Previous research showed an increase of noise-induced symptoms in adolescents. Permanent tinnitus as a consequence of loud music exposure is usually considered as noise-induced damage. The objective was to perform an epidemiological study in order to obtain prevalence data of permanent noise-induced tinnitus as well as temporary tinnitus following noise exposure in a young population. In addition the attitudes and beliefs towards noise and hearing protection were evaluated in order to explain the use/non-use of hearing protection in a young population.A questionnaire was completed by 3892 high school students . The prevalence of temporary and permanent tinnitus was assessed. In addition the ‘Youth Attitudes to Noise Scale’ and the ‘Beliefs About Hearing Protection and Hearing Loss’ were used in order to assess the attitudes and beliefs towards noise and hearing protection respectively.The prevalence of temporary noise-induced tinnitus and permanent tinnitus in high school students was respectively 74.9% and 18.3%. An increasing prevalence of temporary tinnitus with age was present. Most students had a ‘neutral attitude’ towards loud music and the use of hearing protection was minimal (4.7%). The limited use of hearing protection is explained by a logistic regression analysis showing the relations between certain parameters and the use of hearing protection.Despite the very high prevalence of tinnitus in such a young population, the rate of hearing protection use and the knowledge about the risks of loud music is extremely low. Future preventive campaigns should focus more on tinnitus as a warning signal for noise-induced damage and emphasize that also temporary symptoms can result in permanent noise-induced damage. Adolescents and young adults often expose themselves to loud music and excessive noise levels during social and music events Temporary noise-induced tinnitus (NIT) is also a common phenomenon in adolescents as reported prevalence numbers in previous studies vary between 45% and 77% In contrast to the high rate of hearing symptoms after loud music exposure and despite the fact that adolescents claim to be aware of the risks of loud music As mentioned earlier, the prevalence numbers of hearing symptoms after loud music exposure vary between studies. Therefore, the need for an epidemiological study rises. In order to understand adolescents’ behavior concerning the protection of their hearing, the attitudes and beliefs towards noise and HP must be assessed. While several studies analyzed the attitudes and beliefs of young people towards noise The principals of several high schools were contacted by phone with the suggestion to participate in the study. This approach was chosen because this allowed to provide sufficient information concerning the study and to answer all questions. After a positive verbal agreement, all participating schools were sent a written confirmation of participation by e-mail including a copy of the questionnaire. As the study is performed by the administering of a questionnaire, the high school principals were in this case considered as the caretakers of the minors. All questionnaires were administered during class. Students were not at all obliged to complete the questionnaire so the completion of the questionnaire was considered as a silent approval for participation. As such, an additional informed consent was not documented. The approach of the present study was approved by the IRB of the University Hospital Antwerp in May 2011.A cross sectional survey by means of a self-administered questionnaire was performed. Therefore a total of 4800 questionnaires were administered to students of fifteen randomly chosen Flemish high schools . Students of all Flemish provinces and with all educational programs were represented in the sample. The questionnaire assessed following items: demographic and educational information, prevalence of permanent tinnitus and tinnitus characteristics, prevalence of temporary tinnitus and tinnitus characteristics, leisure noise exposure, attitudes towards noise and attitudes towards HP. All categories are explained in more detail in the following sections.Students had to fill in their gender , year of birth and highest obtained educational diploma . Also the highest diploma obtained by their parents was questioned.In the survey the term ‘tinnitus’ was replaced by a more laymen’s term (comparable to ‘ringing in the ears’ in the English language) as ‘tinnitus’ might be an unfamiliar term for adolescents. For our convenience the term ‘tinnitus’ is used throughout the manuscript instead of ‘ringing in the ears’.Firstly, the prevalence of permanent tinnitus was evaluated by a yes/no question (‘Do you constantly perceive tinnitus?’). Secondly, the presence of temporary tinnitus after recreational noise exposure was evaluated by a Numeric Rating Scale (NRS) for loudness going from 0 (no tinnitus present) to 10 . For those students who already perceived a permanent tinnitus a worsening of tinnitus after loud music exposure was evaluated by use of a baseline-NRS  compared to a maximum-NRS (intensity of the loudest tinnitus experienced) after loud music exposure. A tinnitus loudness of 0 indicated the absence of NIT, while a tinnitus loudness >/1 indicated the presence of temporary NIT. The duration of temporary NIT or a temporary worsening of the permanent tinnitus was also assessed. Answer possibilities were: ‘less than thirty minutes’, ‘between thirty minutes and two hours’, ‘between two hours and six hours’, ‘between six hour and one day’, ‘more than one day’ and ‘not applicable’ in case if no tinnitus or no worsening of tinnitus was experienced. Furthermore, in case of temporary NIT, fear of permanent tinnitus was evaluated by questioning whether someone is afraid the tinnitus will not disappear . In a similar way it was evaluated whether one has the feeling the hearing decreases after noise exposure .For the evaluation of leisure noise exposure following situations had to be answered either on a ‘daily basis’, ‘weekly’, ‘monthly’, ‘yearly’ or ‘not applicable’: playing an instrument solo, playing an instrument in a band, listening to PLD’s, and discotheque attendance. The volume setting of PLD’s was assessed with a scale from 0% to 100% of the PLD capacity and music loudness in discotheques had to be rated as “too quiet”, “quiet”, “good”, “loud”, or “too loud”.The Youth Attitudes to Noise Scale (YANS) is an instrument designed by Widén & Erlandsson (2004) The ‘Beliefs About Hearing Protection and Hearing Loss’ (BAHPHL) is originally a 31-item questionnaire developed by the National Institute for Occupational Safety and Health p<0.05.For the statistical analysis a statistical Software package  was used in order to perform independent T-tests and Mann-Whitney U tests for the comparison of the prevalence of temporary and permanent tinnitus per age category and for the analysis of the scores on the YANS and BAHPHL. A step-wise logistic regression model was used to explain the use of HP in adolescents. Nineteen variables were put into the equation: Gender, age, smoking, education level, education level of the mother, education level of the father, temporary tinnitus after loud music exposure, NRS scores for temporary NIT and permanent tinnitus, persistence of NIT, fear of permanent tinnitus, subjective temporary hearing loss, total score on the YANS, total score on the BAHPHL, frequency discotheque visit, rating discotheque loudness, frequency music instrument playing, frequency use of PLD’s and volume settings of PLD’s. The level of statistical significance was defined as p = 0,033) compared to the other age categories. Male respondents reported significantly more permanent tinnitus (20%) compared to female respondents . The overall prevalence of temporary NIT was 74.9% . In most cases tinnitus was perceived bilaterally as shown in 29.7% of students reported to play a musical instrument of which 13.5% on a daily basis. Moreover, 13.5% played in a band, mostly on a weekly basis (8.3%) and only few daily (0.9%). PLD use did not differ between age groups with up to 60% of all students using PLD’s on a daily basis for 30 minutes to one hour in 41.7% of the cases. Furthermore, 35.4% sets the volume settings up to 80% or more of the total PLD capacity.The evaluation of party behavior showed a significant increase of discotheque attendance with age as 4.6% of the 14 year old and 30.4% of the 18 year olds visited discotheques on a daily to weekly basis. When asked to rate music loudness in discotheques, 45.1% reported it to be ‘too loud’, 43.9% considered the levels as ‘loud’ and 10.9% reported them to be quiet or sufficient.p<0.001). A trend for better usage of HP in adolescents with permanent tinnitus was observed, however the influence was only marginal significant . Rating the discotheque loudness as ‘too loud’ had positive influences on the use of HP. Surprisingly, the students experiencing temporary tinnitus used HP to a lesser extent (p = 0004).A logistic regression model was used to determine which factors are involved when it comes to the use of HP when exposed to loud music. Seven variables out of the nineteen showed significant influence on the use of HP: gender, fear of permanent tinnitus, temporary tinnitus after loud music, rating discotheque loudness, permanent tinnitus and the score on the BAHPHL. The results of the logistic regression model are shown in A significant number of the study population (18.3%) reported permanent tinnitus in one or both ears. The prevalence of permanent NIT in this adolescent population was threefold the prevalence found in the study of Widen et al (2004) An overall prevalence of 74.9% of temporary NIT was observed. Such a prevalence of temporary NIT is consistent with previous studies p<0.001) parameter. The lower the score on the BAHPHL, the more positive one was towards HP and the more HP was used. These findings confirm the findings of a recent study by Widén et al. (2013) who suggested that norms regarding HP use play a greater role than the attitudes towards HP and noise The underlying construct of the TPB  were partly followed in the present study. The attitudes towards noise were assessed by the YANS whereas subjective norms and perceived behavioral control can be found in the BAHPHL. Although both scales have intrinsic distinct subcomponents, they are all part of the construct of a certain belief and certain attitudes, in this case beliefs and attitudes concerning noise, the development of hearing loss and the use of hearing protection. As it is believed that beliefs and attitudes are responsible for the eventual ‘actions’  we were mainly interested in the total