Regulation of the p53/SLC7A11/GPX4 Pathway by Gentamicin Induces Ferroptosis in HEI-OC1 Cells.

BACKGROUND: Gentamicin is a commonly used aminoglycoside antibiotic, with ototoxicity as a significant side effect. Ferroptosis, an iron-dependent form of cell death, has been implicated in a variety of disorders. Whether ferroptosis impacts gentamicin ototoxicity is not yet known. The current work used an in-vitro model to examine the influence of gentamicin-induced ferroptosis on cochlear hair cell damage and probable molecular biological pathways. METHODS: House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were treated with different concentrations of gentamicin for 24 hours, with or without ferrostatin-1 pretreatment, to observe gentamicin-induced ferroptosis. The role of p53/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling in gentamicin-induced ferroptosis was explored by pretreating cells with the p53 inhibitor pifithrin-α (PFT-α). We investigated the effect of gentamicin on cells by assessing cell viability. Cellular proteins were isolated and Western blots were performed to detect changes in the expression of p53, SLC7A11, and GPX4. Fluorescence staining was used to assess levels of reactive oxygen species. An enzymatic detection kit was used to detect glutathione, Fe, and malondialdehyde markers. RESULTS: Gentamicin reduced cell viability, glutathione content, and SLC7A11 and GPX4 protein levels, and increased levels of p53 protein, reactive oxygen species, malondialdehyde, and Fe. These effects were largely blocked by pretreatment with ferrostatin-1. Pretreatment with the p53 inhibitor PFT-α prevented the gentamicin-induced reduction in SLC7A11 and GPX4, which alleviated several features of ferroptosis including glutathione depletion, iron overload, and lipid peroxidation build-up. CONCLUSION: Gentamicin induces ferroptosis in the HEI-OC1 cell line, and the mechanism may be related to the p53/SLC7A11/GPX4 signaling pathway.

Sensorineural hearing loss and cognitive impairment: three hypotheses.

Sensorineural hearing loss (SNHL) is a category of hearing loss that often leads to difficulty in understanding speech and other sounds. Auditory system dysfunction, including deafness and auditory trauma, results in cognitive deficits via neuroplasticity. Cognitive impairment (CI) refers to an abnormality in the brain's higher intellectual processes related to learning, memory, thinking and judgment that can lead to severe learning and memory deficits. Studies have established a strong correlation between SNHL and CI, but it remains unclear how SNHL contributes to CI. The purpose of this article is to describe three hypotheses regarding this relationship, the mainstream cognitive load hypothesis, the co-morbidity hypothesis, and the sensory deprivation hypothesis, as well as the latest research progress related to each hypothesis.

TMEM16A inhibits autophagy and promotes the invasion of hypopharyngeal squamous cell carcinoma through mTOR pathway.

Previous studies have indicated that transmembrane protein 16A (TMEM16A) plays a crucial role in the pathogenesis and progression of various tumors by influencing multiple signaling pathways. However, the role of TMEM16A in regulating autophagy via the mammalian target of rapamycin (mTOR) pathway and its impact on the development of hypopharyngeal squamous cell carcinoma (HSCC) remain unclear. Immunohistochemistry and western blotting were used to assess the expression of TMEM16A in HSCC tissues and metastatic lymph nodes. Manipulation of TMEM16A expression levels was achieved in the FaDu cell line through overexpression or knockdown, followed by assessment of its biological effects using cell colony formation, wound healing, transwell, and invasion assays. Additionally, apoptosis and autophagy-related proteins, as well as autophagosome formation, were evaluated through western blotting, transmission electron microscopy, and immunofluorescence following TMEM16A knockdown or overexpression in FaDu cells. Our study revealed significantly elevated levels of TMEM16A in both HSCC tissues and metastatic lymph nodes compared to normal tissues. In vitro experiments demonstrated that silencing TMEM16A led to a notable suppression of HSCC cell proliferation, invasion, and migration. Furthermore, TMEM16A silencing effectively inhibited tumor growth in xenografted mice. Subsequent investigations indicated that knockdown of TMEM16A in HSCC cells could suppress mTOR activation, thereby triggering autophagic cell death by upregulating sequestosome-1 (SQSTM1/P62) and microtubule-associated protein light chain 3 II (LC3II). This study highlights the crucial role of TMEM16A in modulating autophagy in HSCC, suggesting its potential as a therapeutic target for the treatment of this malignancy.

