Hearing impairment was defined as equal to or higher than 20 dB HL. Results of the hearing thresholds were separately reported in the left or right ears and better or worse ears. Logistic regression tests were used to investigate the association between hearing loss and possible risk factors. In this study, all the analyses were conducted using SPSS software (version 21).The present study was carried out on a total of 2019 children. Mean age of the participants was reported as 9.66±1.66 years. Based on low-frequency pure-tone average, the prevalence rates of hearing loss > 15 dB in the right and left ears were reported as 1.94% and 1.68%, respectively. The high-frequency hearing loss > 15 dB in the right and left ears was obtained at 1.14% and 1.04%, respectively. Prevalence rate of hearing loss  in boys was higher than that in girls. There was a strong association between a history of otitis media and sensorineural or conductive hearing loss .In this study, the rate of hearing loss in the participants was approximately 2%. It was concluded that the screening of hearing loss in children is necessary for the identification and management of these children as early as possible. It is recommended to perform further trials to investigate the impact of different causes on childhood hearing impairment. Hearing impairment has adverse effects on a child academic performance, as well as development of appropriate speech, language, and psychosocial abilities . Any typThe hearing screening programs have been used as essential tools to find hearing-impaired children in developed countries. Routine screening of hearing impairment in school-age children should be considered in low- and middle-income countries, as it can be more readily implemented than universal neonatal screening . In chilThe World Health Organization (WHO) estimates the prevalence of mild hearing impairment in the region of the Middle East and North Africa region at 4.5%  and 2.8%  for boys and girls within the age range of 5-14 years, respectively. Corresponding prevalence rates ≥ moderate hearing impairment are reported as 0.8% and 0.5% for boys and girls, respectively . Primary objective of this program is to screen hearing impairment in 100% of neonates in the country. Another focus of the program is to increase the coverage of hearing screening for children within the age range of 3-6 years by 30% in the country . Data onSarafraz et al.  evaluateHajilo et al.  assessedThis cross-sectional study was designed to evaluate the prevalence of hearing loss among the boys and girls of elementary school-age in urban areas in Rasht, northern Iran. Rasht is the capital of Guilan province in Iran with a population of 920,000. The study protocol was approved by the Review Boards of Guilan University of Medical Sciences, Guilan, Iran,  and complied with the principles outlined in the Helsinki Declaration. Sampling was performed using multistage random cluster sampling method. Firstly, 15 schools were randomly selected from a list of elementary schools in the urban areas of Rasht. Then, sampling occurred in classes randomly chosen from each grade in the selected schools. A total of 2100 students were selected from all 6- to 13-year-old children during January and April 2015. Informed consent was obtained from the parents of the participants. Overall, 81 parents would not like to participate in this study; therefore, 2019 children  were included in data analysis. Number of boys and girls participated in this study was proportional to the number of schools for boys and girls.Screening for hearing impairment was conducted by an expert audiologist. A structured questionnaire was distributed to the parents for the determination of the medical and family history of the children and evaluation of their socioeconomic levels. A general medical examination was carried out by two trained interviewers. The ear canals were examined with an otoscope. Foreign bodies, debris, and impacted cerumen were removed. Any children who were diagnosed with acute chronic otitis media or otitis media with effusion were not excluded from the study. The quietest areas in each school with the noise level lower than 45 dB were chosen to perform audiometric tests. A duly calibrated pure-tone audiometer was used with TDH-39 earphones and Audiocups for extra attenuation. The children who failed were referred to Amiralmomenin hospital for otorhinolaryngologic assessment and comprehensive audiometric evaluation. The failure was considered a pure-tone average > 15 dB HL at frequencies 0.5-4.0 kHz. The hearing threshold level was calculated by the average of the frequencies of 500, 1000, 2000, and 4000 Hz. Pure-tone average (PTA) was classified into low-frequency pure-tone average (LPTA) and high-frequency pure-tone average (HPTA), which was the mean hearing threshold within the frequency ranges of 0.5-2 and 4-8 kHz, respectively. As shown in Statistical analysisUsing the expected prevalence of hearing impairment of 9.8% , error mThe results were also reported from subgroup analyses. The data were analyzed using SPSS software (version 21)..A total of 2019 school-age children participated in the present study. Mean age of the participants was reported as 9.66±1.66 years (age range: 6-13 years). In addition, 1046 (51.8%) and 973 (48.2%) subjects were boys and girls, respectively. Most of the children were between 8 and 12 years (86.8%). Both ears of all the cases were screened; therefore, 4038 ears were assessed in this study. Among the children, 42 subjects failed in one or both ears and were referred for comprehensive diagnostic audiometry. Moreover, 27  cases were diagnosed with a hearing loss, which indicated the prevalence rate of hearing loss as 1.2%. The obtained results revealed a lower prevalence of hearing loss in the youngest age group, compared to that of the older groups . There was a positive history of neonatal icterus in 82 children (4.5%). In addition, 20 (1%) subjects were reported with a family history of hearing loss . None of The most common type of hearing loss, unilateral or bilateral, was conductive hearing loss . The most common possible cause identified for ear examination was otitis media with effusion (57.9%) followed by chronic suppurative otitis media (15.8%). Furthermore, 8 children had sensorineural hearing loss .A history of infection (following acute otitis media and meningitis) was observed in 2 subjects. Cause of sensorineural hearing loss could not be discovered for other participants. None of the participants were reported with the mixed type. In addition, one child had conductive hearing loss in one ear and sensorineural hearing loss in the other ear. Binary logistic regression revealed a significant effect for a history of otitis media . In other words, children with a positive history of 8.1 times (95% CI 1.8-36.7) were more likely to have a hearing loss than those with a negative history. However, there was no significant effect for age, gender, family history, or socioeconomic status. When the effects of various factors on conductive and sensorineural hearing loss were separately assessed, the results showed only a strong association between the history of otitis media and sensorineural hearing loss . The highest prevalence of hearing loss in the right and left ear was reported at 500 Hz . The lowest prevalence of hearing loss in the right and left ear was obtained at 4000 Hz . Based on the low frequency of PTA, the hearing level of the right ear in 1.3% of children and hearing level of the left ear in 1.0% of children were ≥ 20 dB. In high-frequency PTA, different degree of hearing loss was observed in 0.7% and 0.6% of the right and left ears, respectively . Means of LPTA and HPTA threshold in the better ear among those tested were 4.7 (range: 0-50) and 4.8 (range: 0-82.5) dB, respectively. Prevalence rates of low- and high-frequency hearing loss in the better ear were 0.8% and 0.4%, respectively. Prevalence rates of low- and high-frequency hearing loss in the worse ear were 1.5% and 1.0%, respectively. Odds ratio of hearing loss at LPTA in students with a history of otitis media to without history was 10.1 with 95% confidence intervals 1.5-56.4 and at the high frequency (HPTA) 15.5 with a 95% confidence interval 2.6-98.7.Hearing impairments among school-age children in developing countries have been widely regarded as a major health problem , 10. MorThe WHO has defined a disabling hearing impairment in children younger than 15 years as a permanent unaided hearing threshold in the better ear > 30 dB HL . AccordiFindings of the present study regarding the prevalence rate of hearing loss are consistent with the rates obtained in a study by Hajilo . The afoLower prevalence of hearing impairment in the present study may be due to the fact that cerumen in the children with cerumen was removed, re-screened, and counted as normal hearing. However, cerumen was regarded as an etiology for conductive hearing loss in previous studies. Another potential reason for this finding may be an increased tendency to preschool assessment throughout the whole country in recent years. In addition, protocols used in these studies differed from the screening protocol of the present study, in which the hearing threshold at frequencies of 500 to 8000 Hz was assessed. On the other hand, the Iran Health Insurance Organization has recently increased universal health coverage to ensure that all people have access to effective health services . TherefoHaemophilus influenza, and 62% of the isolates were type b. The WHO revealed the moderate to high burden of Haemophilus influenza type b (Hib) in the Eastern Mediterranean countries (A multicenter cross-sectional study was conducted by Intakorn et al.  to assesountries . Althougountries -20, whicAccording to the literature, contradictory results are shown regarding the association between hearing loss and gender. Although the result of the present study is consistent with those of previous studies ,21 showiThere was no association between hearing loss and socioeconomic status. The relationship between socioeconomic status and otitis media with hearing loss is controversial, although Saim et al.  proposedThe present study had several strengths. Firstly, the survey was population-based. Secondly, the clinical measurement of hearing impairment was conducted by an expert audiologist. Thirdly, two trained interviewers gathered the data of this study. Reported measures were also included in the survey to allow the researchers to be assured from the results. The present study had also several limitations that need to be considered. Lack of a soundproof room and possible high level of ambient noise could be the challenges of the present survey. Possible causes of hearing impairment presented in this study should be interpreted with caution. Causes of hearing impairment in the children could not be discovered due to the absence of comprehensive history taking, physical examination, and review of medical records. A prospective study is expected to show the true nature of the causes of hearing impairments among school-age children. All the participants were from school-age children in Rasht; accordingly, the results of this study may not be representative of all cases with the national statistics. Therefore, it is required to perform a study with a large sample size to establish a true picture of the situation in Iran. Hearing loss is becoming a public health problem. In this study, the overall prevalence of hearing loss was reported as 1.2% in school-age children. It is recommended to perform the hearing screening of school-age children as a part of medical examinations. This program helps to identify hearing-impaired children in order to refer them for otolaryngological assessment and comprehensive audiological evaluation. It is suggested to carry out another study with a larger sample size to understand the prevalence and causes of hearing impairment among children.


