Screening for functional disability is a promising strategy to identify high-need older adults. We compare 2 disability measures, activities of daily living (ADLs), and life space constriction (LSC), in predicting hospitalization and mortality in older adults.We used the nationally representative National Health and Aging Trends Study of 30,885 observations of adults aged 65 years and older. Outcomes were 1-year mortality and hospitalization. Predictors were ADLs  and LSC (frequency of leaving home).both 3 or more ADLs and who never/rarely left home, 58.4% died. ADL and LSC disability combined was more predictive of 1-year mortality and hospitalization than either measure alone. ADL disability and LSC screens identified overlapping but distinct populations. LSC identified more women (72.6% vs 63.8% with ADL disability), more people who live alone (40.7% vs 30.7%), fewer who were White (71.7% vs 76.2%) with cancer (27.6% vs 32.4), and reported pain (67.1% vs 70.0%).Of respondents, 12.4% reported 3 or more ADLs and 10.8% reported rarely/never leaving home. ADL disability and LSC predicted high rates of 1-year mortality and hospitalization: of those with 3 or more ADLs, 46.4% died and 41.0% were hospitalized; of those who never/rarely left home, 40.7% died and 37.0% were hospitalized. Of those with LSC and ADLs both independently predicted mortality and hospitalization but using both screens was most predictive. Routine screening for ADLs and LSC could help health systems identify those at high risk for mortality and health care use. Translational Significance: Using two distinct screens for functional disability, activities of daily living and life space construction, can help health systems and communities identify older adults at highest risk of death and hospitalization and appropriately target resources to better support them in the community.Older adults with functional decline are at a high risk of poor health care outcomes. After new onset of disability in activities of daily living , adults over the age of 65 have a 56% 2-year mortality rate . Indeed,The impact of functional decline on burdensome, high-cost, health care use has come to the attention of health system leaders. Functional disability increases the likelihood of hospital readmissions , overallThe most common measure of functional disability and decline are the ADLs. First described by Katz, ADL disabilities were identified through observing the sequential loss of domains of ability of older adults over time. They originally contained the six functions of bathing, dressing, toileting, transferring, continence, and feeding , with moLife space constriction (LSC) considers the frequency with which individuals leave their proximal environments and therefore provides a broader perspective of disability . Life spIt is important to assess the independent and cumulative risks of LSC and ADL impairment because disability is the result of a gap between one’s physical/cognitive abilities and environmental and personal supports . TherefoGiven the importance of health system screening for functional disability, we aim to examine the prognostic characteristics of population ADL disability versus LSC screening. Specifically, we aim to examine how LSC performs separate from and in combination with ADL disabilities in predicting hospitalization and mortality and to characterize the populations who would be identified by ADL disability versus LSC screens. We hypothesize that distinct populations will be identified by ADL versus LSC screens, and these measures together will be more predictive of mortality and hospitalization than either measure separately. This work will inform future population health efforts to screen for high-need patients with functional disability and decline.We used data from the National Health and Aging Trends Study (NHATS), a nationally representative survey of Americans aged 65 and older that began in 2011. The sample was drawn from the Medicare enrollment file with oversamples at older ages and of Black enrollees. The survey has been administered annually with a response rate of 71% to for the initial round that individuals entered NHATS (in 2011), and from 86.1% to 94.8% for follow-up surveys . NHATS oWe included respondents from NHATS Waves 1–6 (2011–2016). We limited the sample to individuals who entered NHATS in 2011, with individual respondents observed multiple times. We excluded the 2,798 observations that had missing data for either ADLs or LSC (8.3% of the sample). Given that seven annual waves have been conducted, this allowed us to look forward to the next survey wave for each observation to assess for death and hospitalization.ADLs were defined as the total number of activities that the participant reported receiving assistance with or not doing in the last month: bathing, eating, dressing, toileting, getting out of bed, and walking inside . ADL disLSC can be measured with a single-item assessment . This isMortality in the following year was ascertained by NHATS interviewers during annual surveys. Death was reported by informants during attempts to contact the participant for the annual interview, and assessed via the Medicare Beneficiary Summary File. Given that Medicare claims data are not available linked to NHATS for the third of older adults enrolled in Medicare Advantage plans, hospitalization in the last year was self-reported by participants. However, all NHATS enrollees have a linked Medicare Beneficiary Summary File, meaning that data are available for all NHATS respondents.In order to capture the sociodemographics and health of populations identified through functional screening in this sample, we used additional variables including: demographic characteristics ; proxy respondent status; socioeconomic characteristics ; self-reported illness  a baseline model including just age and gender given that mortality statistics commonly adjust for these factors ; (b) a model that includes age, gender, and the ADL variable; (c) a model that includes age, gender, and the LSC variable; and (d) a model that includes, age, gender, the ADL variable, and the LSC variable. For each model, the area under the receiver operating characteristic (ROC) curve was reported. For the ADL variable, disability was categorized as the count of total ADL disabilities (six levels). For the LSC variable, disability was categorized as leaving the house every day, most days, sometimes, or rarely/never (four levels). Models did not include additional covariates, including comorbidities, given the existing data that demonstrate that function is a better proxy and surrogate for serious illness than comorbidities , this represented a population that was primarily female (56.5%), with a mean age of 77.2 years  from 7,897 individuals. After applying survey weights and adjusting for repeated observations for each individual  as well as categories of LSC . For those with no ADL disability, 1-year mortality ranged from 2.4% to 12.2% across LSC categories, and for those with no LSC disability, 1-year mortality ranged from 2.4% to 21.6% across ADL categories. Those reporting both rarely/never leaving home Rates of hospitalization similarly increased along ADL categories  as well as categories of LSC . The additive effect of ADL disability and LSC measures in predicting hospitalization was less strong than for mortality: the highest hospitalization rate (41.0%) was seen in the population with three or more ADL disabilities who left home. Indeed, among those with three or more ADL disabilities, hospitalization rates were similar across LSC categories (range of 40.0% for those leaving home most days to 42.9% for those leaving home every day). However, among those with no ADL disability, LSC was a strong predictor of hospitalization: within this population, 18.2% of those leaving home every day were hospitalized in the next year, as compared to 32.2% of those who left home rarely/never. For full details of the mortality and hospitalization rates for each combination of ADL and LSC, see p < .05). All functional disability measures performed better than the baseline model. Our sensitivity test that included only the first observation of each individual did not differ from the primary analysis , cancer, pain, and falls; those identified with LSC were socially distinct—they are more likely to be female and to live alone. Given the specific risks of loneliness  and homeWe examined two critical outcomes: death and hospitalization. While our results are statistically significant, it is challenging to interpret the clinical significance of comparisons of ROCs. For this reason, we have highlighted the rates of death and hospitalization for each combination of ADL and LSC measure . HoweverThe ability to leave home and function in social roles, as LSC captures, is extraordinarily important to people . This stigaa065_suppl_Supplementary_MaterialsClick here for additional data file.


