Spatial hearing is critical for us not only to orient ourselves in space, but also to follow a conversation with multiple speakers involved in a complex sound environment. The hearing ability of people who suffered from severe sensorineural hearing loss can be restored by cochlear implants (CIs), however, with a large outcome variability. Yet, the causes of the CI performance variability remain incompletely understood. Despite the CI-based restoration of the peripheral auditory input, central auditory processing might still not function fully. Here we developed a multi-modal repetition suppression (MMRS) paradigm that is capable of capturing stimulus property-specific processing, in order to identify the neural correlates of spatial hearing and potential central neural indexes useful for the rehabilitation of sound localization in CI users. To this end, 17 normal hearing and 13 CI participants underwent the MMRS task while their brain activity was recorded with a 256-channel electroencephalography (EEG). The participants were required to discriminate between the probe sound location coming from a horizontal array of loudspeakers. The EEG MMRS response following the probe sound was elicited at various brain regions and at different stages of processing. Interestingly, the more similar this differential MMRS response in the right temporo-parieto-occipital (TPO) junction in CI users was to the normal hearing group, the better was the spatial hearing performance in individual CI users. Based on this finding, we suggest that the differential MMRS response at the right TPO junction could serve as a central neural index for intact or impaired sound localization abilities. When acting in a multisensory environment, it is predominantly the auditory cues which direct our attention to relevant targets and help us orient in space when the events happen outside the field of view. This skill is of crucial importance for us to become aware of potential pertinent incidents, such as threats. Moreover, spatial hearing enables us to distinguish among different sound sources in complex acoustic environments and to attend only to sounds that interest us. The capacity to follow an auditory stream by virtue of its spatial location is especially important in multi-speaker environments and is referred to as the “cocktail-party effect” . ConsequCochlear implants (CIs) are until now the most successful treatment for patients with severe to profound sensorineural hearing loss as they restore hearing to such an extent that speech recognition is reestablished or considerably improved, enabling verbal communication in numerous deaf and hearing impaired patients . OptimizIn normal hearing people, sound source localization in the horizontal plane relies mainly on the comparison of ILD  and ITD  cues . AlthougTo improve the sound spatial localization performance with a CI, much of the previous work has taken the bottom-up  approach concentrating on implant technology as for example improving coding and stimulation strategies  paradigm that is suitable for both testing and eventually training spatial hearing, and that allows to identify brain regions that are involved in the discrimination of sound locations using high-density electroencephalography (EEG). Previous EEG studies have shown the potential of measuring mismatch negativity (MMN) signals to estimate the spectral discrimination abilities of CI users as an objective evaluation tool of their speech perception . Our appWe investigated the neural correlates that exhibit differential responses (MMRS response) to spatially deviant probe sounds (SAME vs. DIFF) as a potential central neural index of intact audio-spatial discrimination abilities in a group of 17 normal hearing participants. The novelty here is using a modified MMN paradigm in the context of impaired sound localization together with advanced cortical source analysis. We hypothesized that this MMRS response elicited by the probe stimulus in the normal hearing subjects, similar to those in previous sound spatial mismatch studies e.g., , might bSeventeen normal hearing participants  took part in the study. Pure tone audiometry was used to verify normal hearing thresholds in the healthy participants. Hearing thresholds in dB HL for the left and the right ear for 10 frequencies between 0.25 and 8 kHz  were measured. Normal hearing was defined as hearing thresholds ≤25 dB HL for each frequency tested.Additionally, we investigated 13 CI users . Their demographic data such as age, the duration of deafness (DOD), the time of CI use, the brand of the implant and the performance of the not implanted ear as well as their task performance of the MMRS paradigm (percent correct responses) are listed in Normal hearing participants were recruited at the campus of the University of Tübingen. The CI users were recruited through the out-patient clinic at the department of ENT of University Clinics Tübingen (Comprehensive Cochlear Implant Center and Cochlea Rehabilitation Center). All participants reported to have no history of neurological or psychiatric illness. The study was approved by the ethics committee of the Faculty of Medicine Tübingen.The experiment took place in a sound-proof and electrically shielded room. Participants were seated in a chair surrounded by seven loudspeakers all mounted on the same horizontal plane at the approximate height of the participants’ ears, forming a sector of a circle covering 135.0° in steps of 22.5° . The disAuditory stimuli were presented via one out of seven loudspeakers at a time and consisted of a 250-ms broadband frequency signal resembling the sound of a set of keys falling on a solid underground . The louThe task was to indicate the direction of the probe sound with a pointing device that could be rotated around a vertical axis and that was connected to a rotary potentiometer. By pressing a button on the pointer, subjects signaled the validity of the chosen direction. They were asked to bring the pointer back in every trial such that it was pointing toward the central position. Pointer direction was recorded by the computer controlling the experiment and synchronized offline to the EEG recording.A 256-channel high-density EEG system was used for the continuous EEG-recording . Electrodes were placed in an elastic cap . Electrode impedances were kept below 10 kΩ. All channels were recorded against a Cz reference. The data was filtered with a highpass of 0.1 Hz and a lowpass of 120 Hz during EEG data acquisition. The sampling rate was 1000 Hz. Participants were asked to fixate at a central frontal cross and avoid any head and eye movements, as well as eye blinks during the measurement. In order to synchronize the stimulation and the recording, the trigger signal was forwarded to the EEG recording system together with the information of stimulation positions.Electroencephalography-derived cortical source localization results were coregistered to a template head via three fiducial points . Fiducials and the individual head shapes of the subjects were digitized using an electromagnetic 3D-digitizer system .To determine the percentage of correct responses for both SAME and DIFF probe conditions, the direction of the pointer with which subjects indicated the perceived direction of the probe stimulus was compared to the position of the loudspeaker which presented the probe stimulus in each trial. The pointing device provided a voltage depending on the pointing angle, a range around each loudspeaker was defined by an individual calibration procedure. In the calibration procedure a voltage range with a lower and upper limit was acquired for each loudspeaker direction. The individual calibration was used in order to rule out any non-linearities and biases of the subject. When the pointer was directed toward a predefined target area around the loudspeaker through which the probe stimulus was presented, the response was counted as correct. If the device was pointing to a region outside the target area the response was counted as incorrect. The extensions of the non-overlapping target areas for all loudspeaker positions were chosen offline for each recording session such that the overall score of correctly identified stimulus directions was maximal.Electroencephalography-data were analyzed in the MATLAB program  using the open-source Fieldtrip Toolbox .Continuously recorded EEG data were first offline highpass filtered at 1 Hz, down sampled to 200 Hz and then lowpass filtered at 35 Hz. The continuous EEG data were segmented into epochs from 0.55 s before the onset of the first sound stimulus until 2.95 s after it. We then applied a Discrete Fourier Transform (DFT) filter on the trial data (epochs) to minimize the first three harmonics of the 50 Hz line noise. Extremely noisy trials due to facial muscle movement were first manually removed by visual inspection. Afterwards an Independent Component Analysis (ICA) was applied to remove heart and eye movement artifacts. In CI users ICA was also used to reduce CI artifacts . The EEGThe segmented, filtered and artifact-cleaned EEG data were averaged for every subject according to the different trial sorting conditions (SAME and DIFF). Trial numbers for conditions to be compared were equalized by randomly selecting as many trials as there were trials in the condition with the least number of trials. Moreover, the trials in SAME and DIFF conditions were balanced not only to the trial number but also to the adaptor and probe sound positions. Error trials (behavior) were not excluded because this might cause very imbalanced trial numbers across conditions in some participants.http://surfer.nmr.mgh.harvard.edu/). The template cortical mesh decimation (ld factor 10 defining the density of the mesh which lead to 1002 vertices per hemisphere) and surface-based alignment was performed with open-source SUMA – AFNI Surface Mapper  at individual mesh vertices were estimated for each time point resulting in a time course of activation power for each vertex and each experimental condition.Averaged responses for SAME and DIFF conditions were subjected to a cortically constrained “minimum norm estimates (MNE)” approach  for soure Mapper . This ste Mapper . A bounde Mapper  was usede Mapper  includedIn a first analysis, the overall EEG source activity was calculated by computing the mean source activity  across all vertices of the whole cortical mesh for each time point and each condition. In order to obtain the brain activity of all normal hearing subjects the source activity was averaged. A previous EEG auditory azimuth direction deviation study has shown that the mismatch signal peaked within a time window of 100–200 ms after sound stimulus onset , in goodt-tests comparing SAME and DIFF conditions and used non-parametric cluster-based permutation tests  for each vertex. Vertices belonging to the medial wall  