Fishes and marine mammals may suffer a range of potential effects from exposure to intense underwater sound generated by anthropogenic activities such as pile driving, shipping, sonars, and underwater blasting. Several underwater sound recording (USR) devices have been built to acquire samples of the underwater sound generated by anthropogenic activities. Software becomes indispensable for processing and analyzing the audio files recorded by these USRs. In this paper, we provide a detailed description of a new software package, the Aquatic Acoustic Metrics Interface (AAMI), specifically designed for analysis of underwater sound recordings to provide data in metrics that facilitate evaluation of the potential impacts of the sound on aquatic animals. In addition to the basic functions, such as loading and editing audio files recorded by USRs and batch processing of sound files, the software utilizes recording system calibration data to compute important parameters in physical units. The software also facilitates comparison of the noise sound sample metrics with biological measures such as audiograms of the sensitivity of aquatic animals to the sound, integrating various components into a single analytical frame. The features of the AAMI software are discussed, and several case studies are presented to illustrate its functionality. Survival of not only individual animals but of whole populations can be affected by sound –6. The lThe physiological and behavioral impacts of sound on fishes and marine animals can be complicated, and substantial effort is required for their investigation. Investigation of the effects of sound requires measurement of the sound to determine its characteristics such as frequency content and loudness. Several underwater sound recording devices (USRs) have been built to record underwater sound –16. The The purpose of this study was to build a program controlled by an easy-to-use graphical user interface (GUI) that could be used to efficiently post-process underwater sound files recorded by the USRs and other devices. In addition to the features mentioned so far, the Aquatic Acoustic Metrics Interface (AAMI) was designed to implement some of the same sound file editing functions found in other commercial software, as well as offer unique features unavailable in other programs.The main reason underwater sound is measured is to obtain data that can be compared with information that describes biologically important functions, such as, the ability of an animal to hear a sound or to compare the characteristics of the observed sound with physiological or behavioral impact thresholds of exposed animals. For example, an audiogram is a standard way of representing an animal's hearing sensitivity over a frequency range. AAMI makes it easy to directly compare the characteristics of observed sound with the hearing sensitivity of animals of interest by plotting the spectra of the sound sample and the species' audiogram together in a single figure.2.The AAMI program was written in MATLAB  under the Windows operating system . At this time, it has been tested on 32-bit and 64-bit Windows operating systems. To obtain the best user interface presentation, the computer monitor resolution should be at least 1,280 × 1,024 pixels. The MATLAB signal processing toolbox is a component inserted into AAMI to filter and calculate spectrograms and power spectral densities (PSDs) for sound samples. A significant benefit of AAMI is the open source code format, which can be readily modified to meet additional processing, computation, and display needs as they are identified.The operation and primary functions of AAMI are described in this section. Once AAMI is launched, the main window of AAMI pops up . The mai2.1.y-axis and time in seconds on the x-axis. The upper limit for file size is 2 gigabytes (GB); AAMI automatically checks file size and alerts the user if file size exceeds this limit. Depending on the amount of memory in the users' computer, AAMI could become sluggish and may freeze when loading a file larger than 1 GB. In addition, for sequential processing of files, AAMI will automatically load a settings file, which applies default values for some parameters on the scaling factor panel. For each file processed, the user can change the default settings, load another settings file, or save the current settings file under a new name.AAMI can process sound files in .wav, .aif, and .mp3 formats. In the main window, the user can select a sound file for upload. The data source path will be shown in the uppermost white box on the left-hand side of the screen, and the signal is plotted in the time domain with voltage on the 2.2.Scaling factor parameters section of the user interface, provides three options for calculating the scaling factor to convert voltage to physical units:recorder box setting is specifically for sound files recorded from a USR [The om a USR  designedDirectly input scaling factor setting provides a simple way to apply a scaling factor for sound files from any USR. The input scaling factor unit is Pa/V.The Calculate scaling factor setting can be used for audio files obtained from any USR. Two parameters need to be specified: the hydrophone sensitivity in dB or V/Pa units and the receiver gain, entered in dB or x (times) units.The For a signal recorded by USRs and other devices, the typical output unit for the original data file is voltage. AAMI, in the After the user selects one of the above three options, a new window will pop up for entry of the necessary values while the other two methods are disabled .2.3.Subsample component portion of the interface menu.Another important feature of the AAMI program is selection of a subsample of data from a long signal. A data subsample may be selected in one of two ways: by using the computer mouse to select a portion of the signal shown in the interface plot area or by entering a time range in the editing boxes within the 2.4.peakSPL) is the maximum absolute amplitude value in the signal during a specified time interval:peakP is the peak pressure and units are dB re 1 µPa.Peak sound pressure level (rmsSPL) is the log transformed square root of the average square pressure of the signal over a specific time interva:rmsP is the RMS pressure and units are dB re 1 µPa.Root mean square (RMS) sound pressure level (SEL), also referred to as single-strike SEL (ssSEL), is the squared sound pressure integrated over the duration of a signal:P is pressure, refP is reference pressure, Δt is the inverse of sampling frequency, and units are dB re 1µPa2·s.Sound exposure level (cumSEL) is the summation of multiple impulsive or transient signals. SEL is calculated by summing the cumulative pressure squared over the time of the event and normalized to one second. This metrics accounts for both negative and positive pressures. Although not usually the case, when the ssSEL is the same for all impulses cumSEL can be calculated as shown in 2·s.Cumulative sound exposure level /N is the thN root of unity.Fast Fourier transform (FFT) decomposes a signal into its frequency components. AAMI implements the FFT and the inverse transform pair by:Power spectral density (PSD) is the power in the signal per unit frequency over the duration of the signal. AAMI computes the PSD using Welch's method , which iSpectrogram is an image showing the level of energy  across frequencies over time. AAMI uses the short-time Fourier transform (STFT) ,19 to caFor a subsample or the complete sound file, AAMI can calculate the following seven parameters:2.5.An audiogram is a standard way of presenting an animal's hearing sensitivity in functional form as a variable threshold over a frequency range. Currently, AAMI contains a library of audiograms  for a nu2.6.high-pass filter, which removes frequencies lower than a specified value;low-pass filter, which removes frequencies greater than a specified value;bandpass filter, which removes frequencies outside a given range of values.Another important feature contained in AAMI is the ability to filter sound files before processing them. AAMI permits an audiogram to be used as a filter in addition to a selection of other, more common filters given below:2.7.x- and y-axes, and can turn on/off the appearance of text indicating peak values and plot title. Moreover, AAMI can export the working graphic into different types of output files such as .tif, .eps, and .jpg.Users can edit a sound signal or other plots by adjusting the range and scaling for 2.8.AAMI can sequentially batch-process an unlimited number of .wav files using a setting file. As mentioned before, for large files, AAMI might freeze during loading and processing the data. To avoid this situation, break long duration signals into a series of shorter duration files that are less than 1 GB in size and use the AAMI batch-processing capability to process the signal. The batch-processing function loads the .wav files and outputs user-defined plots or data.3.Oncorhynchus tshawytscha) to tidal turbine noise. This species and age of salmon were selected for test because they are an ESA listed species and because they travel through Admiralty Inlet in Puget Sound as both juveniles and adults and could be exposed to tidal turbine and other noise [There is concern for the potential effect of noise generated by tidal turbines on nearby organisms of ecological importance. A laboratory exposure-response study was conducted to investigate the response of juvenile Chinook salmon  sound signals. The data for six 24-h tests were processed. The sound exposure signal, which was on continuously through the 24-h exposure period, was sampled using a USR in 1-min segments to keep file sizes manageable. All the 1-min sound files  plots  plots .The Pacific Northwest National Laboratory animal facilities used in this research are Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-certified; fish were handled in accordance with federal guidelines for the care and use of laboratory animals. The Institutional Animal Care and Use Committee at Battelle-Pacific Northwest Division specifically approved protocols for this study.One of AAMI's unique features was also applied to a 1-min sound sample . An audi4.peakSPL, rmsSPL, ssSEL, and cumSEL The output metrics of AAMI can help drive the standardization of sound analysis with regard to anthropogenic sound sources and their effects on marine life. All of AAMI's design capabilities, functions, and applications make AAMI a powerful tool that is relatively straightforward to employ. AAMI brings together many functions into one source that otherwise required the user to be an expert programmer to generate the metrics, graphs, comparisons, and output elements that AAMI can produce.AAMI has been verified, with internal and external testing, to provide functionality, processing, and computation functionalities that are not available in other audio editing software programs. The AAMI GUI facilitates importing and processing sound signals in various sound file formats. AAMI was constructed to perform common sound file editing functions as well as to provide some unique features customized for use in scientific sound processing. AAMI has the capabilities to read two-channel sound signal recordings, convert the input data from engineering units to physical units  apply user entered calibration information, batch-process sound files, and calculate important acoustic metrics such Furthermore, the pairing of the PNNL-USR  with the


