Adult-onset progressive hearing loss is a common, complex disease with a strong genetic component. Although to date over 150 genes have been identified as contributing to human hearing loss, many more remain to be discovered, as does most of the underlying genetic diversity. Many different variants have been found to underlie adult-onset hearing loss, but they tend to be rare variants with a high impact upon the gene product. It is likely that combinations of more common, lower impact variants also play a role in the prevalence of the disease. Here we present our exome study of hearing loss in a cohort of 532 older adult volunteers with extensive phenotypic data, including 99 older adults with normal hearing, an important control set. Firstly, we carried out an outlier analysis to identify genes with a high variant load in older adults with hearing loss compared to those with normal hearing. Secondly, we used audiometric threshold data to identify individual variants which appear to contribute to different threshold values. We followed up these analyses in a second cohort. Using these approaches, we identified genes and variants linked to better hearing as well as those linked to worse hearing. These analyses identified some known deafness genes, demonstrating proof of principle of our approach. However, most of the candidate genes are novel associations with hearing loss. While the results support the suggestion that genes responsible for severe deafness may also be involved in milder hearing loss, they also suggest that there are many more genes involved in hearing which remain to be identified. Our candidate gene lists may provide useful starting points for improved diagnosis and drug development. Age-related hearing loss is a common disease which is highly heritable, and more than 150 genes are known to underlie human hearing impairment. However, most of these genes are linked to early-onset, severe forms of deafness, and involve variants which are very rare. Investigating age-related hearing loss is challenging because it is so common; there are many genes which may be involved, with multiple contributing variants which may be rare or common. Here, we have obtained audiometric and genomic data from a cohort of older volunteers (aged 55 and over), which includes a vital control group of 99 older people with good hearing. We have taken two approaches to detecting genes and variants implicated in age-related hearing loss, and followed up this work with a second, smaller, cohort. Our study provides multiple candidate genes for further investigation. Hearing loss is a common, complex condition with a strong genetic component. More than 700 genes have been found to underlie Mendelian hearing loss in humans and/or mice (reviewed in ), but laMIR96 [DMXL2 [Identifying genes and variants involved in hearing loss is challenging owing to the heterogeneity of the disease. The inner ear is a complex system with multiple molecular components that need to function and interact correctly to enable normal hearing. Family studies have led to the identification of many variants involved in adult-onset Mendelian hearing loss , but th6 [DMXL2 , but ver6 [DMXL2 ,9 suggesAnother challenge in this field is the complexity of auditory phenotypes. Hearing loss does not have a single pathogenic mechanism, but can result from multiple inner ear pathologies. At present, with few exceptions, accurate diagnosis of the underlying hearing problem is not possible. In addition, many large-scale studies make use of self-reported questionnaires to explore hearing impairment. Although self-reported hearing difficulty is fairly well correlated with overall audiometric thresholds –12, and Here we present our data from a cohort of older adult volunteers having extensive phenotype data, including 99 older adults with good hearing. The cohort was characterised by estimates of Metabolic and Sensory components of age-related hearing loss, calculated from audiograms , then paOur primary cohort consisted of 532 participants; 292 female and 240 male participants, with an overall average age of 72.25 years . 62 women and 187 men reported a positive noise history , Fig 1. There were 159 female participants from the TwinsUK cohort meeting our requirements, including ten dizygotic twin pairs. There were no monozygotic twin pairs, and no other relatedness was reported. The overall mean age was 64.82 years. There were few positive responses to questions about noise exposure in work or leisure activities, so no participants were classified as having a positive noise history. We carried out the same ancestry analysis on these 159 participants, and found they also had a non-Finnish European ancestry, and like the MUSC cohort, they were most similar to the “British in England and Scotland” and “Utah residents with Northern and Western European ancestry” sub-populations from the 1000 Genomes .One twin of each pair was removed from the cohort; where twins were classified into the same category, the removed twin was chosen at random (6 pairs). For 3 pairs, one twin was classified as Older-Normal or Metabolic, with the other either Unclassified or Unselected; in those cases, the Unclassified or Unselected twin was removed. The last pair consisted of one twin classified as Older-Normal and one classified as Metabolic; both were removed for the outlier analysis but for the threshold analysis, one  was chosen at random for removal. After twin removal, there were 49 in the Older-Normal category (average age 61.37 years), 62 in the Metabolic category (average age 66.26 years) and 7 in the Sensory category (average age 68.14 years) , Fig 1.After quality filtering , 938,008In order to validate the exome sequencing, Sanger sequencing was carried out on 114 variants in multiple samples from the MUSC cohort, and individual call accuracy was 94.7% . Only two variants were not validated; the remainder of the incorrect calls were errors in zygosity  .TTN) will not necessarily be an outlier  were compared to the number of variants in the same gene in participants belonging to another group (eg Metabolic). The participants were also compared segregated by sex, because the genetic contribution to adult-onset hearing loss differs by sex ; howevertlier eg . At the DNAH3 is an outlier in metabolic hearing loss in the MUSC cohort  and was also previously reported as associated with hearing loss [MT-CYB is an outlier in the sensory hearing loss groups in the MUSC cohort  but was an outlier in the normal hearing group of the TwinsUK cohort .To investigate these gene lists, findings were compared to a list of 774 genes which are known to underlie hearing loss in humans and/or mice ). These To prioritise the list of candidate genes, a third list of outlier genes was obtained from the TwinsUK cohort. In this case there were not sufficient participants classified as having Sensory hearing loss, and so only two comparisons were carried out ; Table 3PKHD1L1) . The remaining 10 genes were strongly expressed in at least one cell type and age  . For fur and age . Based oer Myo7a .DMD, DUOX2, CELSR1 and ELMO3) and 54 genes linked only to Sensory hearing loss    Tables.  ADGRV1) . The foull types . The Senral wall . We testral wall , which cral wall .KIRREL1 and CCDC171), both the non-segregated alternate allele group and one of the sex-segregated groups exhibited a significant difference in thresholds. In the remaining 38 cases, only one group exhibited a significant threshold difference. One mitochondrial variant  also was found to pass the filter and permutation tests, and was associated with better thresholds in male participants , and ZDHHC6 (n = 3) also had a similar average threshold shape to that seen in the MUSC cohort carriers  but didHADH, also identified in the MUSC cohort. The other three genes were AKR7A3, SCN7A and ME1  had, on average, slightly worse thresholds than homozygous carriers of the reference allele, resembling the thresholds of carriers in the TwinsUK cohort, but the difference was not significant    (S1PR2 and PIEZO1)  , 18 gened ELMO3)  Tables,  ADGRV1) . The thr PIEZO1) .S1PR2, PIEZO1, PKHD1L1, DMD, DUOX2, CELSR1, ELMO3, ARHGAP21, LMO7, UBE3B and ADGRV1. Only 3 of these have been identified in humans; ADGRV1, which is an Usher syndrome type II gene [DMD, which has been associated with congenital hearing impairment as well as muscular dystrophy [S1PR2, variants in which lead to congenital profound hearing impairment [S1pr2 in mice results in early-onset progressive hearing loss [Our candidate gene lists include 11 deafness genes;  II gene , DMD, whystrophy , and S1Ppairment , althouging loss . These pPIEZO1, PKHD1L1, DUOX2, CELSR1, ELMO3, ARHGAP21, LMO7 and UBE3B are all defined as deafness genes through work on the mouse orthologues. Of these, hearing loss caused by mutant alleles of Arhgap21 and Elmo3 have only been reported by the IMPC large-scale phenotyping screen at a single age of 14 weeks (www.mousephenotype.org [Elmo3 homozygous mutants have raised thresholds at low frequencies (https://www.mousephenotype.org/data/genes/MGI:2679007) and Arhgap21 heterozygous mutants exhibit variably raised thresholds across most frequencies tested (https://www.mousephenotype.org/data/genes/MGI:1918685). Mice with a disrupted Ube3b gene display mild hearing impairment at all frequencies at 3 months old, and this impairment was more severe when tested at 6 months old [Pkhd1l1 mutants show early-onset progressive hearing loss [Lmo7 expression in mice results in late-onset progressive hearing impairment [Duox2 have severely raised thresholds [Celsr1 exhibit vestibular defects and misoriented outer hair cells [Piezo1 have recently been reported to exhibit progressive hearing loss [type.org , 27); Elnths old . Pkhd1l1ing loss , while apairment . All theresholds , and micir cells . Finallying loss .SYNE2, FKBP2, and ABCB8 from the main analysis  protein which is upregulated in response to cellular stress such as heat shock, or the accumulation of unfolded protein precursors in the ER [ABCB8 is a mitochondrial ABC transporter which plays a role in cellular viability and is protective against oxidative stress [MADD, which was associated with Sensory hearing loss and has an expression pattern resembling that of Myo7a  compared to women  . The proThe only regression analysis which could be performed using male participants alone from the MUSC cohort was the comparison of variant counts in men classified with Sensory hearing loss versus those classified with Metabolic hearing loss . Some ofB4GALT1 which is protective against cardiovascular disease [GPR75 which reduce the risk of obesity [Gjb6, which protects against hearing loss in mice [Intriguingly, a subset of candidates from both outlier and threshold analyses are associated with better hearing, suggesting that some variants may lead to protection against age-related hearing loss and/or protection against damage from noise exposure. This is not the first report of protective variants. Examples of other protective variants