Beauveria bassiana (B. bassiana) is a famous entomopathogenic fungus that could parasitize on hundreds of insect species, which are being used as an environmentally friendly mycoinsecticide. Nevertheless, the possible effect of genetic diversity of these B. bassiana isolates from different hosts on virulence has not been explored before. In order to explore that issue, we compared the genome sequences among seventeen B. bassiana isolates from 17 different insects using whole genome re-sequencing, with B. bassiana strain ARSEF 2860 as the reference genome.B. bassiana. Among these, two genes with high frequency mutations encode the toxin-producing non-ribosomal peptide synthase (NRPS) protein. Seven genes undergoing positive selection were enriched in the two-component signaling pathway that is known to regulate the fungal toxicity. In addition, the domain changes of three positively selected genes are also directly related to the virulence plasticity. Besides, the functional categorization of mutated genes showed that most of them involved in the biological functions of toxic proteins involved in.There were a total of 10,098 missense mutated genes, 720 positively selected genes were identified in 17 strains of B. bassiana towards hosts during infection process, which provide an insight into the potential effects of natural variation on the virulence of B. bassiana, which will be useful in screening out potential virulence factors in B. bassiana.Based on our data, our results indicate that several mutated genes and positively selected genes may underpin virulence of  Beauveria bassiana (B. bassiana), a well-known entomopathogenic fungus, is belonged to the family Clavicipitaceae, order Hypocreales [B. bassiana exhibits high adaptability to various environmental conditions and thus could parasitize a wide range of insect populations [B. bassiana is actively developed as a biological control tool for various insects in all regions of world [B. bassiana to reduce the occurrence of forest insects in China every year [B. bassiana is thus a generalist with no strict host preference and can be used as an effective and sustainable biological control agent against a myriad of insect pests [ocreales . Owing tulations . Currentof world , 4. Accoery year . Generalct pests .B. bassiana strains from a specific insect host varies frequently [B. bassiana isolated from a given insect showed low virulence against other insects, and showed stronger virulence against these insects after domestication [B. bassiana to hosts has a great plasticity. Genome structure and DNA sequence variation in these isolates could be related to many factors in the biology of this fungus. Recently, the re-sequencing of numerous individuals within pathogen species has been as a forceful tool to explore the genetic mechanism in fungal and fungal-host interaction studies [B. bassiana isolates with different levels of virulence and repellence to the termite, and then some differences were found in the nucleotide sequences of the two isolates of B. bassiana, suggesting a genetic basis for the observed intra-specific differences in their virulence against the termite [B. bassiana with low/high virulence, and finally identified several genes and molecular processes that effect the virulence towards Mosquitoes [B. bassiana isolates isolated from different hosts.Virulence is variable and not universal for a prescribed host and pathogen . Increasequently . It has tication , indicat studies . For exa termite . Valero-squitoes . These sB. bassiana strains by a comparative genomics analysis. Thus, we sequenced the 17 B. bassiana isolates from different hosts compared to the reference genome B. bassiana strain ARSEF 2860. Based on the analysis, understanding the potential factors of genetic variation on the virulence of B. bassiana will improve our methods to use this fungus as an effective and sustainable biological control agent against.In the present study, we aim to gain our knowledge in understanding genomic diversity involved in virulence and host-pathogen interaction for 17 B. bassiana strains were summarized in Table SB. bassiana strain ARSEF 2860 reference genome, with varying 67.30 to 75.30% among different strains  are synonymous and 820,233(29.51%) are nonsynonymous. In addition, a total of 884,811 Indels were obtained, with 423,409 (47.85%) InDel-Ins and 461,402 (52.15%) InDel-Del  in seventeen samples  have been widely applied to characterize potentially adaptive genetic variation [B. bassiana. Among the Indels, the number of InDel-Ins variants  is almost equal to the InDel-Del variants , and most of the variants were located in the upstream region of the genes (79.15%). Furthermore, structural variation is known to be a key component of phenotypic diversity in organisms [Firstly, 17 strains of SEF 2860 , which iariation , revealirganisms . In thisrganisms .B. bassiana isolates. However, significant nucleotide variations were identified in four genes  in all samples, and two of them encoding the common protein non-ribosomal peptide synthase . NRPS is a key mechanism for the biosynthesis of bioactive metabolites in fungi and plays an important role in the production of fungaltoxin. For example, NRP synthetase Pes3 has been found to contribute to Aspergillus. fumigatus virulence, and disruption of pes3 resulted in an augmented virulence phenotype in invertebrate for invasive aspergillosis. NRP synthetase mgoA has a role in the virulence of Pseudomonas. syringae pv. Syringae, and A Tn5 mutant disrupted in mgoA was defective in mangotoxin production and virulence. NRP synthetase gliP was predicted to be involved in gliotoxin production, and mutants with a disrupted gliP locus failed to produce gliotoxin [B. bassiana trains, which may be closely related to the maintenance of the pathogenesis during the evolution process, but the specific virulence effects of each mutation need to be further explored. Furthermore, GO annotations showed that the functions of mutated genes were mainly related to binding, catalytic activity, extracellular region, metabolic process, and nucleic acid binding transcription transporter activity. Mutated genes involved in binding, transport and other biological processes may be associated with increased interaction between virulence associated proteins and hosts when B. bassiana exerts a toxic infection. Additionally, among these groups, the variation involved in the metabolic process may be related to the production of antibacterial compounds, it is very important for B. bassiana to resist microbial competition in the process of killing insects [Secondly, a hierarchical clustering analysis was carried out on the top 50 genes with missense SNPs in 17 samples. The results showed that there was no significant difference in the frequency of missense SNP mutations among different hosts, which revealing the population identity of the seventeen sequenced liotoxin –23. Base insects .B. bassiana were determined using the whole missense SNPs. Three distinct B. bassiana clusters were formed, namely the isolates of S2\S3\S4\S5\S6\S8\S14\S19, the isolates of S2\S9\S10\S13\S15\S18\S19\S20\S21, and the isolates of S7/S11. Previously, the study of Ghikas et al. revealed that seven phylogenetic clusters in B. bassiana that consisted of isolates which had common climate features but did not exhibit any geographic distribution [B. bassiana is not monophyletic, but rather is composed of several clusters that are morphologically identical [B. bassiana. Additionally, the results showed that similar mutations occurred in B. bassiana strains in each cluster during evolution, which indicated a close relationship among B. bassiana isolates isolated different hosts in each cluster, but the relationship between the virulence of B. bassiana isolates in each cluster is not clear and further studies are needed. Furthermore, in the PCA plot, an obvious separation of three clusters was found, which also revealed a clear genetic differentiation among these 17 samples.Thirdly, the phylogenetic relationships and principal component analysis (PCA) between isolates of ribution . Our findentical –27, whicB. bassiana. Finally, a total of 7050 genes with Ka/Ks > 1 and 720 genes with Ka/Ks < 1 are confirmed, respectively. Among genes predicted to be under strong purification selection, there is enrichment for GO categories involving NAD + ADP-ribosyltransferase activity, chromatin, chromatin assembly or disassembly, chitin catabolic process, and actin cytoskeleton. In Salmonella, spv genes can establish successful systemic infection in experimental animals, and SpvB virulence-associated protein has recently been shown to contain the ADP-ribosyltransferase domain, suggesting that NAD + ADP-ribosyltransferase activity is associated with the regulation of virulence [Candida albicans [B. bassiana could secrete several chitin-degrading enzymes, such as Endo chitosanase and Chitinase D [B. bassiana virulence [B. bassiana strains with positive selection were abundantly enriched in this pathway. Increasing studies have revealed the involvement of this pathway in regulating resistance processes and diverse virulence in a wide range of pathogenic bacteria, such as Mycobacterium tuberculosis [Salmonella typhimurium [Staphylococcus aureus [Pseudomonas aeruginosa [B. bassiana strains.In previous study, genes with a signature of positive selection is possible to result in the adaptation to a dynamic environment and may be associated with fungi virulence . Herein,irulence . Chromatalbicans . Earlieralbicans . Chitin tinase D , which airulence . Additioirulence . In the rculosis , Salmonehimurium , Staphyls aureus , Pseudomruginosa . Taken tSalmonella was the direct result of the inactivation of both of the mutS and recD gene products, however, S. typhimurium carrying mutations in either one of these genes behaved similarly to the wild-type strain and without attenuating virulence [Mycobacterium tuberculosis, which could regulate many intracellular metabolic processes and interfere with signaling pathways of the infected host cell [B. bassiana strains may be closely related to its plasticity of virulence. Besides, other genes deletion or genes acquisition may also be critical in virulence evolution. Of course, further study is needed in the future.Finally, a comprehensive protein domain analysis was performed on 720 positively selected genes with missense mutations. Significant differences in 13 domains encoded by 13 protein-coding genes were identified. Among these, three domain proteins involved in the host-pathogen interaction, such as ankyrin repeat domain protein, Metallophos domain protein and MutS domain V that were encoded by gene-7731, gene-5369, and gene-10,314 may affect host-pathogen interactions. For example, Zahrt et al. found that the alteration in virulence on irulence . Ankyrinirulence , 39. Besirulence . The Metirulence . Earlierost cell . In summB. bassiana isolates obtained from different insect hosts. Subsequently, the relationship between stable genetic variation and virulence function shared by 17 strains of B. bassiana in different hosts, and the correlation between mutated genes and virulence effect of B. bassiana were analyzed. A number of genes with stable mutations in these B. bassiana isolates have been established. Among them, some key factors including NRPS mutations, domain changes of ankyrin repeat domain protein, Metallophos domain protein, and MutS domain occurred in B. bassiana isolates may be crucial for the virulence of B. bassiana. Besides, the 720 positive selection genes and the top 50 genes with missense mutations were significantly enriched in some important biological processes, and pathways may also be related to the virulence of B. bassiana against hosts. The study provided the relevant theoretical basis for targeted changes in the virulence effect of B. bassiana. In this case, we think the virulence of B. bassiana might be influenced by natural variation in the process of evolution, which may benefit future molecular investigations of insect-fungus interactions and promote the development of B. bassiana as cost-effective and sustainable mycoinsecticides. However, do these genetic mutations really play a role in virulence or how do it regulate virulence in biological processes, which is interested and will need to further study.According to the plasticity and variability of fungal virulence, we sequenced and compared the genome of seventeen B. bassiana that were obtained from 17 different insects (This study employed seventeen isolates of  insects . All isoB. bassiana was extracted using the CTAB method as the previous report [http://hannonlab.cshl.edu/fastx_toolkit) to filter the low-quality reads (< 10), reads shorter than 50 nt and duplicated reads [B. bassiana strain ARSEF 2860 [Genomic DNA of 17 isolates of s report . DNA coned reads . After tSEF 2860 . The aliSEF 2860 .http://picard.sourceforge.net) was used to discard duplicates, and the obtained BAM files were further processed using SAMtools 1.4.1 (samtools.sourceforge.net) [broadinstitute.org/gatk/) UnifiedGenotyper [http://snpeff.sourceforge.net/) [Picard tools version 1.92 (rge.net) . SNPs anenotyper . After tge.net/) .B. bassiana originates from different hosts. The optimal nucleic acid replacement mode was found by modelfinder. Then a phylogenetic tree was conducted using iqtree with the bootstrapping of 100 replicates [To determine the evolutionary relationship among plicates , and furplicates  on the mB. bassiana isolates were searched using the bidirectional best hit (BBH) method [The positively selected genes among the genomes of 17 ) method . The rathttp://www.genome.jp/kegg/) database to explore main biological functions [http://pfam.xfam.org/) annotation [A hierarchical clustering analysis of the top 50 genes with missense SNPs was conducted by the clustergram function in the Matlab Bioinformatics toolbox with minor changes. GO analysis was performed with Fisher’s Exact Test and classified under three categories: biological process (BP), cellular component (CC), and molecular function (MF) . Meanwhiunctions , and thenotation .Structural variants was determined using BreakDancer (v1.1.2) software , containB. bassiana database using hmmsearch in HMMER (v3.1b) with e-value < 0.00001 [To explore the effect of nonsynonymous SNPs in the positively selected genes on protein domains, we first identify the mutant amino acid sequences using Bioperl. Then, the hidden Markov model (HMM) profile for the NBS domain was applied to search the protein sequences in the  0.00001 .Additional file 1: Table S1. Quality control results of sequencing data of 17 B. bassiana strains. Table S2. Mutation information of the seven positively selected genes that are enriched in the two component systems. Table S3. The formation of the structure variants shared by seventeen B. bassiana isolates genomic sequences. Table S4. The information of the hosts of seventeen B. bassiana isolates. Table S5. The number of genes shared between the 17 isolates presented in each category in Fig.