Valeriana jatamansi Jones is a formula for treating anxiety-related diseases in the clinic, which is composed of Valeriana jatamansi Rhizoma et Radix, Ziziphi Spinosae Semen, Albiziae Cortex and Junci Medulla. The purpose of this study was to explore the anxiolytic properties of this compound in mice.Compound Valerianae Jatamansi Jones , saline, diazepam (2 mg/kg) orally for 10 days and then exposed to elevated maze-plus (EPM) and light–dark box (LDB). The effects of the compound on spontaneous activity were evaluated by locomotor activity test. We further investigated the mechanism of action underlying the anxiolytic-like effect of compound by pre-treating animals with antagonists of benzodiazepine  prior to evaluation using EPM and LDB.Male ICR mice were treated with compound Valerianae Jatamansi Jones  significantly increased entries (P<0.05) into and time spent (P<0.05) on the open arms of the EPM, and number of transitions (P<0.05) and time spent (P<0.05) in the light compartment of the LDB. However, the anxiolytic-like effects of compound were significantly reduced by pre-treatment with flumazenil (P>0.05). In addition, compound Valerianae Jatamansi Jones treatment didn’t affect the spontaneous activity in mice (P> 0.05).Compound Valeriana jatamansi Jones exert anxiolytic action but no sedative effects in mice and that this effect might be mediated by benzodiazepine receptors.The present study supports the hypothesis that compound  Anxiety disorders are the most common and prevalent behavioral disorders that can result in significant impairment of function and quality of life, and they affect 17.2% of the population in the United States. Howeveraleriana jatamansi Jones is used to treat anxiety-related diseases[Valeriana jatamansi Rhizoma et Radix (12g), Ziziphi Spinosae Semen (9g), Albiziae Cortex (9g) and Junci Medulla (1g). The anxiolytic effects of this formulation have been previously established in the rat[Traditional Chinese Medicine has been commonly recognized as a safe and effective means of treating anxiety disorders-8. The cdiseases, and is  the rat. However the rat.Valeriana jatamansi Jones in mice using the elevated plus maze (EPM), light/dark box (LDB) test, and spontaneous activity. The formulation was first evaluated for purity by high-performance liquid chromatography (HPLC). We also examined whether its anxiolytic effects are mediated by GABA(A) receptors through co-administration of the benzodiazepine antagonist flumazenil.Therefore, in this study, we further explored the anxiolytic effects of compound Valeriana jatamansi Rhizoma et Radix was purchased from a commercial source in Yunnan province, China. Ziziphi Spinosae Semen, Albiziae Cortex, and Junci Medulla were purchased from a commercial source in Hebei province, China. The identity of the herbal medicine was confirmed by Professor Shi, a researcher in the Department of Pharmacognosy, Beijing University of Chinese Medicine. Voucher specimens were deposited at the Herbarium of School of Chinese Materia Medica, Beijing University of Chinese Medicine.Valeriana jatamansi Jones has optimum efficacy when the four herbal components were extracted by different solvents. Jatamana Valeriana Rhizome was homogenized to coarse powder, refluxed three times each for 2h in aqueous ethanol , and the combined alcoholic extract was filtered and evaporated (yield 20.7% (w/w)). Albiziae Cortex and Ziziphi Spinosae Semen were also homogenized to coarse powder, infused in 2L of cold distilled water, and extracted for 3h twice by heating. Then, the extracts were concentrated to dryness. The yield of the extracts of Albiziae Cortex and Ziziphi Spinosae Semen were 15.2% and 25.8% (w/w), respectively. Junci Medulla was refluxed for 1.5h in aqueous ethanol  three times, and the combined alcoholic extract was filtered and concentrated (yield 4.5% (w/w)). Based on the original formulation and the herb extraction yield, the four extracts  were mixed with a ratio of 5:3:5:1 to get the compound Valeriana jatamansi Jones.Previous chemical and pharmacological studies in our laboratory demonstrated that the compound 9.80 mg hesperidin reference substance  was transferred into a 25 mL measuring flask and mixed with methanol. Then, the mixture was shaken and filtered through 0.45μm filters. 5g of mixed dry extracts were used to prepare the sample solution and refluxed in an appropriate amount of methanol to a colorless extract and filtered. Then, it was transferred to a 100mL measuring flask and brought up to level with methanol and filtered through 0.45μm filters. 5μL solutions were injected. The samples were chromatographed using an Agilent® C18 chromatographic column , a mobile phase consisting: acetonitrile - 0.3% phosphoric acid , a flow rate of 1mL/min, a column temperature of 35°C, and UV detection at 280nm.Valeriana jatamansi Jones and flumazenil . For oral (p.o.) administration for 10 days, compound Valeriana jatamansi Jones and diazepam were dissolved in saline. Each drug was administered in a volume of 0.4mL/25g body weight. Control animals received vehicle  only. For evaluating the anxiolytic effects of the compound Valeriana jatamansi Jones, the mice were administered the compound Valeriana jatamansi Jones  or diazepam 60 and 30min before the test, respectively. For exploring the anxiolytic mechanisms of the compound Valeriana jatamansi, the mice were also administered the compound Valeriana jatamansi Jones  or diazepam for 10 days described above. On the last day, flumazenil (GABA(A) antagonist) was co-administered  30 min before the test. All of the animal tests were performed on the 10th day of treatment.The following drugs were used in the study: diazepam hydrochloride (2.5mg/tab.), three different concentrations  of the compound ad libitum. Groups of 12–20 mice were randomly assigned to different treatment groups and tested in a counterbalanced order. All experiments were carried out in a quiet room under a dim red light between 8:00 and 14:00. The experimental procedures were approved by the Institutional Animal Care and Use committee of the Institute of Psychology of the Chinese Academy of Sciences and in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and their suffering.Male Imprinting Control Region (ICR) mice weighing 18–22g were purchased from the China Academy of Military Medical Sciences and kept in cages of 5 mice at 22 ± 1°C on a 12-h light/dark cycle (with lights on from 08:00–20:00). Water and food were available Valeriana jatamansi Jones and saline were administered orally 1 h before testing and the positive controls were treated with diazepam 30 min before evaluation in the maze. All tests were carried out on the tenth or last day of treatment. After each trial, the apparatus was cleaned with 30% alcohol.Anxiolytic activity was measured using the elevated plus-maze procedure. The mazThe apparatus (45×21×21cm) consisted of two compartments with one third painted white and two thirds painted black, and these compartments were separated by a divider with a 3.5×3.5cm opening at floor level,13. The Valeriana jatamansi Jones on our measures of anxiolytic effects, the activity of individual mouse was recorded by a computer-based system  for locomotor activity.To rule out any unspecific locomotor effects of the compound The data are expressed as the mean ± S.E.M. The statistical analysis was carried out by a one-way analysis of variance (ANOVA) followed by Student-Newman-Keul’s post-hoc tests using Prism 5.0 . Significance was accepted at a P <0.05.Valeriana jatamansi Jones was used as monarch drug, hesperidin content was selected for the quality control components. The results showed that the hesperidin in the sample produced a well-resolved peak, and the resolution with its adjacent chromatographic peaks was more than 1.5 . No differences were observed in the total arm entries among all groups  and the number of transitions between compartments . Compared with the saline-treated mice, treatment with compound Valeriana jatamansi Jones at doses of 2.4g/kg and 4.8g/kg significantly increased the time spent in the light compartment and the number of transitions between compartments (both P< 0.05). After the mice treated with compound Valeriana jatamansi Jones were co-administered flumazenil , the time spent in the light compartment  compared to the saline group. However, there were no significant differences among saline-treated animals and those treated with compound Valeriana jatamansi Jones at 1.2, 2.4, or 4.8 g/kg (P>0.05).A one-way ANOVA indicated significant differences among the groups in the time spent in the open arms of the elevated plus maze  receptors. The combination of our results and those of a previous study strongly support the idea that compound Valeriana jatamansi Jones produces anxiolytic effects without inducing sedative side effects. These findings may support the development of potent and selective anxiolytic agents.Based on a previous study that showed anxiolytic effects of compound  in rats, we furtValeriana jatamansi Jones. As expected, compound Valeriana jatamansi Jones (2.4 and 4.8 g/kg) significantly increased time spent and entries into the open arms of EPM. Moreover, the anxiolytic effects of the compound Valeriana jatamansi Jones (4.8 g/kg) were similar to those of diazepam (2mg/kg). The main potential confounding factors in this procedure are changes in basal locomotor activity, which can be inferred from the total number of entries. However, the compound Valeriana jatamansi Jones did not alter total arm exploration in the elevated plus maze, suggesting that this drug induces specific anxiolytic-like effects.The EPM is one of the most popular animal models of anxiety,15. It hThe light/dark box test is another widely used animal model of anxiety. It is based on the innate aversion of rodents to bright areas and on the spontaneous exploratory behaviors of rodents in response to novel environments and light. PreviouValeriana jatamansi Jones. These data indicate that the anxiolytic-like effects of compound Valeriana jatamansi Jones may be mediated by benzodiazepine binding site modulation at GABA(A) receptors. However, the EC50 of this formulation is approximately 0.5 mg/mL. As such, it is hard to say whether this is a sufficient potency to produce in vivo neurochemical and behavioral actions, as the repeated oral application of compound by g/kg doses may not result in sufficient brain concentrations. In this study, we intraperitoneally administered flumazenil to mice 30 min before testing[Valeriana jatamansi Jones are mainly mediated via benzodiazepine receptors.GABA (A) receptors are the most prevalent inhibitory neurotransmitter receptors in the brain. Most of them have separate modulatory sites that are sensitive to benzodiazepines. Benzodiazepines receptor agonists are commonly prescribed for the treatment of insomnia, anxiety disorders, and seizures, and these effects can be blocked by the antagonist flumazenil. In a previous binding assay, the bin testing-24. We oValeriana jatamansi Jones, the dose of diazepam was increased to 6 mg/kg in the locomotor activity test. Although this dose of diazepam significantly reduced spontaneous activity counts, none of the three doses of compound Valeriana jatamansi Jones affected spontaneous activity in the locomotor test. As such, we found that compound Valeriana jatamansi Jones induced anxiolytic effects with an absence of sedative effects. This may be because the doses tested were too low to yield sedative effects. In addition, McKernan et al. showed that the GABA(A) receptor α1 subtype mediates the sedative, but not the anxiolytic effects of benzodiazepines[Valeriana jatamansi Jones may be more selective than diazepam, and not act through the α1 subtype but via other GABAA receptor subtypes.Valerian is the main ingredient of this formulation, and it is one of the most popular herbal supplements for the treatment of anxiety and insomnia in Europe. The sedazepines. AccordiValeriana jatamansi Jones that is used is 31g per day. When this human dose is converted into an animal dose , it was equivalent to 4.8 g/kg. Therefore, 4.8 g/kg was chosen as the highest dose tested in this study, which is equal to 934 mg/kg extract of compound Valeriana jatamansi Jones. Unlike the regular formulation extraction process, Valeriana jatamansi Rhizoma et Radix, ziziphi spinosae Semen, Albiziae Cortex, and Junci Medulla were extracted by different solvents and methods. This was because the active constituents of these drugs are all sensitive to water temperature. An orthogonal experimental design method was performed to optimize the extraction process of the four herbs as described previously[9 (34) orthogonal test, the solid–liquid ratio, ethanol concentration, solvent amount, extraction times were determined. The extraction process of Valeriana jatamansi Rhizoma et Radix or Junci Medulla was determined by the content of hesperidin and the total extract. The optimum extraction condition of ziziphi spinosae Semen or Albiziae Cortex was determined by the content of jujuboside A. According to the original formulation and the herb extracting yield, the four extracts  were then mixed with a ratio of 5:3:5:1 to get the compound Valeriana jatamansi Jones.It should be noted that the three doses tested  are related to the original formulation but not to the extract. In the clinic, the dose of the compound eviously. BrieflyValeriana jatamansi Jones for 10 days. Since previous studies have shown that many drugs elicited anxiolytic effects after administration for 7 days[As a positive control drug, diazepam exerted anxiolytic effects after a single dose . However, in the present study, the anxiolytic activity was examined after the mice were given compound r 7 days,29, it ir 7 days. TherefoValeriana jatamansi Jones exert anxiolytic action but no sedative effects in mice and that this effect might be mediated by benzodiazepine receptors.The present study further supports the hypothesis that compound EPM: Elevated plus-maze; LDB: Light–dark box test; GABA: γ-aminobutyric acid; HPLC: High-performance liquid chromatography.The authors declare that they have no competing interests.JSY and MP participated in the pharmacodynamic and mechanic studies drafted the manuscript. HZ, YL and BSZ participated in the design of the study and performed the statistical analysis. JLS and JYG conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/12/223/prepub


