Probing a bullfrog retina with spatially uniform light pulses of correlated stochastic intervals, we calculate the mutual information between the spiking output at the ganglion cells measured with multi-electrode array (MEA) and the interval of the stimulus at a time shift later. The time-integrated information from the output about the future stimulus is maximized when the mean interval of the stimulus is within the dynamic range of the well-established anticipative phenomena of omitted-stimulus responses for the retina. The peak position of the mutual information as a function of the time shift is typically negative considering the processing delay of the retina. However, the peak position can become positive for long enough correlation time of the stimulus when the pulse intervals are generated by a Hidden Markovian model (HMM). This is indicative of a predictive behavior of the retina which is possible only when the hidden variable of the HMM can be recovered from the history of the stimulus for a prediction of its future. We verify that stochastic intervals of the same mean, variance, and correlation time do not result in the same predictive behavior of the retina when they are generated by an Ornstein–Uhlenbeck (OU) process, which is strictly Markovian. The ability to predict or anticipate future events is crucial for the survival of animals. Predicting dynamical inputs can compensate the latency during information transfer and provide predictive information for learning and behavior , between the input and output as a function of a time shift δt between the two signals  should be determined by the predictive dynamics of the retina. However, it is still not clear what kind of information one can extract from Im(δt).Ideally, one would like to quantify and model the predictive properties of a retina. Although the phenomenon of OSR has been discovered for more than 10 years, it is still not clear how to relate OSR to the predictive properties of the retina. In OSR, information of the stimulation is apparently coded into the timing of the pulses. However, when there are fluctuations in the inter-pulse intervals of the stimulation, it is difficult to identify or even produce OSR. Therefore, it is not feasible to make use of OSR in inferring the predictive properties of a retina for the general cases of a non-periodic stimulation which should contain much more information than a purely periodic one. Bialek and Tishby have introduced the idea of predictive information based on the statistical properties of the input and output signal of a data processing system  is measured as a function of the properties of the light pulses, namely, its mean inter-pulse interval 〈τ〉 and correlation time τcor. Our main finding is that the location of the peak of Im(δt) can be shifted from δt < 0 to δt > 0 by an increase of τcor, suggesting that retina has the ability to predict (with some uncertainties) future events in the stimulation when the stimulation is regular enough. However, this ability of prediction can only be observed when 〈τ〉 is in the range of 100–200 ms, similar to that of the OSR phenomenon mentioned above measured in bullfrog retinas. Furthermore, this predictive property of a retina can be used to distinguish signals generated by an Ornstein–Uhlenbeck (OU) process from those generated by a Hidden Markovian model (HMM), with the signals from the HMM process being identified as more predictable by the retina.In this work, we report our experimental results in quantifying the predictive properties of a retina by using the predictive information method mentioned above. With a retina plated on top of a multi-electrode array (MEA) and probed by stochastic light pulses, characteristics of Our experiment is similar to that of Schwartz et al.  is fixed on the MEA by a permeable membrane and perfused with oxygenated Ringer's solution . Each retina preparation can last for 6–8 h for experiments  which illuminates the whole retina after reflected by a 50%:50% beam splitter. A photodiode (Hamamatsu S1223-01) is placed at the other end of the beam splitter to monitor the stimulation. The intervals between pulses are controlled by a computer to produce a train of pulses with different characteristics which will be described in details below.Retinas used in the experiments are obtained under dim red light from bullfrogs which were dark adapted for 1 hour before dissection. A piece of retinal tissue  and (2). Then, the series {τi} is rescaled so to have a standard deviation of 20 ms. An offset is also added to {τi} to obtain the desired mean 〈τ〉. With this method, the correlation of {τi} is not only controlled by Γ. The rescaling of its standard deviation and the addition of offset can also affect the correlation time of the series. The correlation time τcor of the resultant stimulation must then be measured by computing the decay time of its autocorrelation function. Note that when τcor tends to ∞, we will recover the periodic stimulation in OSR. With this stochastic pulse train, we can stimulate the retina using temporal patterns with continuously adjustable 〈τ〉 and τcor.Two types of stochastic intervals are used in our experiments. The first type is generated by a HMM following the idea of Palmer et al. , which iT reverting to a mean value in the long run. We construct the OU stimulation as:The second type of stochastic intervals is generated by the OU process , so the desired mean 〈τ〉 and standard deviation of {τi} (fixed at 20 ms as well) can be adjusted afterwards. Similar to Γ in the HMM stimulation, the correlation time of the OU process can be controlled by T.Identical to the HMM stimulation, Δ is fixed as 1/60 s and ξ is the Gaussian noise with zero mean and amplitude Stimulations constructed from OU process do not only have “first-order” statistics (mean and standard deviation) similar to the HMM stimulations but also have similar half-life decay of autocorrelation and auto-mutual information. The main difference between the OU process and the HMM is that there is no hidden variable in the OU process. Therefore, any differences in the responses from a retina under these two stimulations may imply that the retina can capture “higher-order” signatures  to discriminate between the two processes.Our experiments consist of recording responses of the retina under stimulations with different characteristics. The protocol is to present each set of stimuli continuously for 5 min in a random order, with an inter-experiment resting time of 2–3 min. All the experiments are carried out in a dark room with temperature around 25 °C. In the results reported below, over ten retina samples are used and at least three retina samples  are used to verify each experimental results.Responses from the retina are obtained as extracellular potentials from the 60 channels of the MEA system. Spike sorting is performed through the T-Dist E–M sorting algorithm in Offline Sorter software  in time for OSR. There are strong variations in the recorded responses from different channels of the MEA. As the mutual information between the response recorded by the MEA and the stimulation will be used in this work to quantify the predictive power of a retina, a channel is included for analysis only when its corresponding measurement is significantly (two times) higher than that obtained from its shuffled (time-randomized) version after the bias correction described below. In other words, we exclude channels which record firing patterns that share little information with the stimulation. Less than 25% of the selected units are removed after this validity check. We note that while the deviating performances of the removed channels might signify some different response types, the removal does not affect the conclusion of our statistical tests to be described below. More details of this removal criteria will be given below. Also, because of the finite size of measured data (limited sampling), there will be a bias in the calculation of mutual information. In all mutual information data reported below, the data have been bias corrected by using a method proposed by Strong et al. . DetailsFinally, to validate our findings, we perform the same experiments on more than five retinas to confirm that they give consistent results with what are reported in the current paper.cor between inter-pulse intervals, and the standard deviation of τ. During each experiment reported below, such a pulse train is presented to the retina for 5 min. Figure Figure t between the stimulation . The number of states for the response is then the maximum number of spikes for each channel within a time window of 50 ms. The maximum number of spikes within the 50 ms window is typically 10–15 in our recordings. The mutual information at time shift δt is then given by:p(xi) is the probability of having a state xi and p is the joint probability of the state . Note that the difference k ≡ δt/Δ in time indexes between s and r denotes a shift in time of δt. It can be seen from Figure Im has a peak located at negative δt and it is non-zero for δt > 0. The location of the peak at negative δt indicates that maximum information is shared