did not enter the statistical analysis. Thus, statistically different neighboring vertices in space with pvertex < 0.005 were assigned to a cluster. Each cluster was then characterized by the summed t-values across its vertices. The cluster of the largest absolute t-value was recorded after each permutation  resulting in an empirical null distribution of the test statistics. As significance criterion a pcluster < 0.05  was chosen. Since six time windows were tested, we used a p-value smaller than 0.008  to control the family-wise error rate for the final cluster-of-interest (COI) selection.In order to resolve the potential differential activation of cortical areas for SAME and DIFF stimulation conditions in time, we divided our time window of interest (2100–2400 ms) in the second analysis into six non-overlapping time windows each of 50 ms duration. Given the vertex neighborhood information, we performed two-tailed dependent-samples on tests  providedSignificant clusters obtained from comparing SAME and DIFF conditions in normal hearing participants were used as COIs for the analysis of the source activities in CI users. While in normal hearing participants, COIs could be defined based on statistical different activations between SAME and DIFF stimuli, this is not very feasible for hearing impaired CI users. Due to their auditory impairment, some of the CI users might not have shown any activation differences between SAME and DIFF conditions. Therefore, the same source analysis procedures as in the normal hearing subjects could not be applied. In contrast, we have chosen a different approach. We calculated the inverse solution for each CI user based on the individual leadfield and the pre-stimulus signal noise. Then separate source activity time courses could be extracted for each COI  for each CI user.We reasoned that the cortical differential activation under SAME and DIFF conditions reflected the good performance of the horizontal sound localization task in the normal hearing participants. Therefore, the mean differential time course between SAME and DIFF conditions of the source activity was calculated across 17 normal hearing subjects for each COI. The grand mean differential curve served as COI-specific norm activation to which the differential source activity between SAME and DIFF conditions of individual CI users was compared. To this end, the activity of individual COIs was obtained for each CI user by averaging the source activity across vertices within the corresponding COI. For each COI, samples of the interval ranging from 2100 to 2400 ms of the differential time courses of each individual CI user, and the corresponding norm differential wave of the normal hearing group were subjected to a Pearson’s correlation analysis, resulting in nCI = 10 correlation coefficients per COI. In order to assess whether CI users whose activation patterns strongly deviated from the norm time course would reveal poorer sound localization performance than those CI users with differential wave forms resembling the norm time course, coefficients of individual CI users were entered into a subsequent correlation analysis with individual CI users’ task performance, i.e., total hit rate, as an independent variable. A high correlation value in the analysis thus would indicate the differential COI time course to be a useful predictor for sound localization performance of individual CI users.All normal hearing subjects were confirmed in the pure tone audiometry (hearing thresholds ≤25 dB at the 10 frequencies between 0.25 and 8 kHz). Using the MMRS paradigm subjects were instructed to localize the sound of an auditory stimulus that was preceded by four subsequent and concurrent audiovisual adaptor stimuli originating from the same direction. The fifth probe sound-only stimulus could be either coming from the same direction as the four previous adaptor stimuli or from a position left or right to it. Thus, the chance level for correctly identifying the location of the sound probe is 33.3% assuming that the position of the adaptor stimuli had been perceived.p < 0.001 as revealed by a non-parametric sign-test (paired and two-sided Wilcoxon signed rank test). Comparing percent correct responses for conditions SAME und DIFF, another sign-test revealed a significant performance difference (p = 0.001) for the SAME condition (25%-ile / median / 75%-ile: 97.0 / 99.0 / 99.0%) than for the DIFF condition (25%-ile / median / 75%-ile: 96.0 / 97.0 / 99.0%).Due to malfunction of the pointing device in two participants, the behavioral data of sound localization of only 15 out of 17 normal hearing subjects could be analyzed. All participants showed very good sound localization performance with ≥94% correct responses except one outlier with a score of 84% . The medElectroencephalography overall source activity in the normal hearing participants disclosed a prominent response to the first adaptor stimulus followed by a response decrement for the subsequent three adaptor stimuli. For the probe stimulus, occurring at 2000 ms, its activation revealed a response rebound from the previous activation adaptation . In ordet-tests around the auditory N100 peak (90–110 ms after the corresponding auditory stimulus presentation) and around a secondary peak time window  yielded a significant mean amplitude difference in both the N100 and the secondary time windows. There were significantly stronger activations in the N100  and the secondary  time windows during the fifth stimulus (probe with auditory only) in comparison to the fourth stimulus , however, these activations (fifth probe) were significantly weaker in the N100  and the secondary  time windows comparing to those of the first audiovisual adaptor stimulus. It indicates that dishabituation occurred between the probe stimulus and the fourth audiovisual adaptor stimulus.Since the probe stimulus was presented without any concurrent visual stimulus , it was further tested whether a dishabituation effect could be demonstrated for the fifth as compared to the fourth stimulus, in the SAME condition only. Paired p-values were corrected for multiple comparisons (p < 0.05). Corrections were done at the whole brain level for each 50 ms time windows and for the six time windows tested. As shown in detail in p = 0.002), to the right temporo-parieto-occipital (TPO) junction , spreading to right parietal  and superior and inferior frontal areas . From then on, the significant MMRS areas became bilateral, while C3 became even more spread in the right parietal  and frontal areas and to the supplemental motor areas , the left premotor and supplemental motor areas also showed up . In the last significant time window, the right parietal  and frontal areas  remained similar, the left prefrontal area formed a large cluster together with the left motor and supplemental motor areas . The mean COI time courses for SAME and DIFF of the normal hearing group and the CI user group are displayed in Topographical differences of the cortical activity between SAME and DIFF conditions were assessed in time windows of 50 ms duration from 2100 to 2400 ms with non-overlapping 50 ms steps. p = 0.002) between the SAME (25%-ile / median / 75%-ile: 64.8 / 92.0 / 94.0%) and the DIFF condition (25%-ile / median / 75%-ile: 30.8 / 41.0 / 65.8%).The same MMRS paradigm for sound localization as described for normal hearing subjects was applied in CI users. Due to technical problems, behavioral data of only 10 out of 13 CI users could be analyzed. The performance ranged from 34 to 91%, the median performance was 54.0% with 25 and 75% quantiles of 43.3 and 76.0%. Thereby the sound localization performance to the MMRS paradigm differed greatly between CI users and the normal hearing subjects (who mostly achieved ≥94% correct responses). Comparing percent correct responses for conditions SAME und DIFF , paired r = 0.87, p = 0.0009, aCI(t) = T) and the normal hearing control group (aNH(t)) was extracted and normalized to the source activity of the NH group. In short, singular value decomposition was applied ) and the common activity Clusters differentiating between SAME and DIFF conditions in the normal hearing group were used as COIs for the analysis of source activity of CI users. The differential (DIFF minus SAME) source time courses of each COI were extracted and grand averaged across 17 normal hearing subjects to obtain a “norm” difference curve within the 2100–2400 ms time window. Then such a difference curve was extracted in the same time window in each CI user and was correlated  with the “norm” curve for every COI. The individual correlation coefficients of the CI users were then correlated with their corresponding sound localization task performance. We found one significant correlation in C2, i.e., the right TPO  upon sound location changes were evident in various cortical regions across time. Accordingly, significant EEG MMRS responses could first be detected in the right inferior frontal cortex, the right TPO junction, and then in the right parietal and precuneus regions, and finally in the bilateral frontal areas. These cortical regions revealed in normal hearing subjects were then taken as COIs when the MMRS paradigm was validated in 10 CI users with highly variable sound localization performance. Interestingly, the similarity of the differential MMRS responses between the individual CI user and the mean of the normal hearing group in the right TPO region was positively correlated to the sound localization performance in CI users. Differential MMRS responses in the right TPO junction could thus be used as a neural index for evaluating and monitoring the success of spatial hearing training in hearing impaired patients and CI users.In the sound localization task, all normal hearing subjects achieved a very good sound localization accuracy that was well above chance level. The excellent performance was expected because angles to be discriminated were 22.5°, which is well above the discrimination threshold  for localizing azimuth sound sources . The ratIn our analysis, we contrasted evoked potentials for the fifth (probe) stimuli being either deviant or congruent in terms of spatial location from the previous four audiovisual adaptor stimuli. The differential activation for SAME and DIFF probe stimuli, i.e., MMRS response, was hypothesized to be a central neural index for intact spatial hearing. In normal hearing subjects evoked brain responses of the four adaptor audiovisual stimuli showed a strong response magnitude decrease upon every stimulus repetition, indicating response repetition suppression. The fifth auditory-only stimulus deviating from the previous four stimuli with respect to its location  elicited a strong discrepancy signal in response magnitude. This rebound signal after repetition suppression or stimulus-specific adaptation might correspond