Single-cell transcriptomic atlas reveals increased regeneration in diseased human inner ear balance organs.

Mammalian inner ear hair cell loss leads to permanent hearing and balance dysfunction. In contrast to the cochlea, vestibular hair cells of the murine utricle have some regenerative capacity. Whether human utricular hair cells regenerate in vivo remains unknown. Here we procured live, mature utricles from organ donors and vestibular schwannoma patients, and present a validated single-cell transcriptomic atlas at unprecedented resolution. We describe markers of 13 sensory and non-sensory cell types, with partial overlap and correlation between transcriptomes of human and mouse hair cells and supporting cells. We further uncover transcriptomes unique to hair cell precursors, which are unexpectedly 14-fold more abundant in vestibular schwannoma utricles, demonstrating the existence of ongoing regeneration in humans. Lastly, supporting cell-to-hair cell trajectory analysis revealed 5 distinct patterns of dynamic gene expression and associated pathways, including Wnt and IGF-1 signaling. Our dataset constitutes a foundational resource, accessible via a web-based interface, serving to advance knowledge of the normal and diseased human inner ear.

An essential signaling cascade for avian auditory hair cell regeneration.

Hearing loss is a chronic disease affecting millions of people worldwide, yet no restorative treatment options are available. Although non-mammalian species can regenerate their auditory sensory hair cells, mammals cannot. Birds retain facultative stem cells known as supporting cells that engage in proliferative regeneration when surrounding hair cells die. Here, we investigated gene expression changes in chicken supporting cells during auditory hair cell death. This identified a pathway involving the receptor F2RL1, HBEGF, EGFR, and ERK signaling. We propose a cascade starting with the proteolytic activation of F2RL1, followed by matrix-metalloprotease-mediated HBEGF shedding, and culminating in EGFR-mediated ERK signaling. Each component of this cascade is essential for supporting cell S-phase entry in vivo and is integral for hair cell regeneration. Furthermore, STAT3-phosphorylation converges with this signaling toward upregulation of transcription factors ATF3, FOSL2, and CREM. Our findings could provide a basis for designing treatments for hearing and balance disorders.

TAD evolutionary and functional characterization reveals diversity in mammalian TAD boundary properties and function.

Topological associating domains (TADs) are self-interacting genomic units crucial for shaping gene regulation patterns. Despite their importance, the extent of their evolutionary conservation and its functional implications remain largely unknown. In this study, we generate Hi-C and ChIP-seq data and compare TAD organization across four primate and four rodent species and characterize the genetic and epigenetic properties of TAD boundaries in correspondence to their evolutionary conservation. We find 14% of all human TAD boundaries to be shared among all eight species (ultraconserved), while 15% are human-specific. Ultraconserved TAD boundaries have stronger insulation strength, CTCF binding, and enrichment of older retrotransposons compared to species-specific boundaries. CRISPR-Cas9 knockouts of an ultraconserved boundary in a mouse model lead to tissue-specific gene expression changes and morphological phenotypes. Deletion of a human-specific boundary near the autism-related AUTS2 gene results in the upregulation of this gene in neurons. Overall, our study provides pertinent TAD boundary evolutionary conservation annotations and showcases the functional importance of TAD evolution.

TAD Evolutionary and functional characterization reveals diversity in mammalian TAD boundary properties and function.

Topological associating domains (TADs) are self-interacting genomic units crucial for shaping gene regulation patterns. Despite their importance, the extent of their evolutionary conservation and its functional implications remain largely unknown. In this study, we generate Hi-C and ChIP-seq data and compare TAD organization across four primate and four rodent species, and characterize the genetic and epigenetic properties of TAD boundaries in correspondence to their evolutionary conservation. We find that only 14% of all human TAD boundaries are shared among all eight species (ultraconserved), while 15% are human-specific. Ultraconserved TAD boundaries have stronger insulation strength, CTCF binding, and enrichment of older retrotransposons, compared to species-specific boundaries. CRISPR-Cas9 knockouts of two ultraconserved boundaries in mouse models leads to tissue-specific gene expression changes and morphological phenotypes. Deletion of a human-specific boundary near the autism-related AUTS2 gene results in upregulation of this gene in neurons. Overall, our study provides pertinent TAD boundary evolutionary conservation annotations, and showcase the functional importance of TAD evolution.

