Triclosan (TCS) is a multi-purpose antimicrobial agent used as a common ingredient in everyday household personal care and consumer products. The expanded use of TCS provides a number of pathways for the compound to enter the environment and it has been detected in sewage treatment plant effluents; surface; ground and drinking water. The physico-chemical properties indicate the bioaccumulation and persistence potential of TCS in the environment. Hence, there is an increasing concern about the presence of TCS in the environment and its potential negative effects on human and animal health. Nevertheless, scarce monitoring data could be one reason for not prioritizing TCS as emerging contaminant. Conventional water and wastewater treatment processes are unable to completely remove the TCS and even form toxic intermediates. Considering the worldwide application of personal care products containing TCS and inefficient removal and its toxic effects on aquatic organisms, the compound should be considered on the priority list of emerging contaminants and its utilization in all products should be regulated. Triclosan  phenol) is a synthetic, broad-spectrum antimicrobial agent. It has antibiotic and antimycotic properties . Triclosow value of 4.76 for TCS suggests high sorption potential and it adsorbs onto the settled sewage sludge [The worldwide annual production of TCS in 1998 was approximately 1500 tonnes, out of which about 350 tonnes and more than 450 tonnes were utilized in Europe and USA, respectively ,7. Such e sludge ,11 whiche sludge ,13. Thuse sludge . The cheCurrently, TCS and its degraded byproducts are found throughout the environment, including soil, surface waters, and human breast milk ,16,17,18Similar antimicrobial activity of TCS to antibiotics and its toxicity data demand regular monitoring of its concentration in the environment, along with its safe and regulated use in the consumer products. This article provides a comprehensive literature review on TCS, its occurrence in wastewaters, biosolids, aquatic and terrestrial environment, its removal potential, toxicity levels in humans, wildlife and other aquatic organisms, its bioaccumulation potential and intermediate products. The review also addresses the research gaps in concerns related to long term exposure to TCS.ow values, the hydrophobic substances may adsorb onto settled sludge during primary sedimentation step in WWTP. The different physico-chemical characteristics of TCS governing its removal efficiency in conventional activated sludge treatment plants are given in ow. The high Kow value of TCS is also indicator of its bioaccumulation potential. Another important property governing the removal of organic substances is their volatility. Triclosan is also non-volatile (5.3 × 10−4 Pa at 20 °C) and is moderately soluble in water (10 mg·L−1 at 20 °C). Moreover, it does not hydrolyze easily [c) ≥ 10−3 atm·mol−1·m−3 will easily be removed by volatilization. Hence, the volatilization losses of specific substances during wastewater treatment can be predicted based on Henry’s constant value and Hc/Log Kow ratio [The removal of organic substances, such as TCS after release into environment depends on various physico-chemical properties of the compound. For instance, the sorption of organic compounds on sludge during wastewater treatment processes plays an important role. Depending on their log Ke easily . Normallow ratio .TCS is a chlorinated phenoxyphenol with a pKa of 8.1 and is photodegradable into its photostable phenolate form . The pheGenerally, TCS comes in the form of white powder. TCS has a weak aromatic, phenolic scent as it is a chlorinated aromatic compound. Ever since its invention, TCS has been widely used in numerous consumer products as illustrated in The concentration of TCS recommended by various government agencies to be used in various consumer products is given in According to the FDA monograph for health care antiseptic drug products, which covered antibacterial soap products containing TCS, the recommended limits are up to 1% TCS for use in antiseptic washes and surgical hand scrubs in health care settings . AccordiPlasmodium falciparum and Toxoplasma gondii [TCS possesses a broad range of antimicrobial activity that encompasses several, types of non-sporulating bacteria and a few fungi, such as a gondii ,34. At la gondii . In consStaphylococcus aureus (MRSA) outbreaks in several clinical settings using TCS based products [S. aureus and antibiotic susceptibility or resistance [Studies carried out by FDA found that TCS-fluoride paste prevented tooth deformities, such as gingivitis, tartar and plaque in a way that was superior to fluoride-only toothpastes. Over the last 30 years, TCS has also been successfully used as an antimicrobial agent in hospitals and for other biomedical purposes. The successful control of methicillin-resistant products ,37. Thisproducts . Howeverproducts . Later osistance .The American Medical Association (AMA) has raised concerns about the use of TCS and some other antimicrobial agents in consumer products . The AMAin vitro exposure to increasing TCS quantities and the consequent development of resistant colonies [The overuse of anti-microbial products may lead to increased resistance among bacteria. Considering the published studies, there is a dilemma whether TCS does or does not encourage the development of antibiotic resistance. Triclosan-resistant bacteria can be produced readily by their colonies .et al. [E. coli isolates to TCS. Various studies demonstrated the development of microbial resistance following exposure to TCS [et al. [P. aeruginosa following TCS exposure, resulting in high-level resistance to TCS and the antibiotic, ciprofloxacin. In E. coli, resistance can be attributed to either overexpression of the TCS target enzyme enoyl reductase or to changes in cellular permeability [E. coli resistant to TCS actually possess enhanced growth rates. The intrinsic resistance of P. aeruginosa to TCS can be attributed to: (1) a non-susceptible enoyl reductase; (2) an outer membrane permeability barrier or; (3) pumping of the drug from the cell interior to its exterior [P. aeruginosa. MRSA strains, meanwhile, may or may not show decreased sensitivity to triclosan [et al. [S. aureus strains with decreased sensitivity overproduced the enzyme Fab I by 3–5 fold and moreover, mutations in Fab I were found in the most resistant strains.The mechanism of microbial resistance to TCS has been described by various researchers ,44. Accoet al.  found the to TCS ,47. Reis [et al.  describeeability . The mosexterior . The latexterior ,52 in P.riclosan ,53. Stud [et al.  demonstrFab I among Gram-positive and Gram-negative bacteria and Inh A in Mycobacterium e.g., M. smegmatic and M. tuberculosis) involved in fatty acid biosynthesis are the targets for a number of structurally unrelated drugs, including TCS. For instance, isoniazid an antibiotic used to treat tuberculosis that targets the same enzyme system [Major concern is that the mode of action of TCS and its target site in the microbes is similar to antibiotics. The enzymes enoyl reductase  Act, 1989 (the Act) in the Chemical Gazette of 6 May 2003 [Triclosan possesses broad-spectrum antimicrobial action and has been classified as a Class III drug (compounds with high solubility and low permeability) by FDA . Due to May 2003 . Some siMay 2003 ,72. StudAbsorption, distribution, metabolism and excretion are rapid in the case of TCS in human body. TCS is metabolized to glucuronide and sulfate conjugates (phase II metabolism) and are primarily excreted via urine. These hydrophilic conjugates of TCS limit the bioaccumulation of TCS. Some studies indicated that TCS is comparatively non-toxic to humans and other mammals. Conversely, studies indicated that TCS exposure resulted in contact dermatitis, or skin irritation . A photoTriclosan has been found in urine, plasma, and breast milk of humans ,20,75,76et al. [et al. studies [2O to produce significant quantities of chloroform, a probable human carcinogen [Another area of concern is related to the hypothesis that TCS augments the production of chloroform. A study carried out by Fiss et al.  describeet al.  showed t studies  revealedrcinogen .−1 TCS was associated with induction of the expression of the metamorphic genes in tadpoles, which induced their premature metamorphosis [et al. [The toxic effects of TCS were also studied in various animal models. For instance, its negative effect on the metabolism of thyroidal hormones causes hypothermia and an overall depression of the central nervous system (CNS) of mice . The exporphosis . Similar [et al.  interrelet al. [et al. [Its close structure resemblance to certain estrogens triggered masculinization of secondary characters in rice fishes . A recenet al.  pointed et al. . Applicaet al. . However [et al.  found thet al. [Studies have also demonstrated that TCS accumulates in mice tissue with bioaccumulation factors of 3700–8400 . This daet al.  demonstrScenedesmus valuolatus under realistic exposure conditions [−1 with EC50 values for crustaceans , insects , fish , higher plants (Lemna gibba growth inhibition at 62.5 μg·L−1) and microalgal species . Moreover, the standard test organism, Selenastrum capricornutum (growth inhibition at 4.7 μg·L−1) was reported to be 30-fold more sensitive to TCS than the bacterium Vibrio fischeri (bioluminescence inhibition at 150 μg·L−1) [The structure and the function of algal communities in ecosystems receiving treated wastewater effluent may be affected by TCS contaminated wastewaters . These anditions . Variousnditions ,93,94,95 μg·L−1) . The mic μg·L−1) ,94,97. W μg·L−1) . TCS hasAll toxicity studies on TCS highlight the risks and suggest ban on TCS usage. In consequence, the FDA proposed, for comprehensive assessment of TCS toxicity on human health and animals, to regulate its further usage in consumer products until more information is available. Even though this proposal does not include environmental fate of TCS, this factor should be included in complete profiling of any chemical introduced into consumer products. In this sense, in 2010, more than 80 organizations petitioned EPA to ban TCS usage beyond pesticides. Minnesota has banned sale of any cleaning product (soaps) that contains triclosan on 16 May 2014. This ban makes the most manufacturers to phase out triclosan until early 2017. In 2013, FDA announced that final action on TCS usage in soaps will be taken by 2016 across the world. To complete the North American scenario, in Canada, approximately 1730 products including personal care products, cosmetics and health products containing triclosan were reported in 2011. Some reports indicate that triclosan would be a wide ranging contaminant in Canada. Therefore, from 2015 on, Health Canada is in the process to ban TCS.−1; sea water <0.001–100 ng·L−1; sediment (lake/river/other surface water) <100–53,000 μg·kg−1 dry weight (dw); sediment (marine) 0.02–35 μg·kg−1 dw; wastewater influent 20–86,161 ng·L−1; wastewater effluent 23–5370 ng·L−1; biosolids from WWTP 20–133,000 μg·kg−1 dw; activated/digested sludge 580–15,600 μg·kg−1 dw; pore water 0.201–328.8 μg·L−1 [Incomplete removal of TCS from WWTPs and the applications of TCS laden biosolids into agricultural soils, leads to TCS being distributed in aquatic and terrestrial environment. 