The early phase of salivary gland carcinomas with high-grade transformation (HGT) is extremely rare. We reported one case of adenoid cystic carcinoma (AdCC) with early HGT, herein.The patient was a 27-year-old Japanese woman who suffered from swelling of the left parotid region. Most of this tumor consisted of typical AdCC histology, whereas the central area of this tumor was composed of solid growth component by atypical cells with clear cytoplasm and marked nuclear atypia. Immunohistochemically, this area was strongly and diffusely positive for epithelial membrane antigen, p53, p16, Her-2, cyclin A and cyclin B1. The Ki-67 labeling index of this area was high, entirely different from that of AdCC area.Overall, this area was an early phase of AdCC-HGT. This case is the second case of early AdCC-HGT. We discuss the development of salivary gland carcinoma with HGT.http://www.diagnosticpatology.diagnomx.eu/vx/1598278104895730 Cheuk AdCC-HGT , but theThe patient was a 27-year-old Japanese woman who suffered from the swelling of the left parotid region. She had never admitted to any other hospitals for 9 years, and then, she admitted to our hospital. Computed tomography indicated a 30 × 20 × 25 mm irregular-shaped mass was seen in the left parotid gland. Magnetic resonance imaging showed an irregular-shaped mass, which showed the low intensity with T1-weight imaging and the low and partly high intensity with T2-weight imaging  of AdCC, solid-subtype AdCC and HGT. As three components existed separately, we believe that HGT occurs abruptly in typical AdCC. In the present case, HGT component lacked biphasic differentiation. Recently, Costa AdCC-HGT ; 1) prolAdCC-HGT . TherefoAdCC-HGT . TherefoAdCC-HGT . The preAdCC-HGT . The preAdCC-HGT . HoweverHGT is recognized when a lower-grade malignant neoplasm abruptly transforms to a high-grade ,10, in wet al. indicated that, in the HGT area of AdCC, the Ki-67 labeling index was >50%, and commonly showed the diffuse and strong immunopositivity of p53 protein [et al. indicated that, in AdCC-HGT, p53 gene mutation and overexpression of cyclin D1 and Her-2 were related to the dedifferentiation process [et al. reported 6 cases of HGT-AdCC and suggested that the HGT process was related to p53 gene mutation, overexpression of Her-2 and loss of pRb gene products [Recently, Seethala  protein . Althoug process , the accproducts . In HGT products . In the products . Howeveret al. reported that early phase of AdCC-HGT consisted of the scattered foci of anaplastic carcinoma in the co-/pre-existing low-grade AdCC [et al., one case had only a 20% HGT component in typical AdCC and another case contained only a 10% HGT component, who died of disease [et al.[et al. do not descript on the proportion of HGT components in the main tumor [et al. reported the gradual progression from low-/intermediate-grade AdCC to high-grade AdCC and moreover to AdCC-HGT [Among the salivary gland carcinomas, AdCC frequently showed the HGT. Ide ade AdCC . In seri disease . As medie [et al., we consAdCC-HGT . The dedIn summary the early phase of the AdCC-HGT was derived from abruptly genetic and phenotypic changes. The present case is the first report that an early phase of AdCC-HGT was related to cyclin A, cyclin B1 and p16 overexpression, in addition to p53 gene product accumulation and Her-2 overexpression.Written informed consent was obtained from the patient for publication of this Case Report and any accompanying images.AdCC: Adenoid cystic carcinoma; HGT: High-grade transformation; PAS: Periodic acid-Schiff; EMA: Epithelial membrane antigen; CK: Cytokeratin; CCCNOS: Clear cell carcinoma, not otherwise specified; LGCCC: Low-grade cribriform cystadenocarcinoma; CXPA: Carcinoma ex pleomorphic adenoma.The author declares to have no competing interests.KK analyzed that data and wrote the manuscript as a main contributor. TM and TN conducted the pathological examination. All authors have read and approved the final manuscript.


Chronic acquired neuropathies of unknown origin are classified as chronic inflammatory demyelinating polyneuropathies (CIDP) and chronic idiopathic axonal polyneuropathies (CIAP). The diagnosis can be very difficult, although it has important therapeutic implications since CIDP can be improved by immunomodulating treatment. The aim of this study was to examine the possible abnormalities of nodal and paranodal regions in these two types of neuropathies. Longitudinal sections of superficial peroneal nerves were obtained from biopsy material from 12 patients with CIDP and 10 patients with CIAP and studied by immunofluorescence and in some cases electron microscopy. Electron microscopy revealed multiple alterations in the nodal and paranodal regions which predominated in Schwann cells in CIDP and in axons in CIAP. In CIDP paranodin/Caspr immunofluorescence was more widespread than in control nerves, extending along the axon in internodes where it appeared intense. Nodal channels Nav and KCNQ2 were less altered but were also detected in the internodes. In CIAP paranodes, paranodin labeling was irregular and/or decreased. To test the consequences of acquired primary Schwann cells alteration on axonal proteins, we used a mouse model based on induced deletion of the transcription factor Krox-20 gene. In the demyelinated sciatic nerves of these mice we observed alterations similar to those found in CIDP by immunofluorescence, and immunoblotting demonstrated increased levels of paranodin. Finally we examined whether the alterations in paranodin immunoreactivity could have a diagnosis value. In a sample of 16 biopsies, the study of paranodin immunofluorescence by blind evaluators led to correct diagnosis in 70±4% of the cases. This study characterizes for the first time the abnormalities of nodes of Ranvier in CIAP and CIDP, and the altered expression and distribution of nodal and paranodal proteins. Marked differences were observed between CIDP and CIAP and the alterations in paranodin immunofluorescence may be an interesting tool for their differential diagnosis. Chronic polyneuropathy is a highly prevalent condition with various etiologies, including hereditary, metabolic, toxic or immune-mediated origins. For hereditary polyneuropathies, termed Charcot-Marie-Tooth (CMT) diseases, recent advances in the identification of the responsible genes allow a genetic diagnosis in a growing number of cases CIDP is an immune-mediated disorder of peripheral nerves, with a progressive or relapsing course + channels (Nav), essential for the rapid saltatory conduction of action potentials, are concentrated at nodes of Ranvier in association with ankyrin G, a cytoplasmic protein interacting with cortical cytoskeleton and cell adhesion molecules Myelinated fibers are characterized by highly differentiated domains along the axon, centered by nodes of Ranvier Superficial peroneal nerve biopsies were studied in 12 patients diagnosed with CIDP  and 10 wSince, to our knowledge, the nodal regions have not been explored in CIDP or CIAP, we used electron microscopy to determine how these regions were altered in chronic acquired neuropathies. Ten nodes of Ranvier were examined for either pathology, in longitudinal sections of nerves from two patients with CIDP and two with CIAP. In CIDP myelinated fibers, the abaxonal cytoplasm and the perinodal extensions of Schwann cells were abnormal as well as the paranodal glial loops . The outIn CIAP nodal regions, the abaxonal glial region and Schwann cell microvilli were normal . Some noSince the nodal and paranodal regions were altered in CIDP and CIAP we examined the distribution of Nav that are located at nodes of Ranvier In CIAP nerves, paranodin immunoreactivity was distributed irregularly at paranodes, with areas of weak or absent staining . Nav enrWe then examined the distribution of KCNQ2, a potassium channel subunit present in nodal regions Krox20 gene, which codes for a transcription factor required for the maintenance of the expression of Schwann cell-specific genes. Induced demyelination in adult mice leads to the presence of macrophages degrading Schwann cells, onion bulbs, and abnormally thin myelin sheaths 3±10 103 nodes per mm3 mean ± SEM, n = 3) than in vehicle-treated mice . To determine whether the increased immunofluorescence corresponded to increased levels of proteins, we used immunoblotting  able to bind tamoxifen but not endogenous estrogens, which prevents the nuclear activity of Cre. For inducing Krox20 inactivation, lacZ/floxKrox20, TR26-CreER mice were injected with tamoxifen , in a 10∶1 sunflower oil/ethanol solution, 100 µl twice a day for 5 days. Control mice received only vehicle. Mice were killed and nerves prepared 28 days after this treatment. The total number of mice used was six and all experiments were in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals . Our Institute has the authorization for the use of mice without approval of specific research project.Mice with conditional Krox20 inactivation were produced as described Institut du Fer à Moulin Cell and Tissue Imaging Facility with a Philips CM 100 microscope equipped with a Gatan Orius 832 camera for digitized acquisition. Nodal regions were located at low magnification and extensively studied at higher magnification.Superficial peroneal nerves were obtained from patients for diagnosis purposes, previously to the present study, under local anesthesia, at the lower third lateral part of the leg. One centimeter was fixed in 2.5% (weight/vol) glutaraldehyde in sodium phosphate buffer , post-fixed in 2% (weight/vol) osmium tetroxide in the same buffer and embedded in Epon. Semi-thin sections (0.5 µm) were stained with toluidine blue and examined under light microscope. One centimeter was orientated longitudinally, mounted on a cork disk with tragacanth gum and frozen in isopentane cooled by liquid nitrogen at −160°C. Electron microscopy was carried out on 2 samples from patients with CIAP and 2 with CIDP at the + channel α subunit mouse monoclonal antibody  and rabbit polyclonal anti-sodium channel (PAN-SP19) were from Sigma-Aldrich . Rabbit antibodies for KCQN2 Rabbit antibodies against paranodin (L51) have been described 3) from frozen nerves were homogenized in a buffer containing 10 mM sodium phosphate, pH 7.8, 60 mM NaCl, 1% (vol/vol) Triton X-100, 0.5% (weight/vol) deoxycholic acid, 0.1% (weight/vol) SDS, 10% (vol/vol) glycerol, 25 mM alpha-glycerol-phosphate, 50 mM sodium fluoride, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and Complete protease inhibitor (Roche Applied Science). Insoluble material was removed by centrifugation at 21,000×g for 10 min at 4°C. Protein concentration was determined using microBCA (Pierce). Extracts were in NuPAGE LDS were resolved on 4–12% NuPAGE BisTris gels (Invitrogen), and transferred to nitrocellulose membranes. Membranes were blocked and incubated with antibodies as described Mouse sciatic nerves were dissected out and frozen in isopentane pre-cooled with liquid nitrogen. Samples were stored at −80°C. Tissue fragments , fixed in methanol-acetone (1∶1), washed in phosphate-buffered saline (PBS), blocked in PBS with 30 g/L bovine serum albumin), and incubated overnight at 4°C with the primary antibody in blocking buffer. After washing in PBS with 0.01% (vol/vol) Tween 20, sections were incubated 1 hour at room temperature with Alexa fluor 488 donkey anti-rabbit IgG (Molecular Probes), Cy3 goat anti-mouse IgG (H+L) highly cross-adsorbed, or Cy5 goat anti-chicken IgG (Jackson Laboratories) diluted 1/500 in blocking solution. Sections were washed in distilled water and mounted in Vectashield.Institut du Fer à Moulin Cell and Tissue Imaging facility, using a Leica epifluorescence microscope equipped with a CCD camera (Micromax Roper Scientific), or a Leica SP2 confocal laser scanning microscope . Acquisition features were kept constant for all acquisitions with the same antibodies.Immunofluorescence analysis was carried out at the Images from biopsies of CIAP (8) or CIDP (9) immunostained with paranodin antibodies were coded, randomly mixed and examined by 4 unaware observers who were asked to classify them into 3 categories  according to the intensity and extension of paranodin immunostaining. The proportion of correct identification was determined for each disease.Figure S1Ultrastructure of a node of Ranvier in superficial peroneal nerve from a CIAP patient. A. Low magnification of a longitudinal ultrathin section showing the nodal and paranodal regions. B. Higher magnification of A showing normal perinodal microvilli emanating from the Schwann cells (arrow). C. Higher magnification of A showing paranodal loops with septate-like axoglial junctions (arrowhead). Scale bars: A: 1 µm, B: 0.7 µm and C: 0.5 µm.(3.82 MB TIF)Click here for additional data file.Table S1Clinical, paraclinical, and histological characteristics of CIDP patients.(0.02 MB DOCX)Click here for additional data file.Table S2Clinical and histological characteristics of patients with CIAP.(0.02 MB DOCX)Click here for additional data file.


