Marine microbes must manage variation in their chemical, physical, and biological surroundings. Because they directly link bacterial physiology to environmental changes, TCS systems are crucial to the bacterial cell. This study surveyed TCS systems in a large number of marine bacteria and identified key phylogenetic and lifestyle patterns in environmental sensing. We found evidence that, in comparison with bacteria as a whole, marine organisms have irregular TCS system constructs which might represent an adaptation specific to the marine environment. Additionally, we demonstrate the biogeochemical relevance of TCS systems by correlating the presence of the PMT9312_0717 response regulator protein to phosphate concentrations in the South Pacific. We highlight that despite their potential ecological and biogeochemical relevance, TCS systems have been understudied in the marine ecosystem. This report expands our understanding of the breadth of bacterial TCS systems and how marine bacteria have adapted to survive in their unique environment. Prochlorococcus/Synechococcus response regulator PMT9312_0717 in metaproteomes of the tropical South Pacific. We found that this protein’s abundance is related to phosphate concentrations, consistent with a putative role in phosphate regulation.Two-component sensory (TCS) systems link microbial physiology to the environment and thus may play key roles in biogeochemical cycles. In this study, we surveyed the TCS systems of 328 diverse marine bacterial species. We identified lifestyle traits such as copiotrophy and diazotrophy that are associated with larger numbers of TCS system genes within the genome. We compared marine bacterial species with 1,152 reference bacterial species from a variety of habitats and found evidence of extra response regulators in marine genomes. Examining the location of TCS genes along the circular bacterial genome, we also found that marine bacteria have a large number of “orphan” genes, as well as many hybrid histidine kinases. The prevalence of “extra” response regulators, orphan genes, and hybrid TCS systems suggests that marine bacteria break with traditional understanding of how TCS systems operate. These trends suggest prevalent regulatory networking, which may allow coordinated physiological responses to multiple environmental signals and may represent a specific adaptation to the marine environment. We examine phylogenetic and lifestyle traits that influence the number and structure of two-component systems in the genome, finding, for example, that a lack of two-component systems is a hallmark of oligotrophy. Finally, in an effort to demonstrate the importance of TCS systems to marine biogeochemistry, we examined the distribution of IMPORTANCE Marine microbes must manage variation in their chemical, physical, and biological surroundings. Because they directly link bacterial physiology to environmental changes, TCS systems are crucial to the bacterial cell. This study surveyed TCS systems in a large number of marine bacteria and identified key phylogenetic and lifestyle patterns in environmental sensing. We found evidence that, in comparison with bacteria as a whole, marine organisms have irregular TCS system constructs which might represent an adaptation specific to the marine environment. Additionally, we demonstrate the biogeochemical relevance of TCS systems by correlating the presence of the PMT9312_0717 response regulator protein to phosphate concentrations in the South Pacific. We highlight that despite their potential ecological and biogeochemical relevance, TCS systems have been understudied in the marine ecosystem. This report expands our understanding of the breadth of bacterial TCS systems and how marine bacteria have adapted to survive in their unique environment. A bacterium’s survival is dependent on its ability to respond to changes in its environment. This is especially true for marine microbes, which experience changes in nutrient availability, light, temperature, and community structure that can occur on time scales as short as hours . Two-comCanonically, two-component sensory systems are composed of a single histidine kinase and response regulator protein pair . The hisProchlorococcus, activation of PhoR by low intracellular phosphate stimulates transcription of alkaline phosphatase. Alkaline phosphatase cleaves phosphate from organic matter, providing a source of phosphate that would otherwise be inaccessible  in metaproteomes from the tropical Pacific. We highlight gaps in our knowledge of marine TCS and emphasize the importance of TCS to our overall understanding of marine bacteria.In this study, we surveyed the TCS system genes of 328 diverse marine bacteria, identifying phylogenetic and lifestyle factors that correlate with greater numbers of sensory genes. We compared these marine bacteria to a curated reference collection of 1,152 bacterial genomes, most derived from the GEBA initiative  to 174 (Desulfovibrio inopinatus DSM 10711) and is related to genome size; we found on average 0.902 histidine kinases (HPK) per 100 protein-encoding genes . The number of histidine kinases in each genome ranges from 1 (ng genes . A K-meaP = 3e−15 by Student's t test) , oligotrophs (organisms adapted to low-nutrient conditions), or as organisms that fall between those classifications –12. This– t test) .We compared the TCS system genes of marine bacteria with those of 1,152 reference bacteria . The ref10.1128/mSystems.00317-18.1FIG S1FIG S1, EPS file, 0.09 MB.Phylogenetic breakdown of the marine and reference data sets, showing the diversity of the genomes used in this analysis. Download Copyright © 2019 Held et al.2019Held et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00317-18.4TABLE S2Table S2, XLSX file, 0.2 MB.TCS gene data for the 1,152 reference bacteria, including taxonomic information for each genome. Download Copyright © 2019 Held et al.2019Held et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P = 0.009, F = 6.73].Based on the traditional understanding, the numbers of histidine kinase and response regulator genes are expected to be equal. Indeed, we found the average response regulator/histidine kinase (RR/HPK) ratio in the reference data set to be 0.99 . The RR/To better understand the origin of the high RR/HPK ratio, we examined the locations of the TCS genes in a subselection of marine genomes . GenomesPelagibacter ubique HTCC1016 genome has three modular TCS systems plus one orphan HPK (a KipI family gene) and one orphan RR (RegB). Notably, KipI is known to participate in regulatory networks (Alteromonas sp. Alt199 has 26 orphan histidine kinases (30% of the histidine kinase genes) and 8 orphan response regulators (14% of the response regulator genes). Crocosphaera sp. WH8501 has 8 orphan histidine kinases (15%) and 9 orphan response regulators (17%). It is difficult to ascertain the function of orphan genes due to the lack of experimental evidence associated with them. These systems are ideal for future study.Orphan TCS genes were identified in all seven of the genomes that we examined, consistent with the high RR/HPK ratios described above. For example, the networks . Larger P = 3e−10 by a Student's t test).Hybrid systems occur when the histidine kinase and the response regulator are located on a single protein; here they were identified as genes containing both a histidine kinase HAMP domain and a response regulator receiver domain. In marine bacteria, the proportion of hybrid histidine kinases relative to total histidine kinase content ranged from zero to 61% . Marine Proteobacteria and Cyanobacteria to explore differences among phylogenetically related organisms. Proteobacteria tend to have many histidine kinases , suggesting the possibility of the presence of extra response regulators in the genomes. The RR/HPK ratios of the Cyanobacteria are more variable than those of the Proteobacteria. Prochlorococcus and Synechococcus, for instance, have very high RR/HPK ratios , indicating that there are many extra response regulators in the genome, while Trichodesmium organisms have a low RR/HPK ratio (0.75), indicating the possibility of the presence of extra histidine kinases.Prochlorococcus MIT9312 phosphate-sensing response regulator (PMT9312_0717) in metaproteomes of the tropical Pacific , and M. Saito, Biological and Chemical Oceanography Data Management Office (BCO-DMO), https://www.bco-dmo.org/dataset-deployment/646117 . Download FIG S2, EPS file, 1.0 MB.Hydrographic and pigment data from the METZYME cruise. The distribution of PMT9312_0717 is not well correlated with chlorophyll-a, divinyl chlorophyll-a, temperature, nor salinity in this region but is well correlated with dissolved-phosphate concentrations see . Data arCopyright © 2019 Held et al.2019Held et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the r2 = 0.64)   and C. Pgulation . HoweverTwo-component sensory systems allow bacteria to directly sense their internal and external surroundings and therefore play key roles in bacterial “intelligence” , 19. BecAs in prior surveys, we found that the number of histidine kinases in the genome is largely determined by genome size , 4. Howe9–Pelagibacter, which may rely on simpler regulatory structures (such as one-component systems or riboswitches) which require fewer energetic and nutritional resources  , 11, 19.esources –22. The Synechococcus cyanobacteria supports this notion, where a coastal strain was found to have a more dynamic proteome response to iron scarcity than an oligotrophic strain  higher RR/HPK ratios, (ii) many orphan TCS genes, and (iii) more hybrid histidine kinases than bacteria examined in a reference data set. We discuss each of these observations in turn.Marine bacteria have significantly higher response regulator:histidine kinase ratios (RR:HPK) than bacteria as a whole, suggesting that they have extra response regulator genes. By considering the RR/HPK ratio, we found that approximately 1 in 50 response regulator genes lacks a histidine kinase partner . The ori28–Pelagibacter ubique), suggesting that they play important biochemical roles. The nonmodularity of TCS systems in marine bacteria suggests that the genes are not acquired through HGT but are instead acquired through gene duplication and genetic remodeling  proteins before it reaches a terminal response regulator . The add signals . Hybrid  signals . Marine  systems , concurr systems .Trichodesmium cells  than the Cyanobacteria (0.57 per 100 protein-encoding genes on average)   and C. Tlability –48. ReflProteobacteria and Cyanobacteria  have not been characterized.Notably, the diazotrophic organism el cycle . HoweverProchlorococcus/Synechococcus PhoB-like response regulator protein, PMT9312_0717, in the tropical Pacific Ocean. PMT9312_0717 is one of the most abundant TCS system proteins in this transect. It is an orphan, and its partner histidine kinase is not known. In the metaproteomics analysis, we found that the protein abundance is inversely correlated with inorganic phosphate concentration, particularly at concentrations below 1 μM phosphate such as are encountered near the nutricline  and the analogous Synechococcus sp. WH8102 PhoP protein    but is aTCS systems underpin many of the physiological changes observed in laboratory and field perturbations of marine bacteria. They are involved in nutrient acquisition, detoxification, quorum sensing, and other topical themes in marine microbiology. However, our knowledge of these systems in marine species is limited. Sequence-based identification of TCS systems is difficult because histidine kinases and response regulators share highly conserved catalytic domains. Thus, while it is possible to identify TCS genes, identifying their physiological functions is tricky. Few two-component systems have been experimentally verified in marine species, despite the fact that just a few amino acid substitutions can drastically change physiological function .phoR-phoB two-component genes in Prochlorococcus, while initially hypothesized to be correlated to phosphate availability, cannot be consistently linked to large-scale oceanographic patterns . The. The8). We identified TCS system genes by protein family (pfam) domain annotations . The pfaHistidine kinases are composed of two domains—an HATPase domain and a phosphoacceptor domain—which represent separate entries in the pfam domain database. We tested both the HATPase and phosphoacceptor domains by comparing the number of histidine kinases identified with the results of an extensively curated previously published survey of TCS genes in bacteria . We founResponse regulators are composed of a phosphoreceiver domain (sometimes known as a REC domain) and an output domain. The output domain can vary significantly and determines the biological function of the response regulator , 54. We Hybrid histidine kinases occur when the response regulator phosphoacceptor domain is present on the same protein as the histidine kinase sensory and phosphoacceptor domains. We define them here as genes containing both a HATPase domain  and the response regulator phosphoreceiver domain (pfam00072). Thus, the hybrid histidine kinases are present in both the histidine kinase data and response regulator data for a given genome.https://github.com/naheld/patterns_TCS_sensing_marine_bacteria. A fully executable cloud environment is provided courtesy of the Binder project (https://mybinder.org/). We used the Bokeh (https://bokeh.pydata.org/en/latest/) and seaborn (https://seaborn.pydata.org/) libraries to generate visualizations and perform statistical analyses. For ratio and statistical analyses, genomes containing 0 histidine kinases and/or 0 response regulators were excluded.All analyses were conducted in Python 3.6. The entire data analysis and visualization pipeline, including statistical analyses and machine learning algorithms, can be recreated by accessing the scripts at For K means clustering analyses, we counted the number of histidine kinases, including hybrid genes, and normalized the data by dividing by the number of protein-encoding genes. For human readability, we express this value as the number of histidine kinases per 100 genes. To eliminate the effect of scaling, we subjected the data to unit normalization. We then performed the clustering analysis to identify groups of genomes with similar signaling repertoires in relation to genome size. We used the Silhouette method implemented in the Scikit Learn Cluster module to select the optimal number of clusters  by maximThe locations of TCS genes in the genomes of marine bacteria were examined by plotting the gene starting locations on a linearized depiction of the circular bacterial genome (a number line). The average gene size was calculated for each genome examined. A gene was considered to be an orphan if its start point was farther than four average gene lengths from another TCS gene.in situ particle collection pumps (McLane Labs) on the KM1128 METZYME research expedition. Proteins from the 0.2-uM to 3-uM size fraction were subjected to SDS detergent extraction and in-gel trypsin digestion as previously described (We examined the distribution of response regulator PMT9312_0717 in marine metaproteomes from the METZYME expedition in the tropical Pacific. Analysis of these metaproteomes has been previously described . Brieflyescribed . Peptideescribed . Spectraescribed . Genes wescribed . Peptideescribed . Two uniescribed .


