Jag1 represses Notch activation in lateral supporting cells and inhibits an outer hair cell fate in the medial cochlea.

Notch signaling patterns the cochlear organ of Corti, and patients with the JAG1/NOTCH2-related genetic disorder Alagille syndrome can thus experience hearing loss. We investigated the function of Jag1 in cochlear patterning and signaling using Jag1Ndr/Ndr mice, a model of Alagille syndrome. Jag1Ndr/Ndr mice exhibited expected vestibular and auditory deficits, a dose-dependent increase in ectopic inner hair cells, and a reduction in outer hair cells. Single cell RNA sequencing of the organ of Corti demonstrated a global dysregulation of genes associated with inner ear development and deafness. Analysis of individual cell types further revealed that Jag1 represses Notch activation in lateral supporting cells and demonstrated a function for Jag1 in gene regulation and development of outer hair cells. Surprisingly, ectopic "outer hair cell-like" cells were present in the medial compartment and pillar cell region of Jag1Ndr/Ndr cochleae, yet they exhibited location-dependent expression of the inner hair cell fate-determinant Tbx2, suggesting Jag1 is required for Tbx2 to drive inner hair cell commitment. This study thus identifies new roles for Jag1 in supporting cells, and in outer hair cell specification and positioning.

The Mason-Alberta Phonetic Segmenter: a forced alignment system based on deep neural networks and interpolation.

Given an orthographic transcription, forced alignment systems automatically determine boundaries between segments in speech, facilitating the use of large corpora. In the present paper, we introduce a neural network-based forced alignment system, the Mason-Alberta Phonetic Segmenter (MAPS). MAPS serves as a testbed for two possible improvements we pursue for forced alignment systems. The first is treating the acoustic model as a tagger, rather than a classifier, motivated by the common understanding that segments are not truly discrete and often overlap. The second is an interpolation technique to allow more precise boundaries than the typical 10 ms limit in modern systems. During testing, all system configurations we trained significantly outperformed the state-of-the-art Montreal Forced Aligner in the 10 ms boundary placement tolerance threshold. The greatest difference achieved was a 28.13 % relative performance increase. The Montreal Forced Aligner began to slightly outperform our models at around a 30 ms tolerance. We also reflect on the training process for acoustic modeling in forced alignment, highlighting how the output targets for these models do not match phoneticians' conception of similarity between phones and that reconciling this tension may require rethinking the task and output targets or how speech itself should be segmented.

Documenting and modeling the acoustic variability of intervocalic alveolar taps in conversational Peninsular Spanish.

This study constitutes an investigation into the acoustic variability of intervocalic alveolar taps in a corpus of spontaneous speech from Madrid, Spain. Substantial variability was documented in this segment, with highly reduced variants constituting roughly half of all tokens during spectrographic inspection. In addition to qualitative documentation, the intensity difference between the tap and surrounding vowels was measured. Changes in this intensity difference were statistically modeled using Bayesian finite mixture models containing lexical and phonetic predictors. Model comparisons indicate predictive performance is improved when we assume two latent categories, interpreted as two pronunciation variants for the Spanish tap. In interpreting the model, predictors were more often related to categorical changes in which pronunciation variant was produced than to gradient intensity changes within each tap type. Variability in tap production was found according to lexical frequency, speech rate, and phonetic environment. These results underscore the importance of evaluating model fit to the data as well as what researchers modeling phonetic variability can gain in moving past linear models when they do not adequately fit the observed data.

ZBTB20 is essential for cochlear maturation and hearing in mice.

The mammalian cochlear epithelium undergoes substantial remodeling and maturation before the onset of hearing. However, very little is known about the transcriptional network governing cochlear late-stage maturation and particularly the differentiation of its lateral nonsensory region. Here, we establish ZBTB20 as an essential transcription factor required for cochlear terminal differentiation and maturation and hearing. ZBTB20 is abundantly expressed in the developing and mature cochlear nonsensory epithelial cells, with transient expression in immature hair cells and spiral ganglion neurons. Otocyst-specific deletion of Zbtb20 causes profound deafness with reduced endolymph potential in mice. The subtypes of cochlear epithelial cells are normally generated, but their postnatal development is arrested in the absence of ZBTB20, as manifested by an immature appearance of the organ of Corti, malformation of tectorial membrane (TM), a flattened spiral prominence (SP), and a lack of identifiable Boettcher cells. Furthermore, these defects are related with a failure in the terminal differentiation of the nonsensory epithelium covering the outer border Claudius cells, outer sulcus root cells, and SP epithelial cells. Transcriptome analysis shows that ZBTB20 regulates genes encoding for TM proteins in the greater epithelial ridge, and those preferentially expressed in root cells and SP epithelium. Our results point to ZBTB20 as an essential regulator for postnatal cochlear maturation and particularly for the terminal differentiation of cochlear lateral nonsensory domain.