Sweet syndrome (SS) is an idiopathic autoimmune disease which has been associated with various extracutaneous manifestations. Otologic symptoms secondary to SS are characterized by bilateral, progressive, sensorineural hearing loss, which requires auditory rehabilitation with, for example, cochlear implantation.A 43-year-old woman complaining of bilateral sudden hearing loss visited the Emergency Department of our University. Abrupt onset of fever peaking up to 40°C and vomiting accompanied the hearing loss and other associated symptoms were: tinnitus that sounded like a machine humming, mild dizziness, a painful rash , arthralgia in both the hip and knee joints, and vision loss in the right eye. The patient had no history of autoimmune diseases or surgery.Pure tone audiometry and biopsy on the skin lesion were performed. SS with bilateral sudden sensorineural hearing loss was confirmed.The patient was treated with intravenous prednisolone and topical steroids.After a week of treatment, skin lesions had improved. And 3 months after treatment, the hearing test showed full recovery.This case emphasizes the point that early diagnosis and timely treatment are essential for hearing recovery in patients with SS who have otologic symptoms. The extracutaneous manifestations of SS can affect various organs including the central nervous system, eyes, kidneys, heart, and lungs.,2 As a result of associated autoimmune disorders, SS can affect the inner ear and cause bilateral hearing loss. To date, there have only been 3 reports in the English literature of bilateral progressive hearing loss associated with SS.–5 In these reports, SS is associated with secondary bilateral, progressive, sensorineural hearing loss, which required auditory rehabilitation with, for example, hearing aids or cochlear implants. To the best of our knowledge, there have been no reports of SS occurring simultaneously with bilateral sudden sensorineural hearing loss (SSNHL).Sweet syndrome (SS), also known as acute febrile neutrophilic dermatosis and which is often associated with immunological disease, is characterized by clinical manifestations comprising pyrexia, painful erythematous nodules or plaques, and a dense neutrophilic infiltrate with the absence of leukocytoclastic vasculitis.In the present study, we report a patient with bilateral SSNHL that occurred simultaneously with the diagnosis of SS. The patient immediately received systemic steroids and an intra-tympanic steroid injection, and after 3 months, showed complete recovery of hearing.This study was approved by the institutional review board of Chonbuk National University Hospital Committee, Korea: CUH 2019-03-029. Informed written consent was obtained from the patient for publication of this case report and accompanying images.2A 43-year-old woman visited the Emergency Department (ED) of our University with bilateral sudden hearing impairment. The hearing loss had occurred suddenly with fever and vomiting a day before the visit to the ED and associated symptoms were: tinnitus that seemed like a machine humming, mild dizziness, fever peaking at up to 40°C, a painful rash , arthralgia in both the hip and knee joints, and vision loss in the right eye. The patient had no history of autoimmune diseases or surgery.Upon physical examination, both tympanic membranes were normal, and examination with a tuning fork showed left lateralization for the Weber test, and both ears were positive for the Rinne test. A rash could be seen all over her body, and her eyesight was measured to be 0.2 in the right eye, and 0.9 in the left eye Fig. .Laboratory test results were: high-sensitivity C-reactive protein (hsCRP) 160.12 mg/L, erythrocyte sedimentation rate (ESR) 79 mm/hr, and Seg neutrophil 84.10%. Pure tone audiometry (PTA) was performed on the second day after hearing loss had occurred Fig. . IntraveThere were no remarkable findings from brain magnetic resonance imaging and magnetic resonance angiography, but right orbital cellulitis, as shown by the diffuse soft tissue swelling and enhancement in the right upper and lower eyelids, was found on facial computed tomography (CT). After a week of treatment, there was no change in vision but skin lesions had improved. Follow-up on the hearing test showed an improvement of up to 20 dB in both ears. The patient was discharged after a week of treatment, with follow-up at the outpatient clinic after 1 month. Vision examination performed at the outpatient clinic showed a vast improvement to 1.2 in the right eye, and 1.2 in the left eye. After 3 months, the hearing test showed full recovery: 11 dB in the right ear, and 8 dB in the left ear (Fig. 3 The etiology is thought to be multifactorial and further research is needed. Currently, systemic corticosteroids are considered the criterion standard treatment for this disease and immediate clinical improvement with steroid administration, and immunosuppressants, suggests that the syndrome may be related to an immunological pathogenesis.,4,6 In addition, clinical evidence and laboratory results in recent studies suggest that cytokine effects also have an etiological role. The occurrence of bilateral sudden sensorineural hearing loss is rare compared to unilateral sensorineural hearing loss. In addition, bilateral sudden sensorineural hearing loss is more likely to be due to the patient's general condition rather than to idiopathic causes, with more severe hearing loss and poorer prognosis than with unilateral sudden sensorineural hearing loss. Considering the immunological pathogenesis of SS, bilateral sudden sensorineural hearing loss in SS may be due to systemic conditions which eventually affect the inner ear.SS was originally described by Dr Robert Douglas Sweet in 1964.–5Up to now, research conducted in other countries has reported three cases of progressive bilateral sensorineural hearing loss with SS. All patients from those three cases had a history of chronic SS, and despite many attempts including systemic steroid therapy, multiple steroid sparing agents and cochlear implants, the progress of bilateral sensorineural hearing loss could not be stopped.The case presented here is the first which has shown full recovery of hearing after bilateral sensorineural hearing loss, and which was enabled by early and immediate diagnosis, and appropriate steroid administration.It must be noted that patients in the 3 previous cases showed no signs of sensorineural hearing loss at the time SS was initially diagnosed. It can be hypothesized that autoimmunity had not yet affected the inner ear in the initial phase of SS, and the patients’ condition at some point during the progress of the disease triggered the gradual damage. In comparison, our patient showed bilateral SSNHL from the time of initial diagnosis, with full recovery of hearing after immediate steroid injection.In this case, the patient showed an immediate response to systemic steroids, and symptoms—including hearing loss—were completely reversed. This supports the existing evidence that steroid therapy is a criterion standard for SS and that it is thus associated with an immunological pathogenesis.As most cases up to now did not receive treatment at an early stage of the disease, we suggest that the failure to improve bilateral sensorineural loss in previous cases may be due to delayed treatment. Thus, time-appropriate treatment is crucial for hearing recovery, and patients who are suspected of having SS should undergo a thorough history and physical examination for early diagnosis. Most importantly, when clinical evidence indicates SS, clinicians should not hesitate to perform a hearing test as soon as possible, regardless of symptoms. In addition, patients with SS should be fully warned about the possibility of bilateral hearing loss, and advised to adhere to a periodic examination by an otolaryngologist, even when symptoms are not present.Conceptualization: Yeon Seok You, Eun Jung Lee.Data curation: Yeon Seok You.Investigation: Yeon Seok You.Methodology: Yeon Seok You, Eun Jung Lee.Project administration: Yeon Seok You, Eun Jung Lee.Resources: Yeon Seok You, Sang Woo Park, Seok Kweon Yun.Supervision: Yeon Seok You, Eun Jung Lee.Validation: Yeon Seok You, Eun Jung Lee.Visualization: Yeon Seok You, Sang Woo Park, Seok Kweon Yun.Writing – original draft: Yeon Seok You.Writing – review & editing: Yeon Seok You, Eun Jung Lee.


Pure-tone audiometry (PTA) was performed, paired with a self-report questionnaire. Personal noise dosimetry (PND) was also performed with an additional group of participants. (3) Results: A total of 458 employees were submitted to PTA. The Packaging and Utilities department reported the largest shifts in hearing thresholds . No significant effect of department type on auditory health could be found. PND results were obtained from 39 employees. Packaging and Sales were identified as the most exposed departments. (4) Conclusions: A healthy hearing profile was found for a large proportion of the brewery employees (91.7%). However, NIHL (noise-induced hearing loss) proportions specifically among Bottling and Sales employees were elevated.The health risks of exposure to loud noises are a well-established fact and are widely addressed in modern industries. Yet, in less developed countries, it is thought these hazards receive less attention, both in the workplace and in private life.  A new insight into the field of noise control in the workplace is needed. The objective of this study was to gain insight into the auditory health of brewery employees in a developing country and their exposure to noise at work and in private in order to identify possible groups at risk and key subject areas on which to intervene. This will be attained through two different approaches of the population. At first, audiometric threshold data from a representative sample of the brewing company employees would give insight into the prevalence of NIHL. Secondly, personal dosimetry needed to give information about the possible factors and moments of noise exposure. In the following paper, we start by describing an extensive dataset obtained in a brewery in Nigeria. Next, we present data analyses and possible conclusions.Occupational hazards are a widespread concern in many industries, including the subtopic of noise hazards. Noise exposure brings about noise-induced hearing loss (NIHL) and consequent issues ,2. ExtenAs expressed earlier, two observational studies are described in this paper. At first, audiometry is reported for it gives the best insight into the population that was observed and that was also subject in the second part: personal dosimetry.A cross-sectional study was performed to assess employees’ hearing thresholds. Brewery employees  were categorized by department. For this study, seven departments were distinguished as follows: (a) Packaging, of which Bottling and Canning were considered separately; (b) Utilities, (c) Office, (d) Sales, (e) Brewing, comprising all employees working in and around the brewhouse; and (f) Warehousing, comprising all employees working in shipment and logistics. Randomly selected employees from the full-time employee register were invited to attend the program; an original sample of 250 employees was based on sample size calculations. After completing the informed consent form, subject’s data were anonymized. Participants were given a questionnaire with demographic, employment, and health-related questions. Before the audiometry, otoscopy and tympanometry were performed to identify any abnormalities by two trained students in Clinical Technology supervised by local company doctors. Exclusion criteria from this study were: (a) any middle ear defects, (b) any known hearing defect not due to noise exposure ; and (c) not being a brewing company employee . Results were obtained from 520 participants, adjusted to 458 after exclusion. The analysis was performed using SPSS packages 24 and 25 , focused on the 4 kHz threshold and its surrounding frequencies  as a measure for NIHL. The resulting threshold values for these frequencies were used as a measure for hearing loss and combined with the questionnaire results through further analysis using ANOVA and frequency reports. As the point of interest was the relationship between departments and possible noise hazards, tests were performed to compare the respective department of an employee with his hearing profile. A random subsample of employees from selected departments was invited to take part in a dosimetry study. Given the variation in activity types, particular interest