Colorectal cancer is a very frequent sort of neoplasm among the population, with a high mortality rate. It develops from an association of genetic and environmental factors, and it is related to multiple cell signaling pathways. Cell cultures and animal models are used in research to reproduce the process of disease development in humans. Of the existing animal models, the most commonly used are animals with tumors induced by chemical agents and genetically modified animals. To present and synthesize the main animal models of colorectal carcinogenesis used in the research, comparing its advantages and disadvantages. This literature review was performed through the search for scientific articles over the last 18 years in PubMed and Science Direct databases, by using keywords such as “animal models”, “colorectal carcinogenesis” and “tumor induction”. APC, p53 and K-ras genes. Animals with the APC gene mutation develop colorectal neoplasms, whereas animals with p53 and K-ras genes mutations are able to potentiate the effects of the APC gene mutation as well as the chemical inducers. 1,2-dimethylhydrazine and azoxymethane are carcinogenic agents with high specificity for the small and large intestine regions. Therefore, the two substances are widely used. Concerning the genetically modified animal models, there is a larger number of studies concerning mutations of the Each animal model has advantages and disadvantages, and some are individually efficient as to the induction of carcinogenesis, and in other cases the association of two forms of induction is the best way to obtain representative results of carcinogenesis in humans. Cancer is a sort of disease that can reach most of the organs and tissues of the human body. It is characterized primarily by the disordered growth of cells, often able to metastasize to other regions of the body. According to Petit et al.th most frequent neoplasm among the world’s population, accounting for 694,000 of the 8.2 million cancer deaths in 20122,14 and it is related to multiple signal transduction cascades, important for different types of biological response such as angiogenesis, apoptosis and cell proliferation,Colorectal cancer (CRC) is the 4APC (adenomatous polyposis of the colon) tumor suppressor gene, whereas those who develop hereditary nonpolyposis colorectal cancer have mutations in genes involved in DNA repair and mismatch repair (MMR) genes,CRC may be hereditary or sporadic, accounting for 80% of all patients affected by the disease. The hereditary form is related to two familial syndromes, familial adenomatous polyposis, in which the appearance of multiple intestinal polyps, and hereditary nonpolyposis colorectal cancer is observed. Individuals who develop familial adenomatous polyposis have a mutation in the For studies related to the development, treatment and prevention of colon and rectum tumors, animal models or cell cultures, which are representative of a carcinogenic situation in humans, are used. However, in spite of all the ethical conflicts involved in animal experimentationFor research purposes, the two major animal models of colorectal carcinogenesis are colorectal tumors induced by chemical or environmental agents in rodents, which represent sporadic CRC, and genetically modified mice, which represent the hereditary familial adenomatous polyposis and hereditary non-polyposis colorectal cancer syndromesThis review aims to present and synthesize the main animal models of colorectal carcinogenesis used in the research, comparing its advantages and disadvantages.For the development of this review, a search for scientific articles over the last 18 years in the PubMed and Science Direct databases was carried out, using keywords such as “animal models”, “colorectal neoplasias” and “induced colon cancer”. The search resulted in 89 articles, of which 31 were selected as the most relevant for this review. APC-/+ mutation and 57% the Muc2-/- mutation, which are important in CRC development. However, only 27% of them had alterations in the intestinal mucosa.The two studies showed that diet can directly influence the appearance of colorectal neoplasias, as it promotes reprogramming of intestinal cells, thus representing the spontaneous colorectal cancer model in humans. However, although the authors observed different intestinal neoformations in the animals treated with the hyper caloric diet, it was not possible to evaluate which mutations were responsible for the development of the tumors. Also, the diet may not be considered the best model of colorectal carcinogenesis, since the percentage of animals that develop the neoplasia is small, and the time for this to occur is long.According to Andrade et al. Besides the diet, chemical substances with carcinogenic potential are also used. The two most commonly used tumor inducers in animal models for induction of sporadic CRC are azoxymethane (AOM), which is a direct inducer, and 1,2 dimethylhydrazine (DMH), which is an indirect inducer of carcinogenesis. They are able to represent the mechanisms of development of CRC that occur naturally in humans, being very useful models in studies that aim to study chemopreventive and chemotherapeutic effects of other substancesDMH is a pro-carcinogenic agent for colon cancer, it is activated in the liver and transported to the intestine by bile and blood. Its use promotes the production of free radicals, which are responsible for causing oxidative damage to the DNA of colon and liver cellsAccording to Burlamaqui et al.In 2014, Lahouar et al.Bifidobacteria and an increase in E. coli and C. perfringens species.Aachary et al.Umesalma and SudhandiranDMH was also effective in CRC induction in a study by Youssef et al.Both substances are used for CRC induction in rodents. However, there is controversy over which of the two is the most effective in tumor induction. Burlamaqui et al.Since AOM is an active metabolite of DMH, the mechanism of induction of carcinogenesis promoted by the two substances is very similar. In the studies mentioned, the formation of aberrant crypts foci and intestinal inflammatory processes was observed in the animals treated with both DMH and AOM. However, DMH seems to be a more effective agent because in addition to the pre-neoplastic and inflammatory lesions, it also promoted the development of intestinal adenomas and carcinomas. Thus, from the models induced by exogenous agents it can be concluded that all agents, diet, AOM and DMH are able to induce CRC. But the diet proves to be a slow and low-efficiency model; the AOM is an inducer of mainly aberrant crypts foci and inflammation, and DMH is an inducer of colorectal tumors in more advanced stages, showing to be the most efficient model.APC, DCC , p53 (gene encoding tumor protein p53) and MCC , the oncogenes K-ras, SRC(proto-oncogene SRC) and C-myc , the DNA repair genes hMSH2 (mutS 2 homologue), hMSH6 (mutS 6 homologue), hMLH1 (mutL 1 homologue), hPMS1 (BstNIproline rich protein subfamily 1) and hPMS2 (BstN1 proline rich protein subfamily 2), in addition to CD44 genes (gene encoding CD44 molecule) and COX-2 . Each of them acts differently for the development of colorectal neoplasms, and mutations in two or more of these genes are often related to the malignancy profile of the tumorsMany genes are involved in colorectal carcinogenesis, including tumor suppressors APC> 70%, p53> 60% and K-ras> 40%) and in hereditary familial adenomatous polyposis(APC) and hereditary nonpolyposis colorectal cancer(MMR)Thus, there are innumerable genetically modified animal models that have been developed from the knowledge about the genetic factors involved in the development of the diseaseAPCmin animals are genetically modified that have a mutation in the APC gene. The acronym “min” means multiple intestinal neoplasms, and this is an autosomal dominant mutation, which in homozygous conditions is lethal to animals. Animals that are heterozygous for the mutation develop important anemic conditions at 60 days of life and develop tumors in the large and small intestine. As in familial adenomatous polyposis cases, APCmin animals also develop colorectal adenomas, but they die at 120 days of lifep53 gene rarely develop colorectal tumors. Reed et al.p53 knockout animals did not develop colorectal neoplasias; however, the association of APCmin and p53 knockout mutations promoted an increase in aberrant crypts foci number when compared to APCmin animals. In addition, in 2008 Hu et al.p53 knockout animals with tumor inducer AOM was efficient in inducing carcinogenesis in the colon of the animals, and also to potentiate the action of the AOM. The same could be observed by Sakai et al.p53 knockout animals showed only neoplastic development when placed in contact with the DMH inducer.Knockout animals for APCand p53genes, mice with mutations in codon 12 of the K-ras gene are also used. From a homologous recombination process, one of the K-ras gene alleles is replaced by a mutated K-rasG12D allele in which there is the substitution of a guanine with an adenine at the second base of codon 8. According to Haigis et al.K-rasG12D and APCmin mutations, Calcagno et al.In addition to animals with mutations in the K-rasv12 mutation, in which the substitution of a guanine for a thymine occurs. In 2007, Luo et al.K-rasv12 mutation alone is not capable of inducing tumorigenesis, but once it is associated with mutations in repair genes, such as the MSH2 gene, it promotes and accelerates tumor development. K-rasv12/Msh2-/- animals presented a greater number of tumors, both in the small and in the large intestine, than Msh2-/- animals: ranging from 1.41 to 7.75 tumors per mouse in the small intestine and from 0.13 to 2.7 tumors per rat in the large intestine.Another mutation found in colorectal tumors is the MLH1+/- and 13/18 (72%) of MLH1-/- mice developed tumors, against 1/20 (5%) mice without the mutation. In addition, they also observed that the survival of animals was lower in MLH1-/- animals than in MLH1+/-, 7 and 9.8 months, respectively,These mutations in DNA repair genes are representative of hereditary nonpolyposis colorectal cancer. According to Edelmann et al.APCmin animals, among which the formation of intestinal tumors is observed. The other mutants only become representative models when an association of two or more types of mutation is made, or through the association of a mutation with induction by an exogenous agent, such as diet, for example. All models presented, both induced by exogenous agents and those genetically modified, have advantages and disadvantages (APCmin animals, are more suitable for research because of their ability to develop colorectal neoplasias, without the need to associate two or more induction methods.Among the genetically modified models, the one that best represents the hereditary CRC are the vantages  for the  Animal models are important for studies of the development and pathogenesis of colorectal tumors, as well as for the evaluation of possible risk factors, preventive agents and treatments. As shown, some individual models are representative of CRC in humans and considered to be good animal models for this type of study. However, it is noted that the association of at least two methods of colorectal carcinogenesis induction is required for most models. This is due to the fact that colon cancer is a multifactorial disease that develops by the presence of multiple factors, both genetic and environmental.Each animal model has advantages and disadvantages, and some models are individually efficient as to the induction of carcinogenesis, and in other cases the association of two forms of induction is the best way to obtain representative results of carcinogenesis in humans.


