Bacteriophage MS2 phage-like particles (PLPs) are artificially constructed viral-like particles. The similarity of the particle to a virus allows the particles to be used as a control system for molecular detection and drug delivery systems. The capsid is made up of single chain coat protein dimers (SCCPD) and a singular maturase, and is able to package mRNA within the protein coat due to the dimerization of coat proteins spontaneously forming the capsid structure in the presence of a packaging signal. In addition, the PLPs also have the ability to display what is being packaged on the surface. Currently, it has been shown that PLPs are able to be purified via His-tag affinity, due to fusion of the His-tag onto the SCCPDs. However, if a peptide is displayed at the SCCPD site, the His-tag must be attached elsewhere on the PLP to ensure the PLP can be purified. To address this, I designed a new PLP by plasmid engineering via site-directed mutagenesis to display a peptide on AB-loops within the SCCPD, whilst packaging the corresponding mRNA within the capsid. I purified the PLP via a His-tag attached to the maturase protein. To verify correct particle formation, I ran SDS-PAGE to observe the density of bands corresponding to SCCPD and maturase. I designed a reverse transcriptase polymerase chain reaction and carried out a nuclease protection assay to verify mRNA packaging. Preliminary data of SDS-PAGE has shown the particle has successfully purified, and is correctly forming due to the observed maturase to SCCPD band density ratio on the gel meeting the expected ratio.

Diabetic Cardiomyopathy (DCM) is a cardiovascular complication developed from diabetes mellitus, causing a dilation of cardiac ventricle tissue, ultimately impairing systolic function. Although this condition affects 1.1% of the population and nearly 17% of all diagnosed diabetics, mortality rates are exceeding large, with almost 1 in 3 developing death or heart failure after less than a decade. Therefore, effective therapeutics are required to mitigate this prevailing epidemic. Chloride Intracellular Channels (CLICs) are a family of ion channel proteins, permeable to chlorides, which maintain specific cytoplasmic ion concentrations to regulate necessary homeostatic conditions for cellular function. Previous research from our laboratory demonstrates that the absence of CLIC4, a member of the CLIC family and a mitochondrial-associated membrane ion channel in cardiomyocytes, prevents the alteration of sarcomere integrity and beat rate upon high glucose exposure, which is highly prevalent in DCM. Additional research suggests CLIC4 regulates sarcoplasmic reticulum (SR) mitochondrial calcium signaling in cardiomyocytes, and the absence of CLIC4 prevents the occurrence of diabetic cardiomyopathy. However, the mechanism of CLIC4-mediated cardiac dysfunction is still relatively unknown. Previous investigation demonstrates that dysregulation of Ryanodine Receptor 2 (RyR2) function in the SR of cardiomyocytes, due to its oxidation, could augment cardiac dysfunction upon diabetes via modulating calcium levels. This experiment illustrates that pharmacologically blocking CLIC4 reduces oxidative stress in cardiomyocytes, which we measured via staining of cardiomyocytes with DCFDA. Additionally, we found the association of CLIC4 with RyR2 through immuno-pulldown experiments. We anticipate that CLIC4 regulates the oxidative status of RyR2 and modulates its activity in diabetic cardiomyocytes.