Tinnitus is an auditory phenomenon characterised by the perception of a sound in the absence of an external auditory stimulus. Chronic subjective tinnitus is almost certainly maintained via central mechanisms, and this is consistent with observed measures of altered spontaneous brain activity. A number of putative central auditory mechanisms for tinnitus have been proposed. The influential thalamocortical dysrhythmia model suggests that tinnitus can be attributed to the disruption of coherent oscillatory activity between thalamus and cortex following hearing loss. However, the extent to which this disruption specifically contributes to tinnitus or is simply a consequence of the hearing loss is unclear because the necessary matched controls have not been tested. Here, we rigorously test several predictions made by this model in four groups of participants . Magnetoencephalography was used to measure oscillatory brain activity within different frequency bands in a ‘resting’ state and during presentation of a masking noise. Results revealed that low-frequency activity in the delta band (1–4 Hz) was significantly higher in the ‘tinnitus with hearing loss’ group compared to the ‘no tinnitus with normal hearing’ group. A planned comparison indicated that this effect was unlikely to be driven by the hearing loss alone, but could possibly be a consequence of tinnitus and hearing loss. A further interpretative linkage to tinnitus was given by the result that the delta activity tended to reduce when tinnitus was masked. High-frequency activity in the gamma band (25–80 Hz) was not correlated with tinnitus (or hearing loss). The findings partly support the thalamocortical dysrhythmia model and suggest that slow-wave (delta band) activity may be a more reliable correlate of tinnitus than high-frequency activity. Tinnitus (TI) is the perception of sound in the absence of an external source. Approximately 10 % of the population experience TI permanently, and in around 1–3 % of these people, their quality of life is severely affected by the condition  or magnetoencephalography (MEG): (1) in the presence of hearing loss, TI should be associated with an increase in oscillatory activity in low frequency (<10 Hz) and high-frequency (gamma) bands (25–50 Hz), and (2) altered gamma activity should be maximal at the edge of the hearing loss in primary auditory cortex (the ‘edge effect’). The first prediction can be tested either in a within-subject design (using a manipulation that reduces or eliminates the TI percept) or in a between-group design (that compares people with and without TI). The second prediction cannot be satisfactorily tested using either paradigm because current analysis methods for spatial localisation of EEG and MEG data are too imprecise.N = 10) is consistent with TI-related effects on low-frequency activity in which a normalised measure of delta band activity (1.3–4 Hz) was lower during periods of residual inhibition following a tone stimulus, compared to control periods when a tone stimulus failed to evoke residual inhibition  clinic, Nottingham Audiology Services and NIHR Nottingham Hearing Biomedical Research Unit. Participants with no TI or hearing loss were recruited from the general population. All participants in the TI groups had chronic subjective TI (for at least six months prior to recruitment). Individuals with pulsatile TI, Ménière’s disease, stapedectomy, and neurological disorders were excluded. All of the 52 participants were right handed as assessed by the Edinburgh handedness inventory Oldfield . EthicalN = 22); (2) TI with clinically normal hearing (N = 8); (3) no TI with hearing loss (N = 8); and (4) no TI with clinically normal hearing (N = 14). TI groups were matched for age, hearing loss (pure-tone average between 0.25 and 8 kHz), TI severity, and hyperacusis (Table Participants formed four groups: 1) TI with hearing loss  at standard audiometric frequencies from 0.25 to 8 kHz. Given the age of our participants, clinically normal hearing does not necessarily mean normal auditory processing in the periphery, or absence of hearing loss and deafferentation at higher frequencies. Cochlear status cannot be verified without psychophysical measurement, which is beyond the scope of the current study. Nevertheless, the important factor in the present study is that we attempted to match participants in audiometric profile, which is an improvement on many previously reported TI studies.The individual as well as median hearing thresholds for the four groups are shown in Figure For participants reporting TI, severity of the symptoms was assessed by the Tinnitus Handicap Inventory, Newman et al. . TI handTinnitus Tester software  with 275 radial gradiometers over the scalp at a sampling rate of 600 Hz. An additional 29 reference gradiometers and magnetometers were recorded for ambient noise cancellation . The masker was presented in the MEG system using Presentation  via a TDT System III (Tucker-Davis Technology) and small transducers (Etymotic Research—ER2). Each transducer was placed in specially designed mu-metal casing to protect the MEG sensors from magnetic interference, and the sounds were delivered to the participants via short plastic tubes. The sound level was set to 50 dB SPL for all participants. The recording session comprised 80 trials of 20 s duration as shown in Figure Following the recording, participants indicated their TI loudness on a visual analogue scale (0–10), both during the masker and the silent epochs. The amount of attenuation of the TI was calculated by subtracting the score during masking from the score during silent periods. A positive score indicates some degree of attenuation, while a negative score indicates an increase in loudness. For the MEG analysis, any degree of reported TI attenuation was regarded as ‘masked’ .http://fieldtrip.fcdonders.nl/start)  were rejected. The remaining data were corrected for eyeblinks and heartbeat-related artefacts using an independent component analysis approach implemented in the Fieldtrip toolbox .The preceding sensor-space analyses consider activity across the whole head and do not necessarily reflect the activity of the underlying cortex, but rather the summed activity from multiple sources across variable distances. Furthermore, they are susceptible to various unrelated magnetic fields, which can distort the neural activity of interest. Source-space analyses are preferable because they limit the analysis to activity that originates within auditory cortex. To estimate the source of focal oscillatory activity in various frequency bands, a spatial filtering technique using the adaptive beamforming method, details of which are given elsewhere (Robinson and Vrba z (pZ) values with each z being a normalisation of the estimated signal to the estimated sensor noise . Thus, single-state images of the ratio of source power to noise variance were generated for the masker and silence epochs, which resemble the z-deviate of cortical activity. Volumetric images were generated in 0.5 cm voxels within each frequency band, for the masker and silent epochs separately.To perform a direct test of the predictions made by the thalamocortical dysrhythmia model . Time–frequency wavelet maps were measured for each voxel virtual electrode to obtain an estimate of power over time in each frequency band. The same peak location was then used to compute time frequency maps for the masker epochs in order to observe oscillatory changes at the same location due to the presentation of the masker  power between the ‘TI with hearing loss’ and ‘no TI with normal hearing’ groups (t(34) = 2.33, p = 0.026). However, this was not significant in either the theta band (4–8 Hz) , or in the alpha band (8–13 Hz) . It is interesting to note that t-statistic comparisons between the ‘TI with normal hearing’ and the ‘no TI with normal hearing’ groups and between the ‘TI with hearing loss’ and the ‘no TI with normal hearing’ did not reach significance (p > 0.05). This pattern of null results is not confirmatory, but is at least consistent with the view that a combination of TI and hearing loss is necessary for the observed increase in delta-band activity. There was no difference between the groups in the gamma band for frequencies up to 80 Hz (p > 0.05).One of the main questions is whether the changes in oscillatory activity within different frequency bands are associated with TI or hearing loss. In accordance with the thalamocortical dysrhythmia model, we expected elevated slow-wave activity (in the delta and/or theta band) in ‘TI with hearing loss’ participants compared to the ‘no TI, normal hearing’ controls. Figure The results of spatio-spectral differences between groups in the delta, theta and alpha frequency bands are shown in Figure t(28) = 2.87, p = 0.007]. However, further analysis using a partial correlation that accounted for the degree of hearing loss, failed to reveal a significant relationship between TI attenuation and the difference in delta-band activity .To further investigate the observation made by Weisz et al. , 2007b tity Fig.   = 3.07, p = 0.004] and for theta .The specific predictions made from the thalamocortical dysrhythmia model were assessed using planned pair-wise comparisons. First, we expected elevated slow-wave (delta and theta) activity in the ‘TI with hearing loss’ group compared to the ‘no TI with clinically normal hearing’ controls. An independent t(42) = 2.30, p = 0.27]. Figure and hearing loss is needed to drive the abnormal increases in slow-wave activity. The thalamocortical dysryhthmia model also predicts that gamma activity should be enhanced as a correlate of the TI sensation. The independent t test did not reveal a significant difference between TI with hearing loss and no TI with normal hearing groups .If the slow-wave activity were a consequence of disrupted thalamocortical coherence due to deafferentation per se, then we would expect elevated slow-wave activity in people with hearing loss, even if no TI was reported. However, this was not supported by the data. Collapsing across participants with hearing loss, this contrast did not reach significance for delta . Eleven participants also showed a reduction in oscillatory power in the theta band as a result of TI masking, but this did not reach significance  for the group. For each participant, we extracted the time course of activity at the peak location corresponding to the pseudo-z peak  and performed a time–frequency analysis using a bootstrap technique  appear to be a prominent characteristic of TI. Weisz et al.  propose It has been suggested that gamma activity in the auditory cortex is necessary for the conscious perception of the TI sound  or such pronounced hearing loss that they were unable to hear the masking sound and had to be excluded from the MEG session. We thus acknowledge that this inequality in participant numbers may result in statistical analyses that are potentially underpowered. However, our results initially demonstrate that abnormal oscillatory activity occurs with TI regardless of hearing loss, and we would welcome further replications of the effects reported here.We would like to add a caveat regarding the classification of the ‘TI with clinically normal hearing’ group in this study, to which we also alluded in the introduction. One main purpose of this study was to carefully match groups on hearing level and assess changes in oscillatory power during silence when TI is perceived and when it is attenuated by the masker. However, it is possible that peripheral damage, especially at high frequencies, could be present, but not detected by routine audiometry. For example, Weisz et al.  used theOf the 21 TI participants who experienced TI masking, 7 had normal hearing, 3 of whom also showed an associated reduction in delta activity. This would indicate that TI masking could occur irrespective of hearing loss. The premise of the thalamocortical dysrhythmia model is that the abnormal thalamic dysrhythmia is primarily due to deafferentation, and thus, delta/theta activity should reflect this disruption in ‘communication’. The model proposed by Weisz et al.  also sugIn the light of these findings, we suggest that TI is likely associated some degree of damage to the auditory system and that TI with ‘normal hearing’ based on audiometric thresholds may be a misnomer. We suggest that even small changes in threshold (<20 dB HL), at any audiometric frequencies, can have implications for the onset of TI. Regarding the cortical oscillatory activity associated with TI, our results indicate that slow-wave activity in TI is associated with TI itself rather than deafferentation alone. Whether TI without hearing loss exists is debatable, and it is more likely that all TI sufferers have some degree of hearing loss at much higher frequencies and hence cannot be considered a distinct group. If hearing loss alone were sufficient for abnormal slow wave activity, those with no TI and hearing loss would also exhibit abnormally enhanced slow wave activity. Based on the results of our masking study, it may be that slow-wave delta/theta activity is a neurological correlate of TI. In this respect, it is plausible that a reduction of this abnormal activity might signify a reduction in the level or perceived severity of TI and could potentially be used as a valuable indicator of the course of TI treatment.