Lrrn1 Regulates Medial Boundary Formation in the Developing Mouse Organ of Corti.

One of the most striking aspects of the sensory epithelium of the mammalian cochlea, the organ of Corti (OC), is the presence of precise boundaries between sensory and nonsensory cells at its medial and lateral edges. A particular example of this precision is the single row of inner hair cells (IHCs) and associated supporting cells along the medial (neural) boundary. Despite the regularity of this boundary, the developmental processes and genetic factors that contribute to its specification are poorly understood. In this study we demonstrate that Leucine Rich Repeat Neuronal 1 (Lrrn1), which codes for a single-pass, transmembrane protein, is expressed before the development of the mouse organ of Corti in the row of cells that will form its medial border. Deletion of Lrrn1 in mice of mixed sex leads to disruptions in boundary formation that manifest as ectopic inner hair cells and supporting cells. Genetic and pharmacological manipulations demonstrate that Lrrn1 interacts with the Notch signaling pathway and strongly suggest that Lrrn1 normally acts to enhance Notch signaling across the medial boundary. This interaction is required to promote formation of the row of inner hair cells and suppress the conversion of adjacent nonsensory cells into hair cells and supporting cells. These results identify Lrrn1 as an important regulator of boundary formation and cellular patterning during development of the organ of Corti.SIGNIFICANCE STATEMENT Patterning of the developing mammalian cochlea into distinct sensory and nonsensory regions and the specification of multiple different cell fates within those regions are critical for proper auditory function. Here, we report that the transmembrane protein Leucine Rich Repeat Neuronal 1 (LRRN1) is expressed along the sharp medial boundary between the single row of mechanosensory inner hair cells (IHCs) and adjacent nonsensory cells. Formation of this boundary is mediated in part by Notch signaling, and loss of Lrrn1 leads to disruptions in boundary formation similar to those caused by a reduction in Notch activity, suggesting that LRRN1 likely acts to enhance Notch signaling. Greater understanding of sensory/nonsensory cell fate decisions in the cochlea will help inform the development of regenerative strategies aimed at restoring auditory function.

Pseudomonas-dominant microbiome elicits sustained IL-1ß upregulation in alveolar macrophages from lung transplant recipients.

BACKGROUND: Isolation of Pseudomonas aeruginosa (PsA) is associated with increased BAL (bronchoalveolar lavage) inflammation and lung allograft injury in lung transplant recipients (LTR). However, the effect of PsA on macrophage responses in this population is incompletely understood. We examined human alveolar macrophage (AMΦ) responses to PsA and Pseudomonas dominant microbiome in healthy LTR. METHODS: We stimulated THP-1 derived macrophages (THP-1MΦ) and human AMΦ from LTR with different bacteria and LTR BAL derived microbiome characterized as Pseudomonas-dominant. Macrophage responses were assessed by high dimensional flow cytometry, including their intracellular production of cytokines (TNF-α, IL-6, IL-8, IL-1ß, IL-10, IL-1RA, and TGF-ß). Pharmacological inhibitors were utilized to evaluate the role of the inflammasome in PsA-macrophage interaction. RESULTS: We observed upregulation of pro-inflammatory cytokines (TNF-α, IL-6, IL-8, IL-1ß) following stimulation by PsA compared to other bacteria (Staphylococcus aureus (S.Aur), Prevotella melaninogenica, Streptococcus pneumoniae) in both THP-1MΦ and LTR AMΦ, predominated by IL-1ß. IL-1ß production from THP-1MΦ was sustained after PsA stimulation for up to 96 hours and 48 hours in LTR AMΦ. Treatment with the inflammasome inhibitor BAY11-7082 abrogated THP-1MΦ IL-1ß production after PsA exposure. BAL Pseudomonas-dominant microbiota elicited an increased IL-1ß, similar to PsA, an effect abrogated by the addition of antibiotics. CONCLUSION: PsA and PsA-dominant lung microbiota induce sustained IL-1ß production in LTR AMΦ. Pharmacological targeting of the inflammasome reduces PsA-macrophage-IL-1ß responses, underscoring their use in lung transplant recipients.