between S and R when R lags behind S, confirming our intuition that the retina takes some time to reflect/process the information contained in S in producing R.Mutual information at different time shift δn Figure  and respn Figure  can thenIm in Figure t > 0 indicates that the firing patterns in retina carry some information on the future events in S(t) from its history. In fact, Im is termed predictive information by Bialek and Tishby  is fluctuating around a positive bias below 0.2 bits/s even when δt is much longer than the correlation time of S. One would expect Im to be zero for such a case. This non-physical property of the measured Im originates from the fact that we are computing Im from a finite time series. Bias corrections for finite data mentioned earlier have been applied in Figure Im, randomly shuffled data (either states of stimuli or firing rates) are used to compute the mutual information t. Also shown in the figure is the Im0 = 0.2 bits/s is the baseline value of our experimentally measured mutual information. As mentioned earlier, not all channels are included for analysis. The criteria is based on the difference between Im(δt) and its shuffled version, Im(δt)  is less than two times of that for Similar to the finding of Palmer et al. , the nond Tishby . It can S and R, Figure Im, Im, and Im from data displayed in Figures S and R. To quantify the amount of predictive information extracted by the retina, we define the predictive power based on measured Im as the ratio between the two areas in Figure Pp = a/A, where A and a are the area under the curves Im and Im for δt > 0, respectively. This definition satisfies the intuitive notion that Pp or Pp equals to 1, since the predictive power of a signal for itself is fixed as 1, and will allow the comparison of predictive information between different experiments. A remarkable feature of Figure Im and Im decay symmetrically about δt = 0, Im seems to decay more slowly for δt > 0. Since both R and S are symmetric with respect to time shift, the asymmetry of Im possibly comes from the anticipative nature of the retina dynamics in generating R. To test whether the conventional linear–nonlinear (LN) model  will not contain any information. With this interpretation, Figure cor ≈ 2.5 s.With the normalization introduced in Figure Im during our scan of τcor at fixed 〈τ〉 is that the peak location of the Im shifts from negative δt to positive δt as τcor is increased. Figure tp as a function of tp is the distance of the peak location of Im from the line of δt = 0. The inset of Figure tp and the forms of Im(δt) for τcor = 0.2, 2.0, and 4.0 s. Intuitively, one might expect δtp to be always negative because it will always take time for stimulations just to propagate through the different layers and synapses of the retina. That will be true if the retina is just a passive filter. However, if the retina is actively producing anticipative signals for the incoming events, a peak of Im(δt) at δt > 0 can be its signature.One interesting feature of the measured t but then generated response at time t by marching Equations (1) and (2) forward N steps while using ξi = 0, its most probable value. This gedanken retina is a mathematical construct based on the two equations we used to implement the HMM. Basically, we just pretend that there is an ideally predictive (“gedanken”) retina which can compute the velocity based on the input position. In other words, this “gedanken” retina is anticipating the future of the stochastic input from its present value N step ahead by using the velocity information. With this construction of response, we have implicitly assumed that the “gedanken” retina already “learned” the correct parameters of Equations (1) and (2) from it past experience. Figure N. It can be seen that the Im(δt) indeed has peaks at positive δt, confirming our intuition that a peak of Im(δt) at positive δt indicates anticipative dynamics of the system. Also, the asymmetry of Im(δt) observed in the experiment is well reproduced here. Note that the shift of the peak is larger when N is bigger but the peak value is smaller. That means when the “gedanken” retina is predicting too far into the future, its prediction is less accurate. When comparing our experimental results with different correlation times  from the “gedanken” retina can be higher than that of Im at the same δt. This means that the “gedanken” retina can have a better prediction about the stimulation in the future than by using the information contained in time series of the stimulation, {Si}. This is because {Si} is produced by an HMM. There is a hidden variable vi. The amount of information contained in {Si} can be smaller than that generated by the “gedanken” retina which knows about both variables by Equations (1) and (2). In other words, prediction is possible in this case because the “gedanken” retina can make use of the hidden variable. If this reasoning is correct, prediction from the retina should not be possible if the stimulations are generated from a Markov process.Another remarkable feature of Figure Im obtained from the experiments with the OU process for different correlation times. It can be seen that the peaks of Im from the OU process are all located at δt < 0 and more or less independent of the correlation time of the stimulation. Figure Im which is located at δtp > 0.Experiments with stimulations generated by an OU process, which is a Markovian process with no hidden variables, are carried out to test this latter idea. To generate the stimulations for the experiments, we tune the OU process in such a way that its time scales and fluctuations are similar to the HMM stimulations used in the experiments reported above. Figure cor, the less the amount of encoded information. With a periodic stimulation (infinite correlation time), there is no information encoded. In fact, this carrier wave picture is supported by our finding that both the OSR and the 〈τ〉 for optimal prediction have the same time scale.Although the periodic inputs used in OSR and the stochastic pulses used in this study seem to be quite different, the periodic pulses are in fact a limiting case of the stochastic pulses when the correlation time of the inter-pulse intervals becomes infinite. With this consideration, one can think of the periodic pulses used in the phenomenon of OSR as a carrier of information very much like the carrier frequency in an FM radio signal and the information is being encoded into the deviations (fluctuations) of this carrier period. Therefore, the stochastic pulses (with a fixed mean period) used in our experiments are then encoding information in its deviations from the mean. The amount of information encoded can then be characterized by the correlation time: the longer the τWe have therefore extended the study of anticipative capability of a retina from probing it with period stimulations to stochastic stimulations. Although the responses of the retina induced by these two types of stimulations seem to be very different, they are of the same nature. In the OSR, a clear transient, spontaneous (anticipative) response can be observed after the termination of the periodic stimulations, while there seems to be no clear anticipative responses can be identified after the termination of the stochastic stimulations. However, the results in Figure t) that corresponds the processing or propagation time of the system. To produce an output that is more informative of the stimulus at a targeted time in the future than at the current moment requires the system to filter out variability that is more pertinent to the current stimulus but has less bearing at the targeted time in the future. And, this stronger version of predictive behavior is what we discovered to be exhibited by the retina. Presumably, this predictive capability is implemented in retina through a population of cells and their specially wired circuitries. However, we did not perform experiments to determine the cell types explicitly. According to Schwartz et al.  and (2). For a real retina, information about this predictive mechanism can be revealed by this maximum time shift of the peak of the Im(δt) curve. For moving stimuli, it is relatively known that neural field models  curve could also be maximized at a positive time shift for a stochastic moving bar. It is still less well understood how such an active process is produced in the time domain. Knowledge of this mechanism should be helpful for the understanding of this anticipative dynamics from the physical structure of the networks in the retina.Finally, we would like to point out that, in our experiments, incoming information is coded into time intervals while we are using firing rates of the retina to compute the mutual information between the input and the response. This coding strategy is consistent with the dependency on pulse intervals of firing rate in OSR . The protocol was approved by the Institutional Animal Care and Use Committee of Academia Sinica.KSC: Experiments and data analysis, CCC: Computation Modeling and data analysis; CKC: Article planning and writing.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.