In single-photon emission computed tomography (SPECT), attenuation of photon flux in tissue affects quantitative accuracy of reconstructed images. Attenuation maps derived from X-ray computed tomography (CT) can be employed for attenuation correction. The attenuation coefficients as well as registration accuracy between SPECT and CT can be influenced by several factors. Here we investigate how such inaccuracies influence micro-SPECT quantification.Effects of (1) misalignments between micro-SPECT and micro-CT through shifts and rotation, (2) globally altered attenuation coefficients and (3) combinations of these were evaluated. Tests were performed with a NEMA NU 4–2008 phantom and with rat cadavers containing sources with known activity.125I) and <0.6%  for 1-mm shifts to <4.5% (125I) and <1.7%  with large misregistration (3 mm). Changes induced by 15° rotation were smaller than those by 3-mm shifts. By significantly altering attenuation coefficients (±10%), activity changes of <5.2% for 125I and <2.7% for 201Tl, 99mTc and 111In were induced. Similar trends were seen in rat studies.Changes in measured activities within volumes of interest in phantom images ranged from <1.5% (While getting sufficient accuracy of attenuation maps in clinical imaging is highly challenging, our results indicate that micro-SPECT quantification is quite robust to various imperfections of attenuation maps. Attenctively) . This mectively) . AverageIn clinical SPECT, problems caused by inaccurate attenuation maps have been thoroughly investigated. For example, Tonge et al. ,18 indicThe aim of the present study is to investigate the effects of attenuation map imperfections on the accuracy of micro-SPECT quantification. Two main sources of attenuation map inaccuracies were emulated: (1) misalignments between micro-SPECT and micro-CT images, e.g. as could result from misregistration or animal movement; and (2) global errors in the attenuation coefficients derived from micro-CT data, e.g. as could result from sub-optimal CT calibration.Animal studies involved in this study were conducted following the regulations approved by the Animal Experiment Committee of the Radboud University Nijmegen.The U-SPECT-II/CT system used in the present study . The activity was measured in a dose calibrator. Next, SPECT and fully circular CT (60 kV and 615 μA) were performed. Then, the same procedure was repeated for 201TlCl3, 111InCl3 and Na125I solutions, with activity concentrations of 5.90 MBq/ml, 5.91 MBq/ml and 4.54 MBq/ml, respectively. In addition, 12 small 99mTc sources  weighted by the number of photons detected under each energy E. When the TF for each voxel was estimated, the attenuation was compensated by dividing each voxel value by its corresponding TF [in which HU is the CT image intensity in Hounsfield units, and a tables . In casending TF .For each scan, an attenuation coefficient map was created from the registered CT image by linear scaling as in Equation 2 and then converted to a transmitted fraction map with the non-uniform Chang algorithm as described in Equation 1. To only evaluate the impact of inaccuracies in attenuation maps on the SPECT quantification, we considered these original attenuation maps to be accurate, and thus, we used the SPECT images corrected with these maps as reference images. Meanwhile, quantitative errors, i.e. differences between activities measured in images and in a dose calibrator, were also calculated for each reference image for reasons of comparison.x (towards the right side of the system) or + y (downwards) direction and the maps for animal studies in + x, +y or + z (towards the back side of the system) direction. The distances of shifting in each direction were set to 1 mm and 3 mm. All original TF maps were also rotated anticlockwise by 15° to emulate the consequences of animal movement during scans. All these shifted and rotated TF maps were used for attenuation correction of their corresponding SPECT images. Later on, we globally changed the attenuation coefficients in the attenuation maps by ±10% of the original values. This was to emulate errors introduced by CT itself and/or from converting CT values to attenuation coefficients. TF maps were again calculated with the non-uniform Chang algorithm from these altered attenuation maps and then used for correcting for attenuation in their corresponding SPECT images. As a final test, the TF maps calculated with the +10% attenuation coefficient maps were also rotated anticlockwise by 15° and shifted by 3 mm in the x direction. This way, we introduced a combination of ‘worst-case’ errors. These TF maps were then employed for attenuation correction. All images that were corrected with the abovementioned inaccurate attenuation maps were compared to the reference images.To investigate the effect of misregistration, we shifted the original TF maps for phantom studies in either + 125I, 201Tl, 99mTc and 111In separately.All the above operations were performed for 125I and 0.6% for 201Tl, 99mTc and 111In. When the shifts increased to 3 mm, the changes increased to less than 4.5% for 125I and 1.7% for 201Tl, 99mTc and 111In. With 15° rotation of the maps, the changes were less than 1.4% for 125I and 0.9% for 201Tl, 99mTc and 111In. When the attenuation coefficients were altered by 10%, the activity in the VOIs changed less than 5.2% for 125I and 2.7% for 201Tl, 99mTc and 111In. With the combination of rotation and shifts of the maps and altered attenuation coefficients, the VOI activity changed less than 6.2% for 125I and 3.0% for 201Tl, 99mTc and 111In. The quantitative errors of the reference images are listed in the first data column of Table Three 3 mm × 3 mm × 11 mm volumes of interest (VOIs) were defined in heterogeneous slices of each phantom image, as shown in Figure In addition, the normalized root mean square deviation (NRMSD) was calculated voxel-wise between the reference images and the images corrected with inaccurate attenuation maps:vi is ith voxel inside the phantom chamber in an image corrected with an inaccurate map, and vri is the corresponding voxel in a reference image. n is the number of voxels inside the phantom chamber. The results are listed in Table 125I, 201Tl, 99mTc and 111In, respectively.where 201Tl, 99mTc and 111In imaging (≤5%), while for 125I it was larger (≤10%). Rotation of attenuation maps had almost no effect in homogenous slices for all four isotopes: the line profiles perfectly overlapped the reference profiles. In heterogeneous slices, the profiles slightly deviated (1% to 2%) from the reference profiles only close to the edges of the air chambers. When altering the attenuation coefficients by ±10%, the induced changes were on average about 2% to 3% for 201Tl, 99mTc and 111In, and about 5% for 125I. Unlike shifting or rotating the maps, the changes in quantitation when the attenuation coefficients were altered resulted in global (but not uniform) over- or underestimations.Line profiles through homogenous and heterogeneous slices  were created for each small source. The sources were completely enclosed in the centres of their corresponding VOIs. Activities in the VOIs were measured in the images corrected with different attenuation maps. Comparisons were made between measurements in the reference images and in the images corrected with inaccurate maps. Changes in the measured VOI activities induced by map inaccuracies are plotted in Figure ribed in , are lis.[We previously have shown that quantitative differences between uniform and non-uniform attenuation correction are quite small in micro-SPECT . In this. showed t.,33. Ther125I (worst case approximately 10%), which can be attributed to 125I attenuation that is more prominent.For both phantom and animal experiments, the changes in image quantitation due to strong attenuation map inaccuracies were less than about 5%, except for 125I and about 2% to 3% for 201Tl, 99mTc and 111In. Although sharp attenuation and activity changes exist at the edges of the air chambers in the phantom, the changes in TF maps are rather smooth. As a result, quantification errors due to misregistration stayed also small in these regions. Quantitation changes caused by map rotation were smaller than the changes by shifts, and because the axis of rotation was closely aligned with the axis of the phantom, the changes only existed close to the air chambers. This may be relevant for quantification in e.g. tumours that are close to lungs or in cardiac imaging. These changes of measured activity concentration were consistent with the relative differences between the reference TF maps and the shifted or rotated maps, as shown in Figure In the phantom studies, quantitation changes due to attenuation map shifts were small inside the phantom, but increased close to the outer edge to a maximum of about 5% for With attenuation coefficients globally altered by ±10%, similar global over- or underestimation was observed in both phantom and animal studies. We also examined the relative differences between the reference TF maps and the ones derived from 10% increased attenuation coefficients. Figure 125I and 20% for 201Tl, 99mTc and 111In. Even the largest inaccuracies in attenuation maps investigated here effectively took away only one eighth of the benefit from the attenuation correction. Since accuracy of the attenuation coefficients and alignment between micro-SPECT and micro-CT in practice are usually better than in the worst situations emulated in the present study, effects of attenuation map inaccuracies on micro-SPECT quantification will usually be small.Without attenuation correction, activities were roughly underestimated by 40% for 201Tl, 99mTc and 111In, misalignments up to a few millimetres or in the order of 10° to 15° between micro-SPECT and micro-CT, and/or global attenuation coefficient inaccuracies in the range of ±10% have quite small effects on micro-SPECT quantification. Therefore, we conclude that micro-SPECT quantification is quite robust to imperfections in attenuation maps for most applications.For the more commonly used SPECT isotopes like CT: Computed tomography; NRMSD: Normalized root mean square deviation; SPECT: Single-photon emission computed tomography; TF: Transmitted fraction; VOI: Volume of interest.Freek J. Beekman is a stockholder of and gets honoraria and grant/research support from MILabs. Hugo A. Gratama van Andel is an employee of MILabs. The other authors declare that they have no competing interests.CW designed the study, performed the experiments, analysed and interpreted the data, and drafted and revised the manuscript. HAGvA analysed and interpreted the data and revised the manuscript. PL co-performed the experiments and revised the manuscript. OCB participated in the study design and coordination and revised the manuscript. FJB conceived of and designed the study, participated in its coordination and drafted and revised the manuscript. All authors read and approved the final manuscript.


