The telephone in inter-war Britain was an important tool in both the identification and categorisation of individual hearing loss. Between 1912 and 1981, the British Post Office had control over a nationalised telephone system. Linkage between telephony and hearing has long been noted by historians of sound and science, and Post Office engineers in the inter-war period had considerable expertise in both telecommunications and hearing assistive devices. This article first demonstrates how the inter-war Post Office categorised different kinds of hearing loss through standardizing the capacity of its users to engage effectively with the telephone, and secondly investigates how successful it was in doing so. By utilising the substantial but little used material held by BT Archives, we can trace the development of the Post Office’s ‘telephone for deaf subscribers’ and explore how it was used to manage and standardise the variability of hearing and hearing loss within the telephone system. The ability to hear normally was both defined and moderated by the telephone during the inter-war years in Britain. The telephone was patented by the Scottish/American inventor and teacher of the deaf Alexander Graham Bell in 1876, and it soon became a tool for people with what the Post Office constructed as normal hearing to communicate with each other. It was thus a purely aural device that served to further isolate people with limited hearing from key areas of everyday life. The telephone was domesticated after the First World War had accustomed a generation of soldiers to its use, and it became an essential business tool during the inter-war years. During this period, the telephone was transformed for many users from a luxury item to a necessity, and the ability to use the telephone became a social requisite. For society in general and particularly within the Post Office telecommunications department, the ability to use the telephone (whether amplified or not) meant inclusion in the hearing world. The telecommunications department of the Post Office exemplifies an office hidden behind its role as a cog driving the larger Post Office ‘Government Machine’, with its role in providing a telephone for people with hearing loss ‘marked by opaqueness and discretion’.I begin the first section of this paper with an explanation of the significance of the Post Office’s ‘Artificial Ear’ technology, which was used by the developing field of audiometry to construct normalcy limits and define the zero line  on audiograms for testing hearing.The history of the amplified telephone in the UK has hitherto been almost entirely unexplored. With the exception of some specialised audiological journals, this technology has not been given any attention by the medical or historical professions.The Audible Past is a classic of this kind, and it explores the connections between attempts to make deafness visible and the creation of sound technologies. Sterne explains that deafness was integral to the development of auditory technologies, from telephony to phonography.These so-called deaf subscribers did not wait for the Post Office’s consideration, but rather were actively involved in developing amplified telephone technology.This analysis is relevant to historians working in disability studies who have critiqued medicalised accounts of disability because it demonstrates the role of technology in producing disability. Furthermore, it highlights the agency of the disabled user and prioritises their role in influencing technology. Such an investigation is also relevant to those in medical history who have questioned the loss of the patient’s voice in traditional medical history because this reading allows for a broader interpretation of patients as users.ideal frequency. The final arrangement was measured by an artificial ear that we would think of as more closely resembling a recording device than a replica of a human ear. This allowed for quick and mechanical assessment of the telephone’s transmission quality. Its advantage lay in the fact that the telephone circuit was not interfered with, and it gave comparable results to a ‘human test’ but was 218 minutes faster. The NTC report concluded that: ‘There is thus a saving of 67% in time, in addition to the fact that mechanical testing is of course not nearly so exhausting as speech testing’.The idea of using an artificial ear to test the quality of telephone transmissions predates the Post Office, and can be traced back to its predecessor, The National Telephone Company (NTC). This was the Bell and Edison conglomerate that controlled most of telephony in Britain before the 1880 ruling on the 1869 Telegraph Act mandated a nationalised service – instated in 1911.During the investigation two ears, which were abnormal in that their hearing was known to be below normal, came under observation. In both cases the impedances were found to be abnormal, one giving an exceptionally high value and the other an exceptionally low value of absorption at 1100 cycles per second.The next available report on this subject was produced in 1928, indicating that the Post Office continued using the National Telephone Company system during the intervening 20 years.Understanding impedance (the conversion of vibrations) was important in improving the Artificial Ear’s design, as the electrical impedance of the artificial organ had previously been adjusted using a real ear.as a loss and tried to recover hearing through technology.An artificial ear was considered by the Post Office to be superior to real ears for testing telephone transmission quality for manifold reasons. Firstly, it gave quantitative data ‘for measurement, in absolute units, of the performance of receivers under their working conditions’.The Post Office had total control over the telephone network. State backing also meant that the Post Office was required to work under the financial constraints of the Treasury and act as an arm of the wider government. Due to its position within the Government, the Post Office developed amplified telephone technology according to its changing relationship with the Treasury, whose priorities regarding welfare shifted simultaneously. However, the state and newly enfranchised public expected the Post Office to provide telephones that could be used by people with some hearing loss. Amplified telephony was developed according to, and alongside, the emerging priorities of the welfare state. The Post Office had obtained legalised control over the telephone service in Britain and it was illegal for private companies or individuals to modify or tamper with its apparatus. Crucially, this meant that private hearing aid companies could not attach equipment to Post Office telephones and people with hearing loss could not fit private telephones for use on their lines. As a result, the Post Office was challenged by aspirational users who desired a telephone that could be used by people with less than perfect hearing. For example, between 1928 and 1934 Mr. Horace Buckley, a schoolmaster and war veteran, continually demanded a cheaper amplified telephone for those who had lost their hearing in the First World War and could not afford high telephone rental on a meagre war pension .The first telephone designed specifically for people with limited hearing was advertised by the Post Office in 1924 when a brief description of the ‘Repeater Telephonic 9A’ appeared in a press release that described a telephone ‘for the use of “Deaf Subscribers” who experience difficulty in the use of the standard telephone’.Following sustained complaints from customers, an improved amplified telephone (the Repeater 17A) was released in 1934.The advertisement in British Medical Journal was extensively cited in their report.The Post Office’s understanding of the variability and individuality of hearing limitations was influenced at this point by its collaboration with the medical scientist Dr. Phyllis Kerridge. Her 1935 report on ‘Aids for the Deaf’ in the Those who desired access to telephony in the inter-war years would almost certainly not have recognised the Deaf community and its cultures of the late twentieth century, but less scholarly attention has been paid to those who lost hearing later in life and did not affiliate themselves with the Deaf community. This is in part because there was not an identified community of people with hearing loss, and in part because the stigma surrounding deafness led those with limited hearing to identify as hearing and minimise the significance of their hearing loss. That the stigma attached to hearing loss was high during this time is evident from the rhetoric attached to the advertisements of hearing aids during the inter-war period. Hearing aid companies made exaggerated claims, using vivid language and images to persuade customers of their devices’ effectiveness. The most common trope in such advertisements was to draw on the stigma of deafness to sell their products by emphasising the inconspicuousness and invisibility of the hearing aids.In modern Deaf culture, hearing loss or limitation is not regarded as disabling; rather, the Deaf regard themselves as being defined not by their medical status but through their social and political status.th July that if the matter was not given immediate attention he would take the case up with the Postmaster General.However, the improved amplified telephone (the Repeater 17B) was £1 more  than the older model. This was not acceptable to users who felt they were being increasingly penalised for their hearing. For example, one subscriber (Mr. Mousley) in 1938 refused ‘to pay any additional rental in respect of it’, and threatened in a letter dated 28Mr Mousley now regularly uses special apparatus with which to carry on his normal business conversation. It consists of a headgear receiver connected to a portable valve amplifier, the power being drawn – I am told – from a 2 volt dry battery. The subscriber carries on a conversation apparently without difficulty when wearing the headgear; but in my opinion he is deafer than ever when not utilising this apparatus.The Sales Department therefore allowed Mousley a free-of-charge three-month trial of the improved amplifier. However, their real hope was ‘at the end of that time [to] be able to convince the subscriber that the difficulty that he is experiencing is not due to the service but rather to his affliction’.This dialogue provides an example of what has been described as epistemic injustice of a kind specific to the disabled.BMJ in 1935. Dr. Kerridge reported on ‘Aids for the Deaf’ and explained: ‘Amateur wireless constructors have often designed very satisfactory circuits for themselves or their relatives by the method of trial and error’.… has a quadruple microtelephone instrument, and wears the microphone hidden under his overall. With this help conversation is possible, and he is able to take instructions and keep his job. He uses one battery a week, and finds that the old ones will light his bicycle lamp after they are no good for the hearing aid.It is unclear whether this unusual headgear design was provided by a private company or if it was an invention of Mousley himself.