Parameters and explained variability differed across patients and control as well as between different activities. The models for the patient sample were stronger and particularly high for the document filing task for which kinematics explained 71% of the variance , was used to predict performance in activities of daily living (ADL) of patients suffering from mild cognitive impairment and dementia. 1 patient with dementia of the Alzheimer’s type and 15 healthy, age-matched adults were tested in the standardized ADL of tea making and document filing. The characteristics of the kinematic performance in the RTMT were assessed, and models of multiple linear regression were computed to predict the durations of the ADL. Patients showed increased trial durations (TDs) in the ADL (Cohen’s  Dementia of the Alzheimer’s type [Alzheimer’s disease (AD)] and other types of dementia frequently impair independent living by decrements in the performance of activities of daily living (ADL) and even mild cognitive impairment (MCI) can lead to problems in complex ADL –3. Such Trail making tasks are a common tool in clinical test batteries for the assessment of processing speed, mental flexibility, and executive functions , 24. UntIn this study, we introduced an adaptation of the trail making task A and examined the kinematics in the performance of 11 patients with AD and 15 age-matched control subjects. This reciprocal trail making task (RTMT) allowed a kinematic assessment of performance. In addition, to control for possible influences of impaired motor function we further applied a reciprocal aiming task (RAT), consisting of quick forth and back pointing movements between two marks, with emphasis on speed and accuracy. Patients and controls also performed two typical ADL that required the manipulation of everyday objects and which we have employed in previous studies in stroke patients: document filing  and makip = 0.86) control subjects  and the error frequency (see below).To gather the kinematics of the index finger in the RTMT and the RAT, a passive marker of a Qualisys motion capturing system , incorporating seven Miqus cameras at a frequency of 120 HZ, was attached to the dorsal part of the fingertip. The positional data were postprocessed using MatLab . The raw data were filtered applying a 0.5 s local regression (“loess”) filter.The following parameters were determined for each trial.TD: time needed to execute the task in 1 s. In the case of tea making, the (passive) waiting time when boiling the water was excluded.Frequency: movements per second in 1 Hz. A movement is defined by the transition from one mark to the other in the RAT.Relative activity (RA): relative amount of time with the velocity of at least one hand exceeding 0.05 m/s  exceeding 0.05 m/s in 1 m/s. The mean peak velocity is used to display the general movement speed : each forth and back movement in the reciprocal tasks was extracted, and the velocity plotted against the position (phase plot). In a next step, the ratio of circumference and covered area of this ellipse-like plot (measured ratio) was set into relation to the ratio of circumference  of the TDs in the ADL (Combined = mean z-score of performance in the two ADL) for each group with the parameters of the RTMT, the MMSE score (in the patient group), the count of pathologic scores in the three tests of apraxia (in the patient group), and age; (4) models of multiple linear regression for the TDs of the two ADL (separately) and for the two groups to check for differences in the resulting equations. All multiple linear regression models were adapted stepwise by excluding non-significant factors and factors with a variance inflation factor (VIF) exceeding 5. α was set to 0.05. The threshold for collinearity was set to VIF > 5.The statistical analysis consisted of the following: (1) ohen’s d ]; (2) a p = 0.64). All effect sizes in the kinematic parameters of the RTMT were in the range of 1.24–1.26. Exemplary trajectories in three-dimensional space of a patient (left) and a control subject (right) performing the RTMT are displayed in Figure Two trials of DFin the patient sample were excluded due to the patient failing to execute the task (1× DF) or due to a TD being recognized as an outlier . The patients took an average of 62.6 ± 39.8 s (range: 20–132 s) for DF and an average of 141.0 ± 62.51 s (range: 69–260 s) for TM. The control had average TDs of 30.8 ± 11.2 s (range: 17–55 s) for DF, and 72.9 ± 20.3 s (range: 46–110 s) for TM . The frequency in the RAT was the only factor in the model with β = −0.793 (p < 0.01).The model of multiple linear regression for the TD of RTMT by the parameters of RAT was not significant in the patient group but was significant in the control group with an z-scores of both ADL) by the parameters of RMT were significant for both groups  with an p = 0.01) and MH β = −1.063 (p < 0.01) and in the control model MH β = 0.552 (p = 0.03).The models of multiple linear regression for Combined (mean z-score of the TDs in the ADL (TM and DF), TD = dimensionless TD of the RTMT, and MH = dimensionless MH in the RTMT.The corresponding equations are as follows:The correlations of the predicted values against the original values are displayed in Figure The models of multiple linear regression for the two tasks in the two groups revealed differences concerning the significant factors Table . In bothIn this study, we examined the performance of 11 AD patients and 15 healthy, age-matched controls subjects in 2 ADL (TM and DF), the RAT (maximum motor capacity), and the RTMT (abstract sequencing task). Compared with healthy control subjects, patients revealed increased TDs in both tasks by approximately 100%. Increases of TD in the tea making task have already been observed in healthy elderly (+50% in comparison with young participants)  and in sThe models for Combined by the kinematic parameters of the RTMT revealed strong models for each group. The model for the control group had an The four models for the TDs of the two ADL in the groups differed substantially. On the one hand, the regression analysis for TM did not result in a significant model in the control group. This is interesting, since TM is the ADL with more degrees of freedom  and should therefore be more strongly associated with the cognitive demanding RTMT than DF. The model for DF in the control group was strong with an on times ]. A reasThe model for DF in the patient group was strong with an patients . On the Interestingly, the sum of pathologic scores in the three apraxia tests was not included in the patient models. This could be due to apraxic behavior and resulting errors having a likelihood to be fatal. Future studies could employ logistic regressions to examine possible associations between task success and apraxia scores.The present results indicate several meaningful points. AD had a strong impact on the performance of ADL. The performance in the RTMT differed between healthy controls and AD patients, and this difference appeared to be driven by the cognitive demands of the task for patients. The connection between ADL and RTMT performance is stronger in patients, which is underlined by the different impact of motor capacity on the performance in the RTMT. Analyses of the single ADL in both groups revealed that, if at all possible, predictions for controls are based on motor capacity. The patient models revealed some differences between the two ADL, showing that a generalization of ADL tasks should be done with caution. Nevertheless, TD was a common factor in both ADL models (and the model for Combined), leading to the assumption that the RTMT is covering similar cognitive demands as both tasks, which is very likely the sequencing. But one has to keep in mind that the sequencing in the RTMT is not driven by an ecological goal (like an end product in ADL) and the strong associations, especially in the patient group, could indicate different strategies of action planning in the two groups, meaning that patients appear to plan step by step, similar to a random walk. This could be tested by giving an ADL in which necessary objects are missing.The neurological basis of the observed strong connection between executive functions (RTMT) and ADL could be of two kinds: impairments of synchronous processing or in visual scanning. Lafleche and Albert  suggesteThe application of the RTMT as a clinical tool to assess ADL capability in patients seems to be promising. The model of Combined had no false positives or negatives in our sample. More interesting is the error frequency: only one of the patients was able to execute the task without errors and 13 of the 15 control subjects performed error-free. A combination of both approaches could include the following steps: (1) the error occurrence could indicate if the kinematic analysis is feasible. (2) The Combined model would then give an estimate of the severity of the possible ADL impairment . These assumptions need further support by larger patient samples and a more comprehensive range of tested ADL.Ethics approval was obtained by the ethics committee of the Faculty of Medicine of the Technical University of Munich. All participants gave written informed consent.PG and JH designed the study. SK, TG, and JD-S diagnosed and selected the patients. KL and SK organized the patients’ appointments and their transport. PG, KL, and AA performed the lab testing. PG performed the kinematic and statistical analyses. KL scored the ADL performance. All the authors contributed to the coordination of the study and the final draft of the manuscript.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.