scores of the YANS and BAHPHL because it is the total construct that may underlie the actions and not the subcomponents of the construct. While the YANS focuses more on the attitudes towards noise, the BAHPHL deals particularly with HP use. The experience of a temporary NIHL in about 50% of the cases seems to positively affect the score on the BAHPHL. In the logistic regression model the total BAHPHL score indeed was a highly significant  who concluded that the excessive sound levels at many music events are not demanded nor required by most young people. In addition, it has been found that people would not visit night clubs any less if the intensity levels would be lowered Personal rating of discotheque levels influences HP use as those rating the intensity levels at discotheques as ‘too loud’, intend to more often use HP. Considering that mean sound levels in discotheques vary between 104 and 112 dB Fear of the persistence of tinnitus was also a good predictor for HP use as those fearing tinnitus were more inclined to use HP. In addition, although not significant, permanent tinnitus also seemed to be a motivational factor for HP use. Temporary NIT however, surprisingly, did not raise HP use. A possible explanation is that those students who do not use HP will be more prawn to the experience of temporary tinnitus. Secondly, is it plausible that as students are quite familiar with temporary symptoms, they do not find it necessary to take precautions because of the transient characteristic. In a previous study, young individuals were questioned concerning whether which hearing symptoms apply when noise is too loud and possibly damaging The authors like to point out that, up until now, most preventive campaigns mainly focused on the development of hearing loss as a consequence of frequent exposure to loud music. As a temporary threshold shift or even a mild permanent threshold shift as a consequence of noise exposure, is often not noticed by adolescents, the focus of campaigns on the development of hearing loss might be a less effective approach. As the present study shows a very high prevalence of tinnitus (temporary as well as permanent) in young people we suggest that preventive campaigns should focus more on tinnitus as a warning signal for noise-induced damage. In addition, the fact that also temporary tinnitus or other temporary noise-induced symptoms do not necessarily exclude permanent cochlear or neural damage and that through accumulative noise damage temporary symptoms may evolve into permanent symptoms, should be underlined. In the modern society, where social network sites and smartphones have become so important into the lives of young people, campaigns should use these communication modes in order to achieve adolescents’ attention. In addition, more focus should go to a more interactive way of communicating to make adolescents more aware of the risks of loud music exposure. The authors believe that the personal experience with noise-induced symptoms  is a fast and effective way to rapidly change the beliefs and attitudes and therefore the actions of adolescents. Furthermore, the authors suggest that preventive campaigns should also focus on students younger than 14 years old. As the results have shown an increase of attending social events with loud music and therefore an increase of hearing problems from this age on, educational programs should take place in advance as attitudes and thus beliefs and therefore behaviors have already been formed prior to this age. A better understanding of noise-induced symptoms may result in an increase of HP in a younger population and may prevent permanent hearing loss and tinnitus due to recreational noise exposure.Appendix S1Youth Attitudes to Noise Scale (YANS). Information concerning the YANS questionnaire and the validation of the Dutch YANS including Cronbach’s alpha scores.(DOCX)Click here for additional data file.Appendix S2Beliefs About Hearing Protection and Hearing loss (BAHPHL). Information concerning the BAHPHL questionnaire and the validation of the Dutch BAHPHL including Cronbach’s alpha scores.(DOCX)Click here for additional data file.


Objective. This study examined the patterns of neural activity in the central auditory system in children with hearing loss. Methods. Cortical potentials and mismatch responses (MMRs) were recorded from ten children aged between 9 and 10 years: five with hearing loss and five with normal hearing in passive oddball paradigms using verbal and nonverbal stimuli. Results. Results indicate a trend toward larger P1 amplitude, a significant reduction in amplitude, and latency of N2 in children with hearing loss compared to control. No significant group differences were observed for the majority of the MMRs conditions. Conclusions. Data suggest that the reduced auditory input affects the pattern of cortical-auditory-evoked potentials in children with a mild to moderately severe hearing loss. Results suggest maturational delays and/or deficits in central auditory processing in children with hearing loss, as indicated by the neurophysiological markers P1 and N2. In contrast, negative MMR data suggest that the amplification provided by the hearing aids could have allowed children with hearing loss to develop adequate discriminative abilities. Sensory hearing loss often affects speech perception due to a decreased audibility of the signal as well as decreased temporal analysis ability –3. StudiAuditory neurophysiological functions have been measured in adults and children with hearing loss –11. SensIn children, latency changes in the cortical-auditory-evoked potentials (CAEPs) have been used to document auditory system plasticity and recovery from auditory deprivation following cochlear implantation , 12. ConTo determine CAEPs potential clinical benefits in assessing central auditory functions in children with hearing loss, a clear understanding of the effects of a sensory hearing loss on CAEPs is needed. The main objective of the present study is to explore central auditory neurophysiological functions in children with hearing loss wearing hearing aids. If CAEPs are affected by sensory hearing loss, cortical auditory measures could become neurophysiological markers in clinical audiology for evaluating young children for whom central auditory functions are difficult to assess behaviourally.Two groups of nine-to-ten years old female children participated in the study: five with sensory hearing loss  and five with normal hearing . Participants with normal hearing had auditory detection threshold at 15 dB HL or less between 500 Hz and 8 kHz, bilaterally . All paThree pairs of synthetic stimuli were used for this study: one verbal and two nonverbal pairs. The verbal stimuli consisted of two syllables: /ba/ and /da/. These stimuli were selected from the CD-ROM, Speech Production, and Perception I . The fir(DELL) using Stimaudio (NeuroScan Inc.) and the 2Stim software. They were presented to the right ear through insert-earphone (E-A-RTONE 3A), connected to an audiometer , at 70 dB HL for children with normal hearing and between 85 and 105 dB HL for those with hearing loss (See The stimuli were presented with a computer loss See . Stimuli Dell computer. An electrode located on the forehead (Fpz) served as ground and reference was located at the vertex. Electrode impedance was kept under 7 kΩ for mastoid, central, and frontal regions and below 15 kΩ for ocular and peripheral regions.The cortical responses were digitally recorded using a high-density system, Scan 4.0 software , with SynAmps amplifiers and from 128 Ag/AgCl electrodes. Electrophysiological signals were acquired at a sampling rate of 250 Hz, with an analog online bandpass filtering from 0.1 to 100 Hz using the SynAmps amplifiers running on aThe children were seated in a chair in a double-walled sound-proof booth. Participants watched a movie or cartoon of their choice on a computer monitor with the sound off. They were told to ignore the auditory input and to focus their attention on the movie. Total testing duration was approximately between 90 and 120 minutes.μV). The timeframe of analysis was from −100 ms to 700 ms. Data were baseline corrected to −50 ms. Auditory cortical components were defined as followed: P1 and N1 were the first positive and negative waveforms in the time window of 50–100 ms and 80–120 ms, respectively. They are followed by a positive peak, defined as P2 within the time window of 100–160 ms, and N2, the second negative peak at 200–280 ms. Amplitude values were measured from baseline to peak for each component, and latency values were measured relative to the onset of stimulus presentation.Using BrainVision Analyser program on an IBM computer, the data were corrected for eye movements using Gratton and Coles algorithm . They weMMRs were computed according to the following procedure: ERPs evoked by a standard stimulus were subtracted from ERPs evoked by the presentation of a deviant stimulus for each participant. Responses to standard stimuli that immediately followed the presentation of deviant stimuli were excluded from the standard stimulus average. Two MMRs were observed with the pair 1 kHz pure tone wide-band noise; a first negative peak was measured from 115 to 200 ms and a positive slope was observed from 200 to 330 ms. However, only one prevalent negative response from 115 to 260 ms was observed with the nonverbal and verbal pairs. For each participant, the latency of the most negative or positive peak was measured for the MMRs by using a peak amplitude automatic detection.Statistical analyses were conducted on the amplitude and latency values of the standard sound waveforms because they were better defined and had a clearer morphology compared to those obtained from deviant waveforms. P1, N1, P2, and N2 were observed clearly in children with normal hearing with the three stimuli . By contUsing SPSS software, a two-way ANOVA was performed  with repeated measures on the second factor, for both P1 and N2 latency and amplitude measures Figures .F = 7.85, P < .01]. t-tests were conducted, applying Bonferroni corrections to adjust for multiple comparisons (P < .016). Results revealed only a significant latency prolongation for the verbal /ba/ than the 1 kHz pure tone  and for the nonverbal /ba/ than 1 kHz pure tone .With regard to P1 latency, results revealed a significant effect for the main Type factor only  . Results revealed a significant effect for the Type factor too . However, no significant difference was observed between the three types of stimuli when t-tests with Bonferroni corrections (P < .016) were applied.As pertains to N2 latency, a significant latency reduction was