The western blot results also revealed that TCS induced autophagy via the AMPK/ULK1 and JNK/ERK/p38 pathways independent of mTOR. The immunofluorescence results indicated that TCS up-regulated the expression of the ubiquitin receptors NDP52 and p62 and strengthened the co-localization of these receptors with Salmonella enterica Typhimurium (S. typhimurium) or Candida albicans  in infected MΦ cells. In addition, sub-lethal concentrations of TCS enhanced the clearing of the pathogens S. typhimurium or C. albicans in infected MΦ and in corresponding mouse infection models in vivo. Specifically, we found that a sub-inhibitory concentration of TCS induced autophagy, leading to an imbalance of the intestinal microflora in mice through the analysis of 16s rRNA Sequencing. Together, these results demonstrated that TCS induced autophagy, which enhanced the killing against pathogenic S. typhimurium or C. albicans within mammal cells but broke the balance of the intestinal microflora.Triclosan (TCS) is a broad-spectrum antimicrobial agent, whose well-known antibacterial mechanism is inhibiting lipid synthesis. Autophagy, an innate immune response, is an intracellular process that delivers the cargo including pathogens to lysosomes for degradation. In this study, we first demonstrated that TCS induced autophagy in a dose-dependent manner in non-phagocytic cells (HeLa) and in macrophages (Raw264.7) and  Triclosan (TCS) is a broad-spectrum anti-bacterial agent whose mechanism of action is reported to be blocking fatty acid synthesis  is the key regulator of cell growth, metabolism, survival and the lifespan of organisms, and therefore, it is closely in line with autophagy. mTOR is a negative regulator of autophagy, while AMP-activated protein kinase (AMPK) is a positive regulator. ULK1 is the most upstream autophagy inducer, it plays a role in initiating the autophagy process, and it is directly regulated by mTOR or AMPK. Recent studies show that the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway is also involved in regulating autophagy, and the c-jun N-terminal kinase (JNK) and p38 kinase simultaneously participate in this process  can invade, survive, and replicate in phagocytic and non-phagocytic cells. However S. typhimurium could be selected for autophagy, relying on the accumulation of ubiquitin or autophagy adaptor proteins around the target bacteria  are key members of the innate immune system, providing the first response to infection with various mechanisms, including autophagy. The number of intestinal microflora is large, and the variety is broadly involved in various types of physiological activities. The steady state of the intestinal flora is vital to human health. Intestinal microflora, through a series of physiological effects, affect the intestinal mucosal immune function of dynamic regulation , and Enterococcus faecalis  were isolated from normal mouse feces. S. typhimurium ATCC 14028 and C. albicans SC5314 were kept by our laboratory.The antibodies used in the experiments were purchased from Cell Signaling Technology , the triclosan (TCS) came from Sigma , and the other reagents, unless stated otherwise, were all purchased from Sigma . The pmRFP-LC3 is mammalian expression of rat LC3 fused to mRFP, the ptfLC3 is mammalian expression of rat LC3 fused to mRFP and EGFP, and the pEGFP-LC3 is mammalian expression of rat LC3 fused to EGFP .Intestinal epithelial cells and intestinal macrophages were extracted according to previous studies  or 24-well glass-bottom plates (2 × 105 cells/well) in serum-free and antibiotic-free DMEM medium.TCS was diluted with cell culture medium to concentrations from 0.25 to 32 μM. The cells were seeded into six-well flat-bottom plates (1 × 10For the transmission electron microscopy (TEM) observation, cells were incubated in six-well flat-bottom plates and were treated with TCS at a dose of 8 μM for 180 min. The cells were stained after immobilization. Autophagosome-like vesicles were observed under the TEM .For the fluorescence microscope image collection, the cells needed to be transiently transfected with pEGFP-LC3 or ptfLC3 plasmids. The Raw264.7 cells were incubated in 24-well flat-bottom plates with glass slides. The instructions to complete cell transfection were followed. Then, we replaced the medium with new serum-free DMEM. The cells transfected with plasmids were used in other experiments.S. typhimurium or C. albicans. The images were obtained by an Olympus FV1000 confocal laser-scanning microscope  with a 60x objective lens. Image analyses and exports were performed using a Fluoview ver. 1.7.3.0 .Cells or transfected cells were seeded in 24-well glass-bottom plates. The cells were treated with TCS with or without The cells were treated with TCS as described above, and the total protein was collected by centrifugation and was quantified using the BCA reagent . The images were obtained by a CanoScan LiDE 100 scanner (Canon). Protein blots were measured using Image-J software.S. typhimurium or C. albicans at the respective MOI of 25:1 and 1:1 for 120 and 30 min. The assay of S. typhimurium growth in RAW264.7 cells was performed as previously described , TCS, or TCS and 3-MA . The cells were then exposed to Female BALB/c mice (20 ± 2 g) were kept in conditions of a constant photoperiod  and had free access to food and water. For autophagy observation, mice were injected with TCS from 3.125 to 40 mg/kg for 90 min through the enterocoelia. Then, the mice were sacrificed for liver and spleen harvesting. Western blotting was used for the relevant protein assays.S. typhimurium (1 × 105 organisms in 0.1 ml PBS). After 24 h, the mice were treated with vehicle (0.5% methylcellulose-0.1% Tween 80 in sterile water), TCS, or TCS and 3-MA (24 mg/kg) by intraperitoneal injection once a day for 4 days. Then, the mice were euthanized, and the livers and spleens were collected for homogenization in 5 ml cold PBS. The homogenates were then serially diluted and plated onto LB agar plates. The CFU counting was conducted as described above.For the assays assessing the effect of the bacteria-killing abilities, mice were infected by an intraperitoneal injection of an overnight culture of C. albicans, mice were infected with C. albicans (1 × 107 organisms in 0.1 ml PBS) by oral administration. After 3 days, the mice were randomly assigned to control and treatment groups. C. albicans were treated the same way as those treated with S. typhimurium. The homogenates were then serially diluted and plated onto YPD agar plates. The CFU counting was conducted as previously described.To establish a mouse model that was infected with For the long-term sub-inhibitory concentration animal administration experiment, mice were randomly assigned to control, experiment, and treatment groups. The mice were treated with vehicle, TCS , or TCS (3 mg/day) by oral ingestion of 15 ml and 20 μl of 3-MA (24 mg/kg) by intraperitoneal injection for 30 days.E. faecalis plates were incubated aerobically overnight at 37°C.Representative bacteria in the feces of the mice treated with vehicle, TCS , or TCS (3 mg/day) and 20 μl of 3-MA (24 mg/kg), were counted with differential media on the 30th day. Intestinal bacteria of mice were cultivated with differential media, including MRS medium, MacConkey agar, and Enterococcus faecalis agar, which were purchased from Hopebio Co., Ltd. . The MRS plate was incubated anaerobically for 2–3 days at 37°C, while the MacConkey and 16S rRNA gene high-throughput sequencing was used to determine the structure comparisons of the bacterial species in each of these samples using the Illumina HiSeq platform. PCR primers 515F and 806R targeting the bacterial 16S rRNA V4 region were selected for bacterial community analysis using the Illumina high-throughput sequencing method Sequences were analyzed using Quantitative Insights Into Microbial Ecology (QIIME) software and the UPARSE pipeline. The default settings for Illumina processing in QIIME were used and the UPARSE pipeline was then used to assign taxonomy at 97% similarity via the RDP classifier.http://drive5.com/uparse/). Sequences with ≥97% similarity were assigned to the same OTUs. The Silva Database (https://www.arb-silva.de/) was used based on RDP classifier  algorithm to annotate taxonomic information.Mouse studies were performed in strict accordance with the Institutional Guiding Principles for Biomedical Research Involving Animals. The experiments were approved by the Jilin University Animal Care and Use Committee (no: IZ-2009-008). The gene copies indicated the absolute copy numbers present per unit of dry weight, whereas the normalized copy number based on 16S rRNA was regarded as the abundance. Sequences analysis were performed by Uparse software  improved the ratio of LC3-II/LC3-I both in the livers  were transfected with ptf-LC3 plasmids and were treated with TCS. The GFP-LC3 component is sensitive to lysosomal proteolysis, and therefore, the corresponding GFP fluorescence is quenched once it undergoes proteolysis, whereas the RFP-LC3 component will not be quenched with proteolysis. The results showed that compared with the control groups, the numbers of both the GFP-LC3 and RFP-LC3 puncta in TCS-treated cells increased, and in the merged images, red and yellow puncta were found Figure . This deTo explore whether TCS-induced autophagy via the classical mTOR-dependent pathway, RAW264.7 cells were treated with TCS at different concentrations from 0.25 to 32 μM for 90 min. Western blot assays showed that with the increased concentration, the phosphorylation of mTOR, p70S6K, and 4E-BP1 increased compared to the untreated cells Figure . After tFurthermore, the western blot assay showed that TCS up-regulated the phosphorylation of AMPK and ULK1 in RAW264.7 cells treated with TCS at concentrations from 0.25 to 32 μM for 90 min Figure . After tNext, the cells were treated with TCS at concentrations from 0.25 to 32 μM for 90 min. The western blot assay demonstrated that the levels of phosphorylation of JNK, p38, and ERK were up-regulated in a dose-dependent manner in general Figure . After tTo explore the regulatory relationship among JNK, p38, and ERK in TCS-induced autophagy, the corresponding inhibitors SP600126, SB203580, and PD98059 for JNK, p38, and ERK were used. The results showed that when PD98059 was added, the expression of p-ERK was inhibited, whereas the expression of p-JNK and p-p38 was not affected Figure . When SBS. typhimurium or C. albicans infected cells, and these co-localizations were strengthened by TCS treatment . Furthermore, our western blot results further verified that TCS induced autophagy. The occurrence of autophagy is a complex and conservative process in which mTOR is a negative regulator. However, in this study, the western blot results confirmed that TCS treatment inhibited mTOR activity and the phosphorylation of 70S6K and 4E-BP1, and the pharmacological inhibition assays also indicated that TCS did not regulate the occurrence of autophagy through the mTOR pathway.ULK1 is the initiation molecule of autophagy. AMPK is a positive regulator of ULK1, which activates the phosphorylation of ULK1 to initiate autophagy. In this study, our results showed that different concentrations of TCS induced the phosphorylation of AMPK and ULK1, and the induced intensity of AMPK and ULK1 increased with time. The results suggested that TCS activates the AMPK/ULK1 pathway to induce autophagy. ERK, p38, and JNK are involved in the induction of autophagy, but these three kinases play different roles in different cell types with different inducing factors (Bhui et al., Ubiquitin is a small molecule in eukaryotic cells that tags the protein or pathogen that needs to be degraded by the various ubiquitin-catalyzed enzymes. NDP52 and p62 are ubiquitin receptor proteins that were discovered recently. In this study, the results showed that TCS treatment significantly increased the expression of NDP52, p62, and LC3 and their co-localization with pathogens. These results proved that TCS strengthened the expression of ubiquitin receptor proteins in macrophages to enhance the identification of pathogens.In this study, we established a mouse model of a long-term sub-inhibitory concentration of TCS administration, and the results showed that TCS disrupted the intestinal flora balance. Gaulke et al. also showed that TCS altered the composition and ecological dynamics of microbial