Pde6b-mutant  mice, in which photoreceptors degenerate shortly after eye opening. To unravel metabolic differences emerging before and after eye opening under physiological and pathophysiological conditions, we performed nuclear magnetic resonance (NMR) spectrosco-py-based metabolome analysis of wild type and Rd1 retina and vitreous/lens. We show that eye opening is accompanied by changes in the concentration of selected metabolites in the retina and by alterations in the vitreous/lens composition only in the retinal degeneration context. As such, we identify N-Acetylaspartate as a potential novel vitreous/lens marker reflecting progressive retinal degeneration. Thus, our data can help elucidating mechanisms underlying key events in retinal physiology and reveal changes occurring in pathology, while highlighting the importance of the vitreous/lens in the characterization of retinal diseases.Photoreceptors are the light-sensing cells of the retina and the major cell type affected in most inherited retinal degenerations. Different metabolic pathways sustain their high energetic demand in physiological conditions, particularly aerobic glycolysis. The principal metabolome of the mature retina has been studied, but only limited information is available on metabolic adaptations in response to key developmental events, such as eye opening. Moreover, dynamic metabolic changes due to retinal degeneration are not well understood. Here, we aimed to explore and map the ocular metabolic dynamics induced by eye opening in healthy (wild type) or  Theper se [Metabolites generated in all these conditions can in turn reflect on the composition of structures in close contact with the retina, such as the lens and the vitreous , which aper se . As suchper se ,25,26,27per se ,28, and Rd1Pde6b (Rd1) mutant mouse model, whose severe and early-onset photoreceptor degeneration is known to be aggravated with eye opening, we show that the metabolic composition of the vitreous/lens is affected over time only when a degenerative context is provided, leading us to identify N-Acetylaspartate as a putative degeneration marker.Here, we perform a metabolic analysis of retina and vitreous/lens in postnatal mice prior to as well as shortly after eye opening to pinpoint physiological mechanisms triggered by this key developmental event. By applying the same analysis to the We believe the results of this extensive metabolomic analysis in retina and vitreous/lens at key time-points can help unravelling adaptation mechanisms taking place around eye opening in the healthy retina. Moreover, our data can help shed light on metabolic processes associated with disease onset and progression, thus potentially providing new prognostic markers of retinal degeneration.The Rd1 mutant mouse line is a model of early-onset retinal degeneration that affects primarily rod photoreceptors and shows rapid progression when the animal first opens the eyes at around postnatal day 13 (p13) ,30. The p-value < 0.05) of 19 proteins in the wt condition, while three proteins were found enriched in Rd1 mutant animals  and after (p13) eye opening A. AdditiIn an attempt to simplify the data and get a first overview, we performed a principal component analysis (PCA) of all metabolomics data B. While Next, we focused on the concentration values of the single metabolites C. As seeTaken together, the high-level analysis of the metabolome suggests a higher degree of difference in the vitreous/lens compared to the retina, with the biggest effect being exerted by the phenotype.Following this overview analysis of the data, we set out to simplify their interpretation by performing targeted analysis, all the while keeping some variables separated, e.g., the genotype. To establish the metabolic dynamics of healthy retina and vitreous/lens around eye opening, we first focused on the wt data. As suggested by the overview analysis B, no subp-value in the comparison between time-points, namely Choline, Citrate, Creatine, Acetate, and Glycine, aiming to confirm their differential regulation  in the comparison between p13 and p11 retina, including “glyoxylate and dicarboxylate metabolism”; “glycine, serine, and threonine metabolism”; and “alanine, aspartate, and glutamate metabolism” E. To congulation G. IndeedAltogether, we concluded that in physiological conditions the vitreous/lens metabolome remains largely unvaried, while few changes can be detected in the retina as part of a general metabolic reduction taking place around eye opening.After addressing changes in the metabolite composition of wild type retina and vitreous/lens that occur around eye opening, we next asked what differences are induced in those ocular tissues in the degenerative context of the Rd1 mutation. Whereas in wt conditions the vitreous/lens did not show any substantial change A, the RdThus, our data indicate that in the retinal degeneration context, selected metabolites are differentially represented at p13 compared to p11. Importantly, only in Rd1 mutant mice the vitreous/lens shows significant changes in its metabolite composition around eye opening. Furthermore, specific metabolites undergo the same changes also in the retina, suggesting an initial equilibration of the metabolic content of these two compartments and the loss of the physiological separation between them.In order to dissect changes in the metabolic composition in response to photoreceptor degeneration, we next set out to compare Rd1 mutant and wt conditions, while keeping the other variables independent. Compared to wt, the vitreous/lens samples of Rd1 mutant mice showed a significant reduction in the concentration of few metabolites at p11 , of which N-Acetylaspartate remained downregulated at p13. Moreover, the levels of Putrescine and Sarcosine were significantly altered at p13 only A,C,E. NoSubsequently, we focused on the analysis of degeneration-specific metabolic changes in the retina samples. Prior to eye opening, the degenerative context of the Rd1 mutant mouse determined a significant decrease in the levels of Xanthine and Hypoxanthine, both involved in purine catabolism B,F. SimiThus, we concluded that the vitreous/lens partly reflects the metabolic changes seen in the retina, where an initial decrease in purine metabolism and the subsequent decline in Glucose levels suggest an energy imbalance during degeneration.Finally, we performed metabolic pathway analysis, focusing first on the vitreous/lens. In this eye compartment, we found “alanine, aspartate, and glutamate metabolism” as the main affected pathway at both analyzed time-points, whereas “glycine, serine, and threonine metabolism” was predominantly affected before eye opening A,B.A closer look at the metabolites that mediated the enrichment of the term “glycine, serine, and threonine metabolism” confirmed the different levels in Rd1 mutant condition mainly at p11 C, whereaNext, we assessed which metabolic pathways were deregulated in the retina. As already observed for the vitreous/lens A, the paTaken together, these results highlight changes in the metabolism that are specific to the eye compartment, the time-point and the degenerative state examined, occasionally converging into similar pathways throughout the conditions.Sight is the sense we mainly rely on to interact with the environment. The visual information crosses the lens and the vitreous before it reaches the retina, a complex neural tissue that detects light stimuli with specialized photoreceptor cells. As such, pathologies affecting the retina, and particularly the photoreceptors, are highly debilitating. Though new mutations causing IRDs are routinely discovered and characterized, the mechanisms eventually leading to progressive retinal degeneration are only known partly. Similarly, mechanisms underlying physiological events, e.g., the consequences of eye opening on the ocular metabolome, are also poorly understood. Here, we chose the mouse as a model to study metabolic changes induced by eye opening in a physiological context as well as in response to degeneration, by providing a comprehensive NMR spectroscopy-based metabolome analysis of wild type and Rd1 retina. To investigate how retinal degeneration affects the levels of metabolites outside the retina, we also analyzed the metabolic changes in the adjacent vitreous/lens compartment. Due to the small size of the mouse eye and the relatively small vitreal volume, we combined vitreous and lens and analyzed them together as vitreous/lens samples.Using this experimental setting, we have shown that eye opening under physiological conditions seems to be characterized by an overall decrease in the levels of most metabolites in the retina, particularly of metabolites related to purine metabolism . While to our knowledge such a drive towards a general reduction of metabolite concentrations has not been previously reported, purine levels have been shown to rapidly decrease in response to light exposure . InteresMpc1) display higher consumption of 3-Hydroxybutyrate to compensate for the reduction of Glucose-derived Pyruvate [Conversely, in degenerative conditions of the retina this separation can be partially lost ,33. IndePyruvate . It is aAlternatively, 3-Hydroxybutyrate could concur to the antioxidative stress response of photoreceptors ,49, partIndeed, given the additional presence of extramitochondrial respiratory chain enzymes in the rod disk membranes ,14,15, tAll nucleotide monophosphate levels appeared increased in Rd1 mutant retinas from p11 to p13, although below the significance threshold, while Hypoxanthine was significantly increased. This observation is in direct contrast with what we observed in wt retinas, thereby proving specific for the degenerative context. We speculate that the higher concentration of metabolites belonging to purine and pyrimidine metabolism reflects the shunt of part of the Glucose to the Pentose Phosphate Pathway (PPP), possibly to counteract the formation of reactive oxygen species ,59,61, aFinally, by comparing Rd1 mutant and wt conditions in vitreous/lens, we could identify N-Acetylaspartate as a potential novel marker of photoreceptor degeneration. Together with Acetate, whose levels are also significantly decreased at p11, N-Acetylaspartate is synthesized in the mitochondria from Acetyl-CoA. Low levels of N-Acetylaspartate have been associated to neuronal loss and cellular dysfunction ,65, possAlthough we believe we shed some light on the metabolic interaction (or lack thereof) between vitreous/lens and retina across time and pathophysiological conditions, we are aware that the presence of the lens and the potential contamination of the retina samples with residual vitreous could affect the described readout. However, as only a small proportion of metabolites show similar significant changes in both compartments, we believe we can exclude any substantial interference that this technical issue could bring. Further analyses with specific metabolic assays are needed to consolidate these data and to understand how these different metabolic pathways concur to determine the rapid loss of the photoreceptors seen in the Rd1 mutant model. Additional time-points, e.g., after p13, could help to further pinpoint the