Molecular genetics has proven to be a powerful approach for understanding early-onset hearing loss. Recent work in late-onset hearing loss uses mouse genetics to identify molecular mechanisms that promote the maintenance of hearing. One such gene, Foxo3, is ontologically involved in preserving mitochondrial function. Significant evidence exists to support the idea that mitochondrial dysfunction is correlated with and can be causal for hearing loss. Foxo3 is also ontologically implicated in driving the circadian cycle, which has recently been shown to influence the molecular response to noise damage. In this review, the molecular framework connecting these cellular processes is discussed in relation to the cellular pathologies observed in human specimens of late-onset hearing loss. In bringing these observations together, the possibility arises that distinct molecular mechanisms work in multiple cell types to preserve hearing. This diversity offers great opportunities to understand and manipulate genetic processes for therapeutic gain. One in eight adults has hearing loss, and the likelihood of hearing loss increases as one's age advances . EnvironSensory cells of the mammalian cochlea continuously detect and transmit acoustic information throughout the life of the animal. Outer hair cells amplify acoustic vibrations of the tectorial membrane to promote inner hair cell activation. Inner hair cells detect these vibrations and transmit the information to spiral ganglion neurons via ribbon synapses. Sensory hair cell signaling is powered by differential levels of potassium in separate fluid compartments, called the endocochlear potential. The endocochlear potential is actively maintained by the cells of the stria vascularis, in combination with supporting cells and the cells of the spiral ligament. No regeneration has been reported for lost cells in the adult organ of Corti. Consequently, any cellular losses will persist and accumulate through the lifetime of the mammal.Animal models have elucidated the cellular pathological sequelae of specific inner ear insults . Noise eGiven that a limited number of cell types participate in hearing and the fact that ototoxic insults preferentially affect specific structures in animals, one could imagine designing a suite of therapeutics that each addresses individual kinds of damage. For example, one drug could be designed that protects synapses from glutamate toxicity. Another one could restore the endocochlear potential, and a third one could protect or regenerate outer hair cells. For this thought experiment, it is important to consider the cellular pathologies commonly found in human disease.Qualitative analysis of human postmortem samples reveals cellular pathology similar to animal models. Loss of outer hair cells, especially in the basal turn, is a common finding , along wSurprisingly, quantitative studies have found that human hearing metrics correlate poorly with anticipated cellular pathology. For example, while one might predict that reduced strial function should affect hearing at all frequencies, quantitative studies demonstrate that reduced strial volumes correlate strongly with high-frequency hearing loss and not with general hearing impairment , 27. SimTaken together, these findings draw attention to a basic scientific question: which genes promote the survival and function of the various cells in the cochlea? Do different alleles for these genes govern a propensity to hearing loss, which varies among people? Do different genetic pathways protect different structures? If so, why are combinations of cellular damage common in older individuals with hearing loss? An interlocking network of genes regulates cellular responses to oxidative stress, metabolism, and circadian rhythm. Some genes in this network are implicated in hearing loss. However, there has not been a systematic approach to matching genetic pathways to the structures that they may protect. Once specific protective gene networks are identified, network members could become targets for therapeutics for treating hearing loss.I propose the transcription factor Foxo3 as a candidate member for such a network that protects hearing under stress. Foxo3 is expressed in adult mouse cochlear sensory hair cells and spiral ganglion neurons . In othe Caenorhabditis elegans [Foxo3 is a transcription factor of the winged helix class. It was first identified as a longevity gene in the model system elegans  and is n elegans . Specifi elegans –43. Thes elegans , suggestFoxo3 nuclear localization and transcriptional activity are regulated by a variety of extrinsic signals . When ceFoxo3 expression in the adult mouse cochlea suggests that it acts in sensory cells. Foxo3 protein is detected in the nuclei of spiral ganglion neurons, hair cells, and pillar cells of young adult mice . In contFoxo3 knockout (KO) littermates have identical hearing thresholds. However, Foxo3-KO mice develop high-frequency hearing loss by four months of age, whereas their wild-type littermates do not [ Foxo3-KO. Outer hair cell function persists even while hearing thresholds rise, suggesting dysfunction in inner hair cells, auditory synapses, or spiral ganglion neurons. Myo7a localization in inner hair cells is affected, especially near the stereociliary bundles [Foxo3 appears to protect hearing in mice as they age. As young adults, wild-type and s do not . Surpris bundles . Synapti bundles . These d Foxo3-KO tell us about human hearing loss? First, the fact that Foxo3's location in the cell can change a few hours after nontoxic noise exposure is consistent with the idea that Foxo3 effectors maintain hearing. Second, in the older Foxo3-KO mice, neural components survive yet cease functioning; that is, high-frequency hearing loss develops during adulthood, but hair cells, neurons, and synapses persist. This is striking departure from central neurodegenerative diseases such as Parkinson's, where significant cellular losses precede functional losses. One might wonder if a similar defect impairs human hearing beyond the cellular pathologies discovered in human postmortem samples. Interestingly, around a quarter of postmortem samples from individuals with increased hearing thresholds display no significant cellular losses [What might the data from ther losses , 27. ThuMitochondria power eukaryotic life. The filamentous network of mitochondria is essential for the aerobic generation of ATP via electron transport across membranes, driven by successive redox reactions in the citric acid cycle. Incomplete reduction of the oxygen molecules contained within mitochondrial inner membrane can lead to the generation of superoxides. Cells have evolved multiple layers of enzymatic machinery to reduce superoxides and to detoxify peroxides, their derivative metabolic products. Important mechanisms include the use of glutathione as an electron donor, and mitochondrial enzymes like Sod2 and members of the peroxiredoxin family (reviewed in ). Under α [α coordinates new production of mitochondrial proteins from both the nucleus and mitochondrial DNA (mtDNA) [α, Foxo3 directly interacts with the promoters of Sod2, catalase, and peroxiredoxin to drive their expression [α interactions have been studied in the brains of Alzheimer's patients [α and Foxo3 compete as and collaborate: overexpression of Pgc-1α can suppress Foxo3's ability to promote autophagy [Some of Foxo3's canonical transcriptional targets are key players in cellular processes that mitigate stress damage to mitochondria . Foxo3 cα . Pgc-1α  (mtDNA) . In conjpression , 57. Foxpatients , 59, rodpatients , human apatients , 62, humpatients , 64, humpatients , rodent patients , bovine patients , 67, rodpatients , and celpatients . Importapatients . Foxo3 dpatients . Foxo3-rpatients –73, humapatients –77, humapatients , human cpatients , rodent patients , and rodpatients . It may utophagy . Thus, FMutations that affect mitochondrial function adversely impact hearing as part of a spectrum of neurodegenerative disorders . MutatioFailure of mtDNA integrity is implicated in hearing loss during aging. Mitochondria lack the DNA repair capabilities of the nucleus. As a consequence, mitochondrial DNA accumulates somatic mutations more rapidly than nuclear DNA . NotablyAnimal models are used to determine that acute mitochondrial degradation causes hearing loss. When the cochlear mitochondrial respiratory chain is inhibited with 3-nitropropionic acid applied to the round window, permanent hearing loss ensues . The preα [ Sirt3-KO mice [Genes that decrease mitochondrial oxidation are also implicated in preventing hearing loss from noise. Sirt3 is a mitochondrial deacetylase that regulates enzymatic activity –107. Sirα . Mice ovα . In animα , and treα . Activat-KO mice . Finally-KO mice –115. All-KO mice ).It is clear that decreasing mitochondrial oxidation is important in preventing hearing loss, as seen in genetic models, aging, acute drug studies, and noise damage. In other systems, Foxo3 governs three key elements for mitochondrial health: mitogenesis, autophagy, and the transcription of mitochondrial oxidative stress reduction proteins. Foxo3's expression in sensory cells, such as hair cells, and not in the lateral wall or stria vascularis, suggests that its protective role is restricted to mechanosensory cells rather than the cells that drive the endocochlear potential. Studies of its function in damage paradigms will be integral to understanding its role in this genetic network.The diurnal cycle governs the activity of all life. Every eukaryotic cell maintains an independent circadian cycle using two interlocking Clock mechanisms in a manner that is critical for metabolic regulation. In animals, the central nervous system synchronizes these cycles, like a pacemaker, in response to environmental cues . Clock function causes significant abnormalities in circadian behavior [The canonical cellular circadian cycle relies on transcriptional feedback loops to regulate protein expression. The mechanism requires positive effector proteins to drive transcription and negative effector proteins to inhibit the positive effectors. Protein levels for one positive effector, named Clock in mice, remain constant, whereas levels of another positive effector increase through the night. At the start of the day, the positive effectors together drive transcriptional expression of many genes, including the three negative regulators. It is estimated that 8–10% of transcribed genes receive regulatory input from this mechanism . The negbehavior . Drosophila, recently it was found that some metabolic functions maintain circadian cycles independently of transcription or protein synthesis [While the transcriptional mechanism of circadian cycle genes was first described decades ago inynthesis . NADH anynthesis . In mousynthesis . These oynthesis . Analysiynthesis . When Clynthesis . Clock expression, promoting circadian cyclical activity [ Clock promoter [These data show that the transcriptional Clock machinery interacts with the metabolic Clock mechanism, regulating cellular metabolism, energy use, and oxidative stress response. In mouse liver cells, Foxo3 drives higher levels ofactivity . Foxo3 dpromoter . These fAdministration of gentamicin to humans and animals can induce hearing loss , 123. A  Bdnf levels were investigated after noise exposure. After daytime noise exposure, cochlear Bdnf mRNA is upregulated over 30-fold; however, this effect is not observed after noise exposure at night [A recent report indicates that the circadian cycle also affects the cochlear response to noise, albeit in a different direction . When CBat night . In vivoat night . Howeverat night . These dIt is interesting to contrast noise damage to gentamicin administration with reference to the circadian cycle: for noise, exposure at night incurs greater damage, whereas, for gentamicin administration, exposure during the daytime does. These opposing papers suggest that the cochlear response to these two damage paradigms is intrinsically different and interacts with different gene pathways. It will be interesting to determine Foxo3's role in the cochlear response to hearing insults.The identification of genes involved in early-onset hearing loss has led to a wealth of knowledge about how the cochlea functions . Identif


