A case study is presented of a 17-year-old male who sustained an anoxic brain injury and sensorineural hearing loss secondary to carbon monoxide poisoning. Audiological data is presented showing a slightly asymmetrical hearing loss of sensorineural origin and mild-to-severe degree for both ears. Word recognition performance was fair to poor bilaterally for speech presented at normal conversational levels in quiet. Management considerations of the hearing loss are discussed. Sensorineural hearing loss has been reported with both acute –4 and chThe patient was a 17-year-old male who was in previously good health with no history of hearing loss or significant medical issues. He was found at home and was unresponsive due to carbon monoxide (CO) poisoning. He was sleeping on the family couch in the living room on the first floor. Due to a hurricane-related power failure, a diesel generator was running in the family's connected garage. The patient was later found unconscious on the couch. Other family members were also found unconscious on the scene; not all of the family members survived the CO poisoning. The patient had been unconscious for an unknown period of time and was taken to a shock trauma center by emergency personnel arriving at the scene. He was intubated due to his level of consciousness. Upon arrival at the shock trauma center, a CT scan of the head showed no signs of obvious pathology. A chest X-ray showed evidence of a small focal consolidation on the lower right lobe. There was evidence of mild bilateral calcification suggesting pulmonary edema. On the evening of his admission, he was taken to a hyperbaric oxygen chamber. The day after his admission an MRI was administered which showed some evidence of anoxic brain injury. He was found to have diffuse white matter cytotoxic edema which involved the bilateral frontal, parietal, and temporal lobes. He was later extubated while at the shock trauma center. By the time of his discharge from the shock trauma center he was able to follow some simple commands and was able to pass a PO diet. However, he remained nonverbal. He was discharged when believed to be stable after nine days to an inpatient brain injury rehabilitation facility. Prior to his transfer his auditory status was not formally assessed. Upon admission to the rehabilitation facility he was arousable for short periods of time and was able to respond to yes-no questions with a head nod. Initially he was uncooperative with physical examination. He would attempt to verbalize and was able to follow one-step commands. Overall he had generalized weakness and was flaccid on the left with an increase in tone on the right. His sensation was intact on all four extremities as were deep tendon reflexes. He was diagnosed at admission with gait dysfunction, dysphasia, and generalized weakness. He received intensive physical, occupational, and speech language pathology therapies during his admission where he remained for 23 days. Initially, he appeared to be aphasic. Over the course of his admission his alertness level normalized, and he became more oriented. His speech and language skills reemerged, and he was able to tolerate a regular diet. He was discharged to home after 16 days with 24-hour supervision required. His hearing was never formally assessed during the admission.He subsequently received 13 days of intensive physical therapy, occupational therapy, and speech and language therapy at a day program three days a week. He also received an in-depth neuropsychological evaluation prior to returning to school as well as grief counseling. He was diagnosed at this time with mild mixed aphasia and mild-moderate cognitive deficits. While receiving speech and language pathology services at the day rehabilitation facility, the patient complained of difficulty in hearing to the speech and language pathologist and was referred for an audiological evaluation.The patient was seen for an audiological evaluation 54 days after his exposure to CO. He denied tinnitus. The obtained audiogram is shown in The present patient demonstrated a bilateral mild-to-severe sensorineural hearing loss in the high-frequency region secondary to CO poisoning. The absence of otoacoustic emissions indicates that the impairment in co poisoning is present at the level of the outer hair cells and is not confined to neural levels as has been suggested previously . Absent  The mechanism responsible for hearing loss in CO poisoning has not been definitively established but is likely due to hypoxic effects on tissue . The damAudiological assessment is not routinely offered in cases with brain injury at some rehabilitative centers. This case illustrates the importance of audiological assessment in cases of brain injury secondary to carbon monoxide poisoning. This patient was provided with intensive rehabilitative services without consideration of the presence of the possibility of a communicatively handicapping hearing loss. When he was provided with some assistance to address the hearing loss, some immediate improvement was noted in his ability to respond appropriately to communication.


GPR40 (FFAR1 or FFA1) is a G-protein-coupled receptor (GPCR), primarily expressed in insulin-producing pancreatic β-cells and incretin-producing enteroendocrine cells of the small intestine. Several GPR40 agonists, including AMG 837 and TAK-875, have been disclosed, but no GPR40 synthetic agonists have been reported that engage both the insulinogenic and incretinogenic axes. In this report we provide a molecular explanation and describe the discovery of a unique and potent class of GPR40 full agonists that engages the enteroinsular axis to promote dramatic improvement in glucose control in rodents. GPR40 full agonists AM-1638 and AM-6226 stimulate GLP-1 and GIP secretion from intestinal enteroendocrine cells and increase GSIS from pancreatic islets, leading to enhanced glucose control in the high fat fed, streptozotocin treated and NONcNZO10/LtJ mouse models of type 2 diabetes. The improvement in hyperglycemia by AM-1638 was reduced in the presence of the GLP-1 receptor antagonist Ex(9–39)NH GPR40 is a 7-transmembrane receptor that responds to a plethora of long-chain fatty acids in vivoGPR40 has gained considerable interest as a target for type 2 diabetes from pharmaceutical companies, investigators and the medical community in vivo, including in phase 2 clinical trials of 12-weeks in duration with TAK-875 Previous disclosures have described the discovery of orally bioavailable GPR40 agonists that displayed efficacy in a variety of rodent diabetic models without exhibiting hypoglycemia 50 = 0.12±0.01 µM, n = 26, 29% Emax) compared to the natural free fatty acid ligands, confirming previously published results of partial agonism Characterization of AMG 837 in an assay monitoring calcium flux in CHO cells (a CHO cell line stably expressing GPR40) revealed that it displays partial agonist activity against GPR40 . Interestingly, evaluation of the single enantiomers of this full agonist lead (±)-AM-8596 revealed that the (S)-AM-8596 enantiomer was a partial agonist  while the less potent enantiomer, (R)- AM-8596, displayed the desired properties of full agonism , prompting us to explore this chemical lead for further optimization of its properties.Further examination of our collection of GPR40 agonists synthesized during the discovery of the partial agonist AMG 837 R)-AM-8596 for a trans-1-propenyl group provided AM-8182 that displays a modest increase in potency and maintains full agonism . Three further chemical modifications: exchange of the trans-propenyl group for a cyclopropane, relocation of the ether linkage from a para- to an ortho-orientation on the central phenyl ring, and addition of a 5,5-dimethylcyclopent-1-enyl group to the biphenyl moiety led to the discovery of the potent full agonist AM-1638 (max). Further optimization of AM-1638 resulted in the discovery of AM-6226 .Addition of a fluoro substituent to the terminal aryl ring and exchange of the propynyl moiety of   and GPR43 (FFAR2) up to 10 µM, but had weak activity on the lipid activated receptor GPR120 (EC0 ∼5 µM) . Both ag0 ∼5 µM) .50 = 0.99±0.15 µM (n = 6) and EC50 = 0.94±0.6 µM (n = 3), respectively. Because AM-1638 and AM-6226 had overall similar properties in recombinant-cell expressing GPR40 functional assays and mouse islets, we focused most of the subsequent experiments on AM-1638.The divergent pharmacology displayed by GPR40 full agonists AM-1638 and AM-6226 from AMG 837 motivated us to elucidate these compounds’ effects on isolated primary cells, which would more closely mimic the endogenous receptor expression levels. Mouse islets were isolated and potentiation of insulin secretion at 16.7 mM glucose was tested with different doses of AMG 837, AM-1638 and AM-6226. Both AM-1638 and AM-6226 increased insulin secretion 3–4 fold from that of AMG 837 . The potTo understand the effects on the kinetics of insulin secretion, AM-1638 and AMG 837 were tested at 10 µM in an islet perifusion system in the presence of both 3 mM and 16.7 mM glucose. Under conditions containing 3 mM glucose, neither AMG 837 nor AM-1638 stimulated insulin compared to the vehicle control . In the These results were extended to isolated primary