observed in children with hearing loss comparatively to the latency value of children with normal hearing . The main Group factor failed to reach significance but a trend was observed . For the significant Type factor, a t-tests, with Bonferroni corrections (P < .016), revealed a significant greater amplitude for the 1 kHz pure tone than the nonverbal /ba/  only.Regarding P1 amplitude, results revealed a significant effect for the Type factor only . Results also showed a significant effect for the type factor . t-tests with Bonferroni corrections (P < .016) demonstrated a significant greater amplitude for the 1 kHz pure tone than the verbal /ba/  only.For the N2 amplitude, a significant amplitude reduction was observed in children with hearing loss comparatively to the amplitude value of children with normal hearing . A t-test with Bonferroni corrections (P < .016) demonstrated a significant latency prolongation for the verbal /ba/-/da/ pair as compared with the 1 kHz pure tone and wide-band noise pair  and also comparatively to the nonverbal /ba/-/da/ pairs .With regard to negative MMR latency, results revealed a significant effect only for the main Type factor  , but not for the latency value .Regarding the negative MMR amplitude, results revealed no significant effect for the two main factors nor for the two-way interaction Group × Type. A positive MMR component was observed with the pair of 1 kHz pure tone and wide-band noise . A t-tesThe aim of the present research was to study the patterns of the neurophysiological activity in the central auditory system in children with hearing loss as compared with children with normal hearing. Differential findings were observed with regard to the principal cortical components and the MMR results.P1 amplitude tended to be greater, N1 and P2 components less defined, and amplitude and latency of N2 reduced in children with hearing loss compared with the results of the children with normal hearing. These findings will be discussed according to three factors: the presentation level, the maturation of the central auditory system, and the deficit in the central auditory system.The stimuli were presented between 80 and 105 dB HL for the children with hearing loss and at 70 dB HL for the children with normal hearing. The higher level of stimulus presentation (in dB HL) could have contributed to the large amplitude of P1 and to the shorter latency of N2. Oates et al. (2002) found that the amplitude of the N1 and the P300 was larger and their latency shorter at 80 dB SPL compared to 65 dB SPL in adults with hearing loss .However, the results of the present study were partially in agreement with those results. There were only two indications that the level of presentation could modulate waveform characteristics. In fact, P1 amplitude was larger and N2 latency was shorter in children with hearing loss comparatively to children with normal hearing. The findings indicate that the level of presentation could affect differently the two components.The P1 waveform changes in a complex manner in children. P1 decreases systematically in latency and/or amplitude to reach adult values almost at the age of 14-15 years  or 20 yeIn the present study, all children with hearing loss experienced a period without any stimulation with hearing aids, since their hearing loss was identified between the age of 20 months and 5 years . During Two out of four cortical auditory potential components—N1 and P2—were less defined in children with hearing loss compared to their peers with normal hearing. These two components do not emerge consistently until the age of 8 to 11 years in children with normal hearing , 24, 26.Regarding the N2 maturation in children with normal hearing, N2 amplitude has an initial increase between the age of 5 to 11 years  followedThe greater amplitude of P1 with a concomitant reduction in N2 amplitude and the less well-defined N1-P2 components could also indicate a deficit in central auditory processing. The anomalies have been reported in central auditory late latency components in children with language-based learning problems (LPs) . Albeit Similar patterns of results were obtained in the two groups of children with the negative mismatch response measured in the 150–200 ms window. These results suggest that the auditory system can discriminate sounds, being the verbal or nonverbal, and that this pattern of discrimination can be found in children with hearing loss as well as in children with normal hearing. They further suggest that the amplification provided by the hearing aids could have contributed to get under way the maturational processes, allowing the children to develop adequate discriminative abilities.A positive MMR was measured in the 200–300 ms window with the pair of 1 kHz pure tone and wide-band noise only. Results showed that the amplitude of this positive MMR was significantly smaller in children with hearing loss than that observed in children without hearing loss. This result may simply be related to the fact that children with hearing loss have, as stated above, a smaller N2 amplitude in response to the standard stimuli compared to normal hearing children.The negative MMR was also found to differ according to stimulus type. When the stimuli were simple, (the pair 1 kHz and wide-band noise), the MMR had an earlier latency compared to more complex stimuli, such as the nonverbal and verbal /ba/-/da/. The effect of stimulus type on ERP results has also been reported by other studies , 32. ThoAlthough obtained in a limited number of children and in a restricted age range, these preliminary findings indicate that reduced auditory input early in life has an impact on the development of central auditory functions reflected by the specific patterns of CAEPs. The interaction and the combination of at least two factors, delay in maturation and deficit in the central auditory system, could contribute to the pattern of results obtained in children with hearing loss. The data further indicate that sensory hearing loss affects differently the earlier cortical component P1 compared to the later component N2. Moreover, the findings suggest that CAEPs can be more sensitive markers of the effects of sensory hearing loss than are mismatch responses in children with mild to moderately severe hearing loss. Measuring P1 and N2, as the neurophysiological markers in children with hearing loss, can provide an objective assessment of the maturation of their central auditory system. For well-trained audiologists with CAEPs, results can be easily interpreted. P1 and N2 amplitude measured before and after a given auditory training program may reflect the efficiency of the program and confirm the plasticity of the auditory pathways. Also, with these two neurophysiological components, audiologists may determine whether appropriate stimulation is being provided by a hearing aid or cochlear implant, and based on the findings, they may adjust the auditory training program. However, the CAEPs measures should be adapted before being implanted as an assessment tool in clinics and its cost effectiveness has to be assessed. In the near future, studies will take into account the clinical testing conditions by reducing the number of recording channels  in order to be suitable to clinical equipments and also by developing normative data.


Mice lacking surfactant protein-A  exhibit increased vulnerability to infection and injury. Although many bronchoalveolar lavage (BAL) protein differences between KO and wild-type (WT) are rapidly reversed in KO after infection, their clinical course is still compromised. We studied the impact of SP-A on the alveolar macrophage (AM) proteome under basal conditions. Male SP-A KO mice were SP-A-treated (5 micrograms/mouse) and sacrificed in 6 or 18 hr. The AM proteomes of KO, SP-A-treated KO, and WT mice were studied by 2D-DIGE coupled with MALDI-ToF/ToF and AM actin distribution was examined by phalloidon staining.We observed: a) significant differences from KO in WT or exogenous SP-A-treated in 45 of 76 identified proteins (both increases and decreases). These included actin-related/cytoskeletal proteins , proteins of intracellular signaling, cell differentiation/regulation, regulation of inflammation, protease/chaperone function, and proteins related to Nrf2-mediated oxidative stress response pathway; b) SP-A-induced changes causing the AM proteome of the KO to resemble that of WT; and c) that SP-A treatment altered cell size and F-actin distribution.in vivo SP-A treatment of KO mice, under basal or unstimulated conditions, affects the expression of multiple AM proteins, alters F-actin distribution, and can restore much of the WT phenotype. We postulate that the SP-A-mediated expression profile of the AM places it in a state of "readiness" to successfully conduct its innate immune functions and ensure lung health.These differences are likely to enhance AM function. The observations show for the first time that acute  SP-A, a multi-functional protein, is known to play an important role in host defense. SP-A is a collectin, or collagenous lectin, that can recognize pathogen-associated molecular patterns (PAMP). The recognition and binding of PAMP is complex and may involve binding sites in addition to the C-type carbohydrate recognition domain. Although the direct interaction with pathogens constitutes one aspect of its host defense function, SP-A also plays a role in the clearance of particulate matter, allergens, and debris from the alveolar surface -5. SP-A Klebsiella pneumoniae, Streptococcus pneumoniae, Pseudomonas aeruginosa, Pneumocystis carinii, respiratory syncytial virus, and others [SP-A-/-  mice exhibit increased vulnerability to infection and injury. This has been illustrated with mouse models of pneumonia with organisms including d others ,36-41. Ad others ,42 diffed others  of the Kd others . This main vivo effect of SP-A on the alveolar macrophage proteome under basal or unstimulated conditions, with regards to whether and which groups of proteins SP-A may affect under such conditions. However, based on recent in vitro studies, in response to LPS [A likely source of these host defense deficits in the SP-A KO mouse is the alveolar macrophage, the primary effector cell for innate immunity in the lung. Although macrophages, which are derived from blood monocytes, are found throughout the body, their phenotype may vary depending on their environment. Alveolar macrophages exhibit a unique phenotype  that is e to LPS  or in exe to LPS , it appein vivo the changes in the macrophage proteome in response to SP-A treatment of SP-A KO mice, the functional groups the changed proteins belong to, and whether the