Cystic Fibrosis (CF) is a genetic disorder caused by mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene that leads to production of thick, sticky mucus by airway secretory cells. Gene therapies aim to correct CF by targeting basal cells, but access is limited due to mucus obstruction. Submucosal glands (SMGs), located beneath the airway epithelium, are responsible for producing ~90% of the mucus in the upper and large airways. This study quantifies spatial relationships between SMGs, submucosal ducts, and surrounding vasculature within the intercartilaginous zone (ICZ) to inform models of systemic gene therapy delivery. Samples from de-identified human donors were subjected to immunofluorescence staining and imaging to measure distances between SMGs, ducts, and vasculature. SMGs were isolated to generate organoids, and CFTR function was assessed using a forskolin-induced swelling (FIS) assay with western blot confirmation of protein expression. Mean distances were 14.16 µm between SMGs and the ICZ vasculature and 8.38 µm between submucosal ducts and the ICZ vasculature. Organoids demonstrated functional CFTR activity. Next steps include incorporating vascular components into the organoid model to better simulate systemic delivery. These findings support targeted systemic gene therapy strategies by leveraging SMG biology and spatial organization.

During embryogenesis, macrophages disseminate through complex 3D tissue environments to colonize developing tissues. Previous research showed that early macrophage dispersal in Drosophila is driven in part by BMP signaling, and these BMP-responsive macrophages also upregulate key regulators of developmental pathways (Wnt, Notch, Hippo, FGF, and EGF). However, it is unclear if these pathways directly influence the earliest migratory decisions of macrophages during Drosophila embryogenesis. To identify which pathways regulate early migration, the GAL4-UAS system was used by crossing UAS-RNAi knockdown (KD) lines targeting developmental receptors in macrophages with a macrophage-specific GAL4 driver and marker line. Fixed embryos were imaged using confocal microscopy. Macrophages’ spatial positions were collected with Imaris and analyzed with Kernel Density Estimation to identify migration differences between control and KD embryos. Here, we found that Frizzled 1, Frizzled 2, Notch, Fat, and Heartless receptor KDs led to a loss of anterior-dorsal migration, EGFR receptor KD led to a loss of anterior-ventral migration, and Breathless receptor KD led to a loss of posterior-dorsal migration. These KD embryos also showed a higher density of macrophages along the head-to-pre-germband migration route. These findings indicate that these pathways may influence early macrophage route choice and offer a framework for future studies to better understand the mechanisms guiding macrophage migration during Drosophila embryogenesis.

KAT6A and KAT6B encode lysine acetyltransferases that regulate chromatin accessibility through histone acetylation. De novo mutations in these genes cause rare neurodevelopmental syndromes characterized by intellectual disability, developmental delays, and impaired neuronal maturation. Despite their established roles in neural development, how patient-derived mutations alter early cell-fate decisions and differentiation is still not properly understood. This study investigates whether de novo mutations in KAT6A or KAT6B disrupt the differentiation from induced pluripotent stem cells (iPSCs) to neural progenitor cells (NPCs) and neurons using patient-derived iPSC lines. We successfully confirmed the expression of pluripotency markers in the iPSC lines using immunofluorescence assays (IFAs) prior to differentiating the cells into NPCs. While during the differentiation process, we noted morphological differences between the control, KAT6A, and KAT6B lines, IFAs will be performed to confirm the cellular identity of NPCs. We will further differentiate the NPCs into neurons to model early neurodevelopment. We will perform Western Blot Analysis to determine the expression of KAT6A/B, RNA-sequencing analysis, as well as ATAC-sequencing to determine the effect of mutations in these important chromatin regulators on the gene/protein expression profiles of all three cell types (iPSCs, NPCs, and Neurons) to map the effect of these mutations on early neurodevelopment.

Influenza-A (IAV) is the most common trigger for pneumonia worldwide, a pulmonary infection that is routinely treated with hyperoxia (HO) therapy. Our previous studies showed that IAV infections and HO exposure both lead to downregulation (but not depletion) of stretch-activated TREK-1 K+ channels in the lung, which in mouse pneumonia models, accelerates further lung injury. Interestingly, pharmacological activation of the remaining TREK-1 channels conferred protective effects. However, it remained unknown whether pharmacological activation of TREK-1 also confers protective effects against bacterial lung infections. Using whole-cell patch-clamp and gene/protein expression assays, we established the functional expression of TREK-1 channels in primary human alveolar epithelial cells (HAEC). In a disease-relevant model, infection of HAECs with live Pseudomonas aeruginosa (PA) bacteria resulted in downregulation of TREK-1 gene and protein expression, leading to cell membrane depolarization, increased intracellular Ca2+ levels, activation of the transcription factor NF-kB, and pro-inflammatory cytokine secretion. Notably, pharmacological activation of residual TREK-1 channels with the novel compound ML335 reversed PA-induced cell membrane depolarization and reduced intracellular Ca2+ levels, NF-kB activity, and inflammatory cytokine secretion. Thus, pharmacological activation of TREK-1 channels represents a novel and unifying therapeutic strategy to attenuate inflammation across diverse infectious insults.

This abstract has been withheld from publication.