communities in the gut in adult zebrafish (Gaulke et al., in vitro intestinal epithelial cells and macrophages showed that TCS killed E. faecalis, Lactobacillus, and E. coli by inducing autophagy in the two types of cells. Moreover, the in vivo results showed that the number of Lactobacillus and E. coli decreased, and surprisingly, the number of E. faecalis increased and the shifts of the bacterial community compositions were further corroborated by 16S rRNA gene sequencing. Previous research clarified that Lactobacillus and E. coli are killed by TCS (McMurry et al., E. faecalis has a relatively high-level intrinsic TCS resistance (Zhu et al., Lactobacillus, E. coli, and E. faecalis colonize in different regions in the intestine (Guarner and Malagelada, E. faecalis is mainly distributed in the rectum, where the TCS content is relatively lower compared with other parts of the intestine (Delmas et al., E. faecalis by TCS-induced autophagy may be lower in the rectum. Previous studies demonstrated that a sub-inhibitory concentration of antibiotics leads to the increased expression of bacterial resistance genes and causes their horizontal transmission (Davies et al., E. faecalis was increased by TCS treatment in vivo. Taken together, our study confirmed that a sub-inhibitory concentration of TCS-induced autophagy broke the balance of the intestinal microflora in mice.In this study, our results of in vitro and in vivo (Duran et al., A previous study showed that autophagy acts as a double-edged sword in a variety of diseases (Shintani and Klionsky, in vivo and in vitro. In addition, sub-lethal concentrations of TCS enhanced the clearing of S. typhimurium or C. albicans in infected MΦ and in vivo. Noteworthy, TCS broke the balance of the intestinal microflora and has a carcinogenic tendency.Overall, we demonstrated that TCS induced autophagy in macrophages and non-phagocytic cells LY, CW, and XS participated in the design of this study, and they performed the statistical analysis. LY carried out the study and collected important background information. CW and XS drafted the manuscript. All authors read and approved the final manuscript. CW, XS, YW, and YA carried out the concepts, design, definition of intellectual content, literature search, data analysis and manuscript preparation. ZY completed the additional experiments requested by the reviewers. SL, YL, XfW, and WL provided assistance for data acquisition, data analysis and statistical analysis. XT and XyW carried out literature search, data acquisition and manuscript editing. ZC and ML performed manuscript review. All authors have read and approved the content of the manuscript.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.


Pde6b: the severe rd1 and the milder rd10 models. Compared to normal adult retina, we found increased numbers of γH2AX positive foci in all retinal neurons of the developing retina in both model and control retinas, as well as in wild type untreated retinal explant cultures. In contrast, the 53bp1 staining of the retina differed both in amount and character between cell types at all ages and in all model systems. There was strong pan nuclear staining in ganglion, amacrine, and horizontal cells, and cone photoreceptors, which was attenuated. Rod photoreceptors did not stain unequivocally. In all samples, 53bp1 stained foci only rarely occurred. Co-localization of 53bp1 and γH2AX staining was a very rare event (< 1% of γH2AX foci in the ONL and < 3% in the INL), suggesting the potential for alternate DSB sensing and repair proteins in the murine retina. At a minimum, murine retinal DSB repair does not appear to follow canonical pathways, and our findings suggests further investigation is warranted.Gene editing is an attractive potential treatment of inherited retinopathies. However, it often relies on endogenous DNA repair. Retinal DNA repair is incompletely characterized in humans and animal models. We investigated recruitment of the double stranded break (DSB) repair complex of γH2AX and 53bp1 in both developing and mature mouse neuroretinas. We evaluated the immunofluorescent retinal expression of these proteins during development (P07-P30) in normal and retinal degeneration models, as well as in potassium bromate induced DSB repair in normal adult (3 months) retinal explants. The two murine retinopathy models used had different mutations in  Inherited retinal dystrophies are disorders which lead to visual impairment and in severe forms, to blindness, and have an estimated prevalence of 1 in 4,000  Carroll, . This hain vitro, almost nothing is known about post-mitotic neurons such as photoreceptors (PRs). In these cell types the system appears to differ: in mouse rod PRs, DSBs induced by radiation are insufficiently repaired as measured by quantification of repair foci over time , resulting in the recruitment of checkpoint factors to the DSB  07, p13, p21, p28. Immature rd10 and C57BL/6J were investigated at p14, p18, p24 and p30. At least two immunostaining procedures were performed for each antibody and each investigated mouse tissue of the respective mouse lines and ages. For microscopic analysis, 2–3 sections of each immunostaining per respective mouse line and age were analyzed.RRID:MGI:3803328), rd10 mice (RRID:MGI:3581193) and their respective wildtype strains C3H and C57BL/6J were subjects in this study and were provided by François Paquet-Durand from Tübingen. Animals were housed and bred in the animal facility of the University of Giessen under standard white cyclic lighting, had free access to food and water. Day of birth was considered as post-natal day 0. All procedures concerning animal handling and sacrificing complied with the European legislation of Health Principles of Laboratory Animal Care in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the Justus-Liebig-University (TV-No M_474). All efforts were made to minimize the number of animals used and pain and distress.Three and nine months old wild type C57BL/6J mice  were used in this study. In addition, immature rd1 mice  for 1.5 h at 37°C. Organotypic retina cultures of 2, 4, 6, and 8 days were treated likewise. Working solution of KBrO3 was prepared with pure water. Respective control retinae were treated with plain water only. Subsequently, all treated retinal tissues were briefly rinsed in HBSS and immediately fixed in 4% paraformaldehyde in PBS at room temperature for 45 min. After washing in PBS, all treated retinal tissue was cryo-protected in graded sucrose solutions , frozen, cut, and immunostained with γH2AX antibodies (see next section).To initiate DNA DSBs, whole adult C57BL/6J mouse retina was dissected in Hanks' Balanced Saline Solution , transferred into a small Petri dish (30 mm in diameter), and incubated in 1.5 mM KBrOFor frozen sectioning, immature and adult mouse eyecups and retinal explants were treated as described previously  oil immersion objective at 1,024 × 1,024 pixels and a z-axis increment of 0.3 μm. For analysis of immunolabeled cells and their processes, a stack of 2–12 sections was taken (0.7-μm z-axis step size). Cell processes were reconstructed by collapsing the stacks into a single plane. Brightness and contrast of the final images were adjusted using Adobe Photoshop CS5 .2 was defined in each image in the outer nuclear layer (ONL) and in the inner nuclear layer (INL), and all γH2AX and 53bp1 immunoreactive foci within that square were counted. Only central retinal regions were analyzed. Foci co-localizing 53bp1 and γH2AX immunoreactivity were given as percentage of all γH2AX immunoreactive foci in the respective field. At least three micrographs per time point and mouse line were analyzed. Image stacks 2 μm in depth were taken.Quantification of γH2AX and 53bp1 immunoreactive foci was performed on vertical frozen sections of developing retina of rd1, rd10 mice and their respective wildtype C3H and C57BL/6J. An area of 2320 μmt-test and SigmaPlot 12 software. Error bars indicate SD.Statistical comparisons among different experimental groups were made using a two-tailed Student's We analyzed retinal tissue from two wild type mouse lines, C3H and C57BL/6J, because the subsequently analyzed models with hereditary retinal degeneration are based on the two lines .In the developing mouse retina, defined γH2AX immunoreactive foci were found numerously in the inner (INL) and outer nuclear layers (ONL) Figures . The occQuantification of γH2AX immunoreactive foci in the ONL and INL of immature (p13/p14) and mature (p28/p30) C3H, C57BL/6J, rd1, and rd10 mouse retina revealed highest foci numbers in the ONL (Data not shown). Except for the C3H mouse retina, immature and mature retina showed no significant difference in the number of γH2AX immunoreactive foci in the INL. In the ONL, differences in the number of γH2AX immunoreactive foci were not significant at immature or mature ages. At 4 weeks of age, quantification of γH2AX immunoreactive foci was not possible due to loss of most photoreceptors in the ONL in the rd mouse lines Figures .In addition to the rd1 and rd10 mouse lines, we also analyzed tissue from organotypic retina culture (C57BL/6J) as an alternative model of retinal degeneration.In the developing retina of rd1 and rd10 mice, γH2AX immunoreactive foci were found in the INL and ONL Figures . The occWith the exception of the developing retina at p07, many nuclei of the INL and GCL showed generally high level of immunoreactivity to 53bp1 Figures . At p14 In the developing retina of rd1 and rd10 mice, 53bp1 immunoreactivity was bright and localized in many nuclei of the INL and GCL Figures . At earlFollowing the characterization of the localization of the two proteins separately, we investigated whether γH2AX and 53bp1 are co-localized to the same DNA damage site. Double immunostaining on the developing retina of C3H and rd1 mice showed only very few double labeled foci in the INL and ONL  is an oxidizing agent used as a food additive, which causes kidney damage as a potent nephrotoxic agent, and the mechanism is explained by the generation of oxygen free radicals that induce many DSB and thus cause genomic instability leading to apoptosis . This led to increased numbers of γH2AX immunoreactive foci per nucleus in some photoreceptors. Hence, we were able to show DNA damage detection by immunohistochemistry for γH2AX in all investigated retinal tissues, inclusive of developing, degenerating and mature retinas.DNA damage sensing happens through the appearance of H2AX at the DSB site and its subsequent phosphorylation, yielding γH2AX  or NHEJ (Chapman et al., In summary, we observed different intensities of the 53bp1 immunostaining in specific cell types in the different retinal layers in mouse retinae at all ages from wildtype and retinal degenerating mouse lines, as well as in organotypic retina culture. With little of variation, 53bp1 was characterized by pan nuclear staining in amacrine and horizontal cells, the laterally connecting neurons in the retina. Cones showed 53bp1 immunoreactivity distributed between the nuclear lamina and the two large central chromocenters, which we viewed as pan nuclear staining. In the ONL and INL of the developing retina and in retinal explant culture, γH2AX positive DSBs were found more numerously compared to adult retina. Most interestingly, we could show that the two proteins do not co-localize regularly in repair foci and that in the majority of cases, DNA damage sensing does not seem to result in 53bp1 recruitment, irrespective of the viability state of the retina.In conclusion, our data indicate that DNA double strand breaks are sensed by phosphorylation of H2AX in all neurons of the retina, but this not necessarily leads to the recruitment of 53bp1 to repair foci, indicating the presence of alternative sensing and repair proteins. This observation warrants further investigation into the DNA repair pathway state in post-mitotic neurons of the retina and the central nervous system in general.BM and KS contributed conception and design of the study. BM acquisition, analysis, editing, and interpretation of data for the study. BM drafted the manuscript. KS and NE were revising it critically for important intellectual content. BL read the manuscript critically. BM Agrees to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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. The reviewer SB and handling Editor declared their shared affiliation.