changes taking place specifically at eye opening.Taken together, we can conclude that under physiological conditions the transition to eye opening in the mouse retina is accompanied by very specific changes in the metabolome, which, however, are not mirrored by metabolic changes in the adjacent vitreous or lens. In the presence of an ongoing photoreceptor degeneration, the retina and vitreous/lens metabolomes are uniquely affected, with only a few metabolites being similarly altered in both compartments. Moreover, such changes are mostly specific for the time-point analyzed, suggesting an additional effect of the developmental stage on the pre-existing degenerative background. Importantly, as an exception to the temporal specificity of these metabolic changes, we propose N-Acetylaspartate as a potential marker of retina degeneration that might be assessed via analysis of vitreous or, possibly, aqueous humor samples from patients.We envision that our data could pave the way to new lines of investigation and contribute to a better understanding of basic mechanisms underlying retina physiology and pathology.wtPde6b (Rd1 wt) and Rd1Pde6b (Rd1 mutant) mice (Mus musculus) at postnatal age 11 (p11) and 13 (p13) and with no specific gender bias. The day of birth was considered as p1. All mice were chow-fed and provided with abundance of water, while being kept in a 12 h light/dark cycle. All animal protocols were performed with permission of local authorities (District Government of Upper Bavaria) and in accordance with the German laws on animal welfare (Tierschutzgesetz).Experiments were conducted on Eyes from Rd1 wt and mutant mice were transferred in a dish with Whatman filter paper and Hank’s Balanced Salt Solution  following enucleation. Subsequently, an excision was carefully performed along the limbus to reveal the lens and the attached vitreous, which were both collected in a tube and directly flash-frozen in liquid nitrogen. With the posterior eye cup exposed, the retina was then gently peeled off the underlying retinal pigmented epithelium and processed as the vitreous/lens. Samples were maintained at −80 °C until further processing.Approximately 1 mg of retinal tissue was isolated from both Rd1 wt and mutant mice. The tissues were homogenized manually using Micro-homogenizers, PP , then further subjected to sonication in a Bioruptor bath sonicator  at 4 °C (mainly to shear DNA and get rid of other interfering molecules) after lysing them using the iST Sample Preparation Kit  according to manufacturer’s instructions. The homogenous lysate was incubated with Trypsin/LysC mixture (supplied with the kit) at 37 °C for two and a half hours. The resulting suspension was centrifuged at 10,000 rpm for 5 min and the supernatant containing the peptides was desalted, purified, and redissolved in 10 µL “Load” solution after drying completely in a speed vac.m/z range from 375 to 1600 and performing MS/MS analysis of up to 10 precursor ions. Peptide ion masses were acquired at a resolution of 60,000 (at 200 m/z). High-energy collision-induced dissociation (HCD) MS/MS spectra were acquired at a resolution of 15,000 (at 200 m/z). All mass spectra were internally calibrated to lock masses from ambient siloxanes. Precursors were selected based on their intensity from all signals with a charge state from 2 + to 5 +, isolated in a 2 m/z window and fragmented using a normalized collision energy of 27%. To prevent repeated fragmentation of the same peptide ion, dynamic exclusion was set to 20 s.For reversed phase HPLC separation of peptides on a Ultimate 3000 nanoLC system , 5 μL of the solution were loaded onto the analytical column , washed for 5 min at 300 nL/min with 3% ACN containing 0.1% FA and subsequently separated applying a linear gradient from 3% ACN to 40% ACN over 50 min. Eluting peptides were ionized in a nanoESI source and on line detected on a QExactive HF mass spectrometer . The mass spectrometer was operated in a TOP10 method in positive ionization mode, detecting eluting peptide ions in the Protein identification was performed by MaxQuant 1.5.2.8 software package  . Parent g at 4 °C for 15 min. Extracts were dried under gentle nitrogen stream and reconstituted in 250 μL phosphate buffer (pH 7.4) in deuterated water (D2O) for NMR measurements using a 14.1 T NMR spectrometer. All samples included 0.02 mM of trimethylsilyl-2,3-d4-propionate sodium salt (TSP-d4) as internal standard for quantification.For each group and time-point a total of four to seven samples were used, depending on availability and quality of sampling, and processed as previously described . BrieflyThe collected spectra were processed  and the actual concentration of the internal standard in each sample was calculated using a calibrated electronically synthetized digital signal . Quantification of absolute concentrations of metabolites was carried out in Chenomx and concentrations were corrected for tissue weight. Three spectra (2 from retina and 1 from vitreous/lens) were excluded from analysis due to insufficient quality.Mus musculus KEGG database, after being enriched via global Ancova and analyzed in topology according to relative-betweenness centrality. An example of metabolic network selected from the pathway analysis was further plotted in Cytoscape 3.8.2 (Cytoscape Consortium) [The data were analyzed with MetaboAnalyst  4.0 usinsortium)  and procsortium)  plugin. Following eye enucleation, the eyes were punctured with a needle and incubated in 4% paraformaldehyde (PFA) on ice. After 5 min, the anterior part was excised and the vitreous/lens removed. The posterior eye cup was then allowed to fix in 4% PFA on ice for 45 min and later transferred in a 30% sucrose solution at 4 °C. The next day the eye cup was finally embedded in NEG-50  and frozen at −80 °C. Coronal cryosections (20 µm) were collected serially at the cryostat and incubated at room temperature for 60 min with a blocking solution consisting of 2% BSA, 0.3% Triton X-100 and PBS. The following primary antibodies were diluted in the same solution and incubated overnight at 4 °C: rabbit anti-iba1 , rat anti-Cd11b , mouse anti-GFAP , sheep anti-cGMP . Following several washes in PBS, the sections were incubated for 2 h at room temperature with the following secondary antibodies, all diluted 1:500 in blocking solution: anti-rat Alexa Fluor 488 , anti-rabbit Alexa Fluor 488 , anti-sheep Alexa Fluor 594 , anti-mouse Alexa Fluor 647 , and anti-rabbit Alexa Fluor 647 . The sections were then washed in PBS and counterstained with DAPI  prior to mounting .Images were acquired with an inverted Leica DMI 8 confocal microscope equipped with lasers emitting at wavelengths of 405, 488, 552, and 638, using an objective magnification of 40×. The original images, consisting of multiple z-stacks, were further processed with the open-source software Fiji  prior tot-test [t-test as it utilizes full data to shrink the observed sample variance towards a pooled estimate, resulting in far more stable and powerful inference compared to ordinary t-test. Missing values in the data were imputed from randomly drawn values from a distribution meant to simulate values below the detection limit of the MS instrument [In order to detect changes in protein levels between Rd1 wt and mutant mice, statistical analysis was performed on the acquired LC-MS/MS data. LIMMA moderated t-test  was chosstrument . An in-hstrument  was usedGene ontology analysis was performed using the Functional annotation tool from DAVID Bioinformatics Resources 6.8, NIAID/NIH  ,77.Mus musculus. Furthermore, the biological processes were ranked according to “Enrichment score” defined as follows: Enrichment score =  × Gene count. All the variables are as they are defined in DAVID. Genes involved in metabolic processes (as defined in Uniprot: metabolic process (0008152)) were identified in the entire enriched list of proteins and Log2 (Fold change) of those proteins  was used to assess the overall changes in tissue metabolism.The entire list of enriched proteins in either Rd1 wt or mutant was used as input against a background that contained the entire proteome from For Volcano plots of metabolites, all metabolome data were analyzed in Perseus  via a twp < 0.05 as threshold for significance.Statistical analysis of selected metabolites was performed with GraphPad Prism 9  via unpaired t test with Holm-Šídák correction for multiple comparisons, setting


To detect congenital hearing loss in mice, an objective measure is needed other than mouse behavioral observation. This study aimed to refine the methodology of auditory brainstem response measurement and identify potential congenital hearing loss models in laboratory mouse strains. Mice were anesthetized and fitted with head electrodes. Each mouse underwent four ABR measurements according to four testing conditions: A - no chamber; conventional tone; B - chamber; conventional tone; C - no chamber; short tone; D - chamber; short tone. Potential congenital hearing loss models were identified using 10 mice from each strain  through sound-attenuated ABR measurements with short-tone bursts. Potential congenital hearing loss models exhibited hearing thresholds ≥30 dB in both ears. Data were analyzed for normal distribution and variance homogeneity using the D’Agostino-Pearson/Kolmogorov-Smirnov and F value tests, respectively. One-way analysis of variance (ANOVA), with the Tukey-Kramer test, was used to conduct parametric analysis, and the Kruskal-Wallis/Friedman test, with a Dunn’s test for post hoc analysis, was used to perform non-parametric analysis. The simultaneous use of a sound-attenuating chamber and short-tone bursts provided clearly defined wave patterns, even at lower sound intensities. Inbred strains, especially C57BL/6 sub-strains, constitute suitable congenital hearing loss models. Our study shows that environmental factors should be addressed in animal studies of hearing function. Potential congenital hearing loss models may be found amongst commercially available inbred strains.  