Phalacrocorax carbo) was determined using psychophysical methods (method of constants). Hearing thresholds were derived using pure tones of five different frequencies. The lowest threshold was at 2 kHz: 18 dB re 20 µPa rms. Thresholds derived using signal detection theory were within 2 dB of the ones derived using classical psychophysics. The great cormorant is more sensitive to in-air sounds than previously believed and its hearing abilities are comparable to several other species of birds of similar size. This knowledge is important for our understanding of the hearing abilities of other species of sea birds. It can also be used to develop cormorant deterrent devices for fisheries, as well as to assess the impact of increasing in-air anthropogenic noise levels on cormorants and other aquatic birds.Many aquatic birds use sounds extensively for in-air communication. Regardless of this, we know very little about their hearing abilities. The in-air audiogram of a male adult great cormorant  was determined using psychophysics methods to further our knowledge of hearing sensitivity in aquatic birds in relation to human generated noise. More than 800 out of the 10,000 identified species of birds spend a significant part of their lives in, on or close to water . Many aqThe importance of sound is evident from the well-developed hearing abilities in most birds. Smaller birds have a hearing sensitivity range from 0.25 up to 8 kHz, whereas the frequency range as well as hearing sensitivity of larger birds is lower . It is one of the most efficient of marine predators, foraging daily on several hundred grams of fish caught during long dives  as a function of stimulus levels. There is a problem, however, with this so-called classical psychophysical method  measure of the animal's hearing is to use signal detection theory . The FA rate of the used sessions were 20-31% when all catch trials for each frequency were pooled . The psyROC plots are shown in In F1,6=1.2, P=0.4). If the threshold would have been masked, we would have expected the regression line to have a significant positive slope.Ambient noise levels decreased with increasing frequency . When meF1,195=0.30, P=0.58) nor the FA rate  was seen when the double-blind sessions were compared to the non-double blind sessions (FA rate was 25% for double blind and 30% for non-double blind). Also, the FA rate showed no effect on HIT rate for each individual session . There was a significant increase in HIT rate with increasing stimulus level , while the frequency had no effect on HIT rate .No noticeable difference in neither the HIT rate  housed in Kerteminde, Denmark, at the Marine Biological Research Center (University of Southern Denmark). The bird was caught when 5 months old. Its history prior to being caught is unknown, but throughout its time at the laboratory it has remained very healthy. In 2012, the male was joined by a female great cormorant fledgling. The birds were kept in an outdoor 3×3×2.5 m (length×width×height) aviary with an adjacent 1×1×2.5 m quarantine and a 3×5.5×3 m pool cage (with a water depth of 1 m). The birds were trained twice a day using operant conditioning with positive reinforcement techniques. Their daily diets ranged from 250-400 g of fish , depending on their motivation and weight, as well as the ambient temperature. The birds were caught and kept under Nature Protection Agency Permit SNS-342-00056 and Ministry of Food and Agriculture Permit 2300-50120-00003-09. The experimental work was performed under Permit (2012-DY-2934-00021/BES) from the Danish Animal Experimentation Inspectorate.The experimental bird was a male 6-year-old wild-caught great cormorant  hanging from the ceiling, with a green lamp 20 cm in front of the hoop and a loudspeaker 20 cm above the hoop . The expHearing was measured using the methods of constant stimuli , a G.R.A.S. 12AA amplifier and a half-inch G.R.A.S., Type 26 AC microphone with a G.R.A.S. AA0009 preamplifier. For the ambient noise and signal calibration recordings, the amplifier was set to +20 dB. Prior to measurements, the system was calibrated by recording the signal from a B&K 4230 calibrator .The same recording system was used to measure the ambient noise inside the acoustic chamber at regular intervals, either right before or after the research sessions. To ensure that the microphone itself was not creating too much noise and affecting the noise recordings, a self-noise recording  was made . This wasensuResults from each research session were analysed using MATLAB . Only sessions with a FA rate  of 20-35% were used in the analysis, as an indication that the decision criteria used by the bird was kept relatively constant. The data from all remaining sessions were then pooled for each frequency and the overall HIT rate  was calculated for each intensity level. This data was fitted to a psychometric function using Probit analysis . The fitsensu Chapter 3 in In addition to this classical psychophysical analysis approach, we used signal detection theory to obtain an unbiased measure of the bird's hearing abilities .Melopsittacus undulates), critical ratios (CRs) follow the same linear trend and can be calculated using:e trials .Analysis of acoustic signal and noise levels were made using Matlab . Average ambient noise levels were estimated using Welch's method  with an For exploring the influencing factors of HIT rate, we used repeated measures ANOVA with HIT rate as the dependent variable and stimulus level, frequency, false alarm rate, and double blind treatment as explanatory variables. One-way ANOVA  was used


Hepatitis B virus is a virus that creates significant hepatic and extra-hepatic complications, with widespread prevalence across the community and body systemic involvement, and can impact on hearing performance. This study aims to evaluate hearing loss among individuals with hepatitis B compared with healthy subjects.In this case-control study, 83 HBsAg-positive patients with a 1-year history of disease were selected for pure tone audiometry (PTA) testing, while 108 HBsAg-negative patients were selected as the control group. Subjects in both groups were aged 20–40 years. The threshold was set at 25 db for hearing loss. Final data were analyzed using SPSS software.Significant differences were found between the case group and control group in average PTA and hearing loss. There was also a significant difference between the two groups in average PTA at frequencies of 250, 4,000, and 8,000 Hz, but not at speech frequencies of 500, 1,000 and 2,000 Hz, despite the difference in average PTA.According to significant differences in average PTA between patients with hepatitis B virus and healthy subjects in this study, hearing loss may be attributed to the presence HBV of in the patient group. The impnfection –3.Acute complications of hepatitis B range from fulminant hepatitis to manifestations such as hepatic fibrosis, liver cirrhosis and hepatocellular carcinoma, with approximately 1 million people a year dying from such complications  (necrotizing vasculitis and polyarthritis nodosa (PAN), diseases that involve the arteries, veins, and capillaries and can lead to impaired hearing, is quite clear. In fact, 30% of people with PAN are hepatitis B carriers, and are more susceptible to HBV infection  ,10. The s (LCMV) ,10. Parvs (LCMV) . The relnfection –14. Accolocated in the west of Iran). The project was approved by the Medical Ethical Committee of the University of Medical Science, Ilam, Iran (Ir. medilam. rec.1394.90).This case-control study was conducted at the Nourooz-abad Polyclinic and Reference Lab from March 2012 to September 2013 in the Iranian city of Ilam . otoscope review. After the second screening, 83 and 108 participants were included in the case and control groups, respectively. All participants in both groups were aged between 20 and 40 years. Based on initial examination by otoscopy, no defects in the tympanic or ear infections were observed in either group. Hearing assessment was carried out by an expert using an audiometric devise (OSCILLA SM 96 USA). PTA, and pure tone average for left ear (PTALE) and right ear (PTARE) were performed at frequencies of 250, 500, 1,000, 2,000, 4,000, 8,000 Hz. In addition, speech discrimination score (SDS) at frequencies of 500 to 2,000 and hearing loss percentage (HLP) were estimated. Data were analyzed using SPSS 16.1 and the Kolmogorov-Smirnov test was used to assess the normality of the data. Mann-Whitney and Kruskal-Wallis tests were performed. Hearing loss in this study was classified into five grades according to the American National Standards Institute and American Academy of Otolaryngology. The threshold was set at 25 db for hearing loss.The control group were required to have no history of hepatitis B and a negative HBsAg test. Other inclusion criteria for both groups were age 40 years or younger and a lack of history of physical trauma, autoimmune disease, ear infection, tumor, ear trauma, taking antiviral drugs, measles, mumps, rubella, polyarthritis nodosa, work experience in a noisy environment, and a history of hearing loss or deafness in the family. Participants were also required to have no abnormalities in the ear canal following consisted of 62% males and 38% females. One hundred and eight HBsAg-negative subjects were included in the control group, consisting of 27% females and 73% males, with a mean±SD age of 28±5.5 years. In the investigation of relationship among the average PTA, degrees of hearing loss at frequencies of 250 to 8,000 Hz and age and sex, no significant differences were observed in the control group. There was a significant correlation between left and right PTA with age in the patient group; this difference was observed in the age group 30–40 years (P=0.02). PTA for each frequency in both ears in the case and control groups is presented in  age of 33 ± 6.8 years were selected. The sample ented in .According to A statistically significant difference was observed in total average PTALE and PTARE in both groups . The perMany factors contribute to hearing loss, and almost 10% of people experience hearing loss ranging from mild to severe for various reasons . HearingThe presence of HBV in the cerumen has been demonstrated in patients infected by hepatitis B , and alsAccording to the findings of the present study, the average PTA in both ears of patients aged 30–40 years showed significant differences, which might suggest that the virus has a greater impact on age-related hearing loss in people with hepatitis B infection and prolongs the duration of chronic disease compared with healthy people. However, more studies are needed to prove this hypothesis.By comparing the PTA for each frequency, significant differences can be observed between the two groups at frequencies of 250, 4,000, and 8,000 Hz. Average PTA was greater than the 25-db threshold of hearing loss in patients only at a frequency of 4,000 Hz in both ears . In a st Furthermore, no significant differences in speech discrimination were shown for the two cases and the control groups, similar to previous studies. The highest percentage of hearing loss in the case group was observed in the first grade (26 to 40 decibels), which was less than the control group. Noting that the frequencies of speech include core understanding of speech in daily conversation, any disruption in these frequencies will have a large effect on hearing quality , 19, 20.The findings revealed that there is a relationship between hearing loss and HBV in patients with HBV. Access to a wider statistical population, medical history, and family diseases and investigation of auditory brainstem response (ABR) in future studies may help to clarify the role of hepatitis B in causing hearing loss in individuals infected by HBV, as well as the study of liver enzyme factors and other specific tests. Considering the few studies conducted in this area, hearing loss in some patients with hepatitis B may be attributable to the presence of the virus in the body; although further studies are required.