SP-A-induced changes render the proteome from alveolar macrophages of the KO mice more like that of wild type (WT) mice. SP-A KO mice treated with SP-A and sacrificed at different times post-treatment, as well as WT mice on the same C57BL/6 genetic background were used in this study. The data were subjected to various analytical methods to assess, in an unbiased fashion, the similarities and differences in the proteomes of our experimental groups, as well as, by considering the function of the changed proteins, to obtain insight into underlying mechanisms and the SP-A-mediated functions of the alveolar macrophage. Based on the findings a "proof of principle" experiment was carried out where changes were measured in the actin cytoskeleton.In this study we tested the hypothesis that SP-A exerts a significant impact on the pattern of protein expression by the alveolar macrophage under basal or unstimulated conditions to make the alveolar macrophage ready and competent to subsequently mount a successful innate host defense response in response to various injurious agents. Towards this end we employed two-dimensional difference gel electrophoresis (2D-DIGE) to study The cells from all BAL samples were subjected to total and differential cell counts to exclude any mice with underlying infectious or inflammatory processes. No evidence of inflammation or infection was seen in any of the mice. The total cell counts did not differ significantly from one another and all BAL samples consisted of > 95% macrophages (data not shown).Protein content was the same in all samples (data not shown) and identical amounts of protein (20 μg) were loaded on all gels.Following automatic spot detection and manual editing, 791 protein spots were defined and matched across 2D-DIGE gels from all samples and then subjected to further analysis. A reference gel is shown in Figure When principal component analysis (PCA) was performed on the significantly different spots, the markers for the four gels  were grouped fairly tightly, and each of the experimental groups was well separated from one another Figure . The firA dynamic picture of the changes in expression is afforded by the heat diagram in Figure In Figure Figure Comparison of the KO baseline mice to the KO 6 hr animals (Panel A) revealed 113 significant changes (41 increases and 72 decreases) accounting for 14.3% (5.2% increases and 9.1% increases) of the resolved protein. The largest number of significant changes is seen when KO baseline mice are compared to KO 18 hr. There are 233 significant changes with approximately 2/3 being proteins that are at higher levels in the KO mice (Panel B). The 233 protein spots make up 29.5% of the resolved protein on the gel. Of these, 78 are proteins expressed at increased levels and 155 are proteins expressed at decreased levels in the KO 18 hr mice compared to the KO baseline mice. These proteins make up 9.9% and 19.6% of the resolved protein, respectively. When the KO to KO 18 hr gel and the KO to WT gel are compared, the pattern of changes is similar, suggesting that many of the changes in protein expression resulting from the exogenous SP-A treatment are restoring protein levels to those approximating the levels characterizing the WT phenotype. This trend can be further seen when the WT is compared to the KO 18 hr (Panel D). The number of significant changes is markedly reduced, confirming the similarity of these two groups. In this comparison there are only 88 protein spots that differ significantly, making up 10.8% of total resolved protein. Panels A and D each depict a lower number of changes compared to Panels B and C, indicating that the protein expression profiles, in terms of the magnitude and number of changing spots, are closer to KO (after 6 hr of SP-A treatment) and WT (after 18 hr of SP-A treatment) profiles, respectively. This can be better appreciated in Figure In Additional File We used different approaches as done previously ,42,46 wiOther major processes implicated by our protein list included regulation of inflammation (19 proteins) Table , proteaset al.) the presence of an ARE in their genes has been documented, confirming this potential regulatory mechanism. However, many other proteins that we identified have functions such as those listed above that are typically viewed as Nrf2-regulated. Of significant relevance, Nrf2/Keap1 activity can be regulated by interaction with actin filaments [In addition to the functional categories described above, 21 of the identified proteins (~15-20) have been reported to be regulated by Nrf2. All of these have been reported to either contain antioxidant response elements (ARE) in their genes, or have been demonstrated to be Nrf2-regulated. We did not include proteins that may interact with Nrf2, but are not regulated by it. Nrf2, and its inhibitor, Keap1, are instrumental in regulating proteins that contain an ARE in their promoter regions and have been shown to regulate many antioxidant proteins, a number of cytoprotective enzymes (including Phase 2 detoxifying enzymes), as well as a number of heat shock proteins/chaperones. These proteins help maintain redox balance in the lung by limiting the effects of exogenous and endogenous reactive oxidant species. A number of the identified proteins fall into these categories. In some cases  reduced to approximately 1/2 of that seen in the control cells.Given the relatively large number of actin-related/cytoskeleton proteins that changed in response to SP-A, as "proof of principle" we measured cell size and levels of F-actin. In macrophages that were treated with SP-A for 6 hr and stained with Alexa Fluor 488 phalloidon we examined cell area Figure  and the Macrophages treated with SP-A for 1 hr and their controls Figure , the celin vivo, the impact of SP-A on global protein expression by the alveolar macrophage as a way of gaining insight into molecules and mechanisms affected by SP-A under basal conditions. We thus studied and compared the intracellular proteome of the alveolar macrophage from WT mice and SP-A KO mice, and SP-A KO mice "rescued" with exogenous SP-A and sacrificed at 6 hr, a time point we have previously shown to be sufficient for the same dose of exogenous SP-A to restore tubular myelin formation in KO mice [in vivo treatment of KO mice with SP-A exerted a significant impact on the expression of proteins by alveolar macrophages compared to untreated KO mice; 3) as the time after SP-A treatment of KO mice increased, the proteomic profile more closely approximated that of WT mice; 4) a diverse set of SP-A-dependent functions in the alveolar macrophage, some of which have not been identified in vivo previously; including proteins involved in the regulation of the cytoskeleton; and 5) changes in F-actin and cell size of the alveolar macrophage in response to SP-A.The absence of SP-A in KO mice has been shown to increase susceptibility to infection, compromise bacterial clearance and survival from pneumonia, and adversely affect the response to various other types of lung injury -42,120.  KO mice , and 18 K. pneumoniae in the presence or absence of ozone-induced oxidative stress [In previous studies of BAL proteome analysis, SP-A was found, under both resting conditions and after various perturbations , to have a major impact on BAL protein expression, particularly with respect to proteins involved in host defense or the regulation of redox balance ,42. Prote stress ,28. The The SP-A dose and time points, as noted above, were based on a study where using the same conditions we were able to restore tubular myelin to the lungs of KO mice . MoreoveThe "restoration" of the global pattern of protein synthesis in KO macrophages to that resembling WT macrophages, when exogenous SP-A was given, was remarkable. In 59 out of the 76 (78%) identified proteins the change seen, compared to KO at 18 hr after exogenous SP-A, was consistent with the differences between KO and WT. In other words, when WT levels were lower than KO, the 18 hr SP-A value was lower than KO and the opposite trend was apparent when WT levels were greater than KO. However, this transition from KO towards WT in response to SP-A treatment was complex, because levels of some proteins (~1/3) increased and others (~2/3) decreased in WT macrophages. This raised the possibility that with different proteins SP-A may influence protein synthesis, metabolism, and/or secretion by macrophages to achieve the intracellular levels appropriate for baseline levels in the macrophage.Changes in the proteome of the treated versus untreated mice could be the result of several different regulatory mechanisms. SP-A may act directly on the alveolar macrophage, through different receptors to which it is known to bind ,126-128,An important indirect factor in terms of SP-A regulation of gene expression becomes apparent when we consider one of our previous studies of the BAL proteome of KO mice in which we postulated increased oxidative stress in the absence of SP-A . In the The most common phenotypic trait that has been examined with the SP-A KO mice is increased susceptibility to pneumonia. The simplest explanation for this phenotype is that host defense against specific pathogens is compromised in the absence of SP-A and its opsonic function. Indeed, replacement of SP-A in these models has afforded some restoration of normal host defense function by apparently increasing pathogen clearance ,130. Addin vivo treatment with SP-A under unstimulated, baseline conditions affected the levels of significantly more and diverse macrophage proteins than previously known. These include proteins involved in macrophage motility, phagocytosis, neutralization of reactive oxidant species and other toxins, removal of damaged tissue/proteins from the alveolus, and other functions. These effects begin to be evident by as early as 6 hr after exogenous SP-A treatment. By 18 hr after intratracheal SP-A treatment, the alveolar macrophage proteome of the KO mouse becomes strikingly similar to that of WT animals that have been continually and naturally exposed to SP-A. The diversity of changed proteins broadly indicates that SP-A, under basal conditions, plays a major role in alveolar macrophage "health" in terms of its potential ability to move about and its "readiness" for pathogen elimination and tissue repair.SP-A has previously been shown to exert regulatory effects on the production of several macrophage proteins -21,125. In vitro studies have shown that an intact actin cytoskeleton, as assessed by the use of cytochalasin D, an actin depolymerizing agent, is required for certain SP-A-mediated processes [in vivo is remarkable. Significant changes in protein content