… made himself a valve amplifier set, incorporating a tone control, with which he can hear conversation quite easily […] He finds the tone control satisfactory for clear understanding, and a further advantage is that he can tune out the unpleasant qualities of voices which he disliked in his hearing days.This example gives us insight into the everyday efforts of those trying to use telephone technology to contend with their hearing limitations in this period. It is striking how often such apparatus was characterised by user modification. Another example in this paper was of an amateur wireless constructor who had:This selective hearing and use of hearing aids as a means of control has also been noted in the usage of acoustic aids like ear trumpets, which could be powerfully utilised to signal boredom with a conversation.The development of amplified telephony was marked by tensions between the Post Office’s monopoly and its felt duty to provide a service to citizens with varying hearing needs. The amplified telephone was constructed by the Post Office in a process marked by user input and corresponding design modifications. Improvements to amplified telephony were affected by the complexities of matching individual user needs with the Post Office institutional set up and individuals’ lived experience of hearing conflicted with the Post Office’s desire for standardisation. As a Government department, standardisation was integral to the Post Office’s wider ethos regarding its customers, as providing the same service to all was integral to its democratic position. The aspiration for standardisation was also a built-in component of telephone networks more generally and its pursuit was partially driven by technical necessity. Today, telephony is often used by historians of technology to exemplify how a device can create a network effect because the desirability of the telephone directly correlated to the number of subscribers to the same system.physical attachments. This was an unusual decision because the Post Office operated with a strict blanket ban on any private apparatus on their lines. However, the Post Office did supply amplified telephones for their subscribers with hearing loss throughout the inter-war years, and this was a marked divergence from AT&T’s policy, perhaps indicating a somewhat more inclusive approach towards those with hearing limitations wrought by UK welfare-state ideologies.Mills’ work on AT&T will be used in this final section to provide a transatlantic comparison on the commodification of deafness in the telephone service. Although no single nationalised company in the United States held a state sanctioned monopoly over the telephone service as in Britain, AT&T held a practical monopoly over the telephone system in the US at this time. While AT&T’s monopoly was not legislated by the government, in practice it controlled the telephone service and fought off any competition to maintain its position. One seminal example of its monopolistic powers comes from the 1949–1968 case of the United States versus the Hush-A-Phone company, which centred on the Hush-A-Phone, a device which was attached by the telephone user onto the telephone to improve audibility. This was considered by AT&T to be an illegal attachment that infringed on its monopoly and AT&T went to court to successfully ban the Hush-A-Phone device.normal hearing rather than representing the true variability of hearing ability in the population.AT&T’s specialisation in hearing loss over general telephone lines contrasted with their refusal to provide customers with a telephone system suitable for the deaf, and this became the focus of a widespread campaign in the late 1960s.deafening was appropriated by AT&T as both a useful category and an applied term for telephone engineers. Thirdly, AT&T’s audiometric experiments and surveys on levels of normal hearing were utilised in medicine and used to define the ‘normal’ standard of hearing for the audiograms utilised in hearing tests.The different contexts of nationalisation versus private development meant that the standards in the UK and the US for normal hearing (the zero line of the audiometer) were different until 1964.Designating the standard of normal hearing in a narrow mechanistic fashion using an idealised average resulted in an increased disconnect between the objective measurement of hearing and the subjective correlate. The data used by Post Office engineers in devising the Artificial Ear created the standards that were used to design ‘normal telephones’ in Britain. If users did not have the ability to use the normal telephones, then it followed that they had to use the ‘telephone service for the deaf’. Therefore, these data – fed into the telephone – became the arbiter of a medical condition. Moreover, the National Institute for the Deaf attempted to improve the provision of electric hearing aids sold during the inter-war period by testing them using this machine.Telephony was ultimately used as a tool in the categorisation of disability by the Post Office. The amplified telephone was used by the Post Office to categorise their users’ identity as hearing (could use the standard telephone model), hard-of-hearing (could use the telephone when amplified), or deaf (could not use the telephone even when amplified). Categorisation largely depended on the efficacy of the technology rather than on the telephone user’s level of hearing. Simultaneously, clinicians used the telephone in the form of the audiometer to create standardised levels of normal hearing and defined deviance from that norm as deafness that could be corrected with appropriate hearing aids.During the inter-war years, the state of being deaf or hearing became defined through the ability, or otherwise, to use certain kinds of telephone – both literally in the form of the audiometer and socially through the ability to engage with the telephone. To retain their hearing identity and not be categorised as deaf, with the corresponding stigma that invoked, people with hearing loss engaged with amplified telephones. Through such interactions, telephony was used as a tool in the categorisation of disability and, in turn, telephone users modified the technology to fit their personal needs, experiences and identities. Yet this promise of improvement was not realised in practice because the Post Office’s standard amplified telephone model did not reflect either the significant diversity of users’ hearing or the variability of hearing loss. The standardisation of normal hearing and the categorisation of the deafened was therefore both facilitated and created in line with the priorities of the British Post Office’s telephone system. This analysis demonstrates the fluctuating and contingent thresholds of normalcy construction and reveals how deafness was socially and technologically constructed in inter-war Britain.While a growing number of historians of disability examine the multiple ways in which social contexts shape and define disability and ability, this analysis provides a new perspective on the fluid boundaries between hearing and deafness created by the telephone. This neglected episode of early twentieth-century telephony redefines the relationship between technology, communications, and disability, and broadens our historical understanding of deafness. Science and Technology Studies scholarship has decisively demonstrated that technologies are not neutral, but rather are shaped by the cultures, contexts, and the actors that make them.


It seems that diabetes mellitus (DM) can affect the auditory system due to neuropathy, micro-vascular complications, and hearing cell damage during hyperglycemic states. In the current study, we aimed to compare hearing status in patients with type 2 DM (T2DM) according to their blood-sugar control status.This cross-sectional study was carried out in 104 patients with T2DM attending the diabetic clinics of Guilan University of Medical Sciences within a period of 1 year (2014–2015). One group consisted of 52 patients with poor control and the other consisted of patients with moderate-to-good control (according to glycated hemoglobin [HbA1c] level). All subjects underwent pure tone audiometry (PTA) and distortion product otoacoustic emission (DPOAEs) assessments. A hearing threshold higher than 20 dB and a signal-to-noise ratio ≤3 in each frequency were considered abnormal.In PTA, poorly controlled patients showed more frequent hearing loss compared with the well-controlled group, especially at higher frequencies . Also, patients in the poorly controlled group had worse cochlear function according to the DPOAE test (4 kHz: 32.7% vs 17.3% [P= 0.002] and 8 kHz: 70.6% vs 40.4% [P=0.006]).DM and poor control status of diabetes can affect hearing sensation and cause hearing loss, especially at high frequencies. According to our findings, it seems that diabetic patients with a duration of diabetes >10 years, diabetic complications, poor control status or comorbidities should undergo both endocrine and audiologic follow-up to prevent greater sensory neural hearing loss. The number of people with diabetes mellitus (DM) is growing for a number of reasons including population growth, aging, urbanization, and the increasing prevalence of obesity and physical inactivity. Rational planning and allocation of resources rely on quantifying the prevalence of diabetes and the number of people affected by diabetes, now and in the future. Type 2 DM (T2DM) is a complex disease with both metabolic and vascular complications that affected about 285 million worldwide in 2010, and is predicted to rise to 438 million by the end of 2030 within the 20–70-year age group  who were selected from adult diabetic patients attending the diabetic clinics of our university hospitals during 2014–2015. Patients were selected using a systematic random sampling technique (every other patient who met the inclusion criteria was chosen) and referred to the Ear, Nose, and Throat (ENT) clinic of our hospital for a hearing assessment. All investigations were performed in accordance with the Declaration of Helsinki on biomedical studies involving human subjects, and informed consent was obtained from all participants. This study was approved by the Clinical Research Ethics Committee of Guilan University of Medical Sciences (GUMS).Well-controlled patients were defined as those who had met the following criteria: fasting blood sugar (FBS) ˂130 mg/dl, 2-hour postprandial blood sugar (2hrPPBS) ˂180 mg/dl, and HbA1c ˂7%. Patients aged 20–60 years diagnosed with T2DM according to World Health Organization criteria and with no previous history of ear disease were selected for the study. To rule out the influence of age‑related changes in hearing status, this study was performed in DM subjects aged ˂60 years, and the groups were matched in terms of age. Exclusion criteria were history of chronic ear disease such as chronic suppurative otitis media, history of ototoxic drug intake  in the past 3 months, abnormal otoscopic and tympanometric examinations, heavy smokers (more than one pack per day), occupational noise exposure, severe or uncontrolled DM, heavy alcohol consumption, and neurologic disorders such as multiple sclerosis.Examination procedureA complete detailed medical history  was taken. All subjects underwent biochemical investigations such as postprandial blood sugar (PPBS), FBS, HbA1c, total cholesterol, triglyceride, and low-density lipoprotein (LDL). A clinical and ENT examination was carried out and a history of otologic disease such as tinnitus and hearing loss was taken. An audiological examination including tympanometry and acoustic reflex was performed in all cases to rule out middle-ear disorders. Pure tone audiometry (PTA) was measured at 0.25, 0.5, 1, 2, 4, and 8 kHz and also, high-frequency PTA was measured at 10, 12, 14 and 16 kHz to detect the hearing threshold at each given frequency using an AC40 clinical audiometer  in a sound-isolated room, standardized according to the manufacturer’s instructions. Hearing loss was defined as the threshold of PTA at any frequency >20 dB HL. Speech reception threshold (SRT) and speech discrimination score (SDS) were investigated using one- and two-syllable words of equal stress (Spondees), and thresholds more than 25 dB HL and SDS lower than 75% were considered abnormal.Distortion product otoacoustic emissions (DPOAEs) were obtained using the Madsen Capella DPOAE System. Adequacy of probe fit was inspected prior to the commencement of data acquisition. A series of simultaneous pure tone pairs, of frequencies f1 and f2, at intensities of 65 and 55 dB SPL, respectively, were delivered to the test ear. All primary tones were maintained within ±1 dB of the set intensity. These stimulus intensity levels were chosen based on recommendations concerning optimal results in humans. To achieve optimal DPOAE results, the test frequency ratio (f2/f1) was set at 1.21. For each test ear, a DP-gram that plots intensity of the 2f1-f2 distortion product amplitude (and mean noise floor) against f2 frequency was obtained. To record the DPOAEs, the default protocol recommended by the manufacturer was used. Intensity of the 2f1-f2 distortion product amplitude at frequencies of 0.75–8 kHz was recorded and a signal/ noise ratio (SNR) ≥3 dB was considered as normal result.The main outcome variable was to profile the PTA and DPOAE findings in diabetic subjects. However, there were many variables of interest such as age, gender, glycemic control (HbA1C level), glycemic status (FBS and 2hrPPBS), duration of disease, tinnitus, vertigo and comorbid disease. After data collection, statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) version 19. The Kolmogorov-Smirnov test was used to evaluate normalcy of