Health systems face challenges to accelerate access to innovations that add value and avoid those unlikely to do so. This is very timely to the field of age-related sensorineural hearing loss (ARHL), where a significant unmet market need has been identified and sizeable investments made to promote the development of novel hearing therapeutics (NT). This study aims to apply health economic modeling to inform the development of cost-effective NT.We developed a decision-analytic model to assess the potential costs and effects of using regenerative NT in patients ≥50 with ARHL. This was compared to the current standard of care including hearing aids and cochlear implants. Input data was collected from systematic literature searches and expert opinion. A UK NHS healthcare perspective was adopted. Three different but related analyses were performed using probabilistic modeling: (1) headroom analysis, (2) scenario analyses, and (3) threshold analyses.The headroom analysis shows an incremental net monetary benefit (iNMB) of £20,017 compared to the standard of care due to quality-adjusted life-years (QALY) gains and cost savings. Higher therapeutic efficacy and access for patients with all degrees of hearing loss yields higher iNMBs. Threshold analyses shows that the ceiling price of the therapeutic increases with more severe degrees of hearing loss.NT for ARHL are potentially cost-effective under current willingness-to-pay (WTP) thresholds with considerable room for improvement in the current standard of care pathway. Our model can be used to help decision makers decide which therapeutics represent value for money and are worth commissioning, thereby paving the way for urgently needed NT. Hearing loss currently affects almost 500 million people worldwide and this number is anticipated to rise to 900 million by 2050 as the world’s population ages and the number of individuals with age-related hearing loss (ARHL) increases . This tyThe mainstay of treatment for ARHL includes hearing aids for those with mild to moderate hearing loss and cochlear implants for those with severe to profound deafness . AlthougBiotechnology and pharmaceutical companies have identified this unmet market need and have dedicated sizeable effort and investments in the development of novel approaches to treat ARHL . A betteBecause these therapeutics have the potential to drastically change hearing care pathways in the next 5 years, it is crucial to start planning for their implementation . Early hThis study applies these principles and uses early health economic modeling to assess the potential added value of novel regenerative therapeutics in ARHL compared to the current standard of care.Ethics approval was granted by the University College London Research Ethics Committee 12241/001. Informed consent was obtained from all participants.A state-transition model, following the ISPOR-SMDM Best Practice Guidelines, was created using Microsoft Excel  to assess the potential costs and effects of using novel regenerative hearing therapeutics in adults, 50 or older, with ARHL . This waThe model adopts a cycle length of 1 year and spans the patient’s lifetime until death given the life-long costs and effects of hearing loss. The model is constructed from a healthcare perspective of the National Health Service (NHS) in the United Kingdom and evaluates direct medical costs. A willingness to pay threshold of £20,000 per quality-adjusted life year (QALY) is used to assess the cost-effectiveness (CE) of the intervention . Both con = 24) from the pharmaceutical and biotechnology industry, national hearing charities, otolaryngology, audiology, discovery science, and research funding bodies. The model outcomes were cross validated with relevant literature. No other health economic models on ARHL were found for cross-validation. Additionally, the model was verified for inconsistencies by two independent modeling experts.The model was validated using the AdViSHE validation assessment tool . The conWith no regenerative therapeutic for ARHL having been approved for clinical use, we used a hypothetical regenerative therapy to model different efficacy scenarios. Our model focused primarily on regenerative therapeutics and was not targeted toward a specific gene, cell, molecular therapy or method of delivery. In order to estimate the maximum potential benefit of the therapeutic (headroom), the base case scenario assumed a 100% adherence, uptake and efficacy of the therapy with zero costs. This meant that patients with any form of hearing loss recovered to normal hearing after therapy administration. It also assumed that rates of hearing loss progression in subsequent years were unaffected, meaning that patients could once again develop age-related hearing loss. The model assumed that all patients were eligible for both existing and novel strategies and could receive either a hearing aid, a cochlear implant, or the novel therapy as they developed hearing loss in the model.Transition probabilities were derived from published literature and expert opinion . The modHealth effects were measured using QALYs that factored both length and quality of life into a single measure (SDC 6). Preference based utility measures of health-related quality of life were obtained from published literature . Due to Direct medical costs were assessed in 2018 Pounds (£) and adjusted for inflation using the consumer price inflation indices produced by the World Bank and the UK Office for National Statistics (SDC 7) . Unit coA hypothetical cohort of 1,000 patients entered the model to determine the mean expected costs and effects (QALYs) per patient. Three different but related analyses were performed: (1) headroom analysis, (2) scenario analyses, and (3) threshold analyses. All results were obtained using probabilistic modeling.The headroom analysis explored the maximum potential value of a novel regenerative therapeutic for ARHL. The headroom approach to therapeutic developments relies on the estimation of value-based price-ceilings. Such price-ceilings estimate the commercial opportunities for new products in health care systems . To deliProbabilistic sensitivity analyses (PSA) with 10,000 iterations were used to obtain results for all scenarios to account for uncertainty around parameter estimates . See Tabn × threshold value – Costsn) – , where n = novel therapeutic, NICE threshold value = £20,000/QALY and, c = current treatment . The iNMB represents the added value of an intervention compared to the current standard of care, in monetary terms. iNMB is calculated by using the following formula: iNMB = (QALYreatment . Higher The results demonstrated that compared to the headroom scenario (scenario 1), changing the recovery rates by only one (scenario 2) or two (scenario 3) hearing health states lowers the iNMB to £7,521  and £9,265 , respectively . Since tThe threshold analyses illustrate the ceiling prices of the novel regenerative hearing therapeutics with differing rates of effectiveness and hearing loss severities . The linThe headroom scenario yields an iNMB of £20,017  compared to the current standard of care and show that novel regenerative hearing therapeutics have potential room for improvement in terms of both improved patient outcomes with QALY gains of 0.78 [0.37–1.19] and cost savings of £4,451  per patient. Scenario analyses demonstrate that the iNMB increases with more effective therapeutics and more widespread eligibility criteria. Threshold analysis suggests that the ceiling price of the therapeutic is greater with severe forms of hearing loss compared to less severe states. Both scenario and threshold analyses demonstrate wide confidence intervals which reflects the uncertainty surrounding results.This study shows that there is significant potential room for improvement in the current standard care pathway for patients with ARHL and that novel regenerative hearing therapeutics could become cost-effective in the NHS. This model can be used by both industry and policy makers to evaluate: (1) the maximum price of a novel regenerative therapeutic in ARHL for different levels of effectiveness, (2) the minimum effectiveness required at each pricing target for the therapeutic to remain cost-effective, and (3) the impact of limiting treatment to certain subgroups of patients with hearing loss. Exploring the ceiling price of novel therapeutics at maximum efficacy will also allow pharmaceutical companies to perform feasibility estimates on the potential to recoup research and development (R&D) costs .Given that one in five people in the United Kingdom are estimated to be affected by a hearing disorder by 2035, means that the implementation of novel hearing therapeutics will bring about substantial change to health systems . The devDespite the robustness of our results to a variety of probabilistic sensitivity analyses, this study is subject to limitations; the first of which arises from our limited scientific understanding of ARHL. In addition to ARHL not being well understood, its association and interrelationship with age related neurocognitive decline further complicates our understanding. ARHL is not a single disease entity but a symptom of a range of underlying disease mechanisms and etiologies, resulting in an equal range of potential therapeutic targets within the inner ear and auditory pathways . As suchAnother limitation is that we did not include drug safety profiles for novel hearing therapeutics due to their hypothetical nature. This model assumes that along with being perfectly effective, the novel hearing therapies are free of adverse events which overestimates their potential added value. Finally, this study focused on direct medical costs incurred by the NHS and did not include indirect medical costs associated with hearing loss in either the standard of care or novel hearing therapeutic arms . This haConsultation with our panel of expert stakeholders revealed strong support to move away from the classic classification of hearing loss using pure-tone averages to a classification scheme that incorporates functional impairment. Hearing in Noise Testing (HINT) was proposed as a potential solution moving forward. In order to construct new models using HINT, as opposed to pure-tone averages, further research will be required to delineate both ARHL transition probabilities and utilities in identified HINT states.A growing body of evidence suggests that the use of EQ-5Ds, as recommended by NICE, may not be appropriate for all health conditions such as hearing disorders and vision loss due to the poor responsiveness of the measure to detect change . Our revFinally, this study not only has the capacity to inform current R&D decisions, but also to accelerate decisions in later stages of development once better therapeutic efficacy estimates have been established. As an increasing number of these therapeutics enter into clinical trials, it will be important to incorporate new evidence as it becomes available. This will improve the quality and reliability of the results, allowing for lifecycle HTA of the therapeutics. It will also be vital to conduct similar early health technology assessments in other therapeutic areas of hearing loss such as sudden sensorineural hearing loss and noise and drug induced hearing loss as the field expands .This study presents the first early health economic model for regenerative hearing therapeutics in ARHL and demonstrates a large potential room for improvement in the current care pathway. Novel regenerative hearing therapeutics for ARHL could become cost-effective under current willingness-to-pay thresholds. This model can be used by policy makers and industry to support the development of cost-effective therapies with the largest potential to provide added value to society and will help accelerate the introduction of ground-breaking novel hearing therapeutics in the NHS and around the world.