TMPRSS3 and five had pathogenic variants in genes known to affect the cochlear sensory partition. The mechanism by which variants in TMPRSS3 cause genetic hearing loss is unknown. We used a 500-Hz tone burst to record ECochG responses from an intracochlear electrode. Responses consist of a cochlear microphonic (hair cell) and an auditory nerve neurophonic. Cochlear microphonics did not differ between groups. Auditory nerve neurophonics were smaller, on average, in subjects with TMPRSS3 deafness. Results of this proof-of-concept study provide evidence that pathogenic variants in TMPRSS3 may impact function of the spiral ganglion. While ECochG as a clinical and research tool has been around for decades, this study illustrates a new application of ECochG in the study of genetic hearing and deafness in vivo.Cochlear implantation, a surgical method to bypass cochlear hair cells and directly stimulate the spiral ganglion, is the standard treatment for severe-to-profound hearing loss. Changes in cochlear implant electrode array design and surgical approach now allow for preservation of acoustic hearing in the implanted ear. Electrocochleography (ECochG) was performed in eight hearing preservation subjects to assess hair cell and neural function and elucidate underlying genetic hearing loss. Three subjects had pathogenic variants in  Pathogenic variants in the TMPRSS3 gene cause autosomal recessive non-syndromic hearing loss (ARNSHL) at the DFNB8 and DFNB10 loci1, two loci mapped over 20 years ago in consanguineous families with disparate phenotypes. In the DFNB10 family, the deafness was congenital and severe-to-profound while in the DFNB8 family, it was post-lingual and progressive3. Both families were ultimately found to segregate pathogenic variants in TMPRSS3. The phenotypic differences are consistent with more recent data that show clear correlations with TMPRSS3 variant type and the severity and onset of associated hearing loss7.Hearing loss is the most common sensory deficit in humans. Our understanding of its biology and molecular physiology has been guided in large part by the discovery of genes implicated in both syndromic  and non-syndromic (over 100 genes) forms of hearing loss  could be a substrate of TMPRSS38. However, this channel is not expressed in human cochlear hair cells. More recent data support a role in TMPRSS3 function in localization of the potassium channel Kcnma1, which is critical for maintenance of the resting potential of cochlear inner hair cells and for their survival at the onset of hearing13. Data also show that the function of TMPRSS3 in spiral ganglion neurons is critical for their survival, as demonstrated by Li et al. using siRNA and miR-specific gene suppression14.TMPRSS3 are the most common genetic cause of hearing loss in post-lingually deafened adult cochlear implant (CI) users15. In the same study we also showed that as a group, CI users with deleterious genetic variants in genes expressed in the spiral ganglion or auditory nerve have poorer overall outcomes as compared to other CI cohorts. These observations are potentially important as cochlear implantation has become the standard surgical treatment for persons with severe-to-profound hearing loss. More than 219,000 persons have undergone cochlear implantation since 1985 when the technology was first approved by the FDA. Hybrid cochlear implants were developed more recently as an option for individuals with high frequency hearing loss who do not benefit from conventional hearing aids but are not qualified for traditional cochlear implantations. Hybrid CI users hear high frequencies via electrical stimulation but continue to use acoustic stimulation to hear low frequencies16. This technology has resulted in significant an expansion of CI candidacy.Recently we showed that deleterious variants in electrically evoked neural responses arising from the auditory nerve of CI users17. In addition, electrocochleography (ECochG) has been used in clinical and research practice for decades to measure cochlear hair cell and neural responses from normal hearing and hearing impaired populations in response to acoustic stimulation. The ability to record such responses requires that patients have residual acoustic hearing, precluding applications in CI populations. ECochG recordings have four different and overlapping components: the cochlear microphonic (CM) and the summating potential (SP), generated from cochlear hair cells19; and two neural responses, the compound action potential (CAP) and auditory nerve neurophonic (ANN)22.It has long been possible to use an intracochlear cochlear implant electrode to record 24, monitoring insertion trauma from cochlear implantation26, and monitoring changes in acoustic hearing over time in patients with preserved acoustic hearing in the implanted ear28. Here we propose a novel application of ECochG to study the functional effects of pathogenic variants in TMPRSS3 in humans. We hypothesized, based on our previous work, that these individuals may exhibit abnormal function at the level of the spiral ganglion or auditory nerve15. Results show that, on average, when compared to results obtained from five Hybrid CI subjects carrying pathogenic variants in genes affecting the cochlear sensory partition (Sensory Group), the three Hybrid cochlear implant users with pathogenic variants in TMPRSS3 have ECochG responses that are consistent with a significant reduction in auditory nerve/spiral ganglion function. Results of this study, while seriously underpowered, provide evidence that pathogenic variants in TMPRSS3 could help us refine our understanding of the molecular physiology of hearing and deafness.ECochG has received renewed attention in recent years due to potential clinical applications in CI populations, such as predicting post-operative speech outcomesTMPRSS3 causing DFNB8 deafness (TMPRSS3 Group). Five subjects had pathogenic genetic variants in genes known to affect the cochlear sensory organ including KCNQ4 (required for outer hair cell function), TMC1 (required for mechanotransduction of inner hair cells), MYO6, MYO15A, and LOXHD1 . These five subjects formed the comparison Sensory Group.Table TMPRSS3 Group (±1 s.d.) was 38 ± 41.2% in the electric-only stimulation mode and improved to 55.3 ± 47.7% in the combined acoustic and electric stimulation mode. A two-way repeated measures ANOVA with group (Sensory vs TMPRSS3) as the between-subjects factor and listening mode (electric-only vs acoustic + electric) as the within-subjects factor showed a significant main effect of listening mode . The effect of subject group (Sensory vs TMPRSS3) was not statistically significant (p > 0.05). No significant interaction  was found for this small sample of subjects.Post-operative audiometric thresholds are shown in Fig. 31. The ANN will be a periodic potential with significant energy at twice the stimulus frequency (1000 Hz in this case). We note that neither manipulation results in complete separation of the CM and ANN response32. Figure Figure Figure TMPRSS3 group are small and only one is above the noise floor of the measurement system. To the extent that the ANN/SUM responses reflects neural activity, these results are consistent with a neural  site of lesion for subjects in the TMPRSS3 Group.Figure 28, it is not known whether audiometric thresholds correlate well with suprathreshold CM/ANN amplitudes. We note that subjects in the TMPRSS3 group had the smallest ANN/SUM responses, consistent with a neural/spiral ganglion site of lesion. However, we knowledge that the variance in audiometric thresholds may affect ANN/SUM responses, which led to an alternative analysis shown in Fig. TMPRSS3. Note that for both subject pairs, the CM/DIF responses were almost identical  had ANN/SUM responses that were in or very close to the noise floor of the measurement system. These differences are most likely not attributable to differences in audiometric thresholds.Figure in vivo human electrophysiological application to assess the functional impact of a genetic lesion on the human peripheral auditory system. ECochG has had widespread applications in clinical practice and research studies for several decades33. It has typically been evoked using acoustic stimulation recorded via an electrode placed in the ear canal, on the tympanic membrane or occasionally on the promontory of the middle ear. ECochG amplitudes increase as the position of the recording electrode is moved closer toward the cochlea34. In this study, we recorded the ECochG using an intracochlear electrode, providing the most response possible without resorting to invasive methods. While the technique is not new, the application itself is. To our knowledge, this is the first study where intracochlear measures of hair cell/neural responses from the auditory periphery have been used to assess the functional impact of a genetic lesion on the human peripheral auditory system.In this study, we present a novel 19; and two neural responses, the compound action potential (CAP) and auditory nerve neurophonic (ANN)22. For decades, clinicians have recorded responses using opposite polarity stimuli, adding them to minimize the CM or subtracting them to emphasize responses recorded from the auditory nerve. The addition/subtraction technique we have used to segregate the response of hair cells from the auditory nerve was also used in these earlier studies. The extent to which the two responses can be isolated has been a matter of debate32. However, we argue that while the method we use to separate the hair cell response from the neural response may not be perfect, it provides the most direct measure of the response of the peripheral auditory system to acoustic stimulation that is available to date. This study is novel because, for the first time, we attempt to validate the method for assessing individuals with a known genetic diagnosis.ECochG recordings have four different and overlapping components: the cochlear microphonic (CM) and the summating potential (SP), generated from cochlear hair cells24. Correlations between CM/DIF and ANN/SUM thresholds and audiometric thresholds have also been reported by our group and another group28. There are also research teams who are working on developing methods to use intracochlear recordings to monitor cochlear function and guide insertion of the intracochlear electrode array35. This study, though, is the first to use