Identification of common stria vascularis cellular alteration in sensorineural hearing loss based on ScRNA-seq.

BACKGROUND: The stria vascularis (SV), located in the lateral wall of the cochlea, maintains cochlear fluid homeostasis and mechanoelectrical transduction (MET) activity required for sound wave conduction. The pathogenesis of a number of human inheritable deafness syndromes, age related hearing loss, drug-induced ototoxicity and noise-induced hearing loss results from the morphological changes and functional impairments in the development of the SV. In this study, we investigate the implications of intercellular communication within the SV in the pathogenesis of sensorineural hearing loss (SNHL). We aim to identify commonly regulated signaling pathways using publicly available single-cell transcriptomic sequencing (scRNA-seq) datasets. METHODS: We analyzed scRNA-seq data, which was derived from studying the cochlear SV in mice with SNHL compared to normal adult mice. After quality control and filtering, we obtained the major cellular components of the mouse cochlear SV and integrated the data. Using Seurat's FindAllMarkers and FindMarkers packages, we searched for novel conservative genes and differential genes. We employed KEGG and GSEA to identify molecular pathways that are commonly altered among different types of SNHL. We utilized pySCENIC to discover new specific regulatory factors in SV subpopulation cells. With the help of CellChat, we identified changes in subpopulation cells showing similar trends across different SNHL types and their alterations in intercellular communication pathways. RESULTS: Through the analysis of the integrated data, we discovered new conserved genes to SV specific cells and identified common downregulated pathways in three types of SNHL. The enriched genes for these pathways showing similar trends are primarily associated with the Electron Transport Chain, related to mitochondrial energy metabolism. Using the CellChat package, we further found that there are shared pathways in the incoming signaling of specific intermediate cells in SNHL, and these pathways have common upstream regulatory transcription factor of Nfe2l2. Combining the results from pySCENIC and CellChat, we predicted the transcription factor Nfe2l2 as an upstream regulatory factor for multiple shared cellular pathways in IC. Additionally, it serves as an upstream factor for several genes within the Electron Transport Chain. CONCLUSION: Our bioinformatics analysis has revealed that downregulation of the mitochondrial electron transport chain have been observed in various conditions of SNHL. E2f1, Esrrb, Runx1, Yy1, and Gata2 could serve as novel important common TFs regulating the electron transport chain. Adm has emerged as a potential new marker gene for intermediate cells, while Itgb5 and Tesc show promise as potential new marker genes for marginal cells in the SV. These findings offer a new perspective on SV lesions in SNHL and provide additional theoretical evidence for the same drug treatment and prevention of different pathologies of SNHL.

[Mitochondrial 12S rRNA A1555G mutation associated with nonsyndromic hearing loss in twenty-five Han Chinese pedigrees].

Mitochondrial 12S rRNA A1555AG mutation is one of the important causes of aminoglycoside-induced and nonsyndromic deafness. We report here the clinical, genetic and molecular characterization of 25 Chinese families carrying the A1555G mutation.Clinical and genetic characterizations of these Chinese families exhibited a wide range of penetrance, severity and age-at-onset of hearing impairment. The average penetrances of deafness were 28.1% and 21.5%, respectively, when aminoglycoside-induced hearing loss was included or excluded. Furthermore, the average age-of-onset for deafness without aminoglycoside exposure ranged from 1 and 15 years old. Their mitochondrial genomes exhibited distinct sets of polymorphisms including 16 novel variants, belonging to ten Eastern Asian haplogroups A, B, D, F, G, M, N and R, respectively. Strikingly, these Chinese families carrying mitochondrial haplogroup B exhibited higher penetrance and expressivity of hearing loss. In addition, 7 known secondary mutations and 21 variants resided at the highly conservative residues may enhance the penetrace of hearing loss in these Chinese families. Moreover, the absence of mutation in GJB2 gene suggested that GJB2 may not be a modifier for the phenotypic expression of the A1555G mutation in these Chinese families. These observations suggested that mitochondrial haplotypes and other modifiers may modulate the variable penetrance and expressivity of deafness among these Chinese families.

[Prevalence of common genetic mutations and clinical characteristics analysis in patients at different ages with nonsyndromic hearing impairment].