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the University College London Research Ethics Committee 12241/001. The patients/participants provided their written informed consent to participate in this study.EL designed, collected data, performed the modeling, and wrote the main manuscript. MS, MPS, and YH designed, collected data, performed the modeling, and provided interpretive analysis. RM, MR, and AS provided interpretive analysis and critical revisions. All authors discussed the results and implications and commented the manuscript at all stages.AS advises biotech and pharmaceutical companies in the hearing field on the design and delivery of clinical trials. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hearing aids can be effective devices to compensate for age- or non-age-related hearing losses. Their overall adoption in the affected population is still low, especially in underdeveloped countries in the subpopulation experiencing milder hearing loss. One of the major reasons for low adoption is the need for repeated complex fitting by professional audiologists, which is often not completed for various reasons. As a result, self-fitting procedures have been appearing as an alternative. Key open questions with these digital tools are linked to their effectiveness, utilized algorithms, and achievable end-results. A digital self-fitting prototype tool with a novel quick four-step fitting workflow was evaluated in a study on 19 individuals with moderate hearing loss. The tool was evaluated in a double-blinded, randomized study, having two study aims: comparing traditional audiological fitting with the new self-fitting tool, which can also be used as a remote tool. The main reported results show moderately high usability and user satisfaction obtained during self-fitting, and quasi-equivalence of the performance of the classical audiological fitting approach. The digital self-fitting tool enables multiple sessions and easy re-fitting, with the potential to outperform the classical fitting approach. Hearing impairment is a growing global issue for our society, especially among the elderly, and affordable hearing care is a major public health concern ,2,3. AccOne way to improve hearing is the use of hearing aids (HA) with digital signal processing being worn either behind or in the ears. If correctly fitted (employing the correct setting of appropriate and adequate HA parameters to compensate for the hearing loss in the best possible way), an HA enables those with hearing impairment to listen, communicate, and participate effectively in daily activities ,10,19,20According to a study performed in the United States, the overall satisfaction with hearing aids increased from 74% to 81% between 2008 and 2015 . CriticaThe parameter-fitting (customization) procedure is typically performed by a trained audiologist. At audiology clinics audiologists often give a quick and general tutorial on the basic usage of the HA (that is their priority) and neglect the long-term skills such as cleaning the HAs, adjusting the program, and the use of the telephone . If neceAnother reason for the HA fitting challenge is the growing number of different digital hearing instrument fitting processes, such as fitting for varied situations , as well as the use of different compressions and algorithms, such as echo cancellation, noise reduction, and others ,20,30. SRecently, self-fitting systems have emerged and provided several advantages in terms of enhancing the fitting process . Namely,One example of the self-fitting system has been presented in the Goldilocks software approach ,37,38. IExisting research has already revealed the advantages and disadvantages of self-fitting HAs and smartphone-controlled HAs from the perspective of users ,40.Advantageously, the trial time of using the smartphone app improved the various benefits  and reduced the noticeable barrier (hearing loss stigma) of using an HA . AdditioHowever, one disadvantage encountered by elderly users of HAs is that some are presented with difficulties in using the technology ,36,41. TAccording to the abovementioned studies ,41,43, tTo provide an alternative self-fitting approach and to achieve a complexity reduction with a level of simplicity as described in Valdez, et al. , an imprThe new system, the “Self-Fitting Prototype (SFP)”, built into a hearing aid manufacturer’s fitting software (Phonak TARGET), offers the possibility to complement the conventional fitting performed by the audiologist through self-fitting performed by HA users themselves. The user interface of the self-fitting process is shown in At the top of the interface, users are given instructions about the ideal sound setting, which is to be found by exploring the matrix of cells. At the bottom of the user interface the SFP provides four sound environments, which enable self-fitting with four different kinds of sound: moderate speech, soft speech, loud music, speech in noise. The green buttons on the left are intended for enabling the HA connection and to play the sound of the current sound environment.The main part of the user interface is a two-dimensional grid with cells for fitting the HAs. Vertically, the user can change the loudness, and horizontally the timbre. This is realized by varying the amplification settings. The amount of amplification is varied by selecting different cells in the vertical dimension. Moving upwards increases the amplification, and vice versa. Selecting different cells in the horizontal dimension changes the relation of high-frequency amplification to low-frequency amplification. Moving right increases the weight of high-frequency amplification and decreases low-frequency amplification.After listening to the sound settings of a cell representing a certain fitting parameter, the users can evaluate each cell with one of five symbols  enable fine-tuning, by which the user tries to optimize the HA settings further.Loudness comfort: No sound will be too loud.Sound familiarity: Hearing will not sound strange and should sound “natural”.Overall quality: Relevant sound stimuli will be audible, discriminable, localizable, and recognizable, as well as familiar and not too loud.The objective of the study was to examine the feasibility, benefits, and shortcomings of using a grid-based self-fitting tool for fitting HAs. The primary goal of the study was to collect initial feedback about the usability aspects  of SFP, Participants were recruited through advertisements in local associations for the hard of hearing, the database of a local audiology clinic, through invitation posters and leaflets given at the audiology clinic, social networks, and through the families and friends of colleagues of the local university.The criteria for inclusion into the study were having mild-to-severe sensorineural or mixed hearing loss and being experienced in the use of hearing aids. The participants should be cognitively capable of understanding the instructions and questions of the study, and able to visit the study laboratory and other locations to test the hearing aids in the study. The exclusion criteria were mental illness, including high degrees of distress due to tinnitus ,47,48,49Employing the abovementioned inclusion and exclusion criteria resulted in a study sample of 19 adults with permanent, stable hearing loss. The selected sample was sufficiently large for examining the initial usability of the system—not as a large-scale evaluation. With 19 participants we obtained sufficient data to show a clear first impression of the usability. In the event that the statistical analysis had not provided valid results, we would have invited more people. However, our study complies with the usual recommendations for usability studies .Participants’ ages ranged between 20 and 73 years , and they were well-balanced in terms of gender . They had an average of 2.2 years of experience using HAs (SD = 1.9 years) and had been wearing HAs for at least a year in both ears, except for 2 participants who had only been using an HA in one ear. The reported duration of their hearing problems (M = 13.0 years with SD = 9.8 years) ranged from 3 to 32 years. The participants had a four-frequency average (4FA) hearing loss of M = 43.0 dB HL with SD = 5.8 dB HL, measured across 0.5, 1, 2, and 4 kHz.Based on participants’ audiograms supplied before the study, the majority had binaural hearing loss. Three participants (16%) had no differences in the average hearing loss between the left and right ears, eight participants (42%) had up to 5 dB differences, six participants (32%) had 6–10 dB differences, and two participants (10%) had more than 10 dB difference, with 11 dB and 18 dB differences. Tinnitus was present in 42% of the participants. This heterogeneity of the sample was as expected in a random sample of hearing-impaired subjects, and contributed to the external validity of the study, as it reflects the variability of the population from which the sample was drawn ,52,53,54The participants were asked to rate their experience with the technology using a 5-point scale from no experience to expert. On average, they were moderately experienced in technology use (M = 3.0 with SD = 1.3). Some reported having little to no experience with computers, smartphones, and tablets, whereas some considered themselves very experienced. None of them were technical experts in the sound amplification of HAs.Hearing Aids. The study participants were fitted with a Phonak Audéo V90 312 with an xS receiver, which covers a range from 27 to 55 dB of hearing loss. The participants’ own hearing aids could not be used in the study as they were not compatible with the SFP. The hearing aids with AutoSense OS were programmed using the Phonak Adaptive Digital prescription formula. All hearing aids were fitted to ears with so-called “closed domes” [Twin Hearing Aids. A system of switchable “twin hearing aids” was developed and used for the direct comparison of conventional and self-fitting results. Two agglutinated receiver-in-canal (RIC) hearing aids were mounted on each ear. They were connected to the receiver in the ear canal and to a switch which allowed them to choose a setting. The user could choose to listen through one or the other pair of hearing aids by operating a switch on the switch box. One pair of hearing aids was programmed with the result of the audiologist-driven (AD) fitting, the other one with the patient-driven (PD) fitting. The user could listen to an audio sample and answer a direct comparison question after having switched back and forth between listening through one or the other pair of hearing aids. For the purposes of blinding the subject and the experimenter, the hearing aid settings were labeled “red” and “green”.Booth. The study was conducted in an acoustic modular booth with 180 cm width, 240 cm length and 205 cm height with a sound reduction value of 44 dB. A single loudspeaker was situated at ear height at 1.4 m distance in front of the participant. The sound presentation system was calibrated at the start of each test day.Audio samples. To estimate the subjective qualities of each HA setting, users listened to 10 short audio samples in the same sequence: A very softly ticking clock, a soft flowing river, softly singing birds, a soft female voice, a moderate short dialogue, a moderate male voice, moderate classical music, a loud chirping cricket, loud classical music, and a very loud pneumatic hammer. These audio samples varied in sound pressure level from 34.5 dB to 85.3 dB. The examples were selected such that the audiological outcome of the fitting process could be evaluated by the subject for typical listening conditions , rather difficult conditions , and rather uncomfortable conditions . The selection of audio samples was defined by the results of the study performed by Heller, et al. [, et al. .Self-Fitting Prototype (SFP). The user interface SFP, as seen in The instruments intended to evaluate the usability aspects of the SFP were the User Experience Questionnaire (UEQ) , the quaUser Experience Questionnaire (UEQ). The UEQ is a 26-item questionnaire designed to capture the user’s experience during their interaction with the software. The UEQ contains six different user experience categories [tegories —attractiQuality assessment interview. A short interview was conducted after each participant’s use of the SFP to determine the user experience more precisely. It was used as the dominant qualitative user experience assessment method [t method  to effect