Subjective preferences affect many processes, including motivation, along with individual differences. Although incentive motivations are proposed to increase our limited visual working memory (VWM) capacity, much less is known about the effects of subjective preferences on VWM-related brain systems, such as the prefrontal and parietal cortices. Here, we investigate the differences in VWM capacities and brain activities during presentation of preferred and non-preferred colors. To this end, we used time-frequency (TF) analyses of electroencephalograph (EEG) data recorded during a delayed-response task. Behavioral results showed that the individual VWM capacities of preferred colors were significantly higher than those of non-preferred colors. The EEG results showed that the frontal theta and beta amplitudes for maintenance of preferred colors were higher than those of non-preferred colors. Interestingly, the frontal beta amplitudes were consistent with recent EEG recordings of the effects of reward on VWM systems, in that they were strongly and individually correlated with increasing VWM capacities from non-preferred to preferred colors. These results suggest that subjective preferences affect VWM systems in a similar manner to reward-incentive motivations. As a well-known proverb says, “Everyone to his taste.” there are large individual differences in subjective preferences. Such preferences individually lead to either positive or negative emotions, which seem to influence not only our potential personalities, but also the current behaviors. However, little is known regarding whether subjective preferences directly affect cognitive processing. For example, Are we able to better memorize a preferred visual stimulus? To address this issue, it is useful to measure the visual working memory (VWM) capacity, which refers to our ability to memorize and maintain visual stimuli temporarily  analyses of EEG data. EEG oscillations are thought to reflect the synchronization of a large number of neurons underlying a particular function  took part in this experiment. They reported having normal or corrected-to-normal visual acuity, normal hearing acuity, and normal motor abilities using subjective questionnaires. All participants gave written informed consent, which was approved by the Ethical Committee of the RIKEN (in accordance with the Declaration of Helsinki), r = 255, g = 255, b = 255), red , green , blue , yellow , magenta , cyan , olive , purple , and aqua ]. Each participant completed 90 trials. All possible color combinations were presented twice, with a reshuffling of the right and left positions. We selected the most and least preferred colors for each individual using the number of times each color was selected.Before the EEG experiments, each participant completed a pretest to identify their personal color preferences , which were simultaneously presented at random locations in an invisible 3 × 3 cell matrix for 0.2 s  embedded in an electro cap  in accordance with the extended version of the international 10/20 system. Reference electrodes were placed on the right and left ear lobes. Electrooculography (EOG) measurements were recorded from electrodes above and below the left eye by monitoring eye blinks or vertical eye movements, and from electrodes placed 1 cm from the right and left eyes by monitoring horizontal eye movements. The EEG and EOG data were amplified using Neuroscan equipment . The sampling rate was 500 Hz. The EEG data were filtered in the band-pass range from 0.1 to 50 Hz.We segmented the EEG data into 3 s epochs  for each trial. To reduce or eliminate artifacts, we conducted an infomax independent components analysis (ICA) on the EEG data from the correct trials. ICA components that significantly correlated with vertical or horizontal eye movements in the EOG data were rejected, and the ICA-corrected data were recalculated using a regression of the remaining components. To elucidate the cortical activity with decreasing errors from volume conduction, we applied current source density analysis using the spherical Laplace operator to the voltage distribution on the surface of the scalp , ranging from 0.5 to 40 Hz in 0.5 Hz steps . The TF amplitudes from the ITIs were averaged to generate the baseline amplitudes. The delay-period TF amplitude was calculated by subtracting the baseline data for each trial from each frequency band. The corrected TF amplitude was averaged across single trials for all conditions for each participant.To identify the TF amplitudes during the retention interval, we applied wavelet transforms using Morlet's wavelets having a Gaussian shape in the time and the frequency domains around their center frequency  = 72.69, P < 0.01], but no significant effect of preference  and no significant interaction .Accuracy rates (percentage correct) were higher when we presented fewer objects in both the “preferred” and “non-preferred” condition . A Two-Way analysis of variance (ANOVA) revealed a main effect of the number of objects , but no significant main effects of the number of objects  or preference . There was a significant difference between the “preferred” and “non-preferred” conditions for the larger number of presented objects . The maximum VWM capacity, which was defined by the maximum values among all VWM capacities for the number of presented objects showed significant differences between the “preferred” and “non-preferred” conditions . These results suggest that subjective color-preference affected the available VWM capacity in our experiments.The averaged VWM capacity for each condition is shown in Figure P < 0.05). The theta amplitudes in the frontal and right motor regions were enhanced with increasing number of objects, whereas the occipital alpha amplitudes showed opposite way.To identify the specific pattern of brain activity representing VWM maintenance, we calculated the TF amplitudes from the 60-channel EEG data during the 2 s retention intervals in comparison with the baseline periods. For the theta (the frequency: 4–8 Hz) and alpha (12 Hz) amplitudes, an ANOVA showed main effects of the number of objects in the frontal and parietal regions which electrodes were shown in Figure K values; Kmax) and individual differences in the amplitudes between Kmax and K2 objects. Significant correlations were observed in the averaged theta (4–8 Hz) and alpha (12 Hz) activities (P < 0.05). The theta and alpha correlations were mainly found in the frontal/occipital and occipital regions, respectively. The topographic colored scalp maps for the significant statistical values are shown in Figure r(19) = 0.38, P < 0.05; non-preferred, r(19) = 0.51, P < 0.01], whereas the occipital alpha amplitudes showed negative correlations . Although these statistical values were not enough for multiple comparisons, the frontal theta and occipital alpha oscillations are likely to be involved in the maintenance of VWM, which is similar to our previous findings.Moreover, in order to investigate the VWM-capacity-related activities, we applied correlation analyses between the limitations of individual VWM capacities , P < 0.05; occipital (O1), P < 0.05). In addition, low beta activities (12–20 Hz) under the “preferred” condition were significantly higher than under the “non-preferred” condition, and the enhancements were distributed across both the right lateral frontal areas  and the anterior frontal areas  , where the VWM capacity was found to be significantly different between the preference conditions. For the theta amplitudes (4–8 Hz), significant differences in amplitude were found in the frontal, parietal, and occipital brain regions ; frontal (AF8 electrodes),  Figures . HoweverThe time-course transitions of the different amplitudes between the “preferred” and “non-preferred” conditions on the right frontal electrode (F4) during the maintenance of 2, 4, and 6 items are shown in Figure r(19) = 0.55, P < 0.01]. The frontal areas overlapped the VWM capacity-correlated regions. For the other VWM-capacity-correlated regions, the frontal theta and occipital alpha amplitudes did not significantly correlate with preference-induced increases in VWM capacity .Finally, to identify the brain oscillations reflecting increased VWM capacity under the “preferred” condition, we examined the activity patterns under the “preferred” condition as compared to those under the “non-preferred” condition. Frontal beta  delay-period amplitudes were significantly correlated with the increased VWM capacity in preferred colors vs. non-preferred colors conditions [see Figures per se may affect VWM systems, much like the anticipation of reward.The current study clearly shows that, subjective preferences for visual stimuli affect VWM capacity in individuals. VWM capacity was significantly higher with a subject's favorite color compared to their less preferred colors, particularly under conditions involving high VWM loads. This result is in agreement with previous findings showing that VWM capacity is enhanced by anticipation of high monetary rewards for correct answers, in comparison with low or no monetary rewards  and strongly correlated with individual differences in VWM capacities, as patterns of brain activity directly related to VWM. These VWM-load and VWM-capacity-related brain mechanisms were not overlapped. These results might support the previous fMRI finding about functional dissociations between VWM-load and VWM-capacity-related regions in the fronto-parietal networks , because the chosen colors differed between the participants.  . Therefore, future study should rigorously clarify such effects of the color components on VWM capacity and EEG activities.This study focused on the influence of the color preference on VWM capacity, however, visual stimulus included several features such as shapes. There are possibilities that preferred shapes are better memorized than non-preferred shapes. Unfortunately, it is difficult to judge such preferences of shapes in this study. So, it is necessary to confirm the effects of preference on the VWM capacity by using other visual features in future study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Monaural hearing induces auditory system reorganization. Imbalanced input also degrades time-intensity cues for sound localization and signal segregation for listening in noise. While there have been studies of bilateral auditory deprivation and later hearing restoration , less is known about unilateral auditory deprivation and subsequent hearing improvement. We investigated effects of long-term congenital unilateral hearing loss on localization, speech understanding, and cortical organization following hearing recovery. Hearing in the congenitally affected ear of a 41 year old female improved significantly after stapedotomy and reconstruction. Pre-operative hearing threshold levels showed unilateral, mixed, moderately-severe to profound hearing loss. The contralateral ear had hearing threshold levels within normal limits. Testing was completed prior to, and 3 and 9 months after surgery. Measurements were of sound localization with intensity-roved stimuli and speech recognition in various noise conditions. We also evoked magnetic resonance signals with monaural stimulation to the unaffected ear. Activation magnitudes were determined in core, belt, and parabelt auditory cortex regions via an interrupted single event design. Hearing improvement following 40 years of congenital unilateral hearing loss resulted in substantially improved sound localization and speech recognition in noise. Auditory cortex also reorganized. Contralateral auditory cortex responses were increased after hearing recovery and the extent of activated cortex was bilateral, including a greater portion of the posterior superior temporal plane. Thus, prolonged predominant monaural stimulation did not prevent auditory system changes consequent to restored binaural hearing. Results support future research of unilateral auditory deprivation effects and plasticity, with consideration for length of deprivation, age at hearing correction and degree and type of hearing loss. Monaural hearing induces auditory system reorganization. This occurs in cases of unilateral sensorineural hearing loss, where there is sensory (inner ear) or neural dysfunction, as well as losses in the conductive pathway between the outer and inner ear. In animals, unilateral conductive hearing loss (UCHL) results in structural and functional changes within the auditory system. For example, on the UCHL side in rats, the size of neurons in the anteroventral cochlear nucleus was smaller  hearing loss on sound localization, speech understanding, and cortical organization, both before and after the conductive component of the hearing loss was corrected in adulthood.Written informed consent was obtained from the participant in accordance with the Declaration of Helsinki and guidelines approved by the Human Research Protection Office at Washington University School of Medicine (WUSM).The participant (P1) was a 41 year old female who had a history of unilateral hearing loss in the right ear that had been present since early childhood (probably since birth). Family history included a father and paternal grandmother with mixed unilateral hearing loss. P1’s father had successful stapes surgery in adulthood as a treatment for otosclerosis induced hearing loss. P1 and her family had believed P1’s hearing loss to be sensorineural and non-correctable. A hearing aid was fitted around age 5 but discontinued after a brief trial due to lack of benefit. In adulthood, the participant had a comprehensive audiological evaluation that diagnosed the loss as mixed. P1 consulted an otolaryngologist to discuss treatment options. Audiological air-conduction results  word task , replicating a challenging real-life listening situation. Two 20 sentence lists were administered and an average signal-to-noise ratio (SNR) for 50% accuracy (SNR-50) obtained. Sentences were presented from the front loudspeaker beginning at a +6 dB SNR and adapted to be easier or more difficult based on the participant’s responses. An average of the final 17 SNRs resulted in an SNR-50 for each list.Sentence understanding in noise was evaluated with the Hearing In Noise Test  psychoacoustic task resulted in speech thresholds for quiet and nine competing noise conditions (modified from the task described in Litovsky  and JohnA psychoacoustic measure determined the random spectrogram sound (RSS) that was most different from two others during each trial. RSS are noise-like stimuli created with independent control of temporal or spectral sound parameters, but with all stimuli based on summation across the same 6-octave bandwidth (250–16000 Hz) and with matching intensity and durations . The significance of F-Zstats was determined after multiple comparison corrections based on Monte Carlo simulations  regression analysis method (with a Bonferroni adjustment for multiple comparisons) identified a significant effect of test interval  and follow-up comparisons indicated significantly improved localization compared to baseline at 3 months  and at 9 months . The improvement at 9 months compared to 3 months was not statistically significant (p > 0.05).Figure R-Space sentence understanding in the presence of restaurant noise. The three test intervals are indicated along the x-axis and SNR score along the y-axis. Note that for this measure lower scores indicate better performance. Pre-surgery, P1 had 50% accuracy for sentences presented 3.5 dB softer than the surrounding restaurant noise (60 dB SPL). By 9 months post-surgery, P1 was able to understand sentences at even softer levels (4.9 dB softer than the restaurant noise). P1’s performance improved (1.4 dB) from pre-surgery to the 9-month test interval. The 95% confidence interval for NH adults and noise from the front is ±1.2 dB  included in spectral RSS stimuli  was very similar at all three test intervals (JND 8.8 pre-surgery to 8.2 at 9 months). P1’s performance detecting differences from a standard temporal rate of 30 Hz in the rates (green circles) of temporal RSS stimuli  was similar at pre-surgery (JND 10.8) and at 3 months (JND 10.4). By 9 months P1’s JND for RSS temporal complexity differences had improved to 7.8. P1’s performance on both tasks is similar to that of a group of 20 NH adults, ages 23–62 years, listening bilaterally across four test runs  as purple circles and Localization (Loc) as green triangles. P1’s ratings for both subscales improved over time and were similar to the NH group ratings by 9 months . The rating plots in Panel C are from four subscales in the Qualities domain: Segregation of Sounds (SegSnds) as purple triangles, Identification of Sound and Objects (IdSnd) as green diamonds, Sound Quality and Naturalness (Qlty) as orange squares and Listening Effort (Eff) as purple circles. SegSnds, IdSnd and Qlty ratings were all above nine and comparable to the NH group ratings at all three test intervals. Pre-surgery, Eff was poorer than that of the NH group but improved and was similar to that group’s ratings by 9 months (6.0 pre-surgery to 8.3 at 9 months). In summary, P1’s ratings were poorer than ratings of the NH adults reported by Dwyer et al.  on 6 of F-Zstats). The most affected auditory cortical fields were Te1 and Te2. As shown in the left-most column of coronal sections prior to surgery, the extent of significant activation was limited, bilaterally distributed, but slightly more extensive in the left hemisphere (LH), ipsilateral to the stimulated unaffected left ear. The activated zone was larger bilaterally at 3 months post surgery as shown in the middle column of sections. Activity was even more widely distributed by 9 months as shown in the right-most column of sections. Especially prominent at 9 months was a substantial response distribution contralateral to the stimulated left ear that extended over most of the posterior superior temporal plane and spread more than 1 cm from anterior to posterior. The affected contralateral cortex included all Te auditory cortical fields at 9 months post surgery.The top row of Figure The panels in Figure Corrective surgery for the conductive portion of the hearing loss substantially improved hearing thresholds from 0.25 to 6 kHz. Prior to surgery, P1 could not detect sound in the mid frequencies and perceived loud sounds as soft in the lowest and highest frequencies of the affected right ear. After surgery, air-conduction thresholds were close to P1’s bone-conduction thresholds, closing the air-bone gap. Sensorineural threshold levels remained, as expected, and thus hearing levels were essentially in the mild to moderately-impaired range, reaching 25–35 dB HL at 0.25, 3, 4 and 6 kHz.Source localization significantly improved following surgery. The largest improvement was between pre-surgery and the 3-month post-surgery test interval with stable performance between the 3 and 9-month test intervals. Wilmington et al.  also shoR-Space task, P1’s ability to understand sentences in the presence of restaurant noise improved between the pre-surgery and 9-month post-surgery test intervals. Noise type and location affected Adaptive SRT results. P1’s SRT scores were similar when listening to words in the presence of single talker noise regardless of talker gender. P1’s poorest SRTs were in the presence of MTB noise, as were the greatest improvements. With respect to the noise source, it was not surprising that P1 was able to hear the words at softer levels when noise was towards the affected (right) ear, even after surgery, than when the noise was towards the unaffected (left) ear or from the front. These results for the affected and unaffected ear were similar to Wilmington’s (1994) results using a speech-in-noise task with low and high context sentences and competing babble  was similar to that of NH individuals listening bilaterally  reported by Dwyer et al. , P1’s ratings were between 9–10 and similar to ratings of the NH group  was also changing at the same time, and the two ears were “starting to play nice together”. The result was hearing in stereo, again a notion that P1 had not realized prior to hearing correction. Finally, P1 reported increased feelings of confidence and reduction of effort to hear and communicate in everyday compared to pre-surgery. When questioned 9 months after surgery, the comments from P1 were similar to what had been previously reported. Overall, the descriptions mimic a period of over excitation of sound followed by adaptation and shifting to allow both ears to contribute to improved hearing performance using enhanced binaural abilities.A case study presented by Stange et al.  was of aThere was also evidence of neuroplastic reorganization in auditory cortex that especially involved core and belt regions in the current case. The overall extent and amplitudes of contralateral auditory cortex responses increased after hearing recovery, resulting in increased bilateral activation. Vasama et al.  also fouIn summary, a re-established balance to binaural activity might explain the trend toward improved performance on measures after the stapedotomy procedure for the studied participant. The SSQ results showed that ratings of the participant’s perceived ability were similar to the NH comparison group , handled the review process objectively and no conflict of interest exists.