distribution of the data. We used the Mann-Whitney test or the Chi-square Fisher Exact and Kruskal Wallis tests to compare the data in the groups. To control the effects of various parameters, such as age, and duration of disease for example, we used multivariable analysis. The level of significance was considered to be <0.05.The study included 104 subjects with a mean age of 53.74±4.8 years , among whom 76% were female. The participants were divided into two groups according to HbA1c level (poor control and good control). Age distribution and HbA1c level, FBS and 2hrPPBS according to diabetes's control status are summarized in Using the Chi-square test, the number of males among well-controlled diabetic patients was 15.4% (8/52) compared with 32.7% in the poorly controlled group (17/52); which was statistically significant (P˂0.039).For all frequencies, except 0.25 and 0.5 kHz, most of the patients demonstrated normal hearing status (89.4% in the right ear and 91.3% in the left ear); in addition, at 1- and 2-kHz frequencies, more than 75% of subjects had normal hearing. At higher frequencies, the rate of hearing loss increased. At frequencies of 4 and 8 kHz, this could still be investigated with a conventional PTA test, and 56–59.5% and 43–54.5% of subjects had normal hearing status, respectively. However, at frequencies of 9, 10, 11, and 12 kHz, which are measured by high-frequency PTA, approximately 65%, 70–75%, 85% and 98% of patients demonstrated hearing loss .1c, in the poorly controlled group, hearing loss was more significant at the 8, 9, 10, and 11 kHz frequencies compared with the well-controlled group (P˂0.029) . However, at frequencies of 0.25 and 0.5 kHz in the left ear, two patients in the poorly controlled group and nine patients in the well-controlled group had hearing loss; however, due to the small sample size, this difference cannot be considered significant. According to level of HbAP˂0.029) .In the DPOAE test, most patients (>64–74%) had normal test results at a frequency ˂6 kHz, but at the 8-kHz frequency, approximately 46% of patients were normal and more than 53% were abnormal. The Chi-square test was used and showed a statistically significant difference at frequencies of 0.75, 2, and 3 kHz in the left ear and at 1 kHz in right ear (P˂0.05), and the difference was most pronounced at the 8-kHz frequency in both ears (P˂0.006), indicating that patients in the poorly controlled group had worse cochlear function according to the DPOAE test .To evaluate the association between hearing loss and existence of other complications related to diabetes , patients were divided into two groups: with or without complications. The results showed that at a frequency of 8 kHz in the left ear, 30 subjects among those with diabetic complications and 29 among patients without complications had hearing loss, while at a frequency of 11 kHz, 22 cases with diabetic complications and 29 patients without complications had hearing loss (P˂0.036).In this study, 57 patients (54.8%) had comorbidities such as hypertension, thyroid dysfunction, and rheumatological diseases, for example; of these 24 (42.6%) were in the well-controlled group and 33 (63.5%) were in the poorly controlled group. No significant difference was observed (P˂0.076) . To assess the association between the existence of comorbidities in diabetic patients and hearing loss, patients with and without comorbidities in two separate groups were investigated at different frequencies. The results showed that there was significant difference at frequencies of 10 and 11 kHz in the right ear and at the 8-kHz frequency in the left ear between these two groups . Among all patients, only 20 (19.2%) had tinnitus; 13 of whom were in the poorly controlled group and seven of whom were in the well-controlled group. The Chi-square test was used to analyze the association between tinnitus and glycemic control status. No statistically significant difference was observed between these groups (P˂0.135). In addition, 27 subjects (26%) experienced episodes of true vertigo; 13 of whom were in the well-controlled group with the remaining number in the poorly controlled group, with no statistically significant differences.To assess the effect of the duration of diabetes on hearing status, patients were divided into two groups: those with a known diabetes duration of ≥10 years and those who had been diagnosed for less than 10 years. At frequencies of 10 and 11 kHz, statistically significant differences were observed between hearing loss and duration of illness ≥10 years, in both ears (P=0.0001) .In addition, in order to evaluate the effect of age on hearing status, the patients were divided into two groups: 25–45 years and 45–60 years of age. The results of the DPOAEs tests revealed that for all frequencies except 2 kHz, the greatest hearing loss was seen in the 45–60-year age group in the left ear, and the majority of patients (63.4%) demonstrated hearing loss at the 8-kHz frequency (P˂0.004). These results in the right ear were such that at the 2-, 4-, and 8-kHz frequencies, 25.4%, 28.2%, and 57.7% patients in 45–60-year age group, respectively, demonstrated hearing loss, but the results were not statistically significant.To assess the association between FBS and 2hrPPBS levels and hearing loss, the patients were divided into different groups according to FBS and 2hrPPBS levels: FBS=50–100, 101–150, 151–200, and ˃200 mg/dl and 2hrPPBS ˂200, 200–300, 301–400, and ˃400 mg/dl. At high frequencies (>10 kHz), a statistically significant difference was seen between the different groups. The greatest associations were seen when FBS was 151–200 and ˃200 mg/dl, respectively, and also when 2hrPPBS was 301–400 and ˃400 mg/dl, respectively ,2.Jordao first described the association between DM and hearing loss , which iFor all frequencies except 0.25 and 0.5 kHz, most patients demonstrated normal hearing status, while at frequencies of 1 and 2 kHz, more than 75% of subjects had normal hearing .As the frequency increased, the rate of hearing loss also increased, such that at frequencies of 4 and 8 kHz, this could still be investigated with the usual PTA test, whereby 56–59.5% and 43–54.5% of subjects had normal hearing status, respectively. However, at frequencies of 9, 10, 11, and 12 kHz, which can be measured using high-frequency PTA, approximately 65%, 70–75%, 85%, and 98% of patients had hearing loss, respectively. These results show that high-frequency PTA is more accurate than the usual PTA test for early screening, and it may be helpful for the diagnosis of subclinical cases. Some studies revealed that higher frequencies were affected more in diabetic patients -19, whicThe results of the DPOAE test in the well-controlled and poorly controlled groups showed a statistically significant difference at frequencies of 0.75, 2, and 3 kHz in the left ear and at 1 kHz in the right ear; the difference was most pronounced at the 8-kHz frequency in both ears. This means that patients in the poorly controlled group had worse cochlear function according to the DPOAE test. Lisowska et al. found that the amplitudes of DPOAE were lower in T2DM patients in comparison with the control group. However Park recorded only 8-kHz decreases on DPOAE amplitudes in patients with diabetes compared with the control group ,21. In t1c, such that in the poorly controlled group, at frequencies of 8, 9, 10, and 11 kHz, this hearing loss was more significant than that in the well-controlled group. Panchu et al. reported that the lack of glycemic control reveals a positive correlation with the extent of hearing loss at all frequencies when compared with the good-glycemic control patients  demonstrated statistically significant hearing loss at the 8-kHz frequency. However, a study by Panchu et al. showed that the effect of age on auditory thresholds of diabetic patients was statistically insignificant .The present study revealed that at frequencies of both 10 and 11 kHz, highly statistically significant differences were seen between hearing loss and duration of illness ≥10 years, in both ears. Similar results have not been reported in other studies . Also, aOur study showed that at frequencies of 8 kHz and 11 kHz in the left ear, patients with diabetic complications had statistically significantly greater hearing loss compared with patients without complications. Naini et al. also reported 42% hearing loss at high frequencies in patients with diabetic complications , althougOur results show that there is a significant difference at 10 and 11 kHz frequencies in the right ear and at the 8-kHz frequency in the left ear between patients with and without comorbidities. Swaminathan et al. reported an increased association of hearing loss in people with high total cholesterol, high triglycerides, and high LDL levels , which iAccording to the findings of our study, it seems that diabetic patients with a duration of diabetes >10 years, patients with other diabetic complications, poorly controlled patients or patients with comorbidities should undergo audiologic and endocrine follow-up to prevent greater sensory neural hearing loss. We also propose more comprehensive and detailed studies with a greater sample size to further clarify these associations.


Nevertheless, significant hurdles remain before such technologies can be translated toward clinical use. These include addressing the blood-labyrinth barrier, engineering more specific and effective delivery vehicles, improving surgical access, and validating novel targets. In this review, we both highlight recent progress and outline challenges associated with in vivo gene therapy for human SNHL.Hearing loss is the most common sensory impairment in humans and currently disables 466 million people across the world. Congenital deafness affects at least 1 in 500 newborns, and over 50% are hereditary in nature. To date, existing pharmacologic therapies for genetic and acquired etiologies of deafness are severely limited. With the advent of modern sequencing technologies, there is a vast compendium of growing genetic alterations that underlie human hearing loss, which can be targeted by therapeutics such as gene therapy. Recently, there has been tremendous progress in the development of gene therapy vectors to treat sensorineural hearing loss (SNHL) in animal models  Hearing loss is the most common sensory impairment in humans and currently disables 466 million people across the world; this number is expected to rise to nearly 1 billion by 2050 . It is especially prevalent in the aging population as nearly two-thirds of the U.S. population over the age of 70 years are affected by disabling hearing loss . FurtherThe human inner ear is a small, three-dimensionally complex, fluid-filled structure encased in the densest bone in the body and located deep in the base of skull. Acoustic energy from sound is transmitted to the fluids of the cochlea via vibrations of the tympanic membrane and ossicular chain in the middle ear, producing a traveling wave along the basilar membrane. The length of the cochlea and stiffness of the basilar membrane enables the differentiation of sound frequencies . This in1. Broadly speaking, genetic hearing loss is subcategorized into Mendelian inheritance including both syndromic and non-syndromic cases, or complex inheritance which includes both genetic and environmental factors. Today, there are 115 genes responsible for non-syndromic hearing loss, with 45 autosomal dominant genes, 73 autosomal recessive genes, 5 X-linked genes, and additional loci for modifiers, Y-linked, and auditory neuropathy, respectively. On a global scale, the prevalence of hearing loss is highest in Eastern Europe, Central and South Asia, and Asia Pacific approaching 10%. Regions with lower income and literacy levels also tend to have higher rates of hearing loss  for severe to profound deafness. Nevertheless, neither approach restores the native inner ear sensory epithelium. Given the genetic basis underlying many forms of hearing loss and progress in our understanding of the mechanisms of hair cell regeneration and neuronal synapse repair, targeted modulation of affected genes in specific cell types of the inner ear could be a powerful therapeutic strategy. There have been numerous recent reports highlighting the complexity of genetic hearing loss and both non-viral and viral delivery approaches for therapeutic delivery . In thisThe translation of modern molecular therapy into clinical use is hindered