Noise is the undesired, artificially developing voice of which quality and quantity is impaired. The most important objective effect of noise is on hearing. Noise-related hearing loss (NRHL) is one of the most common causes of hearing loss among adults. NRHL is a health problem known for long years and gained importance together with industrialization in Europe. The lower limit of noise that would lead to hearing loss is accepted as 85 dB SPL according to international standards . HearingThe aim of this study was to investigate noise-related hearing loss in the workers in a noisy workplace, to give educations about hearing loss, to take measures, to investigate the influences of noise on human psychology and prevalence of smoking.This study was conducted in Beypazarı, Ankara, Turkey, in 2015–2016. For this study, the male workers in the same thermal reactor, which had 85 dB SPL or over noise level, were analyzed retrospectively. We evaluated the periodic follow-up results of pure tone audiometry and transient otoacoustic emission (t-OAE). The workers who had hearing loss due to other causes and chronic diseases requiring regular drug use were excluded from the study. Then we performed questionnaire forms to the workers. The results of 89 workers inclusion criteria were analyzed [mean age of 34.0 ± 8.7 yr (21–63 yr)]. The questionnaire form included questions about age, working years, daily working time, whether he received education about hearing protection, using protective equipment, presence of tinnitus and hearing loss, smoking status, anxiety, presence of dysphoria.The protocol was approved by Ethics Committee of Turgut Ozal University (no: 99950669/72). SPSS ver. 17.0  was used for statistical evaluation. Friedman, Mcnemar, Chi-Square tests were used for analyses.The injury caused by noise in outer hair cells leads to permanent hearing loss. These types of losses’ beginning slowly, symmetrically in both ears, from high-pitched frequencies (4000–6000 Hz), speech frequencies are affected later . In our P=0.04).NRHL is more during the first 10–15 yr of noise exposure. If exposure to noise is eliminated, the hearing loss threshold does not progress. An increase in abnormal audiogram frequency was observed as duration of noise exposure increases . In our Smoking could be a risk factor for high-frequency hearing loss in a dose-dependent manner and potentiate the risk together with occupational noise exposure . We coulThe influences of chronic industrial noise on psychological stress symptoms was analyzed  and detected that noise increased these symptoms . We analP=0.007). A significant relationship was not found between using ear protector and hearing loss (P>0.05). This condition results from not using the ear protectors correctly and effectively, the workers’ preferring earplug, which limitedly reduces noise level instead.NRHL is a preventable condition and it does not have a specific treatment. Prevention of noise in its source and in the workers is the two approaches toward preserving hearing. Although prevention of noise in the worker is the last resort, education of the employer and the worker is very important. In our study, we inquired whether the workers received education about the relationship between noise and hearing loss before starting work and whether this influenced using ear protector and hearing loss. Forty-one (46.1%) workers had given education about hearing loss when starting work. Sixty-six (74.2%) workers were using ear protector. While 36 out of 41 workers (87.8%) who received education were using protective equipment, 30 out of 48 (62.5%) who did not receive education were using equipment. Ratio of using protective equipment was significantly higher among the workers who received education about hearing loss (It is important to support industrial audiology with more researches and to improve awareness of workers and employers in order to prevent noise-related Hearing Loss in the Workplace.


This retrospective study included database records from 6558 patients (average age 69.0 years). We employed a multivariate generalised linear mixed model to analyse the probabilities of hearing losses , measured at different frequencies , for nearly all combinations of APHABu subscale scores . We calculated the probability of hearing loss for 28,561 different combinations of APHABu subscale scores (results available online). In general, the probability of hearing loss was positively associated with the combined APHABu score (i.e. increasing probability with increasing scores). However, this association was negative at one frequency (8 kHz). The highest probabilities were for a hearing loss of 45 dB at test frequency 2.0 kHz, but with a wide spreading. We showed that the APHABu subscale scores were associated with the probability of hearing loss measured with audiometry. This information could enrich the expert’s evaluation of the subject’s hearing loss, and it might help resolve suspicious cases of aggravation. The 0.5 and 8.0 kHz frequencies influenced hearing loss less than the frequencies in-between, and 2.0 kHz was most influential on intermediate degree hearing loss (around 45 dB), which corresponded to the frequency-dependence of speech intelligibility measured with speech audiometry.The Abbreviated Profile of Hearing Aid Benefit (APHAB) questionnaire reports subjective hearing impairments in four typical conditions. We investigated the association between the frequency-specific probability of hearing loss and scores from the unaided APHAB (APHABThe online version of this article (doi:10.1007/s00405-016-4385-7) contains supplementary material, which is available to authorized users. In audiology, primary diagnoses of hearing loss are based on pure tone and speech audiometry and on the results of self-reporting questionnaires. Audiometry provides a more or less objective measurement, and questionnaire scores give a subjective evaluation of impairments in hearing.The Abbreviated Profile of Hearing Aid Benefit (APHAB), developed by Cox and Alexander in 1995, consists of 24 questions grouped into four subscales that represent everyday hearing situations , 4. Staru), because hearing problems are assessed without hearing aids. Hearing aids can be subsequently fitted, but there is no need for a second examination. That is, it is not necessary to evaluate differences in scoring between aided and unaided self-perception. Therefore, the APHABu must be given to patients with impaired hearing at an early stage on the diagnostic pathway, along with pure tone and speech audiometry.The first part of the APHAB is used for primary diagnoses of hearing loss; it is designated the unaided APHAB  of hearing loss, the EC and RV APHABu scores increased by approximately 0.2 percentage points, and the AV subscale scores decreased by 0.1 percentage point. No correlation was found between the degree of hearing loss and the BN subscale score. This can be explained by the individual ability for hearing loss compensation. Hearing losses at defined single frequencies can influence the APHABu scores. Neither of those studies demonstrated any relationship between gender, hearing loss, and the APHABu score.Recently, it was demonstrated that there was no dependency on hearing loss represented by seven standard types of audiograms and the APHABu . One thee scores . Signifiu scores and how are the belonging probabilities for each subscale? The aim of this study is to provide a detailed investigation of correlations between the APHAB scores and the probability of having a frequency-dependent hearing loss. In addition, we investigated the probabilities of frequency-dependent hearing loss (with severity grouped in steps of 5 dB) at all octave frequencies, between 0.5 and 8 kHz.These findings due to the opposite question: is the frequency-specific hearing loss dependent on APHABu) was given to the subject before fitting the hearing aid. Thus, the APHAB was used as a primary diagnostic tool in evaluating hearing loss, as described previously . Of these, 3081 were men  and 4118 were women . Some cases . We found no differences in the probabilities of hearing loss between men and women. The distribution of records among different classes of hearing loss based on the three-frequency table .We investigated 6558 cases with a multivariate generalised linear mixed model to determine the probabilities that frequency-specific hearing losses measured at frequencies of 0.5, 1.0, 2.0, 4.0, and 8.0 kHz were associated with different combinations of APHABu were associated with higher probabilities of hearing loss (Tables 3a–l). However, increasing hearing loss severity corresponded to decreasing values of probability. The probability of hearing loss associated with an APHABu score depended on the specific dB-value. However, the probability was lower for the lower frequencies and higher for the higher frequencies, except at 8.0 kHz, for all APHAB subscales in general.We found that, in general, higher scores in the EC, BN, and RV subscales of the APHABu-dependent probability of a 20-dB hearing loss was higher for 4.0 than for 8.0 kHz sounds. Up to a 35-dB hearing loss, increasing EC values were associated with lower probabilities of a hearing loss at 4.0 and 8.0 kHz, but at a 45-dB hearing loss that negative association was only observed for 8.0 kHz sounds. For hearing losses above 60 dB, the probability of a hearing loss was higher for 8.0 kHz than for 4.0 kHz sounds. Above 70 dB, except at 8.0 kHz, the APHABu scores had no detectable influence on the probability of a hearing loss. In the second step of this evaluation , we plotted the probabilities of hearing losses of 20–75 dB (in increasing steps of 5 dB) against the corresponding combinations of all APHABu subscale scores. In the third step of this evaluation, we presented the alterations in probabilities as the dB values increased, by connecting these figures in an animated