this electrophysiological measure of the peripheral auditory system to study Hybrid cochlear implant users who carry a genetic diagnosis.Other investigators have recorded this response from cochlear implant users by placing an extracochlear electrode on the round window at the time of cochlear implant surgery. These investigators have reported significant correlations to post implant speech perception in quietTMPRSS3 would impact spiral ganglion neurons specifically and that we could measure this impact by analyzing neural (ANN/SUM) responses. We anticipated that subjects in the TMPRSS3 group would have smaller neural responses (ANN/SUM) than subjects in the sensory group. While our results are preliminary in nature, they are consistent with that hypothesis , showed variable results in regards to outcomes and a lack of uniform outcome measures for comparison  outperform subjects with pathogenic genetic variants in genes expressed in neural structures 42 and post-mortem spiral ganglion neuron counts46. Only recently has it been suggested that acoustically evoked potentials (ECochG) could correlate with CI speech perception outcomes47. Mixed findings are not surprising as all these evoked potentials reflect a peripheral response while speech perception requires peripheral, central, and cognitive processes. Both peripheral and central measures may be needed to increase the predictive power of electrophysiologic measures48.One may presume that electrophysiological measures of neural function may reflect speech perception outcomes; however, the literature suggests mixed findings. Speech perception outcomes are not necessarily correlated with electrically evoked neural responses31, they may also reflect distortion of the hair cell transduction process32. It seems likely that responses recorded from a normal-hearing animal model will not be identical to responses recorded from an impaired human cochlea. While we cannot argue that we have completely separated hair cell responses from auditory nerve responses, we have argued in our previous study that the derived CM/DIF and ANN/SUM responses are at least biased toward the cochlear hair cells and auditory nerve, respectively27.An important limitation of this work is the small number of subjects, which limits meaningful statistical analysis. As such, our results should be considered proof-of-concept only. Another weakness of this study, as we alluded to previously, is that the method used to isolate hair cell and neural contributions to an acoustic stimulus are not clean. While animal data suggests that the ANN/SUM responses arise from auditory neuronsin vivo recordings, obtained using a nearfield electrode, provide the most direct measure of the response of the peripheral auditory system to acoustic stimulation available to date. While similar recordings could be obtained using surface electrodes, these potentials are much smaller34. Intracochlear ECochG provides a level of specificity about the site-of-lesion in the peripheral auditory system that is not available from behavioral testing. Future studies examining other genetic causes and etiologies of hearing loss are planned and we hope will provide valuable insight into the molecular physiology of hearing and deafness.These limitations notwithstanding, we are encouraged by our results. These 51. One subject (T2) received an S8 electrode array as part of an FDA-approved, investigational trial. This electrode array features six electrodes mounted on a Silastic carrier that is 10 mm in length and, when inserted fully, spans 190–200o of the basal turn of the cochlea53. This study was approved by the Institutional Review Board of the University of Iowa and all research was performed in accordance with University of Iowa guidelines on Human Subjects Research. All subjects signed an informed consent document.Forty-four subjects received a Nucleus Hybrid CI at the University of Iowa Hospitals and Clinics between 2001 and 2016 and underwent comprehensive genetic testing (see below); eight had a confirmed genetic diagnosis and participated in this study (Table https://www.medicine.uiowa.edu/morl/otoscopegenes/ for complete gene list)55. Genetic testing results were discussed at a multidisciplinary meeting with geneticists, bioinfomaticians, graduate students, auditory research scientists and otolaryngologists to determine the likely genetic cause of deafness, if any, for each individual. OtoSCOPE v6 or v7 was used, with the primary difference being the addition of newly discovered deafness genes in v7.Comprehensive genetic testing was performed with the OtoSCOPE® platform using DNA extracted from peripheral blood according to standard practices (see TMPRSS3 (see Table TMPRSS3 Group.Three subjects T1–T3) had pathogenic variants in –T3 had pTMC1, KNCQ4, MYO15A, MYO6 and LOXHD1, genes expressed in the sensory portion of the cochlea  had pathogenic variants in –S5 had p56 at the time of ECochG testing  and a research patch provided by Cochlear Ltd, which allowed us to trigger a signal generator. The acoustic stimulus was a 500 Hz tone burst that was 12 ms long and was presented in both positive- and negative-leading polarities to the implanted ear via an ER-3A insert earphone at a rate of 10 Hz. Tonebursts were presented at the highest level that was still comfortable for the subject. Stimulation levels ranged from 100–110 dB peSPL. ECochG responses were recorded from the most apical electrode in the array. Each ECochG recording was an average of 200 stimulus presentations. Responses were analyzed in the frequency domain. More details regarding the recording and analysis procedures are provided in our previous studySupplementary Information


STRC gene have also been recognized as a major cause of genetic hearing loss. We investigated the frequency of STRC deletions in the Japanese population and the characteristics of associated hearing loss. For CNV analysis, we employed a specialized method of Ion AmpliSeqTM sequencing, and confirmed the CNV results via custom array comparative genomic hybridization. We identified 17 probands with STRC homozygous deletions. The prevalence of STRC homozygous deletions was 1.7% in the hearing loss population overall, and 4.3% among mild-to-moderate hearing loss patients. A 2.63% carrier deletion rate was identified in both the hearing loss and the control population with normal hearing. In conclusion, our results show that STRC deletions are the second most common cause of mild-to-moderate hearing loss after the GJB2 gene, which accounts for the majority of genetic hearing loss. The phenotype of hearing loss is congenital and appears to be moderate, and is most likely to be stable without deterioration even after the age of 50. The present study highlights the importance of the STRC gene as a major cause of mild-to-moderate hearing loss.Sensorineural hearing loss is a common deficit and mainly occurs due to genetic factors. Recently, copy number variants (CNVs) in the  Genetic factors account for 50–70% of SNHL. The major form of inheritance is autosomal recessive, which accounts for 75% of cases. Approximately 100 genes have been recognized as causative factors for SNHL, with the majority of causative alterations in the genes being single nucleotide variants (SNVs) or small insertions/deletions (indels). Recently, copy number variants (CNVs) have also been found to play an important role in many human diseases including neural developmental disorders3. CNVs; i.e., alterations through the deletion, insertion, or duplication of approximately 1 kb or more of a gene, are thought to affect gene expression, variation in phenotype, and adaptation via gene disruption, which may impact disease traits. More recently, CNVs have been recognized as a major cause of SNHL. Shearer et al. reported that CNVs were identified in 16 of 89 hearing loss-associated genes, with the STRC gene being the most common cause of SNHL4.Sensorineural hearing loss (SNHL) is common deficit at birth, affecting approximately 2 in 1,000 birthsSTRC gene is a known deafness-associated gene causing mild-to-moderate hearing loss, and is a part of a large deletion in chromosome 15q15.3 at the DFNB16 locus. The incidence of STRC deletions has been estimated to be between 1% and 5% in deaf populations in previous reports8. However, there have been only a few reports on hearing loss resulting from CNVs including those in the STRC gene. The interpretation of sequence data of the STRC gene is challenging due to the existence of the pseudo-STRC gene (pSTRC), which has 98% homology to the functional STRC gene. The pSTRC arose in a segmental duplication with other genes ; therefore, it is thought to be difficult to detect SNVs or CNVs in this region. Further, the clinical importance of the chromosomal deletion in the region harboring STRC to CATSPER2 is associated with not only hearing loss but also infertility in males, referred to as deafness infertility syndrome, due to the fact that CATSPER2 plays essential roles in sperm motility11.The 12. However, CNV analysis with the NGS dataset remains challenging, and it is thought to be difficult to detect CNVs accurately, especially in case of targeted NGS for hearing loss genes alone.Currently, massively parallel next generation sequencing (NGS) is being widely applied to genetic testing in clinical settings, and is able to allow precise diagnosis through data annotation with specific bioinformatics pipelines. In Japan, genetic testing for SNHL using NGS has been approved for coverage by social health insurance, and is currently being performed in clinical settingsSTRC gene, and confirmed their existence via high-resolution array genomic hybridization (aCGH) analysis of the entire STRC genomic region. The aims of the study were to estimate the prevalence of CNVs in the STRC region in a Japanese deaf population, and obtain a more precise characterization of the clinical features.In the present study, we used the NGS platform to analyze CNVs in the All procedures were approved by the Shinshu University Ethical Committee as well as the respective ethical committees of the other participating institutions . All methods were in accordance with the Shinshu University Ethical Committee for Human Genetic Research guidelines and regulations. Informed consent was obtained from all subjects or parents of the proband for participation in the study.STRC deletions in the normal hearing population, we