However, hearing loss induced by CAPOS has never been characterized to date. Interestingly, the first proband did not manifest any features of CAPOS, except subclinical areflexia; however, the phenotypes of second proband was compatible with that of CAPOS, making this the first reported CAPOS allele in Koreans. This ANSD phenotype was compatible with known expression of ATP1A3 mainly in the synapse between afferent nerve and inner hair cells. Based on this, cochlear implantation (CI) was performed in the first proband, leading to remarkable benefits. Collectively, the de novo ATP1A3 variant can cause postlingual-onset auditory synaptopathy, making this gene a significant contributor to sporadic progressive ANSD and a biomarker ensuring favorable short-term CI outcomes.The etiologies and prevalence of sporadic, postlingual-onset, progressive auditory neuropathy spectrum disorder (ANSD) have rarely been documented. Thus, we aimed to evaluate the prevalence and molecular etiologies of these cases. Three out of 106 sporadic progressive hearing losses turned out to manifest ANSD. Through whole exome sequencing and subsequent bioinformatics analysis, two out of the three were found to share a  The etiologies of ANSD are highly diverse, including hypoxia, infection, kernicterus, cytotoxic oncologic drug, and genetic factors5.Auditory neuropathy spectrum disorder (ANSD) is a type of hearing loss characterized by electrophysiological findings of an impaired or absent response in auditory brainstem responses (ABR), despite evidence of intact outer hair cell function as supported by the presence of cochlear microphonics and/or detectable otoacoustic emission (OAE). Subjects with ANSD have varying degrees of hearing losses; however, they generally present poor speech recognition that is disproportionate to the degree of hearing loss and difficulty hearing in noiseOTOF  and PJVK genes 9. GJB2 and mitochondrial 12SrRNA mutations have also been reported to be related to this phenotype13. Among them, OTOF mutations occupy a major part of prelingual ANSD in many Caucasian populations14. The predominance of OTOF mutations has also been established in Koreans as they were reported to account for up to 85% of the Korean population with prelingual ANSD with normal cochlear nerve15. As for postlingual-onset ANSD, a lot of syndromic forms that cause sensory and motor neuropathy have been documented in adults with ANSD, including Charcot-Marie-Tooth disease17, Friedreich’s ataxia19, deafness-dystonia-optic neuropathy (DDON) syndrome20, autosomal dominant optic atrophy (ADOA)22, and AUNX1 due to mutations in apoptosis-inducing factor24. However, there are not many reported genes related to non-syndromic, progressive ANSD with postlingual onset. Only mutations from DIAPH3 as the cause of AUNA126 have been anecdotally reported from familial ANSD cases, leaving a substantial portion of non-syndromic and sporadic forms of ANSD still unanswered with respect to the molecular etiology. SLC17A8 encoding VGLUT3, if altered, has been shown to cause ANSD in mice28; however, to date, phenotypes of ANSD have not been reported in human subjects with SLC17A8-induced hearing loss30.As for prelingual genetic ANSD, there seems to be less diversity compared with postlingual-onset genetic ANSD. Only a few genes have been associated with prelingual genetic ANSD, including autosomal recessive 37, can fit into the category of ANSD and manifest in a non-syndromic manner.Herein, we aimed to explore the molecular etiology of three unrelated subjects manifesting sporadic, progressive ANSD with postlingual onset. We identified that sensorineural hearing loss (SNHL) in CAPOS syndrome (OMIM #601388), comprising of cerebellar ataxia, areflexia, pes cavus, optic atrophy, and hearing lossAmong the 106 postlingually deafened subjects with moderate or higher degree of sporadic SNHL, at least three  unrelated subjects were documented to have ANSD  in three probands with sporadic ANSD and identified the most convincing causative variant using the bioinformatics filtering process that has previously been describeded Table . CandidaATP1A3 gene , located on chromosome 19q13.2 and classified as ‘pathogenic’ according to CLINVAR (https://www.ncbi.nlm.nih.gov/clinvar/)39, was detected in two (SH191-430 and SH222-518) of the three ANSD subjects   and the Korean Reference Genome Database . However, in the SB284 family, no convincing variants were found.Interestingly, one heterozygous missense variant, c.2452 G > A: p.E818K of the The first proband (SH191-430) was a 24-year-old woman that manifested SNHL when she was a teenager. Her SNHL was compatible with typical ANSD, as shown in Fig. The second proband (SH222-518) was a 6-year-old boy when he first visited the ENT clinic. He successfully passed the newborn hearing screening test, which was performed with automated ABR, suggesting that his SNHL was not congenital. His development had not been noteworthy, except his experience of two acute episodes of neurologic symptoms and signs, including lethargy, instability while sitting, ataxia, dysarthria, visual disturbance with dancing nystagmus, and hearing loss after febrile illness at the age of 3 and 5 years. His SNHL was entirely compatible with ANSD, considering a detectable OAE response without ABR response Fig. . At thatThe third proband (SB284-563) was a 61-year-old female. Her SNHL started in her early thirties. Like the first proband, she did not have any neurologic abnormalities, except hearing loss. She had moderate hearing loss on PTA, 45 dB on the right and 40 dB on the left, with a disproportionately low word recognition score of 20%, bilaterally. In line with this finding, she showed a detectable ABR wave only at 90 dB stimulus on the right ear, despite the presence of a positive OAE response back in 2014, which indicated that her SNHL was compatible with ANSD. Two years later, both her OAE and ABR responses disappeared Fig. .The neurologic features of these three ANSD subjects are presented in Table ATP1A3  underwent cochlear implantation (CI) since they were unable to benefit from hearing aids. The first subject (SH191-430) had the highest difficulty in speech discrimination with hearing aid, especially with background noise. She scored worse on the Korean version of the Central Institute of Deafness (K-CID) test with hearing aid, which decreased from 36% without hearing aid to 0% with hearing aid. Both subjects had CI on the right ear after getting replicable, albeit not perfect, waveforms on trans-tympanic electrically evoked ABR (EABR)  and rapid-onset dystonia Parkinsonism 42. Among various ATP1A3 mutations, two variants, p.D810N and p.E815K, have been found recurrently in more than 50% of patients with AHC, while no distinct mutations have been found in patients with RDP35. A different variant of this gene, p.E818K (c.2452 G > A), which has not been detected in the context of two main diseases associated with ATP1A3, has been reported to cause the third allelic disorder, CAPOS syndrome without exception in several reports37. CAPOS syndrome was first described in 1996 by Nicolaides et al. in a British family43. To date, this single variant has been identified exclusively from all nine unrelated CAPOS families, as described previously with 100% frequency. Unlike the previous two diseases, SNHL with sudden onset and progressive nature is a distinctive disabling feature of CAPOS syndrome. However, there has not been a rigorous study on the type of SNHL thus far.The ATP1A3 led to the diagnosis of CAPOS syndrome. Different variants of this gene have been reported to cause RDP and AHC, without the companion of hearing or visual disturbance35. On the other hand, the first subject, SH191-430, who shared the same p.E818K variant, denied any neurological symptoms, except areflexia that was elucidated only after a rigorous neurological examination by an expert neurologist. This subject would have been classified as non-syndromic without a comprehensive neurologic examination by an expert. This is not surprising considering that there have been some reports showing subjects with CAPOS syndrome exhibiting mild symptoms resulting in recovery with minimal residual ataxia during walking and standing on one foot31. This implicates that extensive history taking and rigorous neurologic examination would be an essential part in the diagnosis of postlingual and progressive ANSD.The second subject, SH222-518, had passed the newborn hearing screening test by automated ABR; however, his hearing impairment was initiated and became prominent with a definite progression through two febrile events. Together with accompanying symptoms and signs, such as ataxia and visual disturbance, the detection of p.E818K in th CAPOS patient (SH222-518) in the literature. This is the first report of a CAPOS allele in Koreans. High mutability of this residue, as shown by frequent de novo occurrence of p.E818K (c.2452 G > A) of ATP1A3 both in Caucasians31 and in Koreans, indicates that the effect of this mutational hotspot is not limited to certain populations but would rather be global. This is noteworthy because other allelic disorder of ATP1A3, RDP, has also been reported to be frequently caused by de novo variants44. Indeed, with the advent of next generation sequencing (NGS) technologies, recent studies using NGS have elucidated the important role of de novo variants in several important neurological disorders47. To date, it is unclear exactly why de novo variants frequently arise in ATP1A3. Recently, the rate of de novo mutations has been reported to be heavily influenced by the age of the father48; however, that study failed to account for all de novo cases. There might be a genetic context in ATP1A3 favoring the occurrence of de novo variants, awaiting further clarification. Our present study also unveils that a phenotypic spectrum related to p.E818K of ATP1A3 can be extended to include nearly non-syndromic postlingual ANSD, not limited to the canonical CAPOS syndrome.Our present study reports the tenth CAPOS family (SH222) and the 27ATP1A3 encodes the α3 catalytic subunit of Na+/K+ ATPase (NKA), which is a membrane-bound transporter using ATP to transport three Na+ outwards in exchange for two K+ into the cell, resulting in a negative transmembrane potential. Regarding the contribution of NKA on hearing, it has mainly been studied in relation to endocochlear potentials (EP). The inhibitors of NKA, such as ouabain, are known to reduce EP50. There are four α isoforms identified in vertebrates52. NKAα1 has been identified in the strial marginal cell, and NKAα2 in the lateral wall fibrocyte of the stria vascularis, which is the source of EP53. Indeed, heterozygous deletion of NKAα1 or NKAα2 in mice resulted in the reduction of EP and progressive, age-dependent hearing loss54. Accordingly, the ATP1A2 gene (encodes NAKα2), which has previously been reported to be the genetic cause of familial hemiplegic migraine type 2 , has recently been reported to be associated with progressive hearing loss with migraine55. Specifically, co-segregation of a novel variant, c.571 G > A, in the ATP1A2 gene with migraine and hearing loss in a family was shown, although the pathogenic potential of the