When analyzing biological datasets, there is an expectation to identify distinct categories among cells, often referred to as cell types. In the context of single-cell RNA sequencing (scRNA-seq), these categories are presumed to be robust and biologically meaningful, based on evolutionary models that predict well-defined and conserved differentiated pathways. However, our study contradicts this expectation by highlighting a surprising fragility in this process. We show that small perturbations, such as the removal of a single cell or minor variation in input data, can lead to significant shifts in dimensionality reduction outcomes and downstream clustering that questions the stability of inferred cell types. Using synthetic data and real scRNA-seq datasets, we introduce a novel method to quantify the global geometry of the data. Our findings suggest that commonly used analysis pipelines exaggerates the stability of cell type assignments, and that careful consideration of robustness is critical when interpreting scRNA-seq results.

Heat shock proteins (HSPs) are chaperone proteins that aid in protein folding and function under stress. Heat Shock Protein 75 (HSP75) is particularly important in resisting apoptosis by protecting against oxidative stress and is often upregulated in cancers, making it a potential therapeutic target in non-small cell lung cancer (NSCLC). Gamitrinib-TPP (GAM) is a small-molecule inhibitor of mitochondrial HSP75 currently in Phase I clinical trials for lymphomas. We hypothesized that GAM's inhibition of HSP75 will induce cell death via apoptosis in NSCLC. To test our hypothesis, we utilized Trypan Blue viability assays, Western blots, caspase activity assays, and confocal microscopy. A high-throughput screening in PC9 cells with HSP75 knockdowns revealed that the absence of HSP75 desensitizes cells to GAM, demonstrating that GAM acts on HSP75. Additionally, Trypan blue cell viability assays showed that GAM significantly reduced cell viability in NSCLC. High-throughput screenings and viability assays using Bax and Bak double knockouts also revealed that GAM functions independently of these proapoptotic proteins. Western blotting confirmed this as we found no change in apoptotic markers after GAM treatment. Our finding that GAM-induced cell death is not necessarily linked to apoptosis prompts further investigation into GAM’s mechanism of cell death and the mitochondrial stress response.

Adipose tissue regulates energy balance through its ability to store lipids and dissipate energy as heat. White adipose tissue (WAT) primarily stores lipids, while brown adipose tissue (BAT) facilitates thermogenesis via mitochondrial uncoupling. Both tissues arise from common progenitors, with differentiation influenced by transcription factors like PPARγ and its co-regulators. The transcriptional repressor TLE3 suppresses thermogenesis in BAT, whereas AES may function as an opposing factor, promoting a thermogenic phenotype. To explore this, 10T1/2 cells are differentiated into brown adipocytes in the presence and absence of rosiglitazone, a PPARγ agonist known to enhance adipogenesis and thermogenic gene expression. Additionally, stable BAT cell lines overexpressing AES or carrying a control vector are used to examine protein expression, with a focus on cold-induced RNA-binding protein CIRBP. Gene expression is assessed through quantitative real-time PCR (qRT-PCR) and normalized using validated reference genes. By evaluating key thermogenic markers such as UCP1, Cidea, and FABP4, these experiments aim to elucidate AES’s role in promoting BAT identity and potentially counteracting TLE3-mediated repression of thermogenesis.

Wound healing is imperative for the prevention of infection and further injury. Chronic wounds, suffered by 9-12 million Americans, can lead to pain, amputation, and in worst cases mortality. Chronic wounds do not move through the repair process well, instead exhibiting a prolonged inflammatory stage. The Coller lab has shown that this stage may be exacerbated by the deficiency of autophagy, a pathway that allows cells to recycle cellular macromolecules. In previous studies done on mice with loss of autophagy, we saw that wounds presented more acute inflammation, NF-kB activation, and a prolonged inflammatory phase. We wanted to determine the type of autophagy important for wound healing. We studied whether loss of only noncanonical autophagy, which involves degradation of extracellular molecules, delays wound healing. We utilized mice, which lack the C terminal WD40 domain of ATG16L1 leading to inactivation of the noncanonical autophagy pathway while retaining canonical autophagy. We wounded mice with loss of noncanonical autophagy and wild type mice and monitored the progression of the wound for up to 15 days of healing. Results show that mice with loss of noncanonical autophagy exhibited slower wound healing than wild type, indicating that noncanonical autophagy likely plays a role in wound healing. Further studies will allow us to identify what drives the transition from inflammatory to proliferative stages of wound healing. This work could lay the foundation for better treatments for chronic wound patients.

Reprogramming female human fibroblasts to induced pluripotent stem cells (iPSCs) has enabled new disease studies and possibilities in regenerative medicine. However, all female hiPSCs display a culture-induced erosion of X-chromosome inactivation (XCI) which impairs differentiation mechanisms. In this process, XIST RNA, which is responsible for silencing the genes on the inactive X chromosome (Xi), is lost, which leads to the Xi re-exhibiting gene expression. The cause of Xi erosion is ultimately XIST loss, but the mechanisms leading to this are still unknown. Recent data has implicated a role of the retrotransposon LINE1 in facilitating the compaction of heterochromatin and has been observed to be highly expressed in iPSCs compared to parental fibroblasts, similar to other XCI epigenetic factors. LINE1 expression is lost throughout cell culture, similar to the loss of XIST in erosion. Considering the process of Xi erosion to be an intersection of the pluripotent state and XCI, LINE1 makes a very compelling candidate to explore in the mechanism of Xi erosion. The goal of this project is to investigate the role of LINE1 in XCI using female iPSCs. In this project, LINE1 expression is investigated in different timepoints being X inactivation, reprogramming, and erosion. Additionally, I seek to understand whether LINE1 transcripts may contribute to erosion. This is an ongoing project that can provide insight into the mechanisms of XCI, Xi erosion, and possibly an understanding of how to prevent erosion.