All three prediction equations over-estimated RMR for both OSI and ORI when measured RMR was ≤5000 kJ·d−1. For measured RMR ≤7000 kJ·d−1 there was statistically significant evidence that the equations overestimate RMR to a greater extent for those classified as obesity susceptible with biases ranging between around 10% to nearly 30% depending on the equation. The use of prediction equations may overestimate RMR and energy requirements particularly in those who self-identify as being susceptible to obesity, which has implications for effective weight management.Predictive equations to estimate resting metabolic rate (RMR) are often used in dietary counseling and by online apps to set energy intake goals for weight loss. It is critical to know whether such equations are appropriate for those susceptible to obesity. We measured RMR by indirect calorimetry after an overnight fast in 26 obesity susceptible (OSI) and 30 obesity resistant (ORI) individuals, identified using a simple 6-item screening tool. Predicted RMR was calculated using the FAO/WHO/UNU , Oxford and Miflin-St Jeor equations. Absolute measured RMR did not differ significantly between OSI versus ORI (6339 vs. 5893 kJ·d Why some individuals remain lean with relative ease (ORI) while others continuously struggle with their body weight, despite living in a similar environment (OSI), is an intriguing question. However, little research has focused on comparing such groups, despite belief that this might yield novel information valuable for developing potential strategies to aid those who continually struggle with their weight. Resting metabolic rate (RMR) or basal metabolic rate (BMR) represents the largest component (60–75%) of total daily energy expenditure in sedentary humans . VariatiObservations from cross-sectional studies tend to show obese individuals have a higher RMR compared to healthy controls because of increased metabolically active mass ,4,5,6,7.Direct and indirect calorimeters are the standard tools for assessing RMR in research settings. However, due to the expense of calorimeters, the time needed to achieve an accurate measurement, and the need for trained personnel to conduct the tests, the measurement of RMR for individual patients/clients is most commonly calculated from prediction equations ,19,20. IWhile some previous research has found prediction equations underestimate RMR , there ip < 0.001). This difference disappeared when RMR was adjusted by fat mass and FFM (5.59 ± 0.97 vs. 5.61 ± 0.13 kJ·min−1) [Due to the way they are generated, predictive regression equations work best in groups of people . When thJ·min−1) . WhetherThe aim of this study was to compare absolute measurement of RMR in individuals specifically identified as obesity resistant or obesity susceptible. In particular, we aimed to compare the ability of three commonly used equations to predict RMR in these two distinct groups in order to identify whether two people of the same sex, age, weight, and height but each from one of these obesity resistant and susceptible groups might be subject to different biases compared to measured RMR.Sixty-two participants were recruited from the general public in Dunedin, New Zealand, via advertisements placed in local newspapers, at supermarkets and in flyers that contained questions designed to specifically target obesity susceptible (OSI) and obesity resistant individuals (ORI). Eligible participants were healthy males or females, aged between 20 and 45 years meeting our criteria as either an OSI  or as an ORI (remains lean with relative ease and can eat whatever they like) as previously defined ,33. Part−1). Of the 76 respondents assessed for eligibility, 11 were excluded from entering the study. Ten were unable to be clearly classified as ORI or OSI and 1 had a previous history of an eating disorder. Thus 13% were neither ORI nor OSI. Two participants were retrospectively excluded from analysis, one due to diagnosis of a genetic condition that would influence study outcomes, and one due to a diagnosis of type 2 diabetes. A further person did not have their RMR measured. Obesity susceptible individuals (OSI) found it difficult to lose but not gain weight, were likely to experience fluctuations in weight (as indicated by self-reported weight history) and had a BMI of 21.6–44.0 kg·m−2. In contrast, ORI had always been lean (as indicated by self-reported weight history), found it difficult to gain but not lose weight and had a BMI of 17.5–27.7 kg·m−2.Participants were excluded if they did not answer positively to any of the screening tool statements or if they answered positively to both an OSI and ORI statement, were smokers, pregnant, lactating, or menopausal, were currently taking stimulants or anti-depressants, had a previous history of an eating disorder, presence of a thyroid disorder or other medical condition/s that affect metabolic rate, or a thyroid stimulating hormone level outside the reference range . All participants provided written informed consent prior to participation.Body weight was measured in the fasting state in light clothing on calibrated electronic scales  that measured to the nearest 0.1 kg. Height was measured to the nearest millimetre using a stadiometer with the head positioned in the Frankfort plane. Waist circumference (WC) was measured at the level of the narrowest point between the bottom of the 10th rib and the border of the iliac crest . Body coResting metabolic rate (RMR) was measured using indirect calorimetry based on best practice methods . The ass2 (26%) and O2/CO2 (16% and 4%) mixtures before testing began each morning and again if more than two tests were carried out in one morning. Volume was calibrated before each test using a standard 3 L syringe. Breakfast was provided to participants following testing.Participants rested for 15 min in a semi-recumbent position and were instructed not to fall asleep. Participants then breathed through a face-mask for 15 min during which time expired gases were collected using a Sensormedics 2900 metabolic cart . Gas analysers were calibrated prior to each test using set O−1) and −1) of ≤ 10% for analysis. If these criteria were not reached (n = 22), values for the lowest CV were used in the analysis. Four female participants  and one male participant (OSI) had a respiratory exchange ratio (RER) value above 1, indicating pre-test protocol violations or measurement inaccuracy and so were excluded from the analysis. The abbreviated Weir equation [−1) and −1) as used previously [The first five min of data collection were discarded and the remaining 10 min used to determine a 4 min period having a coefficient of variation (CV) for equation  was usedeviously :RMR  equation , the OxfParticipants were instructed to wear an Actical accelerometer  on the right hip at waist level for at least seven consecutive days at all times except when sleeping, or engaging in activities potentially harmful to the device . Accelerometer data were scored and interpreted using the MeterPlus Version 4.3 software from Santech, Inc.  . QualityParticipants received detailed verbal and written instructions from a trained researcher on how to collect a weighed four-day diet record (4DDR). All food and beverage intakes were recorded at the time of consumption on non-consecutive days . The completed 4DDRs were analysed using “Kai-culator”  the dietary assessment software developed in the Department of Human Nutrition, University of Otago. The food composition database includes current and previous versions of FOODfiles (2010v2) from Plant and Food Research Ltd and selected recipes calculated from the 2008/09 New Zealand Adult Nutrition Survey . All dieA sensitivity analysis was undertaken to examine the impact of low energy reporting. Participants with an energy intake to RMR ratio (EI:RMR) of <1.06 were classified as low energy reporters (LER) using the Goldberg method as outlined by Gibson .t-tests where the model assumptions were satisfied for continuous variables  and Mann-Whitney U tests otherwise for continuous variables, and Chi-squared (where at least 80% of cells had expected cell counts of 5 or greater) or Fisher’s Exact (otherwise) tests for categorical variables. Ratios of each estimated RMR compared to RMR derived from indirect calorimetry were calculated and modelled for each estimation equation interacting with obesity susceptibility  using linear mixed models, with a random subject effect to accommodate the clustering within individuals who each provided all three ratios. These models also included estimated RMR as a covariate, which was allowed to interact with the six pairs described above . To incorporate potential non-linearities with the continuous derived RMR measure, the addition of a quadratic term was investigated and retained (based on improvements in Akaike Information Criterion AIC), as was the subsequent addition of a cubic term. All 56 participants were used for each model. Due to the sample size, interactions between quadratic and cubic terms and the estimation equation and susceptibility were not investigated. Log-transformations were used for the ratios (as would be anticipated) and this improved model residuals. Restricted maximum likelihood was used for all models. In order to determine whether any differences in these ratios could reflect biases in the equations by susceptibility categories, the equation parameters  were then added to the model as main effects only . All statistical analyses were performed using Stata 14.2  and two-sided p < 0.05 was considered statistically significant in all cases.Appropriate summary statistics were calculated for both the obesity resistant and susceptible groups. These variables were compared between the susceptibility groups using two-sample The characteristics of the study participants are shown in As expected, OSI were significantly heavier, had a higher body mass index (BMI), fat mass and %BF, and a larger WC compared to ORI. The percentage of males and females, age, height and FFM was similar between OSI and ORI. There were no significant differences in the amount of time participants spent participating in various levels of movement intensity or engaging in sedentary behaviours.p < 0.001). The results of the sensitivity analysis revealed that two participants were classified as low energy reporters (LER), defined as EI:RMR <1.06. One LER was an obesity susceptible female and one was an obesity resistant female. Removal of the two LER from the analysis of the dietary intake data did not affect the results and data for the full sample has been reported.No significant differences were observed for absolute energy intake or any of the energy-yielding nutrients expressed as a percentage of total energy intake. When energy intake was expressed relative to body weight, OSI had a significantly lower intake than ORI (p < 0.001). Comparing the number of attempts between the four ORI and 18 OSI participants reporting such attempts, with the number of attempts ranging between 1 and 13, did not find evidence of differences between these groups (Mann-Whitney U p = 0.223). Those in the ORI group were more likely to report having changed their eating habits to gain weight  with a maximum of two attempts reported. Those in the susceptible group had higher lightest (p = 0.020) and heaviest (p < 0.001) body weights and appreciably greater weight ranges  compared to ORI.Those in the OSI group were more likely to have changed their eating habits in an attempt to lose weight  rather than statistically significant for the WHO equation.