human islets cells. Measuring the accumulation of the second messenger inositol phosphate in response to agonist treatment, we observed that both AM-1638 and AM-6226 potently stimulated IP production in human islets with ∼8–10×greater efficacy than AMG 837 . Stimulain vitro by using cells isolated from fetal rat intestines in vitro but the magnitude of effect was greater with AM-1638 . Glucose AUC following improved 15, 23, 35 and 48% at 10, 30, 60 and 100 mg/kg AM-1638, respectively . These e50 values for these agonists on the mouse GPR40 inositol phosphate accumulation assay (AM-1638: EC50 = 12.9±1.4 nM (n = 2); AMG 837: 11.0±0.05 nM (n = 2)).In order to understand the mechanisms underlying the improved glucose clearance, we measured insulin levels in response to AM-1638 treatment. AM-1638 at the higher doses stimulated greater insulin secretion 1.5–2.2 fold greater than AMG 837 . In ordeThe increase in insulin secretion by GPR40 agonists is due to direct stimulation of GPR40 receptors on pancreatic β-cells, but may also be derived from stimulation of the gut peptides GLP-1 and GIP, which subsequently act on the GLP-1 and GIP G protein-coupled receptors found on pancreatic β-cells. AM-1638 and AMG 837 were also tested for stimulation of secretion of the incretins GLP-1 in HF/STZ mice. A single dose of AM-1638 or AMG 837 was administered by oral gavage, and blood GLP-1, insulin and glucose levels were measured at various time-points following dosing. No glucose challenge was performed in order to isolate the effects of the compounds alone. The full agonist AM-1638 differentiated itself from the AMG 837 by exhibiting a markedly greater increase in both plasma GLP-1 and insulin levels . While pIn order to confirm and expand these results to a different rodent type 2 diabetes model, we tested the effects of AM-1638 in the NONcNZO10/LtJ (RCS10) mouse model of diabetes. NONcNZO10/LtJ mice retain an intact leptin pathway but contain polygenic defects contributing to moderate and mature onset obesity and diabetes in vivo. Wild type and GPR40 knockout mice were tested in an OGTT following a single dose of AM-1638 or sitagliptin, which exerts its effects via inhibition of DPP-IV 50 ∼ 5 µM, in vivo was dependent on GPR40. A single dose of AM-1638 at 100 mg/kg elicited an increase in plasma GLP-1 levels in wild type but not GPR40 knockout mice NH2 NH9–39)NH2  E, F, G.9)NH2  can be envisioned which may result in enhanced glucose control. While tachyphylaxis, loss of glucose-dependence in insulin secretion from islets, or potential harmful consequences on the pancreatic β-cell would continue to be a concern, some of these might be less evident on intestinal enteroendocrine cells given their rapid turnover.While here we have reported the engineering and characteristics of potent, full GPR40 agonists, this research opens doors for targeting GPR40 with other classes of ligands. In recent years the plasticity of GPCR ligand-receptor interactions has gained appreciation in vivo may be influenced by the presence of free fatty acids, and vice versa. Future studies should examine the effect of co-administering AMG 837 and AM-1638/6226 with a lipid infusion or a mixed meal. Additionally, the pharmacology of AMG 837 and AM-1638/6226 on islet insulin secretion in vivo as a function of varying serum free fatty acid concentrations and/or different types of free fatty acids is an area of focus.In addition to the enhanced functional activity, AM-1638 and AM-6226 also bind to a distinct site on GPR40 from AMG 837, as determined by competition binding experiments . In factIn conclusion, we have discovered a potent, efficacious class of GPR40 agonists with enhanced efficacy compared to AMG 837. Undoubtedly, these agonists will open new doors of investigation into GPR40 pharmacology and potential therapies for type 2 diabetes.AMG 837 and all AM- compounds were synthesized by the Amgen Therapeutics Discovery Department, South San Francisco, CA. Detailed synthesis and analytical properties are included in the 3H-AMG 837 (specific activity of 52.3 Ci/mmol) and 3H-AM-1638 (31.4 Ci/mmol), were custom synthesized at Movarek. Competition binding assays were carried out in 20 mM Hepes pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) free fatty acid BSA and bound radioligand was collected on 96-well GF/C filter plates.GPR40 aequorin, plasmid titration, and inositol phosphate assays were performed as described http://icr.coh.org). Islet inositol phosphate assays were performed on islets cells that had been dispersed with trypsin. 10,000 islet cells were plated/well of a 96-well plate and were loaded with 3H-myo-inositol (0.5 µCi/well) in RPMI overnight prior to treatment. The next day, response to different stimuli was tested in HBSS containing 10 mM LiCl and 0.1% HSA (w/v) for 1 hour at 37°C. Accumulated 3H-inositol phosphate was measured after capture to SPA beads. Islet perifusion experiments were performed on mouse islets loaded into a Brandel Suprafusion system. Twenty islets were size-matched and loaded per perifusion chamber. Islets were perifused in KRBH containing, sequentially, 3 mM glucose, then 3 mM glucose containing 10 µM drug, 16.7 mM glucose containing 10 µM drug and finally returned to 3 mM glucose containing 10 µM drug. Insulin was measured using an insulin ELISA kit (Alpco). Fetal rat intestinal cells were isolated from e19-day old fetuses of pregnant Sprague-Dawley rats. Briefly, fetal intestines were isolated and digested in HBSS buffer containing 0.5 mg/ml collagenase (Sigma), 0.5 mg/ml of hyaluronidase (Sigma) and 0.05 mg/ml of deoxyribonuclease I (Roche) for 30 minutes at 37°C. The digestion was quenched using HBSS containing 15% FBS. Fetal rat intestinal cells were washed and cultured overnight in DMEM containing 5% FBS in 96-well plates. To test for secretion of GLP-1 and GIP in response to treatments, we incubated cells for 1 hour with treatments in KRBH buffer containing 0.05% BSA (w/v). GLP-1 and GIP1 secreted into the media was measured using ELISA kits specific for GLP-1 (Linco) or GIP (Millipore).Mouse islets were isolated and tested for stimulation of GSIS as described ad libidum access to chow and water. The HF/STZ mice were established as previously described ip to induce beta cell dysfunction. The animals continued on the same high fat diet for 3–6 additional weeks. Animals were then randomized into treatment groups based on fasting blood glucose and insulin measures. NONcNZO10/LtJ (RCS10) male mice (Jackson labs) were used at 12-weeks of age and randomized for treatment based on fasting glucose levels. For OGTT experiments, drugs were formulated in 1% HPMC, 10% HPbCD, 1% Tween-80 and dosed via oral gavage. Glucose was administered at 2 g/kg 1-hour post drug dose by oral gavage.All procedures on animals were approved by the Amgen San Francisco Institutional Animal Care and Use Committee (approved protocols #11–04 and #2007–00106). The Amgen San Francisco vivarium is an AAALAC accredited facility. Animals were housed under a 12-h light, 12-h dark cycle (lights-on 0600 h and lights-off 1800 h) and were allowed 2 (Bachem) were performed with male C57BL/6j (n = 6–7/group) at 26-weeks of age. Ex(9–39)NH2 was formulated in PBS containing 0.1%BSA and dosed ip at 300 µg/kg. Fifteen minutes later, animals were dosed with vehicle or AM-1638 by oral gavage. Glucose was administered by ip injection at 2 g/kg 30-minutes following the AM-1638 or vehicle dose.IPGTT experiments with Ex(9–39)NHFor all glucose tolerance tests, glucose and insulin measurements were taken from tail vein samples at various time-points. Blood glucose values were measured on an Accu-Chek glucometer (Roche Diagnostics), while insulin levels were determined using an ELISA (Alpco).Data were plotted as the mean ± SEM. One-way or two-way ANOVA were carried out using Prism (GraphPad Software) and statistical significance was considered meaningful at p<0.05.Figure S1AMG 837, GW-9508 and TUG-424 are partial agonists. (A) Structure of selected GPR40 agonists. (B) GPR40 agonists were tested in parallel in vitro in an inositol phosphate accumulation assay in A9 cells stably expressing GPR40.(TIF)Click here for additional data file.Figure S2AM-1638 and AM-6226 tested on GPR41, GPR43, GPR120 and PPAR-gamma assays. AM-1638 and AM-6226 were tested against various receptors in cell-based assays. (A) GPR41 (FFAR3) aequorin assay. GPR41 was force coupled to the calcium signaling pathway by co-transfecting the cells with Gqmyri5. (B) GPR43 (FFAR2) aequorin assay. The short chain fatty acid propionate acts as a natural agonist ligand for GPR41 and GPR43. (C) GPR120 aequorin assay. The unsaturated fatty acids α-LNN and DHA act as natural agonist ligands for GPR120. (D) PPAR-gamma luciferase assay using the thiazolidinedione rosiglitazone as a positive control. AM-1638 and AM-6226 also did not activate PPAR-alpha and PPAR-delta in luciferase reporter assays in concentrations up to 10 µM .(TIF)Click here for additional data file.Materials and Methods S13H-AM-1638 and 3H-AMG 837.Chemical Synthesis of AMG 837, AM-1638, AM-6226, AM-8596, AM-8182,  Additional materials and methods describing the chemical syntheses of key GPR40 (FFAR1) agonists studied in this report.(DOC)Click here for additional data file.