was taken in the data that could be obtained from the Sales department. Thus, Sales employees were oversampled compared to other departments. ® 170, Amplivox Ltd., Birmingham, UK) for both ears at frequencies 500 Hz, 1 kHz, 2 kHz, 4 kHz, 6 kHz, and 8 kHz.Pure-tone audiometry was finally performed in a noise-insulating booth (measured levels inside booth were between 33 and 38 dB) at the brewery clinic. Air conduction thresholds were measured on calibrated audiometers  for 24 h while carrying out their daily routine. During the recordings, participants were asked to keep an hourly log of their whereabouts. The recording threshold for the dosimeter was 70 dB, and the value of 35 dB was used to impute the values below that threshold.As previously stated, results were obtained from 520 participants, 458 after exclusion. Due to logistical difficulties, the original sample of 250 was abandoned and oversampling was done in order to maintain statistical power. Mean age was 35 ± 7.38 years with first employment age at 24 ± 4.04 years. Employees worked for 51.0 ± 15.95 h per week. Some extremely high values were reported (maximum = 144 h/week) with 5% above 84 h. It was not possible to find grounds on which to reject these values as outliers due to the self-reporting nature of the data; however, values above maximum (24 × 7 = 168 h) were considered outliers and removed from the analysis. As this variable is not used in any analysis for this article, the influence of this reporting method was not investigated.n = 186, 40.6%, see n = 101, 22%) and Office departments  as well. In the far-right column of n = 399) than female (n = 58) employees attended the program as reported by the participants (1 subject did not report gender). Most (n = 364) stated that they attended post-secondary education. This included advanced courses or even university degrees.Most of the employees were working in the Packaging department at the Bottling line . No other department showed significantly higher hearing levels compared to the profile of Office employees. Furthermore, thresholds for all populations are within the acceptable healthy range (between 0 and 25 dB). A clear association of incidence of age on the hearing threshold was observed, as shown in Hearing thresholds recordings were mapped to the reported department of the participant . Simple To obtain a more detailed impression of the population’s auditive health, BIAP 02/1: Audiometric Classification of Hearing Impairments  was usedp = 0.014, F(6.447) = 193.562), indicating an effect of workplace (current department of employment). Unsurprisingly, the main effect for age was significant as well  = 14.659) because age is a well-known risk factor for hearing loss. The main effect for gender was not significant  = 1.637). Office levels were used as a control group for the ANOVA as it was expected that this group had less exposure to noise. Working in the bottling department showed a significant influence on NIHL development . Bottling employees were more likely to develop NIHL than Office counterparts. Compared to the Office population, Bottling, Utilities, Sales, and Brewing reported a strong yet nonsignificant positive effect on developing NIHL. While Canning and Warehousing employees showed a negative effect  compared to Office personnel, this translates to the fact that canning and warehousing employees presented a better hearing profile. However, these differences were not statistically significant either.In an attempt to find employees at risk for developing NIHL, the 4 kHz hearing threshold was compared to the levels of ambient frequencies. Univariate analysis of variance (ANOVA) was conducted to compare the main effects of the currently occupied department, gender, and age on this difference; results can be seen in p < 0.001). Combined with gender and age, the overall time spent working at each department by a single employee had a significant effect on hearing threshold. Only the Bottling department showed a negative influence on hearing thresholds with a regression coefficient of 0.077 (p = 0.456). Canning showed a negative coefficient of −0.423 (p = 0.057). It was, therefore, not possible to attribute an effect on hearing threshold to a specific department; moreover, the combination with the factors of age and gender makes confounding impossible.A multiple regression  test wasDosimetry results were obtained from 39 participants. Specific demographics for this group were not recorded. This part resulted in 931 h of noise recordings. Due to incomplete or incorrect logging by the participants, about 72 h of this data was incomplete and, therefore, excluded from the analysis. Levels were recorded in dB Leq with frequency weighting A. Results are shown in Loudness is only one of the factors determining the harmfulness of noise. The other main factor is duration of exposure. In order to get insight into this aspect of exposure, dose computation was performed according to NIOSH standards . This anIt was necessary to contextualize the recorded noise levels within the brewery’s safety rules and regulations. Therefore, observations and interviews were conducted on-site. Most sites that reported high noise levels within the brewery required mandatory use of personal protective equipment (PPE). Departments such as Brewing, Packaging, and Warehousing required the use of earplugs. The Utilities building required earplugs to be combined with headphones. The latter reported a low noise level as all duties were performed from an insulated control room. PPE was only mandatory for personnel when crossing the engine rooms. On the other hand, some locations reporting high noise levels are currently out of regulatory scope; the Brewery Canteen and Social Events are not restricted due to the personal nature of these locations. Yet, Social Events are the main work environment for a group of employees: sales representatives. In Nigeria, the main duty of these employees is to visit possible sales locations such as Bars, Clubs, and analogous places. Sales employees do not currently fall under any hearing protection regulations. Yet, in the questionnaire, 48.5% of sales employees reported that they considered PPE’s as necessary, while 51.5% also reported not wearing them when recommended. Regular on-site controls result in high compliance on brewery grounds. Yet, we believe more education and information campaigns would likely result in enhanced self-awareness and personal decision-making for all employees regarding hearing protection in- and especially outside the brewery. Ultimately, overall reports of hearing-related injuries were, however, not above normal recurrence (1–2%). In total, 5% of the employees reported having problems hearing, either sometimes, often, or always.Taking the present study into consideration, there are some limitations and possible improvements worth discussing. First, given the local conditions, it was not possible to perform random sampling within the brewery population. No intentional bias was induced but we cannot exclude that some characteristics of the observed sample may differ in terms of unobserved confounders from the total population. For example, employees not willing to participate may already suffer some form of reduced hearing and may be afraid of possible repercussions if diagnosed. Hierarchy and scarcity of employment are deeply rooted problems in the Nigerian working culture. Overall, as is shown by the estimates in Observations concerning the awareness of employees resulted in a suggestion for better knowledge and information programs. Effort should be put into implementing these into the culture as they may be a solution for what appeared to be one of the main causes of risks around noise ,16. As oSince the publication of Ologe et al.  it seemsWhile not reporting any disconcerting results, this study has given many insights in the populations at risk in the brewery and the way they may be threatened. The setup of this study also taught many lessons about the way these observations could yield better results. This may, therefore, also serve as a pilot for further investigations mainly in terms of risks outside the work environment and a way to raise awareness around the risks of noise in developing countries but also as a reminder for western countries.A healthy hearing profile was measured for a large proportion of all brewery employees. NIHL was diagnosed in 12.9% of all participants, primarily employees from the Bottling department (16.7%), Sales (12.9%), and Utilities (11.5%) departments. The prevalence of NIHL increases with age. The prevalence of NIHL found in these three departments gives cause for concern as it is 10% larger than other departments and way above the prevalence in sub-Saharan Africa  by a sigThe departments showing the highest incidence of NIHL were also the ones reporting the highest noise levels in the audiometric recordings. Fortunately, the observed compliance with the auditory protection equipment rules on site was quite high. Follow-up research is necessary to identify any evolution in the hearing profile. Finally, the high level of Sales employees diagnosed with NIHL could be explained by the high levels recorded at the different social events attended for business reported in this part of the study.


Noise is one of the most common and harmful physical factors in the working environment and has physical and psychological effects on individuals. In this study, the audiometry results of industrial workers were modeled and the effect of noise and other factors on hearing loss was examined.This was a longitudinal study based on the records of workers who had worked over 10 years in the industry and had recorded audiometries since their employment. Data was analyzed through linear mixed models. During each year of noise exposure, hearing loss was 1.9 db at 4000 Hz; 0.059 in low frequencies and 0.62 db in high frequencies. At 8000 Hz the effect of the age at employment on hearing loss was significant (P=0.014). At low frequencies the interaction of smoking and age at employment was significantly related to hearing loss (P˂0.001). This study showed that despite acquaintance with safety measures, workers still face hearing loss in industry and employers should put workers under more surveillance for using protective gear. Smoking might be another risk factor for hearing loss. Nowadays, due to population growth and establishment of large-scale industries; the use of machinery, equipment, processes and chemicals is inevitable. Industrialization introduces various hazards to the workforce and makes the workers face many harmful factors. One of the occupational hazards triggered by technological progress in industrial fields and the extensive use of equipment and machinery is the unpleasant acoustic turmoil, called noise ,2.Among all the physical occupational pollutants, noise has the most emission in many industries. Noise not only causes disease, but can also be an annoyance, cause anger, and interfere with conversations. It can also prevent hearing the warning sounds, cause accidents, and reduce production . Recent Noise is the most common harmful physical factor in work environments in the world. Approximately, 600 million workers are exposed to noise in the work environment .It is estimated that in the United States about 22.4 million workers are exposed to workplace noise and more than 100 million people are exposed to noise from non-occupational situations such as traffic, use of personal devices, and other sound sources .Hearing loss is one of the main occupational diseases in Europe. According to the estimates by the Occupational Safety and Health Organization, 17% of workers in the manufacturing sector are hearing impaired .Noise pollution is not confined to countries that have advanced technology. In many developing countries, noise pollution maybe even more severe; and the proportion of temporary or permanent hearing loss is higher . SeveralThere are no accurate statistics regarding the rate of exposure to industrial noise in Iran, but one can imagine that the extent of the problem in Iran is significant. Naturally, every study examining the effect of noise and factors affecting hearing loss in workers is very important in Iran . EstimatAccordingly, nowadays, to increase productivity and reduce the effects of noise on the human body, noise research has attracted serious attention in many countries . Due to An important point is that hearing loss induced by noise is easily prevented, but it is irreversible