8 μg·L−1 .−1) was found in one of the 22 drinking water samples from Barcelona [Triclosan is commonly detected in aquatic and terrestrial environments ,98,99. Tarcelona . Althoug−1 [−1 with effluent concentrations ranging from 0.027 to 2.7 μg·L−1 [et al. [The efficiency of WWTP for TCS removal has been observed with an average median removal efficiency of 90% ,107. TCS−1 . These r7 μg·L−1 ,108,109. [et al.  studies −1 [−1 are still half of the lowest-observed-effect concentration reported for some fishes. However, taking into account that triclosan is in use since only few years and its derivatives are much toxic and very persistent, the regulations reducing its use seem to be the most accurate decision to prevent environmental consequences.During 1999 to 2000, US Geological Survey detected TCS in 57.6% of streams and rivers sampled, at concentrations ranging from below the detection limit up to 2.3 μg·L−1 . In addi−1 ,107,114.−1 ,115. Deset al. [Triclosan concentrations in sediment increased between the early 1960s after its introduction until the mid-1970s, reflecting steadily increased patterns of its use. Later, an opposite trend was observed from the mid-1970s until the early 1980s, during which period, an additional secondary treatment step was introduced into most WWTPs. However, due to the popularity and increased use of TCS, again from the early 1980s, increase in TCS concentrations was observed until the present time  also repet al.  observedTriclosan that persists in the secondary effluent after activated sludge treatment may be chemically transformed after discharge. In this sense, a disinfecting oxidant, sodium hypochlorite, a source of free chlorine is generally used in US for many purposes and could enter in contact with TCS. It is known to chlorinate the TCS phenol carbons in ortho, or para-positions generating three chlorinated TCS derivative (CTD) intermediate products: ,122,127.et al. [2O [The historical pattern of dioxin photoproducts of TCS and its chlorinated derivatives in sediment cores from the Mississippi river was reported by Buth et al. . Another al. [2O . 2,4-DCP al. [2O . The chl al. [2O . The mecThe repeated exposure to chlorine in water treatment facilities can chlorinate TCS. Chlorinated TCS is discharged from a WWTP, and sunlight can convert it into more toxic dioxins . Accordiet al. [Pseudomonas, Burkholderia or Sphingomonas can degrade them in natural environments to carbon dioxide and chlorine [et al. [Meanwhile, a study by Latch et al.  concludechlorine ,139. Son [et al.  reported [et al. . When TCTCS is readily susceptible to degradation through photolysis in aqueous media with half-life that ranges from <1 h in abiotic conditions, to around 10 days in fresh water bodies. Moreover, its aerial half-life has been estimated to be 8 h based on reaction of TCS with photo-chemically produced hydroxyl radicals. Even though the detected concentrations as of now may not be toxic, but continuous accumulation of TCS and its by-products in the environment could reach the threshold limit which can affect all levels of the animals in the food chain.The ubiquitous use of triclosan and its consequent entry into the environment is of concern due to the effects it could produce if no regulations prevent its accumulation during the next decades. It and its derivatives are already present in measurable quantities, which may potentially affect water quality, impact on ecosystem and human health. Contamination of TCS has been detected in different environmental matrices including terrestrial, aquatic and biosolids resulting from WWTPs. TCS has also been found in drinking waters. There are concerns that the widespread use of TCS in various applications might lead to a preferential selection for microbial resistance to antibiotics. Microbial resistance has become an increasingly serious problem worldwide, and the continued use of biocides including TCS may exacerbate this problem. Increasing accumulation of TCS in the environment was also found to have adverse impacts on the growth of aquatic organisms. Taking into consideration the environmental and health concerns of TCS, more efforts need to be carried out for the understanding of their distribution and fate in various environmental compartments, in particular, wastewater treatment plants and sediments which are the final sinks.


The BTB-KELCH protein Gigaxonin plays key roles in sustaining neuron survival and cytoskeleton architecture. Indeed, recessive mutations in the Gigaxonin-encoding gene cause Giant Axonal Neuropathy (GAN), a severe neurodegenerative disorder characterized by a wide disorganization of the Intermediate Filament network. Growing evidences suggest that GAN is a continuum with the peripheral neuropathy Charcot-Marie-Tooth diseases type 2 (CMT2). Sharing similar sensory-motor alterations and aggregation of Neurofilaments, few reports have revealed that GAN and some CMT2 forms can be misdiagnosed on clinical and histopathological examination. The goal of this study is to propose a new differential diagnostic test for GAN/CMT2. Moreover, we aim at identifying the mechanisms causing the loss-of-function of Gigaxonin, which has been proposed to bind CUL3 and substrates as part of an E3 ligase complex.We establish that determining Gigaxonin level constitutes a very valuable diagnostic test in discriminating new GAN cases from clinically related inherited neuropathies. Indeed, in a set of seven new families presenting a neuropathy resembling GAN/CMT2, only five exhibiting a reduced Gigaxonin abundance have been subsequently genetically linked to GAN. Generating the homology modeling of Gigaxonin, we suggest that disease mutations would lead to a range of defects in Gigaxonin stability, impairing its homodimerization, BTB or KELCH domain folding, or CUL3 and substrate binding. We further demonstrate that regardless of the mutations or the severity of the disease, Gigaxonin abundance is severely reduced in all GAN patients due to both mRNA and protein instability mechanisms.In this study, we developed a new penetrant and specific test to diagnose GAN among a set of individuals exhibiting CMT2 of unknown etiology to suggest that the prevalence of GAN is probably under-evaluated among peripheral neuropathies. We propose to use this new test in concert with the clinical examination and prior to the systematic screening of GAN mutations that has shown strong limitations for large deletions. Combining the generation of the structural modeling of Gigaxonin to an analysis of Gigaxonin transcripts and proteins in patients, we provide the first evidences of the instability of this E3 ligase adaptor in disease. Gigaxonin is a BTB-KELCH protein that plays a central role in sustaining neuron integrity and cytoskeleton architecture. Indeed, recessive mutations in the Gigaxonin-encoding gene are responsible for a devastating neurodegenerative disorder in human, called Giant Axonal Neuropathy (GAN [MIM 256850]) , that leDiagnosed early in infancy, the disease first touches the peripheral nervous system, altering both the motor and sensory tracts in teens, and closely resembles to the most common inherited peripheral neuropathy called Charcot-Marie-Tooth (CMT) diseases. Thus, patients exhibit weakness and severe wasting of the four limbs predominating in distal segments, sensory and motor loss, and reduced deep tendon reflexes. Symptoms evolve towards areflexia, loss of the deep and superficial sensitivity and loss of ambulation. Subsequently, the disease targets the central nervous system, leading to a wide range of symptoms encompassing ataxia, nystagmus, dysarthria and intellectual disability ,3. FatalWhile NF aggregation has been reported in many neurodegenerative disorders, including Alzheimer’s, Parkinson’s diseases and Amyotrophic Lateral Sclerosis, possibly as a results of neuron injury, the disorganization of all classes of IFs is unique to GAN and supports a crucial role of Gigaxonin in sustaining cytoskeleton architecture . In addiHow this low abundance BTB-KELCH protein , prefereTo better understand the disease mechanisms in GAN and provide a specific diagnostic tool able to discriminate GAN from closely related CMTs, we combine here a study on Gigaxonin abundance and stability in disease and a structural modeling of Gigaxonin to a prognostic study on new patients presenting a sensorimotor neuropathy of unknown etiology. We establish that in GAN patients, mutant Gigaxonin levels are greatly reduced in abundance. The quantification of Gigaxonin mRNA reveals nonsense mRNA decay as one of the disease mechanisms in GAN. In addition, the modelization of Gigaxonin structure allows us to map GAN mutations and predict a general destabilization of disease-associated mutants, which is further confirmed by reduced half-lives of mutant Gigaxonins. Finally, we establish that our immunodetection of Gigaxonin constitutes a robust, penetrant and specific new diagnostic test for GAN, circumventing the limitations of gene sequencing and the clinical and histopathological overlap between GAN and the frequent forms of axonal CMTs.Blood samples were collected from patients with written informed consents and under the agreement n° DC-2010-1191 of the Bioethic comittee of the Ministère de l’Enseignement Supérieur et de la Recherche. Numbering of previously reported GAN patients matches publications  for F1-FCell lines, expanded in RPMI 20% FBS, 1% P/S and 2 mM Glutamine (Invitrogen), were lysed and processed for western blotting as previously described in . PrimaryGAN gene were Sanger sequenced using primers described in [http://www.molgen.ua.ac.be/CMTmutations/default.cfm) and Human Gene Mutation Database (HGMD) to exclude polymporphisms in the normal population.All coding exons and flanking intron sequences of the ribed in , the BigGAN gene was analyzed on proband and family genomic DNA by High-resolution custom NimbleGen 135 k CGH microarray (probe spacing of 75 bp for exons and 200 bp for the introns) versus reference DNA . The array also included a genomic backbone probe set with an average probe spacing of 30 kb. DNA samples were labeled (test with Cy3 and reference with Cy5) and co-hybridised to the custom microarray in accordance with the manufacturer’s instructions . The microarray was washed and then scanned on an Axon GenePix 4400A Scanner using GenePix Pro 7 software . Raw data was normalized, LOESS correction applied and the data ratios calculated using DEVA v1.01 Software (Roche NimbleGen). The normalized data was processed using Infoquant Fusion v6.0 software  with analysis call settings of 3 consecutive probes +/- 0.4 Cy3/Cy5 ratios. The arrays were used on affected and unaffected family individuals as well as normal controls.Genomic rearrangement on the i.e. GAN-exon4-5 , together with appropriate positive and negative controls. The