The biosynthesis of 2-oxoglutarate and glutamate are key steps in the biosynthesis of nitrogen compounds and plant development. The reaction catalyzed by cytosolic isoenzyme of NADP+-linked isocitrate dehydrogenase (IDH) is also considered as the main route in the production of 2-oxoglutarate. According to its expression pattern during development, IDH is also involved in other, yet unknown, processes ,2. In ad


War is a known cause of tremendous physical injuries to different body organs, and eyes are not exceptions. War-related no visual acuity (NVA) affects both the victim and the family. Activity of daily living (ADL) can display personal life independency and is considered as a morbidity index. This study was designed to investigate the ADL profile of war survivors with NVA.This cross-sectional study was conducted in 2007 in Iran. In this study, 500 Iranian people with war related NVA were invited to take part in a camp in Mashhad city. ADL was evaluated using Barthel Index and demographic data were collected using a data sheet. Stepwise linear regression was used to determine the associates of ADL.The overall response rate to the invitation was 50%. From the total 250 participants 96.5% were male with a mean age of 43 ± 8 years. Only 8.3% had no dependency in ADL and other 91.7% had some ranges of dependency in at least one of the daily living activities. ADL score was higher in highly educated participants, those younger than 50 years old, those with less co-morbid physical problems (hearing loss) and those with regular physical exercises. According to regression analysis, age and duration of war related NVA were significant predictors of ADL.According to the results, both age and the time passed from war related NVA increase the dependency of people with war related NVA. Different physical injuries - affecting human health - can occur as consequence of war. Loss of visual acuity is by far one of these unwanted consequences of war. Bullets, bursts, burning and chemical agents all can cause severe visual impairment in people participating in wars.1Severe visual impairment can heavily affect people and their family. It decreases social participation.Different measures are used in assessment of being handicap followed by NVA.411131415This study was designed to investigate the ADL profile of people with war related NVA.This is a cross sectional study on Iranian people with war related NVA in 2007. Five hundred people with war related NVA were invited by Martyrs Foundation through provincial offices to take part in a camp in Mashhad city.Participants included 250 out of 500 survivors with NVA who accepted the invitation and came from all the provinces. There was no special exclusion criterion. The total number of people with war related NVA in Iran is 700.For the assessment of ADL, we used Barthel index, which is one of the most famous instruments for ADL study. Barthel index evaluates 10 aspects of daily life including feeding , bathing , grooming , dressing , bowels , bladder , toilet use , transfers (bed to chair and back) , can sit, 10 = minor help , 15 = independent), mobility (on level surfaces) , and stairs . Total score ranges between 0 and 100.16Demographic data were collected through a data sheet. Data gathering team was trained just before the process in the study field. The mean time for each part of the assessment was about 15 minutes. Every data sheet and questionnaire was checked on the site and data cleaning was done in the field of the study.Statistical analysis including stepwise regression, independent sample t test, ANOVA, Chi square and Tukey were done by a professional statistician.Out of 500 people invited for the event, 250 (50%) agreed to participate. The mean age was 43 ± 8 years. The period of NVA ranged from 1 to 29 years, but 79.2% were afflicted to NVA for more than 20 years.Among the participants, 96.5% were male, 80.9% were jobless, 94.4% were married, and 31.1% had an undergraduate degree. The main frequent cause of NVA was explosion (98%). Only 18.7% reported their NVA as their single impairments, and others had at least one other co-morbidity. The most common injury was burst hit to face. Independency in ADL was reported by 8.3%, while 91.7% were dependent at least in one daily life activity. Highly educated participants had higher ADL scores (p = 0.012). Number of injuries was associated with ADL (p = 0.034). Age of 50 years was a marginal level for ADL independency decline. Co-morbid injuries significantly declined ADL score.According to the regression analysis, age and duration of NVA were significant predictors of ADL. According to the results of this study, age and the time passed since NVA both increase the dependency of people with war related NVA.th century due to decrease of birth rate and increase of life span, so that the population of people over the age of 65 during 2010 to 2030 would be significantly higher than previous decades. As the process of ageing continues, most of the fatal factors will change to disability factors. Ageing accompanied by disability makes the situation worst and requires more rehabilitation services to live independently especially in ADL.19The mean age of people in the world has increased within the second half of the 20According to the literature, ADL declines following NVA, and some believe that there is no relation between the cause of NVA and lack of health related quality of life (HRQOL) and ADL independency.2021NVA is a cause of not only morbidity (as presented here), but also mortality. NVA increases the risk of death by 1.4 times, apart from any complication and dependency occurring after NVA. If the functional problems are included in this model, the risk of death for this group will increase 38.1% more than general population. Therefore, prevention is more cost-efficient than service providing, although rehabilitation services can be effective and helpful in current situations.22There is no accurate information about the burden of NVA in developing countries, and nor in Europe.27Similar to other types of NVA, war related NVA is a costly phenomenon. Rehabilitation and care services for one NVA patient may cost annually between 9749 to 26720 dollars.30Some studies presented other classifications. For example, if there were some problems in mobility, bladder control, bowel and bathing, there would be major difficulties in ADL.32According to the results of the current study, 91.3% of the survivors with NVA had some dependency. Ageing decreases ADL. In a study in the United States on people with mean age of 72 years, 32% were dependent for bathing, 37% for clothing, 11% for feeding and toilet use, 10% for bed to chair transfer, 55% for transfer to stairs and 13% for mobility on ground level surfaces. A study conducted by Brench and Horwitz compared two groups of with and without visual impairment old people (over 66 year). They found that visual impairment can reduce ADL and lead to more dependency as compared with those without visual problem, especially in physical and emotional daily living.3133According to this study, the war survivors with NVA face many serious problems in ADL and these problems increase with ageing due to some physical impairments and disabilities.A study that compared ADL in two groups of physically impaired people with and without visual problems found that visually impaired people had significantly lower ADL scores and a periodic rehabilitation was suggested for them. It shows that rehabilitation is really important to NVA people.2237Nowadays, most of the rehabilitation programs aim to improve functional status.38To conclude, age and duration of NVA can deteriorate the dependency of people with war related NVA. The researchers hope that this study can be a baseline for ADL studies on war survivors with NVA. The results can yield intervention studies and increase their independency.