to MMN signal , and migIndeed, investigations of the MMN in relation to spatial hearing have been shown to reveal robust MMN responses to changes in sound location with or without auditory attention involved . The MMRInterestingly, the activity to the 5th probe stimulus was not only increased with respect to the fourth stimulus in the DIFF condition, but also – even though to a much lesser extent - for the SAME condition. This dishabituation can be explained by the fact that in contrast to the previous stimuli the concurrent flashlight was missing in the fifth (probe) stimulus and thus the adapted audiovisual compound stimuli was followed by an auditory-only stimulus. Furthermore, unlike the preceding stimuli, the probe stimulus was relevant for the task, because it needed to be localized and required the participants’ response. The task relevance of the probe sound might be reflected in an increase of deployed attentional resources leading eventually to an increase in neural activation, not only when adaptor and probe stimuli were different, but also when they were coming from the same direction.Sound localization has been shown to be a complex process that involves multiple brain regions . PreviouBy analyzing cortical sources contributing to MMRS responses in subsequent time windows, we revealed the spatiotemporal dynamics of the cortical clusters involved in the MMRS task. The right inferior frontal area showing the significant MMRS response in the first time window (100–150 ms after probe stimulus onset) matches well with the view of inferior frontal generator of MMN response possibly in attentional shift . Then thDuring the later components of the MMRS responses (from 250 ms on), bilateral frontal and right parietal areas are involved in completing the sound localization task. The activation of the supplemental motor areas in medial frontal cortex  can be sWe aimed to find cortical regions which are active during the registration of the sound location change, and relevant for the spatial hearing performance. This is important for the application as a potential evaluation tool for CI users. Sound localization performance was ≥84% in all normal hearing subjects. In contrast, the sound localization performance of 10 CI-users varied within a large range from 34.1 to 91.5% correct responses and reflects the considerable variability of recovered spatial hearing performance in CI users. The CI users presented in the current work are a representative sample for the high variability that is found for spatial hearing after CI implantation. The field of the high variability in performance after the treatment with a CI has to be addressed more extensively in rehabilitation programs and is often described as one of the unsolved “challenges” in implant technology . CI userFactors that have been shown to affect localization performance in having an effect on neuronal cell physiology and functions in CI users are DOD and duration of implant usage as well as the age at implantation . The shoAlthough in the current study the presented CI users differ widely in their age, background of hearing loss, and the sample number is too small to statistically correlate their performance with biographical, etiological factors, our observed tendencies between performance and factors are in line with the relations previously reported . As a fiAs pointed above, using the MMRS paradigm might enable us to carry out studies investigating a multitude of variables contributing to or impeding the recovery of spatial hearing using CI. In the current study, we are addressing the central processing at the cortical level, thus overriding all the potential factors and only examining the MMRS responses as an end result. MMRS could be a potential neural index to objectively assess the spatial hearing capability of an individual. Once this method is established, future studies could be focused on studying one or few factors mentioned above by recruiting a homogenous group within the range of a certain factor of interest and specific experimental manipulations can be implemented to investigate a particular factor in detail.We reasoned that the extent of the similarity  of the differential (DIFF – SAME) activation, i.e., the MMRS response, between individual CI users and the mean of the normal hearing population, could be an indicator of a good sound localization performance of a particular CI user. Indeed, the investigation of the correlation between sound localization performance and a correlation coefficient reflecting the similarity of the MMRS differential curves between individual CI users and the normal hearing group showed that the MMRS response in the right TPO junction could serve as a central neural index for sound localization performance. Our results suggest that the more similar the differential MMRS responses in the right TPO junction between individual CI users and the normal hearing group, the better performance had the CI user. Thus, the spatiotemporal shape of the MMRS response in the right TPO junction could potentially differentiate intact and impaired audio-spatial processing.The right TPO junction identified from our EEG analysis is in a very similar location to the right inferior parietal area that was revealed as sound localization relevant region in previous positron emission tomography (PET) studies , as wellDue to the sensitivity and the large amplitude of the MMRS response elicited in our experiment, we are convinced that the MMRS paradigm will be of considerable value for the diagnosis of central sound localization impairments and for outcome monitoring of trainings of spatial hearing in CI users. Specifically, the activity in the right TPO can be potentially used to probe auditory spatial processing in hearing-impaired patients and CI users. Getting a more detailed picture of the areas involved in the processing of audio-spatial information will help to improve the diagnosis of individual deficits in sound localization and could thus be the key for the understanding of the large variability that has been found for the performance in CI users. In this way therapies and rehabilitation programs can be better adapted for individual hearing-impaired patients. Furthermore, training sound localization with additional teaching visual information as it is implemented in the current protocol might help the CI users to strengthen the auditory spatial attention pathway that might have been weakened due to the prior long-lasting deficit in selective auditory attention.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethics committee of the Faculty of Medicine Tübingen. The patients/participants provided their written informed consent to participate in this study.ES, A-EV, GR, PG, MK, AT, H-OK, CB, and YL: experimental design and reviewing manuscript. ES, A-EV, GR, PG, CB, and YL: data collection. ES, A-EV, GR, CB, and YL: data analysis. ES, CB, and YL: writing manuscript. All authors contributed to the article and approved the submitted version.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. The reviewer DB declared a past co-authorship with one of the authors MK to the handling editor.


Clostridioides difficile (C. difficile). Tetracyclines (TCs) are broad-spectrum antibiotics that are associated with a low risk of development of C. difficile infection (CDI) and are used as photosensitizers (PS) in photodynamic therapy (PDT). We evaluated whether TCs may be useful environmental cleansing agents. We compared the in vitro ability to kill C. difficile of four TCs  combined with PDT using ultraviolet A (UVA). We included chitosan, a cationic material, as a booster to increase the photodynamic bactericidal efficacy of TCs. PDT-induced bactericidal effects were assessed by the number of viable cells and the degree of DNA damage and membrane integrity. To avoid the intrinsic antibacterial activity of TCs at high concentrations, we used low concentrations of TCs (0.05 and 0.1 mg/mL). The bactericidal effect of treatment with chitosan plus PDT was over 100 times higher than that with PDT alone for each of the four TCs. DNA damage measured by ethidium bromide monoazide and real-time quantitative polymerase chain reaction was also greater for PDT plus chitosan treatment than for PDT alone or under control conditions: the threshold cycle (Ct) values for the control, PDT, and PDT plus chitosan were 14.67 ± 0.22, 20.46 ± 0.12, and 25.54 ± 0.17, respectively. All four TCs caused similar levels of severe cell membrane damage during PDT compared with control conditions. These data suggest that PDT combined with any of the four TCs plus chitosan might be an available tool to kill efficiently planktonic form of C. difficile. Surface disinfection in health-care facilities is critical to prevent dissemination of  Clostridioides difficile (C. difficile) is a spore-forming anaerobe that is the causative agent of severe and recurrent pseudomembranous colitis  against C. difficile to identify which was the most appropriate PS.The present study was undertaken in order to determine the capability of TCs to act as an effective photosensitizer (PS) during PDT against The absorbance curves of the four TCs were tested and TC, DXY, MIN, and TGE showed maximum absorption at 355, 350, 345, and 340 nm, respectively .C. difficile in the absence of UVA irradiation was evaluated to assess any inherent inhibition by the antibiotic itself. As shown in C. difficile of the four TCs plus chitosan without UVA was subsequently determined at low TC concentrations (0.05 and 0.1 mg/mL).The bactericidal effect of the four TCs against The bactericidal effect of PDT increased proportional to the concentration of TCs and the duration of exposure to light . In eithThe degree of cellular membrane damage was tested using EMA–qPCR, which can differentiate the DNA from intact and injured bacterial cells. The target gene was the 16S rRNA housekeeping gene. Cellular damage was much higher after treatment with PDT + chitosan than after treatment with PDT alone or under control conditions; the threshold cycle (Ct) values in EMA–qPCR were 14.07 ± 0.22 for the control and 19.45 ± 0.05 for the UVA + chitosan group. In the TCs + UVA groups, the Ct values were 20.46 ± 0.12 to 21.43 ± 0.01. In the TCs + UVA + chitosan groups the Ct values were 24.17 ± 0.08 to 25.54 ± 0.17 . HoweverC. difficile was measured after PDT via fluorescence microscopy by assessing membrane permeability using different-colored fluorescent DNA probes. The control group showed almost green-stained cells, indicating an intact cell membrane. The red cells observed in the control group are considered naturally dead cells. After treatment with UVA for 30 min, cells showed a mixture of green and red fluorescence and the numbers of red cells are increased compared with control group. The same result was also observed in case of chitosan treated