TAM;Bcl-xLMycER model of pancreatic neuroendocrine tumorigenesis (PNET) to assess the role of cathepsin L in Myc-induced tumor progression. By employing a cysteine cathepsin activity probe in vivo and in vitro, we first established that cathepsin activity increases during the initial stages of TAM;Bcl-xLMycER tumor development. Among the cathepsin family members investigated, only cathepsin L was predominately produced by beta-tumor cells in neoplastic pancreata and, consistent with this, cathepsin L mRNA expression was rapidly upregulated following Myc activation in the beta cell compartment. By contrast, cathepsins B, S and C were highly enriched in tumor-infiltrating leukocytes. Genetic deletion of cathepsin L had no discernible effect on the initiation of neoplastic growth or concordant angiogenesis. However, the tumors that developed in the cathepsin L-deficient background were markedly reduced in size relative to their typical wild-type counterparts, indicative of a role for cathepsin L in enabling expansive tumor growth. Thus, genetic blockade of cathepsin L activity is inferred to retard Myc-driven tumor growth, encouraging the potential utility of pharmacological inhibitors of cysteine cathepsins in treating late stage tumors.Motivated by the recent implication of cysteine protease cathepsin L as a potential target for anti-cancer drug development, we used a conditional  Progression of normal cells into malignancies in humans is dependent upon acquisition of a number of functional cancer “hallmarks” including uncontrolled proliferation, suppressed cell death, increased invasion, angiogenesis, and reprogramming of biosynthetic metabolism . HoweverAPCmin-driven model of colon cancer loss of CTS L results in increased tumor multiplicity [in vitro angiogenesis assays [The human cysteine cathepsins are family of 11 proteases, all of which share a conserved active site . Increasiplicity . For exas assays  yet genes assays . Similars assays  but not s assays . Nonethes assays ). It is myc gene, c-Myc, is a highly pleiotropic transcription factor that regulates expression of many, diverse genes [myc oncogene is implicated in driving the relentless growth of many human cancers  via itsview see , 24, angview see , 26 and view see . Each ofgression , a capabpoptosis . Here weCysteine cathepsin proteases, particularly CTS B and L, are frequently upregulated in various types of human cancers . Moreovein vivo is associated with an increase in cysteine cathepsin L, and to determine whether, and at what stage(s) of disease progression, such CTS L might be functionally important. Activation of 4-OHT-dependent c-MycERTAM in the beta-cell compartment of pancreatic islets of the MycERTAM;Bcl-xL model of pancreatic neuroendocrine tumors (PNET) results in synchronous onset and progression of beta-cell proliferation, culminating in highly angiogenic and invasive tumors [in vivo [Deregulated activity of transcriptional factor Myc is implicated in a large number of cancers and often associated with more aggressive tumors. We sought to establish whether Myc-induced tumorigenesis in pancreatic endocrine tumors e tumors , 28. Fol[in vivo .MycERTAM;Bcl-xL islets following 2, 8, 24 hours and 21 days of Myc activation [cathepsin L mRNA expression among the four cysteine cathepsin genes expressed in the MycERTAM;Bcl-xL islets (CACGTG) element at position -458 of the cathepsin L promoter. We reason that the transient induction of CTS B, C and S reflects the Myc-induced recruitment of infiltrating myeloid cells [[Analysis of a beta-cell Myc target gene database obtained from laser-captured L islets . Such red cells [, 32 and d cells [.CTS L mRNA expression in the early stages of Myc-induced tumorigenesis correlates with increased cysteine cathepsin L enzymatic activity, we used DCG-04, a chemical activity-based probe (ABP) specific to the cysteine cathepsins [in vitro and in vivo. Myc was activated in MycERTAM;Bcl-xL mice for 3 or 7 days, corresponding to the early stages of MycERTAM;Bcl-xL PNET tumor progression [Bcl-xL animals to seven-day treatment with TAM , which was administered intravenously into MycERTAM;Bcl-xL mice treatment 7 days. We detected an increase in total cystein cathepsin activity in the hyperplastic islets following Myc activation in vivo  was administered to sections —Myc-ON. or model . MoreoveMycERTAM;Bcl-xL models appear to have similar properties [MycERTAM;Bcl-xL and RT2 models [MycERTAM;Bcl-xL PNET cancer cells are driven by distinct oncogenes, the SV40 antigen and activated c-Myc, respectively.Progression of c-Myc-induced PNET tumorigenesis is accompanied by the recruitment of a variety of inflammatory cells to the neoplastic site, which serve to support tumor growth, angiogenesis and invasion , 32. The2 models . HoweverMycERTAM;Bcl-xL PNET progression, we bred the MycERTAM;Bcl-xL animals into the cathepsin L-deficient background. The resulting MycERTAM;Bcl-xL;Cathepsin L KO  animals were phenotypically similar to CTSL KO littermates [TAM activation. To assess the effect of CTS L-deficiency on the early stages of Myc-induced beta-cell tumourigenesis, Myc;Bcl-xL;CTSLKO and Myc;Bcl-xL animals were subjected to treatment with TAM for the duration of three days. This treatment induced proliferation in the beta-cell compartment of pancreatic islets accompanied by tumor angiogenesis [in vivo administration of a broad-spectrum inhibitor of cysteine cathepsin activity—JPM-OEt [RT2 model is the cell-cell adhesion glycoprotein E-cadherin [MycERTAM;Bcl-xL model, E-cadherin downregulation by Myc correlates with the abrupt loss of intercellular contacts within the islet, macroscopic islet expansion, and local invasion of the pancreas [Myc;Bcl-xL pancreatic islets revealed rapid and sustained loss of cell surface E-cadherin staining within the pancreatic islets following three days of c-Myc activation, independent of cysteine cathepsin activity  animals following three days of Myc-activation revealed that while the Bcl-xL-negative Myc-expressing islets rapidly involute following Myc-activation as described in [MycERTAM;Bcl-xL;CTSLKO islets . While cathepsin L-deficiency had no profound effect on pancreatic tissue pathology in the absence of MycERTAM activation  of tumors collected from the MycERTAM;Bcl-xL mice. We observed no dosage-specific effect of cathepsin L deficiency, as CTSL heterozygous animals developed tumors indistinguishable in their histopathological features from the lesions arising from the CTSL WT background. Intriguingly, we detected a profound deficit in Myc-induced beta-cell proliferation in CTSL-deficient animals after 2-weeks of MycERTAM activation (0.19% of proliferating tumor cells in the CTSLKO mice compared to 21.6% in the CTSL WT lesions)   that wasLKO mice .MycERTAM;Bcl-xL and MycERTAM;Bcl-xL;CTSL-deficient animals subjected to 14 days of Myc-activation in vivo revealed a significant increase in the punctate pattern of LC3 expression in the CTSLKO tumors  in the cathepsin L-deficient background was significantly reduced by ~90% when compared to control tumors with WT CTS L [MycERTAM;Bcl-xL model, deficiency in cathepsin L activity also had a profound effect on late stages of disease progression, manifested by ~97% of growth inhibition and partial collapse of the developed lesions. We conclude that cathepsin L is required for tumor maintenance and progression, rather than tumor initiation, during neuroendocrine tumorigenesis involving Myc-driven proliferation.The distinction between tumor initiating lesions and factors required for tumor maintenance is an important issue in tumor biology. In the constitutive model of PNET progression—WT CTS L . In the Glut-1, induced over-expression of metabolic enzymes, and the use of alternative sources of energy such as glutamine [in vitro and in vivo [Highly proliferative tumor cells require a constant supply of nutrients to support growth, usually achieved by increased uptake of glucose and a high glycolytic rate. C-Myc orchestrates tumor metabolic adaptation by several mechanisms, including upregulation of the glucose transporter lutamine , 46. In  in vivo , 48. On  in vivo , 50. Alt in vivo , 52. Thu in vivo  or knock in vivo  leads toin vivo. We show that deletion of cathepsin L in Myc-driven pancreatic neoplasias results in both a reduction in autolysosomal formation and an increase in tumor cell death. Though our current data does not formally demonstrate a causal relationship between cathepsin L deletion and alteration of autophagy in Myc-driven lesions, or that the increase in tumor cell death detected in late stages of tumor progression in the MycERTAM;Bcl-xL;CTSL-deficient neoplasias is a direct consequence of altered autophagic flux in vivo, it is reasonable to suggest that these events are intertwined [To our knowledge, the work presented herein is the first report demonstrating a link between the c-Myc oncogene and the lysosomal protease cathepsin L in tumor cells ertwined . The exaTAM and constitutive Bcl-xL in their pancreatic β cells pIns-MycERTAM;RIP7-Bcl-xL  mice have been previously described and characterized [TAM was activated in beta-cells in situ by daily i.p. injection of Tamoxifen (TAM) (1mg/mouse/day) dissolved in peanut oil (Sigma). TAM is metabolized in vivo to 4-OHT and has an equivalent effect to 4-OHT when administered to MycERTAM;Bcl-xL mice. The cathepsin L deficient animals were previously reported [In vivo labeling and detection of cathepsin activity with BODIPY 530/550-DCG-04 was performed as described [in vivo, the animals were subjected to 50mg/kg/day injection with the inhibitor JPM-OEt or control vehicle (30% DMSO/70% PBS) in conjunction with TAM injections for the duration of 5 days as previously described [2.This study was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco, protocol number AN076148. The transgenic mice expressing switchable MycERcterized . MycERTA2O2 in tap water for 5 minutes. Avidin/Biotin blocking and blocking of endogenous mouse immunoglobulins in the tissue were performed respectively by Vector Avidin/Biotin blocking kit (SP-2001) and Vector MOM basic kit (Cat. No. BMK-2202), both were used according to the manufacture’s protocols. The primary antibody used: anti-LC3 5F10 mAb . Sections were incubated with ABC reagent VACTASTAIN Elite ABC Standard Kit (PK-6100) and developed with diacylbutyrate (DAB). DAB-stained slides were counterstained lightly with hematoxylin.Briefly, paraffin-embedded sections (10 μm) were deparaffinized and rehydrated in a series of graded alcohols and blocked for endogenous peroxidase activity. Antigen retrieval was performed in 10 mM sodium citrate (pH 6.0). Endogenous peroxidase activity was blocked with 3% HFor the OCT-embedded tissues, 10 μm sections were fixed in 1% paraformaldehyde. The primary antibodies used: anti-mouse