c.571 G > A variant was not confirmed. Contrastingly, the α3 isoform is rather neuron-specific, albeit being co-expressed with ubiquitous α1 in most neurons58. NKAα3 is known to play an important role in the rapid restoration of increased intracellular Na+ concentration after high neuronal activity in the nervous system59. Therefore, disrupted NKAα3 activity leads to a failure in regaining the resting membrane potential after excitatory activity, and it has been proposed to cause paroxysmal neurologic events observed in two main ATP1A3-related syndromes60. Several experimental studies evaluated the pump activity as well as the effect of pathologic variants of ATP1A3. They showed that these mutations caused a reduction in catalytic activity and a failure to generate pump current. This finding is consistent with the inability to bind to K+ ions and a reduction in the Na+ affinity for activating phosphorylation61.ATP1A3 cause hearing loss have not been fully elucidated to date. In a study focusing on the NKAα expression pattern in Organ of Corti and the neuronal element of cochlea, it has been demonstrated that NKAα3 is expressed abundantly in the membranes of type I afferent terminals that are in contact with the inner hair cells, spiral ganglion somata, and medial efferent terminals contacting the outer hair cells50. NKAα1 has been identified in the supporting cells neighboring the inner hair cells, and NKAα2 has not been detected in either the organ of Corti or spiral ganglion50. This expression pattern of NKAα3 in the cochlea has been shown to be compatible with an observation in which perfusion of ouabain, a specific inhibitor of NKAα3 in the cochlea, directly administered through the round window in gerbil, resulted in a reduced or completely abolished compound action potential but did not reduce EP63. This suggests that NKAα3 may have a more direct role in the regulation of signal transmission in the synapses with hair cells and spiral ganglion cells, rather than having a contribution to generate EP. Therefore, it is possible for the alteration of NKAα3 to lead to the clinical and laboratory features compatible with ANSD.Nonetheless, the exact role of NKAα3 in hearing and how mutations in 65. Due to the uncertainties of outcomes after CI, it is important to select patients who are expected to have good results from CI. EABR is widely used to evaluate the integrity of auditory pathway and predict the outcome of CI68. Among the various subtypes of ANSD classified by their predominant lesion, auditory synaptopathy has been reported to have excellent CI results, and EABR is typically present in this subtype71. However, the interpretation of waveforms is somewhat subjective, and preoperative trans-tympanic EABR is not consistently reliable for predicting the outcome of CI72. It is not unprecedented to benefit significantly from CI even in cases without any detectable responses from both preoperative trans-tympanic and postoperative intracochlear EABR tests74. Therefore, genetic diagnosis that enables precise localization of the pathologic site in the auditory pathway has been recognized to be very important.ANSD is a heterogeneous disorder that may result from various etiologies and different lesion sites, ranging from the inner hair cells and synapses to auditory nerve and central cortex. Inherent from such pathologic characteristics of ANSD, the behavioral threshold measures are not consistent with other measures of auditory function, such as ABR and speech understanding scores. Furthermore, it poses a challenge when choosing an appropriate method of auditory rehabilitation and predicting the outcomeOTOF gene, a causative gene of auditory synaptopathy, may cause a dysfunction in Ca2+-triggered exocytosis of a neurotransmitter in the ribbon synapses of the inner hair cells77. Therefore, a direct electrical stimulation of the spiral ganglion cells with CI bypassing the main lesion can restore well-synchronized neural responses that propagate through the intact auditory nerve. Another ANSD with good results on CI is a syndromic ANSD accompanied by autosomal dominant optic atrophy  due to OPA1 variations64. Although the specific function of the OPA1 gene regarding hearing has not been fully elucidated to date, detailed assessment with trans-tympanic electrocochleography and electrically-evoked response following CI indicated that the site of the lesion is at a terminal portion of auditory nerve fibers with the potential to progress proximally at advanced stages11. OPA1 mRNA and protein were found in the hair cells as well as in the neuronal fibers innervating the inner hair cells, ganglion cells of the cochlea, and vestibular organ78. This is significantly overlapped with the expression site of the ATP1A3 protein, NKAα3. Although the function of NKAα3 has not been fully revealed, we can postulate from the expression pattern of this protein that the main pathologic lesion site would be a synapse between the inner hair cells and the afferent nerve fibers, or at most spiral ganglion neurons. If the lesion is mainly the former, then we can expect good CI results, similar to the ANSD associated with OTOF and OPA1. In accordance with this expectation, one (SH191-430) of our two ANSD subjects carrying p.E818K (c.2452 G > A) of ATP1A3 underwent CI and showed excellent performance at 3 and 6 months after CI, although longer observation is needed  out of 106 sporadic cases of postlingual moderate and more degree of SNHL were diagnosed with ANSD. This prevalence is markedly lower when compared with the pediatric group. It is likely that many post-lingual patients with hearing difficulty did not undergo enough audiological and electrophysiological tests to fully confirm ANSD. Moreover, this may be even further underestimated because OAE response, which was once considered a prerequisite, might have been absent or disappeared over time90. In our study, the disappearance of OAE response from the third subject, SB284–563, over a period of 2 years  postlingual-onset ANSD, suggesting that this specific variant of ATP1A3 could be a significant contributor to postlingual-onset ANSD, not only in Koreans but also in other populations. Moreover, an evaluation of postlingual-onset ANSD subjects should require a comprehensive neurologic examination and meticulous history taking to ensure that no episodes of ataxia and visual disturbance triggered by a fever go unnoticed. Molecular genetic testing of this particular variant, p.E818K of ATP1A3, can be helpful to delineate ANSD cases which are related to subtle cases of CAPOS syndrome.In our present study, the single de novo mutation of ATP1A3 can cause progressive ANSD with postlingual onset and with either absence or presence of syndromic features. This confirms that ATP1A3 is an important and prevalent causative gene for progressive ANSD with postlingual onset. The presence of this variant can potentially serve as not only an etiologic biomarker, but also as a prognostic biomarker that allows for the identification of a subset of postlingual-onset ANSD subjects who can significantly benefit from CI, at least during a short-term follow-up period.Here, we showed that recurring This study was approved by the institutional review boards of Seoul National University Hospital (IRBY-H-0905-041-281) and Seoul National University Bundang Hospital (IRB-B-1007-105-402). Informed consent was obtained from all participants in this study. Written informed consent for child participants was obtained from their parents or guardians. All methods in this study were performed in accordance with the relevant guidelines and regulations.et al. were applied; (1) understanding of speech worse than predicted from the degree of hearing loss on their behavioral audiograms; (2) recordable otoacoustic emissions and/or cochlear microphonic; and (3) absent or atypical auditory brain stem responses91.Medical and developmental histories were collected for 106 sporadic patients recruited between 2010 and 2017, who showed postlingual-onset, moderate or higher degree of SNHL. Syndromic cases were excluded. The audiometric evaluation with PTA, OAE, and ABR were carried out for the clinical diagnosis of ANSD. Internal auditory canal MRI and TBCT were also performed to identify anatomically intact cochlear nerve and any other anatomical abnormalities related to hearing loss. Comprehensive neurologic evaluation was performed by a neurology specialist to see if there is any accompanied neuropathy. The diagnostic criteria for ANSD as described by Rapin 92, and were subjected to WES. A SureSelect 50 Mb Hybridization and Capture kit was used for WES, and sequencing was performed using a HiSeq2000. The read length for the paired-end reads was 100 bp.Whole blood (10 ml) was obtained from two probands and, if possible, their siblings and parents for the segregation study. Genomic DNA samples were extracted from the peripheral blood via standard procedures, as previously described93 with the “MEM” algorithm. We used SAMTOOLS version 1.2 for sorting and indexing the SAM/BAM files94. Picard-tools version 1.127 (http://broadinstitute.github.io/picard) was used to mark duplicates. We used the IndelRealigner and BaseRecalibrator from Genome Analysis Toolkit (GATK) version 3.1-395 based on known single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) from the Database of Single Nucleotide Polymorphisms , Mills, and 1000 G gold standard indel b37 sites and 1000 G phase I indel b37 sites for realigning the reads and base recalibration. The targeted gene variants were called using the Unified Genotyper in GATK and also were recalibrated by GATK based on dbSNP138, Mills indels, HapMap, and Omni and filtered at 99.0 truth sensitivity level. Finally, we used ANNOVAR to annotate the variants96.The reads were aligned with the human genome reference sequence (hg19) as proposed by Burrows-Wheeler Aligner, version 0.7.538. In brief, we selected the variants in the coding region and with allele frequencies of <0.01, which were reported in the Exome Aggregation Consortium  and the Korean Reference Genome database . Then, we filtered out the variants reported in dbSNP138, but included flagged SNPs. We chose the variants that were matched with one of the three possible inheritance patterns that could be possible in the sporadic form of genetic hearing loss. They were filtered to discard any variants with false-positive results based on minor allele frequency (MAF) cut-off 97 and with variant allele frequency (VAF) of heterozygosity of less than 0.3; however, the criteria were selectively adjusted to be less than 0.1 when a variant was located in a homologous locus or a gene with a pseudogene or homologous between family members. Finally, the scores of SIFT (http://sift.jcvi.org/), PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) and GERP (http://mendel.stanford.edu/SidowLab/downloads/gerp/) and CLINVAR (https://www.ncbi.nlm.nih.gov/clinvar/)39 were used to assess the pathogenicity of candidates.We went through the filtering process as previously described98. The four STR markers were chosen because they were shown in a previous study to be hypervariable and informative among Koreans98. A definite haplotype was reconstructed based on the information from parental sample and siblings.To exclude non-paternity, genotypes of four unlinked STR markers  from family members were analyzed, using a previously described methodAll data, whether generated or analyzed during this study, were included in this published article (and its Supplementary Information files).Supplementary Figure S1 and S2