group (data was not shown). However, in the groups treated with UVA + TCs + chitosan for 30 min, only red-stained cells were found indicating the presence of only ruptured dead cells , 0.5 mg/mL , and 0.2 mg/mL (TGE), respectively. To elucidate the antimicrobial activity of TCs themselves, we tested PDT of four TCs at low concentrations (0.05 and 0.1 mg/mL).Antibiotic activity against C. difficile, although the bactericidal effects of TC, DXY, and MIN were similar, while TGE was slightly more effective than the other TCs  to assess membrane integrity. The control group showed almost green-fluorescent bacterial cells, indicating intact live cell. Few red cells that observed in control group were considered naturally dead cells. In the UVA-treated group, green cells were mixed with increased numbers of red cells, meaning that this approach is capable of destroying the cell membrane to some extent. In contrast, the groups treated with UVA + four TCs + chitosan showed almost red cells, suggesting the injury or complete disintegration of the cell membrane .C. difficile spore as the more resistant one than vegetative form. However, because the presence of C. difficile spores in the hospital environment is more likely associated with acquisition rather than vegetative cells, it is necessary to investigate in a near future the effect of TCs-based PDT against C. difficile spore by the alteration of killing processes such as elevation of light energy and exposure duration. In this study, we did not study the effect of PDT against C. difficile. The TCs exhibited this bactericidal activity at low concentrations, which could avoid pharmacological side effects. The four TCs studied here did not differ in their photosensitizing and bactericidal activities, but TGE may be preferred as a candidate for PDT. Based on these findings, we propose that TCs might be effective alternative agents for cleaning hospital environments via PDT in the presence of chitosan.In summary, we have shown for the first time the ability of PDT with four TCs combined with chitosan to kill C. difficile KCTC 5009  was obtained from the Korean Collection for Type Cultures . C. difficile was cultivated in strict anaerobic condition at 37°C. The media for C. difficile culture was Clostridium difficile agar .The type strain of TC (Cat. No. T7660), DXY (Cat. No. D34477), MIN (Cat. No. M9511), TGE (Cat. No. PZ002142), chitosan with a molecular weight 50,000–190,000 Da (Cat No. 448869), and ethidium bromide monoazide (EMA) were obtained from Sigma . For the preparation of a stock solution of four TCs, each TC was dissolved in distilled water to 10 mg/mL (1%), filtered with 2 μm filter and refrigerated until use. Chitosan was prepared as described in a previous report . UVA lamA spectrophotometer  was used to measure the absorbance curve of each of the four TCs.In order to assess the anticlostridial activity of the four TCs during PDT, bacterial cells were exposed to various concentration of four TCs for thirty min. To remove remaining antibiotics bacteria were washed with PBS 3 times and serially diluted bacterial suspension were spotted on the media. After incubation for 48 h, viable cells were counted.8 cells/mL. Under UVA irradiation, the cell suspension was exposed to each of the four TCs at concentrations of 0.05 mg/mL or 0.1 mg/mL for 30 min, or to 0.0125% chitosan plus each of the TCs at the same concentrations. These concentrations of TCs were selected to avoid the intrinsic bactericidal activity as determined in our previous study [This experiment was conducted according to the method described by Tegos et al.  and our us study . To evaluate the degree of cellular damage in the irradiated cells, qPCR was conducted after EMA treatment of cells exposed to PDT with chitosan. This experiment was conducted at high concentration of TCs (0.2 mg/mL) for a definite comparison. After treatment of cells with 0.2 mg/mL of each of the four TCs alone or each of the four TCs + chitosan, they were UVA irradiated for 30 min, then each cell suspension was treated with 100 μg/mL of EMA. The cells were incubated in a dark room for 5 min and subsequently exposed to light from a 650-W halogen lamp 20 cm above the tube for 1 min. The tubes were dipped into ice before exposure to light to diminish any elevation of temperature. The procedure for the preparation of DNA and real-time PCR were performed according to the previous study [The membrane damage to bacterial cells during PDT was observed with fluorescence microscopy using fluorescent DNA probes according to membrane permeability . Damage to the bacterial cell membrane was judged a sign of cell death. While membrane-permeable DNA probe SYTO9 in viable cells emits green fluorescence (>505 nm), membrane impermeant propidium iodide (PI) in dead cells emits red fluorescence (>633 nm). The experimental procedure to evaluate the effects of chitosan during PDT, was performed according to the previous study . To compAll values are expressed as the mean ± standard error (S.E.). Statistical differences were evaluated using the student’s t-test.


Age-related hearing loss is prevalent in two-thirds of older adults and reflects progressive impairments in cochlear function leading to impoverished peripheral neural encoding of sound. Research has demonstrated the broader implications of hearing loss for the health and functioning of older adults, particularly with respect to brain aging and dementia. This presentation will summarize current epidemiological and neuroimaging evidence for how hearing loss in older adults affects cognitive load and brain structure/function and relate this contemporary research with previous psychological studies proposing ‘information degradation’ and ‘sensory deprivation’ hypotheses of how hearing may affect cognitive function. Finally, the design of an ongoing NIA-funded randomized controlled trial  that will determine if hearing treatment reduces the risk of cognitive decline, dementia, and brain aging in adults will be discussed. Part of a symposium sponsored by Brain Interest Group.


Supplemental Digital Content is available in the text. We aimed to explore whether higher levels of residential greenness were related to lower odds of disabilities in activities of daily living (ADL) and instrumental activities of daily living (IADL).We included older adults 65 years of age or older from the Chinese Longitudinal Healthy Longevity Survey. Our exposure was Normalized Difference Vegetation Index in 500 m radius around residence. Our outcome was ADL and IADL. We used binary logistic regression and mixed-effects logistic regression to estimate the odds of ADL and IADL disabilities.A total of 36,803 and 32,316 participants were included for the analysis of ADL and IADL, with 71.6% free of ADL disability and 47.3% free of IADL disability. In the logistic regression model, compared with the participants living in the lowest quartile of residential greenness, those in the highest quartile had a 28%  lower odds of ADL disability and a 14%  lower odds of IADL disability. A similar association was found in the mixed-effects logistic regression models. During the follow-up period, 5,004 and 4,880 healthy participants developed ADL and IADL disabilities. Per 0.1-unit increase in baseline annual average Normalized Difference Vegetation Index (NDVI) was related to an OR of 0.95 of developing ADL disability  and IADL disability .Our study suggests that increasing green space is associated with lower odds of ADL and IADL disabilities, which may reduce caregiver burden of long-term care for Chinese older adults. More green space has been linked to better physical health. But there is no evidence on whether higher levels of residential greenness are associated with lower odds of activities of daily living (ADL) and instrumental activities of daily living (IADL) disabilities. To address this evidence gap, our study conducted cross-sectional and longitudinal analysis among 36,803 and 32,316 Chinese older adults, separately. We found that the highest quartile compared with the lowest quartile of residential greenness was associated with a 28% lower odds of ADL disability and 14% lower odds of IADL disability. Our study suggests that more green space might prevent or delay the onset of ADL and IADL disabilities, which has significant implications for reducing caregiver burden of long-term care for Chinese older adults.1 ADL consist of basic tasks in daily life: eating, bathing, dressing, toileting, transferring, and continence, reflecting essential self-care capacity.2 IADL indicate independent skills in a given environment, like shopping, cooking, and taking public transportation. ADL disability (unable to perform one or more of tasks) is more severe than IADL disability and often occurs at a more advanced age.3 Prior estimates of the prevalence of ADL disability among older adults 65 years of age or older varied regionally: 6.4%–18.6% in the United States, 26.6% in Spain, 1.6% in Hong Kong,6 which were less prevalent than IADL disability.7 ADL and IADL disabilities were linked to higher rates of mortality and comorbidities, more health service utilization, and long-term care.10 Furthermore, up to 20% of older adults with ADL disability reported unmet need for ADL assistance around the globe.13Declines in vital capacity, muscular strength, and flexibility are more prevalent among older adults. These physiologic changes can cause assistance in performing activities of daily living (ADL) and instrumental activities of daily living (IADL).16 To our knowledge, there is no study on the effects of residential greenness on ADL and IADL disabilities among older adults. We hypothesized that higher levels of residential greenness were associated with lower odds of ADL and IADL disabilities and might prevent the onset of ADL and IADL disabilities. The potential mechanisms of health benefits of residential greenness on ADL and IADL could be that green space promotes physical activity and social engagement,18 which have been liked to better performance in ADL and IADL.21A number of studies reported benefits of residential greenness on lower mortality rate.22 A cross-sectional study in China showed that about 12.1%–16.8% of older adults 80 years of age or older needed assistance in ADL. However, more than half of them reported unmet needs for assistance in ADL, with higher vulnerabilities observed in rural areas.23 The prevalence of IADL disability was higher, up to 30.1% among older adults 65 years of age or older in Shanghai, China.24 We aimed to generate evidence on whether high levels of residential greenness could reduce odds of ADL and IADL disabilities among older adults, using the Chinese Longitudinal Healthy Longevity Survey (CLHLS).China has the largest number of older population 65 years of age or older, about 148 million in 2017, which is