Bone conduction hearing aids (BCHAs) offer an alternative solution for individuals with outer or middle ear issues who cannot benefit from traditional air conduction hearing aids. However, the phenomenon of “crosstalk,” where sound intended for one ear is mistakenly transmitted to the other ear through bone conduction, presents a challenge. This unintended transmission may limit the benefits of binaural hearing that can be achieved using two BCHAs, such as accurately detecting a sound source's direction. In this article, we present a method to suppress “crosstalk” within the human head using an adaptive algorithm to control two audiometric bone transducers.•Our method involves positioning an error sensor at a location considered close to the cochlea, such as the ear canal or the mastoid, and utilizing an adaptive algorithm to estimate the crosstalk compensation filter. This filter generates an anti-signal, which is then transmitted to one of the two transducers, effectively cancelling the crosstalk.•To verify whether the crosstalk cancellation reaches the cochlea in the inner ear, we provide a procedure for measuring hearing thresholds with and without crosstalk cancellation. This acts as a subjective measure of the efficacy of our crosstalk cancellation method.By leveraging an adaptive algorithm, this approach provides personalized cancellation and has the potential to enhance the performance of binaural BCHAs.  Specifications TableMethod detailsSound can reach the cochlea in the inner ear via two mechanisms: air conduction (AC) and bone conduction (BC). AC sound enters the ear canal, vibrates the eardrum, and sets the ossicles in motion, ultimately transmitting the sound to the cochlea a. In conOne of the limitations of BC hearing aids that can significantly impact the primary binaural cues of interaural phase and level differences (IPD and ILD) is “crosstalk,” or undesirable BC transmission. This phenomenon occurs when BC sounds from the left and right sides get mixed at both cochleae b, resultIn the context of binaural sound reproduction, the “crosstalk” issue encountered in BC is analogous to the challenges faced when using a pair of loudspeakers ,7. With In an effort to address the challenge of “crosstalk,” McLeod and Culling developed a technique based on psychoacoustics to measure the phase and level of BC sounds reaching each cochlea. This approach made use of a pair of bone transducers alongside two insert earphones Although the “two-BT” technique has shown efficacy in reducing crosstalk within the frequency range of 1 kHz to 4 kHz, it encounters limitations when applied to frequencies below 1 kHz In this paper, we detail a method for cancelling low-frequency crosstalk that takes advantage of the “zone of quiet” concept from active noise control (ANC) Our study is built on the assumption that a reduction in crosstalk sound, whether at the mastoid or in the ear canal, as detected by an accelerometer or a probe microphone, respectively, will correspond to a perceptible change at the cochlea. Using the specific methodology presented in this paper, which is focused on achieving cancellation at the respective sensor locations, we designed a hearing threshold experiment under conditions with and without crosstalk cancellation. The purpose of this experiment is to precisely identify the frequencies at which this reduction is most perceptible at the cochlea.In the method detailed in this paper, crosstalk cancellation is aimed at one ear—the ear on the side of the sensor, as illustrated in To implement and evaluate our crosstalk cancellation method, we used a combination of specialized equipment and custom software. We have prepared two tables for a comprehensive overview of the resources utilized. We performed our experiments using the setup illustrated in The placement of bone transducers followed a configuration analogous to a bone-anchored hearing aid (BAHA) setup. Two bone transducers, secured to an elastic headband with 3D-printed custom housing cases for a comfortable fit, were positioned on the user's head at about 50 to 55 mm behind the ear and slightly above the ear canal on the temporal bone. This location is known to be optimal for transmitting sound through bone conduction The placement of the sensor is essential for achieving optimal crosstalk cancellation at the cochlea. In our study, we focused on two potential cancellation sites: the ear canal and the mastoid. We primarily targeted the ear canal, as research has shown that the ear canal sound pressure (ECSP) is subject to change as bone-conducted sounds propagate through the human head ❖Step 1 – Measurement Setup Based on B81 Datasheet: Following the B81 datasheet ❖Step 2 – Frequency Response Measurement: We played the time-stretched pulse (TSP) signal ❖Step 3 – Application of the Kirkeby Algorithm: We represent the left and right B81 impulse responses as The regularization parameter, ❖Step 4 – Applying the Inverse Kirkeby Equalization Filters: We convolved the inverse Kirkeby filters  Estimation of crosstalk compensation (CTC) filter; and (3) Verification of the effectiveness of the CTC filter. Each subsection provides detailed descriptions of its corresponding step. In addition to the text, a video has been included to provide a visual demonstration of the system in operation.The implementation of a unilateral crosstalk cancellation system for bone conduction requires a sequence of steps. For clarity and comprehensibility, these steps have been divided into three subsections: (1) et al. ❖Step 1 – Signal Preparation: We generated a stereo signal, each audio channel of which contained a TSP signal, separated by the length of the TSP signal . The TSP signals in the left and right channels began at 1 s and 5.1 s, respectively, with the output voltages from the audio interface set to −20 dB re 1VRMS  in length. Given the closeness of the transducer and sensor on the user's head, this duration was deemed sufficient  algorithm. We chose this due to the algorithm's simplicity and the proven effectiveness of the FxLMS method for our specific application. It is worth noting that the FxLMS is not the only available approach. For instance, the closed-form solution proposed in ❖Step 1 – Initialize Variables: We set the CTC filter ❖Step 2 – Acquire Input Signals: We utilized white noise filtered by a 224–1122 Hz bandpass as the input signal ❖Step 3 – Filtered-x Signal: The block input signal ❖Step 4 – Update CTC filter coefficients: For each time step (a) Calculating the output of the CTC filter(b) Calculating the error signal (c) Updating filter coefficients: ❖Step 5 – Iterate: We repeated steps 2–4 for a predefined number of iterations (60 s in our case). The change in the CTC filter coefficients becomes negligible at this point : If the crosstalk sound was not successfully reduced or cancelled, it may have been due to the transducers shifting from their original positions. In this case, we reestimated the impulse responses and recalculated the CTC filter. We then repeated Steps 1–3 to verify the effectiveness of the updated CTC filter.In this subsection, our objective is to validate the effectiveness of the CTC filter, which we obtained via computer simulation in the preceding subsection. To perform this verification experiment, we adhered to the following steps:https://youtu.be/D-Oy1AyythQ.The successful implementation of a crosstalk cancellation system in bone conduction applications relies heavily on verifying the effectiveness of the CTC filter. The results of our verification process, which showcase a successful reduction in crosstalk sounds, are depicted in the right panel of Setup, in which we detail the experimental configuration; (2) Stimuli, in which we specify the specific signals used; and (3) Procedure, during which we narrate the systematic steps executed for hearing thresholds measurements.The primary aim of this section is to ascertain if the crosstalk cancellation, specifically designed to take place at the sensor location, indeed extends effectively to the cochlea in the inner ear. We do this by employing pure-tone hearing thresholds, measured in noise conditions both with and without the implementation of crosstalk cancellation. The specifics of this evaluation are distributed across three subsections: (1) The setup for subjective evaluation closely follows the configuration described in the “Experimental Setup” section, but with some distinct differences. After confirming crosstalk cancellation at the sensor location, subjects were instructed to insert earplugs deeply into both ear canals, ensuring that about 75–100 % of the earplug was inside the ear canal, as depicted in This subsection outlines the specific stimuli utilized to measure hearing thresholds under noisy conditions, both with and without crosstalk cancellation. As depicted in RMS. The subject was then presented with three audio intervals, each initiated by a button on the GUI. The button turned green when the corresponding audio was played. The pure tone was randomly assigned to one of the intervals, with noise present in all three. The subject was instructed to listen to all three intervals and identify the one containing the pure tone by clicking the corresponding button. The intensity level was decreased by the step size (4 dB) if the subject correctly identified the interval containing the pure tone in two consecutive trials (2−down). If they failed to do so (1−up), the intensity level was increased by the same step size. After four reversals, the step size was reduced to 2 dB. The procedure continued until a total of 12 reversals were reached, after which we calculated the threshold estimate by averaging the intensity values at the last eight reversals. This process was repeated for all frequencies tested. RMS. This value is lower than the −50.75 dB threshold measured without crosstalk cancellation, suggesting that crosstalk cancellation led to an improvement in hearing thresholds for this individual. Additionally, We estimated the hearing thresholds using a 2−down/1−up adaptive three-interval forced-choice (3IFC) procedure. To facilitate this, we developed a custom-made graphical user interface (GUI) using Python's IPy-Widgets package. The GUI, shown in Participants and Supplementary Methodology: We detail the normal-hearing individuals who took part in our primary study and describe the overall experimental setup. Additionally, to provide context for the earplug insertion depth used in our main experiment, we introduce a supplementary experiment investigating the impact of different earplug insertion depths on the occlusion effect. (2) Experiment Results and Discussion: Here, we present and interpret the findings from the subjective evaluation. This evaluation not only includes results from the primary crosstalk cancellation method but also integrates data from the