relative abundance of the patient’s mRNA was expressed as the fold change to the controls mRNAs. Fold changes were measured as the ratio of the ΔΔCT of each patient to the ΔΔCT of the controls after normalization with HPRT1. Three independent RT-PCR were performed in triplicate for each sample. Accordingly to the manufacturer’s instructions, only fold changes exceeding a value of 2 are considered significantly different.Total RNA from patients and control cell lines was isolated using the RNeasy MicroKit (Qiagen) according to the supplier’s recommendations. For each sample, 1 μg of RNA was used for reverse transcription with oligodT primers and SuperScriptIII (Invitrogen). SYBR green quantitative real-time PCR was performed with LightCycler 480 SYBR Green I Master (Roche) in a two-step cycling protocol on 100 ng of cDNA, using GAN-exon9-11 specific primers (GGGTAGCGAGATGGTAACTTG and CGGATGGAAGGAGTGGTTTAG) and HPRT1 quantitect primers (Qiagen). Carrying a deletion in the GAN exons 10 to 11, F24 mRNA level was determined by another set of primers, t-Test was used to determine statistical significance. Error bars represent standard deviation and p values are reported in the Figure legends.The student’s http://www.sbg.bio.ic.ac.uk/phyre2/) based on the crystal structure of human KLHL11 (3I3N.pdb) [The structural model of the BTB-BACK domain Gigaxonin bound to the N-terminal domain of CUL3 was generated as follows. Residues 8–128 were used from the crystal structure of the BTB domain of Gigaxonin, which also included a partial model for the BACK domain . A compI3N.pdb) . The BTBI3N.pdb) . The KELI3N.pdb) ,21.35-methionine/cysteine (Perkin Elmer) for 45 min. Cells were subsequently washed twice with PBS and incubated with normal DMEM medium, containing serum and antibiotics. Cells were washed with PBS and collected by centrifugation at the beginning of the chase period (0 h) and at 2, 4, 6, 9, 24 hours. Protein extracts were lysed as previously described for lymphoblast cell lines and immunoprecipitation was performed on 60 μg of total proteins using anti Flag antibody  and G-protein . Immunoprecipates were recovered in Laemmi buffer and loaded on two identical gels for autoradiography and immunoblotting . Because immunoblotting of Gigaxonin . R138H, L309R, R477X and R15S correspond to mutations of families F13, F1, F16 and F2, respectively. WT, N-ter and C-ter correspond to the Full-length Gigaxonin, the BTB domain (residues 1 to 223) and the KELCH domain (residues 141 to 597), respectively. 24 hours post transfection, cells were washed twice with PBS, incubated in methionine- and cysteine-free DMEM (Sigma) supplemented with 10% FBS and 1% P/S for 1 h before labeling with 100 μCi of SInherited through a recessive mode of inheritance, GAN is suspected to result from a Gigaxonin loss of function mechanism. We previously showed a dramatic decrease in the abundance of mutant Gigaxonin in few GAN patients using an immunodetection method on immortalized lymphoblast cell lines . To furtTo assess whether the determination of Gigaxonin abundance could contribute to diagnose GAN, we included in the study seven new patients presenting a sensorimotor axonal neuropathy resembling GAN/CMT2 with unknown genetic etiology Table . Some paAmong the seven new patients, five of them, namely patients F23, F24, F26, F28 and F30 present an early (<4 years) onset progressive neuropathy indicative of the typical severe form of GAN. As revealed by the reduction of nerve conduction velocities, they exhibit an axonal motor and sensory neuropathy with muscle weakness/tone, areflexia that evolves to the loss of ambulation and of the deep and superficial sensitivity during childhood. All patients subsequently develop central nervous system impairment encompassing nystagmus, dysarthria and ataxia. Concomitant with these clinical signs, GAN has been shown to induce a wide aggregation of the cytoskeletal IF network both in and out-side the nervous system, and severely affected patients F23, F24, F26, F28 and F30 all exhibit altered keratins (kinky hair) and aggregating NFs in enlarged axons Table .Two additional patients (F27 and F29) present a neuropathy differing from the GAN typical form, with ± late onset, mild central nervous system impairment, extended survival but with the presence of giant axons and NF aggregation that may suggest a milder form of GAN or another related sensori-motor neuropathy called type 2 Charcot-Marie-tooth (CMT) disease. Indeed, we previously identified two moderate forms of the disease with very slow progression, no central nervous impairment and extended survival , or with a late onset at 10 years of age, slow evolution with no central system involvement for patient F13 (with a R138H mutation).The quantification of Gigaxonin in patients suspected of bearing a GAN severe  and mild (F27) forms revealed a considerable diminution of abundance using both normalization methods and that is comprised in the range established for GAN patients with identified mutation  or large deletions, as revealed by our inability to detect the heterozygous mutations in patients F6 and F12 [We previously identified Gigaxonin as the defective protein in Giant Axonal Neuropathy . Develop origins ,15,24. T and F12 ). Hence,GAN gene. Combining systematic sequencing to CGH analysis, we identified all GAN mutations in 5 unrelated patients, encompassing missense, nonsense mutations and a large deletion. Interestingly, our test was able to suspect heterozygosity in a carrier (sister S1 of patient F24) that was further confirmed by the identification of the large GAN deletion on one allele. Noteworthy, patients suspected to bear a mild (F29) but also a severe (F28) form of GAN were excluded by our new test and may be tested for candidate genes in autosomal recessive forms of CMT2 or exome sequencing. Altogether, our study reveals that the clinical evaluation of patients and the histological examination are indeed important but not sufficient to diagnose GAN and differentiate this entity from other frequent peripheral neuropathies. Thus, it is conceivable that the prevalence of GAN is under-evaluated, and that our test will be useful in identifying GAN among related CMTs. Currently being adapted on fresh blood samples, our methodology will enable the community to identify GAN pathogenic variants from targeted diagnosis or following high-speed sequencing analysis, and this in a cost-less and fast manner.We showed in this study that regardless of the type, the position of the mutations or the severity of the disease, all mutated Gigaxonins  display a drastic reduction in their abundance, reaching 85,8% in average of the normal level. The effect was so impressive that we further assessed whether the method may be useful for diagnostic purpose. Indeed, the first phase of the disease progression of typical severe forms of GAN, as well as some GAN atypical mild forms -with no involvement of the central nervous system ,26- presOvercoming the limitation of gene sequencing, the determination of Gigaxonin abundance has proven to be a very important diagnostic tool -specific, reliable and robust- in all GAN families tested so far. Nevertheless, one has to be cautious as some mutations may confer loss of function without necessarily conferring transcript and protein instability. Thus, we propose to determine Gigaxonin abundance prior but in concert with the systematic screening for GAN mutations/deletions in patients, to define in the future the confidence of our methodology as a sufficient diagnostic test for GAN.GAN gene on both alleles display a down regulation of their mRNA would indicate a compensatory mechanism in patients carrying compound missense and truncated mutations: either a stabilization or a enhanced transcription of the truncated and missense mRNA, respectively.We previously determined that Gigaxonin is a new BTB-KELCH protein, predominantly and equally expressed throughout the nervous system but at very low level ,10. A keGAN mutations. Regardless of the stability of the mRNA levels, the majority (85%) of the Gigaxonin mutations are predicted to map to buried surfaces, which could alter the folding of the BTB or KELCH domains, the homodimerization of the protein, and/or the interaction with the Cul3 subunit of the E3 ligase. The other 15% mutations may impair substrate binding, which may indirectly still confer instability. Indeed, as suggested for other BTB-KELCH proteins, many cullin-E3 ligase adaptors are destabilized by (auto)-ubiquitination in the absence of substrates, possibly to avoid constitutive activation of the E3 ligase [The homology modeling of normal Gigaxonin allowed us to predict the structural effects of 3 ligase . AccordiWe have not only developed a new powerful method to diagnose GAN, we have also provided the first evidence that disease-associated mutations confer instability of Gigaxonin in the human pathology. Reconstitution of the E3 ligase activity of the Gigaxonin-Cul3 complex, together with the identification of its partners are now essential to unravel the mechanisms controlled by Gigaxonin in sustaining neuron survival and cytoskeleton architecture. This will shed light onto the role(s) of the BTB-KELCH protein Gigaxonin in Giant Axonal Neuropathy and may contribute in the understanding on how mutations in the UPS contribute to neurodegeneration, as exemplified in Parkinson, Spinocerebellar Ataxia, Angelman syndrome and CMT diseases.The authors declare that they have no conflict of interest.GAN gene in patients and identified all punctual mutations. BAS generated the structural model for Gigaxonin, provided prediction of Gigaxonin’s mutations and contributed to the writing of the manuscript. DM and HH developed and applied the CHG methodology for GAN patients. TS identified patients exhibiting inherited peripheral neuropathy, provided clinical data and contributed to the writing of the manuscript. NK, CH and BC identified patients and provided clinical data. PB designed the study, analyzed the data and wrote the manuscript. All authors corrected the manuscript. All authors read and approved the final manuscript.AB performed the majority of the experiments, analyzed the data and contributed to writing of the manuscript. YTA realized and analyzed some experiments, and helped correcting the manuscript. BR performed a systematic sequencing analysis of the