with a similar pattern were also seen in several proteins involved in the inflammatory regulation (n = 4) and protease/chaperone (n = 5) functional groups.The responses identified exhibited several consistent patterns of protein expression. In one pattern the KO mice and WT mice differed significantly from one another (both increases and decreases) and intermediate responses proceeding in the same direction were observed at the two SP-A replacement time points. This pattern was observed in 17 of the 20 proteins in which KO and WT differed significantly  differed significantly between KO mice and mice that had received SP-A 18 hr earlier, even though no significant differences were observed between KO and WT mice. For those proteins however, in most cases the trend in the WT usually resembled that of the effect seen 18 hr after SP-A. This subset of responses supports a scenario where a robust acute response to SP-A is observed, with some of the proteins exhibiting an overcompensation of expression. For example, a number of protein spots Figure  are incrAlthough statistical techniques such as gene set enrichment analysis or hypergeometric distribution calculations have been used with microarray data to determine whether changes in subsets of genes differ significantly from a reference group or the overall population in a specific group, these cannot be used here. This is because of the relatively small data set examined by gel-based proteomics and the comparatively large numbers of proteins in our subsets that make up a substantial percentage of our overall population or reference group. For example 50% of the identified proteins in our reference group were in our actin-related group and 28% were included in the Nrf2-related proteins, thereby violating the assumption that the analyzed groups be independent of one another and precluding the use of such methods in this study ,140.in vitro were significantly larger than comparable control cells and had lower amounts of F-actin, both at the 6 hr time point used for this proteomic study  and at 1 hr, a time point where we have previously demonstrated an SP-A-dependent enhancement of phagocytosis by alveolar macrophages [in vivo comparisons of SP-A KO and WT mice in a model of pneumonia induced by Klebsiella pneumoniae [in vitro support this scenario [The large number of actin-related proteins with altered levels of expression after SP-A treatment led us to speculate that we would be able to validate the proteomic findings with a preliminary examination of parameters related to actin cytoskeletal dynamics. In fact, macrophages from SP-A KO mice treated with SP-A rophages . As SP-Aeumoniae  and on pscenario .This study was designed to allow us to study restoration of the WT phenotype from KO alveolar macrophages after giving a single dose of SP-A and studying its effect 6 hr and 18 hr later. Remarkably, the dose and sampling times chosen restored levels of many of the proteins we studied. It is likely that additional doses and time points will further restore the phenotype by regulating the expression of additional proteins with different kinetics of translation and secretion. It is also highly likely that SP-A regulation by continually present SP-A (as in the WT state) or by multiple doses of SP-A will differ somewhat from the pattern resulting from a single acute dose, because in those cases a steady-state equilibrium of the SP-A effects on the alveolar macrophage proteome may be attained. Moreover, we had previously proposed that SP-A "primes" the alveolar macrophage  to prepain vivo treatment with SP-A affects, under basal or unstimulated conditions, the expression of diverse groups of proteins in the alveolar macrophage. The group of actin-related/cytoskeletal proteins appeared to be a prominent one, indicating a significant in vivo role of SP-A in alveolar macrophage motility and related functions. This was further substantiated by experiments showing significant changes in cell size and cortical F-actin distribution 1 and 6 hr after SP-A treatment. Furthermore, a significant number of the changed proteins are likely to be Nrf2-regulated indicating a role of SP-A in oxidative stress. We postulate that the SP-A-mediated expression profile of the alveolar macrophage places it in a state of "readiness" to successfully combat various injurious insults. Published comparison studies of SP-A KO and WT mice that showed a poorer outcome in KO in response to various injurious agents support the concept of "readiness." Therefore, SP-A appears to be a critical factor for the "priming" and/or "readiness" of the alveolar macrophage to carry out its innate immune functions effectively and ensure lung health.The observations in the present study show for the first time that an acute This study was conducted using pathogen-free male WT and SP-A KO male mice on the C57BL/6 genetic background. WT mice were obtained from Jackson Laboratories . Breeder pairs of SP-A KO mice had been obtained from Dr. Samuel Hawgood at the University of California, San Francisco and were propagated and raised under specific pathogen-free conditions in a barrier facility at the Penn State College of Medicine . The SP-A total of 16, 25-34 g C57BL/6 WT and SP-A KO mice were used to complete the proteomics study. These were divided into four groups with 4 animals per group: 1) SP-A KO control (baseline) mice that did not receive any treatment; 2) SP-A KO mice that were treated with SP-A and sacrificed 6 hr after treatment; 3) SP-A KO mice that were treated with SP-A and sacrificed 18 hr after SP-A treatment; and 4) WT control (baseline) mice that did not receive any treatment.SP-A was purified from the BAL fluid from normal human lungs obtained from organ donors. The protocol was approved by the Penn State College of Medicine Institutional Review Board. Donor lungs were lavaged with 0.9% saline and the lavage fluid collected and centrifuged at 150 × g for 10 min at 4°C to obtain cell-free BAL. The SP-A was then purified by repeated precipitation with 5 mM calcium chloride. The purity of the preparation was checked by 1D-PAGE with silver stain and by Western blot and determined to be greater than 99 percent pure. LPS content of the SP-A preparation was measured with the QCL-1000 Limulus Amebocyte Lysate assay . The LPS content of a 1 μg sample of SP-A was below the detectable limit of the assay (0.1 EU/ml) or < 500 fg/μg of SP-A.Animals were anesthetized with an intramuscular injection of a mixture of Ketamine HCl  and Xylazine . Mice were suspended from their incisors and intrapharyngeally instilled with 5 μg of normal human SP-A in 50 μL of 0.9% sodium chloride containing 2 mM calcium chloride. Mice were closely watched to ensure that the entire dose was aspirated. We selected a dose of 5 μg of SP-A/mouse based on our SP-A determinations of total BAL SP-A from C57BL/6 mice . We have previously demonstrated that using this dose of SP-A and a 6 hr time point, we were able to detect tubular myelin in the BAL of SP-A KO mice .Mice were anesthetized and subjected to BAL at intervals of 6 hr and 18 hr following treatment with SP-A. We chose 6 hr as our initial time point because we used it successfully in a previous study examining tubular myelin formation . In addiFrozen macrophage pellets were lyophilized until complete dryness and resuspended in 25 μL of standard cell lysis buffer . Protein determinations were done using the Bio-Rad Protein Assay  and the concentration of protein was adjusted to 1 mg/ml for CyDye labeling.http://www.psidev.info/miape/MIAPE_GE_1_4.pdf >) by the Human Proteome Organization Proteomics Standards Initiative  gel an aliquot of 450 μg of sample was diluted with an equal volume of 2X sample buffer  and 1.2% DeStreak reagent) and then brought up to a volume of 450 μl with rehydration buffer (DeStreak™ Rehydration Solution and 0.5% IPG buffer (pH 4-7)). Proteins were focused using the following voltages and times: 14 hr at 0 V (passive rehydration); 6 hr at 30 V (active rehydration); 3 hr at 300 V (step and hold); 3 hr at 600 V (gradient); 3 hr at 1000 V (gradient); 3 hr at 8000 V (gradient); 4 hr at 8000 V (step and hold). The strip was then equilibrated as described above and applied to a 10% polyacrylamide gel (26 cm-w × 20 cm-h × 1 mm thick). For the preparative picking gel a single plate for the gel plate sandwich was treated with Bind-Silane solution  and had reference marker stickers placed on it. After the completion of electrophoresis, the plate that had not been silane-treated was removed from the sandwich and the gel fixed overnight with 30% ethanol, 7.5% glacial acetic acid. The preparative picking gel was then stained with Deep Purple Total Protein Stain  for 2 hr.http://www.psidev.info/files/miape-gi-v1.pdf  currently under development by the Human Proteome Organization Proteomics Standards Initiative Gel images were imported into the Progenesis SameSpots v4.0 program (Nonlinear Dynamics) for analysis and their quality assessed by the program. Image quality control with Progenesis SameSpots v4.0 involves checking images for bit depth, color, manipulation prior to analysis, proper file type, saturation, low dynamic range, and stretched contrast. A reference gel with minimum distortion and streaks was selected from the Cy2 gels. Gel alignment was conducted automatically and then checked manually to ensure correct alignment. Spot detection and spot matching across all the gels were conducted automatically, and then spot matching was checked and manually edited to ensure correct matching. Data from all the spots included in analysis were transported to Progenesis PG240 module of the Progenesis SameSpots v4.0 software for further analysis. Statistical analysis was performed by ANOVA and by t-test to confirm the level of significance among various groups. Differences were considered statistically significant with p < 0.05. Principal components analysis (PCA) was performed and dendrograms plotted in the Statistics module of Progenesis SameSpots v4.0 for the further analysis of the patterns of protein expression and protein clustering among groups.For identified proteins having multiple isoforms, the normalized volumes of all isoforms of a given protein were added together and statistical analysis was performed on the totals using Microsoft Excel.2O for 30 min at 37°C. All of the extracts for each plug were pooled and then dried by lyophilization. Dried extracts were then brought up in 200 μl of dH2O and dried by lyophilization for a series of three cycles to remove any remaining ammonium bicarbonate. The final dried extracted proteins/peptides were brought up in 20 μl of 0.5% TFA and desalted and concentrated using C18 