supplementary occlusion effect experiment. We measure pure-tone hearing thresholds under noise conditions with and without crosstalk cancellation and discuss data concerning the reduction of crosstalk at the sensor location.In this section, we put our crosstalk cancellation method to the test. We aim to confirm whether the cancellation detected at the sensor location effectively reaches the cochlea in the inner ear. This validation process unfolds in two parts: (1) Participants: Seven individuals, five males and two females aged between 21 and 32 years, participated in this study. Each of them had normal hearing, confirmed via pure-tone audiometry, with hearing thresholds all below 20 dB HL. Prior to the experiment, an otoscopic examination was conducted to ensure that no excessive earwax was present, as it could potentially pose a risk when inserting earplugs deeply. All participants provided their written informed consent before the experiment commenced. Participants wore a pair of audiometric bone transducers, as depicted in Supplementary Methodology: Due to the potential occlusion effect from our main study's foam earplugs—an increase in sound perception through BC Our experiment demonstrated substantial crosstalk reduction at both target cancellation locations: the ear canal and the mastoid. As evidenced in the top panel of p = 0.00018), with notable enhancements also at 250 Hz  and 397 Hz . For the probe microphone, the most substantial improvement was at 397 Hz , with significant improvements also at 250 Hz , 315 Hz , 500 Hz , and 1000 Hz .In our hearing threshold measurements, both with and without the anti-noise signal, we found that the hearing threshold improvements, as shown in the bottom panel of In light of our observations regarding hearing threshold improvements  and 794 Hz (1.1 dB). At these two frequencies, some participants actually experienced an increase in hearing thresholds with the addition of anti-noise, suggesting that the anti-noise signal may have inadvertently amplified the crosstalk sound—a result that contradicts our expectations. Such amplification could be due to variations in phase relationships caused by the distance between the sensor and the cochlea in the inner ear.These results suggest that crosstalk cancellation at the cochlea can potentially be achieved by cancelling crosstalk sounds at sensor locations. However, this method's effectiveness may diminish at higher frequencies where the designed phase mismatch at the sensor location does not yield optimal cancellation at the cochlea. Further research is necessary to ascertain whether crosstalk cancellation in bone conduction can genuinely enhance sound localization and improve overall hearing experiences. As our proposed approach demonstrates efficacy mainly at low frequencies, it might be beneficial to integrate this method with a psychoacoustic-based crosstalk cancellation method, such as the “two-BT” method proposed by McLeod and Culling In this study, we proposed and evaluated a method for crosstalk cancellation through bone conduction. Our approach assumes that the cancellation of crosstalk sounds in the ear canal, as measured by a probe microphone, or on the mastoid, as detected by an accelerometer, can effectively reach the cochlea in the inner ear. By employing the FxLMS adaptive algorithm, we demonstrated that sound signals from two audiometric bone transducers could be effectively neutralized at the sensor locations. We also found that participants’ hearing thresholds were improved with the application of crosstalk cancellation. However, the effectiveness of our method was primarily observed at low frequencies. At higher frequencies, due to the distance between the cochlea and the cancellation site, the designed phase mismatch at the sensor location did not result in optimal cancellation at the cochlea. This led to a suboptimal phase relationship between the crosstalk sound and the anti-crosstalk signal, limiting the effectiveness of the cancellation. Future work could consider combining our current approach with a psychoacoustic-based method, which could potentially address the phase mismatch issue and lead to more effective cancellation at the cochlea across a wider frequency range. Additionally, testing our method on people with conductive hearing loss is important. This can help us see where our approach might need improvement and find ways to make it work better.This work involved human subjects. Written informed consent was collected from all participants utilizing a protocol (No. 29–13) approved by the Institutional Review Board of Life Science Research of Chiba University, Chiba, Japan.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Drug-induced ototoxicity resulting from therapy with aminoglycoside antibiotics and loop diuretics is one of the main well-known causes of hearing loss in patients. Unfortunately, no specific protection and prevention from hearing loss are recommended for these patients. This study aimed at evaluating the ototoxic effects produced by mixtures of amikacin  and furosemide  in the mouse model as the hearing threshold decreased by 20% and 50% using auditory brainstem responses (ABRs). Ototoxicity was produced by the combinations of a constant dose of AMI  on FUR-induced hearing threshold decreases, and a fixed dose of FUR  on AMI-induced hearing threshold decreases, which were determined in two sets of experiments. Additionally, the effects of N-acetyl-L-cysteine  on the hearing threshold decrease of 20% and 50% were determined by means of an isobolographic transformation of interactions to detect the otoprotective action of NAC in mice. The results indicate that the influence of a constant dose of AMI on FUR-induced hearing threshold decreases was more ototoxic in experimental mice than a fixed dose of FUR on AMI-induced ototoxicity. Moreover, NAC reversed the AMI-induced, but not FUR-induced, hearing threshold decreases in this mouse model of hearing loss. NAC could be considered an otoprotectant in the prevention of hearing loss in patients receiving AMI alone and in combination with FUR. Ototoxicity as a result of side effects produced by some specific drugs has been known in medicine for many years. Aminoglycoside antibiotics and loop diuretics are the drugs whose activity is closely correlated with the highest incidence of ototoxic effects in humans. Of note, aminoglycoside antibiotics produce hearing loss and vestibular dysfunction due to permanent hair cell loss ,2, whereMany experiments have confirmed that loop diuretics potentiate ototoxic effects in aminoglycoside antibiotics. However, reports are scarce to document the protection from such combinations of drugs. In clinical practice, the co-administration of aminoglycosides  along with loop diuretics  is usually prescribed for patients undergoing continuous ambulatory peritoneal dialysis (CAPD) with peritonitis ,11. In sOxidative stress associated with aminoglycoside antibiotics treatment has been extensively investigated in both animal and clinical studies ,12,13,14Overwhelming evidence indicates that N-acetyl-L-cysteine (NAC), due to its anti-inflammatory effects and free radical scavenging properties, exerted protective effects against kanamycin- , gentamyConsidering molecular mechanisms involved in the development of ototoxicity after treatment with aminoglycosides and loop diuretics, it was crucial to determine the ototoxic effects produced by the mixture of AMI and FUR in an animal model of ototoxicity—the hearing threshold decrease model in mice using auditory brainstem responses (ABRs).To determine the effects of AMI and FUR on hearing threshold decreases, two sets of experiments were conducted on animals to confirm whether or not any difference in ototoxicity existed in relation to the order and application of drugs (AMI + FUR or FUR + AMI). The second aim of this study was to determine whether NAC could reverse or partially alleviate ototoxicity in the hearing threshold decrease model in mice. The application of NAC was important because of the prophylaxis that could be used before the administration of the drugs with ototoxic side effects.20 and hearing threshold decreasing dose of 50%—HTDD50)  reduced in a dose-dependent manner the hearing threshold in experimental animals A,C, whic—HTDD50) .20) and 50% (HTDD50) with one-way ANOVA followed by Holm–Sidak’s multiple comparison tests, which revealed that the doses of AMI significantly differed among the tested groups  and , respectively. More specifically, NAC administered i.p. in a constant dose of 500 mg/kg, which shifted (to the right) the dose–response relationship curves for the AMI-induced hearing threshold decrease of 20% and 50%, respectively (p < 0.05) and by 50% (p < 0.01) compared to AMI+VEH treated animals (p < 0.05) the threshold hearing decreases in experimental animals (p < 0.001 for 20% and p < 0.001 for 50%). In contrast, NAC (500 mg/kg) was added to the mixture of FUR (30 mg/kg) while AMI partially reversed the effects produced by FUR; however, the threshold hearing decrease of 20% for the three-drug combination (AMI+FUR+NAC) did not differ from that for AMI+VEH treated animals  from that of AMI+VEH treated animals  was added to the mixture of FUR (30 mg/kg) with AMI for the threshold hearing decrease by 50%, it did not reverse the FUR+AMI-evoked hearing threshold decrease, but the hearing threshold for the three-drug combination of AMI+FUR+NAC significantly differed   .20) with one-way ANOVA followed by Holm–Sidak’s multiple comparison tests, which revealed that doses of FUR significantly differed among the tested groups . Similarly, one-way ANOVA with Holm–Sidak’s post hoc test revealed that doses of the FUR-induced hearing threshold decrease of 50% (HTDD50) and also considerably differed . NAC administered i.p. in a constant dose of 500 mg/kg, which partially protected the mice from hearing threshold decreases compared to AMI+VEH treated animals, but this effect was not statistically significant for both a 20% and 50% hearing threshold decrease (p < 0.05) the threshold hearing decreases in experimental animals for a hearing threshold decrease of 20%, but not that for a hearing threshold decrease of 50%. In the case of NAC (500 mg/kg), when added to the mixture of AMI (500 mg/kg) with FUR, it did not reverse the effects produced by AMI (500 mg/kg) because the threshold hearing decrease for 20% for the three-drug combination (AMI+FUR+NAC) did not differ for the FUR+AMI treated animals  and NAC, which was additionally added to the mixture of both ototoxic drugs (point C)  was significant at p < 0.001  for a hearing threshold decreased by 20%  for a hearing threshold decreased by 50% (20 and HTDD50 for AMI+FUR (points B) considerably differed from the theoretically calculated additive doses, reducing the hearing threshold in animals (points A). More