NRF2/HO-1 pathway activation by ATF3 in a noise-induced hearing loss murine model.

BACKGROUND: Excessive oxidative stress of the inner ear as a result of high, intense noise exposure is regarded as a major mechanism underlying the development of noise-induced hearing loss (NIHL). The present study was designed to explore the effect and mechanism of activated transcription factor 3 (ATF3) in reduction/oxidation homeostasis of NIHL. METHOD: In vitro and in vivo assays were performed to investigate the functional role of ATF3 in the inner ear. Mice hearing was measured using auditory brainstem response. ATF3 short hairpin RNA (shRNA) was transfected into House Ear Institute-Organ of Corti 1 (HEI-OC1) cells to decrease ATF3 expression. Western blotting and quantitative real-time polymerase chain reaction (RT-qPCR) were performed to quantify ATF3, NRF2, HO-1 and NQO1 expression. Glutathione (GSH) assay was performed to detect intracellular GSH levels. ATF3 immunofluorescence analysis was carried out in cochlear cryosectioned samples and HEI-OC1 cells to localize ATF3 expression. Cell counting kit 8 assay and flow cytometry were performed to analyze cell viability. RESULT: ATF3 was upregulated in noise-exposed cochleae and HEI-OC1 cells treated with H2O2. NRF2 is a key factor regulated by ATF3. NRF2, HO-1, NQO1, and GSH expression was significantly downregulated in shATF3 HEI-OC1 cells. ATF3 silencing promoted reactive oxygen species accumulation and increased apoptosis and necrosis with H2O2 stimulus. CONCLUSION: ATF3 functions as an antioxidative factor by activating the NRF2/HO-1 pathway.

Predictive factors for complications associated with penetrated fish bones outside the upper gastrointestinal tract.

PURPOSE: To investigate predictive risk factors for complications associated with migrating fish bones in the surrounding tissue of upper gastrointestinal tract. METHODS: A retrospective analysis over 12 years was conducted of 45 cases of buried fish bones in the surrounding tissue of upper gastrointestinal tract with complications. Meanwhile, a control group, including 39 cases of prolonged buried fish bones in the surrounding tissue of upper gastrointestinal tract without complications, was set. Patient clinical data were collected and analyzed to predict the risk factors for complications. RESULTS: The results of Chi-square test and univariate analysis both showed a significant difference in length of fish bone (> 2 cm), a history of concurrent medical illness (diabetes mellitus and renal hypofunction), symptoms (medium or heavy pain and dysphagia), and duration of significant symptoms (> 7 days) between the complication group and non-complication group. Multivariate analysis further identified length (> 2 cm), diabetes mellitus, medium or heavy pain, dysphagia, and duration of significant symptoms (> 7 days) as independent risk factors for complications. CONCLUSIONS: The consequences of fish bones migrating outside the upper gastrointestinal tract are various in different people. Awareness should be raised when encountering a patient ingesting a long fish bone, having a history of diabetes mellitus, presenting with significant discomforts, or these discomforts lasting for a long time. This study will help practitioners counsel their patients on the risks and `benefits of surgery versus observation of this condition.

Cochlear NMDA receptors as a therapeutic target of noise-induced tinnitus.

BACKGROUND: Accumulating evidence suggests that tinnitus may occur despite normal auditory sensitivity, probably linked to partial degeneration of the cochlear nerve and damage of the inner hair cell (IHC) synapse. Damage to the IHC synapses and deafferentation may occur even after moderate noise exposure. For both salicylate- and noise-induced tinnitus, aberrant N-methyl-d-aspartate (NMDA) receptor activation and related auditory nerve excitation have been suggested as origin of cochlear tinnitus. Accordingly, NMDA receptor inhibition has been proposed as a pharmacologic approach for treatment of synaptopathic tinnitus. METHODS: Round-window application of the NMDA receptor antagonist AM-101 (Esketamine hydrochloride gel; Auris Medical AG, Basel, Switzerland) was tested in an animal model of tinnitus induced by acute traumatic noise. The study included the quantification of IHC ribbon synapses as a correlate for deafferentation as well as the measurement of the auditory brainstem response (ABR) to close-threshold sensation level stimuli as an indication of sound-induced auditory nerve activity. RESULTS: We have shown that AM-101 reduced the trauma-induced loss of IHC ribbons and counteracted the decline of ABR wave I amplitude generated in the cochlea/auditory nerve. CONCLUSION: Local round-window application of AM-101 may be a promising therapeutic intervention for the treatment of synaptopathic tinnitus.