VEGF A , anti-Meca-32 , anti-Ki67 , anti-murine CD45 , rabbit polyclonal anti-LAMP1 , rabbit polyclonal anti-active-caspase 3 , and anti-cystein cathepsins antibodies—Cathepsin B (AF965), Cathepsin S (AF1183), Cathepsin C (AF1034), and Cathepsin L (AF1515) . All were applied in blocking buffer  for 2–16 hours. Secondary antibodies were from Dako and Molecular Probes. The cells were counterstained with DNA-binding dye—DAPI .Fluorescent images were obtained using Axiovert 100 inverted microscope (Zeiss) or Leica DM5500 microscope with cameras DFC360X (fluorescent detection) and DFC 295 (colorimetric detection). Confocal images were obtained using a LSM 700 (Zeiss). Immunohistochemical analysis for each antigen was performed at least in duplicate. The quantitative analyses of immmunostained sections were performed automatically by Fuji NIH software, with algorithms provided by the Bioimaging and Optics platform at EPFL.Endothelial cell proliferation was quantified in tissue sections by counting randomized fields of endothelial cells (Meca32+) and calculating the percentage of Ki67+ cells. At least three animals were assayed of each genotype all analyses done in duplicate; ten randomized fields per analysis were considered. Statistical significance was assessed using the Student T-test.http://rsb.info.nih.gov/ij) as follows. Ten serial H&E-stained sections of the entire pancreata from the tumor-bearing animals were scanned with a Leica DM5500 at 20x magnification. The tumors were manually outlined with Freehand Selection tool. The software then calculates the outlined area. At least 17 tumors for each genotype were analyzed. Each collected tumor was treated as an independent event. Analysis of BODIFY fluorophore mean intensity was performed with the Fiji Software package. The tumors were manually outlined with Freehand Selection tool. The software then calculates the mean intensity of the selected area. At least eight tumors for each treatment were analyzed.Analysis of size was performed with the Fiji Software package . Protein concentration was established by the Bradford assay. The equal amount of protein lysates were labeled with biotinylated DCG-04 ABP [For escribed  were homG-04 ABP  equally G-04 ABP .These procedures were described in .http://rsb.info.nih.gov/ij) according to algoriphm defined by Bioimaging & Optics platform (PT-BIOP) at EPFL.Data were presented as the means ± standard error of the mean (SEM). The statistical analysis was calculated using GraphPad Prism5 software package. The quantification of tumor proliferation and apoptosis was performed by the Fiji Software package , which increases following administration of 4-OHT (Myc-ON) at the timepoints indicated. Two independent islet purifications are presented for MycERTAM;Bcl-xL animals treated for 7 days. The activity bands corresponding to cysteine cathepsins (CTS) L, B and C are indicated. Molecular weight marker is indicated on the side panel. Note the lack of cathepsin L and cathepsin B activation in samples collected from control Bcl-xL background animals.Cathepsin activity profiles for the pancreatic islets from (TIF)Click here for additional data file.S2 FigMycERTAM;Bcl-xL animals were either untreated or treated daily with TAM for 5 days in conjunction with control vehicle (DMSO) or the broad spectrum cathepsin inhibitor JPM-OEt (JMP). At the times indicated, animals were sacrificed and pancreata isolated, sectioned and stained with anti-VEGF-A antibody (red) and Meca-32 (green). Images collected from two independent animals are presented. The panels are representatives of at least three animals assayed at each data point, immunohistochemical analyses done in duplicate; seven randomized fields per analysis were considered. Scale bars represent 100 μm. (B) Immunohistochemical analysis of E-cadherin in the pancreata collected from the animals described above. The areas positive for E-cadherin expression in inhibitor-treated mice are indicated by arrows. I-islet area is outlined by dotted line. The panels are representatives of at least three animals assayed at each data point, immunohistochemical analyses done in duplicate; five randomized fields per analysis were considered. Scale bars represent 20 μm.(A) Inhibition of cathepsin activity does not interfere with Myc-induced redistribution of VEGF-A in islets. (TIF)Click here for additional data file.S3 FigMycERTAM;Bcl-xL and MycERTAM;Bcl-xL;CTSLKO animals subjected to 3-day-treatment with TAM. . Tissues collected from MycERTAM;CTSLWT islets were used as a negative control for the staining. Three animals were assayed of each genotype; seven randomized fields per analysis were considered. Scale bars represent 50 μm.Immunohistochemical analysis of Bcl-xL expression in pancreatic tissues collected from (TIF)Click here for additional data file.


GJB2) have been reported to be a major cause of non-syndromic hearing loss in many populations worldwide. The spectrums and frequencies of GJB2 variants vary substantially among different ethnic groups, and the genotypes among these populations remain poorly understood. In the present study, we carried out a systematic and extended mutational screening of GJB2 gene in 1067 Han Chinese subjects with non-syndromic hearing loss, and the resultant GJB2 variants were evaluated by phylogenetic, structural and bioinformatic analysis. A total of 25 (23 known and 2 novel) GJB2 variants were identified, including 6 frameshift mutations, 1 nonsense mutation, 16 missense mutations and 2 silent mutations. In this cohort, c.235delC is the most frequently observed pathogenic mutation. The phylogenetic, structural and bioinformatic analysis showed that 2 novel variants c.127G>T (p.V43L), c.293G>C (p.R98P) and 2 known variants c. 107T>C (p.L36P) and c.187G>T (p.V63L) are localized at highly conserved amino acids. In addition, these 4 mutations are absent in 203 healthy individuals, therefore, they are probably the most likely candidate pathogenic mutations. In addition, 66 (24 novel and 42 known) genotypes were identified, including 6 homozygotes, 20 compound heterozygotes, 18 single heterozygotes, 21 genotypes harboring only polymorphism(s) and the wild type genotype. Among these, 153 (14.34%) subjects were homozygous for pathogenic mutations, 63 (5.91%) were compound heterozygotes, and 157 (14.71%) carried single heterozygous mutation. Furthermore, 65.28% (141/216) of these cases with two pathogenic mutations exhibited profound hearing loss. These data suggested that mutations in GJB2 gene are responsible for approximately 34.96% of non-syndromic hearing loss in Han Chinese population from Zhejiang Province in eastern China. In addition, our results also strongly supported the idea that other factors such as alterations in regulatory regions, additional genes, and environmental factors may contribute to the clinical manifestation of deafness.Mutations in Gap Junction Beta 2 ( It can GJB2 gene, which encodes the connexin 26 (Cx26), are a major cause of non-syndromic hearing loss in many populations worldwide/[c.299_300delAT] was found to be the most frequent genotype with a frequency of 30.16% (19/63) in compound homozygotes group. Among the 216 subjects with biallelic pathogenic mutations  genotypes carried pathogenic mutations  and 2 novel genotypes harbored the putative deafness-associated mutations (p.L36P and p.V63L). In addition to the genotypes contained pathogenic mutation(s) mentioned above, there were 22 genotypes did not carry any deafness-associated mutation(s), involving 65.04% (694/1067) cases of this cohort. Of these nonpathogenic genotypes, 21 genotypes were found only harboring polymorphisms including 2 genotypes with silent variants  and 19 genotypes carrying missense variants .GJB2 mutations. The comprehensive history and physical examination including audiological assessment indicated that hearing impairment was the sole clinical phenotype in these subjects, and no other clinical abnormalities was observed in members of their families such as diabetes, muscular diseases, visual loss and neurological disorders. All the 4 subjects did not have a history of exposure to neither aminoglycosides nor other ototoxic drugs. Moreover, there was no evidence that the 4 cases had encountered any other external cause to be responsible for hearing loss. Audiological assessments indicated that they all exhibited profound hearing loss at ages younger than 2 years old, with flat-shaped pattern of audiometric configuration.A genotype-phenotype correlation analysis was performed among 4 probands carrying the 4 putative deafness-associated /[c.109G>A] genotype, showed profound congenital hearing loss. Among this pedigree, the c.293G>C (p.R98P) variant was also detected in III-3, III-9 and IV-3 in heterozygous state. Audiological examinations showed mild hearing loss in III-3 with sloping-shaped pattern, and the III-9 and IV-3 exhibited profound congenital hearing loss.As illustrated in GJB2 is the most common causative gene for hereditary hearing loss in many populations worldwide, and most of the GJB2 sequence variations described to date were localized in the coding region (The Connexin-deafness Homepage. http://davinci.crg.es/deafness). In fact, the GJB2 mutation spectrums and frequencies vary among different ethnic population[GJB2 variants in 1067 Chinese non-syndromic hearing loss subjects from Zhejiang Province in eastern China, and investigated the clinical and genetic characterization of patients with putative GJB2 mutations. A total of 25 different nucleotide changes in the GJB2 gene were identified in this cohort, 34.96% of subjects in the cohort harbored GJB2 pathogenic mutations, which can be classified into 44 genotypes, including homozygotes, compound heterozygotes and single heterozygotes . These results indicate that GJB2 mutations are the important causes of hearing loss in hearing-impairment Han Chinese population.opulation,6. In thGJB2 mutations in Chinese hearing loss patients, which is compatible with other reports[GJB2 gene predominantly occurs in people of East Asian, the incidences of the mutation ranged from 6.7% to 22.5% in several other Chinese deaf cohorts according to previous studies, which were 5.3% and 10.9% in two reported Japanese hearing loss cohorts, 1.5% in a cohort from Mongolia, 5.1% and 6.9% in two cohorts of Korean deafness patients[GJB2 mutations in these populations[http://www.mitomap.org, 2015). It has been well accepted that haplogroup L3 gave rise to two lineages: M and N. In addition, the major haplogroups B and F in East Asian were rising from macrohaplogroup N, and the mitochondrial haplogroups in South Asian were macrohaplogroup M and its derivatives. These results indicate that c.235delC mutation might have arisen after the racial divergence of South and East Asian