Search

In the nucleus, transcriptionally silent genes are sequestered into heterochromatin compartments comprising nucleosomes decorated with histone H3 Lys9 trimethylation and a protein called HP1α. This protein can form liquid-liquid droplets in vitro and potentially organize heterochromatin through a phase separation mechanism that is promoted by phosphorylation. Elucidating the molecular interactions that drive HP1α phase separation and its consequences on nucleosome structure and dynamics has been challenging due to the viscous and heterogeneous nature of such assemblies. Here, we tackle this problem by a combination of solution and solid-state NMR spectroscopy, which allows us to dissect the interactions of phosphorylated HP1α with nucleosomes in the context of phase separation. Our experiments indicate that phosphorylated human HP1α does not cause any major rearrangements to the nucleosome core, in contrast to the yeast homologue Swi6. Instead, HP1α interacts specifically with the methylated H3 tails and slows the dynamics of the H4 tails. Our results shed light on how phosphorylated HP1α proteins may regulate the heterochromatin landscape, while our approach provides an atomic resolution view of a heterogeneous and dynamic biological system regulated by a complex network of interactions and post-translational modifications.

Background: Tracheal adenoid cystic carcinoma (ACC) is a slow growing yet aggressive malignancy with high rates of local recurrence as well as distant metastasis. Tracheal ACC exhibit a low mutation burden along with high mutational diversity, and generally do not respond well to chemotherapeutics. Methods: We present a rare case of primary tracheal ACC initially presenting with nonspecific cervicalgia and globus sensation that was ultimately treated with tracheal resection followed by chemoradiation. Immune profiling of intratumoral T‐cell receptor (TCR) repertoire was subsequently performed using single cell RNA sequencing (scRNAseq). Results: We describe a rare case of primary tracheal adenoid cystic carcinoma highlighting several management principles as well as providing new insights into intratumor T cell populations. Conclusions: Primary tracheal ACC is most commonly treated with surgical resection followed by adjuvant therapy. Further characterization of the tumor immune microenvironment is necessary to better understand ACC disease biology and to identify potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Objectives: Recent immunologic study of the adaptive immune repertoire in the subglottic airway demonstrated high‐frequency T cell clones that do not overlap between individuals. However, the anatomic distribution and antigenic target of the T cell repertoire in the proximal airway mucosa remain unresolved. Methods: Single‐cell RNA sequencing of matched scar and unaffected mucosa from idiopathic subglottic stenosis patients (iSGS, n = 32) was performed and compared with airway mucosa from healthy controls (n = 10). T cell receptor (TCR) sequences were interrogated via similarity network analysis to explore antigenic targets using the published algorithm: Grouping of Lymphocyte Interactions by Paratope Hotspots (GLIPH2). Results: The mucosal T cell repertoire in healthy control airways consisted of highly expressed T cell clones conserved across anatomic subsites (trachea, bronchi, bronchioles, and lung). In iSGS, high‐frequency clones were equally represented in both scar and adjacent non‐scar tissue. Significant differences in repertoire structure between iSGS scar and unaffected mucosa was observed, driven by unique low‐frequency clones. GLIPH2 results suggest low‐frequency clones share targets between multiple iSGS patients. Conclusion: Healthy airway mucosa has a highly conserved T cell repertoire across multiple anatomic subsites. Similarly, iSGS patients have highly expressed T cell clones present in both scar and unaffected mucosa. iSGS airway scar possesses an abundance of less highly expanded clones with predicted antigen targets shared between patients. Interrogation of these shared motifs suggests abundant adaptive immunity to viral targets in iSGS airway scar. These results provide insight into disease pathogenesis and illuminate new treatment strategies in iSGS. Level of Evidence: NA Laryngoscope, 134:3245–3252, 2024 [ABSTRACT FROM AUTHOR]

Objectives: Recent translational scientific efforts in subglottic stenosis (SGS) support a disease model where epithelial alterations facilitate microbiome displacement, dysregulated immune activation, and localized fibrosis. Given the observed immune cell infiltrate in SGS, we sought to test the hypothesis that SGS cases possessed a low diversity (highly clonal) adaptive immune response when compared with healthy controls. Methods: Single cell RNA sequencing (scRNA‐seq) of subglottic mucosal scar in iSGS (n = 24), iLTS (n = 8), and healthy controls (n = 7) was performed. T cell receptor (TCR) sequences were extracted, analyzed, and used to construct repertoire structure, compare diversity, interrogate overlap, and define antigenic targets using the Immunarch bioinformatics pipeline. Results: The proximal airway mucosa in health and disease are equally diverse via Hill framework quantitation (iSGS vs. iLTS vs. Control, p > 0.05). Repertoires do not significantly overlap between individuals (Morisita <0.02). Among iSGS patients, clonality of the TCR repertoire is driven by CD8+ T cells, and iSGS patients possess numerous TCRs targeting viral and intercellular pathogens. High frequency clonotypes do not map to known targets in public datasets. Conclusion: SGS cases do not possess a lower diversity adaptive immune infiltrate when compared with healthy controls. Interestingly, the TCR repertoire in both health and disease contains a restricted number of high frequency clonotypes that do not significantly overlap between individuals. The target of the high frequency clonotypes in health and disease remain unresolved. Level of Evidence: NA Laryngoscope, 134:1757–1764, 2024 [ABSTRACT FROM AUTHOR]