−1 and below) with an under-estimation bias evident for measured RMRs 8000 kJ·d−1 and above. However, the differences between methods and between susceptibility groups by method are no longer evident. Adding physical activity as total hours per day of moderate or vigorous activity did not meaningfully alter these results.After further adjusting for sex, age, height, and weight , the bia−1 and 7000 kJ·d−1. The biases from the Miflin-St Jeor equation continued to differ by susceptibility, although these were again attenuated from the unadjusted analyses, displaying a pattern of greater overestimation for those susceptible where measured RMR was 3000 kJ·d−1 and below and under-estimation when derived RMR was 7000 kJ·d−1 and above.As shown in In the present study, indirect calorimetry was used to measure the RMR of individuals who struggle to maintain a healthy body weight (OSI) compared to individuals who maintain their body weight with relative ease (ORI). In contrast to findings from the majority of previous cross-sectional studies where absolute RMR was shown to be greater in obese versus non-obese controls ,4,5,6,7,Previous research found lower RMRs in lean versus obese participants . In our In the present study, OSI indicated the need to consume smaller amounts to manage their weight   based onBy definition, OSI  are likely to have gained or lost weight in the past and this may be one explanation for the differences in RMR between the two groups in the present study. Previous research suggests RMR is suppressed in conjunction with weight loss, often to a greater extent than would be expected based on changes in body weight/body composition ,13,14. A−1. In addition, across all three equations, there was evidence that when measured RMRs are ≤7000 kJ·d−1, the equations overestimate RMR to a greater extent for those classified as susceptible with biases ranging between around 10% to nearly 30%. We found that adjusting the model of predicted RMR for the components of the equations  greatly attenuated the biases between OSI and ORI, suggesting that further calibration of the equations might effectively remove the clinically significant biases between OSI and ORI, although we have no evidence that this would remove the overall biases of the equations compared to RMR derived from indirect calorimetry. Another possible interpretation is that our observed differences in RMR misestimation between self-reported obesity susceptible and resistant phenotypes can be largely explained by these variables alone without reference to additional mechanisms.Resting metabolic rate is an important issue to consider within the area of weight control, as it is this component that accounts for the greatest percentage of total daily energy expenditure. Clinical nutrition management frequently relies on predicting RMR from equations that use various combinations of anthropometric, age and gender measures. In the present study, important differences were observed between measured and predicted RMR from the selected prediction equations. All three equations overestimated RMR for both OSI and ORI when measured RMR was ≤5000 kJ·dThese findings have important implications for dietetic practice. The Oxford equation has recently been adopted by Dietitians New Zealand and is recommended for calculating RMR in the latest edition of the Clinical Handbook . Meanwhi−1 and 1531 kJ·d−1 respectively. According to the Pennington Biomedical Research Centre calculator [−1). The additional weight gain of the OSI would be 3.9 kg compared to the equivalent ORI. Similarly, for an RMR of 6000 kJ·d−1, the Miflin-St Jeor equation would overestimate by 4% (ORI) and 15% (OSI), equivalent to 262 kJ·d−1 and 896 kJ·d−1, respectively. For a 33 year old male who was 75 kg and 1.78 m, this would lead to weight gains of 1.5 (ORI) and 4.2 (OSI) kg over a year, an additional 2.7 kg gain for the OSI. Similar results apply to the other two equations.To provide some perspective on the magnitude of over-estimation of RMR for OSI and ORI, and subsequent energy imbalance using these popular equations, we present a variety of scenarios. For a person with an RMR of 5000 , the Miflin-St Jeor equation would overestimate RMR by 14% (on average) for an ORI and 31% (on average) for an OSI, equivalent to 717 kJ·dlculator  an energGiven that these equations are overestimating energy requirements, especially among OSI, future research using large samples should focus on developing equations specific to OSI and/or calibrating existing equations, possibly by adding additional components such as percentage body fat. If successful, such approaches would be invaluable for healthcare practitioners and researchers working in this area.An important strength of this study include the use methods considered to be the gold standard for each variable, including DXA to assess body composition, weighed food records to assess dietary intake and accelerometry to provide an objective measure of physical activity. However, some limitations need to be considered when interpreting the results of the current study. Firstly, individual-level matching by age, sex, physical activity or other lifestyle factors for OSI and ORI was not possible. Resting metabolic rate is affected by age and decreases 1–2% per decade after 20 years of age . HoweverIn conclusion, commonly used prediction equations overestimated the RMR of both OSI and ORI, but more so in OSI. This is of concern for OSI using self-monitoring dietary-intake apps for weight loss or following planned energy-restriction programmes prescribed during nutrition counseling, as the weight loss targets may be impossible to achieve using the energy requirements over-estimated by these prediction equations. It is difficult to overcome this issue for individuals using self-monitoring dietary-intake apps, but identification of OSI prior to implementation of planned energy restriction in a nutrition counseling setting could allow a potentially lower energy requirement to be factored in for these clients/patients. Future investigations may explore the development of RMR prediction equations that include calibration for obesity susceptibility. Such equations would be valuable in providing appropriate energy intake targets for weight loss for OSI.


Glossina spp.) are the primary vectors of trypanosomes, which can cause human and animal African trypanosomiasis in Sub-Saharan African countries. The objective of this study was to explore the genome of Glossina morsitans morsitans for evidence of horizontal gene transfer (HGT) from microorganisms. We employed an alignment-free clustering method, that is, batch learning self-organising map (BLSOM), in which sequence fragments are clustered based on the similarity of oligonucleotide frequencies independently of sequence homology. After an initial scan of HGT events using BLSOM, we identified 3.8% of the tsetse fly genome as HGT candidates. The predicted donors of these HGT candidates included known symbionts, such as Wolbachia, as well as bacteria that have not previously been associated with the tsetse fly. We detected HGT candidates from diverse bacteria such as Bacillus and Flavobacteria, suggesting a past association between these taxa. Functional annotation revealed that the HGT candidates encoded loci in various functional pathways, such as metabolic and antibiotic biosynthesis pathways. These findings provide a basis for understanding the coevolutionary history of the tsetse fly and its microbes and establish the effectiveness of BLSOM for the detection of HGT events.Tsetse flies ( Glossina spp.) are the primary vectors of trypanosome parasites; they cause human African trypanosomiasis (or sleeping sickness) and animal African trypanosomiasis (or nagana) in Sub-Saharan African countries. The flies harbour three maternally transmitted endosymbionts, Wigglesworthia glossinidia, Sodalis glossinidius, and Wolbachia pipientis, which influence host physiology. For example, Wigglesworthia provides essential nutrients, such as vitamins, to the host [ Sodalis in the tsetse fly is not clear, it appears to influence various host properties, such as longevity and susceptibility to trypanosome infections [ Wolbachia induces strong cytoplasmic incompatibility, which was also observed in the tsetse fly [ Wigglesworthia [Tsetse flies  in the genomes of laboratory and natural Glossina morsitans morsitans (Gmm) populations [ Wolbachia genome in the Gmm genome via horizontal gene transfer (HGT) events [ Wolbachia-specific sequences from whole-genome Sanger sequencing reads and pyrosequencing data based on nucleotide homology with the complete genome sequences of three Wolbachia strains  [ in situ hybridisation analyses further confirmed the presence of these insertions in Gmm on the two sex chromosomes (X and Y) and the supernumerary B-chromosome [In addition to the parasitism of bacterial organisms themselves, partial genome sequences ofulations . Subsequ) events , 13. Theromosome , 13.HGT elements can be detected by two main methods: phylogeny-based and composition-based methods . The fir Gmm using the alignment-free clustering method BLSOM. Using BLSOM, we detected a number of HGT candidates from diverse origins. In a comparison of the results for HGT from Wolbachia between methods, there was a high level of agreement between BLSOM and BLASTn, a homology-based approach. Based on functional annotation, these potential HGT elements encoded loci in various functional pathways.The objective of this study was to characterise HGT from microorganisms in the genome ofGmm) genome (Accession number CCAG010000000) and all prokaryotic sequences identified to the species level  were obtained from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/). When the number of undetermined nucleotides (Ns) exceeded 10% of the window size (5 kb), the sequence was omitted from the analysis. When the number of Ns was less than 10%, the oligonucleotide frequencies were normalised to the length without Ns and included in the analysis.The tsetse fly (wij) were arranged in the two-dimensional lattice denoted by i  and j . Weight vectors (wij) were set and updated as described previously [http://bioinfo.ie.niigata-u.ac.jp/?BLSOM).G+C% is a fundamental value for the phylogenetic classification of microbial genomes, including viral genomes, but it cannot differentiate a wide variety of genomes. Oligonucleotide composition can distinguish species, even those with the same G+C%, because it varies substantially among genomes; accordingly, it is referred to as a “genome signature” . Multivaeviously , 27. A BTo identify HGT candidates in the tsetse fly genome derived from prokaryotes, two types of large-scale BLSOM were used, that is, Tsetse+Prokaryotes- and Genus-BLSOM, using all genome sequences deposited in DDBJ/ENA/GenBank. A Tsetse+Prokaryotes-BLSOM was constructed with a degenerate