P < 0.05) in male and female. Results from this study demonstrate the usefulness of NHHI questionnaire for evaluating hearing handicap of aged people and it may be a useful adjunct in setting up treatment and determining proper care.Hearing evaluation usually includes hearing threshold assessment, middle ear function, and word recognition tests that lead to an accurate result of peripheral and central auditory system. However, they have some limitations because they cannot fully encompass all aspects of hearing loss problems. Using self-assessment approach, via a questionnaire or telephone survey, is one of the easiest methods to study hearing loss in population. In this research, 60 nursing home residents  ranging from 55 to 85 years with a mean age of 71 ± 5.5 were studied via completing self-assessment questionnaire by the elderly cases (NHHI self-version) and the other one was filled by the nursing home personnel (NHHI staff-version). The effects of the hearing loss level on the self- and staff-version scores indicated that there is a significant relationship between self- and staff-version with hearing loss levels ( Hearing evaluation usually includes hearing threshold assessment, middle ear function, and word recognition tests that lead to an accurate result of peripheral and central auditory system –3. HowevP < 0.05 was considered statistically significant.60 nursing home residents  ranging from 55 to 85 years with a mean age of 71 ± 5.5 were selected. After case history and otoscopy examination, pure tone audiometry test was conducted via Madsen OB822 audiometer to identify hearing loss, and tympanometry test was performed by Interacoustics AT235H tympanometer to rule out any middle ears disorders. Then NHHI questionnaire  was compHearing loss level data is found in The analysis of questionnaire results showed that the average score of self-version part was 33.80% ± 27.01 and the average score of staff-version was 34.54% ± 26.48. These results are listed in P < 0.05). In other words, the severity of hearing loss becomes greater as the questionnaire score goes up and followed by hearing handicap is also increasing in elderly. This relationship is not significant in male group (P > 0.05) however it is significant in the female group (P < 0.05) in the self-version. On the other hand, the relationship between the hearing loss levels and staff-version is significant in both men and women groups (P < 0.05) as can be found in The effects of the hearing loss level on the self- and staff-version scores indicated that there is a significant relationship between self- and staff-version with hearing loss levels  of <0.05 as shown in In testing the relationship between NHHI results and audiological findings by using Chi-square and Pearson's correlation coefficient, the results revealed significantly correlated with each other at a statistically significant level (P < 0.05) between both versions with hearing loss levels and as other studies suggested [Outcomes from a hearing handicap scale can give more information about the patient's understanding of hearing loss effects on daily life. If the resident reports a considerable handicap, then auditory rehabilitation will be suggested. Nursing home staffs that are close to resident can also complete a hearing handicap scale. Results can provide essential information on how others distinguish the resident's hearing loss and audiological rehabilitation necessitate. In the present study, we used NHHI questionnaire and compared its results with PTA findings. Moreover, we investigated the correlation of hearing impairment severity on NHHI (two versions) scores in nursing home elderly. Because of significant Pearson's correlation coefficient results (uggested –19 it caResults from this study demonstrate the usefulness of NHHI questionnaire for evaluating hearing handicap of aged people and it may be a useful adjunct in setting up treatment and determining proper care.In spite of searching for new resources in this field and no newer-revised version of the NHHI questionnaire, references might be somewhat old, but nevertheless they are reliable in this area.


Hearing and touch are genetically related, and people with excellent hearing are more likely to have a fine sense of touch and vice versa. USH2A gene influence touch acuity. Control Usher syndrome cohorts lacking demonstrable pathogenic USH2A mutations showed no impairment in touch acuity. Our study thus provides comprehensive evidence that there are common genetic elements that contribute to touch and hearing and has identified one of these genes as USH2A.In all vertebrates hearing and touch represent two distinct sensory systems that both rely on the transformation of mechanical force into electrical signals. There is an extensive literature describing single gene mutations in humans that cause hearing impairment, but there are essentially none for touch. Here we first asked if touch sensitivity is a heritable trait and second whether there are common genes that influence different mechanosensory senses like hearing and touch in humans. Using a classical twin study design we demonstrate that touch sensitivity and touch acuity are highly heritable traits. Quantitative phenotypic measures of different mechanosensory systems revealed significant correlations between touch and hearing acuity in a healthy human population. Thus mutations in genes causing deafness genes could conceivably negatively influence touch sensitivity. In agreement with this hypothesis we found that a proportion of a cohort of congenitally deaf young adults display significantly impaired measures of touch sensitivity compared to controls. In contrast, blind individuals showed enhanced, not diminished touch acuity. Finally, by examining a cohort of patients with Usher syndrome, a genetically well-characterized deaf-blindness syndrome, we could show that recessive pathogenic mutations in the  In humans many genes have been identified that cause deafness when mutated, but no equivalent genes have been identified that are required for touch. Here, we asked whether genes that influence hearing can also influence touch. Using identical and non-identical human twins it was possible to show that touch performance is substantially influenced by genes. Furthermore, people who have excellent hearing are more likely to have a fine sense of touch and vice versa. Interestingly, people who suffer from congenital deafness have a higher chance of having poor touch performance. In a genetically defined form of human deafness, Usher syndrome type II, a single mutated gene was identified that also impairs touch. Touch and hearing are thus intricately intertwined and there may be other touch/hearing genes waiting to be discovered. Non-syndromic sensorineural hearing loss is commonly caused by single gene mutations, which primarily affect the function of the sensory hair cells that detect movement of the basilar membrane induced by sound. Sensorineural deafness often manifests from birth and some of the responsible genes encode components of the mechanotransduction apparatus of the hair cell that transforms mechanical force into electrical signals All animals are equipped with a range of specialized sensory cells whose prime function is to detect mechanical force. The most familiar of these sensory systems are hearing and touch, but mechanosensory cells also detect important stimuli that are not consciously perceived, for example, changes in blood pressure. We reasoned that since the prime function of different sensory cells is to detect mechanical force they may utilize a common set of mechanosensory proteins for this function. According to this hypothesis genetic variation affecting the function of mechanosensory proteins would be predicted to quantitatively change more than one mechanosensory trait. Genetics has been very successfully used to characterize new molecules that are essential for human hearing NTRK1; MIM:191315, NGFB; MIM:162030, and SCN9A; MIM:603415), mutation of which is known to cause this condition C. elegansStoml3 mutations are causative for impairments in human touch is so far lacking. But assuming that touch sensitivity is a complex genetic trait, it should be possible to detect a heritable component in the normal variation of touch-related traits, as has been shown before for other sensory traits, such as pain sensitivity and hearing In contrast to hearing, virtually nothing is known about the genetics of touch. Indeed there are, to our knowledge, no reported cases of non-syndromic reduced or absent touch sensitivity present from birth in humans. Impaired detection of high frequency vibration (>80 Hz) in humans was recently shown to be associated with pathogenic mutations in the transcription factor c-Maf  and may be due to a failure in the development of specific mechanoreceptors associated with Pacinian corpuscles USH2A gene (MIM:608400), but not in other cases of Usher syndrome. Our study thus provides comprehensive evidence that there are common genetic elements that contribute to touch and hearing and has identified one of these genes as USH2A.Here we show that there is a significant genetic component to touch sensitivity in humans by determining the heritability of touch traits, assessed by quantitative sensory testing, in a classical twin study. In accordance with our hypothesis that there are common genetic factors underlying different mechanosensitive systems, we found that quantitative measures of mechanosensory traits—that is, touch acuity, hearing acuity, and baroreflex function—are positively correlated with each other in a healthy human population. We also examined a cohort of people suffering from congenital hearing loss and found touch sensitivity to be poorer in these individuals compared to a control cohort. To investigate the role of single sensorineural deafness genes in cutaneous touch sensitivity, we assessed touch acuity and sensitivity in people suffering from Usher syndrome. We found touch sensitivity to be impaired in a cohort of individuals carrying pathogenic mutations in the n = 17). The other tests employed in this study were not analyzed by us for test-retest reliability, but data showing good reliability for pure tone audiometry In this study we employed a range of quantitative tests to assess sensory function in a large cohort of volunteers  . In most cases the changes in the sensory trait was best fit by a second order polynomial function, in two cases the age dependence was best described by a linear fit (baroreflex sequence frequency and heat pain threshold), and in one case (baroreflex slope) the data were best fit by an exponential decay function. Here with increasing age the baroreflex sequence slope tends to become shallower, which reflects weaker engagement of the reflex by changes in blood pressure. For subsequent analyses the phenotypes measured were normalized on the basis of the mathematical fits to the mean age of the entire cohort.In general it has been noted that sensory performance decreases with age and is also influenced by sex Of the 13 sensory traits measured, significant sex differences were found for six traits, and in every case women performed better than men when age-matched cohorts were compared . The sen2h of the investigated sensory traits—that is, the component of the variation of the respective trait that can be accounted for by additive genetic effects—was determined in a classical twin study. Of the 100 twin pairs who participated in the study, 38 were monozygotic female pairs, 28 monozygotic male pairs, 25 dizygotic female twin pairs, and 9 dizygotic male twin pairs; no mixed sex twins were recruited. The ages of the twins in our cohort ranged from 18 to 68 years, with a mean age of 29.7±1.14. Zygosity was confirmed using a chip-based method with 9,080 informative autosomal SNPs derived from the so-called Immunochip The heritability For both touch sensitivity traits, vibration detection threshold, and tactile acuity, cross-twin correlations were more than twice as strong in monozygotic twin pairs compared to dizygotic twin pairs . Of the For all three hearing traits the cross-twin correlations were more than twice as strong in the monozygotic twin pairs compared to dizygotic twin pairs, with high overall correlations . The herThe cross-twin correlations for the baroreflex traits were higher in the monozygotic twin pairs than in the dizygotic twin pairs . SignifiThe cross-twin correlations for all temperature sensitivity traits, except cold pain threshold, were higher in the monozygotic twin pairs than in the dizygotic twin pairs . Signifir) was surprisingly low, but ranged from r = 0.21 for VDT and tactile acuity to r = 0.65 for otoacoustic emission strength against otoacoustic emission reproducibility (r = −0.23) as well as between cold pain and heat pain threshold (r = −0.60). We then reasoned that if a group of gene variants positively influences one trait like touch, they may also positively influence another mechanosensory trait in which they also play a functional role . Put simply the question is, If someone has good hearing, are they also more likely to have good touch sensitivity? We made such comparisons for all the phenotypic parameters measured from our twin cohort as well as from an additional cohort of healthy individuals. Significant intermodal correlations between mechanosensory traits were detected between tactile acuity and hearing acuity