after its incidence . TypicalOne of the oldest, simplest, and yet the most reliable audiological tests is pure sound audiometry or PTA. Audiometry is a qualitative and quantitative test of hearing. The device works by creating pure frequency sound signals with different intensities which can help determine the individual’s threshold of hearing at certain frequencies. In this study, the hearing threshold changes have been studied, based on the available recorded periodic audiometries in workers exposed to noise.Although the relation between noise and hearing loss and the necessity of using protective equipment is obvious, but hearing loss is still happening among factory workers in Iran and more research about preventing hearing loss is needed.The statistical models used in this study have not been used before in hearing loss, and can show new dimensions of hearing loss research.The current study is a longitudinal study with repeated measures in which several measurements are done on a particular person at different times. The primary objective in a longitudinal study is evaluating a response variable's changes over time and factors influencing it. There are two important groups of changes in longitudinal studies; one is an individual’s changes over time and the other is variations between individuals. In longitudinal studies, the data have a hierarchical structure at two levels. Repeated measurements form the first level units, and the individuals form the second level units. In such data, observations are not independent, hence appropriate models that take care of the dependencies between observations should be used. One of the most widely used statistical methods for inferring repeated measures is the Multivariate Mixed Analysis of longitudinal data. These models consider the random effects in modelling, model individual changes and render more accurate results. They are called mixed models, because they are a combination of random effects and fixed effects in one model . The general formula for a mixed model is:yi= Xiβ + Zibi+ εibi∼Nqεi∼Nniwhere yi is the ni×1 response vector for observations in the ith group, Xi is the ni×p model matrix for the fixed effects for observations in group i, β is the p ×1 vector of fixed-effect coefficients, Zi is the ni×q model matrix for the random effects for observations in group i, bi is the q ×1 vector of random-effect coefficients for group i, εi is the ni×1 vector of errors for observations in group i, Ψ is the q ×q covariance matrix for the random effects, σ2Λi is the ni×ni covariance matrix for the errors in group i .The current research is a longitudinal study, and has included the audiometries of workers at several time points, and data analysis has been done by Linear Mixed Models.The study was performed on workers of the Shahid Bahonar Ancillary Copper Industries, Kerman, Iran; exposed to noise above 85dB. The Shahid Bahonar Ancillary Copper Industries Co. is the largest supplier of copper products and its alloys. The complex has three main melting and casting, extrusion, and rolling manufactories. From 800 workers, those who were exposed to noise above 85dB entered the study. Workers under 10 years of experience, workers with previous jobs or second jobs exposed to noise, workers with hearing problems from before employment, long-term use of ototoxic drugs, suffering from acute hearing injuries, and workers with tympanic membrane rupture were excluded. However, if the worker had the so-called problems and more than 10 years of experience prior to the incidence of the disorder, their previous audiometries were used. After reviewing the records and applying the exclusion criteria, 273 workers were indicated as eligible to enter the analysis. From these 273 workers, one worker had only one audiometry record and was excluded from the analysis.The random slope model and random intercept model are multilevel regression models. In our analysis, the individuals were considered as groups. There was also the possibility to consider the ear side (right or left) as nested within the individual, because each person had two ear audiometries. The ear side (right or left) analysis was done, and since there was no significant difference, the individuals were considered as groups and each group contained one person measured twice.The random intercept model is a model that allows random intercepts for different groups which is here equivalent to different people. That is, in this model, rather than assuming that all workers’ hearing thresholds were on average the same at the onset and that the thresholds increase with increase of work experience with the same slope (linear model), different people were let to have different hearing thresholds at the beginning which increased with increasing experience with a constant slope .The random slope model allows random slopes of the regression line for different groups which are equivalent to different people. That is, rather than considering the average hearing threshold caused by increase in work experience as the regression line slope for all individuals, it takes into account individual differences in increasing hearing threshold affected by increase in work experience. It is assumed that all individuals have an average initial hearing threshold; however, the impact of increase in work experience is different in hearing thresholds of different individuals.Therefore, the best model allows different baseline hearing thresholds and different line slopes for different workers, and this is the Random Slopes and Intercepts Model. Analysis of variance and how much the model justified the data variances, and comparing the Maximum Likelihood of models, showed that this model explained the data better.Regarding the fact that the hearing loss caused by noise occurring in closed buildings where workers are constantly moving is bilateral, the analysis was performed via the simplest multilevel model and then by entering an additional level for each ear. This analysis showed that the difference between the left and right ear was not statistically significant. So the audiometry tests from the left and right ears were analyzed together.After analysis at different frequencies, the hearing threshold at low frequencies (HTL-L) which is the mean hearing threshold in frequencies 500, 1000, and 2000 Hz, and the hearing thresholds at high frequencies (HTL-H) which is the mean hearing threshold at 4000 and 8000Hz were also studied.It would have been better if the hearing threshold in high frequencies were calculated based on the average hearing thresholds at 3000, 4000, and 6000 Hz; which are mostly damaged by exposure to noise. But, because no measurements were done in the frequencies of 3000 and 6000, the frequencies of 4000 and 8000 Hz were considered instead. Data analysis was performed by Stata 13.Eligible workers who participated in the study were 272 persons, and their average work experience was 14.5±2.27 (mean±SD) years. The average age of the subjects in the first audiometry was 24.97± 3.22 years, and the mean age at the last audiometry was 37.28±5.49 years, and 90% of the workers were under 43 years of age. Between 3 to 7 audiometric measurements were available for each worker. The average number of audiometries was 4.1.One hundred and seven workers (i.e. 39.34% of the workers) were smokers and 245 of them (90%) had normal blood pressure (i.e. ≤140/90 mmHg). Descriptive data of other independent variables are in , and desWorkers participating in the study all had more than 10 years of work experience, and all workers underwent evaluation from the beginning of employment and during their work years. In As it can be seen hearing loss increases with increased job experience. Data analyses based on the Linear Mixed Model with both the constant and random effects are shown in The interaction between years of work experience and age at start of employment was positive and showed that as age at start of employment increased, the slope of hearing loss increased with increase in work experience. The interaction between work experience and smoking was negative in some frequencies and showed that as job experience increased the slope of hearing loss with smoking decreased. This study was a longitudinal research conducted to evaluate the effects of noise on workers hearing, according to sequential audiometries done during more than 10 years of work. Most studies done in this area were cross sectional studies, but this study had a different approach. The main purpose of this study was to describe the changes in hearing threshold levels in a group of workers exposed to noise above 85 dB, for 8 hours a day. Considering the fact that workers in different parts were constantly moving and their workplace was a closed space, bilateral and symmetrical hearing loss was observed in both ears.The relation between hearing loss and aging has been confirmed in other studies ,21. Age In the industry evaluated in this study, the use of engineering control measures to reduce noise intensity was not fully possible in different parts of the plant. Thus, personal hearing protection devices, such as airplugs and airmuffs, had be to used, but their use was very irregular in these workers.Permanent hearing loss due to continued exposure to noise over 85 dB is primarily seen at 4000 Hz and if exposure continues, other frequencies will get involved later, as well. Noise-induced hearing loss is usually bilateral and symmetric in both ears, but because of working conditions, it can be more severe in one ear . In RoghIn a study conducted by Abedi et al. on the hearing loss of airport staff, there were no significant differences between right or left ear hearing loss, confirming the fact that the harmful effects of noise on the hearing system is bilateral and symmetric . Also inBut according to a study conducted on the frequency of hearing loss among drivers of heavy vehicles by Berjis et al., left ear hearing loss was significantly higher than the right ear; probably because the left ear was exposed to a higher intensity and continuous noise from the vehicle's window .The results in The negative interaction coefficient between work experience and initial hearing threshold means that the higher a person's initial hearing threshold baseline, the less is the hearing loss slope with aging. This decrease in the hearing threshold slope of workers with a higher initial hearing threshold is partly related to this fact that these workers are warned about their high hearing threshold at employment and are pursued to use protective and self-care equipment seriously. Thus, probably due to more self-care, their subsequent hearing loss is less than others. However, this effect may also be due to regression toward the mean.The results of this study are in agreement with other studies, suggesting that noise-induced hearing loss mainly affects 4-8k Hz frequencies. Other studies have also shown occupational noise affects at 6kHz . UnfortuIn a study conducted in 2014 on African gold miners, hearing loss at all frequencies in workers who were exposed to noise above 85dB, were higher in 3000 and 4000 Hz and higher in workers of 36 to 45 years of age . The curAnother study done on construction workers exposed to noise in Netherlands revealed 0.54 dB hearing loss per year in 3000, 4000, and 6000 Hz. In this study, particularly in the first decade of exposure to noise, there was a considerable increase in the hearing threshold (30). The current study implies that hearing loss is significantly associated with increased years of work which is in line with Loukzadeh et al.’ study in the tile industry . The resIn a study done by Cantley et al. on different levels of noise pollution at workplace and occupational hearing loss, it was shown that hearing loss caused by noise follows a dose-response pattern, i.e. people who were exposed to noise above 88 dB had a relative risk for hearing loss of 2.29, and those who were exposed to 85-88 dB noise had a relative risk of 1.39, and those who were exposed to noise between 82-84 dB had a relative risk of 1.26 , but in this study we did not have information about other factors and this was a limitation.This study shows that exposure to noise is still causing hearing loss in industries, despite the fact that workers and employers know the adverse effects of noise and have been instructed to use protective gear. Employers should put workers under more surveillence for using protective gear. Smoking should also be considered as a possible risk factor for hearing loss.