The genetic basis of autosomal dominant nonsyndromic hearing loss is complex. Genetic factors are responsible for approximately 50% of cases with congenital hearing loss. However, no previous studies have documented the clinical phenotype and genetic basis of autosomal dominant nonsyndromic hearing loss in Mongolians.TECTA gene, which encodes α -tectorin, a major component of the inner ear extracellular matrix that contacts the specialized sensory hair cells.In this study, we performed exon capture sequencing of a Mongolian family with hereditary hearing loss and identified a novel mutation in The novel G → T missense mutation at nucleotide 6016 results in a substitution of amino acid aspartate at 2006 with tyrosine (Asp2006Tyr) in a highly conserved zona pellucida (ZP) domain of α-tectorin. The mutation is not found in control subjects from the same family with normal hearing and a genotype-phenotype correlation is observed.TECTA gene is a characteristic TECTA-related mutation which causes autosomal dominant nonsyndromic hearing loss. Our result indicated that mutation in TECTA gene is responsible for the hearing loss in this Mongolian family.A novel missense mutation c.6016 G > T (p.Asp2006Tyr) of  Hearing loss is one of the most common sensory disorders in humans. Genetic factors are responsible for approximately 50% of cases with congenital hearing loss ,2. WhileTECTA mutations have been identified in various types of hearing loss, age of onset, progression and frequency involvement in various populations [TECTA can range from mild to severe and have pre or postlingual onset [As one of causative genes of ADNSHL, ulations -13. Thisulations . The α-tulations . Missensulations . PhenotyTECTA gene. This novel mutation causes the aspartate to tyrosine substitution (Asp2006Tyr) in the highly conserved ZP domain of α-tectorin. We further studied the genotype-phenotype correlation of mutation in TECTA gene with control subjects from the same family with normal hearing and confirmed that mutation in TECTA gene is responsible for the hearing loss in this Mongolian family.In this study, we examined the genetic basis of ADSHNL in a Mongolian family with hereditary hearing loss by exon-capture sequencing of known genes associated with hearing loss and identified a novel mutation in Patients of a Mongolian family suffered severe hearing impairment were diagnosed in the Affiliated Hospital of Inner Mongolia University for the Nationalities. Clinical examination showed that none of these patients has any other associated neurological signs, visual dysfunction or diabetes mellitus. Nineteen members of this Mongolian family participated in the study, of which fourteen showed clinical symptoms of hearing impairment. The study protocol was approved by the Committee on Clinical Investigation at the Inner Mongolia University for the Nationalities University. Informed written consent was obtained from all study participants and the study was in accordance with regulations by the Inner Mongolia University for the Nationalities University.Hearing impairment was evaluated by pure tone audiometry (PTA) in a sound-controlled room at frequencies ranging from 500 to 8000 Hz, according to standard protocols. The severity of hearing loss was defined as follows based on the mean PTA using the thresholds measured at 500, 1000, 2000, and 3000 Hz: normal hearing, below 20 dB; mild hearing impairment, 21 - 40 dB; moderate hearing impairment, 41 - 70 dB; severe hearing impairment, 71 – 95 dB; and profound hearing impairment, > 95 dB . The ran21 was randomly fragmented by Covaris  into segments ranging from 200 bp to 300 bp. These DNA fragments were purified and quantified, and then adapters were ligated to both ends. After amplified by ligation-mediated PCR (LM PCR), adapter-DNA fragments were hybridized to the capture-array of BGI  to capture all target genes for 72h. After washing off non-hybridized fragments, captured library which contains the targeted exon sequences of 54 known genes associated with hearing loss was loaded on the Hiseq2000 platform . Raw image files were processed by Illumina base calling Software 1.7 for base calling with default parameters and the sequences were generated as 90bp paired-end reads.Approximately 4 mL of peripheral blood were collected from each study participant and genomic DNA was extracted using the QIAamp DNA Blood Mini Kit . The qualified genomic DNA sample of the affected subject IIIhttp://www.broadinstitute.org/gsa/wiki/index.php/, The Genome Analysis Toolkit), respectively. All variants of the samples were filtered by databases including dbSNP137 (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/snp137.txt.gz), HapMap project (ftp://ftp.ncbi.nlm.nih.gov/hapmap), 1000 Genome Project (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp), and an in-house database of BGI. After filtering, variants were further analyzed by SIFT and PolyPhen to predict the effect of mutation on the function of encoding proteins. Only variants that had significant impact on the function of the target protein were further analyzed.After removing adapter sequences and low quality sequence reads from the raw data, the remaining clean reads were mapped and aligned to the human reference genome (NCBI build 37.1/hg19) using Burrows Wheeler Aligner (BWA). SNPs and indels (insertion and deletion) were called by SOAPsnp and GATK , 14 of whom are willing to participate in this study Figure . The higTECTA  and four synonymous variants  in coding region of TECTA was up to 99%, and the depth of each exon was close to the average median-depth across 23 exons, indicating sequencing in our study is deep enough for identifying all possible variants  with a minor allele frequency (MAF) of 0.005. Only p.Asp2006Tyr was left after filtering, suggesting a novel variant in TECTA.The sequencing coverage of every exon of TECTA gene is known as a causative gene for DFNA8/DFNA12 and DFNB21 hearing loss in humans, and SLC26A4 is related to autosomal recessive hearing loss. The missense mutations p.Asp2006Tyr of α-tectorin and p.Cys282Gly of SLC26A4 are heterozygous, which fits the autosomal dominant inheritance model. These two mutations have not been reported previously based on the public databases. The p.Asp2006Tyr of α-tectorin and p.Cys282Gly of SLC26A4 were predicted to be pathogenic mutations by SIFT and PolyPhen. Thus, p.Asp2006Tyr of α-tectorin and p.Cys282Gly of SLC26A4 were considered as candidate causative mutations of hearing loss in the studied Mongolian family. Therefore, we further screened these mutations in other affected subjects using Sanger sequencing for a genotype phenotype correlation analysis with primers listed in Table The SLC26A4 is also present in controls, ruling out the association of p.Cys282Gly in SLC26A4 with hearing loss in the studied Mongolian family. We then confirmed the presence of the novel missense mutations c.6016 G > T (p.Asp2006Tyr) of the TECTA gene in exon 20 of the rest of all 13 participated subjects with hereditary hearing loss , demonstrating the genotype-phenotype correlation of the mutation in TECTA gene with the hearing loss in the Mongolian family.Sequencing of three patients and three control subjects in the family showed that c.844 T > G (p.Cys282Gly) in TECTA encodes α-tectorin, the major component of extracellular matrix contacting cochlear hair cells and transmitting sounds [g sounds . Mutatiog sounds -13, accog sounds . ADNSHL TECTA. Among these, a novel variant, p.Asp2006Tyr was found to be implicated as the pathogenic missense mutation causing hearing loss. All TECTA mutations described to date have shown significant genotype-phenotype correlation with hearing loss [TECTA is only present in affected members of the family and is not found in any of the control subjects in the family plus a fairly large population sample of the same ethic group. Consistent to previous reports that mutations on the ZP domains are associated with mid-frequency hearing loss, the hearing of affected subjects was predominantly affected at the frequency between 1000 Hz and 2000 Hz. The clinical phenotype of hearing loss in this family displays early onset (as early as 1 year old). However, no correlation between age and severity of hearing impairment has been observed.In this study, we did exon-capture followed by next-generation sequencing to screen 54 known hearing-loss associated genes for mutations in a Mongolian family with hereditary hearing loss. We identified four missense variants  and four synonymous variants  in coding regions of ing loss . In our Y1870C/+, TectaL1820F,G18724D/+ and TectaC1837G/+) display hearing loss in mild-frequency range, phenocopying the clinical observation with affected patients [C1509G/+ and TectaC1619S/+) is more subtle and slightly different from what had been reported in patients [C1509G/+ mouse had increased reverse transduction [C1619S/+ mouse was surprisingly found to have a stable rather than progressive deterioration hearing phenotype [D1619Y/+ will be very useful to investigate how the point mutation identified in current study affects the onset of deafness under various mouse genetic backgrounds.Several recently developed mouse lines with human mutations on Tecta provide extremely useful information for understanding the mechanism of hearing loss in the affected families -19. Micepatients ,19. By cTECTA had been shown within the cytoplasm, suggesting the defect for the mutants to be secreted outside of the cell properly [TECTA without N-terminal secretion peptide signal. Further cellular studies with full-length constructs are necessary to elucidate the exact mechanism of α-tectorin’s role in ADNSHL.This novel variation p.Asp2006Tyr in the ZP domain was a substitution of aspartate with a hydrophilic side chain to tyrosine with a hydrophilic-neutral chain. Previously, various mutations in the ZP domain  of TECTA gene that causes autosomal dominant nonsyndromic hearing loss in a Mongolian family. This expands the mutation spectrum of TECTA gene in nonsyndromic hearing loss. Our study also exemplifies the application of deep sequencing in identifying disease causative mutations from a pedigree.This study documents a novel missense mutations c.6016 G > T (p.Asp2006Tyr) of the The authors declare that they have no competing interests with respect to the present article.HB designed the study and family recruitment, performed experiments, analyzed and interpreted the data, drafted the manuscript and obtained funding. QW designed the study, conducted clinical diagnoses and obtained funding. XY selected and designed the targets capture, performed experiments, analyzed and interpreted the data. T conceived of the study concept, drafted the manuscript, and analyzed and interpreted the data. NN supervised the study design. YL analyzed and interpreted the data. All other authors provided technical assistance, and all authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/15/34/prepub


GJB6-D13S1830) in the GJB6 gene. The remaining hearing impaired relatives were c.35delG homozygotes. The genetic cause of deafness was thus identified in this family. Some noteworthy differences were observed regarding twins' auditory and oral performance after CI. Subsequent follow-up of these children allowed us to conclude that those differences were most likely due to the different environment in which the twins have been living than to their different GJB2/GJB6 genotypes.Individual's hearing performance after cochlear implant (CI) is variable and depends on different factors such as etiology of deafness, age at implantation, and social/family hearing environment. Here we report the case of dizygotic twins, boy and girl, presenting with neurosensorial profound deafness prior CI (age of implantation = 3.5 years old). Both parents have severe/profound deafness, since childhood, and use sign language as primary mode of communication. Clinical and genetic characterization was performed, as well as the assessment of the auditory and oral (re)habilitation after CI, applying a battery of audiological, speech, and language tests. The twin girl and the father were homozygous for the c.35delG mutation in the GJB2 gene, while the twin boy and the mother were compound heterozygotes, both monoallelic for c.35delG and for the deletion del( After cochlear implantation (CI) the individual's hearing performance will vary according to their age at implantation , duratioGJB2 and GJB6 genes, enconding the gap junction beta-2 protein (connexin 26) and the gap junction beta-6 protein (conexin 30), respectively. Mutations in the GJB2 gene are the most frequent in nonsyndromic recessive deafness, accounting for up to 50% of the cases , and different speech perception results after CI. The twin boy presented weaker verbal outcomes and worse level of residual hearing before he was implanted (absence of AEP's). However, both twins' audiograms were very alike after CI, revealing that their hearing thresholds, unlike their auditory and oral performance, were similar.The nonidentical twins here analysed, aged 8 years-old presented different genotypes, c.[35delG]+[35delG] and [c.35delG]+[del(GJB2/GJB6 genotype. The oral outcome of the twin boy improved from the moment he started living in a hearing stimulating environment, which is a strong evidence of this factor's importance in the success of the oral rehabilitation after CI, namely, in DFNB1-associated hearing loss cases.As such, the observed differences in the oral performance are most likely due to the different social context in which the twins have been living and not to their different The remaining twins' affected relatives studied carried the c.35delG mutation in homozigosity. Molecular diagnosis and genetic counselling is thus very important to a family such as this one, namely, to the twins' unaffected half-sister still alive.