specifically, FUR was administered in higher  doses than theoretically expected to produce a hearing threshold decrease of 20% and 50% when combined with a constant dose of AMI  was significant at p < 0.05  for the hearing threshold decreased by 20%  and for a hearing threshold decreased by 50%  a,b. NAC point C) a,b. Withd by 20% a and at d by 50% b. The isd by 20% a and at d by 50% b.20 and HTDD50 values for the mixture of both ototoxic drugs were placed into the Cartesian coordination system to illustrate the interactions between these two drugs (point B) and NAC, which was added to the mixture of both ototoxic drugs (point C)  was significant at p < 0.001  for a hearing threshold decrease of 20%  for a hearing threshold decrease of 50% (20 and HTDD50 for FUR+AMI (point B) considerably differed from the theoretically calculated doses, reducing the hearing threshold in animals by 20%  was significant at p < 0.001  for a hearing threshold decrease of 20% (p = 0.7648) for a hearing threshold decrease of 50%  c,d. NAC point C) c,d. Withe of 20% c and at e of 50% d. The ispoint A; c but notpoint A; d. More spoint A; c. In conpoint A; d. With Se of 20% c but wase of 50% d.Accumulative evidence indicates that some antioxidants  ameliorated the ototoxic effects evoked by aminoglycosides  in various experimental models of ototoxicity ,38,39,40In this study, we conducted two sets of experiments related to the order of drug administration. In the first set of experiments, we determined the effects of constant doses of NAC and FUR on AMI-induced ototoxicity, whereas, in the second set of experiments, we determined the effects of NAC and AMI on the FUR-evoked hearing threshold decrease in experimental animals. In these two sets of experiments, we found evidently that the order of administration of ototoxic drugs was very important and crucial for observed ototoxic effects. NAC alleviated the ototoxicity in AMI-treated animals but not in FUR-treated mice. We confirmed that NAC reversed the ototoxicity evoked by AMI but not that of FUR, suggesting that this compound could be used as a prophylaxis for AMI-induced ototoxicity. In this study, we found that FUR evoked a hearing threshold decrease and was resistant to NAC. Moreover, we confirmed that hearing threshold changes produced by AMI and FUR  were strongly expressed when testing in animals in the model of a hearing threshold decrease of 50% compared to that of 20%. It is important to note that a hearing threshold decrease of 20% was investigated in animals, which received respective treatment 15 min prior to the hearing threshold measurement, whereas the hearing threshold decrease of 50% was detected in the mice receiving the respective treatment 30 min before the hearing threshold measurement. It seems that in this animal model, more time was needed for the drugs to reach their targets in the inner ear in order to diminish the hearing threshold. In both sets of experiments, NAC was injected systemically (i.p.) at 60 min before the hearing threshold detection. It is important to note that this animal model based on the hearing threshold decrease of 20% and 50% was applied in experimental studies to determine the influence of drugs with ototoxic properties on hearing processes and any subtle effects that reduced the hearing threshold by 20% and 50% were investigated. Of note, in this experimental model, each animal was examined twice, the first time before the drug administration (pretreatment threshold), which was considered to be a baseline threshold, and the second examination was conducted after the drug treatment (posttreatment threshold). An experimental evaluation of the hearing threshold confirmed that hearing deficits resulted from drug administration but not from congenital deafness in animals.Another fact related to the selection of constant doses of FUR and AMI, when combined together, needs a short explanation. Previously, we determined the doses of AMI and FUR, which, when used alone, produced by themselves a hearing threshold decrease of 50% in experimental animals. For the combinational experiments, we selected half of the doses of FUR and AMI, which decreased the hearing threshold in animals by 50%. This was the reason to test the effects produced by FUR in a constant dose of 30 mg/kg and AMI in a constant dose of 500 mg/kg, which were approx. their halves of the doses and decreased the hearing threshold by 50% in animals. We used the same AMI and FUR doses to detect the hearing threshold decrease by 20% in animals due to a simple comparison of the results among the respective groups of animals.Isobolographic transformation revealed that all the interactions between AMI and FUR were antagonistic in nature, except for one combination of FUR+AMI in the hearing threshold decreased by 50%, which was additive. The antagonistic interaction between FUR and AMI confirmed at least in part that both drugs concurrently antagonized their own effects and, thus, more drugs were required to produce a hearing threshold decrease of 20% and 50% in animals compared to theoretically calculated , and preGenerally, NAC is able to reverse the effects produced by AMI in terms of the restoration of oxidative potential in cochlear cells. In such a situation, the hearing threshold decrease of 20% and 50% were shifted to higher doses of FUR or the mixture of AMI with FUR to break the protective effects of NAC. Of note, NAC was administered in a constant dose of 500 mg/kg in both sets of experiments, in which doses of FUR or AMI increased proportionally to the observed hearing threshold decreases of 20% and 50%.The main limitation of this study was the acute experimental administration of the drugs . In this study, we evaluated the hearing threshold by 20% and 50% after acute (single) administration of the drugs in combination. Acute vs. chronic administration of the ototoxic drugs are very important in relation to the fact that loop diuretics usually evoke reversible hearing loss due to their influence on sodium, chloride and potassium cotransporters in the cochlea and temporal induction of ions imbalance in the endolymphatic fluid of the inner ears . By contMale (14–21 days old) Albino Swiss outbred mice were used in this study. After adaptation to laboratory conditions, 144 mice were randomly assigned to experimental groups containing 6 animals (24 groups). The experimental protocols and procedures described herein were approved by the Local Ethics Committee (License no. 31/2008) and were in accordance with the ARRIVE guidelines .N-acetyl-L-cysteine  were given intraperitoneally (i.p.) as follows: NAC was given 60 min before AMI and FUR administration, while AMI and FUR were administered i.p. either 15 min (for hearing threshold decrease by 20%) or 30 min (for hearing threshold decrease by 50%) before hearing measurement. The hearing threshold in mice was evaluated in fully anesthetized animals with a combination of xylazine ) and ketamine ).Amikacin , furosemide , and Auditory brainstem responses (ABRs) were collected using a computerized Interacoustics Eclipse EP15 evoked potential unit . The left ear of each animal was stimulated by alternating clicks, and ABRs were recorded via subcutaneous electrodes placed near the ipsilateral pinna, vertex and contralateral pinna, with the ground electrode placed along the trunk. The responses were amplified, filtered, averaged by a computer, and displayed on a computer screen. All the detailed information about the ABRs and their analysis is described elsewhere . Briefly20 and HTDD50) were calculated for AMI, FUR and their combination. To illustrate dose–response effects between the increasing doses of the studied drugs and their corresponding decreases in the hearing threshold, sigmoidal dose–response curves were constructed, from which the HTDD20 and HTDD50 values were calculated. To determine the HTDD20 and HTDD50 values, at least 3 points were plotted, and the curves were best fit to these points, reflecting particular doses of the studied drugs when administered either alone or in combination with their corresponding decreases in the hearing threshold. From a pharmacological viewpoint, to adequately construct each sigmoidal curve, at least 3 drug doses need to be used [20 and HTDD50. From 3 points, it was possible to determine the HTDD20 and HTDD50 values, and this was the reason that only 3 doses of each drug administered alone or in combination with NAC were investigated. Of note, the pretreatment hearing threshold was predicted to be 100%, and any changes in the animals’ hearing threshold after the injection of AMI and FUR were expressed as the % of reduction in the hearing threshold, as described elsewhere [A pretreatment hearing threshold was assessed in each anesthetized mouse before the systemic (i.p.) administration of the drugs either alone or in combination with NAC. Subsequently, the animals, after receiving AMI and FUR (at increasing doses), were subjected to the measurement of posttreatment hearing threshold. Decreases in the hearing threshold (in %) were calculated by comparing the pretreatment with posttreatment hearing thresholds. The percentage of the hearing threshold decrease for the respective doses of a drug mixture (AMI with FUR or FUR with AMI) was linearly assessed, from which the hearing threshold doses decreased by 20% and 50%  and calculated as presented elsewhere [The effects exerted by the mixture of AMI and FUR with NAC underwent isobolographic transformation. Any changes in the hearing threshold were calculated using linear regression analysis. The influence of NAC  on the hearing threshold in experimental mice was assessed by estimating the hearing threshold for animals receiving three drugs , from which the experimentally decreases in the hearing threshold were 20% and 50%  for AMI and FUR were analyzed with one-way ANOVA followed by post hoc Holm–Sidak’s multiple comparison tests. The statistical comparison of isobolographically transformed additive and experimental values was performed with Student’s t-test as presented earlier [20 and HTDD50 values were considered statistically significant if p < 0.05.Experimentally determined hearing threshold decreasing doses of 20% and 50% (HTDD earlier . DiffereNAC reversed the ototoxic effects evoked by AMI in the mouse model of the hearing threshold decrease by 20% and 50%. In contrast, NAC had no impact on the ototoxicity evoked by FUR in experimental animals. Only the isobolographic transformation of interaction should be used in combinatorial studies when evaluating the ototoxic effects produced by drug mixtures, whose side effects contribute to ototoxicity in experimental animals. The evaluation of the hearing threshold decrease by 20% and 50% seemed to be a good experimental model allowing for the detection of any subtle changes in drug-induced ototoxicity in animals.