by the so-called “delivery” challenge. To engineer a precise and effective inner ear delivery system, one must first develop a comprehensive understanding of the anatomic and physiologic barriers that isolate labyrinthine organs from the middle ear space and the brain. The perilymphatic space communicates via the round and oval windows to the middle ear, and via the cochlear aqueduct and cochlear modiolus to the cerebral spinal fluid (CSF) space . The artThe BLB has been compared to the blood–brain barrier (BBB), a complex system of endothelial cells, basement membrane, pericytes, and astrocytes that isolates and protects the central nervous system (CNS). While the complexity of the BBB renders delivery of macromolecule therapeutics challenging, it also presents unique opportunities for drug delivery across the barrier . Novel wUnder physiologic conditions, the intact BLB is thought to maintain ionic homeostasis, isolate from pathogens in the blood, and thereby contribute to the semi immune-privileged nature of the inner ear . By contin vitro, animal studies are required to understand the full panoply of the gene’s effects in the inner ear microenvironment. Furthermore, the long-term therapeutic effects, interactions with other organ systems and organ toxicities can only be characterized in a living host. Historically, in vivo gene delivery experiments have been carried out in zebrafish, birds, and rodents. Both zebrafish and birds have well-characterized genomes and robust regenerative capacity in the inner ear sensory epithelium after injury  correlate with body mass across species, with an estimated volume of 2.3–2.5, 59.4–63, and 191.1–237 μL for mouse, rhesus monkey, and human, respectively . While iGJB2 and SLC26A4 responsible for the majority of cases; most of the remaining cases are inherited in an autosomal dominant pattern, and a small fraction of up to 1.5% are either X-linked or mitochondrial  with mutations in hondrial . Comparein vivo gene therapy depends on many factors including the cell type being targeted, route of administration, and therapeutic potency. Delivery platforms can be broadly classified into viral vectors including adenoviruses (AdVs), adeno-associated viruses (AAVs), and retroviruses including lentiviruses  against TNF-α, known as tumor-penetrating nanocomplexes, was used to actively target primary vestibular schwannoma cultures in vitro . These nin vitro . By engiTo obtain surgical access to the inner ear in patients, two principal approaches employed clinically include intra- or trans-tympanic delivery, where the therapeutic is instilled into the middle ear space and allowed to diffuse into the inner ear; and intracochlear delivery, where the drug is introduced directly into the inner ear via the RWM, the oval window, or a semicircular canal . An effeIntratympanic or transtympanic injections are considered generally low risk procedures to access the middle ear space. In adult patients, this can be done under topical anesthesia in the office, where a small portion of the tympanic membrane is anesthetized and up to 0.7 mL of drug can be instilled through the perforation into the middle ear. While technically simple to perform, the drug must remain in contact with the RWM for a prolonged period of time to allow enough drug to cross the RWM . Equilibvia a stapedotomy.Another approach to access the inner ear is directly through the oval window. Normally, the stapes footplate overlies the oval window, and a mucosal epithelium similar to RWM lines the footplate facing the vestibule . In animA direct method for accessing the RWM can be achieved through a transcanal surgical procedure. With the advent of novel surgical technologies, it is now possible to directly access the round window niche (RWN) through a transcanal approach in the vast majority of patients using an endoscope. Endoscopic ear surgery not only enables superior visualization of complex landmarks within the middle ear  and mastThe RWM is a three-layered structure lined by squamous epithelium sandwiching a connective tissue layer, which serves as a dynamic barrier that protects the inner ear. A locally administered drug must permeate through the RWM to reach the perilymph within the scala tympani. The permeability of the RWM can be influenced by particle size, charge, and concentration , surgicain vitro may need to be placed in the scala media to be effective. In CI, a growing area of investigation involves using the implant electrode as a conduit for intracochlear delivery of drugs that may rescue hair cell or neuronal damage for hearing preservation. This could be achieved either through incorporation of the drug into the electrode, or co-administration of the therapeutic at the time of cochleostomy. Implantation of electrode arrays coated with fibroblasts over-expressing brain-derived neurotrophic factor (BDNF) or neurotrophin 3 (NT3) may have a protective effect on SGNs  over a prolonged period of time is required to drive the drug into perilymph while minimizing traumatic perturbations to the inner ear . In guin2.Since the first human gene therapy treatment in 1990, there have been 2930 gene therapy clinical trials that have been completed, were ongoing, or clinically approved world-wide. Over two-thirds of the trials were conducted in the United States . There iCurrently, over 20 clinical trials for hearing loss therapies are ongoing in the United States with six potential therapeutic molecules; one of these trials involves gene therapy. There are over 80 active trials in Europe, Asia, and Australia with many more candidate drugs being actively investigated. Preliminary results should be available within the next 2 years, and other platforms are currently being tested in early clinical studies with numerous drugs on the horizon. If early results meet the specified efficacy endpoints, the inclusion criteria will likely see an expansion to other patient populations such as children with early or congenital SNHL. On the contrary, if results from these trials do not meet criteria in the treated population, meticulous follow-up studies must be carried out to determine the cause of failure, which may include patient stratification, route of delivery, and measurement of outcomes. Ultimately, the successful translation of a novel therapeutic from the laboratory bench to the otology clinic or operating room require a multidisciplinary approach through the collaboration between molecular biologists, virologists, chemists, biomedical engineers, otologic surgeons, government, and business leaders.As treatments for hearing loss become more personalized, more gene targets that are amenable to targeting will be uncovered. Gene therapy can involve not only the insertion of a transgene through efficient viral transduction, but also silencing of a dominant negative allele through miRNA or siRNAs. Off-target effects will be minimized through enhancing the specificity of therapy. Next-generation CRISPR-Cas systems will be harnessed for precise disruption and editing of DNA or RNA for each patient.The efficacy and specificity of the gene delivery agent will also likely improve. Engineering of the capsid proteins may further improve viral tropism so that viruses can be tuned to preferentially target the cell subpopulation in the sensory epithelium while minimizing off-target effects. Incorporating cell type-specific promoters will enable the precise targeting and gene expression in cellular subpopulations within the cochlea. Simultaneous testing in animal models and humans will highlight differences between the two, and obstacles such as viral immunogenicity can be addressed. Active, or membrane-penetrating delivery platforms may provide a method to traverse cellular tight junctions . This maFuture surgical innovations and technologies could help better detect electrophysiological changes in the inner ear associated with therapeutic interventions. Electrocochleography studies could provide insight into cochlear changes in real time during an implant and predict hearing thresholds postoperatively, and may be utilized to monitor inner ear physiology and minimize the risk of cochlear damage during electrode insertion or infusion of gene delivery vectors . More soPost mortem temporal bone histology has been the gold standard to visualize the pathology underlying hearing loss. However, the advent of intracochlear imaging has been challenging due to the complex three-dimensional anatomy and complete encasement in the bony otic capsule. Future development of tools to non- or minimally invasively assess the function of the inner ear at a cellular resolution will likely revolutionize the way hearing loss is diagnosed and treated. Optical coherence tomography (Current assessments of hearing levels largely rely on indirect measurements such as audiograms, otoacoustic emissions, or auditory brainstem responses. mography , micro omography , and twomography  have themography . While smography . In the mography  and incoYR, LDL, and KMS designed the study and conducted the research and literature search. YR and KMS prepared the manuscript. All authors edited and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Autophagy is a conserved self-degradation mechanism that governs a large array of cellular processes in filamentous fungi. Filamentous insect and nematode mycopthogens function in the natural control of host populations and have been widely applied for biological control of insect and nematode pests. Entomopathogenic and nematophagous fungi have conserved “core” autophagy machineries that are analogous to those found in yeast but also feature several proteins involved in specific aspects of the autophagic pathways. Here, we review the functions of autophagy in protecting fungal cells from starvation and stress cues and sustaining cell differentiation, asexual development and virulence. An emphasis is placed upon the regulatory mechanisms involved in autophagic and non-autophagic roles of some autophagy-related genes. Methods used for monitoring conserved or specific autophagic events in fungal pathogens are also discussed. Autophagy is an evolutionally conserved degradation process much beyond a simply starvation-responsive process considered previously in eukaryotic cells . This seBeauveria bassiana and Metarhizium spp. are a large source of global mycoinsecticides and mycoacricides as alternatives to chemical pesticides [Filamentous entomopathogenic and nematophagous fungi play important roles in the natural control of host populations and have been widely applied for biological control of pest insects and nematodes ,8. As clsticides ,10. Fungsticides . The sucsticides –14. Uponsticides ,16. Nemasticides . The mycsticides , respectsticides ,20. ThisSaccharomyces cerevisiae, Komagataella pastoris (formerly Pichia pastoris) and K. phaffii are model species used in autophagic studies. Up to 42 genes have been found encoding autophagy-related proteins (ATGs) and mostly characterized in the yeasts, as summarized in The yeasts Ascosphaera apis [B. bassiana even possesses four ATG22 homologs, of which two (EJP69073 and EJP65688) are transcriptionally expressed during cell proliferation in host hemocoel [In insect and nematode mycopathogens, the ATG genes involved in different autophagic processes are not always identical with the yeast counterparts, and only those associated with A1C complex are completely conserved in fungi . For insera apis , suggestera apis ,26. In chemocoel  and anothemocoel . This suS. cerevisiae [S. cerevisiae since its homolog is absent in filamentous and other yeast species, such as Hansenula polymorpha, K. pastoris and K. phaffii. This implicates that a novel mechanism might exist in bulk autophagy of the species other than the budding yeast. In addition, selective autophagy required for cellular homeostasis includes mitophagy, pexophagy, ribophagy, reticulophagy and the Cvt pathway [K. pastoris and S. cerevisiae, respectively [S. cerevisiae has been found to interact with ATG9 and play a role in autophagasome formation [S. cerevisiae [B. bassiana, selective autophagy is evidently associated with cellular stress response, development and virulence. Loss-of-function mutation of ATG11 in B. bassiana has been shown to not completely block autophagic process in vacuoles but to abolish pexophagy and mitophagy during growth in vitro and in vivo [Filamentous fungal ATG proteins involved in specific autophagy pathway exhibit a low degree of