Week 10 Summer Undergraduate Research Showcase AMGEN 2- 3:30
Wednesday, August 30 3:30PM – 5:00PM
Location: Online - Live
The Zoom event has ended.
Presentation 1
AISHINI SINGH, Grasiela Torres, Anthony J. Covarrubias
Investigating the role of cell cycle regulator proteins in senescent macrophages
Cellular senescence is characterized by irreversible cell-cycle arrest in response to various stimuli such as genotoxic stress and is an emerging driver of aging. Features of senescence include altered metabolism and secretion of proinflammatory and immune-modulating molecules known as the senescence-associated secretory phenotype (SASP). Prolonged secretion of SASP is thought to contribute to pathogenesis of age-related diseases via chronic low-grade inflammation, ultimately damaging the tissue microenvironment. Targeting senescent cells in vivo is challenging due to lack of biomarker availability, as markers of senescence are not universally expressed by known senescent cell types. The Covarrubias Lab discovered macrophages as a new cell type that become senescent, providing a role for the innate immune system in aging. RNAseq analysis revealed up-regulation of cell cycle regulators that are transcriptionally regulated by senescence marker p53 in senescent macrophages. Thus, to determine the function of cell cycle regulator proteins p53 and p21 in macrophage senescent programming, we took a genetic approach in which cells lacking each protein were generated via CRISPR-Cas9 editing or harvested from knockout mice. These knockouts were then subjected to our lab's in vitro model of senescence and analyzed for effects on various aspects of senescent macrophage biology: cell cycle arrest, beta-gal activity, and expression of senescence-associated genes. p53 KOs lacked senescent characteristics, suggesting that p53 is necessary for macrophage senescent programming. p21 KOs are hypothesized to display similar biological effects. The purpose of this study is to provide insight into the molecular mechanisms and biomarkers driving senescence in resident macrophages.
Presentation 2
SEAN C. PASCOE, Andrew Ramirez, and Aaron S. Meyer
Deciphering Complex Immune Cell Relationships in Systemic Lupus Erythematosus with PARAFAC2
Single-cell data is a crucial tool for understanding cell-level heterogeneity; however, analysis of this data from multiple conditions is difficult because the same cell is not shared across conditions. Such is the case for single-cell RNA-sequencing (scRNA-seq), which profiles the transcriptional responses of individual cells. When scRNA-seq experiments involve cells from multiple conditions, data is often flattened into a 2D matrix, which removes condition-specific information from further analysis. However, the use of tensors, arrays that are not restricted to two dimensions, preserves the structure of the experiment across cells, genes, and conditions. Decomposition of tensors can thus more accurately discriminate condition-level variation from cell-level variation, untangling biological effects throughout conditions. In this study, we use tensor decomposition to identify meaningful patterns in a scRNA-seq dataset from 95 healthy controls and 162 patients with Systemic Lupus Erythematosus (SLE), a heterogeneous autoimmune disease characterized by variation in immune subpopulations and between individuals. Because scRNA-seq provides snapshots of heterogeneous cells across patients, we use PARAFAC2, a tensor decomposition strategy that handles data wherein one dimension (cells) must be aligned before comparison with other dimensions (genes and patients). Using PARAFAC2, we confirm the importance of naïve CD4+ T cell populations in SLE noted by the original publication, while also identifying new distinguishing features of SLE, such as the presence of peripheral blood megakaryocytes. Overall, this study highlights the ability of PARAFAC2 to reveal biological insight in complex clinical data, as well as the value of tensor decomposition in the analysis of multi-condition data.