occurred. It would be necessary to systematically investigate the GJB2 mutations of remaining Asian countries to precisely elucidate the origin of c.235delC mutation[GJB2 deletion/frameshift mutations associated with hearing loss, c.176_191del16 and c.299_300delAT were previously reported in Korean and Japanese hearing loss cohorts[In the present study, we revealed a high prevalence 27.13%) of % of GJB2r reports. Among t patients,14,24–28pulations,29,30. I mutation. Of the s cohorts, and c.5GJB2 was previously reported to be the most prevalent mutation (21%) in a cohort of north Indian population with hearing loss[The c.35delG mutation of ring loss. In partring loss. In factring loss. In thistrans with 35delG in a hearing loss patient of African descent[GJB2 variant we found in this study was the p.R98P mutation which occurred in amino termini and intracellular loop, it is proposed that this mutation may impair the gap junctional intercellular communication in cochlea and finally lead to hair cell dysfunction. More interestingly, we found that the p.R98P variant co-segregates with the phenotype of hearing loss in the WL111 pedigree  were detected among these hearing loss patients, including 4 pathogenic mutations, 7 polymorphisms and 4 unknown variants. To identify putative pathogenic mutation, the unknown variants were further evaluated using following criteria: (1) conservation index greater than 75%, (2) absent in the 203 controls, (3) potential structural and functional alterations, predicted by their locations, PolyPhen-2 and SIFT programs, and (4) pedigree analysis if possible. The p.L36P, p.V43L, V63L and p.R98P variants, which occurred at evolutionarily conserved amino acid residues and absent from the control group, were predicted as pathogenic mutations by PolyPhen-2 and SIFT programs. The p.L36P mutation was first described in n descent. This pun descent. Here, wn descent. Howeverpedigree . But basXenopus oocytes resulted in a dominant-negative effect of the mutation[The p.V37I variant was originally reported as a polymorphism. Subsequ mutation. In gene mutation,40. TakiGJB2 in the large cohort of 1067 hearing-impaired subjects including 24 novel genotypes. Totally, 20.25% of the subjects harbored two GJB2 pathogenic alleles, either homozygotes or compound heterozygotes, and there was a fairly high frequency (14.71%) of single heterozygosity in this cohort. More interestingly, patients with homozygous or compound heterozygous for the same pathogenic mutation exhibit a wide variation in the phenotypic expression of hearing loss. These data strongly supported the hypothesis that other factors may contribute to the clinical manifestation of GJB2-related deafness, such as alterations in regulatory region, additional mutations in other genes such as GJB6 and GJB3, and environmental factors[GJB2 mutations can yield considerable helpful information to those hearing impaired population and their families. Understanding of the cause of hearing loss will effectively promote genetic counseling and implementation of various intervention strategies such as early rehabilitation and fitting of hearing aids, which is particularly important for infants in affected families. In addition, further studies emphasize on the disease associated factors are very important that would enable us to gain a deeper understanding of the complex molecular pathogenic mechanisms of hearing loss.In this report, we found 66 genotypes of l factors,42. AccoS1 FigThe Cx26 protein has four transmembrane domains (M1–M4), connected by two extracellular loops (E1 andE2) and a cytoplasmic loop (CL). The NT and CT denote the N- and C-termini of the protein.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Arabidopsis seedlings in the dark. The IC50 value for FM to inhibit stem elongation of Arabidopsis seedlings grown in the dark was approximately 1.8 ± 0.2 μM. FM-induced dwarfism of Arabidopsis seedlings could be restored by brassinolide (BL) but not by gibberellin (GA). Assessment of the target site of FM in BR biosynthesis by feeding BR biosynthesis intermediates indicated that FM interferes with the side chain hydroxylation of BR biosynthesis from campestanol to teasterone. Determination of the binding affinity of FM to purified recombinant CYP90D1 indicated that FM induced a typical type II binding spectrum with a dK value of approximately 0.79 μM. Quantitative real-time PCR analysis of the expression level of the BR responsive gene in Arabidopsis seedlings indicated that FM induces the BR deficiency in Arabidopsis.The plant steroid hormone brassinosteroids (BRs) are important signal mediators that regulate broad aspects of plant growth and development. With the discovery of brassinoazole (Brz), the first specific inhibitor of BR biosynthesis, several triazole-type BR biosynthesis inhibitors have been developed. In this article, we report that fenarimol (FM), a pyrimidine-type fungicide, exhibits potent inhibitory activity against BR biosynthesis. FM induces dwarfism and the open cotyledon phenotype of  Brassinosteroids (BRs) are plant-specific polyhydroxysteroids that are structurally similar to cholesterol-derived animal steroid hormones and the insect molting hormones ecdysteroids. The identification of BR-deficient and BR-insensitive mutants provided conclusive evidence that BRs are potent growth-promoting plant hormones. BRs play important roles in plant growth, development and responses to environmental cues ,2. BRs aAs a key player involved in the control of plant architecture, the biosynthetic pathway of BRs is believed to be a good target for engineering a high yield of crops . Crop plArabidopsis CYP85A1 and CYP85A2 were found to catalyze the C-6 oxidation reaction [The establishment of the biosynthetic pathways  17] and and17] areaction . These oThe inhibition mechanisms of P450 have been studied in considerable detail . The nitBased on these observations, we conducted a chemical screening of new BR biosynthesis inhibitors among the 14-α demethylase (CYP51)-inhibiting fungicides. We found that fenarimol -4-(2-propoxyphenoxymethyl)-dioxolan-2-ylmethyl]-1H- triazole}, a potent inhibitor of BR biosynthesis developed in our laboratory .,20.ArabiM  vs. the concentration of FM  analysis. The concentrations of FM used were 1 and 3 μM, at which FM promisingly induces BR-deficiency-like phenotypes in Arabidopsis , which are down-regulated with BR stimulation through a negative feedback mechanism , and BSSdet2 and wild-type plants treated with brassinazole showed increased expression of photosynthesis genes in the dark, which is a phenomenon known as “de-etiolation in the dark” by BR deficiency . Yu. Yu41]. Small molecules targeting the biosynthesis of BRs are useful tools in discerning the functions of BRs . They ca3 and campesterol were purchased from Wako Pure Chemical Industries, Ltd. . Brassinolide, castasterone, and teasterone were purchased from Burashino Co., Ltd. . Campestanol was prepared by reduction of campesterol using a method as described previously [Fenarimol, GAeviously . All of Arabidopsis (Columbian ecotype) were purchased from Lehle Seeds . The seeds used for assay were sterilized in 1% NaOCl for 20 min and washed with sterile distilled water. Seeds were sown on a 1% solidified agar medium containing half Murashige and Skoog salt in agripots , with or without chemicals. Plants were grown under 16 h light (240 μE/m2s) and 8 h dark conditions in a growth chamber. For the dark condition, agripots were wrapped in four layers of aluminum foil. The biological activities of the test compounds were measured 5 days after sowing the seeds. Stock solutions of all of the chemicals were dissolved in DMSO to a designated concentration and applied to growth media at 0.1% (v/v).Seeds of The methods for total RNA isolation, cDNA synthesis, and RT-PCR have been previously described . ACT2 anArabidopsis full-length cDNA was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT , Japan [SacI-PstI and was inserted into the pCold-GST expression vector. All of the constructed plasmids were transferred to the BL21 star (DE3) strain of E. coli . The transformed cell was incubated in 10 mL of Luria broth containing 100 μL/mL of chloramphenicol overnight at 37 °C. Then, 10 mL of pre-culture was incubated in 1000 mL of Luria broth containing 100 μL/mL of ampicillin at 37 °C.), Japan ,44. The ), Japan . The DNAThe expression and purification of recombinant CYP90D1 were performed as described previously . The purBinding of FM to CYP90D1 was measured by optical difference spectroscopy of purified recombinant CYP90D1 using a Shimadzu UV3100 spectrophotometer as we previously described . Data ob


The objective of the present study was to examine the associations between various anthropometric measures and metabolic syndrome and hearing impairment in Asian women.We identified 11,755 women who underwent voluntary routine health checkups at Yeungnam University Hospital between June 2008 and April 2014. Among these patients, 2,485 participants were <40 years old, and 1,072 participants lacked information regarding their laboratory findings or hearing and were therefore excluded. In total 8,198 participants were recruited into our study.hearing loss were 65.8% and 73.4%, respectively. The WHR had the highest AUC and was the best predictor of metabolic syndrome and hearing loss. Univariate and multivariate linear regression analyses showed that WHR levels were positively associated with four hearing thresholds including averaged hearing threshold and low, middle, and high frequency thresholds. In addition, multivariate logistic analysis revealed that those with a high WHR had a 1.347–fold increased risk of hearing loss compared with the participants with a low WHR.The AUROC value for metabolic syndrome was 0.790 for the waist to hip ratio (WHR). The cutoff value was 0.939. The sensitivity and specificity for predicting metabolic syndrome were 72.7% and 71.7%, respectively. The AUROC value for hearing loss was 0.758 for WHR. The cutoff value was 0.932. The sensitivity and specificity for predicting Our results demonstrated that WHR may be a surrogate marker for predicting the risk of hearing loss resulting from metabolic syndrome. Metabolic syndrome is a well-known risk factor for cardiovascular diseases including atherosclerosis and hypertension (HTN) and is diagnosed according to five criteria: increased waist circumference (WC), high triglyceride level, low high-density lipoprotein (HDL) cholesterol level, high blood pressure, and high fasting glucose level . ObesityHearing loss is a major public health problem that is associated with a low quality of life ,3. The pThe WHR is a well-known indicator of metabolic disturbance that is related to the amount of visceral fat, and a high WHR is associated with the