currently under a rapid increase.25Established in 1998, the CLHLS aimed to investigate the determinants of healthy longevity among Chinese older adults. The CLHLS used a multistage, stratified sampling design to recruit the participants from 631 randomly selected cities and counties, 22 out of 31 provinces in China. The sample sites represent about 85% of the Chinese population. This survey included only the participants 80 years of age or older in 1998 and 2000 and expanded to the participants 65 years of age or older since 2002. The CLHLS conducted follow-up surveys among the survivors and recruited new participants in 2000, 2002, 2005, 2008, 2011, 2014, and 2018. Extensive data on determinants of health have been collected, including demographic and socioeconomic characteristics, lifestyle, ADL, IADL, cognitive function, psychological status, and chronic diseases. Extra data regarding causes of death, health service utilization, and health status before death were collected from the family members of deceased participants. More details on study design and data quality could be found elsewhere.2.5 values . We included 36,803 participants for the cross-sectional analysis. We additionally excluded those who were lost follow-up after the baseline survey  and died before the follow-up survey  for the longitudinal analysis .Our study utilized 2000, 2002, 2005, 2008, and 2011 waves of the CLHLS for the analysis of Normalized Difference Vegetation Index (NDVI) and ADL. The total sample size of five pooled waves consisted of 39,225 participants. We excluded the participants who were missing NDVI values (n = 353), were younger than 65 years (n = 545), and were missing particulate matter (PM)2.5 values . We included 32,316 participants for the cross-sectional analysis. We additionally excluded those who were lost follow-up after the baseline survey  and died before the follow-up survey  for the longitudinal analysis .Because the variables used for coding IADL were available since 2002, we used 2002, 2005, 2008, and 2011 wave, including 34,342 participants, for the analysis of NDVI and IADL. We excluded the participants who were missing NDVI values (n = 17), were younger than 65 years (n = 545), and were missing PM26 NDVI is equal to the ratio of the difference between the near-infrared region and red visible reflectance to the sum of these two measures. NDVI ranges from −1.0 to 1.0, with larger values indicating more green space.28We calculated NDVI, a satellite image-based vegetation index, to reflect residential greenness. The plants absorb red visible light during the process of photosynthesis, while leaves reflect near-infrared light to scatter extra heat.30 Due to the temporal resolution of 16 days at MODIS, we measured two NDVI values for January, April, July, and October from 2000 to 2014 to reflect seasonal variation in greenness.Based on participants’ residential addresses, we obtained NDVI values from the Moderate-Resolution Imaging Spectro-Radiometer (MODIS) in the National Aeronautics and Space Administration’s Terra Satellite.We calculated baseline NDVI in the 500 m radius around residential addresses of participants. Baseline NDVI was the annual average NDVI value at the year of study entry. In addition, we categorized baseline annual average NDVI into quartiles and calculated their 0.1 unit of values.31 ADL had a continuous scale of zero to six with each question scored zero (without assistance) or one (with assistance). Higher ADL scores indicated more ADL disabilities. In our analysis, we dichotomized ADL scores to zero, defined as free of ADL disability, as the reference group, and one to six, defined as with ADL disability.ADL assessed self-care capacity by using six self-reported questions: “Do you need assistance in bathing/dressing/toileting/transferring/eating/continence?”31 We scored each activity zero (able to do without help) or one (need help). Ranging from zero to eight, we dichotomized IADL scores to zero, defined as free of IADL disability, as the reference group, and one to eight, defined as with IADL disability.IADL assessed independent living skills including eight activities: visiting neighbors, shopping, cooking, washing clothes, walking 1 km, lifting 5 kg, crouching and standing up three times, and taking public transportation.2.5, and Mini-Mental State Examination (MMSE). We also generated a variable of time to reflect the number of years for each follow-up survey since entering the cohort for the longitudinal analysis.We measured a range of baseline characteristics, including age, sex, ethnicity, marital status, geographic region, urban/rural residence, education, occupation, financial support, social and leisure activity, smoking status, drinking status, physical activity, annual average PM25 We dichotomized sex to males and females. We divided ethnicity into Han Chinese and ethnic minorities . We categorized marital status into married and not married at the time of interview . We categorized the participants into seven geographical regions based on their residential addresses: Central China , Eastern China , Northeastern China , Northern China , Northwestern China (Shaanxi Province), Southern China , and Southwestern China (Chongqing and Sichuan Provinces). Residence was dichotomized to urban and rural area.Age was the difference between the interview dates and birth dates.mahjong, listening to the radio or watching TV, and participating in organized social activities, with each scored zero or one.32 We evaluated smoking status by asking “smoke or not at present.” We evaluated drinking status and physical activity using similar questions. We used the adapted Chinese version of MMSE to reflect cognitive function. MMSE scores ranged from 0 to 30, with higher scores indicating better cognitive function. Based on participants’ residential addresses, we obtained the estimates of ground-level concentrations of PM2.5 from Atmospheric Composition Analysis Group.33 We used annual average PM2.5 at baseline year to indicate air pollution levels.We divided education into groups of formal education (≥1-year schooling) and no formal education. We dichotomized occupation to professional work  and nonprofessional work . We assessed financial support depending on whether participants relied on their own work and retirement wage  or received financial assistance from other family members . We took into consideration seven activities into social and leisure activity index, including gardening, personal outdoor activities excluding exercise, raising poultry or pets, reading, playing cards or 2.5, and MMSE at baseline. Firstly, we used binary logistic regression models to assess the association between annual average NDVI and ADL disability at baseline. We stratified the analysis by age, sex, marital status, urban/rural residence, education, occupation, financial support, social and leisure activity index, smoking status, drinking status, and physical activity. Secondly, we applied mixed-effects logistic regression models to explore the relationship between annual average NDVI at baseline and ADL disability among the participants with follow-up surveys. Annual average NDVI was measured at baseline. The outcome ADL disability was repeatedly measured at baseline and subsequent follow-up surveys. The regression models were also adjusted for the variable of time elapsed. In addition, about half of the participants died or were lost before the follow-up survey. We conducted a sensitivity analysis on the participants with/without follow-up surveys using the binary logistic regression, to see how possible informative censoring may influence the association between residential greenness and ADL disability. Furthermore, we used binary logistic regression to test whether higher levels of annual average NDVI at baseline were related to lower odds of developing ADL disability among healthy participants who were free of ADL disability at baseline. We stratified the analysis by age group. We used the same statistical analysis to explore the association between NDVI and IADL disability.We used binary logistic regression and mixed-effects logistic regression models to examine the associations between residential greenness and ADL disability, adjusted for age, sex, ethnicity, marital status, geographic region, urban/rural residence, education, occupation, financial support, social and leisure activity, smoking status, drinking status, physical activity, annual average PMWe calculated odds ratios (ORs) and 95% confidence intervals (CIs) to estimate the magnitude of the associations. We reported the results of quartiles and per 0.1-unit increase in baseline annual average NDVI. We plotted the cubic splines with three knots to examine nonlinearity of the associations. We used STATA 14.0  for statistical analysis.The CLHLS study was approved by the Institutional Review Board, Duke University (Pro00062871) and the Biomedical Ethics Committee, Peking University (IRB00001052-13074). All participants signed a written informed consent.Table http://links.lww.com/EE/A57). Our sensitivity analysis found a similar association between baseline annual average NDVI and ADL disabilities among those without follow-up surveys . Cubic splines . The difference in health effects could also be caused by the difference in time spent in green space, access to health care, baseline health status, and socioeconomic status. Health effects of green space differed by socioeconomic status in our study are in line with prior studies. An observational study of 40 million population in the United Kingdom reported that income-related health inequalities were smaller in more green areas.There are several limitations to our study. Firstly, although NDVI reflects overall greenness, it could not indicate exposure to a specific type of vegetation. We also had no information on activity patterns of participants. How these differences could influence the association between residential greenness and ADL and IADL disabilities is unclear. Secondly, there may be concerns in informative censoring in the longitudinal analysis. Our mixed-effects logistic regression models only included the participants with follow-up surveys, consist of only about half of the total sample. Those without follow-up surveys who were excluded from the longitudinal analysis were more vulnerable. However, our sensitivity analysis reported similar protective effects on ADL and IADL disabilities, compared with the longitudinal analysis. Thirdly, reverse causation relationship among residential greenness, ADL disability, and IADL disability is possible. In our study, the older adults with ADL or IADL disability live in the less green area  than those without ADL or IADL disability at baseline. This indicates that older adults with ADL disability probably may be more likely to live in urban areas, where has more