Presentation 3
TRUMAN C. NATIVIDAD, Benny Mosquieria, Katie Wilhem, Olivia Hung-Jhen Chen, D’Juan T. Farmer
Investigating the Presence and Development of a Novel Meningeal Population Using Mouse and Zebrafish
The calvaria is the bony skeleton that encases and protects the brain. The meninges that sit just below the calvaria have been implicated as a regulator of calvaria development, but our comprehension of this interaction is limited. Previously, using single-cell genomic technologies in mice, we identified the transcriptional signature (Ctgf+, Foxc1+, Matn4+, Nppc+) for the population of meninges that lies closest to the calvaria, termed the periosteal dura. We postulate that this population of cells acts as a critical regulator of calvaria development. Single-cell analysis in zebrafish suggests that the periosteal dura’s transcriptional signature might be conserved across vertebrate species, but a spatiotemporal analysis of the periosteal dura remains necessary. To address this gap, we performed in situ analysis in zebrafish using RNAscope for periosteal dura enriched genes (i.e. ctgfa, hyal4) in combination with a broad fluorescent reporter for the meninges (foxc1b:gal4; UAS:mCherryNTR). We identified ctgfa+mCherry+ cells below the calvaria, consistent with the anticipated location of the periosteal dura. We also used recently analyzed scATAC-seq data from mouse skullcaps to identify candidate periosteal dura-specific enhancers. By isolating, cloning, and injecting enhancer constructs into zebrafish embryos, we hope to identify green fluorescent protein reporters that specifically label the periosteal dura for future live-imaging experiments. We similarly engineered and injected ctgfa and ctgfb promoter constructs to detect ctgf+ cell locations throughout zebrafish meningeal development. The presented results lay a critical foundation for designing future experiments to elucidate the periosteal dura's development, function, and relationship with the calvaria.
Presentation 4
HANYI JIA, Calvin M. Burns, Katherine K. Zhang, and S. Joseph Endicott
Comparative Analysis of the Drugs that Activate Chaperone-Mediated Autophagy
Chaperone-mediated autophagy (CMA) is the most selective pathway of lysosomal proteolysis, selecting individual cytosolic proteins for degradation. CMA negatively regulates the abundance of proteins associated with the pathogenesis of Alzheimer's, Parkinson's, fatty liver disease, cancer, and atherosclerosis. CMA has recently been found to degrade proteins that negatively affect lifespan, and the CMA-activating drugs has become attractive therapeutic targets for age-associated diseases. Several recent studies have identified novel compounds or repurposed clinically relevant drugs as CMA activators. However, the results of these studies have never been replicated and there are no side-by-side comparisons to determine the relative efficacy of CMA activating small molecules, and this study aims to address this knowledge gap. Using both a fluorescent CMA reporter and western blotting for CMA degradation targets, we evaluated several putative CMA activating drugs at five doses each, in two cultured cell lines. Similar to previous studies, class I PI3K inhibitors, such as buparlisib, and mTOR inhibitors, such as Torin2 strongly activated CMA at micromolar doses. Interestingly, RARα inhibitors, which allegedly activate the transcription of lysosomal genes, had weaker effects than buparlisib at much lower doses and showed no differences in the expression of any of the lysosomal proteins measured. This means to explore the potential of the RARα inhibitors, its distinct pathway of influencing CMA needs to be discovered. Future studies will further explore the possible therapeutic viability of these, and other possible CMA-activating drugs.
Presentation 5
ALYSSA M. PAMA, Frances D. Nicklen, Kyle J. Chen, and Daniel T. Kamei
Application of Dextranase to Improve the Performance of Lateral-Flow Immunoassays Integrated with Polyethylene Glycol-Dextran Aqueous Two-Phase Systems
Although the lateral-flow immunoassay (LFA) has seen widespread success as a point-of-care (POC) device for COVID-19, it is limited by its low sensitivity. To address this challenge, our laboratory has utilized polymer-salt aqueous two-phase systems (ATPSs) to preconcentrate target biomarkers into the smaller volume phase before applying that phase to the LFA. While polymer-salt ATPSs are effective at increasing LFA sensitivity, these ATPSs are temperature sensitive, making them less viable for use at the POC as they need to operate within a wide range of temperatures. Since polymer-polymer ATPSs are less dependent on temperature, we explored the integration of the polyethylene glycol (PEG)-dextran ATPS with the LFA. Hydrophilic biomarkers partition preferentially into the more hydrophilic dextran-rich bottom phase of this ATPS, but unfortunately, the dextran-rich phase is viscous and thus flows poorly on paper, resulting in little to no binding at the detection zone of an LFA. We hypothesized that dextranase could be used to cleave dextran polymers and thus increase the fluid flow rate of the dextran-rich phase, thereby improving overall LFA performance. The ability of dextranase to improve the fluid flow of the dextran-rich phase was examined by conducting flow studies on paper. An LFA for modified latex nanoparticles was then developed, and using this LFA, we demonstrated that the incorporation of dextranase could allow the identification of the target that initially was undetectable. The incorporation of dextranase is therefore critical for this workflow, and future work will involve varying the method of dextranase incorporation.