overproduction of adverse adipokines and insulin resistance . TherefoThis was a single center-retrospective study. We identified 11,755 women who underwent voluntary routine health checkups at Yeungnam University Hospital between June 2008 and April 2014 . Partici2), WHR, WHtR, WC (cm), body shape index (BSI), body adiposity index , visceral fat area , systolic blood pressure (mmHg), diastolic blood pressure (mmHg), and fasting glucose (mg/dL), triglyceride (mg/dL), and HDL cholesterol (mg/dL) levels.The subjects arrived at the hospital after an overnight fast. Clinical and laboratory data collected during the health examination included age, sex, hearing thresholds, BMI  by the enzymatic method . DM was defined as a self-reported history of a DM diagnosis or a fasting glucose level of ≥126 mg/dL. HTN was defined as systolic blood pressure of ≥140 mmHg, diastolic blood pressure ≥90 mmHg, self-reported history of HTN, or use of anti-hypertension drugs. Metabolic syndrome was defined according to the National Cholesterol Education Program Adult Treatment Panel III guidelines [Weight, height, WC, and hip circumference were measured by well-trained medical professionals. Standing height was measured with the subject facing directly ahead with shoes off, feet together, arms by the sides, and heels, buttocks, and upper back in contact with the wall using a SECA 225 . Weight was measured using a GL-6000-20 scale . WC was measured at the midpoint between the bottom of the rib cage and the top of the iliac crest. Hip circumference was measured over the widest part of the gluteal region. BMI was calculated by dividing the total body weight in kilograms by the square of the participant’s height in meters. WHR and WHtR were calculated as WC divided by hip circumference or height. BAI and BSI were calculated using equations described previously ,14:BAI=VFA was measured using multi-frequency bioimpedance analysis .®, USA) and headphone . Testing was conducted as per the recommended standard audiometry procedures using automatic mode, except when respondents could not physically press the response button, or when there were difficulties using automatic mode. Manual mode was carried out using the down-10 up-5 method [Audiologic evaluation was performed by a specialist practitioner with training/supervision provided by a certified audiologist, which included on-site visits to ensure quality control. Air-conduction pure-tone thresholds were obtained in a soundproof booth using an audiometer  or MedCalc version 11.6.1.0 . Categorical variables are expressed as numbers and percentages, and continuous variables are expressed as the mean ± the standard deviation or standard error. We calculated the sensitivity, specificity, cutoff values, and probability of area under the receiver operating characteristic curve (AUROC) to predict metabolic syndrome or hearing loss using variable anthropometries indices. The best cutoff risk point was derived from the maximum of the Youden index in the AUROC. Correlation analysis was performed to assess the strength of the relationship between variable anthropometric indices and VFA or numbers of metabolic syndrome components.2 test or Fisher’s exact test were used to assess the strength of association between age and the presence of metabolic syndrome or hearing loss. Multivariate analysis was performed using age, DM, HTN, and WHR. The level of statistical significance was set at P < 0.05.Linear regression analysis was used to determine whether the WHR contributes to the hearing threshold, which is a continuous variable expressed as dB HL. The patients were divided into low and high WHR groups according to the cutoff value for hearing loss. Changes in hearing thresholds according to the two WHR groups were analyzed using an independent t-test for univariate analysis and analysis of covariance for multivariate analysis. Logistic regression analyses were used to determine whether the WHR contributes to hearing loss. Pearson’s χThe mean age of the participants was 54.7 ± 9.9 years  for WHR . The cutThe AUCs of WHR were 0.698  for elevated fasting glucose level, 0.727  for elevated blood pressure, 0.688 (0.677–0.698) for elevated triglyceride level, and 0.643 (0.632–0.653) for decreased HDL cholesterol . For eleUnivariate and multivariate linear regression analyses showed that WHR levels were positively associated with four hearing thresholds including PTA and Low-Freq, Mid-Freq, and High-Freq thresholds .P < 0.001). The Low-Freq threshold was 20.0 ± 0.1 dB HL in those with a low WHR and 28.5 ± 0.2 dB HL in those with a high WHR (P < 0.001). The Mid-Freq threshold was 21.0 ± 0.1 dB HL in those with a low WHR and 32.5 ± 0.3 dB HL in those with a high WHR (P < 0.001), and the High-Freq threshold was 23.5 ± 0.2 dB HL in those with a low WHR and 40.4 ± 0.3 dB HL in those with a high WHR (P < 0.001). All hearing thresholds in participants with a high WHR were significantly elevated compared to those in participants with a low WHR. In multivariate analysis, the PTA was 23.8 ± 0.16 dB HL (mean ± standard error) in participants with a lower WHR than the cutoff value for hearing loss  and 25.3 ± 0.2 dB HL in those with a higher WHR than the cutoff value for hearing loss  (P < 0.001). The Low-Freq threshold was 22.7 ± 0.2 dB HL in those with a low WHR and 24.2 ± 0.2 dB HL in those with a high WHR (P < 0.001). The Mid-Freq threshold was 24.8 ± 0.2 dB HL in those with a low WHR and 26.5 ± 0.2 dB HL in those with a high WHR (P < 0.001). The High-Freq threshold was 29.1 ± 0.2 dB HL in those with a low WHR and 31.8 ± 0.3 dB HL in those with a high WHR (P < 0.001). All hearing thresholds in participants with a high WHR were significantly elevated compared to those in participants with a low WHR. In addition, univariate logistic regression analyses showed that the participants with a high WHR had a 5.308–fold increased risk of hearing loss compared with the participants with a low WHR . Multivariate analysis revealed that those with a high WHR had a 1.347–fold increased risk of hearing loss compared with the participants with a low WHR .Differences in hearing thresholds according to the WHR are shown in P < 0.001), and the prevalence of hearing loss in these groups was 11.0%, 27.9%, 56.3%, and 84.4%, respectively (P < 0.001).The numbers of participants in the 40–49, 50–59, 60–69, and ≥70-years-old groups were 2,828, 2,994, 1,534, and 842, respectively, and the WHR in these groups was 0.88 ± 0.03, 0.92 ± 0.04, 0.97 ± 0.04, and 1.00 ± 0.05, respectively. The prevalence of metabolic syndrome in the 40–49, 50–59, 60–69, and ≥70-years-old groups were 5.1%, 14.4%, 27.0%, and 36.0%, respectively (P < 0.001), 1.495– , 1.670– , and 3.271–  fold increased risk of hearing loss compared to participants with a low WHR. Multivariate analyses performed using DM, HTN, and age revealed that those with a high WHR in the 40–49, 50–59, 60–69, and ≥70-years-old groups had a 1.814– , 1.231– , 1.440– , and 3.377–  fold increased risk of hearing loss compared to participants with a low WHR.All hearing thresholds in participants with a high WHR were significantly elevated compared to those in participants with a low WHR . In addiIn this study, we investigated the predictive effect of WHR compared with WHtR, WC, BMI, BSI, and BAI with respect to metabolic syndrome or hearing loss. We found that WHR had the highest AUC for prediction of metabolic syndrome or hearing loss and was highly correlated with VFA measured by bioimpedance analysis and numbers of metabolic syndrome components. A High WHR was associated with elevated hearing thresholds including the PTA and Low-Freq, Mid-Freq, and High-Freq thresholds. This result suggests that WHR in Asian women is associated with the prevalence of hearing impairment results from the metabolic syndrome.r = 0.901). These results suggest that WHR is a good indicator for predicting metabolic syndrome or hearing loss.First, we established the best predictive index for predicting metabolic syndrome or hearing loss among WHR, WHtR, WC, BMI, BSI, and BAI. Analyses using AUROC showed the AUCs for predicting metabolic syndrome or hearing loss among these indices, and we identified cutoff values Click here for additional data file.S2 FigThere were 5,324 participants in Daegu Metropotian City, 2,243 in Gyeongsangbuk Province, 451 in Gyeongsangnam Province, 24 in Seoul Metropolitan City, 22 in Gyeonggi Province, 18 in Ulsan Metropolitan City, 11 in Pusan Metropolitan City, 11 in Kangwon Province, 8 in Chungcheongnam Province, 6 in Chungcheongbuk Province, 5 in Daejon Metropolitan City, 2 in Jeollanam Province, 2 in Jeollabuk Province, 2 in Jeju Province, 1 in Inchon Metropolitan City, 1 in Kwangju Metropolitan City, and 13 in other countries. No data was available regarding the place of residence for 54 participants.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


Ancylecha fenestrata. Correspondingly, A. fenestrata has a rather complex ear morphology including cuticular folds covering the anterior tympanal membrane. The position-dependent tuning might contribute to sound source localization in the habitats. Acoustic orientation might be a selective factor for the evolution of morphological structures at the bushcricket ear and, speculatively, even for frequency fractioning in the ear.A primary task of auditory systems is the localization of sound sources in space. Sound source localization in azimuth is usually based on temporal or intensity differences of sounds between the bilaterally arranged ears. In mammals, localization in elevation is possible by transfer functions at the ear, especially the pinnae. Although insects are able to locate sound sources, little attention is given to the mechanisms of acoustic orientation to elevated positions. Here we comparatively analyse the peripheral hearing thresholds of three species of bushcrickets in respect to sound source positions in space. The hearing thresholds across frequencies depend on the location of a sound source in the three-dimensional hearing space in front of the animal. Thresholds differ for different azimuthal positions and for different positions in elevation. This position-dependent frequency tuning is species specific. Largest differences in thresholds between positions are found in  For orientation, directional cues of sensory modalities are of pivotal importance. Sound waves are highly directional, although reflections and diffraction reduce their usage for localization. Ears themselves have directional characteristics and processing of directional information takes places at an early stage of auditory networks in the central nervous system . TypicalSound source localization is a main task in acoustically communicating insects. The threshold sound pressure levels of afferents and interneurons typically