Three of every 1,000 newborns experience hearing loss; one of these three exhibits congenital deafness (3). Common consequences of congenital deafness include speech and communication disorders, delay in language understanding, educational disadvantage, and social segregation and stigmatization (4); thus, early detection is essential for improving the quality of life for affected children.Hearing loss is the most common sensory disorder, affecting approximately 70 million people worldwide (1). Genetic hearing loss can be classified as syndromic or non-syndromic, depending on whether it is related to other clinical issues. Many pediatric cases are non-syndromic (5); these can be categorized as autosomal recessive (75%-80%), autosomal dominant (15%-25%), or X-linked or mitochondrial  (6).Environmental causes, such as trauma, toxicity, or premature birth, contribute to 40%-50% of hearing loss cases; 50%-60% are caused by genetic factors (7). Mice and humans share remarkable similarities with respect to the auditory system; using mouse models allows histopathological and ultrastructural studies, which are not feasible in human patients (8). Mutant mouse models of hereditary hearing loss have helped to identify genes involved in the inner ear’s development or function; this has allowed better understanding of the genetic basis of hearing (9), as well as the genetic and physiological mechanisms underlying early- and late-onset human deafness (10).Mouse models are vital to understanding of hearing loss. Large numbers of mice can be screened easily for hearing phenotyping of mouse mutants; such screening led to the identification of several deafness genes, each of which contributed to understanding human hearing impairments (4); during presentation of sound stimulus, the brain elicits electric signals, known as auditory evoked potentials (AEPs), of which ABRs comprise the earlier portion (0-12 msec) (4). ABRs comprise several waves and peaks, typically identified by Roman numerals, of which waves I, III, and IV are the most clinically significant (11). ABRs can be measured non-invasively, via electroencephalography (EEG), and used to assess a subject’s hearing threshold. They are interpreted by measurements of peak and inter-peak latencies, calculated from pre-recorded wave patterns; visual scoring is the most common interpretation method and can be subjective (13).A standard test to evaluate hearing loss in humans and animals is the automated measurement of auditory brainstem responses (ABRs)―electrical potential signals emanating from the brain scalp upon presentation of a sound stimulus Hearing assessments in mice can be performed quickly by placing two electrodes on the head of the mouse to perform EEG; however, assessing whether detected hearing impairments are congenital requires repeated ABR measurements because deafness cannot be detected via contiguous behavioral observation of the mouse. The ABR measurement system consists of precision apparatus that need careful adjustments to detect peaks with extremely short latencies and avoid increased background noise during ABR measurement, and average 1,000 events to produce accurate waveforms. In this study, we focused on methodological aspects of hearing loss research; specifically, we investigated the effect of background noise on the accuracy of ABR measurement. Using the optimized conditions, we identified several laboratory mouse strain subgroups, including potential congenital hearing impairment models.All experimental protocols were approved by the Committee for Animal Experimentation of the School of Science and Engineering at Waseda University (permission #09A11) and were performed in accordance with the laws of the Japanese government. Adequate measures were taken to minimize pain or discomfort.To measure the effect of background noise on ABR measurement accuracy, 10 randomly selected ICR male mice  weighing 20-30 g, were used at the age of 8 weeks.ad libitum.The animals were singly housed in individually ventilated cages , in a room with controlled temperature (22℃ ± 2℃), humidity (60% ± 5%), and light (12/12 h light/dark cycle with lights on at 08:00). The light intensity at the surface of the cages was approximately 100 lux. All mice were on a standard rodent diet , and food and water were provided Next, to identify sources of mouse model candidates for congenital hearing loss, we selected 10 male mice from each of the following strains: C57BL/6JJmsSlc , C57BL/6JJcl , C57BL/6NCrl , Balb/c , CH3 , ICR , and ddY .Mice were tested at 8 weeks of age, and their body weights ranged between 20 and 30 g. Mice were randomly selected from each strain.All mice remained in the same environmental conditions as described.In admission, this experiment was approved by the Waseda University laboratory animal ethical review board.(4). ABRs were measured with a 16-kHz tone burst stimulus and recorded with an ABR recording system . This system is widely used MF1 Multi-Field Magnetic Speakers  were attached to the mice, and ABR was measured in closed fields. We also used the tube without attaching it before the MF1 Multi-Field Magnetic Speakers cone.To calibrate the speakers, the maximal sound pressure of the frequencies used was set to 90 db. The sound pressure measurement amplifier, NA-42, and the capacitor microphone, UC-54 , was used to measure real sound pressure. An attenuator was used to attenuate the sound pressure.Two types of tone burst were used for this experiment. The conventional tone burst , consisted of a 5-msec duration with a sound gate（Cos2Gate）of 1 msec. The tone burst duration was 3 msec, and the following sampling time was 5 msec. A newly programmed tone burst  consisted of a 1-msec duration with a sound gate (Cos2Gate) of 0.1 msec. Therefore, the tone burst duration was 0.6 msec, and the following sampling time was 9.9 msec. Both stimuli presentation timing was 21 times/sec, and the configuration number average was 500. Our program was developed using the SigGen RP device  .The hearing threshold was calculated by averaging the responses of 1,024 sweeps at each intensity level: 30 dB, 40 dB, 50 dB, 55 dB, 65 dB, 75 dB, and 90 dB (7 steps). The sound to be measured at each level was defined using the BioSig software (Tucker-Davis Technologies). All mice were subjected to measurements in the same way.All ABR measurements were performed inside a sound-attenuating chamber , which was covered by double 0.06-mm aluminum foil and sound-absorbing material. The chamber size was outer 420 mm × 425 mm × 420 mm and inner 240 mm × 300 mm × 240 mm (L × W × H). The chamber’s function as a Faraday cage was confirmed by the interruption of the mobile phone. On the other hand, sound reduction performance is D-40. The Architectural Institute of Japan’s "D value" corresponds to the Dr value of the Japanese Industrial Standard.To determine the effect of background noise on ABR measurement accuracy, we used four testing conditions:A. RF field chamber absent; conventional tone burstB. RF field chamber present; conventional tone burstC. RF field chamber absent; new tone burstD. RF field chamber present; new tone burstEach mouse was anesthetized and tested once under each test condition. Each trial lasted approximately 10 minutes.The sound stimulus was presented through a small speaker , which, according to the testing conditions, was placed either inside or outside the chamber. Because waveforms over 75 dB are almost identical, only a 90 dB sound level was used for this experiment.Next, to identify the strains of mouse model candidates for congenital hearing loss, all mice were tested under testing condition IV from RF field chamber and new tone burst.Each mouse was tested once for each of the following sound levels: 90 dB, 75 dB, 65 dB, 55 dB, 50 dB, 40 dB, and 30 dB. Each trial lasted approximately 10 minutes, and the sound was presented through a small speaker  placed inside the chamber.Following analysis of the hearing threshold of each strain, strains that exhibited a hearing threshold ≤ 30 dB in both ears, with clearly defined wave patterns, were classified as “competent mice,” because congenital hearing loss could be excluded in such mice.Each wave pattern was analyzed by measuring the length between top and bottom of the wave peak. Lengths of the maximum and minimum latencies, 1 msec before the first wave occurred, were also measured; these were defined as “Until wave I.” Thereafter, any wave pattern occurring when the amplitude was greater than “Until wave I” was recognized as an effective wave pattern .All data are represented as mean ± SEM (standard error of the mean). GraphPad Prism version 6.03  was used for statistical analysis. Data were analyzed for normal distribution and homogeneity of variance using the D’Agostino-Pearson/Kolmogorov-Smirnov and F value tests, respectively. One-way analysis of variance (ANOVA), with the Tukey-Kramer test, was used to conduct parametric analysis, and the Kruskal-Wallis/Friedman test, with Dunn’s test for post hoc analysis, was used to perform non-parametric analysis.As the sound volume decreased, all wave patterns became attenuated. For a 30-dB sound, identification of all wave patterns was only possible under C and D conditions.Under testing condition A, no waves were identified; for condition B, there was a clear wave I, but subsequent waves were increasingly difficult to identify; under testing conditions C and D, wave I could be identified, but subsequent waves could only be identified if the RF field chamber was used.Use of the RF field chamber significantly attenuated the noise until the occurrence of wave I, but did not have a similar effect for subsequent waves . For wave I, a high wave pattern amplitude was achieved by using either the RF shield chamber or the short tone .For wave II, a higher amplitude was achieved by simultaneous use of the short tone and the RF shield chamber . For waves III and IV, a higher amplitude was achieved by simultaneous use of the shorter tone and RF shield chamber , but isolated use of either the chamber or the eshort tone did not provide a similar result .The highest accuracy of ABR measurement was achieved under condition D, indicating simultaneous use of both conditions is necessary for an optimal result.Identifying strains of mouse model candidates for congenital hearing loss shows the honey bee records present hearing thresholds for 10 mice  of each strain . Table 1Within C57BL/6SLC, C57BL/6JCRL, and C3H/CLEA strains, no competent mice were detected; all subjects demonstrated a hearing threshold >30 dB .Therefore, these subgroups of strains can be a source of model mice for congenital hearing loss. Within the C57BL/6JCLEA and BALB/cSLC strains, only one and two subjects, respectively, were classified as competent mice, indicating that these subgroups of strains may also serve as congenital deafness models. Conversely, most mice within the ICR/SLC (60%) and ddY/SLC (50%) strains were classified as competent mice, indicating that those subgroups of strains rarely contain models for congenital deafness.(16), hearing threshold is typically determined through ABR measurements. However, environmental and methodological factors are likely to affect those measurements’ quality. For example, anesthetizing mice has been shown to decrease ABR measurement accuracy (17).In mouse studies, which have been crucial to understanding the mechanisms of hearing and hearing loss (18). Therefore, when measuring ABR, it is reasonable to hypothesize that background noise, undetectable by humans, may influence ABR measurement accuracy. We investigated the influence of background noise on ABR measurement by comparing the accuracy of measurements performed with and without an RF field sound-attenuating chamber, which allowed us to isolate the animals from environmental electromagnetic waves.Mice hear ultrasonic frequencies, which are not detectable by the human ear (19). In our study, we programmed a new, shorter tone burst with a 1-msec duration and 0.1-msec sound gate, to investigate how this might affect ABR measurements’ accuracy.ABRs are typically measured via presentation of a tone burst with 5-msec duration and 1-msec sound gate. A previous study found that increasing the stimulus rate, through presentation of shorter tone bursts, could improve threshold assessment Our results show that using an RF field chamber increases measurement accuracy for wave I; however, this enhancement effect decreases for subsequent waves. Notably, the simultaneous use of the RF chamber and the new, shorter tone burst, allowed clear separation of waves I-V, even for lower sound intensities.