examined 152 normal control individuals using the same CNV analysis with the NGS dataset as used for the hearing loss subjects. The age range of each individual was 20–30 years. Ear examination and pure-tone audiometry were performed and showed normal results.One thousand twenty-five  Japanese subjects  from unrelated and non-consanguineous families were identified from 67 otolaryngology clinics in 28 prefectures across Japan between February 2012 and October 2015. All subjects had presumed non-syndromic bilateral SNHL. Clinical information and blood samples were obtained for each proband and for all consenting affected and unaffected relatives. Collected data included (1) pure-tone audiograms, and behavioral audiometry or auditory brain stem responses (ABR); (2) medical history, including onset of hearing loss, and progression of hearing loss; and 3) temporal bone imaging (computed tomography and/or magnetic resonance) if done. Hearing levels were classified based on the better hearing ear as normal, <20 dB; mild hearing loss, 21–40 dB; moderate hearing loss, 41–70 dB; severe hearing loss, 71–95 dB; and profound hearing loss, >95 dB. To clarify the prevalence of TM Custom Panel , per with the manufacturer’s instructions, for 68 genes reported to cause non-syndromic hereditary HL . The sequence data were mapped against the human genome sequence (build GRCh37/ hg19) with a Torrent Mapping Alignment Program. After sequence mapping, the DNA variant regions were piled up using the Torrent Variant Caller plug-in software. After variant detection, their effects were analyzed using ANNOVAR software.Amplicon libraries were prepared using an Ion AmpliSeqTM sequencing that utilizes multiplex PCR-based targeted genome enrichment14. The depth of coverage information for each amplicon included in the barcode/amplicon coverage matrix file was used for copy number analysis. Normalization of coverage data depth was performed after considering the relative value of each amplicon. After normalization and removal of outlier data, the normalized relative read depths of amplicons were sorted based on chromosome position order and separated based on chromosome and gene identity, which were then plotted as a graph using conventional spread sheet software (Microsoft ExcelTM) , and probe intervals varied depend on the design platform due to highly homology of pseudogenes. We used the same DNA samples as used for amplicon resequencing, and quality assessment was also performed. Ten microliters of genomic DNA solution (0.5 µg of DNA) were fragmented, labeled with cyanine-3 for reference DNA samples and cyanine-5 for subjects, and then hybridized. Scanning of the array was carried out per the manufacturer’s recommended protocols. Scanned aCGH data were analyzed using CytoGenomics software version 3.0.6.6 (Agilent Technologies) , and in 3 of the 264 cases with autosomal dominant inheritance (1.14%). We identified duplications (3 copies) of STRC in 19 subjects (1.85%). It was unclear whether the 3 STRC copies were pathogenic or had any impact on phenotypes. We also found 27 subjects with STRC heterozygous deletions defined as carrier deletions. The frequency of carrier STRC deletions was 2.63%  in the hearing loss cohort, which was identical  to that in the normal hearing controls.As shown in Table GJB2 being the most commonly identified (39%) among all subjects across all forms of inheritance. CNVs implicated in homozygous deletions (2 copies) in STRC accounted for 5% (17/395) of all subjects  in the subjects with mild-to-moderate SNHL  and all had failed. Among the other individuals, hearing loss was identified at wellness checkups during elementary school years, or they became aware of hearing loss around the ages of 6 to 12. A distortion product otoacoustic emission  test has been undertaken by several individuals, and showed an absence of response indicating SNHL. The average hearing levels ranged from 28.8 to 60.8 dB, although the age at which the audiogram was taken varied  showed homozygous deletions in the STRC gene, not including the CATSPER2 gene.We performed array CGH for 17 probands in whom homozygous deletions in the STRC and CATSPER2 deletions detected using array CGH analysis.Supplementary Fig. STRC genomic region leading to SNHL in 17 of 1,025 Japanese deafness cases , and heterozygous deletions in 27 subjects (2.63%), indicating that the frequency of CNVs in this region was 4.29% (44/1025). Francey et al. studied 659 probands with bilateral SNHL and identified 7 homozygous and 10 heterozygous deletions in the STRC region using SNP genotyping array; among 10 probands with heterozygous deletions, SNVs or interstitial deletions were identified on the trans-allele in 4 probands, which were defined as compound heterozygotes of CNV and SNV resulting in hearing loss5. Shearer et al. identified causative mutations in STRC including homozygous deletions, gene conversions, and heterozygous deletions in-trans leading to missense changes, in 37 out of 686 hearing loss patients (5.4%) through analysis of NGS read data in CNV detection4. Vona et al. also reported homozygous or heterozygous STRC deletions as a cause of hearing loss in 9 probands among 94 SNHL probands using whole-genome aCGH7; among 9 probands with heterozygous deletions, SNVs were identified on the trans-allele in 4 probands. In the present study, 17 out of 27 STRC deletion carriers exhibited mild-to-moderate hearing loss. We inferred that 17 carriers might have a causative SNV on the other allele in the STRC, suggesting the presence of SNV and CNV as a compound heterozygote. A limitation of our study was that we focused on identification of CNVs alone and detected gene gains or deletions in the STRC genomic region; indeed, it was likely to be more challenging to detect SNVs due to low reliability of NGS read data of the region with highly homologous pseudogenes.In the present study, we identified homozygous deletions in the STRC region might vary among different populations, or the results could be influenced by the different CNV detection methods used such as SNP array or NGS. In the present study, we undertook CNV analysis using NGS read data as a first screening step followed by confirmation with aCGH, which is thought to be a robust method for CNV detection. Whole-genome array CGH designs are based on principles described in several publications and cover targeted regions including individual disease-related genes. For the STRC gene and its surrounding region, only a few probes lay across the whole-genome CGH array. We therefore designed a custom array CGH with a high density of probes for deafness-associated genes including the STRC genomic region. We found a false-positive result in only one sample in which CNV analysis with NGS read data showed a homozygous deletion, while aCGH revealed a normal copy number. Therefore, our present findings regarding the frequency of CNVs in the STRC region in the largest cohort studied to-date, while preliminary, are thought likely to be accurate for subjects with SNHL. We were able to identify gene deletions or duplications in the STRC region, but could not detect gene conversions or balanced rearrangement. Thus, the frequency of STRC variations causing SNHL is expected to be much higher if conversions could be detected.The prevalence of CNVs in the STRC deletions was 2.63% (4/152) in the normal hearing controls, which was identical  to that in the hearing loss cohort. The frequency of carrier mutations in the GJB2 gene, which is the most prevalent cause of autosomal recessive deafness, is reported to be approximately 2% in the normal hearing population16. Although the frequency of heterozygous GJB2 mutations is similar to that of heterozygous STRC deletions, the frequency of hearing loss due to homozygous STRC deletions is reported to be much lower than that due to homozygous/compound heterozygous GJB2 mutations. One possible reason for this discrepancy is that the detection method using NGS dataset and custom aCGH employed in this study was fundamentally challenging; however, the observed deletions were reliable. The basis for the relationship between carrier frequency and prevalence of hearing loss with STRC deletions is currently unclear.The frequency of carrier STRC deletions in 3 probands (17.6%) with autosomal dominant-appearing inheritance among the 17 probands showing all forms of inheritance, indicating pseudodominant inheritance. As shown in Fig. STRC, and segregation analysis via aCGH showed that her father (YSU5044) had a homozygous deletion and that her mother (YSU5045) was a heterozygous carrier. Due to higher carrier frequency of deletions in the STRC region , it was possible to identify homozygous deletions in STRC even in families showing autosomal dominant inheritance. On the other hand, in our cohort, mutations in the GJB2 gene, which is the most prevalent cause of autosomal recessive deafness, were found to follow pseudodominant inheritance in 15 (10.1%) of 149 probands showing all forms of inheritance. The carrier frequency of the SNVs in GJB2 was previously reported to be 6.7% in Japanese bilateral SNHL populations16.Interestingly, we found causative homozygous GJB2, SLC26A4, and CDH23, which cause severe-to-profound congenital SNHL17. Most of the mutations in these genes are SNVs or small insertions/deletions (indels). The major genetic causes of mild-to-moderate SNHL in recessive inheritance remain unclear. We showed that homozygous deletions in the STRC region were identified in 17 of 398 (4.3%) mild-to-moderate SNHL subjects, and our results suggested that STRC deletions were the second most common cause of mild-to-moderate SNHL after SNVs in GJB2.Approximately 70% of hereditary hearing loss patients exhibit autosomal recessive inheritance and are affected with severe-to-profound congenital SNHL. The major causative genes with recessive inheritance are STRC gene encodes the large extracellular structural protein stereocilin, which is expressed in the outer hair cells of the inner ear. Stereocilin is associated with the formation of tip link connectors between stereocilia and connections between outer hair cells and the tectorial membrane. Stereocilin-null mutant mice show progressive hearing loss, and their hair bundle stiffness appears decreased despite no apparent structural abnormalities20. In the present study, we observed that hearing loss did not progress to a severe