C. elegans genome comprises three ceramide synthase genes; hyl-1, hyl-2, and lagr-1. HYL-1 function is required for synthesis of ceramides and sphingolipids containing very long acyl-chains (≥C24), while HYL-2 is required for synthesis of ceramides and sphingolipids containing shorter acyl-chains (≤C22). Here we show that functional loss of HYL-2 decreases lifespan, while loss of HYL-1 or LAGR-1 does not affect lifespan. We show that loss of HYL-1 and LAGR-1 functions extend lifespan in an autophagy-dependent manner, as knock down of the autophagy-associated gene ATG-12 abolishes hyl-1;lagr-1 longevity. The transcription factors PHA-4/FOXA, DAF-16/FOXO, and SKN-1 are also required for the observed lifespan extension, as well as the increased number of autophagosomes in hyl-1;lagr-1 animals. Both autophagic events and the transcription factors PHA-4/FOXA, DAF-16, and SKN-1 have previously been associated with dietary restriction-induced longevity. Accordingly, we find that hyl-1;lagr-1 animals display reduced feeding, increased resistance to heat, and reduced reproduction. Collectively, our data suggest that specific sphingolipids produced by different ceramide synthases have opposing roles in determination of C. elegans lifespan. We propose that loss of HYL-1 and LAGR-1 result in dietary restriction-induced autophagy and consequently prolonged longevity.Ceramide and its metabolites constitute a diverse group of lipids, which play important roles as structural entities of biological membranes as well as regulators of cellular growth, differentiation, and development. The  The complexity of sphingolipid metabolism and the biological functions it affects are vast. Each class of sphingolipid has been generally thought of as entities, being regulated and acting in the same way, however, by virtue of their structural diversities each individual molecular sphingolipid species may have distinct regulatory functions in specific cellular pathways. Mammals contain six ceramide synthases, CERS1-6 (formerly named Lass1-6), which are all differentially expressed and show substrate specificity towards subsets of fatty acyl-CoAs, characterized by chain length and degree of saturation and hydroxylation , lagr-1(gk327/gk331), and hyl-1(ok976);lagr-1(gk327) animals, which can be relieved by injection of C16 ceramide sphk-1(ok1097)) have an elevated germ line death baseline compared to wild type worms and are hypersensitive to radiation-induced apoptosis, effects which are not observed in lagr-1(gk327);sphk-1(ok1097) animals hyl-2(gnv1/tm2031) animals are sensitive to anoxia while hyl-1(gk203/ok976) animals are more resistant hyl-1(ok976) animals while C24-26 ceramides are less abundant. In contrast, hyl-2(gnv1) animals contain less C20-22 ceramides and sphingomyelins but more with C24-26. Tedesco et al. hyl-1 extended lifespan and reduced mRNA levels of both hyl-1 and hyl-2, while hyl-1 deletion mutants (hyl-1(ok976/gk203)) showed no alterations in lifespan and an increased level of hyl-2 mRNA.Several model organisms have been utilized to address sphingolipid metabolism, each offering different advantages. In hyl-1 and lagr-1 results in a lifespan extension, which depends on autophagy and the transcriptions factors PHA-4, DAF-16, and SKN-1. We also find that hyl-1;lagr-1 animals have an altered sphingolipid profile and that the extended lifespan can be normalized by sphk-1 RNAi, indicating the composition and/or levels of certain sphingolipids are key effectors in the lifespan extension.In the present study we have further examined the role of ceramide synthases in longevity and find that simultaneous deletion of C. elegans lifespan we obtained strains carrying a loss of function allele of each of the genes encoding a ceramide synthase. After backcrossing we made all possible combinations of ceramide synthase mutant strains, resulting in a collection of ceramide synthase mutants; hyl-1(ok976), hyl-2(ok1766), lagr-1(gk331), hyl-1;lagr-1, and hyl-2;lagr-1 . In accordance with previous findings, we found that hyl-1;hyl-2 mutants are unviable and cannot be obtained by genetic crossing hyl-2 and hyl-2;lagr-1 animals live significantly shorter than wild type animals  and skn-1(RNAi) N2 animals  N2 animals  N2 animals . These findings indicate that hyl-1;lagr-1 longevity is elicited independently of IIS and that hyl-1;lagr-1 animals are more susceptibility to functional loss of DAF-16. We also found that the life span extension of hyl-1;lagr-1 animals is independent of the AMP-dependent kinase AMPK, as knock down of aak-2, the catalytic subunit of AMPK sphk-1 decreases hyl-1;lagr-1 lifespan to the same level as sphk-1(RNAi) N2 animals  and lagr-1(gk331) mutations into DA2123, a transgenic strain expressing GFP-tagged LGG-1. Under control conditions, hyl-1;lagr-1 animals have a consistently increased level of autophagy compared to wild type worms  was lower in hyl-2 and hyl-2;lagr-1 animals while increased in hyl-1;lagr-1 to increase as they do in hyl-2 animals ”, “Increased lifespan (mutant)”, and “No change (ctrl N2)” ” . The cortrl N2)” . Among ttrl N2)”  are oppo animals . These oSaccharomyces cerevisiae extended chronological lifespan LAG1 from C. elegans not only complement Lag1 function in S. cerevisiaeC. elegansC. elegans and show that deletion of hyl-1(ok976) or lagr-1(gk331) does not affect lifespan (hyl-2(ok1766) significantly shortens lifespan of C. elegans. Interestingly, we consistently find that deletion of both lagr-1 and hyl-2 further reduces lifespan, while deletion of both lagr-1 and hyl-1 significantly extends longevity compared to wild type C. elegans  animals results in L1 arrest (data not shown). In contrast to our observations, Menuz et al. ok976) extends lifespan significantly, while deletion of hyl-2(ok1766) did not affect lifespan under normal conditions, but was required for survival under anoxic conditions hyl-1;lagr-1 animals is independent of insulin signaling  can regulate the longevity and autophagy, the genetic tractability of hyl-1(ok976) IV, RB1498: hyl-2(ok1766) X, VC765: lagr-1(gk331) I, DA1116: eat-2(ad1116) II. Double mutants: FE0007: hyl-1(ok976) IV;lagr-1(gk331) I, FE0008: hyl-2(ok1766) X; lagr-1 (gk331) I. Transgenic strains: DA2123: adIS2122 , FE0015: hyl-1(ok976) IV;lagr-1(gk331) I; adIS2122, BC10421: dpy-5(e907) I; sEx10421[rCesC09G4.1::GFP+pCeh361], VB2360: svEX798-800 , BC10482: dpy-5(e907) I; sEX10482[lagr-1::GFP]. All single mutant strains were obtained from the Caenorhabditis Genetics Centre (CGC) and unless otherwise noted generation of the remaining strains listed were conducted in our lab. Generating a hyl-1;hyl-2 strain was also attempted but worms homozygous for both mutations were not attained and we later confirmed by RNAi that worms lacking both HYL-1 and HYL-2 arrest development and die. The three single mutant strains were out-crossed seven times to N2 Bristol wild type worms.Standard procedures were used for culturing Transgenic L4 worms expressing GFP fusions were mounted in M9 buffer containing Tetramisol (10 µM)  on a 2% agar pad on a microscope slide and covered with a coverslip and analyzed by confocal microscopy on an LSM 510 META microscope . Primary image analysis was performed using LSM Image Browser .All clones were from the Julie Ahringer RNAi library All lifespan analyses were conducted at 20°C on worms non-starved for at least two generations. For the RNAi lifespan assays, 3-day-old synchronized worms were transferred to gene-specific RNAi bacterial plates. The worms were counted and moved to new plates every second day during the reproductive period and afterwards only moved to new plates every forth day. Lifespan is defined as the time from day one of adulthood to the time when they were scored as dead (i.e. no longer responded to gentle prodding with a platinum wire). Worms that “exploded”, were bagged, or went missing were censored the day the event was observed. For the OP50 lifespan assays , synchroThe level of autophagy was investigated using an LGG-1::GFP translational reporter hyl-1;lagr-1 were allowed to feed for 30 min on the bead-plates after which they were transferred to eppendorf tubes with 1 ml of 10 mM levamisole  and centrifuged for 2 min at 2000 g, room temperature. The supernatant was removed and ∼30 µl of resuspended worms were transferred to a freshly made agarose pad (2% agarose in M9). The amounts of ingested beads were photographed at 1000× using a Leica DMI 6000 B microscope and quantified using NIH ImageJ freeware NGM plates were seeded with a 500∶1(vol:vol) blend of OP50 bacteria and Fluoresbrites Multifluorescent microspheres, 0.2 µm  22 hours prior to the assay. L4 larvae of N2 and Five non-starved L4 worms of each strain were transferred to NGM plates seeded with OP50 and filmed three times 15 consecutive seconds using a Leica DMI 6000 B microscope with Infinity Capture Software. Pumping rates were counted by playing the recordings at one fourth of the normal speed and mean pumping rate calculated from the fifteen measurements of each genotype.Brood size assays were performed at 20°C by picking late L4s to fresh NGM plates and transferring them to new plates every 24 hours for 4–5 days. After this worms were left on the same plates for 3 days. Worms that “exploded”, were bagged, or went missing were omitted from the analysis. All progeny plates were incubated at 20°C for about two days before the number of developed progeny was scored.Synchronized L4 worms grown at 20°C were picked to fresh plates and subjected to heat shock at 35°C for one hour after which the worms were counted. Worms were scored dead when they no longer responded to gentle prodding with a platinum wire. The worms were continuously subjected to heat stress one hour followed by scoring.tbb-2 gene (encoding the C. elegans orthologue of the human β-tubulin). Primers for qRT-PCR were designed using Primer Express version 2.0 . Primer sequences can be obtained upon request. Statistical analyses were