RNA Sequencing and Gene Ontology Analysis in Acute Invasive Fungal Sinusitis.

BACKGROUND: Acute invasive fungal sinusitis (AIFS) is an aggressive and dangerous disease of the paranasal sinuses with high morbidity and mortality. The immune response at the level of the nasal mucosa, the site of entry, has not been previously evaluated. OBJECTIVE: To evaluate differential gene expression in the sinonasal mucosa of AIFS patients as compared to control patients using RNA sequencing. METHODS: Sinonasal tissue samples were prospectively obtained from consenting patients undergoing surgery between November, 2020 and November, 2021. RNA extraction and sequencing were performed and differential expression was analyzed to detect transcriptional differences between patient groups. RESULTS: Tissue samples were collected from 4 patients with active AIFS diagnoses, 2 patients with recovered AIFS, 1 patient with a diagnosis of non-invasive fungal ball, and 4 healthy controls. 255 genes were differentially expressed in AIFS patients as compared to control patients. Specific Gene Ontology (GO) biological processes that were identified as differentially expressed in AIFS patients as compared to controls included the following: 1. GO:0007155 (cell adhesion), 2. GO:0030199 (collagen fibril organization) and 3. GO:0001525 (angiogenesis). CONCLUSION: Transcriptional differences were noted between AIFS and control patients in sinonasal tissue samples. Future work is necessary to determine causes of the differential gene expressions between AIFS and control patients, specifically those who are immunosuppressed, or with preexisting non-invasive forms of fungal sinusitis, to guide treatment and prevention strategies.

Cell-type identity of the avian utricle.

The avian utricle, a vestibular organ of the inner ear, displays turnover of sensory hair cells throughout life. This is in sharp contrast to the mammalian utricle, which shows limited regenerative capacity. Here, we use single-cell RNA sequencing to identify distinct marker genes for the different sensory hair cell subtypes of the chicken utricle, which we validated in situ. We provide markers for spatially distinct supporting cell populations and identify two transitional cell populations of dedifferentiating supporting cells and developing hair cells. Trajectory reconstruction resulted in an inventory of gene expression dynamics of natural hair cell generation in the avian utricle.

Spatiotemporal dynamics of inner ear sensory and non-sensory cells revealed by single-cell transcriptomics.

The utricle is a vestibular sensory organ that requires mechanosensitive hair cells to detect linear acceleration. In neonatal mice, new hair cells are derived from non-sensory supporting cells, yet cell type diversity and mechanisms of cell addition remain poorly characterized. Here, we perform computational analyses on single-cell transcriptomes to categorize cell types and resolve 14 individual sensory and non-sensory subtypes. Along the periphery of the sensory epithelium, we uncover distinct groups of transitional epithelial cells, marked by Islr, Cnmd, and Enpep expression. By reconstructing de novo trajectories and gene dynamics, we show that as the utricle expands, Islr+ transitional epithelial cells exhibit a dynamic and proliferative phase to generate new supporting cells, followed by coordinated differentiation into hair cells. Taken together, our study reveals a sequential and coordinated process by which non-sensory epithelial cells contribute to growth of the postnatal mouse sensory epithelium.

Transcriptomic characterization of dying hair cells in the avian cochlea.