Nasopharyngeal Carcinoma Subtype Discovery via Immune Cell Scores from Tumor Microenvironment.

Background: Nasopharyngeal carcinoma (NPC) is one of the most prevalent cancers with a poor prognosis. Immunotherapy, especially immune checkpoint blockade (ICB), is becoming a potential therapeutic choice for NPC patients. Thus, the identification of patients who could benefit from immunotherapy is clinically significant. Methods: The NPC expression profiles from GSE102349 were used to calculate the cell scores of the tumor microenvironment (TME). The consensus clustering method was utilized to identify the potential molecular subtypes among NPC samples. The hub genes were selected from subtype-specific genes by bioinformatics analysis. Machine learning models, including random forest (RF) and support vector machine (SVM) algorithms, were constructed to predict the immune subtype. Results: In the present study, we identified two TME subtypes among NPC patients. Patients with the S1 subtype have higher levels of immune cells, immune checkpoint genes, and prognosis. Using expression data profiles of NPC patients, we constructed machine learning models for predicting TME subtypes of NPC patients. This model consists of 8 genes (LCK, CD247, FYN, ZAP70, SH2D1A, CD3D, CD3E, and CD3G). Among them, LCK, FYN, SH2D1A, and CD3D were associated with better prognoses. Among the two constructed models, SVM exhibited a higher area under curve (AUC) of 0.977, when compared with RF (AUC = 0.966). The web server based on the constructed machine learning models will contribute to the identification of NPC patients likely to benefit from ICB therapies. Conclusions: This study identified NPC subtypes and provided an accurate model to select individuals who are most likely to respond to ICB.

Expression patterns of Ca(V)1.3 channels in the rat cochlea.

Although Ca(V)1.3 channels are known to be essential for neuronal excitation and signal transduction in the auditory system, their expression patterns in the cochlea are still not fully understood, particularly in the regions where non-sensory cells are located. We performed immunohistochemistry, western blotting and reverse transcription-polymerase chain reaction (RT-PCR) to identify the expression and distribution of Ca(V)1.3 channels in the rat cochlea. Immunohistochemistry revealed that Ca(V)1.3 channels were localized in the outer hair cells (OHCs), inner hair cells (IHCs), limbus laminae spiralis, spiral ganglion cell, spiral ligament (SL), and stria vascularis (STV). The results of RT-PCR and western blotting demonstrated Ca(V)1.3 channels had a tissue-specific expression pattern. Ca(V)1.3 mRNA and protein were intensively expressed in the basilar membrane and spiral ganglion while moderate level of Ca(V)1.3 channels was observed in SL and STV. Our study preliminarily revealed the expression patterns of Ca(V)1.3 channels in the rat cochlea, providing a theoretical basis for further research on the role of Ca(V)1.3 channels in the periphery auditory system.

Correlation of PDCD5 and apoptosis in hair cells and spiral ganglion neurons of different age of C57BL/6J mice.

This study examined the expression pattern of programmed cell death 5 (PDCD5) in cochlear hair cells and spiral ganglion neurons (SGNs) and its association with age-related hearing loss in mice. Sixty C57BL/6J (C57) mice at different ages were divided into four groups (3, 6, 9 or 12 months). PDCD5 expression was detected by using immunohistochemistry, real-time PCR and Western blot. Morphological change of the cochleae was also evaluated by using immunoassay. The results showed that the expression of PDCD5 had a gradual increase with ageing in both protein and RNA levels in C57 mice, as well as gradually increased apoptosis of cochlear hair cells and SGNs. In addition, we also found that caspase-3 activity was enhanced and its expression was enhanced with ageing. It is implied that overexpression of PDCD5 causes the increase in caspase-3 activity and the subsequent increase of apoptosis in cochlear hair cells and SGNs, and thereby plays a role in the pathogenesis of presbycusis. Thus, PDCD5 may be a new target site for the treatment and prevention of age-related hearing loss.

CircSOX9 acts as a molecular sponge of miR-485-3p to promote the progression of nasopharyngeal carcinoma.