To evaluate the correlation between genetic mutations and the age in nonsyndromic hearing impairment (NSHI) and the clinical characteristics of NSHI, 215 patients with NSHI were enrolled between April 2006 and April 2012. All patients were divided into four groups according to ages of hearing loss onset and clinic presentation (0-3, 3-6, 6-18 and 18+ years). The mutations of GJB2 and mitochondria DNA (mtDNA) 1555G/C1494T were screened from peripheral blood samples in each age group. The prevalence of mutations and the age ratio were obtained. The study showed that 18.14% of all patients were found to have GJB2 mutations and 11.16% were found to have mtDNA A1555G/C1494T mutations. The prevalence of GJB2 mutation in adult group (5.26%) was lower than juvenile group who sought medical attention at 0-18 years of age (22.36%), while the prevalence of mtDNA A1555G/C1494T in adult group (31.48%) was higher than juvenile group (4.97%). Significant differences in the prevalence of GJB2 (χ2=7.108, P=0.008) and mtDNA A1555G/C1494T (χ2=20.852, P=0.000) were observed in both of two groups. The prevalence of GJB2 mutations between adult and juvenile groups according to ages of hearing loss onset was statistically significant different (0%, 20.10%, respectively, and P=0.023), while the prevalence of mtDNA A1555G/C1494T mutations was not different (14.29%, 11.34%, respectively, and P=0.698). The onset age of 66.67% of patients with GJB2 mutations was less than 1 year old, while the onset of patients with mtDNA A1555G/C1494T mutations could be found at any age group. Different standardizations of hearing loss could also show different results. These data strongly suggest that most of GJB2 mutations are found in congenital deafness and mtDNA A1555G/C1494T mutations mainly represent acquired deafness, which can be induced or aggravated by aminoglycoside antibiotics in all age groups and should be tested mainly ranging from 4 kHz to 8 kHz. Both newborn hearing screening and genetic testing are important to find early deafness.

Inhibition of histone methyltransferase PRMT5 attenuates cisplatin-induced hearing loss through the PI3K/Akt-mediated mitochondrial apoptotic pathway.

This study aimed to evaluate the therapeutic potential of inhibiting protein arginine methyltransferase 5 (PRMT5) in cisplatin-induced hearing loss. The effects of PRMT5 inhibition on cisplatin-induced auditory injury were determined using immunohistochemistry, apoptosis assays, and auditory brainstem response. The mechanism of PRMT5 inhibition on hair cell survival was assessed using RNA-seq and Cleavage Under Targets and Tagment-quantitative polymerase chain reaction (CUT&Tag-qPCR) analyses in the HEI-OC1 cell line. Pharmacological inhibition of PRMT5 significantly alleviated cisplatin-induced damage to hair cells and spiral ganglion neurons in the cochlea and decreased apoptosis by protecting mitochondrial function and preventing the accumulation of reactive oxygen species. CUT&Tag-qPCR analysis demonstrated that inhibition of PRMT5 in HEI-OC1 cells reduced the accumulation of H4R3me2s/H3R8me2s marks at the promoter region of the Pik3ca gene, thus activating the expression of Pik3ca. These findings suggest that PRMT5 inhibitors have strong potential as agents against cisplatin-induced ototoxicity and can lay the foundation for further research on treatment strategies of hearing loss.

[Spectrum and frequency of mitochondrial 12S rRNA variants in the Chinese subjects with nonsynrdomic hearing loss in Zhejiang Province].

Mitochondrial DNA (mtDNA) mutations are one of the important causes of deafness. In particular, the 12S rRNA gene is the hot spots for mutations associated with both aminoglycoside ototoxicity and nonsyndromic deafness. In this report, a total of 318 Chinese pediatric hearing-impaired subjects were recruited from otology clinics in the Zhejiang Province, China. These subjects underwent clinical, genetic evaluation and molecular analysis of 12S rRNA gene. Mutational analysis identified 34 variants in the 12S rRNA gene in this cohort. The incidences of the known deafness-associated 1555A>G, 1494C>T and 1095T>C mutations were 9.1%, 0.6% and 1.25% in this cohort, respectively. Other mtDNA variants were evaluated by structural and phylogenetic analysis. Of these, the 839A>G and 1452T>C variants could confer increased sensitivity to aminoglycosides or nonsyndromic deafness as they were not present in 449 Chinese controls and localized at highly conserved nucleotides of the 12S rRNA. However, other variants appeared to be polymorphisms. These data further support the idea that mitochondrial 12S rRNA is one of major targets for aminoglycoside ototoxicity. These data have been providing valuable information to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside antibiotic therapy, and eventually to decrease the incidence of deafness.

Astaxanthine attenuates cisplatin ototoxicity in vitro and protects against cisplatin-induced hearing loss in vivo.