include the N352S variant in  disease  and prot in mice . IdentifAll human subjects research was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained in this study, which was approved by the Medical University of South Carolina (MUSC) Institutional Review Board (for the MUSC cohort) and Guys & St Thomas’ Trust (GSTT) Ethics Committee (for the TwinsUK cohort).All mouse experiments were carried out in accordance with UK Home Office regulations and the UK Animals (Scientific Procedures) Act of 1986 (ASPA) under UK Home Office licences, and the study was approved by the Wellcome Sanger Institute Ethical Review Committee.The primary cohort consisted of 532 volunteers enrolled in an ongoing longitudinal study of age-related hearing loss at MUSC, dating from 1987, described in detail in Dubno et al, 2013 . NotablyFor the follow-up cohort, we selected 159 participants from the TwinsUK study based on age (55 years and older), self-reported ethnicity (“White”), and availability of both exome and pure-tone audiometry data. The pure-tone audiometry data collection has previously been described [Phenotype cohorts were formed based on selection criteria to define individuals with representative metabolic or sensory hearing losses, as well as normal hearing, to enable comparisons between specific phenotypes. Audiograms were classified into one of three main categories . These rejected audiograms are referred to as “Unselected” below. Third, a set of simple rules (below) using the metabolic and sensory estimates were applied to classify cases into the four remaining categories.Using this approach, the Older-Normal category was defined by cases with summed metabolic + sensory estimates that were <20 dB HL, with <10 dB difference in the estimates between ears. The Metabolic category was selected from the remaining cases  with metabolic estimates ≥20 dB, ear asymmetries in the metabolic estimate ≤15 dB, sensory estimates <20 dB, and metabolic > sensory estimates. The Sensory category was selected from the remaining cases  with sensory estimates ≥15 dB, ear asymmetries in the sensory estimate ≤20 dB, metabolic estimates <25 dB, and sensory > metabolic estimates. Finally, the remaining cases  were less clearly representative of metabolic or sensory hearing loss, and were labelled Unclassified. After classifying the audiograms based on these rules, all audiograms in each category were reviewed by eye ; a few anomalous cases were removed and a few cases were added based on consistency with a category. There were a total of 1–10 manual additions or removals for each category  Figs.Libraries for exome sequencing of the MUSC cohort were prepared using the Agilent SureSelect X2 Target Enrichment System (version 5) and the Agilent SureSelect Human All Exon V5 kit, which included 5’ and 3’ UTRs. DNA was sheared using the Covaris S220 focused ultrasonicator. Libraries were sequenced on the Illumina HiSeq 2500.The exome sequencing of the Twins UK cohort has been previously described . BrieflyFor both cohorts, fastq files were aligned to GRCh38 using Hisat2.0 , followihttps://gatk.broadinstitute.org/hc/en-us/sections/360007226651-Best-Practices-Workflows). BCFtools calls were filtered using vcfutils [After read alignment, genomic variants were called using three callers; GATK HaplotypeCaller ,62, BCFtvcfutils , and Frevcfutils   and 281,261 (TwinsUK) variants passed these quality filters, and were then tested for excess heterozygosity using the R HardyWeinberg package ,69 to idMitochondrial variants were called using GATK Mutect2  and filtGenomic and mitochondrial variants were annotated using the Ensembl Variant Effect Predictor (VEP) v100 . AnnotatChosen variants from the MUSC cohort were resequenced using Sanger sequencing . Primers for Sanger sequencing were designed using primer3 , and seqFour comparisons were carried out: Older-Normal hearing to all hearing loss (including Unselected and Unclassified participants); Older-Normal hearing to Metabolic hearing loss; Older-Normal hearing to Sensory hearing loss; and Metabolic hearing loss to Sensory hearing loss. 12176 genes  had at least one variant called in one sample, and were assessed in each analysis. For each comparison, a linear regression was carried out on the total number of variants per gene per group. In the first three comparisons, the number of variants in the Older-Normal hearing group was used to predict the expected number of variants in the hearing loss group, and in the fourth comparison, the number of variants in the Metabolic hearing loss group was used to predict the expected number of variants in the Sensory hearing loss group. The residuals (the difference between the observed and predicted variant load for each gene) were used to determine the outlier genes. Briefly, the first (Q1) and third (Q3) quartile and the interquartile distance D (Q3-Q1) were calculated for each regression’s residuals, and outlier genes were defined as those with residuals > Q3 + 6D and those with residuals < Q1 – 6D . Hypergewww.mousephenotype.org) . This list is an update of that reported in our previous study [The list of deafness genes consists of those genes known to underlie hearing impairment in humans or in mice, and was manually compiled and curated from the literature. It includes all the genes listed in the Hereditary Hearing Loss Homepage (hereditaryhearingloss.org/) and genes which, when mutated, result in altered hearing thresholds in mutant mice, as reported by the International Mouse Phenotyping Consortium , expression levels were averaged. The expression of each gene was plotted in 12 different cell types, as defined by the original experiments. Eleven marker genes were plotted for comparison . These are known marker genes for their cell types, with the exception of Gm525, which was chosen based on its fibrocyte-specific expression at P30 [Gene expression in the mouse inner ear was assessed using single cell RNAseq data obtained from the gEAR portal (gear.org ). Datasegear.org , P15 [85gear.org  and P30 gear.org ), and exn at P30 .Wildtype mice from the C57BL/6N strain were used for the Madd expression study. Animals were collected, fixed, embedded in paraffin wax and sectioned as described in , with thTo assess each individual variant, audiograms were plotted with participants separated into groups by genotype and sex. Variants with fewer than 5 people/group in all alternate allele groups were excluded. For each group, the average thresholds at each stimulus frequency were compared to the reference group, testing for a difference of 20dB or more. This is a greater average threshold difference than that seen between those reporting a positive noise history and those reporting a negative noise history . A maximgithub.com/moraglewis/ThreADD.We carried out 20,000 permutations for each variant to assess the likelihood that those stimulus frequencies which passed the filter were observed by chance. For each permutation, individuals from the cohort were assigned randomly to groups of the same number and sex as the original alternate allele and reference groups, and the relevant stimulus frequencies tested using the same settings as before. If more than 1000 random shufflings produced a similar result (i.e. p > 0.05), the variant was rejected. This was carried out automatically, and the scripts can be found at The final p-values, derived from the permutation testing, represent the likelihood that the differences between the alternate and reference allele groups arose by chance. For the MUSC cohort, which had 41 variants passing the permutation test, we corrected for multiple testing using the Benjamini-Hochberg correction.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S1 FigPopulation principal component analysis for the MUSC  and TwinsUK  cohorts, along with data from participants from different genetic ancestries in the 1000 Genomes project . The glo(PDF)Click here for additional data file.S2 FigSee (PDF)Click here for additional data file.S3 Fighttp://umgear.org) was plotted for each of the ten genes. Expression was normalised to Hprt . Marker genes included for comparison are Myo7a (hair cells), Fgf8 (inner hair cells), Slc26a5 (outer hair cells), Sox2 (non-sensory cells), S100b (inner pillar cells), Hes5 (Deiters’ cells), comparison , Met(intermediate cells), Cldn11 , Slc26a4 (spindle and root cells) and Gm525 (fibrocytes). Two sets of plots are presented; the first set show expression in organ of Corti cell types and the second show expression in lateral wall cell types.Single cell RNAseq data from the gEAR ((PDF)Click here for additional data file.S4 Fighttp://umgear.org) was plotted for each of the 29 genes associated with Metabolic (M) or Sensory (S) hearing loss. Expression was normalised to Hprt . Marker genes included for comparison are Myo7a (hair cells), Fgf8 (inner hair cells), Slc26a5 (outer hair cells), Sox2 (non-sensory cells), S100b (inner pillar cells), Hes5 (Deiters’ cells), comparison , Met(intermediate cells), Cldn11 , Slc26a4 (spindle and root cells) and Gm525 (fibrocytes).Single cell RNAseq data from the gEAR ((PDF)Click here for additional data file.S5 FigImages of the cochlear duct  at ages from E14.5 (where no staining is visible) to P4. At least three mice were examined at each age. Brown shows where Madd is present (visible from E16.5 and older); hair cells are marked with arrowheads. Scale bar = 20μm.(PDF)Click here for additional data file.S6 FigHADH was identified in both cohorts. For the remaining 3 variants identified in the TwinsUK cohort, the audiograms of MUSC carriers are shown on the left. Two audiograms are shown for each variant in each cohort; the thresholds from the left ear are shown on the left, and those from the right ear on the right. Numbers and average ages of each group are listed on the graph. The symbols at the top of each graph mark which groups passed the criteria for each stimulus frequency compared to the relevant reference group . Error bars are standard deviation.For the 41 variants identified in the MUSC cohort, audiograms of TwinsUK carriers are shown on the right where available. The variant in (PDF)Click here for additional data file.S7 FigA) Schematic showing the classification process and numbers at each stage. B) Plot of the sensory estimate against the metabolic estimate for each well-fit case, with the blue/red shading indicating the magnitude of each estimate. The small dots are the Unclassified cases. C) shows the mean audiograms for the cases assigned to each category (error bars are standard error of the mean).(PDF)Click here for additional data file.S8 FigA) Schematic showing the classification process and numbers at each stage. B) Plot of the sensory estimate against the metabolic estimate for each well-fit case, with the blue/red shading indicating the magnitude of each estimate. The small dots are the Unclassified cases. C) shows the mean audiograms for the cases assigned to each category (error bars are standard error of the mean).(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.