Circular RNA (circRNA) plays a vital role in the occurrence and development of nasopharyngeal carcinoma (NPC). However, the role of certain specific circRNAs in NPC are still unknown. In this study, collect tumor samples and adjacent normal tissues from clinical NPC patients and detect the expression of circSOX9 by qRT-PCR. Use nucleoplasmic separation analysis, RNase R digestion assay and FISH to detect the characteristics of circSOX9. After knocking down circSOX9, clone formation experiment and transwell assay were used to detect the proliferation and invasion ability of nasopharyngeal carcinoma cells HONE1 and CNE2, and western blot was used to further detect the level of epithelial-mesenchymal transition (EMT). Use the database to screen for possible downstream target genes and verify them with dual-luciferase experiments. Bioinformatics analysis showed that circSOX9 was significantly up-regulated in NPC, and its expression level was positively correlated with the malignant progression of cancer. Data from function gain or loss studies showed that decrease of circSOX9 inhibited the invasion and proliferation of HONE1 and CNE2 cell lines. Further analysis proved that miR-485-3p was the downstream target of circSOX9. The luciferase test showed that by acting as a molecular sponge of miR-485-3p, circSOX9 promotes the proliferation and invasion of NPC cells, while miR-485-3p can target the expression of SOX9. In conclusion, circSOX9 acts as an oncogene in the progression of NPC through miR-485-3p/SOX9, indicating that circSOX9 can be used as a potential therapeutic target and predictive marker for nasopharyngeal carcinoma.

New Target of Oxidative Stress Regulation in Cochleae: Alternative Splicing of the p62/Sqstm1 Gene.

We investigated oxidative stress and antioxidant response in the p62/Sqstm1-Keap1-Nrf2 pathway in C57BL/6 mice cochleae during age-related hearing loss (ARHL) and noise-induced hearing loss (NIHL), and the function of full-length and variant p62 in the regulation of Nrf2 activation. Groups of young (2 months), old (13-14 months), control, and acoustic trauma (AT) mice were examined cochlear damage and oxidative stress as follows: auditory brainstem response and hair cell counts; malondialdehyde (MDA) levels measured by assay kit and 7,8-dihydro-8-oxoguanine (8-oxoG) detected by immunohistochemistry. Full-length and variant p62 were examined for expression in cochleae, hippocampus (HIP), and auditory cortex (AC) using immunoblotting. Keap1-Nrf2 pathway activation was based on immunoblotting of nuclear Nrf2 and quantitative real-time PCR of Nrf2 target genes HO-1/NQO-1. The oxidative function of full-length and variant p62 was examined in HEI-OC-1 cells by flow cytometry. The results showed hearing loss, and cochlear hair cell loss was associated with MDA accumulation and 8-oxoG expression during ARHL and NIHL. Nrf2 showed no obvious changes in nuclear protein. Expression levels mRNA for HO-1 and NQO1 were lower in old mice and mildly greater in AT Mice. The expression of p62 splicing variant lacking the Keap1-interacting region was greater than full-length p62 in cochleae. However, the expression of p62 splicing variant was lesser than full-length p62 in HIP and AC. For HEI-OC-1 cells, overexpression of full-length p62 decreased ROS levels induced by H2O2. Oxidative stress is closely related to ARHL and NIHL. Changing the ratio of full-length to variant p62 protein expression may be a new target to reduce the level of oxidative stress in cochleae.

Comparisons of Supine Roll Test and Alternative Positional Tests in HC-BPPV Lateralization.