TMPRSS3 gene, which can lead to prelingual (DFNB10) as well as postlingual deafness (DFNB8). TMPRSS3 mutations can be classified as mild or severe, and the phenotype is dependent on the combination of TMPRSS3 mutations. The combination of two severe mutations leads to profound hearing impairment with a prelingual onset, whereas severe mutations in combination with milder TMPRSS3 mutations lead to a milder phenotype with postlingual onset. We characterized a Chinese family (number FH1523) with not only prelingual but also postlingual hearing impairment. Three mutations in TMPRSS3, one novel mutation c.36delC [p.(Phe13Serfs⁎12)], and two previously reported pathogenic mutations, c.916G>A (p.Ala306Thr) and c.316C>T (p.Arg106Cys), were identified. Compound heterozygous mutations of p.(Phe13Serfs⁎12) and p.Ala306Thr manifest as prelingual, profound hearing impairment in the patient (IV: 1), whereas the combination of p.Arg106Cys and p.Ala306Thr manifests as postlingual, milder hearing impairment in the patient , suggesting that p.Arg106Cys mutation has a milder effect than p.(Phe13Serfs⁎12). We concluded that different combinations of TMPRSS3 mutations led to different hearing impairment phenotypes (DFNB8/DFNB10) in this family.Autosomal recessive hearing impairment with postlingual onset is rare. Exceptions are caused by mutations in the Autosomal recessive nonsyndromic hearing impairment (ARNSHI) is the most common type and accounts for ~80% of cases of inherited hearing loss. A large majority of recessive mutations are associated with congenital, nonprogressive, and severe-to-profound hearing loss . We concluded that different combinations of mutations led to different hearing impairment phenotypes in this family.This study identified one Chinese family with ARNSHI (number FH1523), in which affected individuals had two different hearing impairment phenotypes: prelingual and postlingual. We performed large-scale mutational screening of 129 known deafness-related genes and identified three disease-segregating mutations in theFamily FH1523 is a four-generation Chinese family with prelingual and postlingual nonsyndromic sensorineural hearing loss. DNA samples were obtained from seven members of family FH1523  and 260 ethnicity-matched controls. A detailed medical history was obtained through a questionnaire. Computed tomography (CT) of the temporal bone was performed in the index patient (IV: 1). A physical examination, otoscopy, and pure tone audiometric examination (at frequencies from 125 to 8000 Hz) were performed to identify the phenotype. The hearing loss range was described depending on the pure tone audiometry (PTA) parameters: low frequency, 125–500 Hz; mid frequency, 1-2 kHz; and high frequency, 4–8 kHz. Prelingual and postlingual hearing loss was classified according to the onset of prominent hearing loss. Prelingual hearing loss is present before speech develops and usually begins before 3 years of age, whereas postlingual hearing loss occurs after the development of normal speech , and c.316C>T (p.Arg106Cys) mutations was tested in seven family members , including the four whose gDNA had been subjected to the 129 deafness-associated genes and next-generation sequencing (NGS) analysis, using polymerase chain reaction (PCR) (primer sequences available on request) followed by bidirectional Sanger sequencing of the amplified fragments . Sequence alterations were identified by alignment with the TMPRSS3 GenBank sequence  using the GeneTools software. In addition, a total of 260 ethnicity-matched controls with normal hearing underwent mutation screening by direct sequencing.The segregation of the NP_001243246.1 (H. sapiens), XP_001105841.2 (M. mulatta), XP_001137100.3 (P. troglodytes), XP_001179855.1 (B. taurus), XP_853682.3 (C. lupus), NP_001157248.1 (M. musculus), NP_001101089.1 (R. norvegicus), XP_425558.3 , and XP_001340422.5 (D. rerio).Multiple sequence alignment was performed according to a HomoloGene program with default settings and the sequenceshttp://www.ncbi.nlm.nih.gov/homologene?cmd=Retrieve&dopt=MultipleAlignment&list_uids=56985)(http://swissmodel.expasy.org/workspace/). This study used the automatic modeling approach to apply the complete protein sequence of human TMPRSS3, including its 454 amino acids and its mutation, which are available in the NCBI GenBank (NP_001243246.1) in the FASTA format. Data obtained by the homology models were visualized using Swiss-PdbViewer 4.1.Three-dimensional (3D) modeling of the human wild type, p.Ala306Thr and p.Arg106Cys, was performed using SWISS-MODEL, an automated homology modeling program (Family FH1523 is a four-generation Chinese family with ARNSHI  and inclFor her affected grandfather (II: 4) and grandfather's four cousins , hearing impairment was postlingual, late onset (in the second or third decades of life), and progressive according to memory description. Their hearing progressed gradually with advancing age. Their previous audiograms were absent due to living in the isolated village of China. They developed normal speech ability and now have oral communication capacities with the help of lip reading. They complained of having trouble detecting sharp voices since their 20s to 30s. We predicted that although low-frequency hearing was initially normal in the affected subjects of the second generation, their hearing would ultimately deteriorate at all frequencies. Flatter audiogram configurations were observed at ages older than 60 years , whereas the audiogram of II: 2 (57 years of age) was ski-sloped, and the threshold of 250 Hz was 30–35 dBHL, that of 500 Hz was 35–40 dBHL, that of 1 kHz was 75 dB, and that of 2 kHz to 4 kHz was greater than 100 dBHL  (rs181949335), c.36delC [p.(Phe13Serfs∗12)], and c.316C>T (p.Arg106Cys) (rs139805921). Of these, two variants, c.916G>A (p.Ala306Thr) and c.316C>T (p.Arg106Cys), were reported previously as a pathogenic mutation, whereas c.36delC [p.(Phe13Serfs∗12)] was novel and had not been found in the dbSNP databases.We sequenced all the coding exons plus ~100 bp of the flanking intronic sequence of 129 deafness genes in two affected  and two unaffected members of family FH1523 . Three variants leading to amino acid change were detected in TMPRSS3 were identified in three affected family members of the second generation . Compound heterozygous p.(Phe13Serfs∗12) and p.Ala306Thr mutations of TMPRSS3 were found in one affected family member of the fourth generation (IV: 1). In addition, TMPRSS3 heterozygous variant p.Ala306Thr was found in two normal hearing family members , whereas heterozygous variant p.(Phe13Serfs∗12) was found in one normal hearing family member (III: 4)  in family FH1523 were genotyped to identify the mutations. Compound heterozygous p.Ala306Thr and p.Arg106Cys mutations of(III: 4) , which i∗12) was possible. The age at onset and severity of the hearing impairment in IV: 1 [p.Ala306Thr and p.(Phe13Serfs∗12)] were much earlier and worse, respectively, than those in II: 2, II: 3, and II: 5 (p.Ala306Thr and p.Arg106Cys). Comparison of the audiograms of IV: 1 (6 years of age) and II: 5 (57 years of age) revealed that II: 5 had a better hearing threshold at 250 Hz and 500 Hz. This result suggests that p.Arg106Cys mutation has a milder effect than p.(Phe13Serfs∗12).Because all the patients in family FH1523 had the p.Ala306Thr mutation in common, a comparison of the phenotypic effect of the p.Arg106Cys and the p.(Phe13SerfsThree amino acid substitutions occurred in evolutionarily conserved regions across different species  and wereThe Ala306Thr molecular model covered the target sequence of TMPRSS3 (residues 217–451) and was constructed based on the crystal structure of FAB DX-2930 in complex with human plakallikrein (PDB ID: 4ogyB). The sequence identity between the target and template was 41.67%. Using Swiss-Pdb Viewer 4.1, the mutation was predicted to perturb the amino acid side chain due to the substitution of alanine to threonine at position 306. The wild-type alanine forms two salt bridges with threonine at position 254, whereas the mutant-type threonine forms an extra salt bridge with valine at position 291. The salt bridge differences will likely disturb the interaction made by the wild-type residue .The Arg106Cys molecular model covered the target sequence of TMPRSS3 (residues 71–109) and was constructed based on the crystal structure of C5B-6 (PDB ID: 4e0sB). The sequence identity between the target and template was 43.59%. Using Swiss-Pdb Viewer 4.1, the mutation showed that the wild-type and mutant amino acids differed in size. The mutant residue was smaller, a feature that might lead to the loss of interactions . TMPRSS3 mutations, p.Ala306Thr, p.Arg106Cys, and p.(Phe13Serfs∗12), are the cause of hearing impairment in this family.These data, together with the clinical presentation of the affected siblings and consistent autosomal recessive inheritance of the mutations in the affected and unaffected members, indicate that the TMPRSS3 mutations are good candidates for residual hearing preservation by cochlear implantation, which is in accordance with the evaluation of cochlear implantation outcomes reported by Chung et al.  identified in this study is a novel mutation located within exon 2 which lead to a truncated protein and is close to the reported mutation c.36dupC (p.Phe13fs) [The frameshift mutation c.36delC [p.(Phe13SerfsPhe13fs) . It has  TMPRSS3 such as homozygous p.Arg106Cys would be very subtle or even close to normal.The missense mutation p.Arg106Cys located within exon 4 causes the negatively charged residue arginine to change to a neutral cysteine at position 106 and is close to the reported mutations p.Asp103Gly and p.Arg109Trp, which are located in the LDLRA domain and are predicted to be involved in the binding of TMPRSS3 with extracellular molecules. Moreover, this mutation introduces a more hydrophobic residue at position 106 and can result in the loss of hydrogen bonds and/or disturb correct folding. This mutation was firstly identified in 1.1%  Japanese patients with late-onset hearing loss . FurtherAll affected patients share one common mutation, c.916G>A (p.Ala306Thr), a mutation previously identified in German, Dutch, and Korean deaf patients that substitutes alanine by threonine at amino acid position 306 , 8, 23. Low-frequency hearing preservation is very useful in auditory rehabilitation. Patient IV: 1 underwent cochlear implantation using the AOS insertion technique, and her residual low-frequency hearing was well preserved, adding to her ability to hear in noisy environments and appreciate the quality of music. TMPRSS3 mutations, c.916G>A, c.36delC, and c.316C>T, as disease-causing mutations. Mutations in TMPRSS3 have previously been described to cause autosomal recessive progressive hearing impairment with postlingual onset (DFNB8) as well as severe-to-profound prelingual hearing impairment (DFNB10). Interestingly, the hearing impairment of affected individuals in this family varied due to different combinations of mutations. Our results indicated that patients with TMPRSS3 mutations are good candidates for residual hearing preservation via cochlear implantation. Therefore, screening for TMPRSS3 in ARNSHI patients with a postlingual, progressive, and ski-slope audiogram is necessary for efficient genetic diagnosis and intervention.This study examined the clinical and genetic characteristics of a nonconsanguineous Chinese family (number FH1523) with autosomal recessive hearing loss. Through multiple deafness gene capture and next-generation sequencing, we identified three129 deafness genes list includes the information of NCBI Reference, OMIM, Description, CDS bps, # of Exons, Covered Region, inheritance pattern and syndromic or not