are higher for stimuli from contralateral than for stimuli from ipsilateral ,9. Howev2.2.1Mecopoda elongata, (ii) Stilpnochlora couloniana and (iii) Ancylecha fenestrata. All species were reared to the adult stages in large cages with fresh leaves , seedlings and fish food (M. elongata). Animals were kept in a humid environment at 25°–28°C at a 12 L:12 D cycle. M. elongata (Mecopodinae) was bred in the laboratory for several generations, whereas A. fenetrata and S. couloniana (Phaneropterinae) were reared in first or second generation from commercially obtained nymphs.Three different bushcricket species were tested: (i) For morphological investigations, the length of the tympana  and the maximum opening width of the acoustic spiracle were determined. The structures were photographed with a Leica camera (2048×1028 px) attached to a dissecting microscope. Measuring was done with the software IMAGEJ.Neural tracing of the tympanal nerve revealed the number of sensory cells in the crista acustica (for details see ). Basica2.2Adult animals were mounted upright on a narrow (3 mm width) metal holder with wax. The animals had a body width of 6.9–8.2 mm (at the thorax) and thereby concealed the metal holder. The femur of the foreleg was fixed 90° from the longitudinal axis of the body with a small wire (1 mm diameter) and the tibia with the tympanal organ was free in the sound field. The distance between tibia (ear) and the median body plane was 14–18 mm. Regarding the direction of the sound stimulation, the longitudinal body axis of the animal was orientated to 0° azimuth and 0° elevation . The holFor the sound stimulation, a loudspeaker (Dynaudio DF21) was mounted on a half of a modified bicycle rim 25 cm distance to the animal. By tilting the rim, different angular positions around the centre where the animal was placed could be obtained. Sound source positions in three main planes in respect to the body orientation were analysed: in the horizontal plane (azimuth), the loudspeaker could be moved to archive test angles of i110°, i80°, i60° and i30° on the ipsilateral side, c30°, c60° and c80° on the contralateral side as well as the frontal position 0°; . In the pike2 (Cambridge Electronic Design).For recording, a small cut was made in the dorsal femur of the left foreleg. Care was taken not to damage any trachea and the tympanal nerve was placed on a hook electrode. A second electrode was inserted close to the recording electrode in the haemolymph of the femur. The nerve was kept moist with application of small droplets of locust ringer  and sealpike2 software. Each stimulus had a duration of 50 ms  with a pause of 200 ms and was repeated five times. After a time delay of 400 ms, stimuli with the next intensity were presented. In pilot tests, the approximate thresholds for ipsilateral stimuli had been determined. For the experiments stimuli 10–15 dB sound pressure level (SPL) below and up to 20 dB SPL above the expected threshold were tested with 5 dB increments. Calibration of the SPL (rel. 20 μPa) was done on a continuous sound with a calibrated XL2 sound analyser (NTI Audio Instruments) and 1/4 inch calibrated microphones .Acoustic stimuli comprising pure sine waves between 4 and 40 kHz were generated with the Sx,y,z) coordinates  and interpolated with Origin 9.0 (Originlab).For data analyses, thresholds were determined visually as the sound intensity at which neuronal activity was seen in response to at least four of the five stimulus repetitions. For averaging threshold, SPL data were transferred to sound pressure (rel. 20 μPa) and thereafter transformed back to dB SPL for visualization. The mean threshold data were also depicted in contour graphs, whereby the threshold is colour coded. The contour was created from . Reduction in threshold or increase in threshold was colour coded for each frequency and depicted in a space diagram for each species.pike2. The sound-evoked activity 60 ms after stimulus onset was compared with the background activity of the nerve determined in a 60 ms window before the stimulus . The resulting values were plotted as percentage of the background activity for different stimulus angles.For the calculations of the suprathreshold response strength, the modulus of the recording trace was calculated in S3.3.1M. elongata, but occluded by the pronotum in S. couloniana and A. fenestrata. Mecopoda elongata and S. couloniana have open tympana, whereas in A. fenestrata a pronounced cuticular flap covers the anterior tympanum. All three species possess a typical arrangement of auditory receptor cells, with A. fenestrata exhibiting by far the highest number of sensory units .The auditory systems of the investigated species exhibit species-specific morphological characters . The ope3.2M. elongata is rather flat from 4 kHz up to 40 kHz with a minimum at approximately 8 kHz. The threshold curve of S. couloniana has a pronounced minimum at 8 kHz, but is about 7 dB more sensitive with a threshold of about 35 dB SPL. The most-sensitive hearing was recorded from A. fenestrata with a broad minimum range from 8 kHz up to 16 kHz with a threshold of approximately 30 dB SPL. The thresholds of males and females were similar, except for a few frequencies in two of five loudspeaker positions in S. couloniana .Extracellular recordings of the tympanal nerve activity during pure-tone stimulation from a stimulus direction of i80°  show species-specific hearing thresholds . The thr3.3Mecopoda elongata has relatively high thresholds for all loudspeaker positions . The response of S. couloniana was similar (data not shown). However, for A. fenestrata, a nonlinear increase with azimuthal position-dependent factors is seen at higher stimulus intensities (d), whereas for vertical positions only a linear increase is found (c).Since hearing properties are not reflected only in the threshold, suprathreshold responses of the sound-induced tympanal nerve activity have been calculated . The response to stimuli is plotted as a percentage above the baseline activity. Suprathreshold responses at a stimulus frequency of 20 kHz up to 25 dB above threshold were depicted for two species . Data oft slopes a,b. The ensities d, whereais found c.Figure4.Mecopoda elongata has a rather broad spectrum from 7 to 70 kHz correlating to the broad hearing threshold, although for M. elongata also a tuning of the hearing threshold to frequencies of 15–16 kHz has been reported [S. couloniana and A. fenestrata also match to the peak frequencies of the spectrum of the respective calling songs. Nevertheless, such matches have to be considered cautiously, as the behaviourally important frequency domains are not known. Filtering in the habitat might influence the signal and additional processes like parasite pressure might influence the hearing threshold [We investigated peripheral hearing thresholds of three species of bushcrickets in respect to different loudspeaker positions. Firstly, the results show species specific hearing thresholds. Species-specific hearing thresholds have been reported for a number of tettigonid species. Typically, thresholds correlate roughly with the peak frequency of the intraspecific calling songs ,38–40. Treported ,41. The hreshold .Secondly, the hearing thresholds of all three tested species depend on the sound source location. As early as 1940, Autrum showed that the threshold for ipsilateral sound sources is lower than for contralateral sound sources . Since tLocalization of elevated sound sources in vertebrates can be explained by transfer functions of sound waves along structures like the pinnae which guide and filter sound waves . The resM. elongata, whereas in others the pronotum covers the opening, like in S. couloniana and A. fenestrata. Other species can even actively close the spiracle [Tettigoniids have distinct morphologies of the auditory system. Our results indicate that morphology is important, as the three species have distinct auditory spaces. Typically, tettigoniids have four sound entrances : the ipsspiracle .A. fenestrata. The slits and the spiracles are responsible for coding directionality [Furthermore, the tympanal organ in the tibia is differently structured. The tympana can be openly exposed, as in the Phaneropteridae or covered by cuticular flaps . The cutionality ,52. Modiionality .All these peripheral structures, including the animal's body shape, might influence hearing and coding of auditory space. We have investigated the tuning of the afferents of one ear in the three species, whereby care has been taken to disturb the hearing system as little as possible. The animal holder including the recording electrode was relatively small and differences in thresholds are likely to be caused by attenuation and diffraction at the animal's body.Here we showed that in the auditory space the peripheral tuning is affected by the position. Thus, the thresholds of some frequencies are changed, whereas others are relatively unaffected. The tympanal membranes vibrate in complex modes in relation to sound input ,53 and pA. fenestrata) even had a nonlinear increase above threshold which might amplify differences between frequencies depending on the sound source position. In the suprathreshold range also additional factors like intensity fractioning of afferents [The suprathreshold responses of the afferents typically showed a linear increase for all frequencies, carrying forward the position-dependent neuronal excitation into the central nervous system. Some responses (in fferents  or non-mfferents  will afffferents .All these differences might provide cues for the animals for the sound source location in a three-dimensional space. It has been shown, that tettigoniids, can behaviourally locate a sound source in three dimensions ,16. PhysAncylecha fenestrata with a covered anterior tympanum shows the largest differences. Although we did not manipulate the hearing system, the different responses indicate the importance of these structures. In some species, morphological asymmetries have been detected in the hearing system, for example, in the spiracle size [The three species seem to have position-dependent differences in hearing thresholds, correlating to the different morphologies. cle size ,60. For cle size . For theSpeculatively, the position-dependent frequency tuning of hearing might also be a driving force for the frequency fractioning and tonotopy in the tettigoniid hearing organ. While many insects have the possibility of frequency discrimination , the tetIn conclusion, species-specific tuning depending on frequency and position has been found in the peripheral hearing threshold. This study opens possibilities of analysing the contribution of the morphological characters for frequency-dependent diffraction of sound waves or to correlate physiology with behaviour.