Cortisol Sensitizes Cochlear Hair Cells to Gentamicin Ototoxicity Via Endogenous Apoptotic Pathway.

BACKGROUND: The widespread use of aminoglycosides is a prevalent cause of sensorineural hearing loss. Patients receiving aminoglycosides usually have elevated levels of circulating stress hormones due to disease or physiological stress; however, whether the stress hormone cortisol impacts aminoglycoside-mediated injury of cochlear hair cells has not been fully investigated. METHODS: House Ear Institute-Organ of Corti 1 (HEI-OC1) cells with or without cortisol pretreatment were exposed to gentamicin, we investigated the effect of cortisol pretreatment on gentamicin ototoxicity by assessing cell viability. Molecular pathogenesis was explored by detecting apoptosis and oxidative stress. Meanwhile, by inhibiting glucocorticoid receptors (GR) and mineralocorticoid receptors (MR), the potential roles of receptor types in cortisol-mediated sensitization were evaluated. RESULTS: Cortisol concentrations below 75 µmol/l did not affect cell viability. However, pretreatment with 50 µmol/l cortisol for 24 hours sensitized hair cells to gentamicin-induced apoptosis. Further mechanistic studies revealed that cortisol significantly increased hair cell apoptosis and oxidative stress, and altered apoptosis-related protein expressions induced by gentamicin. In addition, blockade of either GR or MR attenuated cortisol-induced hair cell sensitization to gentamicin toxicity. CONCLUSION: Cortisol pretreatment increased mammalian hair cell susceptibility to gentamicin toxicity. Sensitization was related to the activation of the intrinsic apoptotic pathway and excessive generation of reactive oxygen species. Cortisol may exacerbate aminoglycoside ototoxicity.

Exploring the Connectivity of Neurodegenerative Diseases: Microglia as the Center.

Degenerative diseases affect people's life and health and cause a severe social burden. Relevant mechanisms of microglia have been studied, aiming to control and reduce degenerative disease occurrence effectively. This review discussed the specific mechanisms underlying microglia in neurodegenerative diseases, age-related hearing loss, Alzheimer's disease, Parkinson's disease, and peripheral nervous system (PNS) degenerative diseases. It also reviewed the studies of microglia inhibitors (PLX3397/PLX5622) and activators (lipopolysaccharide), and suggested that reducing microglia can effectively curb the genesis and progression of degenerative diseases. Finally, microglial cells' anti-inflammatory and pro-inflammatory dual role was considered the critical communication point in central and peripheral degenerative diseases. Although it is difficult to describe the complex morphological structure of microglia in a unified manner, this does not prevent them from being a target for future treatment of neurodegenerative diseases.

Potential molecular mechanisms of Erlongjiaonang action in idiopathic sudden hearing loss: A network pharmacology and molecular docking analyses.

Background: Idiopathic sudden hearing loss (ISHL) is characterized by sudden unexplainable and unilateral hearing loss as a clinically emergent symptom. The use of the herb Erlongjiaonang (ELJN) in traditional Chinese medicine is known to effectively control and cure ISHL. This study explored the underlying molecular mechanisms using network pharmacology and molecular docking analyses. Method: The Traditional Chinese Medicine System Pharmacological database and the Swiss Target Prediction database were searched for the identification of ELJN constituents and potential gene targets, respectively, while ISHL-related gene abnormality was assessed using the Online Mendelian Inheritance in Man and Gene Card databases. The interaction of ELJN gene targets with ISHL genes was obtained after these databases were cross-screened, and a drug component-intersecting target network was constructed, and the gene ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes, and protein-protein interaction networks were analyzed. Cytoscape software tools were used to map the active components-crossover target-signaling pathway network and screened targets were then validated by establishing molecular docking with the corresponding components. Result: Erlongjiaonang contains 85 components and 250 corresponding gene targets, while ISHL has 714 disease-related targets, resulting in 66 cross-targets. The bioinformatical analyses revealed these 66 cross-targets, including isorhamnetin and formononetin on NOS3 expression, baicalein on AKT1 activity, and kaempferol and quercetin on NOS3 and AKT1 activity, as potential ELJN-induced anti-ISHL targets. Conclusion: This study uncovered potential ELJN gene targets and molecular signaling pathways in the control of ISHL, providing a molecular basis for further investigation of the anti-ISHL activity of ELJN.

