Beauveria bassiana is an entomopathogenic fungus and is a rare cause of keratitis. We present a case of fungal keratitis caused by B. bassiana that was diagnosed by in vivo confocal microscopy and in vitro corneal cultures. In addition, we determined the temperature- and drug-sensitivities of the isolated strain of B. bassiana.B. bassiana. The keratitis responded poorly to systemic and topical voriconazole and to natamycin ointment. However, it was resolved after changing the natamycin to micafungin combined with surgical debridement. The isolated strain was sensitive to itraconazole, miconazole, micafungin, voriconazole, and resistant to flucytosine and fluconazole. It was moderately sensitive to amphotericin B, and natamycin. After 7 days in culture, the isolate grew small white colonies at 25°C, very small colonies at 35°C and 37°C.A 59-year-old Japanese man with a 2-month history of keratitis was examined by slit-lamp biomicroscopy, in vivo confocal microscopy, and histology and cultures of corneal scrapings. The corneal scrapings were used to determine the minimal inhibitory concentrations of different antifungal drugs and also to determine the temperature-sensitivity. In vivo confocal microscopy and histological examinations showed filamentous fungal keratitis. The characteristics of the fungal growth indicated that the keratitis was caused by B. bassiana should be helpful in the treatment of B. bassiana keratitis. Therapeutic surgery may be helpful for mycotic keratitis poorly responsive to medical therapy alone.The drug-sensitivity and temperature-sensitivity profiles of  Beauveria bassiana is a fungus that is distributed worldwide. It can be isolated from soil, insects, and mites. It is an entomopathogenic fungi and is used as a biological control agent for insect pests as alternative supplements to chemical insecticides[B. bassiana is considered to be non-pathogenic to vertebrates, a small number of patients with keratitis caused by B. bassiana have been reported[B. bassiana that was diagnosed by in vivo confocal microscopy and in vitro corneal cultures. In addition, we determined the temperature- and drug-sensitivities of the isolated strain of B. bassiana.cticides–3. Althoreported–13. We pThe patient was a 59-year-old Japanese man who noted a corneal opacity in his right eye in October, 2009. However, he did not seek treatment for the opacity for about two months. He visited a private ophthalmological clinic on December 11, 2009, because his right eye felt irritated and his vision had decreased. The patient was a farm worker, but did not use entomopathogenic fungi as a biological control agent. He had no systemic diseases and no signs or symptoms of immunosuppression during the clinical course. He was diagnosed with infectious keratitis and was treated with topical gatifloxacin and erythromycin eye ointment for two weeks. Because the signs and symptoms did not improve, he was referred to the Ehime University Hospital on December 28, 2009.On the initial examination, his best-corrected visual acuity was 20/500 OD and 20/16 OS. Slit-lamp examination of the right eye showed a grayish stromal infiltrate with a dry texture and indistinct margins  p.o., topical 0.1% voriconazole hourly, and natamycin eye ointment 5 times/day. Because the lesion did not respond despite the intensive antifungal therapy, the isolate from the corneal scrapings was sent to the First Laboratory in Medical Mycology Research Center, Chiba University for identification and to determine the minimal inhibitory concentrations (MICs) to different antifungal drugs  was changed to topical 0.1% micafungin (hourly) along with topical 0.1% voriconazole. During the next 3 months, surgical debridement was also performed 3 times and the infectious lesion slowly improved. Finally, the infectious lesion healed but with mild scar formation (Figure To try to determine the cause of the slow clinical course and poor response to antifungal agents, the isolate was grown on Sabouraud dextrose agar at 25°C, 35°C, and 37°C for up to 7 days at the Clinical Laboratory of Ehime University Hospital as described in detail. The isoBeauveria are rare pathogens in humans, and only 11 cases of keratitis due to Beauveria species have been reported[B. bassiana keratitis have not been determined. The course of our non-necrotic keratitis case was slow-growing, there was no clinical improvement after treatment with voriconazole even though voriconazole was the preferable antibiotic according to the sensitivity tests. The cases of B. bassiana reported by Low et al., Kisla et al., and Tu et al., were also slow-growing with late penetration into the corneal stroma which is consistent with our case[The entomopathogenic fungi belonging to the genus reported–13. Becaour case, 7, 12. our case–8, 12.B. bassiana. Tu et al. and Figueira et al. reported that both of their strains were sensitive to posaconazole and voriconazole but resistant to amphotericin. These sensitivities are similar to that of our strain of B. bassiana[et al. showed that the drug sensitivity of their Beauveria species was similar to our strain[et al. also had a successful treatment with topical voriconazole, although they also treated the eye with topical miconazole and oral itraconazole which their strain was sensitive to[Our antifungal susceptibility tests showed that the isolated strain was sensitive to micafungin and voriconazole and moderately sensitive to natamycin. There have been several reports on the drug sensitivity of bassiana, 12. Ther strain. In addiitive to.et al.[B. bassiana strain in our case could not grow at temperatures of 35°C or higher and thus did not penetrate into the corneal stroma. This temperature sensitivity might be one reason why B. bassiana is a rare pathogen in humans, and why the infections in humans are limited to the body surface such as the cornea.The results of the temperature sensitivity profile supported the microbiological results reported by Figueira et al.. The temet al., 18. ThuB. bassiana to produce chitinase which lyses not only chitin but also keratin and collagen. This would then enable it to adhere and penetrate the cornea[B. bassiana has 3 different cell forms and can adhere to both hydrophobic and hydrophilic surfaces. This would enhance its adherence to the corneal surface along with the permissive avascular immune environment of the central cornea[Another reason for the infection of the cornea may be the ability of e cornea, 12. B. l cornea, 12. In Micafungin is a echinocandins which blocks fungal cell wall beta-glucan synthesis, thus the different actions of voriconazole and micafungin may have a synergetic effect. It is also possible that the surgical debridement might have worked in our case because it removed antigenic and infectious elements and necrotic tissues. This supports the results of earlier studies that surgical debridement or keratectomy is required for mycotic keratitis that respond poorly to medical therapy, 20. FurB. bassiana keratitis. The drug-sensitivity and temperature sensitivity profiles of B. bassiana should be helpful in diagnosing and treating B. bassiana keratitis. Therapeutic surgery may also be helpful for mycotic keratitis poorly responsive to medical therapy.We report a case of Written informed consent was obtained from the patient for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Diabetic retinopathy (DR) is a multifactorial progressive disease of the retina and a leading cause of vision loss. DR has long been regarded as a vascular disorder, although neuronal death and visual impairment appear before vascular lesions, suggesting an important role played by neurodegeneration in DR and the appropriateness of neuroprotective strategies. Upregulation of vascular endothelial growth factor (VEGF), the main target of current therapies, is likely to be one of the first responses to retinal hyperglycemic stress and VEGF may represent an important survival factor in early phases of DR. Of central importance for clinical trials is the detection of retinal neurodegeneration in the clinical setting, and spectral domain optical coherence tomography seems the most indicated technique. Many substances have been tested in animal studies for their neuroprotective properties and for possible use in humans. Perhaps, the most intriguing perspective is the use of endogenous neuroprotective substances or nutraceuticals. Together, the data point to the central role of neurodegeneration in the pathogenesis of DR and indicate neuroprotection as an effective strategy for treating this disease. However, clinical trials to determine not only the effectiveness and safety but also the compliance of a noninvasive route of drug administration are needed. Diabetic retinopathy (DR) is a multifactorial progressive disease of the retina with high social impact and with an extremely complex pathogenesis that involves a variety of different cells, molecules, and factors. Metabolic changes in the diabetic retina result in altered expression pattern of a number of mediators including growth factors, neurotrophic factors, cytokines/chemokines, vasoactive agents, and inflammatory and adhesion molecules, resulting in vascular lesions and cell death –4. In thThe present review focuses on the possible causes and the effects of neuronal damage in DR and discusses possible approaches based on neuroprotection for the treatment of this disease. In particular, with this review of the literature we invite the reader to consider the following hypotheses: (i) neuronal damage directly induced by diabetic stress may be responsible for functional abnormalities in visual function; (ii) a central role in linking neuronal damage to early microvascular damage  may be played by VEGF expressed and released by damaged neurons; (iii) therapeutic strategies based on neuroprotection will be useful in preventing or arresting DR development.There is evidence that death of neurons appears before that of vascular cells in the retina of both humans with DR and experimental animal models of DR , while rThe neural cell types suffering alterations in early DR have been studied in more detail in different rodent models of DR. A number of investigations reported increased apoptosis of retinal ganglion cells , 35–38. In addition to changes in the cell bodies , defects in the processes of retinal neurons are also evident in the retinas of rodent models of DR, and they are represented mainly by abnormal swellings observed in ganglion cell axons , 50 and The damage to retinal neurons in early phases of DR is likely to be responsible for the functional deficits observed both in animal models of DR and in patients with diabetes. For instance, in diabetic db/db mice, retinal thinning and ganglion cell apoptosis have been found to correlate with pattern ERG alterations . This asTogether, these data document the important role that neurodegenerative phenomena may play in the retina of a diabetic subject before the classical signs of DR are recognizable and support the view that a neuroprotective strategy should be considered for the treatment of patients before the appearance of vascular lesions.A logic consequence of the observation that neurons are damaged in DR is the search for the possible causes of this fact. A knowledge of the mechanisms that are likely to affect neuronal survival in the diabetic retina is an essential prerequisite to design therapeutic strategies aimed at reducing the impact of diabetes on retinal neurons.AGEs are late products of nonenzymatic glycation. The deposition of hyperglycemia-induced AGEs in retinal blood vessels plays an important role in the onset and development of DR. AGEs and their receptors (RAGEs) have been detected in virtually all cellular types of the diabetic retina . The AGEGlutamate is the major excitatory neurotransmitter in the retina and glutamate excitotoxicity is the process damaging