Hearing loss can affect approximately 15% of the pediatric population and up to 40% of the adult population. The gold standard of treatment for hearing loss is amplification of hearing thresholds by means of a hearing aid instrument. A hearing aid is an electronic device equipped with a topology of only three major components of aggregate cost. The gold standard of hearing aid fittings is face-to-face appointments in hearing aid centers, clinics, or hospitals. Telefitting encompasses the programming and adjustments of hearing aid settings remotely. Fitting hearing aids remotely is a relatively simple procedure, using minimal computer hardware and Internet access.This project aimed to examine the feasibility and outcomes of remote hearing aid adjustments (telefitting) by assessing patient satisfaction via the Portuguese version of the Satisfaction With Amplification in Daily Life (SADL) questionnaire.The Brazilian Portuguese version of the SADL was used in this experimental research design. Participants were randomly selected through the Rehabilitation Clinical (Espaco Reouvir) of the Otorhinolaryngology Department Medical School University of Sao Paulo. Of the 8 participants in the study, 5 were female and 3 were male, with a mean age of 71.5 years. The design consisted of two face-to-face sessions performed within 15 working days of each other. The remote assistance took place 15 days later.The average scores from this study are above the mean scores from the original SADL normative data. These indicate a high level of satisfaction in participants who were fitted remotely.The use of an evaluation questionnaire is a simple yet effective method to objectively assess the success of a remote fitting. Questionnaire outcomes can help hearing stakeholders improve the National Policy on Hearing Health Care in Brazil. The results of this project indicated that patient satisfaction levels of those fitted remotely were comparable to those fitted in the conventional manner, that is, face-to-face. The prevalence of hearing loss in Brazil has been identified as 50%, specifically, individuals with permanent hearing loss of more than 41 decibels hearing level (dB HL) in the municipality of Minas Gerais [The treatment of sensorineural hearing loss typically consists of the use of hearing aids (HA), and in cases of profound hearing loss, cochlear implants are used ,3. An HAFitting an HA demands specific technical knowledge of the electroacoustic characteristics of the device in relation to the patient’s hearing loss and, in Brazil, must be performed by an audiologist . The fitAfter receiving an HA, it is essential to monitor the patient in order to understand changes that affect the auditory system  and thusCox and Alexander  describeTelemedicine has evolved in many health care areas. With a greater coverage area and lower operating costs, it is fast becoming a suitable method for assessment, diagnosis, and rehabilitation of various conditions. In an early work with hearing rehabilitation through telemedicine, Wesendahl  reportedWith regard to telemedicine nationally, a study conducted by the University of Sao Paulo described the effectiveness of video conferencing for transmitting video-laryngoscopic images . While iA study of remote HA fittings  listed tThe National Policy on Hearing Health Care in Brazil was established through Ordinance #2.073/04 GM (September 2004). Ordinance #402 (February 2010) of the Ministry of Health established the program nationwide . Telemedicine in a structured format has aimed to quantify how to expand and strengthen strategies in family health. The Brazilian Federal Board of Audiology issued Resolution #366 (April 2009) that defined the lawful exercise of Telehealth in audiology with the use of information technology in order to “assist, promote education and conduct health research”. According to official data, the government has become the largest purchaser of HAs in Brazil, as shown in 2, according to the 2010 census.Ordinance SAS/MS #58 specifies that HAs must be dispensed through centers accredited by the Unified Health System (SUS), where professionals must “perform diagnosis and rehabilitation of hearing loss in all age groups spanning neonates to geriatrics, and perform consulting ENT, neurological, pediatric audiological evaluation”. Additionally, they must “ensure rehabilitation through clinical treatment in otolaryngology; selection, fitting and provision of an HA and speech therapy”. The Ministry of Health has a list of 139 accredited centers to serve the population, which was 190,732,694 inhabitants divided into 8,514,876,599 kmOur research describes a pilot study conducted with 8 patients fitted remotely through telemedicine, using the Brazilian Portuguese version of the SADL as a tool for measuring subjective satisfaction, with the goal of improving hearing health policies in Brazil.This research protocol was approved by the Ethics Committee for Analysis of Research Projects under #0293/11. The data collection was completed between June and October 2012.The Brazilian Portuguese version of the SADL was used see ; howeverAdditionally, Question 3 required a change as hearing aids were provided free of charge. The original question “Are you convinced that obtaining your hearing aids was in your best interests?” was changed to “Are you convinced that the received devices was the best option?” The documents used in this research are presented in The Windows operating system was used for the data collection in this study because it has a large set of commercial applications available and is the largest PC platform used in Brazil. A broadband Internet connection was provided by the specialized unit (SU) and the remote unit (RU). The SU had a trained audiologist, who provided support and scientific training for the audiologist at the RU, thus acting as a facilitator.The digital HAs donated and used this study were developed by researchers at the Medical School University of Sao Paulo, manufactured by Politec Saude . Only BeParticipants were randomly selected through the Rehabilitation Clinical (Espaco Reouvir) of the Otorhinolaryngology Department Medical School University of Sao Paulo based on the following criteria: (1) male and female individuals aged between 18 and 90 years, (2) with either no obstruction of the external auditory canal or middle ear pathology, or an absence of any neurological or psychological impairment, (3) individuals with no prior HA experience, (4) bilateral sensorineural hearing loss of varying degrees , (5) postlingual hearing loss, and (6) native Brazilians. It was important to include participants with no prior experience with HAs as long-term fitted subjects would have had difficulty answering Question 10 of the SADL, which relates to amplification. Furthermore, participants with no prior experience of amplification could make a judgment based solely on the amplification fitted in this study.We used an experimental research design. The following procedures were done face-to-face with the participants: (1) interview and otoscopy by otolaryngologist, (2) impedance and audiological measurements by audiologist, (3) agreement between patient and professional on the HA fitting, (4) earmold impressions, and (5) initial programming procedures with patient.The following elements were remote (telefitting): (1) presence of an audiologist in RU, (2) presence of an audiologist in the SU (as the facilitator), (3) remote aid adjustments and changes to fitting data based on patient audiogram and subjective feedback, and (4) verification of patient satisfaction using the SADL questionnaire.The SADL questionnaire was used as an interview schedule, that is, read aloud and completed by a trained interviewer for this purpose. The sessions were described as face-to-face (F) and remote assistance (R). There were two face-to-face sessions (F1 and F2) done within 15 working days of each other. The remote assistance (R) was 15 days after F2. This 15-day delay was justified so that patients would have adequate time and experience with the device and thus be able to respond accordingly to the SADL. At F1, patients agreed and signed the informed Consent Form (Chart 7). In the initial fitting sessions, the HAs were programmed through the easyFIT application, and the SU audiologist provided the necessary guidance to the patient. The face-to-face sessions followed the basic scheme described in The SU had an audiologist trained to fit the HA Mini Retro C through easyFIT while the audiologist at RU had no specific training for the Mini Retro C, nor for easyFIT. Both units were supervised by an otologist, while 2 information technology professionals offered the technological support, one for each side.For the remote (telefitting) sessions , all patThe SU audiologist then remotely accessed the fitting application easyFIT and began procedures to check the HA settings. The patients were questioned subjectively about various aspects of their HA usage, which allowed the audiologist at SU to make adjustments remotely. In all cases, adjustments were required. Remotely, the SU audiologist recorded the new settings in the HAs and updated patient information in easyFIT. Additionally, situational/environmental advice was provided to the patient remotely before the SU audiologist ended their interaction with the patient.Finally, the RU audiologist administered the SADL. The methodology of the questionnaire was explained, and each question was read aloud. This allowed for participants with poor literacy to fully understand all questions posed. Frequent pauses and repetitions were allowed for so that the patient had time to think about the answers. The session ended with the signing of Terms of Donation (TD - Chart 7) of HAs, again read aloud, so that patients with poor literacy were made aware of issues around the need to service HA quarterly from the date of signing the TD, as well as warranty periods, etc.In the initial telefitting session on September 18, 2012, 30 random measurements of Internet speed between the SU and RU were conducted . ThroughThe results of the SADL from this study compared to four other studies are provided in Telefitting can help improve hearing health policies in Brazil by gradually expanding the current 139 accredited centers to centers closer to patients’ homes as Basic Services Units, or health clinics, so the patient can receive adjustments to their HAs in their homes, with greater comfort and a greater chance of success in hearing rehabilitation. The participants in our study had a mean age of 71.5 years and had to travel an average of 8 miles (12.8 km) from home to RU. Despite missing official data, it is possible to speculate that all the government-run health centers have at least one PC and probably an Internet account, which highlights the possibility of performing telefitting supported by an SU. There are 537 Basic Services Units (ambulatory level) throughout the city of Sao Paulo alone. Furthermore, it may not be necessary to have the hardware component HI-PRO, since it could be replaced by an application installed over the Internet. A universal programming cable for all HA manufacturers could be used instead of standard cables for a particular manufacturer. In general, most clinics have to operate with a large number of programming cables for various manufacturers.The 2010 Brazilian Census reports that between 2005 and 2008, Internet access increased by 75.3% or 56 million users, due to various factors such as PCs and notebooks as well as high-speed Internet connections being more accessible and available at a lower cost. These factors, combined with other applications, can promote the use of telemedicine. Swanepoel et al  have higIn this study, applications and user-level information technology were used, which although limited, allowed for the fitting of HAs in 8 patients. Nevertheless, if more advanced technology were used , the possibility of conducting real-time orthoscopic reviews would be increased. In addition, traffic-encrypted data over the Internet, access from other accredited PCs on the network, as well as integration with other applications, would ensure that management costs are kept to a minimum. There would also be a reduced number of patient visits to the clinic and a database of valuable patient information to allow for remote HA adjustments.In a more comprehensive telemedicine approach, it would be beneficial to include video training for the audiologist, as well as detailed training on the equipment and troubleshooting for complex fittings. An online tutor can assist in immediate cases , while full online courses on anatomy and physiology of hearing, interpreting audiometry, among others may be available.Other questionnaires that could be used include the IOI-HA questionnaire, adapted to Brazilian Portuguese. It consists of seven questions, with a closed set of five different responses, and is thus easier to apply compared with SADL. The HHIE adapted to Brazilian Portuguese is structured into 25 questions, with a closed set of three possible answers on which Aiello et al  report tOne can standardize the application of a satisfaction questionnaire, after the initial HA fitting and before the end of the warranty period. Thus, two questionnaires could record satisfaction levels at two significant times in the hearing rehabilitation process.This study was one of the first of its kind with regard to adjusting HA settings via the Internet in Brazil. Furthermore, it creates a baseline for future research in this area of remote audiology and telefitting. However, our sample size of participants was small and within a limited geographical area. In addition, a limited number of applications were utilized.In this study, certain applications and features were used to perform remote adjustments and fitting of HAs in 8 patients without injury. In the more comprehensive telemedicine approach, it would be necessary for the RU audiologist to have additional training and support. Further studies with a larger sample population should be conducted to explore the reproducibility of the results recorded here.For implementation and public policy, we recommend the Windows platform be replaced by an open platform  in order to reduce costs and promote the development of local solutions. The Internet services should ideally be linked to attributes such as stability, availability, speed, absence of risk, and confidentiality of patient data must be protected.Furthermore, investigations conducted with the Brazilian Portuguese version of IOI-HA and APHAB questionnaires may be valuable. Although recording patient satisfaction through questionnaires provides valuable information about the use of HAs, the information derived is not entirely sufficient to assess the quality of overall hearing health. Bevilacqua et al  stated tA key step is to monitor satisfaction with technical issues by use of key performance indicators described by Kaplan and Norton , as a syRemote HA adjustments (telefitting) have proved effective for these 8 patients, as indicated by their dynamic responses in SADL. Results were comparable to those of patients fitted in the conventional manner . Thus, the use of telefitting can be seen as an effective method to improve service delivery of hearing health in Brazil.


Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is currently classified into clinical subtypes, including typical and atypical forms  and distal acquired demyelinating symmetric neuropathy (DADS)). The aim of this study was to elucidate the patterns and severity of breakdown of the blood-nerve barrier (BNB) in each CIDP subtype.We evaluated the effects of sera obtained from patients with typical CIDP, MADSAM and DADS and control subjects on the expression levels of tight junction proteins and transendothelial electrical resistance (TEER) value in human peripheral nerve microvascular endothelial cells (PnMECs).The sera obtained from the patients with the three clinical phenotypes of CIDP decreased the amount of claudin-5 protein levels and TEER values in the PnMECs. In addition, the sera obtained from typical CIDP patients more prominently reduced claudin-5 protein levels and TEER values in the PnMECs than did that obtained from the MADSAM and DADS patients. Furthermore, the severity of BNB disruption after exposure to the sera was associated with higher Hughes grade, lower MRC score, more pronounced slowing of motor nerve conduction in the median nerve and higher frequency of abnormal temporal dispersion.Sera derived from typical CIDP patients destroy the BNB more severely than those from MADSAM or DADS patients. The extent of BNB disruption in the setting of CIDP is associated with clinical disability and demyelination in the nerve trunk. These observations may explain the phenotypical differences between CIDP subtypes. Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a rare autoimmune-mediated neuropathy thought to constitute a group of heterogeneous disorders involving a wide range of clinical phenotypes, variable clinical course and differing responses to immunotherapy Various previous reports have demonstrated that the pathological breakdown of the blood-nerve barrier (BNB), which allows for the entry of immunoglobulins, cytokines and chemokines into the peripheral nerve system (PNS) parenchyma, is a key event in the disease process of CIDP ○C for 30 minutes just prior to use. Cerebrospinal fluid (CSF) samples obtained from the 25 patients with CIDP were analyzed with respect to the protein level in the CSF, the IgG index and/or CSF/serum albumin ratio (Q Alb). The clinical and electrophysiological data for all CIDP patients were analyzed. The clinical parameters included the Hughes functional grading scale The study protocol was approved by the ethics committee of Yamaguchi University and Chiba University. All patients consented to participate and written informed consent was obtained from each subject. Serum was collected from a total of 25 CIDP patients with t-CIDP (n = 12), MADSAM (n = 10) and DADS (n = 3) in the initial progressive phase of the disease or at relapse, without either corticosteroid or intravenous immunoglobulin (IVIg) treatment, diagnosed at Chiba University Hospital or Yamaguchi University Hospital. All patients fulfilled the diagnostic criteria for CIDP based on the guidelines reported by the EFNS/PNS 2010 ○C with 5% CO2/air. The cells were maintained for either 24 hours to measure the transendothelial electrical resistance (TEER) value or 48 hours to extract total proteins.Immortalized human peripheral nerve microvascular endothelial cells (PnMECs), termed “FH-BNBs”, were generated previously in our laboratory We purchased polyclonal anti-claudin-5 and anti-occludin antibodies from Zymed . Polyclonal anti-actin antibodies were purchased from Santa Cruz .After boiling, aliquots containing equal amounts of protein (15 µg) were separated via SDS-PAGE . The proteins were then transferred onto nitrocellulose membranes , as previously described 6 cells/insert) on collagen-coated Transwell inserts , and the TEER value for each insert was calculated following treatment with each type of medium  for 24 hours by subtracting the blank from each reading. Each condition was tested in triplicate for each experiment.The TEER values in the cell layers were measured using a Millicell electrical resistance apparatus , according to the manufacturer's instructions. The cells were seeded  of conduction block is involved in the pathophysiology of MADSAM via the up-regulation of inflammatory cytokines and adhesion molecules. Therefore, further studies to clarify the association between BNB damage and cellular immunity in the setting of MADSAM are required.Based on the hypothesis suggested by the finding of electrophysiological studies, the conduction block in the nerve trunk noted in patients with MADSAM is thought to always be accompanied by focal breakdown of the BNB in vitro BNB model The clinical and electrophysiological features of MADSAM and multifocal motor neuropathy (MMN) are very similar, although MADSAM can be distinguished from MMN by the presence of overt sensory involvement, infrequency of anti-GM1 IgM autoantibodies and responsiveness to steroid treatment In conclusion, the present findings suggest that the severity of BNB breakdown differs depending on the clinical phenotype of CIDP, and may be associated with both the clinical disability and demyelination in the nerve trunk. Our data imply that measurements of the degree of the BNB breakdown would be useful diagnostic biomarkers for predicting both the clinical phenotype and course of CIDP. However, because this was retrospective, further prospective, large-scale studies are required to validate our findings. The accumulation of further data regarding the molecular mechanism(s) responsible for the BNB impairment observed in patients with CIDP may also lead to the development of improved or novel treatments for CIDP.The study was approved by the ethics committee of Yamaguchi University.Not commissioned; externally peer reviewed.


Metabolic gene clusters (MGCs), physically co-localized genes participating in the same metabolic pathway, are signature features of fungal genomes. MGCs are most often observed in specialized metabolism, having evolved in individual fungal lineages in response to specific ecological needs, such as the utilization of uncommon nutrients  or the production of secondary metabolic antimicrobial compounds and virulence factors . A flurry of recent studies has shown that several MGCs, whose functions are often associated with fungal virulence as well as with the evolutionary arms race between fungi and their competitors, have experienced horizontal gene transfer (HGT). In this review, after briefly introducing HGT as a source of gene innovation, we examine the evidence for HGT's involvement on the evolution of MGCs and, more generally of fungal metabolism, enumerate the molecular mechanisms that mediate such transfers and the ecological circumstances that favor them, as well as discuss the types of evidence required for inferring the presence of HGT in MGCs. The currently available examples indicate that transfers of entire MGCs have taken place between closely related fungal species as well as distant ones and that they sometimes involve large chromosomal segments. These results suggest that the HGT-mediated acquisition of novel metabolism is an ongoing and successful ecological strategy for many fungal species. The ability to respond to dynamic ecological pressures, such as shifts in nutrient availability or biological interactions, is a defining characteristic of many successful species and often requires evolutionary innovation. Horizontal gene transfer or HGT—the transfer of genetic material from one organism to another through a process other than reproduction—is one source of innovation that can result in the rapid acquisition of genes that contribute to ecologically important traits , are all likely barriers to rampant HGT in eukaryotes  that was horizontally acquired from the yeast Zygosaccharomyces bailii, a common wine fermentation contaminant  MGCs responsible for the fungal production of gliotoxin, sirodesmin and related mycotoxins. Patron et al. , which are not essential for normal growth but may carry genes for specialized functions that are beneficial in certain conditions Covert, . An extrAlternaria arborescens contains 209 genes including ten putative MGCs  MGC required for causing disease on pea plants. Genes in the PEP cluster also have a different GC-content and codon bias compared to genes on essential chromosomes and may have been acquired via HGT , an ongoing community sequencing initiative sponsored by the Joint Genome Institute, aims to sequence at least two genomes from each of the more than 500 fungal families, including genomes representative of all the major fungal lifestyles and morphologies; similarly, the yeast 1000 project, recently funded by the National Science Foundation (http://www.nsf.gov/awardsearch/showAward?AWD_ID=1442148), aims to decode the genomes of all ~1000 yeast species belonging to the subphylum Saccharomycotina. Such projects promise to not only serve as comprehensive surveys of fungal metabolism but also provide unprecedented opportunities for evaluating and quantifying the impact that processes, such as HGT-mediated gene innovation, have had on the generation of fungal biodiversity.These outstanding questions on the prevalence and consequences of HGT of fungal MGCs, as well as the ecology and mechanisms driving transfer events, require systematic tests of hypotheses about the process that often require integrative data from diverse facets of fungal biology, from genomics to chemistry, and from ecological lifestyles to evolutionary history. Fortunately, the ever larger number of fungal genomes sequenced and the increasing importance given to the sampling of ecologically and taxonomically important groups, coupled with novel functional genomic technologies, is quickly bringing remarkable amounts of data to bear on these questions. For example, the 1000 fungal genome project (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cochlear neuropathy resulting from unsafe noise exposure is a life altering condition that affects many people. This hearing dysfunction follows a conserved mechanism where inner hair cell synapses are lost, termed cochlear synaptopathy. Here we investigate cochlear synaptopathy in the FVB/nJ mouse strain as a prelude for the investigation of candidate genetic mutations for noise damage susceptibility. We used measurements of auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) to assess hearing recovery in FVB/nJ mice exposed to two different noise levels. We also utilized confocal fluorescence microscopy in mapped whole mount cochlear tissue, in conjunction with deconvolution and three-dimensional modeling, to analyze numbers, volumes and positions of paired synaptic components. We find evidence for significant synapse reorganization in response to both synaptopathic and sub-synaptopathic noise exposures in FVB/nJ. Specifically, we find that the modulation in volume of very small synaptic ribbons correlates with the presence of reduced ABR peak one amplitudes in both levels of noise exposures. These experiments define the use of FVB/nJ mice for further genetic investigations into the mechanisms of noise damage. They further suggest that in the cochlea, neuronal-inner hair cell connections may dynamically reshape as part of the noise response. Hearing dysfunction arises when the auditory system fails to properly convey acoustic signals to the brain. Of the many human conditions which fall under this description, the sensorineural hearing loss otherwise known as auditory neuropathy spectrum disorder, may be the most insidious and difficult to treat. The many different forms of neuropathy observed within the cochlea can be thought of as organized around the particular sensory element causing the dysfunction. Specific points at which signal conveyance may be disrupted include synaptic loss, which is termed cochlear synaptopathyCochlear synaptopathy is one of the pathologies caused by acoustic overexposures. Noise-induced hearing neuropathies have been reported as causing up to 90% of hearing deficits amongst the elderlyDiagnosis of cochlear synaptopathy as a result of auditory exposure in patients is particularly difficult. Hearing disorders are commonly assessed with auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) tests, but patients may have normal thresholds for both. Other methods of diagnosis, such as word recognition scoring and speech perception in noise scoring have also been usedMouse models remain the predominant method by which to assess the anatomical pathologies of noise damage, and it will be interesting to apply the power of mouse genetics to this issue. Most studies of noise damage have been performed on the CBA/CaJ mouse lineage, which otherwise maintains good hearing into old age141516Here we correlate changes in hearing measures with changes in pre-and post-synaptic markers in noise-exposed FVB/nJ mice. We use two noise exposure conditions, one that establishes a reduction in peak 1 amplitude without significant synaptic losses as well as one where both sequelae are observed. We analyze synaptic components