OBJECTIVE: The purpose of the study was to evaluate the efficiency of the supine roll test (SRT) and alternative positional tests (APTs) including the bow and lean test (BLT), pseudo-spontaneous nystagmus (PSN), and lying down nystagmus (LDN) to identify the affected side in horizontal canal benign paroxysmal positional vertigo (HC-BPPV). METHODS: In our prospective study, we performed a testing profile (PSN, BLT, LDN, SRT) on 59 HC-BPPV patients using videonystagmography. We compared the accuracy and sensitivity of these tests in HC-BPPV lateralization. Data from 30 healthy patients were collected as the control group. RESULTS: When performing positional tests, the elicited nystagmus coinciding with Ewald's second law was defined as a "positive response". In 44 patients with geotropic nystagmus, the rates of positive response in LDN, PSN, and BLT were 22/44 (50%), 19/44 (43%), and 18/44 (41%), respectively, while in 15 patients with apogeotropic nystagmus, the positive response rates of these three tests were 10/15 (66.7%), 9/15 (60%), and 4/15 (27.00%), respectively. The sensitivity of LDN (54.38%) was higher than that of PSN (47.37%) and BLT (38.60%) but lower than that of SRT (89.47%). Notably, the accuracy rate of PSN (71.8%) was higher than that of the other APTs. In 6 patients with symmetrical nysgtamus during the roll test, 5 patients showed a positive response in both LDN and BLT (83.34%), whereas 4 patients showed a positive response in PSN (66.67%). CONCLUSION: All positional tests are helpful for determining the affected side of HC-BPPV, but SRT carries the highest accuracy of lateralization followed by PSN.

Downregulation of Cav3.1 T-type Calcium Channel Expression in Age-related Hearing Loss Model.

OBJECTIVE: Age-related hearing loss (AHL), characterized by degeneration of cochlea structures, is the most common sensory disorder among the elderly worldwide. The calcium channel is considered to contribute to normal hearing. However, the role of the T-type voltage-activated calcium channel, Cav3.1, remains unclear in AHL. Here, we investigate the age-related change of Cav3.1 expression in the cochlea and D-gal-induced senescent HEI-OC1 cells. METHODS: Cochleae from C57BL/6 mice at 2 months and 12 months of age were assessed. Senescence in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells was induced by D-gal treatment. The immunofluorescence technique was employed to investigate the distribution of Cav3.1 in vivo and in vitro. Quantitative assessment was achieved by Western blotting and real-time PCR. RESULTS: In comparison with 2-month-old animals, 12-month old C57BL/6 mice exhibited great loss of hair cells and elevated auditory brainstem threshold. The Cav3.1 was located in hair cells, spiral ganglion cells, lateral walls, and the expression of Cav3.1 protein and mRNA decreased in the aged cochleae. D-gal-induced senescence assay confirmed the down-regulation of Cav3.1 expression in senescent HEI-OC1 cells. CONCLUSION: Our results show that age-related down-regulated expression of Cav3.1 in the cochleae is associated with AHL and may contribute to the pathogenesis of AHL.

A Comparison of Triamcinolone Acetonide Econazole Cream and Nystatin Suspension in Treatment of Otomycosis.

OBJECTIVES/HYPOTHESIS: To compare the efficacy and adverse effects of triamcinolone acetonide econazole cream and nystatin suspension in the treatment of otomycosis, and to determine the clinical features, predisposing factors, and etiology of otomycosis. STUDY DESIGN: A prospective study. METHODS: A prospective clinical trial was conducted on 786 patients diagnosed with otomycosis. The study population was randomly divided into two treatment groups of triamcinolone acetonide econazole cream (TAEC) and nystatin suspension in a 1:1 ratio. After clearing all fungal deposits in the external auditory canal, the antimycotic drugs were locally applied for at least 2 weeks. The efficacy and adverse effects were compared between the two antifungal reagents by statistical analysis. Meanwhile, patient clinical data were collected to find out the clinical features, predisposing factors, and etiology. RESULTS: Pruritis was the most common symptom and Aspergillus niger was the leading fungal pathogen. There was high association (44.5%) of otomycosis with a history of unclean ear picking. The cure rate was 97.6% in the TAEC group and 73.5% in the nystatin group (P < .01). Treatment with TAEC resulted in 2.4% of patients complaining of discomforts (irritant dermatitis, otalgia, or headache) versus 59.8% of patients complaining discomforts treated with nystatin (P < .01). The residue rate of antifungals was 1.9% in the TAEC group and 89.9% in the nystatin group (P < .01) at the end of treatment. CONCLUSIONS: Thoroughly cleaning of the external auditory canal followed by local use of TAEC under endotoscope is an effective, convenient, and well-tolerated treatment for otomycosis. LEVEL OF EVIDENCE: 1 Laryngoscope, 131:E1640-E1646, 2021.

Expression of α2-Na/K-ATPase in C57BL/6J Mice Inner Ear and Its Relationship with Age-related Hearing Loss.