tetranucleotide composition for all 5 kb sequences derived from tsetse fly genome sequences of longer than 5 kb plus 5,600 identified prokaryotes for which at least 10 kb of sequence was available from DDBJ/ENA/GenBank. The degenerate tetranucleotide composition was the composition of degenerate sets in which a pair of complementary tetranucleotides was added . To obtain more detailed phylotype information for the prokaryotic sequences, Genus-BLSOM was constructed for each phylum derived from 5,600 identified prokaryotes.1), which were mapped to Tsetse+Prokaryotes-BLSOM by identifying the lattice point with the minimum Euclidian distances in the multidimensional space (2). For every lattice point at which tsetse fly genomic segments were mapped to prokaryotic territories, the most abundant phylum was identified, and the mapped tsetse fly genomic segments were tentatively assumed to belong to the phylum. Finally, when the most abundant phylum in more than 40% of the segments derived from a single tsetse fly contig was the same, the tsetse fly contig was assigned to this phylum by BLSOM. To identify the phylogenetic origin of the tsetse fly genomic segments that were mapped to the prokaryotic territories on Tsetse+Prokaryotes-BLSOM, they were successively mapped on Genus-BLSOM (3). Similar stepwise mappings of tsetse fly genomic segments on BLSOMs constructed with sequences from more detailed phylogenetic categories  were conducted.For tsetse fly contigs of longer than 5 kb , a 5 kb window with a 1 kb step was used to obtain 303,250 segments , Step 1,al space , Step 2.us-BLSOM , Step 3. Wolbachia, a local BLASTn search was conducted with the contig sequences of the tsetse fly that exceeded 5 kb against the NCBI nonredundant nucleotide database. When more than 1 kb of the sequence showed similarity with Wolbachia with top-hits and an E-value threshold of 1 × 10−5, the contigs were considered HGT candidates derived from Wolbachia.To detect HGT candidates derived fromhttp://www.genome.jp/tools/kaas/) [http://www.genome.jp/kaas-bin/kaas_org).The sequence fragments with prokaryote origins were functionally annotated using KEGG (Kyoto Encyclopedia of Genes and Genomes) mapping  with thes/kaas/) . KEGG Orn = 758), followed by the phyla Bacteroidetes (n = 370), Alphaproteobacteria (n = 90), and Gammaproteobacteria (n = 23)  clustered with reads from prokaryotes and thus were HGT candidates according to Tsetse+Prokaryotes-BLSOM . These s(n = 23) . We did  Bacillus (n = 239). The second most highly represented origin was the class Flavobacteria (n = 187), which we were unable to classify to the genus level owing to coclustering with genome sequences that lacked genus information, followed by the genera Staphylococcus (n = 134), Enterococcus (n = 83), Wolbachia (n = 56), Polaribacter (n = 37), and Listeria (n = 31). The HGT candidates associated with the genera Wigglesworthia and Sodalis, which are common endosymbionts of the tsetse fly, were not detected using BLSOM. Most tsetse flies are heavily infected with Wigglesworthia; its abundance reaches over 99% in natural Gmm populations [ Wigglesworthia may support the high quality of tsetse fly genome sequences, since some genome data are contaminated by symbiont genomes, which can lead to the false-positive detection of HGT events [ Wigglesworthia and the tsetse fly was recent, as suggested by its genome features [We performed further characterisation of donor sequences to the genus level using Genus-BLSOM . The resulations . The laceria n = . The HGTfeatures . Nonethefeatures  and thei Bacillus suggests that there was a strong association between the tsetse fly and Bacillus in the past. Members of the genus Bacillus are ubiquitous in nature and have been isolated from diverse environments such as water, soil, plants, animals, and air [ Bacillus thuringiensis, have been well studied as agents of biological control of arthropods [ Bacillus species, including B. thuringiensis, to the adult Gmm and found that the mortality of Gmm was depending on the bacterial species [ Bacillus species may have infected Gmm persistently without adverse effect on the hosts and served as HGT donors. In fact, in a microbiota analysis of one tsetse fly species, Glossina fuscipes fuscipes, the bacteria belonging to the genus Bacillus were found dominant in a culture-dependent manner [The high frequency of HGT candidates from the genus and air . Some spthropods . Kaaya a species , indicatt manner .Blattabacterium strain Bge) in the omnivorous German cockroach (Blattella germanica) suggested that it plays roles in nutrient supply to the host, amino acid catabolism, and nitrogen excretion [Coleomegilla maculata), and coccinellid beetle (Adonia variegata) induce male-killing [ Wolbachia, Rickettsia, Arsenophonus, Spiroplasma, and Cardinium [Adalia bipunctata) infected with two symbionts, Rickettsia and Spiroplasma [ Wolbachia in the tsetse fly and HGT elements from Wolbachia in the tsetse fly genome [In contrast, there is no report on the relationship between the tsetse fly and Flavobacteria, which was identified as a second dominant donor of HGT candidates in this study . Flavobaxcretion . Flavoba-killing , 39, in ardinium . Hurst ey genome , 13 may  Wolbachia. For 38 contigs, we detected sequence homology with Wolbachia sequences based on the criteria described earlier. Of these 38 contigs, we identified 36 as HGT candidates from Wolbachia using BLSOM, while we assigned the remaining two contigs to the genus Rickettsia using BLSOM. Accordingly, we observed a high level of agreement (36/38 = 94.7%) between the two approaches, indicating that the analytical sensitivity of BLSOM is at least comparable to that of BLASTn. These results also suggest that the genetic elements derived from Wolbachia were recently introduced to the tsetse fly genome. Based on a comparison between the BLSOM and BLASTn results, we observed 20 contigs that were only identified as HGT candidates using BLSOM. There may be as-yet unidentified Wolbachia strains, explaining the failure to identify HGT candidates based on sequence homology. In fact, for short regions  of four contigs identified as HGT candidates from Wolbachia only using BLSOM, we detected sequence homology with a Wolbachia endosymbiont of Gmm (Accession number AWUH01000121) with an E-value of <1 × 10−5.We performed a BLASTn analysis to detect HGT candidates derived from Wolbachia that were presumably obtained by HGT have been detected in the genomes of multiple arthropod species, including the adzuki bean beetle  [Drosophila ananassae) [Nasonia spp.) [Acyrthosiphon pisum) [Aedes aegypti and Aedes mascarensis) [Monochamus alternatus) [ Wolbachia-derived HGTs that exhibit low sequence similarity with known Wolbachia strains, it provides an alternative method with which to explore the mechanisms of HGT. The application of BLSOM to an increasing number of eukaryotic genomes will reveal the diversity and frequency of Wolbachia-derived HGTs in other arthropods, including vectors of medical and veterinary importance.Genetic elements related toinensis) , 46, a fanassae) , parasitia spp.) , the pean pisum) , two mosarensis) , 50, andernatus) . Some HGernatus) , but furn = 11,524) to the KEGG pathway. The KO included 317 biological pathways. The predicted pathways were mainly related to “Metabolic pathways” (193 molecules), “Biosynthesis of secondary metabolites” (75 molecules), and “Biosynthesis of antibiotics” (50 molecules). These results suggested that the HGT candidates have the potential to affect a large number of metabolic activities; however, further analyses are essential to demonstrate the active transcription of HGT-acquired genes using transcriptomics or gene-specific reverse transcription-PCR; such analyses can provide initial evidence for the functional importance of HGT-acquired genes [ Wolbachia-derived gene in the Aedes albopictus C6/36 cell line [We mapped all of the HGT candidates identified using Tsetse+Prokaryotes-BLSOM n = 11,4 to the ed genes , 52, 53.ell line . Moreoveell line . Analyse Wolbachia, a well-known symbiont of the tsetse fly. In addition, using BLSOM, we identified HGT candidates from bacteria that have not previously been associated with the tsetse fly. We observed the HGT candidates from diverse bacteria such as Bacillus and Flavobacteria, suggesting a strong past association between these taxa. In a comparison between BLASTn and BLSOM results for the detection of HGT candidates from Wolbachia, the analytical sensitivity of BLSOM was at least comparable to that of the sequence homology-based approach. Furthermore, BLSOM can be used to detect HGT elements from organisms with low similarity with currently available sequences. These data obtained using BLSOM provide a basis for understanding the coevolutionary history of the tsetse fly and its microbes.We investigated the use of BLSOM to detect HGT candidates in the tsetse fly genome. Using BLSOM, we successfully detected a number of HGT candidates from diverse bacterial origins. The HGT candidates represented 3.8% of the tsetse fly genome. The predicted donors of these HGT elements included


Dear Editor,An 83-year-old female patient complaining of constipation was referred to our institutionfor elective enema with barium contrast, which showed diffuse irregularity in themucosal folds of the colonic loops and signs of extravasation of the contrast mediuminto the abdomen and pelvic cavity .After t,2. The sites most commonly affected are the sigmoid colon and therectum.Colorectal perforation is a serious complication of a barium enema. Although its exactoccurrence is difficult to establish, some studies indicate a mean incidence of0.02–0.23% among the exams performed, with a mortality rate of up to 50%. In such high-riskcases, the use of water-soluble contrast should be considered.Etiologically, colorectal perforations cause by enema administration can be divided intothose that are iatrogenic and those that are secondary to weakness of the colorectalwall. Iatrogenic perforations can occur as a result of forced introduction of thecatheter into the anterior rectum wall, balloon hyperinflation, or excessive hydrostaticpressure during contrast injection. Perforations secondary to colorectal wall weaknessoccur in patients with a history of inflammatory bowel disease, acute diverticulitis, orobstructive colorectal processes, as well as in those who have recently undergone asurgical procedure, are of advanced age, or are on corticosteroid therapy, any of whichmake these patients more susceptible to perforation during the administration of theenema,4.The symptoms of colorectal perforation are variable, depending on the location and sizeof the lesion, and can initially manifest as abdominal pain progressing to peritonitis,sepsis, and shock. However, in fewer than 10% of cases, patients are asymptomatic in thefirst days after the examination, and the radiologist can be the first to suggestperforation, as was the case in the patient described here.In cases of colorectal perforation in which the patient is stable, the puncture is small,and there is no fecal matter in the gastrointestinal tract or retroperitoneum,conservative treatment is adopted. Otherwise, exploratory laparotomy isnecessaryAlthough barium enema is a routine examination, it should be performed with caution. Incases of perforation resulting from the examination, treatment should be initiated earlyand should be tailored to the type of injury, as well as to the clinical status of thepatient, thus reducing the morbidity and mortality associated with the condition.