(17) that measuring ABR in anesthetized mice reduced the accuracies of peak and interpeak latencies as well as thresholds; they recommended using awake mice. However, using awake mice involves significantly longer recording times due to myogenic noise. Improving the measurement methodology by reducing background noise and presenting shorter tone bursts may avoid the detrimental effects of anesthesia, making accuracy comparable to that obtained in awake mice.Van Looij et al. reported Typically, clear wave patterns can be obtained with an 80-dB sound, whereas those obtained at 10-20 dB are largely indistinct. When the resulting ABR wave patterns are unclear, the measurements must be repeated several times, which is time-consuming, burdensome to the subject, and yields a lower amplitude of detected wave patterns. Simultaneous use of a sound-attenuating chamber and a shorter tone avoids these issues.ABR waveform does not oscillate significantly with the different anesthetic regimens used.For example, since our basic experiments have shown that inhalation anesthesia, such as isoflurane, causes less ABR and delays in waveform than celiac anesthesia, such as pentobarbital, we use anesthesia that has less effect on waveform and the mouse’s body.Further studies are needed to investigate how this methodology can be transposed into the clinical setting for applying ABR measurements of human patients.(8). While several suitable models are available to study age-related hearing loss (7), identifying good models for congenital hearing loss requires different testing procedures since the onset of hearing loss is early in a mouse’s life.More than 400 hearing-impaired mutant mouse models have been identified amongst laboratory mice populations (20). Conversely, in laboratory mice, it mostly occurred among C57BL/6 sub-strains, whereas 20% of the BALB/C mice revealed a normal hearing threshold; the outbred strains were both albino. Notably, some strains  are less likely to exhibit congenital hearing loss and are more suitable for studying hearing function.We investigated the hearing threshold and ABR pattern of several commonly used mouse strains during early adulthood (eight weeks). Within each strain, mice with normal or close to normal hearing were classified as “competent mice,” while those not meeting this criterion were regarded as exhibiting potential congenital hearing loss. A greater proportion of “competent mice” was found within the closed colony outbred strains, indicating that potential congenital hearing loss models are more easily found among inbred strains. Interestingly, congenital deafness in many species is often associated with the loci of white pigmentation (22). Those previous studies concluded that inbred strains are good models of age-related hearing research and identified the genes contributing to this condition (23); however, our results suggest that some strains may be suitable congenital hearing loss models, although further studies are needed to consolidate this view and investigate underlying mechanisms.Although unrelated to coat pigmentation, our results regarding hearing in young adulthood are consistent with previous, ground-breaking research performed in older mice, which showed that several inbred strains exhibit hearing impairments from middle age, unlike F1 hybrids This study does not allow reference to the functional analysis of mouse genetic background and deafness-related genes. However, it is of great help in selecting mice for ABR measurements.Environmental factors, such as background noise, exert an influence on ABR measurements’ accuracy. Improving measurement methodology by addressing these kinds of factors will lead to enhanced use of animal models of hearing loss, with consequent understanding of human hearing pathologies.A several inbred, commercially available mouse strains are potential models for congenital hearing loss; further research is needed to clarify mechanisms involved in early-onset hearing loss.NoneThis work was supported by the Asama Institute (grant number AIGNO0001).The authors thank Mr. Shinji Kume from the Bio Research Center Co., LTD. for his valuable comments.


Resting metabolic rate (RMR) tends to decline with aging. The age-trajectory of decline in RMR is similar to changes that occur in muscle mass, muscle strength and fitness. However, while the decline in these phenotypes have been related to changes of mitochondrial function and oxidative capacity, whether lower RMR is associated with poorer mitochondrial oxidative capacity is unknown. In 619 participants of the Baltimore Longitudinal Study of Aging, we analyzed the cross-sectional association between RMR , assessed by indirect calorimetry, and skeletal muscle maximal oxidative phosphorylation capacity, assessed as post-exercise phosphocreatine recovery time constant (tau-PCr), by phosphorous magnetic resonance spectroscopy. Linear regression models were used to evaluate the relationship between tau-PCr and RMR, adjusting for potential confounders. We found that independent of age, sex, lean body mass, muscle density and fat mass, higher RMR was significantly associated with shorter tau-PCr, indicating greater mitochondrial oxidative capacity. In conclusion, higher RMR appears to be associated with a higher mitochondrial oxidative capacity in skeletal muscle. This association may reflect a relationship between better muscle quality and greater mitochondrial health.


During COVID-19 pandemic, protective measures such as social distancing and face masks posed a challenge in daily communication, in this context the elderly are one of the most at risk categories as widely exposed to hearing loss. This article focuses on how the COVID-19 pandemic affected verbal communication, especially on those people that even in normal conditions present an increased difficulty in speech perception. Special attention has been paid to hearing aids and cochlear implant users, these devices indeed can be affected by a speech intelligibility reduction and could be uncomfortable if used together with face masks. Possible alternatives and solutions will be proposed to reduce the negative impacts of face coverings on communication, to enhance speech intelligibility and to manage wearability of hearing rehabilitation devices. The current COVID-19 pandemic has forced worldwide mandatory adoption of social distancing measures and face masks to limit the diffusion of the SARS-CoV-2 infection via droplets. These actions are crucial to protect the most vulnerable part of the population currently facing the highest number of victims, the elderly people [Even though the implementation of these precautions should be considered essential, their impact on the communicative skills is dramatic. Moreover, two thirds of patients over 70 years old present hearing loss, making this population at high risk for communicative issues even in normal conditions . NoteworFacial masks are indeed a barrier that impair speech production and perception, make lip reading impossible and facial expressions not visible: consequently, any additional clue to hearing perception is in the end missing. In addition, face masks make it difficult to read emotions from facial expression  and commThe impact of the pandemic in terms of diagnostic delay for multiple pathologies and surgical treatment , logistiEven patients using hearing aids or cochlear implants face severe difficulties due to the above-mentioned barriers that might lead to limited use of the devices, which may negatively impact these patients’ health.Although some limits are unbreakable, others can be bypassed with modern technology.Facial masks behave like a low-pass filter, attenuating the medium–high frequencies of the speaker’s voice with a reduction from 3 to nearly 12 dB (depending on the mask type) . More imOld age is in itself a risk factor to social isolation  that mayHearing loss, strictly associated with aging, increases these risks and negatively impacts the quality of life of these subjects; auditory impairment is associated with social isolation and loneliness , cognitiNegative effects of hearing loss can be minimized by auditory rehabilitation with hearing aids or cochlear implants , 14. In The benefit has proved effective and should be recommended even in mild hearing loss . ConcernCOVID-19 pandemic has negatively affected older adults, especially for those with hearing impairment reducing their communicative skills, favoring social isolation and compromising their mental health . For theFace masks are considered to be essential barriers to reduce the diffusion of droplets while breathing or speaking. The usefulness is non-negligible and it is a cost-effective solution. Nonetheless, in some conditions, the necessity to communicate might force both the clinician and the patient to give up on their use, but alternatives must be considered for safety reasons.The simplest way to effectively communicate with a hearing-impaired patient using face masks is to write the sentences down or on a computer screen, allowing the patients to read them and reply. Despite the safety of this method, time spent for each patient greatly increases, and it cannot be used for speech rehabilitation and therapy, because no auditory information is provided.However, in this context, the clinician should remind that other options are available to protect him/herself and the patients. Face masks with clear windows are available in the market and provide the same protection of the classic face masks enabling lip reading.Although their value as barriers is limited, even transparent shields can be considered when both lip reading and observation of facial expression are required, such as in speech therapy. A double barrier can be achieved in this setting if the speech therapist uses both a transparent wearable shield and another, wider transparent screen on the desk. In these conditions, providing an appropriate and continuous change of air can reduce the high density of droplets in the air.In most situations, it is not possible to avoid the use of face masks; consequently, it may be useful to improve the way we interact especially with the elderly people to avoid some of the limitations imposed by face coverings. Specifically, it is important to use a more sustained tone and a lower pitch of voice, because face masks damage high-frequency transmission in speech . In geneAnother coping strategy is to simplify as much as possible the message to reduce the required cognitive effort. In addition, slowing down the rhythm of the eloquium without “chanting” syllables (i.e. repeating the words dividing them in syllables) and repeating the information several times (while keeping the structure of the sentence unchanged) should be considered useful strategies especially in elderly people.Unfortunately, face coverings impair communication far beyond the acoustic of speech transmission. As highlighted by a recent survey, wearing face masks impacts content of communication, feeling of