Primary tracheal adenoid cystic carcinoma: A case report and analysis of the tumor immune microenvironment using single cell RNA sequencing.

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.

Association between Estrogen Exposure and Idiopathic Subglottic Stenosis.

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.

Inflammatory Cytokine Elaboration Following Secondhand Smoke (SHS) Exposure Is Mediated in Part by RAGE Signaling.

The receptor for advanced glycation end products (RAGE) is a key contributor to immune and inflammatory responses in myriad diseases. RAGE is a transmembrane pattern recognition receptor with a special interest in pulmonary anomalies due to its naturally abundant pulmonary expression. Our previous studies demonstrated an inflammatory role for RAGE following acute 30-day exposure to secondhand smoke (SHS), wherein immune cell diapedesis and cytokine/chemokine secretion were accentuated in part via RAGE signaling. However, the chronic inflammatory mechanisms associated with RAGE have yet to be fully elucidated. In this study, we address the impact of long-term SHS exposure on RAGE signaling. RAGE knockout (RKO) and wild-type (WT) mice were exposed to SHS using a nose-only delivery system (Scireq Scientific, Montreal, Canada) for six months. SHS-exposed animals were compared to mice exposed to room air (RA) only. Immunoblotting was used to assess the phospho-AKT and phospho-ERK activation data, and colorimetric high-throughput assays were used to measure NF-kB. Ras activation was measured via ELISAs. Bronchoalveolar lavage fluid (BALF) cellularity was quantified, and a mouse cytokine antibody array was used to screen the secreted cytokines. The phospho-AKT level was decreased, while those of phospho-ERK, NF-kB, and Ras were elevated in both groups of SHS-exposed mice, with the RKO + SHS-exposed mice demonstrating significantly decreased levels of each intermediate compared to those of the WT + SHS-exposed mice. The BALF contained increased levels of diverse pro-inflammatory cytokines in the SHS-exposed WT mice, and diminished secretion was detected in the SHS-exposed RKO mice. These results validate the role for RAGE in the mediation of chronic pulmonary inflammatory responses and suggest ERK signaling as a likely pathway that perpetuates RAGE-dependent inflammation. Additional characterization of RAGE-mediated pulmonary responses to prolonged exposure will provide a valuable insight into the cellular mechanisms of lung diseases such as chronic obstructive pulmonary disease.

ZEB1 promotes non-homologous end joining double-strand break repair.

Repair of DSB induced by IR is primarily carried out by Non-Homologous End Joining (NHEJ), a pathway in which 53BP1 plays a key role. We have discovered that the EMT-inducing transcriptional repressor ZEB1 (i) interacts with 53BP1 and that this interaction occurs rapidly and is significantly amplified following exposure of cells to IR; (ii) is required for the localization of 53BP1 to a subset of double-stranded breaks, and for physiological DSB repair; (iii) co-localizes with 53BP1 at IR-induced foci (IRIF); (iv) promotes NHEJ and inhibits Homologous Recombination (HR); (v) depletion increases resection at DSBs and (vi) confers PARP inhibitor (PARPi) sensitivity on BRCA1-deficient cells. Lastly, ZEB1's effects on repair pathway choice, resection, and PARPi sensitivity all rely on its homeodomain. In contrast to the well-characterized therapeutic resistance of high ZEB1-expressing cancer cells, the novel ZEB1-53BP1-shieldin resection axis described here exposes a therapeutic vulnerability: ZEB1 levels in BRCA1-deficient tumors may serve as a predictive biomarker of response to PARPis.

Localizing Hormone Receptor Expression to Cellular Compartments in Idiopathic Subglottic Stenosis.

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.

A Chemical Biology Primer for NMR Spectroscopists.

Among structural biology techniques, NMR spectroscopy offers unique capabilities that enable the atomic resolution studies of dynamic and heterogeneous biological systems under physiological and native conditions. Complex biological systems, however, often challenge NMR spectroscopists with their low sensitivity, crowded spectra or large linewidths that reflect their intricate interaction patterns and dynamics. While some of these challenges can be overcome with the development of new spectroscopic approaches, chemical biology can also offer elegant and efficient solutions at the sample preparation stage. In this tutorial, we aim to present several chemical biology tools that enable the preparation of selectively and segmentally labeled protein samples, as well as the introduction of site-specific spectroscopic probes and post-translational modifications. The four tools covered here, namely cysteine chemistry, inteins, native chemical ligation, and unnatural amino acid incorporation, have been developed and optimized in recent years to be more efficient and applicable to a wider range of proteins than ever before. We briefly introduce each tool, describe its advantages and disadvantages in the context of NMR experiments, and offer practical advice for sample preparation and analysis. We hope that this tutorial will introduce beginning researchers in the field to the possibilities chemical biology can offer to NMR spectroscopists, and that it will inspire new and exciting applications in the quest to understand protein function in health and disease.

Turnaround Time of Plasma Next-Generation Sequencing in Thoracic Oncology Patients: A Quality Improvement Analysis.