Fusarium verticillioides  is an important plant pathogen that causes seedling blight, stalk rot, and ear rot in maize (Zea mays). During infection, F. verticillioides produce fumonsins B1 (FB1) that pose a serious threat to human and animal health. Recent studies showed that Set1, a methyltransferase of H3K4, was responsible for toxin biosynthesis in filamentous fungi. However, to date, the regulation of FvSet1 on FB1 biosynthesis remains unclear. In the current study, we identified only one Set1 ortholog in F. verticillioides (FvSet1) and found that the deletion of FvSET1 led to various defects in fungal growth and pathogenicity. More interestingly, the FvSET1 deletion mutant (ΔFvSet1) showed a significant defect in FB1 biosynthesis and lower expression levels of FUM genes. FvSet1 was also found to play an important role in the responses of F. verticillioides to multiple environmental stresses via regulating the phosphorylation of FvMgv1 and FvHog1. Taken together, these results indicate that FvSet1 plays essential roles in the regulation of FB1 biosynthesis, fungal growth and virulence, as well as various stress responses in F. verticillioides. Fusarium verticillioides  is an important plant pathogen that causes seedling blight, stalk rot, and ear rot in maize (Zea mays), which is one of the most common diseases of affecting maize worldwide  and 10-μL of protease inhibitor cocktail . After homogenization with a vortex shaker, the lysate was centrifuged at 14,000×  at 4 °C . The res at 4 °C . GAPDH (F. verticillioides. The results of the current study demonstrate that FvSet1 plays an important role in FB1 biosynthesis, fungal growth and virulence, and environmental stress responses in F. verticillioides. These results present a relationship between FvSet1 and FB1 biosynthesis in F. verticillioides. However, investigations are still required for elucidating the mechanisms by which H3K4me regulates secondary metabolism.In summary, we genetically and biochemically characterized the functions of FvSet1 in


KRAS, BRAF, and PIK3CA mutations. A statistically significant interaction was found between LINE-1 methylation level and tumor location in colorectal cancer-specific mortality analysis (Pinteraction = 0.011). The association of LINE-1 hypomethylation with higher colorectal cancer-specific mortality was stronger in proximal colon cancers  than in distal colon cancers  or rectal cancers . Our data suggest the interactive effect of LINE-1 methylation level and colorectal cancer location on clinical outcome.Colorectal tumors arise with genomic and epigenomic alterations through interactions between neoplastic cells, immune cells, and microbiota that vary along the proximal to distal axis of colorectum. Long interspersed nucleotide element-1 (LINE-1) hypomethylation in colorectal cancer has been associated with worse clinical outcome. Utilizing 1,317 colon and rectal carcinoma cases in two U.S.-nationwide prospective cohort studies, we examined patient survival according to LINE-1 methylation level stratified by tumor location. Cox proportional hazards model was used to assess a statistical interaction between LINE-1 methylation level and tumor location in colorectal cancer-specific mortality analysis, controlling for potential confounders including microsatellite instability, CpG island methylator phenotype, and  BRAF and PIK3CA mutations change along the bowel subsites , a and 20) , 66 wereP values were two-sided. Our primary hypothesis testing was a statistical interaction of tumor LINE-1 methylation level and colorectal cancer location in colorectal cancer-specific mortality analysis. Overall mortality was a secondary outcome. The statistical interaction was assessed by the Wald test on the cross-product term of tumor LINE-1 methylation level as a continuous variable and colorectal cancer location as an ordinal variable  and all al colon , and recKRAS (mutant vs. wild-type), BRAF (mutant vs. wild-type), and PIK3CA (mutant vs. wild-type). A single analysis model could assess the prognostic association of tumor LINE-1 hypomethylation in each stratum of colorectal cancer location, using a reparameterization of the interaction term  as previously described [P = 0.05 as a threshold, to select variables for the final model. For cases with missing information in any of the categorical covariates , we included these cases in the majority category of a given covariate to minimize the number of variables in the multivariable Cox models. We confirmed that excluding the cases with missing information in any of the covariates did not substantially alter results (data not shown). The proportionality of hazards assumption was assessed by a time-varying covariate, using an interaction term of colorectal cancer-specific survival and tumor LINE-1 methylation level (P = 0.97).For analyses of colorectal cancer-specific mortality, deaths as a result of other causes were censored. The Kaplan-Meier method was used to describe the distribution of colorectal cancer-specific survival, and the log-rank test for trend was performed to assess a linear trend in survival probability across the ordinal categories  by simple Bonferroni correction for multiple hypothesis testing. To assess associations between categorical variables, the chi-square test was performed. To compare mean age, an analysis of variance assuming equal variances was performed.CI, confidence interval; CIMP, CpG island methylator phenotype; HPFS, Health Professionals Follow-up Study; HR, hazard ratio; LINE-1, long interspersed nucleotide element-1; MSI, microsatellite instability; MSS, microsatellite stable; NHS, Nurses’ Health Study; PCR, polymerase chain reaction; SD, standard deviation.www.genenames.org. Gene names are italicized, and gene product names are non-italicized.We use HUGO (Human Genome Organisation)-approved official symbols for genes and gene products, including BRAF, CACNA1G, CDKN2A, CRABP1, IGF2, KRAS, MLH1, NEUROG1, PIK3CA, RUNX3, and SOCS1; all of which are described at


Techniques and biomaterials for reconstructive middle ear surgery are continuously and steadily developing. At the same time, clinical post-surgery results are evaluated to determine success or failure of the therapy. Routine quality assessment and assurance is of growing importance in the medical field, and therefore also in middle ear surgery. The exact definition and acquisition of outcome parameters is essential for both a comprehensive and detailed quality assurance. These parameters are not the audiological results alone, but also additional individual parameters, which influence the postoperative outcome after tympanoplasty. Selection of patients and the preoperative clinical situation, the extent of the ossicular chain destruction, the chosen reconstruction technique and material, the audiometric frequency selection and the observational interval are only some of them. If these parameters are not well documented, the value of comparative analyses between different studies is very limited. The present overview aims at describing, comparing, and evaluating some of the existing assessment and scoring systems for middle ear surgery. Additionally, new methods for an intraoperative quality assessment in ossiculoplasty and the postoperative evaluation of suboptimal hearing results with imaging techniques are available. In the area of implant development, functional elements were integrated in prostheses to enable not only good sound transmission but also compensation of occurring atmospheric pressure changes. In combination with other components for ossicular repair, they can be used in a modular manner, which so far show experimentally and clinically promising results. Since the first reports about the reconstruction of the sound conduction apparatus by Wullstein and Zöllner , 2], th, th2], tIn a first step, biological and disease-specific influence factors will be discussed that have an impact on the postoperative hearing result. The consideration of those parameters plays an important role in the description and stratification of the study population because only in that way, comparison between the different studies is possible and applicable. Afterwards, the evaluation parameters that may determine the success of middle ear surgeries, will be in the focus. Only by selecting and describing the evaluation parameters in detail, the results may be analyzed and interpreted regarding their significance. The lack of acknowledged national and international standards makes it difficult to compare different studies so that meta-analyses cannot be performed effectively. In addition, new developments of intra- and postoperative quality assessment and assurance will be presented and evaluated. Finally, some new developments of middle ear prostheses and new achievements in ossiculoplasty will be dealt with.In the last years, increasing efforts regarding quality assessment and assurance have been integrated in reconstructive middle ear surgery. Besides the clinical and audiological outcome parameters after tympanoplasty also patient-related aspects such as the health-related quality of life (HRQoL) play an increasing role. In order to describe the quality in a comprehensive and differentiated way, its influence factors and their documentation must be transparently determined. The assessment of success or failure of tympanoplasty for every individual patient seems to be manageable, well understandable, and communicable. But already when considering the individual patient, the intraoperative site or the postoperative hearing result do not allow a complete statement about the outcome. The more existing assessment methods are applied, the more extensive and valid is the possible statement or an extrapolation to future results and the comparability with other series. Figure 1 The process chain of quality assessment for tympanoplasty already starts with the description of the preoperative situation, includes the actual surgical intervention, and ends long time after final wound healing with the clinical status at least one year after surgery Figure 2 . It mustWith regard to clinical as well as scientific questions, mainly functional parameters are applied internationally to assess the surgical outcome after middle ear reconstruction. Additionally, pre- and postoperatively the inflammation situation and the incidence of recurrences or the necessity of surgical revision are mentioned.Under functional aspects, the pure tone audiometry prevailed as the most important psycho-acoustic measurement instrument, which represents also a basis for correlation with other outcome parameters because of its confirmed validity . For comSimilar recommendations regarding the quality criteria and a minimal standard of outcome communication in clinical studies issued by the committee of German-speaking audiologists, neuro-otologists, and otologists  do not exist, they are currently elaborated. In the German as well as international literature, diverging frequency ranges including 4 and 6 kHz and the entire measured frequency range are found. Speech audiometry is another audiometric procedure that is only rarely applied for description of functional results after tympanoplasty. For speech audiometry, again, no international standard exists regarding the measurement methods and the documentation of the results. The most recent recommendations of the AAO–HNO include speech audiometry as measurement procedure and request the depiction of the word recognition score (WRS) together with pure tone audiometry (PTA) in a combined graph, a so-called scattergram” . GeneralA literature research identified more than 160 publications on the key word of surgery for hearing improvement between 2005 and 2015; they were evaluated regarding the application of the AAO-HNS recommendations of 1995. It could be revealed that only in 59% the PTA (hearing threshold and ABG) was given as average with standard deviation, so 41% of the trials could not be included in a meta-analysis and statistical statements are of limited value. The frequency range was described in 85% of the studies, which means that 15% of the publications did not define this parameter. The recommended test frequencies of 0.5, 1, 2, and 3 kHz were applied in 47% of the studies. Speech audiometry was only performed in 4%, while the follow-up interval to assess the outcome amounted to less than 6 months in 60% . F. F21]. FMore than 20 years after formulating a recommendation of a minimal quality standard regarding the reporting of the outcome of hearing results, their application in the study landscape shows a high need of improvement. At the same time, it must be feared that the extended recommendations from 2012 to include speech audiometry will only be realized reluctantly, also because of the mentioned measurement difficulties. The efforts made to find a consensus in the German speaking and European ENT-specific societies regarding a minimally standardized reporting of results should be promoted vigorously. Only in that way, it may be assured in the international discourse that established and validated audiometric measurement methods and parameters , maximal understanding of polysyllables, or others) will be further taken into consideration and are not sacrificed to politically motivated rapid decisions only because they had never been defined as quality parameters.For several years now, measurement instruments on the assessment of the health-related quality of life (HRQoL) as outcome parameter become more and more important , 23], , . This deIt is basic for an implementation of the HRQoL in clinical and scientific routine to carefully