Patients with otitis media (OM) encounter significant functional hearing impairment with conductive, or a combined hearing loss and long-term sequelae involving impaired speech/language development in children, reduced academic achievement and irreversible disorders of middle and inner ear requiring a long time therapy and/or multiple surgeries. In its persistent chronic form, Otitis media (COM) can often only be treated by undergoing ear surgery for hearing restoration. The persistent inflammatory reaction plays a major role, often caused by multi-resistant pathogens in the ear. Herein, we present outcomes of patients implanted with currently the only FDA approved active Middle Ear Implant Vibrant Soundbridge (VSB), suffering from persistent COM.The study enrolled 42 patients, treated by performing middle ear (ME) surgery to different extents and implanted with the VSB to various structures in the ME. Included were 17 children and 25 adults that had recurrent and/or persisting OM and significant hearing loss. Preoperative and postoperative patients' audiometric data were evaluated and the benefit with VSB assessed using the Glasgow Benefit Inventory for adults and pediatric cohorts. The microbial spectrum of pathogens was assessed before and after surgery, exploring the colonization of the otopathogens, as well as the intestinal microbiome from individually burdened patients.The mean functional gain is 29.7 dB HL (range from 10 to 56.2 dB HL) with a significant improvement in speech intelligibility in quiet. Following VSB implantation, no significant differences in coupling were observed at low complication rates. Postoperatively patients showed significantly increased benefit with VSB compared to the untreated situation, including less otorrhea, pain, medical visits, and medication intake, with no recurrent OM and significant bacterial shift in otopathogens. The analysis of the intestinal microbiome displayed a high abundance of bacterial strains that might be linked to chronic and persistent inflammation.Functional ear surgery including rehabilitation with a VSB in patients suffering from COM present to be safe and effective. The successful acceptance accompanied by the improved audiological performance resulted in significant benefit with VSB, with a shift in the ear pathogens and altered microbiome and thus is a great opportunity to be treated. Pseudomonas aeruginosa and Staphylococcus aureus . There was no significant differentiation in the groups between the different coupling methods, nor within the adult and children groups compared to the WRS result at the first fit (Table The Word recognition score (WRS) before and after implantation at 65 dB SPL in a quiet situation was used to assess the comparison between age groups. The mean unaided WRS averaged for adults was 5.95%  and 76% of the children’s group (n = 13) participated in answering the questionnaires of the GBI/GCBI. All participants showed high levels of benefit (scores > 0) after ear surgery and implantation of the VSB with a total benefit of 53 ± 6  for adults and Glasgow Children’s Benefit Inventory (GCBI) for children were used to assess the patient’s benefit from VSB, 84% of the adult’s group (Staphylococcus aureus (24%) and Pseudomonas aeruginosa (19%), followed by Proteus mirabilis (9%)  and β-hemolytic Streptococci (5%). Moreover, pathogens of the lower intestine, next to the prominent Pseudomonas and Proteus could be detected in our patients, such as Corynebacterium striatum, Enterobacter cloacae (5%) and Klebsiella oxytoca (5%). Moreover, ubiquitary Acinetobacter Iwoffi (4%) and Origella urethralis (5%) could be detected.Presurgery, patients with severe, therapy-resistant COM display mainly two prominent pathogens: 9%) Fig. A. Less pStaphylococcus aureus (13%) could be detected, whereas less prevalent, we found Streptococcus pneumonia (4%), β-hemolytic Streptococci (4%), Pseudomonas aeruginosa (4%), Proteus mirabilis (4%), Corynebacterium striatum (4%) and Enterobacter cloacae (4%), again elucidates, that many persistent inconvenient pathogens origin from the lower intestine.After surgery and VSB-Implantation in a one or two step procedure, perioperative antibiotic treatment and postoperative care, the microbial colonization in patients displays a distinct different pattern Fig. B. After Pseudomonas aeruginosa (patient 1) and Proteus mirabilis (patient 2) persistence, presenting a severe progressive destructive COM that is very difficult to deal with. Therefore, we performed gut microbiome analysis, aiming to gain a deeper understanding of the microbial composition of these selected patients. The gut consortium was dominated by phyla Firmicutes and Bacteroidetes represented by genera Oscillospira, Ruminococcus, Gemmiger, Clostridium and Bacteroides, Prevotella, Parabacteroides, Alistipes accordingly  and its perioperative treatment, seems to be an elegant and effective therapy solution for both, the hearing ability and the recurrent local inflammation.The causes of a chronic progressive therapy-resistant OM are multifactorial and represent a challenge in the treatment and restoration for every patient suffering from recurrent inflammation. This is often associated with chronification resulting in significant hearing impairment and otorrhea due to microbial ME infections. Moreover, wearing conventional hearing aids in the external auditory canal, often aggravate the local situation, leading to a The favorable results from this study confirm the long term safety and the audiological benefit with VSB treatment , 52, 53.The objective outcomes in terms of Word Recognition Score (WRS) showed significant improvements after surgery independent of coupling modality applied, which was also accompanied by subjective outcomes indicated by high patient satisfaction and confirm other publications for children , 54, 55 At the same time, there was a decrease in the number of unpleasant otorrheas caused by bacterial infections. Prior to therapy, often the affected patients could not benefit from conventional hearing aids and had a tremendous lack of social interaction due to their hearing deficit. A poor fit of hearing aids resulting in recurring bacterial colonization and infections was the major reason why hearing aids were not used.Corynebacterium, Dolosigranulum with the species Dolosigranulum pigrum, and Moraxella with the species Moraxella lincolnii occurred more often in healthy children’s nasopharynx [There are theories on the mismatch between normal commensal and coexisting pathogenic bacteria and, consequently, an alternate immune response that then leads to chronic infections . A studyopharynx . Patientopharynx . A standStaphylococcus aureus and Pseudomonas aeruginosa. 10 out of the 42 patients suffered from an infection with S. aureus, while 8 out of the 42 had Pseudomonas aeruginosa, which are both well-known in COM and often develop resistance against most antibiotic remedies. Essentially, the therapy of COM and CSOM (chronic suppurative OM) face two main difficulties: first, the development of multi-resistant bacteria, e.g., 3MRGN and 4MRGN P. aeruginosa and, second, the formation of biofilms that allow pathogens to evade conventional antibiotic therapy [S. aureus and P. aeruginosa are both known to exhibit biofilms and create shielded microenvironments within the mucus layer [Haemophilus influenzae, Streptococcus pneumoniae and S. aureus and hearing loss was 10 dB worse compared to patients with negative bacterial results [S. pneumoniae and S. aureus could be detected in our study population before surgery and a decrease of both species could be achieved afterward. Hence, patients have a dual benefit due to an improvement of chronic inflammation and hearing.Pathogens in the ME are key players in the destructive process of COM and are sometimes hard to overcome. The presented patients showed a common spectrum of pathogens  dominate therapy , 60. S. us layer . To guarWe were able to increase the colonization with normal commensal bacteria after surgery by more than 30% compared to pre-surgical conditions and achieved a suitable state for VSB installation. This increase of healthy commensal bacteria underlines the hypothesis that chronic inflammation is affected by a re-establishment of microbial balance. Nevertheless, 37% still presented pathogens after surgery. Noteworthy, postoperative all patients had an intact tympanic reconstruction or a fat obliterated radical cavity with a closed outer ear canal and none of the patients suffered from otorrhea. These changes might be evaluated as a possible result of the therapy as a whole including surgery and antibiotics, and further studies, investigating the microbial shift after other surgeries without VSB are necessary and ongoing.In the end, therapeutical options are yet limited. Antibiotic treatment and aural toilet are still standard procedures to solve local infections and there is an urgent need to expand the range of possibilities and look for modulating factors beyond the aural area.Bacteroides, Bifidobacteria, Faecalibacterium, Clostridium and Gemminger were highly abundant. Butyrate is a short chain fatty acid, known to play a crucial role in the modulation of anti-inflammatory responses, intestinal barrier function, [Several studies indicated a correlation between the microbial composition of upper respiratory tract and ME infection , 61. A munction,  as well unction, . Still, Our study is limited by its relatively small sample size. However, our observations warrant further studies with larger numbers to obtain more conclusive and deeper understanding of the microbial signaling and interactions between the respiratory tract and the intestinal microbiotic composition. It appears necessary to eliminate negative effectors of chronic inflammation, such as bacterial colonization and any probable gut–dysbiosis, to achieve a long-lasting attempt of the supplemental hearing aid systems like VSB and to avoid future re-operations. As mentioned before, non-compliance by not wearing hearing aids is higher, when there is still an unsolved inflammatory condition. Studies reported the impact of intestinal bacteria and their products on health and well-being in the context of chronic inflammation –69. PartPatients suffering from a long-term history of COM, benefit significantly from undergoing surgery and hearing rehabilitation with a Vibrant Soundbridge. This is reflected in a significant hearing improvement and benefit, including less otorrhea, pain, doctor visits and medication use, with less or no recurring OM plus a significant shift in the composition of the bacterial colonization in the ear, specifically the origin of the lower intestine. Further insights into the network of the ear–respiratory–gut axis may reveal potential probiotic candidates as well as key metabolic pathways involved in disease development.