COVID-19 Physician Burnout: Louisiana's Workforce Vulnerability and Strategies for Mitigation.

Background: Louisiana is historically one of the lowest-performing states in terms of health outcomes in the United States. Hurricane Katrina led to a decrease in available health care resources, with a larger impact on resources for those living below the poverty line. Subsequently, the coronavirus disease 2019 (COVID-19) pandemic has been shown to have had disproportionately large impacts on minority communities, uninsured populations, and rural communities-all of which are found in Louisiana. Methods: This review focuses on the unique challenges of health care in Louisiana, the influence of COVID-19 on physician burnout, and methods of reducing work exhaustion for those in the health care field. Results: A national survey showed that physician satisfaction decreased from June 29, 2021, through September 26, 2021, compared to before the pandemic. A critical component in the provision of the essential services of public health is the ability to build and sustain a clinically skilled and diverse physician workforce. Maintaining well-being and retaining the physician workforce are prerequisites to the equitable provision of access to health care services. Conclusion: Maintaining one's own wellness is critical to occupational sustainability, particularly when unique stressors such as those encountered during the COVID-19 pandemic are present. The future of a vital health care system depends on physicians maintaining healthy habits and seeking help when burnout symptoms are recognized, both at the individual and institutional level.

Semantic richness effects in isolated spoken word recognition: Evidence from massive auditory lexical decision.

While known to influence visual lexical processing, the semantic information we associate with words has recently been found to influence auditory lexical processing as well. The present work explored the influence of semantic richness in auditory lexical decision. Study 1 recreated an experiment investigating semantic richness effects in concrete nouns (Goh et al., 2016). In Study 2, we expanded the stimulus set from 442 to 8,626 items, exploring the robustness of effects observed in Study 1 against a larger data set with increased diversity in both word class and other characteristics of interest. We also utilized generalized additive mixed models to investigate potential nonlinear effects. Results indicate that semantic richness effects become more nuanced and detectable when a wider set of items belonging to different parts of speech is examined. Findings are discussed in the context of models of spoken word recognition. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Specification of neuronal subtypes in the spiral ganglion begins prior to birth in the mouse.

The afferent innervation of the cochlea is comprised of spiral ganglion neurons (SGNs), which are characterized into four subtypes (Type 1A, B, and C and Type 2). However, little is known about the factors and/or processes that determine each subtype. Here, we present a transcriptional analysis of approximately 5,500 single murine SGNs collected across four developmental time points. All four subtypes are transcriptionally identifiable prior to the onset of coordinated spontaneous activity, indicating that the initial specification process is under genetic control. Trajectory analysis indicates that SGNs initially split into two precursor types (Type 1A/2 and Type 1B/C), followed by subsequent splits to give rise to four transcriptionally distinct subtypes. Differential gene expression, pseudotime, and regulon analyses were used to identify candidate transcription factors which may regulate the subtypes specification process. These results provide insights into SGN development and comprise a transcriptional atlas of SGN maturation across the prenatal period.

A Nesprin-4/kinesin-1 cargo model for nuclear positioning in cochlear outer hair cells.

Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, Nesprin-4 (encoded by Syne4) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how Nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of Nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of Nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between Nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

Cochlear Development; New Tools and Approaches.

The sensory epithelium of the mammalian cochlea, the organ of Corti, is comprised of at least seven unique cell types including two functionally distinct types of mechanosensory hair cells. All of the cell types within the organ of Corti are believed to develop from a population of precursor cells referred to as prosensory cells. Results from previous studies have begun to identify the developmental processes, lineage restrictions and signaling networks that mediate the specification of many of these cell types, however, the small size of the organ and the limited number of each cell type has hampered progress. Recent technical advances, in particular relating to the ability to capture and characterize gene expression at the single cell level, have opened new avenues for understanding cellular specification in the organ of Corti. This review will cover our current understanding of cellular specification in the cochlea, discuss the most commonly used methods for single cell RNA sequencing and describe how results from a recent study using single cell sequencing provided new insights regarding cellular specification.

Using acoustic distance and acoustic absement to quantify lexical competition.