Hearing loss is the most common communication disorder affecting about 1-7/1000 births worldwide. The most affected areas are developing countries due toextensively poor health care systems. Environmental causes contribute to 50-70% of cases, specifically meningitis in sub-Saharan Africa. The other 30-50% is attributed to genetic factors. Nonsyndromic hearing loss is the most common form of hearing loss accounting for up to 70% of cases. The most common mode of inheritance is autosomal recessive. The most prevalent mutations associated with autosomal recessive nonsyndromic hearing loss (ARNSHL) are found within connexin genes such as GJB2, mostly in people of European and Asian origin. For example, the c.35delG mutation ofGJB2 is found in 70% of ARNSHL patients of European descentand is rare in populations of otherethnicities. Other GJB2 mutations have been reported in various populations. The second most common mutations are found in theconnexin gene, GJB6, also with a high prevalencein patients of European descent. To date more than 60 genes have been associated with ARNSHL. We previously showed that mutations in GJB2, GJB6 and GJA1 are not significant causes of ARNSHL inpatients from African descents, i.e. Cameroonians and South AfricansIn order to resolve ARNSHL amongst sub-Saharan African patients, additional genes would need to be explored. Currently at least 60 genes are thought to play a role in ARNSHL thus the current approach using Sanger sequencing would not be appropriate as it would be expensive and time consuming. Next Generation sequencing (NGS) provides the best alternative approach. In this review, we reported on the success of using NGSas observed in various populations and advocate for the use of NGS to resolve cases of ARNSHL in sub-Saharan African populations. It is lglobally . Hearingglobally . It affeglobally . Prevaleglobally  which arglobally . This isglobally .pathophysiology refers to the component of the hearing system which is non-functional. This can be any of the outer, middle or inner ear components. Conductive hearing loss refers to the non-functionality of the outer or middle ear. Sensorineural refers to hearing loss due to non-functionality of the inner ear. When both the inner and outer or middle ear is not functioning, it is referred to as mixed hearing loss; severity is determined by measuring the threshold needed for sound to be perceived. It is determined by generating an audiogram. The higher the threshold where sound is perceived, the more severe the loss of hearing; onset is either pre-lingual, which is before the development of speech as seen in congenital cases, or post-lingual. If the hearing loss in both ears then it is referred to as bilateral and unilateral if only one ear is affected.There are various classifications used to describe the clinical manifestation of hearing loss. The American Speech-Language-Hearing Association (ASHA) classifies hearing loss according to the following characteristics : pathophWe reviewed the available studies on the aetiology and genetics of hearing loss in Africa, including our previous publications on this topic that have shown the non-implication in Africansof the genes most commonly associated with ARNSHL among patients of European and Asian descent: GJB2 and GJB6. In this article, we review the success of using NGS as done so by other research groups in various populations and advocate for the use of NGS to resolve cases of ARNSHL in sub-Saharan African populations. This will provide rapid results which will contribute towards building a genetic profile on ARNSHL amongst sub-Saharan Africans.® and Google Scholar® (August 2014). Only publications in English, were retrieved and included in the manuscript.The present article is a systematic review of most recent publications of hearing loss with focus on relevant articles on aetiology of hearing loss in sub-Saharan Africa and those that involved NGS to study the causes of hearing loss. The key word used was hearing loss, aetiology, genetics, Africa. We used the following search engines: PubMedThe causes of hearing loss can either be genetic or environmental. In developing communities, the environment contributes significantly more to the incidence of congenital hearing loss than in the developed world . This isSpecifically in Africa, an infection which contributes to cases of hearing loss in infants and young children is that of bacterial meningitis. In a study in Kenya  and manyThere has been some investigation into genes that are thought to be candidates for differential susceptibility of certain individuals to noise-induced hearing loss (NIHL) while others seem unaffected . A more There is high variability in the genes causing hearing loss as well as their causative mutations. To date, there are over 60 genes implicated in cases of hearing loss .The manifestation of the hearing loss may be syndromic whereby there are other clinical features associated with the loss of hearing. On the other hand, it might be nonsyndromic whereby hearing loss is the only observed symptom. Nonsyndromic hearing loss accounts for up to 75% of cases of putative genetic origin . SyndromMYO7A, CDH23, and USH1C to name a few. MYO7A has been implicated in various populations as harbouring causative mutations for ARNSHL [This syndrome is characterised by hearing loss and retinitispigmentosa. It accounts for 50% of cases of combined deafness and blindness worldwide. It has three clinical subtypes classified according to the severity of the hearing loss . The mosr ARNSHL . There ir ARNSHL .This is an autosomal recessive syndrome with symptoms such as sensorineural hearing loss andpartially defective iodine organification, a process of adding iodine to thyroglobin for the production of thyroid hormone. It accoGJB2 and GJB6 [GJB2 [This is a congenital disorder characterised by profound hearing loss, hyperkeratosis (thickening of the skin) and erythrokeratoderma   is the most common type of hearing loss . The mosScreening of GJB2 has been conducted in other populations  such as GJB2 has been investigated in various countries. It was only implicated in Ghana with p.R43W being the most common mutation for this population [GJB2 mutations. Wonkamet al reported two cases of KID syndrome due to mutations in GJB2 and illustrated that these cases have the most common mutation in the global population [In Sub-Saharan Africa, pulation , South Apulation  and Kenypulation  did not pulation . In addipulation . Togethepulation , 48, 49,GJB6 has also been investigated in other populations and its variants are yet to be identified outside of the European Caucasian population [GJB6 mutations are not of significance in sub-Saharan African populations and that there is little evidence to suggest that rare causative mutations might be harboured by this population group [GJB6 to try and uncover any rare point mutations [GJB6 genetic variation is not a significant factor amongst sub-Saharan African patients with ARNSHL.pulation , 50–55. on group . We alsoutations . The resGJA1 in hearing loss with initial reports being subsequently discredited as the researchers had initially failed to distinguish between the gene from the pseudogene; no pathogenic mutations in GJA1 coding region in Africans from Cameroon and South Africa [There has been some contention on the role of h Africa .Outside of connexin genes, there have been mutations reported in various other genes. Due to the number of different genes and mutations that have been implicated in hearing loss, it has become imperative that methods that are able to interrogate many gene positions at the same time be employed to find the relevant variants in African populations. Thus, the obvious choice becomes sequencing, especially using next-generation sequencing (NGS).Massive parallel sequencing allows for the interrogation of multiple genes at the same time. This can be whole genome sequencing where by the entire 3 billion base pairs of the individual's genome are sequenced. With most monogenic disorders having been caused by exonic mutations, whole exome sequencing is often favoured. Moreover, exonic regions represent only 1% of the entire genome which makes this interrogation both time and financially feasible as compared to whole genome sequencing . This inThe use of consanguineous families also helps narrow the search for such mutations as one has to look for homozygosity and the close relatedness of the family members’ helps in distinguishing between variations and disease related mutations. Other approaches involve using unrelated individuals whom present with the same clinical symptoms and comparing their captured variants to decide whether or not they are significant .The use of gene mapping has long been a standard principle to try and identify the gene associated with a disease. In 2010 Walsh Et al. used homozygosity mapping and whole exome sequencing to identify the causative gene on locus DFNB82 in a Palestinian family with nonsyndromic hearing loss . The regIn the Middle East, 30 individuals from 20 consanguineous families were selected for whole-exome sequencing analysis . The famThe drawback of using Whole exome sequencing is that you select for the entire exome which will also carry genes not related to your disease of interest. One might also enrich for areas of less importance while losing genes which could be of interest or importance. The filtering of variants leaves a few variants to be interrogated. There is also the risk of losing rare variants. If one does not have family members’ or parents’ DNA to also work with, the efficiency is also negatively affected. A better approach would be to select for genes or specific areas of interest to screen rather than the entire exome, especially if associations for the disease have already been made.When the genes associated with a particular disorder have already been identified, targeted exome sequencing could be the most appropriate subsequent step in clinical settings. This involves using DNA chips and arrays with the desired genes already selected to capture DNA from those selected regions . This fiGenes associated with ARNSHL have been identified in many populations around the world . These rThe developers of the tool demonstrated the sensitivity and specificity of their tool by screening three positive controls and one negative control . Upon vaMYO7A, SLC26A4 and CDH23. Twenty fourmutations were identified in 28 families. Eleven of these mutations were novel MYO7A mutations [Another next generation sequencing tool has been developed called OtoSeq . It targutations .GJB2, SLC26A and a mitochondrial mutation in MT-RNR1. They report 45 novel recessive mutations and three novel dominant mutations. They also put emphasis on the fact that17.4% of these novel mutations were found in less commonly screened genes advocating for more genetic testing to be done on these genes [GJB2, SLC26A4, USH2A, GPR98, MYO15A, COL4A5 and CDH23 [This method of targeted enrichment and massively parallel sequencing has been adopted by other research groups to resolve cases hearing loss. Amongst them investigators of the Chinese population, Yang et al, have demonstrated the success of using targeted exome sequencing. Their technique used exonic data from 79 deafness genes . The 125se genes . Amongstse genes . Amongstnd CDH23 . Eighty nd CDH23 . More thThese are a few examples of the use of NGS to uncover mutations responsible for cases of hearing loss across various population groups. Upon the identification of such mutations, there can be a step towards population specific diagnostic tool development. There is very little data available on the genetic profile of hearing loss patients in Sub-Saharan Africa; with the current investigatory route being NGS in resolving this matter, it is the only way forward.