healthcare resources but less green space. But our analysis of baseline annual average NDVI and development of ADL disability among the healthy participants indicates that the potential reverse association shall not greatly bias the association between residential greenness and ADL disability. Additionally, socioeconomic status is unlikely to confound the association between residential greenness and ADL disability. Our study found that the older adults living in greener area have lower socioeconomic status.Our study has several strengths. To our knowledge, this is the first study assessing the effects of residential greenness on ADL and IADL disabilities among older adults. The protective effects of residential greenness observed in our study add to the mechanism of residential greenness and overall mortality and also provide evidence for preventing ADL and IADL disabilities in further research. In addition, our study included a large size of nationally representative sample, from 22 provinces in Mainland, China. Furthermore, our study used both the cross-sectional and longitudinal designs, which showed consistent findings. We used aerial satellite-derived measurements, which take into consideration the seasonal variation of greenness, for the exposure assessment. This more objective measurement of greenness exposure by using NDVI and mixed analysis provided stronger evidence on the relationship among residential greenness, ADL disability, and IADL disability.Our study showed that higher levels of residential greenness were associated with lower odds of ADL and IADL disabilities among older adults. These protective effects indicated that more green space might help prevent or delay the onset of ADL and IADL disabilities. Our findings have public health implications for reducing caregiver burden of long-term care for the large and ongoing increase group of Chinese older adults.We would like to acknowledge Chen Bai, Chengcheng Qiu, Anran Tan, Jingyu Tong, and Longkai Zang for their contribution in geocoding residential addresses; Yinq-Rong Chern for NDVI data preparation.The authors declare that they have no conflicts of interest with regard to the content of this report.This study was supported by Bill & Melinda Gates Foundation (Grant Number: OOP1148464), and NCI (Grant Number: R00 CA201542). The data analyzed in this paper were provided by the Chinese Longitudinal Healthy Longevity Study (CLHLS) which has been jointly supported by National Natural Sciences Foundation of China (71490732) and the National Key R&D Program of China (2018YFC2000400).Data used in this study are available upon request from Center for Healthy Aging and Development Studies, National School of Development, Peking University. Computer code will be made available upon request.


Recently, dynamic text presentation, such as scrolling text, has been widely used. Texts are often presented at constant timing and speed in conventional dynamic text presentation. However, dynamic text presentation enables visually presented texts to indicate timing information, such as prosody, and the texts might influence the impression of reading. In this paper, we examined this possibility by focusing on the temporal features of digital text in which texts are represented sequentially and with varying speed, duration, and timing. We call this “textual prosody.” We used three types of textual prosody: “Recorded,” “Shuffled,” and “Constant.” Recorded prosody is the reproduction of a reader’s reading with pauses and varying speed that simulates talking. Shuffled prosody randomly shuffles the time course of speed and pauses in the recorded type. Constant prosody has a constant presentation speed and provides no timing information. Experiment 1 examined the effect of textual prosody on people with normal hearing. Participants read dynamic text with textual prosody silently and rated their impressions of texts. The results showed that readers with normal hearing preferred recorded textual prosody and constant prosody at the optimum speed (6 letters/second). Recorded prosody was also preferred at a low presentation speed. Experiment 2 examined the characteristics of textual prosody using an articulatory suppression paradigm. The results showed that some textual prosody was stored in the articulatory loop despite it being presented visually. In Experiment 3, we examined the effect of textual prosody with readers with hearing loss. The results demonstrated that readers with hearing loss had positive impressions at relatively low presentation speeds when the recorded prosody was presented. The results of this study indicate that the temporal structure is processed regardless of whether the input is visual or auditory. Moreover, these results suggest that textual prosody can enrich reading not only in people with normal hearing but also in those with hearing loss, regardless of acoustic experiences. Dynamic text presentation is used in everyday life, such as in electronic advertisements and TV tickers. Text scrolling within a fixed speed and direction is often used to show a larger amount of information in a limited space. In addition, text flashing is sometimes used to catch the audience’s attention. The presentation rate of dynamic text is an important factor for readers, as they cannot control the speed in most conventional dynamic text presentation. When the presentation speed is too high, the reading performance becomes poor . On the Though dynamic text is useful, conventional dynamic text cannot convey complex paralinguistic information, such as emotions, intonations, speaker’s speed, and duration that can be conveyed by spoken language. While reading silently, we often have a subjective experience of inner speech, which resembles overt speech . Hirose This paper addresses three questions. First, we focus on whether impressions of reading are affected, as they are with prosody, by adding visual timing information  to the written language. We call this “textual prosody.” The temporal structures in conventional dynamic text presentation  do not convey timing information. For our purpose, we adopted a special dynamic text presentation format to enable textual prosody. In this format, the letters are statically displayed, but the contrast of letters changes dynamically . For exaOur second research question was whether information conveyed by textual prosody is stored visually or auditorily. Although texts are presented visually, they may convey temporal information as prosody. Normally, auditory prosody information is initially processed through the listener’s ears and stored auditorily. On the other hand, textual prosody is initially processed through the reader’s eyes. It is not clear whether the information conveyed by textual prosody is stored visually or auditorily. To determine this, we examined the characteristics of textual prosody using an articulatory suppression paradigm. Articulatory suppression is a research tool that is often used to explore phonological processing in reading . In the Finally, we asked whether the effect of textual prosody depends on acoustic experiences in daily life. People with hearing loss exhibit problems in learning to read as a result of the difficulties they face in developing spoken language . There sSign language is often used as a means of communication in people with hearing loss. Both spoken and signed languages are acquired naturally. Sign language possesses all the linguistic complexity and levels of structure of spoken language . Spoken In this study, therefore, we examined whether textual prosody can affect impressions of reading  of participants with hearing loss in addition to those of participants with normal hearing. If textual prosody can enrich people’s reading, especially in people with hearing loss, it should be a useful tool to convey speech speeds and timing information visually.We conducted three experiments in which we manipulated the textual prosody of visually presented language and asked readers to judge their impressions of readability, favorability, or emotionality . The textual prosody was manipulated in three ways: recorded, shuffled, and constant prosody. Recorded prosody reproduces utterance speed and duration in correspondence with letters. Shuffled prosody is inappropriate in the sense that the timings of pauses do not match the boundaries of sentences or words. Constant prosody has constant presentation speed and has no prosody. In Experiment 1, we examined the effects of textual prosody, showing visual timing information in readers with normal hearing. If textual prosody affects the impression of reading, the scores of the impressions would be higher/lower than when there is no textual prosody. In Experiment 2, we examined how textual prosody is stored with an articulatory suppression paradigm. If representation of textual prosody is stored in the articulatory loop, scores of impressions of reading under articulatory suppression condition should be worse than under the no suppression condition. In Experiment 3, we examined whether textual prosody affects the impressions of readers with hearing loss. Although people with hearing loss have experience with prosody information in sign language and lip reading, they have less auditory experience. If textual prosody affects the impressions of readers with hearing loss, the scores of the impressions would be higher in recorded prosody than in constant or shuffled prosodies.In this experiment, we presented text to readers with normal hearing at various presentation speeds and with various textual prosodies (timing information of texts). It is assumed that textual prosody may enrich the impression of reading because it offers more paralinguistic information visually. However, prosodic processing occurs even when people read texts silently . If readThis experiment was conducted with two groups to confirm the reproducibility of the results. The first group comprised 29 female college students who volunteered to participate in the experiment. The participants were either in their first or second year in college, and their mean age was 18.62 years (SD: 0.81). The second group comprised 23 female college students who volunteered to participate in the experiment. The participants were either in their second or third year in college, and the mean age of participants was 19.57 years (SD: 0.65). There were no duplicate participants between the first and second groups. The sample size of each group was determined based on prior studies . There wWe adapted a special dynamic text presentation format to present textual prosody described in the introduction section. To measure, record, and present reading positions, we utilized a computer program “Yu bi Yomu” on tablet devices . In thisWe also created three types of textual prosodies  for each presentation speed using the software. One