develop and apply the measurement methods. Measurements of the quality of life must be sharply delimited from patient interviews that inquire about symptoms and possible impairments by means of own item lists. They can exclusively contribute to the practical documentation of complaints. In order to find scientifically sound, reliable conclusions, the application of psychometric measurement instruments that meet all quality criteria  of a standardized measurement procedure, are essential. Beside statistical evaluation, only those allow a comparability of data and can contribute to the assessment of outcome parameters.Currently it is still quite easy to keep an overview about the studies on surgical hearing improvement that include HRQoL data in addition to clinical and audiometric outcomes. With the continuous development of new measurement instruments for other diseases, the HRQoL data situation, also for tympanoplasty, will increase in the future.Beside the actual reconstruction of the conductive apparatus, many factors influence the postoperative hearing outcome. Undoubtedly, the postoperative ventilation of the middle ear is one of the most relevant factors. The underlying middle ear pathology, its extent, the presence of residual ossicles, the status of the middle ear mucosa, and other factors additionally influence independently the postoperative acoustic middle ear function. Studies encompassing statements on the postoperative hearing outcome often do not mention those factors in detail. One can only speculate about the reasons. The stratification based on too many factors easily reduces a study population of more than 100 patients to single-digit values so that no reasonable statistical calculations can be made. On the other hand, experimental as well as clinical results of the literature confirm that some factors have a definite influence on the hearing outcome and thus they should be included in the description of the patient population and the discussion of the hearing results. The publication of results after tympanoplasty pursues three objectives: (1) the assessment of the surgical method or a reconstruction technique or prosthesis, (2) the comparability of the results with other case series and studies, and (3) the determination of the prognostic success . This isAlready the classification presented by Wullstein in 1956 . As indiAnother instrument that was described by Black as “SPITE criteria” encompasses five dimensions, which have to be assessed by the surgeon based on a questionnaire . The sinClearly more frequently, the application of the Austin classification  is foundIn the most recent version, “smoking” is mentioned as additional risk factor  Table 3. In compIn contrast to other instruments, the MERI assesses the extent of different pre- and intraoperative risk factors with a numeric value. The sum of those values leads to the risk that increases with higher scores and thus allows a prognosis of the probable success of tympanoplasty. So the MERI is comparable to the modern HRQoL measurement instruments leading to a more frequent application also due to its easy handling and significance.The development of the described assessment systems was merely empirical and all considered influence factors were taken into account because of clinical observations. In 2001, Dornhoffer and Gardner were the first to perform a multivariate analysis correlating the MERI influence factors with the hearing outcome. They were able to show in 200 patients that the malleus handle, the condition of the middle ear mucosa, the surgical technique, the revision situation, and otorrhea had a good correlation with the hearing result . So thisThe malleus handle represents a preferred position for the coupling of middle ear prostheses . ExperimPathological changes of the middle ear mucosa may develop in the context of chronic inflammatory processes or after the mucosal trauma of middle ear surgery. Morphological changes such as granulations, fibrosis, scars, tympanosclerotic plaque, or bone formations are possible , 54], , . Those iThe necessity to perform revision surgery seems to be obvious as negative influence factor on the postoperative hearing ability and is confirmed by numerous studies , 43], , , 51], , , 57], , , 59], , , 61], , , 63]. S. S63]. SThe intention and advantages of the risk assessment of tympanoplasty are not only a differentiated counseling and better information of the patients. They are extremely important for a differentiated description of a study population and comparative evaluation of study results. They oblige the authors to prospectively determine and thus exactly define the influence parameters that are found in their patient population, which is difficult or impossible to realize in a retrospective study design, on which most of the articles are based. A consequent application of the assessment instruments in the context of clinical studies would be desirable because the patient population is similar in the therapeutic endpoint with the term of “tympanoplasty” but it comprises different disease entities with different prognostic starting points . For thiDuring and after a tympanoplasty, the surgeon has only limited possibilities to assess the quality of the reconstruction he has performed or to identify the reasons for failure. For the intraoperative assessment and improvement of the acoustic quality of reconstruction, several solutions were described that are different with regard to their practicability and importance. A measurement system that consists of a combination of electromagnetic stimulation and laser vibratiometry, allows a fine tuning of the reconstruction in real-time and seems to be very promising. To assess an unclear postoperatively residual conductive hearing loss, imaging procedures to identify the origin can be successfully applied.The wish to retrieve information about the expectable hearing improvement already during surgery, is understandable. If ossiculoplasty is performed under local anesthesia, the patient himself can give direct feedback about the acoustic result of the ossicular reconstruction. Further manipulation of the prosthesis such as change of the coupling, length or position between the ossicular remnants or the reconstructed eardrum may lead to a subjectively optimal outcome for the patient. In case of surgeries performed under general anesthesia, this possibility is not available. The surgeon must then rely on his experience and knowledge about the biomechanics of the middle ear.In order to add an objective reconstruction quality assessment to those subjective factors, several strategies were pursued. The starting position for its implementation is rather unfavorable. The stimulation of the prosthetic and ossicular vibration must be possible with a defect or reconstructed eardrum and at the same time meet the requirements of sterile precautions of the surgical intervention. The acoustic stimulation via insert phones can be realized in a sterile way with some effort, but acoustic stimulation is not possible with a defect or forward-folded eardrum. Even the selection of the measurement system and parameters determining the quality of the reconstruction is subject to narrow basic conditions. The measurement system should react without time delay and thus display changes directly – in real-time – at the reconstruction. Time-consuming measurements of acoustically evoked potentials are not suitable.The combination of a mechanical stimulation and an optical measurement (LDV) of the ossicular vibration seems to be most appropriate for the requirement of real-time monitoring. The quality of the coupling of stapes prostheses could be successfully investigated with such an experimental measurement system .®  or or68] or® , 70], ®  are clinThe mechanical stimulation of the ossicular chain, which is independent from the status of the eardrum, uses a method of hearing aid development where a magnet is placed on the umbo and driven electromagnetically by a coil , 73], , , 75], , . Even exIt must be taken into account that the best hearing result that can be achieved by means of real-time feedback can only be identified by comparative transmission measurements. The wish to discover a technically exact and generally defined transmission function of the middle ear must remain unfulfilled because sound transmission through the middle ear corresponds to an individual characteristic curve. Under stimulation of the eardrum with 94 dB SPL, the frequency-specific ossicular vibration (measured on the stapes footplate) varies of ±10 dB, which corresponds to a corridor of the transfer function of 20 dB . In otheAfter surgery, audiometric data such as tone audiometry with determination of the postoperative ABG, absolute and as difference regarding the preoperative original findings, are taken as standard reference (see chapter 2.1). Significantly more rarely, speech audiometry is applied. Additionally clinical data on the status of the eardrum (intact/defect), sometimes the ventilation , and the inflammation are assessed, unfortunately not in a standardized way and thus they are individual.The evaluation of a postoperatively remaining conductive hearing loss is often problematic in cases of closed and/or reconstructed eardrum because a prosthetic reconstruction cannot be assessed visually. Generally, beside the complete dislocation of the prosthesis with complete transmission loss, also partial dislocation and changes of the coupling at the prosthesis-ossicle-interface are possible reasons. Of course, other reasons may also be disease-related, biological, and thus independent from the prosthesis, factors that cannot be influenced by the surgeons. The gold standard to find the origin and if possible a causal therapy is the surgical exploration , 83]. B. B83]. BExperimentally, RT and CBT provided statements about coupling and the spatial orientation of titanium prostheses . After iBeside sound transmission, the middle ear has the function of adjusting static pressure variations. The middle ear has 2 working ranges for which it is functionally equipped: the nano-area for sound transmission for hearing and the macro-area for the compensation of atmospheric pressure changes. Regarding the compensation of atmospheric pressure changes, the eardrum, the ligaments and the joints of the ossicles, and the annular ligament play a decisive role. After prosthetic middle ear reconstruction, those elements partly do no longer exist. Because of favorable properties of the material, titanium prevailed among the alloplastic materials during the last years. Beside the excellent acoustic transmission characteristics and a very good biocompatibility, the possibility to develop particularly filigree design, is of major significance. This property allows integrating functional elements in the middle ear prostheses. The aim of such functional elements is the compensation of pressure variations . GeneralThe extraordinary significance of the malleus handle (manubrium) for the postoperative hearing results was already mentioned in chapter 2.2.1. Efforts have been undertaken to reconstruct a completely destroyed manubrium. Several approaches to replace the manubrium, have been described , 98], , , 100]. . 100]. IExperimental measurements with temporal bone specimens reveal that there is nearly no difference of the middle ear transfer function between a malleus-TORP-footplate reconstruction and a MRP-TORP-footplate reconstruction Figure 7 . In the In order to avoid prosthesis head plate dislocation from the tympanic membrane or to achieve long-term stability in this area without negative effects on the acoustic transmission properties, modifications of the rigid connection between the prosthesis strut and the head plate or the head plate itself are useful. A modification of the head plate to which 0.33 mm high spikes are fixed in direction of the eardrum, revealed experimentally a clear stabilization against lateral displacement forces . Under e® . . 103]. I® . Long-teThe integration of a joint in the prosthesis strut is another possibility to avoid the rigid coupling between the eardrum and the footplate for compensation of atmospheric pressure variations and thus protection of the inner ear and of dislocations , 108]. . 108]. TExperimental investigations with titanium total prostheses with a silicone-enhanced micro-joint that was integrated in the prosthesis stem, the feasibility of this approach could be confirmed. Bionically following the example of the birds’ columella, the prosthesis stem deviates from the vertical axis in cases of static pressure increase. The integration of a joint in a silicone mantle provides the desired reset effect so that a decrease of the pressure leads to a restoration of the original position. In this way, compared to the articulated connections in the area of the prosthesis plate that have a ball-joint with friction, there is a significant advantage: resilient joint with reset effect. There is no difference in transmission properties of the bended prosthesis in the dynamic range of sound transmission compared to rigid TORP. Under increased pressure (negative pressure in the middle ear) the transmission can be compared to the one of the intact middle ear. Currently, the silicone enclosure of the joint represents a manufacturer-related obstacle. With regard to the acoustic and static performance, however, this approach is very promising .® , , 106], , 122]..122].The authors declare that they were supported by Heinz Kurz Medical Technique Company during the last 3 years in the context of travelling grants and performed contract research.