Enlarged vestibular aqueduct (EVA) syndrome is a common congenital inner ear malformation characterized by a vestibular aqueduct with a diameter larger than 1.5 mm, mixed or sensorineural hearing loss that ranges from mild to profound, and vestibular disorders that may be present with a range from mild imbalance to episodic objective vertigo. In our study, we present the case of a patient with unilateral enlarged vestibular aqueduct and bilateral endolymphatic hydrops (EH). EH was confirmed through anamnestic history and audiological exams; EVA was diagnosed using high-resolution CT scans and MRI images. Therapy included intratympanic infusion of corticosteroids with a significant hearing improvement, more evident in the ear contralateral to EVA. Although most probably unrelated, EVA and EH may present with similar symptoms and therefore the diagnostic workup should always include the proper steps to perform a correct diagnosis. Association between progression of hearing loss and head trauma in patients with a diagnosis of EVA syndrome is still uncertain; however, these individuals should be advised to avoid activities that increase intracranial pressure to prevent further hearing deterioration. Intratympanic treatment with steroids is a safe and well-tolerated procedure that has demonstrated its efficacy in hearing, tinnitus, and vertigo control in EH. In adults, the vestibular aqueduct presents a diameter of 0.4–1.0 mm, with a mean value of 0.62 mm .Clinical presentation includes audiological and vestibular symptoms that often mimic those of other middle and inner ear disorders such as otosclerosis , 5 and eDiagnosis of EVA syndrome is radiological. Computed Tomography (CT) scan shows the bony labyrinth anatomy, and an axial CT with 1.5-mm sections generally provides the best view of the vestibular aqueduct from the vestibule to the posterior surface of the petrous bone , 18. MagNo treatment protocol for EVA syndrome was demonstrated to be uniformly successful in halting the progression of the disease; cochlear implantation is the optimal solution for hearing loss restoration when profound hearing loss is present .Intratympanic corticosteroid treatment for inner ear diseases by direct injection in the middle ear has gained wide popularity in the last years –25, presMany audiovestibular symptoms found in EVA syndrome are in common with other inner ear disorders such as EH; differential diagnosis is therefore important for a correct diagnostic and therapeutic management of these patients. In this paper, we describe the case of a patient with a history of bilateral EH and a radiological diagnosis of EVA in the left ear, along with a detailed description of the diagnostic workup and therapeutic approach.A 39-year-old man was admitted to the ENT department of our institution with a four-year history of fluctuating bilateral SNHL, associated with acute objective vertigo, nausea, and vomit (4–8 episodes/year); the vertigo attacks, lasting from 15 minutes to three hours, were often accompanied by headache. The patient had no history of acoustic trauma and/or noise exposure and had a previous glycerol test positive for EH.After admission, patient underwent a complete ENT examination with otoscopy, Pure Tone Audiometry (PTA), Acoustic Immittance Test, Transient Evoked Otoacoustic Emissions (TEOAEs), Distortion Products Otoacoustic Emissions (DPOAEs), Tympanometry, Cervical Vestibular Evoked Myogenic Potentials (cVEMPs), and caloric test.f1, f2 [f2 > f1]) and two intensity levels . cVEMPs were tested with the binaural simultaneous stimulation method, using an Amplaid MK22 polygraph . The electrodes were positioned as indicated by Colebatch et al. [PTA was carried out in a soundproof room and the pure tone thresholds for each side were measured at frequencies of 125, 250, 500, 750, 1000, 2000, 3000, 4000, 6000, and 8000 Hz; Air-Bone Gap (ABG) was measured at frequencies of 250, 500, 1000, 2000, and 4000 Hz. A standard 226 Hz tone tympanometry probe was performed to exclude external and middle ear pathologies. TEOAEs and DPOAEs were recorded in a sound attenuated chamber with an ILO-92 instrument . TEOAEs were evoked through 80–85 dB SPL stimuli, with a stimulation rate less than 60 stimuli per second, delivered through a probe inserted into the external auditory canal. DPOAEs were recorded with two acoustic stimuli (pure tones) at two frequencies . Targeted imaging of the vascular and nervous structures of the pontocerebellar angle were performed using axial 3-dimensional heavily T2-weighted images (3D TSE T2 WIs) and TSE T1 weighted images (TSE T1 WIs) with a slice thickness of 0.5 mm and 3 mm, respectively. Coronal T2 WIs were obtained using orthogonal planes to the long axis of the internal auditory canal and with oblique parasagittal and paracoronal planes (MPR reformatted images—slice thickness ranging between 0.4 mm and 3 mm).PTA revealed a threshold of 95.9 dB and an ABG of 42.5 dB in the left ear and a threshold of 97.70 dB and an ABG of 17.5 dB in the right ear . TEOAEs,Under local anesthesia , patient was treated with bilateral intratympanic prednisone (5 mg/mL) once a day for three consecutive days, followed by 7 days of treatment suspension and additional 3 days of injections, using a 25-gauge spinal needle inserted in the posteroinferior portion of the tympanic membrane. Pure tone audiometry, TEAOEs, DPOAEs, tympanometry, VEMPs, and caloric test were repeated after 8 days and one, three, and six months. PTA values one month after the first injection were 86.8 dB with an ABG of 28.75 dB in the left ear and 62.7 dB with an ABG of 12.5 dB in the right ear . ThreshoThe causes of EVA syndrome are currently unknown. Different authors have hypothesized a blockade of inner ear development during the fifth week of embryonic life, when its growth is maximal, and an abnormal communication between the subarachnoid space and the inner ear .Clinical manifestations in EVA syndrome are variable, suggesting that it may be related not only to anatomical abnormalities of the inner ear, but also to the physiology of the auditory and vestibular systems. In this case report, there are two important aspects to consider that, if missed, could lead to an incorrect diagnosis: the patient had a long-time history of bilateral hearing fluctuation and episodes of vertigo, suggestive for a diagnosis of bilateral EH. However, fluctuation in hearing can also be found in EVA patients, often following relatively minor head trauma; such fluctuations however are not usually associated with vertigo attacks . In thisAlthough EVA is a congenital disorder, some authors proposed that hearing loss in EVA syndrome is acquired as it has been reported to be triggered by minor head trauma . There iThe overall incidence of vestibular alterations in patients with EVA syndrome ranges from 12 to 86% . Emmett In the comparison of MRI and CT scan for the diagnosis of an EVA syndrome, current literature suggests that both techniques are complementary for identifying structural alterations , 32. MRIThis patient was treated with intratympanic injection of corticosteroids with a partial hearing restoration, more evident in the right side, and an improvement in vertigo symptoms. As expected, the larger benefits in hearing restoration following corticosteroid treatment were seen in the ear contralateral to EVA. One of the first reports regarding the effects of intratympanic treatment of steroids for EH showed an 80% improvement in vertigo . AfterwaIn the literature, to the best of authors' knowledge, there is only one case report of a patient with EVA syndrome and EH, in which the authors hypothesized that the two conditions may be due to a common primary dysfunction of inner ear fluid homeostasis . AlthougPatients with a diagnosis of EVA, in the presence of serviceable hearing, should be advised to avoid contact sports or activities that increase intracranial pressure to prevent hearing loss or further hearing deterioration. Intratympanic treatment with steroids is a safe and well-tolerated procedure that has demonstrated its efficacy in hearing, tinnitus, and vertigo control in EH.