Computational and animal studies suggest that stimulus amplitude, and relatedly, spatial stimulation profiles, of coordinated reset pulses can have a major impact on the degree of desynchronization achievable. In this study, we transform the tone spacing of aCR into a scale that takes into account the frequency selectivity of the auditory system at each therapeutic tone’s center frequency via a measure called the gap index. Higher gap indices are indicative of more loosely spaced aCR tones. The gap index was found to be a significant predictor of symptomatic improvement, with larger gap indices, i.e., more loosely spaced aCR tones, resulting in reduction of tinnitus loudness and annoyance scores in the acute stimulation setting. A notable limitation of this study is the intimate relationship of hearing impairment with the gap index. Particularly, the shape of the audiogram in the vicinity of the tinnitus frequency can have a major impact on tone spacing. However, based on our findings we suggest hypotheses-based experimental protocols that may help to disentangle the impact of hearing loss and tone spacing on clinical outcome, to assess the electrophysiologic correlates of clinical improvement, and to elucidate the effects following chronic rather than acute stimulation.Acoustic coordinated reset (aCR) therapy for tinnitus aims to desynchronize neuronal populations in the auditory cortex that exhibit pathologically increased coincident firing. The original therapeutic paradigm involves fixed spacing of four low-intensity tones centered around the frequency of a tone matching the tinnitus pitch,  Network dynamics—occurring at multiple levels and spatio-temporal scales—play a crucial role in several physiologic and pathophysiologic domains . ChronicTinnitus, or the perception of sound in the absence of any external stimuli , affectsAbnormal neuronal synchrony has also been demonstrated to play an important role in a number of other brain disorders, including Parkinson’s disease (PD) , migrainThe CR approach was initially developed for the treatment of PD, essential tremor, and epilepsy . SustainAs shown computationally, anti-kindling can robustly be induced in networks with STDP regardless of whether CR stimulation is administered directly to the soma or through synapses . SubsequBy design, effective CR stimulation requires phase resetting stimuli to be delivered to different subpopulations of a synchronized population of neurons . AccordiFor acoustic stimulation of the inner ear, auditory filters provide an equivalent concept to spatial stimulation profiles for electrical stimulation of neuronal tissue . DiffereIn psychoacoustics, the bandwidth of an auditory filter in human hearing is approximated by the corresponding equivalent rectangular bandwidth (ERB). An ERB describes a rectangular band-pass filter that passes the same amount of energy as its corresponding auditory filter . In thisOn the whole, the findings presented herein are a re-analysis of the data acquired from 18 subjects with tinnitus from  who wereThis study was approved by the Ethics Committee of Cologne University’s Faculty of Medicine. Written informed consent was obtained from all subjects according to the Declaration of Helsinki and Good Clinical Practice. Participants were 18 individuals with subjective bilateral chronic tonal tinnitus . Individuals with pulsatile, buzzing, roaring, or hissing tinnitus and subjects with a history of auditory hallucinations, Ménière’s disease, middle ear disorders, and diagnosed neurological or mental disorders, as well as individuals taking CNS-acting medication or using hearing aids, were excluded. The mean age was 45.89  years, and the mean tinnitus duration was 9.83 (±7.08) years. Otoscopic examination was performed in all participants. Additional details can be found in .fT) vary across studies  was calculated for each subject as the average of thresholds at 500, 1,000, 2000, 4,000, and 8,000 Hz.Extended high-frequency air conduction audiometry was performed in all subjects, ranging from 125 Hz to 16 kHz, with thresholds measured in dB HL. While estimates of the frequency distribution of the tinnitus pitch match  . Startinregular aCR, two tones are placed below noisy aCR stimulation, prior to each cycle, four frequencies 1.4⋅fT) . For thebability  in the fThe frequency span for regular aCR stimulation reads  article . For botThe first part of each experimental session consisted of a 10-min period of silence . The parN. Its value in Hz is given byIn the present study, we used a frequency scale based on the equivalent rectangular bandwidth (ERB) of the auditory filter, as determined from masking experiments using notched noise or spectrally rippled noise with human listeners . The avecy in Hz . This eq5,000 Hz . The edg5,000 Hz . From th5,000 Hz :ERB(h)=relative gap between For subjects with normal hearing, neighboring ERBs of CR tones do not overlap for tinnitus frequencies n 560 Hz . With inn 560 Hz . To quanmean relative gap between all pairs of neighboring ERBs, ERB spangap indexFor regular aCR stimulation, the a1, b4] . As an ar ranging from 0.92 to 0.99, depending on which tone pair is assessed), regardless of whether the relative gap is negative, zero, or positive. Further, as shown in g (r = 0.99). This motivates the use of the gap index as a summary index of the spacing of the ERBs of all four CR tones, rather than the behavior of individual pairwise ERBs.As shown in gap countc and the gap index g (r = 0.94).In addition, we determine the number of gaps . As shown in r = −0.98). Thus, the overlaps of the CR tone ERBs with the tinnitus ERB are also captured by the gap index.With this the The advantage of using the gap index, a single, integral spacing measure for all CR tones  is that the gap index reflects the spacing of CR tone ERBs in their entirety and enables a comprehensive correlation to different clinical outcome measures. It also captures the relationship of CR tone ERBs to the tinnitus ERB.relative gap defined by To determine the mean gap between all pairs of CR tone frequencies cn) see .mean relative gap between all pairs of neighboring CR frequencies Stimulation Protocols and Symptom Scoring). To determine the gap index for noisy aCR stimulation, for each realization of noisy aCR cycles we consider the percentage ERB span consisting of gaps in between the CR tone ERBs and average over all  possible realizations, where the set of realizations is given bygap index for noisy aCR readsr ranges from 0.93 to 0.98 depending on which tone pair is assessed) and mean relative gap to gap index for noisy aCR in r = 0.999).With this, the gap countr = 0.97).For noisy aCR stimulation, we additionally determine the H . Supplemrelative overlap between the tinnitus mean relative overlap between all three realizations of a CR frequency mean relative overlap between the tinnitus r ranges from −0.97 to −0.95 depending on which tone pair is assessed). The mean relative overlap vs. gap index for noisy aCR is shown in r = −0.97). As is the case with regular CR, the gap index once again represents a single measure that captures multiple relationships among the noisy aCR tone ERBs and between the noisy aCR tone ERBs to the tinnitus ERB.In addition, we describe the spatial CR tone arrangement by using the tinnitus According to the American Speech-Language-Hearing Association (ASHA), the hearing thresholds relevant to our study can be classified with the following descriptors: normal hearing (0–15 dB), slight hearing loss (16–25 dB), mild hearing loss (26–40 dB), and moderate hearing loss (41–55 dB) . Due to To illustrate how the range of possible gap index values for aCR stimulation depends on tinnitus frequency mulation . The maxHowever, the gap index not only depends on the average hearing loss in the vicinity of the tinnitus frequency On average 26 dB hearing loss:1) Lower-frequency hearing loss (“HL 26 dB LF”): 2) Higher-frequency hearing loss (“HL 26 dB HF”): 3) Tinnitus-centered hearing loss (“HL 26 dB TC”): For pairment :1) LowerAs shown in 1) The audiogram of every tinnitus patient was linearly interpolated on a logarithmic frequency axis.2) The frequencies of the tones used for regular CR stimulation and for noisy aCR stimulation were computed for every tinnitus patient, and the hearing thresholds at the frequencies of the stimulation tones were interpolated from the audiometric thresholds.3) The edges of the ERB-wide frequency intervals centered at the frequencies of CR tones were estimated using 4) The gaps between the ERBs centered at the neighboring CR tones were calculated using 5) The values of the gap index were calculated using a) Notably, if the tinnitus frequency was the same in both ears, the gap index for both ears was calculated with the mean values of the linearly interpolated audiograms of both ears.b) However, if the tinnitus frequency was different between the ears, the gap index was initially calculated for each ear separately . Then, the two indices for the ears were averaged for the final gap index.To compute the distances between adjacent ERBs and the overall gap index , above fAll statistical analyses were performed using VAS-L score during stimulation was chosen as the primary outcome variable. This was motivated by several factors. Firstly, the most pronounced clinical effect seen in r = −0.659 for regular aCR. The gap index and 5-PTA had an r = −0.705 for noisy aCR. This indicates that higher 5-PTA is correlated with lower gap indices, as would be expected due to broadening of ERBs with increasing threshold. Because the 5-PTA is a function of the patient’s underlying hearing loss and cannot be modified when selecting CR parameters, we chose the gap index as the more preferred predictor variable in this study over 5-PTA. To that end, we calculated the residual variance of 5-PTA toward the response variable of gap index for both regular aCR and noisy aCR. Residual variance is the unexplained variance between 5-PTA and the gap index. This method enabled us to correct for multicollinearity between these predictor variables. Of note, patient age was not included in the regression models, as hearing loss has a known significant impact on cochlear tuning, while age may have a smaller impact  were included in the models with the VAS-L during therapy as the primary outcome variable.Additionally, we chose to perform secondary analyses that were exploratory in nature and therefore had no formal hypotheses. The secondary analyses were performed with the outcome variables of VAS-L after stimulation, as well as VAS-A during and after stimulation. Furthermore, the mean relative overlap and mean relative gap were also assessed as part of the secondary analyses. As in the primary analysis, the 5-PTA was residualized in all secondary analyses to account for multicollinearity.y-axis in each subfigure of x-axis represents the residual of the gap index after removing the linear effects of the residual variance of 5-PTA. The slope of each plot is the same as the unstandardized beta coefficient (B) for the gap index for each multiple linear regression, the values of which can be found in As shown in As shown in As shown in As shown in Some additional findings from observing tone spacing relationships are described herein. Notably, only for regular aCR do the pairwise relative gaps increase with increasing tone frequency, as seen in Coordinated reset was developed computationally to lead to desynchronization of populations of neurons with abnormal degrees of coincident firing . HoweverThis study aims to shed light on the impact of tone spacing on clinical efficacy of regular and noisy aCR, as measured by the changes