Hearing impairment is one of the most frequent chronic conditions. Persons with a hearing impairment (PHI) have various experiences during their ‘journey’ through hearing loss. In our previous studies we have developed a ‘patient journey’ model of PHI and their communication partners (CPs). We suggest this model could be useful in internet-based pre-fitting counseling of a person with hearing disability (PHD).A randomized controlled trial (RCT) with waiting list control (WLC) design will be used in this study. One hundred and fifty eight participants with self-reported hearing disability ) will be recruited to participate in this study. They will be assigned to one of two groups (79 participants in each group): (1) Information and counseling provision using the ‘patient journey’ model; and (2) WLC. They will participate in a 30 day (4 weeks) internet-based counseling program based on the ‘patient journey’ model. Various outcome measures which focuses on hearing disability, depression and anxiety, readiness to change and acceptance of hearing disability will be administered pre (one week before) and post (one week and six months after) intervention to evaluate the effectiveness of counseling.Internet-based counseling is being introduced as a viable option for audiological rehabilitation. We predict that the ‘patient journey’ model will have several advantages during counseling of a PHD. Such a program, if proven effective, could yield cost and time-efficient ways of managing hearing disability.ClinicalTrials.gov Protocol Registration System NCT01611129. The term ‘patient journey’ refers to the experiences patients go through during their disease and treatment. It is believed that understanding the ‘patient journey’ can help the clinician to gain an insight into the unique experiences of patients. In the last decade, with increased focus on patient-centered treatment approaches in healthcare, studies of the ‘patient journey’ have become popular. The ‘patient journey’ has been studied in various conditions including: Parkinson’s disease , locked-et al. argued that this model could help the clinicians during history taking to understand at what stage the patient might be and then to tailor the way they speak to them [Figure  to them .There is a range of interventions focusing on the psychosocial needs of people with acquired hearing loss. For example, counseling-based aural rehabilitation ; active et al. it was identified that PHI mainly use their life experiences to describe their hearing help-seeking and hearing rehabilitation process rather than the interactions with clinicians [PHI go through various unforeseen consequences during their ‘journey’ through hearing loss. In a recent study by Laplante-Lévesque inicians . It is binicians . This isThe ‘patient journey’ model considered above corresponds to the stages of change in the ‘transtheoretical model of change’ ,21. TherMoreover, considering the above, it is reasonable to expect that the ‘patient journey’ model can be used as a counseling tool to create awareness and educate PHI and their CPs during initial consultations. This may result in various advantages such as decreased emotional and social problems, decreased anxiety and depression, increased acceptance of hearing disability, and may positively facilitate stages of change through hearing disability, which are important factors in determining the success of audiological enablement/rehabilitation.et al. suggested that online support groups in the form of a subscription distribution list can help people with hearing loss [et al. used daily internet-based communications (in the form of Emails) in addition to the usual audiological service provided to people with hearing loss [et al., in a randomized controlled study evaluated the use of a rehabilitative online education program with discussion groups for hearing aid users [In recent years there has been an increase in the use of the internet for seeking health-related information. Cummings ing loss . Laplanting loss . Their sid users . They obConsidering the increased use of the internet by the general population, which increases flexibility for patients to participate in such programs in their own time and preferred place, and in the interest of reducing clinicians’ contact time, we will employ internet-based counseling in this study.This paper describes the design of a randomized controlled trial to evaluate the effectiveness of the ‘patient journey’ model in the internet-based pre-fitting counseling of persons with hearing disability.The study is aimed at testing the hypotheses that, during the initial consultation of the PHD:(1) Use of the ‘patient journey’ model as an internet-based counseling tool will result in decreased hearing-related emotional and social problems.(2) Use of the ‘patient journey’ model as an internet-based counseling tool will result in decreased depression and anxiety.(3) Use of the ‘patient journey’ model as an internet-based counseling tool will result in increased readiness to change (or positively facilitate stages of change through hearing disability).(4) Use of the ‘patient journey’ model as an internet-based counseling tool will result in increased acceptance of hearing disability.Ethical approval has been received from the Research Ethics Committee, College of Human and Health Sciences, Swansea University. A study advertisement will be made (in the United Kingdom) through various sources including local/national newspapers, hearing loss charity websites and mailing lists, inviting those with acquired hearing disability who have not yet had their hearing aids and who have internet access, to participate in this study. They will be given an information sheet describing the study and directing them to the website. In the first stage, interested participants will complete an informed consent form and the online questionnaires and the first 158 participants showing scores of over 20 in the Hearing Handicap Questionnaire (HHQ) will be recruited to participate in the study . The HHQ scores can range from 12 to 60, from no hearing disability to a severe degree of hearing disability. In this study a wide range of people with hearing disability  will be selected. However, a minimum score of over 20 was deemed appropriate to avoid the floor effect. The participants recruited will be randomized  to one of the two groups: (1) Information and counseling provision using the ‘patient journey’ model; and (2) Waiting list control (WLC). In addition, in the beginning of the study an Email with clear information about the study will be sent to willing participants to ensure that they have the time to participate in the study. Data will be collected using the various outcome measures discussed below pre and post counseling sessions. Figure Inclusion criteria:▪ Age over 18 years▪ Noticing symptoms of hearing disability▪ Access to internetExclusion criteria:▪ Already using hearing aids▪ HHQ score 20 or below▪ Those with additional disabilities  which may affect individuals’ ability to participate in an internet-based programGroup one will be given ‘patient journey’ counseling which is based on previous studies ,8,26, thEach stage will contain a short video  which briefly explains the tasks. However, the information presented in the video is also available in text to make sure equal access is provided to those who may find it difficult to understand speech in the video. Having information in different formats  would help in ensuring that the information is presented in an interactive manner and that it engages the users in the activity. Throughout this process, the participants will be advised to reflect (a simple guide for reflection will be provided) and maintain notes. This reflection exercise is to explore the activity limitations and participation restrictions they have and also to explore the ways in which they can overcome them. In addition, there will be an Email contact with the researcher  with whom they can communicate, update their progress with the intervention and seek further assistance.The second group, which serves as the WLC, will complete the questionnaire at the beginning of the study and after 30 days will be provided with the same intervention as the other group. During the 30 days waiting period, the WLC will be advised to read generally about hearing loss and its treatment.http://ClinicalTrials.gov Protocol Registration System and the registration number is NCT01611129 (Study ID Number FAS-IT-03).The study protocol has been registered in the The main outcome measure used will be the Hearing Handicap Questionnaire (HHQ) and the secondary outcome measures include the Hospital Anxiety and Depression Scale (HADS), the University of Rhode Island Change Assessment Scale (URICA) and the Hearing Disability Acceptance Questionnaire (HDAQ).et al. [HHQ is an instrument which has 12 questions scored on a 5-point Likert scale  and provides a measure of personal and social effects - emotional distress and discomfort, social withdrawal, and general restriction of participation. HHQ was developed on the basis of a questionnaire by Hétu et al.  and an uet al. . HHQ haset al. .HADS is an instrument for screening for anxiety and depression . The HADet al. which investigated the usage of the URICA scale in adults with acquired hearing impairments seeking help for the first time, showed good construct, concurrent and predictive validity of the scale [URICA is a stages of change measure consisting of four sub-scales: pre-contemplation, contemplation, action and maintenance ,33. The he scale .et al. [HDAQ has been designed based on the Tinnitus Acceptance Questionnaire (TAQ) which was developed by Westin et al.  in SwedeHHQ is the primary outcome measure used for the power calculation. The results from a previous study suggest that the mean scores of HHQ for those with mild hearing loss who are seeking help for the first time are expected to be about an average of 26, with a standard deviation of 8 . HHQ hasDue to the nature of the study, it is not possible to blind the participants and the clinician. However, the allocation of participants to study groups will be done randomly using computer-generated numbers and with the help of another person . However, the participants will be given unique reference numbers to keep the participants’ personal details blinded while analyzing the results.Considering that the participants do not have any direct contact with the researcher , co-intervention cannot be avoided. However, participants will be requested not to participate in any interventions which may influence the results during the beginning of the study and will be questioned about this at the end by being asked a similar question after outcome measures have been collected. These notes may help us while analyzing the results. In relation to compliance with the intervention protocol, participants will be encouraged to complete the task after each session throughout this process. A reminder Email will be sent on a regular basis advising the participants about the recommendations made regarding intervention. Moreover, attempts will be made to classify the Email interactions with participants to understand at what phase each participant might be, so that the suggestions and recommendations made will be tailored accordingly.Data will be collected via online questionnaires administered through the secure internet-based counseling protocol system one week pre intervention, one week post intervention and six month follow-up. To reduce attrition rate at follow-up, reminders will be sent by Email. However, if the participants decide not to participate further in the study, the reasons for their withdrawal will be recorded. Analysis of covariance will be made for T1 and T2 data with initial T0 measures  as the continuous predictor. Any missing data at T1 and T2 will be imputed with an assumption that values are missing completely at random (MCAR) and using an appropriate imputation method .It is well documented that only a small percentage of people with hearing loss seek help and uptake amplification devices . MoreoveStudies indicate that participating in counseling-based aural rehabilitation would bring additional benefit in reducing perceived hearing disability than hearing aids alone . A recenInternet-based rehabilitation programs have recently started coming in to use and studies have shown promising results ,25. ThisWhilst we expect to reduce perceived hearing disability through this intervention, there are very small chances that the opposite could happen in some cases. This is because thinking about the future challenges related to hearing problems may be overwhelming for some participants. For this reason, care must be taken during this process to boost positive aspects and to reduce negative aspects associated with their hearing problems. However, if proven effective, such a program could be a cost-effective and time-efficient program to help hundreds and thousands of people with perceived hearing difficulties.Whilst efforts will be made to provide internet-based information in an interactive manner, no particular accommodation has been made for people with other disabilities such as visual impairment, learning disability, dementia, and so on, who may have difficulty understanding the information presented online. Moreover, one of the pre-requisites for the participants is having internet access. These criteria may result in the exclusion of some groups of people from this study. Moreover, one of the main predicted problems is in recruiting study participants, which will be managed by using appropriate advertisement methods.Participant recruitment is complete and the trial is running during November to December 2012.ACE: Active communication education; ANOVA: Analysis of variance; CP: Communication partner; HBM: Health belief model; HHQ: Hearing Handicap Questionnaire; HADS: Hospital Anxiety and Depression Scale: HDAQ, Hearing Disability Acceptance Questionnaire; MAR: Missing at random; PHD: Person with hearing disability; PHI: Person with hearing impairment; RCT: Randomized controlled trial; SRMI: Sequential regression multiple imputation; TAQ: Tinnitus Acceptance Questionnaire; URICA: University of Rhode Island change assessment scale; WLC: Waiting list control.The authors declare that they have no competing interests.All the authors took part in the design of the study. VM is responsible for the overall operational aspects of the study including data collection, analysis and writing-up. TL, DS, JR and GA provided input to study design, development of internet-based counseling protocol and supervision of the study. All authors participated in the preparation of, and approved for publication, the final manuscript.