performed by unpaired two-tailed t-test using GraphPad Prism version 6.0 (GraphPad Software).Total RNA was extracted from four independent synchronized worm populations of each strain as described (51). cDNA was synthesized from 500 ng total RNA as previously described (52). Quantitative real-time PCR (qRT-PCR) was performed on an ABI PRISM 7700 RealTime PCR-machine  or Stratagene MXPro 3000  using 2× SYBR Green JumpStart™ Taq ReadyMix™ and Sigma Reference Dye  as described by the manufacturer. PCR reactions were performed in 25 µl reactions containing 1.5 µl diluted cDNA. Reactions were incubated at 95°C for 2 min followed by 40 cycles of 95°C for 15 s, 60°C for 45 s and 72°C for 45 s. All reactions were performed in duplicates and normalized to the level of the 2O. All centrifugation steps were carried out at 20°C, 1000 rpm, 1 min. Liquid was aspirated to 600 µL and 550 µl were transferred to an eppendorf tube. Samples were flash frozen in liquid nitrogen and stored at −80°C. Lipid extraction was carried out at 4°C. Approximately 1000 worms were aliquotted (∼100 µl) mixed with 100 µl 150 mM ammonium acetate before adding 990 µl chloroform/methanol (2∶1). Samples were vortexed at 4°C for 45 min and centrifuged 2 min . The lower organic phase was transferred to a new tube and subjected to vacuum evaporation. Lipid extracts were dissolved in 100 µl chloroform/methanol (2∶1).Synchronized L4 worms were washed twice with 10 mL 0.9% NaCl and incubated end-over-end for 15 min to ensure intestinal emptying. Worms were washed trice with 10 mL 150 mM ammonium acetate in HLipid extracts were analyzed by normal-phase liquid chromatography using a PVA-SIL column (YMC Europe GmbH) interfaced with a nanoflow ion source Triversa NanoMate  and a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). Cer, HexCer and SM species were monitored in negative ion mode by recording FT MS analysis using a target mass resolution of 100,000. Lipid species were quantified by extracting their peak intensities and using ALEX software as previously described All statistical analyses were performed using GraphPad Prism version 6.0 (GraphPad Software), except for the Principal Component Analysis (PCA), which was performed using Markerview software (AB SCIEX).Statistical analyses were performed by Gehan-Breslow-Wilcoxon tests. The Bonferroni method was used to correct for multiple comparisons and P-values equivalent to a significance level of 0.05 were considered statistically significant. Worms that “exploded”, were bagged, or went missing were censored the day the event was observed. Further statistical lifespan details can be found in Statistical analysis was performed by unpaired two-tailed t-test (with Welch’s correction if variances were significantly different). The Bonferroni method was used to correct for multiple comparisons and P-values equivalent to a significance level of 0.05 were considered statistically significant. N used for analysis is the total number of worms observed for each treatment.Statistical analyses were performed by unpaired two-tailed t-test (with Welch’s correction if variances were significantly different).Statistical analyses were performed by one way analysis of variance followed by Dunnett’s multiple comparisons test.Statistical analysis was performed by Log-rank (Mantel-Cox) tests. Some animals were censored as they crawled of the plate or ruptured, however they are incorporated in the data set up until the time point they were censored.Statistical analyses were performed by one way analysis of variance followed by Dunnett’s multiple comparisons test and P-values below 0.05 were considered statistically significant. Results are from two biological experiments.hyl-1 and hyl-1;lagr-1 Click here for additional data file.Figure S2Lifespan analyses of all ceramide synthase mutants. Cumulative lifespan analyses of N2, hyl-1, hyl-2, lagr-1, hyl-1;lagr-1, and hyl-2;lagr-1 grown on OP50 at 20°C. (A) hyl-1 displays no change in lifespan compared to N2 (P = 0.3592). (B) hyl-2 displays a shortened lifespan compared to N2 (P<0.0001). (C) lagr-1 displays no change in lifespan compared to N2 (P = 0.1651). (D) hyl-1;lagr-1 displays an extended lifespan compared to N2 (P<0.0001). (E) hyl-2;lagr-1 displays shortened lifespan compared to N2 (P<0.0001). (F) Table summarizing the depicted lifespan analyses. aSome animals were censored as they crawled of the plate, ruptured, or died as a “bag of worms”, however they are incorporated in the data set up until the day they were censored. The number of individual trials is in parentheses. Statistical analyses were performed by Gehan-Breslow-Wilcoxon tests using GraphPad Prism version 6.0 (GraphPad Software). bThe Bonferroni method was used to correct for multiple comparisons and P-values below 0.01 are considered statistically significant equivalent to a significance level of 0.05. Cumulative statistics is shown in this table as experimental animals subjected to the same treatment behaved similarly between trials.(PDF)Click here for additional data file.Figure S3Expression levels of longevity and autophagy genes in wild type N2 and in hyl-1;lagr-1 animals. Total RNA was harvested from N2 worms at the L4 stage. The expression level of the indicated genes was quantified using qRT-PCR, and normalized to tbb-2 mRNA and shown in arbitrary units (a. u.). Mean ± SEM is shown, number of independent experiments ranged from 3 to 9. (*) P≤0.05. Only the expression of pha-4 and atg-12 in hyl-1;lagr-1 animals is significantly changed relatively to wild type animals.(PDF)Click here for additional data file.Figure S4LGG-1::GFP-positive puncta in hypodermal seam cells of wild type and hyl-1;lagr-1 animals. Representative micrographs of young L4 N2 (DA2123) or hyl-1;lagr-1 larvae expressing GFP-tagged LGG-1 in hypodermal seam cells. A yellow arrow head indicates a hypodermal seam cell, while blue arrow heads indicate LGG-1::GFP positive puncti. Using fluorescence microscopy, LGG-1::GFP positive puncti were counted in 3–10 seam cells were counted in each of 23–45 animals and averaged. Scale bar: 20 µm.(PDF)Click here for additional data file.Figure S5Effect of 3-methyladenine and Concanamycin A on autophagy. Transgenic animals expressing LGG-1::GFP were treated with 3-MA (1 mM) or Concanamycin A (50 nM) for 24 hours after they reaching the young L4 larval stage. Bars represent mean number of LGG-1::GFP-containing puncta per seem cell in non-starved wild type and hyl-1;lagr-1 worms grown at 20°C. The number in each bar indicates the total number of seam cells observed. N used for analysis is the total number of worms observed for each treatment (the number of worms examined ranged from 12 to 21). Mean ± SEM is shown. (**) P≤0.01 and (****) P≤0.0001.(PDF)Click here for additional data file.Figure S6Ingestion of fluorescent beads in hyl-2 and in hyl-2;lagr-1 animals. Quantification of fluorescent beads in the pharynx and the anterior part of the intestine following a feeding period of 30 minutes. Fluorescence intensities were normalized to the level in wild type animals. Mean ± SEM is shown, n = number of worms analyzed.(PDF)Click here for additional data file.Figure S7Overview of all Cer, HexCer, and SM species detected in hyl-1;lagr-1. (A) Top: Relative abundance of all Cer species detected in hyl-1;lagr-1. Bottom: Zoom of low abundance Cer species detected in hyl-1;lagr-1. (B) Relative levels of all HexCer species detected in hyl-1;lagr-1. (C) Top: Relative abundance of all SM species detected in hyl-1;lagr-1. Bottom: Zoom of low abundance SM species detected in hyl-1;lagr-1.(PDF)Click here for additional data file.Figure S8Overview of all Cer, HexCer, and SM species detected in the five different ceramide synthase mutants. (A) Top: abundance levels of all Cer species detected in ceramide synthase mutants. Bottom: Zoom of low abundance Cer species detected in ceramide synthase mutants. (B) Top: Relative levels of all HexCer species detected in ceramide synthase mutants. Bottom: Zoom of low abundance HexCer species detected in ceramide synthase mutants. (C) Top: Relative levels of all SM species detected in ceramide synthase mutants. Bottom: Zoom of low abundance SM species detected in ceramide synthase mutants. (D) Relative levels of all C33, C34, and C35 sphingolipid species detected to have significantly different levels in one or more ceramide synthase mutant compared to N2. (E) Relative levels of all C38 sphingolipid species detected to have significantly different levels in one or more ceramide synthase mutant compared to N2. (F) Relative levels of all C39 sphingolipid species detected to have significantly different levels in one or more ceramide synthase mutant compared to N2. (G) Relative abundance of all C40 sphingolipid species detected to have significantly different levels in one or more ceramide synthase mutant compared to N2. (H) Relative levels of all C41 sphingolipid species detected to have significantly different levels in one or more ceramide synthase mutant compared to N2. (I) Relative levels of all C42 sphingolipid species detected to have significantly different levels in one or more ceramide synthase mutant compared to N2. (J) Relative levels of all C43 sphingolipid species detected to have significantly different levels in one or more ceramide synthase mutant compared to N2. (K) Relative levels of all species significantly altered according to lifespan changes (e.g. Oppositely regulated in long-lived and short-lived strains). Statistical analyses were performed by one way analysis of variance followed by Dunnett’s multiple comparisons test using GraphPad Prism version 6.0 (GraphPad Software). Two technical replicates of two biological replicates were analysed for each strain. (*) P≤0.05, (**) P≤0.001, and (***) P≤0.0001. Mean ± SD is shown.(PDF)Click here for additional data file.Figure S9Multivariate analysis by principal component analysis of sphingolipidomic data segregated according to lifespan changes. (A) Score plot projecting the first (D1) and second (D2) principal components show a clear separation of the strains when grouped according to the following lifespan changes: “Decreased lifespan ”, “Increased lifespan ”, and “No change ”. D1 and D2 account for 100% of the total sample variance. (B) Loading plot depicting the sphingolipid species contributing the most to the total sample variance. Red stars denotes the species significantly altered when comparing hyl-1 (no lifespan change) to hyl-1;lagr-1(increased lifespan). (C) Relative abundance of the four species contributing the most to the score plot separation. (D and E) The 5 species which contribute most to the separation and are significantly altered in hyl-1;lagr-1 compared to hyl-1 (denoted by red stars in B).(PDF)Click here for additional data file.Figure S10Expression patterns of the ceramide synthase genes at the L4 stage. (A) HYL-1 shows expression in the body wall muscles, the pharyngeal muscles PM3 and PM5, and unidentified cells in the pharynx. (B) HYL-2 shows expression in the body wall muscles and the nervous system. (C) LAGR-1 shows expression in the pharyngeal muscles PM3-5 and in pharyngeal nerves. Scale bar: 80 µm.(PDF)Click here for additional data file.Table S1P-values from the t-test of hyl-1 and hyl-1;lagr-1 of the Principal Component Analysis.(XLSX)Click here for additional data file.