level in the patients even after 50 years of age. SNHL appearing at birth is detectable via NHS; however, only 7 of 17 subjects were diagnosed with hearing loss through NHS. Indeed, NHS has been implemented since the year 2000 in Japan. NHS is not required by the law, and is provided based on the parents’ request. Therefore, we inferred that hearing loss could have been detected in all cases if NHS was universally applicable.The STRC gene and the pseudo-STRC (pSTRC) gene, which has 98% homology to the functional STRC gene. The pSTRC gene arose in a segmental duplication with other genes . CATSPER2 is involved in sperm motility, and is responsible for driving the hyperactivated motility that is essential for fertilization10. Zhang et al. reported three unrelated Iranian families with SNHL and male infertility caused by a long deletion involving four genes  at chromosome 15q15.39. In the present study, we found a homozygous long deletion involving the STRC and CATSPER2 genes in 15 of 17 cases (88.2%). Consequently, patients with a STRC deletion might also possess a CATSPER2 deletion that could be involved in hearing loss and male infertility. This is important information that should be shared during genetic counseling.One region of chromosome 15q15.3 contains the STRC using NGS read data from the platform used for the current social health insurance-based genetic testing in Japan as a first screening step, followed by confirmation using custom aCGH. We clarified the prevalence of causative STRC deletions in a Japanese population, and our data indicate that these were the second most common cause of mild-to-moderate hearing loss, which was congenital and non-progressive in the population.In summary, we performed CNV analysis in Supplementary information


Activation of cGMP phosphodiesterase (PDE) by activated transducin α subunit (Tα*) is a necessary step to generate a light response in vertebrate photoreceptors. PDE in rods is a heterotetramer composed of two catalytic subunits, PDEα and PDEβ, and two inhibitory PDEγ subunits, each binding to PDEα or PDEβ. Activation of PDE is achieved by relief of the inhibitory constraint of PDEγ on the catalytic subunit. In this activation mechanism, it is widely believed that Tα* binds to PDEγ still bound to the catalytic subunit, and removes or displaces PDEγ from the catalytic subunit. However, recent structural analysis showed that the binding of Tα* to PDEγ still bound to PDEα or PDEβ seems to be difficult because the binding site of PDEγ to PDEα or PDEβ overlaps with the binding site to Tα*. In the present study, we propose a novel activation mechanism of PDE, the trapping mechanism, in which Tα* activates PDE by trapping PDEγ released reversibly and spontaneously from the catalytic subunit. This mechanism well explains PDE activation by Tα* in solution. Our further analysis with this mechanism suggests that more effective PDE activation in disk membranes is highly dependent on the membrane environment. Briefly, after absorption of light, light-activated visual pigment catalyzes the exchange of GDP for GTP on the α subunit of transducin (Tα) to produce a GTP-bound active form of transducin (Tα*). Tα* then activates cGMP phosphodiesterase (PDE). PDE is a heterotetrameric protein composed of two catalytic subunits of similar amino acid sequence (PDEα and PDEβ showing >70% sequence identity) and two inhibitory subunits (PDEγ), and therefore is in the form of PDEαγβγ.  Each catalytic subunit has an active site to hydrolyze cGMP to GMP. Tα* binds to inhibitory PDEγ, and relieves its constraint on the active site in the catalytic subunit. This activation of PDE causes hydrolysis of cGMP, leads to closure of cGMP-gated cation channels situated in the plasma membrane of the outer segment, and induces a hyperpolarization of the cell.In the vertebrate photoreceptors, an enzymatic cascade, the phototransduction cascade, is responsible for generation of a light response4. However, this mechanism seems to be difficult based on the recent structural studies on the PDEγ·PDEα complex and the PDEγ·Tα* complex: most of the amino acid residues in the C-terminal region of PDEγ, from Asp-63 to Ile-87, are in contact with Tα*5, and almost the same region, from Leu-60 to Ile-87 in PDEγ, is in contact with the catalytic site of PDEα or PDEβ6. These observations suggest that PDEγ utilizes the same region to bind to Tα* and to the catalytic site of PDEα or PDEβ, and that Tα* and the catalytic subunit cannot bind to this region simultaneously.In the activation process of PDE by Tα*, it is widely believed that Tα* directly binds to PDEγ still bound to the catalytic subunit, and removes or displaces PDEγ from the active site of a catalytic subunit1 in Fig. 2) from the catalytic subunit to activate PDE.  In the novel mechanism . In the present study, therefore, we determined KD1 and KD2, and examined whether one can explain PDE activation at various concentrations of Tα* using an equation formulated for the trapping mechanism. The result reasonably explained PDE activation caused by addition of various concentrations of Tα* in solution.These considerations led us to examine a novel mechanism of PDE activation in vertebrate photoreceptors Fig. . In the ism Fig. , PDEγ is1 in Fig. To make sure that Tα* binds much more effectively to free PDEγ than to PDEγ still bound to PDEcat, we measured the binding of recombinant free PDEγ and that of purified PDE to Tα* with the Surface Plasmon Resonance (SPR) method. For this, we used the guanosine 5′-O-(γ-thio) triphosphate (GTPγS)-bound form of Tα (Tα-S*) as Tα*, and immobilized it on the surface of an SPR sensor chip as the common binding target of free PDEγ and PDEγ still bound to PDEcat. Figure 7. When these non-catalytic sites are empty, which is most likely with our purified PDE used, Tα* physically removes PDEγ from PDEcat upon activation8. Thus, on injection of PDE, we could expect that PDEcat is removed from PDEγ that has been associated with immobilized Tα-S* on the sensor chip. Then, we could expect that the binding signal of PDE is almost the same as that of recombinant PDEγ of the same concentration, which is not the case in Fig. 2 in Fig. We measured the binding signals using a running buffer that did not contain cGMP throughout our study. It is well known that PDEcat has one or two non-catalytic cGMP binding sitesThe binding signal of PDE in Fig. D1 and KD2 are the dissociation constants of the PDEγ·PDEcat complex and of the PDEγ·Tα* complex, respectively  membranes, we examined both cases in the followings.According to the reaction scheme shown in Fig. ely Fig. ; [PDEcatD1 of the PDEγ·PDEcat complex, first we measured the PDE activity using purified PDE at diluted low concentrations  and close to the reported value of <10 pM obtained with purified bovine PDE previously4. It should be mentioned here that freed PDEγ is completely freed from PDEcat. If freed PDEγ is removed from the active site but is still attached to PDEcat, dilution will not induce the increase in the relative PDE activity. It is because dilution does not affect re-binding of PDEγ to PDEcat in this case.To determine KD1 of the PDEγ·PDEcat complex in ROS membranes was 54 pM, and the range was 40–60 pM (broken curves for KD1 of 40 and 60 pM). Postulated elution of freed PDEγ in the dark in ROS membrane suspensions was examined with washing membranes, and the resultant increase in PDE activity was observed with repetitive washes  for the PDEγ·Tα-S* complex.To measure Kons Fig.  using thD2 was determined in the reversed configuration: PDEγ was immobilized and Tα-S* was perfused .  were ~8 times different (0.73 nM/5.6 nM = 1/7.7). Although immobilizations of Tα-S* and PDEγ were designed not to affect the binding site seriously (see Methods), immobilization seemed to affect KD2 slightly. We, therefore, concluded that KD2 is 0.73–5.6 nM, which is consistent with the values of 0.1–33 nM reported previously utilizing various methods for the measurement10.Obtained values of KD1 of the PDEγ·PDEcat complex for purified PDE . PDE activity at a given Tα-S* concentration is expressed as the % of the full PDE activity, and the summarized result is shown in Fig. D1 in solution  were used to measure the PDE activation at various concentrations of Tα-S*. The activity was measured similarly as in Fig. 12, in 20 μM rhodopsin membranes, we obtained almost a full PDE activity that is observed in trypsin-treated ROS membranes . Although KD2 values in 1.5 and 20 μM rhodopsin membranes  were not in the KD2 range we observed in Fig. 13 and PDE14 contents are 1/10 and 1/270, respectively, of that of rhodopsin. As a result, at 0.3 mM Tα* (full Tα activation) and 22.2 µM PDEcat, and with KD values in membranes (KD1 = 55 pM and KD2 = 56.5 pM), we obtained the result that 93% of PDEcat is activated in membranes. This high activation of PDE at these Tα* and PDEcat concentrations was not obtained with use of KD values we obtained in solution (KD1 = 5 pM and KD2 = 4.5 nM), and the PDEcat activation in this case was only ~11%. The result showed that PDE activation is different between in solution and in membranes, and that one cannot apply the KD values obtained in solution directly to the study on PDE activation in membranes.In Table 1 in Fig. 2 shown in Fig. D1, Fig. D2, Fig. In the activation mechanism of photoreceptor PDE, it has been generally believed that Tα* binds to PDE, and removes or displaces PDEγ from the catalytic site of PDEcat  molar ratio14 of 1/270: the molar ratio of 15% of Tα-S* to PDEcat in ROS membranes is ~2:1. It is possible that there could be two types of Tα*. One type binds to membranes tightly and the other loosely, and the latter re-binds to the membrane effectively when the membrane concentration is high. We speculate that the loosely-bound Tα* becomes soluble rather easily at low membrane concentrations to increase KD2 and to reduce the maximum PDE activation by reducing the effective Tα* concentration. At high membrane concentrations, this type of Tα* re-binds effectively to the membranes to contribute significantly to activate PDEcat and to lower KD2. In fact, Tα has been known to be differentially lipidated with 65% of unsaturated and 30% of saturated C12 or C14 fatty acids15.In the fitting of PDE activation in ROS membrane suspension, best-fitted KD1 and KD2, both determined experimentally  and KD2 is higher (4.2 nM) in solution than those in membranes, which would