In the co-culture group, the activity of all three stages of GCs showed significant (P<0.01) changes, and they had a strong (P<0.01) correlation with culture time; further, the intracellular lipid deposition of hierarchical GCs was significantly different (P<0.01) between the two methods. Moreover, after co-culture, in pre-hierarchical GCs, the expression of SREBP, CYP11 and 3βHSD was promoted (P<0.01). In hierarchical GCs, the expression of ACC, SREBP, STAR, CYP11, 3βHSD and CCND1 was promoted at 48 h, but they were inhibited (P<0.05) at 96 h. In F1 GCs, the expression of ACC, FAS, SREBP, CYP11, BCL2 and CAS3 was inhibited (P<0.01). The results indicate that goose TCs had complex and time-dependent effects on the biological function of GCs at each corresponding stage, and the effects were distinct in the different stages. In addition, DNL, steroidogenesis, proliferation and apoptosis in hierarchical and F1 GCs might have some synergistic relationships in the effects of TCs on GCs. Furthermore, we speculated that TCs might play an important role in the differentiation and maturation of GCs during follicular development.Granulosa cells (GCs) play a critical role in follicular development, which cannot be separated from the assistance of theca cells (TCs). In the present study, we used a transwell system to develop three stages of goose GCs  The granulosa cell (GC) is a part of the follicle, and it is important for follicular growth and development . The theIn mammals, GCs and TCs together constitute the steroid synthesis system of follicles, and these roles in synthesis are their main physiological functions ,8. FolliIn contrast with mammals, the effects of TCs on GCs have specific avian characteristics ,19. For in vivo. We hope this present work will lay a theoretical foundation for the study of mechanisms of follicular development in poultry.Obviously, the effect of TCs on GCs is important for follicular development in poultry. However, there are few reports on the specific mechanisms of TCs effects on GCs at different developmental stages of follicle. Therefore, we developed a noncontact transwell co-culture system of GCs (with TCs at same stage) from goose (an important and relatively low-yielding poultry species) pre-hierarchical, hierarchical (F4–F2) and F1 follicle stages ; fetal bThe healthy laying maternal line of Tianfu meat geese  was used in the present study. All geese were grown under natural conditions of light and temperature at the Experimental Farm for Waterfowl Breeding at Sichuan Agricultural University , and the geese were provided with unlimited access to feed and water. Individual laying cycles were recorded for each goose, and all geese in the same laying cycle were killed by cervical dislocation 7–9 h before oviposition. All the experiments were carried out in Sichuan Agricultural University , and all procedures in the present study were approved by the Laboratory Animal Operation Standard and Welfare Management Committee, Sichuan Agricultural University .Pre-hierarchical, hierarchical (F4–F2) and F1 follicles  from eac6 per well with 2.5 ml of Dulbecco’s modified Eagle medium (DMEM) F-12 (HyClone) containing 10% FBS (Gibco). The corresponding stages of TCs were seeded in transwell insert wells  at 1 × 106 per well in 1.5 ml of DMEM/F12 medium containing 10% FBS. Both cell types were cultured at 37°C under an atmosphere of 5% CO2 in humidified air. After the cells adhered (6–8 h), the culture medium was changed. In the co-culture group, two types of cells from the same follicular stage were placed in the same culture plate, and then the co-culture began (set as 0 h). For the GCs in the mono-culture group, we used a transwell insert well with no cells but containing 1.5 ml of culture medium as the control (The three stages GCs were seeded in six-well plates (Corning) at 1.2 × 10 control .The activity of mono-cultured and co-cultured GCs was measured every day for 7 days by MTT (Amresco) assays, which were performed as described previously ,26. At 4−△△Ct method ..35]. In Subsequently, we detected the intracellular lipid content of GCs at three stages and found that co-culture had no significant effect on lipid deposition in F1 and pre-hierarchical GCs at 48 and 96 h A,B,D, whACC and FAS) in F1 GC, but it had no significant effect on DNL in pre-hierarchical GCs, indicating that TCs had different effects on GC DNL at different stages. Combined the results of The mechanism of DNL in GCs has an important effect on follicles and is closely related to follicle proliferation, apoptosis and steroidogenesis  27,36].,36.27,36SREBP, CYP11, and 3βHSD) in GCs, suggesting that the effect of the TC steroid pathway on GCs existed before selection. At 96 h, 3βHSD (a key gene in progesterone synthesis) expression in GCs in co-culture was significantly higher than it was in mono-culture. Combining these data with what is found in the literature [in vivo developmental process [3βHSD , suggesting that there might be some synergy and interaction between the anti-apoptotic, apoptotic, DNL and steroidogenesis pathways in F1 GCs. Moreover, after co-culture, the hierarchical GC gene expression of CCND1  of promotion followed by inhibition. This confirmed that TCs had a complex and time-dependent effect on hierarchical GCs [In addition, we analyzed expression patterns for GC functional pathways. In F1 GCs, the genes n trends –6, and tof CCND1 , ACC (FiND1 (ACC , and genND1 (ACC  also shoical GCs ,49 and fical GCs ,36,50,51in vitro. The effects are complex, time-dependent and unique per different follicle stage. In addition, DNL, steroidogenesis, proliferation and apoptotic pathways in hierarchical and F1 GCs may have some synergistic relationship in the process of TCs regulating GCs. Furthermore, we speculate that TCs may play an important role in the differentiation and maturation of GCs during follicular development.In summary, the present study shows for the first time that three stages of goose follicle TCs have certain effects on the cell activity, lipid metabolism, steroidogenesis, proliferation and apoptosis of GCs


There is an increasing global recognition of the negative impact of hearing loss, and its association to many chronic health conditions. The deficits and disabilities associated with profound unilateral hearing loss, however, continue to be under-recognized and lack public awareness. Profound unilateral hearing loss significantly impairs spatial hearing abilities, which is reliant on the complex interaction of monaural and binaural hearing cues. Unilaterally deafened listeners lose access to critical binaural hearing cues. Consequently, this leads to a reduced ability to understand speech in competing noise and to localize sounds. The functional deficits of profound unilateral hearing loss have a substantial impact on socialization, learning and work productivity. In recognition of this, rehabilitative solutions such as the rerouting of signal and hearing implants are on the rise. This review focuses on the latest insights into the deficits of profound unilateral hearing impairment, and current treatment approaches. Hearing loss is the third leading cause of disability globally , with apProfound unilateral sensorineural hearing loss, often termed single-sided deafness (SSD), refers to clinically-unaidable hearing, as defined by severe-to-profound hearing thresholds with a poor word recognition ability . AcquireSpatial perception is multisensory and multifaceted. The auditory system plays a particularly important role, helping to map where we are in space by continuously sensing auditory events. The constructs of auditory space are complex, depending on our interaction with signals that are dynamically changing in terms of frequency spectrum, level and time. The more complex the listening environment, the more these signals interact and overlap. Therefore, hearing is not just about sound detection or awareness, but also about managing these complex interactions to provide meaning to those signals. Our auditory system is quite sophisticated in wholly managing the acoustic information presented to our ears in dynamically changing environments. Human listeners are able to rapidly process this information to identify and orient to acoustic stimuli, selectively attending to signals of importance while suppressing competing signals. This is all seemingly accomplished quite simply and effortlessly across numerous everyday environments, while allowing listeners to be highly functional.Spatial hearing is dependent on the processing of monaural and binaural hearing cues A,B. WhilSounds arriving at the ears from different locations in space allow listeners to take advantage of spatially separated sounds to improve the signal-to-noise ratio (SNR). For example, the ear closest to the signal benefits from the shadow created by the head to block noise that would otherwise mask the signal. Furthermore, the integration of the input from the two ears results in a summation of the signals leading to perceived enhancement. In addition to boosting the target signal, a reduction of competing noise occurs with binaural hearing . This isThese essential interaural hearing cues are unavailable to those with SSD. The reduced ability to discriminate ILDs and ITDs results in primary deficits in speech understanding in noise and localization ,27. As mNormal hearing listeners are highly accurate in their ability to localize sound in space, whereas this is largely disrupted in monaural listeners ,36 due tAsymmetry in hearing has been reported to result in a reduced quality of life comparable to , or exceProfound sensorineural unilateral hearing loss that is sudden in onset should receive swift medical work-up. Depending on etiology, sudden hearing loss may also be accompanied by ringing in the ear and vestibular symptoms. Where indicated, imaging studies should be considered to rule out retrocochlear pathology as the origin of hearing loss. In some cases, intratympanic or oral steroids can result in a partial or even full recovery of hearing, but the success of such interventions is time dependent . FurtherTraditional hearing rehabilitative options have relied on rerouting the acoustic signal from the deafened ear to the normal or better hearing ear for processing. Contralateral routing of signal (CROS) hearing aids have long been used to reroute the signal of interest to the better hearing ear. This is achieved by placing a microphone behind or in the deaf ear to collect the sound and transmit it wirelessly to a receiver worn in the better ear . CROS heRerouting primarily benefits the unilateral listeners by reducing the negative effects of the acoustic head-shadow , by imprCochlear implants (CI) are an emerging treatment option for SSD , providing direct electrical stimulation to the deafened ear via an electrode placed in the impaired cochlea. Because they provide direct input to the impaired ear, independent stimulation of each ear may provide some binaural hearing benefits not realized with rerouting solutions. Studies of performance in individuals with SSD consistently report improved speech perception in the deaf ear, hearing in noise and localization compared to untreated ,56,57,58At present, the CI cannot restore access to ITD cues . AlthougIn traditional CI candidates, the duration of deafness has been shown to be an important predictive factor of hearing outcomes and benefit ,73,74. SPoor spatial hearing, characterized by impaired localization and speech perception in noise, is a common sequela of profound UHL. Much has been learned about the importance of spatial hearing for functional listening abilities. The functional deficits associated with the loss of access to binaural hearing cues have a cascading effect on the unilaterally impaired listener, with high reports of handicap and disability. Evidence suggests that treatments, which reroute the signal from the impaired ear to the normal ear, are successful at reducing the negative effects of the acoustic head-shadow and improving hearing in noise when the signal of interest is located at the deaf side, but are not successful in restoring access to binaural cues. Evidence suggests that the electrical stimulation of the deafened ear by a CI may provide some increased access to these cues, but does not result in the full restoration of binaural hearing abilities. Longitudinal studies are necessary to see how CIs will evolve as treatment solutions in this population. New and/or evolving treatment approaches will be necessary to restore binaural hearing abilities in unilaterally deafened individuals.


Age-related hearing loss is highly prevalent among persons with dementia (PwDs) and is associated with an increased risk of neuropsychiatric symptoms. However, few use hearing aids and disparities exist. PwDs and, in particular, minority older adults, have some of the lowest rates of hearing aid use. Recent federal legislation created the designation of over-the-counter hearing aids, which will debut by 2020-2021, and represents an opportunity to advance accessibility. This presentation will share estimates of hearing aid use among community-dwelling PwDs from two cohorts, where hearing aid use ranges from 7-11% among African Americans versus 33-45% among whites. To explore this gap, the presentation will share findings from semi-structured interviews with care partners of PwDs and hearing loss around barriers and facilitators of hearing care, including device usability. With growing understanding of sensory health, a changing hearing care landscape represents a critical opening to increase access to hearing care for PwDs. Part of a symposium sponsored by the Alzheimer’s Disease Research Interest Group.


Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from  Salpingoeca rosetta reveal unusual rhodopsin topology comprising 8 transmembrane helices (TMs) and suggest that TM0 plays a crucial role in the enzymatic photoactivity.Rhodopsin phosphodiesterase (Rh-PDE) hydrolyzes both cAMP and cGMP in a light-dependent manner. Structural and functional analyses of the Rh-PDE from  All microbial rhodopsins share a seven transmembrane topology, in which all-trans retinal chromophore is bound to a conserved lysine residue via Schiff base in the transmembrane helix (TM) 7. Most microbial rhodopsins function as light-driven ion pumps that actively transport various ions1 . Some microbial rhodopsins function as light-gated ion channels (channelrhodopsins4), which are used as optogenetic tools in neuroscience6. Enzyme rhodopsins are a group of newly discovered microbial rhodopsins7, found in eukaryotes such as fungi, green algae, and choanoflagellates. Enzyme rhodopsins comprise the N-terminal rhodopsin domain and C-terminal enzyme domain, connected by a linker, and function as light-activated enzymes. Two types of enzyme rhodopsins were initially discovered, the histidine kinase rhodopsins8 (HKRs) and rhodopsin guanylyl cyclase9 (Rh-GC). While HKRs function as ATP-dependent light-inhibited guanylyl cyclase10, Rh-GC from Blastocladiella emersonii (BeGC1) induces a rapid light-triggered cGMP increase in heterologous cells (~5000-fold) and could be utilized as an optogenetic tool to rapidly manipulate cGMP levels in cells and animals12.Microbial rhodopsins are photoreceptive membrane proteins with an all-13 (Rh-PDE), a type of enzyme rhodopsin, was discovered. Rh-PDE derived from Salpingoeca rosetta (SrRh-PDE) was first identified and exhibits light-dependent hydrolytic activity for both of cAMP and cGMP, whereas it lacks pump or channel activity. Rh-PDE is constitutively active in the dark, and the increase in its light-dependent hydrolytic activity is up to twofold14. Moreover, four Rh-PDE homologs were recently discovered in Choanoeca flexa15. These organisms belong to the choanoflagellates, a group of free-living unicellular and colonial flagellate eukaryotes, considered as the closest living relatives of animals. In response to sudden darkness, Rh-PDEs trigger the coordinated, polarized contraction of C. flexa cells, which results in colony inversion. Since the light-dependent phosphodiesterase activity of Rh-PDE is complementary to that of photosensitive adenylyl cyclase17 (PAC) and Rh-GC, SrRh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations and cell signaling.In 2017, rhodopsin phosphodiesteraseSrRh-PDE consists of the long N-terminal region, transmembrane domain (TMD), including the rhodopsin domain, C-terminal PDE domain, and 76-residue linker region connecting the TMD and PDE domain. The rhodopsin domain of SrRh-PDE shares relatively low sequence identity with other microbial rhodopsins (about 20%)18. Moreover, as in the other enzyme rhodopsins, SrRh-PDE is predicted to have eight transmembrane helices, in which the extra helix TM0 is at the N-terminus of the rhodopsin domain19. To date, the structures of the TMDs and the full-length enzyme rhodopsins have not been determined. Thus, the existence and functional role of TM0 have remained unclear. While the structure of the PDE domain of SrRh-PDE was determined19, little is known about the overall domain architecture and photoactivation mechanism of the full-length Rh-PDE. Here, we present crystal structures of SrRh-PDE–TMD with and without the linker region, at 3.5 and 2.6 Å resolutions, respectively. The structures and mutational analyses revealed a 8-TM topology and the functional mechanism of the extra TM. We also determined the crystal structure of the PDE domain with the linker region, at 2.1 Å resolution. Based on these three structures, we propose a model of the full-length Rh-PDE structure and the mechanism of the light-dependent enzyme activation, with the support of HS-AFM observations and computational modeling of the linker region.20. In total, 271 datasets were merged with the KAMO software21. The structure was determined at 2.6 Å resolution by molecular replacement, using the structure of bacteriorhodopsin  . Crystals were obtained with the lipidic cubic phase (LCP) method . TM1–7 adopt a canonical rhodopsin-like topology with covalently linked retinal at Lys296 in TM7, as in the other microbial rhodopsins. Moreover, one extra transmembrane helix (TM0) is observed at the N-terminus, as previously predicted19 Fig. . TM0 is 19 showed that the PDE domain in Rh-PDE itself forms a dimer, as in other PDEs25, suggesting that the full-length Rh-PDE also functions as a dimer. In the Rh-PDE–TMD structure, the two protomers in the asymmetric unit form a dimer. The interfacial interactions occur in the intracellular halves of TM1 and TM7 of each molecule, whereas two monoolein molecules fill the dimer interface at the extracellular half  and ChR227, which are representative microbial rhodopsins. Rh-PDE has 24.4% and 19.3% sequence identities with BR and ChR2, and the Rh-PDE structure superimposes well on those of BR and ChR2   of Rh-PDE with bacteriorhodopsinly) Fig. . The ECLtrans retinal (ATR), as in the other microbial rhodopsins  of all-ins Fig. . Asp85 ains Fig. , which i292 Fig. . MoreoveC. flexa Rh-PDE 3, and it is surrounded by aromatic residues. These residues play a critical role in the exclusive 13-cis isomerization of the retinal chromophore via steric hindrance. Notably, in the vicinity of the β-ionone ring, Trp189 in BR or Phe230 in ChR2 is replaced with Glu271 in Rh-PDE, which is conserved over all Rh-PDE homologs. The hydrophilic glutamic acid flips and does not participate in the β-ionone ring recognition, thus weakening the hydrophobic interactions around it.The retinal polyene chain interacts with the hydrophobic residues in the rhodopsin domain of Rh-PDE, as in BR and ChR2 Fig. . Notably13, which is an intermediate value between those of BR (568 nm28) and ChR2 (480 nm3). The rhodopsin absorption maximum depends on the energy gap between the ground and excited states; that is, the interaction between the retinal and protein29. The absorption maximum will be red-shifted when the energy gap is small, while blue-shifted when the gap is large. The energy at the ground state greatly depends on the counterions around the RSB. Asp292 in Rh-PDE (corresponding to Asp212 in BR) directly hydrogen bonds with the RSB . TM0 exists on the exterior of the rhodopsin domain, and is located between TM2 and TM3 , as in the crystallization construct  faces the cytosol, and is predicted to have no secondary structureuct Fig. . We firsuct Fig. . The Δ32uct Fig. . Therefouct Fig. . The wilTo examine the functional role of TM0, we next truncated TM0 (Δ76) and ECL0 (Δ88) and performed the functional analyses. Both mutants showed no rhodopsin-related absorption in the DDM micelles Fig. , and theThe above Rh-PDE–TMD structure revealed the architecture of the enzyme rhodopsin, but the TMD and PDE domain are connected by a linker region (residues 311–386). To obtain structural insight into the full-length Rh-PDE, we determined the 3.5 Å resolution crystal structure of Rh-PDE–TMD with the linker region  Fig. . While t19 and PDE924 , which have similar dimer interfaces   simulations. The aMD simulations were performed with boosting of the dihedral potential (aMDd), total potential (aMDT), and both  to test the stability of the model. The final structures of the aMDd and aMDT simulations showed stable α-helices with RMSD values of 3.65 and 3.12 Å over all Cα atoms, respectively  observations. We detected two major structures, containing large and small dimer rods Fig. –3. Since35, which is found in bacterial membrane sensors and transmit conformational changes in the periplasmic ligand-binding domain to cytoplasmic signaling modules. Although the linker region does not contain the HAMP motif, it may dimerize and transmits the conformational change of the TMD to the PDE domain, as the HAMP domain.Finally, we combined the three structurers of the TMD–Linker, Linker–PDE, and aMDdual into the full-length model using the program, PyMOL Fig. . The aMD13 . The high monomer ratio in the equilibrium may be associated with the weak dark activity of Rh-GC.Rh-PDE forms a dimer in the crystal and supposed to form a dimer under the physiological condition, while Rh-GC catalytic domain is a monomer in the crystal and guessed to be in a monomer/dimer equilibrium under the physiological condition39. Although HeRs share low sequence identity with the other rhodopsins and adopt the inverted membrane topology, the crystal structures revealed that HeR comprises seven TMs with the retinal chromophore, as in the other rhodopsins42. Unlike these rhodopsins, the enzyme rhodopsins have long N-termini, and are predicted to have 1–2 additional transmembrane helices43, while the existence of TM0 and its function still have remained to be elucidated. The current Rh-PDE structures revealed that TM0 certainly exists and plays key roles in the stable retinal binding to TM1–7 and photoactivation  gene (encoding aa 33–316) and Rh-PDE (TMD–Linker) gene (encoding aa 33–378) were subcloned into the pEG BacMam vector, with a C-terminal GFP-His8 tag and a tobacco etch virus (TEV) cleavage site, and both genes were expressed in HEK293S GnTI− cells44. Cells were collected by centrifugation  and disrupted by sonication in buffer A  supplemented with 100 μM all-trans retinal (Sigma). The membrane fraction was collected by ultracentrifugation , and solubilized for 1 h at 4 °C in buffer B , 0.2% cholesteryl hemisuccinate, CHS (Sigma)). Insoluble materials were removed by ultracentrifugation . The supernatant was incubated with TALON Metal Affinity Resin (Clontech) for 30 min at 4 °C. The resin was washed with buffer C , 0.01% CHS), and eluted with buffer C containing 200 mM imidazole. The eluate was treated with TEV protease and dialyzed against buffer D . The protease and cleaved GFP-His8 tag were removed with TALON resin, and the protein was further purified by SEC on a HiLoad 16/600 Superdex 200 pg column , equilibrated with buffer E . The peak fractions were collected and frozen until the crystallization.The Rh-PDE (full) gene (NCBI Gene ID: 16078606) was synthesized after human codon optimization (GenScript)45. Aliquots of the protein-LCP mixture were dispensed onto 96-well glass plates and overlaid with the precipitant solution, using a Gryphon LCP . Crystals of Rh-PDE (TMD) were obtained in 29–35% (w/v) PEG400, 100 mM Na-citrate, pH 5.0, 100 mM KSCN and 10 mM ZnCl2, and crystals of Rh-PDE (TMD–Linker) were obtained in 25% (w/v) PEG500DME, 100 mM Na-citrate, pH 5.0, 100 mM NaK-tartrate. All crystals were incubated at 20 °C for 1 week in the dark. The crystals were harvested using micromounts (MiTeGen) and were flash-cooled in liquid nitrogen without any additional cryoprotectants.The purified protein was concentrated to 10 mg /mL and mixed with monoolein (Nu-Chek Prep) in a 2:3 protein to lipid ratio (w/w)2 (width × height) micro-focused beams and an EIGER X 9 M detector (Dectris). Small wedge data sets  were collected manually or automatically with the ZOO system20, an automatic data collection system developed at SPring-8. The loop-harvested microcrystals were identified by raster scanning and subsequently analyzed by SHIKA46. The collected images were processed with KAMO21 with XDS47. 