This study elucidates the importance of paramagnetismin BNO semiconductors and provides fundamental insight into its photophysics.The work herein paves the way for tailoring of its optoelectronicand photochemical properties for solar fuel synthesis.A family of boronnitride (BN)-based photocatalysts for solar fuelsyntheses have recently emerged. Studies have shown that oxygen doping,leading to boron oxynitride (BNO), can extend light absorption tothe visible range. However, the fundamental question surrounding theorigin of enhanced light harvesting and the role of specific chemicalstates of oxygen in BNO photochemistry remains unanswered. Here, usingan integrated experimental and first-principles-based computationalapproach, we demonstrate that paramagnetic isolated OB As a result of this doping,BNO exhibits semiconducting and magnetic properties,14 which can be exploited in photocatalysis.4 In a recent study,15 we investigatedoxygen doping in BN and presented a route to readily tune and predictthe relative oxygen content and optical band gap concomitantly usinga design of experiments approach. Detailed statistical analysis revealeda strong inverse relationship between the oxygen content and bandgap, which experimentally validated the computational results of Wenget al.9 However, the role of differentchemical states of oxygen in band gap narrowing in BNO remains unclear,and an understanding of these mechanisms would lead to the designof improved semiconductors.The optoelectronic properties of boronnitride (BN) have been increasinglystudied to explore the application of the material in fields suchas photocatalysis, photovoltaics and electronics, sensing, and quantumemission.which chemical state in BNO contributes tored-shifted optical band gaps and enhanced photochemistry? Our hypothesis is twofold. First, we postulate that paramagnetic,isolated OB3 states are responsible for lowering the band gap to the deep visible regionin BNO. We predict that a higher concentration of paramagnetic, isolatedOB3 states would lead to enhanced light harvesting. Second,we claim that adjacent OB3 centers yield diamagnetic O–B–O species (formed by placing OB3 centers adjacent to each other) akin to those in boron oxide,which cause undesired blue-shifted band gaps, unlike the paramagnetic,isolated OB3 states.In the work herein, we delve deeperinto the fundamental photochemistryof BNO and aim to shed light on the origins of the visible-range bandgaps previously observed. The underlying research question framingthis study is: 15 Through a combination of room temperature X-band electronparamagnetic resonance (EPR) spectroscopy and UV–vis diffusereflectance (DR) spectroscopy, we present an inverse correlation betweenthe magnitude of the paramagnetic OB3 signatures and thecorresponding apparent band gaps. The experimental trends were substantiatedby density functional theory (DFT) simulations of BNO nanosheets:the oxygen dopant introduces a localized state in the band gap ofthe original undoped boron nitride and significantly red-shifted light-harvestingcapability with the presence of multiple paramagnetic OB3 states. Using a combination of spectroscopic techniques (X-ray photoelectronspectroscopy (XPS), near-edge X-ray absorption fine structure (NEXAFS),UV–vis DR, and X-band EPR), supported by DFT simulations, weexperimentally show that the presence of O–B–O sitesfrom adjacent OB3 states leads to diamagnetic states andundesired blue shifts in the absorption spectrum, relative to paramagnetic,isolated OB3 states. Our results provide fundamental insightinto the photophysics of BNO and show that among the chemical statesof oxygen in BNO, paramagnetic, isolated OB3 sites appearto have a significant influence on the optoelectronic and photochemicalproperties of BNO.Here, we address our researchquestion and hypotheses using a combinedexperimental and first-principles-based computational approach. Ourexperimental characterization was previously conducted on a largeBNO sample set, spanning a range of oxygen contents (2–14 atom%) and apparent band gaps (1.50–2.90 eV).3BO3, ACS reagent, 99.0%, Sigma-Aldrich) and hexamethylenetetramine (HMTA) in varying molar ratios  were addedto 100 mL of deionized water at 90 °C under rapid stirring toform a boric acid-HMTA complex in solution. The solution was allowedto evaporate overnight until the resulting white powder was collectedand subsequently dried for 24 h at 90 °C in a drying oven. Thedried material was transferred to an alumina boat crucible (approx.1.4 g), which was placed in a horizontal tubular furnace. The samplewas initially maintained at ambient temperature for 30 min under pureammonia flow, with the flow rate set to 250 mL min–1 to establish an ammonia-rich atmosphere. Caution: ammonia is a toxicgas. A risk analysis must be performed accompanied by the deploymentof appropriate safety measures prior to using this gas. Once this step was complete, the ammoniaflow rate was set to a chosen flow rate , and the sample was heated from ambient temperatureto a set temperature , with a ramp rateof 10 °C min–1. This steady-state temperaturewas maintained for 3 h, after which the samples were allowed to naturallycool to approximately 600 °C under the same ammonia flow rate.At this point, the ammonia flow was shut off, and inert argon gaswas flowed through at a rate of 100 mL min–1 overnightuntil the furnace had cooled to room temperature. Upon completionof the synthesis, either light brown or yellow powders were obtained,which we refer to as BNO.In a typical synthesis, a reactionmixture  of boric acid (H3H3O6]n), using an adaptedmethod of Bertoluzza et al.16 Briefly,3.0 g of boric acid  was weighed and transferred to a glass reagent bottle.This bottle was placed in a drying oven at 90 °C for 24 h withthe lid open to release water and promote dehydration to form orthorhombicmetaboric acid. After 24 h, the lid was sealed to prevent hydration,and the oven temperature was increased to 140 °C. This temperaturewas maintained for a further 24 h to yield monoclinic metaboric acid.The second step involves the reaction of monoclinic metaboric acidwith hexamethylenetetramine (HMTA) . The synthesis conditionswere matched to those for the synthesis of BNO exhibiting the highestOB3 intensity. Then, 40 mmol of monoclinic metaboric acidand 20 mmol of HMTA were thoroughly mechanically mixed and transferredto an alumina boat crucible (approx. 1.4 g), which was placed in ahorizontal tubular furnace. The sample was initially maintained atambient temperature for 30 minutes under pure ammonia flow, with theflow rate set to 250 mL min–1 to establish an ammonia-richatmosphere. Caution: ammonia is a toxic gas. A risk analysis mustbe performed accompanied by the deployment of appropriate safety measures prior to using this gas. Oncethe degassing was complete, the ammonia flow rate was set to 50 mLmin–1, and the sample was heated from ambient temperatureto a set temperature of 800 °C, with a ramp rate of 10 °Cmin–1. This steady-state temperature was maintainedfor 3 h, after which the samples were allowed to naturally cool toapproximately 600 °C under the same ammonia flow rate. At thispoint, the ammonia flow was shut off, and inert argon gas was flowedthrough at a rate of 100 mL min–1 overnight untilthe furnace had cooled to room temperature. Upon completion of thesynthesis, a light yellow/white powder was obtained, which we referto as m-BNO.The first stepinvolves the synthesisof monoclinic metaboric acid polymeric precursor (n1/ against photon energy(hν). We consider BNO and m-BNO as direct bandgap (n = 0.5) semiconductors based on the literature.17UV–vis DR spectroscopy was conductedusing a Shimadzu UV-2600true optical double beam UV–vis spectrophotometer equippedwith an integrating sphere. The integrating sphere has an InGaAs detectorwith a detection range of 220–1400 nm. Spectral bandwidth wasset to 5 nm, and barium sulfate (BaSO+ X-ray Photoelectron Spectrometer equipped with a MXR3 AlKα monochromated X-ray source (hν = 1486.6eV). The samples were initially ground and mounted onto an XPS sampleholder using a small rectangular piece of conductive carbon tape.The X-ray gun power was set to 72 W (6 mA and 12 kV). Survey scanswere acquired using 200 eV pass energy, 0.5 eV step size, and 100ms (50 ms × 2 scans) dwell times. All of the high-resolutioncore level spectra  were obtained usinga 20 eV pass energy and 0.1 eV step size. Any charging effect in thecore level was mitigated using a dual-beam flood gun that uses a combinationof low-energy electrons and argon ions.XPS was employedto determine therelative elemental composition of the samples and the chemical statesof the elements. This was conducted using a Thermo Scientific K-α18 Samples were ground as powders and mounted ontocarbon tape. We used the 400 lines/mm Pt gratings of the beamline’splane grating monochromator with an exit slit width of 50 μm,which leads to an energy resolution of 50 and 200 meV at the B andO K-edges, respectively. B and O K-edge spectra were recorded in totalelectron yield (TEY) mode at room temperature under ultrahigh vacuum.Near-edge X-ray absorption finestructure (NEXAFS) spectroscopy experiments were carried out on thebeamline B07 at the Diamond Light Source Synchrotron, U.K.20 Listed are the basis sets utilized: boron: B_6-21G*_pople,21 nitrogen: N_6-31d1G_gatti_1994,22 oxygen: O_8–411_muscat_1999,23 and hydrogen: H_5-11G*_dovesi_198424 . Overall, the model considers a single hBN layer as it is not currentlypossible to model the exact and complex structure of the actual material.The XRD patterns of the material suggest little crystallinity andtherefore a structure closer to amorphous BN (aBN) than hBN. However,in aBN, the BN layers are “arranged” randomly from oneanother. Hence, considering porous BN as a collection of single BNlayers seems like a reasonable approximation. This approach was alsoadopted in ref  consistingof atom-centered Gaussian orbitals with s, p, or d symmetry. The ground-stategeometry was optimized for each BNO slab using the global hybrid exchangeB3LYP functional to describe electronic exchange and correlation.This functional provides a qualitatively correct correction for electronicself-interaction and thus reproduces the energy, electronic structure,and band gap of self-localizing magnetic states in 2D systems.