method . We inquSFP-Questionnaire. Based on a preliminary study on SFP usability (n = 8), we wanted to address some usability concerns specific to the tool. Since the UEQ only addresses general usability issues, we developed an SFP-Questionnaire  participants compared the two HA settings directly. Using a comparative 9-step rating scale they reported whether loudness, familiarity, and overall quality of the “red” or “green” setting was higher for each of the audio samples.Environmental sounds questionnaire. Participants were asked to listen to surrounding sounds as they walked around in the vicinity of the lab, in the building and outside for 20 min, with the new HA setting. They were asked to pay attention to male, female, and children’s voices, any music they might hear, their perception of the surrounding noisy and quieter environments. Afterwards, they filled in a questionnaire, evaluating those sound experiences. Note that the uncontrolled nature of the environmental sounds does decrease the reliability of the HA settings’ evaluations by users. Such an approach upgraded the methodological approach successfully implemented in previous research [research , where iPhonological speech test. A series of 20 monosyllables was played from the speaker at a 60-dB sound level, measured at a distance of 1.4 m from the speaker. Participants were asked to repeat each monosyllable immediately, exactly as heard. The researcher noted whether each participant’s reply was correct or incorrect. Based on this response, the number of correctly recognized words was calculated. The methodological approach of the phonological speech test derived from the recommendations for the constructions of multilingual speech test [ech test .Researcher observations. One researcher was tasked with observing and recording all user comments, complaints, and behavior during self-fitting to provide a log of user behavior and experience as a source for further research and development [elopment . The lisAudiologist-driven (AD) fitting using the Phonak Target application and its evaluation.Patient-driven (PD) fitting of hearing aids using the SFP and its evaluation.First direct comparison between AD and PD settings using comparative perceptual ratings of audio samples.Each participant was invited to the lab for two sessions, approximately one week apart. The first session consisted of three parts, separated with 20-min breaks :AudiologFirst, we report on the general usability results as gauged by the UEQ. The average category results across the 19 participants are reported in Aiming to investigate the usability results further, we present a brief and selective summary of the quality assessment interview. The bulk of the relevant information was given as answers about the positive and negative aspects of the app. Regarding positive aspects, there was a clear pattern of users valuing the idea of self-fitting without an audiologist’s help, as 14 out of 19 (74%) participants mentioned this explicitly as a positive aspect. A related benefit, mentioned by five (26%) participants, was adjusting settings according to their current environment. One user did not report any positive app qualities. Regarding negative aspects of the app, six (32%) users mentioned having trouble in the learning process—some elements of the app were counterintuitive or not understood. Secondly, issues with the quickness and smoothness of the app due to slow response and loading times were present for five (26%) participants. An additional five (26%) participants did not report any negative app qualities.The next group of specified usability feedback was obtained from the answers to the SFP-Questionnaire . Four itIn the process of evaluating sounds during the PD fitting, according to the SFP-Questionnaire, many participants (53%) found it somewhat difficult to determine whether they liked a particular sound setting or not, whereas the remainder found it relatively easy or very easy. Moreover, determining why a certain sound was disliked was sometimes relatively difficult (15%), or somewhat difficult (15%), although it was easier for the rest. When asked about confidence in their final cell choices, 53% had slight doubts about making ideal choices, and 47% thought they chose optimally.In terms of mental effort during PD fitting, the majority of the participants reported that the tasks required some effort or minimal effort (73%), with only one person reporting a significant amount of effort required, according to the SFP-Questionnaire. Memorizing the sounds of previously tried cells was mostly relatively easy (53%) or somewhat difficult (32%). Most participants decided on the next cell choice with relative ease (82%), while also not choosing the next cells randomly (79%).The final group of findings about usability originated from researcher observations (see Measures above). Eighteen out of 19 participants had comments and questions during the short tutorial phase and required explanations from the audiologist. Ten participants required an additional explanation about the functionality of the tuning grid and its two axes. After all direction was provided and self-fitting began, some misunderstandings and small issues were still present, but did not seem to endanger self-fitting completion for most participants. Seven participants were reluctant to listen to sounds on the extremes of each dimension, whereas five participants thought the starting (middle) cell represented an ideal setting. Nine participants needed encouragement to try more cells, and six participants commented on long cell response times. In summary, learning the functionality of the SFP took some effort and detailed instruction, whereas in the self-fitting process, users seemed more confident and relatively effective, completing the exercises with little outside help.The perceptual ratings of loudness, familiarity, and the quality of sounds with PD settings and AD settings are reported in The first and second comparative perceptual ratings of the loudness, familiarity, and quality of sounds with PD and AD settings are reported in When sound settings were compared directly, no category evaluations yielded a significantly different result. However, a slight pull toward higher loudness, familiarity, and quality scores of AD settings was apparent, as is clear from the exclusively negative Mean scores shown in The comparisons between environmental sound evaluations of PD and AD hearing aid settings are reported in Additionally, we present the average proportions of correct answers to the phonological speech test for each method. The AD fitting scored slightly better; users averaged 87.1% correctly replicated monosyllables, whereas the PD fitting resulted in 80.8% correct answers.The goal of this study was to determine the viability of a tool with a grid-based interface (with loudness and timbre as the two dimensions) as a means of the independent fitting of hearing instruments by users themselves. In terms of its usability, the primary focus of the study, the SFP fitting approach proved to be usable and effective overall; according to researcher observations, all participants were able to complete the self-fitting procedure after they were properly instructed on how the tool worked. According to the UEQ, participants found the app stimulating, novel, and attractive, as they reported appreciation for the potential benefits of fitting with no outside help. Interestingly, they did not report having trouble discriminating between the subtle differences in sound experiences of adjacent cells and did not perceive the self-fitting procedure as mentally demanding.This is a remarkable difference compared to conventional fitting. In an AD fitting the user is not educated to do the fitting actively him/herself. Instead, the user answers questions from the audiologists about perception while the audiologist operates the fitting software to adjust the hearing aid settings based on the descriptions of the user. The PD fitting involves the user more actively in the fitting process, in order to better adjust the hearing aids to the users’ hearing needs.However, some aspects of the SFP’s usability require improvement. The process of learning how the tool works was commonly lengthy according to researcher observations. The short tutorial that had been designed did not result in a sufficient level of understanding, as some aspects of the tool , were still misunderstood. Optimizing the learning curve and the intuitiveness of all tool features is an important issue moving forward, as many, primarily elder HA users, are not particularly experienced with computers, tablets, and smartphones.The second usability aspect, which users were not satisfied with, was some lack of smoothness and the response time of the app, with cell response times being the main issue. Considering that the process of HA fitting typically requires establishing different levels of amplification across a frequency range, it is reasonable to expect the fitting tool to contain features and algorithms which are relatively complex. This can influence the app’s responsiveness negatively or increase the incidence of bugs, decreasing the perceived dependability and effectiveness of the tool. Conceptual simplicity in self-fitting tools of the future is, therefore, desirable.A third, potentially challenging, usability aspect of patient-driven fitting may be its complete dependence on the person’s experience as opposed to the audiologist-driven fitting, which is based largely on the hearing loss data and audiological models and expertise on how to adjust the sound for mitigation of the consequences of hearing loss. Indeed, many participants reported they had some trouble deciding whether they liked a certain sound experience (53%), whereas some had trouble establishing what exactly was the reason (30%). Understandably, 53% of our sample had slight doubts about their final choices of HA settings. Considering that the self-fitting process was a complete novelty to the participants, and that they did not fully trust their adjustments, this outcome might be expected.The secondary goal of the study was to compare the audiological outcomes of the self-fitting process versus the conventional audiologist-driven fitting process. Participants’ ratings revealed the perceived loudness, familiarity, and quality of the two HA settings to be similar, as the average differences were categorically less than 1 unit on a 9-step Likert scale. The only significant rating difference was in the loudness category, in which the AD setting was evaluated consistently as louder. Although we do not report the HA amplification data explicitly, it is likely that the AD setting, on average, provided more gain, as was the case in Boymans and Dreschler . Future We have substantiated that the self-fitting approach based on a grid interface is a reasonable alternative to the audiologist-driven fitting approach, both in terms of its usability and audiological outcomes. It promises to be an addition to conventional HA fitting, and a convenient solution, providing more freedom in personal preferences in adjustments, not depending on additional visits to audiologists. The study also highlighted areas of possible improvement, where the learning process, responsiveness of the app, and user confidence should be given additional attention in future endeavors. As an argument against these shortcomings, we emphasize that only one iteration of self-fitting was performed in the study, whereas, in the intended use of the app, users would be able to complete multiple iterations of the self-fitting procedure. As users improve their mastery of the functionality of the app and grow to become confident in self-fitting through continued use of the tool, we expect them to work towards even better audiological outcomes and satisfaction with hearing aids.Additionally, more research on the viability of self-fitting tools with a larger sample size would be welcome, in order to establish the challenges of this approach accurately. Importantly, both quantitative, as well as qualitative research methods should be employed, considering the novelty and complexity of the self-fitting process.