of these types of textual prosodies is “recorded.” This type uses the tracing mode. One of the authors traced the sentences using pauses and changing the tracing speed as if talking. The tracing movement is assumed to exhibit similar speed and timing as that of their actual or inner speech. The software “Yu bi Yomu” can record a video of how the letters appear and disappear and replay it without barely visible letters at initial presentation. Here, we expected that if the user traces sentences as if talking and replays it, the movement of contrast change might play a role similar to prosody in speech, such as speed and timing. The author read texts with the intention that the meaning of the text would be conveyed correctly. For example, the author paused according to the syllables or large units of text or at the boundaries of some clauses. He/she also read the words that might convey emotions  or might be important slower than other words. Only a single reader’s tracing was used as recorded prosody because the more averaged the tracing of multiple readers, the more often the stimulus was displayed at a constant speed. Recorded prosody is appropriate in the sense that the timings of pauses matches the boundaries of a sentence or word. The second type of prosody is “shuffled.” This type randomly shuffles the time course of speed and pauses in the recorded type. Accordingly, pauses occur while presenting a letter, or in the middle of a syllable, a word, or a large unit of text. Shuffled prosody is irrelevant in the sense that the timings of pauses do not match the boundaries of sentences or words. The acceleration of speed is non-zero in recorded and shuffled prosody. The third type of prosody is “constant,” in which the presentation speed is constant and the acceleration of speed is zero. This was achieved in an automatic mode.We varied the presentation speed, i.e., 3, 6, or 12 letters/second . This was because previous studies  showed tWe used four types of Japanese plain text. One was called “Thank you”; it conveys gratitude . The second one was called “Telegram”; it is a typical telegram that celebrates success on an examination . The third, “Weather forecast,” presents sentences typical of weather reports on TV news . The forth, “Earthquake warning,” warns of an imminent earthquake and is very familiar to Japanese people (“This is an emergency earthquake flash report. Please beware of a strong shake.”). Japanese text stimuli are in The software was run on a tablet computer (Apple iPad) and connected to a projector . The texts were presented on the projector. The participants were divided into two groups and observed the text stimuli. They sat in three rows, at distances of 2–5 m from the screen. The visual angle of a letter was about 1–2.5°. All participants reported that the stimulus texts were adequately visible.This experiment was conducted for each text. The nine stimuli (three speeds × three prosody types) per text were presented. It was confirmed that the impression of static text did not change before and after reading the dynamic text repeatedly . Thus, tThis experiment was conducted with two groups. The data of the two groups were merged because both the groups displayed similar tendencies. Each condition was repeated twice, and the means were calculated as rating values. We also merged the values of the four texts because the tendencies of data in the four texts were similar. A two-way within-subject Analysis of Variance (ANOVA), with prosody type and presentation speed as factors and the rating value as the dependent value, was conducted for each judgment. The degree of freedom was corrected by Greenhouse-Geisser correction when the Mauchly’s sphericity test was found to be significant. When the interaction was significant, we tested simple main effects, using Bonferroni corrections.The overall results are shown in In general, the impression of reading at 6 LPS was the highest among readers with normal hearing. This finding is consistent with the optimum reading speed reported by Except for emotionality, the effect of textual prosody was not so clear at 12 LPS, that is, under fast text presentation conditions. The pauses of textual prosody were relatively shorter and the overall speed was faster  at 12 LPS. The differences between the three textual prosodies may become smaller at 12 LPS because the absolute amount of duration and speed change was the smallest in faster conditions. It is assumed that this is the reason why the effect of textual prosody was not clear at 12 LPS. On the other hand, the rating values in recorded prosody tended to be higher than those in shuffled prosody at 3 and 6 LPS. It is assumed that the effect of recorded prosody was relatively stronger because the absolute duration and speed change were larger at lower speeds. The differences between the textual prosody types were smaller than those observed between the three conditions in presentation speeds.The rating values of 3, 6, and 12 LPS for each textual prosody in In sum, for readers with normal hearing, some enhancement of impression of reading by textual prosody was observed. For recorded prosody, the degradation of impressions at the slow presentation speed could be somehow alleviated. When the presentation speed was appropriate at 6 LPS, presentation with recorded and constant prosody was preferred. When the temporal structure of recorded presentation was shuffled, participants’ evaluations generally were low, which means that the mere presence of acceleration is not sufficient to cause the enhancement by textual prosody and that an appropriate temporal structure is required.Experiment 1 shows that readers with normal hearing prefer text to be presented at the rate of 6 LPS and that textual prosody can affect their impressions of reading. The characteristics of textual prosody, however, are not clear. For example, it is not apparent whether textual prosody is converted to visual or to auditory representations. In this experiment, we examined whether textual prosody is stored as visual or auditory information using an articulatory suppression paradigm. Articulatory suppression prevents the articulatory loop selectively . If suppAlthough we used three types of prosodies, only recorded prosody is useful and should be utilized because it pauses according to the syllables or large units of text or at the boundaries of some clauses. Constant and shuffled prosody do not need to be stored because constant prosody has no specific prosody and shuffled prosody is irrelevant. Thus, it is assumed that only recorded prosody should be affected by articulatory suppression and that the impressions may be impaired if textual prosody is stored in the articulatory loop. In contrast, if recorded prosody is stored visually and not stored in the articulatory loop, storing the prosody should be easy and the impressions may not be impaired even when participants are articulatory suppressed. We examined this prediction using articulatory suppression and the results revealed the characteristics of textual prosody. Though memory tests are normally used with articulatory suppression, it could not be used because we had presented the same texts repeatedly. We then measured readers’ impressions of texts with articulatory suppression.Forty-four female college students voluntarily participated in this experiment as a part of their classwork. Sample size was determined by the sample used in Experiment 1. The mean age of the participants was 19.00 years (SD: 0.64). No participants had hearing loss. This experiment was performed in compliance with the Declaration of Helsinki. The protocol was approved by the Ethics Committee of Hakodate Junior College . This study was carried out in accordance with the recommendations of Provisions of Experiments, Ethics Committee of Hakodate Junior College with written informed consent from all participants.There were two suppression conditions: no suppression and articulatory suppression. In the former condition, participants read the text stimuli silently and had no disturbance. In the latter condition, participants were asked to read text stimuli silently and to repeat “a, i, u, e, o, …” simultaneously.Text stimuli and three types of textual prosodies were the same as those used in Experiment 1. The presentation speed was fixed at 6 LPS because Experiment 1 showed the impressions of reading were the most positive at that speed.The software was run on a tablet computer (Apple iPad) and connected to a projector. Texts were presented on the projector. The participants were divided into two groups and each observed the text stimuli. The visual angle of a letter was about 0.5–2.5°. All participants reported that the stimulus texts were visible well. In one group, an articulatory suppression condition was assigned to the first half of the trials and a non-suppression condition was assigned to the latter half. The order of the suppression conditions was reversed in the second group. In this experiment, we did not include repetition because articulatory suppression was cumbersome for the participants and caused fatigue in them. This experiment included 24 trials  and took about 30 min. Other procedures were the same as those in Experiment 1.We merged the values of the four texts because the tendencies of data in the four texts were similar. A two-way within-subject ANOVA, with prosody type and articulatory suppression as factors and the rating value as the dependent value, was conducted for each judgment. The degree of freedom was corrected by Greenhouse-Geisser correction when the Mauchly’s sphericity test was found to be significant. When the interaction was significant, we tested simple main effects using Bonferroni corrections.The results are shown in Results showed that impressions of readability and favorability tended to be worse under articulatory suppression condition. But the effect of articulatory suppression was significant only for recorded prosody in favorability judgment. We predicted that reading texts with textual prosody requires phonological coding if the suppression impairs the impression of reading at least one condition, and that only recorded prosody would be affected by articulatory suppression. Readers with normal hearing had some difficulties storing the information of recorded prosody in the articulatory suppression condition, and as a result, the impression of the prosody worsened. This indicates that a part of textual prosody, although it is visual, is stored in the articulatory loop. The effect, however, was marginal.Although the articulatory non-suppression condition in this experiment is exactly that of the 6 LPS condition in Experiment 1, the rating values were lower, and the effect of prosody type was smaller in Experiment 2. The reason for these results may be that Experiment 2 focused only on the optimum