Cyclin D1 was proved to be regulated by MCM7–MAPK signaling pathway. Clinically, high expression of both MCM7 and cyclin D1 exhibited a relatively high sensitivity and specificity to predict worse outcome of HCC patients. Taken together, our results suggest that MCM7–cyclin D1 pathway may participate in cancer progression and serve as a biomarker for prognosis in HCC.DNA replication is a central procedure of cell proliferation, whereas aberrant DNA replication is indicated to be a driving force of oncogenesis. Minichromosome maintenance complex component 7 (MCM7) plays an essential role in initiating DNA replication. To investigate the potential oncogenic properties and prognostic value of MCM7 in hepatocellular carcinoma (HCC), we conducted immunohistochemistry staining of MCM7 in 153 HCC samples and found that MCM7 high expression level was associated with worse overall survival (OS) of HCC patients. Mechanistically, knockdown of MCM7 significantly inhibited cellular proliferation  At the initiation stage of this process, the minichromosome maintenance (MCM) complex is considered as a critical first step of formation of pre-replication complex that melts origin DNA and unwinds replication forks.10 In almost all eukaryotes, the MCM complex is composed of six highly conserved MCM proteins, namely MCM2–7 (minichromosome maintenance complex components 2–7).11 Several MCM proteins are recently found to be tightly associated with tumorigenesis.20 Among of them, MCM7, an important subunit of the presumed heteromeric MCM helicase, is reported to be involved in tumor formation and progression, and is considered as a potential biomarker in a variety of human malignancies.21 In HCC, elevated expression of MCM7 was found in nearly half of tumor samples, and significantly correlated with intrahepatic metastasis and vascular invasion.20 In addition, MCM7 expression was also observed to be inhibited by sorafenib and other anti-HCC therapies.23 Although accumulating evidence has suggested a critical role of MCM7 in liver carcinogenesis, the molecular mechanisms underlying pro-HCC functions and prognostic implication of MCM7 remain to be elucidated.Enabling replicative immortality, as an important hallmark of cancer cells,The present study aims to demonstrate whether MCM7 plays a key role in promoting HCC progression and to explore underlying molecular mechanisms. Furthermore, we are also interested in evaluating the prognostic and predictive values of MCM7 along with its downstream signaling molecules in HCC patients.http://kmplot.com/analysis/) suggested that higher MCM7 mRNA expression was significantly correlated with a worse overall survival (OS) time in breast, gastric and lung cancer patients , higher frequency of vein invasion (P=0.015) and more advanced tumor stage (TNM staging: P<0.001 and BCLC staging: P=0.026).Exploration using publicly available data sets (P=0.023)  . We furtP=0.020, .P⩽0.036, P⩽0.018, Similarly, consistent association between MCM7 expression level and clinical outcome was observed in HCC patients who were stratified by demographic and clinicopathological characteristics. Interestingly, the death risk of low MCM7 expression on OS was found to be smaller in those HCC patients with less malignant characteristics, such as low AFP level (< 200 ng/ml), small tumor size, single tumor number and early BCLC stage . Our data showed that downregulation of MCM7 significantly suppressed cell viability in HepG2 and SMMC-7721 cells . In addi11 However, the effect of MCM7 modulation on cell cycle in HCC cells has rarely been addressed. In the present study, we detected cell cycle status in HCC cells with MCM7 downregulation by flow cytometric assay. As shown in The essential role of MCM7 in DNA replication has been demonstrated by previous studies.24 To find out the regulatory pathway that is affected by MCM7 and consequently causes G1 arrest, we screened gene expression profile including 16 G1/S-regulatory genes using the real-time reverse transcription PCR (qRT-PCR) measurement. The expression of cyclin D1 (CCND1) was shown to decrease at approximately twofold in HCC cells with MCM7 downregulation . First, these data confirmed a significant positive correlation between the expression level of MCM7 and CCND1 in HCC cell lines , ERK3, ERK4, ERK7, c-Jun N-terminal kinase 1 (JNK1), JNK3 and p38gamma were suppressed by MCM7 downregulation in both HepG2 and SMMC-7721 cells   . Our datP<0.001) . Based oP<0.001, . In addi disease .in vitro and tumorigenicity in vivo. Mechanistically, MCM7 upregulated cyclin D1 expression by modulating MAPK signaling pathway activity, thereby affecting cell cycle progression. Furthermore, a positive association between MCM7 and cyclin D1 expression was found in mouse model and human tumor tissues. Our data revealed that the combination of MCM7 and cyclin D1 had a highly prognostic value in HCC patients. Our studies presented robust evidence to support that MCM7 and its downstream target cyclin D1 are potential biomarkers for HCC prognosis.In the present study, we found that high expression of MCM7 was associated with poor prognosis of the HCC patients. The upregulation of MCM7 enhanced the proliferation of HCC cells 8 DNA replication, the crucial central event during cell proliferation, is observed to be more pronounced in cancer cells than in normal cells. A number of proteins participating in DNA replication are abnormally expressed in cancer cells. For example, MCM7, a replication factor that binds to DNA double strand at replication origins in the late G1 phase and forms the pre-RC complex during cell cycle progression,27 is highly expressed in various cancers and can be used as a potential proliferation marker.29 Zhou et al.20 reported that MCM7 was positive in 42 of 87 HCC tumors (48.2%) but negative in all noncancerous tissue. Consistently, we also observed that IHC index of MCM7 in HCC tumor was significantly higher than that in paired normal liver tissue. Although the positive immunostaining of MCM7 in cancer has been frequently observed, the oncogenic role of MCM7 in cancer development remains unclear. The first study on the molecular function of MCM7 was focused on genome analysis of prostate cancer.17 Researchers found that DNA copy number of MCM7 was increased several fold, and associated with prostate cancer relapse and metastasis. Subsequent studies have suggested that increases in MCM7 copy number and protein level were the driving force in skin and esophageal cancer models.30 Liu et al.31 recently reported that knocking down of MCM7 inhibited proliferation and invasion of MHCC-97H cells . In this study, we further confirmed this result in both MCM7-knockdown HCC cells (HepG2 and SMMC-7721) and murine xenograft models. The mechanistic investigations revealed that knockdown of MCM7 resulted in the cell cycle arrest of HCC cells with the subsequent inhibition of tumor growth, suggesting that MCM7 might be a potential target for cancer therapy.Enabling replicative immortality is a hallmark of cancer.32 Overexpression of cyclin D1 and its accumulation in cancer cell nuclei can result from DNA damage and chromosomal abnormalities. Given the critical role of MCM7 in DNA replication licensing and causing chromosome instability,33 and the strong association between MCM7 and cyclin D1 expression in both human HCC tissue and murine xenograft tumor, it is reasonable to hypothesize that MCM7 acts as a regulator of cyclin D1. In fact, we previously reported that MCM7 depletion could prevent cyclin D1 expression in untransformed human dermal fibroblasts.34 In this study, as expected, we also found that knockdown of MCM7 significantly inhibited cyclin D1 expression in HCC cell lines, indicating an essential role of MCM7 in regulation of cell cycle.We identified cyclin D1 as a downstream target of MCM7 involved in cell cycle regulation. It has been widely accepted that cyclin D1 is a mitogenic sensor for the cell cycle machine and it also acts as an oncogene during cancer development.35 There are seven classes of MAPK signaling cascades, and three of them are essential for liver carcinogenesis, including ERK, JNK and p38 signaling cascades.36 Although Li et al.37 have observed simultaneous expression of MCM7, cyclin D1 and MAPKs in breast cancer cells, it was unclear whether MCM7 induced cyclin D1 expression through a MAPK-dependent mechanism in HCC. In the present study, inhibition of MCM7 expression suppressed the activities of all three MAPK signaling cascades. However, only two of them, ERK and p38, but not JNK signaling cascade, were found to be involved in the regulation of mitogen-induced cyclin D1 expression in HCC cells, consistent with previous studies.38 A possible explanation for this observation is that JNK signaling cascade is generally responsible for the apoptotic response induced by DNA damage, but not the mitogen-induced cell proliferation.39 Taken together, the above evidence suggests that by activating MAPK pathways, especially the ERK and p38 signaling cascades, MCM7 may possess a significant role in the regulation of cyclin D1 and the subsequent cancer progression.Great efforts have been made over the past decade to explore the signaling pathways involved in the regulation of cyclin D1. The MAPK signaling pathway can induce multiple biologic events, such as cell proliferation, differentiation and survival, as well as stimulate cyclin D1 transcription in human cell lines.14 colorectal cancer,15 ovary cancer,16 prostate cancer17 and lung cancer.18 Here we confirmed that MCM7 expression could be an independent survival predictor for HCC. The higher MCM7 protein level was correlated with a worse prognosis. Interestingly, we found a statistically significant association between them only in patients with early stage or less aggressive cancer. Recently, Liu et al.40 evaluated the prognostic values of MCM7 in 494 patients with non-small-cell lung cancer (NSCLC). They found that patients with higher MCM7 expression level had a significant poorer OS compared with those with low MCM7 expression in early stages of NSCLC (P<0.00001). Similarly, Fujioka et al.41 also reported that MCM7 was related to poor prognosis in patients with stage I lung adenocarcinoma. A possible explanation for above findings is that MCM7, as a regulator controlling when DNA replication can begin, contributes to oncogene-driven