Using phonological neighborhood density has been a common method to quantify lexical competition. It is useful and convenient but has shortcomings that are worth reconsidering. The present study quantifies the effects of lexical competition during spoken word recognition using acoustic distance and acoustic absement rather than phonological neighborhood density. The indication of a word's lexical competition is given by what is termed to be its acoustic distinctiveness, which is taken as its average acoustic absement to all words in the lexicon. A variety of acoustic representations for items in the lexicon are analyzed. Statistical modeling shows that acoustic distinctiveness has a similar effect trend as that of phonological neighborhood density. Additionally, acoustic distinctiveness consistently increases model fitness more than phonological neighborhood density regardless of which kind of acoustic representation is used. However, acoustic distinctiveness does not seem to explain all of the same things as phonological neighborhood density. The different areas that these two predictors explain are discussed in addition to the potential theoretical implications of the usefulness of acoustic distinctiveness in the models. The present paper concludes with some reasons why a researcher may want to use acoustic distinctiveness over phonological neighborhood density in future experiments.

Elucidating complex triplet-state dynamics in the model system isopropylthioxanthone.

We introduce techniques for probing the dynamics of triplet states. We employ these tools, along with conventional techniques, to develop a detailed understanding of a complex chemical system: a negative-tone, radical photoresist for multiphoton absorption polymerization in which isopropylthioxanthone (ITX) is the photoinitiator. This work reveals that the same color of light used for the 2-photon excitation of ITX, leading to population of the triplet manifold through intersystem crossing, also depletes this triplet population via linear absorption followed by reverse intersystem crossing (RISC). Using spectroscopic tools and kinetic modeling, we identify the reactive triplet state and a non-reactive reservoir triplet state. We present compelling evidence that the deactivation channel involves RISC from an excited triplet state to a highly vibrationally excited level of the electronic ground state. The work described here offers the enticing possibility of understanding, and ultimately controlling, the photochemistry and photophysics of a broad range of triplet processes.

Assessment of auditory and vestibular damage in a mouse model after single and triple blast exposures.

The use of explosive devices in war and terrorism has increased exposure to concussive blasts among both military personnel and civilians, which can cause permanent hearing and balance deficits that adversely affect survivors' quality of life. Significant knowledge gaps on the underlying etiology of blast-induced hearing loss and balance disorders remain, especially with regard to the effect of blast exposure on the vestibular system, the impact of multiple blast exposures, and long-term recovery. To address this, we investigated the effects of blast exposure on the inner ear using a mouse model in conjunction with a high-fidelity blast simulator. Anesthetized animals were subjected to single or triple blast exposures, and physiological measurements and tissue were collected over the course of recovery for up to 180 days. Auditory brainstem responses (ABRs) indicated significantly elevated thresholds across multiple frequencies. Limited recovery was observed at low frequencies in single-blasted mice. Distortion Product Otoacoustic Emissions (DPOAEs) were initially absent in all blast-exposed mice, but low-amplitude DPOAEs could be detected at low frequencies in some single-blast mice by 30 days post-blast, and in some triple-blast mice at 180 days post-blast. All blast-exposed mice showed signs of Tympanic Membrane (TM) rupture immediately following exposure and loss of outer hair cells (OHCs) in the basal cochlear turn. In contrast, the number of Inner Hair Cells (IHCs) and spiral ganglion neurons was unchanged following blast-exposure. A significant reduction in IHC pre-synaptic puncta was observed in the upper turns of blast-exposed cochleae. Finally, we found no significant loss of utricular hair cells or changes in vestibular function as assessed by vestibular evoked potentials. Our results suggest that (1) blast exposure can cause severe, long-term hearing loss which may be partially due to slow TM healing or altered mechanical properties of healed TMs, (2) traumatic levels of sound can still reach the inner ear and cause basal OHC loss despite middle ear dysfunction caused by TM rupture, (3) blast exposure may result in synaptopathy in humans, and (4) balance deficits after blast exposure may be primarily due to traumatic brain injury, rather than damage to the peripheral vestibular system.

Corrigendum: Characterizing Adult Cochlear Supporting Cell Transcriptional Diversity Using Single-Cell RNA-Seq: Validation in the Adult Mouse and Translational Implications for the Adult Human Cochlea.

[This corrects the article DOI: 10.3389/fnmol.2020.00013.].

The influence of the number of part-set cues on order retention.