Determining the primary site of metastatic cancer with confidence can be challenging. Pathologists commonly use a battery of immunohistochemical (IHC) stains to determine the primary site. Gene expression profiling (GEP) has found increasing use, particularly in the most difficult cases. In this pilot study, a direct comparison between GEP and IHC-guided methods was performed.Ten archived formalin-fixed paraffin embedded metastatic tumor samples for which the primary site had been clinically determined were selected. Five pathologists who were blinded to the diagnosis were asked to determine the primary site using IHC and other stains selected from a panel of 84 stains. Each pathologist was provided patient sex, biopsy site and gross sample description only. Slides were digitized using ScanScope®XT at 0.25 μm/pixel. Each evaluating pathologist was allowed to provide a diagnosis in three stages: initial (after reviewing the H&E image), intermediate (after reviewing images from the first batch of stains) and final diagnosis (after the second batch of stains if requested). GEP was performed using the only FDA-cleared test for this intended use, the Pathwork Tissue of Origin Test. No sample information was provided for GEP testing except for patient sex. Results were reported as the tumor tissue type with the highest similarity score.In this feasibility study, GEP determined the correct primary site in 9 of the 10 cases (90%), compared to the IHC-guided method which determined the correct primary site for 32 of 50 case evaluations . The five pathologists directing the IHC-guided method ordered an average of 8.8 stains per case (range 1 to 18). GEP required an average of 3 slides per case (range 1 to 4).Results of the pilot study suggest that GEP provides correct primary site identification in a higher percentage of metastatic cases than IHC-guided methods, and uses less tissue. A larger comparative effectiveness study using this study design is needed to confirm the results.http://www.diagnosticpathology.diagnomx.eu/vs/1749854104745508The virtual slide(s) for this article can be found here:  Determining the primary site of metastatic cancer with confidence can be challenging; for 3–5% of cancer cases there is no clinically evident primary site -7. PathoIn some cases, however, the primary site cannot be identified with certainty using conventional IHC evaluation. In a review and meta-analysis of published studies of IHC accuracy that were adequately blinded, four studies representing a total of 308 tumor samples reported average accuracy of 67% on metastatic samples [In recent years, gene expression profiling (GEP) tests and tests using microRNA markers have been developed to aid in the diagnosis of difficult-to-diagnose tumors -11. ThesRecent reports discuss the need to coordinate the use of available methods of identifying primary site to optimize patient care ,13. HoweOne of the GEP tests for primary site identification is the Tissue of Origin Test  -17. We cA web interface was developed which linked to a digital microscopy database to facilitate and standardize immunohistochemical evaluation by evaluating pathologists. Histology slides were digitized and the images were provided to evaluating pathologists for histopathological evaluation. Using digital pathology and secure web access provided a controlled means to allow access to only the stains requested by each pathologist and maintained the blinding of the pathologist.The methods being compared have inherent differences that can confound a comparison – GEP is objective, independent of clinical history, based on algorithmic applications to a dataset, and is a one-time test, whereas histopathologic evaluation is subjective, reliant on clinical history and often performed in consultation with other pathologists . Stains This publication describes the novel methods used to conduct the study, results of the pilot project, and provides information needed for conducting a more comprehensive study.Archived human formalin-fixed paraffin-embedded (FFPE) specimens (blocks) containing metastatic tumors with a clinically established primary site were used. Samples were coded and both the evaluating pathologists and the laboratory performing the GEP at Pathwork Diagnostics were blinded to the primary site. The study was conducted under an Institutional Review Board-approved protocol. The overall study design and workflow is illustrated in Figure 2 of tumor tissue for GEP, and (c) at least 15 unstained slides for IHC staining and analysis. (iv) H&E stained slides were evaluated by a board certified pathologist to verify tumor content. All specimens were estimated to contain ≥ 60% non-necrotic tumor tissue (tumor and stroma) in the first and last H&E stained slide and (v) found to be consistent with the reported histology on quality review by a board-certified pathologist. If the number of stains ordered exceeded the number of slides that were cut initially, additional slides were prepared from the block, and the last slide was stained with H&E to verify that at least 60% tumor content remained.The selection criteria were as follows. (i) The sample represented a FFPE metastatic tumor with a known primary site as determined by review of the medical records. The determination of primary site was based on all available clinical information, but was not accepted when the diagnosis was made exclusively using immunohistochemistry (IHC) or special stains. (ii) All tumor samples were selected from a panel of 15 tumor tissue types covered by the Tissue of Origin Test. These types include: bladder (BL), breast (BR), colorectal (CO), gastric (GA), testicular germ cell (GC), kidney (KI), hepatocellular (LI), non-small cell lung (LU), non-Hodgkin’s lymphoma (LY), melanoma (ME), ovarian (OV), pancreas (PA), prostate (PR), thyroid (TH) and sarcoma (SC). (iii) The FFPE tissues submitted on unstained slides or paraffin blocks were checked for adequacy of tissue required for the study. Samples had to be sufficient to produce at least 25 5-μm-thick (+/− 1 μm) sections: (a) the first and last sections for staining with hematoxylin and eosin (H&E) stain, (b) eight unstained slides (USS) containing no less than 1 mmAll stains were performed in a CLIA-certified laboratory by a histotechnologist with more than 20 years of IHC staining experience. All IHC stains were performed on a Ventana Benchmark ® LT automated immunohistochemical stainer. Relevant controls were included with each batch of stains. Prior to study initiation, a panel of 84 stains was agreed upon by all investigators, and made available for the study. This included 73 IHC stains and 11 histochemical stains (Table All H&E slides and IHC stained slides were digitized using a Whole Slide Imaging (WSI) system . All slides were scanned at 0.25 μm/pixel resolution, and the images saved in the password-protected database (Spectrum version 10.2.2.2314) provided by Aperio® on a web-accessible server. All digitized images were reviewed by a board-certified pathologist for quality assurance.Five board-certified pathologists with a wide range of experience (3 to 30+ years post pathology training) and from a diverse set of institutions  were selected for evaluating the cases. All evaluating pathologists confirmed previous use of digital pathology.A web-accessible user interface was designed  for evaluating pathologists to access the images in the Spectrum database, order stains as needed, and record diagnoses and associated confidence levels. This information was collected at the following points: a) after review of the H&E slide alone b) after review of the first batch of stains, and c) after review of the second batch of stains. Each evaluating pathologist (EP) had secure password-protected access to the interface. All 10 cases were evaluated by each EP. One H&E image was provided to all EPs as the starting point for each case. Each pathologist had access only to IHC stains that they had ordered; if a stain was ordered by more than one pathologist, requestors were provided with a copy of the same digital image. This added control and reduced variability in interpretation that might be attributed to the staining procedure or tumor heterogeneity.The EPs were blinded to the clinical history and tissue of origin for the samples. They received only the patient’s sex and gross sample description for each case, including biopsy site, given in Table 2 in area. To increase the percent tumor in the submitted sample, tumor tissue was microdissected (scraped) from the slides and placed into vials for RNA extraction. Total RNA was isolated using the Agencourt® FormaPure Kit . The total RNA was processed to prepare labeled cDNA for hybridization to Pathchip® microarrays manufactured by Affymetrix  with a two-cycle amplification method using the RampUP Kit . A positive/negative total RNA control was run with every amplification batch. The microarrays were washed and stained using the GeneChip® Hybridization Wash and Stain kit in a GeneChip Fluidics Station FS450Dx, and scanned with a GeneChip Scanner 3000Dx (Affymetrix). Microarray data files (CEL) that passed data verification [The specimens were processed for the Pathwork® Tissue of Origin Test at Pathwork Diagnostics Laboratory (PWDL) as described previously . Unstainication  were anaIt should be noted that in clinical practice, a PWDL pathologist provides an interpretation of the test results along with a confidence level. This is based on test performance information derived from analyses of results from the clinical validation study , as wellTen metastatic specimens were selected from among common solid malignancies representing nine different primary sites, and four metastatic sites, as shown in Table Five pathologists reviewed 10 cases each, for a total of 50 case reviews. The average number of stains ordered by an EP in the first round was 7.06. For 21 of 50 42%) of the case reviews the EPs ordered a second round of stains with an average of 4.2 stains for each of these cases. From among the 442 total stains ordered by all the EPs for all the cases, an average of 22 unique stains per case was ordered by the group of five EPs. None of the EPs ordered histochemical stains other than IHC stains. The number of stains ordered for each case by individual EPs is shown in Table 2% of theThe diagnoses of primary site reached by each EP and the predictions of primary site by the GEP test are shown in Table The variation in the total number of stains requested by all 5 EPs per case may be indicative of relative diagnostic complexity. Using this measure, case 3 was the simplest, and all EPs provided a final diagnosis without ordering a second round of stains. One EP diagnosed the case with as few as 3 stains. The average number of stains used by all EPs was 5 . Case 6 was the most complex: no EP required fewer than 8 stains, and the average across all EPs was 12.2 . For both cases, all EPs and the GEP test correctly determined the primary site.The GEP test determined the correct diagnosis in 90% (9/10) of cases. The range of highest SS was 24.4 to 97.6 Table . The aveWe have devised and tested a novel approach for directly comparing a GEP test to special stain evaluation using multiple evaluating pathologists. The study design reduced subjectivity of the pathologic diagnosis and minimized the variables between the two approaches being compared. The use of a single central laboratory to perform IHC staining and digital pathology to provide the results to multiple participating pathologists eliminated the need for shipping slides from one site to another for pathologist evaluation, thus enabling more efficient study logistics and timelines. The whole slide imaging system created a digital replica of the entire content of a glass microscope slide on the computer, closely emulating traditional viewing of a slide with a conventional microscope . EPs couAll 5 EPs and the GEP test provided a final diagnosis on all 10 cases. Case 3 was of very low morphological difficulty and all EPs provided a final diagnosis that matched the reference diagnosis while ordering the smallest number of stains . Cases 4 and 10 were incorrectly diagnosed by all 5 EPs and warrant further discussion. The patient information provided to all the EPs and the GEP testing laboratory is shown in Table The PWDL pathologist interpreting the GEP results favored a non-small cell lung origin, since a prediction of ovarian was implausible in males and the pattern of scores was most consistent with a non-small cell lung origin. The full value of GEP and other novel molecular evaluations in oncology will most likely be achieved by a judicious incorporation into the final pathologist consultation report .The amount of tissue used by each testing method is an important consideration in evaluating tissue-based diagnostics. GEP used an average of three slides per case, whereas the EPs used an average of nine slides per EP per case. In this sample set, molecular testing required less tissue and reduced the risk of tissue depletion. For some cases  EPs ordered a large number of stains (average 11 slides per EP per case); whereas in cases 2, 3, 5 and 10 they ordered fewer IHC stains (average 6 slides per EP per case). The GEP test used 3 slides on average for both sets.In this pilot study GEP performed favorably (90% accuracy) compared to histopathologic evaluation (average 64% accuracy) by 5 EPs. It is interesting to note that the average accuracy seen in this study is very similar to the average 67% accuracy reported in the previous meta-analysis . In thisThis pilot study of 10 samples found important differences in accuracy between two methods. In the 10 metastatic samples reported here, GEP identified the correct primary site more often than the IHC-guided methods used by the pathologists participating in the study. This study design will be applied to a larger set of samples to provide a statistically powered assessment of the comparative effectiveness of the GEP and IHC-guided methods.(EP): evaluating pathologist; (FFPE): formalin-fixed paraffin-embedded; (GEP): gene expression profiling; H&E: hematoxylin-and-eosin; (IHC): immunohistochemistry; (SS): Similarity Score; (TOO): tissue of origin.CRH, AK and AME were recipients of research grants from Pathwork Diagnostics for the performance of this study. WDH and RP are employees and stock holders of Pathwork Diagnostics.CRH was the Principal Investigator. CRH and AK participated in the conception, study design, sample identification, results interpretation, and correlation with clinical information and manuscript writing. AME participated in the sample identification, data management, results interpretation, and manuscript writing. RP and WDH participated in the conception, study design, conduct of the study, data management, results interpretation, and manuscript preparation. All authors read and approved the final manuscript.


Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia and is responsible for significant economic losses to the pig industry. To better understand the mode of action of a commercial, adjuvanted, inactivated whole cell vaccine and the influence of diversity on the efficacy of vaccination, we investigated samples from vaccinated and non-vaccinated pigs experimentally infected with either a low (LV) or a highly virulent (HV) M. hyopneumoniae strain. Non-vaccinated and sham-infected control groups were included. Lung tissue samples collected at 4 and 8 weeks post infection (PI) were immunohistochemically tested for the presence of T-lymphocytes, B-lymphocytes and macrophages in the bronchus-associated lymphoid tissue (BALT). The number of M. hyopneumoniae organisms in bronchoalveolar lavage (BAL) fluid was determined using quantitative PCR at 4 and 8 weeks PI. Serum antibodies against M. hyopneumoniae were determined at 0, 2, 4, 6 and 8 weeks PI.M. hyopneumoniae organisms in the BAL fluid was measured at 4 weeks PI for the HV strain and at 8 weeks PI for the LV strain. Vaccination reduced the number of organisms non-significantly, though for the HV strain the reduction was clinically more relevant than for the LV strain. At the level of the individual pigs, a higher lung lesion score was associated with more M. hyopneumoniae organisms in the lungs and a higher density of the investigated immune cells in the BALT.The immunostaining revealed a lower density of macrophages in the BALT of the vaccinated groups compared to the non-vaccinated groups. The highest number of M. hyopneumoniae is reduced by vaccination. The M. hyopneumoniae replication in the lungs is also reduced in vaccinated pigs, though the HV strain is inhibited more than the LV strain.In conclusion, the infiltration of macrophages after infection with  Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia (EP) in pigs. This disease is characterized by chronic non-productive coughing and poor growth rate and feed conversion ratio [on ratio . The dison ratio .M. hyopneumoniae strain [Q1t within a herd and even within a pig, different strains of M. hyopneumoniae can be present [The infection pattern and the severity of the disease in pig herds can be influenced by many factors such as management practices, housing conditions and the virulence of the e strain ,4. Basede strain ,6. Recen present ,8. In a  present . The dif present .M. hyopneumoniae strains also seems to influence the efficacy of vaccination, with better results for some strains [M. hyopneumoniae infection are significantly reduced after vaccination [The diversity between  strains . Previoucination . Howevercination ,13.M. hyopneumoniae are cell-surface proteins, which vary between strains [The exact mechanisms of protection after vaccination are not yet fully understood, although both mucosal antibodies and cell-mediated immunity may play a part . The pre strains ,8. There strains .M. hyopneumoniae are macrophages, but T-lymphocytes, B-lymphocytes and to a lesser extent neutrophils are also present [Little is known about the effect of different strains on the pig's immune system. Meyns et al.  showed t present . Macroph present -18.M. hyopneumoniae strain was compared and the influence of vaccination on this process was studied.In the present study, the infiltration of lung tissue with mononuclear cells after infection of pigs with a highly and low virulent The results of the scoring of the immunostaining are presented in Table At 4 weeks PI, the density of macrophages in the BALT of the non-vaccinated groups was higher compared to the vaccinated groups, though this difference was only significant for the HV strain. At 8 weeks, no significant differences were observed for the macrophages though the score in the non-vaccinated groups remained higher than in the vaccinated groups.M. hyopneumoniae organisms was highly variable between individual animals, even in animals belonging to the same group. This resulted in few significant differences between groups, though in some cases high numerical differences were seen . No causal relationship can be deducted from our data, though we suggest the hypothesis that more M. hyopneumoniae organisms cause a higher infiltration of immune cells and this causes more tissue damage leading to more severe lung lesions. Meyns et al. [M. hyopneumoniae, the induction of immune cells and the severity of the lung lesions at the level of the individual pig. It should be noted that the data from qPCR showed much stronger correlations with the microscopic and macroscopic lung lesions compared to IF suggesting that qPCR is more suitable to investigate the number of organism that IF.Though it is well known that the outcome of s et al.  observeds et al.  found a M. hyopneumoniae was not associated with severity of the lung lesions at 4 weeks post-infection, but a high titer at later time points (6 and 8 weeks PI) was associated with a higher density of immune cells and a higher microscopic lung lesion score at 8 weeks PI. This not only confirms the absence of a link between serum antibody titer and protection against infection [The titer of serum antibodies against nfection , but it M. hyopneumoniae is reduced by vaccination. The growth of M. hyopneumoniae organisms in the lungs is also reduced in vaccinated pigs, though the HV strain is inhibited to a higher extent than the LV strain. This indicates that future studies should focus on the interaction between macrophages and lymphocytes after infection with different strains and the influence of vaccination on this interaction.Although this study was performed on a limited number of samples, some interesting observations were made. Our results suggest that the infiltration of macrophages in BALT after infection with M. hyopneumoniae and PRRSV free farm, were injected at one week of age with either a one-shot commercial, adjuvanted, whole-cell vaccine (v)  or PBS (nv). At three weeks of age, piglets were weaned and transported to the experimental facilities of the Faculty of Veterinary Medicine, Ghent University, Belgium. After one week, 40 pigs were inoculated with a highly virulent (HV) M. hyopneumoniae isolate (F7.2 C), 40 with a low virulent (LV) M. hyopneumoniae isolate (F13.10B) and 10 with sterile medium. This resulted in five groups: nvLV, nvHV, vLV, vHV and a non-vaccinated non-infected control group. Half of the piglets in each group were euthanized at 4 weeks post inoculation (PI), the remaining half at 8 weeks PI. The immunofluorescence scoring of M. hyopneumoniae, the histopathological scoring (microscopic lung lesion score) and the macroscopic lung lesion score determined in Villarreal et al. [Samples were obtained during a previously described trial . The stul et al.  can be fFor each group, lung tissue samples from five randomly selected pigs were investigated at 4 and 8 weeks PI, except for the control group, from which only two pigs were selected. Formalin fixed and paraffin embedded lung samples collected for histopathological examination for the study of Villarreal et al.  were exaTen images were taken per sample by standard light microscopy at a magnification of 40× , covering the whole surface area of the slide. A visual score according to the density of stained cells in the BALT was assigned ranging from 0 (no to a few stained cells) to 3 . All images were scored by the same person and this person was blinded to the groups.M. hyopneumoniae organisms in the lung was estimated by performing quantitative PCR (qPCR) on the BAL fluid as described previously [M. hyopneumoniae DNA was made in PBS.The number of eviously . BrieflyM. hyopneumoniae in the serum using the IDEXX M. hyo. ELISA . The serum antibody titer is estimated by the sample-to-positive (SP) ratios calculated from the optical density (OD)-values: (ODsample - average ODneg control)/(average ODpos control - average ODneg control) [At 0, 2, 4, 6 and 8 weeks PI, blood samples were taken from all pigs and tested for the presence of antibodies against control) .One-way analysis of variance (ANOVA) was used to analyze the results from the scoring of immunostaining, qPCR and the serum antibody titer between groups at the same time point. When the homogeneity of variances test was passed, Bonferroni was used as post hoc test, when it failed, Dunnett T3 was used. Data from qPCR was transformed to the logarithm prior to analysis to normalize the data. Correlations between macroscopic lung lesion score, microscopic lung lesion score (histopathology), IF, immunostaining, log qPCR and serum antibody titer on the level of the individual pig were determined by calculating the Spearman's rho correlation score (ρ) both for the data 4 and 8 weeks PI. Correlations were also calculated for these three parameters and the antibody titer at and before euthanasia for both 4 and 8 weeks PI. The statistical results were considered significant when p-values were ≤ 0.05. All analyses were performed with SPSS 17.0 for Windows .The authors declare that they have no competing interests.KV, DM, IV, FP and FH participated in the conception and design of this study. KC contributed in the conception and design of the immunostaining. KV and IV participated in the acquisition of data. KV and SBM participated in the analysis and interpretation of data. KV, DM and FH drafted the manuscript. All authors read and approved the final manuscript.