conservation . During revisiae . Of thos pathway . In sele pathway  and is h pathway . ATG11 a pathway . In the  pathway . In spit pathway , ATG19 iectively ,34, but ectively . ATG39 iectively . Howeverormation . As a nerevisiae  and exis in vivo  althoughOverall, many of yeast ATG homologs, particularly those receptors, are distinct or absent in the genomic databases of insect and nematode mycopathogens ,38–40. TΔATG mutants in B. bassiana [Arthrobotrys oligospora [Interest in unveiling the prominent role of autophagy in the life cycles of insect and nematode mycopathogens is increasing in the postgenomic era. Effective methods have been explored to monitor autophagic events in these mycopathogens. The acidophilic dye monodansyl cadaverine (MDC) has been used as an indicator of acidic autophagosomes to examine whether the stained structures accumulate in the vacuoles of bassiana  or at thigospora . Due to igospora .in vivo hyphal bodies of B. bassiana [Metarhizium robertsii, another important insect mycopathogen [Highly conserved ATG8 is localized on the membrane of autophagosomes to be translocated into vacuoles and considered as a molecular marker for autophagic tracking due to its essentiality for preautophagomal structure formation and autophagosome maturation in eukaryotic cells ,44. Fluobassiana  and in tpathogen .B. bassiana [M. robertsii, autophagic bodies in the vacuoles of hyphal cells stressed by starvation are also well visualized via TEM [B. bassiana propagation within host hemocoel [in vivo sample of small size.Transmission electron microscopy (TEM) is the most effective method that allows for observation of various autophagic structures, such as phagophores, autophagosomes and autophagic bodies . This mebassiana . In M. r via TEM . Recentlhemocoel . This suin vivo propagation of fungal cells and efficiency of in vitro mass-production [The biological control potential of a fungal insect or nematode pathogen depends on not only the virulence or pathogenicity as an indicative ability to invade the host but also cell tolerance to environmental adversity and the asexual development that is critical for oduction . Thus, tB. bassiana may make use of autophagy to mobilize and recycle intracellular stored nutrients. This role is evidenced with abolishment of autophagic process in the absence of some ATG genes, such as ATG1, ATG5 and ATG8 [ΔATG mutants germinated as well as the wild-type conidia on rich medium but suffered germination defects in response to nutritional starvation on water agar and host cuticle. Additionally, deletion of ATG11 in B. bassiana resulted in a block of selective autophagy during conidial germination on oligotrophic substrata and significant germination defects under nutrient deficient conditions [During conidial germination on scant media or oligotrophic insect cuticle, and ATG8 ,41. The nditions . Similarnditions , in whicnditions .K. pastoris grown in an oleic acid-based medium and then shifted into a glucose-based medium [B. bassiana, both pexophagy and mitophagy are evidently involved in cell response to carbon shift [A plenty of fatty acids and lipids are used as carbon sources by insect mycopathogens during their infection to host through cuticular penetration . Upon end medium . In B. bon shift .B. bassiana, ATG1 and ATG8 are functionally different in antioxidant response since total activity of superoxide dismutases (SODs) decreased by 50–70% in absence of ATG8 but was not affected in absence of ATG1 [ATG1 or ATG8 in Aspergillus niger [ATG1 and ATG8 in S. cerevisiae resulted in enhanced SOD activities [B. basssiana to oxidative stress, and mechanistically different from their homologs in the antioxidant response of both mentioned fungi.Autophagy plays an important role in scavenging damaged organelles and proteins in the response of mammal cells to oxidative stress . In B. b of ATG1 . This cous niger . Similartivities . ApparenB. bassiana through pexophagy rather than mitophagy [Reactive oxygen species (ROS) causing oxidative damage to mitochondria can be scavenged by selective autophagy for mitochondrial homeostasis . Interesitophagy  althoughB. bassiana, normal autophagy is required for asexual development and morphogenesis. For instance, deletion of ATG1, ATG5 or ATG8 has been shown to greatly reduce the yields of aerial conidia as infective propagules or submerged blastospores as an index of in vivo dimorphic transition rate [M. robertsii [A. oligospora [Autophagy has been widely linked to cell differentiation in many filamentous fungi . In B. bion rate ,41. Autoobertsii  or in thigospora .B. bassiana [B. bassiana due to conidial size enlarged in absence of ATG5 [in vitro and in vivo life cycles of insect and nematode mycopathogens.Moreover, some ATG genes may regulate conidiation via different pathways. For example, a conidial protein (BbCP15) is required for conidiation due to the role of its acting as a downstream target of ATG1 instead of ATG8 in bassiana . ATG5 is of ATG5 . These fM. robertsii, ATG8 is essential for the formation of appressoria that initiate cuticular penetration in the course of host infection [ATG1, ATG5 or ATG8 resulted in blocked autophagy and attenuated virulence in B. bassiana [M. robertsii [A. oligospora [Magnaporthe grisea, a phytopathogenic fungus [Fungal virulence is a pleiotropic phenotype linked to an array of cellular processes and events. In nfection . Deletiobassiana ,41, M. robertsii  or A. oligospora . These sc fungus . The limB. bassiana, the ATG1 kinase may induce autophagy in response to starving cues [Sordaria macrospora (a filamentous ascomycete), a bZIP transcription factor required for vegetative growth and fruiting-body development represses transcriptional expression of ATG4 and ATG8 [A. oligospora by amino acid starvation in a manner absolutely depending on transcriptional regulation of GCN4 which activates a set of genes required for amino acid biosynthesis [ATG1 and ATG2 during the in vitro blastospore formation of B. bassiana [in vivo transcriptomic analysis has uncovered that all ATG genes are expressed during B. bassiana propagation in host hemocoel, including ATG4, ATG8 and ATG10 regulated by alternative splicing [B. bassiana, as unveiled by blocked autophagy in absence of Pil1B, restored autophagy in absence of Pil1A and opposite changes in transcript levels of many ATG genes in the mutant strains [In eukaryotes, autophagy is a precisely regulated self-degrading process. The target of rapamycin (TOR) pathway is considered to be a main regulator of autophagy and can inhibit autophagy via phosphorylation of ATG13, a regulator of ATG1 complex . The TORing cues . Transcrand ATG8 . Fungus-ynthesis . G-protebassiana , suggestsplicing . In addiin vitro and in vivo life cycles of entomopathogenic and nematophagous fungi, in which many ATG genes remain to be functionally explored. The previous studies restricted to several conserved ATG genes have unveiled that their roles in autophagic events are not necessarily similar to those learned from model yeasts or phytopathogenic fungi. We speculate that insect and nematode mycopathogens could have evolved a distinct autophagy-regulatory network that warrants their adaptation to entomopathogenic or nematophagous lifestyle, which could have originated from different evolution histories. In classic insect mycopathogens, for instance, the Beauveria/Cordyceps lineage is considered to have evolved insect pathogenicity 130 million years earlier than the Metarhizium lineage from plant affinity or pathogenicity [B. bassiana and Metarhizium spp [Autophagic events exert comprehensive effects on the genicity –40,60. Pzium spp . So do t


Haemaphysalis longicornis.Three main enzymes including cathepsin B, cathepsin D and acid phosphatase are involved in vitellin degradation, which is a major biochemical event of the embryonic development and can provide nutrients and metabolites for tick embryos. In the present study, the mRNA expression profiles and enzymatic activity of cathepsin B, cathepsin D and acid phosphatase were investigated during embryonic development in the tick The results revealed that all three enzymes were expressed throughout embryonic development. Both cathepsin B and acid phosphatase transcripts were accumulated during the first four days. Cathepsin B reached its highest expression on day 5, whereas the peak expression of acid phosphatase and cathepsin D occurred on day 11. The highest activity of cathepsin B was observed on the first day of egg development, whereas cathepsin D reached its highest activity on day 13. Acid phosphatase activity increased gradually during the first five days and then remained stable until the end of egg development.Three enzymes were expressed and activated in eggs, and also presented different dynamic changes with the development of embryos. The profiles of both mRNA expression and enzymatic activity of these enzymes indicate that they are controlled orderly and play multiple roles during embryonic development in ticks. Ticks are obligatory hematophagous ectoparasites of wildlife, domestic animals and humans. They are notorious vectors that can transmit various pathogens  among arthropods , 2. WithRhodnius prolixus, cathepsin D is an essential regulator for yolk protein degradation [Periplaneta americana [In ticks, vitellin (Vn) is a phosphorylated heme-lipoglycoprotein and its degraded products provide the source of amino acids, carbohydrates, heme, and other nutrients for embryonic development and unfed larva . Regulatradation . Acid phradation . It has mericana ; this isRhipicephalus (Boophilus) microplus, have suggested that some enzymes may play different roles in Vn degradation in vitro [Compared to insects, the functional roles of three enzymes in Vn degradation in ticks and the regulatory mechanisms are still poorly understood. Previous studies which were only based on one species of tick, in vitro –23. HoweHaemaphysalis longicornis is widely distributed in Australia, New Zealand, Korea, Japan and 17 provinces of China [Theileria spp., Babesia gibsoni, Rickettsia and Coxiella burnetti [H. longicornis infested sheep in Hunterdon County, New Jersey, USA, suggesting an invasion of H. longicornis which presents a significant threat to human and animal health in the USA [H. longicornis and its hormone regulation during ovary development [H. longicornis, finding that Vn from H. longicornis was composed of eight subunits with molecular weights of 112, 103, 80, 78, 71, 68, 62 and 52 kDa, respectively [The hard tick of China . Moreoveburnetti . Recentl the USA . In our elopment . Furtherectively .H. longicornis, aiming to reveal the existence and dynamic changes of three enzymes during embryonic development in tick H. longicornis.In the present study, we investigated the mRNA expression profiles and enzymatic activity of cathepsin B, D and acid phosphatase during embryonic development in H. longicornis ticks were collected on vegetation in Xiaowutai National Natural Reserve Area in China by flag dragging and reared on domestic rabbits, Oryctolagus cuniculus, as described by Liu et al. [Free-living u et al. . The rab® ND-1000 Spectrophotometer  and RNA integrity was evaluated by agarose gel electrophoresis. RNA samples were stored at -80 °C until use. The complementary DNA (cDNA) was synthesized from 4 μg of total RNA by reverse transcription reaction using TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix , which excluded genomic DNA contamination. The cDNA samples were stored at -20 °C until use.Total RNA from each developmental stage (100 mg of eggs) was extracted using the AxyPrep™ Multisource Total RNA Miniprep Kit  following the manufacturer’s protocol. The RNA concentration and purity were determined using a Nano DropH. longicornis. The genes chosen for this study are presented in Table H. longicornis, respectively. For control actin gene, primers were designed using the β-actin sequence (AN AY254898) from H. longicornis. All primers were designed with Primer Premier 5 considering a theoretical optimal annealing temperature of about 60 °C, with a melting temperature (Tm) difference between the forward and reverse primers not greater than 5 °C, guanine and cytosine percentages between 40–60%, primer lengths of 18–22 bp and free of dimers and hairpins.Primers were designed using the information from the GenBank sequences for different kinds of enzymes in ®-T1 Simple Cloning Vector  using pEASY®-T1 Simple Cloning Kit . The recombinant plasmid