Aim of this cross-sectional study was to investigate the association between PDFF over the supraclavicular fat region as a proxy of BAT proportion and resting metabolic rate (RMR). In addition, the association between the single nucleotide polymorphism (SNP) rs1421085 at the 2) were included in the analysis. The RMR was measured by indirect calorimetry. The magnetic resonance imaging (MRI) was used for the measurement of visceral and subcutaneous adipose tissue  volumes and for the measurement of adipose tissue PDFF.Anthropometric, clinical, and lifestyle data from 92 healthy adults , no association was found between supraclavicular PDFF (p = 0.346) and gluteal PDFF (p = 0.252), respectively, and RMR, whereas statistically significant evidence for a negative association between delta PDFF  and RMR (p = 0.027) was obtained. No statistically significant evidence was observed for per FTO rs1421085 variant is not associated with either RMR or PDFF. Further studies are needed to elucidate the effect of BAT on RMR.Supraclavicular PDFF as a surrogate marker of BAT presence is not a determinant of RMR under basal conditions. In the present study, the  Understanding the determinants that influence resting metabolic rate (RMR) as the main component of energy metabolism is fundamental in obesity research. Main determinants of RMR variability include fat-free mass (FFM), sex, age, and body temperature –3. In paThe FM consists of white adipose tissue (WAT), composed of lipid-storing white and – to a poorly defined, but much lower extent - lipid-burning “beige” adipocytes, and brown adipose tissue (BAT) in specific depots , 9. WherOver the last decade, functional imaging provided evidence for the existence of BAT in adult humans , where Bfat mass and obesity associated (FTO) locus and preadipocyte differentiation  the association between supraclavicular PDFF, as a surrogate marker for the presence of BAT, and RMR as well as (II) associations between the Recruitment of study participants started in October 2013 at the Else Kroener-Fresenius Centre for Nutritional Medicine in Munich, Technical University of Munich, Germany. Adults with a body mass index (BMI) equal to or above 18.5 kg/m² were included. Participants were excluded if an acute inflammatory state, a history of severe diseases, surgeries within the last three months or physical limitations were reported. Pregnant and breast-feeding women and persons on specific medications such as beta-blocker were also excluded. Written informed consent was obtained before participation in the study. The study protocol and the standard operating procedures were approved by the ethical committee of the Technical University of Munich, Germany (Number 2719/10 S).Height was measured in a standing position without shoes using a stadiometer  and reported to the nearest 0.1 cm. Body composition, including FFM and FM, was assessed using bioimpedance analysis . Measurements were performed before indirect calorimetry in light clothing and with voided bladder, with subtraction of 1.0 kg for clothes. The BMI was calculated as quotient of weight in kg and squared height in m (kg/m²). Blood pressure and heart rate were obtained using a validated electronic device  according to the recommendations of the American Heart Association .2) and carbon dioxide (VCO2) expiration resulting from substrate oxidation, while participants were fully awake and motionless. The respiratory quotient (RQ) was calculated as VCO2/VO2 ratio. For RMR calculation, the following equation was used: RMR  = [3.941 × VO2 (ml/min) + 1.106 × VCO2 (ml/min)] × 1.44 . The RMR measurement was performed with the following requirements: overnight fast (10 to 12 hours), no smoking, and abstinence from physical activity the day before. The 30-minutes measurement started after an acclimatization time of five minutes. The flow rates were assessed as recommended by the manufacturer. The Quark RMR software analyzed oxygen consumption . For PDFF analysis, a semi-automatic segmentation tool based on Matlab  was used. Weight data at the time of MRI measurement was self-reported. Detailed data analysis of MRI measurement was performed as previously described . The proFTO polymorphism rs1421085 was carried out by the company Eurofins  with Cycle Sequencing Technology (dideoxy chain termination/cycle sequencing) using Applied Biosystems 3730xl DNA Analyser. Call rate was calculated with 96.1% showing a high quality of the genotyping procedure. Calculated minor allele frequency (MAF) of the risk C allele was 43%. Hardy-Weinberg equilibrium (HWE) was fulfilled.Deoxyribonucleic acid (DNA) was isolated from EDTA full blood samples according to a standard protocol following manufacturer`s protocol . Polymerase chain reaction (PCR) was used for amplification of DNA. Sequencing of the FTO genotype and PDFF and RMR. Due to time intervals between the RMR and MRI measurement, all statistical analysis were adjusted for the body weight difference (“weight change”) between the time of RMR and MRI measurement. RMR is known to be significantly associated with age, sex, and FFM  and percentages (%). For comparison of variables, the unpaired student t-test was conducted for normally distributed variables, otherwise Mann-Whitney U-test was used. Analysis of variance (ANOVA) were conducted to compare more than two variables. Regression analysis was calculated for associations between different variables. Additive, as well as dominant and recessive linear models were used to identify associations between the  and FFM , 26, 27. and FFM .In total, 92 participants  were included in the analysis Table 1.Supraclavicular PDFF was positively associated with RMR, if data was unadjusted , however significance disappeared after adjustment in model 2 and model 3  Table 2.Evidence for a significant association between VAT and RMR was observed in the statistical models 1 and 2  Table 2.To exclude effects of overweight and obesity on the relationship between PDFF and RMR, a sub-group analysis of participants with normal weight  was performed. Evidence for a statistically significant negative association between gluteal PDFF and RMR was obtained in the unadjusted model 1  as well as in the full adjusted model 3  Table 3.FTO genotypes and the RMR were seen in the additive as well as in the dominant or recessive genetic models , respectively. No evidence for any significant association between the FTO SNP rs1421085 and the adipose tissue variables under investigation was obtained, independent of the genetic model . Moreover, the negative association between delta PDFF and RMR remained statistically significant after adjusting for age, sex, weight change, FFM and FM. However, the negative correlation disappeared after exclusion of persons with overweight and obesity. These results were in contrast to our hypothesis as we assumed (I) the lower the supraclavicular PDFF, the higher the content of BAT and beige fat cells (II) resulting in a higher delta PDFF and in a higher RMR. The associations may indicate that the adipose tissue composition of the supraclavicular region might not be relevant for energy expenditure, at least under thermoneutrality. Inverse correlations between outdoor temperature and BAT activity were found in different studies, in which BAT was detected a PET-CT , 30. Morutrality  leading The significant difference between supraclavicular and gluteal PDFF as demonstrated in this study is in line with other studies , 14 indiFrom previous studies, it is well known, that FFM is the major determinant of RMR , 26, 34,FTO SNP rs1421085 was of interest. No association was detected between the rs1421085 obesity C risk allele and PDFFs. However, this negative finding should be dealt with caution as the sample size was limited and, therefore, the number of homozygous C risk allele carriers was rather small. Another explanation might be that the effect of the FTO risk genotype may only become apparent when BAT is metabolically active, e.g. after activation by cold exposure. As MRI-derived PDFFs do not reflect current BAT activity, an association between the FTO genotype and BAT activity is not excluded and may require further studies.Based on the findings of Claussnitzer et al. , the invThe present analysis investigated the relationship between PDFF as a proxy of BAT and RMR in humans. However, PDFF is not specific for the presence and the proportions of brown or beige adipose cells in a defined adipose tissue depot. A low PDFF in the supraclavicular adipose tissue may suggest a large proportion of less lipid-loaded brown fat cells, but suffers from partial volume effects and cannot reflect the real volume of brown adipocytes in the human supraclavicular fossa comprising a heterogeneous mixture of brown and white adipocytes . In addiFTO SNP rs1421085. Our results did not provide evidence for a significant association between supraclavicular PDFF and RMR. In addition, our analysis did not reveal an association between supraclavicular PDFF and the FTO genotype. Future studies on the association between BAT and RMR should consider the activation of BAT.In conclusion, our analysis is the first to investigate the associations between PDFF, as a surrogate marker for BAT and beige fat cells, and RMR and the The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethical committee of the Technical University of Munich. The patients/participants provided their written informed consent to participate in this study.HH, DK, and CHo contributed to conception and design of the study. TD and DJ organized the database. TD, WM, CHe, and DJ performed data collection and analysis. TD and SB performed the statistical analysis. TD, SB, and CHo wrote the manuscript. All authors contributed to the article and approved the submitted version.Else Kroener-Fresenius-Foundation, Bad Homburg, Germany, and the Helmholtz cross-programme topic “Metabolic Dysfunction”.DK receives grant support from Philips Healthcare.The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