tumorigenesis.12 Altered expression of MCM7 is to be considered as an early event during tumorigenesis. Higher MCM7 expression is always associated with high proliferative capacity in the early stage of cancer.42 In contrast, in late-stage cancer patients, many confounding factors, including tumor size, vascular or lymphocyte invasion, can affect the prognostic values of MCM7 that might produce a nonsignificant result.Accumulating evidence has supported MCM7 as a proliferation marker, comparable to the existing ones such as Ki-67 or proliferating cell nuclear antigen (PCNA) in multiple malignancies, including lymphoma,Considering the regulatory relationship between MCM7 and cyclin D1, we further studied the prognostic value of the combination of MCM7 and cyclin D1 in HCC patients. As expected, the prognostic sensitivity of the combination was higher than either MCM7 or cyclin D1 alone. The moderate sample size in our study limited the validity of some stratified analyses. Although the diagnostic power for MCM7 and cyclin D1 in combination is rather modest, our present study offers evidence for the future investigation of MCM7 and cyclin D1 as prognostic markers in HCC management. Therefore, a large prospective validation study needs to be performed before their application in the clinic.in vitro and in vivo. In addition, our study indicates a joint effect of MCM7–cyclin D1 molecular markers in predicting the prognosis of HCC patients. Our data also shed light on the potential of MCM7 as a therapeutic target for HCC.In summary, MCM7, a key factor involved in DNA replication initiation, was shown to be overexpressed in mostly HCC tumors and significantly associated with a poor prognosis of HCC patients. Mechanistically, MCM7 promotes HCC cell proliferation via upregulating MAPK–cyclin D1 pathway both A total of 153 patients, including 101 males and 52 females, with an average age of 53.5 years (range: 24–80 years) were consecutively enrolled in this study. The clinicopathological characteristics of patients, including age, gender, hepatitis virus infection status, cirrhosis, Child–Pugh score, serum AFP level, tumor size, tumor number, tumor differentiation grade, TNM stage, capsule integrity and venous invasion are summarized in μm thickness. Sections were stained with antibodies as described in other study.43 The detailed protocols and scoring system are shown in The expression levels of MCM7 or cyclin D1 in tissue samples were determined by using IHC assay. Briefly, tissue samples were fixed in 10% buffered formalin, embedded in paraffin and sectioned at 4 Two HCC cell lines (HepG2 and SMMC-7721) were transfected using lentiviral vectors (Lv-shRNA-MCM7 and Lv-shRNA-Control) according to the manufacture's instruction . The detailed protocols are shown in The intercellular expression levels of genes and proteins were determined by using western blotting and qRT-PCR methods, respectively. The detailed protocols are provided in in vitro cell proliferation of HCC cell lines was measured using the MTT method. Cell colony formation assay was used to assess the effect of MCM7 knockdown on the reproductive potential of HCC cells. The detailed protocols are provided in The Cell cycle progression was determined by flow cytometry of propidium iodide (PI)-stained cells on a flow cytometer . Meanwhile, cell apoptosis was determined by flow cytometry of Annexin V-APC and PI -stained cells according to the manufacturer's instructions. The detailed protocols are provided in in vivo was approved by the institutional animal care and use committee (IACUC) at College of Medicine, Xi'an Jiao Tong University. The detailed protocols are provided in The protocol of tumorigenicity assay P-value of <0.05 was considered significant.Statistical analyses were performed using PASW Statistics 19 . The detailed statistical methods used in this study are shown in


Cervical spine abnormalities can affect the ear vessels and or nerves with different mechanisms. Ear dysfunctions following cervical spine injuries can be manifested as hearing loss, vertigo, or tinnitus. Usually, cervical spine injuries can cause pain and Range of Motion (ROM) limitation. The major objective of this study was to determine which cervical ROM limitation was accompanied with higher level of hearing loss.In this cross-sectional study, 42 volunteers (20 women and 22 men) with cervical spine injury and pain participated after giving their informed consent. Audiometry, tympanometry, and pure-tone threshold of individuals were taken in frequencies from 250 to 8000 Hz in all cases. The ROM limitation in flexion, extension and rotation was recorded.About 53% of participants had bilateral hearing loss. In 40.48% (n=17) of cases, rotation to the left was limited. Flexion and extension motion were restricted on 23.8% (n=10) and 30.95% (n=13) of the participants, respectively. There was no statistically significant relationship between sex and hearing loss but a significant correlation was observed between hearing loss and ROM limitation of rotation to the left in men.According to the present study, the likelihood of hearing loss was high in patients with cervical left rotation limitation, and that the incidence of hearing loss following the cervical spine injuries was more in men. It seems that left Rotation limitation can be used as a predictor to diagnosis of hearing impairment following the cervical spine injuries . The neck consists of seven vertebrae and is most prone to spinal injuries after the lumbar area. The cervical spine is divided into the upper and lower parts. The upper neck consists of the occiput and the first and second vertebrae, and the lower neck consists of the third to seventh vertebrae . PreviouCervical spine diseases usually cause pain and patients usually avoid moving and suffer from limitation of movement. Meanwhile, the inflammatory nature of many diseases may cause adhesion in the soft tissue and decrease the range of physiological movements of the cervical spine. There are four general categories of physiological movements for the cervical spine as follows: flexion, extension, bending, and rotation .All studies on the incidence of hearing loss in cervical spine injuries and diseases only examined the presence of an injury and its effects on auditory system  . The majStudy Participants were recruited from Rheumatology and Orthopedic outpatient clinics at Hazrat-e Rasool Hospital. Finally, this cross-sectional study was performed on 42 patients who suffered from cervical injury and neck pain, and volunteered to participate in this study with informed consent. The present study was approved by the Ethics Committee of Iran University of Medical Sciences. The patients underwent audiometry tests after the doctor diagnosed and confirmed their cervical spine injuries . A questionnaire including general information on age, sex, and duration of neck pain was completed for all patients.3; Static compliance of 0.2–1.5 m℧; and sound pressure of ±50 daPa . The statistical methods comprised ANOVA, independent t test and the Chi-square test, at significance level of α≤0.05.This study was performed on 22 men with the mean(SD) age of 41.1(13.86) years and 20 women with the mean(SD) age of 45.21(11.75) years. Almost 50% of the participants were over 45 years old. Tinnitus, vertigo, and dizziness were respectively observed in 50%, 48.5%, and 48% of the participants. About 53% of cases had bilateral hearing loss. The limitation of Flex, Ext and, left Rot movements was respectively observed in 23.8% (n=10), 30.95% (n=13) and 40.48% (n=17) of patients. The examination of the cervical movements did not show any limitation at right Rot.The Chi-square test did not show a significant difference between the two sexes in terms of the presence or absence of limitation in cervical Flex, Ext, and Rot movements. The limitation in cervical Flex, Ext, and Rot movements did not show a statistically significant relationship with the reduced static compliance of both ears. There was no statistically significant relationship between cervical movement limitation and vertigo/dizziness. However, there was a statistically significant relationship between limitation of left Rot movement and hearing loss only in men .No statistically significant correlation was found between sex and hearing loss when the studied frequencies were divided into two categories, speech frequencies (500–2000 Hz) and high frequencies (4000–8000 Hz). The statistical analysis of the above frequencies and presence/absence of movement limitation showed a significant difference in left Rot limitation (except the range 500–2000 Hz) (P<0.05). Most patients in this study were using sedatives and rehabilitative treatment to relieve the cervical pain; however, the amount and type of drugs were not recorded precisely.The present study showed that among patients with cervical spinal diseases, those with left Rot limitation had higher odds of hearing loss than those who did not, and that the prevalence of hearing loss following spinal diseases was more among men than among women. It seems that left Rot limitation can be used as a predictor of hearing impairment following the cervical spine diseases . Sensorineural hearing loss can be caused by the inflammation of the blood vessels and nerves, the involvement of the middle ear bones, ligament injuries, or the effects of ototoxic drugs for relieving pain  in patients with cervical injuries .Öztürk et al. study showed bilateral hearing loss in patients with cervical disorders . The preThe movement limitation is one of the earliest symptoms of cervical spine injuries. Although various studies revealed the effect of cervical injuries on hearing threshold , in thisMovement limitations of cervical spine mostly seen in people over 35 years and for different reasons . As the The results showed no significant differences between mean hearing thresholds of men and women with limitations in Flex and Ext movements and those without limitations. However, there was a significant difference between mean hearing threshold of men with limitations in left Rot movements and those without limitations . As mentIt has been shown that women had more favorable hearing threshold than men, and their right ears hearing responses were better than those of the left ears . The preIn this study, vertigo and dizziness were observed in many patients. Vertigo and hearing loss are mostly caused by dysfunction of the upper cervical vertebrae, and this is a common problem in the fields of ear, nose, and throat, and neurological diseases . It seemOne of the limitations of this pilot study relates to ROM limitation which was recorded only as presence or absence. Therefore, future studies are recommended to check degree of ROM and how it affects the level of hearing loss. In addition, further studies should be performed to clarify whether hearing loss occurs after movement limitations in patients with muscular diseases (not bone diseases) of the neck (such as trigger point).Based on the results, in the event of injury to the cervical spine following degenerative diseases, the left Rot limitation could predict the occurrence of hearing impairment  among the various mobility restrictions that arise in the neck.