be the reason why PDE activation by Tα-S* is not so efficient in solution . This result seems to indicate that the trapping mechanism can be applied also to PDE activation in membranes.We further examined whether we can expect sufficiently large PDE activation at a level of a single photon response with the trapping mechanism. Using Eq.  and expeD2 in ROS membranes were not determined experimentally and further we are not sure how we can apply KD1 and KD2 values to the activation of PDE in membranes. Tα* and PDE in a disk membrane are undoubtedly situated at certain orientations on the disk membrane, which probably increases the chance of encounter of Tα* to PDEγ. Additionally, membrane proteins  are localized only in disk membranes while soluble proteins (Tα-S* and freed PDEγ) are in an aqueous phase in our measurement in ROS membrane suspensions. It is not certain whether we can appropriately apply Eq.  in this study were performed in accordance with the institutional guidelines and all experimental protocols were approved by Osaka University Graduate School of Frontier Biosciences . ROS membranes were prepared as described previously using a stepwise sucrose density gradient18. Obtained ROS membranes were frozen in liquid nitrogen and stored at −80 °C until use. To calibrate the concentration of ROS membranes, the amount of rhodopsin in an aliquot of the membranes was quantified spectrophotometrically with assuming that the molar absorption coefficient of frog rhodopsin is 40,000 M−1 cm−1 at 500 nm. All of these manipulations were carried out in complete darkness with the aid of an infrared image converter .All experiments with frogs , and a 0–1 M NaCl gradient in an elution buffer  containing 0.005% (v/v) Tween 20 was applied. Eluted fractions at 0.47–1 M NaCl containing purified PDE were concentrated using a Spin-X UF column . Then, the buffer containing purified PDE was changed to a potassium gluconate buffer , pH 7.5) containing 0.005% (v/v) Tween 20 using a Superdex 200 PC 10/300 GL column . The resultant purified PDE solution was concentrated using a Spin-X UF column, and stored at −80 °C until use. An aliquot of purified PDE was subjected to SDS-PAGE and the gels were stained with Oriole Fluorescent Gel Stain Kit (Bio-Rad) to assess the purity of PDE and also to quantify its amount using bovine serum albumin as a molar standard. Purity of PDE was almost 100% and the molar ratio of 2PDEγ/PDEαβ was 1.01 ± 0.01 .Crude PDE and crude Tα-S* were extracted basically as described previously19. Briefly, a Blue Sepharose 6 Fast Flow column  and a DEAE Sepharose Fast Flow column  were connected in tandem in this order for the purification. Then, a solution of crude Tα-S* supplemented with 2 mM MgCl2 was loaded on the column equilibrated with the elution buffer. The column was washed with the elution buffer sufficiently to remove unbound proteins, and then the Blue Sepharose column and the DEAE Sepharose column were separated: frog Tβγ bound to the Blue Sepharose column, and most of Tα-S* passed through this column and bound to the DEAE Sepharose column. Thus, Tα-S* bound to the DEAE Sepharose column was eluted using a 0–1 M NaCl gradient in the elution buffer. Tα-S* was then concentrated using a Spin-X UF column . The buffer was changed to K-gluc buffer containing 0.005% (v/v) Tween 20 using a Superdex 75 PC 3.2/30 column . Purified Tα-S* was stored at −80 °C until use. Purity and the concentration of Tα-S* were assessed with SDS-PAGE, and the purity was almost 100%. All of the manipulations for extraction and purification were performed at 4 °C.Tα-S* was purified from crude Tα-S* according to the method reported previously21. Purified recombinant PDEγ was lyophilized, dissolved in K-gluc buffer and stored at −80 °C until use.DNA sequence of frog PDEγ (GenBank Accession Number AB578858.1) was inserted into NdeI/BamHI sites of expression vector, pET-3a (Novagen). PDEγ was expressed in E. coli BL21(DE3) pLysS strain (Novagen) after induction with IPTG for 3 hr at 30 °C. Purification of expressed PDEγ was carried out based on the method described previously22, and therefore the binding to PDEγ. Frog Tα also contains 8 thiol groups (NM_001090561.1), and our Tα-S* showed almost a single component in the binding to PDEγ (see below).Proteins were immobilized on the SPR sensor tip according to the protocol described by Biacore. To immobilize Tα-S* on the sensor chip, Tα-S* was first biotinylated at its thiol groups. For this purpose, 3.3 μl of 2 mM EZ-Link Maleimide-PEG2-Biotin (Thermo Fisher Scientific) was added to 100 μl of 13 μM of purified Tα-S* in K-gluc buffer without DTT, and the mixture was incubated for 1 hr on ice. After the incubation, 0.35 μl of 1 M DTT was added to reduce and deactivate the non-reacted maleimide group of Maleimide-PEG2-Biotin. Then, the buffer was changed to K-gluc buffer to remove the deactivated Maleimide-PEG2-Biotin using a Zeba Spin Desalting Column (Thermo Fisher Scientific). Purified biotinylated Tα-S* was immobilized on a streptavidin (SA) sensor chip  through the streptavidin-biotin interaction. There are 8 thiol groups in bovine Tα (NCB Accession # NM_181022.2). However, it is known that only one group is chemically modified by N-ethylmaleimide in the GTP-bound form of Tα and that this modification does not affect PDE activationRecombinant PDEγ was immobilized at its lysine amino groups on a carboxymethylated dextran (CM5) sensor chip  using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Unreacted NHS-ester was blocked with ethanolamine after the immobilization. There are 8 lysine residues in PDEγ, and all of them are at the region outside of the major binding site of PDEγ to Tα-S* and to PDEcat, but four of them are near the possible binding site of PDEγ to Tα*and PDEcat. However, these four did not seem to affect the binding (see below).The binding of a protein to the immobilized protein was measured using Biacore X100  at 25 °C. The buffer used was K-gluc buffer containing 0.005% (v/v) Tween 20. Binding signals were stored and processed in Biacore X100. We used two ways to record the binding, one with binding and dissociation both terminated before their completion Figs  and the D2. The programs used are designed to include one of the crucial effects, mass transport effect (MTL)23. For the analysis of binding signals of PDEγ to immobilized Tα-S*, we used Heterogeneous Ligand with MTL program with assuming that there are at least two populations of Tα-S* immobilized differently depending on which thiol site was immobilized. However, our analysis indicated that the binding of PDEγ to immobilized Tα-S* consisted of only one major component (>98%). Unfortunately, the binding signal of Tα-S* to immobilized PDEγ was not analyzed with this program, and instead, 1:1 binding with MTL program was used. However, as shown in Fig. When necessary, the binding data were analyzed by BIAevaluation software  to determine KD1, PDE activity was measured using purified PDE or ROS membranes at various concentrations of PDE (≤40 nM), both in the light without GTP. The activity was measured with the pH assay method using a combination glass microelectrode  as described previously24. At time 0, 5 mM cGMP was added to initiate the hydrolysis. All measurements were performed at room temperature. To measure the full PDE activity, PDEγ bound to PDEcat was digested with trypsin  for 5 min at room temperature, and the digestion was terminated by adding trypsin inhibitor at a final concentration of 0.5 mg/ml. Then, the full PDE activity measurement was initiated with adding 5 mM cGMP.To determine K12. To estimate the concentration of transducin at different concentrations of ROS membranes, we assumed that molar ratio of transducin to rhodopsin13 is 1/10 . In both types of preparations, solution and membrane suspension, full PDE activity was measured after trypsin digestion as described previously12 to determine the relative PDE activity (% max).PDE activities at various concentrations of Tα-S* were measured with the pH assay method in a solution and in a ROS membrane suspension. In a solution, Tα-S* of known concentration was added to 15 nM purified PDE in K-gluc buffer in the light. At time 0, 5 mM cGMP  was added to initiate cGMP hydrolysis. In a ROS membrane suspension, first 5 mM cGMP was added to purified ROS membranes containing 1.5, 10 or 20 μM rhodopsin in the dark, and the membranes were illuminated to activate rhodopsin fully. Then, GTPγS of known concentration was added to the membranes to initiate the cGMP hydrolysis. In the measurement in ROS membrane suspensions, concentrations of GTPγS were set so as to limit the amount of Tα-S* by the amount of GTPγS addedsupplementary infomation


Social isolation is a critical public health issue that socially isolated individuals are at increased risk for mortality and deteriorated health. Those who acquire hearing loss in later life experience difficulties with communication, potentially leading to social isolation. However, less is known about the social consequences of age-related hearing loss, and few studies have assessed the influence of environmental factors on hearing loss and social isolation. The aims of this study are to examine: (1) the association between hearing loss and social isolation of older adults over time, and (2) the moderating effects of perceived neighborhood social cohesion and disorder on this relationship. We analyzed 2,080 community-dwelling Medicare beneficiaries aged 65 or above from Round 1 to 3 of National Health and Aging Trends Study. We conducted random coefficient models, entering hearing loss as a random coefficient. Older adults with hearing loss were less socially isolated than those without hearing loss. However, the effect of hearing loss on social isolation varied depending on perceived neighborhood social cohesion. Older adults with hearing loss who reported high neighborhood cohesion had significantly lower social isolation compared to those without hearing loss, while those with hearing loss who perceived low social cohesion had significantly higher social isolation. Our findings suggest neighborhood social cohesion can serve as a potential protective factor for older adults with hearing loss. This poster will propose neighborhood-level interventions that could supplement other services for those with hearing loss, such as assistive devices that are rarely covered by health insurance.