Although stereotactic radiotherapy (SRT) for vestibular schwannoma has demonstrated excellent local control rates, hearing deterioration is often reported after treatment. We therefore wished to assess the change in hearing loss after SRT and to determine which patient, tumor and treatment-related factors influence deterioration.2. Primary outcomes were loss of functional hearing, Gardner Robertson (GR) classes I and II, and loss of baseline hearing class. These events were used in Kaplan Meier plots and Cox regression. We also calculated the rate of change in Pure Tone Average (PTA) in dB per month elapsed after radiation—a measure we use in linear regression—to assess the associations between the rate of change in PTA and age, pre-treatment hearing level, tumor size, dose scheme, cochlear dose, and time elapsed after treatment (time-to-first-audiogram).We retrospectively analyzed progression of hearing loss in patients with vestibular schwannoma who had received stereotactic radiosurgery (SRS) or fractionated stereotactic radiotherapy (FSRT) as a primary treatment between 2000 and 2014. SRS had been delivered as a single fraction of 12 Gy, and patients treated with FSRT had received 30 fractions of 1.8 Gy. To compare the effects of SRS and FSRT, we converted cochlear doses into EQDThe median follow-up was 36 months for 67 SRS patients and 63 months for 27 FSRT patients. Multivariate Cox regression and in linear regression both showed that the cochlear V90 was significantly associated with the progression of hearing loss. But although pre-treatment PTA correlated with rate of change in Cox regression, it did not correlate in linear regression. The time-to-first-audiogram was also significantly associated, indicating time dependency of the rate of change. None of the analysis showed a significant difference between dose schemes.We found no significant difference between SRS and FSRT. As the deterioration in hearing after radiotherapy for vestibular schwannoma was associated with the cochlea V90, restricting the V90 may reduce progression of hearing loss. The association between loss of functional hearing and baseline PTA seems to be biased by the use of a categorized variable for hearing loss. An increasing number of studies have shown excellent local control after stereotactic radiotherapy (SRT) for vestibular schwannoma. –3. ThereWhile progression of hearing loss is often reported after SRT –9, thereAnother area of uncertainty is whether FSRT yields better hearing preservation rates than SRS. A recent systematic review of clinical trials concluded that the evidence for better hearing preservation in vestibular schwannoma patients treated with FSRT is inconclusive . This maMany studies have analyzed hearing loss with a time-to-event analysis, using the preservation of functional hearing or preservation of the baseline hearing class as outcome variable. The hearing classifications used most commonly are the Gardner Robertson (GR) classification, and the classification of the American Academy of Otolaryngology – Head and Neck Surgery (AAO-HNS). Patients retaining GR class I or II, which correspond to AAO-HNS classes A or B, are considered to have preserved functional hearing. The reported rates of long-term functional hearing preservation after irradiation differ widely, from 31 to 94% [Although the term functional hearing is an attractive concept, the limits of audiological functioning are not well defined by the limits of GR class I and II. Even when hearing loss exceeds 50 dB hearing may still be serviceable in the presence of good speech perception, but a drop in speech discrimination below 70% impedes audiological function at any level of pure tone average (PTA).As better identification of factors that influence hearing loss would help physicians to optimize treatment planning and to achieve better hearing outcomes, we wished to identify factors that influence the rate of change in hearing after SRT. Hypothesizing that associations predicting the change in hearing after SRT might be identified through the use of continuous variables such as PTA rather than a dichotomous variable such as functional hearing classes, we used the change in PTA over time in our analysis, alongside the time-to-event analysis to analyze preservation of functional hearing class and preservation of GR baseline class.The purpose of our study was to assess the change in hearing after SRS and FSRT in vestibular schwannoma patients, and to investigate patient, tumor, and treatment-related factors that influence hearing loss.This study was performed as a retrospective, single-institution study. The study protocol was approved by our local Medical Ethics Review Board.We reviewed all patients with unilateral vestibular schwannoma who had been treated with stereotactic radiotherapy at the Department of Radiation Oncology at Erasmus University Hospital between 1 January 2000 and 31 December 2014. The diagnosis of vestibular schwannoma had been based on the clinical presentation and the typical radiological appearance of a cerebellopontine angle (CPA) tumor upon gadolinium-enhanced magnetic resonance imaging (MRI). We excluded the following patients: those who had had previous interventions for vestibular schwannoma, those with neurofibromatosis type 2, and those who had not been audiometrically assessed less than 6 months before treatment. Unlike many other studies, we included all patients who had had measurable hearing before treatment; this enabled us to investigate the effect of treatment irrespective of pre-treatment hearing. We defined measurable hearing as PTA ≤ 100 dB and Speech Discrimination Score (SDS) > 0%, which corresponds to GR classes 1 through 4. Ninety-four patients of the 225 who had been treated were eligible for inclusion.All patients had been discussed in a multidisciplinary tumor board attended by a radiation oncologist, a neurosurgeon, an otolaryngologist and a specialized nurse practitioner. Treatment with SRT had been recommended for tumors touching or displacing the brain stem or cerebellar peduncle, and for tumors that progressed during wait and scan policy. The choice of technique had then been based on tumor size. Tumors extending up to 3 cm in the CPA cistern were preferably treated with SRS, whereas larger tumors were treated with FSRT. In the early years of the study period, FSRT was preferred to SRS for patients with functional hearing, independent of tumor size. Later on, this practice was abandoned on the basis of new publications , 6, 18, Pure tone and speech audiometry had been performed in all patients less than 6 months before treatment and up to 10 years after its completion. We calculated PTA on the basis of the masked bone conduction responses at 500, 1000, 2000 and 4000 Hz. As the maximum phoneme score of “Consonant Vowel Consonant” words in percentages had been used for the SDS, we then classified according to the GR hearing classification . GR hearDuring treatment, patients receiving SRS had been were immobilized using the invasive Brown-Roberts-Wells frame , and with the relocatable Gill-Thomas-Cosman frame (Radionics) for FSRT. Until March 2014, treatments were delivered with a conventional LINAC  using 6 MV photons. From March 2014 onwards, SRS treatments were delivered with Cyberknife  using a thermoplastic mask for immobilisation.Using the immobilization system, a computerized tomography (CT) planning scan was acquired. These images were fused with MRI and this dataset was used for target delineation. The planning target volume (PTV) was defined as the area of contrast enhancement on T1-weighted MRI with no margin for SRS and with 2 mm margin for FSRT. The organs at risk considered were cochlea, brainstem, trigeminal nerve, optic nerves, optic chiasm, pituitary gland, and eyes. The cochlea was delineated on CT. The method of delineation was discussed with a neuroradiologist. All delineations were performed and reviewed by two of the authors . For single-dose treatments, a dose of 12 Gy was prescribed at the 80% isodose surrounding the PTV. For fractionated treatments, a dose of 54 Gy was prescribed at the 100% isodose with the 95% isodose surrounding the PTV. FSRT was delivered in 30 daily fractions of 1.8 Gy over a period of 6 weeks.After treatment, patients were followed with MRI, audiometry and clinical evaluation. A post-treatment MRI and audiogram after SRT were planned at 12 months and thereafter at 2, 3, 5, 7 and 10 years. Not all patients complied with the full follow-up schedule.In the current study, our primary outcome was the progression of hearing loss after treatment. We compared three different ways to define hearing deterioration. First, deterioration was defined in the usual way, as loss of functional hearing (baseline GR classes I or II). Next, it was defined as loss of the baseline GR hearing class (class GR I to IV). In our third analysis, hearing loss was not categorized, but progression was measured as the rate of change in PTA in dB per month. This rate was calculated by dividing the difference between the PTA of the pre-treatment audiogram and each post-treatment audiogram by the number of months that had elapsed since SRT.We evaluated the relationship between progression of hearing loss with patient, tumor, and treatment-related factors: age, pre-treatment hearing level, tumor volume, tumor diameter in the CPA, fractionation scheme, and cochlear doses.2) [To compare SRS and FSRT, we use the linear quadratic formula to convert cochlear doses into equivalent 2-Gy-fraction doses (EQD2) :2 is the dose in 2-Gy-fractions that is biologically equivalent to the total dose D that was actually administered in fractions of d Gy [2. Next, we sought to establish the equivalent dose delivered with SRS. We could then calculate the volume of the cochlea that had received this EQD2 in both the FSRT and SRS treatments.where EQD of d Gy . The α/β of d Gy . We measOne-way ANOVA analysis was used to evaluate the differences between the FSRT and the SRS populations. The relationship between the change in PTA and the change in SDS was tested with the Wilcoxon Signed Rank test.After categorizing hearing according to the Gardener Robertson grading, we used the Kaplan Meier method to assess the preservation of useful hearing and the preservation of baseline hearing class. Factors that could be predictive of an increase in GR class were evaluated with the Cox proportional hazard model for univariate and multivariate analysis.p-value. Relationships with p < 0.05 were considered to be significant.The change in PTA is a continuous outcome measure that allows univariate