In environmental noise control one commonly employs the A-weighted sound level as an approximate measure of the effect of noise on people. A measure that is more closely related to direct human perception of noise is the loudness level. At constant A-weighted sound level, the loudness level of a noise signal varies considerably with the shape of the frequency spectrum of the noise signal. In particular the bandwidth of the spectrum has a large effect on the loudness level, due to the effect of critical bands in the human hearing system. The low-frequency content of the spectrum also has an effect on the loudness level. In this note the relation between loudness level and A-weighted sound level is analyzed for various environmental noise spectra, including spectra of traffic noise, aircraft noise, and industrial noise. From loudness levels calculated for these environmental noise spectra, diagrams are constructed that show the relation between loudness level, A-weighted sound level, and shape of the spectrum. The diagrams show that the upper limits of the loudness level for broadband environmental noise spectra are about 20 to 40 phon higher than the lower limits for narrowband spectra, which correspond to the loudness levels of pure tones. The diagrams are useful for assessing limitations and potential improvements of environmental noise control methods and policy based on A-weighted sound levels. Health effects considered by the World Health Organization include annoyance, sleep disturbance, and cardiovascular disease ,2. ExposNoise exposure for the exposure-response relations is represented by an A-weighed sound level . The A-weighted sound level is related to health effects of environmental noise, but is only a limited representation of direct perception of noise. A quantity that is more closely related to the direct perception of noise is the loudness level. However, while annoyance caused by environmental noise is related to the loudness of the noise , it alsoThe A-weighting curve for rating noise was originally derived from an equal-loudness contour for pure tones, the 40 phon Fletcher-Munson curve , so equanot suggest that the loudness level is a better indicator of environmental noise than the A-weighted sound level is, as ‘corrected’ A-weighted levels may be just as good as loudness levels.For assessing limitations and potential improvements of current noise control methods based on the A-weighted sound level, it would be useful to have a global picture of the ranges of loudness levels that occur in practice, for various types of environmental noise such as traffic noise, aircraft noise, and industrial noise. A relevant question is: how much does the loudness level vary in practice at constant A-weighted sound level? The analysis presented in this note provides an answer to this question. It should be noted that we do A distinction should be made between outdoor levels and indoor levels. The exposure-response relations for the health end-points of environmental noise are based on outdoor levels, measured or calculated at the most-exposed façade of a dwelling. The façade levels are an approximation for the ‘true’ noise exposure of people. A possible improvement of this approximation is to take into account also indoor levels, since noise-related health effects may originate to a large extent from indoor noise levels. For sleep disturbance indoor levels in bedrooms play a role. Therefore, this note also considers indoor levels, calculated from outdoor levels and a typical spectrum of façade insulation. However, the effect of façade insulation on environmental noise annoyance is a complex issue, since various non-acoustic factors also play a role, such as expectations of people with respect to the insulation, and the opening and closing of windows. A further complication of indoor levels is the spatial variations in LF sound fields in rooms, with pronounced regions of high and low sound levels due to standing waves .The article is organized as follows. Section 2 describes standard methods for calculating loudness, both loudness of pure tones and loudness of broadband noise spectra. Section 3 presents the analysis of loudness levels calculated for various spectra of environmental noise, including pure tones in Section 3.2, band-limited noise in Section 3.3, and broadband noise representative of traffic noise, aircraft noise, and industrial noise in Section 3.4. Conclusions are presented in Section 4.2.The international standard ISO 226:2003 , which iEnvironmental noise usually consists of sound signals with a broad spectrum, covering both the low frequency region, say between 20 Hz and 200 Hz (various definitions of the LF region exist), and the higher frequency region up to about 5 or 10 kHz. The A-weighted spectrum of noise emitted by road vehicles  has the highest levels in the frequency bands around 1 kHz. With increasing distance from the source, however, the spectrum gets a stronger LF signature, due to frequency-dependent air absorption and screening attenuation by obstacles such as noise barriers and buildings –31. A noThe calculation or prediction of the loudness of a noise signal with a broad spectrum is a complex problem. The theory of psychoacoustics describes the human hearing system as a set of auditory band-pass filters, corresponding to a set of contiguous frequency bands that are called critical bands . Each crThere exist two standard methods for calculating loudness of (steady) broadband noise , a methoThe ANSI standard is based on a recently developed loudness model ,40. The The DIN standard is based on a loudness model developed by Zwicker . The sta3.This section presents an analysis of loudness levels calculated for various noise spectra. Pure tones are considered in Sections 3.1 and 3.2. Band-limited noise spectra are considered in Section 3.3. Broadband noise spectra that are representative for various types of environmental noise sources in practice are considered in Section 3.4.3.1.inverse contours of 3.2.N), which is related to loudness level (LN) by:Lp is the sound pressure level and K is a constant. K has been chosen such that the loudness level for 60 dB is 60 phon, i.e., the value for 1 kHz. This gives K = 0.063 . Consequently, the 0.3 power law corresponds to LN = Lp for 1 kHz, in agreement with the definition of the loudness level of a tone as the sound pressure level of an equally loud 1 kHz tone.The ISO 226:2003 loudness levels and loudness values in 3.3.In Section 2 it has been indicated that the standard ISO 226:2003 is an updated version of the older ISO 226:1987 standard. Since the ANSI S3.4-2007 standard agrees with ISO 226:2003 for pure tones, the ANSI standard has been used for the results presented in the remainder of this article for band-limited noise spectra and broadband noise spectra.A spectrum of environmental noise usually covers a wide frequency range, rather than a single 1/3-octave band, so it is important to include the effect of bandwidth on loudness levels in this study. In the remainder of this section the bandwidth of a simple spectrum is varied in a systematic way, while in Section 3.4 more realistic broadband spectra are considered.i.e., the ratio of the spectrum bandwidth to the critical bandwidth. With decreasing frequency, 1/3-octave bands become narrower while the critical bandwidth levels off at 100 Hz below a frequency of 500 Hz. For C–A values larger than 15 dB the effect of increasing the bandwidth from one to three 1/3-octave bands is small :m is the mass of the wall per unit area, Z is the impedance of air, and ω is the angular frequency of the transmitted sound wave. For ω >> 2Z/m the transmission loss increases by 6 dB per octave. For example, for m = 50 kg/m2, the expression yields a transmission loss of 12 dB at 10 Hz and about 50 dB at 1 kHz. Sound transmission through the façade of a house depends not only on the walls, but also on various structural elements and openings in the façade , which limit the façade insulation spectrum at high frequency. Consequently, façade insulation spectra in practice show large variations [The strong frequency dependence of façade insulation can be understood from the expression for the transmission loss smission ,45,46. Hriations –52, depeI, are indicated in the legend of I decreases. This is shown graphically in I decreases linearly with C–A, except for negative values of C–A. Values of the broadband A-weighted façade insulation, denoted with the symbol outdoor noise: the spectrum shapes shown in outdoor A-weighted sound levels of 20, 40, 60, and 80 dB. Only in this way the