interpersonal connection and willingness to engage in conversation . All theFor this reason, it is important to have a more accurate communication awareness when addressing old people, especially those with hearing loss.In those cases, the use of gestures and written language provides visual information that can be a helpful source of guidance to enhance auditory comprehension. Also, the use of eyebrows or upper cheeks mimic movements can be helpful in enhancing the emotional messages connected to the speech . Only a The concomitant use of hearing aids and/or cochlear implant and personal protective equipment can be challenging and uncomfortable especially in the elderly people. Nonetheless, their use should be stimulated, even in the current conditions, since it is easier than it seems.Modern retro-auricular hearing aids have now reached excellent levels of miniaturization and they can be used without problem when glasses and face masks are also present. In-the-ear hearing aids can be even better in these conditions. For cochlear implants users, single-unit processors are available,they are easier to manage, and there is no conflict with glasses or face masks; even retro-auricular sound-processor can be used, for example, it can be clipped directly on clothes, or using silicone laces, customized coupling, or anchoring systems to the auricle. These strategies can prove particularly useful in patients who need an oxygen mask or helmet. For these patients, it may also be advisable to use the appropriate moisture protections to guarantee the correct functioning of the processor. In addition, the sound-processors can be set to use signal LEDs to show if the device is working correctly, making it easy to check if the patient can hear.One of the greatest challenges for hearing aids and/or cochlear implants users is to discriminate language in noise. The goal is not easy, especially for the elderly people. In addition, FFP3 masks combined with a face shield in noisy environments can cause a speech intelligibility reduction of up to 70% .In general, fitting parameters for both hearing aids and cochlear implants should be set to improve speech intelligibility enhancing the high frequencies (those reduced by face masks). In addition, higher levels of volume and specific microphone directionality can be set to compensate for sound attenuation.There are also several instruments to reduce the problem of background noises that impairs speech intelligibility such as magnetic induction and wireless systems. As a rule, these systems are based on a transmitter that can be a wearable microphone or a connectable device that transmits the hearing input directly to the hearing aid or the cochlear implant, thus limiting the background noise coming from the surrounding environment. The advantage of these systems are that they limit the negative influence of distance, environmental noise and reverberation with the chance of a direct communication with healthcare staff or relatives that can be even distanced by a glass or a window.The magnetic induction system is a quite “old” but efficient technology used with telephone coil (T-coil). If the hospital is equipped with this technology, the patient can receive the signal from a microphone or a screen directly to the hearing aid or the cochlear implant, without the addition of any device.Most recently, more and more hearing aids and cochlear implants are available with an integrated wireless receiver that can be directly connected to a wireless microphone (transmitter). This technology enables a connection to telephones, smartphones, multimedia players, stereos, TVs, PCs and tablets without the need of additional devices. The connection is efficient not only for phone calls and FaceTime (R), but also to take advantage of all the Voice Over features for automatic reading of what is displayed on the screen. This may be useful easily to communicate both with the family and with the healthcare staff, even by phone or to support short-distance communication.In addition, hearing aids and cochlear implant companies have also developed apps for mobile phones, supported by Bluetooth(R) 4.0 or later. These apps permit changing programs or volume, controlling battery life, getting help finding your sound processor if it gets lost and starting wireless streaming all without having to deal with a separate controller but only using the phone.Magnetic induction and wireless connections are efficient even if obstacles are present  or when the receiver (hearing aid/cochlear implant) is meters away from the transmitter , thus allowing to avoid close contacts.All the abovementioned solutions are easy to manage even for inexperienced people and can be extremely helpful to communicate in complex environments and to avoid patient isolation from family members and healthcare providers.Face masks and social distancing are mandatory in the current pandemic time. Limited hearing input can be extremely harmful with possible psychological and cognitive consequences that should be prevented.Although enhancing hearing performance is not without economic and care costs, the impact on the elderly people is non negligible, and we should all realize that most issues can be faced with communication-awareness and limited personal effort.The healthcare providers should not give up on the use of hearing aids and cochlear implants since the psychological support is necessary for the patients and also informed consent is based on the clear communications and understanding of the messages.Moreover, a correct use of the available technology (i.e. wireless systems) can be useful to promote social distancing, without giving up on an efficient oral communication with high-quality hearing input.


CDKL5-related disorders (CDD) is an epileptic encephalopathy resulted of gene mutations of CDKL5. This study aimed to explore the development process of CDD and to expand its mutation spectrum.Clinic datawas collected about three infantile epileptic encephalopathy cases diagnosed at Xinhua Hospital Affiliated to Shanghai Jiaotong University, School of Medicine. Next generation sequencing technology was used to find three de novo mutations of CDKL5. We searched published literatures about CDKL5 in pubmed and made an analysis about our clinic data and the related literatures.The three patients were all girls. Their average onset age of seizures was around 2 months, and all of them have intractable epileptic seizures, severe intellectual disability, and hypotension. Among them, two presented infantile spasm and high arrhythmia in EEG, and the other manifested clonic seizure and broad epileptiform discharge in EEG. Extracerebral space widening in cranial MRIs was demonstrated in two cases. Visual evoked potential was abnormal in two cases. Seizures were resistant to all kinds of antiepileptic drugs (AEDs). Gene tests showed three de novo mutations of CDKL5: one was a truncated mutation , which was pathogenic according to the ACMG guide, the other two were missense mutations  and a frameshift mutation (c.362-362insG(p.Ala122GlyfsTer7), which were likely pathogenic according to the ACMG.All three de novo mutations are first reported. Based on the combined related literature and the manifestations observed, we diagnosed the three children as CDKL5-related disorders, and concluded that the de novo CDKL5 mutations are the reason for their epilepsy. Cyclin-dependent kinase-like 5  belongs to the serine/threonine kinase protein family. It is composed of a conserved N-terminal catalytic domain and a long variable C-terminal extension, which may have a regulatory role . It is bPatients were chosen from children who were diagnosed with epileptic encephalopathy and received therapy at our hospital. All of them signed the informed consent. Clinic data containing their medical history, blood tests, cranial MRI, electrophysiological changes, therapy procedures and prognosis were collected. Next generation sequencing technology was used to determine the CDKL5 mutations after obtaining their parents’ permission. By collecting studies related to CDKL5 in PubMed, we performed a retrospective analysis of their data.The patients were all girls. Except Patient 1 whose mother had a history of unhealthy pregnancies with three drug-induced abortions before her and diabetes mellitus during early pregnancy, the other two were the first pregnancy of their mothers. All the patients were delivered full-term, without any history of suffocation rescue. However, patient 2 showed a smaller gestational age with a birth weight of 2450 g. There was no record of defects in their family history. The average age of onset in the three girls was 2 months.Focal secondary generalized tonic-clonic seizures were first presented in patient 1 at 2 months, and were verified by video-EEG. Later, the seizures transformed to infantile spasm at 18 months, as shows in Fig. Patient 2 first displayed general clonic seizures at 2 months and developed infantile spasms 1 month later. EEG presented hypsarrhythmia in the interictal phase and a series of spike-slow wave discharges when the seizures occurred leading to a diagnosis of infantile spasms, as shows in Fig. Patient 3 presented with generalized clonic seizures several times a day when she was 80 days old and synchronized electroencephalogram displayed broad epileptiform discharges, as shows in Fig. During hospitalization, a series of tests were completed, including blood routine, biochemical, etiological examination, inflammatory factors, cerebrospinal fluid examination, cranial MRI, electroencephalogram (EEG), auditory/visual evoked potential (AEP/VEP), and so on. Cerebrospinal fluid (CSF) and blood tests were normal in all three patients. Except for CMV infection in patient 1, We did not find any infectious factors. Wider anterior sulcus of bilateral frontotemporal lobes were found in patient 2 and 3, but this was not specific. The cranial MRIs are enclosed in Fig. CDKL5 protein is widely expressed in the brain and plays important roles in cell proliferation, neuronal migration, axonal outgrowth, dendritic morphogenesis, and synapse development , 8. TypiIntellectual disability occurs in almost every child with CDD –4, 9–11,The CDKL5 gene is located in the short arm of the X-chromosome at position 22 (Xp22), and contains 20 coding exons; its importance was emphasized first when Montini et al. first discovered it in patients in 1998 . Its firThe CDKL5 inheritance pattern is XD, and almost all patients reported are girls. The reason may be that boys carrying the mutation are miscarried or die before diagnosis. Recently there have been several papers reporting the CDKL5 mutation in both genders –22. LianCDKL5 protein is an important protein expressed in the brain and its mutation results CDKL5-related disorders (CDD) representing typical characters including infantile-onset refractory epilepsy, hypotonia, developmental delay, intellectual disability, and visual impairment. Its inheritance type is XD and most patients are girls. In our three patients, the clinical manifestations coincided with CDDs and their de novo mutations were the reasons for their epilepsy. Next we will collect more data to determine the gender differences.