H9N2 influenza virus is endemic in many Asian countries and is regarded as a candidate for the next human pandemic. Knowledge of the induction of inflammatory responses and toll-like receptors (TLRs) in chickens infected with H9N2 is limited. Here, we show that H9N2 induces pro-inflammatory cytokines such as transforming growth factor-beta 3; tumor necrosis factor-alpha; interferon-alpha, -beta, and gamma; and TLR 1, 2, 3, 4, 5, 7, and 15 in trachea, lung, and intestine of infected chickens. In the lung, TLR-15 was dominantly induced. Taken together, it seems that H9N2 infections efficiently induce inflammatory cytokines and TLRs in trachea, lung and intestine of chickens. Influenza A viruses circulate in aquatic birds such as gulls and shorebirds, and are in evolutional stasis . Some avH9N2 influenza virus (hereafter referred to as H9N2) was first isolated from diseased chickens in China in 1994. Since then, this virus has been found in many countries including Korea, India, and Pakistan -7. In EuH9N2 can infect humans. In 1999, two cases of mild influenza were reported in two children infected with H9N2  and in 2Inflammatory cytokines are suggested to be involved in pathogenesis in humans, pigs, and horses -16. InfeToll-like receptors (TLRs) are important for eliciting innate immunity in animals by playing an essential role as pattern recognition receptors that detect infectious pathogens by recognizing the conserved molecular structures known as pathogen associated molecular patterns -19. The The current study was undertaken to investigate the inflammatory cytokines and TLRs induced in H9N2-infected chickens, to further the understanding the early pathogenesis and immune responses in H9N2 infection.The H9N2 representative, A/Chicken/Korea/S21/2004, was grown in 10-day-old fertilized eggs. The experiments were performed in a BSL-3 facility approved by the Korean government. Specific pathogen free (SPF) chickens (White Leghorn) that were 3-4 weeks o1d were purchased from a local farm. Animal work was approved by internal animal ethics committee at Chungnam National University.n = 5 per group) were intranasally (i.n.) infected with 0.5 mL of 106 log 10 egg infectious dose 50/mL (log10EID50/mL) of A/Chicken/Korea/S21/2004 and tissues of the trachea, lung, and intestine were collected after chickens were euthanized by cervical dislocation. The collected tissues (0.5 g) were homogenized in 1 mL of phosphate buffered saline (PBS) containing ampicillin and penicillin, and were centrifuged to obtain the supernatant. Virus in the supernatant was determined by log10EID50/mL as described previously [Chickens (eviously . The virn = 5 per group) that were infected i.n. with 0.5 mL of 106 EID50/mL of A/Chicken/Korea/S21/2004 using TRIzol reagent . Chickens were euthanized by cervical dislocation and tissues samples (0.5 g) of the trachea, lung and intestine were collected in tubes. One milliliter of TRIzol reagent was added to tubes containing tissues and incubated at room temperature for 5 min. Chloroform (200 μL) was added and the solution was mixed by vortexing for 15 s and centrifuged for 15 min, 12 000 rpm, 4°C. The upper RNA-containing band was collected and mixed with 500 μL of isopropanol  in a new 1.5 mL tube. Each sample was centrifuged for 10 min, 10 000 rpm, 4°C, and the RNA-containing pellet was washed with 100 μL of 75% ethanol in water by centrifuging for 5 min at 10 000 rpm and 4°C. The washed RNA was resuspended in 50 μL of diethyl pyrocarbonate-treated water.Total RNA was collected from chickens . Each sample was incubated for 5 min at 25°C, 60 min at 42°C, and 15 min at 70°C. SYBR Green-based real-time PCR was performed using a Roto-Gene 6000 apparatus  and SensiMix Plus SYBR  based on recommendations of the manufacturer. A duplicate of each sample was run. A total volume of 20 μL containing 2 μL cDNA, 10 μL SYBR mixture, and inflammatory cytokine-specific   was added to a total volume of 9 μL in a 0.05 mL tube. The mixture was reacted for 5 min at 70°C prior to incubation for 5 min at 4°C. Then, each sample received 4 μL of 25 mM MgClP-value < 0.05 was considered statistically significant.Statistical analysis was performed using the Statistical Product and Services Solutions, version 10.0 . ANOVA analysis was performed by comparing infected data to uninfected data. A n = 5 per group) were exposed to the representative H9N2 to establish that the virus was capable of infection. The viral titers were determined by log10EID50/mL in tissue samples from trachea, lung, and intestine collected on day 1, 2, and 4 post-infection (pi). H9N2 replicated well in all tissues  on day 1, 2, and 4 pi. In trachea, the inflammatory cytokines  were induced to higher levels in the H9N2-infected chickens than in the uninfected chickens (Figure To determine inflammatory cytokines in tissue of trachea, lung, and intestine of H9N2-infected chickens, quantitative real-time PCR was performed using primers specific for chicken cytokines such as TGF-β3, TNF-α, IFN-α, IFN-β, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, and IL-10, and mRNA of trachea, lung, and intestine of chickens (s Figure . In lungs Figure . In intes Figure . The tiss Figure . In lungs Figure . In intes Figure .We determined the induction of TLRs in trachea, lung, and intestine tissue in those chickens examined in the experiment summarized in Figure H9N2 is endemic in many countries including Korea and China, and is regarded as one of the candidates for the next human pandemic. We studied mRNA expression of inflammatory cytokine and TLRs in H9N2-infected chickens to understand the early inflammatory responses and pathogenesis of the infection. The data demonstrate that H9N2 infects trachea, lung, and intestine tissue, and induces the production of inflammatory cytokines and TLRs in these tissues.Our data showed that cytokine inductions increased as viral titers increased in tracheas and lungs of chickens infected with H9N2. In tracheas, the higher inductions of TNF-α and INF-γ were made on 4 days pi when viral titer peaked. In lungs, TNF-α, INF-β, and INF-γ were induced higher on 4 days when viral titer peaked. Previous studies suggest that cytokines may be involved in both clearance of virus and pathological tissue damage. A study on a chicken macrophage cell line infected with H9N2 showed that inflammatory cytokines such as IL-1β and IL-8 were up-regulated . HoweverWhen we determined the induction of TLRs in trachea, lung, and intestine tissue, TLR-4, TLR-15, and TLR-5 were dominantly induced in trachea, lung, and intestine, respectively. The results suggest that the different tissues can respond to H9N2 using the different TLRs and that tissue specific expression of TLRs can be induced. TLR-15 was induced higher in lungs, but it was not induced in tracheas of chickens infected with H9N2 influenza virus. Targeting theses TLRs for therapeutic purpose can be one of ways to defend chickens from the infections of avian influenza viruses. The recent study showed that the pre-stimulation of TLR-2 and TLR-4 by their ligand could increase the resistance to highly pathogenic H5N1 influenza viruses in mice . InformaWhen we determined the kinetics gene expression of inflammatory cytokines or TLRs some genes increased as time elapsed, but others did not. In trachea, the induction of TNF-α was similar on 1, 2 or 4 days pi, while the induction of INF-γ increased as time lapsed. In trachea, TLR-4 was induced highest on 1 day pi, rather than 4 days pi, while in intestine, the induction of TLR-5 was induced highest on 4 days pi rather than 1 day pi.In conclusion, infection of chickens with H9N2 may induce the production of inflammatory cytokines in trachea, lung, and intestinal cells by inducing different TLRs.The authors declare that they have no competing interests.NTN and JSL carried out the experiment on cytokines and toll-like receptors in the infected chickens and drafted the manuscript. BMS and YMK helped the animal experiment. HSK preformed the statistical analysis. SHS conceived the study, and participated in its design and coordination. All authors have read and approved the final manuscript. All authors read and approved the final manuscript.


