cultured cells compared to control cells. We also noted repression of differentiation markers, such as ALPi, IHH, and PTCH1, in co-cultured colonoids. HBMMSCs show promise for promoting mucosal healing in IBD patients by enhancing epithelial regeneration. Yet, further research is needed to understand the repair mechanisms and cellular pathways influenced by HBMMSCs.Item Plant growth-promoting bacteria in lettuce for resilience against enteropathogens(2024) Lee Sang, Marissa; Klair, Diksha; Micallef, Shirley A.Introduction: Observe the role of Plant Growth Promoting Bacteria (PGPB) obtained from the phyllosphere, rhizoplane, and roots of Bok choy for antagonistic responses and resilience to biotic stressors, Salmonella Typhimurium and E. coli 2705C in vitro and in lettuce. Purpose: Unravel the potential of PGPB root colonization for resilience to enteric pathogens in Romaine lettuce. understanding the responses of PGPB to enteric pathogens for the enhancement of plant growth and contributions to food safety Methods: Rhizobacteria (n=35) were isolated from the roots of bok choy (Brassica rapa subsp. chinensis) grown at the University of Maryland TerpFarm. Bacterial isolates were taxonomically classified using full-length 16S rRNA gene sequencing. Lettuce seed (Rogue d’Hiver) was inoculated 3- and 6- days post germination with single strain suspensions of 7 log CFU/plant. All the isolates were further screened for their potential to enhance food safety, utilizing both in vitro and vivo assay. Leaf populations of Salmonella Typhimurium were enumerated by direct plate counting on Tryptic Soy Agar 24 h after leaf inoculation with 5 log CFU. An antagonistic assay was conducted by inoculating PGPB onto TSA plates and applying a drop of E. coli 2705C and Salmonella Typhimurium to observe potential inhibitory effects. Results: From the Bok choy leaves and rhizoplane a total of 7 and 4 unique genera were classified, respectively. Intriguingly, all eight endophytes from the root tissue exclusively belonged to the genus Pseudomonas, indicating its dominance, and suggesting influence over other symbiotic microorganisms in plant roots. Through in vitro assay, 3 phyllosphere endophytes created a zone of inhibition around E. coli 2705C and Salmonella Typhimurium, indicating their antimicrobial activity. The reduction in Salmonella populations on lettuce treated with three rhizoplane isolates (Pseudomonas fluorescens, Priestia magaterium, Lysinibacillus xylanilyticus) and two root endophytes (Pseudomonas spp.), was slightly greater compared to uninoculated plants. Significance: Our research highlights the prospect of leveraging microbial interactions to achieve sustainable and safe crop production during environmental challenges.Item Evaluating the effect of flavonoids in tomato exocarp on Salmonella enterica(2024) Pineau, Olivia; Hopper, Adam; Micallef, ShirleyIntroduction: The exocarp of tomato fruit are known to produce high levels of flavonoids, compounds with antimicrobial effects. The purpose of this study was to investigate flavonoid levels in peels of different tomato cultivars and their potential antimicrobial effects on Salmonella Newport and Montevideo. Methods: Phytocompounds were extracted from 20 mg of lyophilized exocarp of tomato fruit ‘Black Icicle’, ‘Midnight Roma’, ‘Dixie Red’, ‘Indigo Rose’, ‘Inigo Cherry’, ‘Heinz-1706’ and ‘Charger’ in 5% methanol for flavonoid and microbial analysis. Total flavonoids were measured using a colorimetric assay at 510 nm, quantified using catechin standard curve and expressed as mg catechin equivalent (CE)/g. Flavonoid finger-printing was performed on a UV-Vis spectrophotometer with a scanning range set to 240-400 nm. Extracts were inoculated with log 6 CFU/mL Salmonella Newport or Montevideo, incubated with shaking at 35°C and sampled at 2, 8 and 20 h post-inoculation. Bacteria were enumerated by serial dilution. Microbial data were analyzed statistically. Results: Salmonella counts declined over time in all cultivar extracts (p<0.01) and ‘Midnight Roma’ yielded the lowest counts.. No significant differences in Salmonella counts were detected within each time point by cultivar. ‘Indigo Rose’ had higher total flavonoids (85.38 mg CE/g), than ‘Charger’ (17.98 mg CE/g, p<0.01), ‘Heinz-1706’ (20.61 mg CE/g, p<0.01) and ‘Black Icicle' (27.25 mg CE/g, p<0.05). Flavonoid scan data showed cultivar differences. Significance: Flavonoid concentrations differed by cultivar. Salmonella decline in peel extracts over a 20 hour period was cultivar dependent.Item Antimicrobial Resistance in Urban Agriculture Environments(2024) Zeng, Qingyue; Lam, Kevin; Salcedo, Autumn; Konsen, Derek; Blaustein, Ryan; Blaustein, RyanBiological soil amendments of animal origin (BSAAOs) are widely used in urban agriculture to improve soil health. Although BSAAO use is regulated in the United States due to food safety risks, impacts on antimicrobial-resistant (AMR) bacteria are not well established. We aimed to explore impacts of BSAAOs, among other factors, on AMR bacteria in leafy vegetable production environments across urban farms and community gardens in the Washington D.C. area. Samples of leaf tissue (LT), root zone soil (RZS; amended soil in crop beds), and bulk soil (BS; soil along site perimeter) were collected and analyzed for concentrations of total heterotrophic bacteria (THB). Isolates of AMR strains and coliforms were further screened for multi-drug resistance (MDR) phenotypes. Across-site variation in AMR bacteria in RZS and LT samples broadly reflected land use history and crop management and within-site variation demonstrated effects of specific amendment sources, as well as vegetable type and variety. Overall, our findings demonstrate that promoting soil bacteria activity in the urban landscape has potential implications for managing AMR.Item The Impact of Lipid Accumulation on Mitochondrial Metabolism in the Liver(2024) Fadlaoui, Ayeesha; Sunny, Nishanth E.High rates of new lipid synthesis (de novo lipogenesis) and dysfunctional mitochondrial metabolism are central features of non-alcoholic fatty liver disease (NAFLD). However, it is not clear whether sustained induction of de novo lipogenesis will alter the activity of mitochondrial oxidative networks during progressive severity of NAFLD. We tested the alterations in mitochondrial metabolism in livers with high rates of de novo lipogenesis. Male mice (C57BL/6J) were reared on normal chow with either regular drinking water (NW) or 30% fructose in drinking water (FW) to induce lipogenesis, for 2-wks. Liver mitochondria were isolated from fed and overnight fasted (~16-hrs) mice. Mitochondria were allowed to respire at 37C for either zero or 10-min. Changes in mitochondrial metabolites were determined via gas chromatography-mass spectrometry (GC-MS). Plasma samples were used for GC-MS based targeted metabolomics. Liver tissue was utilized to profile lipogenic gene expression. After 2-wks of treatments, lipogenic gene expression in the liver (Acc, Fasn, Elovl6) was significantly higher in the FW group (p≤0.05). While plasma β-hydroxybutyrate levels were induced by overnight fasting in both the groups, the fed-to-fasted fold change was significantly higher in the FW group compared to the NW group (p=0.001). Furthermore, levels of mitochondrial TCA cycle intermediates (pyruvate, citrate, fumarate) were induced with fasting in FW compared to NW (p≤0.07) after 10-mins of mitochondrial respiration. Taken together, these results suggest that the mitochondrial oxidative function is higher in livers sustaining higher rates of de novo lipogenesis.