to self-reports of tinnitus loudness and annoyance during and after stimulation. The existing tone paradigms were transformed into an ERB-scale, which allowed for an analysis of the impact of aCR spatial stimulation profiles on clinical outcome. The gap index—or fraction of the target spectral range uncovered by stimulation—was introduced as a measure of spacing between adjacent auditory filters. We carefully analyzed several possible metrics, as described in the Methods, and found the gap index to be a useful composite measure that captures multiple relationships, both among the therapeutic tones and also between the therapeutic tones and the tinnitus frequency.To that end, several multiple linear regression models were developed, using tinnitus duration, the gap index, and residual variance of 5-PTA (after controlling for multicollinearity with the gap index) as predictor variables. The primary outcome variable was VAS-L during therapy, with additional secondary analyses performed on the response variables of VAS-L after therapy and VAS-A during and after stimulation with the two CR paradigms. The gap index was found to be the primary predictor variable for the primary outcome variable of VAS-L during regular aCR and during noisy aCR. This suggests that, as the gap index becomes smaller, i.e., the ERBs corresponding to the CR tones have less space between them, there is a decrease in the response to treatment, with higher symptom scores during and after stimulation. Thus, higher gap indices—and thus loosely spaced CR tones—are associated with a more pronounced treatment response in the acute stage. These results hold true when controlling for both tinnitus duration and 5-PTA.When examining VAS-L after regular aCR, only the duration was significant. When examining VAS-A after regular aCR, both gap index and duration were significant. The results of these exploratory analyses suggest that having had tinnitus for longer duration had a possible negative impact on therapeutic efficacy for the acute after-effect. In contrast, in a prospective, randomized, single-blind, placebo-controlled 12-week proof of concept study in 63 patients with chronic tonal tinnitus, up to 50 dB hearing loss and tinnitus duration characteristics comparable to the sample considered here, it was shown that tinnitus duration was not a confounding factor for patients treated with aCR stimulation for 4–6 hours per day . Hence, Of note, acute desynchronizing effects (obtained during stimulation) are not necessarily predictive of long-term desynchronizing effects emerging after cessation of stimulation. For instance, a theoretical study in spiking neuronal networks with STDP receiving periodic stimulation revealed complex and counterintuitive stimulus-response relationships between acute (de)synchronization during stimulation and sustained, long-lasting (de)synchronization emerging as time evolves after cessation of stimulation . In partThere are some important differences between regular and noisy aCR that are worth mentioning. First, regular and noisy aCR differ slightly with respect to acute after-effects. In the original These electrophysiologic differences suggest a likely underlying mechanistic difference. The two protocols also differ with respect to long-term effects. While noisy aCR does have an acute on-effect, this effect is not long-lasting. After cessation of stimulation, only regular aCR has a lasting off-simulation effect, while noisy aCR does not . BecausefT around which to place the therapeutic tones, whether using fixed spacing or ERB-based spacing. Each of these considerations is discussed in detail in the Additional studies are required to validate the findings of this acute effects clinical study. To that end, there are several important considerations discussed herein that impact the interpretation of these results and the design of future experiments. Firstly, the ERB model itself utilizes several assumptions, which may limit the generalizability of this approach to specific subgroups of tinnitus patients. Secondly, various cochlear nonlinearities can impact the frequency specificity and thus the frequency-to-place match of auditory tonotopy which are unaccounted for in the existing ERB-transformation of CR tones. Thirdly, peripheral estimations of frequency selectivity are reasonable but imperfect representations of cortical tuning, i.e., the ERB model represents the behavior at the level of the basilar membrane more closely than it may represent the auditory cortex in full, which is more difficult to probe non-invasively. And finally, ascertaining an accurate pitch match is a necessity of aCR in order to determine the correct Auditory filter widths tend to broaden in individuals with cochlear deficits , with thNotably, there is a weak correlation between threshold elevation on audiometry and the widening of the filter bandwidth, as there is variability on an individual level in the degree of broadening and in the asymmetry of the filter, possibly due to varying patterns of cochlear damage . This poPrimary Analysis. This involves regressing the two predictor variables and using the resulting residual variance in the overall multiple linear regression instead of one of the original predictors. However, this is a statistical estimation of the residual variance of the 5-PTA, and as a result, it is not possible within the framework of this re-analysis to definitively eliminate the relationship of hearing loss to each of these measures of tone spacing. In the future, additional experiments would require a within-subject approach to vary the tone spacing on the frequency axis  while holding the underlying hearing thresholds constant in order to eliminate the effect of hearing loss on the ERB spacing arrangements. Ultimately, on account of these limitations, direct measurements of auditory filters may be required in individual patients rather than estimations based on tinnitus pitch and hearing thresholds. In the Overall Findings.Furthermore, the analysis was done on previously acquired data. As a result, it was not possible to manipulate the spacing of CR tones independently of the hearing thresholds, for which reason the 5-PTA demonstrated multicollinearity, i.e., high intercorrelation, with the gap index, mean relative gap, and mean relative overlap. In each of these multiple linear regressions, the 5-PTA variable was residualized to help reduce the effect of multicollinearity, as described in Based on the results of this study, we hypothesize that a larger gap index—and thus more loosely spaced CR tones—may improve the ability of tinnitus patients to respond positively to aCR acutely. Follow-up studies would involve the direct measurement of the ERBs of the auditory filters centered at the therapeutic tone frequencies, such as with one or more of the “rapid” methods described above. A first experiment may involve traditional, fixed spacing aCR compared head-to-head with aCR with some specified larger gap index between the ERBs of the therapeutic tones, on both an acute and chronic stimulation basis. Furthermore, our study at present was limited to the analysis of self-reported questionnaire data. Subsequent experiments should consider electrophysiologic measures of desynchronization as well, to assess the power changes that may result from different ERB-based aCR paradigms.fT may themselves to some extent mask the tinnitus frequency. High-frequency CR tones may in theory also decrease the efficacy of lower-frequency CR tones. The inverse could be seen in excess upward spread of masking. To parse out these effects, follow-up experiments in tinnitus subjects may involve pairs of tones above and below the tinnitus frequency. The distance between the CR tones themselves and the distance between the CR tones and fT could be varied systematically in order to determine degree of subjective tinnitus relief as well as degree of electrophysiologic desynchronization resulting from various spacing configurations. Considering as well that CR tones may be susceptible to the effects of forward masking, in which a probe tone is masked by a preceding masker tone, the temporal spacing of CR—in addition to frequency spacing—may also be an avenue of further exploration.As a result of the afore mentioned cochlear nonlinearities and the related effect of stimulus level used to probe the auditory system’s frequency selectivity, it is evident that the desynchronization ability of individual CR tones may depend on more than simply the degree of overlap or gap between the corresponding ERBs. Upward and/or downward spread of masking, as well as lateral suppression, may have additional effects on CR efficacy, dependent on the magnitude and perhaps directionality of the individual inter-tone effects. As an example, if in fact there is pathologic downward spread of masking in SNHL, then higher frequency tones above R2 and p-value as the original analysis, as shown in It should be noted that when reversing the residualization procedure in the methods, such that the residual variance of the gap index is calculated toward the 5-PTA, the model yields the same adjusted fT. If normal hearing is more favorable, rather than inherently larger gap indices, we could provide a normal hearing subject with a relatively high fT, such as on the order of 8 kHz, regular CR tones (in which the predicted gap index would be low for this patient). If the acute effect is favorable with significant tinnitus suppression during therapy, it may be that the absence of hearing impairment, rather than the tone spacing, is associated with clinical improvement. Further, if we were to provide wider tone spacing to this subject  and the patient does not have clinical benefit, again this would suggest that the absence of hearing impairment is associated with improved outcomes, rather than loosely spaced tones on the ERB scale. The same effect should be observable throughout the fT range. However, due to the fact that the gap span increases with increasing fT, we hypothesize that the difference between regular CR tone spacing and the mimicked hearing impairment spacing would be greatest when fT is on the higher end of the spectrum .As shown in fT, hearing loss below the fT, and hearing loss in the vicinity of fT, thereby directly measuring outcomes with differently shaped audiograms, as was done in In an additional related experiment, one could test a patient with moderate hearing loss. With hearing loss, the gap index tends to decrease. To compensate for hearing impairment, we could increase the spacing and thereby the gap index. If the patient has a clinical reduction in tinnitus loudness during CR, this could mean the tone spacing is the more salient predictor than the hearing loss. In each of these cases, whether normal hearing or moderate hearing loss, we could provide the aCR tone spacing expected in the opposite scenario to differentiate the effects of hearing threshold and tone spacing. Furthermore, it would be of immense utility to test patients whose audiograms demonstrate hearing loss above the Overall, the results of this study are a first step in the clinical optimization of tone spacing for aCR in order to obtain maximal anti-kindling and long-term therapeutic benefit. Broader spatial stimulation of CR tones, as indicated by higher gap indices, may result in improved relief from tinnitus loudness and annoyance in the acute stage of therapy. Additional studies are required to determine electrophysiologic correlation of these findings, to assess the impacts of chronic stimulation, and to correlate measured rather than estimated auditory filter widths with symptom and electrophysiologic data.