Pyroptosis and degenerative diseases of the elderly.

Pyroptosis is a recently described mechanism of programmed cell death mediated by proteins of the gasdermin family. Widely recognized signaling cascades include the classical, non-classical, caspase-3-dependent gasdermin E and caspase-8-dependent gasdermin D pathways. Additional pyroptotic pathways have been subsequently reported. With the rising prevalence of advanced age, the role of pyroptosis in the degenerative diseases of the elderly has attracted increased research attention. This article reviews the primary mechanisms of pyroptosis and summarizes progress in the research of degenerative diseases of the elderly such as presbycusis, age-related macular degeneration, Alzheimer's disease, intervertebral disc degeneration, and osteoarthritis.

Application and prospect of quasi-targeted metabolomics in age-related hearing loss.

Age-related hearing loss (ARHL) is a common sensory deficit in the elderly, which seriously affects physical and mental health. Therefore, understanding its underlying molecular mechanisms and taking interventions to treat ARHL are urgently needed. In our study, cochlea of 4-week-old C57BL/6 mice as the Youth group (n = 6) and 48-week-old cochlea as the Old group (n = 6) were subjected to quasi-targeted metabolomics analysis by Ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). In total, 208 differential metabolites were identified in 12 cochlea samples, which highlighted the following discriminant compounds: tryptophan, piperidine, methionine, L-arginine, histamine, serotonin, acetylcholine, and 4-aminobutyric acid. Differentially expressed metabolites were identified which were involved in KEGG pathways related to the digestion and absorption of oxidative stress associated amino acids, Synaptic vesicle cycle of serotonin, Pantothenate and CoA Biosynthesis. These findings are a first step toward elucidating the pathophysiological pathways involved in the etiology of ARHL and provide the possibility to further explore the mechanisms of ARHL using metabolomic analysis.

Tumor necrosis factor-α mediated inflammation versus apoptosis in age-related hearing loss.

An almost universal phenomenon occurring during aging is a state of chronic, low-grade, sterile inflammation. Inflammation is a crucial contributor to various age-related pathologies and natural processes in aging tissues. Tumor necrosis factor-α (TNF-α), a master regulator of the immune system, plays an important role in the propagation of inflammation. Recent research has found correlations between hearing loss and markers such as TNF-α. However, the intrinsic molecular mechanism by which TNF-α influences aging individuals' increased risk of hearing loss remains unclear. In this study, we found that TNF-α expression gradually increased with age in DBA/2J mice. We then used recombinant TNF-α to upregulate TNF-α levels in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and found that low concentrations of TNF-α could activate the nuclear factor kappa B (NF-κB) transcriptional response to mediate hair cell survival, while high concentrations of TNF-α could activate the Caspase-3 cascade to mediate hair cell apoptosis, which preliminarily confirmed that a TNF-α mediated signaling pathway plays an important role in the pathogenesis of age-related hearing loss.

A newly identified mutation (c.2029 C > T) in SLC26A4 gene is associated with enlarged vestibular aqueducts in a Chinese family.