slide show .Some interesting details emerged from this analysis. We found that the APHABThe statistical dispersion of the probabilities for hearing loss at test frequencies 4.0 and 8.0 kHz is decreasing for a hearing loss from 20 dB to a value about 35–40 dB. The probabilities for hearing loss at the test frequency 2.0 kHz have a wide spreading for a possible middle-degree hearing loss around 45 dB. The probability of a high-degree hearing loss is highest at test frequencies 4.0 and 8.0 kHz. But the belonging probabilities found in our study are on a very low level in these cases.Although investigations of the relationships between audiometric and self-reported measures began decades ago, some questions in this field remain unanswered. The association between the probability of hearing loss and questionnaire scores has only been investigated in evaluations of screening-instruments, like the HHIS-E or MAT , 12, to u scores. The similarity between genders, both in distribution and average age, was consistent with findings in previous studies [In this study, we did not discover any significant differences between genders in how the probability of hearing loss was associated with APHAB studies , 8.u values. Thus, the reader might use these original data to predict pure-tone thresholds in all octave frequencies between 0.5 and 8 kHz. In addition, Tables 3a–l (Online resource 1–12) might allow the reader to introduce the APHABu in diagnoses of subjects that receive statutory accident insurance. The original data could be used further to compare the results of the APHABu scores for each subscale to the probability of the corresponding hearing loss, measured with audiometry. This information could enrich the expert’s evaluation of the subject’s hearing loss, and it might help resolve suspicious cases of aggravation and simulation.The tables generated by this study give the probabilities that certain frequency-specific hearing losses might occur in patients that show a specific range of APHABu scores of the AV subscale and the probabilities of hearing loss, based on pure-tone thresholds, could be a method for measurement of subjective hyperacusis or recruitment phenomenon. High scores in the AVu should correspond to a severe hearing loss, particularly at high frequencies, and our tables can provide the associated probabilities of hearing loss. Our results have demonstrated more or less stringent associations between a possible frequency-specific hearing loss and all APHABu subscale scores. However, these probabilities are not equal at all frequencies, and the frequency-specific probability of hearing loss varies for different degrees of hearing loss. The probabilities we presented  might enhance our understanding of the association between subjective speech intelligibility and hearing loss. Clearly, hearing losses in 0.5 and 8.0 kHz sounds are less possible than for sounds at the frequencies in-between, and the importance of 2.0 kHz sound was highest for a probability of an intermediate degree of hearing loss (around 45 dB). These findings corresponded to the frequency-specific dependency of speech intelligibility measured with speech audiometry .u, and a sharp threshold slope (dead region) was observed at high frequencies. This result pointed out the importance of high frequencies for speech intelligibility. In these cases, the score for the ECu subscale had little or no influence on the probability of the associated hearing loss. The widespread dispersion of the influences of 4.0 and 8.0 kHz on a mild hearing loss and the influence of 2.0 kHz on a moderate hearing loss could also be explained this effect.We found that high frequencies had limited influence, and frequencies below 4.0 kHz completely lacked influence on the probability of hearing losses of 65 dB and above. This finding could be explained by two components. First, only a relatively few number of subjects (5.9%) had profound hearing loss Table . Thus, iu could be used by ENT surgeons and audiologists in primary measurements. In addition, the APHABu could be used as a method for measuring the benefit of hearing aids after fitting and for diagnosing subjects that receive statutory accident insurance. Due to the wide use of the APHAB in over 90% of all severe cases of hearing loss in Germany, future studies should supplement these results with determinations of the sensitivity and specificity of the APHAB.This study provided additional information on how the APHABIn a broader view, a future fourth step of evaluation could be developed. The data we provided could be compiled in a computer program or a smart phone application that could calculate the predictions of different types and degrees of hearing loss, based on the patient’s answers on the APHAB questionnaire. An app like this would provide general practitioners a forecast of their patients’ hearing loss and indicate the urgency of scheduling an examination by an ENT specialist and/or audiologist.Supplementary material 1 Supplementary material 2 (PDF 3029 kb)Supplementary material 3 (PDF 3035 kb)Supplementary material 4 (PDF 3033 kb)Supplementary material 5 (PDF 3045 kb)Supplementary material 6 (PDF 3022 kb)Supplementary material 7 (PDF 3009 kb)Supplementary material 8 (PDF 3032 kb)Supplementary material 9 (PDF 3030 kb)Supplementary material 10 (PDF 3017 kb)Supplementary material 11 (PDF 2969 kb)Supplementary material 12 (PDF 2946 kb)Supplementary material 13 (JPEG 149 kb)Supplementary material 14 (JPEG 170 kb)Supplementary material 15 (JPEG 179 kb)Supplementary material 16 (JPEG 186 kb)Supplementary material 17 (JPEG 177 kb)Supplementary material 18 (JPEG 174 kb)Supplementary material 19 (JPEG 179 kb)Supplementary material 20 (JPEG 179 kb)Supplementary material 21 (JPEG 162 kb)Supplementary material 22 (JPEG 150 kb)Supplementary material 23 (JPEG 149 kb)Supplementary material 24 (JPEG 149 kb)Supplementary material 25 Below is the link to the electronic supplementary material.


Hey2 gene mutations in both humans and mice have been associated with multiple cardiac defects. However, the currently reported localization of Hey2 in the ventricular compact zone cannot explain the wide variety of cardiac defects. Furthermore, it was reported that, in contrast to other organs, Notch doesn’t regulate Hey2 in the heart. To determine the expression pattern and the regulation of Hey2, we used novel methods including RNAscope and a Hey2CreERT2 knockin line to precisely determine the spatiotemporal expression pattern and level of Hey2 during cardiac development. We found that Hey2 is expressed in the endocardial cells of the atrioventricular canal and the outflow tract, as well as at the base of trabeculae, in addition to the reported expression in the ventricular compact myocardium. By disrupting several signaling pathways that regulate trabeculation and/or compaction, we found that, in contrast to previous reports, Notch signaling and Nrg1/ErbB2 regulate Hey2 expression level in myocardium and/or endocardium, but not its expression pattern: weak expression in trabecular myocardium and strong expression in compact myocardium. Instead, we found that FGF signaling regulates the expression pattern of Hey2 in the early myocardium, and regulates the expression level of Hey2 in a Notch1 dependent manner. Drosophila homologue, dHey, is a member of a subfamily of hairy-related basic helix-loop-helix (bHLH) transcription factors2, that are implicated in cell fate determination and boundary formation3. Hey2 mutations in both humans and mice cause a variety of cardiac morphogenetic defects, as well as a cardiomyocyte maturation defect. In humans, non-synonymous sequence changes in Hey2 correlate with atrioventricular septal defects and other cardiac defects5, and Hey2 duplication contributes to both congenital heart defects and neurodevelopmental defects6. Similarly, Hey2 knockout mice display defects including atrioventricular valvular defects, pulmonary stenosis, Tetralogy of Fallot, tricuspid atresia, and abnormal cardiac hemodynamics9, indicating that Hey2 is an essential regulator of cardiac morphogenesis and cardiac function. Furthermore, genome-wide association study associated Hey2 mutation with Brugada syndrome, a rare cardiac arrhythmia disorder in humans10.Hey2, together with Hey1, HeyL and their presumptive Hey2 mutation in both humans and mice, the current knowledge of the Hey2 expression pattern—that Hey2 is expressed in the compact zone of the myocardium—cannot fully explain this broad range of congenital defects. It is unclear how Hey2 enrichment in the ventricular compact myocardium could contribute to atrioventricular morphogenesis and semilunar valvular morphogenesis. Using an RNAscope, an assay that enables single mRNA molecule detection and is compatible with immunofluorescent staining, we were able to determine the cell type- and developmental stage-specific expression pattern of Hey2 at the level of single mRNA molecule. Specifically, we found that, in addition to the previously reported expression in the ventricular compact myocardium and interventricular septum, Hey2 is also expressed in the endocardial cells of the atrioventricular canal (AVC), the outflow tract (OFT), and at the base of trabeculae, as well as in pro-epicardial cells and epicardial cells. The expressional level and pattern of Hey2 examined by the RNAScope were also consistent with the expression pattern determined by the Hey2CreERT2 knock-in mouse line.Despite profound cardiac defects caused by Hey2 functions in cell fate specification and cardiomyocyte maturation. Gridlock/Hey2 is involved in adjudicating an arterial versus venous cell fate decision during the assembly of the first embryonic artery in zebrafish11. In mice, cardiac specific Hey2 knockout hearts display ectopic atrial gene expression13. Hey2 null cardiomyocytes displayed abnormal mitochondria, abnormal accumulation of glycogen particles, and disorganized