Msh4-Msh5 and Mlh1-Mlh3, act in meiosis to promote interference-dependent crossing over. Mlh1-Mlh3 also plays a role in DNA mismatch repair (MMR) by interacting with Msh2-Msh3 to repair insertion and deletion mutations. Mlh3 contains an ATP-binding domain that is highly conserved among MLH proteins. To explore roles for Mlh3 in meiosis and MMR, we performed a structure−function analysis of eight mlh3 ATPase mutants. In contrast to previous work, our data suggest that ATP hydrolysis by both Mlh1 and Mlh3 is important for both meiotic and MMR functions. In meiotic assays, these mutants showed a roughly linear relationship between spore viability and genetic map distance. To further understand the relationship between crossing over and meiotic viability, we analyzed crossing over on four chromosomes of varying lengths in mlh3Δ mms4Δ strains and observed strong decreases (6- to 17-fold) in crossing over in all intervals. Curiously, mlh3Δ mms4Δ double mutants displayed spore viability levels that were greater than observed in mms4Δ strains that show modest defects in crossing over. The viability in double mutants also appeared greater than would be expected for strains that show such severe defects in crossing over. Together, these observations provide insights for how Mlh1-Mlh3 acts in crossover resolution and MMR and for how chromosome segregation in Meiosis I can occur in the absence of crossing over.Crossing over between homologous chromosomes occurs during the prophase of meiosis I and is critical for chromosome segregation. In baker’s yeast, two heterodimeric complexes,  Saccharomyces cerevisiae is initiated by the induction of approximately 140−170 SPO11-dependent double-strand breaks (DSBs) that are located throughout the genome . In this pathway the presence of one crossover decreases the likelihood of another nearby  colocalization between the two foci; however, as pachytene progresses, this colocalization gradually disappears  complexes, sappears . The preMsh4-Msh5 interference pathway and the actions of Sgs1 helicase, which may play a role in promoting crossing over, as well as serve as an anticrossover factor by removing aberrant recombination intermediates , the MSH proteins n errors . In the sh2-Msh3 . Mlh3 coMlh3 in DNA MMR and meiosis by analyzing the phenotype of eight mlh3 ATPase mutants. Our data suggest that ATP hydrolysis by both Mlh1 and Mlh3 is important for both meiotic and MMR functions. In meiotic assays these mutants showed a roughly linear relationship between spore viability and genetic map distance. To further analyze the role of Mlh3 in meiosis, we analyzed crossing over on four chromosomes in mlh3Δ mms4Δ cells and observed a strong decrease in crossing over at all intervals, but higher spore viability than would be expected for strains that show such strong crossover defects. Together these observations provide insights for how Mlh1-Mlh3 acts in crossover resolution and MMR, and for how chromosome segregation in Meiosis I can occur in the absence of crossing over.In this study we investigated the role of S. cerevisiae strains were grown at 30° in either yeast extract-peptone, 2% dextrose media, or minimal selective media (SC) containing 2% dextrose, sucrose, or galactose  using the single-step integration vector pEAI254 as a template. pEAI254 contains the SK1 MLH3 gene with a KANMX4 selectable marker inserted 40 bp downstream of the stop codon  as a template , and mutant derivatives pEAE374 (GAL10-mlh3-E31A), pEAE375 (GAL10-mlh3-R96A), and pEAE376 (GAL10-mlh3-G97A) were transformed into EAY1269 .The isogenic SK1 strain EAY1062 [ion rate . For thehttp://www.molbio.uoregon.edu/∼fstahl/), which uses the formula of 2 test with P-values < 0.05 considered statistically significant. The genetic intervals measured in this study , and strains were analyzed for reversion to Lys+  versions of the respective media for 26 hr. Appropriate dilutions were made, and cells grown in sucrose only were plated on uracil, lysine dropout SC agar plates containing 2% sucrose, and uracil dropout SC agar plates containing 2% glucose. Cells grown in sucrose and galactose were plated on uracil, lysine dropout SC agar plates containing 2% sucrose and 2% galactose, and uracil dropout SC agar plates containing 2% glucose. Using Mlh1-Pms1 are functionally asymmetric. For example, the Mlh1 subunit of the yeast Mlh1-Pms1 complex displayed a much greater affinity for ATP compared to the Pms1 subunit, and an ATP hydrolysis mutation in MLH1 (mlh1-E31A) conferred a much greater effect on MMR than the equivalent mutation in PMS1 . Mutations also were identified that severely disrupt ATP binding [e.g., mlh1-N35A  and binding mlh3-N35A, and six other mutations that map within or near motifs identified in the GHKL family of ATPases, of which the MLH proteins are members [lys2::14InsE-A allele , ranging from 3.2 to 6.7-fold greater than wild-type levels. mlh3-K83A strains showed a wild-type phenotype . We showed previously that overexpressing Mlh3 using the GAL10 promoter conferred a high mutator phenotype in the lys2::14InsE-A, reversion assay with mutation rates more than 1000-fold greater than wild-type. This phenotype was similar to that seen in wild-type strains overexpressing Mlh1  conferred null phenotypes in the meiotic assays, and one mutation, mlh3-K83A, conferred a wild-type phenotype. Three mutations, mlh3-E31A, mlh3-R96A, and mlh3-G97A, conferred intermediate phenotypes  and MMR  compared with Y55 mlh3Δ strains [92% . However, three mlh3 hypomorph mutants, mlh3-E31A, -R96A, -G97A, displayed null phenotypes in MMR, but intermediate meiotic phenotypes, as measured in meiotic spore viability and crossover assays (Mlh1 (Mlh3 functions are more easily disrupted for MMR.It is important to note that five of the eight r assays  and 5. Tmlh3 mutants displayed a relationship where spore viability decreased progressively with map distance (R2 = 0.87). Consistent with this we observed that wild-type spore viability was significantly greater than that seen in mlh3-E31A, -R96A, and -G97A (P ≪ 0.001). This pattern is in contrast to the pattern observed in msh4/5 mutants, where crossing over could be decreased to approximately 50% of wild-type levels  without an apparent defect in spore viability, after which point spore viability and crossing over decreased linearly . Such a relationship might be expected if Mlh1-Mlh3 acts late in crossover resolution because a decrease in Mlh3 function would be expected to cause a random loss in crossing over, thus not assuring that all obligate crossovers would take place.As shown in mlh3 spore viability and map distance data support a roughly linear relationship, we more closely examined the phenotype of two mutants, mlh3-G97A and mlh3-R96A. These mutants show a relatively large difference in genetic map distance but a negligible difference in spore viability (P > 0.5). We attempted to detect any difference in phenotype conferred by these mutants by making double mutants with msh5 alleles. When mlh3-R96A was combined with msh5-T423A, very little change in spore viability or map distance was observed compared with single mutants  and between mlh3-G97A msh5-D423A and mlh3-R96A msh5-D423A (P < 0.01) were statistically significant. This analysis confirms that mlh3-G97A confers a more severe defect compared with mlh3-R96A, as predicted if the pattern seen for msh4/5 mutants did not hold for the mlh3 mutants. Consistent with these observations, mlh3-G97A conferred a mild nondisjunction phenotype, as measured by an excess of 4, 2, 0 viable spore tetrads compared with 3 and 1 viable tetrads . In addition, three meiotic joint molecule resolvase complexes have been identified: Mus81-Mms4, Yen1, and Slx1-Slx4  in crossing over compared with wild type  reduced crossing over by 49%, mms4yen1 by 39%, and exo1mms4yen1 by 86%. Strikingly, crossover levels decreased roughly 20-fold in mlh3mms4slx4yen1sgs1 cells  and viability (∼40%) as well as high levels of aberrant recombination events  and viability (45%) whereas mlh3Δ displayed greater levels of spore formation  and viability . mlh3Δ mms4Δ displayed significantly greater sporulation  and viability  compared to mms4Δ. In addition, mlh3Δ mms4Δ mutants showed gene conversion levels that were similar to wild-type but lower than mms4Δ alone  despite large reductions (6- to 17-fold) in crossing over. Such reduced levels should yield crossover levels below the obligate number (16) required to segregate all yeast homologs. If we assume that crossover levels decrease to similar extents across the length of a single chromosome, then only chromosome VII would appear to have at least one crossover in mlh3Δ mms4Δ. This calculation is based on high-resolution genotyping of meiotic spore progeny performed by mlh3Δ mms4Δ.As indicated previously, spore viability in mlh3Δ mms4Δ, both of which assume achiasmate chromosome disjunction mechanisms. The first suggests that the high spore viability is due to distributive disjunction, which is defined as the process in which “two nonhomologous chromosomes that lack homologs or two homologs that have failed to recombine, disjoin at meiosis I”  Chromosome disjunction in mlh3Δ mms4Δ is facilitated by Zip1, a synaptonemal complex protein that promotes homology-independent centromere pairing (Zip1 promotes centromere pairing in both nonhomologous chromosomes and nonexchange homologous chromosomes, providing a mechanism for nonexchange chromosomes to be held together until the first meiotic division, possibly by promoting correct spindle orientation (Msh4-Msh5 acts to facilitate disjunction in mlh3Δ mms4Δ by promoting homolog pairing. Consistent with this idea, Msh5 has been shown to act in early steps in homolog pairing in mice and Sordaria (A second explanation is that homologous pairing mechanisms are taking place in


Silurus glanis (Siluridae) and the South American bronze catfish Corydoras aeneus . The non-invasive auditory evoked potential (AEP) recording technique was utilized to determine hearing thresholds at 10 frequencies from 0.05 to 5 kHz. Neither auditory sensitivity nor shape of AEP waveforms differed between normally pigmented and albinotic specimens at any frequency tested in both species. Silurus glanis and C. aeneus showed the best hearing between 0.3 and 1 kHz; the lowest thresholds were 78.4 dB at 0.5 kHz in S. glanis (pigmented), 75 dB at 1 kHz in S. glanis , 77.6 dB at 0.5 kHz in C. aeneus (pigmented) and 76.9 dB at 1 kHz in C. aeneus . This study indicates no association between albinism and hearing ability. Perhaps because of the lack of melanin in the fish inner ear, hearing in fishes is less likely to be affected by albinism than in mammals.Pigmentation disorders such as albinism are occasionally associated with hearing impairments in mammals. Therefore, we wanted to investigate whether such a phenomenon also exists in non-mammalian vertebrates. We measured the hearing abilities of normally pigmented and albinotic specimens of two catfish species, the European wels  W  or ocular albinism (OA) depending on loss of melanin in the skin (hair) and eyes versus just in the eyes. Partial albinism describes diseases such as the Waardenburg syndrome, piebaldism and the Tietz–Smith syndrome, in which only part of the skin (hair) lacks pigmentation  occasionally show hearing anomalies  thresholds compared with normally coloured specimens . In miceThere are some indications for a relationship, but no general linkage, between OCA and hearing impairments in humans  neuromasts and taste buds.Sox10 zebrafish mutants. Mutations of this gene cause Waardenburg syndrome in humans.Silurus glanis (Siluridae) and the South American