BACKGROUND: The enlarged vestibular aqueduct (EVA), associated with mutations in the SLC26A4 gene, characterized by non-syndromic hearing loss, is an autosomal recessive disorder. Here, we intended to investigate genetic causes of hearing loss in a Han Chinese man. METHOD: First, whole-exome sequencing was performed to identify the gene mutations responsible for hearing loss in the proband. Sanger sequencing was used to verify the candidate mutations detected in the family. Next, we collected blood samples and clinical data from the three-generation pedigree. Finally, SLC26A4 mRNA and protein expression levels were detected by qPCR and western blotting. RESULT: The proband suffered from bilateral progressive sensorineural hearing loss with EVA. The sequence analysis of SLC26A4 revealed that the proband and his sister both harbored a compound heterozygous mutation of c.2168A > G/c.2029C > T, inherited from their father and mother respectively. c.2029C > T mutation has not been recorded in the relevant literature previously. Relative mRNA levels of the SLC26A4 gene in individuals carrying a compound heterozygous mutation were significantly lower compared to a heterozygous mutation. SLC26A4 protein levels of 293t cells which transfected with recombinant plasmids [GV219-SLC26A4-mut (c.2029C > T) and GV219-SLC26A4-mut (c.2168A > G/c.2029C > T)] were significantly lower than normal control recombinant plasmids (GV219-SLC26A4-wt). CONCLUSION: This study found a novel heterozygous mutation c.2029 (exon17) C > T compound with c.2168 (exon19) A > G in the SLC26A4 gene in a patient with EVA. The c.2029 (exon17) C > T mutation is proved to be pathogenic. This finding broadens the spectrum of variants in SLC26A4 gene.

Prevalence and prognosis of otorhinolaryngological symptoms in patients with COVID-19: a systematic review and meta-analysis.

OBJECTIVE: A systematic review and meta-analysis were performed to evaluate the prevalence and prognosis of otorhinolaryngological symptoms in patients with the diagnosed coronavirus disease 2019 (COVID-19). METHODS: A systematic search of PubMed, Embase, Web of Science, and Google Scholar databases was performed up to August 19, 2020.We included studies that reported infections with COVID-19 and symptoms of otolaryngology. The retrieved data from the respective studies were evaluated and summarized. The study's immediate result was to assess the combined prevalence of otorhinolaryngological symptoms in patients with COVID-19. However, the secondary result was to determine the exacerbation of COVID-19 infection in patients with otorhinolaryngological symptoms. RESULTS: Fifty-four studies with 16,478 patients were included. Olfactory dysfunction, sneezing and sputum production were the 3 most prevalent otorhinolaryngological symptoms in patients with COVID-19. The pooled prevalence amongst the prevalent symptoms was 47% (95% CI 29-65; range 0-98; I2 = 99.58%), 27% (95% CI 11-48; range 12-40; I2 = 93.34%), and 22% (95% CI 16-30; range 2-56; I2 = 97.60%), respectively. The proportion of severely ill patients with sputum production and shortness of breath was significantly higher among patients with COVID-19 infections (OR 1.66 [95% CI 1.08-2.54]; P = 0.02, I2 = 51% and 3.29 [95% CI 1.57-6.90]; P = 0.002, I2 = 49%, respectively). Subgroup analysis showed no statistically significant differences between the incidence of otolaryngology symptoms in severely ill patients and non-severely ill patients (OR 1.43 [95% CI 1.12-1.82]; P = 0.07 I2 = 53.1%). In contrast, the incidence of shortness of breath in severely ill patients was significantly increased (3.29 [1.57-6.90]; P = 0.002, I2 = 49%). CONCLUSION: Our research shows that otorhinolaryngology symptoms in patients with COVID-19 are not uncommon, which should attract otorhinolaryngologists' attention.

Asthma does not influence the severity of COVID-19: a meta-analysis.