or killing neurons expressing NMDA or AMPA receptors due to excessive receptor stimulation and calcium influx secondary to altered levels of glutamate in the extracellular space. Elevated glutamate levels have been observed in experimental models of diabetes –84, wherThe polyol pathway is activated in the presence of elevated intracellular glucose levels and it is linked with the progression of DR . The ratPoly(ADP ribose) protein (PARP), a nuclear enzyme involved in the regulation of a variety of cellular events, is another potential inductor of retinal damage in DR. Indeed, PARP is activated in diabetic retinas causing DNA damage and oxidative stress –100, whiHyperglycemia and diabetes also increase the function of the hexosamine biosynthetic pathway, which results in many changes in both gene and protein levels, contributing to the pathogenesis of DR . In partActivation of PKC is known to be implicated in several pathologic changes occurring in DR, such as basement membrane thickening, extracellular matrix expansion, vascular permeability, apoptosis, angiogenesis, leukocyte adhesion, and cytokine activation . SimilarA considerable increase of oxidative stress is known to occur in the diabetic retina , 106 andThe most well-known function of RAS is regulation of blood pressure as well as of fluid and electrolyte content in the body. The classical RAS is a systemic hormonal system, but local RASs have been identified in a number of organs, including the eye and the retina . In DR, A considerable body of evidence from animal models and patients shows that DR is a chronic low-grade inflammatory disorder with participation of inflammatory mediators . DysreguER stress results from an impairment of the folding capacity of the ER causing accumulation of unfolded proteins in the ER lumen and activation of the unfolded protein response (UPR). ER stress contributes to increased oxidative stress as well as inflammation and apoptosis may occur upon failure of UPR to resolve the ER stress. This condition is likely to be involved in the pathogenesis of different neuronal diseases in brain and retina, including the early stages of DR . DisruptInsulin provides trophic support for retinal neurons via phosphatidylinositide 3-kinase/Akt and P70S6 kinase pathways –133. Thi in vitro studies in retinal Müller cells suggest that PEDF could increase glutamine synthetase expression and prevent downregulation of glutamate transporter in these cells, thereby limiting glutamate excitotoxicity in the diabetic retina [Pigment epithelium-derived factor (PEDF) has been described as a multifunctional protein with neuroprotective, antiangiogenic, antivasopermeability, anti-inflammatory, and antioxidative effects . Its levc retina , 140.NTR) may be present in DR. Indeed, in the retina of streptozotocin-treated rats, the upregulation of NT-3 and NT-4 and of their receptors TrkA and TrkB is associated with the progression of DR [NTR receptor [NTR-mediated secretion of tumor necrosis factor-α by Müller cells [Alterations in retinal levels of neurotrophins (NTs) and their receptors  is a photoreceptor-secreted glycolipoprotein that is essential for photoreceptor survival . IRBP leThe neuropeptide somatostatin (SST), together with its receptors, is expressed in the retina, where it is involved in a variety of functions . A downr ex vivo ischemic retinas not only undergo cell death but also express and release VEGF, which, in turn, is likely to bind to its receptors expressed by endothelial cells [ ex vivo ischemic models of the retina [Observations of the brain after ischemia suggest that brain damage, in addition to the activation of death pathways, also stimulates protective mechanisms to counteract the expansion of the injury. Although the damaging effectors in the end prevail, the evidence suggests that concomitant self-protective mechanisms are elicited, implying that the central nervous system possesses intrinsic cytoprotective mechanisms that are likely to be mediated by chemical signals derived from the injured brain itself . VEGF maal cells , 159. Thal cells , while ial cells . In addial cells . Togetheal cells . Increasal cells , while Val cells . Repeateal cells . Furtheral cells  and to pal cells . Finallyal cells . Therefoe retina , 159, mae retina , 169. Ine retina  and incre retina , as welle retina .Improvements in diabetes care and management have been crucial in lowering the incidence and severity of DR. One limiting factor that has hampered the investigation of new drugs for DR is the low sensitive endpoints in the clinical trials, which require large sample sizes as well as a long duration (about 5 years) to achieve a sufficient statistical power to detect potential treatment effectiveness. Best corrected visual acuity (BCVA) and derived variables are the only endpoints that have served as the basis for regulatory approval of retinal drugs. However, BCVA captures only a small portion of visual function and patients with good BCVA may have difficulties with daily activities such as reading or driving. Therefore, there is now widespread recognition of the need for improved endpoints for DR research.Since neurodegeneration is an early event in the pathogenesis of DR that could participate in the development of microvascular impairment , 66, theAmong the methods for assessing the functional impairment, the mfERG is the gold standard. This is a noninvasive technique providing a topographic measure of retinal electrophysiological activity. The use of mfERG has provided compelling evidence suggesting a direct link between neural dysfunction and vascular abnormalities in DR. Thus, a delayed mfERG implicit time predicts the development of early microvascular abnormalities , 172. InThe current method for exploring the morphological changes in retina is the SD-OCT, which provides an anatomical image of the retina and complements the functional information obtained with mfERG. The reduction in the thickness of the ganglion cell layer and of the nerve fiber layer are the main parameters detected in DR and they have been observed even before any microvascular abnormality appears in the fundoscopic examination . HyperreIn recent years a noninvasive instrument to assess mitochondrial function using flavoprotein autofluorescence has been developed. This method is based on the fact that the retina is particularly susceptible to oxidative stress because of high energy demands and light exposure. Before apoptosis, mitochondria exhibit impaired electron transport by energy generating enzymes in the respiratory chain, causing increased percentages of flavoproteins in the chain to be oxidized and rendered capable of absorbing blue light and emitting green autofluorescence . Since oThere are several therapeutic strategies based on the main pathogenic mechanisms involved in retinal neurodegeneration that, theoretically, could be implemented . HoweverThe use of eye drops has not been considered an appropriate route for the administration of drugs aimed at preventing or arresting DR because of the general assumption that in this way the drugs do not reach the posterior chamber of the eye . However, there is emerging evidence showing that several topically administered compounds are able to reach the retina in pharmacological concentrations, at least in animal models –181. In Adherence to ophthalmic treatments has a unique set of challenges compared to oral medications . It mustPathologic conditions, including those characterizing the diabetic status, are known to affect retinal levels of neuroprotective agents, thus impairing the balance between neurotoxic and neuroprotective factors and resulting in damage to the neural retina. In this respect, many different substances have been tested for their neuroprotective properties in models of retinal stress similar to that in DR. Since a comprehensive review of all the substances employed in experimental studies is virtually impossible, we will briefly consider the potential use in DR of endogenous neuroprotective substances or of phytochemicals with antioxidant and anti-inflammatory properties.Insulin, IGF-1, PEDF, SST, pituitary adenylate cyclase activating peptide (PACAP), glucagon-like peptide- (GLP-) 1, and NTs are potential neuroprotective factors for the treatment of DR.Subconjunctival insulin administration restores prosurvival signaling and reduces the rate of retinal cell death in streptozotocin-induced diabetic rats . In this in vitro studies in retinal Müller cells suggest that PEDF decreases glutamate excitotoxicity induced by the diabetic milieu by increasing the expression of glutamine synthetase and by preventing glutamate transporter downregulation [PEDF is a peptide with neurotrophic properties produced by retinal pigment epithelium cells and found to have neuroprotective effects towards photoreceptors . In addigulation , 140. Figulation . Taken tgulation  and a PEgulation  have beegulation . Furthergulation .1–sst5) are expressed in the retina, where they are involved in multiple functions [2 expressed by bipolar cells may control glutamate release in the retina [The neuropeptide SST and its five receptor subtypes  as effective molecules in the management of retinal complications in diabetes, although further investigations using human clinical studies are needed to confirm the beneficial effects of phytochemicals in the treatment of DR.Given that diabetic murine models do not develop advanced vascular abnormalities, the experimental studies mentioned above have demonstrated the usefulness of neuroprotective agents to prevent the development of retinal neurodegeneration and initial vascular leakage. Although it could be reasonable to speculate that neuroprotection of the retina will be useful at any stage of DR, further evidence demonstrating this issue is needed. In particular, more work both in animal models, using sufficiently large numbers of animals to have statistical power, and in the clinics is necessary to provide conclusive demonstration that neuroprotection not only preserves neurons from death or damage but also results in actual functional improvements.Alterations of the neurosensory retina and retinal vasculature are integrally linked, and the study of the interactions between blood vessels and the neurosensory retina is crucial to understand the pathogenesis of DR. Neurovascular coupling is the intrinsic physiological mechanism by which neural activity is coupled to blood flow and metabolism, thus enabling the retina to regulate blood flow in response to neural activity or metabolic demands. Indeed, the increased delivery of oxygen in response to the increased activity of retinal neurons is a consequence of the coupling between neural activity and the vasculature. The neurovascular coupling in the retina is altered in diabetes .Visual stimulation is a powerful modulator of retinal and optic nerve blood flow . Since t2 consumption and, therefore, is particularly susceptible to hypoxia caused by compromised blood flow. The loss of this vascular response could starve the retina of needed oxygen and glucose, putting neurons at risk and contributing to retinal pathology. The improvement of functional hyperaemia could be a useful therapeutic target. In this regard, it has been demonstrated that blocking inducible nitric oxide synthase with systemic administration of aminoguanidine recovers flicker-evoked vasodilation in diabetic retinas [The retina has a high metabolism and O retinas .Among the mediators of the hemodynamic impairment of the neurovascular coupling that occurs in diabetic patients, endothelin seems to play a significant role. It should be noted that endothelin 1 (ET-1), a potent vasoconstrictor, is overexpressed in endothelial cells in the setting of DR and enhances glutamate-induced neurotoxicity in retinal neural cells , 231. InSince the extracellular accumulation of glutamate plays a key role in