NF2 gene mutations. The knowledge of biomarkers associated with VS would be of significant value to maximize outcomes of hearing preservation in these patients.Vestibular schwannomas (VSs) are benign tumors composed of differentiated neoplastic Schwann cells. They can be classified into two groups: sporadic VS and those associated with neurofibromatosis type 2 (NF2). VSs usually grow slowly, initially causing unilateral sensorineural hearing loss (HL) and tinnitus. These tumors cause HL both due to compression of the auditory nerve or the labyrinthine artery and due to the secretion of different substances potentially toxic to the inner ear or the cochlear nerve. As more and more patients are diagnosed and need to be managed, we are more than ever in need of searching for biomarkers associated with these tumors. Owing to an unknown toxic substance generated by the tumor, HL in VS may be linked to a high protein amount of perilymph. Previous studies have identified perilymph proteins correlated with tumor-associated HL, including μ-Crystallin (CRYM), low density lipoprotein receptor-related protein 2 (LRP2), immunoglobulin (Ig) γ-4 chain C region, Ig κ-chain C region, complement C3, and immunoglobulin heavy constant γ 3. Besides, the presence of specific subtypes of heat shock protein 70 has been suggested to be associated with preservation of residual hearing. It has been recently demonstrated that chemokine receptor-4 (CXCR4) is overexpressed in sporadic VS as well as in NF2 tumors and that hearing disability and CXCR4 expression may be correlated. Further, the genetic profile of VS and its relationship with poor hearing has also been studied, including DNA methylation, deregulated genes, growth factors, and  Vestibular schwannomas (VSs), previously termed acoustic neuromas, are non-malignant tumors composed of Schwann cells of the vestibulocochlear nerve , arising from either the internal auditory canal (IAC) or the cerebellopontine angle (CPA). They can be classified into two groups: sporadic VS and those associated with neurofibromatosis type 2 (NF2) . Most VSThe mortality rate of VS ranges from 0.2 to 1% . Due to Diagnosis of VS may be either from cranial MRI performed for unrelated complaints or, usually, due to unilateral hearing loss or tinnitus , 9.Audiological tests including audiometry and auditory brainstem response are not reliable predictors of CPA pathology . As mostApproximately three of each of the four VSs exhibit no growth, leading to an observation strategy (wait and scan). Mean growth is about 2–4 mm/year in growing tumors . The heaAs more and more patients are diagnosed and need to be managed, we are more than ever in need of searching for biomarkers associated with these tumors, in order to help with the choice of selecting between a “wait-and-scan” approach and surgery  aimed atDue to the lack of biopsy sampling without the destruction of the organ, little is known about the cellular and molecular correlates in inner ear pathology .In recent years, considerable progress in proteomics has been enabled by modern technology. By a shotgun proteomics approach, the identification of proteins with high sensitivity is enabled . Mass spPerilymph, an extracellular fluid of the inner ear, is found within the scala tympani and vestibuli of the cochlea. During an apoptotic or necrotic episode inside the inner ear, the proteins that are secreted can be found at high concentrations in this fluid . The knoIn 2011, Lysaght et al. identified 15 proteins from perilymph specimens (selected by comparing VS and cochlear implant samples) with differential expression and biological function. They suggested the use of this list in future research focused on distinguishing between better vs. worse hearing in patients with VS see  (20).CRYM gene mutations cause autosomal-dominant hearing loss due to changes in the intracellular localization and the inability to bind to T3, which may lead to an altered K+ recycling  or nicotinamide adenine dinucleotide phosphate (NADPH)-regulated thyroid hormone binding protein is located within the cytoplasm, where it promotes transcription of the thyroid hormone triiodothyronine (T3) , 21. CRYecycling , 22.LRP2 gene (Low density lipoprotein receptor-related protein 2 (LRP2) or megalin is a trans-membrane receptor protein, which can be found in certain epithelial cells such as those of the ear. LRP2 has the ability to bind several ligands, being essential in the process of endocytosis of different elements such as sterols, lipoproteins, hormones, and vitamin binding proteins. Two well-known conditions, Donnai-Barrow and facio-oculo-acoustico-renal (FOAR) syndromes , both asRP2 gene .On the other hand, of the 91 commonly identified perilymph proteins of patients with VS on an individual level, Rasmussen et al. described four proteins that were significantly associated with tumor-related deafness: Immunoglobulin (Ig) γ-4 chain C region, Ig κ chain C region, complement C3, and immunoglobulin heavy constant γ 3. These 91 proteins were identified in 12 out of 15 samples they used in the study , 24.Moreover, alpha-2-HS-glycoprotein, a suggested inflammatory and immunological intermediary in perilymph, was suggested to be associated with deafness in patients with SVs. It was also discovered in samples from VS patients in 2017 , and altHeat shock proteins (HSPs) are stress proteins, which mediate cell survival under critical environmental conditions . IncreasHSP90 is the most important chaperone for cellular stress. It is involved in pathological processes, such as cancer development , and itsn = 18) experiencing complete loss of residual hearing loss after cochlear implantation, whereas only one of the patients with preserved residual hearing showed HSP90 in perilymph. The upregulation of HSP90 in the perilymph may therefore induce the migration of macrophages and leukocytes, resulting in cochlear inflammation. However, despite the cellular changes observed, the authors could not detect a significant difference in HSP90 expression in patients with VS compared with patients without tumor  MRI has been reported in various diseases, including SNHL, and VS –33. An iKim et al. demonstrated a correlation between a higher cochlear signal on 3D FLAIR images and hearing loss in patients with VS . The corIpsilateral inner ear alterations, including endolymphatic hydrops (EH) and acidophilic-staining precipitate, have been observed in temporal bone histopathological studies from patients with VS , 39. In A large number of cytokines are produced by tumors  includinChemokine receptor-4 (CXCR4) is implicated in several pathological processes, including autoimmune disease, infection, and tumor development , 51. CXCNF2 gene in VS (C-X-C Motif Chemokine Ligand 12 (CXCL12), a ligand for CXCR4, cooperates with metastatic cells to CXCL12-expressing organs. The Ras/Raf/MEK and the PI3K/Akt/mTOR pathways can be activated by CXCL12 binding to the CXCR4 receptor. In a similar way, the loss of Merlin , 53, a tne in VS , leads tRecently, Breun et al. have described that CXCR4 could play a role in the pathogenesis of both sporadic and NF2-associated VS. In their study CXCR4 was overexpressed in these tumors, with no significant differences found between the two groups. CXCR4 mRNA expression increased with the degree of hearing loss when compared with the control group, with the results lacking statistical difference .Although tumor extension may be related to hearing impairment in VS , there iNF2 gene mitotic recombination  = 43 and 17 dB for patients with methylated and unmethylated TP73 genes, respectively]. A genome-wide methylation analysis in VS also showed a trend toward hypomethylation in several miRNAs and coding genes, including alternative transcripts, opening a window to possible therapeutic targets  protein were overexpressed in VS patients with poor hearing . They hypothesized that a younger age of onset of VS could explain the younger age of onset of hearing loss (and tinnitus) .Halliday et al. proposed a genetic severity score  in order to predict morbidity for NF2 subjects in certain dimensions including hearing status . AccordiNF2 gene mutation have also been related to hearing loss in subjects with VS. Most of these potential markers of hearing loss are not routinely available for the clinician. On the other hand, recent findings on imaging, especially delayed intravenous gadolinium-enhanced high-resolution MRI, and 3D non-contrast heavily T2-weighted sequences are promising in terms of therapeutic management of patients with VS showing signs of EH. The precise knowledge of biomarkers associated with hearing loss in patients with VS would be useful to minimize morbidity and to maximize outcomes of hearing in these patients.So far, no reliable methods are able to predict the evolution of hearing loss in subjects with VS. Several markers such as perilymph proteins have been associated with tumor hearing loss. Also, specific subtypes of HSP70 have been correlated with hearing outcomes. Cytokines produced by VS, especially CXCR4 expression, have been related to hearing impairment. DNA methylation, deregulated genes, growth factors, and All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In July 2018 the active transcutaneous bone conduction hearing implant received FDA approval in the US (for patients 12 years and older with conductive and/or mixed hearing loss or single-sided deafness), reflecting the current trend of moving away from percutaneous hearing solutions towards intact skin systems.To critically assess the current literature on safety, efficacy and subjective benefit after implantation with an active transcutaneous bone conduction hearing device.Literature investigation was performed by electronic database search including PubMed and Cochrane Central Register of Controlled Trials, and manual search of relevant journals and reference lists of included studies.Randomized controlled trials, clinical controlled trials and cohort studies, case series and case reports investigating subjective and objective outcomes.Retrieved literature was screened and extracted by two reviewers independently. Subgroup analysis of indications  and participant ages (pediatric vs. adults) was conducted on patients with active transcutaneous bone conduction devices. Sensitivity analysis was performed to test the stability of the results in meta-analysis.39 citations reporting on pre- and postoperative audiological results, speech performance in quiet and in noise, localization testing as well as subjective outcomes were included in this systematic review. Functional gain as well as word recognition score outcomes could be further investigated via meta-analysis. All outcomes reported and summarized here reflect beneficial audiological performance and high patient satisfaction, accompanied with a low complications rate  for the indications of conductive and mixed hearing loss as well as in individuals suffering from single-sided deafness for all age groups of subjects who underwent active transcutaneous bone conduction hearing device implantation.A limiting factor of this systematic review was the Level of Evidence of the reviewed literature, comprising 2a/3a studies (cohort studies and case-control studies). Furthermore, the reporting standards, especially in outcomes such as word recognition scores in quiet and in noise, vary across study cites from various countries, which impedes comparisons. Last but not least, no other comparable other device was retrieved as the active transcutaneous bone conduction hearing device is the only available at the moment.The device’s transcutaneous technology results in a minor event incidence rate of one in 9.9 person-years and a major incidence rate of one in 148.9 person-years. Based on the audiological outcomes, high patient satisfaction as well