Across seven experiments, the present article examined the influence of the number of part-set cues on order retention, as assessed by both reconstruction of order and serial recall tests. Historically, part-set cueing facilitation occurs when half of the items are provided as valid part-set cues on tests of order memory. Using a variety of list lengths (10 or 16), numbers of cues (0-14), and types of cues (full or partial), the present experiments showed disparate effects of the number of part-set cues on reconstruction and serial recall tasks. On reconstruction tests, a minimum number of full cues was required before part-set cueing facilitation was produced and the magnitude of facilitation increased as the number of cues increased. Generally, partial cues did not influence order retention until almost the entire list was provided as partial cues. On serial recall tests, part-set cueing facilitation was only evident with a few full cues. In contrast, part-set cueing impairment was the norm when many partial cues were provided. These results were largely consistent with predictions of the retrieval strategy disruption hypothesis, as well as with an anchoring account of part-set cueing for order.

Identification and characterization of key long non-coding RNAs in the mouse cochlea.

The auditory system is a complex sensory network with an orchestrated multilayer regulatory programme governing its development and maintenance. Accumulating evidence has implicated long non-coding RNAs (lncRNAs) as important regulators in numerous systems, as well as in pathological pathways. However, their function in the auditory system has yet to be explored. Using a set of specific criteria, we selected four lncRNAs expressed in the mouse cochlea, which are conserved in the human transcriptome and are relevant for inner ear function. Bioinformatic characterization demonstrated a lack of coding potential and an absence of evolutionary conservation that represent properties commonly shared by their class members. RNAscope®  analysis of the spatial and temporal expression profiles revealed specific localization to inner ear cells. Sub-cellular localization analysis presented a distinct pattern for each lncRNA and mouse tissue expression evaluation displayed a large variability in terms of level and location. Our findings establish the expression of specific lncRNAs in different cell types of the auditory system and present a potential pathway by which the lncRNA Gas5 acts in the inner ear. Studying lncRNAs and deciphering their functions may deepen our knowledge of inner ear physiology and morphology and may reveal the basis of as yet unresolved genetic hearing loss-related pathologies. Moreover, our experimental design may be employed as a reference for studying other inner ear-related lncRNAs, as well as lncRNAs expressed in other sensory systems.

Spectrum of genes for inherited hearing loss in the Israeli Jewish population, including the novel human deafness gene ATOH1.

Mutations in more than 150 genes are responsible for inherited hearing loss, with thousands of different, severe causal alleles that vary among populations. The Israeli Jewish population includes communities of diverse geographic origins, revealing a wide range of deafness-associated variants and enabling clinical characterization of the associated phenotypes. Our goal was to identify the genetic causes of inherited hearing loss in this population, and to determine relationships among genotype, phenotype, and ethnicity. Genomic DNA samples from informative relatives of 88 multiplex families, all of self-identified Jewish ancestry, with either non-syndromic or syndromic hearing loss, were sequenced for known and candidate deafness genes using the HEar-Seq gene panel. The genetic causes of hearing loss were identified for 60% of the families. One gene was encountered for the first time in human hearing loss: ATOH1 (Atonal), a basic helix-loop-helix transcription factor responsible for autosomal dominant progressive hearing loss in a five-generation family. Our results show that genomic sequencing with a gene panel dedicated to hearing loss is effective for genetic diagnoses in a diverse population. Comprehensive sequencing enables well-informed genetic counseling and clinical management by medical geneticists, otolaryngologists, audiologists, and speech therapists and can be integrated into newborn screening for deafness.

An optimized protocol for retina single-cell RNA sequencing.

Purpose: Single-cell RNA sequencing (scRNA-seq) is a powerful technique used to explore gene expression at the single cell level. However, appropriate preparation of samples is essential to obtain the most information out of this transformative technology. Generating high-quality single-cell suspensions from the retina is critical to preserve the native expression profile that will ensure meaningful transcriptome data analysis. Methods: We modified the conditions for rapid and optimal dissociation of retina sample preparation. We also included additional filtering steps in data analysis for retinal scRNA-seq. Results: We report a gentle method for dissociation of the mouse retina that minimizes cell death and preserves cell morphology. This protocol also results in detection of higher transcriptional complexity. In addition, the modified computational pipeline leads to better-quality single-cell RNA-sequencing data in retina samples. We also demonstrate the advantages and limitations of using fresh versus frozen retinas to prepare cell or nuclei suspensions for scRNA-seq. Conclusions: We provide a simple yet robust and reproducible protocol for retinal scRNA-seq analysis, especially for comparative studies.