TCERG1L (rs893507) to be associated with hearing loss in 390 non-cranial irradiated, cisplatin-treated children with cancer. These results were replicated in two independent, similarly treated cohorts  . Modulating TCERG1L expression in cultured human cells revealed significantly altered cellular responses to cisplatin-induced cytokine secretion and toxicity. These results contribute to insights into the genetic and pathophysiological basis of cisplatin-induced ototoxicity.In children with cancer, the heterogeneity in ototoxicity occurrence after similar treatment suggests a role for genetic susceptibility. Using a genome-wide association study (GWAS) approach, we identified a genetic variant in  Cisplatin is a highly effective chemotherapeutic agent for an important subset of childhood cancers that depend on this drug for curation. However, the occurrence of irreversible hearing loss that occurs in ~50% of cisplatin-treated children is a serious clinical challenge3. Young age at cancer diagnosis, high total cumulative dose (TCD) of cisplatin, cranial irradiation, and/or subsequent carboplatin use might increase the risk of developing hearing loss6. Understanding the biology of cisplatin-induced hearing loss and identifying risk factors that could predict ototoxicity is highly relevant as children are at a critical stage of their speech and language development, with the added risk of experiencing social, emotional, or vocational difficulties related to hearing loss. This ultimately impacts development and quality of life during treatment but also later in life7.Survival probabilities for pediatric cancer have increased tremendously over the past decades8. Therefore, we performed a GWAS in a discovery cohort of 390 cisplatin-treated, non-cranial-irradiated European children with cancer (n = 168 (43.0%) with hearing loss, Supplementary Table 9. This cohort was assembled by the European initiative of the PanCareLIFE (PCL) group (http://www.pancarelife.eu/)10. Cases were defined as having deleterious hearing loss according to Muenster ≥2b after the aforementioned treatment and were compared to subjects with Muenster 0–2a  of non-cranial irradiated, cisplatin-treated children  with hearing loss) (Supplementary Tables 9. Rs893507 showed evidence of replication (P = 0.01), resulting in a combined OR of 2.77, adjusted for age at diagnosis, sex, cisplatin TCD, and principal components 1–4  gene  GWAS analysis identified eight suggestive loci  as well as after stratification , adjusted for age at diagnosis and sex. Next, a potential cisplatin dose–response effect with TCERG1L was estimated. By modeling cisplatin dose as a continuous variable, effect modification (P = 0.04) was observed. After stratification, a dose–response effect was neither observed for 360–480 mg/m2 (P = 0.9), nor for >480 mg/m2 (P = 0.1) as power was lost. Future studies with larger sample sizes are needed to accurately model the effect of cisplatin dose.Although a specific cisplatin threshold dose associated with ototoxicity has not been found previouslyTCERG1L) was not associated with age-related hearing loss in a general population , and other hearing loss (P = 0.2) (http://big.stats.ox.ac.uk/variant/10:133013187-T-C). This suggests that rs893507 (TCERG1L) is specific for cisplatin-induced hearing loss.A possible association between the SNP and age-related hearing loss was evaluated. By contrast, rs893507 TCERGL was notTCERG1L gene, a paralog of TCERG1, is a transcription elongation regulator that has been described to be involved in the pathogenesis of cancer and non-cancer-related diseases, but it has not previously been associated with chemotherapy-induced hearing loss. Previous studies revealed an association with inflammatory bowel disease, as well as with colon cancer predisposition suggesting that TCERG1L influences immunological pathways17. TCERG1L is expressed in the human brain, gut, thyroid, stem cells, adenoid, and tonsils, (http://biogps.org/#goto=genereport&id=256536) (https://www.genecards.org/cgi-in/carddisp. pl? gene=TCERG1)20, and more importantly also in human cochlear inner and outer hair cells21 as well as murine cochlear inner hair cells20.The TCERG1L silencing and overexpression experiments in vitro, to examine the effect of TCERG1L expression on cisplatin cytotoxicity and inflammatory response in cultured human cells. We observed that modulating TCERG1L expression significantly altered cell viability in response to cisplatin treatment, where TCERG1L overexpression and silencing, respectively, protected and sensitized cells to cisplatin toxicity. Overall, modulation of TCERG1L expression significantly shifted the cisplatin CC50 fourfold  expression in response to cisplatin treatment by RNA-seq in various mouse tissues  carriership was associated with cisplatin-induced hearing loss in non-cranial-irradiated, cisplatin exposed childhood cancer survivors of the PCL second replication cohort but that the significance was lost after 553 cranial-irradiated subjects were added to the analyses  in a cohort of this size can be accounted for by the observation that toxicity-associated pharmacogenomic variants tend to have a larger effect size22. Nevertheless, establishing worldwide collaborations to perform future studies with even larger sample sizes remains the ideal strategy to discover additional genome-wide significant variants that predict adverse drug reactions in childhood cancer patients. In the discovery cohort and second replication cohort, we were unable to perform a robust assessment of ototoxic co-medications and their potential influence on the strength of the TCERG1L genetic association because retrospective patient data did not capture complete details of these co-medications. Furthermore, eQTL evidence for the effect of rs893507 on gene-expression levels was not found, possibly due to limited sample sizes in these tissues. However, eQTLs are highly tissue-specific  thus the absence of an eQTL between rs893507 and TCERG1L remains equivocal. In addition to a genetic association, we were able to functionally implicate TCERG1L in the development of cisplatin-induced inflammatory response and toxicity. We observed an inverse correlation of TCERG1L with cellular responses to cisplatin suggesting a plausible biological link between our association results and the mechanism of cisplatin-related hearing loss in non-cranial-irradiated subjects. The functional data are consistent with rs893507 having a deleterious effect on TCERG1L function.In this work, we report a genetic variant associated with cisplatin-induced hearing loss stemming from childhood cancer treatment. Our genetic discovery showed genome-wide significance upon further study in additional patient cohorts including two independent international cohorts. In total, our study included 770 patients which is a relatively large cohort considering that childhood cancer is very rare. The strength of association for rs893507 genetic regulation of the genetic variant associated with cisplatin-induced hearing loss.In conclusion, the combined results of this study suggest that cisplatin-treated, non-cranial irradiated childhood cancer patients with a genetic intronic variant (rs893507) in 24. Inclusion criteria for this study were patients: (1) diagnosed with cancer before the age of 19 years; (2) initially treated with cisplatin, as a single platinum drug during childhood cancer treatment, or switched from cisplatin to carboplatin during treatment; (3) did not receive cranial or inner ear radiation; (4) completed their chemotherapy treatment; (5) had at least one pure tone audiometric evaluation available within two years after completion of chemotherapy; (6) had their biomaterial  available for DNA extraction; and (7) no baseline hearing loss. Patients with hearing loss before the start of chemotherapy and patients with initial treatment of carboplatin were excluded. Patient data  was collected retrospectively from medical records at participating institutions in Europe. Patients were enrolled after approval had been obtained from local review boards and written informed consent was obtained from patients, parents, or legal guardians. The PCL study was 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, EK-1447/14; 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, MEC-2014-633; Medisch Ethische Toetsingscommissie, 2015_202 9.The discovery cohort  consists of childhood cancer patients from the PCL consortium, a multicenter cohort of childhood cancer patients and survivors across Europe designed to address ototoxicity, fertility impairment, and quality of life. In the current study, 390 cisplatin-treated, non-cranial irradiated patients were included. Subjects were diagnosed and treated for childhood cancer in Europe. A detailed description of the PCL discovery cohort is availablen = 192) treated with cisplatin from the Canadian Pharmacogenomics Network for Drug Safety (CPNDS), a national research and patients care network established to reduce serious adverse drug reactions in children 25 (http://cpnds.ubc.ca/). Participants in this replication cohort fulfilled all inclusion criteria of this study as described above. In addition, cases of cisplatin-induced hearing loss have a bilateral hearing loss only, and unilateral hearing loss cases with normal hearing in the contralateral ear are not considered cisplatin-induced. Demographic, diagnostic, audiological, and treatment-related data were retrieved from medical records. The study was approved by the research ethics board of each of the participating institutions of the enrolled patients and written informed consent was obtained from each patient or their legal guardian9.The first replication cohort  consisted of childhood cancer patients (2). Patients were enrolled after approval was obtained from local review boards and written informed consent was obtained from patients, parents, or legal guardians9.An independent second replication cohort  consisted of childhood cancer survivors from the PCL cohort. Participants were enrolled both retrospectively and prospectively . Eligibility criteria were: (1) no participation in the discovery PCL cohort (D); (2) age at diagnosis <19 years; (3) treatment with cisplatin or cisplatin and carboplatin; and (4) at least one pure tone audiometric evaluation available within 5 years after the end of chemotherapy. Exclusion criteria were (1) non-consent; (2) hearing loss before the start of platinum treatment; and (3) cranial irradiation. Identified case-patients  were matched 1:1 with controls  for sex, age at diagnosis (tolerance 5 years), and cumulative cisplatin dose (tolerance 50 mg/mA lookup of the GWAS hits was performed in the discovery cohort of the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) to evaluate a possible association between the SNP and age-related hearing loss. This cohort on age-related hearing impairment consisted of 9,675 subjects from the general population who were all 45 years or older at the time of the study. Phenotypes were examined that represented low/mid  and high-frequency hearing loss (at 