–6 electronsper unit cell. A commensurate grid of k points inreciprocal space was selected according to the Pack-Monkhorst method(using a shrinking factor 24 for the primitive cell as a reference).The value for the shrinking factor used was dependent on the unitcell size, whereby doubling the cell length both in the a and b lattice directions halving the grid of k points in the reciprocal space. Further, a Gilat net of48 was used to calculate the density matrix and Fermi energy for higheraccuracy. A slab model  was usedto simulate the surface; in the third direction, the wave functiondecays to zero at an infinite distance from the surface.For the calculations, a pruned  grid consisting of 99radial points and 1454 angular points (the XXLGRID option implementedin CRYSTAL17) was adopted due to the ability of converging the integratedcharge density to an accuracy of about 10a and b) for the stoichiometric BN slab are a = b = 2.519 Å and γ = 120°. A nitrogenatom was placed in the unit cell with fractional coordinates , and boron was placed with fractional coordinates . For BNO, a supercell of 2 × 2 was used, and thenitrogen in the origin was replaced with an oxygen atom; within thissupercell, a ratio of 4:3:1 of boron, nitrogen, and oxygen, respectively,was used. This concentration of oxygen corresponds to 12.5 atom %.Due to the paramagnetic nature of this material, a spin-polarizedcalculation was performed.TheB3LYP-optimized 2D lattice parameters , , , and . Within this supercell, a ratio of 4:3:1 of boron, nitrogen,and oxygen, respectively, was adopted again. In this scenario, however,the oxygen atoms were placed such that they were adjacent to eachother and not isolated.a and b plane and through the y,z plane and plotted using CRYSPLOT.25For OH-passivated BNO, a supercell of3 × 3 was defined. Thehydroxyl group was placed vertically over a boron, and then the atomiccoordinates of the system was optimized, while the hydroxyl grouppassivating a nitrogen was also placed vertically on a nitrogen atomand then also optimized. Total electron charge and spin densitieswere calculated for the ideal BNO slab looking above on the 3 sites.The calculations were performed usingthe spin-unrestricted brokensymmetry Kohn–Sham formalism. The magnetization of the cellis equal to the number of OB3 centers formed throughoutthe lattice by alternate nitrogen atoms being substituted with oxygen.The substitution of O for N in the formation of isolated OB3 centers introduces an unpaired electron. This gives rise to a paramagneticradical signal that can be observed using X-band EPR spectroscopyin ambient conditions.26 We normalizedthe intensity of the radical signal to the sample mass to allow forvalid comparison across the sample set. The intensity of the normalizedEPR signal is proportional to the number of spins in the system.27 Since the spins in BNO originate from radicalsin isolated OB3 centers, the intensity of the normalized EPR signalis proportional to the radical content, and hence the number of paramagnetic,isolated OB3 states. We show the BNO samples with the highest andlowest specific paramagnetic OB3 content in 15 We note that characterization analyses of the samples can be foundin this former work (including XRD patterns). While the relative oxygencontent of both samples is within 7 atom % of each other (samples10 and 27 in Table S1), the OB3 radical content is around 40 times lower in the BNO sample, exhibitingthe minimum EPR signal intensity (Table S1). This suggests that the doped oxygen atoms are forming diamagneticstates, identified below as O–B–O species. The correspondingabsorption spectra for the same BNO samples are shown in We focus first on the characterizationof the 27 BNO samples usedfor this study. Here, we link the EPR patterns to the type of O-containingsite and investigate any correlation with the apparent band gap. Basedon our hypothesis, a schematic illustration of an “ideal”BNO nanosheet is shown in 3 intensity, as measured by EPR, exhibited an absorptionedge of 696nm, corresponding to a deep visible-range apparent band gap of 1.78eV; this apparent band gap was among the lowest measured among the27 BNO samples screened in this study . The BNO sample with the lowest paramagneticOB3 intensity exhibited a significantly lower absorptionedge of 440 nm, corresponding to an optical band gap of 2.82 eV. Thisobservation suggests that a larger proportion of paramagnetic, isolatedOB3 states leads to lower apparent band gaps, as visualizedby the scatter plot presented in 3 content. The experimentaldata was regressed with a least-squares line of best fit (red linein R2 = 0.77), with mostpoints clustered within a 95% confidence interval . As the specificparamagnetic OB3 intensity increases beyond 100 au g–1, implying a greater free radical content, the apparent band gapof BNO appears to plateau at a constant value of 1.5–1.6 eV.This could suggest that beyond a certain concentration of isolatedOB3 sites, the increasing presence of free radicals does not decreasethe apparent band gap of BNO further but may potentially influencethe charge carrier dynamics, carrier mobility, or perhaps conductivity,which would improve photophysical properties for optoelectronics applications.Another explanation could be the influence of varying relative atomiccontents of residual carbon from the HMTA precursor in the differentsamples. Carbon could be incorporated as isolated atoms within theBN structure and/or as “graphene islands” within thehBN layers. While both could exist a priori, we do not see patternsfrom graphene using the characterization techniques we have employed.In addition, structural features  mayplay a role. Disentangling the contributions of carbon and oxygenas well as structural features on the physical–chemical propertiesof BN materials remains a challenging task.28 Nevertheless, we observe a noticeable inverse relationship betweenthe apparent band gap and specific paramagnetic OB3 content. Thissupports the notion that the proportion of oxygen chemical states,namely, paramagnetic isolated OB3 sites, may have the most significantinfluence on the band gap in BNO materials compared to other chemicalstates of oxygen. This experimentally supports the first part of ourhypothesis.The BNO sample with the highest specific paramagneticOB3 content and band gap with the first-principles DFTcalculations in 32). The calculations provide a fundamental insight into the photochemistryof the oxygen chemical states in BNO. The DFT-simulated total- andpartial density of states (TDOS and PDOS) plots for BNO systems withdifferent chemical environments of oxygen are presented in 103 state withoutthe formation of O–B–O sites  groups as another chemical state of oxygen on the bandgap of BNO . This shows that the paired radicals,now on the bridging boron atom, likely predominantly participate inbonding as opposed to conjugation throughout the BNO system. Thislack of conjugation could explain the larger band gaps. Therefore,the combined DFT results from S2 confirmthat paramagnetic isolated OB3 sites are a unique chemicalstate of oxygen in BNO that leads to red-shifted band gaps to thedeep visible region.As shown above, the radical, derived from the paramagnetic, isolatedOBO system 2d is fro3 states, is from a boronatom neighboring the oxygen atom. This suggests that the radical isoccupying a boron orbital. We explore this further by constructingmolecular orbital (MO) diagrams from first principles, using linearcombination of atomic orbitals (LCAO), for an isolated OB3 and NB3 state  e′ and oxygen2p(y) e′, they are assumed to be weakly interactingin comparison to the boron 2s e′ interaction and the oxygen2p(y) e′, and so they are ignored. Examiningthe results, NB3  the radical from isolated OB3 states occupies a nonbonding boron 2p orbital, which further supportsthe DFT results in z orbitals.In lts, NB3 3a exhibi3 center 3b, which3 sites. Theelectron charge density is concentrated about the boron atom . Equally, the spin density is localizedabout the boron atom, as seen in x–y plane, such thatone is looking at the BNO sheet from above. To elucidate the locationof the spins, we examined the spin density in the y–z plane  asthe precursor, the control was synthesized using monoclinic metaboricacid ([B3H3O6]n), a polymeric boron precursor with an abundance of O–B–Ospecies. All other synthesis conditions were kept the same. The controlsample is labeled and referred to as m-BNO. The formation of monoclinicmetaboric acid was confirmed through Fourier transform infrared spectroscopy(FT-IR), in agreement with the literature16 . Using X-ray photoelectronspectroscopy (XPS), the relative oxygen contents and atomic compositionsof BNO and m-BNO were found to be virtually identical, with valuesof 10.6 and 10.5 atom %, respectively . Importantly, the same oxygen content betweenthe two samples allows us to ascribe variations in the magnetic andoptoelectronic properties to the chemical states of oxygen.We now turn our attention to the second part of our hypothesissurrounding the effect of O–B–O states. To compare thebehavior of OBK near-edge X-ray absorption fine structure(NEXAFS) spectra (S8). The near-edge absorption spectra representelectronic excitations from O 1s or B 1s orbitals into unoccupiedstates above the Fermi energy. Dipole selection rules only allow transitionsinto p-like states overlapping with the respective atoms, which aredepicted in Figure S8. Neither orbitaloverlap nor lifetime broadening of excited states is considered inthe calculations; therefore, the height and width of the calculatedprojected DOS do not necessarily correspond to that of the NEXAFSspectra. We focus first on the O K-edge spectra, where we observea distinct broad peak at 532.4 eV (π1*) in the OK-edge spectrum for BNO , which are observed above the absorption edge (around533 eV) are associated with transitions into the p states above 4eV, which dominate the DOS plots of both BNO and m-BNO in Figure S8. Notably, in the O K-edge spectra ofBNO or m-BNO (4*), whichwould correspond to π* (B–O) transitions in boron oxide(B2O3).34 This confirms thatboth BNO and m-BNO remain BN materials despite oxygen doping, andwe have not formed an oxide.To identify the chemical states in BNO and m-BNO, we recorded Oand B  spectra 4a,b and  spectra 2 and S8.K-spectra.In the DOS calculations shown in Figure S8, the unoccupied p states have nonzero DOS above 0.8 eV for BNO andabove 4.3 eV for m-BNO. We would therefore expect the first resonanceobserved for m-BNO to appear at photon energies approximately 3.5eV higher than for BNO. For the latter, we observe distinctive peaksat 191.6 eV (π1*), 192.0 eV (π2*),192.6 eV (π3*), and 193.2 eV (π4*), which can be mapped onto the features in the B-projected pDOSfor BNO between 0.8 and 4.5 eV . π1* is associated with the π* transitionsin the BN3 state in BNO and m-BNO, confirming the formationof a BN material . π2*, correspondsto π* transitions in the BN2O state, leading to isolatedOB3 centers.36We now turn our attention to the B 5*), i.e., 2.6 eV above the lowest energy peakseen for BNO and in reasonably good agreement with the shift of 3.5eV expected from the calculations. Additional features at higher photonenergies are assigned to the pDOS states of m-BNO above 4.3 eV. Weaksignals of π1*, π2*, π3*, and π4* can also be seen in these spectra,highlighting the fact that the layer is not pure. The NEXAFS spectrain 3 centers are the dominant chemicalstate of oxygen in BNO, and (ii) O–B–O species are thedominant state in m-BNO.The B-K-edgespectrum of m-BNO spectrum shows a new peak at 193.9eV  spectroscopy, we presented an inverse correlationbetween the magnitude of the paramagnetic OB3 signaturesand the corresponding apparent band gaps over a set of 27 BNO samples.The DFT simulations provided additional evidence that paramagnetic,isolated OB3 states appear to be the sole chemical stateof oxygen that can facilitate light harvesting in the deep visibleregion in BNO. We developed a first-principles theoretical frameworkusing molecular orbital (MO) theory and linear combination of atomicorbitals (LCAO) to gain fundamental insight into the chemical bondingand location of the radical in the paramagnetic, isolated OB3 sites. The MO diagrams predicted that the radical occupies a nonbonding2p orbital on the boron atom. This agrees with DFT simulations, illustratingthe electron charge- and spin density to be localized about the boronatom in a pz orbital in the paramagnetic OB3 sites. Thus, we conclude that the radical from paramagnetic, isolatedOB3 sites occupies a nonbonding boron 2pz orbital. A synthesis routewas developed to produce m-BNO, a control sample, with the same relativeoxygen content as BNO but O–B–O sites instead of isolatedOB3 sites. Using NEXAFS spectroscopy, we confirmed thepresence of paramagnetic isolated OB3 sites in BNO andthe absence of such states in m-BNO, which only appeared to exhibitdiamagnetic O–B–O sites. The contrasting chemical andmagnetic states in BNO and m-BNO yielded significant red- and blue-shiftedlight-harvesting behavior, respectively. The study herein not onlyprovides valuable insight into the photochemistry of BNO at the fundamentallevel but also brings to light the importance of paramagnetism intailoring and optimizing the optoelectronic and photochemical propertiesof BNO.Toconclude, we have demonstrated through a combined experimentaland first-principles-based computational approach that paramagneticisolated OB