with r = 0.16 (p<0.05) and tactile acuity and EOAE reproducibility with r = −0.16 (p<0.05). Additionally, we noted a significant intermodal correlation between EOAE strength and baroreflex sequence frequency  +measured male value. After the raw data were adjusted in this way and intermodal correlations tested again, the only statistically significant correlation that remained was between tactile acuity and hearing acuity with r = 0.15 (p<0.05) . As a prerequisite for an intermodal correlation, one should observe strong correlations between different measures of one sensory system, such as between tactile acuity and vibration detection threshold . This was indeed the case as all measures of one sensory system showed a significant correlation with each other, although in many cases this correlation coefficient (cibility . Notablerequency . There w p<0.05) . It is p(p<0.05) . The facp<0.001; t test) in the control cohort . In both cases it appeared plausible from the distribution of individual values that the difference was primarily due to the presence of a subset of individuals with an exceptionally poor touch performance in the hearing impaired cohort . Comparing these data from the young cohort to our data from the hearing impaired individuals revealed that both measures of touch sensitivity were significantly different from each other .We asked if touch sensitivity might be affected in some forms of hereditary hearing loss. We tested touch sensitivity in a cohort of individuals aged between 14 and 20 years who were recruited for this study at a school for the hearing impaired. In total, 39 individuals were assessed for tactile acuity and 29 of these individuals were also tested for vibration detection threshold. All the participants suffered from severe congenital hearing impairment or hearing loss. It has been estimated that in around 70% of individuals suffering from severe hearing impairment from birth, there is an underlying genetic lesion d cohort . Thus ofUSH2A mutations (n = 18), individuals with only one identified USH2A mutant allele (n = 18), as well as individuals with clinically diagnosed Usher syndrome type 2 in which no genotype has been determined (n = 29). Interestingly, we observed that the mean tactile acuity threshold was significantly elevated in patients proven to carry compound heterozygous or homozygous pathogenic mutations in the USHA gene; thus tactile acuity was 1.88±0.14 mm (n = 19) compared to 1.63±0.02 mm for controls  (n = 17) was also significantly elevated, , we found no evidence of impaired vibration sensitivity  and found that tactile acuity in this mixed cohort was slightly attenuated .Our data so far strongly suggested that genetic factors that influence hearing may also influence touch. Ideally one would like to identify such genes by measuring touch performance in patients with single gene mutations that cause deafness and decrease touch sensitivity. We decided to recruit patients with Usher syndrome to participate in our study. Usher syndrome is characterized by early onset deafness with late onset retinitis pigmentosa leading to tunnel vision and blindness and can be classified into three clinical sub-types, with type 1 (USH1) typically being the most severe and type 3 (USH3) the least severe. There are nine known Usher genes, mutations in which cause the disease  t test) . In addisitivity . This Ussitivity . We analUSH2A mutations (n = 15), we also routinely tested temperature sensitivity traits. We found that warmth and cold detection thresholds as well were not significantly altered in these patients compared to the control cohort . When we examined temperature traits in patients with two or just one genotyped USH2A mutant allele (n = 32), we noted that this cohort performed significantly better in the warmth detection task ; all other temperature traits did not differ from controls. It should be noted that the effect on warmth detection was only apparent in a mixed cohort in which the second mutation was not always known; thus mutations in genes other than USH2A might conceivably cause this effect. Usher type 2 patients for which the underlying mutation was not known showed normal performance in the temperature detection task (unpublished data).In Usher patients with compound heterozygous or homozygous pathogenic l cohort . Heat pan = 57) was recruited at an occupational training center for blind people and the cause of blindness was sometimes presumed to be genetic but in many individuals had been caused by accidents or other non-genetic causes. The severity of visual impairment varied in the tested individuals, but was in all cases so severe that the test persons were using the Braille system to read. In agreement with previous studies of tactile acuity we found that the mean acuity of the index finger was significantly better in the blind group p<0.001, t test)  . In the  t test) . The enhUSH2A or Usherin, mutations in which are associated with poor touch acuity as well as congenital hearing loss and adult onset blindness. Patients with Usher syndrome in which the underlying mutation was not known did not show reduced touch performance. As well as identifying a gene that influences both hearing and touch acuity, we can conclude that there is probably a larger set of as yet unidentified genes that influence both touch and hearing. The functional characterization of such genes will help in the molecular characterization of normal and abnormal touch sensation.We show here using two psychophysical measures that touch performance varies considerably in the normal human population. Importantly, we could show in a classical twin study that a large part of touch performance variability (between 27% and 52%) can be accounted for by genetic factors. We also confirmed and extended published work showing that human performance traits that depend on mechanosensory systems (e.g. hearing and baroreflex sensitivity) are also partly genetically determined 2h = 0.27) and vibration detection threshold (2h = 0.52), are heritable .Classical twin studies are used to estimate the heritable component that contributes to phenotypic variation in complex traits. We show that two measures of touch sensation, tactile acuity , USH1C (MIM:605242), CDH23 (MIM:605516), PCDH15(MIM: 605514), SANS (MIM: 607696), USH2A, VLGR1(MIM: 602851), WRHN (MIM: 607928), USH3A(MIM: 606397), and PDZD7(MIM: 612971) USH2A  USH2A . USH2A iUSH2A is an extraordinarily large gene consisting of 72 exons, which can encode a protein with a length of 5,222 amino acids USH2A gene and so it is conceivable that mutations in this gene may have differential effects on protein products expressed in different tissues . Most patients in which one pathogenic USH2A mutation has been identified are probably also carriers of a second mutation in the same gene USH2A mutations also exhibited impaired touch sensitivity, although this difference was not statistically significant. Interestingly, many individuals with Usher syndrome type II in our study did not exhibit impaired touch acuity, although previous analyses of large populations of these patients have provided estimates that the majority (up to 70%) may carry pathogenic mutations in the USH2A gene USH2A was known performed on average better in the touch acuity test than controls . Finally blood samples were taken. Audiometry testing was carried out by trained personnel of the Clinic for Audiology and Phoniatrics of the Charité–Berlin. All other testing was carried out by the same investigator. The entire testing procedure for one twin pair typically lasted about 4 h.Vibration detection thresholds were determined using the CASE IV system  Tactile acuity was determined with a two interval forced choice grating orientation determination test using the Tactile Acuity Cube. In the tactile acuity test a transformed-rule up and down method was applied Audiometry was carried out in the Klinik für Audiologie und Phoniatrie, Charité–Universitätsmedizin Berlin employing the standard procedures for clinical use. For hearing acuity the pure tone thresholds in decibels  at 0.5, 1, 2, and 4 kHz were determined using a ST36 Audiometer (Maico) and the mean calculated. The otoacoustic emissions were measured using an OAE (Otodynamics). Otoacoustic emissions were evoked by 1 ms clicks spanning a frequency range from 0–6 kHz. The measured parameters were the reproducibility of the frequency distribution of consecutively evoked emissions in percentages and the overall intensity of the emissions in dB (SPL).Studies were conducted with the participant in a supine body position. Five-minute recordings were obtained after 10 min of rest. Blood pressure (BP) was measured in the left arm by automated oscillometric device (Dinamap) as well as continuously by Finapres (Ohmeda) BP monitor attached to the middle finger of the right hand. The participant's right hand was kept at heart level. ECG was recorded continuously. Data were analog-digital converted (both channels at 1 kHz), peak detection , and subsequent analyses were done using the PV-wave software . Sequences of at least three coupled minimum steps of 0.5 mmHg BP changes and 5 ms RR–interval changes with minimum correlation coefficients of 0.85 were detected and their slopes taken as the baroreflex sensitivity in ms/mmHg. Blood pressure levels were allowed for by regression for both baroreflex sequence slope and frequency before analysis of heritability or phenotypic correlations.Temperature sensitivity was determined using the TSA-II System  according to the manufacturer's instructions. The thresholds were determined using the ascending method of limits. A peltier thermode (3 cm×3 cm) was placed in the middle of the volar forearm. The baseline temperature for all four tests was 32°C and the temperature change rate was 0.5°C/s. In the temperature change detection tests, the test persons indicated when they felt a change in skin temperature. For warming and cooling ramps, the mean of four thresholds was calculated. In the temperature pain threshold tests, the test persons indicated when a rising or falling temperature became painful. Here the mean of three thresholds was calculated.2h) estimates were calculated by structural equation modeling using the Mx software Heritability  Immunochip array Figure S1Age dependence of touch sensitivity traits. The vibration detection threshold (A) and the tactile acuity (B) showed strong age dependence, with a lower sensitivity in older participants. Solid lines are regression lines. Equations of the regressions are listed in (EPS)Click here for additional data file.Figure S2Age dependence of hearing traits. Performance in all three traits deteriorates with increasing age, especially hearing acuity (A). Solid lines are regression lines. Equations of the regressions are listed in (EPS)Click here for additional data file.Figure S3Age dependence of baroreflex traits. The baroreflex sequence slopes showed a strong age-dependence decrease (A), whereas the baroreflex frequency seems to be unaffected by age (B). Solid lines are regression lines. Equations of the regressions are listed in (EPS)Click here for additional data file.Figure S4Age dependence of temperature sensitivity traits. All temperature traits showed age dependence, with lower sensitivity in older participants. Solid lines are regression lines. Equations of the regression are listed in (EPS)Click here for additional data file.Figure S5ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.Sex comparison of sensory traits. Tactile acuity, otoacoustic emission reproducibility and strength, baroreflex sequence frequency, as well as the cold and warmth detection thresholds showed a significant difference in the sex comparison , whereas the other traits did not. Sensitivity or parameter magnitude was higher in female participants in all cases. (EPS)Click here for additional data file.Figure S6ns, not significant * p<0.05; ** p<0.01; *** p<0.001. False discovery rates for p value cutoff at 0.05 for (1) intramodal correlations: 0.004 and (2) mechanosensory intermodal correlations: 0.28. Values were corrected for age before analysis.Cross-correlations between the investigated sensory traits with sex-dependent values adjusted for sex. Only one intermodal significant intermodal correlation between tactile acuity and hearing acuity remains after sex correction. (EPS)Click here for additional data file.Figure S7USH2A mutations . Mean cold and warmth detection thresholds were not significantly different in the USH2A defective cohort compared to controls, but heat pain thresholds were significantly lower in this cohort. ns, not significant, * p<0.05.Temperature sensitivity of patients suffering from Usher syndrome type II with compound heterozygous or homozygous pathogenic (EPS)Click here for additional data file.Table S1Trait–age correlations and regressions used for age correction.(PDF)Click here for additional data file.Table S2Sex comparison of sensory traits.(PDF)Click here for additional data file.Table S3Summary of cross-twin correlations and heritability estimates of investigated sensory traits.(PDF)Click here for additional data file.Table S4USH2A gene of the people tested for touch sensitivity and the corresponding tactile acuity thresholds.Individual mutations in the (PDF)Click here for additional data file.Table S5Conversion table for just noticeable difference to peak-to-peak amplitude in µm.(PDF)Click here for additional data file.