Salivary gland choristoma is an extremely rare middle ear mass and is hypothesized to be caused by second branchial arch developmental anomalies. We present a 14-year-old girl with Dandy–Walker syndrome and conductive hearing loss. Middle ear exploration revealed a large middle ear mass with absent incus and stapes and displaced facial nerve. The mass was completely excised with histological confirmation of salivary gland choristoma. Her hearing was improved with bone-anchored hearing aids (BAHA). As facial nerve involvement is common, physicians should consider partial excision to avoid facial nerve palsy. Hearing restoration can be achieved with OCR or BAHA. A choristoma is a congenital, benign overgrowth of mature tissue found in an abnormal location of the body. On extremely rare occasions, benign salivary gland tissue can be present in the middle ear cavity. Presenting symptoms include conductive hearing loss, ear fullness, tinnitus, otalgia, and otorrhea , 2. ThesWe present a unique clinical case from our institution that is in line with the Surgical Case Report (SCARE) criteria along with a thorough literature review of all salivary gland choristomas, focusing on congenital and middle ear malformations, facial nerve involvement, treatments, and outcomes . StatistA 14-year-old female with a history of Dandy–Walker syndrome, hydrocephalus with ventriculoperitoneal shunt placement, and recurrent otitis media presented to our academic center with left hearing loss and aural fullness. Tympanostomy tubes were placed in each ear 5 years prior. Her mother states the hearing loss was present since birth and denies any otorrhea, otalgia, aural bleeding, dizziness, vertigo, or familial hearing loss. Audiogram showed normal hearing on the right and a moderate-severe rising to moderate mixed hearing loss with 25–50 dB air-bone gap on the left. Word recognition scores were excellent bilaterally . TympanoOn examination, the left tympanic membrane had a shallow retraction pocket in the pars flaccida and a white retrotympanic mass suspicious for a possible secondary-acquired cholesteatoma. The decision was made to perform an exploratory tympanotomy and removal of the mass.Under continuous facial nerve monitoring, tympanomeatal flap was elevated by a senior otology surgeon who uncovered a white, smooth, and mildly pulsatile mass. Compression of the external jugular veins and increased vagal pressure did not expand the mass. Mild compression of the mass did not cause blanching. Electrical stimulation of the mass at the inferior pole of the tumor and marching superiorly showed no electromyography (EMG) response. Bone removal from the canal floor was required to gain better access to the tumor. The tumor was first mobilized from its infracochlear position followed by the superior pole using curved dissectors. Total tumor removal was possible without any facial nerve stimulation.After complete tumor removal, middle ear inspection revealed malformed long process of the incus without a stapes superstructure or oval window niche for reconstruction. An inferiorly displaced tympanic segment of the facial nerve was identified and was able to be electrically stimulated.Pathology showed a 1 cm polypoid mass of respiratory sinus mucosa lined with pseudostratified columnar ciliated epithelium and scattered goblet cells, consistent with a salivary gland choristoma of the middle ear. There was no dysplasia or malignancy identified. On 2-week postoperative visit, she had no facial nerve weakness, and her aural fullness had improved, much to her satisfaction. Although ossicular chain reconstruction was not possible intraoperatively due to nonvisualized oval niche, the patient showed promising hearing response to a bone-anchored hearing aid (BAHA).Salivary gland choristoma in the middle ear cavity is an extremely rare condition with fewer than 50 cases ever reported , 2. TherSalivary gland choristoma of the middle ear is hypothesized to form from malformation of the second branchial arch prior to the fourth month of gestation , 6. SaliAlthough the abnormal deposition of tissue is often isolated to the middle ear, a unique developmental syndrome may be present , 6. SeveCT imaging and otomicroscopic appearance are usually sufficient to presumptively diagnose middle ear pathology before making the first incision. However, due to the rarity of middle ear salivary choristoma, it is often misdiagnosed as cholesteatoma given its white, retrotympanic appearance  or otoscMalignant transformation of salivary choristoma is rare , and manp=0.0027, The major limiting factor for complete surgical excision is facial nerve involvement. Cases involving the facial nerve had a significantly lower complete excision rate than those in which the facial nerve was spared . In our present case, OCR was not attempted due to inaccessibility of the oval window niche. However, bone-anchored hearing aids (BAHA) can provide a viable alternative to hearing enhancement as our patient showed encouraging response on BAHA demo.Ossicular chain reconstruction has been attempted both during initial removal  and stagSalivary gland choristoma is an extremely rare diagnosis of a middle ear mass. Radiographic imaging and careful middle ear exploration are needed to rule out vascular and intracranial tumors. As facial nerve involvement is common, physicians may consider partial excision to avoid facial nerve palsy. Hearing restoration can be achieved with ossicular chain reconstruction or BAHA.


Limited studies have assessed the relation between telomere length and risk of hearing loss; moreover, they have reported equivocal associations. In the first case-control study, the subjects were chosen from the general population of Zhejiang province in order to assess the association between leucocyte telomere length and risk of hearing loss from 2016 to 2018. A total of 817 cases (55.93 ± 8.99 years) and 817 age-, sex- and residential city-matched controls (55.91 ± 9.03 years) were included for analysis. In the multivariable models, individuals in the top quartile of relative telomere length (RTL) had an odds ratio (OR) for hearing loss of 0.53  compared to those in the bottom quartile, and specifically, the OR was 0.45  in females. In females, the risk of hearing loss decreased by 46% as RTL doubling increased; the standard deviation of RTL was associated with a 29% decrease in hearing loss risk. Additional analysis showed significant difference between participants in the female mild hearing loss group and corresponding controls. These results suggest that telomere length is associated with hearing loss in the general population, particularly in females with mild hearing loss. Telomere length might be a potential predictive biomarker of hearing loss at early stage. Hearing loss, a disease included in the ear, nose, and throat medical specialty, has become the most common sensory disorder in humans . Data frTelomeres, repetitive DNA sequences (TTAGGG repeats) extending from a few bases to 20 kilobases in length ,8, are sHowever, limited evidence exists in domestic and foreign databases ,16,17 thA case-control study using a multistage stratified cluster random sampling method was conducted in the Zhejiang province from 2016 to 2018. Data from the following seven healthcare centers in Zhejiang was collected: one center in Quzhou (Jiangshan city), one in Jiaxing, one in Huzhou, one in Lishui, and three in Hangzhou . Thereafter, we randomly selected 1008 individuals with hearing loss, and randomly matched each control with each case by age (±1 year), sex, and residential city. Finally, 1008 controls were selected for this study; all subjects agreed to provide the relevant information and consented to its use in future research. Furthermore, given that prevalence of hearing loss increases with age, and that shortening of telomere length is believed to be coupled to the aging process, we selected a study population comprising only middle-age and older individuals (>40 years).The study was approved by the Institutional Review Board of Hangzhou Normal University . All subjects provided written informed consent and the entire study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments, and according to local government policies.n = 11; i.e., OD  260/OD280 < 1.60 or OD260/OD280 > 2.00) were excluded. After adjusting the DNA concentration of all samples to 10 ng/uL with 10 mmol/L Tris-ethylenediaminetetraacetic acid buffer solution (pH = 8.0), real-time quantitative-polymerase chain reaction (RT-qPCR) was performed to measure the relative telomere length (RTL), using the ratio of the telomere repeat copy number with the copy number of the b-globin (single copy gene) ), median (interquartile range), or proportions. Because of the case-control study design, the paired p = 0.004) , who were matched for age (±1 year), sex, and residential city. The average age was 55.93 (8.99) years among the cases and 55.91 (9.03) years among the controls. Except for participant sex and lead concentration, the other variables were significantly different between the case and control groups. Notably, compared with the control group, participants with hearing loss had shorter RTL  . P for trend = 0.002). Among the common chronic diseases, hypertension  and diabetes  were found to have a significant positive correlation with hearing loss. However, no significant association was found between presence of hyperlipidemia or hypercholesterolemia and hearing loss . Furthermore, as the focused variable—RTL—we modeled it as a continuous variable (per doubling or per SD increase in RTL). The per SD increase values for RTL was significantly associated with a 21% decrease risk of developing hearing loss , and the per doubling increase in RTL was significantly associated with a 34% decrease in the risk of hearing loss (P for trend = 0.011); RTL doubling was associated with a 46% decrease in the risk of hearing loss ; the per SD increase values for RTL was significantly associated with a 29% decrease in the risk of hearing loss .ing loss . Furthering loss . The resn = 543), moderate (n = 220), and (extremely) severe . As p = 0.004).In this study, among all the participants included (41–89 years old), the scatter plot showed that RTL negatively correlated with age , which aThe present province-wide, population-based case-control study identified the association between hearing loss and telomere length, and reports that longer RTL was associated with a lower risk of developing hearing loss. Furthermore, we report that the association remained significant even after adjusting for hypertension, hyperlipidemia, diabetes, hypercholesterolemia and cadmium in blood . MoreoveIn vivo, repeated DNA sequences (TTAGGG) of telomere are made by the telomerase enzyme during cell division. However, telomerase activity is repressed critically in most physiological, mature somatic cells, except for germline cells, stem cells, and lymphocytes ,26. The This study reports that the risk of hearing loss increases significantly in people with shorter telomeres. Subjects in the top quartile of RTL had significantly less risk of developing hearing loss as compared to those in the bottom quartile who had a relatively shorter telomere length. When modeled continuously with covariate adjustment, per doubling (or per SD 0.374]), RTL was associated with a 33% (or 18%) decrease in the risk of hearing loss. Oxidation damage, including cochlear DNA damage, caused by ROS plays a causal role in the development of hearing loss , and the4), RTL wn = 54). This aspect, therefore, needs additional studies for verification. In particular, for the study of hearing loss and telomere length, three cross-sectional studies have assessed the association between RTL and the risk of hearing loss; two studies reported an inverse association ,17, wherOur study had the following strengths and limitations. First, we adjusted the data to negate the effects of sex, age, health (common chronic diseases) and environmental exposure  as much as possible in the study design and analysis. Second, the RTL measured in peripheral blood cells may not accurately reflect the ongoing cellular aging in different tissues ,9, and tIn conclusion, to the best of our knowledge, this is the first case-control study to assess the association between hearing loss and telomere length at an epidemiological level. We found a strong and independent association between hearing loss and telomere length in the general population after adjustment for some potential confounders, particularly, mild hearing loss in females. Hence, telomere length in individuals who manifest the early symptoms of hearing loss may help prevent subsequent deterioration and hearing can be salvaged with appropriate management. Given the findings obtained in this study, further studies conducted among various ethnic cohorts are needed to verify the usefulness of telomere length as a biomarker to predict future risk of hearing loss, and to explore the possibility of a causal relationship between the two.