The diagnosis of large vestibular schwannomas (VS) with retained useful hearing has become increasingly common. Preservation of facial nerve (FN) function has improved using intraoperative EMG monitoring, hearing preservation remains challenging, with the recent use of cochlear nerve action potential (CNAP) monitoring. This prospective longitudinal series of VS with useful hearing operated on using a retrosigmoid approach included 37 patients with a mean largest extrameatal VS. diameter of 25 ± 8.7 mm (81% of Koos stage 4). CNAP was detected in 51% of patients, while auditory brainstem responses (ABR) were present in 22%. Patients were divided into two groups based on the initial intraoperative CNAP status, whether it was present or absent. FN function was preserved (grade I–II) in 95% of cases at 6 months. Serviceable hearing (class A + B) was preserved in 16% of the cases, while 27% retained hearing with intelligibility (class A–C). Hearing with intelligibility (class A–C) was preserved in 42% of cases when CNAP could be monitored in the early stages of VS resection versus 11% when it was initially absent. Changes in both the approach to the cochlear nerve and VS resection are mandatory in preserving CNAP and improve the rate of hearing preservation. When vestibular schwannoma (VS) tumors are too large for either a wait-and-scan strategy or radiosurgery; the main therapeutic option is tumor resection ,2. HowevIntraoperative electrophysiological facial nerve monitoring (IOFNM) is routinely performed to avoid the onset of a severe nerve conduction block, guiding the degree of VS resection to limit post-operative facial nerve palsy to 5% or less ,7,8.Synchronized auditory brainstem response (ABR) was the only positive predictive factor described in a recent study, with a five times greater chance of hearing preservation than desynchronized ABR . To presCochlear nerve compound action potential (CNAP) monitoring is an intraoperative, continuous, near-real-time monitoring of cochlear nerve (CN) function through acoustic stimulation in the external ear canal. It is recorded using a specially designed CNAP electrode placed on the CN ,11,12. IIn this study, we discuss our preliminary experience with intraoperative CNAP monitoring during resection of medium/large VS. Tips and pitfalls in obtaining CNAP are reported.A prospective monocentric longitudinal study was conducted between September 2018 and July 2019. Inclusion criteria were surgery for medium/large VS (Koos 2 to 4) in patients with serviceable hearing (classes A and B) and useful hearing with intelligibility (class C) according to the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) classification . InformeTumors were staged according to both Koos classification  and the IOCNM was performed using Neuropack . Electrodes were positioned with one active electrode in front of the tragus of the operated ear , one electrode at the contralateral mastoid, one reference electrode at the vertex level, and one neutral electrode (ground) on the ipsilateral shoulder. Stimulation was performed using intra-ear headphones. CNAP monitoring did not extend surgical time.CNAP data could be collected very rapidly (3–5 s). The stimulus used was rarefaction clicks at 95 dB HL and 11 Hz, and the amplitude and latency of N1 were obtained  18,19].,19.18,19ABR were measured from the beginning of the intervention, and continuously during tumor dissection if present. Per acquisition, 1000 stimuli were given (90 s), and a minimum of two acquisitions were necessary to verify good reproducibility. The presence, amplitude and latency of wave V were considered. In the case of an increase in wave V latency by more than 0.4 ms or wave loss, the surgical procedure was transiently interrupted.IOFNM was performed using an NIM 3 response device . If the amplitude response after a supramaximal stimulation (2 mA) decreased by more than 40% or if a very significant adhesion was present, dissection of the facial nerve from the tumor was interrupted and the tumor resected around it leading to different degrees of resection .After having opened the dura and retracted the cerebellum through RS approach it was, in most cases of large VS, difficult or even impossible to identify the facial and cochlear nerves at the brainstem, so blind mapping of the posterior VS surface was performed using both facial nerve monitoring and the ball-electrode for CNAP monitoring from the Inomed device A. The fiWhen the facial nerve was displaced superiorly, this surgical strategy was not possible as the cochlear and facial nerves are diametrically separated by a piece of tumor. In those cases, VS resection could be interrupted to preserve normal postoperative facial function, leaving a tumor remnant of greater importance along the facial nerve. Then, the IAC was drilled to expose and remove the intrameatal part of the VS, which is relatively straightforward depending on the extension at the fundus and the texture of the tumor. At this time, the ball-electrode was moved to ensure the integrity of the CN and compare the CNAP responses between the surface on the VS, the fundus, and the brainstem C. As for3; Partial resection (PR), mostly preoperatively planned, if the tumor residue was greater than 0.5 cm3 , or number (percentage). Statistical analysis was performed using Prism . Mann-Withney, In total, 37 patients were included (25F/12M) in this study with an average age of 43 ± 13.1 years (19–71). Two patients had neurofibromatosis type 2 and two others had been treated previously by radiosurgery. The largest mean diameter of extrameatal VS was 25 mm ± 8.7 mm (12–46) with 34 (92%) Koos 3 and 4 VS and 32 (86%) Sterkers 3 and 4 VS . The tumn = 28; 76%), rapid tumor growth of >2 mm/year , or slow tumor growth of ≤ 2 mm/year with recent trigeminal neuralgia .Preoperative PTA was 32 ± 18.7 dB (3–70) with an average SDS of 90 ± 16% (50–100). Hearing was classified as class A for 20 (54%) patients, class B for 11 (30%) patients and class C for 6 (16%) patients. Eight patients (22%) had preoperative synchronized ABR. Facial nerve function was normal for all patients. VS resection was performed because of brainstem compression symptoms  or absent  at the brainstem after CN identification , once CNAP was recorded, average values of N1 latency and amplitude were 4 ± 0.8 ms (2–6) and 12 ± 18 µV (2.1–5.5), respectively. CNAP at the brainstem was still present at the end of VS dissection in the CPA for 12 (63%) patients, and after IAM drilling and intrameatal tumor resection for 8 (42%) patients . The surIn Group 2 (n = 18), CNAP was not recorded for a number of presumed causes listed by order of frequency: CN unidentified or interrupted before recording in 8 cases (44%), unknown reasons with no recording at the fundus of the internal auditory canal (IAC) with intact CN  in 4 (22%), presumed vascular damage as CNAP was not found from the brainstem to the IAC fundus and associated with complete hearing loss in 3 (17%), no CNAP together with ABR loss during the surgical approach before CN identification in 2 (11%), and technical problems as ABR were present, CN intact, and hearing unchanged in the last case.Among the 8 (22%) patients with preoperative synchronized ABR, 5 had CNAP at the brainstem at the time of CN identification . At the Hearing with intelligibility  was retained for 10 (27%) patients and serviceable hearing (hearing class A and B) for 6 (16%) patients .When hearing with intelligibility was preserved, CNAP at the brainstem was recorded at the end of surgery for 7 of these 10 (70%) patients (See example In the case of postoperative anacusis (n = 27), CNAP was no longer present at the brainstem at the end of surgery in all cases but one, who experienced a sudden hearing loss 4 h after surgery, presumably due to cochlear ischemia . In GrouSuspected causes of hearing loss during tumor removal in Group 1 (11 patients) were: microvascular damage  with absence of CNAP during surgery, serviceable hearing was retained in only 11% (2/18) of cases and there were no additional cases with class C hearing.p = 0.06, Fisher’s test).Postoperative hearing with intelligibility was achieved less frequently in Group 2 than in Group 1, although not significantly different  for 30 (81%) patients at discharge, and for 35 (95%) patients  6 months after surgery. Only one grade III and one grade VI were still present at 6 months. Postoperative facial nerve function was not different between the two groups .p = 0.90, VS resections were classified as TR, NTR, STR, and PR for 21 (57%), 7 (19%), 5 (13%), and 4 (11%) patients, respectively. No statistically significant difference was found between the two groups  approach, in an attempt to preserve hearing together with a stable high rate of postoperative normal facial nerve function . It is nIn many institutions, small VS are resected routinely under intraoperative ABR monitoring, but the effects of surgical manipulations on hearing are observed with a delay, which precludes any reversible maneuvers. At variance, as CNAP is rapidly recorded (every 3–5 s) providing immediately reliable information on CN function, tumor dissection can be adapted to these real-time changes.In the present study, reporting intraoperative auditory monitoring during medium/large VS  resections, ABR and CNAP were recorded in 24% and 51% of cases, respectively, which compares favorably to the seminal paper by Yamakami et al.  focused The reasons why hearing can be lost during tumor resection from the CN differ from those for the facial nerve. Cochlear monitoring has changed and improved gestures and surgical strategy for separation of the CN from the tumor . We perfCNAP monitoring is of primary importance during resection of the residual intracanalar tumor. After drilling of the porus, the ball is placed on the CN so that the residual tumor can be removed in a single piece under CNAP monitoring after resection of the vestibular nerves and separation from both cochlear and facial nerves C,D.A particular cause of hearing loss is of vascular origin, accounting for at least 20% of hearing loss in our study, either by coagulation or spasm of the internal auditory (labyrinthine) artery. Accordingly, we observed that, after coagulating this artery by necessity to stop its bleeding in one case, the CNAP signal suddenly disappeared 15 min later at the fundus and did not recover . CoagulaFinally, in case of useful preoperative hearing, a near-/subtotal VS resection through a retrosigmoid approach could be guided by CNAP monitoring as is already performed using IOFNM to preserve the hearing and facial functions with the minimal tumor residue . In caseThe main limitation of this study is its low power, due to the small sample studied.Despite the lack of better results for hearing preservation, our early experience of CNAP monitoring during surgery for the largest VSs through a RS approach has convinced us that this innovation is a major advance in the field of VS surgery allowing atraumatic dissection of the CN with a significant increase in hearing preservation when CNAP are still present. For these large VSs, care should be taken to maintain CNAP during the approach to the CN, and then to guide the degree of VS resection under the control of both facial and cochlear nerve monitoring.


