Rett syndrome is an intellectual and developmental disorder that leads to severe physical and cognitive impairment and is caused by loss-of-function mutations in the X-linked DNA-binding protein methyl-CpG-binding protein 2 (MECP2). Our lab has observed that mutant neurons with loss of MECP2 function have significantly more DNA damage than wild type neurons due to impaired DNA repair mechanisms, and this subsequently drives other cellular Rett phenotypes like increased senescence, decreased dendritic branching, and mitochondrial dysfunction. Recent data suggests that double stranded breaks (DSB) are induced at the promoters of neuronal early response genes for their expression. However, this topic has not been explored in relation to MECP2. While MECP2 is known to be an epigenetic regulator, not much is known about its role in DNA damage and repair. We are interested in investigating the involvement of MECP2 in the activation and expression of neuronal genes and associated DNA damage. We are studying the effect of repeated neuronal activation on gene expression and genomic DNA damage in wild type and mutant neurons. We hypothesize that the repair of activity-induced DSB is impaired in mutant neurons and that this further affects their gene expression during repeated stimulation. Overall, understanding the role of MECP2 in neuronal activity-induced damage will help us identify potential therapeutic targets for Rett Syndrome while furthering our knowledge about the mechanism of DNA damage and neuronal excitation.

Purpose: To conjugate anti-GPC3 antibodies to lipid microbubbles and demonstrate selective binding to GPC3+ hepatoma cells in vitro. Methods: Anti-GPC3 antibody was incubated with oYo-link dibenzocyclooctyne (DBCO) under ultraviolet (UV) light to chemically cross-link DBCO groups to antibody. Lipid microbubbles with surface azide groups were incubated with the anti-GPC3-DBCO antibodies for two hours to conjugate the antibodies to the surface of the azide bubbles. Anti-GPC3 microbubbles were then incubated with HepG2/c3a GPC3+ presenting cells for four hours and imaged pre and post-wash to visualize cell binding. Results: SDS-PAGE showed a molecular weight shift in the antibody heavy chain after DBCO conjugation, confirming successful linkage. DLS revealed uniform ~1 μm size for both azide and antibody-conjugated microbubbles. Post-wash imaging revealed peripheral microbubble binding to HepG2/c3a cells, indicating specific targeting. Conclusion: Anti-GPC3 microbubbles selectively bound GPC3+ cells, supporting a modular platform for generating antibody-targeted microbubbles. Background/Clinical Relevance: GPC3 is overexpressed in certain hepatocellular carcinoma subtypes and can guide treatment decisions. Current diagnosis relies on biopsy, but is limited by sampling bias and tumor heterogeneity. Antibody-labeled microbubbles visible via ultrasound offer a noninvasive alternative for identifying tumor subtypes.

Direct Reprogramming shows promising results for regenerative medicine; however, the conversion efficiencies for many specific lineages remain low. Previous studies have shown that plant Methyl Binding Proteins (MBDs) use Alpha Crystallin Domains (ACDs) to recruit complex members to specific genomic loci. Furthermore, ChIPseq analysis of Zinc Finger-ACD fusion proteins have revealed larger peaks at binding sites, suggesting that ACDs facilitate targeted oligomerization. This study aims to fuse ACD-containing proteins to transcription factors (TFs) for improved efficiency in direct reprogramming. To test the binding abilities of the TF-ACD fusion, Myc was fused with ACDs, transfected in Human Embryonic Kidney (HEK) cells, and activated through Doxycycline (Dox). CUT&RUN and RNAseq demonstrated that ACDs enhanced TF localization at target sites and upregulated downstream genes. These results suggest that the reengineered TF may enhance lineage conversion efficiency, offering considerable potential for regenerative medicine. Ongoing experiments involve fusing ACDs to neuronal transcription factors NGN2 and BRN3A to assess reprogramming efficiency from fibroblasts to neurons.

Direct reprogramming is the process of converting from one cell type into a very distantly related cell type without the use of an intermediate proliferative stem-cell-like stage. Direct cardiac reprogramming involves the conversion of fibroblasts into cardiomyocytes via the forced expression of three transcription factors, Gata4, Mef2c, and Tbx5 (GMT). Previous studies have shown that cardiac reprogramming can be enhanced by using varying transcription factors, small molecule compounds, and biophysical cues. However, whether the immune microenvironment may play a role in the direct conversion process remains unknown. To investigate the effect of the immune cells on induced cardiac reprogramming, cardiac fibroblasts were reprogrammed in the absence or presence of polarized macrophages. We found that anti-inflammatory (M2-like) macrophages promoted reprogramming while inflammatory (M1-like) macrophages inhibited this process. Further epigenetic analysis revealed that M2-like macrophages decrease heterochromatin mark H3K9me3 levels compared to non-polarized (M0-like) and M1-like macrophages, which may facilitate the reprogramming process. Taken together, our findings provide novel insights into the mechanisms of induced cardiac reprogramming, which has potential implications in heart tissue regeneration and drug discovery.

Prions are proteins that cause other proteins to misfold and aggregate. Past studies have shown that protein aggregation is a major contributor to the symptoms of neurodegenerative disorders. It has been confirmed that many of these proteins, such as amyloid beta and tau, form β-sheets in a specific orientation, leading to fibril formation. Despite the understanding that prion aggregation plays a role in neurodegenerative disorders, the folding mechanisms and structures of many prions remain unknown. Therefore, this presentation will use yeast prions as the model organism for mammalian prions to study the mechanism of fibril formation in different types of prions. In this project, we will apply similar techniques to investigate the folding of New1, a variant of yeast prion. New1 is hypothesized to exhibit parallel β-sheets between amino acid positions 40–110. This project will create mutant New1 constructs and analyze the interactions within each mutant using electron paramagnetic resonance (EPR) scans to identify the amyloid core and the type of folding involved in New1 aggregation. The data is expected to show regions within the binding area that display stronger interactions, indicated by a lower single-line ratio in the EPR scan. Understanding the mechanisms by which prions fold will aid in the development of treatments and potential cures for neurodegenerative conditions such as Alzheimer’s and Parkinson’s disease.