via three-dimensional reconstructions of deconvolved confocal microscopy stacks. The volumes of pre-synaptic ribbons and post-synaptic receptor patches are each dynamically regulated in both conditions two weeks post-exposure. In particular, ribbons appear to exist in multiple size distributions which are differentially affected in response to each noise condition. The precise positioning of synapses on the inner hair cell also appears affected. These data provide an anatomical correlate for reductions in peak 1 amplitude in the absence of synaptic loss, and define conditions where FVB/nJ mice may be used to understand the genetic basis of noise damage.In order to assess the effects of acoustic over-exposure on the FVB/nJ mouse model, we first needed to determine the amount of noise exposure necessary to produce significant but reversible hearing threshold shifts. We chose 105 dB of 8–16 kHz single octave noise as our acoustic exposure for these experiments, varying the length of exposure and assessing temporary and permanent threshold shifts. We found that the FVB/nJ mouse strain is comparably susceptible to acoustic over-exposure relative to what has been reported for CBA/CaJ mice1819202121Acoustic over-exposure may result in cochlear synaptopathy. One signature of cochlear synaptopathy is a loss of peak 1 ABR waveform amplitudes without loss of ABR threshold5We compared average numbers of synapses per inner hair cell in FVB/nJ mice exposed to either acoustic over-exposure or to untreated controls. Immunofluorescence and confocal microscopy were utilized to characterize the pre- and post-synaptic markers for inner hair cells mapped to specific frequencies within the cochlea . Marker We wanted to quantitatively analyze synaptic components of inner hair cells after acoustic over-exposure. Automated identification of synaptic components could enable analysis of component volume and position as well as number. In order to automate the statistical analysis of these components, we employed a pair determination algorithm published previouslyTo test the algorithm, a human observer counted paired synaptic components on projected views of confocal stacks of inner hair cells for both 12 kHz , orange We analyzed the volume information for both synaptic components to identify dynamic changes after acoustic over-exposure. For 12 kHz IHCs, when Ctbp2 volumes are plotted against Gria2 volumes, the distributions may not appear greatly affected, although statistically they are very different .Synaptic component dynamics are even more pronounced at the 24 kHz region. Recall that while both treatment conditions reduce peak 1 measures , in the We wanted to determine if the synaptic components that displayed the most changes were located on the pillar or modiolar side of the inner hair cell. To this end, the positions of paired synaptic components within stacks were rotated and projected onto the habenular-cuticular and modiolar-pillar position axes of inner hair cell optical sections . AlignmeWe wondered if synaptic clusters would display segregation along a modiolar and pillar axis2425It is interesting to see which ribbons are preferentially affected by acoustic over-exposure. A clustering analysis was performed to examine the effects of treatment on outlier (larger) components in both sub-populations of ribbons  and GriaLarge ribbon prevalence is particularly dynamic in 24 kHz IHCs  in this We quantified the effects of acoustic over-exposure on outlier ribbons within 12 kHz and 24 kHz IHCs . An anal528Acoustic exposure can permanently alter the ability to hear in background noise, a pathology called auditory neuropathy in humans. In animals, acoustic exposure can affect the high-frequency suprathreshold responses in the ABR, which may reflect compromised signaling ability. These changes can correlate with the loss of inner hair cell synapses, called cochlear synaptopathy, but they also occur in its absence. Here we correlate changes in synaptic component volumes with the presence of reduced suprathreshold responses in FVB/nJ mice, which occur in the absence of synaptopathy. Our use of deconvolution enables a more precise estimation of synaptic component volumes, revealing a previously unseen complex dynamic. These studies also set a baseline for analyzing noise damage in a congenic mouse model that should be amenable to later genetic analyses.Cochlear synaptopathy in humans remains an under-reported problem which continues to impact the lives of people of all ages. Noise induced cochlear synaptopathy is a specific kind of auditory injury which deeply impacts the lives of those affected, with particular negative quality of life consequences in older workers and military veterans. In mouse models of noise induced hearing damage, specifically CBA/CaJ, it has been shown that inner hair cell synapses can be compromised5From the threshold shift data in 56Acoustic overexposures in CBA/CaJ mice have been consistently shown to induce differential effects on synapse integrity1The synaptic component sizes and positions analyzed in this paper were obtained from confocal stacks after deconvolution using the iterative maximum likelihood estimation package included with the AMIRA FEI software suite. Deconvolution is important because fluorescence microscopy has artifacts: significant numbers of fluorescence photons are scattered by tissue outside of the image plane. This problem is dependent on wavelength and penetration depth. It is also tissue dependent, due to the variable scattering cross-section of support and membrane proteins, a particular problem for whole mount samples. The total photon scattering convolves the acquired data with a point spread function to make objects appear larger. Unfortunately, convolution is non-linear, in that smaller bright objects appear similar in volume in raw data stacks to medium sized but dimmer objects at any particular thresholding level. The data must be deconvolved from the point spread function plane-by-plane due to the changing nature of the stack depth and scattering centers. In this way we gained more accurate positional and volumetric data for analysis.et al.Deconvolved confocal imaging provides the ability to assay fine structure dynamics of most of the ribbon synapse populations. Through averaging in deconvolved optical stack reconstructions, we are able to assay small, medium, and large ribbon populations independently, something not possible from serial electron microscopy, pure confocal, or super-resolution imaging experiments. We pair this with algorithmic synapse identification based on pre- and post-synaptic element proximity. Thus, we are able to do an analysis of fine structure in each ribbon volume distribution. Ribbon volumes in particular are interesting because auditory nerve fiber spontaneous rates may be related to ribbon synapse sizeBy plotting synapse anatomical domain volumes against each other  we show The changes we observe in resizing dynamics are also shown to occur in concert with changes in position of synapses after noise exposure. We utilized Bayesian cluster analysis to determine the amount of positioning fidelity inherent to synapses arranged about the cuticular-habenular axis before and after acoustic over-exposures. In the control cases for both 12 kHz and 24 kHz, population clusters are observed to segregate roughly parallel to the cell body axis. Following noise exposure, positioning is altered for both frequency regions examined. These changes do not correlate with ABR peak 1 amplitudes reductions. The noise exposure seems to disrupt synapse position along the soma of the inner hair cells, however we are unable to determine if modiolar local synapses are affected differently compared to pillar synapses at this time.The positional dynamics of resizing of the anatomical correlates are interesting to consider. The 12 kHz  and 24 kMean ribbon sizes offer an additional metric for comparison of FVB/nJ to CBA/CaJ. Comparison of means in FVB/nJ  with meaPostnatal day 1 congenic FVB/nJ mice were purchased from Jackson Laboratories, raised in our facility and used for characterization of the effects of acoustic overexposure. All mice were randomized for treatment condition. Both males and female mice were used for all experiments. The numbers of mice used per condition were 20 mice for controls, 5 mice for 30 minute noise treatment condition, and 3 mice for the 60 minute treatment condition. They were pretested for auditory function and then noise exposed at P60. Animals were kept for 14 days post treatment, re-tested, and sacrificed. All experiments were performed in accordance with the Department of Health and Human Services and were approved by the University Committee on Animal Resources at the University of Rochester Medical Center.Noise exposure conditions were a single octave of 8–16 kHz band Gaussian noise at 105 dB SPL for either 30 or 60 minutes. Mice were placed into individual triangular mesh wire cages, 12 cm × 5 cm × 5 cm, in an asymmetric plywood box. The ceiling of this enclosure was modified to connect with a mounted JBL2250HJ compression speaker and a JBL2382A bi-radial horn. The entire system was enclosed within a soundproof booth and was driven using a TDT RX6 multifunction processor and dedicated attenuator. This was controlled using TDT TPvdsEx sound processing software.Auditory testing was conducted using a Smart EP Universal Smart Box (Intelligent Hearing Systems). P60 FVB/nJ mice were tested for hearing thresholds via ABR and DPOAE responses. Mice  were anesthetized by intraperitoneal injection using a mixture of ketamine (80 mg/kg) in a sterile acepromazine/saline mixture (3 mg/kg). A 10B+ high-frequency transducer probe was placed within the external auditory canal. Auditory brainstem response stimuli were either 5 ms full tone spectrum clicks or 5 ms single tone pips presented at 8, 12, 16, 24 and 32 kHz. Each set of frequency and amplitude measurements were averaged for 512 scans with electrical responses being reported via a 3-electrode system. The three needle electrodes (Grass) were inserted at the vertex (ground) and on each side beneath the pinna. ABR thresholds were determined by the last trace corresponding to a correct latency and waveform by visual identification for a particular stimulus. The individual scoring thresholds was blinded to condition and time point. Threshold response determinations were performed by collection of ABR response using 75–15 dB SPL single tones or a click stimulus. ABR amplitude progressions were taken by reporting the ABR peak 1 voltages for these measurements. Amplitudes and latencies of ABR peak 1 were identified using IHS Software analytical tools. Peak 1 was identified as the first peak in the waveform, provided it occurred within 2.4 msec of the stimulus presentation.Distortion product otoacoustic emissions (DPOAE) measurements were determined by measuring amplitudes of the distortion product response. Evoked emissions were recorded at frequencies for paired tones of f1 and f2 with f1/f2 = 1.2 and f1 level = f2 + 10 dB. Averages of 32 sweeps were used in reporting 5 dB f1 and f2 steps with f1 starting with 20 dB and ending with 75 dB. 3 dB was used as the DPOAE threshold. The value at 3 dB was calculated from a linear interpolation of the surrounding points.Primary antibody were used as follows: rabbit anti-Myosin7a , mouse anti-Ctbp2 , and mouse anti-Gria2 . Secondary antibodies were all purchased from Jackson Laboratories and were used as follows: goat anti-mouse 488 (1:500), goat anti-mouse 594 (1:500), donkey anti-rabbit 647 (1:200).°C. Decalcification was carried out in EDTA solution (0.1 M) at 4 °C on a rocking platform for 5 days. Synapse analysis were performed on whole mount preparations by microdissection as described elsewherehttp://rsbweb.nih.gov/ij/).Cochleae were removed via dissection and had their stapes removed. The round window membranes were opened and cleared of tissue and a small hole was placed at the tip of the cochlea to permit fluid exchange. Cleared organs were fixed in 4% paraformaldehyde for overnight at 4 Confocal imaging was performed using an Olympus FV 1000 laser scanning confocal microscope. Visualization and projections were done using FIJI and ImageJ 64. Visual quantification of pairing in synaptic structures was done by counting unpaired Ctbp2+ ribbons and Gria2+ receptor patches in each optical stack. Counting was performed using the ImageJ 64 “cell counter” plugin and up to 2 stacks were counted per organ.The synaptic component sizes and positions analyzed in this paper were obtained from confocal stacks after deconvolution using the iterative maximum likelihood estimation package included with the AMIRA FEI software suite. Algorithmic pair determinations on high-resolution reconstructions were performed as described previously33How to cite this article: Paquette, S. T. et al. Noise exposure modulates cochlear inner hair cell ribbon volumes, correlating with changes in auditory measures in the FVB/nJ mouse. Sci. Rep. 6, 25056; doi: 10.1038/srep25056 (2016).