Hair cells in the cochlea can be damaged by various causes. Damaged hair cells can lead to additional destruction of parts of the auditory afferent pathway sequentially, which is called secondary degeneration. Recently, researches regarding cochlear implants have been actively carried out for clinical purposes; secondary degeneration in animals is a much more practical model for identifying the prognosis of cochlear implants. However, an appropriate model for this research is not established yet. Thus, we developed a secondary degeneration model using an ototoxic drug. 35 gerbils were separated into four different groups and kanamycin was applied via various approaches. ABR was measured several times after drug administration. SGCs were also counted to identify any secondary degeneration. The results showed that outer and inner HCs were damaged in all kanamycin-treated groups. Twelve weeks after kanamycin treatment, the round window membrane injection group showed severe subject differences in hair cells and SGC damage, whereas the gelfoam group showed consistent and severe damage in hair cells and SGCs. In this study, we successfully induced secondary degeneration in hair cells in a gerbil model. This model can be used for various purposes in the hearing research area either for treatment or for preservation.  Social impacts of hearing loss have increased in many aspects more than ever, since the prevalence of hearing loss surges in accordance with the aging process of our modern society. Noise, ototoxic drugs, infections, aging, and other diseases are responsible for cochlear end organ damage during our lifetimes. In many cases, the loss of cochlear hair cells is the main contributor to loss of sound perception. Cochlear hair cell damage can subsequently progress towards the proximal part of the auditory pathway including the nerve fiber, spiral ganglion cells (SGCs), and cochlear nucleus, which is also known as secondary degeneration . This seSpecifically, this degeneration had been considered to be highly dependent on the status of the inner hair cell (IHC) , 7, 8. SEven after severe hearing loss, the degree of secondary degeneration on the remaining SGCs is very critical for hearing rehabilitation in the area of cochlear implant which is a cutting-edge modality for profound sensorineural hearing loss patients nowadays , 13. CurAminoglycoside is a widely used class of antibiotics which also has ototoxicity that can induce permanent damage to the organ of Corti (OC) . ParticuAs described above, delaying and attenuating the timing of secondary degeneration are important for hearing rehabilitation. To investigate the therapeutic methods which can delay or prevent secondary degeneration, a stable and consistent secondary degeneration model is essentially needed. For this reason, the purpose of this study is to establish a consistent secondary degeneration model by approaching different drug treatments of kanamycin.N = 12), kanamycin at round window niche soaked in gelfoam , kanamycin injection through the round window membrane (RWM) , and control (N = 6). For the drug administration and hearing measurements, gerbils were anesthetized with zolazepam  and xylazine . All procedures were approved by the Institutional Animal Care and Use Committee of Dankook University (DKU-15-006).Female Mongolian gerbils (40–45 g) at 6–8 weeks of age were included in the experiment and were divided into four different groups: kanamycin via percutaneous injection  sulphate was diluted in normal saline (150 mg/ml) and administered in three different ways . For theAuditory brainstem responses (ABRs) were measured to investigate the changes of hearing threshold before and after drug administration. The evoked response signal-processing system  was adopted for ABR measurement. Animals were anesthetized with zolazepam  and xylazine  and were placed in a soundproof chamber. Then, needle electrodes were inserted to the vertex (as response) and ventrolateral sides of both pinnae (as reference and ground). Tone stimuli with 4, 8, 12, 16, and 32 kHz were generated from 90 dB to 10 dB in 5 dB steps and average waveforms were generated from 1024 responses. Hearing thresholds were measured before and 1, 4, and 12 weeks after drug administration.Animals were sacrificed after 4 and 12 weeks of drug administration for the histological analysis. Cochleae were harvested and fixed in 4% paraformaldehyde for 24 hours at 4°C. After fixation, the samples were decalcified with 0.5 M ethylenediaminetetraacetic acid (EDTA) for a week. Cochleae from 6 gerbils in each group were embedded in paraffin and sectioned from the apex to the basal turn to quantify the number of SGCs at 4 and 12 weeks after drug administration. Four micrometers of sectioned samples was stained with hematoxylin and eosin (H&E).http://rsb.info.nih.gov/ij/). Samples were sectioned as midmodiolar plan to represent the overall place of the cochlea. SGCs in 10000 square micrometers at four different parts of the cochlea  were counted and compared with the control group. Three or more sectioned images at each part with a 50 um interval were averaged and examined by polarizing microscopy using a BX51-P microscope .The number of SGCs was counted using ImageJ software  and MyosinVIIa . After mounting on a slide, images were taken with a confocal microscope . 40x magnification was used and p value was <.05.Statistical analysis was performed using the Statistical Package for the Social Sciences  software. Two-way analysis of variance (ANOVA) with Bonferroni post hoc test was adopted and significant differences were determined when the F = 193.1, p < .0001). In the KP group, post hoc t-test revealed that statistical significance exists at 12, 16, and 32 kHz . As with the KG group, hearing thresholds were significantly elevated at all test frequencies . Similarly, the KI group showed a significant difference at all test frequencies  (We tested different delivery techniques (single application) to induce hearing loss and evaluated the hearing outcome. ABR was measured at three time points in all groups, one, four, and twelve weeks after the drug administration. Hearing threshold shifts were observed in all groups at any given time points . At the  < .001) .According to the results of this part, using the local delivery techniques to induce a substantial hearing threshold shift with a single application was possible. Threshold changes of the KG and KP groups showed increased hearing threshold shifts at higher frequencies due to the closer location to the round window where the drug is presumably delivered. In the KI group, most of the deterioration in the hearing threshold was observed; however, the invasiveness of the delivery technique cannot be disregarded.F < 326.9, df < 3, p < .0001). Post hoc t-test revealed that all three groups showed statistically significant threshold shifts at all the tested frequencies . These threshold shifts were constant until 12 weeks after the drug administration while the KP group showed a little recovery at 4 kHz (p < .01).At the 4-week time point, hearing thresholds of all kanamycin injected groups were statistically different from the control  and nerve fibers connected to them. Thus, we investigated the status of the HC and nerve fibers by immunostaining with whole mount preparation. The status of HCs and nerve fibers at the apex, middle, and base parts of the cochlea, which represents 8, 16, and 32 kHz, was identified. After twelve weeks of drug administration, the KG and KI groups showed a total loss of HCs and a partial loss of nerve fibers at three selected parts of the cochlea. In the case of the KP group, we found that the status of the nerve fiber and HCs was preserved . These rAccording to the immunostaining results, kanamycin causes damage not only to the HCs in the OC, but also to the auditory nerve fibers. To investigate the degree of degeneration in the OC and the possibility of additional degeneration of the auditory ascending pathway, the status of the OC within the sectioned images was identified. Four locations in the cochlea were selected as representative areas Figures  and 5. AAfter twelve weeks of drug administration, still, the status of the OC was intact at all locations in the control and KP groups. In the KG group, damage to the OC was extended to the upper parts of the cochlea, but the high middle part of the OC was undamaged. In the KI group, the status of the OC was varied depending on the subject. One subject showed a total loss of the OC at all the selected parts, whereas two subjects showed an intact status of the OC. These results suggest that the kanamycin solution injected through the RWM would have leaked out if the perilymph was not well flushed.The status of SGC was also observed for identifying additional damage in the auditory pathway. Similar to the OC, SGCs at four locations were counted and compared with the control group Figures  and 4. IKanamycin is a well-known ototoxic agent, and it is a widely used model to mimic human sensorineural hearing loss with various delivery methods in an animal model , 26–29. The ototoxicity of kanamycin is a well-known issue throughout the clinical and animal research. It is reported that kanamycin induces production of reactive oxygen species and these attack the cochlear hair cells, which is an irreversible injury, resulting in a hearing disorder . We did not explore the mechanism of kanamycin toxicity at the HC and SGC in this study. Nevertheless, we might expect that the same ototoxic mechanism would be involved in this study.The Mongolian gerbil is a well-established animal model for hearing research . Since tWe applied various ways of drug administration to find the most appropriate way that can induce secondary degeneration. According to our results, the RWM injection showed a more dramatic change in the OC within a short time than the gelfoam group. The number of SGCs was severely decreased at four selected parts of the cochlea at 4 weeks after kanamycin treatment through the RWM injection. However, a prominent subject difference also existed with this approach. During treatment, the KM solution could not disperse well because of the pressure inside the scala tympani, and it also leaked out right after injection using a cannula. Together with these, we considered that KM RWM injection is not an appropriate way to create a secondary degeneration model . OtherwiDrug delivery agents have been studied in the otology research area for ototoxic or therapeutic purposes. It has been reported that gelfoam increases the effect of a drug itself by allowing the drug to be retained longer at the target area. Abbas and Rivolta 2015) used aminoglycosides with a gelatin sponge and reported a more significant change in the hearing threshold after 2 weeks of treatment than with KM alone  used ami, 37. TogFor generating a secondary degeneration model for diverse purposes, various methods would be applicable in animal research. A recent study that investigated KM toxicity reported that KM did not damage adult spiral ganglion neurons . In thisAs seen in We induced secondary degeneration of HCs in a gerbil model through diverse drug delivery approaches in this study. High concentrations of kanamycin application with gelfoam on the RWM caused severe HC loss and this extended to degeneration of the auditory nerve and SGCs. This model can be used for various purposes in the hearing research area either for treatment or for preservation. Furthermore, this model would be applicable for research regarding cochlear implants.