and multivariate linear regression analysis of the monthly rate of PTA increase. We used the step-backward method to optimize both multivariate models, this method removes factors from the model that are not significant, until only significant factors remain. The factors removed are rated non-significant, without reporting a hazard ratio (HR), regression coefficient (RC) or We performed all analyses using the statistical programs R (version 2.13.0) and IBM SPSS (version 23).Between 01 January 2000 and 31 December 2014, 225 patients had been treated for vestibular schwannoma with either technique. Ninety-four patients fulfilled the criteria for inclusion in this study. Patients were excluded due to the absence of an audiometric assessment taken less than 6 months before treatment or due to a pre-treatment speech-discrimination score equal to zero.Sixty-seven patients had been treated with SRS and 27 with FSRT. Our treatment algorithm caused differences between the two groups Table . Patientr = − 0.752, p < 0.0001).Figure Categorization of hearing according to the Gardner Robertson classification showed that the patients’ distributions over the hearing classes differed between dose schemes. Table Before radiotherapy, 57 out of 94 patient had had hearing class I or II. Preservation of GR hearing class I or II had not differed significantly between the two treatment groups Fig. , Table 2Univariate Cox regression showed that loss of functional hearing was significantly associated with the V90, pre-treatment PTA, and age. After multivariate regression, pre-treatment PTA and V90 remained significant factors.Preservation of baseline GR class did not differ significantly between the two treatment groups Fig. . At 1 yeUnivariate Cox regression showed a significant association of loss between baseline GR hearing class and the V90. Pre-treatment PTA was significantly associated with loss of baseline hearing class only after multivariate regression. Age did not show an association.Figure To compare results between patients despite the different follow-up times, we included the time elapsed after treatment as a variable in the analysis. This was useful, as the follow-up schedule had not been adhered to for all patients. As the greatest change occurred shortly after treatment, we only included patients whose first audiogram had been taken within 2 years of follow up. As the date of audiometry clustered around the scheduled follow-up dates, we used 21 months as our cut-off for inclusion in this analysis, i.e., between the 18-month and 24-month follow up. Seventy-seven patients had had an audiometric assessment within the first 21 months . This sustains our hypothesis that the rate of hearing deterioration decreases over time after treatment—a finding that is in agreement with others, who also described a rapid decline during the initial period after treatment  was intended to make the different fractionation schedules comparable between the groups, readers interpreting our results should remember that this is just a model.The groups of patients who had received FSRT and SRS differed in several respects, such as tumor size, PTV margin and dose distribution. The two latter factors may influence the V90, specifically when the intracanalicular tumor component is located close to the cochlea. As our use of the linear-quadratic approach , we aimed to make the two schemes we had applied comparable. When interpreting these results, it should be realized that this approach has some limitations. For large fraction doses such as 12 Gy, the calculations may be less certain than for low doses per fraction. Another point that deserves special attention is the α/β selected to perform the EQD2 calculations. While the value of 2 was chosen on the basis of the published literature, it could also be argued that 3 could be also have been used, and that these different values may have had some small impact on the results . Last buOur results confirm that hearing deteriorates after SRT for vestibular schwannoma. In our patient population, the most rapid decline had occurred shortly after treatment.We found that the effects of FSRT and SRS on hearing change had not differed significantly.The cochlear V90 was associated with progression of hearing loss. Restricting the V90 during radiotherapy treatment planning may help to reduce progression of hearing loss.Better baseline hearing does not protect against changes in PTA. Although a protective effect may be apparent when hearing was evaluated dichotomously, it was not observed when the PTA was evaluated as a continuous variable.


The aim was to develop a diagnostic questionnaire for damp phlegm pattern and blood stasis pattern in coronary heart disease patients (CHD-DPBSPQ).  The standard procedures of questionnaire development were carried out to develop and assess CHD-DPBSPQ. The patients were assessed using the CHD-DPBSPQ, CHD-DPPQ, and CHD-BSPQ. Four methods were used to select the items on the CHD-DPBSPQ in a pilot study based on data from a Guizhou tertiary grade A hospital. Cronbach's alpha and the split-half reliability, test-retest reliability, content validity, criterion validity, construct validity, and convergent validity were determined in a validation study using a nationwide sample. P < 0.01). The root mean square error of approximation (RMSEA) was equal to 0.05 . Convergent validity was demonstrated with a moderate correlation.  After item selection, the CHD-DPBSPQ contained 15 items in two domains: the phlegm domain (9 items) and the blood stasis domain (6 items). For the CHD-DPBSPQ, the alpha coefficient was 0.88, the split-half coefficient was 0.90, and the intraclass correlation coefficient was 0.83. The range of the item-level content validity index (I-CVI) was 0.71 to 1.0 and that of the scale-level content validity index/average  was 0.97. The domain scores on the CHD-DPBSPQ were in close relation to the scores on a questionnaire for damp phlegm pattern in coronary heart disease patients (CHD-DPPQ) and a questionnaire for blood stasis pattern in coronary heart disease patient (CHD-BSPQ) ( The CHD-DPBSPQ is a reliable and valid instrument. Currently, coronary heart disease (CHD) is the most common type of cardiovascular disease (CVD) and is a major cause of death and disability among adults worldwide , 2. CHD Damp phlegm pattern and blood stasis pattern (DPBSP) is commonly found in CHD patients. Along with changes in lifestyle habits, DPBSP is becoming increasingly common –9. TurbiTCM syndromes can be studied using measures such as scales. In recent years, some researchers have developed diagnostic questionnaires for CHD with stable angina (syndrome involving both phlegm and blood stasis) –15. HoweTherefore, we aimed to develop and assess a damp phlegm pattern and blood stasis pattern questionnaire for patients with coronary heart disease (CHD-DPBSPQ) based on a series of standard and systematic procedures of instrument development.To develop the CHD-DPBSP diagnostic questionnaire, we used standard procedures for developing and validating the CHD-DPBSPQ –29. The All the items on the CHD-DPBSPQ were drafted from systematic reviews, the Delphi method and analytic hierarchy process (AHP) –33. UltiA pilot study was adopted to identify items for the CHD-DPBSPQ. Recruited from a Guizhou tertiary grade A hospital, all of the CHD patients with DPBSP provided informed consent prior to their participation. The eligibility criteria are as follows: (1) diagnosis of CHD according to the Chinese guidelines for diagnosed with CHD based on the Chinese guidelines for the diagnosis and treatment of patients with chronic stable angina published in 2007 ; (2) diaFour methods were employed to select the items for the CHD-DPBSPQ: (1) the coefficient of variation: if the standard deviation (SD) of every item was less than 0.9, the item was discarded; (2) EFA: if the correlation coefficient was less than 0.4 after factor rotation, the item was deleted; (3) alpha reduction: if Cronbach's alpha coefficient was apparently increased if one item was removed, that item was marked; and (4) correlation analysis: if the item had a proportion less than 0.4, the item was abandoned –39. If aA multicentre dataset was used to assess the CHD-DPBSPQ. Patients from Guangdong Provincial Hospital of Traditional Chinese Medicine, Hunan University of Traditional Chinese Medicine, Hubei Provincial Hospital of Traditional Chinese Medicine, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Guizhou Provincial Hospital of Traditional Chinese Medicine, Second Affiliated Hospital of Wenzhou Medical University, Changzhou City Hospital of Traditional Chinese Medicine, and Tianjin University of Traditional Chinese Medicine were enrolled. All patients provided informed consent for participation. The inclusion criteria were as follows: (1) diagnosis of CHD on the basis of the Chinese guidelines for the diagnosis and treatment of patients with chronic stable angina published in 2007 ; (2) diaTwo TCM doctors with over 20 years of experience in CVD performed the assessments, and the doctors explained the questionnaire to each patient. The questionnaire included a demographic portion, followed by the CHD-DPBSPQ, CHD-DPPQ, and CHD-BSPQ. The demographic portion collected data on age, sex, ethnicity, marital status, occupation, and other diseases. The CHD-DPPQ and CHD-BSPQ were used to evaluate the criterion validity. The CHD-DPBSPQ was used for diagnosis by two experienced TCM doctors, and these two doctors could independently differentiate between the syndromes. If their diagnoses were inconsistent, diagnosis was made by a third doctor (associate professor or higher). Terwee et al.  believedSPSS version 22.0 and Amos software 22.0 were used for data analysis , 42. Thiα. After the arrangement of odd and even numbers, split-half reliability was calculated using Pearson's correlation coefficients. Test-retest reliability was assessed by adopting the intraclass correlation coefficient (ICC) and its 95% confidence interval.Internal consistency reliability, split-half reliability, and test-retest reliability were all evaluated , 43. TheValidity was evaluated as content validity, criterion validity, construct validity, and convergent validity. The content validity of the questionnaire was assessed using the item-level content validity index (I-CVI) and the scale-level content validity index (S-CVI). When more than 6 experts assessed this questionnaire, the value of the I-CVI was not less than 0.78 –46. We hUltimately, 103 CHD patients with DPBSP were involved in the pilot study. The age of the patients ranged from 46 years to 91 years , namely, taut and slippery pulse, viscous stool, wiry pulse, uneven pulse, and taut and uneven pulse. Thus, CHD-DPBSPQ ultimately contained 15 items in 2 domains—the phlegm syndrome domain and the blood stasis syndrome domain. (1) The phlegm syndrome domain included chest distress, sleepiness, physical heaviness, obesity, sticky mouth, abdominal fullness, anorexia, greasy tongue fur, and slippery pulse. (2) The blood stasis syndrome domain included chest pain, cyanotic lips, dim complexion, dark purple tongue, petechiae or ecchymosis on the tongue, and sublingual vein cyanosis.Of the 20 items, 5 items were removed from the original pool based on the selection criteria (N = 729) was measured for the first time, with 81 of the patients measured a second time.To further improve the demographic composition of the sample, data were collected from the clinical population of 8 hospitals in China. The demographic characteristics of the 729 total participants are shown in The CHD-DPBSP had high internal consistency and retest reliability. The mean score on the CHD-DPBSPQ was 17.4, the mean score on the phlegm domain was 9.7, and the mean score on the blood stasis domain was 7.7 . ① The CP < 0.01). ③ The model fit for the scale was tested using CFA   and the Previous instruments for the assessment of CHD-DPBSP have not been widely adopted; our group formulated and validated TCM outcomes on the basis of standard development and validation procedures , 28, 40.