Exposure to secondhand smoke (SHS) during fetal development results in negative postnatal effects, including altered organ development, changes in metabolism, and increased risk of respiratory illness. Previously, we found the induction of intrauterine growth restriction (IUGR) dependent on the expression of the receptor for advanced glycation end-products (RAGE) in mice treated with SHS. Furthermore, antenatal SHS exposure increases RAGE expression in the fetal lung. Our objective was to determine the postnatal effects of antenatal SHS treatment in 4- and 12-week-old offspring. Pregnant animals were treated with SHS via a nose-only delivery system (Scireq Scientific, Montreal, Canada) for 4 days (embryonic day 14.5 through 18.5), and offspring were evaluated at 4 or 12 weeks of age. Animal and organ weights were measured, and lungs were histologically characterized. Blood pressure and heart rates were obtained, and RAGE protein expression was determined in the lungs of control and treated animals. We observed the following: (1) significant decreases in animal, liver, and heart weights at 4 weeks of age; (2) increased blood pressure in 4-week-old animals; and (3) increased RAGE expression in the lungs of the 4-week-old animals. Our results suggest an improvement in these metrics by 12 weeks postnatally such that measures were not different regardless of RA or SHS exposure. Increased RAGE expression in lungs from 4-week-old mice antenatally treated with SHS suggests a possible role for this important smoke-mediated receptor in establishing adult disease following IUGR pregnancies. [ABSTRACT FROM AUTHOR]

Objective: Idiopathic subglottic stenosis (iSGS) is a rare, recurrent, fibroinflammatory disease affecting the larynx and proximal trachea. Given it occurs primarily in adult females, estrogen is speculated to play a central pathophysiological role. This study aimed to evaluate relationships between estrogen exposure, disease progression, and recurrence. Methods: North American Airway Collaborative (NoAAC) data of adults with iSGS obstructive airway lesions, who underwent index endoscopic airway dilation, were used to identify associations between estrogen exposure, disease characteristics, and time to recurrence (TTR), and interventions were analyzed using Kruskal–Wallis test and Pearson coefficient. Cox proportional hazards regression models compared hazard ratios by estrogen exposure. Kaplan–Meier curves were plotted for TTR based on menopausal status. Results: In all, 533 females had complete estrogen data (33% premenopausal, 17% perimenopausal, 50% postmenopausal). Median estrogen exposure was 28 years. Overall, there was no dose–response relationship between estrogen exposure and disease recurrence. Premenopausal patients had significantly shorter time from symptom manifestation to diagnosis (1.17 vs. 1.42 years perimenopausal vs. 2.08 years postmenopausal, p < 0.001), shorter time from diagnosis to index endoscopic airway dilation (1.90 vs. 2.50 vs. 3.76 years, p = 0.005), and higher number of procedures (1.73 vs. 1.20 vs. 1.08 procedures, p < 0.001). Conclusions: We demonstrate premenopausal patients may have a more aggressive disease variant than their peri‐ and postmenopausal counterparts. However, it is unclear as to whether this is related to reduced estrogen in the peri‐ and postmenopausal states or the age‐related physiology of wound healing and inflammation, regardless of estrogen. Level of Evidence: 3 Laryngoscope, 134:825–830, 2024 [ABSTRACT FROM AUTHOR]

Objectives: Idiopathic subglottic stenosis (iSGS) is an unexplained progressive fibrosis of the upper airway. iSGS almost exclusively affects women; as a result, female hormones (estrogen and progesterone) have been proposed to participate in the pathogenesis of iSGS. Our aim was to localize cell‐specific gene expression of estrogen receptors (ESR1 and ESR2) and progesterone receptor (PGR) using an established iSGS single‐cell RNA sequencing (scRNAseq) cell atlas. Study Design: Ex vivo molecular study of airway scar and healthy mucosa from iSGS patients. Methods: An established scRNAseq atlas consisting of 25,974 individually sequenced cells from subglottic scar (n = 7) or matched unaffected mucosa (n = 3) in iSGS patients was interrogated for RNA expression of ESR1, ESR2, and PGR. Results were quantified and compared across cell subsets, then visualized using Uniform Manifold Approximation and Projection (UMAP). Confirmatory protein assessment of endocrine receptors in fibroblasts from iSGS patients (n = 5) was performed via flow cytometry. Results: The proximal airway mucosa in iSGS patients demonstrates differential expression of endocrine receptors (ESR1, ESR2, PGR). Within airway scar, endocrine receptors are primarily expressed by fibroblasts, immune cells, and endothelial cells. Fibroblasts show strong ESR1 and PGR expression, while immune cells possess RNA for both ESR1 and ESR2. Endothelial cells predominantly express ESR2. Epithelial cells in unaffected mucosa express all three receptors, which are all reduced in airway scar. Conclusions: scRNAseq data localized endocrine receptor expression to specific cell subsets. These results provide the foundation for future work interrogating how hormone‐dependent mechanisms promote, sustain, or participate in iSGS disease pathogenesis. Level of Evidence: NA; Basic science Laryngoscope, 133:3506–3511, 2023 [ABSTRACT FROM AUTHOR]