Cochlear implants are the treatment of choice for auditory rehabilitation of patients with sensory deafness. They restore the missing function of inner hair cells by transforming the acoustic signal into electrical stimuli for activation of auditory nerve fibers. Due to the very fast technology development, cochlear implants provide open-set speech understanding in the majority of patients including the use of the telephone. Children can achieve a near to normal speech and language development provided their deafness is detected early after onset and implantation is performed quickly thereafter. The diagnostic procedure as well as the surgical technique have been standardized and can be adapted to the individual anatomical and physiological needs both in children and adults. Special cases such as cochlear obliteration might require special measures and re-implantation, which can be done in most cases in a straight forward way. Technology upgrades count for better performance.Future developments will focus on better electrode-nerve interfaces by improving electrode technology. An increased number of electrical contacts as well as the biological treatment with regeneration of the dendrites growing onto the electrode will increase the number of electrical channels. This will give room for improved speech coding strategies in order to create the bionic ear, i.e. to restore the process of natural hearing by means of technology. The robot-assisted surgery will allow for high precision surgery and reliable hearing preservation. Biological therapies will support the bionic ear. Methods are bio-hybrid electrodes, which are coded by stem cells transplanted into the inner ear to enhance auto-production of neurotrophins. Local drug delivery will focus on suppression of trauma reaction and local regeneration. Gene therapy by nanoparticles will hopefully lead to the preservation of residual hearing in patients being affected by genetic hearing loss. Overall the cochlear implant is a very powerful tool to rehabilitate patients with sensory deafness. More than 1 million of candidates in Germany today could benefit from this high technology auditory implant. Only 50,000 are implanted so far. In the future, the procedure can be done under local anesthesia, will be minimally invasive and straight forward. Hearing preservation will be routine. Cochlear implants are electronical stimulus prostheses for the functional replacement of the inner ear. Because of the rapid technical development and the good results, they could be established as standard therapy for sensory deafness.Cochlear implantation requires an interdisciplinary team and a quality-controlled concepts that reaches from the indication to the life-long control and that is fixed in the AWMF guideline on cochlear implant .Current cochlear implant systems are partially implantable and equipped with a multitude of additional functions similar to hearing aids for sound pre-processing and noise elimination. The intracochlear position of the electrode allows for a differentiated stimulation of the hearing nerves and thus the transmission of different perceptions of pitches. This simulation of the frequency organization of the inner ear leads to the fact that complex sound signals such as speech are transformed into a differentiated neuronal stimulation pattern of the hearing nerve, which is the base for speech understanding with a cochlear implant.Today, indications are bilateral sensory hearing loss and deafness in children as well as in adults, single-sided deafness as well as high-frequency hearing loss. Cochlear implants are indicated when a sufficient speech and communication ability (use of the telephone) or speech development are not possible or cannot be expected with alternative methods.The surgical technique has been standardized and can be applied in all patients. Generally, a transmastoid procedure with posterior tympanostomy and insertion of the electrode through the round window membrane is favored. The fixation of the implant in the bone as well as the secure fixation of the electrode near the cochlear represent crucial elements for a low-complication procedure. Cochlear implant surgery with preservation of hearing is standard today and allows treatment of patients with residual hearing.Beside the functional control of the implant, the intraoperatively assessed electrophysiological parameters allow an adjustment of the systems, especially in children, based on objective parameters. The subsequent hearing and speech training aims at speech acquisition and speech recognition. The life-long follow-up comprises also technological upgrades and the treatment of complications beside medical and technical controls.Generally, postlingually deafened patients achieve an open speech understanding and may use the phone. In children, an early implantation after onset of deafness usually leads to a nearly normal speech development.The complication rate is rather low. Implant failures occur in about 2–4% of the patients, medical complications are observed in about 4% of the implanted people. Re-implantations can usually be performed without any problems. The patients benefit from a technological upgrade. Future developments focus on the bionic ear aiming at the restoration of hearing by simulation of the physiological hearing process by means of the technology. For this purpose, electrodes are developed with a significantly higher number of electrically separated channels. By surface functionalization and additional biological therapies, the regeneration of the hearing nerves with dendrites growing onto the electrode as well as avoiding further degeneration of spiral ganglia cells will be achieved. This may lead to significantly better speech processing strategies that even allow for a tonal hearing, for example of music. Telemedical concepts provide new types of patient care with active involvement of the patients, automated technological implant control, remote care, programming, and technological upgrades. Universal hearing implants will become possible due to multimodal stimulation with integrated intracochlear mechanical or optoacoustic actuators. These implants allow for an individually optimized hearing rehabilitation and can be readjusted at any time in cases of progredient hearing loss. The use of robotic systems will lead to a relevant increase of precision and improved hearing preservation. So-called closed loop systems with measurement of the EEG signal will allow an automated adaptation of the implant system to different hearing situations. Fully implantable hearing systems are currently developed and will make possible the so-called invisible hearing to overcome the stigma of hearing impairment.Cochlear implants are electrical prostheses that trigger auditory sensations via a direct electrical stimulation of the hearing nerve. They replace the function of the inner hair cells that have the role of biological microphone. Hereby, a technical simulation of the natural hearing process is performed with tonotopic presentation of the frequencies along the basilar membrane on different parts of the hearing nerve Figure 1 . In compFirst attempts of hearing rehabilitation were performed by Djourno and Eyries in Paris at the end of the 1950ies . In the Even if the initial systems showed a high failure rate, the adaptation of the technology of the cardiac pacemaker led rapidly to a relevant improvement of the reliability of cochlear implants. The transcutaneous transmission substituted soon the percutaneous connector system that was often related to complications. Because of the technical and surgical difficulties despite good channel separation, the intraneural stimulation  was not further developed .Current systems insert intracochlear electrodes in the scala tympani, which allows reliable stimulation of the hearing nerve