Communication is fundamental for social participation with communication difficulties often leading to social isolation and depression. Nevertheless, everyday communication is often hindered either by internal factors such as sensory loss, or by external factors including the background noise that commonly occurs in places where people meet, such as restaurants, schools, and railway stations. In such adverse conditions, working memory and executive functions have been proposed to play a critical role in communication. Thus, the role of cognition in hearing is a central theme in the field of Cognitive Hearing Science and has crystalized as one of the main themes of this research topic. This is reflected in papers reporting the role of cognition in hearing in persons with varying sensory and cognitive status and varying degrees of language knowledge, over the lifespan. Another theme represented in this topic is rehabilitation in the form of amplification and training. Importantly, the broad remit of the research topic is reflected in papers addressing cognition and communication in children with sensory and cognitive issues as well as adults and children who are profoundly deaf and use sign language to communicate. Apart from the impressive number of empirical studies, there are several theoretical contributions to the field.Heinrich et al. report a study showing, in line with previous work, that individual differences in sensory and cognitive skills explain variance in the ability of older listeners with mild sensorineural hearing loss to process speech. However, they also show that the relative explanatory value of these skills depends on the linguistic demands of the particular speech test, with hearing sensitivity being more important at the phoneme level and cognition at the sentence level. Further, they reported associations between self-reported aspects of auditory functioning and speech intelligibility. Smith and Pichora-Fuller compared performance on the reading span test (RS), which is a well-established measure of working memory delivered visually, and the Word Auditory Recognition and Recall Measure (WARRM), a newer measure of working memory with auditory delivery, which they propose is more ecologically valid. WARRM performance was better and more varied than RS performance in all groups tested  and the authors suggested that this pattern of performance indicates WARRM may be a useful clinical test. However, no consistent pattern of correlations was found between the two cognitive measures and measures of the ability to understand speech in noise. Smith and Pichora-Fuller suggest that there is a need for a more consistent approach to determine in more ecologically relevant conditions associations between working memory and speech understanding. During speech comprehension, encoding of new memories may be hampered by interference from established memories; this is known as proactive interference. Ellis and Rönnberg studied whether the ability to suppress such interference was associated with speech recognition in noise in older hard-of-hearing adults. In line with previous work on individuals with normal hearing, they did find an association, but only when hearing was unaided. They suggested that the cognitive flexibility reflected by performance on their cognitive task is a key factor in listening ability.The observation of consistent correlations between cognitive skills and the ability to understand speech under adverse conditions has played an important role in driving the field of Cognitive Hearing Science. In particular, it has been reported repeatedly that working memory explains variance in the ability to recognize speech in noise above and beyond differences in hearing thresholds. In the current research topic, Kidd and Humes used an auditory working memory task to determine differences in the ability of older and younger listeners to keep track of who said what. They found that although older listeners were slower, they were almost as accurate as younger listeners. However, older listeners did not benefit from consistent mapping of target speaker and location in the same way as younger listeners. Doherty and Desjardins investigated how amplification influenced auditory working memory performance in hard-of-hearing listeners who were fitted with hearing aids for the first time. They found that amplification improved working memory and the overall pattern of results suggested that this was due to perceptual benefit rather than cognitive change. Moradi et al. used a gating paradigm to determine whether background noise influences how much of the auditory signal is needed before identification of its linguistic content is achieved. Results showed that more auditory signal was required in noise and that this effect was modulated by both working memory and executive function. DiDonato and Surprenant investigated how speech manner influences the ability of older and younger listeners to remember auditory information with ecological relevance. They found that older listeners could remember medical information better when it was presented clearly rather than conversationally, even in background noise. The electrophysiology study by Petersen et al. investigated how working memory indexed by neural oscillations in a low frequency  band, is influenced by increasing stimulus degradation and working memory load, in hard-of-hearing individuals. In line with previous work in individuals with normal hearing thresholds, performance decreased and alpha power increased with greater stimulus degradation and working memory load. However, at the highest levels of degradation and working memory load, alpha power dropped for the participants with the greatest degree of hearing loss, suggesting a breakdown in an important neural mechanism that may support listening in noise.Experimental approaches are adopted by another set of studies studying the role of cognition in communication. Keidser et al. found that performance on the CSC Test (CSCT) was influenced by some of the manipulated parameters  and that there was no consistent relation between CSCT and RS. Further, there was no relation between CSCT and a novel speech comprehension test presented in noise. Using the Auditory Inference Span Test (AIST), a sentence-based test which involves storage and processing of the message, Rönnberg et al. showed that, even when audibility is relatively well-maintained, processing of a spoken message becomes harder for listeners with normal hearing thresholds when noise level increases, but only when the noise is speech-like. This suggests that speech-like background reduces CSC. Lin and Carlile used a version of AIST to investigate the listening costs associated with shifts in spatial attention during conversational turn-taking in listeners with normal hearing thresholds. They found that listening costs were dependent on load and cognitive complexity but not on the nature of the spatial shift.If cognitive resources are consumed during listening in noise as indicated by the association between working memory and listening performance, fewer resources, or less cognitive spare capacity (CSC) will be available for higher level processing of the message. The research topic includes a set of studies investigating this phenomenon. In line with previous work, Souza et al. investigated the role of working memory in speech intelligibility in noise with hearing aid signal processing. The data corroborated previous results showing that individuals with low working memory capacity may benefit more from signal processing that better retains the signal envelope. Neher studied whether working memory and executive function were related to speech recognition in noise performance with hearing aid signal processing as well as preference for different hearing aid fittings in older hearing aid users. His study found that working memory was related to performance with directional microphones while executive function was related to preference for noise reduction.Hearing aid signal processing is designed to improve speech understanding. It is important to determine whether this is actually the case and at the same time identify any contingent cost in terms of cognitive function. In this research topic, Ferguson and Henshaw reviewed three auditory training studies and conclude that training which combines auditory and cognitive demands is most likely to benefit hard-of-hearing adults in real-world listening situations. Henshaw et al. argue that training benefit is dependent on uptake, engagement and adherence. Their study showed that uptake was associated with extrinsic motivation  while engagement and adherence were influenced by both intrinsic , and extrinsic  motivations.Kilman et al. studied the amount of disturbance perceived by hard-of-hearing listeners and listeners with normal hearing thresholds when attending to a target talker against a multitalker background. Speech was either native or non-native. Results showed that hard-of-hearing participants were particularly disturbed by native speech masked by native babble. Hygge et al. investigated how nativeness of speech influenced the ability to recognize and recall speech in different levels of background noise. They found that recall was more sensitive than recognition to both factors and thus a better indicator for the acoustics of learning.An atypical language model can lead to particular involvement of working memory and executive function in language processing. Hirshorn et al. assessed the impact of language experience on predictors of reading comprehension in deaf readers. They found that while English phonological knowledge best predicted reading comprehension in oral deaf individuals, free recall was a better predictor in deaf native signers. Marshall et al. investigated the relationship between working memory and language in deaf signing children who were either native or non-native users of British Sign Language compared to hearing children. The non-native signers performed less accurately than both the native signers and the hearing children. Further, vocabulary predicted working memory, suggesting that the good language skills resulting from early acquisition are important for development of working memory.Because profoundly deaf individuals do not have access to sound, reading and other academic skills may develop differently from those of individuals with normal hearing. Sandgren and Holmström discuss the clinical challenge of assessing language impairment in bilingual children and present work suggesting that measuring executive function may be a useful approach. In a mini-review Lyberg-Åhlander et al. discuss their recent work investigating how children's listening comprehension is influenced by speaker voice quality and background noise, as well as the child's own cognitive capacity. They highlight risk of underachievement when speech is delivered in a dysphonic (hoarse) voice, especially when the task is simple or the child's capacity is stretched. In another mini-review, Sandgren et al. summarize their work on referential communication showing that while children with sensorineural hearing impairment are active and competent conversational partners, their conversational strategies are distinct from those of their peers with normal hearing, even when the listening situation is optimized.A number of the papers in the research topic report studies investigating cognitive aspects of language development in children with disabilities. In a perspective article, Lemke and Scherpiet discuss communication from an aging perspective, and the psycho-social