α coefficients (0.77–0.88) and split-half reliability coefficients (0.81–0.90). Terwee et al. considered that the Cronbach's α showed a good internal consistency range, from 0.70 to 0.95 [The 15 items on the CHD-DPBSPQ were empirically selected by four methods using a sample population from a Guizhou tertiary grade A hospital. The CHD-DPBSPQ was verified with national data, showing that it is a reliable and valid tool for research and clinical trials. (1) The internal consistency of the domains was expressed by Cronbach's  to 0.95 , and the to 0.95 .K) was 0.65 (>0.60), which showed good validity [(2) The content validity of the questionnaire was evaluated by seven experts. A standard value of I-CVI greater than 0.78 was recommended by Lynn ; the scovalidity . The S-Cvalidity . The corvalidity . The RMSvalidity . The corvalidity . In shorThe purpose of our study is to develop and validate the CHD-DPBSPQ, a questionnaire intended for use in the clinical practice of TCM. However, the questionnaire is not applicable for diagnosing CHD and all TCM syndromes of CHD. Thus, the diagnosis of CHD was first confirmed in patients recruited for our study according to the Chinese guidelines for the diagnosis of CHD, which were based on the Chinese guidelines for the diagnosis and treatment of patients with chronic stable angina published in 2007 . The CHDThere were a few limitations. (1) The patients were retested again 1–7 days after the first investigation. Thus, test-retest reliability could have been overestimated due to the short time interval. (2) No pathological data were provided for the diagnosis of CHD-DPBSP. (3) The pilot study enrolled patients from a Guizhou tertiary grade A hospital. The CHD-DPBSPQ should be further assessed throughout every province in the country. (4) The gold standard adopted was the CHD-DPPQ and CHD-BSPQ developed in China, which do not include the internationally recognized scale for CHD.This questionnaire also has some strengths. First, compared with one published scale , our queA DPBSP questionnaire was developed for patients with CHD. The 20 items were successfully reduced to 15 items during the selection process, a quantitative stage of questionnaire development. The CHD-DPBSPQ showed good reliability and validity and is feasible for diagnosing DPBSP in CHD patients. We recommend the application of the CHD-DPBSPQ for diagnosing DPBSP in Chinese CHD patients.


ILDR1 gene has been identified in proband and in his younger brother. Sanger sequencing confirmed that proband’s father and mother are carrying this mutation in a heterozygous manner. This mutation has not been identified in 100 normal healthy control individuals. This mutation (c.427delT) causes frameshift (p.Tyr143Ilefs∗19) which leads to the formation of a truncated ILDR1 protein of 162 amino acids instead of the wild type ILDR1 protein of 546 amino acids. ILDR1 associated hereditary hearing impairment is very rare and this is the first report of identifying a loss-of-function mutation in ILDR1 gene associated with hereditary hearing impairment in Chinese population. Our present study also emphasized the significance of rapid, accurate and cost-effective screening for the patient with hereditary hearing impairment by targeted next generation sequencing.Hereditary hearing impairment is one of the major and common birth defects in Chinese population. Non-syndromic sensorineural hearing loss (NSHL) is the most common types of hereditary hearing impairment. Genotypically and phenotypically NSHL is extremely heterogenous and follow either autosomal dominant or autosomal recessive or X-linked mode of inheritance. Presently, 127 genes have been identified to be associated with both syndromic and (NSHL). Here, we studied a Chinese family with moderate and profound hearing impairment. The proband is a 30-year old Chinese man. The proband was born with normal hearing and at the age of 5-years, the proband was first noticed with hearing impairment. Gradually and progressively the proband was presented with loss of hearing in his both right and left ears at the age of 30 years. The clinical symptoms, age of onset or progression to loss of hearing was similar in both the proband and his younger brother. The proband’s parents are phenotypically normal and non-consanguineous. Clinical diagnosis of the proband and his younger brother has been done by classical pure tone audiogram (PTA). Computed Tomography (CT) found no abnormality in bilateral external ear, middle ear and inner ear. Targeted next generation sequencing was performed with a panel of 127 genes reported to be associated with hereditary hearing impairment. A novel homozygous single nucleotide deletion (c.427delT) in exon 4 of  GJB2, SLC26A4, MYO7A, OTOF, CDH23, TMPRSS3, and TMC1. Different populations identified with different founder mutations underlying the phenotype of ARNSHL (Hereditary hearing loss (HL) is the major and most common sensorineural disorders with an incidence of 1/1000 live birth world-wide . Howeverf ARNSHL .loss-of-function mutation of ILDR1 gene was causing very rare non-syndromic autosomal recessive deafness type 42 (DFNB42). ILDR1 gene is located in chromosome 3. ILDR1 gene is consisting of 8 exons. Human ILDR1 gene encodes the immunoglobulin-like domain containing receptor 1 (ILDR1), a predicted type 1 transmembrane protein. ILDR1 protein showed a tissue specific expression and it is highly expressed in prostate, testes, pancreas, and kidney tissues. However, in mouse and zebrafish, ILDR1 is playing a major role in the development of auditory hair cells, semicircular canal and tricellular tight junction  is really very challenging. So, we customize a gene panel and performed targeted next generation sequencing for the screening of the patients with hereditary non-syndromic sensorineural hearing loss SNHL; .ILDR1 gene. ILDR1 associated hereditary (HL) is a very rare disorder and only 20 mutations of ILDR1 gene has been reported so far. In Chinese population, we first report the “loss-of-function” mutation in ILDR1 gene causing hereditary (HL) with an autosomal recessive mode of inheritance. Our present study also describes the significance of targeted next generation sequencing for rapid, accurate and cost-effective approach for screening of the patient with hereditary hearing impairment.In our present study, we identified two patients with hereditary (NSHL). Targeted next generation sequencing and sanger sequencing identified a novel homozygous single nucleotide deletion in exon 4 of the Our present study included a Han Chinese family. The proband is a 30-year old man of non-consanguineous Chinese parents . ProbandClinical diagnosis of the proband and proband’s younger brother has been done on the basis of complete medical history, family history and detailed physical examination. Proband and his younger brother’s auditory test has been performed with otoscopic examination and pure tone audiometry (PTA) followed by the brainstem evoked response audiometry (BERA). In addition, hearing threshold of proband and his younger brother were evaluated or estimated by the air-conduction pure-tone average thresholds ranging from 250 to 8000 Hz. However, PTA, BERA and hearing threshold were also evaluated for proband’s father and mother and no abnormality was found.In PTA test, hearing level was categorized into five groups; viz., normal (<20 dB), mild (20–40 dB), moderate (41–70 dB), severe (71–90 dB), and profound (>90dB). Here, hearing threshold was calculated based on the average of the right and left ears. In order to understand the structural abnormality of the temporal bone, computerized tomography (CT) scan was performed. Vestibular function analysis was done by caloric stimulation.Proband and his family members denied to have any previous potential causes of acquired hearing loss. In addition, audiological history of this family has been obtained to understand the age of onset, gradual progression rate and presence of other related audiological symptoms. Four family members  underwent a clinical otorhinolaryngological examination. Moreover, tympanogram was performed and middle-ear associated causes of (HL) were excluded. In addition, other ophthalmological and pedo-neurological examinations was performed.This study was approved by the ethics committee of the Gansu Provincial Maternity and Child-care Hospital, LanZhou, 730050, China, in accordance with the recommendations of the Declaration of Helsinki. Written informed consent has been obtained from all the participant of this study in accordance with the Declaration of Helsinki.The proband  was presented with bilateral hearing loss and admitted to our hospital. Proband’s detailed and comprehensive medical report showed that there is no abnormality in vestibular organs. Caloric stimulation test also supported it. In contrast with other NSHL cases, here the proband’s speech perception ability was not impaired. PTA test result revealed that the hearing perception level were 60, 80, 90, 95, and 100 dB for the right ear and 70, 80, 85, 95, and 100 dB for left ear for stimulation at 0.125, 0.25, 0.5, 1, and 2 kHz