Sensory hair cells are prone to apoptosis caused by various drugs including aminoglycoside antibiotics. In mammals, this vulnerability results in permanent hearing loss because lost hair cells are not regenerated. Conversely, hair cells regenerate in birds, making the avian inner ear an exquisite model for studying ototoxicity and regeneration. Here, we use single-cell RNA sequencing and trajectory analysis on control and dying hair cells after aminoglycoside treatment. Interestingly, the two major subtypes of avian cochlear hair cells, tall and short hair cells, respond differently. Dying short hair cells show a noticeable transient upregulation of many more genes than tall hair cells. The most prominent gene group identified is associated with potassium ion conductances, suggesting distinct physiological differences. Moreover, the dynamic characterization of >15,000 genes expressed in tall and short avian hair cells during their apoptotic demise comprises a resource for further investigations toward mammalian hair cell protection and hair cell regeneration.

Greater epithelial ridge cells are the principal organoid-forming progenitors of the mouse cochlea.

In mammals, hearing loss is irreversible due to the lack of regenerative potential of non-sensory cochlear cells. Neonatal cochlear cells, however, can grow into organoids that harbor sensory epithelial cells, including hair cells and supporting cells. Here, we purify different cochlear cell types from neonatal mice, validate the composition of the different groups with single-cell RNA sequencing (RNA-seq), and assess the various groups' potential to grow into inner ear organoids. We find that the greater epithelial ridge (GER), a transient cell population that disappears during post-natal cochlear maturation, harbors the most potent organoid-forming cells. We identified three distinct GER cell groups that correlate with a specific spatial distribution of marker genes. Organoid formation was synergistically enhanced when the cells were cultured at increasing density. This effect is not due to diffusible signals but requires direct cell-to-cell contact. Our findings improve the development of cell-based assays to study culture-generated inner ear cell types.

American Neurotology Society, American Otological Society, and American Academy of Otolaryngology - Head and Neck Foundation Guide to Enhance Otologic and Neurotologic Care During the COVID-19 Pandemic.

: This combined American Neurotology Society, American Otological Society, and American Academy of Otolaryngology - Head and Neck Surgery Foundation document aims to provide guidance during the coronavirus disease of 2019 (COVID-19) on 1) "priority" of care for otologic and neurotologic patients in the office and operating room, and 2) optimal utilization of personal protective equipment. Given the paucity of evidence to inform otologic and neurotologic best practices during COVID-19, the recommendations herein are based on relevant peer-reviewed articles, the Centers for Disease Control and Prevention COVID-19 guidelines, United States and international hospital policies, and expert opinion. The suggestions presented here are not meant to be definitive, and best practices will undoubtedly change with increasing knowledge and high-quality data related to COVID-19. Interpretation of this guidance document is dependent on local factors including prevalence of COVID-19 in the surgeons' local community. This is not intended to set a standard of care, and should not supersede the clinician's best judgement when managing specific clinical concerns and/or regional conditions.Access to otologic and neurotologic care during and after the COVID-19 pandemic is dependent upon adequate protection of physicians, audiologists, and ancillary support staff. Otolaryngologists and associated staff are at high risk for COVID-19 disease transmission based on close contact with mucosal surfaces of the upper aerodigestive tract during diagnostic evaluation and therapeutic procedures. While many otologic and neurotologic conditions are not imminently life threatening, they have a major impact on communication, daily functioning, and quality of life. In addition, progression of disease and delay in treatment can result in cranial nerve deficits, intracranial and life-threatening complications, and/or irreversible consequences. In this regard, many otologic and neurotologic conditions should rightfully be considered "urgent," and almost all require timely attention to permit optimal outcomes. It is reasonable to proceed with otologic and neurotologic clinic visits and operative cases based on input from expert opinion of otologic care providers, clinic/hospital administration, infection prevention and control specialists, and local and state public health leaders. Significant regional variations in COVID-19 prevalence exist; therefore, physicians working with local municipalities are best suited to make determinations on the appropriateness and timing of otologic and neurotologic care.

Third-generation bisphosphonates for cochlear otosclerosis stabilizes sensorineural hearing loss in long-term follow-up.