ZipTips . Tips were wetted with 10 μl of 100% acetonitrile and equilibrated with 10 μl 0.1% TFA (pH < 4). Samples were then drawn into ZipTip columns by aspirating for 10 cycles and then washed twice with 10 μl 0.1% TFA. Peptides were then eluted from the column twice with 10 μl of 50% acetonitrile, 0.1% TFA. The ZipTip procedure was performed four times for each extract to maximize peptide yield and the elutions were pooled, lyophilized and resuspended in 2 μl of 50% Acetonitrile, 1% TFA for MALDI plate spotting.We have used this procedure in previous studies for other types of protein samples ,46,143 bPeptides were analyzed by MALDI-ToF/ToF mass spectrometry  in the Mass Spectrometry Core at the Penn State University College of Medicine. All of each ZipTip cleaned samples (1 μl at a time) was applied onto a 384-well MALDI plate  and then 0.7 μl of 2 mg/ml ACH cinnamic acid in 60:40 (acetonitrile: water) was spotted on each well containing peptide. All 13-calibration wells on the MALDI plate were spotted with (1:12 diluted) 4700 calibrant. Autolytic trypsin peptides were also used to internally calibrate the spectra to an accuracy of 20 ppm. Using the GPS Explorer 3.0 software (Applied Biosystems), the MS and MS/MS data were submitted to the MASCOT search engine using the NCBI non-redundant database and mouse taxonomy for identification. The search parameters included: trypsin digestion with a maximum of three missed cleavages; fixed modifications, carbamidomethylation; variable modifications, carbamylation, acetylation, deamidation, oxidation; peptide mass tolerance, 0.15 Da. MASCOT confidence interval scores of > 95% combined with a ProteinPilot score of greater than 61 were considered as a positive protein identification. An image of the reference gel is shown in Figure 5/mL and 1 mL (105 AMs) plated to cover glasses  in 12-well tissue culture plates and allowed to adhere for 90 min at 37°C. The medium was then removed and replaced with 1 mL of fresh AM medium and the cells were cultured overnight at 37°C. AMs were treated with SP-A for either one or six hours by adding 5 μL (5 μg) of a 1 μg/μL human SP-A stock solution in 10 mM Hepes (pH 7.5) directly to the 1 mL of medium from the overnight culture.AMs were collected from the BAL fluid of male KO mice as described earlier. AMs were washed 1X with AM medium (RPMI-Hepes modification containing 2 mM L-glutamine and 1:100 antibiotic antimycotic solution). AMs were then resuspended in AM medium at 1 × 10After incubation with SP-A, AMs were washed 1X with PBS at 37°C, fixed with 3.7% paraformaldehyde in PBS for 10 min at 37°C and permeabilized with 0.5% Triton in PBS for 5 min. After permeabilization, cells were washed with PBS, incubated for 30 min in staining solution  and again washed with PBS 3X for 10 min. Cover glasses containing the AMs were mounted to slides using PBS containing 80% glycerol and 0.3% n-propyl gallate, and then sealed using nail polish.AMs were visualized using a Nikon TE-2000 PFS fluorescent microscope with a 60X (1.4 NA) phase contrast lens with a 1.5× tube lens in place and the images captured using a Photometrics Coolsnap HQ2 digital camera (0.07 μm/pixel) and saved as TIFF files. Exposure time (200 ms) was kept constant for all images acquired. Nikon NIS-Elements AR (version 3.0) software was used to do both the image acquisition and data analysis. AMs were analyzed by manually drawing a border for each cell as well as copying an area of equal size to a location immediately adjacent to each cell to be used for background subtraction. Data were then collected for area (cell size), mean fluorescence intensity per pixel of cell area, and the sum of the fluorescence intensity for all of the pixels within a cell. Statistical analysis was performed using Minitab and Excel.The authors declare that they have no competing interests.DSP and JF designed the study. DSP interpreted data and prepared the manuscript. JF assisted with data interpretation, and participated in manuscript preparation. TMU treated mice, collected samples, ran gels, prepared mass spec samples, evaluated mass spec data, did preliminary analysis of proteomic data, and participated in the writing of the manuscript. OAQ prepared the cell samples for the microscopy experiments, collected images, and performed the image analyses. CMY participated in design of actin experiments and interpreted actin data. All authors read and approved the final manuscript.MIAPE: Gel Electrophoresis. File containing Minimum Information About a Proteomics Experiment-Gel Electrophoresis in the format recommended by the Human Proteome Organization Proteomic Standards Initiative.Click here for fileMIAPE: Gel Informatics. File containing Minimum Information About a Proteomics Experiment-Gel Informatics in the format recommended by the Human Proteome Organization Proteomic Standards Initiative.Click here for fileProtein names and cross references to accession numbers and categories. File containing a table that has the gel numbers and names of all identified proteins. It contains both NCBI GI numbers and Swiss Prot Accession numbers for all proteins, as well as the designation for the functional group(s) to which each protein was assigned. Reference numbers listed in the table are from the reference list in the manuscript.Click here for fileValues for all identified alveolar macrophage proteins with note of significant changes. File containing a table that gives normalized volumes for all proteins for each individual group +/- SD and indicates comparisons between groups that were significantly different.Click here for file


The organ of Corti and the vestibule in inner ears also suffer from degeneration caused by environmental stresses including noise and heavy metals, resulting in impairments of hearing and balance. In this review, we introduce impairments of hearing and balance caused by genetic and environmental factors and focus on impairments of SGNs and the vestibule in inner ears as the pathogeneses caused by these factors.Impairments of hearing and balance are major problems in the field of occupational and environmental health. Such impairments have previously been reported to be caused by genetic and environmental factors. However, their mechanisms have not been fully clarified. On the other hand, the inner ear contains spiral ganglion neurons (SGNs) in the organ of Corti, which serve as the primary carriers of auditory information from sensory cells to the auditory cortex in the cerebrum. Inner ears also contain a vestibule in the vicinity of the organ of Corti—one of the organs responsible for balance. Thus, inner ears could be a good target to clarify the pathogeneses of sensorineural hearing losses and impaired balance. In our previous studies with  It has been reported that about 250 million people worldwide suffer from hearing losses. About 30 % of people with congenital hearing loss are syndromic and the remaining 70 % are non-syndromic. In addition, most elderly people develop age-related (late-onset) hearing loss –3. In geInner ears have been analyzed in order to clarify the pathogeneses of sensorineural hearing losses. The inner ears contain the organ of Corti and stria vascularis (SV). The SV is essential for maintenance of endolymph potential. The organ of Corti contains two kinds of sensory cells [inner hair cells (IHCs) and outer hair cells (OHCs)] and plays an important role in mechanotransduction, by which sound stimuli are converted into electric stimuli. Auditory information from the sensory cells is transferred to spiral ganglion neurons (SGNs) as the primary carriers and is eventually transferred to the auditory cortex in the cerebrum , 2. The Ret and Ednrb) and by environmental stresses [low frequency noise (LFN) and heavy metals].Impairments of hearing and balance—both major problems in the field of occupational and environmental health—are caused by the intricate interplay of genetic, aging and environmental factors –3. HowevRET has been shown to be essential for the development and maintenance of the enteric nervous system (ENS)  mice [Ednrb/EDNRB cause syndromic hearing loss due to congenital defects of melanocytes in the stria vascularis of the inner ear [Ednrb homozygously deleted mice [Ednrb(−/−)-mice] developed congenital hearing loss , which is caused by mutations in the transcription factor Sox10 , cytokin mutated  and Ednr−)] mice  have beenner ear , 32–35. c-Ret-KIY1062F/Y1062F-mice developed severe congenital deafness with neurodegeneration of SGNs on postnatal day (P) 8-18, while c-Ret-KIY1062F/Y1062F-mice showed morphology of SGNs comparable to that in WT mice on P2-3 [c-Ret-KIY1062F/Y1062F-mice developed normally at least until P3 after birth, when Y1062 in c-Ret of SGNs from WT mice is unphospholylated. However, in c-Ret-KIY1062F/Y1062F-mice, phosphorylation of Y1062 is no longer maintained by P8–P18, when Y1062 in c-Ret of SGNs from WT mice exhibits significant phosphorylation [Ednrb(−/−)-mice with congenital hearing loss showed a decreased number of SGNs -mice did not show typical apoptotic signals and did not involve disturbance of hair bundles of IHCs and OHCs [Ednrb(−/−)-mice were improved markedly by introducing an Ednrb transgene under the control of the dopamine beta-hydroxylase promoter (Ednrb(−/−); DBH-Ednrb-mice). Neurodegeneration of SGNs was restored by introducing constitutively activated RET also in the case of c-Ret-mediated hearing loss. Thus, our results indicate that c-RET and EDNRB expressed in SGNs could be molecular targets in the prevention of hearing impairments.Phosphorylation of Y1062 in c-Ret has been shown to mediate several biological responses, including development and survival of neuronal cells , 37. In  on P2-3 . Phoshor on P2-3 . Thus, irylation . Furtherrylation . On the and OHCs , 36. TheExposure to noise is recognized as one of the major environmental factors causing hearing loss . Noise cOur studies provide direct evidence that c-RET and EDNRB expressed in SGNs are novel targets for hearing loss. These studies underline the importance of considering the activity as well as the expression of the target molecule in order to elucidate the etiologies of hereditary deafness. In addition, environmental stresses, including exposure to noise and heavy metals, can cause impairments of hearing and balance that are affected intricately by aging and genetic factors. Information obtained in previous studies prompts further investigation of the influence of environmental stresses on the impairment of hearing and balance with consideration of aging and genetic factors to develop new diagnostic, preventive and therapeutic strategies against impairment of hearing and balance.