impact of sensory and cognitive decline. Wingfield et al. discuss the Ease of Language Understanding (ELU) model  as one of the few attempts to offer a fully encompassing framework for language understanding. They identify its strengths and point out avenues for future work.Finally, two perspective articles round off the research topic. Altogether, the articles in this research topic demonstrate the crucial role of cognition, including working memory and executive functions but also cognitive flexibility and cognitive load, in communication under adverse conditions, in different modalities, and over the lifespan.MR prepared the first draft of the editorial, and CS and MR contributed equally to completion of the final version.This work was supported by funding from the Swedish Research Council to the Linnaeus Centre HEAD, Swedish Institute for Disability Research, Department of Behavioral Sciences and Learning, Linköping University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tinnitus includes all the sounds perceived by the patient, without any external stimulus that affects all aspects of life; there is no cure for most patients. Given that tinnitus and hearing of the ear are common complaints of patients in clinics and due to the impossibility of cure at the present time, it is necessary to set up common causes of tinnitus to solve social problems and present optimal solutions. The purpose of this study was to determine the causes of tinnitus in patients referred to ENT Clinic of Imam Khomeini Hospital in Urmia, 2012-2013.This is a cross-sectional analytic study that was performed on 184 patients with tinnitus referred to ENT clinic of Imam Khomeini Hospital, Urmia. After examination, audiometry performed and questionnaires of tinnitus patients were completed. The results were analyzed by statistical SPSS software.The study included 111 males and 73 females and the mean age was 50.6 years. 168 patients (91.3%) had non-pulsatile tinnitus and the rest had pulsatile tinnitus. The mean hearing loss in patients was 31.4 dB, 79.9% were of sensory neurons kind, and 12.5% of patients did not show any hearing loss in audiometry. In total, 90.2% of the cases were detected; most causes of tinnitus were noise (19.6%), ototoxicity (16.8%) and presbycusis (16.3%). The most common causes of tinnitus were noise with 31.5% in males and ototoxicity with 27.4% in females. Between age and hearing loss of patients, there was a significant relationship , but there was no significant relationship between gender and degree of hearing loss.The most common cause of tinnitus (NIHL) is quite predictable, except by accident, you can completely avoid by reducing noise levels on the source and ear protection. By reasonable prescription of ototoxic medications particularly antibiotics can reduce the prevalence of tinnitus that causes sleep disorders and concentration problems, depression and anxiety and help the public health. Tinnitus includes all the sounds perceived by the patient, without any external stimulus . The souIn general, in most of the books tinnitus is classified as either subjective or objective. Objective tinnitus can be heard by the patient and the person who examines, whereas subjective tinnitus can be heard only by the patient, which makes it difficult to assess; in addition, causes of subjective tinnitus is less known and there is no cure for most patients with chronic tinnitus . TinnituTrauma , presbycusis, Meniere’s disease, ear infections, neurological disorders (MS), musculoskeletal disorders (otosclerosis) and neoplasms (schwannomas and stybolar) are other causes of SNHL  and are non -pulsatile tinnitus. Causes of pulsatile tinnitus include increased benign intracranial pressure, glomus tumor, atherosclerotic disease of the carotid artery, malformations and arterial venous fistulas of the head and neck, Eustachian tube dysfunction and etc. . InfectiIn a study in 2010 with the aim of estimating the prevalence of auditory cues and to estimate the prevalence of exposure to hearing damage causes and analyze the relationship among these factors in a group of healthy young men were studied. 839 males, 22-19 years old, participated in this study. Data were collected by questionnaires and audiometry. The prevalence of tinnitus, sensitivity to sound and hearing defects were 32.2%, 15.5% and 14.5%, respectively. Contact with noise in 21.4% and loud music in 15.5% of participants were reported. People who had experienced tinnitus after exposure to noise was at higher risk for hearing loss in high frequencies, tinnitus and noise sensitivity. Those who listened to loud music had a higher prevalence of tinnitus but no hearing impairment . A studyThis is a cross-sectional analytic study on 184 patients with tinnitus referred to ENT clinic of Imam Khomeini Hospital, Urmia. The clinical examination including otoscopy was performed by a physician and the patient’s pure-tone audiometry and speech was performed by 822 Madsen OB audiometers, then the questionnaire that contains information such as the description of the profile and history of tinnitus, medical conditions and treatments as well as job history is completed. And any of their previous medical records were used to complete the questionnaire. For patients with pulsatile tinnitus for diagnosis of BIH, fundoscopy (see foot edema) and LP (CSF pressure above mmH2O200) and for carotid artery Doppler ultrasonography for ACAD and in cases where tinnitus and hearing loss was unilateral or asymmetric (suspected CPA pathology) MRI of the brain was performed with and without injection. Finally, by collecting information based on clinical findings and other assessments the cause of patient’s tinnitus was determined and treated according to the cause. Data used for this study were collected using a questionnaire and audiometry. And finally, after collecting data diagrams and frequency was used to answer research questions. Also to respond the hypothesis, the Chi-square test for comparison of qualitative variables and ANOVA test for comparison of mean hearing loss in terms of dB in groups were studied. For the above analysis SPSS16 software was used. This is a cross-sectional analytic study that was conducted on 184 patients with tinnitus referred to ENT clinic of Imam Khomeini Hospital, Urmia during the six months during 2012-2013. The study included 111 men and 73 women, mean age 50.6 years, age range 18 to 90 years. Tables  and 1BOf the patients, 168 patients (91.3%) had non-pulsatile tinnitus and 16 patients had pulsatile tinnitus. The most buzzing area was the left ear with 35.9% and the lowest percentage was feeling the buzz in head with 7%. Average hearing loss of dB patients was 31.4%, 79.9% had sensorineural hearing loss and 50.5% had hearing loss in the high frequencies. There were not hearing loss in 23 cases. Remarkably, there was a deaf person with tinnitus. 12.5% of patients showed no hearing loss in audiometry and 42.9% of patients showed normal hearing loss (less than dB 25). Tables Each patient’s tinnitus after examination, audiometry and questionnaire, which contains information such as the description of the profile and history of tinnitus, medical conditions and treatments as well as occupational exposure history, was determined. In total, 90.2% of patients were diagnosed and for 18 patients (9.8%) containing 16 patients with non-pulsatile tinnitus and 2 patients with pulsatile tinnitus did not find any known cause. Most causes of tinnitus were noise, including the noise of work, military or history of being in front and recreation noise (19.6%), ototoxicity (16.8%) and presbycusis (16.3%). Ear infections are also made up 9.8% of patients that the chronic central otitis was accounted for half of the cases. Causes of pulsatile tinnitus include glomus tumor, Eustachian tube dysfunction, and malformations of arterial venous of the head and neck. In all of the causes examined in any compact cerumen disease, neurocifilis, and palatal myoclonus were not diagnosed as causes of tinnitus Tables  and 8.Noise with 31.5% and presbycusis with 13.5% showed up most causes of tinnitus in males. Also, in females ototoxicity with 27.4% and presbycusis with 20.6% were the causes of tinnitus. Figure 11 shows frequency of causes of tinnitus in patients by gender. In terms of noise 35 of 36 cases were male and 26 patients had a loss in the high frequencies. Noise caused 43.7 dB hearing loss in high frequencies. Acoustic trauma in 11 of 12 cases loss was just at high frequencies with the mean of 35.8dB. Ototoxicity and presbycusis also caused 49.8 and 57.5dB loss at high frequencies, respectively. There was a significant relationship between age and hearing loss of the tinnitus patients. , meaning that the prevalence of hearing loss increases with age. But there was no significant difference between sexes and hearing loss. That is, the average hearing loss in men was 31.9dB and in women was 30.2dB. Between the degree of hearing loss and tinnitus quality, there was a significant relationship . This means that the people who had non-pulsatile tinnitus the prevalence of hearing loss was higher than the people who had pulsatile tinnitus (32.9dB versus 13.1dB). Also there is a significant relationship with the reliance index of 95% between hearing loss and the cause of tinnitus , so that the highest average cases of hearing loss was otoscleroses (70dB) and ear infections (49.2dB) and the lowest cases of hearing loss was pulsatile tinnitus (13.1dB) and acoustic trauma (17.5dB).According to previous studies, the main causes of tinnitus are NIHL, presbycusis and ototoxicity . DependiWhile the deleterious effects of continuous noise and percussion in the military and battlefield environments on public health, including the health of hearing devices are well known, harmful effects of noise pollution on human health is so serious that warnings regarding the daily noise exposure including traffic, noise at work and listen to music at high intensity levels are raised; most social groups due to lack of adequate knowledge cannot understand that long listening to loud music cause hearing loss and tinnitus. We all know that young workers often simply do not realize the consequences of such a fall in the hearing that we can show tinnitus and hearing loss in terms of acoustic to population at risk to have a better understanding and awareness of the problems and motivated them to use ear protection when noise increases. By raising the level of awareness, providing information and research results to medical centers and create jobs and employment protection, deleterious effects caused by noise can be prevented. Thus, one of the useful measures will be widespread and public education. Irrational prescribing of ototoxic drugs without indication especially antibiotics, aspirin and NSAIDs (the most common drugs used in this study were ototoxic) either outpatient or inpatient departments increased incidence of tinnitus that with rational prescribing of these drugs for patients can reduce the prevalence of tinnitus that cause sleep disorders and concentration problems, depression and anxiety, and help the public health community. Also it is recommended for patients who are treated with ototoxic drugs, evaluated for early detection of hearing loss by audiometry. Although NIHL is the most common cause of tinnitus but little research has been done in this area. Because people with NIHL were almost all male, aged 20 to 60 years there are suitable possibilities for epidemiological studies. These studies have guidelines for solving this annoying and more or less untreatable problem.