Salmonella enterica serovar Typhimurium continues to be a major cause of foodborne illness worldwide and pork can serve as a source of infection. Co-infection of S. enterica with Lawsonia intracellularis, a common intestinal pathogen of swine, has been found as risk factor for increased S. enterica shedding. The objective of this study was to investigate if vaccination against L. intracellularis could lead to decreased S. Typhimurium shedding. To test this hypothesis, pigs were challenged with either S. Typhimurium or S. Typhimurium and L. intracellularis, with and without L. intracellularis vaccination (n = 9 per group). A non-challenged group served as a negative control. Vaccination decreased the shedding of S. Typhimurium in co-infected animals by 2.12 log10 organisms per gram of feces at 7 days post infection. Analysis of the microbiome showed that vaccination led to changes in the abundance of Clostridium species, including Clostridium butyricum, in addition to other compositional changes that may explain the protection mediated against S. Typhimurium. These results indicate that vaccination against L. intracellularis in co-infected herds may provide a new tool to increase food safety by helping to prevent S. enterica without the need for antibiotics. Salmonella enterica is a leading cause of foodborne illness worldwide1. In the United States, the Centers for Disease Control and Prevention has estimated that S. enterica is responsible for over 1 million cases of foodborne illness per year, and is the leading cause of death due to foodborne illnesses2. It has been estimated that the economic losses due to salmonellosis in the United States exceed $3 billion per year3.4. Pigs frequently carry S. enterica, and efforts in the United States to reduce the incidence of salmonellosis have mainly remained ineffective. The prevalence of S. enterica in pigs is approximately 52.6%5 and the incidence of salmonellosis in humans has not been reduced appreciably since 19966. Some believe that control of S. enterica in swine may not be possible using current technologies, there are many sources of infection including feed, the environment, and carrier animals8.Past studies have estimated that levels of human salmonellosis due to pork consumption vary; in the United States around 6% of outbreaks can be attributable to pork while in western European countries pork has been found to account for 44% of casesS. enterica during pork production11. However, these are not particularly amenable targets for developing improved control strategies. The source of S. enterica that contaminates pork products are the animals themselves10 and novel intervention strategies are needed.Factors such as stresses associated with transportation to slaughter plants, pathogen transmission during the comingling of pigs during lairage at processing plants, and introduction of new gilts into breeding herds contribute to increased shedding and transmission of L. intracellularis is a common porcine intestinal pathogen and is prevalent in pig production sites worldwide, with prevalence ranging from 48 to 100% in different swine producing countries12. In the United States, it has been estimated that L. intracellularis is present in more than 90% of swine farms13. L. intracellularis causes porcine proliferative enteropathy (PPE) which more commonly occurs in post-weaned pigs and leads to decreased weight gain, diarrhea and is often subclinical. Transmission of this organism also occurs by the fecal-oral route and lesions are marked by a thickening of the mucosa of the ileum and colon14.S. enterica herd prevalence of 52.6% in US swine, it is reasonable to assume that co-infection with both pathogens occurs frequently. The association between L. intracellularis infection and increased shedding of S. enterica was first demonstrated by Beloeil et al.15, who performed an epidemiological study and found a statistically significant association between seroconversion to L. intracellularis and increased prevalence of pigs shedding S. enterica. Furthermore, we have observed that experimental co-infection of pigs with L. intracellularis and S. Typhimurium leads to increased colonization of pigs by S. Typhimurium16. Because of this association between L. intracellularis infection and S. enterica, we hypothesized that vaccination against L. intracellularis would decrease shedding of S. enterica in co-infected animals and thus potentially become a new tool to aid in controlling S. enterica shedding. The importance of the gut microbiome has been established for the maintenance of health, as well as colonization resistance to S. enterica18, and it is known that both S. enterica and L. intracellularis can alter the composition of the gut microbiome19. Thus, we further investigated the swine gut microbiome to understand how vaccination and co-infection with L. intracellularis may disfavor or favor S. enterica colonization, respectively.With the S. enterica serovar Typhimurium strain 798 was used for challenge and prepared by overnight growth in Luria-Bertani (LB) broth19. The PHE/MN1-00 strain of L. intracellularis was grown in McCoy cells for challenge of animals following previously a described protocol20.The nalidixic acid resistant S. Typhimurium alone , (2) challenged with both S. Typhimurium and L. intracellularis , (3) challenged with S. Typhimurium and vaccinated against L. intracellularis , (4) challenged with both S. Typhimurium and L. intracellularis and vaccinated against L. intracellularis , and (5) non-infected control (Control). To minimize potential differences in microbiome composition prior to the start of the study, animals were obtained from the same herd and co-housed prior to the enrollment in the study. Additionally, animals were randomized in to treatment groups to minimize any potential confounding factors of sex or of which sow gave birth to them. Each group was comprised of nine pigs divided into three separate BSL2 large animal isolation rooms with three animals per room  at three weeks of age. Twenty-one days post vaccination, two groups of pigs were challenged with a pure culture of 2 × 109 L. intracellularis (strain PHE/MN1-00) organisms per pig. One week post challenge with L. intracellularis, all pigs except the non-challenge controls were challenged orally with 1 × 108 S. Typhimurium (strain 798) organisms per pig. Fecal samples from pigs were obtained on the day of L. intracellularis challenge, on the day of S. Typhimurium challenge, two days post S. Typhimurium challenge and weekly thereafter until 49 days post challenge with S. Typhimurium.The animal protocol #1411-31993A used was approved by the University of Minnesota Institutional Animal Care and Use Committee and all experiments were performed in accordance with relevant guidelines and regulations. In this study, a total of five treatment groups were used: (1) challenged with S. enterica shed in feces of animals, a most probable number enrichment method was used as previously described22. Briefly, one gram of feces was suspended in 9 ml tetrathionate broth (TTB), diluted in triplicate five-fold dilutions and incubated at 41 °C for 48 hours. One hundred μl of each dilution was then transferred to 900 μl Rappaport-Vassiliadis R10 broth and incubated for 24 hours at 41 °C. Cultured dilutions were then inoculated onto XLT4 agar plates containing 100 μg/ml of Nalidixic Acid (NA) to quantify the challenge strain which was NA resistant. A duplicate inoculation was performed onto XLT4 agar without NA to quantify any other indigenous S. enterica strains the pigs could harbor. Colonies with typical S. enterica morphology were confirmed by PCR using primers specific for the gene invA (Forward Primer: ACAGTGCTCGTTTACGACCTGAAT and Reverse Primer: AGACGACTGGTACTGATCGATAAT)23.To quantify the amount of L. intracellularis, antibodies against L. intracellularis were investigated in serum samples of animals. Blood samples were collected at different timepoints of the study and analyzed with the immunoperoxidase monolayer assay24.To assess the efficacy of vaccination and confirm infection with 25. Primers Meta_V1_27F: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGAGTTTGATCMTGGCTCAG and Meta_V3_534R: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATTACCGCGGCTGCTGG were used in an initial reaction to amplify the V1–V3 variable region followed by a second PCR reaction to add index and flowcell adapters. Both reactions used the KAPA HiFidelity Hot Start Polymerase. Sequencing was performed using the Illumina MiSeq with paired end 300 base pair reads at the University of Minnesota Genomics Center. DNA sequence data is available at the University of Minnesota Digital Conservancy (conservancy.umn.edu).For microbiome analysis, DNA was extracted from fecal samples using the MoBio PowerSoil DNA extraction kit. DNA quantity and 260/280 ratios were assessed by Nanodrop. The V1–V3 region of the 16 SrRNA gene was amplified following a dual indexing approach26 and assembled using FLASH27. Homopolymers and ambiguous sequences were removed with the trim.seqs command of Mothur28 and chimeras were removed with usearch61 in QIIME (version 1.9.1)29. Closed reference operational taxonomic unit (OTU) picking was performed with NINJA30 and sequences that did not map to the Greengenes database (version 13_8) underwent de novo picking and assigned taxonomy using uclust31 in QIIME. OTUs identified using closed-reference picking and de novo OTUs assigned to 16S taxonomy were combined into one OTU table for analysis. Low abundance and rare OTUs  were filtered out. Samples were rarefied to 9000 sequences per sample to calculate alpha and beta diversity indices. Weighted UniFrac distance32 was used to evaluate beta diversity differences between samples. The Chao1 index was used to estimate alpha diversity richness and the Simpson index to evaluate evenness in addition to richness. The Wilcoxon Rank Sum test was used to assess significant differences between treatments as well as Analysis-of-similarities (ANOSIM). For differential abundance testing, DESeq 2 was used with the phyloseq package (version 1.19.1) in R33, using a non-rarefied OTU table with samples having less than 7,000 sequences being discarded. DESeq 2 was used as it has been found to be the most sensitive method of differential abundance testing with small sample sizes34. OTUs with differential abundance resulting from pairwise comparisons with p values below 0.05 corrected for multiple comparisons are reported. The Spearman’s rank correlation was used to test for correlation between OTUs of interest and the level of S. Typhimurium shed by animals.Paired end sequences were quality filtered with trimmomaticS. Typhimurium between treatments over time, most probable number (MPN) quantification values were used in a linear mixed model with log10 MPN as the response, treatment, day, and the treatment/day interaction as fixed effects, barn as a fixed block effect, and pen, pig, and day within pen as random effects. Reported are the least square means for treatment by day, and pairwise comparisons between treatments for each day, with p-values corrected for multiple comparisons. To test for differences in the number of animals shedding per group, the N-1 variation of the Chi-square test was used.To test for differences in shedding of S. Typhimurium. At this time point, the Sal Law group shed 2.94 log10S. Typhimurium organisms per gram of feces, while the Sal Law Vac group shed 0.82 log10S. Typhimurium organisms per gram of feces . L. intracellularis vaccination did not have a significant impact on S. Typhimurium shedding when animals were singly challenged with S. Typhimurium. Although not statistically significant, the co-infected vaccinated group was the group that shed the least amount of S. Typhimurium at the timepoints of 2, 7, 14 and 21 days post challenge  compared to 100% of pigs shedding S. Typhimurium in all other challenged groups. The trend of having less positive animals in the Sal Law Vac group persisted at other time points, although the differences were not always statistically significant. At the last time point of the study (49 dpi), all challenged groups had a similar number of animals shedding S. Typhimurium (from 40% to 50%) and a similar quantity of S. Typhimurium.There were significantly less pigs actively shedding ps Table . Only 38L. intracellularis infection, antibodies against L. intracellularis were measured in serum of animals. Animals in the Sal Law Vac group had an earlier seroconversion as compared to animals in the Sal Law group, and more animals were positive for antibodies in serum at earlier timepoints in the Sal Law Vac group  compared to the Sal Law group . At the last timepoint of the study both groups had similar number of animals with anti L. intracellularis antibodies in serum . There were also animals that developed antibodies in the Sal Vac group demonstrating that the vaccine was effective in eliciting an immune response in the animals. No animals in the Sal and Control groups had antibodies in serum against L. intracellularis.To control for the efficacy of vaccination and of S. Typhiumrium or L. intracellularis led to alterations in the composition of the swine gut microbiome36. To determine if vaccination with the live oral attenuated vaccine against L. intracellularis would lead to changes in the microbiome, we looked for community composition differences between treatment groups using the weighted UniFrac distance32. At 7 days post challenge with S. Typhimurium, which was when the greatest difference in S. Typhimurium shedding between groups was observed, treatment had a significant impact on the grouping of samples as confirmed by ANOSIM  all groups had very similar Simpson indices  , these