Cortical areas involved in sensory analysis are also believed to be involved in short-term storage of that sensory information. Here we investigated whether transcranial magnetic stimulation (TMS) can reveal the content of visual short-term memory (VSTM) by bringing this information to visual awareness. Subjects were presented with two random-dot displays (moving either to the left or to the right) and they were required to maintain one of these in VSTM. In Experiment 1, TMS was applied over the motion-selective area V5/MT+ above phosphene threshold during the maintenance phase. The reported phosphene contained motion features of the memory item, when the phosphene spatially overlapped with memory item. Specifically, phosphene motion was enhanced when the memory item moved in the same direction as the subjects' V5/MT+ baseline phosphene, whereas it was reduced when the motion direction of the memory item was incongruent with that of the baseline V5/MT+ phosphene. There was no effect on phosphene reports when there was no spatial overlap between the phosphene and the memory item. In Experiment 2, VSTM maintenance did not influence the appearance of phosphenes induced from the lateral occipital region. These interactions between VSTM maintenance and phosphene appearance demonstrate that activity in V5/MT+ reflects the motion qualities of items maintained in VSTM. Furthermore, these results also demonstrate that information in VSTM can modulate the pattern of visual activation reaching awareness, providing evidence for the view that overlapping neuronal populations are involved in conscious visual perception and VSTM. Visual short-term memory (VSTM) is a major process subserved by working memory see . In VSTMThe emerging view is that working memory is supported by the coordinated recruitment of brain systems that are involved in processing sensory input and in action-related functions  were recruited for the experiment. An inclusion criterion was the ability to perceive moving phosphenes and three subjects were excluded for this reason. All subjects gave informed consent before participating in the study which had been approved by the local ethics committee and were treated in accordance with the declaration of Helsinki.TMS was delivered by means of two Magstim Super Rapid machines  via 70 mm figure-of-eight-shaped coils. The sites for stimulation were located using a functional method typically used in studies investigating phosphenes  and whicThe motion stimuli (presented with a 17-inch (800 × 600) CRT monitor) appeared within an imaginary square that subtended 10° by 10° of visual angle at a viewing distance of approximately 57 cm. The stimuli consisted of 200 white dots (with a size of 1 pixel each), placed at random positions on a black background. The dots moved at a speed of either 2 or 3 pixels per frame (frame duration: 16 ms). The motion direction was either to the left or right. The stimuli were presented in the lower quadrant of either left or right hemifield, with center of the stimulus at an eccentricity of 10° of visual angle from both the vertical and horizontal meridian .The motion stimuli were followed by a display (1000 ms) indicating whether the subject should hold in their memory the speed and direction of the first stimulus (indicated by the number “1”), the second stimulus (“2”) or no stimulus (“0”). After a 3-s delay , a TMS pulse was applied either over the left or right V5/MT+ . After the TMS pulse, subjects were asked to verbally provide (within a 2-s response period), a rating of phosphene appearance using the phosphene scale of There were a total of 128 “Maintenance and TMS” trials, i.e. trials on which subjects were asked to maintain a motion stimulus , and on which TMS was applied over either the left or right V5/MT+ . Whether the motion stimuli at the start of the trial appeared in the left or right lower quadrant, and whether subjects were asked to maintain the first or second motion stimulus in VSTM, was counterbalanced. There were a total of 64 “spatially congruent”  and 64 “spatially incongruent”  TMS trials.after the presentation of the two motion stimuli that no maintenance was required (and that the VSTM task would not be performed). The maintenance cue was presented after stimulus presentation to ensure that there would be no fundamental differences in the encoding of the target stimulus in the “Maintenance” and “No maintenance” trials.There were a total of 64 “No maintenance and TMS” trials. On these trials, subjects were informed by a cue There were a total of 64 “Baseline phosphene” trials on which no visual motion stimuli were presented before phosphene induction. These trials were used as the baseline measure for subjects' phosphene.There were a total of 32 “Maintenance and No TMS” trials on which subjects performed the VSTM task but no TMS was applied. This condition was included in order to be able to determine the possible impact of TMS on the VSTM task.At the start of the session, subjects performed a block of 32 trials on the VSTM task without any TMS to obtain a measure of their performance without TMS . The left and right V5/MT+ were then localised. At this stage, three subjects who could not perceive moving phosphenes were excluded from the study. The 64 “baseline phosphene” trials were carried out in two blocks of 32 trials . The rest of the conditions were run in eight blocks of 24 trials. All the blocks contained 16 “Maintenance and TMS” trials and 8 “No maintenance and TMS” trials. The trials were counterbalanced in such a way that in each block, on half of the trials the left V5/MT+ was stimulated and on the other half the right V5/MT+ was stimulated. The location of the motion stimulus (i.e. whether it appeared on the left or right lower quadrant) was also counterbalanced.A further experiment was performed to investigate whether any impact of VSTM maintenance on phosphene perception is restricted to phosphenes induced from the motion-selective V5/MT+ region, or whether phosphenes induced from regions which are not motion- selective are also affected, possibly reflecting a general effect of VSTM maintenance on visual perception. This possibility was tested by examining the impact of VSTM maintenance on phosphenes induced from the left and right lateral occipital cortex, a region associated with object processing  e.g. .As previously done by At first we examined whether the mere presentation of motion stimuli (without any maintenance) affected subjects' phosphene judgments. This was accomplished by comparing phosphene ratings in the “No maintenance and TMS” condition  to phosphene ratings in the “baseline phosphene” condition .t(8) = 0.43; p = 0.68) or whether the phosphene appeared in the other hemifield (t(8) = 0.41; p = 0.69). The mean phosphene ratings were as follows: Baseline phosphene, 2.09; Phosphene location spatially overlapping with the memory item, 2.14; and Phosphene location not spatially overlapping with the memory item, 2.12.This analysis revealed no significant differences, regardless of whether the phosphene appeared in the same spatial location as the memory item ; and b) whether the memory item spatially overlapped with the phosphene location. The “maintenance and TMS” trials were thus divided into four types:1)Memory item motion direction congruent with baseline phosphene; spatial overlap.2)Memory item motion direction congruent with baseline phosphene; no spatial overlap.3)Memory item motion direction incongruent with baseline phosphene; spatial overlap.4)Memory item motion direction incongruent with baseline phosphene; no spatial overlap.For trials involving VSTM maintenance, only trials on which subjects performed correctly in the speed discrimination task were included in the analysis. The maintenance conditions were divided into the following categories as a function of (a) whether the motion direction of the memory item was congruent with the direction of phosphene motion  = 18.93 p = 0.0001). Pairwise comparisons revealed that, relative to the baseline condition, phosphene ratings were affected when the memory item and the phosphene spatially overlapped. Specifically, in the memory item/phosphene spatial overlapping condition, there was a decrease in phosphene rating relative to the baseline measure (i.e. phosphene ratings in the control runs) when the motion direction of the memory item was incongruent with the direction of phosphene motion (t(8) = 4.39; p = 0.002). This indicates that subjects reported their phosphene to contain motion less frequently than was the case in the baseline condition. In contrast, there was an increase in the phosphene rating when the direction was congruent with the direction of phosphene motion (t(8) = 3.95; p = 0.004). This in turn indicates that the subjects reported their phosphene to contain motion more frequently than in the baseline condition.Direction congruent; no spatial overlap: t(8) = 0.36; p = 0.72; Direction incongruent; no spatial overlap: t(8) = 0.66; p = 0.53). When there was no spatial overlap between the memory item and the phosphene, subjective ratings were not significantly different from the baseline condition  in each of the four trial types (described on page 9) were Trial type 1: 2; Trial type 2: 3.3; Trial type 3: 2.9; and Trial type 4: 3.6. In the control condition, in which subjects did not engage in maintenance, there were on average 2.9 of such trials.t(8) = 1.45; p = 0.18).Direction congruent, no spatial overlap: 84%; Direction incongruent, no spatial overlap: 87%; Direction congruent, spatial overlap: 85%; and Direction incongruent, spatial overlap: 79%. Subjects' mean accuracy in the VSTM task in the absence of TMS (No TMS condition) was 89%. A repeated-measures ANOVA with trial type as the main factor revealed a significant main effect (F(4) = 3.01; p = 0.03). Pairwise comparisons indicated that, relative to the No TMS condition, VSTM performance was impaired in the “Direction incongruent; spatial overlap” condition (t(8) = 2.75; p = 0.025). Furthermore, performance in the “Direction congruent; spatial overlap” condition was significantly higher than in the “Direction incongruent; spatial overlap” condition (t(8) = 3.25; p = 0.011). Other comparisons were not statistically significant.The mean accuracies in the VSTM task as a function of trial type were As in the analysis of Experiment 1, at first we examined whether the mere presentation of motion stimuli (without any maintenance) affected subjects' phosphene judgments. This was accomplished by comparing phosphene ratings in the “No maintenance and TMS” condition  to phosphene ratings in the “baseline phosphene” condition .t(8) = 0.19; p = 0.85) or whether the phosphene appeared in the other hemifield (t(8) = 0.46; p = 0.66). The mean phosphene ratings were as follows: Baseline phosphene, 0.97; Phosphene location spatially overlapping with the memory item, 0.98; and Phosphene location not spatially overlapping with the memory item, 0.99.For LO phosphenes, this analysis revealed no significant differences between the conditions, regardless of whether the phosphene appeared in the same spatial location as the memory item (1)Spatial overlap between the phosphene and the memory item.2)No Spatial overlap between the phosphene and the memory item.For trials involving VSTM maintenance, only trials on which subjects performed correctly in the speed discrimination task were included in the analysis. The maintenance conditions were divided into two categories as a function of whether or not the phosphene spatially overlapped with the memory item. Because LO baseline phosphenes do not contain motion, the trials could not be divided as a function of whether the motion component of the baseline phosphene was congruent or incongruent with the motion direction of the memory item. There were thus two trial types:F(2) = 0.16; p = 0.85), indicating that LO phosphene were not influenced by the motion qualities of the memory item.F(2) = 1.21; p = 0.32).The mean accuracies in the VSTM task as a function of trial type were as follows: spatial overlap, 88%; no spatial overlap, 86%. Subjects' performance in the VSTM task in the absence of TMS was 91%. A repeated-measures ANOVA found no significant differences between the conditions ; in contrast, phosphene motion was weakened when the motion in the memory item was to the opposite direction as what was contained in the baseline V5/MT+ phosphene (i.e. motion towards the fovea). The nature of this maintenance/phosphene appearance interaction demonstrates that V5/MT+ is involved in the maintenance of motion information. Importantly, these interactions only occurred when the phosphene spatially overlapped with the memory item, demonstrating that the VSTM holds information in a retinotopic fashion.In Experiment 2, phosphenes induced from the lateral occipital region were not affected by phosphene motion even when there was a spatial overlap between the phosphene and the memory item. This is an expected finding, given the neuroimaging evidence that motion processing does not specifically engage the LO region unless the motion represents a cue for object recognition . The lacOur findings are consistent with the sensory recruitment hypothesis, according to which the neural mechanisms that are involved in the short-term storage of visual information are also closely associated with those areas that are involved in its sensory analysis . Recent active maintenance of visual information whereas this is not the case in the visual priming paradigms previously used in conjunction with TMS. Recent fMRI evidence suggests that this difference is important: in an fMRI study by That TMS seems to preferentially activate neurons engaged in VSTM maintenance (and facilitate VSTM performance, cf. The shape, motion and intensity of TMS-induced phosphenes have been used to investigate questions such as the role of feedback connections in visual awareness , neural In summary, our results demonstrate that V5/MT+ activity reflects the motion attributes of stimuli held in VSTM. Furthermore, that TMS could bring these motion attributes to conscious perception indicates that VSTM and visual awareness rely on the same neuronal representations.