myofibrils based on transmission electron microscope analysis8. Expression levels of β-MHC and ANF genes in the Hey2 knockout are increased8. The phenotypes of Hey2 knockout heart indicate that Hey2 might play roles in cardiomyocyte differentiation and maturation.14. The facts that Hey2 is expressed in the cardiomyocytes of the compact myocardium and that Hey2 disruption results in a larger and wider sarcomere suggest that Hey2 might repress cardiomyocyte maturation and might serve as a marker for less differentiated and/or less mature cardiomyocytes. Previously, single cell lineage tracing studies revealed that asymmetric distribution of Hey2 during cardiomyocyte oriented cell division might contribute to the differential expression of Hey2 in compact and trabecular cardiomyocytes15. However, the signaling pathways that regulate the Hey2 expression pattern and its asymmetric distribution in a perpendicular oriented cell division during trabecular initiation are unknown. To identify the signaling pathways that regulate the differential expression levels of Hey2 in the compact zone and the trabecular zone, we assessed the expression of Hey2 in various mutants that are defective in several pathways implicated in trabeculation and compaction, including Nrg1/ErbB2/4 and Notch1. We found that these mutants showed decreased expression levels of Hey2, but displayed normal enrichment and repression of Hey2 in the compact zone and trabecular zone, respectively. FGF ligand stimulation changes the expression pattern of Hey2 in the ventricles temporally in both in vivo and ex vivo models, resulting in more trabeculation. We further found that FGF2 regulates the expression level of Hey2 in a Notch1 dependent manner.Previous work has suggested that trabecular cardiomyocytes, which take the major responsibility for pumping during the early stages of cardiac development, are more differentiated than the cardiomyocytes of the compact zonein situ hybridization (ISH) studies using whole embryos or tissue sections demonstrated that Hey2 is expressed in the compact zone, AV cushion, and ventricular septum16. However, harsh treatment in traditional ISH protocols prevents successful simultaneous immunofluorescence staining, which is necessary to accurately identify the cell types that express Hey2. In this study, we used the RNAscope, which can detect single mRNA molecules and allows for ISH and immunofluorescence staining in the same samples15, and a Hey2CreERT2 knockin mouse line17, to determine the expression level and pattern of Hey2 and found a broader expression pattern of Hey2 than previously reported. In both left and right ventricles, Hey2 is enriched in the outer compact zone (OCZ), and is weakly expressed in the inner compact (ICZ) and trabecular zones  , which is relatively greater than the Hey2 expression in endocardial cells that are not adjacent to the cardiomyocytes, which contain 3.20 dots (n = 6)  using E9.5 hearts from Hey2CreERT2 knockin embryos, and consistently Hey2 was strongly expressed in the compact zone and in the endocardial cells of the AVC and OFT  Fig.  and outfVC) Fig.  at E9.5. 6) Fig. . We alsoOFT Fig. , and weaOFT Fig. . Howeverhed Fig. , which mHey2 is highly expressed in the outer compact zone, endocardial cells of the AVC and OFT, and weakly expressed in ventricular endocardial cells in E10.5 hearts  and that Hey2 disruption leads to the valvular morphogenesis defects previously reported in Hey2 knockouts16. In addition to its expression in the aforementioned endocardial cells, we also found that Hey2 is weakly expressed in pro-epicardial cells and epicardial cells  overexpression activated Hey1 but not Hey2 expression in the heart using the NICD transgenic line20. Furthermore, Notch2 knockout mice displayed normal expression patterns of Hey1 and Hey2 in the myocardium21 and Rbpjk global knockout embryos did not alter the expressional level of Hey2 in the heart based on Q-PCR22. These reports indicate that Notch signaling does not regulate Hey2 expression in the heart. However, it is possible that Notch signaling regulates Hey2 expression in some cell types such as endocardial cells that cannot be detected by whole heart RNA quantification and traditional ISH. To test this possibility, we examined the expression of Hey2 in different mutants via RNAscope coupled with immunofluorescence staining. We used Nfatc1Cre/+, which is specifically expressed in the endocardial cells23, to mediate Notch1 deletion, and found reduced NICD expression . The difference in expression of Hey2 between control and knockout hearts was not as large as expected, which might be due to late expression of Cre in the Nfatc1Cre/+ mouse . These data suggest that Notch signaling mildly regulates the expression level of Hey2, but not its expression pattern in the myocardium.Echocardiographic studies of one Fig. , indicat2.5 Fig. . We thenles Fig. . We founuts Fig. , while iuts Fig. . The Nkxpes Fig. . ConsistHey2 in the compact zone. Previous work has shown that global deletion of the receptors or ligand of the Nrg1/ErbB2,4 pathway abrogates trabecular formation28. We used the Nkx2.5Cre/+ line to delete ErbB2 . Previous work has shown that Notch1 signaling regulates cardiomyocyte differentiation through Nrg1/ErbB2,424. We hypothesized that Nrg1/ErbB2,4 might regulate Hey2 expression in the compact zone. We examined the expression of Hey2 in the Nkx2.5Cre/+; ErbB2fl/fl heart and found that the Hey2 expression level was slightly reduced  to delete Notch1. qRT-PCR and western blot analyses indicated that the deletion of Notch1 is efficient  from vel Fig. . ROSA26Cent Fig. . We stimd33 Fig. . We thenses Fig. . FGF2 stMEF Fig. . These dHey2, Hey1, and HeyL comprise a subfamily of mammalian hairy/enhancer of split-related basic helix-loop-helix genes with a putative Drosophila homologue, dHey34, and a zebrafish homologue, Gridlock11. Hey2 displays a spatiotemporal expression pattern and is expressed in the somite, heart, craniofacial region, and nervous system at early embryonic stages34. Detailed examination of Hey2 expression in mouse heart shows that Hey2 is expressed in the ventricular septum and compact zone but not in the atrium at all. In this study, we used RNAscope and Hey2CreERT2 knockin mouse line to further characterize Hey2 expression level and pattern and found that Hey2 is also expressed in the endocardial cells of the AVC, OFT and at the base of trabeculae  at Albany Medical College and performed according to the NIH Guide for the Care and Use of Laboratory Animals.Mouse strains In Situ hybridization (ISH) and immunofluorescence staining (IFS) were performed according to the protocol of the RNAscope® Chromogenic (RED) Assay (Cat. No. 310036), which can detect single mRNA molecules15. Briefly, 24 hours after fixation, the embryos were frozen embedded in OCT compound. Sections of the frozen embedded samples were processed following the step-by-step protocol of the kit. The expressional level of mRNA in each cell was based on the number of mRNA molecules or signal intensity detected using the confocal scanned pictures, and three sections for each genotype were quantified. The number of mRNA molecules in cells at comparable locations was also quantified, and only the control and knockout samples that came from the same litter and underwent the same experiments were quantified and compared.Hey2 molecules. For each sample, we quantified 3 sections, and for each section, 12 cells at 3 different regions per layer were quantified. The mean value of the 36 cells in each layer was presented. Only control and mutants that came from the same litter and underwent the same experiments were quantified and compared. Each experiment was repeated three times.Cardiomyocytes at different layers starting from the outer compact layer, to the inner compact zone and trabecular zone were selected to quantify the number of Notch1fl/fl; ROSA26CreERT2. Cultured MEFs were infected treated with 4-OHT as indicated in different experiments for 36 hours, and then starved with serum free medium overnight. The cells were then stimulated with FGF2 ligand for 15 minutes. After the stimulation, the cell lysate was harvested. Lysates from MEFs of different genotypes and treatment were processed for western blot. Antibodies used for western blots were listed above. The experiments were repeated three times and quantification data was shown in the figure.Mouse embryonic fibroblasts (MEF) were isolated from E13.5-E14.5 embryos with the genotype of 59. The following primary antibodies were used: BrdU , PECAM , MF20 ), NICD , ErbB2 , ESR , Endomucin , p-Akt  and p-Erk .Immunohistochemistry (IHC) and Immunofluorescence (IF) were performed as previously publishedex vivo culture protocol was developed by Dr. C.P. Cheng and was utilized as reported36. Briefly, embryos lacking the head and tail were cultured in medium containing FGF2 at 2ng/gram body weight or vehicle for 2 hours. Then, the embryos were fixed and used for RNAscope.The The following systems were used: for confocal imaging, Zeiss LSM880-META NLO confocal microscope equipped with Airyscan; for color imaging, Zeiss Observer Z1 with a Hamamatsu ORCA-ER camera; for fluorescence imaging, Zeiss Observer Z1 with an AxioCam MRm camera. All histology analysis and immune-fluorescent staining analysis were quantitated in a blinded way by at least two investigators.Data is shown as mean ± standard deviation. An unpaired, two-tailed student’s t-test or linear regression comparison as specified were used for statistical comparison. A P-value of 0.05 or less was considered statistically significant.Supplemental Dataset