4 and 8 kHz) based on pure tone audiograms.11. Audiogram assessors were blinded to patient characteristics, treatment factors, and genetic data. Grading was based on the worst ear, determined at the first measurement available after the end of treatment. In the current study, patients who had deleterious hearing loss grade ≥2b according to Muenster were considered cases. Patients with Muenster grade 0−2a were assigned to the comparison group. A comparison between subjects with Muenster grade 0 and subjects with Muenster grade ≥2b was not adequately powered due to a lack of sufficient grade 0 subjects.The main endpoint of this study was hearing loss following cisplatin treatment. Pure tone audiometry (up to 8000 Hz) was performed in all patients after the end of platinum treatment. For all cohorts, the results of audiological examinations were graded according to Muenster classification  was used for DNA genotyping. A stringent quality control protocol containing multiple filters was applied to clean the genetic data  were performed using the Michigan Imputation Server with default settings (https://imputationserver.sph.umich.edu/index.html).In the PCL discovery cohort, DNA was isolated from blood and saliva samples. Blood samples were stored at ≤−20 °C and shipped on dry ice, and saliva was stored and shipped at room temperature. The salting-out method was applied to extract genomic DNA. The Illumina Infinium© OmniExpress array. Genotyped variants underwent stringent quality control procedures, for which the QCTOOL (version 2), GTOOL (version 0.7.5), and PLINK (version 1.90) were used. Genetic variants with a call rate of <95%, a minor allele frequency of <1% in both cases and controls, and variants deviating from HWE genotype distributions 28. Sixty samples (31%) of non-European ancestry were included. Imputation was performed using SHAPEIT229 and IMPUTE230, using the Phase 3 1000 Genomes Project samples as a reference .Within the CPNDS cohort, all blood and saliva samples were genotyped by use of the Illumina Infinium® PCR31.In the PCL replication cohort, genotyping calls of variants replicating in the Canadian replication cohort were validated by using TaqManP < 5 × 10−8) were unlikely. We therefore decided to pursue variants with suggestive levels of association (P < 1 × 10−5) for replication.Measurements were taken from distinct samples. For the GWAS analyses, we used logistic regression models including age at diagnosis (linear term), sex, total cumulative cisplatin dose (linear term), and four principal components using rvtests  were calculated using the R package PredictABEL. P < 0.01  were considered statistically significant. Only variants replicating in the Canadian first replication cohort were candidates for replication in the PCL second replication cohort. Association of the variant with cisplatin-induced hearing loss was examined using logistic regression, assuming an additive effect of the minor allele. Adjustments were made for sex, age at diagnosis, and cumulative cisplatin dose. Data from the discovery and replication cohorts were combined and examined using meta-analytic approaches in R version 3.5.1, package “rmeta”33. An inverse-variance meta-analysis was used.For replication, firstly, the Canadian childhood cohort GWAS data were usedTCERG1L on cisplatin cytotoxicity and cisplatin-induced inflammatory cytokine production. We used HeLa cells as a model of human cultured cells . To determine the impact of gene knockdown on general cell viability in the presence and absence of varying concentrations of cisplatin in HeLa cells, MTT -2,5-diphenyltetrazolium bromide) cell viability assays were used. The cisplatin-induced inflammatory response was assessed by interleukin-8 (IL-8) enzyme-linked immunosorbent assays (Thermofisher). Knockdown experiments were all performed using pre-designed TCERG1L siRNA  according to the manufacturer’s specifications. siRNA transfection was performed with Lipofectamine RNAiMAX according to the manufacturer’s protocol (Thermofisher). Real-time quantitative PCR (RT-qPCR), using TCERG1L specific primer-probe mix  showed 70% silencing efficiency of TCERG1L compared to the non-targeting siRNA control. Overexpression experiments were all performed using ectopic transfection of pCMV6TCERG1L  using JetPrime reagent (PolyPlus) according to the manufacturer’s instructions.Functional validation experiments were performed to determine the effect of knockdown or overexpression of TCERG1L overexpression experiments cells were first seeded at 1.5–3 × 106 cells in 10 cm dishes for 24 h prior to transfection. Transfected cells were then trypsinisized and seeded into either 96-well plates, with 5000 cells per well, or 24-well plates, with 70,000 cells per well. Newly transferred cells were allowed to grow for another 24 h prior to cisplatin treatment. Treatment with cisplatin proceeded for another 48 h prior to supernatant collection and cell viability assays. For TCERG1L silencing experiments, cells were treated as above or seeded directly into 24-well plates prior to transfection. Cell viability curves were generated in Prism7 using non-linear curve fits normalized response and compared using Extra sum-of-squares F-test. Relative IL-8 secretion was compared using a two-tailed Student’s t-test.For TCERG1L and IL-6 induced by cisplatin treatment across various mouse tissues, we made use of a publicly available dataset at the NCBI gene expression omnibus (GEO) under accession number GSE11716734. This dataset comprises total RNA from liver, kidney, spleen, and lung tissue specimens from 6-month-old female mice in a pure C57Bl6/J genetic background, which were collected after 4 h following cisplatin (10 mg/kg) or saline IP injection and sequenced using the HiSeq 4000 Illumina RNA-seq platform35. The raw data files were downloaded and subjected to our in-house generated data analysis pipeline. Sequence adaptors were removed from the sequence reads using Trimmomatic version 0.39 and the trimmed reads were subsequently aligned to the mouse genome using Star version 2.7.0f with gencode.vM20.annotation.gtf and GRCm38. p6.genome.fa as annotation and genome (http://gencodegenes.org/mouse/release_M20.html). Read counts for each gene were obtained using FeatureCounts and log-fold changes and false discovery rates were quantified using EdgeR version 3.24.3. Visualization of the relative fold changes  across the various organs was performed in GraphPad Prism version 8.4.2 .For the expression changes of Further information on research design is available in the Supplementary InformationReporting Summary


Cleft palates are one of the most common congenital malformations. Because of the loss of Eustachian tube function, middle-ear ventilation is reduced. The aim of this study was to determine if middle-ear effusions were present at birth or at the three-month audiological evaluation.A total of 53 children with a cleft palate were included. Data review included the results of newborn hearing screening, microscopic findings, a tympanometry, a free field audiometry and intra-operative findings.A total of 58.4 per cent of patients had a median, 26.4 per cent had a bilateral, 11.3 per cent had a unilateral and 3.8 per cent had a limited soft palate cleft. Newborn hearing screening showed a pass in 83.1 per cent of newborns bilaterally. The first ear microscopy showed a bilateral middle-ear effusion in 90.6 per cent of cases. During cleft surgery, bilateral paracentesis was performed in all cases, and in 90.6 per cent middle-ear effusion was sucked out.The majority of children with a cleft palate do not present with middle-ear effusion at birth. It develops within several days or weeks of life. This is treated by insertion of ventilation tubes to improve the sound conduction to the inner ear.5 The use and the management of ventilation tubes is an issue of debate among specialists.9Cleft palate is one of the most common congenital craniofacial malformations with an incidence of 1 in 700 newborns.17 According to Szabo et al., 82 per cent of the newborns with a cleft palate pass the newborn hearing screening test. The onset of middle-ear effusion is not specified.18 Viswanathan et al. describe a hearing loss in ‘early infancy’.19 Flynn et al. showed a significantly higher prevalence of middle-ear effusion in patients with cleft palate at 1, 1.5, 3 and 5 years of age1 compared with patients without cleft palate. Chen et al. stated that middle-ear effusion in a cohort of 319 children can be measured with tympanometry at the age of 9 months.10 They also reported on ethnic variations showing populations of Asian and native North American descent have the highest incidence, Caucasian populations have intermediate incidence and African populations have the lowest incidence of cleft palate.10 However, in all these publications the earliest data on the middle ear are based on investigation one month after birth in the cleft palate clinic.In the literature, few data are available on the period of time when middle-ear effusion in children with cleft palate occurs for the first time. An incidence of 71.9 per cent up to 97 per cent within the first months is reported.24 The physiological function depends on three different pivotal points, called hypomochlia: the pterygoid hamulus, Ostmann's fatty tissue25 and the medial pterygoid muscle.26 A sufficient tubal function depends on the complete integrity of these three hypomochlia.27The function of the Eustachian tube depends on the tensor veli palatini muscle. Its activity is almost isometric. One main action of the tensor veli palatini muscle is to actively open the Eustachian tube.28 This device is used for automated auditory brainstem response - screening throughout Germany. With such a soft level of the auditory stimulus, a regular, positive automated auditory brainstem response screening (‘pass’) implies a normal tube function. This is also the case in measurements using transient evoked otoacoustic emissions (TEOAE). Hence, if middle-ear function is not normal, screening automated auditory brainstem response will be abnormal and TEOAE will be absent.In Germany, newborn hearing screening has been mandatory since 2009. The automated auditory brainstem response screening with the Beraphon®  at 35 dBnHL shows a high specificity and sensitivity.The aim of this study was to determine if middle-ear effusion was present at birth or at the three month audiological evaluation in the joint cleft clinic. This retrospective study included data of newborn hearing screening, the audiological