frequencies, respectively, . ComputeThe proband’s younger brother  was presented with bilateral hearing loss and admitted to our hospital. He was not identified with any abnormality in vestibular organs. His speech perception ability was quite well. The PTA test for proband’s younger brother (II-2) revealed that the hearing threshold 55, 60, 85, 95, and 100 dB for the right ear and 60, 75, 85, 95, and 100 dB on the left ear at 0.25, 0.5, 1, 2, and 4 kHz frequency range, respectively, . The CT Both these patients are clinically diagnosed with (NSHL). The patient’s parents are phenotypically normal .ILDR1 locus. The procedure for preparation of libraries was consistent with standard operating protocols published previously (DNA samples obtained from the proband (II-1), his younger brother (II-2), his father (I-1) and his mother (I-2) were sequenced using target exome-based next-generation sequencing. Roche NimbleGen’s  custom Sequence Capture Human Array was used to designed to capture targeted sequence, covering all exons and flanking sequence (including the 100 bp of introns) of 127 genes which is associated hereditary hearing impairment . The deteviously . Accordieviously . Then, wILDR1 was used.Sanger sequencing has been used to validate this homozygous mutation identified by targeted next generation sequencing by using the following primers: F-5′-AGGCGCGGAGCCTTATGCCCAG-3′, R-5′-GAACCGGCCGCTTAGGGGCCGG-3′. The reference sequence; NM_175924 of ILDR1 gene (∗19) ILDR1 protein with 162 amino acids compared with the wild type ILDR1 protein of 546 amino acids. Sanger sequencing confirmed that proband’s younger brother also carrying this homozygous mutation. The family segregation analysis was performed by sanger sequencing and identified that proband’s father and mother also carrying the heterozygous ILDR1 c.427delT mutation. This mutation was not present in the Human Genome Mutation Database (HGMD) or in the 1000 Genomes database. This mutation was also not detected in 100 normal Chinese healthy control individuals. These findings lead us to suggest that this novel mutation found in the proband in this Chinese family may be the cause of the disease.Targeted next generation sequencing and Sanger sequencing identified a homozygous novel single nucleotide deletion (c.427delT) in exon 4 of DR1 gene . This muloss-of-function mutation in ILDR1 gene in both the proband and proband’s younger brother. This homozygous mutation in ILDR1 gene is inherited in the proband and his younger brother from their unaffected parents.In the present study, we identified a Chinese family with (NSHL). In this Chinese family, proband and proband’s younger brother were presented with(NSHL). Targeted next generation sequencing identified a novel homozygous ILDR1 associated NSHL is classified as Deafness, autosomal recessive, 42 (DFNB42). DFNB42 [MIM# 609646] is a very rare form of autosomal recessive hereditary hearing impairment  in pairment .ILDR1 associated ARNSHL is a rarest type of hereditary (HL). As it is inherited in an autosomal recessive manner, pathogenic mutation in ILDR1 gene is causing ARNSHL either in homozygous condition or with compound heterozygosity. In addition, it has been reported that majorly ILDR1 gene associated ARNSHL is predominantly occur in consanguineous families . Accession number: SB, CL, SD, and YN designed the project. JA, JY, XL, YW, YxW, and SC performed patient workup. XW, BX, GX, HL, SX, and QH were involved in the genetic analysis. SB, YN, CL, and SD drafted the manuscript. SB, YN, JA, and JY approved the final version to be published and agreed to be accountable for all aspects of the work.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.


Arsenic (As) pollution in drinking water is a worldwide health risk for humans. We previously showed hearing loss in young people who live in areas of As-polluted drinking water and in young mice orally treated with As. In this study, we epidemiologically examined associations between As levels in toenails and hearing in 145 Bangladeshi aged 12–55 years in 2014. Levels of As in toenails, but not those in urine, were shown to be significantly correlated with hearing loss at 4 kHz , 8 kHz  and 12 kHz  by multivariate analysis with adjustments for age, sex, smoking and BMI. Our experimental study further showed a significant association between As levels in inner ears and nails  in mice orally exposed to As, suggesting that As level in nails is a suitable index to assess As level in inner ears. Taken together, the results of our study suggest that As level in nails could be a convenient and non-invasive biomarker for As-mediated hearing loss in humans. Exposure to arsenic (As) via drinking water is a health risk for humans –3. PreviLevels of toxic elements in noninvasive biological samples including nails, hair and urine have been determined to investigate associations with hearing loss in humans , 14. A uIn our previous study using pure tone audiometry (PTA), an association of oral exposure to As via drinking water with hearing loss in young people was shown by multivariate analysis with adjustments for confounders including age, smoking, sex and BMI . HoweverIn experimental studies, oral exposure to As has been shown to cause several health risks including carcinogenesis  and cardIn this study, we epidemiologically and experimentally investigated whether As levels in nails are reliable as non-invasive indexes for As-mediated hearing loss in humans and whether As levels in nails are associated with As levels in inner ears in mice.2) and used definitions of underweight (< 18.5 kg/m2), normal weight (18.5–24.9 kg/m2) and overweight (≥ 25 kg/m2) set by the WHO , where SGtarget is a population mean SG [As levels in biological samples including toenails and urine were measured by inductively coupled plasma mass spectrometry  as described previously , 22, 23. mean SG . In thisU test and Steel-Dwass test were used to detect significant differences in hearing levels between the groups because the hearing levels were discontinuous variables. In multivariate analysis, binary logistic regression analysis was performed with adjustments for age, sex, smoking and BMI as confounding factors. We used the method in a previous study [All statistical analyses were performed by JMP software (version 11.0.0). In univariate analysis, the Mann-Whitney us study  to categIn our experimental study, Spearman’s correlation coefficients were used to evaluate the association between As levels in inner ears and those in nails. Values of p < 0.05 were considered statistically significant.Concentrations of As (mean ± SD) in toenails and urine were 1.38 ± 1.17 μg/g and 90.27 ± 103.04 μg/L, respectively . In thisWe next performed logistic regression analysis with adjustments for age, sex, smoking history and BMI to determine the risk of hearing loss in subjects with high As in biological samples . In thisWe finally performed an experimental study to determine the correlation between As levels in nails and inner ears in mice. After exposure of mice to As via drinking water for 2 months, we measured As levels in nails and inner ears from the exposed group and the control group. We found that there was a significant correlation  between As levels in inner ears and nails .In our epidemiological study, As levels in toenails were shown to be significantly correlated with hearing loss at 4, 8 and 12 kHz in humans by multivariate analysis with adjustments for age, sex, smoking and BMI. Our experimental study also showed that As level in nails is a reliable index to assess As level in inner ears. Thus, the results of our combined study suggest that As level in toenails is a possible biomarker associated with hearing loss at 4, 8 and 12 kHz in humans.The levels of an element in nails and hair are indexes reflecting chronic exposure status, while those in blood and urine samples are regarded as indexes of acute exposure , 33. In The association between As levels in hair and hearing loss in humans aged 12–55 years was also analyzed in this study . As leveR2 values [Age and smoking are known to be strong confounders for hearing loss. In this study, multivariate analysis showed that age was significantly associated with hearing loss at 4 kHz , 8 kHz  and 12 kHz . Smoking also had significant associations with hearing loss at 4 kHz  and 12 kHz . We then used the McFadden’s Pseudo 2 values  to deter2 values . In thisThe major route of exposure to As in the subjects in this study is not clear, but in our experimental study in which mice were orally exposed to As, there was a correlation between As levels in nails and inner ears. In our epidemiological study, we found a significant correlation  between As levels in toenails and duration of drinking tube well water . TherefoThis pilot study has several limitations. First, we used an ipod with headphones as the screening method for the field work in Bangladesh, since there is no clinical audiometer in rural areas. In addition, we did not perform otoscopy and tympanometry to check middle-ear problems. Second, our cross-sectional analysis was useful for determining the association between As levels in biological samples and hearing loss, but a the causal relationship could not be established. Cohort studies will be needed to determine the causality. Third, there was no information about noise background, though the subjects lived in rural areas and did not have portable music players with earphones. Fourth, the sample size of the pilot study in Bangladesh was small. Additional study is needed to analyze the association between As and hearing loss with consideration of the above limitations in larger sample sizes in other areas.In conclusion, our combined experimental study and epidemiological study showed that As levels in nails were significantly associated with hearing loss in humans and that As levels in nails were significantly associated with those in inner ears in mice. Our epidemiological study also suggests the possible thresholds of As ranging from 0.30 μg/g to 0.70 μg/g in toenails that increase the risk for hearing loss in humans. Exposure to As is a worldwide health risk for humans. Our study provides new information that As level in toenails is a reliable index to predict As-mediated hearing loss in humans living in As-polluted areas.S1 Supporting Method(DOC)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(DOC)Click here for additional data file.S1 FigCorrelation between As levels in toenails and duration of drinking tube well water (years) was determined by spearman correlation coefficients.(TIF)Click here for additional data file.