P<0.001). We also demonstrated that HEY2 expression was inhibited by miR-137. In clinical samples, HEY2 expression was reversely associated to miR-137 expression. Furthermore, overexpression of HEY2 increased cell viabilities, colony formation and cell migration, whereas knockdown of HEY2 resulted in the opposite phenotypes. Collectively, our data suggest HEY2 as a promising biomarker for unfavorable outcomes and a novel therapeutic target for the clinical management of HCC.HEY2, a bHLH transcription factor, has been implicated in the progression of human cancers. Here, we showed that HEY2 expression was markedly increased in HCC, compared with the adjacent nontumorous tissues. High HEY2 expression was closely correlated with tumor multiplicity, tumor differentiation and TNM stage. Kaplan-Meier analyses revealed that HEY2 expression was significantly associated with poor overall and disease-free survival in a training cohort of 361 patients with HCC. The prognostic implication of HEY2 was validated in another cohort of 169 HCC patients. Multivariate Cox regression model indicated HEY2 as an independent factor for overall survival in HCC (Hazard ratio = 1.645, 95% confident interval: 1.309-2.067,  The incidence and mortality of hepatocellular carcinoma (HCC) has not been improved in the last decade worldwide . NumbersHEY2, also known as HESR2, HRT2 and CHF1), belongs to of the HEY family of basic helix-loop-helix (bHLH) transcription factor /2.1×102 test was performed to analyze the correlation between HEY2 expression and clinicopathological parameters. Kaplan-Meier analysis (log-rank test) was utilized for survival analysis. Cox proportional hazards regression model was used to identify the independent prognostic factors. P<0.05 (two-tailed) was considered statistically significant.Statistical analyses were performed using the SPSS 19.0 software . Wilcoxon matched paired test was used to determine the significance of HEY2 expression in fresh HCC and normal liver tissues. χ


Colorectal perineuriomas are uncommon benign mucosal-based proliferations of mesenchymal cells that express perineurial markers, often associated with colonic crypts displaying a serrated/hyperplastic architecture. The vast majority of cases arise distal to the splenic flexure and have been described as sessile polyps. Using molecular analysis, BRAF mutations have been demonstrated in the serrated crypt epithelium. We report a new case of perineurioma presenting as a pedunculated polyp in the transverse colon, with prominent hemosiderin deposits in the uninvolved lamina propria that separated the perineurial proliferation from the surface epithelium, a previously unreported histological finding. By using immunohistochemistry, we demonstrated the presence of BRAF V600E mutated protein in the serrated crypt epithelium. In addition, a review of the literature on colorectal perineurioma is provided.A 5 mm pedunculated polyp was removed from the transverse colon of a 42 year old man who presented with epigastric pain, weight loss and rectal bleeding. A proliferation of uniform plump spindled cells expanded the lamina propria and separated serrated colonic crypts. The epithelial component closely resembled microvesicular hyperplastic polyp. Immunohistochemical stains for epithelial membrane antigen (EMA), glucose transporter 1 (GLUT1) and collagen IV were positive in the stromal proliferation. A mutation-specific monoclonal antibody directed against BRAF V600E showed positive cytoplasmic staining in the serrated crypt epithelium but not in the perineurial proliferation. Conspicuous hemosiderin deposition was seen in the inflamed lamina propria between the perineurial proliferation and the surface epithelium.Although the majority of colorectal perineuriomas occur in the sigmoid colon and rectum and are described as sessile polyps, colorectal perineurioma can present as a pedunculated polyp proximal to the splenic flexure as described in this case. Conspicuous hemosiderin deposition can be seen in the superficial lamina propria. BRAF mutations are limited to the serrated crypt epithelium. Colorectal perineuriomas, first described as benign fibroblastic polyps in 2004 by Eslami-Varzaneh et al.,  are beniA 42 year old man with a history of epigastric pain for two years, weight loss and an episode of rectal bleeding was referred for colonoscopy after gastroscopy revealed only antral gastritis. Physical examination was unremarkable except for the presence of mild epigastric tenderness. Serum lipase levels, liver function tests and full blood count were essentially normal. A pedunculated polyp, 5 mm in diameter, was removed from the transverse colon and sent for histopathological evaluation. No other endoscopic abnormalities were identified.On histological examination, a proliferation of uniform, plump spindled cells in the lamina propria surrounded and separated serrated and nonserrated crypts that resembled those of microvesicular hyperplastic polyps Fig.  and b. AImmunohistochemical staining with a monoclonal epithelial membrane antigen (EMA) antibody, using clone E29  at an antibody concentration of 1:500, showed weak positive staining in the stromal proliferation Fig.  while boColorectal perineuriomas, although uncommon, are probably underrecognized lesions . These bUsing a PubMed search and reference lists of published articles on colorectal perineurioma / fibroblastic polyp, a total of 157 reported cases were found . A furthThe mean age at diagnosis for published cases was 60 years (range 35 – 87 years). A slight female predominance was noted (F:M = 1.3).For those cases where the reason for the colonoscopy was indicated, 51 of 74 (69%) were for routine screening, 3 of 74 (4%) for screening in a patient with previous colorectal carcinoma, 10 of 74 (13%) for gastrointestinal bleeding or occult blood in the stools, 7 of 74 (9%) for abdominal pain and one each for diarrhoea, change of bowel habit and thickening of large bowel wall on computed tomography scan. Although the perineurioma might have been responsible for some of these presenting symptoms and signs, the associations might have been purely coincidental.Almost three quarters of reported cases occurred in the sigmoid colon or rectum. Less than 15% of cases were located proximal to the splenic flexure. The average size was 4.1 mm (range: 1 – 30 mm). Most of the polyps were described as small sessile polyps –9 but GrThe typical histological appearance was that of an intramucosal proliferation of plump, uniform spindle cells that filled the lamina propria causing separation, distortion and entrapment of colonic crypts. The spindled cells had pale eosinophilic cytoplasm with indistinct cell borders within a fine collagenous stroma. The cells often had a concentric arrangement around crypts or glands. A thin zone of uninvolved, mildly inflamed lamina propria separated the perineurial proliferation from the overlying surface epithelium in many cases. The presence of hemosiderin deposition has not been specifically described in colorectal perineuriomas, although conceptually, it can occur in any pedunculated polyp. It has been described in hyperplastic polyps  as an inHemosiderin deposition in the lamina propria was reported as a common finding in inflammatory myoglandular polyps , polyps Mild disorganization of the muscularis mucosae with occasional thin bundles extending towards the surface was reported . No signThe diagnosis of perineurioma can be suspected with reasonable confidence on morphology alone in typical cases with associated serrated epithelium but the demonstration of expression of at least two perineurial markers is recommended for accurate diagnosis . It is pThe immunohistochemical features of previously reported colorectal perineuriomas, including the current case, are summarized in Table The current case is the first reported colorectal perineurioma where the BRAF V600E mutated protein was demonstrated by immunohistochemical detection. Interpretation of the BRAF V600E immunohistochemical stain was however not straightforward due to moderately intense nuclear staining in lesional and non-lesional colonic epithelial cells. Difficulties in the interpretation of immunohistochemistry for BRAF in serrated lesions of the colon have been highlighted by other authors .Ultrastructural features of perineuriomas include parallel, elongated spindled cells with collagen-rich intervening matrix, long thin cytoplasmic processes with frequent pinocytotic vesicles and junctional complexes and a discontinuous basal lamina .Eight gastrointestinal cases from four studies showed perineurial features on ultrastructural examination –10, 12. BRAF mutations in 63% of 22 cases of perineurioma  [BRAF mutations occur only in the epithelial component and not in the stromal component came from a study by Groisman et al. who demonstrated BRAF mutations (using direct sequencing) in 5 of 8 serrated perineuriomas while none of 12 cases of non-serrated perineuriomas harboured a BRAF mutation [Agaimy et al. demonstrated p.V600E tecture) . NotablyBRAF mutations which led them to suggest that there was a strong indication of epithelial-mesenchymal interactions in BRAF positive serrated polyps and the possibility of epithelial mesenchymal transformation occurring in a proportion of serrated polyps. Further studies are necessary to assess clonality and gene alterations in the perineurial stromal component of colorectal perineurioma.At this stage the exact nature and origin of the perineurial proliferation is unclear. It has been suggested that the perineurial stromal component might be derived from modified pericryptic fibroblasts as a consequence of a yet poorly understood epithelial-stromal interaction . PerineuIn conclusion, although the majority of colorectal perineuriomas occur in the sigmoid colon and rectum and are usually reported as sessile, colorectal perineurioma can present as a pedunculated polyp proximal to the splenic flexure as demonstrated in this case. The polyp showed conspicuous hemosiderin deposition in the overlying uninvolved lamina propria and, by using immunohistochemistry, we demonstrated BRAF mutated protein restricted to the serrated crypt epithelium.