evaluation results and the clinical findings.The study protocol was approved by the local ethics committee (number: 20190927 01). This retrospective study included 53 children who had an appointment at the joint cleft clinic between July 2015 and December 2015 and were still under revision in 2020. According to the algorithm of the joint cleft clinic, all of them had a newborn hearing screening, an audiological evaluation within the first three months of life, a lip repair at the age of six months or a palatoplasty at one year of age. During surgery of the lip or in absence of a cleft lip at the age of six months, a microscopic examination of both ears was performed including a paracentesis and, if necessary, the insertion of ventilation tubes.Data evaluation of the charts included the results of newborn hearing screening, the clinical findings at the first appointment within the first three months, the results of tympanometry and free field audiometry as well as the intra-operative microscopic findings. The newborn hearing screening was performed by using the Beraphon MB 11 hearing screening device  or by measurement of the transient evoked otoacoustic emissions (TEOAE)  within the first 14 days of life. In Germany, the TEOAE were performed with different devices. Generally, automatic brainstem evoked response audiometry was carried out with Beraphon device. The statistic was calculated using the chi-square test.In the group of 53 children, there were 58.4 per cent (31) with a median, 26.4 per cent (14) with a bilateral, 11.3 per cent (6) with a unilateral and 3.8 per cent (2) with a soft cleft palate. A total of 41.6 per cent (22) were female and 58.4 per cent (31) were male. The palatal malformation was part of a Pierre Robin sequence in 11 children. The newborn hearing screening was performed in three children by means of transient evoked otoacoustic emissions and in 50 cases by Beraphon. A total of 83.1 per cent (44) of the 53 children passed the hearing screening bilaterally.p = 0.28, chi quadrat).Bilateral middle-ear effusion was detected in 90.6 per cent (48) of the children during the first clinical evaluation session at the age of 3 months. The tympanometry showed a bilateral type B curve in 86.8 per cent (42) and a bilateral type C configuration in 13.2 per cent (6). The free field audiometry showed a median reaction at 48 dB HL (20–75 dB). In 73.6 per cent (39) of the children with a ‘pass’ in the newborn hearing screening, a middle-ear effusion was seen. Only 9.5 per cent (6) with a regular newborn hearing screening showed a ventilated middle ear. In 16.9 per cent (9), a newborn hearing screening ‘fail’ with middle-ear effusion was observed. There was no significance between the regular newborn hearing screening and the middle-ear effusion at the first investigation  of patients, a middle-ear effusion was sucked out. The middle-ear effusion was classified as ‘serous’ in 27.1 per cent (13), ‘seromucoid’ in 45.8 per cent (22) and ‘mucoid’ in 27.1 per cent (13) of cases. In all 48 children with a middle-ear effusion, a ventilation tube was inserted. Children with an aerated middle ear did not receive a tube.The post-operative follow up with a first appointment six weeks after tube insertion showed normal free-field reactions between 20 and 30 dB in all children. The tympanometry confirmed a patent ventilation tube with high volumes. In the study group, no sensorineural hearing loss was found.In 2020, all of the children had a five-year follow up. All patients had intact tympanic membranes with tympanosclerosis bilaterally. All pure tone audiograms showed hearing levels between − 5 and 20 dB HL bilaterally. In 92.5 per cent (49), the tympanometry showed a type A tympanogram and in 7.5 per cent (4), it showed a type B tympanogram.30 The presence of middle-ear effusion especially influences perception of high frequencies. Therefore, the observation of children with risk factors for a middle-ear effusion is necessary.31Sufficient pantonal hearing is required for regular speech development. The most frequent reason for peripheral hearing loss in children is middle-ear effusion causing a conductive hearing loss.27 The pressure equalisation is mainly based on the tube function realised by action of the tensor tympani and tensor veli palatini muscle.19 A disturbance in the tube ventilation leads to a negative pressure in the middle-ear cavity followed by a serous effusion.31 If aeration of middle-ear spaces is reduced for a longer period of time, changes in the middle-ear mucosa induce a mucoid character of these effusions. Intra-operative findings of mucoid effusions are a clear sign of a chronic ventilation problem.The compliance of the tympanic membrane ossicle complex is based on the middle-ear ventilation.32 Ventilation tubes improve the tympanic membrane middle-ear ossicle complex compliance by drainage of the middle-ear effusion.34 However, ventilation tubes are known to induce tympanosclerosis and can also lead to a chronic otitis epitympanalis or mesotympanalis.35 Nevertheless, in order to enable a normal speech development, a chronic middle-ear effusion as well as a tympanosclerosis or chronic epitympanic or mesotympanic otitis should be treated. In this study, all patients showed hearing levels between − 5 and 20 dB HL bilaterally in the pure tone audiograms at the 5-year follow up. Indeed, all of the 106 tympanic membranes had signs of tympanosclerosis. Looking at the tympanometry approximately five years after ventilation tube insertion, the majority of the study group presented a good compliance of the tympanic membrane ossicle complex.Newborns with a cleft palate have a predisposition to develop middle-ear effusions because of missing tubal function. In the literature, there is a long-standing debate about the use of ventilation tubes in these patients.1 Most of the publications report on first investigations at 3 to 12 months after birth.11 Most newborns get their newborn hearing screening during the first days at the maternity ward. The hypothesis that we sought to verify in this study was that the occurrence of middle-ear effusion in patients with cleft palate is not inborn but develops during the early days or weeks of life. A newborn hearing screening result pass (using automated auditory brainstem response or TEAOE) requires sufficient middle-ear function. Otherwise, the stimulus will be attenuated by poor middle-ear function or, for the same reason, the otoacoustic emissions generated by the outer hair cells cannot be reflected to the outer ear canal properly. Thus, a regular newborn hearing screening is sign of a regular compliance of the tympanic membrane middle-ear ossicle complex.Very little is known about the point in time when middle-ear effusions occur in patients with cleft palate.et al. reported (82 per cent).18 Therefore, it can be assumed that tubal function and middle-ear ventilation are intact at the time of newborn hearing screening. On examination at the first clinical appointment within the first three months of life, the microscopy and the tympanometry show an abnormal tube ventilation with middle-ear effusion in 90.6 per cent and 86.6 per cent of cases, respectively. The free field threshold at a median of 48 dB HL gives further evidence of an existing hearing loss. During intra-operative microscopy, data on the earliest performed procedure at 6 months of life (lip repair) and latest performed at 12 months of life  showed that 90.6 per cent of the children had a middle-ear effusion. In 27.1 per cent of the cases, middle-ear effusion was already ‘mucoid’, which indicates the presence of a long-standing ventilation problem. This might be a clinical sign that tube function changes over the time. However, in this cohort no tympanometry was performed with the newborn hearing screening to confirm a middle-ear effusion objectively, which is a limitation of the study. The regular newborn hearing screening during the first 14 days of life is therefore an indirect sign of a ventilated middle ear.Poor tube ventilation with middle-ear effusion is a typical symptom of cleft palate patientsData in the literature are based on investigation at one month after birth in the cleft clinicIf middle-ear function is not normal, screening automated auditory brainstem response will be abnormal and transient evoked otoacoustic emissions will be absentIn 44 of 53 children (83.1 per cent), a regular post-natal bilateral newborn hearing was measuredMiddle-ear effusion was detected in 90.6 per cent (48) of children at the age of 3 monthsMiddle-ear effusion with tube ventilation disorder due to the cleft palate is not congenital as it develops later in lifeThe data from this study show that 83.1 per cent of the newborns with cleft palate pass the bilateral newborn hearing screening successfully. These data are within the same range that Szabo The results of this study may be explained based on the following theory. Intrauterine, the middle-ear spaces are filled with amniotic fluid. The first scream and swallowing of the newborn produce high pressures in the upper aerodigestive tract. Referring to the mechanism of Politzer's manoeuvre, this pressure may achieve a middle-ear ventilation. In the following weeks and months, the middle ears fill up with fluid and effusion is detected at the first clinical appointment at the joint cleft clinic within the first three months while the newborn hearing screening was performed earlier under quite normal middle-ear conditions. The middle-ear effusion with tube ventilation disorder because of the cleft palate is not congenital as it develops later in life.Sufficient middle-ear ventilation is based on normal activity of the tensor tympani and tensor veli palatini muscle. Accordingly, the development of poor tube ventilation with middle-ear effusion is a typical symptom of cleft palate patients. So far, little is known about the point in time of development of poor middle-ear ventilation in these patients. The data of this study show there is a regular newborn hearing screening in 83.1 per cent which is an indirect sign of tubal patency shortly after birth. Within 3 months, at the first appointment in the joint cleft clinic, 90.6 per cent of the children show a middle-ear effusion confirmed by microscopy and tympanometry. The following poor tubal function leads to a middle-ear effusion with the effect of conductive hearing loss. In conclusion, middle-ear effusion with tubal ventilation dysfunction because of cleft palate seems to develop within the first weeks of life.