Hearing screening can be used to detect hearing loss, but its value for identifying employees with work functioning difficulties is unclear. The objective of this study was to assess the association between the hearing status measured with an occupational hearing-in-noise screening test, Listening Effort (LE), and Need For Recovery (NFR) in employees of a manufacturing company, and to examine whether these associations depend on the perceived noise level at the workplace.Employees of coatings and paints manufacturing company were included. Their hearing status was assessed with an occupational hearing-in-noise screening test. An online survey was used to assess their LE, NFR, and the perceived noise level at the workplace. Responses from 143 employees were analyzed (mean age = 53 years) using hierarchical multiple regression analysis with the outcomes LE and NFR.Regression analysis—with adjustments for gender, age, educational level, health status, pace/amount of work, job variety, and work pleasure—revealed that hearing status was significantly associated with LE, but the interaction between hearing status and the perceived noise level was not. Hearing status nor the interaction between hearing status and the perceived noise level was significantly associated with NFR.The results confirm that poorer hearing is associated with higher LE, but not with higher NFR. These associations were unrelated to the perceived noise level at the workplace. Therefore, the value of occupational hearing screening appears to be early identification of hearing loss in employees, but not identification of work functioning difficulties. Hearing loss is a common condition in the working population, with higher prevalence with higher age and in populations that are exposed to occupational noise –4. In thIn line with earlier studies that indicate that high NFR is a predictor of negative work implications , 12, heaIn some workplaces, occupational hearing screening is applied to detect hearing loss in an early stage . This seSeveral hearing tests have been proposed for screening purposes. Otoacoustic emissions (OAEs) objectively measure the outer hair cell function, pure tone audiometry evaluates the detection of sounds in a quiet environment, and speech-in-noise tests measure the ability to understand speech in a noisy environment . Since dMany researchers have investigated the reliability and validity of speech-in-noise screening tests for identifying hearing loss, which is also the case for the OEC , 21. HowIn an earlier study, the outcome of a speech-in-noise test was significantly associated with LE, but not with the NFR . This stThis cross-sectional and observational study was conducted at a coatings and paints manufacturing company. We analyzed the outcome of an online survey and the outcome of an internet-based hearing screening. This screening is routinely administered at the company as part of a voluntary administered health check. The ethics Committee of the Academic Medical Center declared that no formal approval of the detailed protocol was required according to the Dutch Medical Research Involving Medical Subjects Act (No. W18_369 # 18.421).With consent of the company’s management, information about this study was provided in the period from December 2020 to February 2022 at the company’s intranet and in the coffee rooms of the factory workers. Also, employees who visited the occupational physician of the company for a routinely health check received information about the study. Participation was voluntary; employees who were interested in the study received an informed consent form and an online survey that could be accessed after providing consent. This survey was hosted by Castor, a highly secured, cloud-based electronic data capture platform [Employees could participate in the study regardless of their position in the company. Eligible employees were 40–68 years, spoke Dutch fluently, and completed the informed consent form. The informed consent included permission to complete the survey, to request the OEC outcome at VeiligheidNL, and to use the outcomes for this study.NFR was the primary outcome measure of the study, which was assessed with the NFR-scale score of the Questionnaire on the Experience and Evaluation of Work version 2.0 (QEEW2.0) . This scLE was the secondary outcome of the study, which was assessed with the Amsterdam Checklist for Hearing and Work (ACHW). Employees were asked how much effort and concentration it takes to fulfill six hearing activities at their workplace, specifically detecting sounds, distinguishing sounds, communicating in quiet, communicating in noise, localizing sounds and being exposed to loud sounds. According to Van der Hoek-Snieders et al. (2020), we calculated a sum-score of the items. This score can range between 0 and 18.The ability to understand speech in noisy situations was assessed with the OEC; an internet-based speech-in-noise hearing screening that is hosted by VeiligheidNL . The stiThe workplace noise intensity perceived by the employees was assessed by a visual analogue scale ranging from 0  to 100 (very noisy).Since NFR is a complex construct that is influenced by personal and work-related factors , 26, we The work-related factors include the pace and amount of work, job variety, and work pleasure. These factors were measured with the three scales of the QEEW 2.0. These scales consist of statements measured on a five-point Likert scale. The sum-score of the scales can be converted to a scale score ranging from 0 to 100 with higher scores representing more unfavorable scores. A higher score indicates higher pace and amount of work, less job variety, and less work pleasure. The scale pace and amount of work consists of six statements, for example “Do you have to hurry”. The scale job variety consists of 4 statements, including “Do you have enough variety in your work”? The scale work pleasure consists of five statements, including “I enjoy my work”.Descriptive statistics was generated to report the characteristics of the study population. Distributions of all variables were examined and checked on normality. To gain insight into possible multicollinearity, we computed Pearson correlation coefficients between all dependent, continuous variables and the outcome measures and bi-serial correlation coefficients between all dependent, dichotomous/ordinal variables and the outcome measures. Correlations between the dependent variables were allowed if they were lower than 0.60.Hierarchical multiple regression analyses were conducted to assess the relationship between understanding speech in noise and NFR (primary outcome measure) and LE (secondary outcome measure). For both outcomes, predictor variables were included in three blocks (forced entry). For each block, we calculated the change in amount of variance in the outcome variable that is explained by the dependent variables and the contribution of the individual predictors.The blocks of independent variables were the same for the primary and secondary outcome measure. The first block included the possible confounders. A main effect of hearing status was added in the second block. In the third block, an interaction term was added, respectively the interaction between hearing status and the perceived noise level. A significance level of 0.05 was used.In total, 180 employees were interested to participate in the study of which 8 did not respond on the study information and 29 could not be included because they were aged below 40. This resulted in a study population of 143 employees. Table The majority (75%) of the study population was male, mean age was 53, and their educational level varied from primary school to university. Most employees (89%) reported their health condition to be very good or good, and the others reported their health condition to be fair (10%) or bad (1%). Most employees (65%) used hearing protection at work. Only a few employees (4) were hearing aid users.A normal distribution was confirmed for all continuous variables. The mean outcome of the hearing screening was -16.0 . The outcome of the hearing screening was pass for 98 (68.5%) employees and fail for the other 45 employees (31.5%). Considering the best ears only, the mean outcome of the hearing screening was -17.0  and the outcome of the hearing screening was fail in 19 employees (13.3%).r = 0.39, p < 0.01), but not with NFR .A normal distribution was confirmed for all variables. Table Gender, age, educational level, health status, pace/amount of work, job variety, and work pleasure were entered as predictors in Block 1 and as control variables in Block 2.The results of the hierarchical multiple regression analysis are presented in Table p < 0.01), but not for the second block  nor the third block . The percentage explained variance for all three blocks was 27%.Regarding the outcome NFR, the change in explained variance was significant for the first block , but not for the first block  nor the third block (R square change = 0.015 p = 0.14). The model including the possible confounders explained only 3% of the variance. The second model, after inclusion of hearing status, explained 10% of the variance. The third model, after inclusion of hearing status and the interaction between hearing status and the perceived noise level, explained 12% of the variance.For the outcome LE, the change in explained variance was significant when the second block was included (R square change = 0.07, Many researchers have assessed the reliability and validity of speech-in-noise screening tests for identifying hearing loss, but it was unclear if poorer screening outcomes are also associated with higher LE and higher NFR. We found that hearing status was significantly associated with LE, but not with NFR in a population of employees of a manufacturing company. The associations did not depend on the perceived noise level at the workplace.r = 0.20, and r = 0.69 in the earlier studies, Ref. [The finding that poorer hearing is associated with higher LE during the performance of auditory job tasks is in line with earlier studies in clinical populations , 9. It ses, Ref. , 9, respes, Ref. , such asA non-significant association was found between hearing status and NFR. Earlier, mixed results were presented regarding the association between the ability to understand speech in noise and NFR , 10, 28.Although it is assumed that the impact of poorer hearing on listening effort may be greater in noisy work environments , 10, we Some study limitations should be noted. There is a risk for selection bias, since employees voluntarily participated in this study. For example, feeling insecure about the hearing status might have been a reason to not participate in the study. Also, because there is currently no validated questionnaire available that measures LE during hearing related job activities, we used a non-validated questionnaire. Finally, we controlled for a broad spectrum of confounders. Nevertheless, we are not sure that we controlled for all relevant confounders since NFR is a complex construct. For example, the cognitive load and physical demands of employees’ job might have been relevant.Considering the moderate association between hearing status and LE, the OEC is expected to inadequately predict subjective listening difficulties at the workplace at individual level. The predictive value of the OEC for high NFR is expected to be even poorer. Although the OEC is an appropriate instrument to assess employees’ ability to understand speech in noisy environments , 21, theOur results confirm that poorer hearing is associated with higher LE, but not with higher NFR. These associations were unrelated to the perceived noise level at the workplace. Therefore, the value of occupational hearing screening appears to be primarily in early identification of hearing loss in employees, but not in the identification of subjective listening difficulties and/or difficulties with work participation. Future research should investigate the value of occupational hearing screening for identifying hearing loss as a hidden cause of work participation difficulties.