Astaxanthine (AST) has important biological activities including antioxidant and anti-inflammatory effects that could alleviate neurological and heart diseases, but its role in the prevention of cisplatin-induced hearing loss (CIHL) is not yet well understood. In our study, a steady interaction between AST and the E3 ligase adapter Kelch-like ECH-associated protein 1, a predominant repressor of nuclear factor erythroid 2-related factor 2 (NRF2), was performed and tested via computer molecular docking and dynamics. AST protected against cisplatin-induced ototoxicity via NRF2 mediated pathway using quantitative PCR and Western blotting. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential revealed that AST reduced ROS overexpression and mitochondrial dysfunction. Moreover, AST exerted anti-apoptosis effects in mouse cochlear explants using immunofluorescence staining and HEI-OC1 cell lines using quantitative PCR and Western blotting. Finally, AST combined with poloxamer was injected into the middle ear through the tympanum, and the protection against CIHL was evaluated using the acoustic brain stem test and immunofluorescent staining in adult mice. Our results suggest that AST reduced ROS overexpression, mitochondrial dysfunction, and apoptosis via NRF2-mediated pathway in cisplatin-exposed HEI-OC1 cell lines and mouse cochlear explants, finally promoting cell survival. Our study demonstrates that AST is a candidate therapeutic agent for CIHL.

Comprehensive Identification of Potential Crucial Genes and miRNA-mRNA Regulatory Networks in Papillary Thyroid Cancer.

BACKGROUND: Thyroid cancer is the most common endocrine malignancy, with a recent global increase of 20% in age-related incidence. Ultrasonography and ultrasonography-guided fine-needle aspiration biopsy (FNAB) are the most widely used diagnostic tests for thyroid nodules; however, it is estimated that up to 25% of thyroid biopsies are cytologically inconclusive. Molecular markers can help guide patient-oriented and targeted treatment of thyroid nodules and thyroid cancer. METHODS: Datasets related to papillary thyroid cancer (PTC) or thyroid carcinoma (GSE129562, GSE3678, GSE54958, GSE138042, and GSE124653) were downloaded from the GEO database and analysed using the Limma package of R software. For functional enrichment analysis, the Kyoto Encyclopedia of Genes and Genomes pathway analysis and Gene Ontology were applied to differentially expressed genes (DEGs) using the Metascape website. A protein-protein interaction (PPI) network was built from the STRING database. Gene expression, protein expression, immunohistochemistry, and potential functional gene survival were analysed using the GEPIA website, the Human Protein Atlas website, and the UALCAN website. Potential target miRNAs were predicted using the miRDB and Starbase datasets. RESULTS: We found 219 upregulated and 310 downregulated DEGs, with a cut-off of p < 0.01 and ∣log FC | >1.5. The DEGs in papillary thyroid cancer were mainly enriched in extracellular structural organisation. At the intersection of the PPI network and Metascape MCODEs, the hub genes in common were identified as FN1, APOE, CLU, and SDC2. In the targeted regulation network of miRNA-mRNA, the hsa-miR-424-5p was found to synchronously modulate two hub genes. Survival analysis showed that patients with high expression of CLU and APOE had better prognosis. CONCLUSIONS: CLU and APOE are involved in the molecular mechanism of papillary thyroid cancer. The hsa-miR-424-5p might have the potential to reverse the processes of papillary thyroid cancer by modulating the hub genes. These are potential targets for the treatment of patients with papillary thyroid cancer.

The Role of the Reactive Oxygen Species Scavenger Agent, Astaxanthin, in the Protection of Cisplatin-Treated Patients Against Hearing Loss.

Emerging evidence of significant hearing loss occurring shortly after cisplatin administration in cancer patients has stimulated research into the causes and treatment of this side effect. Although the aetiology of cisplatin-induced hearing loss (CIHL) remains unknown, an increasing body of research suggests that it is associated with excessive generation of intracellular reactive oxygen species (ROS) in the cochlea. Astaxanthin, a xanthophyll carotenoid, has powerful anti-oxidant, anti-inflammatory, and anti-apoptotic properties based on its unique cell membrane function, diverse biological activities, and ability to permeate the blood-brain barrier. In this review, we summarize the role of ROS in CIHL and the effect of astaxanthin on inhibiting ROS production. We focus on investigating the mechanism of action of astaxanthin in suppressing excessive production of ROS.

Safety and Immunogenicity of a Nonadjuvant Human Papillomavirus Type 6 Virus-like Particle Vaccine in Recurrent Respiratory Papillomatosis.