without relevant complications .The cochlear implant systems of today are generally conceived as two component systems. The external speech processor is used for sound recording, sound pre-processing, and transformation of the acoustic information into a logical sequence of electrical impulses  and it is the sender of the FM signal for a transcutaneous transmission and power supply of the implant per induction by a sending coil. The implant that is located under the skin, contains the receiver coil for reception of the FM signal, a demodulator for extraction of the electrical pulses, an electrode carrier with different intracochlear electrode contacts for transmission of the electrical impulses to the hearing nerve and telemetric measurement systems. Current cochlear implant systems dispose of a broad spectrum of signal pre-processing including directional microphones, beam former, noise elimination, and acoustic scene analysis, and of a mostly wireless interaural connection of the systems in cases of bilateral and bimodal treatment. In this way, both speech processors can be synchronized. Telemetric measurement systems record electrophysiological data with the implant itself such as electrode impedances, measurement of acoustically and electrically evoked potentials Figure 3 . Some syDifferent electrode systems are available for the individual cochlear implantation Figure 4 . GeneralSpecial electrodes are available for malformations and for ossified cochleas . Those arrays distribute the electrode contacts on 2 electrode carriers that are inserted into the first and second turns via 2 cochleostomies ..11].The preservation of the residual hearing ability in the context of high-frequency hearing loss allows the use of electroacoustic stimulation. In the future, new treatment concepts for presbyacusis might be developed , 13], , .The indication requires standardized diagnostics that beside general age-independent examinations include additional elements for adult and children Table 1 .Because of the small dimensions of the structures, high resolution imaging, especially of neuronal structures, is most important. Since often malformations are the cause of pediatric deafness, an exact assessment and description is essential for the therapeutic approach. The verification of inner ear structures requires special sequences and section planes so that for example all 4 nerves may be identified in the internal auditory meatus  Figure 9 .Residual hearing: It is very important to assess the hearing ability in order to estimate the benefit for speech understanding as well as the potential for hearing and language development. While the focus is placed on psychoacoustic procedures in adults, the diagnostics in children are supported by objective measurements. If required, also follow-up examinations are indicated, for example in cases of substantial residual hearing and progredient hearing loss (see chapter on limiting cases).Generally, the function of the hearing nerve may be verified with the so-called promontory test. By means of a needle electrode that is either placed transtympanically on the promontory or by means of auditory canal electrodes, stimulation currents of different frequencies between 50 and 1600 Hz may be applied that trigger an auditory sensation in the patient. With decreasing frequencies, the dynamic range narrows due to then dominating pain sensation. Auditory fatigue or a missing hearing impression may indicate a neural or central deafness. An objective functional test is possible by recording electrically evoked brainstem potentials. Despite the low sensitivity, this method is a valuable diagnostic procedure for pediatric patients. Further objective procedures are methods of functional imaging with identification of an increased activity in the area of the auditory cortex under electrical stimulation. Those are for example the positron emission tomography (PET) , functioCT scan and CBT further allow sizing of the cochlea, especially of its length. This is the basis for selection of the electrode carrier and for determination of the insertion depth in the context of individualized cochlear implantation .One relevant pillar of the etiological clarification is the genetic diagnosis. About 50–60% of pediatric hearing impairment are due to a genetic predisposition. They are classified into non-syndromic and syndromic types of hearing loss, i.e. generally the hearing loss is a symptom in the context of a syndrome.Currently, more than 100 so-called deafness genes are known. The most frequent expression is the mutation on the gene locus GJB2 that leads to a disorder of the connexin molecule (Connexin 26), a gap junction protein. The consequences are disorders of the ionic homeostasis of the hair cells that are irreversibly damaged by a potassium intoxication , 20]..20].The difference is made between autosomal recessive, autosomal dominant, X-linked, and mitochondrial, genetically caused hearing losses. Autosomal recessive types often reveal deafness already at birth and are entitled DFNB (e.g. DFNB1 with connexin 26 disorders). Autosomal dominant types often have a postnatal onset and are progredient (DFNA). X-linked hearing losses are located on the X chromosome and only appear in males (DFN). Mitochondrial hereditary hearing impairment is passed on by maternal genes.Generally, the patients who deafened postlingually, dispose of an acoustic memory. They benefit highly from cochlear implantation. The important inter-individual variability reveals further prognostic factors. Those are the duration and the time course of deafening, the presence and the use of residual hearing by hearing aids as well as cognitive abilities and their impairment, for example in the context of pre-dementia  .The following audiometric limit values are applied today for adults:Mean hearing threshold in the audiogram (250–4000 Hz) >75 dB HLSpeech understanding in the Freiburg monosyllabic test <45% at 65 dB under best-aided condition (hearing aids)The last mentioned value is based on the ability to use the telephone. Phone calls are possible with an understanding of monosyllables >50%.The measurement of speech understanding in noise provides an additional criterion for the evaluation of communication disorders under difficult acoustic conditions  , 23]..23].th year of life) it is called post-lingual deafness. If hearing loss is detected during the phase of language acquisition, it is the case of a peri-lingual deafness.The origins of deafness in children are manifold. The difference must be made between congenital and acquired causes as well as the time of deafness . If the onset is observed before language acquisition, the term of pre-lingual deafness is applied, after final language acquisition .The maturation processes in the field of the hearing system have to be observed. Initially, slightly increased hearing thresholds may improve or normalize in the context of maturation. In cases of higher-grade sensorineural hearing loss, however, this cannot be observed .Cochlear implantation in children should be performed immediately after indication. Generally, it may be done after the 6th month of life in the first year. Bilateral implantation should be performed in cases of bilateral deafness, if possible simultaneously or otherwise as sequential implantation with a short time interval in order to use the sensitive phase for the development of binaural hearing , 29]..29].Hearing aid fitting beside unilateral cochlear implantation is indicated in cases of asymmetric hearing with unilateral sensory hearing loss and useable hearing ability in the contralateral ear. Narrow controls of the residual hearing as well as the hearing success in this ear must be performed so that progredient hearing loss or development failure of bilateral hearing are detected in time and the point of then necessary sequential cochlear implantation of the second ear may be correctly chosen , 30], , .If maturation is delayed, as it may be expected