IntroductionAlopecia areata (AA) is an autoimmune disorder causing hair loss, including eyebrows, eyelashes, and body hair, primarily due to melanocyte impact. Though the precise AA melanocyte hearing loss mechanisms are not fully clear, it's speculated that cochlear melanocyte inflammation could disrupt endolymph production, which is necessary for sound signal transmission. Cochlear melanocytes maintain crucial potassium ion levels, which are pivotal for hearing. The potential AA-melanocyte-hearing loss link underscores the need to monitor auditory and cochlear function and consider interventions for AA-related hearing challenges. The study aimed to assess auditory and cochlear function using OAE and audiometry measurements to correlate disease severity and duration with OAE outcomes.Materials and methodsIn this study, we included 32 patients diagnosed with AA; the control group consisted of 29 healthy volunteers. We collected data on the patient's age, gender, onset age, family history, and disease duration. Audiological and otological evaluations were conducted, including pure tone audiometry (PTA), speech discrimination test (SD), and otoacoustic emission (DPOAE) measurements at frequencies of 500, 1000, 2000, 4000, 6000, 8000, and 10000 Hz. The patients were divided into two groups based on age: 18-25 and over 25 years old, and all parameters were compared. To examine differences between the right and left ears, gender, and age groups, we initially tested the variables for normal distribution using the Kolmogorov-Smirnov and Shapiro-Wilk tests. An independent sample t-test was conducted to compare the means for normally distributed variables.ResultsThere were statistical differences at the 5% significance level in the mean DPOAE values of the 1 KHz SNR and 6 KHz SNR variables. According to the Mann-Whitney U test results, a significant difference was found in the gender-based DPOAE value at 2 kHz SNR (p=0.041), which was lower in men than women. Although there were no significant differences in the audiological parameters based on age, significant differences were found in the otoacoustic emission values. Variables, including 4 kHz DP1 (p=0.049), 500 Hz SNR (p=0.045), and 1 kHz SNR (p=0.023), differed significantly between age groups, with these values being lower in patients over 25 years old.ConclusionOverall, our study contributes to the growing body of evidence supporting an association between AA and auditory dysfunction, emphasizing the need for comprehensive assessment and management of hearing-related issues in individuals with AA. Alopecia areata (AA) is an autoimmune disorder characterized by hair loss primarily affecting hair follicles. Various studies have reported a prevalence of 12% for coexisting autoimmune diseases with AA, including thyroid diseases, psoriasis, pernicious anemia, and vitiligo. Compelling evidence suggests that autoimmune targeting of follicular melanocytes plays a significant role in the development of AA [The mechanisms underlying melanocyte damage in the skin can also impact other organs, such as the inner ear. For example, vitiligo, a condition that affects all active melanocytes, has been associated with auditory problems. Studies indicate that in vitiligo patients, hyperacusis ranges from 4% to 37%. The loss of epidermal melanocytes is believed to contribute to the development of vitiligo, and a similar loss of melanocytes in the inner ear can lead to sensorineural hearing impairment, compromising their essential functions and increasing susceptibility to environmental factors -4.Interestingly, evidence suggests a potential link between AA and the inflammation of melanocytes in the cochlea. This inflammation is proposed as a mechanism connecting AA to hearing loss. The exact mechanisms underlying how melanocytes in AA contribute to hearing loss are not fully understood. However, it is speculated that inflammation and damage to melanocytes in the cochlea could disrupt their essential role in producing endolymph, the fluid necessary for transmitting sound signals to the auditory nerve. Melanocytes in the cochlea maintain high concentrations of potassium ions (K+) in the endolymph, which are vital for proper hearing function .Melanocytes play a crucial role in the transduction and modulation of auditory signals in the inner ear. Previous research has suggested that melanocytes may serve practical and structural functions within the auditory system, which can be impaired in vitiligo. Recent studies have explored the potential association between AA and sensorineural hearing loss, proposing a hypothesis of autoimmune-mediated loss of follicular melanocytes -6.It is plausible that AA affects hearing function by influencing melanocytes in the inner ear through a comparable mechanism. Therefore, this study aims to establish a link between AA and sensorineural hearing loss, further investigating the relationship between these conditions ,7.Our research was conducted as a prospective case-control study following the approval of the Adiyaman University Faculty of Medicine Ethics Committee N 2020/8-5 on 22.09.2020.For this investigation, a cohort of 32 patients diagnosed with alopecia areata (AA) was enrolled alongside a control group comprising 29 individuals of sound health. The study encompassed comprehensive data collection pertaining to the patient's age, gender, age of onset, familial medical history, and duration of the ailment. Thorough audiological and otological assessments were executed, as were pure tone audiometry (PTA), speech discrimination tests (SD), and otoacoustic emission (DPOAE) measurements across a spectrum of frequencies: 500, 1000, 2000, 4000, 6000, 8000, and 10 000 Hz (DP1 and SNR). Stratification of patients was performed based on age, dividing them into two categories: 18-25 years old and above 25 years old, with subsequent comparative analysis of all parameters.To ascertain distinctions between the right and left auditory channels, gender influences, and age divisions, a preliminary assessment of variable normality was executed via the Kolmogorov-Smirnov and Shapiro-Wilk tests. An independent sample t-test was performed for normally distributed variables to assess and compare means.Exclusion criteria were rigorously applied, excluding individuals with histories of otologic diseases, familial susceptibility to hearing loss, use of systemic ototoxic drugs, presence of chronic systemic disorders, prolonged exposure to intense noise, a history of cranial trauma, prior diagnosis of autoimmune conditions, type B tympanogram (indicative of irregular middle ear function), air-bone conduction threshold discrepancies exceeding 5 dB, interaural threshold divergence of ≥15 dB, external auditory canal disorders, and other otological diseases.Historical information regarding type 1 diabetes was collected. Also, essential blood tests were administered to gauge serum vitamin B12 levels, thyroid-stimulating hormone (TSH) levels, and concentrations of anti-thyroglobulin and anti-thyroid peroxidase antibodies. Only participants within the defined normal range for these parameters were included in the study.Dermatological evaluationA dermatology specialist at the same hospital conducted a comprehensive dermatological assessment of all patients. This encompassed an examination of various factors, including age, gender, disease duration, pattern and extent of alopecia , classification of alopecia type , severity of alopecia, and frequency of disease episodes. Furthermore, concomitant dermatological conditions like atopy and vitiligo were assessed.Otologic and audiometric evaluationA thorough otomicroscopic and tympanometric assessment was carried out by an otolaryngology specialist on participants from both the AA and the control groups. This meticulous evaluation aimed to identify any middle ear pathologies, with individuals displaying such conditions being subsequently excluded from the study.The audiological evaluation encompassed pure tone audiometry and distortion product otoacoustic emissions (DPOAE) measurements, all administered by an expert audiologist within an acoustically controlled environment. Pure tone audiometry involved the determination of air conduction thresholds spanning frequencies from 250 Hz to 8,000 Hz for each ear, alongside bone conduction threshold levels ranging from 500 Hz to 4,000 Hz. DPOAE measurements were executed at 0.5 kHz, 1 kHz, 2 kHz, 4 kHz, 6 kHz, 8 kHz, and 10 kHz for both ears. The resulting data were scrutinized alongside signal-to-noise ratio (SNR) values, thereby facilitating a comprehensive evaluation of otoacoustic emissions.No significant differences between the AA and control groups were observed in the audiometry parameters (p > 0.05) between the healthy volunteers and AA patients at 1,000 Hz, 2,000 Hz, 4,000 Hz, and 8,000 Hz frequencies. However, there were statistically significant differences at the 5% significance level in the mean DPOAE values of the 1 KHz SNR and 6 KHz SNR variables (p=0.03 and p=0.027). According to the Mann-Whitney U test results, a significant difference was found in the gender-based DPOAE value at 2 kHz SNR (p=0.041), with this value being lower in men than women . This comprehensive statistical examination encompassed a range of factors, including the localization of the disease , the type of alopecia, the severity of the condition, the frequency of its occurrences, and the presence of nail involvement.The results of this in-depth analysis indicated that there were no noticeable distinctions observed in terms of inner ear involvement between the groups (p > 0.05). This suggests that the various parameters studied, such as the specific manifestations and characteristics of alopecia, did not appear to significantly impact inner ear function as assessed by DPOAE measurements. Therefore, this study suggests that while there may be associations between AA and auditory abnormalities in some cases, inner ear involvement, as indicated by DPOAE measurements, did not seem to be strongly influenced by the diverse aspects of the alopecia condition analyzed.Although there were no significant differences in the audiological parameters based on age, significant differences were found in the otoacoustic emission values. Variables, including 4 kHz DP1 (p=0.049) and 1 kHz SNR p=0.023), differed significantly between age groups, with these values being lower in patients over 25 years old by the independent test. Also, according to the Mann-Whitney U test results, the variable 500Hz SNR demonstrates a significant difference across age groups at a 5% significance level, which is also lower in patients over 25 years old , particularly emphasizing higher frequencies. Regrettably, the significance of high-frequency SNHL often goes unnoticed, potentially presenting solely as tinnitus initially . In a stThere are fewer number of manuscript that show the gender difference in hearing loss during AA. Research indicates that there might be differences in the pattern of hearing loss between genders in AA patients. For instance, males might exhibit more frequent involvement in specific frequency ranges or more pronounced sensorineural deficits than females ,16. GendThe presence of cochlear dysfunction in individuals with AA suggests that the presumed autoimmunity targeting follicular melanocytes in AA may also affect the melanocytes in the inner ear. This observation strengthens the relationship between sensorineural hearing loss and autoimmunity. Furthermore, this study in the literature represents the first documented evidence of hearing loss in AA using DPOAE.