Background: This study introduces a new framework based on an artificial neural network (ANN) for testing whether social determinants are major determinants of association among diabetes mellitus, visual impairment and hearing loss in a middle-aged or old population. Methods: The data came from the Korean Longitudinal Study of Aging (2014–2016), with 6120 participants aged 45 years or more. The association was divided into eight categories: one category for having no disease, three categories for having one, three categories for having two and one category for having three. Variable importance, the effect of a variable on model performance, was used to evaluate the hypothesis based on whether family support, socioeconomic status and social activity in Y2014 are among the top 10 determinants of the association in the year 2016 (Y2016). Results: Based on variable importance from the ANN, brothers/sisters cohabiting (0.0167), voluntary activity (0.0148), income (0.0125), family activity (0.0125), parents alive (0.0121), leisure activity (0.0095) and meeting with friends (0.0092) in Y2014 are the top-10 determinants of comorbidity in Y2016. Conclusion: The findings of this study support the hypothesis, highlighting the importance of social determinants for the effective management of the comorbidities of the three diseases.  Diabetes mellitus, visual impairment and hearing loss are the leading causes of disease burden in the world ,3,4,5,6.Then, is there a strong association (or comorbidity) among the three diseases above, i.e., diabetes mellitus, visual impairment and hearing loss? What determines the association? Is it true that “social determinants of health”  calls “structural determinants of health such as socioeconomic capital, policy, status and value systems” ) are majHypothesis 1.Social determinants are major determinants of association among diabetes mellitus, visual impairment and hearing loss in a middle-aged or old population.Hypothesis 2.The association among diabetes mellitus, visual impairment and hearing loss is very strong in a middle-aged or old population.In this context, this study develops a framework based on an artificial neural network (ANN) (1) to identify major determinants of association among all eight combinations of diabetes mellitus, visual impairment and hearing loss (as examples of chronic diseases in a middle-aged or old population) and (2) to test whether the association is very strong. The ANN, the central model of this study, has been known for its performance comparable or superior to those of traditional methods such as  logistic regression and the random forest, regarding the prediction of chronic diseases ,12,13,14Data came from the Korean Longitudinal Study of Aging (KLoSA) in Y2014 and Y2016. The data were publicly available and de-identified. The KLoSA is designed to create nationally representative longitudinal data on Koreans aged 45 years or more, which help to trace their characteristics over time and to develop socioeconomic policies for these rapidly growing populations. This biennial survey involves a multistage stratified sampling based on geographical areas and housing types across the nation. It uses computer-assisted personal interviewing and covers a wide range of demographic, socioeconomic and health-related topics. The panels in the 1st, the 5th and the 6th waves for Y2006, Y2014 and Y2016 consisted of 10254, 8387 and 7893 subjects aged 45 or more, respectively. Among these 7893 subjects, 1773 subjects were excluded in this study, given that they lacked demographic, socioeconomic or health-related information. The final sample of this study consisted of 6120 subjects aged 53 or more (75% of whom were older than 60 as in Y2014). Despite the censoring of participants by the listwise deletion, there were no significant differences between the Y2016 sample and the analytic sample on the measures of interest. Diabetes Mellitus, visual impairment and hearing loss in Y2014 and Y2016: The KLoSA question on diabetes mellitus in Y2014 and Y2016 was “Since the last survey, have you ever been diagnosed by a doctor as diabetes mellitus? 1. Yes. 5. No.” (C011) The inquiry on visual impairment (or hearing loss) in Y2014 and Y2016 was “Do you experience difficulty in daily activity because of visual impairment (or hearing loss)? 1. Yes. 5. No.” (C089 (or C092)). Disease–disease association in Y2016: The association among diabetes mellitus, visual impairment and hearing loss in Y2016 was divided into eight categories: (1) “0” for having no disease; (2) “1”, “2” and “3” for having diabetes mellitus only, visual impairment only and hearing loss only, respectively; (3) “4”, “5” and “6” for having diabetes mellitus and visual impairment, diabetes mellitus and hearing loss, and visual impairment and hearing loss, respectively; and “7” for having all three diseases. This approach, called “powerset methods”, changed a “multi-label” classification design (with three dependent variables) to its “multi-class” counterpart (with eight categories of one dependent variable). Here, “multi-label” means “many (dependent) variables” whereas “multi-class” means “many categories” of one dependent variable. Demographic, socioeconomic and health-related factors in Y2014: The following independent variables were also included in this study: (1) demographic factors, i.e., gender, age, marital status , the number of children alive, the number of brothers and sisters cohabiting, parents alive ; (2) socioeconomic status including educational level , personal income , health insurance (Medicare and Medicaid) and economic activity (employed and unemployed); (3) social activity, i.e., monthly frequencies of meeting with friends, religious activity, friendship activity, leisure activity, family activity, voluntary activity and political activity; (4) health-related factors such as subjective health , poor and very poor), BMI, smoker , drinker  and drug/medication intake (yes and no); and (5) other determinants, i.e., religion , residential type (apartment and other), region , life satisfaction for economic status (0 to 100) and life satisfaction for overall life (0 to 100). Six popular machine learning methods were compared for the prediction of the association to check the reliability of the ANN: ANN, logistic regression, decision tree, naïve Bayes, random forest and support vector machine. A decision tree consists of (1) internal nodes (each meaning a test on an attribute (or independent variable)), (2) branches (each denoting an outcome of the test) and (3) terminal nodes (each representing a class label (or dependent variable)). A naïve Bayesian classifier is a predictor based on Bayes’ theorem. A random forest creates many training sets, trains many decision trees and makes a prediction with a majority vote (“bootstrap aggregation”). A support vector machine makes a prediction by maximizing a margin among hyperplanes separating the data. The ANN of this study includes one input layer, two hidden layers and one output layer with 91,800 neurons as data units in the input layer, 15 in each hidden layer and 8 in the output layer. The number of neurons in the input layer, 91,800, comes from the multiplication of 30 and 3060, which are the numbers of attributes and observations in the training set, respectively. Neurons in the input or previous hidden layer are combined with the weights in the next hidden or output layer . Then, the weights in the output layer and its previous hidden layers are adjusted based on how much they contributed to the loss of the ANN, i.e., a gap between the actual and predicted class labels . Initially, the weights are set as small random numbers around 0 and the feedforward and backpropagation algorithms iterate until certain criteria are met for the accurate prediction of a class label [The association among the three diseases with the eight categories in Y2016 served as the dependent variable of the models. Diabetes mellitus, visual impairment and hearing loss in Y2014 and the demographic, socioeconomic and health-related factors in Y2014 served as the independent variables of the models. The data on 6120 participants were divided into training and validation sets with a 50:50 ratio. The models were built (or trained) based on the training set with 3060 observations, and then, the models trained were validated based on the validation set with 3060 observations. Accuracy, a ratio of correct predictions among 3060 observations, was introduced as a criterion for validating the models trained. Variable importance from the ANN, an accuracy gap between a complete model and a model excluding a certain variable, was used for testing the two hypotheses of this study: (1) Hypothesis 1 was evaluated based on whether family support , social activity  and socioeconomic status  in Y2014 are among the top-10 determinants of the association in Y2016, and (2) Hypothesis 2 was evaluated based on whether diabetes mellitus, visual impairment and hearing loss in Y2014 are among the top-5 determinants of the association in Y2016 (This can be considered one way of testing the correlation among the three diseases over time.). Finally, the greater “accuracy decrease” leads to the greater variable importance. This derivation is in a similar context but in the opposite direction of its random-forest counterpart: The variable importance from the random forest is a mean-impurity gap between a complete model and a model excluding a certain variable . The greater “mean-impurity increase” leads to a greater variable importance. Python 3.52 was employed for the analysis on December 2018. According to the variable importance from the ANN , brotherThe ANN might be reliable with its accuracy being similar with that of the logistic regression for the prediction of the association among diabetes mellitus, visual impairment and hearing loss in a middle-aged or old population. Based on the variable importance from the ANN, brothers/sisters cohabiting, voluntary activity and income in Y2014 are the top-3 determinants of the association in Y2016. Indeed, family activity, parents alive, leisure activity and meeting with friends in Y2014 are among the top-10 determinants of the association in Y2016. There exists a positive linkage between a pair of diabetes mellitus, visual impairment and hearing loss through the damage of small blood vessels, which is affected by age, BMI, exercise, marriage, self-rated health and the quality of life. This study uses an ANN for testing (1) whether social determinants are major determinants of association among diabetes mellitus, visual impairment and hearing loss in a middle-aged or old population and (2) whether the association among the three diseases is very strong in the middle-aged or old. Indeed, this study satisfies desirable conditions such as (3) nationally representative longitudinal data for Koreans aged 45 years or more; (4) 30 demographic, socioeconomic and health-related determinants for the association; and (5) a new approach called “powerset methods”, which changes a “multi-label” classification design (with three dependent variables) to its “multi-class” counterpart (with eight categories of one dependent variable). The results of this study do not support Hypothesis 2: diabetes mellitus, visual impairment and hearing loss in Y2014 are out of the list for the top-10 determinants of the association in Y2016 (This might indicate that the association among the three diseases might not be as strong as expected.). However, the findings of this study support Hypothesis 1, and the ANN puts more focus on various social activities  than the random forest (friendship). As explained in the Methods Section, the feedforward and backpropagation algorithms with constant learning  iterate in the ANN until certain criteria meet for the accurate prediction of a class label. This unique process of the ANN might lead to its distinctive outcomes from other machine learning methods including the random forest. In addition, this study draws the following policy implications, given that family support , social activity  and socioeconomic status (income) in Y2014 are found to be among the top-10 determinants of the disease–disease association in Y2016. Firstly, the promotion of family support and social activity among those aged 60 or more might be needed to prevent the comorbidities of diabetes mellitus, visual impairment and hearing loss in the population in Korea. As a matter of fact, family support and social activity among Koreans aged 60 or more was found to be still low, and economic burden was reported to be a major reason for the result ,18. In tSecondly, Korea’s social policy needs to be updated to improve the socioeconomic status of the old and to prevent the comorbidities of diabetes mellitus, visual impairment and hearing loss in the population in the nation. Korea has recently experienced an abrupt rise in the one-person family   and a suThis study had some limitations. Firstly, this study used a weak form of the longitudinal design because of constraints on memory capacity: The association among diabetes mellitus, visual impairment and hearing loss with the eight categories in Y2016 (Wave 6) served as the dependent variable of the models, while the three diseases in Y2014 (Wave 5) and the demographic, socioeconomic and health-related factors in Y2014 (Wave 5) served as the independent variables of the models. Considering a stronger form of the longitudinal design, e.g., 2012–2016 , brings too much burden on the computer. One solution for this issue is to employ a smaller number of determinants for the disease association; this might be an interesting topic for future research. Indeed, improving the memory capacity and employing the data in all six waves with a strong form of the longitudinal design is expected to improve the accuracy of the ANN much more. Secondly, this study did not consider the levels and durations of diabetes mellitus, visual impairment and hearing loss, which might be important factors for the association among the three diseases. Thirdly, this study used the KLoSA question on diabetes mellitus in Y2014 and Y2016, which might be misleading to some individuals with the diagnosis of diabetes mellitus prior to Y2014: They might consider the inquiry to be about a new diagnosis only, answering “No” to the inquiry ). Refining the question is expected to improve the quality of the research on the topic. Fourthly, expanding this study to other chronic diseases and other determinants of association such as health utility usage might add a great contribution to this line of research. Fifthly, this study did not consider the possible mediating effects among variables. Sixthly, a subgroup analysis, e.g., 45–54, 55–64, 65–74 and 75+ in age, might have offered more insight on the major determinants of the association among the three diseases. Finally, this study considered six popular machine learning methods . Expanding this study to other machine learning methods such as the AdaBoost, the k-nearest neighbors, the convolutional neural network and the recurrent neural network might be a good topic for further research.For preventing the comorbidities of diabetes mellitus, visual impairment and hearing loss in the middle-aged or old populations, the promotion of family support, social activity and socioeconomic status might be needed.