Objective: To assess long-term hearing outcomes in patients treated with third-generation bisphosphonates for otosclerosis-related progressive sensorineural hearing loss (SNHL). Study Design: Retrospective case series review. Methods: We performed a retrospective case series review of patients with otosclerosis and progressive SNHL. Patients were treated with either risedronate or zoledronate after a diagnosis of otosclerosis with a significant SNHL component. Bone conduction pure tone threshold averages (BC-PTAs) and word recognition scores (WRS) before and after bisphosphonate administration in long-term follow-up was analyzed. Significant change in BC-PTA was defined as greater than 10dB or between 4% and 18% in WRS based on binomial variance. Results: Seven patients were identified and 14 ears met inclusion criteria. Three patients were female and the mean age was 48.3 ± 10.3 years. The mean duration between treatment with bisphosphonate administration and long-term post-treatment follow-up audiometry was 87.6 ± 18.3 months, with a range of 61.6 to 109.1 months and median of 89.2 months. Analysis using BC-PTA and WRS demonstrated that 11 ears remained stable while 2 improved and 1 worsened. No patient experienced any major complication as the result of bisphosphonate therapy. Conclusion: Treatment with third-generation bisphosphonates is associated with stability in otosclerosis-related sensorineural hearing over 5- to 9-year period. These results suggest that such medications may prevent the progression of SNHL in patients with otosclerosis. Level of Evidence: 4 (Case series).

Relationship between Surgically Treated Superior Canal Dehiscence Syndrome and Body Mass Index.

Objective Examine the association between body mass index (BMI) and superior canal dehiscence (SCD) among patients who have undergone surgical repair for superior canal dehiscence. Study Design Retrospective comparison study. Setting Neurotology tertiary care center. Subjects and Methods Retrospective review of consecutive adult patients evaluated at our institution for SCD syndrome between November 2006 and August 2015. A control group who underwent imaging within the same period for reasons other than SCD was also included. Patient demographics, weight, and height were examined. We performed multiple subgroup analyses to investigate the relationship of BMI, surgery vs no surgery, and correlation between patient BMI and SCD size. Results Of the 268 patients with SCD, 99 underwent surgery; 96 of these patients had complete medical records and were eligible for inclusion. Eighty-eight patients were noted to have arcuate eminence defects, and the mean BMI of this surgical cohort was 28.09 ± 5.26 kg/m2. Nonsurgically treated patients with SCD with available data (n = 94) had a mean BMI of 27.97 ± 6.95 kg/m2. A control group of 204 patients who underwent computed tomography for non-SCD-related causes was analyzed, of whom 155 had available data with a mean BMI of 27.91 ± 6.38 kg/m2. Conclusion We demonstrate that adult patients who undergo surgery for SCD are not obese (mean BMI <30), and size of dehiscence poorly correlates with BMI. Our observations call into question the proposed theory that patient weight is a risk factor for the development of symptomatic SCD involving the arcuate eminence.

Profiling Specific Inner Ear Cell Types Using Cell Sorting Techniques.

Studies of specific tissue cell types are becoming increasingly important in advancing our understanding of cell biology and gene and protein expression. Prospective isolation of specific cell types is a powerful technique as it facilitates such investigations, allowing for analysis and characterization of individual cell populations. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and limited cell markers. In this chapter, we describe methods for selectively labeling and isolating different inner ear cell types from the neonatal mouse cochlea using fluorescence-activated cell sorting.

Improvement in word recognition following treatment failure for sudden sensorineural hearing loss.

OBJECTIVES: Patients with sudden sensorineural hearing loss (SSNHL) may have word recognition scores (WRS) that correlate with pure tone average (PTA). We hypothesize that there is a subset of patients with SSNHL who have improved WRS despite stable PTA. METHODS: Retrospective case review at a tertiary otolaryngology practice. RESULTS: We identified 13 of 113 patients with SSNHL whose WRS increased despite overall stable pure tone averages. There was an observed average improvement in WRS by 23.8 points in this patient cohort at follow-up, with mean initial PTA in the affected ear at 48.7 dB. CONCLUSIONS: We identify a novel cohort of SSNHL patients that have failed treatment as measured by PTA, but who have increased WRS over time. These data have implications for patient counseling and lend insight into the pathophysiology of SSNHL.