Beauveria bassiana and Metarhizium anisopliae applied as seed treatments for their endophytic establishment in the common bean. Endophytic colonization in sterile sand:peat averaged ca. 40% higher for fungus treatments and ca. six times higher for volunteer fungi , relative to sterile vermiculite. Colonization by B. bassiana and M. anisopliae was least variable in sterile vermiculite and most variable in sterile soil:sand:peat. The impact of soil sterilization on endophytic colonization was assessed in a separate experiment using six different field-collected soils. Soil sterilization was the variable with the largest impact on colonization , while the fungal isolate used to inoculate seeds explained 8.4% of the variance. Under natural microbial soil conditions experienced by common bean farmers, seed inoculations with B. bassiana and M. anisopliae are unlikely to yield predictable levels of endophytic colonization.The common bean is the most important food legume in the world. We examined the potential of the fungal entomopathogens  An important reason for focusing on seeds is that they are more likely to be adopted by growers because they are easier to use than the other endophyte-related treatments involving foliar sprays, soil drenches, or stem injections involving fungal entomopathogens.Seed treatments with biological control agents provide an environmentally friendly alternative to the use of chemical inputs . Among t studied  and have studied . Seed tr studied . Still, −1, roughly a third of the yield achieved in the US and Canada , respectively.22.1B. bassiana and M. anisopliae were obtained from commercial products available in Colombia and from the collections of entomopathogenic fungal cultures maintained by the International Center for Tropical Agriculture (CIAT), the Colombian Corporation for Agricultural Research (CORPOICA), and the Colombian Oil Palm Research Center . Monosporic cultures were prepared following procedures described by 8 conidia m L−1 in maize oil , which we used as a sticker to adhere conidia to the seed surface . Plates were incubated in the dark at 26 ± 2 °C for 24 h after which three groups of 100 randomly selected conidia were evaluated to assess percent conidial germination. Conidia were deemed germinated when the length of the germ tube exceeded half the diameter of the conidia.Eleven isolates/strains each of  surface . A germi2.2Phaseolus vulgaris cv. Calima seeds were obtained from plants grown at CIAT. Prior to their inoculation, they were surface-sterilized by immersion in 0.1% Triton X-100 for 2 min, 0.5% sodium hypochlorite for 2 min, and 70% ethanol for 2 min, followed by three rinses with sterile distilled water. To assess the effectiveness of the sterilization method, three randomly selected seeds from 15 or 45 seeds used in the screening and full experiments, respectively , were individually pressed onto ¾-strength PDA media plates . Trays were arranged randomly in plant growth chambers at 25 ± 2 °C, 47% relative humidity (RH) and 12:12 photoperiod . Plants were watered daily with ca. 8 ml of sterile tap water starting the third day after inoculation (dai) until 16 dai, then every other day with 50 ml of tap water until 30 dai. Plants were then watered with 100 ml of tap water every other day for the remainder of the experiment.Groups of 15 seeds  or 45 seeds  were transferred into sterile 100 x 20 mm Petri dishes and air dried in a laminar flow hood for approximately 1 h before inoculation. Surface sterilized seeds were immersed in 1 × 102.43 of each sterilized potting substrate sample was suspended in 9 ml of sterile 0.1% Triton X-100 and vortexed for 3 min, followed by plating 100 μl of 10−1 and 10−2 dilutions of this suspension onto ¾-strength PDA and PDA media plates amended with penicillin (100 mg L−1), streptomycin (200 mg L−1) and tetracycline (50 mg L−1). Antibiotics were filter-sterilized through a 0.2 μm filter Nalgene syringe filters with 25-mm surfactant-free cellulose acetate . Plates were incubated at 26 ± 2 °C in darkness and were checked after 10 d for any fungal growth. No fungal growth was observed in any of the samples.To validate the effectiveness of the potting substrate sterilization methods (discussed below) in eliminating fungal spores, 1 cm2.5B. bassiana isolates and 11 M. anisopliae isolates. The fourth experiment  evaluated the effects of substrate and dai on common bean plant colonization with two selected isolates of each fungal species, and with four replicates over time. The fifth experiment  evaluated the effect of soil sterilization on endophytic colonization achieved by seed treatments with two fungal entomopathogens. For this experiment, soil samples were collected from six bean production fields in Colombia . This experiment was conducted twice.Five separate experiments were conducted as completely randomized designs with factors varying among experiments. Three experiments  were run to evaluate the endophytic potential of 11 2.6M. anisopliae isolates/strains: CIAT 001, CIAT 014A, CIAT 042, CIAT 053, BioMa® , and Metarhiplant® WP . The second experiment consisted of six B. bassiana isolates/strains: CIAT 359, CIAT 405, Bovetrópico® WP , Micosis® WP , Beauveriplant® WP , and Mycotrol® O . The third experiment used five isolates each of B. bassiana  or M. anisopliae . Screening experiments were designed to eliminate isolates that showed no potential on a first experimental round. Based on results from screening experiments, the two best Beauveria and Metarhizium isolates were selected for full experiments, which were repeated four times (see below).Three separate screening experiments were conducted in a growth chamber to evaluate the ability of the 22 fungal entomopathogen isolates/strains to endophytically colonize common bean plants. The first experiment used six 3) for each sterilization batch. After sterilization, vermiculite was cooled for at least 72 h and moistened with 1300 ml of sterile tap water prior to filling the trays and planting the seeds. Each treatment tray held five bean seeds. Evaluations for endophytic colonization were conducted 8 and 14 dai.Vermiculite  with particle size <0.7 mm was used as a potting substrate for all screening experiments. Vermiculite was sterilized in an autoclave for 20 min at 121 °C using 800 g (0.0012 m2.72. The six root fragments were plated separately from the leaf and stem fragments on two 60 mm diameter Petri dishes containing ¾-strength PDA media plates amended with penicillin (100 mg L−1) streptomycin (200 mg L−1) and tetracycline (50 mg L−1). Antibiotics were filter-sterilized as described above. The plates were incubated at 26 ± 2 °C in darkness, and monitored for the presence of B. bassiana and M. anisopliae for up to 14 d. Presence of fungal endophytes other than B. bassiana or M. anisopliae was also recorded.Germination trays containing the seedlings were transferred from the growth chambers to the laboratory, where the plants were carefully uprooted. Individual plants were washed under running tap water for approximately 1 min and secondary roots were carefully removed manually, leaving only the taproot. Each plant was divided into two sections, the above ground part and the below ground part. The latter was obtained by cutting approximately 3 cm above the root crown and therefore includes 3 cm of stem. Both plant sections were introduced into a Fisherbrand™ 50 ml polypropylene centrifuge tube  to be bulk surface-sterilized following the 2.8B. bassiana  and two M. anisopliae isolates  were selected. The isolates were used to evaluate the effect of three sterile potting substrates on endophytic colonization of common bean seedlings resulting from seed inoculations (as previously described).Based on the highest levels of endophytic colonization in the screening experiments (data not shown) two −1, and was collected from a CIAT experimental field. The soil was mixed with sand (3:1), resulting in an additional 25% sand. The soil:sand:peat had an additional 20% peat . The substrates were sterilized in an autoclave under the same conditions as in the screening experiments. Each fungal entomopathogen-inoculated seed was sown into one of the three substrates in a 50 cm3 germination tray cell . Trays were randomly placed in growth chambers at 25 ± 2 °C, 47% RH and 12:12 photoperiod . The plants were grown for 16 d before being individually transplanted into one liter (10 cm diameter × 12 cm height) disinfected pots filled with the same substrate, moistened with 50 ml of tap water before transplantation. The substrate used for transplanting was steam-sterilized at 82 °C for 6 h in 1 m3 batches in a media steamer cart using a steam generator. The plants were maintained in the plant growth chambers under the same conditions as before transplantation.The three sterilized potting substrates were: a mixture of soil:sand (3:1); a mixture of soil:sand:peat (3:1:1); and vermiculite. The soil used was a silt loam soil  with an organic matter content of 32 g kgEndophyte colonization evaluation was conducted at 14 , 30 , and 60  dai, based on the developmental growth stages proposed by CIAT . Plants B. bassiana and M. anisopliae had dropped severely. We suspected a problem with sterilization of the substrate used for transplanting and decided to change the substrate sterilization method in the remaining two replicates. Therefore, substrates used for transplantation were steam-sterilized for 6 h with two cycles using an SST-60 electric soil sterilizer  at 82 °C. The total volume of the sterilizer is 0.4 m3 (½ yd3) and is equally divided into five compartments. Four of the five compartments were filled with soil:sand and were moistened with 32 L of tap water before initiating the sterilization process. The fifth compartment was filled with peat moistened with 12 L of tap water before initiating the sterilization process. Substrates were cooled for 72 h before the second sterilization cycle. Treatments and procedures were the same as described for the two previous replicates.The experiment was replicated four times. At the time of evaluating the second repetition, the colonization by 2.9B. bassiana (Mycotrol® O) or M. anisopliae (CIAT014A). Mycotrol® O was selected due to its commercial availability in many countries. Five soils were collected from localities where common beans were being grown (Colombian Department in parenthesis): Colón , Gama , San Gil , Río de Oro , Dabeiba . The sixth soil was collected at the same CIAT experimental field in Palmira  used for the “full experiments” (see above). For collecting the soils, one soil sample (4 kg from the top 10 cm) was randomly collected. Each soil was tested with and without sterilization. The sterilization treatment for each soil  consisted of three cycles in an autoclave at 121 °C for 20 min. Between the first and second cycle, a period of 24 h was allowed to pass, and between the second and third cycle, 48 h. Colonization data were collected 14 dai using the same protocol outlined for the screening experiments.A fifth experiment was conducted using six soils and seed inoculated with 2.10We fit mixed models using the R package, lme4 , to a da3In preliminary analyses for each experiment, we found a clear ordering of the main effects of fungus and media. However, this ordering differed greatly among experiments, and we found no covariate or other factor to explain why. Thus, rather than focus on reporting main treatment effect differences averaged over all experiments (mean responses), which makes little sense given the large experiment-to-experiment variation, we found it better to focus on which variables explained the largest proportions of the variance, through a variance decomposition approach. We found that using this approach for each planting media separately provided the most interpretable results, since this decomposition required only second order interactions to produce models without over-dispersion.B. bassiana nor M. anisopliae were recovered as endophytes in any plants that were not inoculated with them . Preliminary screening experiments in vermiculite with 22 fungal isolates suggested that B. bassiana is generally more successful at colonizing bean seedlings than M. anisopliae, with the top seven isolates belonging to the former species (data not shown). When considering ease of cultivation in addition to colonization success, the screening experiments identified B. bassiana isolates CpBb0417 and Bv012 and M. anisopliae isolates CpMa1107 and CIAT014A as potential candidates for seed inoculations. However, within plant colonization was never high, averaging 27 ± 4.4%, 21 ± 4.4%, 10 ± 0.5%, and 6 ± 2.1% for plants inoculated with CpBb0417, Bv012, CpMa1107, and CIAT014A, respectively.Neither Four subsequent experiments tested the effect of growth medium and sampling day on endophytic colonization by our treatment fungi and any other fungal endophytes naturally-occurring in our samples . We refeColonization by treatment endophytes was least variable in vermiculite and most variable in soil:sand:peat . EvaluatAs a percentage of the total variance for each planting medium , the fungal isolate used to inoculate seeds explained 41.4% of the total variance in vermiculite, but less than 1% of the total variance in soil:sand and soil:sand:peat . This isB. bassiana (Mycotrol® O) and M. anisopliae (CIAT014A)  . As suspIAT014A) , accountIAT014A) . By contIAT014A) . These r4B. bassiana, Lecanicillium sp., L. lecanii, M. anisopliae, M. brunneum, M. robertsii, and Purpureocillium lilacinum  and planted the seeds in the field. Negative effects on Sesamia calamistis (Lepidoptera: Noctuidae), were ascribed to B. bassiana, even though no attempt was made to determine whether B. bassiana had become endophytic. Using maize seed (Zea mays) coated with M. anisopliae conidia, followed by field planting, Solanum lycopersicum), B. bassiana conidia and seed germination in sterile vermiculite, followed by replanting in sterile potting medium. Endophytic colonization by B. bassiana in sorghum following seed dressing, foliar or soil sprays was influenced by the substrate used, i.e., sterile soil, non-sterile soil, or vermiculite (B. bassiana for seed dressings of opium poppy (Papaver somniferum), which resulted in endophytism when seeds were germinated in sterile vermiculite and then transferred to sterile potting medium. Vicia faba) in conidial suspensions of B. bassiana or M. anisopliae, and planted the seeds in sterile soil:sand, with positive endophyte recovery. V. faba and common bean seeds in B. bassiana and M. anisopliae conidial suspensions, planting the seeds in a sterile soil:manure mixture. Endophytism was positive for B. bassiana and negative for M. anisopliae. Corchorus capsularis) in B. bassiana conidial suspensions, followed by planting in sterile soil, resulting in positive endophytism. B. bassiana. Gossypium hirsutum) in conidial suspensions of B. bassiana or Purpureocillium lilacinum, with positive endophytism when seeds were grown in non-sterile potting medium; when seeds were directly planted in the field, endophytism was assumed, but not experimentally determined. Similarly, B. bassiana or P. lilacinum and planted seeds in non-sterile potting medium, where endophytism was assumed based on previous work, but not experimentally determined. B. bassiana as inoculants for oilseed rape (Brassica napus) and the filmcoated seeds “was not evaluated further due to the suppressive effect of non-sterile soil observed with the encapsulated fungus.” Metarhizium pingshaense, followed by planting in peat compost (authors don’t mention whether it was sterile or not), and reported positive endophytic recovery from roots.Most endophyte seed-related experiments with other plants use sterile substrates, and some studies do not experimentally assess endophytism. miculite : “Seed iB. bassiana when conidia are in contact with non-sterile soil, and these effects could be due to antagonistic soil microbial activity , which would then allow to investigate the mechanism responsible for improved establishment and hopefully improve our approach to their use as effective endophytic biological control agents. Simultaneous plant inoculations with different species or genera of fungal entomopathogens might also yield interesting findings. Finally, it is important to consider more in-depth statistical analyses such as a variance decomposition approach, which should aid in gaining a better understanding of the experimental results, rather than the usual focus on main treatment effects (mean responses).