retinal neuron death, treatments addressed at reducing glutamate levels seem appropriate. In this regard, substances targeting the glutamate receptors, such as memantine, have shown beneficial effects on neurodegeneration and vascular abnormalities in streptozotocin-induced diabetic rats . MemantiSeveral potential therapies using endothelial precursor cells have been explored. However, the usefulness and safety of these approaches for DR remain to be determined. Regarding retinal development and transplantation strategies, the last decade has seen an enormous progress in the research for photoreceptor replacement and regeneration . RestoraThe advances in both retinal imaging and functional assessment will allow detecting early changes and will accelerate drug discovery and delivery strategies to improve visual prognosis in diabetic patients. In addition, this new approach will encourage the implementation of personalized treatments. In this regard, OCT angiography and confocal adaptive optics scanning ophthalmoscope (AOSLO) are two new technologies that will play a key role in the near future for the diagnosis and monitoring of treatments in early stages of DR. OCT angiography proved to be capable of showing 10 micron-thick blood vessels, revealing the areas of vascular nonperfusion and identifying microexudates that were not otherwise visible on clinical examination and fundus photography. Moreover, OCT angiography allows appreciation of spatial relationships of fundus vessels and makes it possible to separately visualize the large retinal vessels as well as the superficial and the deep capillary plexus . AdaptivFrom the therapeutic point of view, as previously mentioned, when the early stages of DR are the therapeutic target it would be inconceivable to recommend an aggressive treatment such as intravitreal injections. There is experimental evidence that topical administration is effective for the treatment of early stages of DR , 202. ThIn conclusion, the central role of neurodegeneration in the pathogenesis of DR is a solid basis for proposing neuroprotection as an effective strategy for preventing or arresting DR. However, clinical trials to determine not only the effectiveness and safety, but also the compliance of a noninvasive route of drug delivery, as well as a standardization of the methods for monitoring neurodegeneration, are needed. In addition, neuroprotection in the setting of DR should be contemplated as an early treatment; therefore, this exciting experimental approach seems to be still far from acceptance in clinical practice.


Interestingly, nucleolin has been shown to mediate the anti-apoptotic effect of HSP70, a protein found to prevent ototoxicity and whose polymorphisms have been associated with susceptibility to NIHL. Increase in nucleolin expression has also been associated with the prevention of apoptosis in cells undergoing oxidative stress, a well-known metabolic sequela of noise exposure. To assess the potential role of nucleolin in hearing loss, we tested down-regulation of nucleolin in cochlear sensory cells HEI-OC1 under oxidative stress conditions and report increased sensitivity to cisplatin, a chemotherapeutic drug with ototoxic side effects. Additional SNPs were found with suggestive association (p < 5 x 10-4), of which 7 SNPs were located in genes previously reported to be related to NIHL and 43 of them were observed in 36 other genes previously not reported to be associated with NIHL. Taken together, our GWAS data and in vitro studies reported herein suggest that nucleolin is a potential candidate associated with NIHL in this population.Noise-induced hearing loss (NIHL) is the most significant occupational health issue worldwide. We conducted a genome-wide association study to identify single-nucleotide polymorphisms (SNPs) associated with hearing threshold shift in young males undergoing their first encounter with occupational impulse noise. We report a significant association of SNP rs7598759 (p < 5 x 10 Noise exposure is a primary cause of hearing loss with a broad range of secondary non-auditory effects including cognitive impairment, sleep disturbance and cardiovascular disease . Noise-iIn the inner ear, noise exposure affects the lateral wall, organ of Corti and afferent neurons resulting in hearing impairment categorized as either temporary or permanent hearing threshold shifts. Although there are morphological and functional differences associated with either form of hearing impairment, the molecular basis for that difference is not well defined. However, it is well established that the permanence of hearing loss resides principally in the irreversible loss of cochlear hair cells in the organ of Corti , 4, 5, wSupporting the role of oxidative stress and apoptosis in hearing loss, administration of antioxidants in animal models exposed to noise and inhibition of apoptotic signaling pathways, respectively, have shown efficacy in preventing cochlear hair cell loss –12 and iThe susceptibility of individuals to NIHL results from both environmental factors that increase physiological stress, inflammation and oxidative stress , 6, 14; Here, we report the results of a GWAS identifying a novel susceptibility locus associated with NIHL in 41 individuals selected amongst 314 age-controlled subjects during their first encounter with occupational noise. We then validate the potential role of nucleolin, the top candidate gene containing a significant SNP in our study, by developing a HEI-OC1 cochlear cell with silenced nucleolin, then experimentally testing its susceptibility to oxidative stress conditions and to ototoxic drug cisplatin.The protocol for the collection of saliva samples and the identification of biomarkers of susceptibility to NIHL by GWAS was reviewed and approved by the Institutional Review Board at Naval Medical Center, San Diego. The use of de-identified saliva samples for DNA analysis was further reviewed by the Institutional Review Board at Harvard and received a non-human subject approval. All volunteers enrolled in the study provided written informed consent for saliva collection and its use for the identification of genetic markers. Samples were de-identified, anonymized and an identification number was randomly assigned to each subject. At the time of the saliva collection, samples were labeled with the randomized identification number used throughout the analysis.Prevention of Noise-Induced Hearing Loss Using the Antioxidant Supplement, N-acetylcysteine: Impulse Noise Study (NMCSD.2007.0013)”. Briefly, subjects were in good health as determined by enlistment criteria, without previous exposure to military noise. For admission into the study, subjects were required to have normal hearing in both ears (no hearing threshold greater than 25 dB HL (Hearing Level) at any of the 1, 2, 3, 4, 6 kHz standard audiometric test frequencies and no more than 30 dB SPL (Sound Pressure Level) asymmetry between ears at 8 kHz). Subjects were required to have normal tympanometry on baseline and on the day of final hearing testing, 5 weeks after pre-exposure audiogram. Recruits with abnormal tympanograms consistent with middle ear pathology and those with prior history of head trauma were excluded. The average age of the study participants was 19.82 years old.Subjects were randomly selected from a pool of volunteer participants enrolled in the placebo arm of the Marine Recruit Study “During their training period, subjects underwent 10 days of small arms training with an M16 rifle, which commonly generates an approximate 157 dB peak SPL impulse noise . T. T36]. TWe constructed stable clones expressing shRNA targeted against nucleolin (sh-Ncl). In comparison to shRNA scramble control clones (sh-Scr), nucleolin expression in silenced sh-Ncl clones was significantly reduced . The dowPrevious susceptibility studies have identified NIHL associated genes mainly in pathways that regulate oxidative stress response and potassium channels. Our results show that novel candidate genes in these pathways are associated with NIHL. In particular, the results reported here suggest that nucleolin, a multifunctional phosphoprotein involved in the modulation of cell proliferation and protection against apoptosis , 41, is 2O2-exposed rat cardiomyocytes prevents apoptosis  wh whKCNQ3 tinnitus , while ts [KCNQ4  and mighs [KCNQ4 . In addis [KCNQ4 .KCNMB2, is a subunit of large-conductance calcium-activated potassium channels previously reported in linkage disequilibrium with rs4603971, another SNP in suggestive association with hearing loss [KCNMB2 for hearing loss susceptibility. Of interest, both candidate genes KCNQ3 and KCNMB2 as well as KCNJ10 previously mentioned, appear to be associated with epilepsy [KCNJ10 has been thus far associated with both epilepsy and hearing loss [Another candidate gene, ing loss . In addiing loss , the α-sing loss  and whosing loss . Furthering loss , an infling loss . Our resepilepsy , 66, 67,ing loss .GPR6 and encodes a G protein-coupled receptor. Interestingly, this receptor shows a high affinity for sphingosine-1-phosphate [The third SNP, rs4374858, is located within 20 kb of gene hosphate , a sphinhosphate .Many of the SNPs identified in this study, including genome-wide significant SNP rs7598759 in nucleolin, are located within introns. The means by which these polymorphisms might contribute to altering the pathways in which the associated proteins are involved is unknown, although we can speculate that they could affect gene transcript levels, as previously reported . This miDoes nucleolin dysregulation confer a predictive outcome for permanent threshold shift in humans? In practice, recovery from temporary threshold shift in human subjects varies from minutes to days. For example, human ears show significant recovery within hours from a 4-hour noise exposure at 100 dBA and full recovery within one week . In moreMany of the SNPs identified in this study are in genes that can potentially modulate apoptotic pathways and contribute to either temporary or permanent hearing loss. Indeed, although the loss of hair cells following activation of apoptotic signals clearly contributes to permanent hearing loss, it is not yet clear whether transient activation of these same pathways will contribute to temporary hearing loss . Until fIn the placebo arm of the overall Marine recruit study, 35% of subjects experienced a threshold shift when exposed to occupational noise, which is well above the anticipated 16% from noise exposure studies conducted in other occupational settings worldwide . LookingWe posit that strong environmental conditions, leading to exercise-induced inflammation and fatigue, contribute to NIHL susceptibility under certain occupational conditions. In this context, the state of inflammation and fatigue of an individual becomes a relevant risk factor of noise exposure in the work environment. Physiologic measures of stress, inflammation and oxidative stress were not evaluated in this study. However they are important factors to be explored in further studies to help place noise exposure in context with the genetic susceptibility to NIHL.Intensive exercise or prolonged sleep restriction periods similar to Marine recruits training might result in altered hearing threshold shifts. Several reports have shown a significant increase in expression of inflammation markers C-reactive and IL-6 proteins , 77 undeThere is also growing evidence that hearing loss is a risk factor in inflammation-driven diseases such as diabetes  and demeWSF1 (p < 7 x 10−4) identified in another impulse noise study [To address the question of whether different sets of genes will have an effect on the susceptibility to either impulse or continuous noise, we compared the SNPs genotyped in our study to previously reported SNPs associated with NIHL. Of the 46 SNPs within 23 candidate genes previously reported associated with NIHL , 15 SNPsse study . In contβ < 0.63), challenging its objective interpretation. However, the results of in vitro experiments confirm the main finding of this study supporting the potential role of nucleolin in NIHL susceptibility.Although the small sample size is a limiting factor, many of the results shown here converge with previously reported data and add more weight to previously identified polymorphisms associated with NIHL. Estimation of the power of this study, which depends on the NIHL prevalence and genetic model selected, is highly variable  goat anti-rabbit IgG Texas-Red conjugate used as antibody specific control. Cells were further counterstained with (A) DAPI to show nuclear profile and (C) phalloidin Alexa 488 conjugate to show actin cytoskeletal features. (D) The merge panel clearly shows nuclear and actin profiles and absence of non-specific staining. White scale bar represents 20 μm.