increase of indoor loudness with increasing LF parameter C–A, at constant outdoor A-weighted noise level, is taken into account. It should be noted that the indoor values of C–A were calculated for the indoor spectra (to show the enhanced indoor LF content), but the corresponding outdoor values of C–A follow from the legends of indoor loudness level with C–A at constant outdoor A-weighted sound level. The slopes of the indoor curves in First, the loudness level of pure tones or single 1/3-octave bands increases with C–A for A-weighted sound levels above 60 dB and decreases with C–A below 60 dB (see Sections 3.1−3.3). Second, an increase of the bandwidth of the spectrum causes an increase of the loudness level (see Section 3.3), but since the increase is larger at small values of C–A than at large values, the effect of a bandwidth increase is also a decrease of the slopes of the curves see . This excf. Loudness levels calculated for other broadband noise spectra (not shown here), including the spectra shown in The area below 20 dB and 20 phon in the graph is shown in light gray, for two reasons: (i) sound levels below 20 dB are not relevant for most practical situations, and (ii) the accuracy of loudness calculation standards is limited below 20 phon.4.The effects of bandwidth and LF content on the loudness level of environmental noise have been analyzed in this article. In The effect of bandwidth on the loudness level at constant A-weighted sound level has been shown in The effect of LF content on the loudness level of broadband environmental noise has been shown in The analysis presented here may be used as a starting point for assessing limitations and potential improvements of environmental noise control methods based on A-weighted sound levels or loudness levels. A possible approach is to apply specific weightings, or penalties, for certain acoustic parameters, such as LF content, bandwidth, and tonality. Ultimately, such weightings or penalties should be based on noise annoyance studies, but considering the effect of the acoustic parameters on loudness may be helpful, since loudness is related to annoyance but is a less complex quantity than annoyance is.


Cochlear implantation can facilitate the development of communication skills in children with profound hearing loss. The objectives of our study were to determine the average ages at suspicion and diagnosis of hearing loss, amplification, intervention, and performing the cochlear implantation and to investigate the effects of the parents’ level of education and economic circumstances on the age of the child at cochlear implantation.The parents of 96 children with profound sensorineural hearing loss who had received a cochlear implant at Amir-Alam Cochlear Implant Center between 2008 and 2010 were asked to complete a survey. The survey included demographic information, and birth, medical, and hearing loss history of their child. Study data were obtained through the patient database in the Cochlear Implant Center and interviews with the parents. The mean times between the age of the children at diagnosis of hearing loss and amplification, beginning the rehabilitation program, and performing the cochlear implantation were 4.05 (±0.86), 2.59 (±0.9), and 25.43 (±1.45) months, respectively; delays that were statistically significant (P≤0.004). In 47.9 percent of cases, the parents were the first people to suspect the occurrence of hearing loss in their child. Statistical analysis indicated that the age at cochlear implantation decreases as the educational level of the parents increases (P≤0.003). There was also a significant difference between parents’ economic circumstances and the age of cochlear implantation (P<0.0001). There is still a remarkable delay between the diagnosis of hearing loss and aural rehabilitation in hearing-impaired children. Parents’ levels of education and economic circumstances have a noticeable effect on the age of cochlear implantation in hearing-impaired children.  The consequence of late identification of hearing loss manifests as poor auditory, language, cognition, and psychosocial skills, which prohibits academic achievement and ultimately results in poor vocational prospects . In addition, delay in the diagnosis of hearing loss can be a cause of anxiety and stress for the family of hearing impaired children . NewbornEarly identification of hearing loss is the most important step for attaining successful communication outcomes in deaf children and leads, in turn, to earlier intervention and decreases in the age at which cochlear implantation is performed . SimilarSeveral studies have considered the factors that influence the age at diagnosis of hearing loss and any consequent intervention in various parts of the world. Van der Spuy and colleagues, in their study in South Africa, reported significant delays in the diagnosis of hearing loss and intervention. These delays were attributed to inadequate support services for early intervention in this country . A study by Gopal and colleagues mentioned the long time lapse between identification of hearing loss and the fitting of hearing aids . Watkin Ozcebe and colleagues suggested that poor socioeconomic circumstances and a low level of knowledge in a family contribute to late identification of hearing loss and intervention . On the In terms of treatment outcomes, the results of several studies suggest that children who are diagnosed early and receive early amplification, intervention, and cochlear implantation, may acquire developmental skills equivalent to their hearing peers -14. SeviGiven the importance of early identification of hearing loss for the development of communication skills and the role of family in diagnosis and intervention in hearing loss, the purpose of this study was to: 1) determine the average ages at suspicion and diagnosis of hearing loss, amplification, intervention, and cochlear implantation; and 2) investigate the effect of parents’ level of education and economic circumstances on the age at which cochlear implantation is performed in deaf children. The results of this study can inform experts about the diagnosis and treatment of hearing loss in Iran. Moreover, a survey of the ages at which the various stages of intervention are performed and the cause of delays between them is a key to resolving the problems related to the performance of cochlear implantation in Iran.ParticipantsOur study sample consisted of 96 children under 6 years of age with profound sensorineural hearing loss  months) who had received cochlear implantation in Amir-Alam Cochlear Implantation Center in Tehran between 2008 and 2010 and their parents. ProcedureThe parents of the deaf children who had undergone cochlear implantation all gave their informed consent to be included in the study. Data were collected from a review of the cochlear implantation center patient database and interviews with the parents. A 31 item survey designed for the interviews with the parents included: demographic information (7 items), the child’s birth history (11 items), medical history (2 items) and hearing loss history (11 items).Data analysisP<0.05 was considered to indicate a significant difference between the data. Significant differences between the ages of the children at the different stages of intervention studied were determined by Student’s independent t-test. The effect of the parents’ level of literacy and economic circumstances on the age of the child at cochlear implantation was examined using the Wilcoxon test. Student’s independent t-test was used to determine the effect of family history of hearing loss and the effect of the sex of the children on the ages at which they were diagnosed and received the various interventions. Significant differences between the number of family members and the studied ages were analyzed using Pearson's correlation test. Data analysis was performed using SPSS 16.0 and P=0.002), 4.05 (±0.86) (P<0.0001), 2.59 (±0.9) (P=0.004), 25.43 (±1.45) months (P<0.0001), respectively , 9.35 (±5.79), 13.41 (±6.11), 16 (±6.37), 41.25 (±11.12) and 42.15 (±11.00) months, respectively. Consequently, the average delays between the ages at suspicion and diagnosis, amplification, beginning the rehabilitation program, and cochlear implantation operation were 2.62 (±0.84) .According to the parent interviews, 52.1% of the parents had poor economic circumstances, 45.8% had moderate economic circumstances and 2.1% had good economic circumstances . StatistThe distribution of the parents’ levels of education is shown in P<0.0001) and diagnosis (P=0.012) compared to those without such family history.Risk factors associated with hearing loss were reported in 51% of cases. Hyperbillirubinemia (26%), family history of congenital sensorineural hearing loss (26%), and pre- and post-term births  were the most frequently reported risk factors. Statistical analysis revealed that deaf children with a family history of congenital sensorineural hearing loss had lower mean ages of suspicion (P≥0.319). Moreover, there were no significant differences between the sexes of the deaf children and the studied ages (P≥0.077).The number of children in a family ranged 1 to 7. There were no significant differences between the number of family members and the ages at diagnosis and during the various stages of rehabilitation program  was observed. Also, parents’ levels of education and economic circumstances had a remarkable effect on the age at cochlear implantation. Our results highlight the importance of age at diagnosis of hearing loss and its effects on age at cochlear implantation, since early implantation has substantial effects on the improvement of communication skills in deaf children. More importantly, the main clinical point of this study is that it is possible to minimize the age at cochlear implantation via increasing the level of public knowledge regarding hearing loss, cochlear implantation, and providing support services for families.