Rhizopus homothallicus is an emerging pathogen that causes pulmonary mucormycosis.R. homothallicus in a 54-year-old type 2 diabetic patient. The organism was isolated from bronchoalveolar lavage fluid and preliminarily identified by fungal morphology and finally by sequencing of the internal transcribed spacer region. We report a case of pneumonia caused by  Mucormycosis may be associated with cavitary lung lesions against a backdrop of poorly controlled diabetes or other immunosuppressed states. Pulmonary mucormycosis may have variable clinical and radiological presentations. Therefore, strong clinical suspicion and prompt management can address the high fatality associated with the disease. The following ITS sequence has been submitted to GenBank under accession number OP019829:1 ttctcgcatc gatgaagaac gtagcaaagt gcgataatag tgtgaattgc atattgtgaa61 tcatcgagtc tttgaacgca gcttgcactc tatggttctt ccatagagta cgcctgcttc121 agcatcataa caatcccaca cataaaaatt tttttttatg tggttatggg caattctttc181 atagtatgga atcgcctaaa aaattctagg tataggtgct tgaataaagg atcatatctc241 caatccattt tttgggagac caaggaaaca ggattgggcc accgacatac cctcatatat301 tctgaagt caggtgggac tacccgctga acttaagcat atcaataagRhizopus, Absidia, Rhizomucor, Mucor and Cunninghamella, species of the order Mucorales, are found in the soil and cause opportunistic infections. The known predisposing factors in patients with mucormycosis are uncontrolled diabetes, neutropenia, carcinomas, immunosuppressive therapy, deferoxamine therapy and voriconazole prophylaxis .Rhizopus species are the most commonly isolated fungi from the patients of mucormycosis. Apophysomyces elegans, Apophysomyces variabilis and Rhizopus homothallicus are reported to be emerging species [et al. first reported infections due to R. homothallicus in pulmonary infection patients [ species . Chakrabpatients . There apatients . The incpatients .Rhizopus in the body causes extensive vessel thrombosis and tissue necrosis, as a result of which the fungus disseminates to other organs [Our patient was diagnosed with sepsis and DKA and was at high risk of developing mucormycosis. In diabetic ketoacidosis (DKA), hyperglycaemia and low pH make phagocytes dysfunctional, with impaired chemotaxis and defective intracellular killing of the pathogen . Mucoralr organs .Pulmonary mucormycosis has been described in patients with associated immunosuppression, such as neutropenia or graft-versus-host disease, whereas rhino-orbital disease is typically reported in patients with uncontrolled diabetes . The patMicroscopy and culture of the bronchoalveolar lavage fluid remain the gold standard to establish a diagnosis of pulmonary mucormycosis. The first-line drug is liposomal amphotericin B; however, the clinical team, on assessing the risk/benefit in view of the nephrotoxicity of the drug, initiated posaconazole. The patient succumbed to the disease due to delay in reaching to the healthcare facility and also a delay in initiating antifungal treatment, as fungal infection was not suspected at the outset. Early surgical treatment, appropriate antifungal therapy, and control of predisposing factors are of great importance in the treatment of such cases . HoweverThe internal transcribed spacer sequence showed maximum similarity to sequences KU926333 and MK351861, as shown in


The limits were 2.5–5.6 and 12.0–21.0 (ng g−1) for SAs and TCs by HPLC–DAD, respectively. The obtained accuracy rates were in the ranges of 83.07–86.93% and 86.90–91.19%, respectively, for SAs and TCs, with precision rates lower than 9.54%. Concerning the occurrence of antibiotics, the positive samples constituted 57.6%, 75%, and 77.7% of the Egyptian, Saudi Arabian, and Libyan samples, respectively. Notably, SAs antibiotics were the most prevalent in the Egyptian and Saudi Arabian samples; in contrast, TCs were the most dominant in Libya. Calculated parameters of risk assessment, concerning the aggregated exposure to SAs and TCs, showed no potential adverse effects from the exposure to contaminated honey in studied countries.Several studies worldwide have reported contamination of bees’ honey by antibiotics, which may pose a hazard to consumers’ health. The present study was thus established to: (1) introduce a validated multi-residue method for determining sulfonamides (SAs) and tetracyclines (TCs) in honey; and (2) characterize the potential risk due to the exposure to SAs and TCs in honey samples from Egypt, Libya, and Saudi Arabia. SAs and TCs were simultaneously extracted using solid-phase extraction and matrix solid phase dispersion methods. SAs and TCs were screened using HPLC–MS/MS and HPLC–DAD. The results confirmed detection limits for SAs and TCs by HPLC–MS/MS of 0.01 and 0.02–0.04 (ng gThe online version contains supplementary material available at 10.1007/s10653-022-01258-0. Honey is a natural sweet substance that is produced from the nectar collected from flowers by honeybees. The honey food matrix is very complicated, in which over 300 chemical substances have been identified  and tetracyclines (TCs) are the most important antibiotics used in commercial apiculture, with broad-spectrum effects against bacteria  for antibiotics in honey in Egypt, Libya and Saudi Arabia. However, some countries, such as Belgium, Switzerland, and the UK, have established action limits for antibiotics in honey, which generally lie between 10 and 50 µg/kg for each antibiotic group  and solid-phase extraction (SPE)  to introduce a multi-residue method for determining SAs and TCs in bees’ honey using either HPLC-diode array detector (DAD) or HPLC–MS; (2) quality assurance of the proposed methods of analysis; (3) to study the occurrence of SAs and TCs residues in bees’ honey from three Arabian countries ; and (4) to assess the potential risk due to the exposure to SAs and TCs in the studied samples.Different samples of honey were collected randomly from three Arabian countries with different environments in different seasons.Eighty-four samples of bees’ honey were collected from within 3 countries, Egypt, Libya, and Saudi Arabia, as follow: from 7 regions in Egypt , 8 regions in Libya , and 6 regions in Saudi Arabia . Figure S1 shows the geographic locations of sampling in the three countries. The collected honey samples were pure, unfiltered and unprocessed based on the obtained information from the beekeepers or honey suppliers to supermarkets in each of the studied regions. The collected samples were of different categories belonging to the following diverse floral origins: clover, citrus, thyme, sider, harmal, talh and summra (Table S1). The collected samples were directly extracted and stored in 250 mL fine plastic containers duly labeled with identification code, name and date of collection. The samples were then stored in a dry dark place at 20 °C until further analysis to avoid the laboratory conditions having an effect on the chemical composition and physical properties of the honey samples Abdalla, .Diatomaceous earth or Celite (approximately 400 meshe) was obtained from Sigma-Aldrich Corporation . SPE cartridges, 6 mL/200 mg of HLB , were purchased from Waters Oasis Co., . In addition, 0.45 µm nylon filters, 13 mm in diameter , were obtained.Standards of sulfamethazine (SMT), sulfamethoxazole (SMX), sulfadimethoxine (SDM), tetracycline hydrochloride (TC), oxytetracycline hydrochloride (OTC), and chlortetracycline hydrochloride (CTC) were obtained from Sigma-Aldrich.−1) were prepared as follows: 10 mg of individual standard was accurately weighed, dissolved in a small amount of methanol, diluted to 100 mL with methanol (HPLC-grade), and then stored at − 20 °C. From these stock solutions, working solutions were freshly prepared by gradient dilution with methanol (HPLC-grade).Stock solutions of antibiotics  followed by 5.0 mL of methanol (5%) containing 2% acetic acid. Subsequently, the cartridge was air dried under suction for 5 min. The analyte was sequentially eluted from the cartridge into a 30 mL rotary round—bottomed flask using 5.0 mL of methanol (absolute), followed by 5.0 mL of methanol (5%) containing 2% NH4OH. The extract was evaporated, under vacuum at 38 °C to dryness using a rotary evaporation system. The dried residue was re-suspended in 1.00 mL of methanol (HPLC grade) followed by filtration through a 0.22 µm filter before being subjected to HPLC analysis. Twenty microliters (10 µL of each of extract 1 and extract 2) were injected into the HPLC system for analysis.The supernatant of the honey extract from the above procedures was passed through an Oasis HLB 200 mg cartridge  for the SPE clean-up. The cartridge was sequentially washed with 5.0 mL of methanol (5%) . The mobile phase gradient program is applied as shown in Table S2. The system consisted of a vacuum solvent degassing unit, a quaternary gradient pump, an automatic sample injector, and DAD. The LC separation column was an Eclipse XDB-C18 , Eclipse; USA. A Phenomenex  guard column (3.0 × 4 mm) of the same material was also used. Mobile phase A was composed of ACN (HPLC grade) containing 0.01 M oxalic acid and 0.1% formic acid, and mobile phase B was 0.03 M oxalic acid, pH 2.3. The flow rate and sample run time were 1.0 mL min−1, pH 2.6), while mobile phase B was HPLC grade acetonitrile and formic acid (99.9:0.1 v v−1). The gradient program for separation is shown in Table S1. The column was maintained at 25 °C with a flow rate of 0.2 mL min−1. The injection volume was 20 µL.The HPLC–MS/MS system used was an Agilent 1260 series for high-performance liquid chromatography-tandem mass spectrometry  equipped with a variable wavelength UV detector. Positive electrospray ionization (ESI) mode was used to detect SA and TC antibiotics. The SA and TC determination was conducted following the method described by Ahmed et al. . Specifi−1.Conditions of the mass spectrometry (MS) analysis: The used MS detector was a Thermo Finnigan LCQ Duo ion trap mass spectrometer , equipped with a heated capillary interface and ESI. Ultra-pure nitrogen gas was used for drying and nebulizing. Spray voltage was set to 4.5 kV and the capillary voltage was maintained at 3500 V. Drying gas temperature was set at 350 °C, and the flow rate was 10.0 L minr2 were obtained.The linearity of response to antibiotic pure standards was examined for HPLC–DAD and HPLC–MS/MS using concentration ranges of 50–1000 ng and 1–100 ng, respectively with five concentrations for each antibiotic. Each concentration was prepared in triplicate for each antibiotic. Calibration curves for SAs and TCs were created by plotting the peak area against the antibiotic concentration (ng) using Microsoft Excel 2010. Linearity equations and The DL and QL were determined using equations of the calibration method . Honey samples were spiked with the six antibiotics at a concentration of 50 µg kgPrecision (repeatability) was estimated by calculating the relative standard deviation rate (RSD %) for each antibiotic accuracy using three replicates.Exposure assessment of SAs and TCs in honey was based on the mean detected concentrations in samples. The estimated daily intake (EDI) of SAs and TCs as µg/day was calculated using the following equation:−1. Calculated in the same way, the daily consumption of honey in Libya is 9.8 g day−1 per capita , poultry meat (29.31 g), eggs (19.8 g), fresh milk (180 g), and fish (97.88 g) CAPMAS, . The cor−1 bw day−1), respectively, according to the FAO/WHO . We have used the assigned MRLs by the Swiss authorities, which are 50 and 20 µg/kg for SAs and TCs, respectively  and hazard index (HIS), as well as the adversity-specific hazard index (HIA).The new approaches for risk characterization were performed following the equations presented by Goumenou and Tsatsakis  for singS was calculated according to the following equation:HQn refers to the number of food items in which SAs and TCs might be found (Five food items previously mentioned above in the exposure assessment section). The equation numerator represents the consumed amount of honey multiplied by the MRL, while the denominator refers to the sum of the calculated values of the other five food items representing the whole diet. The calculated CF value for SAs antibiotics was 0.0037, while it was 0.0014 for TCs.Here: n chemicals (3 SAs and 3 TCs) in a specific food item (honey), the source related HIS was calculated based on the following equation:Regarding the risk characterization for a mixture of n represents the number of chemicals in the mixture.Here, A was calculated according to the following equation:HIHere, we considered the sum of the three SAs and the sum of 3 TCs because antibiotics from the same family have the same mode of action.R2 values, for both SAs and TCs, were between 0.978 and 1.00. These results confirmed the validation of both