PURPOSE: Genomic analysis of plasma cell-free DNA has become a widespread tool for advanced non-small-cell lung cancer care. Whereas accuracy has been reported on widely, its usefulness is also tied tightly to its turnaround time (TAT), which is not well studied. METHODS: We studied the TAT of commercial plasma next-generation sequencing (NGS; Guardant360) for 533 results from 461 patients at our center between August 2016 and October 2019. The study received institutional review board approval as a quality improvement study; therefore, the results of the test and clinical setting were not analyzed. RESULTS: TAT from blood draw to result (median of 9 days) was slightly longer than the TAT from laboratory receipt to result, a median of 7 days. Testing volume at our center increased three-fold over the time of the study. During this period, clinical TAT decreased from an initial median of 12 days to a median of 8 days in 2018, but more recently the median increased slightly to 9 days. During the most recent 12 months, 231 (95%) of 247 cases resulted within 14 days from blood draw, with delayed results usually because of billing, whereas 44 cases (18%) resulted within 7 days of blood draw. Studying 92 cases drawn in the most recent 3-month period, the median time of result receipt was 4:01 pm Eastern Time/1:01 pm Pacific Time; 39 results (43%) were returned after 5:00 pm Eastern Time. CONCLUSION: In a large single-institution experience, we find that plasma NGS results can routinely be expected within 2 weeks, but uncommonly result within 1 week, supporting the need for new strategies to incorporate plasma NGS into the initial genotyping of advanced non-small-cell lung cancer.

Conodipine-P1-3, the First Phospholipases A2 Characterized from Injected Cone Snail Venom.

The phospholipase A2 (PLA2s) superfamily are ubiquitous small enzymes that catalyze the hydrolysis of phospholipids at the sn-2 ester bond. PLA2s in the venom of cone snails (conodipines, Cdpi) are composed of two chains termed as alpha and beta subunits. Conodipines are categorized within the group IX of PLA2s. Here we describe the purification and biochemical characterization of three conodipines (Cdpi-P1, -P2 and -P3) isolated from the injected venom of Conus purpurascens Using proteomics methods, we determined the full sequences of all three conodipines. Conodipine-P1-3 have conserved consensus catalytic domain residues, including the Asp/His dyad. Additionally, these enzymes are expressed as a mixture of proline hydroxylated isoforms. The activities of the native Conodipine-Ps were evaluated by conventional colorimetric and by MS-based methods, which provide the first detailed cone snail venom conodipine activity monitored by mass spectrometry. Conodipines can have medicinal applications such inhibition of cancer proliferation, bacterial and viral infections among others.

Structural plasticity of mini-M conotoxins - expression of all mini-M subtypes by Conus regius.

The mini-M conotoxins are peptidic scaffolds found in the venom of cones snails. These scaffolds are tightly folded structures held together by three disulfide bonds with a CC-C-C-CC arrangement (conotoxin framework III) and belong to the M Superfamily of conotoxins. Here, we describe mini-M conotoxins from the venom of Conus regius, a Western Atlantic worm-hunting cone snail species using transcriptomic and peptidomic analyses. These C. regius conotoxins belong to three different subtypes: M1, M2, and M3. The subtypes show little sequence homology, and their loop sizes (intercysteine amino acid chains) vary significantly. The mini-Ms isolated from dissected venom contains preferentially hydroxylated proline residues, thus augmenting the structural reach of this conotoxin class. Using 2D-NMR methods, we have determined the 3D structure of reg3b, an M2 subtype conotoxin, which shows a constrained multi-turn scaffold. The structural diversity found within mini-M conotoxin scaffolds of C. regius is indicative of structural hypervariability of the conotoxin M superfamily that is not seen in other superfamilies. These stable minimalistic scaffolds may be investigated for the development of engineered peptides for therapeutic applications. DATABASES: Sequences are available in GenBank under accession numbers MF588935-MF588952. Structural data are available in the RCSB protein database under the accession code 6BX9.

In vivo and in vitro testing of native α-conotoxins from the injected venom of Conus purpurascens.

α-Conotoxins inhibit nicotinic acetylcholine receptors (nAChRs) and are used as probes to study cholinergic pathways in vertebrates. Model organisms, such as Drosophila melanogaster, express nAChRs in their CNS that are suitable to investigate the neuropharmacology of α-conotoxins in vivo. Here we report the paired nanoinjection of native α-conotoxin PIA and two novel α-conotoxins, PIC and PIC[O7], from the injected venom of Conus purpurascens and electrophysiological recordings of their effects on the giant fiber system (GFS) of D. melanogaster and heterologously expressed nAChRs in Xenopus oocytes. α-PIA caused disruption of the function of giant fiber dorsal longitudinal muscle (GF-DLM) pathway by inhibiting the Dα7 nAChR a homolog to the vertebrate α7 nAChR, whereas PIC and PIC[O7] did not. PIC and PIC[O7] reversibly inhibited ACh-evoked currents mediated by vertebrate rodent (r)α1ß1δγ, rα1ß1δε and human (h)α3ß2, but not hα7 nAChR subtypes expressed in Xenopus oocytes with the following selectivity: rα1ß1δε > rα1ß1δγ ≈ hα3ß2 >> hα7. Our study emphasizes the importance of loop size and α-conotoxin sequence specificity for receptor binding. These studies can be used for the evaluation of the neuropharmacology of novel α-conotoxins that can be utilized as molecular probes for diseases such as, Alzheimer's, Parkinson's, and cancer. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'