Persons with a hearing impairment have various experiences during their ‘journey’ through hearing loss. In our previous studies we have developed ‘patient journey’ models of person with hearing impairment and their communication partners (CPs). The study was aimed to evaluate the effectiveness of using the patient journey model in the internet-based pre-fitting counseling of a person with hearing disability .The study employed a randomized controlled trial (RCT) with waiting list control (WLC) design. Even though we had intended to recruit 158 participants, we only managed to recruit 80 participants who were assigned to one of two groups: (1) Intervention group; and (2) WLC. Participants from both groups completed a 30 day internet-based counseling program (group 2 waited for a month before intervention) based on the ‘patient journey’ model. Various outcome measures which focus on self-reported hearing disability, self-reported depression and anxiety, readiness to change and self-reported hearing disability acceptance were administered pre- and post-intervention.The trial results suggest that the intervention was not feasible. Treatment compliancy was one of the main problems with a high number of dropouts. Only 18 participants completed both pre- and post-intervention outcome measures. Their results were included in the analysis. Results suggest no statistically significant differences among groups over time in all four measures.Due to the limited sample size, no concrete conclusions can be drawn about the hypotheses from the current study. Furthermore, possible reasons for failure of this trial and directions for future research are discussed. Hearing impairment is one of the most common chronic conditions in adults and can have physical, mental and social consequences for both persons with hearing impairment and their communication partners (CPs). Despite these consequences only a small percentage of those with hearing impairment tend to seek help and uptake audiological interventions . VariousThere are a range of audiological interventions focusing on different aspects of hearing impairment, such as hearing status, psychosocial needs and supporting enablement at workplace. Even though there is information provision to persons with a hearing impairment before and after the hearing tests, generally most counseling programs are post hearing aid fitting with an emphasis on both technological and psychosocial aspects. The focus of pre-fitting counseling program is to support the person with hearing disability in terms of social and emotional needs, modify attitudes and motivation, and to provide information about choice of interventions . A recenPerson with hearing impairment may have various experiences during their ‘journey’ through hearing loss. The experiences patients go through during a disease/acquired health condition and treatment are referred to as ‘patient journey’. It has become common in recent years to study the patient journey of various chronic conditions -23. In oThis paper presents the results of a randomized controlled trial (RCT) which intended to evaluate the effectiveness of using the ‘patient’ journey model in the internet-based pre-fitting counseling for person with hearing disability . In addiThe study employed a RCT design with Waiting List Control (WLC). Figure inclusion criteria included: age over 18 years; noticing symptoms of hearing disability; access to internet. Also, the exclusion criteria included: already using hearing aids; HHQ scores 20 or below; and those with additional disabilities  which may affect individuals’ ability to participate in an internet-based program. We had initially planned to recruit 158 participants based on the sample size calculation , HADS , URICA  and HDAQ . There was also no interaction effect seen between groups among these scores.Table The study presents the results of a randomized controlled trial which was aimed at evaluating the effectiveness of the ‘patient journey’ model in the internet-based pre-fitting counseling of person with hearing disability. The trial results suggest that the intervention was not feasible. No significant difference was found between treatment and control groups over time. Due to limited sample size no concrete conclusions can be drawn about the hypotheses from the current study. However, the cross sectional data obtained in the beginning of the study  could be very useful in better understanding the characteristics of this unique population. Furthermore, there are mixed findings about benefits of pre-fitting counseling programs in the literature and the effects seen seem to be generally small -11.It is not uncommon that the clinical trials fail, but they often do not get reported and this selective reporting has caused bias in published reports . StudiesBy considering the observations made before, during and after the current trial we propose the following may be some of the possible reasons for the study failure. We can broadly categorize them into issues related to recruitment and retention of participants.Firstly, due to study design chosen , the researcher did not have any direct contact and/or control over the participants’ behavior. We can assume that in some cases family members of those with hearing disability may have registered online for the study on behalf of the participant even if the person with hearing disability had limited knowledge and skills of internet use. This means the person with hearing disability can only access internet with support of family members. Even though it is not possible to completely avoid this some measures may have helped reduce such instances. For example, telephone calls to potential participants before the start of the counseling program to make sure they are able to use internet and have the time to participate in the study may have helped. In addition, advertising the study in audiology and Ear, Nose and Throat departments and registering them only if they meet the entry criteria may also be a useful approach to adopt. However, it is important to note that internet-based recruitment strategies for studies on hearing impaired population have been successful in few countries including Sweden, Netherlands and Germany ,46-49.Secondly, it appears that the internet-usage in older adults in the UK is limited when compared to other developed countries. We noticed that a high number of participants rarely or never logged into online counseling protocol system and also did not respond to the emails sent directly to their inbox. This is also supported by a recent study done in the UK which suggests that the internet use in younger adults i.e., 50–62 years) and older adults  is only 60.9% and 29.8% respectively [–62 yearsThirdly, counseling materials did not seem to be popular with everyone. This is evident from reports of those participants who withdraw and also from the fact that only half of those who completed the pre- and post-counseling questionnaires actively took part in the counseling program. This may due to the fact that having ‘self-reflection’ as the core concept may not appeal to everyone. Also, many participants may have expected to see some technical details related to hearing loss and hearing aids in the counseling program. This may suggest that it may be appropriate to use information focusing on verities of aspects while developing a counseling program. This issue was considered while developing the counseling materials and the study design. However, decision was made to use only the concept of patient journey to evaluate its effectiveness rather than the effect from composite materials. This issue may pose a dilemma to researchers as to whether they want develop a counseling program which appeals to majority of the population or alternatively find the individuals who may like the aspects of self-reflection and study the effectiveness of the patient journey independently. Furthermore, it is likely that the self-reflection could be unexpected by the persons with suspected hearing problems who are looking for information, rather they may be expecting some action .Lastly, the current study population may be considered as a challenging group as they are not actively seeking help due to various factors . The mean age of participants in the study sample was about 62 years. Given that the typical age for first consultation with hearing healthcare professional and also first fitting of hearing aids is approximately 70 to 74 years and with many of these adults experiencing hearing difficulties for an average of 10 years prior to that ,51, the In summary, despite following the structured approach to conducting clinical trial the current study failed. To some degree this clinical trial had failed even before it got started due to recruitment issues. However, we had anticipated this as one of the potential threats to our study as reported in the study design publication .There are limited studies focusing on pre-fitting counseling, so efforts are needed to develop and evaluate such programs as the significant number of those with hearing difficulties does not seek professional help. Further research is necessary to evaluate the use of ‘patient journey’ model in the pre-fitting counseling and also to test the proposed hypotheses. Considering the limited use of internet in some countries especially in older adults it might be appropriate to look into other methods of counseling provision  and it is worth investigating the user preferences towards treatment mode. Also, as not much is known about the population at early stages of hearing disability who may not seek help actively, research is need to understand their population characteristics and also help-seeking behavior. For this reason, it may be useful to include other measures related to motivation, self-efficacy, etc. which may provide some information about the characteristics of the study population. Also, recruitment of participants using on-line strategies has been criticized by some researchers suggesting that it may not be representative of the general population ,53 whichThere are some suggestions made in the literature about front loading the process  while conducting clinical trials which may be a useful approach to adopt . FurtherNo concrete conclusions about the hypotheses can be drawn from the current study due to limited sample size. However, the study has helped us identify various issues which may have resulted in the study failure. It appears that evaluating the pre-fitting counseling program is challenging and researchers should take caution while designing clinical trials to avoid some of the common mistakes some of which are reported in this paper.CP: Communication partner; HHQ: Hearing handicap questionnaire; HADS: Hospital anxiety and depression scale; HDAQ: Hearing disability acceptance questionnaire; RCT: Randomized Controlled Trial; URICA: University of Rhode Island Change Assessment Scale; WLC: Waiting list control.The authors declare that they have no competing interests.VKCM was the main researcher who collected data, analysed results and written up the paper. JR, GA and TL assisted in designing the study, planning the analysis and provided assistance in writing up the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6815/14/3/prepub