We present new dispersion and hydrogen bond corrections to the PM6 method, PM6-D3H+, and its implementation in the GAMESS program. The method combines the DFT-D3 dispersion correction by Grimme et al. with a modified version of the H+ hydrogen bond correction by Korth. Overall, the interaction energy of PM6-D3H+ is very similar to PM6-DH2 and PM6-DH+, with RMSD and MAD values within 0.02 kcal/mol of one another. The main difference is that the geometry optimizations of 88 complexes result in 82, 6, 0, and 0 geometries with 0, 1, 2, and 3 or more imaginary frequencies using PM6-D3H+ implemented in GAMESS, while the corresponding numbers for PM6-DH+ implemented in MOPAC are 54, 17, 15, and 2. The PM6-D3H+ method as implemented in GAMESS offers an attractive alternative to PM6-DH+ in MOPAC in cases where the LBFGS optimizer must be used and a vibrational analysis is needed, e.g., when computing vibrational free energies. While the GAMESS implementation is up to 10 times slower for geometry optimizations of proteins in bulk solvent, compared to MOPAC, it is sufficiently fast to make geometry optimizations of small proteins practically feasible. Dispersion and hydrogen bonded corrections to the PM6 method  such as However, computing this vibrational free energy contribution can be complicated by the presence of one or more imaginary frequencies in the vibrational analysis . The source of these imaginary frequencies are usually numerical errors amplified by a flat potential energy surface and the imaginary frequencies often correspond to low lying frequencies that make a significant contribution to the vibrational entropy. Thus, these numerical problems can introduce a significant error in the binding free energy.Preliminary calculations suggested that one of the sources of the imaginary frequencies in PM6-DH+ calculations using MOPAC could be solved by using different geometry optimization algorithms. To test this we implemented a new variant of PM6-DH+, called PM6-D3H+, in the GAMESS program  to allowThe energy model PM6-D3H+ has three contributions d-orbitals for elements past neon but the associated integral code has not yet been implemented. Physical constants in the semi-empirical part of the GAMESS source code were updated, to match those in the current version of MOPAC. All semi-empirical methods in GAMESS uses a finite difference scheme for gradient evaluation.E(PM6) is the molecular PM6  energy, s6 is set to unity, α was set to its default value. s8 and the scaling parameter sr,6 of the atomic cut-off radii used in the dispersion damping function are fitted parameters as in standard DFT-D3  is the third generation dispersion correction developed by rAB is the donor–acceptor distance for the given hydrogen bond geometry, with A and B being the two possible acceptor/donor electronegative atoms, either oxygen or nitrogen. CA and CB are adjustable parameters and refer to either CN and CO. CN and CO are re-parametrized as part of this work as described below.E(H+) is a slightly modified version of the third-generation hydrogen bonding correction, H+, by fgeom is defined as θ is the angle defined by atom A, atom B and the hydrogen  implementation differs slightly from the one originally proposed by The analytical gradient is done using internal coordinates from the energy model  and the Tryptophan-cage (PDB: 1L2Y) using PM6-D3H+ were also carried out. We used the first structure available in each of the downloaded structures. For comparison, we performed two-body Fragment Molecular Orbital (FMO)  geometry−4 Hartree/Bohr.Calculations were performed in either in the gas phase or in bulk solvent using a polarizable continuum to model the solvent . For solAll MOPAC calculations were done with MOPAC2012 . GeometrTimings was carried out on either a 8 core Intel(R) Xeon(R) CPU X5560 @ 2.80 GHz or 24 core AMD Opteron(tm) Processor 6172 @ 2.1 GHz machine.CN and CO. The parameters for H+ are parameterized to minimize the root-mean-square deviation (RMSD) between the interaction energies for PM6 with dispersion correction only (PM6-D3) for a subset of structures from the S22 and S66 data sets , plus the H+ term and the estimated CCSD(T)/CBS reference interaction energy. The CN and CO parameters are then scanned in ranges from −0.2 to 0.0, around the original optimum. A global optimum was found at CN = −0.11 and CO = −0.12, with a RMSD of 1.11 kcal/mol, as seen in Because we use a different dispersion energy function than in the previous DH+ model and make modification to the original hydrogen bonding correction model, it is necessary to determine new optimum values for the This was done using both two and three-body dispersion, but including three-body dispersion did not make any substantial difference in the resulting optimum, and the default was set to two-body for PM6-D3H+, because of the extra computational time associated with three-body. The computational cost becomes a time consuming issue for protein-sized molecules. The final set of parameters for both dispersion and hydrogen bond correction terms can be seen in Overall, the accuracy of PM6-D3H+ is very similar to PM6-DH2 and PM6-DH+, with RMSD and MAD values within 0.02 kcal/mol of one another. The main difference is that the maximum error for PM6-D3H+ is 1.42 and 0.36 kcal/mol smaller than for PM6-DH2 and PM6-DH+, respectively. The maximum error for PM6-DH2, -DH+, and -D3H+ were observed for the S66-19, S66-60, and S66-65 dimer, respectively and, in general, we did not notice any particular dimer that resulted in unusually large errors for all three corrections. All interaction energies can be found in Next, we test PM6-DH3+ on two sets of molecules not in the training set. All structures from the S22 and S66 data sets were optimized with PM6, and PM6-DH+ using MOPAC or PM6 and PM6-D3H+ using GAMESS to test how well the methods reproduce the reference MP2/cc-pVTZ geometries and to compare the optimization algorithms in GAMESS and MOPAC.−4 Hartree/Bohr and an RMS gradient less than 5/3 × 10−4 Hartree/Bohr. These convergence criteria are five times higher than the default and are chosen because we have found that for large systems these criteria can lead to significantly faster convergence without affecting the structure or final energy significantly. See supporting information for GAMESS examples of input files. For complex 58 in the S66 set it was necessary to re-compute the Hessian every 20 steps to obtain convergence and in the case of complex 22, 51, and 58 it was necessary to skip the projection of translational and rotational degrees of freedom from the gradient to obtain convergence, which was done by settings the keyword PROJCT=.F. in $Force. For 11 of the complexes . This is not the case for PM6 and thus a result of the D3H+ energy correction.For the GAMESS optimizations we used the default (quasi Newton–Raphson) geometry optimizer and defined convergence as having a maximum gradient component less than 5 × 10exes see  it was nFor the MOPAC optimization we used the LBFGS geometry optimizer because we found that this is the only optimization algorithm that can be practically applied to optimization of large systems. Using eigenvector following leads to termination of the geometry optimization and the following error message: “TRUST RADIUS NOW LESS THAN 0.00010 OPTIMIZATION TERMINATING”. Based on the output the convergence criterion for the LFBGS optimizer appears to be a change heat of formation of less than ca 0.1 kcal/mol during several consecutive optimization steps. For PM6, this convergence test was not passed after ca 200 geometry optimization steps for complex 10 and 17 from the S22 set and 29, 53, and 54 from the S66 set. For PM6-DH+, this convergence failed after ca 140 geometry optimization steps for complex 11 from the S22 set and 53, 54 and 60 from the S66 set. In all these cases MOPAC terminates the geometry optimization after the mentioned number of steps with the message: “A FAILURE HAS OCCURRED”.The results are summarized in Furthermore, MOPAC optimized geometries tend to have a significantly larger RMS gradient, compared to GAMESS. This leads to significantly more imaginary frequencies in a subsequent vibrational analyses compared to those obtained with GAMESS. In the case of MOPAC 54, 17, 15, and 2 geometries result in 0, 1, 2, and ≥3 imaginary frequencies, while the corresponding numbers for GAMESS are 82, 6, 0, and 0 . Using t−4 Hartree/Bohr is used, but lowering the convergence criterion further does not remove the imaginary frequencies. In the sixth case, complex 16 in the S66 set , we believe the imaginary frequency is due to highly symmetric hydrogen configuration, with switching torsion angles (atomic definition of ψ). Since this only affects structures with highly symmetric hydrogen bonds it is unlikely to cause problems in most applications. We note that the PM6-DH+ method has the same problem.For four of the six cases where a GAMESS optimization leads to a structure with a single imaginary frequency a convergence criterion of 10 angle ψ  which is2X…H cf.  and 2, wi cm−1. Similarly, the lowest real frequencies for these five cases are all <40 cm−1.In the remaining four cases where a GAMESS optimization leads to a structure with an imaginary frequency the cause is most likely an extremely flat potential energy surface for the corresponding degrees of freedom: all imaginary frequencies are <31In summary, the PM6-D3H+ method as implemented in GAMESS offers an attractive alternative to PM6-DH+ in MOPAC in cases where the default geometry optimizer fails to find a converged structure and the LBFGS optimizer must be used and a vibrational analysis is needed e.g., when computing vibrational free energies.In this section we test the applicability of the PM6-D3H+ method, combined with the PCM for bulk solvation as implemented in GAMESS, to geometry optimization of large systems such as proteins and compare to corresponding calculations performed using MOPAC.We optimize the proteins Chignolin (1UAO) and Trp-Cage (1L2Y), which are two small proteins with 138 and 304 atoms, respectively. We optimize the structures using PM6-DH+ in MOPAC , and PM6The results are summarized in iN) are listed in −4 aus. This required 205 and 298 additional optimization steps for Chignolin and Trp-cage, respectively.The number of imaginary frequencies computed for the optimized protein geometries (The relative speedup from running in parallel in solvent is shown on Recent studies by Gilson  and GrimIn this paper we address this problem by developing the PM6-D3H+ method and implementing it in the GAMESS program. The method combines the D3 dispersion correction devloped by Grimme and co-workers with a modified version of the H+ hydrogen bond correction developed by Korth and co-workers. Overall, the accuracy of PM6-D3H+ is very similar to PM6-DH2 and PM6-DH+, with RMSD and MAD values within 0.02 kcal/mol of one another. The main difference is that the maximum error for PM6-D3H+ is 1.42 and 0.36 kcal/mol smaller than for PM6-DH2 and PM6-DH+, respectively.Geometry optimizations of 88 complexes result in 82, 6, 0, and 0 geometries with 0, 1, 2, and ≥3 imaginary frequencies using PM6-D3H+ implemented in GAMESS, while the corresponding numbers for PM6-DH+ implemented in MOPAC are 54, 17, 15, and 2 –S4. ThisFurthermore, the numerical stability of the method could be increased by changing the definition of some of the dihedral angles used in the hydrogen bond correction term. However, this appears only to be an issue for very symmetric systems which is unlikely to occur in large heterogenous systems such as proteins.The PM6-D3H+ method as implemented in GAMESS offers an attractive alternative to PM6-DH+ in MOPAC in cases where the LBFGS optimizer must be used and a vibrational analysis is needed, e.g., when computing vibrational free energies.While the GAMESS implementation is up to 10 times slower for geometry optimizations of proteins in bulk solvent, it is sufficiently fast to make geometry optimizations of small proteins practically feasible.10.7717/peerj.449/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.449/supp-2Supplemental Information 2Click here for additional data file.