instruments in the analysis of SAs and TCs. These results are in agreement with our previous studies  for TCs which were lower than the DLs for DAD by 250 (SDM)–650 folds (TC). The QLs of MS/MS for SAs and TCs were also markedly lower by values ranged between 239 (OTC) and 500 (SMT) times less than those of DAD.Significant differences in the values of DLs and QLs were markedly observed between the DAD and MS/MS detectors. The DLs for MS/MS ranged between 0.01 ng g−1 and 21.5–30 ng g−1, respectively) were also notably lower than the permissible limits assigned by the Swiss authorities . Therefore, HPLC–DAD, in the proposed method of current study, could recover the trace concentrations of antibiotic-contaminated honey and can be used for the periodical inspection programs for SAs and TCs in honey.The QLs of DAD for SAs and TCs  proved their selectivity for SAs and TCs compounds and the easier distinction between them and other potential interfering compounds. Data in Table  proved tOwing to chemical differences between SAs and TCs, significant differences in accuracy rates were found following the two methods of extraction Table . When us18 as a MSPD. Meanwhile, MSDP with diatomaceous earth exhibited lower recovery rates with TCs (45.48–50.20%). Because diatomaceous earth mainly consists of 80–90% silica, 2–4% alumina, and 0.5–2% iron oxide, Al and Fe ions may negatively affect the extraction of TCs, resulting in low accuracy.In contrast, the extraction of honey samples using MSDP by diatomaceous earth proved highly capable of extracting SAs with accuracy rates of 84.65%, 86.93%, and 83.07% for SMT, SMX, and SDM, respectively. These results are in agreement with the work of Zou et al.  who repoNotably, the precision and repeatability values of the two applied methods were quite accepted, RSD rates were markedly lower than the recommended value (15%) assigned by the EU directive  concerniBased on the obtained data in Table −1. SMT and SMX showed high incidence in all Egyptian samples (100%), while SDM was not detected anywhere. The examined samples from the regions of Dakahlia, Gharbia, Menofia and Qalyubia contained multiple antibiotics from both SAs and TCs groups. The highest averages of the detected concentrations of SMT and SMX were scored for Gharbia samples and Giza samples, respectively as 23.5 and 86.9 µg kg−1. This means that SMX was the most used antibiotic from the SAs group. On the other hand, the highest averages of the detected TCs were scored by samples from Dakahlia (OTC: 38.72 µg kg−1 and TC: 131.75 µg kg−1) and Qalybia (CTC: 58.18 µg kg−1). This revealed that TC could be the most used member of the TCs group followed by CTC then OTC. In terms of frequency, of positivity, the antibiotics were found in the following order: SMT = SMX > TC > CTC > OTC > SDM.Data in Table Notably, 57.6% of samples (19 out of 33) were positive for antibiotics. In terms of the frequency of positive samples, the tested regions can be arranged in the following order: Giza > Gharbia = Menofia > Qalyubia > Dakahlia > Ismailia.−1).To the best of our knowledge, only one Egyptian study, by Abd Alla , determi−1 (Swiss MRL). The detected concentration of OTC did not exceed 40 µg kg−1, while the elevated average of TC and CTC levels were within the ranges of 74.83–239.36 µg kg−1 and 50.42–322.24 µg kg−1, respectively. The highest detected concentrations of TC and CTC were recorded for the samples from Tarhounah (269.35 µg kg−1) and Khoms (462.48 µg kg−1), respectively. The results showed that the frequencies of TC congeners were in the following order: CTC > TC > OTC.The overall use of antibiotics in Libyan apiculture can be deduced from the data presented in Table . The resNotably, 75% of samples (18 out of 24) were positive for antibiotics and 87.5% of the positive samples had concentrations over the Swiss permissible limits. Moreover, 66.6% of samples contained multiple TCs congeners, which means that Libyan beekeepers used TCs as a mixtures or cocktails. Briefly, the tested regions in Libya can be arranged, in terms of the frequency of positive samples, as follows: Tarhounah = Khoms = Gheryan = Msallata > Bani Walid > Tripoli > Al Aziziyah > Wadi al Hayaa.−1). On the other hand, for TCs, TC exhibited the highest detected concentrations of over 20 µg kg−1 (the Swiss MRL) for the samples of Abha and Al Bahah which ranged between 64.74 and 151.06 (µg kg−1), respectively. CTC was only detected in two samples from Riyadh with elevated values above the Swiss MRL of 30.99 and 33.60 µg kg−1. Notably, SDM and OTC were not found in any of the examined samples  were the most abundant antibiotics found at a rate of 78.5% (11 out of 14 positive samples). TC and CTC were the third and fourth most common after SMT and SMX. Notably, all of the detected levels of SAs were markedly lower than the Swiss legislated limit  for each antibiotic detected in honey were markedly lower than the critical value of 1 for all samples from the three countries. This indicated that the aggregated exposure level was lower than the ADI; consequently there were no significant risks associated with the consumption of contaminated honey samples with the detected levels of SAs and TCs at the studied sites. The highest values of HQS were recorded for TC and CTC in Libyan samples  followed by Saudi Arabian samples  and then Egyptian samples .Data of Table S) for mixtures of antibiotics (3 SAs and 3 TCs), it was noted that SAs had higher HIS values than TCs. The highest HIS values of SAs were scored for Libyan samples, followed by Saudi Arabian samples and then those from Egypt, being 0.338, 0.168, and 0.156, respectively. However, notably, HIS values for the SA and TC groups were markedly lower than 1, indicating the safety of the studied samples. Similarly, the calculated values of the adversity-specific hazard index (HIA) for the SA and TC groups were also located within safe ranges. These findings revealed that there were no potential adverse effects from the synergistic action of the antibiotics.Concerning the source-related hazard index , when compared with the Egyptian population. As well, it should be taking into account that the maximum detected concentrations of antibiotics, such as in samples from Giza and Dakahlia (Egypt), Khoms, Tarhounah, and Masallata (Libya), and Al Bahah (Saudi Arabia), could be ingested through honey.In the present investigation, quality assurance of the extraction and screening methods for both HPLC–DAD and HPLC–MS/MS against SAs and TCs revealed high accuracy and selectivity, and good sensitivity. Regarding the survey on the occurrence of SAs and TCs, the results confirmed that SAs antibiotics were dominant in the Egyptian and Saudi Arabian samples, while TCs were the most prevalent in Libya. Although a number of samples had elevated concentrations of SAs and TCs, over the Swiss MRL, the risk assessment study revealed no potential risk of the detected concentrations to consumers’ health. Meanwhile, it should be noted that chronic exposure to antibiotics could represent a potential risk to public health, particularly for those samples with the highest concentrations. Further efforts are needed to limit human exposure to antibiotics through the control of antibiotic application, inspection programs, and encouraging the use of natural alternatives to antibiotics in the production of foods of animal origin. In addition, authorities such as the European Union and US FDA have to set MRLs of these drugs in bees’ honey.Supplementary file1 (DOCX 839 KB)Below is the link to the electronic supplementary material.


Glaucoma is a neurodegenerative condition in which increased intraocular pressure (IOP) causes progressive degeneration of retinal ganglion cells (RGCs), the neurons projecting from the retina to secondary visual areas of the brain. Raised IOP results in apoptosis of RGCs because of damage to RGC axons at the optic nerve head. Axonal damage triggers disruption of RGC mitochondria, metabolism, and active transport. It also disturbs the anatomic distribution of excitatory connections in RGC targets, including the superior colliculus and the thalamic dorsal lateral geniculate nucleus (dLGN). However, the timeline of the processes linking initial damage to RGC death is not well characterized. It is especially unclear at which point functional changes begin in excitatory synapses between RGCs and target structures like the thalamocortical (TC) neurons in the dLGN. To solve this conundrum, After establishing the age of onset of IOP elevation at approximately seven months in D2 mice, the authors first measured anterograde axonal transport along RGCs to the dLGN using injection of fluorescently tagged cholera toxin (CTb-594) into the eye. D2 mice showed a progressive decline of anterograde transport, which was significant in the nine-month age group. Similarly, D2 mice showed progressive loss of glutamatergic RGC presynaptic terminals in the dLGN, as indicated by a decreased density of immunolabelled vGLUT2 puncta. This effect was significant by nine months of age, was severe by 12 months, and occurred to a greater extent in areas of the dLGN with impaired axonal transport as determined by the CTb-594 assay.r), and the AMPA/NMDA ratio, which indicates the relative activations of postsynaptic AMPA receptors (AMPARs) and NMDA receptors (NMDARs). They discovered a progressive increase in the PPR, indicating a falling Pr, and coincident decreases in both the AMPAR and NMDAR components of EPSCs, but no changes in the overall AMPA/NMDA ratio. These findings point toward a presynaptic locus of effect for the reduction in synaptic transmission, indicating that the release probability in RGCs terminals progressively decreases with age, but finding no evidence that postsynaptic sites on TCs change in receptor composition. Using a reduced stimulus to evoke single-axon synaptic vesicle release from RGCs (single fiber EPSC), the authors found that the EPSC amplitude recorded in TCs was unchanged. However, the ratio of the single fiber EPSC to the maximal EPSC was increased in the 12-month-old group, suggesting that TC neurons in D2 mice receive overall less inputs than the controls. In summary, the effects observed in both spontaneous and evoked synaptic transmission converge onto a pathologic change in transmitter release at the RGC to TC synapse.Smith and colleagues then sought to investigate changes in functional synaptic transmission at the RGC-TC synapse using whole-cell electrophysiology in acute slices. Spontaneous EPSCs (sEPSCs) in TCs did not display changes in amplitude but had a decreased frequency at nine months of age. This effect suggests that a proportion of synapses had been lost as opposed to individual synapses being individually weakened. Next, the authors assessed the paired pulse ratio (PPR), which relates inversely to presynaptic vesicle release probability (PTCs are known to be affected in the context of elevated IOP, with defects in intrinsic excitability and soma atrophy . To inveFinally, even if the axonal defects are well advanced in 11- and 12-months D2 mice, the density of RGC somas in the central and peripheral retina was unchanged, showing that at this stage pathology has not yet caused cell loss. RGC degeneration is rather a late event in the D2 mouse model, starting after 15 months of age .In conclusion, this study uses an elegant combination of anatomic and physiological approaches to describe the progressive dysfunction of the retinogeniculate synapse in the D2 mouse model of glaucoma . Some ofAs emphasized in the paper’s Discussion, some points remain to be answered considering these results. For example, it is still unclear if decreased vGLUT2 staining is caused by degenerating RGC-to-TC synapses or by defects in axonal transport. In addition, knowing if dendritic remodeling in TCs is followed, or alternatively preceded, by a loss of postsynaptic markers will be key to understanding the pathologic processes operating at retinogeniculate synapses. To conclude, the authors highlight an interesting paradox: while this study shows decreased synaptic transmission between RGCs and TCs, they reported increased TC excitability in D2 mice in previous work . Smith aThis study adds unprecedented value to glaucoma research. Further work describing cellular and molecular mechanisms behind retinogeniculate synapse dysfunction will be essential to fully understand the pathologic underpinnings of this disease.