864 and 74 datasets of the TMD and TMD–Linker were indexed with the consistent unit cell parameters. For TMD, 271 datasets were finally merged after hierarchical clustering based on correlation coefficients of intensities and outlier rejections implemented in kamo.multi_merge and XSCALE. For TMD–Linker, 58 datasets were merged after outlier rejections. The structure of TMD was determined by molecular replacement using Phaser48. The search model was prepared using HHpred49 and phenix.mr_model_preparation50 using the structure of BR (PDB ID: 1M0M) with a sequence identity of 22%. The resultant structure (TFZ score of 3.3) was further improved and refined using phenix.mr_rosetta51, phenix.autobuild52, jelly body refinement using REFMAC553, phenix.refine, and manual rebuilding using COOT54. The TMD–Linker structure was determined by molecular replacement using the TMD model. The final model of the TMD contained residues 49–310 in mol A, residues 47–310 in mol B, 14 monoolein molecules and 16 water molecules. The final model of the TMD–Linker contained residues 43–320 in mol A, residues 44–353 in mol B and 4 monooleinv molecules. Figures were prepared using CueMol (http://www.cuemol.org/ja/) and PyMOL (Schrödinger).X-ray diffraction datasets were collected on beamline BL32XU at SPring-8, with 10× v15 (for TMD) or 5 × 5 (for TMD–Linker) μm55, as implemented in the ASTRA software package (Wyatt Technology). The UV extinction coefficient of the protein was calculated by PROTPARAM and assumed to be 1.909. The refractive index increments (dn/dc) of the protein and the detergent were assumed to be 0.185 and 0.132, respectively.The instrument set-up for the SEC–MALLS experiment consisted of a Shimazu HPLC system connected in series with a Shimadzu SPD-10Avp UV absorbance detector, a Wyatt DAWN HELEOS 8+ light-scattering detector, and a Shodex RI 101 refractive index detector. Analytical SEC was performed at 4 °C on a Superdex 200 increase 10/300 column equilibrated with buffer, containing 20 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 0.01% LMNG. Elution was monitored in-line with the three detectors, which simultaneously measured UV absorption, light scattering, and refractive index. A 658-nm laser was used in the light-scattering measurement. Molecular masses were calculated using the three-detector method13. HEK293T cells were transfected with the pcDNA3.1-Rh-PDE variant plasmids by the calcium phosphate method. The DMEM/F-12 media contained 0.5 μM all-trans-retinal, penicillin and streptomycin. The cells were harvested after 24 h and washed in buffer A . The cells were resuspended with buffer A and homogenized using a Potter-Elehjem Grinder (Wheaton) and a syringe with a 27 G needle. The syringe was filled and drained five times while stirring the homogenate. Samples were kept in the dark before measurement for at least 2 hours. Catalytic activity was measured at room temperature in 100 μL buffer A with 1.6–1.8 μg (in the case of cGMP) or 16–18 μg (in the case of cAMP) of total HEK293 cell protein, in a 1.5 ml sample tube. The sample was illuminated with a xenon lamp  through a Y52 sharp cut filter  (7 mW mm−2). Reactions were initiated by adding cyclic nucleotides . Aliquots were taken out at different time points, and the reactions were immediately terminated by adding 100 μL of 0.1 N HCl and the samples were frozen in liquid nitrogen. After thawing, the samples were centrifuged to remove the membranes and denatured proteins. Nucleotides  were separated by HPLC (Shimadzu Systems) with a C18 reversed-phase column (Waters), using 100 mM potassium phosphate (pH 5.9), 4 mM tetrabutylammonium iodide, and 10% (vol/vol) methanol as the eluent. Nucleotides were monitored at 254 nm. Data were evaluated with LabSolutions (Shimadzu). Peak areas were integrated and assigned to the educt cyclic nucleotide, based on the retention time of the corresponding standard compound.Phosphodiesterase activity was measured in vitroProtein separation was performed on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and immunoblotting (BIO-RAD) was carried out according to standard protocols. The RHO 1D4-tag was detected by an anti-RHO 1D4-tag antibody (The University of British Columbia) diluted to 1:5000 and a HRP-conjugated anti-mouse antibody  diluted to 1:10,000 was used as secondary antibody. The chemiluminescent signal was detected by ECL Plus Western Blotting Detection Reagents  and quantified with an Image Quant LAS4000 image analyzer . Source data are provided as a Source Data file.l-Glutamine and Phenol Red (Wako), containing 10% (vol/vol) fetal bovine serum (FBS) and penicillin–streptomycin. The cells were co-transfected with the plasmid carrying the Rh-PDE variant and the pGloSensor-22F cAMP vector (Promega), by using Lipofectamine 2000 (Invitrogen). Changes in the intracellular cAMP concentration of the HEK293 cells were measured by the GloSensor assay (Promega). The transfected cells were incubated with or without 0.5 μM all-trans-retinal . Before measurements, the culture medium was replaced with a CO2-independent medium, containing 10% (vol/vol) FBS and 2% (vol/vol) GloSensor cAMP stock solution (Promega). The cells were then incubated for 2 h at room temperature in the dark. The intracellular cAMP level was observed by monitoring the luminescence, using a microplate reader (Corona Electric) at 27 °C. The cells were treated with 3.5 μM forskolin (Wako), a direct activator of adenylyl cyclase, to elevate the intracellular cAMP level. The cells were illuminated with a xenon lamp  through an interference filter (510 nm). The light intensity was adjusted to 2.28 µW/mm2, and measured by an LP1 power meter .HEK293 cells were purchased from JCRB Cell Bank and cultured in E-MEM media with 8 tag and a TEV protease cleavage site, and expressed in Rosetta2 (DE3) cells. Cells were collected by centrifugation  and disrupted by sonication in buffer A . Debris was removed by centrifugation  and the supernatant was incubated with Ni-NTA resin (Qiagen) for 30 min at 4 °C. The resin was washed with buffer A and eluted with buffer A containing 200 mM imidazole. The eluate was treated with TEV protease and dialyzed against buffer B . The protease and His8 tag were removed with Ni-NTA resin, and the protein was further purified by SEC on a HiLoad 16/600 Superdex 200 pg column , equilibrated with buffer B. The peak fractions were collected and concentrated to 10 mg/mL for the following crystallization.The PDE domain of Rh-PDE was subcloned into a modified pEG-CGFP-BC vector, with a C-terminal HisAliquots of the protein were dispensed onto 96-well sitting drop plates (Swissci) and overlaid with the precipitant solution, using a Gryphon LCP . Crystals were obtained in 29% (v/v) PEG400, 50 mM calcium acetate, pH 5.0, and 200 mM NaCl. All crystals were incubated at 4 °C for 1 week in the dark. Crystals were harvested using micromounts (MiTeGen), supplemented with 15% ethylene glycol as an additional cryoprotectant, and flash-cooled in liquid nitrogen.2 (width × height) micro-focused beams and an EIGER X 9 M detector (Dectris). One hundred twenty-eight small wedge data sets  were collected manually from different potions of a few large crystals (100–300 μm), each of which was broken in cryoloops to have multiple orientations. The collected images were processed in the same way as TMD. In total, 109 datasets were indexed, and finally 64 datasets were selected through hierarchical clustering by using the correlation coefficients of the normalized intensities between datasets47 and outlier rejections by kamo.multi_merge. The structure was determined by molecular replacement, using the previously reported PDE domain structure (PDB ID: 5VYD) as the search model with Phenix Phaser48. The resultant structure was refined using phenix.refine56 and manually rebuilt with Coot54. The final model of Rh-PDE (Linker–PDE) contained residues 380–708, 1 magnesium ion, 1 zinc ion, 2 PEG molecules, 2 ethylene glycol molecules, and 168 water molecules. Figures were prepared using CueMol (http://www.cuemol.org/ja/).X-ray diffraction data sets were collected on beamline BL32XU at SPring-8, with 10 × 15 μmThe full-length Rh-PDE was purified as described above, using buffer C containing 0.03% DDM and 0.006% CHS instead of 0.1% LMNG and 0.01% CHS, and buffer E containing 0.03% DDM and 0.006% CHS instead of 0.01% LMNG and 0.001% CHS.57. The lipid fractions including the full-length Rh-PDE were deposited on a mica substrate that had been chemically modified with 0.1% (3-aminopropyl)triethoxysilane . After 3 min incubation, the sample was washed with the buffer solution without the detergent. The HS-AFM observation was also performed in the non-detergent buffer at a room temperature. All HS-AFM images and movies were processed by plane-fit background subtraction and Gaussian noise-reduction filters.A laboratory-built HS-AFM operated in the tapping mode was used to image the full-length Rh-PDE. The solubilized proteins were deposited on a freshly cleaved mica substrate and incubated for 5 min. After the incubation, the residual proteins were thoroughly washed with a buffer solution containing detergent . The HS-AFM observations were performed in the same buffer, at room temperature. The full-length Rh-PDE reconstituted in a lipid bilayer was also observed. The lipid was composed of asolectin from soybean , and the details of the reconstruction procedure are described elsewhere58. Rosetta 3.1131 was used for ab initio modeling of the linker region, and 3/9-mer fragments were picked using RamaScore. The best structure from the total of 5000 structures was used for the following MD simulations.The secondary structure of the Rh-PDE linker region (residues 311–386) was predicted with the PSIPRED 4.0 server59. The net charge of the simulation system was neutralized through the addition of 150 mM NaCl. The simulation system was 96 × 96 × 96 Å3 and contained 84,698 atoms. The molecular topologies and parameters from the Charmm36 force field60 were used for the protein and water molecules.A periodic boundary system, including explicit solvent, was prepared with VMD61. The systems were first energy minimized for 1000 steps with fixed positions of the non-hydrogen atoms, and then for another 1000 steps with 10 kcal mol−1 restraints for the non-hydrogen atoms. Next, equilibrations were performed for 0.01 ns under NVT conditions, with 10 kcal mol−1 restraints for the heavy atoms of the protein. Finally, equilibrations were performed for 0.5 ns under NPT conditions with 1.0 kcal mol−1 restraints. In the equilibration and production processes, the pressure and temperature were set to 1.0 atm and 300 K, respectively. Constant temperature was maintained by using Langevin dynamics. Constant pressure was maintained by using the Langevin piston Nosé–Hoover method62. Long-range electrostatic interactions were calculated by using the particle mesh Ewald method63. The cMD run was performed for 150 ns, to calculate the average total and dihedral potential.Molecular dynamics simulations were performed with the program NAMD 2.1334 were performed for 150 ns, boosting the aMDd, the aMDT, and aMDdual. The dihedral energy threshold Edihed was set at 304 kcal/mol above the average dihedral energy of the cMD, and the dihedral energy boost factor αdihed was set to 60.8 kcal/mol. The total energy threshold Etotal was set at 13,500 kcal/mol above the average dihedral energy of the cMD, and the dihedral energy boost factor αtotal was set to 13,500 kcal/mol.aMD simulationsFurther information on research design is available in the Supplementary InformationPeer Review FileSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Reporting Summary