(TIF)Click here for additional data file.S2 FigThis figure demonstrates the results of the principal component analysis to identify stratification in order to avoid bias. Eleven sub-populations from 1000 Genomes were tested to stratify the NIHL study population. Principal component 1, defined as axis of the linear least square fit of the tested population and principal component 2, which is defined as second axis of the linear least square fit orthogonal to principal component 1, show the largest genetic variance between ASW (African ancestry in Southwest USA), CEU (Utah residents with Northern and Western European ancestry), CHB , CHD (Chinese in Metropolitan Denver), GIH (Gujarati Indians in Houston), JPT (Japanese in Tokyo), LWK , MEX (Mexican ancestry in Los Angeles), MKK , TSI , YRI (Yoruba in Ibadan).(TIF)Click here for additional data file.S1 Table2 < 0.8). Linkage disequilibrium and distances between pairs of SNPs were calculated from either the 1000 Genomes [Genome-wide significant (peak) SNP rs7598759 is in strong linkage disequilibrium (LD) with the three suggestive SNPs (D' > 0.9) although some the correlation are low due to lower allele frequency (r Genomes  or HapMa Genomes  based on(XLSX)Click here for additional data file.


Sensorineural hearing loss occurs due to damage to the inner and outer hair cells of the peripheral auditory system. Hearing loss can cause decreases in audibility, dynamic range, frequency and temporal resolution of the auditory system, and all of these effects are known to affect speech intelligibility. In this study, a new reference-free speech intelligibility metric is proposed using 2-D neurograms constructed from the output of a computational model of the auditory periphery. The responses of the auditory-nerve fibers with a wide range of characteristic frequencies were simulated to construct neurograms. The features of the neurograms were extracted using third-order statistics referred to as bispectrum. The phase coupling of neurogram bispectrum provides a unique insight for the presence (or deficit) of supra-threshold nonlinearities beyond audibility for listeners with normal hearing (or hearing loss). The speech intelligibility scores predicted by the proposed method were compared to the behavioral scores for listeners with normal hearing and hearing loss both in quiet and under noisy background conditions. The results were also compared to the performance of some existing methods. The predicted results showed a good fit with a small error suggesting that the subjective scores can be estimated reliably using the proposed neural-response-based metric. The proposed metric also had a wide dynamic range, and the predicted scores were well-separated as a function of hearing loss. The proposed metric successfully captures the effects of hearing loss and supra-threshold nonlinearities on speech intelligibility. This metric could be applied to evaluate the performance of various speech-processing algorithms designed for hearing aids and cochlear implants. Performing listening tests with real subjects is a complex and time consuming process. It is also often difficult to measure the benefit of hearing aids which are intended to improve speech intelligibility for people with hearing loss. Speech intelligibility is reduced not only by the hearing loss, but also by environmental noise, room reverberation, speed of the speech by a speaker, distance from the speaker, intrinsic distortions within the auditory system including decreased frequency and intensity resolution at high presentation levels, central auditory nervous system dysfunction, and the nonlinear distortions introduced by the hearing aids. The assessment of speech intelligibility under adverse conditions has led to the development of several measurements and prediction procedures. The subjective scores could be predicted by replacing the human listener with a reliable model of the auditory system. A model that can predict the speech intelligibility scores for a given people with hearing loss would be an important tool for hearing-aid fitting or development of hearing-aid algorithms.Objective measurements of speech intelligibility are calculated from original and distorted acoustic signals using a number of mathematical formulas. The articulation index (AI) , speech The responses of a population of model auditory-nerve (AN) fibers can be represented as a “neurogram”, in which the spike counts for AN fibers tuned to different characteristic frequencies (CFs) are plotted as a function of time. The features are extracted from the neurogram to estimate the intelligibility score. For example, in STMI , the joiIn general, the second-order statistical measures, such as the autocorrelation function and power spectrum, work well for signals having a Gaussian  probability density function. For many decades, the estimation of the power spectrum of discrete-time deterministic or stochastic signals has been a primary tool for digital signal processing applications, including radar, sonar, communication, speech, biomedical, geophysical, and imaging systems –15. In pIn this study, the proposed method is motivated by the above-mentioned applications of HOS in the field of signal processing. Also, most of the existing acoustic signal property-based speech intelligibility metrics such as the AI, SII, STI, SRMR, and STOI use information mainly related to the second-order statistics of the signal. However, it is well-known that most auditory processing stages respond in a nonlinear way. As the auditory periphery is the sole conduit for acoustic information to reach the higher auditory centers, the inclusion of nonlinear transformation of acoustic signals in the model of the auditory periphery is necessary to determine how changes or deficits in certain underlying mechanisms in the healthy or impaired ear may affect perception. In this study, an auditory-nerve (AN) model developed by Zilany and colleagues , 25 has When a complex broadband sound is analyzed in the cochlea of a normal ear, the result is a series of bandpass-filtered signals, each corresponding to one position on the basilar membrane. Each of the band-passed signals can be considered as a slowly varying envelope (ENV) superimposed on a more rapid temporal fine structure (TFS). Both ENV and TFS information are represented in the timing of neural discharges, although TFS information depends on phase locking to individual cycles of the stimulus waveform . Thus, tThe following section describes the computational procedure of the proposed method. In addition, a brief description of the computational model of the auditory periphery is provided.Zilany and colleagues developed a computational model of the auditory periphery, and the responses of the model were validated against a wide range of physiological responses of AN fibers from the literature . The AN IHC ≤ 1 affects IHC transduction function and 0 ≤ COHC ≤ 1 affects the control path output  stands for the expectation operation, and * represents the complex conjugate.The HOS are spectral representations of moments and cumulants that are defined for deterministic signals and random processes. In this study, the features related to the third order statistics of the signal, referred to as the bispectrum, were considered. The bispectrum is the Fourier transform of the third order cumulant sequence of a stationary random process and is given by , 12B . The frequency is normalized by the Nyquist frequency to be between 0 and 1. The range of frequency indicates the extent of phase coupling between frequency components  is said to be quadratic phase coupled if and only if f3= f1+ f2 and ϕ3= ϕ1+ ϕ2. The phase relations are easily investigated by the quadratic phase coupling. The power spectrum cannot explain the phase relations between harmonic components whereas the bispectrum is suitable for detecting the phase relations in many applications [The bispectrum is a complex-valued function of two frequencies  is a symmetric function. For real processes, there are 12 symmetry areas in the bispectrum. The bispectrum in the triangular area (Ω) is calculated by the conditions, f2≥ 0, f1≥ f2, and f1+ f2 ≤ 1. The triangular area can describe the whole bispectrum as shown in B(Mave) and phase entropy (pe) [f1m, f2m) [p1, p2) [H1), the sum of logarithmic amplitudes of diagonal elements (H2), and the first-order spectral moment of amplitudes of diagonal elements in the bispectrum (H3). All the features were calculated in this work. However, the magnitude of some features did not change consistently as a function of hearing loss. This study only considered the H1, H2, and H3 features, because the magnitude of these features either increased or decreased consistently as a function of the degree of hearing loss.In this study, the features of the bispectrum were used to develop the proposed metric. It has been reported in the literature that different types of features can be extracted from bispectrum. These features include mean magnitude (opy (pe) , the wei1m, f2m) , and the[p1, p2) . Entropy[p1, p2)  such as H1, H2, and H3 are defied as:The features Two standard speech databases, Texas Instrument and Massachusetts Institute of Technology (TIMIT)  and NortThe NU6  databaseIHC) and outer hair cell (COHC) were adjusted to account for one-third and two-third of the threshold shift, respectively, for each CF. Both parameters were set to 1 for normal hearing and to 0 for complete impairment. The ENV and TFS neurograms were constructed using the output of the synapse model. The bispectrum was then estimated from the neurograms to predict intelligibility scores. The complete procedure of the proposed method is illustrated in The input to the AN model was the speech signal from TIMIT or NU6. The speech token was resampled at 100 kHz, as required by the AN model . In thisMave, f1m, f2m, p1, p2, H1, H2, and H3 [To estimate bispectrum, the direct FFT-based method implemented in the higher order spectral analysis (HOSA) Matlab toolbox  was empl, and H3 , 45–47 oH1, H2, and H3) in the bispectrum was relatively constant for two extreme conditions  irrespective of the input speech token, as shown in It was found that the magnitude of three features  for each of two phoneme groups  as well as from the neurograms of words (panels B and D). The results are shown for 100 samples (randomly chosen) in all cases. The upper panels  show the result for ENV neurograms, and the lower panels  represent the responses from the TFS neurogram. The results are shown for speech signals presented at a conversational speech level of 65 dB SPL.H3 for profound hearing loss (in quiet) was comparable to the values estimated under noisy condition (at an SNR of -100 dB) for listeners with normal hearing, which allowed the same normalization to be applied to estimate speech intelligibility under noisy conditions. Also the magnitude of the bispectrum features consistently changed for people with hearing loss under