K+ cycling in the cochlea is critical to maintain hearing. Many sodium-potassium pumps are proved to participate in K+ cycling, such as Na/K-ATPase. The α2-Na/K-ATPase is an important isoform of Na/K-ATPase. The expression of α2-Na/K-ATPase in the cochlea is not clear. In this study, we used C57BL/6 mice as a model of presbycusis and implemented immunohistochemistry staining and quantitative real time-PCR, and the α2-Na/K-ATPase expression pattern was confirmed in the inner ear. It was found α2-Na/K-ATPase was expressed widely in cochlea and its mRNA and protein expression was gradually reduced with aging (4-, 14-, 26- and 48-weeks old mice). We suspected that, the down-regulation of α2-Na/K-ATPase expression might be associated with the remodeling of K+ cycling, degeneration of morphological structure and decrease of hearing function in aging C57 mice. In conclusion, we speculated that the reduction of α2-Na/K-ATPase might play an important role in the pathogenesis of age-related hearing loss.

Differential Expression of LaminB1 in the Developing Rat Cochlea.

OBJECTIVES: To explore the temporal expression pattern of LaminB1 in the cochlea of postnatal rat, and whether LaminB1 is associated with cochlear development. MATERIALS AND METHODS: Sprague-Dawley rats ranging from postnatal day 0 (p0) to 21 (p21) were used. The tissues of stria vascularis (STV) including spiral ligament, spiral ganglion cell (SGC), and basilar membrane (BM), including the organ of Corti, were dissected, respectively. Immunofluorescence, quantitative real-time polymerase chain reaction, and western blot were applied to detect the expression of LaminB1 in individual cochlear tissues at both mRNA and protein levels. RESULTS: Immunofluorescence revealed that LaminB1 was localized in the outer hair cells, inner hair cells, Kolliker's organ, Reissner's membrane, SGC, STV, and spiral ligament. The intensity of staining surrounding the scala media decreased during cochlear development. The expression of LaminB1 mRNA and protein in STV, SGC, and BM was at a maximum level at p0 but gradually declined to a minimum level at p21. CONCLUSION: Our research provided direct evidence that LaminB1 was expressed in the developing cochlea and developmentally regulated in cochlear tissues, suggesting a possible role of LaminB1 in cochlear development. Our result provided a theoretical basis for further study about the physiological function of LaminB1 in the peripheral auditory system.

Sleep Deprivation Modifies Noise-Induced Cochlear Injury Related to the Stress Hormone and Autophagy in Female Mice.

A lack of sleep is linked with a range of inner ear diseases, including hearing loss and tinnitus. Here, we used a mouse model to investigate the effects of sleep deprivation (SD) on noise vulnerability, and explored the mechanisms that might be involved in vitro, focusing particularly corticosterone levels and autophagic flux in cells. Female BALB/c mice were divided into six groups [control, acoustic trauma (AT)-alone, 1 day (d) SD-alone, 1d SD pre-AT, 5d SD-alone, and 5d SD pre-AT]. Cochlear damage was then assessed by analyzing auditory brainstem response (ABR), and by counting outer hair cells (OHCs) and the synaptic ribbons of inner hair cells (IHCs). In addition, we measured levels of serum corticosterone and autophagy protein expression in the basilar membranes by ELISA kits, and western blotting, respectively. We found that SD-alone temporarily elevated ABR wave I amplitude, but had no permanent effect on hearing level or IHC ribbon numbers. Combined with AT, the number of synaptic ribbons in the 1d SD pre-AT group was significantly higher than that in the AT-alone group, whereas the 5d SD pre-AT group showed more severe synaptopathy, and a greater loss of OHCs after 2 weeks than the other experimental groups exposed to noise. Correspondingly, the levels of corticosterone in the AT-alone group were higher than those of the 1d SD pre-AT group, but lower than those of the 5d SD pre-AT group. The 1d SD pre-AT group showed a marked elevation in the expression of microtubule-associated protein 1 light chain 3B (LC3B), whereas the AT-alone group exhibited only a mild increase. In contrast, the levels of LC3B did not change in the 5d SD pre-AT group. Experiments with HEI-OC-1 cells and cochlear basilar membrane cultures showed that high-concentrations of dexamethasone, and the inhibition of autophagy, aggravated cellular apoptosis induced by oxidative stress. In conclusion, noise-induced synaptopathy and hair cell loss can be mitigated by preceding 1d SD, but will be aggravated by preceding 5d SD. These findings may be attributable to corticosterone levels and the extent of autophagy.