OBJECTIVES: To assess the safety and immunogenicity of a nonadjuvant human papillomavirus (HPV) type 6 L1 virus-like particle (VLP) vaccine in recurrent respiratory papillomatosis (RRP) in local Chinese patients. METHODS: Patients with RRP who had undergone surgical treatment before intramuscular administration of an escalating dose of HPV type 6 L1 VLPs (1, 5, and 25 µg at 4 weekly intervals) as part of their treatment were followed up for more than 10 years. Efficacy was assessed by detecting the vaccine-induced type-specific antibody titer, calculating the intersurgical interval, and observing recurrence or remission of papillomas after receiving the vaccine. RESULTS: Nonadjuvant HPV vaccine was generally well tolerated, with no serious vaccine-related adverse episodes. It induced seroconversion for each vaccine-related HPV type. At week 12 (4 weeks after injecting 25 µg), the vaccine-induced type-specific antibody titer was significantly high. Analysis of all patients found a significant increase in the intersurgical interval and decrease in the scores. One patient (16.7%; female) experienced complete remission. Five patients (83.3%) (two males and three females) experienced partial remission. In total, complete or partial remission was achieved in six (100%) patients. CONCLUSIONS: Administration of nonadjuvant HPV type 6 L1 VLPs vaccine to RRP was generally well tolerated and highly immunogenic.

[Juvenile onset respiratory papillomatosis: risk factors for severity].

OBJECTIVE: This study was designed to explore the risk factors associated with severity of juvenile onset recurrent respiratory papillomatosis. METHOD: A retrospective study was conducted to study determinants of severe forms of juvenile recurrent onset respiratory papillomatosis. The patients were separated into different groups based on the onset age, the first recurrence of age, the first recurrence of period, gender and incision of tracheal respectively. The relationship among the lesion severity score,the involvement of the subregion, operation period and the next operation period were also explored. RESULT: It was observed that some children who recurred before 4 years old required more surgery, shorter operation period(the average, longest or shortest operation period) than those elder children, the differences were statistically (P=0. 029, 0. 003, 0. 010, 0. 039, respectively). The severity score of lesion was correlated positively with the involvement of the subregion and negatively with operation period (r=0. 914, -0. 451, respectively). Some children who diagnosed before 4 years old had to endure more severity score and shorter operation period than those older children, the differences were statistically (P= 0. 036, 0. 000, respectively). 8 cases accepted incision of tracheal, they accepted more surgery too. But the differences in the onset age, the first recurrence of age, and the operation period were not statistically. CONCLUSION: The results showed that the clinical course of juvenile onset recurrent respiratory papillomatosis was closely related to the first recurrence age and period, while the severity of disease was associated to the onset age and the involvement of the subregion.

[Function study of non-syndromic deafness associated mitochondrial 12S rRNA A839G mutation].

OBJECTIVE: To investigate the correlation between nonsyndromic deafness and mitochondrial 12s rRNA A839G mutation. METHODS: According to the clinical manifestations of mitochondrial DNA sequencing and analysis to find and determine family containing mitochondrial 12s rRNA A839G mutation. Harvested its family members blood and transferred their lymphocytes into lymphoblastoid cell lines, followed by cells cultured, cell doubling experiment, susceptibility testing, cellular oxygen consumption rate experiment, ROS and mitochondrial membrane potential experimental tests were progressed to explore the correlation between the A839G mutation and nonsyndromic deafness. RESULTS: The mitochondrial 12s rRNA A839G mutation pedigrees were determined through the full sequence detections of the Mitochondrial DNA, further phylogenetic analysis showed that 839 point conservative index (CI) up to 78.6%; in RPMI-galactose medium containing A839G gene mutant cell line, the doubling time was significantly longer than the control group, and the difference was significant (P = 0.033). The effect to cell lines containing the A839G mutation of aminoglycoside drugs was not obvious. When compared with the control group, cell lines containing the A839G mutation significantly reduced cellular oxygen consumption rate(P = 0.033); compared with the control group, the ROS levels of cell lines containing the A839G mutation appeared more substantial elevated with significan difference (P < 0.01). The mitochondrial membrane potential of cells of experimental group was significantly reduced than the control group. CONCLUSION: The present study proved that the mitochondria 12s rRNA A839G mutations affect the function of the mitochondrial respiratory chain at the cell level, which might reduce the growth rate of the mutant cell lines, result in hearing.

[Levels of serum IL-4, IFN-gamma, IL-32 in children with juvenile-onset recurrent respiratory papillomatosis].