speed, that is, 6 LPS. Participants might rate their impressions as higher at the optimum speed, as compared to the less optimum speeds in Experiment 1. On the other hand, the rating values in Experiment 2 were not very high at the optimum speed. This may be because the participants were unable to compare their impressions to the less optimum speed and a floor effect may have occurred. Thus, we could hardly observe the effect of prosody (except for the effect of shuffled and constant prosody in readability) in this experiment.Experiment 1 indicated that textual prosody could enrich impressions of dynamic texts in readers with normal hearing. This finding raises the question of whether textual prosody could enrich impressions of reading in people with hearing loss. Some previous studies investigating speech production found that prosodies of people with hearing loss are different from those of people with normal hearing e.g., . HoweverTwenty-six  people with hearing loss participated in this experiment. The sample size was determined based on Experiment 1. Their mean age was 23.36 years (SD: 3.40), and their mean schooling length was 15.04 years (SD: 1.32).All participants had physical disability certificates and relatively severe hearing loss. Self-reported impairment types were as follows; 23 were sensorineural, 1 was conductive, 1 was neurogenic, and 1 was unclear. Twenty-two of the participants had hearing loss from birth; out of these, four had hearing loss that subsequently worsened. The hearing capacities of both ears were as follows: 22 participants were at ≥ 100 dB, 2 were at ≥ 90 dB, 1 was at ≥ 80 dB, and 1 was at ≥ 70 dB.Information about their communication methods was obtained using a multiple-choice question; 100% used sign language, 88.5% used lip reading, 82.7% used spoken language, 69.2% used writing, and 69.2% used acoustic aids. They were also asked to rate their sign language and written communication skills on a four-point scale with the following options: “very good,” “good,” “bad,” and “very bad.” In terms of sign language, 10 reported with “very good” and 16 with “good.” On written communication, 8 reported with “very good,” 13 with “good,” and 4 with “bad.”The participants were paid ¥5,000 (roughly $50). All participants rated the texts “Thank you” and “Telegram,” while only seventeen  rated “Weather forecast” and “Earthquake warning” because of sufficient time to read four texts. This experiment was performed in compliance with the Declaration of Helsinki. The protocol was approved by the Ethics Committee of Hakodate Junior College . This study was carried out in accordance with the recommendations of Provisions of Experiments, Ethics Committee of Hakodate Junior College with written informed consent from all subjects.The variables, text stimuli, and procedures were the same as in Experiment 1. Note that participants were divided into three groups for “Thank you” and “Telegram” and into two groups for “Weather forecast” and “Earthquake warning.” The order of trials for each text was randomized and different for each group.We merged the values of the four texts because the tendencies of data in the four texts were similar. A two-way within-subject ANOVA, with prosody type and presentation speed as factors and the rating value as the dependent value, was conducted for each judgment. The degree of freedom was corrected by Greenhouse-Geisser correction when the Mauchly’s sphericity test was found to be significant. When the main effect was significant, we tested it using Bonferroni corrections.The results are shown in Performances of people with hearing loss might depend on their nature of hearing impairment. Most participants in this study had sensorineural hearing impairment. The roots of sensorineural hearing loss are located in the inner ear, the vestibulocochlear nerve, or the central auditory processing center . Among rA two-way within-subject ANOVA was conducted for participants with sensorineural hearing loss from birth for each judgment. The degree of freedom was corrected by Greenhouse-Geisser correction when the Mauchly’s sphericity test was found to be significant. When the main effect was significant, we tested it using Bonferroni corrections. The main effects of speed and the main effects of prosody type were observed in all judgments. However, the interactions of speed and prosody type were not significant for all judgments. We did not conduct ANOVA for participants who were sensorineural hearing loss not by birth, because of their small number. However, 6 LPS tended to be preferred, and recorded and constant prosody tended to have more positive impressions for readability and favorability. Both recorded and constant prosodies were more positive at 6 LPS, and the difference between prosodies was not clear.As for the effect of presentation speed, readers with hearing loss reported the highest evaluations at 6 LPS –6. The rConcerning the effect of textual prosody, the full group of with hearing loss  and the p2) were larger than those in Experiment 1) and the advantage of recorded prosody was observed. It is assumed that people with hearing loss from birth would have more experience of visual languages  and would utilize it to process textual prosody. Further, it is assumed that visual information is more important for people with hearing loss from birth because they cannot rely on auditory information. Thus, they may extract more information from the visual language.These results suggest that textual prosody affects both readers with normal hearing and those with hearing loss. It is also suggested that, contrary to our prediction, readers with hearing loss from birth are sensitive to textual prosody  for both readers with normal hearing and hearing loss. There are three possible explanations for this. First, the effect of textual prosody was large at low presentation speeds because the differences in prosody types are relatively large at lower speeds than at higher speeds. Second, 12 LPS was too fast for readers to integrate the prosodic cues. Third, recorded prosody could support sentence processing when the processing of visually presented text is difficult because of slowness in presentation speed .When texts were presented at 6 LPS, readers with sensorineural hearing loss from birth had more positive impressions in recorded prosody, although readers with normal hearing had positive impressions in both recorded and constant prosodies. There are at least three possible reasons. First, when texts have no prosody (constant prosody), it appears that normal hearing readers could construct appropriate prosodies and prefer them. Readers with sensorineural hearing loss from birth, however, could not construct appropriate prosodies by themselves. As a result, they depended on textual prosody information (recorded prosody) and have positive impressions with it than constant prosody. Second, more positive impressions of recorded prosody in readers with hearing loss could also be due to more experience with visual language . They could apply this experience to process textual prosody. Third, the recorded prosody in this study was based on the reading of only one person. Thus, the recorded prosody was not always favored because the timing or the speed may be idiosyncratic to this particular individual.Experiment 2 showed that articulatory suppression could affect the impression of recorded prosody. It indicated that recorded prosody should be stored in the articulatory loop and that prosody information may be converted to auditory representation even when the prosody is visual. The results also indicate that the temporal structure of texts is processed regardless of whether the input is visual or auditory. Under these conditions, readers with hearing loss also might use the articulatory loop or might retain their information by using different means to store the textual prosody information. Further study is required to address these possibilities.In sum, the results of this study indicate that the temporal information is processed regardless of whether the input is visual or auditory. The results also demonstrated that textual prosody could enrich the reading not only of people with normal hearing but also of those with hearing loss, regardless of acoustic experiences. Although people with hearing loss have less acoustic experience than people with normal hearing, they may be able to utilize their experience of visual language, such as sign language, lip reading, and closed caption for processing textual prosody. Furthermore, textual prosody could be a useful tool to convey speech speeds and timing information visually.We should note that the enrichment of impressions was moderate in size of effect and unstable among reading contents and conditions, especially in readers with normal hearing. However, for recorded prosody, the degradation of impressions at 3 LPS could be somehow alleviated. The ratings of recorded prosody at slow speed were not as high as those of constant prosody at 6 LPS. Recorded prosody may support text processing when the processing does not go smoothly by giving readers clues to the structures of sentence. For example, multimedia situations where texts, music, and/or images are presented simultaneously  should be difficult for readers to concentrate text processing. Textual prosody, however, may engage readers’ attention and enrich text impressions under the multimedia situations. Textual prosody can achieve novel and people-friendly impressions of reading in digital books, TV or films.We have demonstrated the effectiveness of textual prosody (varying the speed and the timings of pause) in digital text presentation. However, there are also other types of dynamic text presentation. For example, scroll display, in which texts move from left to right, is often used on TV news or electronic message boards. It is difficult for scroll display to convey prosodic information, resulting in visual processing difficulties such as visual masking  and motiSome studies have tried to express prosodic information with written text. For example, Some issues must be considered. First, our discussion is based merged results based on four text stimuli. In the All datasets presented in this study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of Hakodate Junior College and Ethics committee of Aoyama Gakuin University. The patients/participants provided their written informed consent to participate in this study.MU, KM, and JW conceived, designed the experiments and wrote up the study. MU performed the experiments and analyzed the data. KM and JW contributed the software. All authors contributed to the article and approved the submitted version.KM and JW were employed by Communication Science Laboratories, Nippon Telegraph and Telephone Corporation. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.