in cases of increased hearing thresholds and prolonged inter-peak latencies in brainstem audiometry, control examinations in narrow intervals and probatory hearing aid fitting seems to be appropriate.Often hearing improvement is observed due to maturation of the peripheral auditory system and the central hearing pathway so that a repetition of the examination of the residual hearing after some weeks or months is indicated. If no significant improvement of hearing is observed, cochlear implantation should be performed in the second year of life at the latest.In this context, the so-called perisynaptic audiopathy must be mentioned summarizing disorders of the inner hair cells, the synapsis to the afferent nerve fibers as well as true auditory neuropathy with damage of the afferent neuron (auditory neuropathy spectrum disorder). This term encompasses different pathophysiological disorders of stimulus transmission and stimulus forwarding in the peripheral auditory system. Only in the context of true auditory neuropathy a relative contraindication for a cochlear implant system exists.The typical constellation of the findings is described in Table 2 In cases of particular urgency such as for example threatening cochlear obliteration by labyrinthitis and meningitis require immediate, early implantation.Limiting cases describe patients with a relatively good hearing and a comparable poor speech understanding, e.g. often in cases of Menière’s disease, fluctuating hearing ability, or combined hearing loss with a bone conduction threshold that is difficult to determine. In such cases, the exact differential diagnosis is relevant with application of objective measurement procedures including electrocochleography. If after optimized hearing aid fitting and sufficient observation no improvement of speech understanding is observed, cochlear implantation is indicated.If malformations or obliterations lead to a missing hearing and language development in children or missing hearing impression in post-lingually deafened patients, a central-auditory implant, generally an auditory brainstem implant (ABI), is indicated in cases of bilateral disorder . This alThe surgical technique is largely standardized and may be applied in all age groups and special cases. Generally a transmastoid surgical approach with posterior tympanostomy is performed comprising the following steps :Retroauricular incision  Figure 3  - Cochlear monitoring During insertion of the electrode, residual hearing can be monitored by measuring cochlear microphone potentials outside and inside the cochlea. Critical changes of the Cochlear Microphonics amplitude indicate an impairment of the inner ear function. By repositioning the electrode, a permanent hearing loss might be avoided , 35], ,  Figure . Appropriate wound closure in several layers to securely cover the implant.The intraoperative control of the electrode position by radiography or CBT is the standard in order to identify insertion failures in time and to correct them in the same session Figure 4 . Furtherth month of age within the first year of life in cases of congenital deafness. Only in cases of particular urgency such as the risk of obliteration in the context of labyrinthitis, the implantation should be performed earlier. The implantation should include both sides to allow the development of binaural hearing with the ability of directional hearing and improved speech understanding in noise. Hereby, the simultaneous implantation should be preferred if it is possible from an anesthesiologic point of view. Compared to sequential implantation, the following advantages should be mentioned:Because of the immaturity of the organs, the implantation should be performed generally from the 6Only one hospital stayOnly one anesthesia for surgerySimultaneous activation of the hearing system for the development of binaural hearingThe disadvantage is the prolonged anesthesia and the duration of surgery with the associated risk for example of increased blood loss. If sequential bilateral cochlear implantation is performed, the interval should be rather short in order to keep the auditive deprivation of the second ear as low as possible and thus to achieve a similar hearing ability for both ears. If the intervals are longer, poorer hearing results in the later implanted ear and poorer bilateral and binaural hearing performance are observed  Hearing preservationb) Obliteration of the cochleac) Malformationsd) Re-implantationsThose cases require special surgical techniques, large experience of the surgeon, special equipment and special implants.To preserve residual hearing, a so-called soft-surgery technique has to be applied. Generally, the insertion is performed through the round window membrane. The electrode is carefully inserted to a pre-determined depth and then fixed  for every single electrode contact. Then the loudness between the contacts is balanced, the dynamic range is defined, and the speech processing strategy is selected. The strategy is an algorithm according to which the acoustic signal is transformed – completely or partially – into a defined sequence of electrical pulses that are then transmitted to the hearing nerve via the electrode. The aim is a possibly physiological activation pattern of the hearing nerve.The single electrode contacts are allotted to different frequency ranges in the form of frequency bands . So-called NRT based maps allow an approximation of the profile over the complete electrode carrier as soon as a T and C level can be psycho-acoustically determined on an electrode contact. Additionally, electrically evoked brainstem potentials and EEG signals are applied . In ordeTelemedical procedures allow remote fitting. Hereby, the patient is connected with an interacting center via a data transmission line. The specialist in the cochlear implant center is able to observe the patient and talk to him directly. A direct access to the implant is possible by a specialist on site or via an interface that the patient may control himself. In this way, fine tuning, especially with regard to the domestic setting, technology checks, and upgrades of the software may be performed.Via the telemedical connection, daily implant controls are possible. This allows early detection of increased impedances as sign of a labyrinthitis onset. Remote care is particularly important for the life-long follow-up .Self-fitting by the patient will also be possible in the future. The patient will be enabled to optimize single parameter settings due to his subjective hearing experience by applying certain interactive procedures, as soon as the first fitting is complete. This procedure will be increasingly used after first fitting and hearing experience.To support hearing and speech acquisition, especially in children, specific rehabilitation measures are suitable. This is also true for adults who experience only slow progress or who benefit from a more intense therapeutic approach because of unfavorable prognostic factors such as long-term deafness. Significantly better hearing results may be achieved in this way .After implantation, the surgeon is responsible to organize and perform a life-long follow-up. It refers to the technical check-up as well as the settings of the implants. Furthermore, regular updates of the software and hardware are necessary since they make progress of the implant technology useable for the patients. Besides, medical complications and functional failure can be detected and addressed.See chapter 5.2.The outcome is assessed and documented by means of standardized test procedures.The thresholds with cochlear implant are measured. They should amount to values between 20 dB and 30 dB over the whole frequency spectrum. The consistent amplification over all electrode contacts is important for a good hearing result.For the assessment of speech understanding, age-dependent test procedures are available. They register the speech understanding for numbers and monosyllables as well as the