OBJECTIVE: Previous studies have reported a correlation between coronavirus disease-2019 (COVID-19) and asthma. However, data on whether asthma constitutes a risk factor for COVID-19 and the prevalence of asthma in COVID-19 cases still remain scant. Here, we interrogated and analyzed the association between COVID-19 and asthma. METHODS: In this study, we systematically searched PubMed, Embase, and Web of Science databases for studies published between January 1 and August 28, 2020. We included studies that reported the epidemiological and clinical features of COVID-19 and its prevalence in asthma patients. We excluded reviews, animal trials, single case reports, small case series and studies evaluating other coronavirus-related illnesses. Raw data from the studies were pooled into a meta-analysis. RESULTS: We analyzed findings from 18 studies, including asthma patients with COVID-19. The pooled prevalence of asthma in COVID-19 cases was 0.08 (95% CI, 0.06-0.11), with an overall I2 of 99.07%, p < 0.005. The data indicated that asthma did not increase the risk of developing severe COVID-19 (odds ratio [OR] 1.04 (95% CI, 0.75-1.46) p = 0.28; I2=20%). In addition, there was no significant difference in the incidence of asthma with age in COVID-19 infections [OR] 0.77(95% CI, 0.59-1.00) p = 0.24; I2=29%). CONCLUSION: Taken together, our data suggested that asthma is not a significant risk factor for the development of severe COVID-19.

miR-146a enhances regulatory T-cell differentiation and function in allergic rhinitis by targeting STAT5b.

BACKGROUND: MicroRNA (miR)-146a, as an important immune regulatory factor with an anti-inflammatory effect, plays a crucial role in regulatory T-cell (Tregs) differentiation and function in allergic rhinitis (AR). The present study aimed to investigate the regulatory mechanism employed by miR-146a to control Treg differentiation and function in AR. METHODS: Expression of miR-146a and STAT5b in peripheral blood mononuclear cells (PBMCs) and nasal mucosa from patients with AR was detected by qPCR and Western blotting. Tregs were quantified by flow cytometry in miR-146a knockdown or STAT5b knockdown PBMCs. FOXP3, IL-10, and TGF-ß levels were detected by Western blotting or ELISA in miR-146a knockdown or STAT5b overexpressing PBMCs, as well as in STAT5b knockdown PBMCs overexpressing miR-146a. The effect of miR-146a on STAT5b was observed by luciferase assay and knockdown experiments. RESULTS: Levels of miR146a and STAT5b in the nasal mucosa or PBMCs were significantly lower in the AR group than in the control group. There were significantly fewer Tregs in miR-146a knockdown or STAT5b knockdown PBMCs compared to control PBMCs. Expression of FOXP3, IL-10, and TGF-ß was decreased in the miR-146a knockdown group but increased in the STAT5b overexpression group. In contrast, miR-146a overexpression increased the levels of these factors, but knockdown of STAT5b significantly inhibited this effect. Luciferase assay and knockdown experiments showed that miR-146a bound directly to STAT5b. CONCLUSIONS: miR-146a enhances Treg differentiation and function in AR by positively targeting STAT5b.

Biocompatible perovskite quantum dots with superior water resistance enable long-term monitoring of the H2S level in vivo.

The application of perovskite quantum dots (PQDs) in biomedical fields such as bioimaging and biosensing has been limited owing to their instability in the physiological environment. Herein, PQDs are innovatively encapsulated into nano-micelles composed of a polyethylene glycol-polycaprolactone (PEG-PCL) block copolymer, which allows the preparation of biocompatible PQDs (bio-PQDs) with excellent water resistance. Due to the benefits of extraordinary water resistance and biocompatibility, these bio-PQDs are capable of real-time and long-term quantitatively monitoring the H2S level in living cells as well as in zebrafish.

Tissue-specific expression atlas of murine mitochondrial tRNAs.