Down-regulation of Cav1.3 in auditory pathway promotes age-related hearing loss by enhancing calcium-mediated oxidative stress in male mice.

In this study, age related Cav1.3 expression in cochlea and auditory cortex of C57BL/6J male mice was evaluated. It was found that the expression of Cav1.3 in cochlea decreased with aging whereas this phenomenon was not observed in neuron of auditory cortex. The correlation between decreased expression of Cav1.3 and age-related hearing losses was studied in vitro, after Cav1.3 was knocked out, the rate of apoptosis of hair cells increased after being subjected to ROS stresses, accompanied with enhanced senescence. Further, Cav1.3 knock down also interfered with the electrophysiology of hair cells. The effect was further confirmed in vivo, after Cav1.3 knocked down by injection of AAV, hearing impairment was observed in C57BL/6J male mice subjected to senescence and this was accompanied by increased loss of hair cells in cochlea. The effect was further confirmed in 3D organ culture, increased loss of hair cells after Cav1.3 was knocked down under ROS stresses.Mechanistically, Cav1.3 knock out resulted in decreased intracellular calcium which subsequently reduced the inactivation of ROS from complex I, and finally resulted in increased intracellular ROS and enhanced senescence.Collectively, these findings confirmed that Cav1.3 could protect cells in auditory pathway from oxidative stresses, and decreased expression of Cav1.3 in auditory pathway could contribute to hearing losses by enhancement of calcium-mediated oxidative stress.

Expression Patterns and Implications of LaminB1 in Rat Cochleae.

LaminB1, a major component of the nuclear lamina, is a potent regulator of cellular proliferation and senescence and also known to be essential for neuronal migration and brain development. However, the expression patterns of LaminB1 in the rat cochleae are still not fully revealed. Utilizing immunofluorescence, Western blotting, and quantitative real-time PCR, we identified the distribution and expression of LaminB1 in the rat cochleae. Immunofluorescence staining indicated that LaminB1 was mainly localized in the auditory hair cells (HCs), spiral ganglion cells (SGC), stria vascularis (STV, including spiral ligament), Reissner's membrane (RM), and limbus laminae spiralis (LLS). Western blotting analysis illustrated that the distribution of LaminB1 in rat cochleae was characterized by tissue specificity. The LaminB1 protein was expressed more in SGC and basilar membrane (BM) than in STV. Meanwhile, the mRNA expression of LaminB1 displayed difference in cochlear tissues. These observations preliminarily revealed the expression patterns of LaminB1, providing a theoretical basis for further study on the role of LaminB1 in auditory function.

Lessons Learned From 35 Cases of Laryngeal Foreign Bodies Undergoing Misdiagnosis in Pediatric Population.

OBJECTIVES: To present 35 cases of laryngeal foreign bodies (FBs) in pediatric population undergoing misdiagnosis so as to draw on our lessons to improve early diagnosis. METHODS: A retrospective analysis over 15 years was conducted of 35 cases of laryngeal FBs undergoing misdiagnosis in children. Meanwhile, a control group, including 42 cases of laryngeal FBs without misdiagnosis in children, was set. These patients' clinical data were collected and analyzed to identify the risk factors for misdiagnosis. RESULTS: The results of chi-square test and univariate analysis both showed a significant difference in time elapsed between discomforts and admission, witnessed foreign body (FB) aspiration history, biphasic stridor, aphonia, roentgenologic findings, and type and size of FBs between the misdiagnosed group and control group. Multivariate analysis further identified delayed doctor visits, unwitnessed FB aspiration history, nonspecific symptoms, and negative roentgenologic manifestations as independent risk factors for misdiagnosis. CONCLUSIONS: Diagnosis of laryngeal FBs, especially small, thin, and radiolucent FBs, remains a challenge. We emphasized the importance of timely doctor visits, careful clinical history inquisition, and prompt performance of radiographic or endoscopic examinations for diagnosis.

Elevated expression of yes­associated protein is associated with the malignant status and prognosis of laryngeal squamous cell carcinoma.