noisy conditions. Thus, the behavioral results were compared to the predicted scores in quiet and under noisy conditions for listeners with and without hearing loss.It was observed that the magnitude of Ymax and Ymin are the values of the feature in the bispectrum of response for listeners with normal-hearing and profound hearing loss, respectively. The magnitude of Ymax and Ymin  for the features  is provided in To obtain speech intelligibility scores between 0 and 1, the magnitude of a feature was normalized by the corresponding feature values for two extreme conditions . The score was computed using the following equation:This section describes the behavior of different bispectrum features as a function of hearing loss. In addition, the performance of the proposed method is evaluated and compared to the subjective scores from behavioral studies as a validation of the proposed metric.H1, H2, and H3) computed from the bispectrum of neural responses for different groups of phonemes. The phonemes were taken from TIMIT database, and the responses are shown for six phoneme groups . The phonemes were presented at 65 dB SPL. The normalized scores  from the TFS bispectrum did not show any substantial change from completely unimpaired to mild hearing loss. This effect can be explained primarily by audibility, because ENV cues are more prominent in higher CFs [It is clear that the scores for both ENV and TFS responses consistently declined for all phoneme groups as a function of the degree of hearing loss. Although the dynamic range of predicted scores was approximately same for both ENV and TFS responses, the normalized score from the ENV bispectrum showed a gradual roll-off with varying degree of hearing loss. On the other hand, the magnitude of normalized features  for both ENV and TFS responses changed as a function of hearing loss, which enables the proposed metric to predict speech intelligibility as a function of hearing loss. If hearing thresholds of the listeners are within 10–15 dB of the normal hearing, most behavioral studies treat them as normal listeners [H1, H2, and H3) declined consistently as a function of the degree of hearing loss, and the results were comparable to each other. In this study, the feature H3 estimated from the ENV bispectrum was chosen for all subsequent analysis, because the result using H3 reflected the representative behavior of normalized scores for all three features, and the scores from the ENV responses declined more gradually than the scores from the TFS responses as a function of the degree of hearing loss. To test the statistical significance of predicted scores, the level of significance (p) was calculated by using a pair wise (two adjacent hearing loss-profile) t-test for all phoneme groups. The level of significance was found to be less than 0.01 (p<0.01) for all cases.Overall, it is clear that the normalized magnitude of features  from TIMIT database and words from NU6 database. The scores were predicted for sound levels at 65, 85, and 95 dB SPL as a function of hearing loss, and the results are shown in In general, speech intelligibility is decreased even for listeners with normal hearing when speech is presented at higher-than-normal levels (above about 80 dB SPL) . DependiTo validate the proposed metric, the subjective speech intelligibility scores from a behavioral study by Studebaker et al.  were comOHC and CIHC) were adjusted to match the average hearing loss profile of the listeners who participated in the experiment. Scores were estimated using the proposed method and NSIM for 200 words from the NU6 database in quiet (clean) and under speech-shaped noise at SNRs of -1, 5, and 12 dB with a sound presentation levels of 69, 79, 89, and 99 dB SPL. The mean scores are shown in The audiogram of the hearing-impaired group was described by pure-tone hearing levels of 13, 15, 23, 40, 53, and 52 dB at the octave frequencies of 250, 500, 1000, 2000, 4000, and 8000 Hz, respectively. The parameters of the AN model  was also comparable to that of predicted scores (~0.15–0.8) for the proposed metric. However, the dynamic range of NSIM scores (~0.3–0.7) was lower than the range of subjective scores. Although a function could be applied to extend the dynamic range, a metric with a dynamic range comparable to that of behavioral responses may indicate a plausible strategy employed by the human auditory system to generate those responses.The linear regression coefficient for both metric was found to be same (~0.92). The root-mean-square error (RMSE) between the behavioral scores and the scores predicted by the fitted function was found to be 7.82 and 7.74 for the proposed metric and NSIM, respectively. The solid line indicates the linear fit, and the dotted lines represent the 95% confidence interval (C.I.) computed for 200 predicted intelligibility scores. All of the data fall within the confidence interval, meaning that the subjective scores can be reliably estimated using the proposed metric.H3) only. The mean subjective and predicted scores were computed across 200 NU6 words for each condition. For SII and the proposed method, the best fit between the subjective and predicted scores was achieved by a sigmoid rather than a linear function, which is described by the following equation:For the proposed method, the parameters were found to be c = 89, d = 3, m = 10, and k = 4.1, and for SII, the best fit was obtained for c = 92, d = 0.9, m = 10, and k = 4.8. However, the best fit for the STOI scores were achieved by a linear equation as in  with theIn general, the predicted scores progressively declined as the more noise was added to the original signal (in quiet), consistent with the subjective scores observed in the behavioral study. However, the dynamic range of subjective scores (~0–0.94) and SII scores (~0.1–0.96) was substantially higher than the dynamic range of scores for the proposed method (~0.3–1.0) and the STOI (~0.6–1.0). It is clear that the predicted scores using the proposed method fell within the 95% confidence interval of the fitted function, which indicates that the subjective scores could be predicted reliably using the proposed method. However, some of SII scores fell outside the 95% confidence interval of the fitted function. The (RMSE) between the subjective scores and the scores predicted by the fitted functions was found to be 8.14, 19.16, and 13.76 for the proposed method, SII, and STOI, respectively.In this section, the effects of the degree of hearing loss, noise, audibility, and sound presentation level on the bispectrum  are presented in order to illustrate how the proposed metric works. The distribution of predicted scores across phonemes and subjects is also described.As mentioned earlier, the bispectrum is a method to detect the presence of quadratic phase coupling among different frequency bands in a signal. To study how the distribution of phase coupling of a clean signal changes as a function of the degree of hearing loss, the contour maps of the bispectrum for a typical speech word (such as /use/ taken from NU6) are shown in The bispectrum was calculated after simulating the ENV responses of the AN model as a function of hearing loss. The bispectrum contour maps for listeners with normal hearing and profound hearing loss are shown in the panels A and B, respectively. The presence of phase coupling was indicated by the peaks in the bispectrum of the signal. It is clear that the extent (range of frequencies) and magnitude of phase coupling degraded substantially in the case of profound hearing loss as the signal is inaudible in most frequencies. For other intermediate profile of hearing loss, the feature values fell within these two extreme cases . Thus thIn order to illustrate the effect of noise on phase coupling for acoustic signal in the time domain (i.e. without using AN model responses), the bispectrum was calculated for the same word (/use/) under clean and noisy conditions  of supra-threshold nonlinearities incorporated in the AN model was successfully captured by the bispectrum features. This indicates that the predicted scores for the listener with hearing loss not only reflect the effect of audibility of the signal but also takes into account the effect of nonlinearities at higher presentation levels. It is to be noted that the magnitude of features at 100 dB SPL for the listener with flat 70 dB hearing loss was lower than the respective features at 65 dB SPL for a listener with normal hearing , and thuTypically, there is more variability in behavioral responses across subjects with hearing loss than for subjects with normal hearing , 62. HowIn general, it is assumed that any objective speech intelligibility measure having the same dynamic range and a linear relationship with the subjective scores would indicate a plausible strategy employed by the human auditory system to produce the behavioral responses. In this regard, it is obvious that although the proposed metric had a linear relationship with the subjective scores for listeners with hearing loss , the besPerceptual studies of speech generally use human subjects, and thus a specialized neural mechanism is thought to be responsible for speech perception. However, the AN model used in this study was largely based on the physiological data from animal studies, especially from cat. It is well-known that physiological properties of many neurons show strikingly similar properties in unrelated species such as rodents, cats, and bats, suggesting a more general auditory neural mechanism common to mammals including human. Thus, it is likely that although the predicted scores are based on the simulated neural responses for nonhuman species, a great deal can be learned about the neural processing of speech by human auditory system.OHC and CIHC, were chosen such that approximately two-thirds of the threshold shift measured in the audiogram can be attributed to OHC impairment and one-third to the IHC impairment. However, there is substantial variability across individuals [In order to model each audiogram, the values of two parameters, Cividuals . As a fu1, H2 and H3 estimated from the neurogram of sentences varied substantially depending on the length of the sentences (results not shown). Thus the proposed reference-free metric could not be extended to predict intelligibility for sentences.In the literature, the subjective speech intelligibility score is measured using phonemes, words, or sentences . HoweverThis study proposed a reference-free metric to predict speech intelligibility for people with hearing loss and also for listeners with normal hearing in quiet and under noisy conditions. For this, the responses of AN fibers were simulated to construct neurograms, and then bispectrum features were computed to estimate speech intelligibility. In this study, three features from the bispectrum were identified whose magnitude was relatively constant  for a particular profile of hearing loss, and these features consistently changed as a function of signal-to-noise ratio. Thus unlike other full reference-based metrics, the responses to clean signals were not required to predict speech intelligibility using the proposed method. The presence or deficit of supra-threshold nonlinearities captured in the neural responses for listeners with normal hearing or hearing loss was also reflected in the phase coupling information of the neurogram bispectrum. Although the dynamic range of feature values for TFS responses was larger compared to the feature values from ENV responses, the normalized score using ENV responses showed a relatively more gradual declination with hearing loss. The performance of the proposed metric was compared to the subjective scores from a behavioral study, and the results showed a good fit with a small error suggesting that the subjective scores can be estimated reliably using the proposed neural-response-based metric.