Mammalian mitochondrial tRNA (mt-tRNA) plays a central role in the synthesis of the 13 subunits of the oxidative phosphorylation complex system (OXPHOS). However, many aspects of the context-dependent expression of mt-tRNAs in mammals remain unknown. To investigate the tissue-specific effects of mt-tRNAs, we performed a comprehensive analysis of mitochondrial tRNA expression across five mice tissues (brain, heart, liver, skeletal muscle, and kidney) using Northern blot analysis. Striking differences in the tissue-specific expression of 22 mt-tRNAs were observed, in some cases differing by as much as tenfold from lowest to highest expression levels among these five tissues. Overall, the heart exhibited the highest levels of mt-tRNAs, while the liver displayed markedly lower levels. Variations in the levels of mt-tRNAs showed significant correlations with total mitochondrial DNA (mtDNA) contents in these tissues. However, there were no significant differences observed in the 2-thiouridylation levels of tRNALys, tRNAGlu, and tRNAGln among these tissues. A wide range of aminoacylation levels for 15 mt-tRNAs occurred among these five tissues, with skeletal muscle and kidneys most notably displaying the highest and lowest tRNA aminoacylation levels, respectively. Among these tissues, there was a negative correlation between variations in mt-tRNA aminoacylation levels and corresponding variations in mitochondrial tRNA synthetases (mt-aaRS) expression levels. Furthermore, the variable levels of OXPHOS subunits, as encoded by mtDNA or nuclear genes, may reflect differences in relative functional emphasis for mitochondria in each tissue. Our findings provide new insight into the mechanism of mt-tRNA tissue-specific effects on oxidative phosphorylation.

DNA methyltransferase 1 inhibits O6-methylguanine-DNAmethyl-transferase-mediated cell growth and metastasis of hypopharyngeal squamous carcinoma.

OBJECTIVE: To explore the role of DNA methyltransferase 1 (DNMT1) in development and progression of hypopharyngeal squamous carcinoma. DESIGN: A total of 32 hypopharyngeal squamous carcinoma biopsy samples and 20 normal tissue specimens were collected. Immunohistochemical staining, quantitative real-time polymerase chain reaction, and Western blot were performed for expression analysis. The mRNA and protein expression in the specimens and subcellular localization were analyzed. hypopharyngeal squamous carcinoma cells (FaDu) were used for small interfering RNA of DNMT1, and proliferation, cell cycle, and apoptosis were determined in the transfected cells. Furthermore, metastatic ability and methylation status of O6-methylguanine-DNAmethyl-transferase (MGMT) promoter was assessed. RESULTS: Our results showed that DNMT1 was overexpressed, while MGMT was down expressed in hypopharyngeal squamous carcinoma. DNMT1 overexpression and MGMT down expression were significantly associated with poorly differentiated tumors, lymph node metastasis, and clinical stage. DNMT1 and MGMT were majorly distributed in the nucleus. Furthermore, knockdown of DNMT1 inhibited proliferation and metastasis, induced apoptosis and G1 phase arrest in FaDu cells, and upregulated MGMT expression to reverse methylation status of MGMT promoter. CONCLUSIONS: This study for the first time demonstrated the clinical value and the role of DNMT1 and MGMT in the biological function of hypopharyngeal squamous carcinoma. This work suggested that DNMT1 might serve as a potential therapeutic target for patients with hypopharyngeal squamous carcinoma.

Serum CA 19-9 Level is Correlated to the Clinical Characteristics and Chronic Complications of Patients Newly Diagnosed with Type 2 Diabetes Mellitus.

AIM: This study investigated the relation of serum carbohydrate antigen 199 (CA 19-9) levels to the clinical characteristics and chronic complications of patients newly diagnosed with type 2 diabetes mellitus (T2DM). METHODS: A total of 371 patients newly diagnosed with T2DM and 133 healthy people with consecutively matched age were compared. The 371 patients with T2DM were divided into four groups by quartiles based on their serum CA 19-9 levels, in which clinical characteristics and chronic complications, such as diabetic retinopathy (DR), diabetic nephropathy, and macrovascular complications were compared. Logistic regression analysis was used to evaluate the risk factors of DR. RESULTS: Among the 371 patients newly diagnosed with T2DM, 60 had elevated CA 19-9 levels (16.17%). The frequencies of elevated serum CA 19-9 were 24.39% (30 of 123) for females and 12.10% (30 of 248) for males, in which the values for females were higher than those for males (P<0.01).Differences were observed among the serum CA 19-9 levels, hemoglobin A1c (HbA1c), and DR (P<0.05). Logistic regression analysis showed that serum CA 19-9 levels, fasting blood glucose (FBG) and fasting C-peptide (FC-P) were risk factors for DR (P<0.05). CONCLUSIONS: Serum CA 19-9 levels were correlated with HbA1c and DR in patients newly diagnosed with T2DM. The elevated serum CA 19-9 levels, high FC-P, and FBG levels were important risk factors for DR in patients newly diagnosed with T2DM.