was used to transform the competent Escherichia coli DH5α cells and sent to Invitrogen  for sequencing. The resulting sequences were analyzed by a BLASTn search in GenBank and by using Clustal W method in the DNANAN V6 software .Positive polymerase chain reaction (PCR) products were purified and cloned into pEASY® Top Green qPCR SuperMix (TransGen Biotech) following the manufacturer’s instructions. The qPCR assays were conducted in 96-well polypropylene plates  in a 20 μl reaction volume comprising 1 μl of cDNA template, 0.4 μl of each 10 μM primer  and 7.8 μl of H2O. The thermal cycling program was as follows: 94 °C for 30 s, followed by 40 cycles of 94 °C for 5 s and 60 °C for 30 s. The primers with high amplification specificity were verified by different peaks observed in corresponding melting curves. Each plate contained triplicate reactions for each DNA sample. Melting curves were also traced after each assay to confirm that the fluorescence signal was retrieved from specific PCR products and to ensure the absence of primer dimers. The relative gene expression levels for each gene in each sample were calculated as Ct and transformed into relative values (RQ) by the 2−ΔΔCT method, where ΔΔCT =  sample −  control [Gene amplification was performed by qPCR in a Mx3005P qPCR system  using TransStartrpm for 10 min at 4 °C and the supernatant was transferred to a new tube for testing. The total protein concentration was determined according to Bradford [Eggs (1 g) were ground with a mortar and pestle in liquid nitrogen and dissolved in 20 mM sodium acetate buffer (pH 5.0). The homogenate was then centrifuged at 13,000× Bradford  using boThe activity of cathepsin B and D was assessed using Activity Fluorometric Assay Kits  following the manufacturer’s instructions. The Cathepsin B Activity Assay kit utilizes the preferred cathepsin B substrate sequence RR labeled with amino-4-trifluoromethyl coumarin (AFC). Samples that contain cathepsin B cleave the synthetic substrate RR-AFC to release free AFC. The released AFC can be quantified using a fluorometer or fluorescence plate reader at Ex/Em = 400/505 nm. The Cathepsin D Activity Assay kit utilizes the preferred cathepsin D substrate sequence GKPILFFRLK(Dnp)-D-R-NH2 labeled with 4-methylcoumarin-7-amide (MCA). Samples that contain cathepsin D cleave the synthetic substrate to release fluorescence, which can be quantified using a fluorometer or fluorescence plate reader at Ex/Em = 328/460 nm. The Acid Phosphatase Activity Assay kit uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate, which turns yellow (λmax = 405 nm) when dephosphorylated by acid phosphatase.P < 0.05.Six biological replicates, representing different batches of separated spawning events, were performed for mRNA expression and enzymatic activity assessment. Data in all groups were analyzed using SPSS 12.0 software. The differences in mRNA expression and enzymatic activity among samples were compared by ANOVA. The differences were considered statistically significant when Non-specific amplification was performed to confirm each primer pair using the Tm shown in Table F = 100.02, P < 0.0001). However, cathepsin B mRNA expression steadily decreased, along with the progress of embryonic development  = 39.66, P < 0.0001)  Fig. b.F = 269.59, P < 0.0001)  Fig. a.Fig. 3EF = 21.08, P < 0.0001)  Fig. b.F = 140.08, P < 0.0001). It reached highest expression on day 11 and quickly reduced until the end of development  = 17.05, P < 0.0001) (Fig. Unlike cathepsin B and D, acid phosphatase activity increased significantly in the first five days, and then remained stable at high levels until the end of egg development (ANOVA, F8,5 = 17.05Spodoptera exigua [Dipetalogaster maxima [Blatella germanica [R. prolixus [Bombyx mori [Musca domestica [Culex quinquefasciatus [Enzymes for protein hydrolysis in insect eggs are controlled scrupulously and orderly to ensure appropriate degradation of yolk proteins. These can be categorized into four types, namely cysteine proteases such as cathepsins B  and L 3, serine a exigua , Dipetalr maxima , Blatellermanica , R. prolprolixus , Bombyx byx mori , Musca domestica  and Culeasciatus .H. longicornis, we observed both mRNA expression and activity of these three enzymes during egg development from a very early stage until hatching. The results showed that all three enzymes were expressed during egg development, which agrees with that observed in insects in other studies [To evaluate the dynamic changes of cathepsin B, cathepsin D and acid phosphatase during Vn degradation and embryonic development in  studies , 16, 19. studies .Helicoverpa armigera and Seriola lalandi, in which cathepsin B showed the highest expression on the first day and gradually decreased during embryonic development [M. domestica [Fundulus heteroclitus [Phenacoccus solenopsis, the expression level was relatively low at the egg stage [H. longicornis is consistent with the results in He. armigera, which showed a steady decline during egg development [Cathepsin B is a kind of proteinase in cytolysosome, which plays a crucial role in yolk utilization in oviparous animals and is able to cleave peptide bonds of their substrates . In thiselopment , 36. Theomestica . The exproclitus , whereasgg stage . Neverthelopment .B. mori is also first increased and then subsequently decreased [R. prolixus [Cathepsin D belongs to lysosomal aspartic protease, which is nearly ubiquitously expressed. This enzyme participates in several physiological processes such as protein degradation, autophagy, programmed apoptosis regulation and enzymatic precursor activation . The mRNecreased . The actprolixus .P. americana [R. microplus [Acid phosphatases are known as hydrolases which are often associated with degradation of proteins. They usually described as lysosomal markers . Our resmericana  and R. microplus , in whicB. germanica and B. mori [P. americana, pH acidification might be a possible mechanism for proenzymes activation during embryogenesis [Pisaster ochraceus, researchers detected differences of egg proteins in cortical granule populations [D. maxima, where both pro-cathepsin D and cathepsin D were detected at all reproductive stages, but the highest cathepsin D activity was found at early and late atresia [Dermacentor andersoni completed an extension of germ band around yolk on day 5, while on day 11, the differentiation of leg segments became clear and palp endites have begun to fuse. As such, we speculate that in addition to the roles in these two stages of Vn degradation, these three enzymes also participate in tissue differentiation and organ formation during embryonic development, which results in the difference between mRNA expression and enzymatic activity.The reproduction and development of ticks highly rely on Vn degradation during embryogenesis. Various proteolytic enzymes are responsible for utilization of yolk proteins during embryonic development in hematophagous arthropods and insects, including acidic cysteine proteases such as cathepsins B  and L 3, serine  B. mori , 42, whiogenesis . Moreoveulations . Our res atresia . It is n atresia , embryosD. maxima, R. microplus and R. prolixus, acid phosphatase has been proved to cooperate with aspartyl protease to degrade Vn during embryogenesis [P. americana [M. domestica showed similar activity patterns during embryogenesis, the inhibition of acid phosphatase still did not affect yolk protein hydrolysis by cathepsin [In arthropods, the interaction effect between acid phosphatase and other hydrolases on Vn degradation during embryogenesis has been mentioned. In ogenesis , 16, 21.mericana . Conversathepsin . UnfortuH. longicornis. All three enzymes were not only expressed and activated in eggs but also presented dynamic changes with the development of embryos. Their transcription levels and activity profiles demonstrated for the first time in H. longicornis, that the three enzymes were existent and had different change patterns during egg development, indicating they were orderly controlled and played various roles in Vn degradation. However, further studies are required to explore the functions of these enzymes in Vn degradation during embryogenesis and their interaction. This will contribute to selecting the best potential molecular target and figuring out protective antigen for controlling tick life-cycle and disease transmission.In this study, we investigated the mRNA expression and enzymatic activity of cathepsins B, D and acid phosphatase during embryonic development in


Survival rates after childhood cancer now reach nearly 80% in developed countries. However, treatments that lead to survival and cure can cause serious adverse effects with lifelong negative impacts on survivor quality of life. Hearing impairment is a common adverse effect in children treated with cisplatin-based chemotherapy or cranial radiotherapy. Ototoxicity can extend from high-tone hearing impairment to involvement of speech frequencies. Hearing impairment can impede speech and language and neurocognitive development. Although treatment-related risk factors for hearing loss following childhood cancer treatment have been identified, the individual variability in toxicity of adverse effects after similar treatment between childhood cancer patients suggests a role for genetic susceptibility. Currently, 12 candidate gene approach studies have been performed to identify polymorphisms predisposing to platinum-induced ototoxicity in children being treated for cancer. However, results were inconsistent and most studies were underpowered and/or lacked replication.We describe the design of the PanCareLIFE consortium’s work packages that address the genetic susceptibility of platinum-induced ototoxicity.As a part of the PanCareLIFE study within the framework of the PanCare consortium, we addressed genetic susceptibility of treatment-induced ototoxicity during and after childhood cancer treatment in a large European cohort by a candidate gene approach and a genome-wide association screening.This study included 1124 survivors treated with cisplatin, carboplatin, or cranial radiotherapy for childhood cancer, resulting in the largest clinical European cohort assembled for this late effect to date. Within this large cohort we defined a group of 598 cisplatin-treated childhood cancer patients not confounded by cranial radiotherapy. The PanCareLIFE initiative provided, for the first time, a unique opportunity to confirm already identified determinants for hearing impairment during childhood cancer using a candidate gene approach and set up the first international genome-wide association study of cisplatin-induced direct ototoxicity in childhood cancer patients to identify novel allelic variants. Results will be validated in an independent replication cohort. Patient recruitment started in January 2015 and final inclusion was October 2017. We are currently performing the analyses and the first results are expected by the end of 2019 or the beginning of 2020. Genetic factors identified as part of this pan-European project, PanCareLIFE, may contribute to future risk prediction models that can be incorporated in future clinical trials of platinum-based therapies for cancer and may help with the development of prevention strategies.DERR1-10.2196/11868 Survival outcomes after childhood cancer have improved considerably over the last decades, now reaching approximately 80%. This marked increase is a result of advanced diagnostic and treatment procedures, improved stratification options, and optimized supportive care . NeverthHearing loss is frequently encountered in childhood cancer patients and survivors treated with platinum derivatives such as cisplatin and carboplatin -9. StudiEven though hearing loss is not a life-threatening disorder, it is a serious adverse effect of treatment, especially in children at ages before and during language acquisition. It can cause distress, anxiety, and depression leading to problems with speech development, neurocognitive functioning, school performance, and social life ,9,12-14.Apart from platinum compounds, several other risk factors for hearing loss during and after childhood cancer therapy have been identified. These include a high platinum dose, renal dysfunction, young age at diagnosis, concomitant use of other potentially ototoxic drugs, and cranial irradiation. However, in total these factors only partially explain the interindividual variability in ototoxic responses to platinum . This