Idiopathic Sudden Sensorineural Hearing Loss (ISSHL) is an otologic emergency, and an early prediction of prognosis may facilitate proper treatment. Therefore, we investigated the prognostic factors for predicting the recovery in patients with ISSHL treated with combined treatment method using machine learning models.We retrospectively reviewed the medical records of 298 patients with ISSHL at a tertiary medical institution between January 2015 and September 2020. Fifty-two variables were analyzed to predict hearing recovery. Recovery was defined using Siegel’s criteria, and the patients were categorized into recovery and non-recovery groups. Recovery was predicted by various machine learning models. In addition, the prognostic factors were analyzed using the difference in the loss function.There were significant differences in variables including age, hypertension, previous hearing loss, ear fullness, duration of hospital admission, initial hearing level of the affected and unaffected ears, and post-treatment hearing level between recovery and non-recovery groups. The deep neural network model showed the highest predictive performance . In addition, initial hearing level of affected and non-affected ear, post-treatment (2-weeks) hearing level of affected ear were significant factors for predicting the prognosis.The deep neural network model showed the highest predictive performance for recovery in patients with ISSHL. Some factors with prognostic value were identified. Further studies using a larger patient population are warranted.Level 4. Idiopathic Sudden Sensorineural Hearing Loss (ISSHL) is an otological emergency that occurs within a 72 -h window and is characterized by unilateral or bilateral hearing loss of ≥30 decibels at three consecutive audiometric frequencies. Although the cause of ISSHL has not been precisely identified, it is thought to be related to viral infection, vascular impairment, or autoimmune diseases.8The purpose of this study was to overcome the limitations of previous studies, analyze larger number of patients with ISSHL, and predict hearing recovery using the Deep Neural Network (DNN) model. In addition, we identified factors that had prognostic value.In this study, we retrospectively reviewed the medical records of 524 unilateral ISSHL patients who had received inpatient treatment at a tertiary medical center, between January 2015 and September 2020. Sixty-nine patients with sudden hearing loss due to other conditions, such as Meniere’s disease, head trauma, meningitis, and central lesions, and twenty-two patients who could not be followed up after treatment were excluded. Among 443 patients with ISSHL, 94 patients in the oral steroid group and 41 patients in the ITSI group were excluded from the analysis to reduce bias due to differences in treatment methods. Finally, 298 patients with ISSHL treated with the combined treatment method  were included in the analysis .Figure 1Patients’ age, height, weight, sex, Body Mass Index (BMI), history of smoking and alcohol consumption, history of otological diseases , current underlying diseases , initial otological symptoms accompanying hearing loss , duration of hospital admission, delay from symptom onset to treatment, laterality, and pure tone audiometry results  were extracted from medical records and investigated. This study was approved by the institutional review board of the Pusan National University Yangsan Hospital . Obtaining informed consent was waived due to the retrospective study under the permission of the institutional review board.The patients with ISSHL in the combined treatment group were treated with oral steroids and ITSI simultaneously or with additional salvage ITSI after the oral steroid treatment. The choice of treatment was based on the initial hearing level, early hearing level after treatment, comorbidities, and preference of the patient.2 + 5% CO2, twice daily) were used during inpatient treatment, and lipoprostaglandin  was administered for 5 days. The oral methylprednisolone  was administered for 10 days and was then tapered to half the dosage over the next 4 days. ITSI was administered either daily or once every 2 days. The status of the patient’s tympanic membrane was checked prior to injection; the injection was not administered if the patient had otitis media, adhesions, or perforation of the tympanic membrane. The ear canal and tympanic membrane were anesthetized using an ointment  for 10 min, and 0.5–0.7 cc of dexamethasone  was injected into the anteroinferior quadrant of the tympanic membrane using a 1-cc syringe connected to a 26-gauge spinal needle.In all patients, Gingko biloba  and carbogen inhalation therapy , decision tree, support vector machine, random forest, boosting, and DNN. A DNN is an artificial neural network composed of multiple hidden layers between the input and output layers. Neurons in the input or previous hidden layers are combined with the weights of the next or previous hidden layers, and the weights of the output and previous hidden layers are adjusted according to their contribution to the loss function. The machine learning methods are described in the Appendix Asupporting information file, and the information on the number of layers and nodes in the DNN, and the hyperparameter tuning values are listed in S1 Table. Variable importance, a loss function gap between a full model and a model excluding a certain variable, was used for selecting the prognostic factors. The greater the difference in loss between the two models, the higher the variable importance. Statistical analyses were conducted using R 4.0.5  and Python 3.7 software.The The average age of all included patients was 51.7 ± 14.3 years, and the proportions of males and females were almost the same. Continuous variables, such as age, weight, height, BMI, duration of hospital admission, treatment delay from symptom onset, and hearing thresholds before and after treatment, are summarized in In all patients, the initial average hearing thresholds of the affected and unaffected ears were 76.4 ± 28.3 dB and 21.8 ± 23.7 dB, respectively. The average hearing thresholds of the affected ear at 2-weeks and 3-months post-treatment were 55.8 ± 36.6 dB and 50.4 ± 30.2 dB, respectively. The initial and post-treatment hearing thresholds of the affected ear and the initial hearing threshold of the non-affected ear were significantly better in the recovery group than in the non-recovery group .Significant differences in specific variables were evaluated between the two groups. Patients in the recovery group were significantly younger than those in the non-recovery group. The duration of hospital admission was significantly longer in the non-recovery group than in the recovery group. There were significantly more patients with a history of hearing loss, hypertension, and dizziness as an accompanying symptom in the non-recovery group. In contrast, the number of patients with ear fullness as an accompanying symptom was significantly higher in the recovery group.Treatment outcomes based on Siegel’s criteria were as follows: CR, PR, SI, and NI were achieved in 73 (24.5%), 44 (14.8%), 82 (27.5%), and 99 (33.2%) patients, respectively . The recThe performance of various machine learning methods in predicting the prognosis of ISSHL is summarized in To identify the significant prognostic factors for recovery from ISSHL, the variable importance, which is the difference in loss function between models based on the presence or absence of a specific variable, was calculated in the DNN method. If the value of the variable importance becomes negative, it can be judged as a significant factor for predicting the prognosis. According to the analysis, initial hearing level of the affected ear and non-affected ear, post-treatment (2-weeks) hearing level of the affected ear, smoking, tinnitus, laterality, and BMI were significant factors for predicting the prognosis of ISSHL  and was considered the most useful for prognostic prediction. Initial hearing level of affected and non-affected ear, post-treatment (2-weeks) hearing level of affected ear were significant prognostic factors. Our prognosis prediction model could help design an appropriate treatment plan for patients with ISSHL. Further studies with more patients, more analyzed variables, and randomly assigned treatment methods should be undertaken.Conceptualization: UTW and HML. Data curation: TWU and SBY. Formal analysis: TWU, SWC and HML. Funding acquisition: HML.dd Methodology: TWU, SJO and HML. Project administration: HML and IWL. Advising: SKK and IWL. Visualization: TWU and HML. Writing-original draft: TWU and HML Writing-review & editing: TWU and HML.Research institute for Convergence of biomedical science and technology (30-2017-015), Pusan National University Yangsan Hospital, Yangsan, Republic of Korea.This study was supported by The authors declare no conflicts of interest.