TMC1) can cause both DFNA36 and DFNB7/11 hearing loss. More than thirty DFNB7/11 mutations have been reported, but only three DFNA36 mutations were reported previously. In this study, we found a large Chinese family with 222 family members showing post-lingual, progressive sensorineural hearing loss which were consistent with DFNA36 hearing loss. Auditory brainstem response (ABR) test of the youngest patient showed a special result with nearly normal threshold but prolonged latency, decreased amplitude, and the abnormal waveform morphology. Exome sequencing of the proband found four candidate variants in known hearing loss genes. Sanger sequencing in all family members found a novel variant c.1253T>A (p.M418K) in TMC1 at DFNA36 that co-segregated with the phenotype. This mutation in TMC1 is orthologous to the mutation found in the hearing loss mouse model named Bth ten years ago. In another 51 Chinese autosomal dominant hearing loss families, we screened the segments containing the dominant mutations of TMC1 and no functional variants were found. TMC1 is expressed in the hair cells in inner ear. Given the already known roles of TMC1 in the mechanotransduction in the cochlea and its expression in inner ear, our results may provide an interesting perspective into its function in inner ear.Mutations in the transmembrane channel-like gene 1 ( Among these genes, eight of them are inherited in both autosomal dominant and recessive patterns, such as TMC1 (http://hereditaryhearingloss.org/).Hearing loss is the most common sensory disorder affecting one in 1000 births TMC1 was identified as a causative gene for both autosomal dominant (DFNA36) and autosomal recessive (DFNB7/11) non-syndromic hearing loss by Kurima and colleagues in 2002Tmc1 gene and found the p.M412K mutation in a hearing loss mouse model named Bth which was arisen in a large-scale ENU mutagenesis program TMC1 have been reported in DFNB7/11 families. TMC1 is identified as a common gene associated with non-syndromic hearing loss with a frequency up to 6.6% in Turkey Bth mouse mutation in the Tmc1 gene, which may have similar function consequences. However, the mutation orthologous to p.M412K in murine Tmc1 has yet to been found in human hearing loss family since it has been reported in 2002 TMC1 through sequencing the whole exome of the proband, which is important and beneficial to discover the pathologic mechanism of DFNA36 hearing loss caused by TMC1 mutation.The study was approved by the Committee of Medical Ethics of Chinese People's Liberation Army (PLA) General Hospital. All the samples were analyzed under the appropriate ethical approvals, and written informed consents were obtained from all subjects or caregivers. Next of kin, care takers or guardians consented on the behalf of minors/children participants whose capacity to consent was compromised.Family 1304 were ascertained from the Department of Otolaryngology, Head and Neck Surgery, at the Institute of Otolaryngology of PLA, Chinese PLA General Hospital. Members of this family were interviewed by a team of experienced ear, nose and throat doctors, and physicians to identify either personal or family medical evidence of hearing loss, tinnitus, vestibular symptoms, use of aminoglycosides, and other clinical abnormalities. Audiometric evaluations included pure tone audiometry using Madsen Orbiter 922 audiometer (Denmark), auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE) using SmartEP of Intelligent Hearing system (USA). The audiological data were evaluated based on the criteria established by European Working Group on Genetics of Hearing loss GJB2, SLC26A4 and mitochondrial DNA A1555G, were all excluded. As a comparison group, 100 unaffected individuals of matched geographical ancestry were recruited in this study.A total of 51 unrelated autosomal dominant hearing loss families were chosen as the other affected set for further analysis. In these families, mutations in common genes associated with hearing loss, such as Peripheral blood samples were obtained and genomic DNA was extracted according to standard procedures. Qualified genomic DNA samples (6 ug) of the proband were sheared by sonication. Then the fragment of each shared genomic DNA sample was hybridized to the SureSelect Biotiny lated RNA Library (BAITS) for enrichment.The enriched libraries were loaded on the HiSeq 2000 platform to be sequenced. Raw image files were processed by Illumina Pipeline v1.6 for base-calling with default parameters and the sequences of each individual were generated as 90 bp paired-end reads. Then the sequenced raw data was aligned to the NCBI human reference genome (NCBI 36.3) using SOAPaligner Insertions and deletions (indels) in the exome regions were identified through the sequencing reads. We aligned the reads to the reference genome by Burrows-Wheeler Aligner (BWA0.5.8) TMC1 was found to be co-segregated with the hearing loss in family 1304. As the candidate gene, the variant in TMC1 was screened in 100 unaffected individuals geographically matched. In addition, 51 unrelated autosomal dominant hearing loss families without GJB2, SLC26A4 and mitochondrial DNA A1555G mutations were chosen as another affected set for further analysis. For this cohort, exon16, exon19 and their flank sequences containing all the four mutations associated with dominant hearing loss were sequenced applying Sanger sequencing. Primer pairs were designed using the online Primer 3.0 software and synthesized by Invitrogen  in Family 1304 was a six-generation pedigree with 222 members, 35 of whom suffered hearing loss . This faThe proband (V:6) was a 23-year-old female suffering from hearing loss accompanied by tinnitus when she first visit the outpatient clinic in the year of 2005. She showed a severe hearing loss at 2 kHz and 4 kHz and a moderate hearing loss at 1 kHz and 8 kHz. Seven years later, the threshold increased about 15–30 dB HL at 1 kHz–8 kHz in the right ear, 10–15 dB HL at 2 kHz–8 kHz in the left ear . The thrTMC1), p. R382C (ESRRB), p.S385P (ESPN) and p.D2E (WFS1) located in the known hearing loss genes, which may attribute to the phenotypes of the family. Then these four variants were sequenced in all available members of family 1304 by Sanger sequencing. It was found that the mutation of c.1253T>A  in TMC1 gene completely co-segregated with the phenotype and all 18 patients were heterozygous on this site  to predict the biophysical consequences of this substitution and found that this variant is likely to be deleterious. Alignments of the amino acid sequences of TMC1 in human species, mouse, rat, macaque, dog, pig and chick as well as with human TMC2 and mouse Tmc2 showed that the mutation is located in a highly conserved position, which is homologous to the p.M412K mutation in the Bth mouse inherited in autosomal dominant pattern  from four DFNA36 hearing loss families have been reported Notably, for the first time, our study reported a dominant mutation of TMC1 is predicted to encode a transmembrane protein containing at least six membrane spanning domains, a cytoplasmic N- and C-termini, and a large cytoplasmic loop named TMC domain TMC1 indicates that this region should be important for the proper function of the protein.Tmc1 of Bth mouse model since it was found in 2002 Bth mouse showed progressive loss of the Preyer reflex from around P30 with appeared normal structure of middle ear and inner earTmc1 is expressed in both outer and inner hair cells from early stage of development Tmc1Bth allele implicated that Tmc1 was the component of the mechanotransduction channel in auditory hair cells of the inner ear Tmc1 reduced calcium permeability and single-channel currents TMC1 should act as a pore-forming subunit of the transduction channel and be involved in determining permeation properties It's noteworthy that, for the first time, we have found a family in human with the mutation orthologous to p.M412K in TMC1 showed a prolonged latency and abnormal waveform morphology for wave I to V, which was most likely a direct and causative link, although the mechanism was still obscure. The identification of the p.M418K in TMC1 in family 1304 makes the Bth mouse an excellent animal model to study the mechanism for autosomal dominant hearing loss caused by TMC1 mutation.To summarize, we found ABRs of the patients carrying the p.M418K mutation in Figure S1The distribution of per-base sequencing depth in target regions for each sample. Y-axis indicated the percentage of total target region under a given sequencing depth.(TIF)Click here for additional data file.Figure S2Cumulative depth distribution in target regions for each sample. X-axis denotes sequencing depth, and y-axis indicated the fraction of bases that achieves at or above a given sequencing depth. From the figure above, we can see about 75.50% of target region bases obtains at least 20x fold coverage, that is to say, about 75.50% of target region was covered by more than 20 reads. And about 89.10% of target region achieved at least 10x.(TIF)Click here for additional data file.Table S1Primer sequences for p.M418 in exon16 and p.D572 in exon19.(DOCX)Click here for additional data file.Table S2Summary of effective datafor exome sequencing. * The region near target refers to flanking region within 200 bp of target regions. ** Total effective reads is the same meaning as the unique mapped reads which was stated in the pipeline above. Here the effective reads consist of two parts: i) the reads have only one best hit in the alignment. These reads comes from the unique region of genome ii) the reads have multiple best hits on the genome (the number of hits between 1 and 20), and they were randomly aligned onto the target regions. These reads mainly come from low complex genomic region, such as repetitive sequences, and account for about 4% of total effective reads. *** Target regions used here refer to genomic regions that the Exome array actually covered. The aggregate length of target is about 37.8 Mb.(DOCX)Click here for additional data file.Table S3Summary of SNPs in Exome Sequencing for each Sample. * Consensus genotype with quality score of at least 20. ** Intronic SNPs within 4 bp of exon/intron boundary. *** 5' UTR refers to 200 bp upstream of initiation codon, 3'UTR is defined as 200 bp downstream of termination codon.(DOCX)Click here for additional data file.Table S4Summary of Indels in Exome Sequencing for each Sample.(DOCX)Click here for additional data file.