bronze catfish Corydoras aeneus , were studied. These two species represent two different groups of catfishes. Silurus glanis has a large free swimbladder and four Weberian ossicles  and auditory sensitivity in fishes exists, we compared the auditory evoked potentials (AEP) and hearing abilities in albinotic and pigmented individuals of two species of fishes possessing hearing specializations. We chose species having excellent hearing sensitivities  because we hypothesize that slight changes in hearing caused by albinism may affect hearing specialists to a higher degree than species having low hearing sensitivities. Two catfish species, namely the European wels ossicles ;, wherea ossicle ;. Based  ossicle , we expeC. aeneus from Ruinemans Aquarium, Montfoort, the Netherlands, and S. glanis albinos from Hornbach, Brunn am Gebirge, Austria) and a fish hatchery    and 2.S. glanis and 25±1 °C for C. aeneus. A 12 h:12 h L:D cycle was maintained. Silurus glanis were fed frozen food  and live earthworms, C. aeneus were fed frozen chironomid larvae and artificial food . Standard length (SL) was measured as ‘standard length 2’ following Silurus specimens ranged from 152 mm SL and 35 g body weight to 215 mm SL and 85 g, Corydoras specimens ranged from 32.7 mm SL and 1.53 g to 45.7 mm SL and 3.34 g.Fish were kept in planted aquaria with a sand bottom equipped with roots and clay or plastic tubes as shelters. Only external filters were used in order to provide a quiet environment. The temperature was maintained at 20±1 °C for Hearing thresholds were obtained using the AEP recording technique developed by μg g−1 for S. glanis normal, 3.57–5.78 μg g−1 for S. glanis albino, 0.41–0.62 μg g−1 for C. aeneus normal and 0.55–0.85 μg g−1 for C. aeneus albino. Water temperature during recordings was adjusted to 20±1 °C for S. glanis and 25±1 °C for C. aeneus.Hearing thresholds were determined at 0.05, 0.07, 0.1, 0.3, 0.5, 0.8, 1, 2, 3, 4 and 5 kHz. Test subjects were mildly immobilized with Flaxedil  diluted in a Ringer solution. The dosage applied was 2.12–3.39 S. glanis had black eyes and a high variability in body pigmentation. Some individuals were dark brown with numerous black and only few silvery whitish blotches and dots, whereas in other specimens, the number of whitish blotches was much higher, covering most of the brown body   . All catThe AEP of pigmented and albinotic groups of each species did not differ in the overall shape of waveforms, latency, amplitudes or in the number of positive or negative peaks  and 4.S. glanis were the lowest between 300 and 1000 Hz; the lowest threshold was 78.4 dB at 500 Hz in pigmented S. glanis and 75 dB at 1000 Hz in albinos . Comparison of hearing thresholds at each frequency with an unpaired t-test showed no significant differences between normally coloured and albino fish (P>0.05).Hearing thresholds of  albinos , Table 1C. aeneus, auditory sensitivity was the highest between 300 and 1000 Hz. The maximum sensitivity was 77.6 dB at 500 Hz in pigmented and 76.9 dB at 1000 Hz in albinotic C. aeneus . Comparing hearing thresholds at each frequency with an unpaired t-test showed no significant differences between the normally pigmented and the albino group (P>0.05).In . aeneus , Table 1Wv/Wv mouse mutants showed lower or no endocochlear potentials. Significant differences have been observed in the ABRs of normal and albinotic humans  cats  cats . In geness) cats ;. LatencHearing impairment and pigmentation disorders frequently occur together in many syndromes in mammals ;. The arAs noted above, comparative studies on hearing abilities in normally pigmented and albinotic specimens revealed widely differing results in AEPs and hearing thresholds. A main reason for these inconsistencies may be the term ‘albinism’, which is much too sketchy to describe the heterogeneous reasons for defects in pigment production in vertebrates.Besides the general differentiation between OA and OCA , there aOCA is a group of four autosomal recessive disorders (OCA1–OCA4) caused by either a complete lack or a reduction of melanin biosynthesis in the melanocytes, resulting in hypopigmentation of the hair, skin and eyes . StudiesSox10 gene results in abnormalities in ear development and lack of body pigmentation, whereas the eyes remain normally pigmented.In fish that lack a cochlea and a stria vascularis, melanocytes – in particular their absence in the inner ear – may play a less crucial role than in the mammalian ear. This may explain why our study animals did not exhibit pigmentation-related differences in hearing abilities. Nonetheless, hypopigmentation could potentially co-occur with hearing disorders in zebrafish mutants. Astyanax jordani and its eyed ancestor A. mexicanus, did not differ. Today, A. jordani is not regarded as a valid species, but as a blind, depigmented, cave-dwelling form of A. mexicanus (A. mexicanus (Fish lacking pigmentation have been tested for hearing by exicanus . In factexicanus  in diffeexicanus ;. AccordIn summary, based on our current results, we assume that albinism is not associated with hearing disorders in fishes.Siluris glanis has one large free swimbladder and four Weberian ossicles, whereas C. aeneus has paired, tiny, bony, encapsulated swimbladders and a single ossicle, which should result in better high-frequency hearing in S. glanis (S. glanis is quite low at high frequencies compared with other species with a well-developed swimbladder and ossicles. At 3 kHz and at higher frequencies, S. glanis exhibited higher hearing thresholds than all but one catfish species with free bladder tested so far, which belonged to the families Ariidae, Auchenipteridae, Heptapteridae, Malapteruridae, Mochokidae and Pseudopimelodidae (The order of catfishes comprises more than 3000 species . Catfish. glanis . While telodidae , Ictalurelodidae ; and PimC. aeneus are similar to its congenerics Corydoras paleatus and Corydoras sodalis (C. paleatus having a lower high-frequency hearing sensitivity than the two other species.As expected, the hearing abilities of the callichthyid  sodalis ;, with CS. glanis and the callichthyid C. aeneus fit the concept that hearing abilities in catfishes depend on the swimbladder size and the number of Weberian ossicles, as stated by In summary, the hearing abilities of the silurid


Spontaneous tegmen tympani defects are rare with even rarer bilateral cases. The symptoms are nonspecific; hence, a high index of suspicion is required to prevent serious intracranial complications. We present a case of spontaneous bilateral tegmen tympani defects with associated meningoencephalocoeles in a 54-year-old male who presented with the signs and symptoms of severe meningitis. After careful workup which included a lumbar puncture, CT and MRI scans, both defects were repaired using a middle fossa approach. The patient made an uneventful recovery with complete cessation of otorrhoea and improvement in his hearing. CSF leaks from skull base defects are classified as either spontaneous with no obvious cause or secondary due to trauma, surgery, tumours, infection, and inflammation , 2. PatiA 54-year-old man presented to the emergency department with agitation and seizures following a generalized febrile illness and a progressively worsening headache over 24 hours. He was treated with ceftriaxone IV, metronidazole, and acyclovir. A contrast CT scan of the brain showed no focal central lesion but features in keeping with bilateral chronic middle ear and mastoid disease. A lumbar puncture showed no evidence of raised intracranial pressure (ICP). CSF microscopy supported the diagnosis of meningitis, and on culture streptococcus sensitive to ceftriaxone and amoxicillin was grown. He responded to a three-week course of appropriate systemic antibiotics and made a complete recovery. On reviewing his history, he had been able to produce clear rhinorrhoea on leaning forward for, years and this tested positive for beta-2 transferrin. He had a left conductive hearing loss due to a middle ear effusion for several years. He had a grommet inserted in 2008, which was later removed due to persistent clear otorrhoea. CSF analysis at the time was inconclusive, and a CT scan of the temporal bones reportedly showed no abnormality. In 2000, he had a generalized seizure for which no cause could be found, and he has been on phenytoin ever since. He has type 2 diabetes mellitus for which he is on insulin. There was no past history of head trauma, CNS infections, neurologic, and otologic surgery. A High resolution CT and MRI scans of the petrous temporal bones were performed confirming bilateral tegmen tympani dehiscence, bilateral meningoencephalocoeles and bilateral middle ear and mastoid effusions. A middle fossa approach was used to repair both defects starting with the left. Six months later, the contralateral side was repaired. A 20 × 10 mm defect in the tegmen with substantial herniation of brain tissue was noted on the right, and an 8 × 2 mm defect with encephalocoele was present on the left. A strip of cortical bone harvested from the bone flap was placed over the defects which were then covered with a fascia lata graft. Tisseel was used to secure the graft in place. The recovery period was uneventful. CSF leakage ceased after surgery, and his hearing improved bilaterally.A defect in the tegmen tympani can result in CSF otorrhoea in the presence of a tympanic membrane perforation or a ventilation tube, conductive hearing loss, and aural fullness with an intact tympanic membrane. The findings of clear, watery, and pulsatile middle ear fluid at the time of myringotomy for conductive hearing loss secondary to a middle ear effusion are well documented , 8. If tLess commonly, a middle ear mass due to the prolapse of a cephalocoele may be the initial presentation , 10. A sThere is still much controversy as to what causes spontaneous tegmen tympani defects with various theories being postulated. Cases presenting in the paediatric population associated with anomaly of the inner ear and congenital hearing loss are thought to be caused by abnormal embryologic development, which results in gaps within the skull base , 7, 11. Recognition of a tegmen tympani defect is important because of the potential of developing meningitis and other intracranial complications. The index of suspicion should be higher in cases of recurrent meningitis . Any obvAs seen in this case, a CT of the brain done prior to performing a lumbar puncture may fail to identify skull base defects. High resolution CT images of the temporal bones with 1 mm slices scanned in both axial and coronal planes are recommended . MRI is In our case, even though there were clinical features of a CSF leak in 2008, CT scan of the temporal bones failed to show the defect. Studies have shown that high resolution CT can identify the associated skull base defect in most but not all cases of CSF leak . We can For hearing preservation and with the relative anterior location of the tegmen, a middle cranial fossa approach was used to repair both defects. This approach seems to be the consensus in patients with tegmen tympani defects and serviceable hearing . The optA spontaneous tegmen tympani defect is a rare but an important diagnosis given the potential for catastrophic intracranial sepsis. Diagnosis is based on clinical signs and symptoms such as clear rhinorrhoea/otorrhoea and conductive hearing loss. However, certain patients can present in extremis with meningitis or other intracranial complications. High resolution CT with 1 mm slices is essential for locating the defect. MRI scan is helpful to detect herniation of a meningoencephalocoele  and asso