Transient, afferent input-dependent, postnatal niche for neural progenitor cells in the cochlear nucleus.

In the cochlear nucleus (CN), the first central relay of the auditory pathway, the survival of neurons during the first weeks after birth depends on afferent innervation from the cochlea. Although input-dependent neuron survival has been extensively studied in the CN, neurogenesis has not been evaluated as a possible mechanism of postnatal plasticity. Here we show that new neurons are born in the CN during the critical period of postnatal plasticity. Coincidently, we found a population of neural progenitor cells that are controlled by a complex interplay of Wnt, Notch, and TGFß/BMP signaling, in which low levels of TGFß/BMP signaling are permissive for progenitor proliferation that is promoted by Wnt and Notch activation. We further show that cells with activated Wnt signaling reside in the CN and that these cells have high propensity for neurosphere formation. Cochlear ablation resulted in diminishment of progenitors and Wnt/ß-catenin-active cells, suggesting that the neonatal CN maintains an afferent innervation-dependent population of progenitor cells that display active canonical Wnt signaling.

A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

Intranasal myiasis: a rare complication of transnasal skull base surgery.

A 57-year-old woman with a history of atypical intracranial meningioma had undergone multiple craniotomies and endoscopic skull base procedures over several years. She presented most recently with nasal discharge consisting of intranasal larvae. Isolated organisms from the nasal cavity and maxillary sinus were identified as blow fly larvae (Calliphoridae family). The patient was treated with transnasal debridement and antibiotic therapy. The organisms were successfully eradicated and she is free from further signs of infection. Intranasal myiasis is an unusual complication of anterior skull base surgery.

Effect of obesity and medical comorbidities on outcomes after adjunct surgery for obstructive sleep apnea in cases of adenotonsillectomy failure.

OBJECTIVE: To evaluate the effect of body mass index (BMI, calculated as weight in kilograms divided by height in meters squared) and medical comorbidities on outcomes after lingual tonsillectomy and supraglottoplasty performed for obstructive sleep apnea syndrome (OSAS) caused by lingual tonsillar hypertrophy and occult laryngomalacia. DESIGN: Retrospective case review series SETTING: Academic tertiary referral center PATIENTS: Children with persistent OSAS after adenotonsillectomy who underwent surgery to correct obstruction at the level of the lingual tonsils and/or supraglottis identified on sleep endoscopy. INTERVENTIONS: All children underwent lingual tonsillectomy, supraglottoplasty, or both. MAIN OUTCOME MEASURES: Change in polysomnographic parameters, including apnea-hypopnea index (AHI), number of nighttime apneas, and lowest oxygen saturation level. RESULTS: We analyzed the medical records of 84 children with persistent OSAS after adenotonsillectomy who underwent either lingual tonsillectomy (n = 68), supraglottoplasty (n = 24) or both (n = 8). Compared with children with lingual tonsillar hypertrophy, children with occult laryngomalacia were younger, had lower BMI, and were more likely to have a medical comorbidity. Overall, both operations significantly improved the AHI; however, children with comorbidities had significantly higher postoperative AHIs after supraglottoplasty than those without, and overweight children had significantly higher postoperative AHIs after lingual tonsillectomy than those of normal weight. The BMI z-score and age had direct, though weak, correlations with postoperative AHI among all children undergoing either technique of adjunct airway surgery. CONCLUSIONS: Lingual tonsillar hypertrophy and occult laryngomalacia are 2 important causes of residual OSAS after adenotonsillectomy. However, they tend to affect distinct populations of children, and though appropriate surgical correction can improve AHI, cure rates are significantly worse for overweight children undergoing lingual tonsillectomy and for children with medical comorbidities undergoing supraglottoplasty.