suPrerequisites for a satisfactory approach to identification of genetic determinants of platinum-induced hearing loss would be adequate numbers of research subjects and well-documented clinical and treatment data. The multinational PanCareLIFE (PCL) study provides a unique opportunity to investigate preexisting and novel genetic markers for treatment-related hearing loss in CCS . PCL is This study is part of the PCL project and addresses the genetic susceptibility of platinum-induced hearing loss. Specifically, the aims were to identify clinical and genetic risk markers in a large cohort of CCS and identify additional genetic risk markers of hearing loss by genome-wide association screening (GWAS) in a carefully characterized subgroup.In total, 8 work packages (WPs) are included in the PanCareLIFE study, of which 5 are scientific WPs. In this study, WP5 and WP4b will be addressed.WP5 aimed to identify clinical and genetic risk markers in a large cohort of CCS. For eligibility in the PCL WP5 genetic study, the following inclusion criteria were applied: (1) patients were younger than age 18 years at cancer diagnosis; (2) patients had been treated for cancer with cisplatin, carboplatin, or both; (3) patients were off therapy and had at least one pure tone audiometric evaluation available after the end of chemotherapy; and (4) patients had provided biomaterial  for DNA extraction . SubjectWP4b used a subset of research subjects from WP5 and aimed to identify additional genetic risk markers of hearing loss by GWAS with better control of nongenetic risk factors. An additional set of inclusion criteria was imposed to reduce potentially confounding factors and focus on the influence of cisplatin. These were (1) cancer treatment included upfront cisplatin, either cisplatin as the single platinum drug throughout the entire course of treatment or change from cisplatin to carboplatin during treatment, and (2) no radiotherapy administration to the brain or inner ear . ResearcThe PanCareLIFE study has been approved by the local ethics committees: Kantonale Ethikkommission Bern, 362/2015; Comitate Etico Regionale, 507REG2014; Ethical Committee University Hospital Brno, June 11, 2016; Ethics Committee Fakultni Nemocnice v Motole, Prague; De Videnskabsetiske Komiteer Region Hovedstaden, H-1-2014-125; Ethikkommission Medizinische Universität Graz, 27-015 ex 14/15; Ethikkommission der Universität Ulm, 160/17; Ethikkommission der Universität zu Lübeck, 14/181; Ethik-Kommission der Ärztekammer Westfalen-Lippe und der Westfälischen Wilhelms-Universität Münster, 2014-619; Medische Ethische Toetsings Commissie Erasmus MC; Medisch Ethische Toetsingscommissie, 2015_202. Informed consent was obtained from the patient or their the legal representatives.International Classification of Disease –coded diagnosis and date of diagnosis (for each tumor). Treatment-related data included information on platinum treatment  and potentially ototoxic comedication . The data were stripped of all identifiers and assigned a unique PCL-ID number, rendering the data pseudonymous for the investigators of this study. Data providers sent their data to the PCL data center in Mainz , which also collected and archived the genetic, clinical, and audiological data from the lab and the audiometry center.CCS were recruited through an institutional network from several countries in Europe. The participating institutions are referred to as data providers. Each data provider collected retrospective demographic, diagnostic, treatment, and audiometric data from their medical record files and registries. Diagnostic data included the The main outcome of this study was hearing function following platinum treatment in pediatric cancer survivors. Diagnosis of hearing loss was based on pure tone audiometry performed at frequencies of 250, 500, 1000, 2000, 4000, 6000, and 8000 Hz . Data prACYP2, LRP2, NFE2L2, OTOS, TPMT, SOD2, SLC22A2, GSTP1, ABCC3, and SLC16A5 [Detailed methods for DNA collection and genotyping are described elsewhere . For the SLC16A5 ,38,40-43 SLC16A5 . The Inf SLC16A5 .P<1*10–7) was assessed to identify potential genotyping errors. Samples with gender mismatches, familial relationships, and extreme heterozygosity were removed to ensure sample quality. After the quality control, principal components were calculated in order to adjust for population heterogeneity and technical confounders in all subsequent analyses [A stringent quality control protocol was applied where multiple filters were used to ensure the quality of the genetic data prior to either imputations or analysis. The quality control procedure is described elsewhere . To remoanalyses . Imputatanalyses . The refanalyses . This paanalyses  and Geneanalyses .P value threshold of P<5*10–8 for the GWAS analysis, a cohort of 574 patients was considered sufficient to detect an odds ratio of at least 2.8 with a statistical power of 80% in the design of the study.To estimate the number of cases required for the GWAS analyses, a sample size calculation was performed. Assuming a risk allele frequency of 0.2, a case to control ratio of 1:1, and a P<1*10–6 was used to identify relevant SNPs that could be important but did not reach genome-wide significance (P<5*10–8). All statistical analyses were performed by investigators of WP4b and WP5 in close collaboration with the Biostatistical Support Group of UMC Mainz and Ulm.For both candidate gene and GWAS analyses, genetic profiles from children who were treated with cisplatin and have hearing impairment were compared to those of children treated with cisplatin who did not develop hearing impairment. Relationships of categorical data were compared using the chi-square and Fisher exact tests. Comparison of distribution between groups with continuous data was tested with the Mann-Whitney and Kruskal-Wallis tests. Standard logistic regression models adjusting for age at diagnosis, gender, total cumulative cisplatin dose, and principal components were employed to calculate odds ratios with 95% confidence intervals in order to assess the risk of hearing loss. Principal component analysis, a common tool that has been widely used for the combined analysis of correlated phenotypes in genetic linkage and association studies, was used to correct for population stratification by modeling ancestry differences between cases and controls. Bonferroni correction was used in the candidate gene analysis to adjust for multiple testing. In the GWAS, a suggestive significance threshold of For both the WP4b candidate gene approach and WP4b GWAS, replication analysis is planned within an independent Canadian cohort from the Canadian Pharmacogenomics Network for Drug Safety (CPNDS).Study participants were recruited through a network of 14 institutions from 7 countries: Switzerland, Italy, Czech Republic, Denmark, Germany, Austria, and the Netherlands. The data providers and number of patients per data provider are shown in Patient recruitment started in January 2015 and final inclusion was October 2017. We are currently performing the analyses and the first results are expected by the end of 2019 or the beginning of 2020.This paper describes the design of the PCL ototoxicity genetics study aiming to identify clinical and genetic risk markers in a large and heterogeneous cohort of CCS and identify additional genetic risk markers of hearing loss by GWAS with better control of nongenetic risk factors in a more homogenous subcohort of CCS. Data were collected from 1124 CCS from 7 different European countries. Some of the previously identified genetic variants for hearing loss were validated by a candidate gene approach. In addition, the first international GWAS of cisplatin-induced hearing loss sets out to identify novel allelic variants in the largest European cohort assembled for such a genome-wide pharmacogenetics association study so far.For this study, a subcohort was recruited consisting of patients who were treated with cisplatin and did not receive cranial irradiation, a well-known independent risk factor for sensorineural hearing loss. From 14% to 27% of children who received radiotherapy without ototoxic chemotherapy suffered from high-frequency hearing loss ,52. ThisAppropriately sized cohorts are required to identify genetic determinants of platinum-induced hearing loss. The many associations that are tested in a GWAS require a very low significance threshold to prevent an inflated genome-wide type I error. This reduces the probability of identifying SNPs with small effect size, unless sample sizes are large enough to achieve sufficient power to identify such SNPs. The large combined cohort within the PCL consortium is expected to provide adequate statistical power.Many classification systems of drug-induced hearing loss have been developed—Brock grading system , AmericaThe availability of a large European set of clinical, audiometric, and genetic data provides the PCL consortium excellent opportunities for further collaboration, including replication studies in independent transatlantic cohorts or meta-analyses. In order to validate findings from the initial discovery cohort, it is standard practice to include an independent replication cohort. A collaboration with the CPNDS for replication of results of this study has been initiated. The patients enrolled in our replication cohort were recruited from 11 hospitals and health care centers in Canada. Hearing loss in the CPNDS cohort was originally graded according to the CTCAE . ApplyinThe PanCareLIFE ototoxicity studies have some limitations. As a result of missing or unclassifiable audiograms, some patients cannot be included due to a missing phenotype. Because many of the patients with missing audiograms might have good hearing function, they might therefore no longer be followed up for audiometric testing, As a consequence, the risk of ototoxicity based on the results of this study might be an overestimation of the true risk. Currently, the International Late Effects of Childhood Cancer Guideline Harmonization Group is developing recommendations for audiological monitoring in CCS. The guideline unifies existing recommendations and provides optimum follow-up practices, which is important for consensus on the frequency and timing of audiological evaluations after childhood cancer .In summary, our paper described the design of a genetic susceptibility study that addresses an important late effect of cancer therapy . Identification of genetic risk factors may assist in the development of more accurate prediction models that can be incorporated in future clinical trials of platinum-based therapies for cancer. Increased knowledge of nongenetic and genetic risk factors of cisplatin-induced hearing loss may contribute to the development of preventive methods to improve quality of life in CCS.


Hearing impairment is a risk factor for dementia but the mechanism underlying this association is unknown. We investigated the relationship between hearing and cognitive performance and brain β-amyloid deposition in 252 adults aged 67-88 years (37% black race) without dementia from three U.S. communities. Global cortical standardized uptake value ratios (SUVRs) were calculated from florbetapir-positron emission tomography scans, with elevated SUVR defined as >1.2 . Air conduction hearing threshold levels for the frequencies of 0.5, 1, 2 and 4 kHz were obtained by pure tone audiometry and averaged for the better-hearing ear to yield a pure tone average (PTA) in decibels hearing level (dB). A composite cognitive score was created from ten neuropsychological tests summarized using latent variable methods. Multivariable-adjusted linear and Poisson regression with robust standard errors were used to estimate the average difference in cognitive scores and prevalence of elevated SUVR, respectively, by hearing impairment status. In analyses adjusted for age, sex, education and APOE ε4 status, hearing was not associated with elevated SUVR [prevalence ratio per 10 db increase (worse hearing) = 0.94 ]. Results did not differ by race. In contrast, each 10 db increase in hearing impairment was associated with 0.08 standard deviation  lower cognitive score, after adjustment for demographic and cardiovascular factors. Poorer hearing is associated with poorer cognitive performance but not with amyloid deposition, suggesting that the mechanism underlying the relationship between hearing and cognition may be independent of Alzheimer’s-related pathologic brain changes.