as the low complication rate, the authors recommend the active transcutaneous bone conduction hearing device as a safe and effective treatment for patients suffering from hearing loss within the device’s indication criteria (conductive and/or mixed hearing loss or single-sided deafness). Population—Subjects of any age, gender or ethnicity. Intervention—Implantation of the active transcutaneous bone conduction implant by either surgical approach. Comparators—n/a. Outcomes—Data regarding safety, efficacy, quality of life and subjective outcomes with the device. Efficacy outcomes were divided into audiological/performance outcomes, including preoperative and postoperative hearing thresholds, functional gain, speech perception in quiet and noise, speech recognition thresholds, sound localisation; and subjective outcomes determined by questionnaires, patient-oriented scales of improvement and satisfaction scales. Study design—All study designs were included. Letters, editorials and systematic reviews with no original data, animal, in-vitro and laboratory studies were excluded.In 2018, the WHO reported that around 466 million people worldwide (34 million children) suffer from disabling hearing loss, defined as hearing loss greater than 40dB in the better hearing ear in adults and 30dB in the better hearing ear in children. Advances in medicine and technology have led to many new treatment options for all different types as well as severities of hearing loss and include hearing aids, medical intervention via prostheses and surgically implanted medical devices such as cochlear, middle ear, or, as currently reviewed, bone conduction implants. While the majority of patients with moderate to severe hearing loss can be supplied with conventional hearing aids, some patients either do not benefit enough from hearing aids or cannot wear them due to anatomical or skin-related issues. In these cases, implantable hearing devices fill a clinical need and active transcutaneous bone conduction implants (atBCI) may serve as a valuable solution for adults and children with moderate to severe conductive (CHL) and mixed hearing loss (MHL), as well as those affected by single-sided deafness (SSD). Surgical and technical details as well as information about the device’s indications have been previously published by the authors .The meta-analysis for 30 CHL subjects revealed a weighted functional gain of 39.48 dB SPL –21Fig 3Fig 3.Investigating the mixed hearing loss group (C/MHL), the mean FG resulted in 29.08 dB SPL (n = 58), 18, 13.In the outcomes reporting on 10 subjects with SSD, the average weighted functional gain was 28.94 dB SPL  , 10, 18 Speech tests used included the Italian bisyllables, Freiburger monosyllables, Mainzer monosyllables, Göttinger Kindersprachtest, Dantale (Danish), Oldenburg sentence test, dissyllabic Fournier and Spanish bisyllables words and numbers lists. The preoperative and postoperative results can be seen in the The meta-analysis outcomes for reported mean word recognition scores including SD at 65 dB SPL in the CHL group resulted in an improvement of 56.73% (n = 57)(F/U range: 2–5 months)  26, 6, , 39. OutSpeech in noise tests used included the QuickSIN and BKB-SIN with four-talker babble noise, Italian bisyllables, Freiburger monosyllables, Dantale II (Danish), Oldenburg sentence test, words and numbers lists. The preoperative and postoperative results reported can be seen in the Results for speech in noise tests could not be compared directly in a meta-analysis due to the individual test set-up and inconsistency in reporting for the different studies. However, an improvement in speech understanding in noise was observed with the atBCI in all studies for subjects with CHL or MHL  23, 8, , 38, 21.Mean aided signal-to-noise ratio (SNR) values in a total of 54 individuals with CHL and MHL were found to range from +2.9 dB to -6.1 dB SNR, compared to + 11.5 to -3.8 SNR unaided , 42, 43.For single-sided deaf subjects, an improvement in speech understanding in noise was observed with the atBCI , 12, 13.Sound localization ability is usually investigated with white noise presented at a level of 65 dB SPL from certain, usually randomized angles (α) of -90°, -45°, 0°, 45°, or 90°. The ability to localize sound is then calculated as the angle detection error , which is the difference, Δαi, between the actual angle and the detected angle , 33, 38.Sixteen publications with 215 subjects reported on subjective outcomes after atBC implantation  27, 7, , 38, 21.The Abbreviated Profile of Hearing Aid Benefit (APHAB) was the most frequently used questionnaire and was administered in seven publications evaluating 71 subjects , 13, 21.The Glasgow Benefit Inventory (GBI) was reported in five studies in 47 subjects , 44, 13.A modified BBSS (Bern Benefit in Single-Sided Deafness) questionnaire was also used in two studies to evaluate the subjective outcome in conductive and mixed hearing loss patients (n = 18), 43. On Outcomes in children (subjects 18 years or younger) were reported in six publications. For children with CHL or MHL the average functional gain was 34 dB for 77 implantations , 20, 14.Baumgartner et al. reported a significant improvement in warble tone thresholds from preoperative testing to 3-month postoperative testing . PreoperVyskocil et al. reported on two pediatric patients (14 and 17 years old) suffering from conductive hearing loss due to microtia and atresia . Mean WRSubjective outcomes in children were reported by Baumgartner et al., investigating the Hearing Device Satisfaction Scale (HDSS) in children aged 5 to 17 years (n = 12) . OutcomeSafety of the device was assessed by collecting information on complications during surgery and adverse events in the postoperative period  Tables. Out of 286 ears under investigation, 259 reported no complications (90.6%). Minor complications in 22 ears resulted in a 7.7% rate over a cumulative period of reported mean follow-up of 12.7 years (mean: 11.7 months ± 4.5) , 14, 46.Substantial and stable benefit for patients with C/MHL and SSD who underwent active transcutaneous bone conduction device implantation was shown in 39 citations. Benefit was defined in terms of hearing thresholds and speech recognition in quiet, as well as speech discrimination and functional gain. Averaging the studies reporting all indication groups, a weighted functional gain of 31 dB could be achieved (C/MHL/SSD). A mean weighted benefit in word recognition score at 65dB of 52.1% was found with the atBCI for all subject groups (C/MHL/SSD), with the SSD group still performing well but not as high as the other hearing loss types (38.3% WRS improvement). The conductive hearing loss group benefitted the most, with a score of 56.7%. Speech understanding in noise also improved significantly. The comparison with other, non-transcutaneous and non-active devices has shown similar results in quiet and noise with a ceiling effect, although their functional gains are different in the low and high frequencies .The device’s transcutaneous technology avoids several complications found in percutaneous bone conduction implants including skin reaction, growth of skin over the abutment, implant extrusion, and wound infection , 34, 46.The currently reviewed device is the only available active transcutaneous system. Other active bone conduction devices that utilize a percutaneous screw still have to battle high, especially skin-related complication rates. This led to the development of bone-anchored systems that use transcutaneous magnetic coupling to enable sound conduction (screw-based transcutaneous device). Despite being a closed-skin option that aims to avoid skin complications, reports of skin irritations, like edema and erythema and even necrosis seem to be a common occurrence –61. ImplNonetheless, the limitations of this systematic review need to be emphasized in regard to the reporting standard, especially in outcomes such as word recognition scores in quiet and in noise. Test apparatus and language varies across study sites from various countries and may impede outcome comparisons. Furthermore, the Level of Evidence of the reviewed literature, comprising 2a/3a studies (cohort studies and case-control studies) reduces the strength of outcome variables. For this reason, a meta-analysis, was performed to overcome this, when possible. In addition, no other active transcutaneous bone conduction hearing implant is available at the moment and therefore no comparative evidence could be extracted. Last but not least, long term outcomes remain unknown and must be further investigated to check the maintenance of functional gain / WRS as well as potential future long-term complications.The atBCI is very well tolerated up to 36 months following implantation, improves audiometric thresholds and intelligibility for speech in quiet and noise, and gives satisfaction to patients with mixed and conductive hearing loss as well as those with SSD. The improved audiological benefit with the active transcutaneous bone conduction implant reviewed here is furthermore reflected in the high levels of subjective satisfaction reported by users via several questionnaires  and the remarkably low complication rate.To extend the evaluation of the atBCI, more data and an even longer follow-up is required.The only active transcutaneous bone conduction technology presented here avoids several well-known complications of the percutaneous bone conduction implants. The complication rate reported was low, with one minor event in 9.9 person-years. The low overall incidence rate with the atBCI is also reflected in the ‘severity of events’, with the remarkably low major incidence rate of one in 148.9 person-years. Based on the reviewed outcomes it can furthermore be concluded that the Bonebridge is an effective solution for adults and children suffering from conductive and/or mixed hearing loss as well as single-sided deaf subjects, with the advantage of an intact skin condition after implantation.Based on the audiological outcomes, high patient satisfaction and low complication rates, the authors conclude that this active transcutaneous bone conduction implant is a safe and effective treatment for patients suffering from hearing loss within the device’s indication criteria.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.