Hearing impairment has been suggested to increase the risk of falls. However, most previous studies were conducted in an older population without classification of the fracture regions. This study aimed to delineate the risk of each fracture type in all age populations.The Korean National Health Insurance Service-National Sample Cohort was collected from 2002 to 2013. A total of 4,854 severe hearing-impaired and 1,354 profound hearing-impaired participants were matched for age, group, sex, income group, and region of residence with 19,416 and 5,416 control participants, respectively. The fracture diagnosis was based on the International Classification of Disease (ICD-10) codes as follows: distal radius fracture (S525), hip fracture , and spine fracture . Crude (simple) and adjusted hazard ratios (HRs) for each fracture associated with severe or profound hearing impairment were analyzed using the Cox proportional hazard model.The severe hearing-impaired group had an increased risk of distal radius fracture, hip fracture, and spine fracture compared with the control group . The profound hearing-impaired group had an increased risk of hip and spine fracture .The risk of distal radius fracture, hip fracture, and spine fracture was increased in the severe hearing-impaired group compared with the control group. Fractures are prevalent injuries that occur across all age groups. For example, the incidence of distal radius fracture was estimated to be approximately 278 per 100,000 people and 709 per 100,000 women in Europe . This elVarious factors increase the risk of fractures according to types of fractures including age and osteoporosis, the latter of which is a leading cause of fracture . More thHearing impairment has been reported to increase the risk of falls, although there have been conflicting reports in the literature. A meta-analysis demonstrated a 1.72 pooled odds ratio of falls in hearing-impaired elderly individuals (95% CI = 1.07–2.37) . HoweverThe hypothesis of this study was that hearing impairment may elevate the risk of fractures in all age groups besides the elderly population. In addition, severe hearing impairment may impact fracture risk irrespective of the fracture site. To evaluate these hypotheses, we conducted a longitudinal follow-up study on the risk of fracture, including distal radius, hip, and spine fractures, in severely or profoundly hearing-impaired populations across the lifespan.The ethics committee of Hallym University (2014-I148) approved the use of these data. Written informed consent was exempted by the Institutional Review Board.This national cohort study relied on data from the Korean National Health Insurance Service-National Sample Cohort (NHIS-NSC). The Korean National Health Insurance Service (NHIS) selects samples directly from the entire population database to prevent non-sampling errors. Approximately 2% of the samples (one million) were selected from among the entire Korean population (50 million). These selected data can be classified at 1,476 levels  using randomized stratified systematic sampling methods via proportional allocation to represent the entire population. After data selection, the appropriateness of the sample was verified by a previous study . The detBecause all Korean citizens are recognized by a 13-digit resident registration number from birth to death, exact population statistics can be determined using this database. It is mandatory for all Koreans to enroll in the NHIS. All Korean hospitals and clinics use 13-digit resident registration numbers to register individual patients in the medical insurance system. Therefore, the risk of overlapping medical records is minimal, even if a patient moves from one place to another.Of the 1,025,340 cases with 114,369,638 medical claim codes, we included participants who were registered as hearing-impaired individuals by the Ministry of Health and Welfare. Of these participants, those who were registered with other disabilities  in the Ministry of Health and Welfare were excluded. They were divided into two groups based on the grade of impairment: severe hearing impairment  and profound hearing impairment (hearing threshold ≥ 90 dB in both ears). In Korea, to be registered as a hearing-impaired person, an individual must be evaluated three times by a pure tone audiometry test (PTA) and once by an auditory brainstem response test. The average hearing threshold of the PTA was calculated as follows: (500 Hz + 2*1000 Hz +2* 2000 Hz + 4000 Hz)/6.The hearing-impaired participants were matched 1:4 with the participants (control group) who had never been diagnosed with a hearing impairment or with other disabilities among this cohort from 2002 through 2013. The matching was performed according to age group, sex, income group, and region of residence. To prevent selection bias when selecting the matched participants, the control group participants were sorted using a random number order and were then selected from top to bottom. It was assumed that the matched control participants were assessed at the same time as each matched hearing-impaired participant (index date). Therefore, the control group participants who died before the evaluation period of the matched hearing impairment participant were excluded. The hearing-impaired participants for whom we could not identify enough matching participants were excluded . Among them, the participants who had a history of fracture  before the index date were excluded . Finally, 1:4 matching resulted in the inclusion of 4,854 severe hearing-impaired participants and 19,416 control participants as well as 1,354 profound hearing-impaired participants and 5,416 control participants .The age groups were classified using 5-year intervals: 0–4, 5–9, 10–14…, and 85+ years old. A total of 18 age groups were defined. The income groups were initially divided into 41 classes . These groups were re-categorized into 11 classes (class 1 [lowest income]-11 [highest income]). Region of residence was divided into 16 areas according to administrative district. These regions were regrouped into urban  and rural  areas.Fracture history was evaluated using ICD-10 codes. Distal radius fracture was selected as Fracture of lower end of radius (S525). Hip fracture was chosen as Fracture of head and neck of femur (S720), Pertrochanteric fracture (S721), or Subtrochanteric fracture of femur (S722). Spine fracture were defined as Fracture of thoracic vertebra (S220) or Fracture of lumbar vertebra (S320).To analyze the hazard ratio (HR) for fracture  in each severe or profound hearing loss group, the Cox proportional hazard model was used. In this analysis, crude (simple) and adjusted  models were used. Bonferroni correction was used in this analysis to adjust the expected value of a wrongly declined null hypothesis. Two-tailed analyses were conducted, and P values less than 0.05 were considered to indicate significance. The results were statistically analyzed using SPSS v. 21.0 .The general characteristics of age, sex, income, and region of residence were exactly matched between the severe or profound hearing impairment and control groups . The incThe HR for distal radius fracture was higher in the severe hearing impairment group than the control group in both the crude and adjusted models  . The HR According to the age subgroups, in the < 60-year-old group, the HR for distal radius fracture and spine fracture in the severe hearing impairment group was higher than that the control group  . The HR The severe hearing impairment group demonstrated a higher risk of distal radius, hip, and spine fractures than the control group in the present study. The profound hearing impairment group also had a higher incidence of hip and spine fractures than did the control group. The older age of the severe hearing impairment group than profound hearing impairment group might result in high crude fracture rate and the risk of radius fracture in severe hearing impairment group. Approximately 59.4% and 37.6% were 60 or older in severe hearing impairment group and profound hearing impairment group, respectively. This increased fracture risk was present in both the < 60-year-old and ≥ 60-year-old subgroups. Few previous studies reported the risk of fracture in the hearing-impaired population according to age and fracture region.Increased accidental traumatic injuries in hearing-impaired subjects could elevate fracture risk. Hearing-impaired subjects may be susceptible to falls due to decreased spatial orientation and speech perception . To suppCombined vestibular dysfunction could contribute to the increased risk of falls in hearing-impaired subjects . The labDecreased cognitive function due to the elevated cognitive load in hearing-impaired subjects could elevate the risk of falls and fracture . EquilibOsteoporotic changes in the auditory system and in other bones, including the skeletal bones and spine, could result in fracture susceptibility in hearing-impaired subjects. Low cochlear capsule demineralization has been found to correlate with hearing loss in subjects with bone disorders such as osteogenesis imperfecta, Paget’s disease, and otosclerosis –29. In aThe present study used a representative large population across the lifespan. The validity of the sampling procedure was verified based on a previous study . There wSevere hearing impairment increased the risk of distal radius, hip, and spine fractures across the lifespan. Both the < 60-year-old and ≥ 60-year-old subgroups had an increased risk of spine fractures compared with the control groups.S1 Table(DOCX)Click here for additional data file.