Role of TGF-ß1-mediated epithelial-mesenchymal transition in the pathogenesis of tympanosclerosis.

The present study aimed to explore the role of TGF-ß1-mediated epithelial-mesenchymal transition (EMT) in the pathogenesis of tympanosclerosis. Sprague Dawley rats were injected with inactivated Streptococcus pneumoniae suspension to establish a rat model of tympanosclerosis. The rats were sacrificed 8 weeks after the model was established. H&E and von Kossa staining was used to observe the morphological changes of middle ear mucosa. Western blotting was used to detect the expression of TGF-ß1 and EMT-associated proteins in the mucosa samples. Middle ear mucosal epithelial cells of rats were collected to establish a primary culture. The cultured cells were stimulated with TGF-ß1 and the expression of EMT-associated proteins was detected by western blotting and immunofluorescence. In addition, the cells were treated with TGF-ß receptor type I/II inhibitor and the expression level of EMT-associated proteins was detected by western blotting. Sclerotic lesions appeared on 72.4% of tympanic membranes, and marked inflammation, inflammatory cell infiltration and fibrosis were found in the middle ear mucosa of rat models of tympanosclerosis. In middle ear mucosa of rats with tympanosclerosis, the expression of mesenchymal cell markers increased and that of epithelial cell markers decreased compared with the control group. TGF-ß1 stimulated the activation of the EMT pathway in middle ear mucosal epithelial cells, resulting in an increased expression of fibronectin and N-cadherin. In addition, a decreased expression level of EMT-associated proteins was observed when TGF-ß1 was inhibited. In conclusion, the present study indicated that TGF-ß1-mediated EMT may play an important role in the pathogenesis of tympanosclerosis.

Mutations of MAP1B encoding a microtubule-associated phosphoprotein cause sensorineural hearing loss.

The pathophysiology underlying spiral ganglion cell defect-induced deafness remains elusive. Using the whole exome sequencing approach, in combination with functional assays and a mouse disease model, we identified the potentially novel deafness-causative MAP1B gene encoding a highly conserved microtubule-associated protein. Three novel heterozygous MAP1B mutations (c.4198A>G, p.1400S>G; c.2768T>C, p.923I>T; c.5512T>C, p.1838F>L) were cosegregated with autosomal dominant inheritance of nonsyndromic sensorineural hearing loss in 3 unrelated Chinese families. Here, we show that MAP1B is highly expressed in the spiral ganglion neurons in the mouse cochlea. Using otic sensory neuron-like cells, generated by pluripotent stem cells from patients carrying the MAP1B mutation and control subject, we demonstrated that the p.1400S>G mutation caused the reduced levels and deficient phosphorylation of MAP1B, which are involved in the microtubule stability and dynamics. Strikingly, otic sensory neuron-like cells exhibited disturbed dynamics of microtubules, axonal elongation, and defects in electrophysiological properties. Dysfunctions of these derived otic sensory neuron-like cells were rescued by genetically correcting MAP1B mutation using CRISPR/Cas9 technology. Involvement of MAP1B in hearing was confirmed by audiometric evaluation of Map1b heterozygous KO mice. These mutant mice displayed late-onset progressive sensorineural hearing loss that was more pronounced in the high frequencies. The spiral ganglion neurons isolated from Map1b mutant mice exhibited the deficient phosphorylation and disturbed dynamics of microtubules. Map1b deficiency yielded defects in the morphology and electrophysiology of spiral ganglion neurons, but it did not affect the morphologies of cochlea in mice. Therefore, our data demonstrate that dysfunctions of spiral ganglion neurons induced by MAP1B deficiency caused hearing loss.