Shiraia fruiting body, which was used clinically on various skin diseases and developed as a photodynamic therapy agent against cancers. The fruiting bodies may harbor a diverse but poorly understood microbial community. In this study, we characterized the bacterial community of Shiraia fruiting body using a combination of culture-based method and Illumina high-throughput sequencing, and tested the involvement of some companion bacteria in fungal HA production using the fungal–bacterial confrontation assay. Our results revealed that the bacterial community in the fruiting body was dominated by Bacillus and Pseudomonas. Some Pseudomonas isolates such as P. fulva, P. putida, and P. parafulva could stimulate fungal HA accumulation by Shiraia sp. S9. The bacterial treatment of P. fulva SB1 up-regulated the expression of polyketide synthase (PKS) for HA biosynthesis and transporter genes including ATP-binding cassette (ABC) and major facilitator superfamily transporter (MFS) for HA exudation. After the addition of live P. fulva SB1, the mycelium cultures of Shiraia sp. S9 presented a higher HA production (225.34 mg/L), about 3.25-fold over the mono-culture. On the other hand, B. cereus was capable of alleviating fungal self-toxicity from HA via down-regulation of HA biosynthetic genes or possible biodegradation on HA. To our knowledge, this is the first report on the diversified species of bacteria associated with Shiraia fruiting bodies and the regulation roles of the companion bacteria on fungal HA biosynthesis. Furthermore, the bacterial co-culture provided a good strategy for the enhanced HA production by Shiraia.Hypocrellin A (HA) is a natural red perylenequinone pigment from  Shiraia bambusicola P. Hennings is a pathogenic fungus of bamboos, and its fruiting body has been used in traditional Chinese medicine for the treatment of vitiligo, stomachache, psoriasis, and rheumatic pain  twigs at their intermediate developing stage from June to July 2016 in Tianmu mountain of Hangzhou, China, and collected from three plots (approximately 100 m2/plot). Each sample contained 30 fruiting bodies. All samples were immediately stored in sterilized sealed bags on ice. In order to observe the structural features of the fruiting body, the samples were cut into thin slices and observed with a light microscope . The paraffin wax sections of fruiting bodies were made according to the method reported by The fruiting bodies were picked from the bamboo (2 (w/v) for 3 min and washed with sterile water. Fungi derived from the fruiting body were obtained according to the method described by Shiraia sp. through the observation of its morphology as well as the ITS rDNA sequence (GenBank Accession No. MF062656.1) (see section “Results”). The strain has been deposited in China General Microbiological Culture Collection Center (CGMCC) with the deposition number CGMCC16369 and was cultured routinely and stored at 4°C on PDA medium.Fresh fruiting bodies were sterilized by immersion in 0.1% HgClThe extraction and determination of HA were based on the methods described in our previous report . HA contThe bacteria were isolated from 15 fruiting bodies (5 fruiting bodies per replicate) by using surface sterilization method . The int2O). The reaction was performed under the following conditions: 3 min at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at 56°C, and 1 min at 72°C by using T-100 thermal cycler . Sequencing of the amplified product was carried out by Sangon Biotech, Co., Ltd. . The phylogenetic tree of bacteria was constructed by using phylogeny server1 and the neighbor-joining method.Total DNA of bacteria were extracted from the overnight bacteria culture, and DNA integrity and purity were confirmed using the Agilent 2100 Bioanalyzer . The 16S rDNA was amplified using the conserved bacterium-specific primers, 27F (5′-AGAGTTTGATCATGGCTCAG-3′)/1492R (5′-TACG GCTACCTTGTTACGACTT-3′) . The PCRThe essential biochemical characteristics including Gram character, glucose utilization, starch hydrolysis, citrate utilization, nitrate reduction, oxidase, catalase, and gelatin liquefaction of bacteria have been analyzed and tested .2O. All reactions were performed under the following conditions: 3 min at 95°C, followed by 32 cycles of 30 s at 95°C, 30 s at 56°C, and 15 s at 72°C. A final 5-min extension step at 72°C was also performed. The PCR products were confirmed using 2% (w/v) agarose gel. The DNA band with the correct size was excised and purified using a PCR Clean-up System . After purification, the PCR products of 16S rDNA V3–V5 regions were quantified by Pico Green analysis . The libraries were then sequenced using Illumina MiSeq platform   .Raw sequence data (NCBI Accession No. PRJNA541247) were sorted based on sample-specific barcode tags and then the primers and tag sequences were trimmed from sorted sequences. The resultant sequences were processed initially using the Trimmomatic  and FLASin vitro confrontation bioassay between the fungus S9 and bacteria was conducted using the reported method by An Shiraia sp. S9 in the mycelium cultures. A small piece (5 mm × 5 mm) of the strain from PDA plate was dug and transferred into a 150-ml Erlenmeyer flask containing 50-ml PDB broth at 28°C on a rotary shaker at 150 rpm for 4 days. The single colony (approximately 2 mm diameter) of bacterium was inoculated in LB broth (without agar) at 37°C on a rotary shaker at 200 rpm for 12 h. Then, bacterial cells were added into the cultivation medium of Shiraia at a final density of 100 cells/ml. After 8 days of co-cultures at 28°C on a rotary shaker at 150 rpm, the mycelia were harvested and dried to constant weight in a 60°C oven to evaluate the fungal biomass and HA contents.The co-culture was established between the live bacterium and 5 spores/ml) in a 1.5-ml reaction tube, which was cultured in the dark on a rotary shaker at 150 rpm and 28°C for 8 days. To further analyze the bacterial effect on the MIC value of HA, bacterial cells (400 μl of 0.75 × 103 cells/ml) were added to the test system. To test the bacterial degradation of HA, the bacterial cells (400 μl of 0.75 × 103 cells/ml) in LB mixed with 60 mg/L HA in a 5-ml reaction tube were incubated in the dark on a rotary shaker at 150 rpm and 28°C according to the description of 3 cells/ml) were incubated in LB with HA at 0–100 mg/L in a 5-ml reaction tube on a rotary shaker at 150 rpm and 28°C for 24 h in the dark. The bacterial cell density was measured using a UV-VIS spectrophotometer  at 600 nm.HA  was dissolved in absolute ethanol at 1 g/L as stock solution. The minimum inhibitory concentration (MIC) of HA on fungal growth was determined by broth dilution susceptibility testing method . HA at 0After 24-h co-culture with live bacteria (SB1 and No. 1), total RNA of the fungal mycelia was extracted using RNAprep pure Plant Kit . The primers of target genes  for HA bt-test and one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison tests were applied for experimental results. All results were expressed as mean ± standard deviation (SD), with p < 0.05 being considered statistically significant.All treatments consisted of triplicate independent repeats (10 plates or flasks per replicate). Student’s S. bambusicola presented irregular pinkish tubercles surrounding branches of the host bamboo  was initially classified as Shiraia sp. based on hit taxon strain with the ITS rDNA sequence data in the Basic Local Alignment Search Tool (BLAST) server3 compared to Shiraia sp. SUPER-H168 (GenBank AccessionNo. EU267793.1) with a similarity of 94%. A phylogenetic tree of S9 based on ITS rDNA sequences is shown in Shiraia.Six red pigment-producing fungi were isolated from the fruiting body, and the strain S9 was chosen on the basis of higher hypocrellin yield on PDA plate . The mycuid 4 in . Simultauid 4 in   and Pseudomonas (approximately 20%). Bacillus spp. (14 strains) showed the average similarities to B. cereus (96–98%), B. tequilensis (97%), B. subtilis (97%), B. safensis (98%), and B. anthracis (97%). In addition to the Bacillus isolates, six bacterial strains were identified as Pseudomonas spp., displaying high similarities to the species of P. putida (96–97%), P. fulva (97–99%), and P. parafulva (96%). Three bacterial isolates , Paenibacillus (5.85%), Pseudomonas (4.37%), Sphingomonas (3.75%), Janthinobacterium (2.89%), Alkaliphilus (1.78%), and Luteibacter (1.67%) at the genus level, which were partly consistent with those found in fruiting body by using culture-dependent approach , P. fulva (No. 11), and P. parafulva (No. 20) exhibited the capacity to stimulate the secretion of red pigments from the mycelium  is shown in Aeromonas hydrophila (No. 13) and Microbacterium oxydans (No. 28) have no obvious effects on pigment secretion, while other Bacillus isolates suppressed the accumulation of pigment remarkably  in medium (Due to the limitation of the wild sources of the fruiting bodies of oduction . In the oduction . The hypoduction . The livbviously . In submbviously . It was n medium .PKS), O-methyltransferase (Omef), monooxygenase (Mono), multicopper oxidase (MCO), FAD/FMN-dependent oxidoreductase (FAD), zinc finger transcription factor (ZFIF), major facilitator superfamily (MFS), and ATP-binding cassette transporter (ABC) were analyzed by using qRT-PCR after 24 h of the co-culture. The expressions of all tested genes except FAD gene were up-regulated by SB1 in the co-culture, about 5.49-, 1.88-, 2.18-, 2.53-, 3.78-, 3.41-, and 1.95-fold of the mono-culture control, respectively , Omef (2.43-fold), MCO (2.09-fold), ZFIF (4.34-fold), and ABC (1.81-fold).To investigate the regulation mechanism of live bacterium on HA biosynthesis, some HA biosynthesis-related genes  includinectively . On the P. fulva SB1 were applied to the cultures of fungus S9. The lower concentration at a density of 100–400 cells/ml showed no retardation on fungal growth but promotion on HA biosynthesis  of bacterial ynthesis . The maxynthesis . The relynthesis  and thenynthesis  was simiynthesis . When SBynthesis . The higynthesis . Total HShiraia growth was observed , followed by Pseudomonas (20%) and Staphylococcus species (10%). On the other hand, Illumina sequences provided more rapid and discriminatory analysis on the bacterial communities including 723 bacterial OTUs that belonged to 30 bacterial phyla, 84 classes, 149 orders, 244 families, and 364 genera (Shiraia fruiting bodies have high abundance taxa of Proteobacteria and Firmicutes (Bacillus and Pseudomonas with higher frequency based on both methods (culture-based and Illumina high-throughput sequencing), whereas members of Paenibacillus and Sphingomonas were common in high-throughput sequencing data but rare in cultures, indicating a method-driven discrepancy   of g bodies . Differeg bodies . Among t4 genera . Our resrmicutes , S9. Allcrepancy  or the pcrepancy . The domal fungi . The mosectively . Althougd roots) . Our resd roots)  and the d roots) . On the adiation . The stradiation . Howeveracterium . Our resulva SB1 . The posShiraia fruiting body. Despite many bacterial isolates from various fruiting bodies (Pseudomonas strains (DJ35 and DY22) from fruiting body of A. bisporus were reported to be potent mushroom growth-promoting bacteria due to their production of indole acetic acid (IAA) and cellulase  into thiophene derivative, the principal ingredient of truffle aroma  for membrane damage and cell death of host plant cells  in the solid cultures  of the control . The addition of the same strain induced the production of new pimarane diterpenoids, libertellenones A–D in cultures of the marine-derived fungus Libertella sp. (Fusarium tricinctum with the bacterium B. subtilis 168 trpC2 (Since HA has great potential in PDT for cancers and skin diseases , extensicultures  and aboucultures . Thus, mcultures , elicitacultures , and molcultures . In our cultures , the higcultures . In the  control . Bacteriella sp. . An enha68 trpC2 . Compare68 trpC2 , bacteri68 trpC2 , suggestS. bambusicola by using culture-dependent and -independent approaches. The data revealed that the predominant bacteria are Bacillus and Pseudomonas. It is an interesting finding that the associated bacteria from some Pseudomonas strains exhibited the capacity to stimulate HA biosynthesis in host Shiraia fungus, whereas some Bacillus strains suppressed fungal HA production significantly. These results revealed some physiological roles of the associated bacteria on the regulation of metabolites of host fungus in the fruiting bodies. To our best knowledge, this is the first report to show that live bacterium could regulate the biosynthesis of hypocrellins in Shiraia. Although the mechanism of the elicitation or inhibition on HA biosynthesis needs further investigation, the present study successfully provided a novel co-culture method for HA production. With the optimization of co-culture conditions or using the combined eliciting strategies, we believe that HA production in a large-scale culture of Shiraia could be improved greatly.In summary, this study presented the first assessment of the diversity of bacteria communities inhabiting in the fruiting body of JW and YM were the recipients of funds, conceived the experiments, and prepared the manuscript. YM and LZ undertook experiments and data analysis. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.