A receptor-mediated sedative-hypnotic actions restrict use. In this study, we defined the sedative-hypnotic and steroidogenesis inhibiting actions of etomidate and four phenyl-ring substituted etomidate analogs -etomidate) that possess negligible GABAA receptor modulatory activities.Cushing’s syndrome is an endocrine disorder characterized by the overproduction of adrenocortical steroids. Steroidogenesis enzyme inhibitors are the mainstays of pharmacological treatment. Unfortunately, they produce significant side effects. Among the most potent inhibitors is the general anesthetic etomidate whose GABA− 1 min− 1) of either etomidate or an etomidate analog.In the first set of experiments, male Sprague-Dawley rats were assessed for loss of righting reflexes (LoRR) after receiving intravenous boluses of either etomidate (1 mg/kg) or an etomidate analog (40 mg/kg). In the second set of experiments, rats were assessed for LoRR and their abilities to produce adrenocortical and androgenic steroids after receiving 2-h infusions . Rats that received etomidate or isopropoxy-etomidate had significant reductions  in plasma testosterone concentrations whereas those that received naphthalene-etomidate had significant increases (1400%) in plasma dehydroepiandrosterone concentrations. Neither etomidate nor any etomidate analog significantly affected plasma androstenedione and dihydrotestosterone concentrations.Our studies demonstrate that the four phenyl-ring substituted etomidate analogs form a novel class of compounds that are devoid of sedative-hypnotic activities and suppress plasma concentrations of adrenocortical steroids but vary in their effects on plasma concentrations of androgenic steroids. Cushing’s syndrome is a rare endocrine disorder that is characterized by the overproduction of adrenocortical steroids and associated with significant morbidity including insulin resistance, hypertension, immunosuppression, cardiovascular disease, neurocognitive deficits, and early mortality –6. It isAlthough several classes of drugs can be utilized in the treatment of Cushing’s syndrome, steroidogenesis enzyme inhibitors are the mainstays of pharmacological management . SteroidA receptor [A receptor modulatory activity and resultant sedative-hypnotic actions of etomidate are undesirable side effects that have led to recommendations that etomidate administration be restricted to highly monitored hospital environments such as an intensive care unit [R)-ethyl 1-ethyl)-1H-imidazole-5-carboxylate (dimethoxy-etomidate), a phenyl ring substituted etomidate analog that retains the potent 11β-hydroxylase inhibitory activity of etomidate but is essentially devoid of the drug’s GABAA receptor modulatory and sedative-hypnotic actions [A receptor modulatory activity , and Glycine-HCl were purchased from Sigma-Aldrich Chemical Company . Isoflurane was purchased from Patterson Veterinary . Heparin and saline were purchased from Hospira . EDTA-containing vacutainer tubes were purchased from Becton Dickinson . Etomidate was purchased from Bachem . Enzyme-linked immunosorbent assay kits for corticosterone were purchased from IDS , whereas those for aldosterone, dehydroepiandrosterone (DHEA), and testosterone were purchased from Enzo Life Sciences , and those for androstenedione and dihydrotestosterone were purchased from MyBioSource . Dimethoxy-etomidate, (R)-ethyl 1-(1-(3-isopropoxyphenyl)ethyl)-1H-imidazole-5-carboxylate (isopropoxy-etomidate), (R)-ethyl 1-ethyl)-1H-imidazole-5-carboxylate , and (R)-ethyl 1-ethyl)-1H-imidazole-5-carboxylate -etomidate) were custom synthesized by Aberjona Laboratories  and their structures confirmed by nuclear magnetic resonance spectroscopy.ACTHAll studies were conducted with the approval of and in accordance with rules and regulations of the Institutional Animal Care and Use Committee at the Massachusetts General Hospital, Boston, Massachusetts (Protocol # 2013 N000130) and the principles outlined in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. Adult male Sprague-Dawley rats (300–500 g) were purchased from Charles River Laboratories  and housed with a 12–12 light-dark cycle with light switched on at 7 AM. Rats were caged in our animal care facility, which is maintained at 22.5 °C, and allowed free access to food and drinking water until the day of study. Rats were housed socially (i.e. with other rats) whenever possible with access to enrichment materials (wooden block and plastic tube). Each rat was weighed on the day of study. All studies were performed in our laboratory at the Massachusetts General Hospital and carried out at room temperature. During infusions, animal core temperature was monitored and maintained at a target temperature of 36–37 °C using a heat lamp. At the end of study, all animals were euthanized by our veterinary staff in the animal care facility by carbon dioxide asphyxiation or in our laboratory under propofol anesthesia using intravenous KCl as recommended under American Veterinary Medical Association guidelines.− 1. An etomidate intravenous bolus dose of 1 mg kg− 1 was chosen for context with the expectation that it would produce loss of righting reflexes in all rats [For experiment 1, our goal was to test all etomidate analogs for sedative-hypnotic activity at the highest attainable intravenous bolus dose that was reliably non-lethal. That dose was determined in pilot studies to be 40 mg kgall rats . On the − 1 min− 1) given the limitations in etomidate and analog aqueous solubility and infusion volume constraints in rats. On the day of study, rats were block randomized into 6 rats per treatment group -etomidate group). Two 24-gauge intravenous catheters were placed in each rat  under brief isoflurane (2–3%) anesthesia to allow the reliable intravenous administration of drugs and test compounds and to draw blood. Catheter patency was maintained with a heparin lock flush. Animals were then allowed to recover for at least 2 hours prior to study. To avoid potential interference with our steroid assays by Captisol [For experiment 2, our primary goal was to assess the impact of 2-h etomidate and etomidate analog infusions on steroidogenesis. Our secondary goal was to determine whether such infusions produce sedation/hypnosis. For these studies, we used the highest attainable dose  or at the end of a 2-h etomidate or etomidate analog infusion (Experiment 2), each rat was assessed for sedation/hypnosis using a loss of righting reflex assay . In thisIn Experiment 2, the in vivo steroidogenic actions of etomidate and etomidate analogs were assessed in male rats (six per group) using a hormone stimulation test Fig. . For thep < 0.01.All individual data points are expressed as the mean ± the S.E.M. of six independent measurements. Group size was chosen based on previous experience . Statistn = 6 rats) exhibited loss of righting reflexes. This loss of righting reflexes lasted for 3.4 ± 0.3 min. In contrast, none of the rats that received an etomidate analog bolus  exhibited loss of righting reflexes.All rats that received an etomidate bolus . In contrast, none of the 24 rats that received a 2-h infusion of any of the four etomidate analogs  exhibited loss of righting reflexes.Consistent with our previous studies , 29, 30,In contrast to their similar inhibitory actions on stimulated plasma concentrations of the two adrenocortical steroids , etomidate and the four etomidate analogs varied in their impact on stimulated plasma concentrations of the androgenic steroids testosterone Fig. a and dehBoth naphthalene-etomidate and naphthalene (2)-etomidate increased hCG-stimulated plasma DHEA concentrations in rats from a vehicle control value of 750 ± 330 pg/mL to values of 11,600 ± 2600 pg/mL and 2800 ± 590 pg/mL, respectively. In the case of naphthalene-etomidate, this increase (1400%) reached statistical significance. hCG-stimulated plasma DHEA concentrations in rats that received etomidate (940 ± 230 ng/mL), dimethoxy-etomidate (770 ± 230 ng/mL), and isopropoxy-etomidate (1270 ± 570 ng/mL) were not significantly different from those in the vehicle control group  unlike etomidate, are essentially devoid of sedative-hypnotic activities; (2) similar to etomidate, suppress plasma concentrations of adrenocortical steroids; and (3) vary in their effects on plasma concentrations of androgenic steroids.In the current manuscript, we report the results of animal studies aimed at further defining the pharmacology of dimethoxy-etomidate – a potential lead compound for the development of new drugs to treat Cushing’s syndrome – and three new etomidate analogs that also contain bulky phenyl-ring substituent groups and are essentially devoid of GABASteroid biosynthesis is primarily mediated by enzymes belonging to the cytochrome p450 and hydroxysteroid dehydrogenase superfamilies. The structures of the enzymes that comprise each of these superfamilies are highly homologous, explaining the low selectivities that most exogenous ligands (e.g. steroidogenesis inhibitors) possess for superfamily members . For exaA receptor positive modulatory actions thought to mediate the sedative-hypnotic effects of etomidate [As expected, we found that all rats that received a 1 mg/kg etomidate bolus or 2-h etomidate infusion exhibited loss of righting reflexes, an animal correlate for sedation/hypnosis. In contrast, none of the rats that received a 40-fold higher bolus dose of an etomidate analog (i.e. 40 mg/kg) or a 2-h etomidate analog infusion lost their righting reflexes. Such a lack of sedative-hypnotic activity among the etomidate analogs is consistent with previous electrophysiological work showing that these analogs are essentially devoid of the GABAtomidate , 38, 39.3 (11β-hydroxylase) and 334 Å3 (18-hydroxylase), which are only modestly larger than the molecular volume of etomidate (269.7 Å3) [3 and 52.7 Å3, potentially introducing steric hindrance and reducing the binding affinity to these enzymes [Consistent with previous studies, we also found that etomidate reduces plasma concentrations of corticosterone and aldosterone , 29, 30.69.7 Å3) . The add enzymes . Such a Although etomidate and all four etomidate analogs significantly reduced plasma concentrations of both adrenocortical steroids, only two compounds (etomidate and isopropoxy-etomidate) significantly reduced those of any androgenic steroid. This reduction was limited to testosterone, whose plasma concentrations were reduced by 90% (etomidate) and 57% (isopropoxy-etomidate). In contrast, naphthalene-etomidate actually increased plasma concentrations of the androgenic steroid DHEA by more than an order of magnitude without significantly affecting those of the other androgenic steroids. Unfortunately, the mechanisms responsible for these actions cannot be unambiguously determined from our data. However, previous in vitro studies indicate that etomidate can inhibit 17β-hydroxysteroid dehydrogenase leading to suppression of testosterone synthesis.In conclusion, we have defined a novel class of steroidogenic enzyme inhibitors consisting of etomidate analogs that contain bulky phenyl ring substituent groups. Compounds in this class are devoid of sedative-hypnotic activity and inhibit stimulated plasma adrenocortical steroid concentrations, but differ in their effects on stimulated plasma androgenic steroid concentrations. They provide a proof-of-concept for the development of non-sedating etomidate analogs to treat Cushing’s syndrome as well as other pathologies whose clinical courses may be improved by altering steroid biosynthesis.