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Bdellovibrio bacteriovorus (B. bacteriovorus) is a predatory bacterium that preys on other gram-negative bacteria. In order to survive and reproduce, B. bacteriovorus invades the periplasm of other bacterial cells creating the potential for it to act as a “living antibiotic”. In this work, a comparison was made between the rates of predation of B. bacteriovorus in vitro and in vivo. In vitro, the behavior of B. bacteriovorus was examined in the presence of prey. In vivo, the behavior of B. bacteriovorus was examined in the presence of prey and a living host, Caenorhabditis elegans (C. elegans). C. elegans were infected with Escherichia coli (E. coli) and treated with B. bacteriovorus. In previous studies that analyzed B. bacteriovorus in vitro, a decrease in concentrations of bacteria has been observed after introduction of B. bacteriovorus. In vivo, B. bacteriovorus were found to not have a net reduction of E. coli but to reproducibly raise the level of fluctuations in E. coli concentrations.
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The Mohawk (mkx) gene functions as a transcriptional repressor for tendon morphogenesis during embryonic development. Previous research showed that mkx KO mice overexpressed the osteogenic gene Runx2. Runx2 plays a role in recognition and long-term immune memory. A study showed Runx2 KO mice had a significantly lower number of CD8 T cells specific to lymphocytic choriomeningitis virus (LCMV) and CD8 memory precursor T cells. To determine the direct effects of Mohawk expression on the immune system, development, acute response, and immune memory of innate, B and T cells were compared between WT and mkx KO mice after LCMV infection. Paired t-test analyses were performed between KO and WT data. We first found significantly higher numbers of granulocytes and dendritic cells in the periphery but lower numbers of B cells in the bone marrow and T cells in the thymus of KO mice. When analyzing immune response, we observed a significantly high number of activated CD8 T cells that proliferated in the KO mice in response to the infection. Next, we found no difference in cytokine production for TNF and IFNγ which shows Mohawk does not impair acute immune response. Finally, we found no significant difference between WT and KO mice in the CD8 T cells' ability to make an immune memory. In the present study, we found that, fewer immune cells continued their maturation. However, Mohawk expression did not impact their acute response or ability to become memory cells once the T cells matured and became activated. Rather, T cells specific for LCMV were present in higher numbers in mkx KO mice. Further research will study the impact Mohawk has on both B and T cell memory.
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Most protein-coding mRNAs in eukaryotes must undergo a series of processing steps so they can be exported from the nucleus and translated into protein. Cleavage and polyadenylation are vital steps in this maturation process. Improper cleavage and polyadenylation results in variation in the 3′ UTR length of genes, which is a hallmark of various human diseases. Previous data have shown that the majority of 3’UTRs of mRNAs from the nematode Caenorhabditis elegans terminate at an adenosine nucleotide, and that mutating this adenosine disrupts the cleavage reaction. It is unclear if the adenosine is included in the mature mRNA transcript or if it is cleaved off. To address this question, we are developing a novel method called the Terminal Adenosine Methylation (TAM) assay which will allow us to precisely define whether the cleavage reaction takes place upstream or downstream of this terminal adenosine. The TAM Assay utilizes the ability of the methyltransferase domain (MTD) of the human methyltransferase METTL16 to methylate the terminal adenosine of a test mRNA transcript prior to the cleavage reaction in vivo. The presence or absence of methylation at the terminal adenosine will then be identified using direct RNA sequencing. This project focuses on 1) preparing the chimeric construct that positions the MTD on the mRNA cleavage site of a test mRNA transcript, and 2) testing the functionality of this construct in vitro and developing a transgenic C. elegans strain expressing it. The TAM assay has the potential to be a valuable tool for elucidating the role of the terminal adenosine in cleavage and polyadenylation.
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