Background: A large number of undiscovered fungal species still exist on earth, which can be useful for bioprospecting, particularly for single cell oil (SCO) production.Mortierella is one of the significant genera in this field and contains about hundred species. Moreover,M. alpina is the main single cell oil producer at commercial scale under this genus.Methods: Soil samples from four unique locations of North-East Libya were collected for the isolation of oleaginous Mortierellaalpina strains by a serial dilution method. Morphological identification was carried out using light microscopy  and genetic diversity of the isolatedMortierella alpina strains was assessed using conserved internal transcribed spacer (ITS) gene sequences available on the NCBI GenBank database for the confirmation of novelty. The nucleotide sequences reported in this study have been deposited at GenBank (accession no.MZ298831:MZ298835). The MultAlin program was used to align the sequences of closely related strains. The DNA sequences were analyzed for phylogenetic relationships by molecular evolutionary genetic analysis using MEGA X software consisting of Clustal_X v.2.1 for multiple sequence alignment. The neighbour-joining tree was constructed using the Kimura 2-parameter substitution model.Results: The present research study confirms four oleaginous fungal isolates from Libyan soil. These isolates  were discovered and reported for the first time from diverse soil samples of district Aljabal Al-Akhdar in North-East Libya and fall in the class:Zygomycetes; order:Mortierellales.Conclusions: Four oleaginous fungal isolates barcoded as MSU-101, MSU-201, MSU-401 and MSU-501 were identified and confirmed by morphological and molecular analysis. These fungal isolates showed highest similarity withMortierella alpina species and can be potentialistic single cell oil producers. Thus, the present research study provides insight to the unseen fungal diversity and contributes to more comprehensiveMortierella alpina reference collections worldwide. Mortierellales contains nearly 100 described species and theMortierellaceae family contains about 13 genera.M. alpina is one of the main single cell oil producing species/arachidonic acid producing at commercial scale underMortierella genus. . Most of these oil accumulating microorganisms are species of yeast and fungi. The comprehensive nuclear ribosomal deoxyribonucleic acid (DNA) molecular phylogeny analysis reported that the order genus. .Wang etrotocol .Mortierella species are ubiquitous, saprophytic and belong to zygomycetes class. The polyunsaturated fatty acids (PUFA) production potential makes these fungi unique and significant to oil producing industries. Modern internal transcribed spacer based taxonomical classification , colony diameters were documented. Individual colonies of fungi that showed varying morphologies were picked up and identified bymycokeys 3.0 version. The morphological features of four fungal isolates were compared with distinguished monographs precisely with II Subgenus:Mortierella; 2. Section ALPINA Linnem. Mucorineen-Gatt.Mortierella: 35. 1941 monograph  medium after one-point inoculation in 9-cm petri dishes and incubation at 25°C for 5-7 days . The samnograph  to assesDirect microscopic identification was performed by using distilled water (wet mount technique) in which, a clean glass slide was labelled in the middle portion by marker and a drop of sterilized distilled water was put in on the marked middle portion, aerial spores and vegetative hyphae of the fungal isolate taken with the help of sterilized inoculation needle and distributed evenly within the water drop. Subsequently the glass coverslip was carefully added on the preparation in such a way that there was no air bubble formed. Same procedure was applied with lactophenol solution for identification of distinguished structures and prepared slides were examined under a light microscope at 40× magnification . The sporangiophore, sporangium and sporangiospores, shape and size, developmental pattern, mature and immature sporangiospores, intercalary chlamydospore were measured and documented . Pure cuTotal genomic DNA (gDNA) was extracted according to the standardized protocol . ITS andClustal_X v.2.1 and neighbour joining based phylogenetic tree was constructed usingMega  X software version 16.04.4  was extracted directly from the mycelia of fungal isolates, using Genomic DNA reparation Kit . Step by step protocol of gDNA isolation includes 1. 500 μL of Fungal Lysis Buffer added into 1.5 mL micro-centrifuge tube that contains the 1 cm agar cube of pure fungal culture. 2. 3 μL of Proteinase K solution added. Vortex to mix and spin down briefly. 3. The tubes were incubated at 56° C for overnight and centrifuged the lysate at 14,000 to 16,000×g for 10 minutes. 4. Transferred ~500 μL of supernatant to a new 1.5 mL micro-centrifuge tube, which contains 500 μL of isopropanol. The tube was inverted several times to mix gently. 6. Centrifuged at 14,000 to 16,000×g for 10 min and the supernatant was discarded. 7. 1 mL of 70% ethanol was added, centrifuged again at 14,000 to 16,000×g for 5 min and the supernatant was discarded. 8. The pallet was air dried for 3 min, resuspended with 50 μL TE Buffer and incubated at 56°C for < 1 hr. 9. Optical density (OD) was measured reading using spectrophotometer  and the nucleic acid was diluted to 15 to 25ng/μL and 2 μL of diluted nucleic acid was used as DNA template for PCR. 10. PCR mix was prepared according to manufacturer’s instructions and 2 μL of DNA template was added with each 23 μL of PCR mix into 0.2 mL tube or 96-well plate. 11. PCR cycle protocol was run on thermocycler and ~700bp PCR products were checked on 1% agarose gel  and sequenced by ABI3100 sequencer. 12. After the sequencing results were ready, the reads were trimmed off with quality value (QV) < 20, after that the forward and reverse sequencing results were aligned. 13. The obtained sequences were compared against the earlier submitted NCBI database using the BLAST algorithm  to verifMortierella genus. Colonies of oleaginous fungal isolates after seven days of incubation at 25°C on PDA, were sporulating, fast growing, producing a concentric pattern, had flower-shaped radial growth, and were yellowish to whitish in color as depicted inM. alpina  isolate are given inOn the basis of morphological and cultural characteristics, the fungal isolates were confirmed and belong toMortierellales as depicted inM. alpina genomes sequences submitted in GenBank NCBI  as shown inM. alpina species. The ITS sequences of these fungal isolates were deposited in GenBank with accession number ofMZ298831:MZ298835.In the ITS sequences analysis based on BLASTn , MSU-101, MSU-201, MSU-401 and MSU-501 isolates were fall within the orderMortierella alpina fungal strains reference collections and describes the diversity of these strains with known strains to date as shown inMortierella isolates add on to a large contribution of fungal diversity collections all over the world but still there is a plenty of room for more comprehensiveM. alpina collections from Libya and this is the limitation of the present study. Thus, further research work needs to be carried out in future so that the hiddenMortierella fungal diversity and their SCO production potential can be harnessed.The present study added on theM. alpina isolates were conserved except some identified polymorphic sites. Furthermore, the interpretation was made that the variability is present in ITS1 and ITS2 regions, as there was no polymorphic site in the 5.8 rDNA region. Thus, it was evident that the ITS region could be used to confidently discriminate betweenM. alpina and other closely related species. These researchers also highlighted that NJ (Neighbour-Joining phylogenetic tree) tree analysis provides precise genetic diversity betweenM. alpina strains to come out with significant interpretation and conclusion.Chen and Ho (2008) reported the significance of internal transcribed spacer region  for the genetic characterization and these strains and found that the 5.8 rDNA regions ofMortierella are potentialistic producers of C18 and C20 PUFAs (polyunsaturated fatty acids) such as γ-linolenic acid and arachidonic acid.M. alpina species is quite famous for the production of single cell oils as describes and reported by multiple scientist’s time by time  gel image.jpg-Mortierella alpina novel strain-MSU-201_Front view.jpg-Mortierella alpina novel strain_MSU-101.jpg-Mortierella alpina novel strain_MSU-101_Back view.jpg-Mortierella alpina novel strain_MSU-201_Back view.jpg-Mortierella alpina novel strain_MSU-401_Back view.jpg-Mortierella alpina novel strain_MSU-401_Front view.jpgMortierella alpina novel strain_MSU-501_Back view.jpg-Mortierella alpina novel strain_MSU-501_Front view.jpg-Net of hypha with branching and chlamydospore, MSU-501.jpg-Net of hypha with branching and septation, MSU-501.jpg-Phylogenetic tree MSU-101 and MSU-401.jpg-Phylogenetic tree_MSU-201 and MSU-501.jpg-Terminal chlamydospores with papillate ornamentation and hyphal segment remaining at the distal end, MSU-501.jpg-Terminal chlamydospores.jpgThis project contains the following underlying data:Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the M. alpina strains from Al-Jabal Alakdar (Northeast of Libya). The novel strains sequences submitted in NCBI GenBank with specific barcode  (accession no. MZ298831: MZ298835) and cultures preserved at Culture Collection Center of University. Thus, the research work is accepted and recommended for indexing.The research study addresses and discusses the novel strains isolation, identification and genetic diversity assessment by standard protocols. The conserved internal transcribed spacer regions amplified and probably gives idea about the specific region in internal transcribed spacer for loci in terms of bioprospecting and exploitation of these novel strains for single soil production. The methodology discusses the isolation, identification and molecular characterization ofIs the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Molecular Biology, BionanotechnologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. M. alpina strains to produce single cell oil and describes the isolation, identification, and molecular characterization ofM. alpina strains from al-Jabal al-Akhḍar (Northeast of Libya) by standard protocols. The conserved internal transcribed spacer regions amplified, and assessment of genetic diversity carried out in this research work.The research study documented four novel The novel strains sequences submitted in NCBI GenBank with specific barcodes  (accession no. MZ298831: MZ298835) were given. However, further research work in connection with single cell production can be done and set a benchmark for bioprospecting in the future. I hope in future the work will be carried out in this direction. As far as the present research study is concerned, the novel strains are described, organized, and documented, thus accepted for indexing.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Genetics & GenomicsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Comments:Mortierella alpina novel strains isolated from Libya. Justification on the research work shown is manuscript is profound and significant since the research manuscript describes valuable information onM. alpina strains from Al-Jabal Alakdar (Northeast of Libya). Stepwise flowsheet and methodology discuss the isolation, identification and molecular characterization ofMortierella alpina strains are well known and documented for the potential of single cell oil production (SCO) at large scale. So, the conserved internal transcribed spacer regions amplification and assessment of genetic diversity carried out in this research work will give insight on future standard protocols and further work in this field for exploitation and production of arachidonic acid/poly-unsaturated fatty acids production. The novel strains sequences submitted in NCBI GenBank with specific barcode  (accession no. MZ298831: MZ298835) and cultures preserved at Culture Collection Center of University. Further research work in connection with single cell production can be done and set a benchmark for bioprospecting in future. The work was carried out by a PhD candidate and will facilitate the career growth for the candidate for future opportunities and prospects.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Cell and cancer biology; plant biotechnology; industrial microbiologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.