Aedes albopictus and Culex pipiens. The pathogenicity and virulence against the two species of mosquitoes were evaluated by using 30 isolates of Beauveria bassiana, an entomopathogenic fungus isolated from Korea that has high thermotolerance and UV-B tolerance. Regarding pathogenicity, 23 isolates were pathogenic to Ae. albopictus and 12 isolates were pathogenic to Cx. pipiens; Ae. albopictus adults were more susceptible to B. bassiana than Cx. pipiens adults. Among the isolates, 6 isolates that were simultaneously pathogenic to the two species of mosquitoes were used to evaluate virulence and conidia productivity. B. bassiana CN6T1W2 and JN5R1W1 had higher virulence than the other isolates, and they were more virulent in Ae. albopictus than inCx. pipiens. The conidia productivity of B. bassiana JN5R1W1 on millet grain medium was higher than that of B. bassiana CN6T1W2. Based on these results, B. bassiana JN5R1W1 was selected as the most efficient isolate for the simultaneous control of the two mosquito species. B. bassiana JN5R1W1 can be used effectively in the development of fungal insecticides to simultaneously control Ae. albopictus and Cx. pipiens adults with similar distribution areas.This study was conducted to determine the optimal entomopathogenic fungus for the simultaneous control of the adults of two mosquito species,  Aedes albopictus (Skuse) (Diptera: Culicidae), transmits many viral diseases, such as yellow fever, dengue and chikungunya. It is also believed to have the potential for Zika transmission among humans due to the host capacity of the Zika virus  (Diptera: Culicidae), is a temperate Eurasian nocturnal mosquito that has established populations throughout temperate North America  and Lysinibacillus sphaericus  were transferred to sterile PCR tubes and immediately placed in a thermal cycler adjusted to 45 °C. Aliquots (20 μL) were removed after 4 h and plated (without spreading) on Sabouraud dextrose agar  with 0.5 μg/mL of benomyl (95% active ingredient) (SDA-B)  from each isolate was plated (without spreading) on SDA-B. Conidia were immediately exposed to irradiances of 0.2 J/cm2 in a UV irradiation chamber  at 25 °C  was sprayed into each cylinder using an SD tower sprayer  capped with netting material. To evaluate the pathogenicity of the fungus, 10 mosquito adults aged 1–2 days were placed in the cylinder, and 1 mL of a conidial suspension . Millet grains for the production of conidia were prepared according to a previous report . The conidia on the millet grains were collected daily and counted using a haemocytometer.Conidia were produced in Petri dishes on Italian millet grains . The lethal time for the treatments was analysed with probit analysis. Data were expressed as the mean ± standard error (SE), and statistical significance was set at the conventional α < 0.05 level.B. bassiana isolates used in this study showed low thermotolerance but high UV-B tolerance  showed 100% mortality against both species of mosquito adults. Regarding lethality, all isolates were found to be slightly more pathogenic to Ae. albopictus than to Cx. pipiens adults. The LT50 values of B. bassiana CN6T1W2 and JN5R1W1 in Cx. pipiens were 4.3 and 4.4 days, respectively, which were lower than the values for the other 4 isolates, which were all more than 5.2 days. B. bassiana CN6T1W2 and JN5R1W1 also showed the lowest LT50 values for Ae. albopictus at 4.8 and 4.5 days, respectively. The LT90 values of B. bassiana CN6T1W2 and JN5R1W1 were also significantly lower than those of the other isolates. Thus, B. bassiana CN6T1W2 and JN5R1W1 were found to be the most virulent for both species of mosquitoes at the same time.The virulence of 6 isolates of nt Table . MoreoveB. bassiana that were simultaneously pathogenic to the two species of mosquitoes was evaluated on millet grain medium. All the isolates showed conidia productivity of 1.6–2.3 × 109 conidia/g at 7 days after inoculation; there was no significant difference in productivity  and 4.4 days (Cx. pipiens). Although there are few report of entomopathogenic fungal virulence on Ae. albopictus and Cx. pipiens adults, similar report has shown that M. anisopliae showed 3.1–4.1 days against Ae. aegypti adult   and Cx. pipiens (B) adults showing sporulation of B. bassiana.


The evolution of the various hearing adaptations is connected to major structural changes in nearly all groups of vertebrates. Besides hearing, the detection of acceleration and orientation in space are key functions of this mechanosensory system.The symposium “show me your ear – the inner and middle ear in vertebrates” held at the 11th International Congress of Vertebrate Morphology (ICVM) 2016 in Washington, DC (USA) intended to present current research addressing adaptation and evolution of the vertebrate otic region, auditory ossicles, vestibular system, and hearing physiology. The symposium aimed at an audience with interest in hearing research focusing on morphological, functional, and comparative studies. The presented talks and posters lead to the contributions of this virtual issue highlighting recent advances in the vertebrate balance and hearing system. This article serves as an introduction to the virtual issue contributions and intends to give a short overview of research papers focusing on vertebrate labyrinth and middle ear related structures in past and recent years. With the increasing application of high resolution and noninvasive computer tomography since the 1980s, 3D visualization and their respective analyses became essential for anatomical studies of the middle and inner ear.The vertebrate ear combines two main functions: balance and hearing. The vestibular part of the inner ear is the organ of equilibrium detecting head and body movements during locomotion . The semicircular canals (SCs)  of the vestibular system detect angular acceleration, while utricule and saccule detect linear acceleration  of elasmobranchs  of the lateral line receptors and the labyrinth  differs anatomically from the derived amniote ear  in showing large and bulky stapedial bones (relative to braincase size) capable of the detection of substrate‐borne vibrations  is the tympanum (tympanic membrane) overlying an air‐filled middle ear cavity with ossicles transmitting the sound to the oval window of the inner ear  are able to close their external acoustic meatus with a meatal muscle, possibly for protection against mechanical damage or their own sound  of nonmammalian amniotes, sounds bouncing off the skin surface are transmitted to the cochlear duct Manley, .In the 19th century, Georges Cuvier  describeDiarthrognathus), which possess both functional jaw joints (primary and secondary) at the same time , incus (anvil), and stapes (stirrup) is laterally restricted by the ectotympanic ring and completely detached from the mandible Allin, . Complexe Allin, . The sege Allin, . Even ife Allin, , 1973. De Allin,  conductee Allin,  and Fleie Allin, .1.2The inner ear (labyrinth) of vertebrates comprises two main functions: (a) balancing of the individual using the ampullae of the three SCs and the sensory field of utriculus and sacculus, and (b) hearing ability with the sensory hair cells of basilar papilla of the lagena of nonmammalian amniotes . The inner ear is composed of a membranous system (membranous labyrinth) filled with endolymphatic fluid enclosed inside a bony endocast (bony labyrinth), filled with perilymphatic fluid. Both fluids differ in their concentration of potassium and sodium  and early tetrapods, which remains unossified during embryogenesis (Clack, In jawless vertebrates, the number of SCs varies Retzius, . The vess Clack, . The rous Clack, , which es Clack, .In the 16th century, Bartholomeus Eustachius discovered the cochlear part of the ear, which was denoted by Gabriel Falloppio as “cochlea” in 1561 Gitter, . PolitzeEven though Hyrtl  doubted The shape and number of coils of the mammalian cochlea is known to have a strong phylogenetic signal Ekdale, . MonotreThe evolution of the amphibian inner ear on its way to amniotes is controversially discussed Lombard, , 1976. T1.3Since the expanding availability of noninvasive microcomputed tomography anatomical investigations of the middle and inner ear region of extant and extinct vertebrates are of still growing interest to the scientific community addressing complex questions. For example, the evolution of the huge diversity of the membranous inner ear labyrinth morphology, the function of the vestibular system for detecting acceleration and deceleration of the head and related transmission of sensory signals, the evolution of the mammalian coiled cochlea for enhanced hearing capability, and many more. Recently conducted studies focus on phylogenetic signals to contribute to the tree of life and reveal ecological adaptations. The goal of the symposium held at the 11th ICVM was to exchange newly acquired insights, new technical approaches, and exchange of information addressing anatomical meaning of the bony structure of inner and middle ear related structures in the system of vertebrates.There are no Conflict of interests.All authors drafted the manuscript, and gave final approval for publication.