It is increasingly appreciated that cochlear pathology is accompanied by adaptive responses in the central auditory system. The cause of cochlear pathology varies widely, and it seems that few commonalities can be drawn. In fact, despite intricate internal neuroplasticity and diverse external symptoms, several classical injury models provide a feasible path to locate responses to different peripheral cochlear lesions. In these cases, hair cell damage may lead to considerable hyperactivity in the central auditory pathways, mediated by a reduction in inhibition, which may underlie some clinical symptoms associated with hearing loss, such as tinnitus. Homeostatic plasticity, the most discussed and acknowledged mechanism in recent years, is most likely responsible for excited central activity following cochlear damage. The mammalian auditory system falls broadly into two pieces, the auditory periphery and the central auditory system. The auditory periphery, which comprises the sound receptors, performs an acoustoelectric transformation. The electrical signals are then sent to the central auditory system for further processing, and eventually, the sensation of sound occurs. In pathological conditions, hearing impairment usually develops as a result of receptor dysfunction. For example, the great majority of acquired sensorineural hearing loss is caused by damage to hair cells in the cochlea. Risk factors for this kind of hearing loss include administration of ototoxic drugs, aging, and overexposure to noise. In fact, among the various manifestations of hearing pathologies confined to the auditory periphery, hearing loss is the most common one.Pathology of the cochlea as a cause of hearing loss has been investigated comprehensively in recent years. However, the role of the central auditory system in hearing loss is still not fully understood. Except in rare cases where the brain and cochlea are both impaired by certain agents, brain changes are considered to be a response to an altered input from cochlea, rather than being directly caused by chemical or environmental factors. These neural changes in the central auditory system, that is, auditory neuroplasticity, have been observed in a broad range of brain behaviors during development and maturation –3. AuditIt has long been appreciated that tinnitus appears to persist in the patients diagnosed with acoustic neuroma and in cases where the auditory nerve is transected. Indeed, recent animal experiments have confirmed this by showing an overexcited state in the auditory brainstem after acoustic overstimulation, regarded as a behavioral sign of tinnitus , 6. HoweThe aim of this review is to assess how peripheral pathologies are associated with different damage agents and especially how pathologies of sensory cells influence neurons in the central auditory pathways. Responses to different damage agents are compared, and some commonalities and correlations among various hearing pathologies are discussed.Important parallels have been drawn between this topic and the current state of research on neuroplasticity in the visual system. Vision and hearing are two major human sensations and share many similar structures and functions. Visual deprivation brings about changes in response properties of neurons in the visual circuits. For example, when one eye is covered, the spiking responses of visual cortical neurons are shifted in favor of the untreated eye, and visual acuity in the blocked eye is reduced –13. CentSeveral studies have demonstrated that unilateral cochlear ablation can enhance the responsiveness of neurons in the central auditory system. In cochlea-ablated neonatal gerbils, stimulation to the nonoperated side resulted in lower response thresholds, greater peak discharge rates, and reduced minimum response latency in inferior colliculus (IC) neurons, despite response patterns comparable to those of normal animals . These cThe organ of Corti functions as a receptor to interpret sounds received and to transform them into electrical signals. Hair cells are the main elements in the organ of Corti that participate in this process. It is not difficult to understand that any external damage that leads to certain pathological alterations in the hair cells would impair signal transmission and therefore lead to a series of changes in the central auditory circuits. Even minor injuries to subcellular structures within the organ of Corti should be considered.Cisplatin is known to have ototoxic side effects, acting especially on the organ of Corti. Hair cells are the primary target, especially OHCs. Tinnitus is a common consequence of cisplatin chemotherapy. Increased spontaneous activity in the dorsal cochlear nucleus (DCN) was shown to be a contributing factor in the etiology of tinnitus . CisplatCompared with cisplatin, carboplatin preferentially damages inner hair cells (IHCs) at relatively low doses  and has Ribbon synapses are responsible for synchronous auditory signaling and transmitter release, and they have been implicated in temporal resolution –50. It hNoise exposure is known to result in insults to hair cells , impaired hearing sensitivity, and elevated hearing thresholds. Although it is likely that the characteristics of the noise, such as the frequency, intensity, and duration, determine the varying patterns of these effects, an overview is still of great value. Changes in the properties of neurons following noise-induced hearing impairment are often regarded as the mechanism underlying dysfunction of the integrated and sophisticated central auditory system. There has been much research regarding neuronal hyperactivity in recent decades. High-intensity acoustic overstimulation  substantially reduced the amplitude of CAP and increased the hearing threshold by about 15 dB at 1 kHz compared with a preexposure group , indicatOne possible explanation for the enhanced activity in the auditory nuclei is that the profile of inhibition weakens after acoustic trauma . For exaThere are many models and results that can be used for comparison. Aminoglycoside antibiotics, known for causing “classical” ototoxicity, were used frequently in models of deafness in early research. Studies with this model found increased evoked c-Fos expression, indicating enhanced neuronal activity, and a marked decrease in GABA release from the central nucleus of the IC . Due to Presbycusis, hearing loss as a consequence of aging, is characterized by a loss of hair cells, and downregulation of inhibition, both glycinergic and GABAergic, is found in the CN , 84, IC Recently, an alternative model has been developed with salicylate administration and confirmed in humans . SalicylAs the suggested mechanisms vary, no firm conclusion can yet be drawn. One of the most inclusive and plausible explanations is the theory of homeostatic plasticity, briefly, the ability of a neuronal network to maintain its present state . When thAn imbalance in synaptic strength represents another possible mechanism, as cochlear damage may be followed by downregulation of inhibition. Basically, this refers to the tendency of a neuronal network to stabilize the total synaptic strength , 97. WitRecently, much importance has been attached to the role of ion channels in the pathology of the hyperactive auditory brain. Researchers have found that exposure to excessive noise causes reduced activity in the voltage-gated potassium channel Kv7, which induces DCN hyperactivity and leads to the development of tinnitus. Manipulations that increase Kv7 activity or reduce the activity of another type of voltage-gated potassium channel, the HCN channel, can help to prevent tinnitus-associated hyperactivity , 109. ThBased on the research reviewed, it seems likely that specific insults to the peripheral auditory system, including cochlear ablation, selective IHC or OHC loss, and noise-induced mixed and incomplete IHC and OHC injuries, result in a reduction of input from the cochlea, thereby giving rise to hyperactivity in the central auditory circuits. A good example of this process is found in tinnitus, which may be associated with neuronal hyperactivity and is likely a common consequence of various kinds of cochlear damage. From an evolutionary perspective, hyperactivity in the brain may be a maladaptive response to reduced input, indicating that the system needs to become more sensitive to the reduced input to obtain more information and thereby remain balanced and stable. This dysfunctional neural state might contribute to some brain pathologies with auditory dysfunction, as indicated in a recent review suggesting that hyperactivity in the auditory brain is closely related to tinnitus and hyperacusis . Despite