Previous studies have demonstrated that elevated yes­associated protein (YAP) expression is associated with tumor aggression and poor prognosis in various types of human cancer. However, the clinicopathological significance and the prognostic value of YAP in laryngeal squamous cell carcinoma (LSCC) is unknown. The aim of the present study was to identify the expression pattern and prognostic significance of YAP in patients with LSCC. YAP mRNA and protein expression levels were examined in fresh and archived LSCC samples using the reverse transcription­quantitative polymerase chain reaction, immunohistochemistry (IHC) and western blotting. The association between YAP expression levels with the malignant status and prognosis of patients with LSCC was analyzed. Upregulated protein and mRNA expression levels of YAP were detected in LSCC tissues compared with paired healthy surgical margin tissues. Positive expression of YAP was identified in 84/121 (69.4%) LSCC tissues and in 4/30 (13.3%) healthy surgical margin tissues by IHC. Positive YAP protein expression was significantly associated with clinical stage, TNM classification, lymph node metastasis and differentiated degree. Patients with positive YAP expression exhibited a significantly decreased overall survival time compared with patients with negative YAP expression (P=0.0002). Multivariate analysis indicated that the level of YAP expression was an independent prognostic factor for poor survival in patients with LSCC (P=0.012). In conclusion, the expression level of YAP was significantly increased in LSCC and associated with the malignant status of LSCC. Therefore, YAP may represent a novel biomarker for predicting the prognosis of patients with LSCC.

Aminoglycoside ototoxicity in three murine strains and effects on NKCC1 of stria vascularis.

BACKGROUND: After establishing a murine model of aminoglycoside antibiotic (AmAn) induced ototoxicity, the sensitivity of AmAn induced ototoxicity in three murine strains and the effect of kanamycin on the expression of Na-K-2Cl cotransporter-1 (NKCC1) in stria vascularis were investigated. METHODS: C57BL/6J, CBA/CaJ, NKCC1(+/-) mice (24 of each strain) were randomly divided into four experimental groups: A: kanamycin alone; B: kanamycin plus 2, 3-dihydroxybenzoate; C: 2, 3-dihydroxybenzoate alone; and D: control group. Mice were injected with kanamycin or/and 2, 3-dihydroxybenzoate twice daily for 14 days. Auditory brainstem response (ABR) was measured and morphology of cochlea delineated with succinate dehydrogenase staining. Expression of NKCC1 in stria vascularis was detected immunohistochemically. RESULTS: All three strains in groups A and B developed significant ABR threshold shifts (P < 0.01), which were accompanied by outer hair cell loss. NKCC1 expression in stria vascularis was the weakest in group A (A cf D, P < 0.01) and the strongest in groups C and D (P < 0.05). CBA/CaJ mice had the highest sensitivity to AmAn. CONCLUSIONS: Administration of kanamycin established AmAn induced ototoxicity. Kanamycin inhibited the expression of NKCC1 in stria vascularis. 2, 3-dihydroxybenzoate attenuated AmAn induced ototoxicity-possibly by enhancing the expression of NKCC1. Age related hearing loss did not show additional sensitivity to AmAn induced ototoxicity in murine model.

Localization of NKCC1 in the cochlea and morphology of the cochlea in NKCC1-knockout mice.

The distribution of the Na-K-2Cl co-transporter (NKCC1) in the cochlear K+ cycling pathway in cochlea and cochlear histological changes in the NKCC1 knockout mice were investigated. By using immunohistochemistry and toluidine blue staining, the localization of NKCC1 in cochlea of the C57BL/6J mice and the cochlear histological changes in the NKCC1 knockout mice were observed. It was found that the NKCC1 was expressed mainly in the stria marginal cells and the fibrocytes in the inferior portion of the spiral ligament in the adult C57BL/6J mice. Subpopulation of the fibrocytes in the suprastrial region and the limbus was also moderately immunoreactive. While in the cochlea of the NKCC1 knockout mice, Reissner's membrane was collapsed and scala media disappeared, accompanied with the loss of inner hair cells, outer hair cells and the support cells. The tunnel of Corti was often absent. All the findings suggested the localization of NKCC1 in the cochlea was closely correlated with cochlear K+ cycling. Loss of NKCC1 led to the destruction of the cochlear structures, and subsequently influenced the physiological function of cochlea.