To the Editor: Recent reports from Afghanistan have focused on the current status of war- and refugee-related anthroponotic cutaneous leishmaniasis (ACL), caused by Leishmania tropica, in Kabul City, refugee camps, and Fayzabad City (Badakhshan Province) , caused by Leishmania parasites in Giemsa-stained smears obtained directly from skin lesions. Although sporadically confirmed by culture and PCR, ZCL and ACL are usually differentiated by specific clinical aspects, especially of skin lesions: dry lesions characterize urban ACL; wet lesions, often superinfected and disseminating, characterize rural ZCL.Regional epidemiologic reports, when available from Afghan or international health authorities, usually mention cutaneous leishmaniasis cases without further elaboration. To evaluate the current threat and specific epidemiology of cutaneous leishmaniasis in Mazar-e Sharif, medical entomologic and epidemiologic field investigations were conducted in June and October 2005. Results show that ZCL, ACL, and visceral leishmaniasis (VL) are endemic to Mazar-e Sharif. Data from patients seeking treatment from March 21, 2004, through March 20, 2005, at the Balkh Province Leishmaniasis Center, Mazar-e Sharif, showed that of 3,958 cases, 3,782 (95.5%) were ZCL, 174 (4.4%) were ACL, and 2 (0.05%) were VL; the number of unreported cases during this time is unknown. A sharp increase (4.4- to 5.4-fold) in ZCL cases was found when data from July 21 through August 20 and from August 21 through September 20, 2004,  were compared with data for the same periods in 2005 . ZCL chiefly affects farmers, nomads, and refugees. By sex, cases occurred in 1,991 (52.6%) male and 1,791 (47.4%) female patients. By age group, the rate for ZCL in young children was similar to that for other age groups: 1,167 (30.9%) cases in children <4 years of age, 1,218 (32.2%) cases in children 5–14 years of age, and 1,397 (36.9%) cases in persons >15 years of age. In the leishmaniasis center, cutaneous infections are confirmed by seeing In terms of seasonal patterns in Mazar-e Sharif, ZCL peaks in mid-October and most ACL cases occur during mid-February . ObservaRhombomys opimus, the main animal reservoir of L. major  and sticky traps placed in areas that were heavily infested with R. opimus. Phlebotomus papatasi, the principal vector of ZCL (The Mazar-e Sharif area, especially near the airfield, is semidesert, although extensive farming of seasonal crops is possible because of irrigation systems built in the 1980s. Irrigation canals are usually several kilometers long and 1.5–2 m deep and wide. Earth removed during canal construction is deposited as embankments that attract large numbers of the great gerbil, Our data suggest that foci of high-density enzootic ZCL occur in northern Afghanistan, especially in the Mazar-e Sharif area. ACL may also pose future problems because of its epidemic urban transmission potential (


Laetiporus sulphureus is an edible wood-rotting basidiomycete fungus whose fruiting bodies contain substances with verified therapeutic evidences and large amounts of α-(1 → 3)-glucan which is used as an effective inducer of microbial α-(1 → 3)-glucanases. However, production of mature fruiting bodies of this species under artificially controlled conditions has not been reported until now. Here, we provide the first report of successful initiation and development of L. sulphureus fruiting bodies in large-scale experiments. Twelve Laetiporus strains were isolated from a natural habitat. A synthetic log production system with a substrate composed of a mixture of sawdust enriched with organic and inorganic additives was developed. It was found that shocking the fungus mycelium with cold water or low temperature was the only suitable method for forced fruiting of L. sulphureus strains. Primordia of two strains were initiated already after 5–6 days from induction, and after another 2 days, they began to develop into fruiting bodies. Carpophores appeared fastest on substrates with high organic supplementation (40–45 %) and a low moisture content (40 %). The resulting mature fruiting bodies reached a weight of 200–300 g. The method of cultivation presented in this paper opens the way to commercial production of this valuable basidiomycete. Laetiporus sulphureus (Bull.: Fr.) Murrill belonging to Basidiomycotina, Aphyllophorales, Polyporaceae is a parasitic and saprobic fungus growing alone or more typically in large clusters on decaying logs, stumps and trunks of many deciduous and coniferous tree species. It is widely distributed in Europe, Asia, and North America. Its striking yellowish or orange-colored shelf-like fruiting bodies appear usually in summer and fall, but very often are not formed until years after the fungus is well established, so when present, they indicate significant internal defect. L. sulphureus causes a reddish brown cubical heart rot, with thin areas of white mycelium visible in the cracks of wood -d-glucan. Their cell wall contains up to 88 % of the dry matter of this glucan, whereas in other fungi it is present in an amount of 9–46 % -glucan can be used as inexpensive and safe raw material to obtain an alternative inducer of α-(1 → 3)-d-glucanases  in Trichoderma harzianum and Paenibacillus curdlanolyticus . The purity and concentration of the DNA samples were evaluated using ND-1000 . Polymerase chain reaction amplifications (PCR) followed the protocol of White et al. , alder 20 %), aspen 10 %) and poplar (10 %) sawdust. Oak sawdust and the mixture of sawdust were blended at a ratio of 1:1 by volume. The ratio of small to medium to large (chips) sawdust fraction in the substrate was 3:5:2. In order to optimize the composition of the experimental substrate, it was enriched with an organic and an inorganic additive. The organic additive comprised a mixture of materials of agricultural origin with a composition as shown in Table 0 % and p0 %, aspeL. sulphureus fruiting: (1) injection, through a filter, of 300 mL sterile water at 10 °C into the bag; (2) incubation the bag at 2–4 °C for 24 h; (3) cut-off the top of the bag; (4) incision of the side surfaces of the bag . The bags with the substrate were then kept at 23 ± 1 °C and 65–68 % relative humidity with a 10 h photoperiod. Adequate ventilation was provided to prevent an increase in CO2 concentration. Fruiting bodies were harvested from the substrates when mature. Biological efficiency (BE %) was calculated using an equation reported by Stamets  were tested for production of fruiting bodies in a cultivation experiment.Twelve strains of L. sulphureus develop on dying trees, mainly during late spring and early summer . Analogous results have been reported by Habijanič and Berovič , the mycelia of these fungi overran completely the exposed L. sulphureus mycelium  proved to be useless due to infections of the exposed substrate surfaces with fungi from the genera ium Fig. b. The efion Fig. c, while ion Fig. d–f. Out L. sulphureus fructification. Fruiting bodies of the LAE 12 and LAE 01 strains were obtained only on substrates containing at least 30 % of the mixture of organic supplements. However, the number of bags in which carpophores were formed depended on the amount of the applied organic additive. The highest percentage of substrate-filled bags in which there appeared fruiting bodies of both L. sulphureus strains was recorded when the supplementation reached 45 %; in the remaining combinations, this proportion was lower. It was further observed that, at this level of organic supplementation, in the case of strain LAE 01, carpophores appeared on all the substrates with a moisture content of 50 and 55 %, while in strain LAE 12—on substrates with a moisture content ranging from 40 to 55 % . For this reason, only one flush was obtained due to the infection at the carpophore fracture site. The BE calculated for the combinations for which fruiting bodies appeared on all the substrates, ranged from 15 to 21 %. It can be assumed that after minor modification of culture technology, larger fruiting bodies could be obtained.L. sulphureus could be produced on a large scale on a bed of sawdust under controlled conditions. In the literature, there are only a few studies on procedures for obtaining immature fruiting bodies in in vitro conditions. Ershova et al.  wood was used as a substrate. After inoculation with the agar mycelium, the substrate was incubated in a constant-temperature cabinet at 21 °C. The stumps overgrown with mycelium were then transferred to experimental plantations in Pribaikal’e (Irkutsk region). However, also in this case there is no direct evidence that the method allows to obtain fully mature fruiting bodies.The natural plantation method of growing L. sulphureus on an artificial substrate. The strains of L. sulphureus used in this study were isolated from a natural habitat and grew on enriched sawdust. Two of the strains fruited successfully. High levels of organic supplementation of the substrate promoted fruiting body formation and simultaneously reduced the time required for fruiting to 6 days after induction. The optimum moisture content of the substrate was 40 %. An increase in the water content resulted in prolonged fruiting run time. Our future studies will focus on further optimization of cultivation conditions of L. sulphureus and improving the technology of fruiting body production to increase the yield and biological efficiency of the fungi.In conclusion, we provide the first report of successful large-scale production of mature fruiting bodies of