The objective of the study is to provide some implications for rehabilitation of hearing impairment by investigating changes of neural activities of directional brain networks in patients with long-term bilateral hearing loss. Firstly, we implemented neuropsychological tests of 21 subjects , and these tests revealed significant differences between the deaf group and the controls. Then we constructed the individual specific virtual brain based on functional magnetic resonance data of participants by utilizing effective connectivity and multivariate regression methods. We exerted the stimulating signal to the primary auditory cortices of the virtual brain and observed the brain region activations. We found that patients with long-term bilateral hearing loss presented weaker brain region activations in the auditory and language networks, but enhanced neural activities in the default mode network as compared with normally hearing subjects. Especially, the right cerebral hemisphere presented more changes than the left. Additionally, weaker neural activities in the primary auditor cortices were also strongly associated with poorer cognitive performance. Finally, causal analysis revealed several interactional circuits among activated brain regions, and these interregional causal interactions implied that abnormal neural activities of the directional brain networks in the deaf patients impacted cognitive function. A meta-analysis in adults  proposedFirstly, we constructed the individual specific virtual brain using resting-state fMRI data of each participant. Then we generated auditory stimulating signals, which were composed of the sum of sines and cosines of various frequencies with range from 500Hz to 2000Hz. To simulating a real environment, we presumed that the auditory signals were decayed in the transmission process from the cochlea to the primary auditory cortex (BA 41), and the weakened amplitudes were different according to the normal hearing subjects and the deaf patients respectively Figure  and 1b. The results are shown in Figure To further verify the influences of long-term bilateral hearing loss on cerebral cortices, we exerted directly the auditory stimulating signals to BAs 41L and 41R Figure  and 2b, We studied the interregional synchronous or asynchronous entropy connections  among those brain regions that presented significant changes  of activated strengths in the deaf patients utilizing causal analysis method . ResultsWe found several causal circuits among those brain regions whose activity strengths presented significant changes through analyzing Figures Mini-mental state examination (MMSE) and Wechsler adult intelligence test  were useWe investigated the relationships among the hearing levels, neuropsychological testing scores, and activity strengths of significantly activated brain regions by using Pearson's correlation analysis. Cognitive performance scores in MMSE and recall presented strongly negative correlation with the left hearing levels of participants ; on the contrast, testing scores in stroop color-word test A (SCWTA) presented positive correlation with the left hearing levels . Both activated strengths in BAs 41L and 41R displayed significant negative correlations with SCWTA testing scores . Lower SCWTA performance scores and higher MMSE testing scores indicate better cognitive abilities. Activated strengths in BA 41L was positively related with language processing scores , similarly, Activated strengths in BA 41R was positively related with MMSE testing scores . Activated strengths in BAs 41L, 41R, and 42R presented strongly positive correlations with the left hearing levels  and the right hearing levels of participants .In this study, we investigated changes of neural activities of directional brain networks in patients with long-term bilateral hearing loss. Our main findings are described as follows. Compared with normal hearing subjects, patients with long-term bilateral hearing loss presented weaker brain region activations in the primary auditory cortex  and the language network; enhanced neural activities in the DMN. In addition, we also found some significant correlations among the hearing levels, neuropsychological testing scores, and activated strengths in the primary auditory cortex. Poorer cognitive performance scores were always associated with higher hearing levels and weaker activated strengths in BAs 41L and 41R. Previous study also demonstrated that hearing loss was independently associated with poor cognitive performance scores , 16.We also found reduced neural activities in BAs 22R and 43R of the language network. Moreover, neuropsychological tests revealed a significant difference in language processing scores between the deaf patients and the controls. Previous studies , 18 indiSeveral brain regions associated with cognitive processes in the DMN displayed enhanced activations. These areas included the dorsal frontal cortex (BA 8) involving the management of uncertainty ; the rigIt is worth noting that patients with bilateral hearing loss presented more changes in the right cerebral hemisphere than those in the left. Additionally, the left hearing levels presented strongly correlation with MMSE and SWCA testing scores. One possible reason is that long-term hearing loss contributes to complex causal interactions among brain regions, and these causal circuits lead to more changes in the right cerebral hemisphere than in the left. Another possible reason is that the right cerebral hemisphere is more sensitive to hearing loss than the left. Transmission of auditory signals is lateralized to the contralateral hemisphere. Therefore, this finding suggests that left hearing loss may affect the cerebral cortex more than the right hearing loss does, as shown in previous study .In a word, long-term causal interactions were main reasons that contributed to the decline of the deaf patients’ cognitive abilities. Our findings imply that early clinical interference is important for the deaf patients, especially, left cochlear implant might have more advantages than the right for patients that had bilateral post-lingual deafness.The protocol of this prospective study was approved by the institutional Ethics Committee of Taishan Medical University, and all participants signed informed consent forms prior to the experiment. A total of 21 subjects  were recruited from Taian. Specially, 10 patients had long-term bilateral hearing loss with tinnitus. Hearing loss was acquired due to traumatism in one patient, deafness was caused by infection in two patients, and hearing loss in others was unknown cause. All were right-handed and none of them had a history of medical, strokes/cerebrovascular ischemia. Demographic characteristics of participants are shown in Table No subjects were excluded because of excessive head movement (greater than 2.5 mm or 2.5 degree) during imaging.MRI data of 21participants were acquired using a GE Discovery MR 750 3-T scanner. Each subject underwent an 8.06-minute scan during a conscious resting-state. Functional images were collected axially using an echo-planar imaging sequence (echo time (TE) = 2000 ms; repetition time (TR) = 30 ms; matrix size = 64 × 64 × 41, voxel size 3.4375 mm×3.4375 mm×3.2 mm). A three-dimensional T1-weighted structural image was also acquired for each subject using a magnetization prepared gradient echo sequence .http://www.fil.ion.ucl.ac.uk/spm/software/spm8/) and in-house C++ codes. Slice timing and motion corrections were first executed, and then processed functional images and structural ones were normalized to the standard brain template from the Montreal Neurological Institute (MNI 152) by applying nonlinear registration. The normalized images were smoothed using a 3D isotropic Gaussian kernel of full-width at half-maximum 8mm. Running segmentation for structural images generated white matter, gray matter, and cerebrospinal fluid images. Finally, the following procedures were applied: the removal of linear and quadratic trends; regress out covariates including six realignment parameters, white matter, and cerebrospinal fluid; band-pass temporal filter. To contain more frequency components, the frequency range of band-pass temporal filter was from 0.0039Hz to 0.1039Hz.Data were preprocessed using spm8 (Our method consists of two steps: (1) We constructed the individual specific virtual brain using resting-state fMRI data of each participant. These data had been preprocessed in the section data preprocessing. Each virtual brain comprised of an effective network that was constructed utilizing the entropy connectivity method , and eveThe chi-squared test and two-sample t-test were performed to explore group differences in sex, age, educational levels, hearing levels, the activated strength of brain region, and neuropsychological testing scores. Statistical analyses were conducted with software , and a P value of less than .05 was considered to indicate a significant difference for any single analysis.