Most adults who acquire hearing loss find it to be a disability that is poorly corrected by current prosthetics. This gap drives current research in cochlear mechanosensory hair cell regeneration and in hearing restoration. Birds and fish can spontaneously regenerate lost hair cells through a process that has become better defined in the last few years. Findings from these studies have informed new research on hair cell regeneration in the mammalian cochlea. Hair cell regeneration is one part of the greater problem of hearing restoration, as hearing loss can stem from a myriad of causes. This review discusses these issues and recent findings, and places them in the greater social context of need and community. Worldwide, half a billion people have significant hearing loss, defined by the World Health Organization as auditory thresholds greater than 40 decibels (dB) in both ears . This laThe majority of individuals who acquire hearing loss as older adults lived for decades with oral language; for them its impact is disabling. Their hearing loss correlates with higher levels of morbidity, depression, and social isolation . HearingHearing loss may be classified as conductive or sensorineural. Sound waves enter the outer ear and are transmitted to the inner ear, the cochlea, through the auditory ossicles of the middle ear. Injuries, infections, or biological processes that impact middle ear function cause conductive hearing loss, whereas processes that impact cochlear function cause sensorineural hearing loss. This review will focus on causes of sensorineural hearing loss and recent progress in cochlear regeneration.Information about sound is encoded by the cochlea and transmitted to the brain, and hearing loss occurs when this neural transmission is reduced or interrupted. Sound waves enter the cochlea through the oval window and travel through the perilymph fluid column, which acts as a frequency filter. Higher frequency sounds have a shorter wavelength and impact the basal cochlea, whereas lower frequency sounds have longer wavelengths and impact apical regions. Outer hair cells (OHCs) respond to and amplify vibrations to increase shear forces along the tectorial membrane, which stimulates the inner hair cells (IHCs). Hair cell (HC) activity is continuous and energetic. It is sustained by a unique ionic differential between the endolymph, which is high in potassium and low in sodium, and the perilymph, which is more similar to extracellular fluids. Called the endocochlear potential, this electrical gradient is maintained by a potassium recycling chain, where potassium excreted by HCs is taken up by neighboring supporting cells, and then transported to the cells of the stria vascularis. Strial cells lining the endolymph restore potassium to the endolymph. Loss of OHCs or reduced strial activity both impact the activity of IHCs and increase hearing thresholds.IHCs encode sound information and transmit it to Type I spiral ganglion neurons (SGNs) through changes in the activity of specialized ribbon synapses. Type I SGNs comprise over 90% of total SGNs and receive input from IHCs; the more rare Type II SGNs innervate OHCs. Each IHC signals to up to 30 SGNs, depending on frequency and species, and each SGN receives auditory input from a single synapse. Type I SGNs are classified into three groups by gene expression, which encode auditory signals with low, medium, or high spontaneous discharge rates . They prHearing loss may arise from a myriad of causes, including aging, genetic variation, infection, and exposure to ototoxins or noise. It is intuitive that different kinds of insults could impact the chain of auditory transmission in different ways, resulting in the same impairment through different means. Cochlear cells express a number of proteins uniquely required for hearing, and genetic variants in those proteins can cause non-syndromic hearing loss. Other proteins may be necessary for hearing, and also function in other organs. Variants in those genes can cause syndromic disease, in which hearing loss is only one aspect. More than 120 genes for non-syndromic hearing loss have been identified, along with 55 genes for syndromic hearing loss . For famInfectious pathogens can drive hearing loss. As a sequela from disease, this may occur to either children or adults. Perinatal or prenatal infection by so-called TORCH pathogens, which include rubella, cytomegalovirus, and other viruses, is associated with childhood hearing loss . Adults Platinum-based chemotherapies and certain antibiotics are ototoxic and cause hearing loss (reviewed in ). OtotoxNoise exposure is a significant cause of hearing loss, estimated to affect 10 million adults in the United States . TraumatOver thirty years ago, two groups serendipitously discovered that birds spontaneously regenerate damaged auditory HCs ,49. A tiWhat signaling pathways regulate avian regeneration? Gap junction communication  is requiAtoh1 is negatively regulated by NOTCH signaling [Other investigations have shown that HC differentiation and supporting cell proliferation are further regulated independently. To initiate differentiation into HCs, supporting cells or precursor cells must express the basic helix-loop-helix (bHLH) transcription factor ATOH1 . In expeignaling . ATOH1 aignaling  and HES ignaling . The expignaling . During ignaling . Inhibitignaling , indicatignaling . HC specignaling . In contignaling . Prolifeignaling , and is ignaling . In addiignaling ,75, suggSpontaneous HC regeneration is also observed in cold-blooded animals, including fish . In addiIn pursuit of therapeutics to restore hearing from drug or noise damage, investigators have focused on the cellular events that could regulate the generation of new HCs from supporting cells in the mammalian cochlea. Here the mouse has become a commonly used model system for human hearing loss. Mice are less expensive to maintain than larger rodents such as guinea pigs. They can be maintained as congenic lines with well-described damage characteristics. Finally, they may be manipulated genetically to assess gain and loss of function of candidate pathways, permanently mark cell types, or perform cell-specific ablation studies. Like other rodents, mice are born before the onset of hearing, which occurs around two weeks of age . Interestingly, the immature neonatal mouse cochlea retains a limited ability for regeneration, both in vitro  and in vLgr5 at birth [Cochlear progenitors may be isolated, purified, propagated in sphere culture, and differentiated into HCs, supporting cells, neurons, and glia. Using these methods, multiple labs have found that supporting cell populations which include interphalangeal cells, the inner pillar cell, and the third Deiter cell  have robat birth ,89, and at birth ,89 or byat birth . The abiat birth ,92. Thisat birth .Progenitors for cochlear regeneration can also be studied in organ culture or in vivo, by stimulating regeneration through HC ablation, activating candidate pathways, or combining multiple approaches. Cell-specific ablation may be achieved through genetic expression of the human diphtheria toxin receptor (DTR), followed by injection of diphtheria toxin . HC lossAtoh1 expression; however, it concomitantly promotes the expression of the ATOH1 antagonists such as Hes and Id family members [ERT2 mouse line to label supporting cells appears to promote both proliferation and hair cell conversion through WNT and NOTCH-dependent processes [Many early efforts on understanding supporting cell to HC transdifferentiation focused on the effects of manipulating NOTCH signaling on supporting cells in the cochlea (see  for a co members ,101. Thi members . It can rocesses . Maintairocesses . Howeverrocesses , indicatrocesses , and suprocesses . Not surrocesses . Taken tAtoh1 transcription [WNT signaling was also implicated early in the specification of the otic placode ,108 and cription . Analysicription . Co-exprcription , suggestcription . As withcription . WNT actcription . As the cription . Thus, wInvestigations into mammalian cochlear regeneration also assess the effects of signaling pathways not implicated by studies of chick and fish. For example, SHH specifies ventral otic structures, including the cochlea, during the otic vesicle stage of development . At lateERBB2 signaling is an additional candidate pathway under investigation in mammals. ERBB2 is one of four members of the EGF receptor family, all of which are expressed by mammalian supporting cells throughout life . The famERT2 mouse line, which may be predisposed to HC differentiation [Hes1 have been introduced into the cochleae of guinea pigs after noise exposure [While progress has been made towards a better understanding of HC differentiation in the immature mouse cochlea, the problem of hearing restoration remains. Several groups have assessed the effects of driving ATOH1 expression or blocking NOTCH activity in the cochlea after noise damage, using different approaches. Gene therapy, consisting of intra-labyrinth injection of adenoviruses driving expression of the HC determinant Atoh1, has been proposed as means of restoring hearing in deafened adults . A clinintiation . The othntiation . Lastly,exposure . This inexposure . Moreoveexposure .Additional hurdles remain to the development of HC differentiation from supporting cells in adults. Adult supporting cells have significant transcriptional differences from neonatal supporting cells ,106, sugLastly, restoring hearing after noise damage may require additional approaches beyond assessing pathways that promote HC differentiation. Since the discovery of spontaneous HC regeneration and hearing restoration in birds after noise or drug damage, research efforts have improved our understanding of the cellular events that underlie this response. In particular, the progenitor cells and signaling pathways that enable the development of new HCs are becoming better understood. Efforts to extend these findings from bird and fish to the mammalian cochlea highlight the similarities and differences of these systems. Prior to the onset of hearing, the mammalian cochlear cells are plastic in their specification, but this capacity changes with maturity. There remain significant hurdles to overcome in pursuit of therapeutic solutions, especially for noise damage where it is clear that HC loss alone does not explain the loss of function. Furthermore, investigations will be needed to address hearing loss from other causes, especially infections.It should be noted that any discussion on hearing restoration must occur with the understanding that this is not something desired by all individuals with hearing loss. In fact, there are communities of adults with hearing loss who view hearing restoration as cultural genocide ,139. Thi