Undergraduate Research Day 2024

Permanent URI for this collectionhttp://hdl.handle.net/1903/31825

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Subject: search.filters.subject.Cell Biology and Molecular Genetics

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    Novel Computational Methods for the Comparison of Leishmania Genomes and Transcriptomes
    (2024-04) Klimes, Daniel; Belew, Trey; El-Sayed, Najib
    Leishmania is a genus of protozoan parasites and the causative agent of Leishmaniasis. Twenty-one infectious species have been identified, with many previously being investigated by DNA and RNA-sequencing. However, significant genetic variation has prevented the comparison of this data across Leishmania species. Gene orthology grouping fails to compare over 70% of Leishmania genes’ expression across multiple species because of this variation. Here, we describe a novel method to reliably compare the genomes and transcriptomes of multiple Leishmania. This method produces a hybrid genome from the genetic sequence of any Leishmania species, while containing the rich sequence annotations of the L. major reference genome. In a demonstrative analysis, our approach allowed the comparison of 87% of genes in L. major against 5 other Leishmania species. Significant genetic variation was found to prevent comparison of many virulence-implicated genes, including surface antigens (e.g. amastins), cysteine proteases, and HSP-83. Within genes that could be compared, parasite transcriptomes segregated primarily by developmental stage (promastigote and amastigote). Transcriptomes secondarily separated by species. Genes upregulated in infection across all Leishmania were identified and included surface amastins, methyltransferases, and an apoptosis inhibition factor. Downregulated genes showed significantly greater sequence conservation than upregulated genes and comprised paraflagellar, flagellar, and HSP-70 proteins among others. The transcriptome differences between the clinical manifestations of leishmaniasis were additionally characterized. Taken together, these results provide much needed insight into the shared Leishmania pathway of infection and survival.
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    Identifying Difficidin in the Supernatant of Bacillus velezensis using High Resolution Mass Spectrometry
    (2024) Nasisi, Benjamin; Jenkins, Conor; Jermain, Madison; Winkler, Wade
    Difficidin is a naturally occurring extracellular antibiotic produced by Bacillus velezensis. The dfn operon encodes for the difficidin biosynthesis proteins. This operon (dfn) is transcriptionally regulated by intrinsic transcription termination sites that are spread throughout the 70 kilobase operon and that limit dfn expression. LoaP is a specialized transcription elongation protein that promotes readthrough of these terminators. We hypothesize that LoaP associates with RNA polymerase to promote readthrough of the termination sites; however, the LoaP regulatory mechanism is still unknown. We have constructed different bacterial strains containing mutations in LoaP. Yet, we do not have an assay for measuring the impact of these mutations on the regulation of the dfn operon. This study seeks to address this problem by developing a method to quantify difficidin. Specifically, we will establish a mass spectrometry-based detection assay to identify the levels of difficidin under various conditions. Three strains - wild-type, ΔloaP, and Δdfn - were utilized to test native levels, low levels, and absence of difficidin, respectively. Strains were grown overnight to an OD600 of 2 in 150 mL of rich medium (LB broth). Cells were pelleted and extraction of difficidin was performed by incubation of the supernatant at 4°C overnight with Amberlite XAD-16 resin. Metabolites were then eluted from the resin using 8 mL of methanol and analyzed by mass spectrometry. Data for the three strains were compared against each other to identify the retention time of difficidin. The WT strain had an intense cluster of peaks at a retention time of 17-19 minutes. These peaks completely disappeared in the Δdfn strain. The disappearance of a peak in the Δdfn strain that is present in the WT strain is a strong indication that difficidin has been identified. Further tests using the fragmentation of the metabolites at the retention time determined are required to confirm whether difficidin has been identified by mass spectrometry.
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    Engineering HeLa Cells to Better Support Rhinovirus C Infection In Vitro
    (2024) Swaminathan, Divya; Goldstein, Monty; Pankow, Alec; Scull, Margaret; Scull, Margaret
    Human rhinoviruses (RVs) are non-enveloped, positive-sense, single-stranded RNA viruses that target the respiratory tract for infection, leading to common cold-like symptoms, bronchiolitis, or, in some cases, pneumonia. RVs are classified into three phylogenetically distinct groups: RV-A, RV-B, and RV-C, with RV-C being identified most recently, in 2006. However, while RV-A and RV-B viruses have been studied for decades in HeLa cells, RV-C does not naturally infect conventional cell lines and thus, it has been difficult to propagate and study in the lab. Further, no assay currently exists to quantify infectious RV-C particles, such as a plaque assay or limiting dilution assay. It has been demonstrated that cadherin-related family member 3 (CDHR3) is a critical cellular entry receptor for RV-C, and further, that RV-C may depend on the stimulator of interferon genes (STING) for viral genome replication. To investigate the roles of these proteins in RV-C infectivity, inducible human STING (hSTING) was transfected into HeLa cells that stably express CDHR3. Elevated STING expression was then confirmed in these cells after induction with Doxycycline (Dox). Ongoing studies seek to further explore how the expression of additional ciliated cell-specific host factors - such as Prominin-1 (PROM1) - in HeLa cells impact virus infection in this cell line. The hypothesis is that the combined expression of CDHR3, PROM1, and STING in HeLa cells will enhance entry and replication, facilitating amplification of RV-C in the laboratory and aiding detection of infection via antibody-mediated staining or enhanced cytopathic effects. These engineered HeLa cells would thereby constitute a novel cell line that is more amenable to the establishment of a plaque assay or limiting dilution assay to quantify infectious virus.