A negative association between resting metabolic rate (RMR) and lifespan is the cornerstone of the rate of living and free-radical damage theories of aging. Empirical studies supporting a negative association of RMR to lifespan may arise from the correlation between RMR and both daily energy expenditure (DEE) and thermoregulatory activity energy expenditure (TAEE). We screened 540 female mice for higher and lower DEE and measured RMR in the resulting 324 (60 %). We then selected 92 mice in which there was no link between residual from the regression of RMR against body mass (BM) and residual of DEE against BM to separate the effects of these traits. Lifespan was not significantly related to body mass, DEE and TAEE, but significantly negatively related to RMR. Fat-free mass (FFM) and fat mass (FM) were both significantly positively related to RMR. After removing the effect of FFM on RMR, the association between RMR and lifespan remained significantly negative; however, after statistically removing the effect of FM on RMR, the significant association between RMR and lifespan disappeared. We conclude that the negative association between RMR and lifespan is primarily due to the effect of FM, with FM positively related to both RMR and mortality and hence RMR negatively to lifespan. In 40 additional screened mice, greater FM was also associated with greater oxidative damage to DNA.The online version of this article (doi:10.1007/s11357-014-9731-3) contains supplementary material, which is available to authorized users. The idea that there is a negative relationship between the resting metabolic rate (RMR) and lifespan is at least 100 years old and probably originated with Rubner who observed in 1908 that larger, longer lived animals had lower metabolic rates—and in particular that the product of their metabolism (per gram) and lifespan was essentially constant (Rubner 1908). These data formed a cornerstone of the rate of living theory Pearl  and lateOne problem with diagnosing the effects of RMR on lifespan is that RMR is typically associated with other features of energy balance. For example, in our previous study of the effects of metabolism on lifespan in mice  of these for further screening of RMR. This initial screening involved plotting DEE against body mass and fitting a linear regression to the data using the least squares fit procedure. We then calculated the residuals to the fitted regression line and selected for inclusion those individuals with the highest and lowest residuals (top and bottom 30 %). Both variables were tested for normality using the Kolmogorov–Smirnov test prior to fitting the regression model, and the generated residuals and the plotted data were examined for any indication of non-linearity that might invalidate making a linear fit. Because a fitted regression is most strongly influenced by the data that are in close proximity to the regression line, eliminating 40 % of data that sat closest to the fitted regression line reduced the significance of the relationship between DEE and body mass. This procedure therefore reduced the covariance due to body mass differences between the individuals, which is a major factor influencing metabolic rates and food intake. In the selected 324 individuals, we measured the RMR based on measurements of oxygen consumption in the thermoneutral zone using indirect calorimetry , we plotted the residual RMR (to body mass) against the residual DEE (to body mass) and fitted a regression to the data. We sequentially eliminated 30 more individuals from this regression until it was no longer anywhere near to significance (P > 0.1). In total, we eliminated 192 individuals in this process. We retained a total of 132 individuals in which there was no association between residual RMR and the residual DEE estimated from the food intake. In this manner, we could then explore the association between RMR and lifespan uncontaminated by the covariance effects of RMR with both DEE and BM. We calculated the difference between RMR and DEE and called this the thermoregulatory and activity energy expenditure (TAEE). In 40 of these animals, we sought associations between energetic measures and measures of oxidative stress, while the remaining 92 were used in lifespan studies. There was a significant repeatability of body mass, RMR and residual RMR when comparisons were made between measurements at the screening age of 10 weeks and when they were 9–11 months old . We screened an initial population of 540 individually housed MF1 mice aged 10 weeks for daily food intake as a proxy for daily energy expenditure (DEE) see “” and retKlebsiella sp. in the sentinels, but not from the rooms where these animals were kept.All experiments were authorised by a local ethical review committee and carried out under UK Home Office regulations. All animals were maintained in specific pathogen-free facilities monitored using sentinel animals analysed at regular intervals. During the study, we had sporadic records of mouse parvovirus and Our aim in this work was to screen a large population of mice for aspects of their energy balance and then select from this population a subset where the covariances between components of balance were not significant. The overall selection process is summarised by the flow diagram in Fig. Mus musculus L.: outbred strain MF1) were purchased from Harlan UK Ltd., Oxon, UK, at 7 weeks of age, in six separate batches. After 1 week of acclimation, they were housed individually in shoebox cages (440 × 120 × 130 mm) with sawdust and paper bedding. The lights were maintained on a 12:12-L/D photoperiod (lights on at 0700 hours), and the ambient temperature was regulated at 21 ± 1 °C. Animals received pellet rodent food  and water ad libitum. After the acclimation period, we estimated the total DEE by measuring food intake over a 2-week period at 8–10 weeks of age. DEE and BM (per batch) were tested for normality using Kolmogorov–Smirnov tests and were both found to be normally distributed. Estimated DEE from food intake was positively related to body mass for each batch. Analysis of the distribution of residuals in relation to BM and visual inspection of the plot did not indicate the relationship to be non-linear. To minimise the influence of body mass effects, we selected 60 % of the individuals (n = 324) that had the highest 30 % and lowest 30 % residual intakes relative to the regression of intake on body mass. We eliminated the animals with the central 40 % residual values (n = 216). In the selected 324 individuals, we measured the RMR based on measurements of oxygen consumption in the thermoneutral zone using indirect calorimetry , we plotted the residual RMR (to body mass) against the residual DEE (to body mass) and fitted a least squares regression to the data. We sequentially eliminated 30 more individuals from this regression until it was no longer near to significance (P > 0.1). In total, we eliminated 192 individuals in this process. We retained a total of 132 individuals in which there was no association between residual RMR and the residual DEE estimated from the food intake.A total of 540 virgin female mice  in groups of four animals per cage. When the animals were aged 11 months, they were transferred to individual cages (440 × 120 × 130 mm) and kept for 2 weeks for acclimation. They then had their individual food intake, RMR and DEE measured using the doubly labelled water technique. We performed these measurements to confirm that there was still no relationship between the traits that had been used to select them when they had been aged around 4 months. In addition, we used an independent measure of daily energy expenditure (doubly labelled water) because of the known potential problems with equating food intake and total daily energy expenditure. Food intake and respirometry measurements were performed in the same way as described for the screening stage. Ninety two of these individuals were subsequently involved in a longevity screen and were kept in groups of four animals per large cage. When animals died, the groups were joined to form new groups of four to ensure that lifespan was not correlated with group size. The remaining 40 animals were used in laboratory assays for measurements of oxidative stress and antioxidant protection.This process of selection generates a subset of animals where there was no relationship between the residual RMR and the residual DEE at the age of about 4 months, which was confirmed when the animals were 11 months old using an independent method to measure the DEE. There was similarly no relationship at this age between RMR and TAEE. This selection process was the major strength of the current study because it uniquely allowed us to diagnose the effect of RMR on longevity uncontaminated by the covariance of RMR with either total DEE or TAEE.n = 40) and a subsample of 53 animals kept for longevity had their body composition measured using dual-energy X-ray absorptiometry DXA instruments GE (previously Lunar) PIXImus2 Series Densitometers installed with software version 1.46.007 . DXA scans and analyses were carried out as instructed by the manufacturer. Mice were weighed and then anaesthetised by 3.5 % isoflurane  inhalation for the duration of the X-ray scanning (~3 min). Lunar PIXIMUS 2.10 software was used to calculate total lean mass, total fat mass, total bone mineral density and total bone mineral content in the region of interest  using a previously described protocol . Mortality rate comparisons performed using Kaplan–Meier analysis of survival revealed no difference between the two groups  with no difference in mortality rate between the two groups of DEE . Also, we found no difference between mean lifespan for comparison between low and high TAEE animals  and no difference in mortality rate between the two groups . The comparison between RMR longevity curves confirmed that animals with low RMR  lived longer than mice with high RMR  by about 10 %. Kaplan–Meier analysis of survival revealed a significant difference in mortality rate between the two groups .In the selected sample of 92 individuals that were retained for lifespan analysis, there was no significant relationship between lifespan and body mass : F1,90 = 58.0, P < .001, R2 = 0.39, b = 0.39), but especially by differences in body composition. Fat-free mass (FFM), which accounts for the larger part of body mass, is directly related with RMR, and fat mass (FM) has a smaller but similarly positive contribution to RMR . In this sample, the negative relationship between lifespan and RMR was confirmed . To discriminate the effect of FM and FFM on RMR and consequently their putative effects on lifespan, we used the residuals from two separate analyses. In the first, we performed a regression analysis of RMR on FM , but there was no effect of fat-free mass on longevity . We repeated the analysis using soft lean tissue mass obtained by excluding bone from fat-free mass, and we got the same results (not shown here).RMR is known to be affected not only by variations in body mass, an effect we also confirmed . The relationship between body mass and markers of oxidative stress  was significant only for DNA damage (Table n = 40). Of the antioxidant enzyme activities  there was a significant negative relationship with body mass for GPx; no significant correlation was found for the antioxidant adsorbent test (OXY)  Table . Due to P = 0.15). Prior studies have also shown positive effects of physical activity interventions on average survival time in rodents Below is the link to the electronic supplementary material.