OBJECTIVE: To explore the relationship between serum IL-4, IFN-gamma, IL-10 levels and the aetiology of juvenile-onset recurrent respiratory papillomatosis. METHOD: Serum IL-4, IFN-gamma, IL-32 levels of 15 JORRP children were detected by use of enzyme-linked immunosorbent assay (ELISA) and compared with those of healthy control group. RESULT: Serum IL-4 levels were significantly higher in the JORRP children (P<0.01): (524.65 +/- 147.77)pg/ml in the JORRP children and (213.27 +/- 87.48) pg/ml in the healthy control group. Serum IFN-gamma levels were significantly lower in the JORRP children (P<0.01): (2.87 +/- 0.84) pg/ml in the JORRP children and (10.63 +/- 5.09) pg/ml in the healthy control group. Serum IL-32 levels were significantly lower in the JORRP children (P< 0.01): (2.47 +/- 1.60) pg/ml in the JORRP children and (9.08 +/- 2.66) pg/ml in the healthy control group. CONCLUSION: 1) While the concentration of Th2 like cytokine IL-4 in children with JORRP was higher than that in control group, the concentration of Th1 like cytokine IFN-gamma in children with JORRP was lower than that in controls, indicating that the polarization of Th1 /Th2 T cell in children with JORRP; 2) The polarization of Th1/Th2 T cell may cause the reduction of the serum IL-32 as a proinflammatory role in host immunity system that could not eradicate HPVs because of lacking enough inflammatory stimulation.

[Analysis of genotype-phenotype correlation for GJB2 in 221 non-syndromic deafness probands and their pedigrees].

OBJECTIVE: To assess the possible genotype-phenotype correlation for GJB2. METHODS: Retrospectively analyzed GJB2 gene mutations with non-syndromic hearing impairment (NSHI) patients and their families audiological data. Individuals were grouped, according to non-truncated mutant (non-truncating, NT) and truncating mutations (truncating, T), into T/T group, T/NT group and NT/NT group. And according to whether they carry 235delC, grouped into 235delC/235delC group, 235delC/Non-235del group and Non-235delC/Non-235delC group. RESULTS: Grouped according to whether the truncation mutants:Fisher exact statistical analysis showed that the degree of hearing loss among the three groups did not meet the random distribution (P = 0.003) , T/T group was significantly higher than T/NT group (P = 0.000) and NT/NT group (P = 0.000) on the degree of hearing loss. Grouped according to whether they carry 235delC mutation: degrees of hearing loss among the three groups were statistically significant differences. Respectively pairwise comparisons (Fisher exact test) found 235delC/235delC group was significantly higher than 235delC/Non-235delC on the degree of hearing loss group (P = 0.001) and Non-235delC/Non-235delC group (P = 0.000), 235delC/Non-235delC group higher than Non-235delC/Non-235delC group (P = 0.033). In GJB2 mutations homozygous and compound heterozygous mutation genotype:G109A/G109A, 235delC/512insAACG, 299delAT/G109A and 235delC/G109A degree of hearing loss caused by genotype was significantly lower than 235delC/235delC group. CONCLUSIONS: 235delC homozygotes have significantly more hearing impairment, when compared with 235delC/non-235delC compound heterozygotes. People with two non-235delC mutations have even less hearing impairment. Patients with non-truncation mutants (G109A) suffer from lighter hearing loss than truncation mutations(235delC, 299delAT).

[Peri-operative management on juvenile recurrent respiratory papillomatosis].

OBJECTIVE: To investigate the safety of peri-operative management on children with juvenile recurrent respiratory papilloma (JORRP). METHODS: A retrospective analysis was conducted on preoperative assessment, anesthesia methods and options, operative procedure, and postoperative airway maintenance in 28 JORRP children aged from ten months to seven years old. A total of 148 times of surgery was performed on these 28 children. RESULTS: One hundred and nine JORRP children graded one and two-degree dyspnea underwent surgery within 24 hours and were intubated successfully in the first attempt after intravenous induction. Thirty-nine emergency operations were performed in the children graded three and four-degree dyspnea, 35 of them were intubated successfully in the first attempt after inhalation induction and 4 succeeded in the second attempt. No complications occurred in 129 JORRP children postoperatively, 17 children suffered from mild dyspnea and relieved after oxygen inhalation, 2 children were intubated and sent to intensive care unit because of postoperative hypoxemia. All JORRP children got through the peri-operative period safely. The quality of pronunciation in 101 children improved markedly and 35 suffered from slight hoarseness on the 1st postoperative day. Three children had the tracheal tube of tracheostomy removed after receiving five, four and three operations respectively. Nineteen children were followed up for 2 - 5 years. Among them, one child did not relapse 3 years after surgical management.One child suffered from laryngostenosis postoperatively. No death occurred. CONCLUSION: Complete preoperative preparation, rational anesthesia methods, careful operative procedure and airway maintenance after surgery could increase the safety for children with recurrent respiratory papilloma.