animals were still considered in microbiome analysis since they shed very low levels and at only one of the several timepoints of the study. We additionally analyzed data with and without these animals and saw no differences (data not shown). To investigate compositional differences in the microbiome that may have led to the observed reduction in shedding of S. Typhimurium due to L. intracellularis vaccination, we focused on the time point of seven days post challenge with S. Typhimurium, when L. intracellularis vaccination led to the greatest reduction in shedding. At this time point, several significant (p < 0.05) differences in the abundance of OTUs at the genus level were observed comparing the different treatment groups  increase in the abundance of the genera Coprococcus, Lachnospira and Blautia and a decrease in Lactobacillus, Treponema, Fibrobacter and YCR22  had both a significant (p < 0.05) increase and decrease in abundance in the Sal group compared to the Control group  and a significant increase in Lachnospira compared to Controls  and a significant enrichment of Streptococcus and Lactobacillus, which was not observed with the Sal group compared to Controls . The abundance of sequences related to C. butyricum were significantly negatively correlated to S. Typhimurium shedding levels at 7 days post S. Typhimurium challenge  animals to non-vaccinated dually challenged animals , the Sal Law Vac group had a significant decrease in abundance of S. Typhimurium challenge, we found a significant decrease in abundance of an OTU that closely matches Collinsella aerofaciens, two OTUs that closely match Prevotella copri and another OTU pertaining to the genus Dorea (p < 0.05) and an enrichment of Epulopiscium in the Sal Law Vac group  or when comparing the effect of co-infection  to non-infected animals (Control), suggesting that these changes are specific to the effect of vaccination on co-challenge  positively correlated to the level of S. Typhimurium shedding in pigs at 7 dpi  virulence genes of S. Typhimurium, which are important for cell invasion50. Depletion of butyrate producing Clostridium also can promote S. enterica infection by allowing for aerobic expansion in the gut51. In this study, S. Typhimurium alone did not cause a disturbance in the abundance of Clostridium, however co-challenge led to a decrease in its abundance (Fig. S. Typhimurium colonization and shedding mediated by dual challenge with S. Typhimurium and L. intracellularis. Vaccination against L. intracellularis led to an increase in the abundance of Clostridium compared to co-infection without vaccination. This increase included a 4.27 log2 fold increase in the abundance of sequences that match to Clostridium butyricum (Fig. C. butyricum was given its name due to its capacity to produce large amounts of butyrate and non-toxigenic strains are widely used as probiotics in Asia52. The decrease of Clostridium, and C. butyricum in particular, could potentially benefit S. enterica serovars other than Typhimurium, since different serovars can have similar mechanisms of infection with the use of SPI-1 genes18. Therefore, the increase in Clostridium and C. butyricum abundance due to L. intracellularis vaccination in co-infected animals potentially could reduce the shedding of different serovars, another desired quality of a S. enterica vaccine. This possibility should be confirmed with more directed experiments.In contrast to the possible pro-inflammatory effects of nce Fig. . This decum Fig. . C. butyL. intracellularis, a common pathogen in swine, is able to reduce the level and prevalence of S. Typhimurium shedding. The effect of vaccination was dependent upon challenge with L. intracellularis, which suggests that apart from changes in the microbiome an immune response may also be involved in these observations. The use of vaccination against L. intracellularis to control S. enterica is a novel and promising new tool that is much needed for controlling S. enterica in pig herds as well as improving food safety, and may be an alternative to the use of antimicrobials.This study demonstrated for the first time that vaccination against Supplementary Information


Hearing loss is one of the most disabling impairments. Using a hearing aid as an attempt to improve the hearing problem can positively affect the quality of life for these people. This research was aimed to assess satisfaction of hearing impaired patients with their hearing aids regarding the employed technology and style. This descriptive-analytic cross-sectional research was conducted on 187 subjects with hearing loss who were using a hearing aid. The subjects were over 18 years of age and were using a hearing aid for at least 6 months. The Persian version of Satisfaction with Amplification in Daily Life (SADL) questionnaire was the instrument which was used for assessing satisfaction with the hearing aid. Cronbach’s alpha was calculated to be 0.80 for instrument reliability.A significant difference was observed among satisfaction subscales’ mean scores with hearing aid technology. Also a significant difference was observed between the total satisfaction score and the hearing aid model. With respect to the analysis of satisfaction with the hearing aid and its style, cost and services was the only subscale which showed a significant difference (P=0.005).Respondents using hearing aids with different technology and style were estimated to be quite satisfied. Training audiologists in using more appropriate and fitting hearing aids in addition to using self-reporting questionnaires like SADL for estimating patients’ social condition and participation in their life can essentially change their disability condition and countervail their hearing loss. WHO 2013) estimated that 360 million people in the world are suffering from disabling hearing loss 3 estimat.Under 25% of individuals who can improve by hearing aids are real users and this rate is lower in developing countries .The aim of using a hearing aid is amplifying signals that make sounds audible for hearing-impaired people. Basically, all hearing aids use analogue technology to amplify sounds . AlthougAdvancements in digital technology and the rising speed of speech signal processing have ensued current developments in existing features of modern hearing aids. However, hearing aid users still have complaints hearing speech signals in noisy environments and while talking on the phone .Digital hearing aids are more flexible for fitting and include more complex processing . They alThis research was an analytical and cross-sectional study. The participants were hearing-impaired individuals who were referred to an audiology clinic in the south of Bushehr province, Iran. The inclusion criteria for participation in the study were: referring to a clinic in the last two years, over 18 years of age ,and at least a 6 months’ experience in using hearing aids. There was no exclusion criteria to participate in this research. The population size included 187 people all of whom consented with the research procedures. The population under study included 100 male and 87 female participants aged between 18 to 90. Initially, audiology evaluations were performed for the subjects. Then, they filled the questionnaire . Subject evaluations included two phases: 1. Audiological Evaluation Including:Otoscopy for examining ear appearance and pure tone audiometry was performed by a calibrated audiometer in an acoustic room. In this evaluation audio absolute thresholds for Air Conditions  and Bone Condition  were determined. Speech audiometry evaluated Speech Recognition Thresholds (SRT) and word Recognition Score (WRS). Immitance audiometry was used for the evaluation of the accurate function of the sound transmission system to the inner ear.2. Satisfaction Assessment Instrument: A standard Persian version of SADL and a questionnaire for demographic characteristics were used for data gathering. The questionnaire was validated through face and content validity by 5 experts. Cronbach’s alpha calculated 0.80 for reliability of the data gathering instrument . The SADThis project was conducted under the ethics committee of Ahvaz Jundishapur University of Medical Sciences . All respondents declared and signed their consents formally. The data was analyzed by descriptive statistics (mean and standard deviation) and inferential statistical  in SPSS.One hundred males and 87 females (range: 18 to 90 years old) were evaluated in this study. Demographic characteristics of the participants are presented in As seen in The majority of the participants (79.20%) used a hearing aid 8-16 hours per day. 14.40% and 6.40% of subjects were using their hearing aid respectively as long as 4-8 and 1-4 hours daily. Most respondents had been using their current hearing aid for 1 to 10 years. 107 (57.21%), 22 (11.76%), and 58 (31.03%) subjects were using digital, programmable, and analogue hearing aids respectively. Satisfaction assessment results with hearing aid based on technology is shown in ANOVA test showed a significant difference in SADL subscales for different technologies of hearing aids. In the cost and services subscale, significant differences were seen between participants who used a digital hearing aid and the other two groups. In the personal image subscale, significant differences were observed between subjects with a digital hearing aid and those with analogue and programmable hearing aids (using LSD post hoc test).In the global score, a significant difference was observed between people who used digital hearing aids and those with analogue hearing aids. Satisfaction level with hearing aids with different technologies was estimated at the same level. A maximum level of satisfaction was in the positive effect subscale where a high degree of respondents’ satisfaction was observed. A minimum level of satisfaction was observed in the negative features. Users were estimated relatively satisfied in the other two subscales.Fifty (26.73%) respondents were using ITE types of hearing aids and 137 (73.27%) were using BTE ones. Results of assessing the satisfaction level based on the model of hearing aids are shown in Nineteen (10.16%) subjects were using a hearing aid binaurally and 168 (89.84%) were using it monaurally. Results of assessing the satisfaction level with a hearing aid based on the style of hearing aids are shown in Our analysis demonstrated that users with binaural style of hearing aids were significantly more satisfied in the Cost and Services subscale. Other subscales showed no significant difference.173 (92.51%) subjects were suffering from sensorineural hearing loss and 14 (7.49%) were suffering from mixed hearing loss. No significant difference was observed in the satisfaction level of respondents with different kinds of hearing loss. In addition, satisfaction level showed no significant difference between the two genders.Subjects were estimated to be relatively highly satisfied based on their mean global score in this study. This result is in agreement with the results of the studies by Cox and Alexander, Viega et al , and Carvalho -16.In the present work, no significant difference was seen in satisfaction level with hearing aid between the respondents’ sex and age, which is similar to Uriarte et al's studies . In HospThe result of the present study showed less satisfaction in age groups in comparison with Kochkin's research . AlthougSignificant differences were seen between all subscales of satisfaction considering different technologies: people with digital hearing aids were estimated significantly more satisfied in cost and services, personal image, and negative features subscales. Also, patients with analogue hearing aids were estimated significantly less satisfied in positive effects subscale and global satisfaction. Yet, all respondents were estimated to be satisfied with their hearing aids, which is similar to Vuchrialho et al and Uriarte et al's findings ,20. TheyMoreover, a significant difference was observed between different models of hearing aids in the global score as well as the positive effect, the cost and services, and the personal image subscales in this study. Dillon et al and Kochkin reported a correlation between high satisfaction in the personal image subscale with ITE hearing aids, which supports our findings ,23. In tThe positive effect subscale showed the highest mean among other subscales indicating the high satisfaction of hearing-impaired people with hearing aids in their social life. Considering the hearing aid's sound quality, only few subjects reported dissatisfaction with acoustical specifications and psychological effects of their hearing aid. This result confirms the outcomes of Cox and Alexander as the developers of the study's questionnaire .In this study, satisfaction level with digital hearing aids was estimated to be higher than other types of hearing aids. However, these types of hearing aids impose higher costs on the users. Establishing policies in order to remove access financial barriers to these types of hearing aids need to be studied. Since the prevalence of hearing loss as well as the need for its rehabilitation is growing, these rehabilitation services need to be supported by social security and retirement funds. These organizations must specify which groups are in priority for using these resources and which patients gain more advantage with hearing aids. Given the lower satisfaction level with their hearing aids among illiterate subjects in this study, more counselling meetings for these patients and spending more time for instructing these customers in using their hearing aid is recommended. Besides, providing an educational protocol for using amplification in daily life can lead to better results.