Tympanoplasty is a standard procedure to repair tympanic membrane perforation. The aim of this study is to evaluate the results of tympanoplasty (hearing improvement and tympanic membrane closure rate) in patients suffering from chronic perforation of the tympanic membrane by considering the prognostic factors.In a prospective study, based on the results of tympanoplasty with temporal graft fascia in 60 patients in the ENT department of the Medical Science University of Tabriz, we evaluated prognostic factors, such as age, sex, smoking, size, and site of perforation, for the outcome of this surgery.The rate of surgical success- integration of the graft- was 93.3%. Improvement of hearing, as demonstrated through audiometry, occurred in 93% of cases. We did not find any factors to be statistically signiﬁcant to affect surgical outcome.Even by considering the influence of different factors on the results of a tympanoplasty operation, according to the statistical results of this study, there is not a significant difference in the results of the operation, neither in the health of the tympanic membrane after surgery nor in hearing development.  Chronic otitis media (COM) is defined as an inflammation of the middle ear with signs of infection lasting three months or longer. Chronic suppurative otitis media (CSOM) is defined as a chronic inflammation of the middle ear and mastoid cavity, which presents with recurrent ear discharges or otorrhoea through a tympanic perforation. The presence of a persistent tympanic perforation and middle ear discharge differentiates CSOM from other forms of chronic otitis media . CSOM has been known since pre-historic time. Contributing factors are: low living conditions, poor personal hygiene, and diet . PerforaMany factors have been investigated to determine their effect on the tympanic membrane closure rate and hearing improvement. Various studies on tympanoplasty have been conducted, which show that success rate and criteria for success vary from author to author. Some studies demonstrated that surgical outcome depends on several factors including size and location of the perforation, ossicular status, type of surgical technique, graft type and function of the eustachian tube. However, according to many authors, surgical outcome is independent of factors that are deemed as relevant -10. It is difficult to compare these studies because of differences in age, definition of success, the method used, and the experience of the surgeon. Thus, the factors affecting tympanoplasty must be studied depend of the conditions of each study.A total of 60 patients who underwent tympanoplasty operations between June 2013 and September 2014 were reviewed in this study. The age of the patients was between 18 to 49 years. Underlying diseases such as diabetes or immunodeficiency, cholesteatoma and erosion ossicular, presence of sensorineural hearing impairment, definitive diagnosis of tympanosclerosis, revision cases were the exclusion criteria for the study. Patient evaluation was undergone through proper history noting and thorough examination. Examination was performed radiologically if needed. For example, if there was a suspected retraction pocket, the patient was evaluated with computer tomography. Examination could also be performed audiologically and finally examined under microscope. The operation method used on all patients was the same. The post auricular approach, underlay technique, and harvesting of the temporalis fascia as the graft material was used in all the tympanoplasty procedures and the surgeon and its assistant were stable in all surgeries. One week after the operation, the status of the surgical wound was evaluated and packing was removed. Patients were followed up postoperatively up to 3 months. During the follow-up, the condition of the wound and the tympanic membrane was noted. An audiogram was performed on the 12th weeks to assess the outcome. Data were analyzed using the chi-squared tes and t-test on a spss statistical package . A P<0.05 was the level of significance.For the 60 tympanoplasties, the male to female ratio was 1:2 and the age ranged between 18 to 49 years . The distribution of patients by age and sex is shown in The perforation sites were anterior maleolar , posterior maleolar, , and central . The most common perforations were small (less than 50% of the tympanic membrane). 30% of patients were smokers and 70% were non-smoker.The overall graft success rate was 93.3% (56 of 60 patients). Based on the univariate analysis, size of perforation (P=0.08), site of the perforation (P=0.26), smoking (P=0.36), age (P=0.36) and sex (P=0.143) no statistical signiﬁcance was observed as prognostic factors .The average air–bone gap improvement for all 60 tympanoplasty procedures was 18.8 dB±5.62 SD. Serviceable hearing (air–bone gap<20 dB) was achieved in 93.3% of the 60 tympanoplasties postoperatively. The mean preoperative air–bone gap was 28.41 dB±5.4 SD and the mean postoperative air–bone gap was 9.56 dB ± 6.07 SD. The inﬂuence of the prognostic factors on hearing improvement in the 60 tympanoplasties is shown in Tympanoplasty is a surgical technique for the treatment of patients with chronic otitis media (COM). The goal of tympanoplasty is to eradicate disease and restore the function of the middle ear .In order to repair tympanic membrane perforation, various materials including areolar connective tissue, fascia, prechonderium, periosteum, skin, fat, veins, and alloderm mucosa are used as a graft ,11. In tThe mean air-bone gap improvement in our study was 18.8 dB ± 5.62 SD and air-bone gap was significantly decreased postoperatively (P=0.002). Karela et al indicated hearing improvement in 91.5% of cases and stated that myrangoplasty is a procedure that can be successful in many cases, regardless of age, gender, location, and size of the perforation . However, there was no statistically significant correlation between sex and success rate, which was similar in other studies ,6,21 .Em0% vs 40%Based on several studies, the highest hearing loss occurs with the perforation of the large central and the lowest hearing loss occurs with the perforation of the anterior central. Larger perforations produce more pronounced losses; therefore, the size of perforation is an important factor for hearing loss. However, below 10% of the tympanic membrane, perforation size does not influence hearing ,22. In oZhang and colleagues demonstrated that after myrangoplasty for small perforation of the tympanic membrane (less than 50%) ABG is minimum (average 5.5dB) and most ABG average 10.5 dB) was after closure of large perforations (more than 50%) . Lee and.5 dB wasKarela and et al, examined the outcome of myrangoplasty and hearing improvement and stated that the size and location of the perforation has no effect on the tympanic membrane closure rate and hearing improvement . Other sYung et al.'s study concluded that a large central, central maleolar, and central posterior perforation show the most hearing loss and that posterior inferior perforations cause greater hearing loss than anterior inferior perforations . Ahmad aThere are several studies examining the effects of the location of the perforation on the surgical outcome. Jurovitzki stated that an anterior perforation was less favorable than perforations seen in other locations . GonzaleControversy exists in the results of tympanic membrane closure rate and hearing improvement in smoker and non-smoker patients. In a study of 132 patients, performed by Cantrell, success rate in non-smokers was 92.5% and 43.7% in smokers .Becavarovski, in the study of 74 tympanoplasty surgeries, showed that delayed complications of surgery, such as severe atelectasis or delayed perforation after 6 months, was 20% in non-smokers and 60% in smokers (P<0.5) .Onal et al reported tympanic membrane closure rate to be 78.7% and 47.4% in non-smokers and smokers, respectively (P=0.008) ( Other studies have not shown that smoking is a prognostic factor for graft integration, but did demonstrated that it had negative effects on the long-term results of surgery .In our sEven though many different factors can influence the results of a tympanaplosty operation, according to the statistical results of the study, there is no significant difference in the outcome of the operation, neither in the health of the tympanic membrane after surgery nor in hearing development. However, more studies on more samples in various centers sould be conducted in order to make an acceptable conclusion.