Biology subjects

Thurman, A. L.

Publications and source records attributed to Thurman, A. L..

4 recordsLinked to original sources

bioRxiv2021

Topography-dependent gene expression and function of common cell archetypes in large and small porcine airways

The small airways of humans are affected early in several lung diseases. However, because they are relatively inaccessible, little is known about the epithelial cells that line these airways. We performed a single cell RNA-seq census of small and large airways of wild-type pigs and pigs with disrupted cystic fibrosis transmembrane conductance regulator (CFTR) gene. The sequencing data showed that small airway epithelia had similar major cell types as large airways but no ionocytes; moreover, lack of CFTR expression had minimal effect on the transcriptome. Small airway epithelial cells expressed a different transcriptome than large airway cells. Quantitative immunohistochemistry showed that small airway basal cells participate in epithelial barrier function. Finally, sequencing data and in vitro electrophysiologic studies suggest that small airway epithelia have a water and ion transport advantage. Our data highlight the archetypal nature of basal, secretory, and ciliated airway cells with location-dependent gene expression and function.

genomics↗

bioRxiv2021

Performance of a scalable extraction-free RNA-seq method

RNA sequencing enables high-contents/high-complexity measurements in small molecule screens performed on biological samples. Whereas the costs of DNA sequencing and the complexity of RNA-seq library preparation and analysis have decreased consistently, RNA extraction remains a significant bottleneck for RNA-seq of hundreds of samples in parallel. Direct use of cell lysate for RNA-seq library prep is common in single cell RNA-seq but not in bulk RNA-seq protocols. Recently published protocols suggest that direct lysis is compatible with simplified RNA-seq library prep. Here, we evaluate the performance of a bulk RNA-seq library prep protocol optimized for analysis of many samples of adherent cultured cells in parallel. We combine a low-cost direct lysis buffer compatible with cDNA synthesis ("in-lysate cDNA synthesis") with Smart-3SEQ and examine the effects of calmidazolium and fludrocortisone-induced perturbation of primary human dermal fibroblasts. We compared this method to normalized purified RNA inputs from matching samples followed by Smart-3SEQ or Illumina TruSeq library prep. Our results show that whereas variable RNA inputs for each sample in the in-lysate cDNA synthesis protocol result in variable sequencing depth, this had minimal effect on data quality, measurement of gene expression patterns, or generation of differentially expressed gene lists. We found that in-lysate cDNA synthesis combined with Smart-3SEQ RNA-seq library prep allows generation of high-quality data when compared to library prep with extracted RNA, or when compared to Illumina TruSeq. Our data show that small molecule screens using RNA-seq are feasible at low reagent and time costs.

genomics↗

bioRxiv2020

Lentiviral vectors transduce lung stem cells without disrupting plasticity

Life-long expression of a gene therapy agent likely requires targeting stem cells. Here we ask the question: does viral vector transduction or ectopic expression of a therapeutic transgene preclude airway stem cell function? We used a lentiviral vector containing a GFP or cystic fibrosis transmembrane conductance regulator (CFTR) transgene to transduce primary airway basal cells from human cystic fibrosis (CF) or non-CF lung donors and monitored expression and function after differentiation. Ussing chamber measurements confirmed CFTR-dependent chloride channel activity in CF donor cells. Immunostaining, quantitative real-time PCR, and single-cell sequencing analysis of cell-type markers indicated that vector transduction or CFTR expression does not alter the formation of pseudostratified, fully-differentiated epithelial cell cultures or cell type distribution. These results have important implications for use of gene addition or gene editing strategies as life-long curative approaches for lung genetic diseases.

molecular biology↗

bioRxiv2020

HSP90 inhibition modulates NFB signaling in airway goblet cell metaplasia

Goblet cell metaplasia and mucus hyper-production are key features of chronic muco-obstructive lung diseases such as asthma, chronic bronchitis, and cystic fibrosis. Various mechanisms lead to goblet cell metaplasia in the airways; the driving mechanism for goblet cell metaplasia in a specific patient may be unknown. We recently found that heat shock protein 90 (HSP90) is important for both IL-13- and IL-17- induced airway goblet cell metaplasia. HSP90 interacts with multiple clients that are important in goblet cell metaplasia including Akt, Jak/STAT, IRS, Notch, and various kinases involved in NF{kappa}B signaling. Here, we used a targeted phospho-proteomic approach to identify candidate HSP90 clients modulated by the HSP90-inhibitor geldanamycin. NF{kappa}B family members were enriched amongst the top candidate targets of HSP90 inhibition in IL-13 an organotypic model of human airway epithelia. We hypothesized that HSP90 inhibition modulated goblet cell metaplasia by interfering with NF{kappa}B signaling. We used transcription factor activation, nuclear translocation, and phospho-specific immunofluorescence assays to investigate how IL-13 exposure and HSP90 inhibition modulated NF{kappa}B. We found that HSP90 inhibition prevented goblet cell metaplasia by non-canonically blocking NF{kappa}B p100/p52 function in human airway epithelia. NF{kappa}B modulation via its interaction with HSP90 is a pharmaceutically feasible therapeutic target for goblet cell metaplasia; this approach may enable treatment of patients with chronic muco-inflammatory lung diseases with both known or unidentified disease-driving mechanisms.

cell biology↗