understanding of sentences in quiet and in defined noise.Generally the Freiburg speech intelligibility test for monosyllables is used in adults. Comparative evaluations regarding the preoperative hearing status as well as the development of the speech understanding over the time may be documented. In addition, speech tests in noise such as the Oldenburg sentence test (OLSA) or the HSM sentence test allow for the determination of speech understanding under difficult hearing conditions.In the context of children, speech development is documented. In order to take into account that the test results depend on the age and to perform comparative evaluations, the scale of the CAPs (categories of auditory performance) was developed . These CThe total hearing situation in cases of bimodal and bilateral cochlear implantation can be assessed by free-field testing. Often the patients are either bimodally treated (cochlear implant and hearing aid) or bilaterally (2 cochlear implants) or have a hybrid system for electroacoustic hearing in one ear and a hearing aid in the contralateral ear . The various hearing situations have to be assessed separately and the percentage of the different hearing modalities  regarding the total hearing situation must be evaluated.In general, stable hearing results are achieved after 6–12 months. About 70% obtain an open speech understanding. However, those results are highly diverse . For children, a comparison with normally hearing children can be made regarding hearing and speech development. Usually, early implanted children (1st year of life) achieve very good speech development scores that nearly correspond to those of normally hearing children, especially in quiet environments. In noise, however, poorer scores are observed, which reveals that a cochlear implant does not make a child normally hearing but that it is hearing impaired. Under the aspect of the overall development, it can be summarized that, compared to normally hearing people, deficits remain even in cases of early implantation that impair the cognitive development of the brain. This is mainly due to the close interrelation between the hearing system and other brain areas and functions  Figure .In cases of early implantations, about 2/3 of the children may visit regular schools . The proIn summary, the hearing efforts of cochlear implant patients is significantly higher compared to normally hearing people. This means that a higher percentage of the cognitive capacity is used for hearing and thus the cognitive load increases. The remaining cognitive capacity for the actual learning process is hereby clearly restricted.Hybrid systems for electroacoustic stimulation generally lead to a clear improvement of speech understanding especially in noise, to a better directional hearing as well as a better tonal hearing (e.g. music). It is crucial to preserve residual hearing in low frequencies to use the specific advantages of acoustic hearing and to combine them with electric hearing. Limit values for the useable residual hearing in low frequencies amount to about 60 dB at 500 Hz , 13], , . The eleThe objective of bilateral cochlea implantation is an improved speech understanding in noise as well as directional hearing. Both parameters can be achieved in both ears under comparable conditions. Children may develop binaural hearing in this way (see chapter 3.4)  Figure .In cases of unilateral deafness, the cochlear implant may lead to a significant improvement of speech understanding in noise as well as directional hearing and tinnitus suppression. Early implantation in children may induce the development of binaural hearing , 11]. H. H11]. HDevice failure occurs in about 2–4% of the cases. In children they are more frequently observed than in adults which is mainly due to a higher incidence of external forces. The continuous technical improvement of the implant, in particular since the introduction of titanium cases by nearly all manufacturers, a clear reduction of the cumulative failure rate  could be achieved . Beside When a device failure is observed and confirmed, re-implantation should be performed as soon as possible. This is especially true for children with implant in only one ear , 62]..62].Medical complications may occur during or after surgery.Intraoperative complications mainly occur as damage of the facial nerve, the sigmoid sinus, the internal carotid artery, or the ossicular chain. Further complications are injuries of the external wall of the auditory canal, the tympanic membrane, and the dura.In general, they can be avoided by an adequate surgical technique. A low complication rate reflects a high quality standard of cochlear implantation and sufficient training due an adequate minimum number of surgeries performed per year , 45], , , 64] , these factors may be addressed by a precise insertion of the electrode. Beside the selection of suitable trajectories, the optimal insertion of the electrode as well as the 3-dimensional control of the other parameters play a crucial role  Figure .The improved electrode-nerve interface allows new and better possibilities of speech processing strategies. Those are algorithms that translate the acoustic signal into a logical sequence of electrical pulses for the cochlear implant system. In terms of a significant improvement of the electrode-nerve interface with a higher number of electrically separated channels, other speech coding strategies can be applied that aim at an increase of the transmitted information, a spectral contrasting, and a simulation of physiological stimulation patterns of the hearing nerve. Suitable modelling of the individual electric distribution in the cochlea allows for the best combinations of electrode contacts for stimulation.The use of objective parameters, especially of acoustically evoked compound action potentials of the hearing nerve as well as the centrally located auditory evoked potentials, currently so-called closed-loop systems are being developed, in which additional registration electrodes are placed for example above the auditory cortex. The implant serves at the same time as sensor (theranostic implant).The obtained EEG signals can be used for fine tuning of speech processing with selection of appropriate parameters and for the support in difficult acoustic situations. This is of high importance especially for children. Because of the higher complexity of speech coding algorithms, it becomes more and more important also in adults and it will substitute the currently manual programming.The increasing number of patients that are treated with residual hearing makes it necessary to adequately use the acoustic residual hearing in combination with the electrical hearing. For this purpose, electrode systems with integrated mechanical actuators or an optoacoustic fiber actuator are developed. They can be adjusted to the individual functional state of the inner ear and allow an optimized usage of the cochlear reserve.In order to achieve a synchronized stimulation of the cochlea for the acoustic and electrical stimulus, it is necessary to timely harmonize both stimuli. Additionally, the mechanical stimulus should be coupled ideally into the cochlea to avoid coupling problems. Hereby, the use of multimodal stimulation systems is suitable; here, a mechanical stimulator may be also an integral part of the electrode system. Further, actuators on electromagnetic or piezoelectric basis are possible. Those so-called electromechanical stimulations open new possibilities for the use of the cochlear reserve and may adjust to a changing residual hearing so that ideally re-implantation of the patient can be avoided.Stimulation with high-energy optical short pulses via laser systems leads to a stimulation of the hair cells in the inner ear. The basis is the so-called optoacoustic effect based on thermo-elastic expansion. The applied laser pulse leads to a short-term temperature increase of the biological tissue and thus the development of a mechanical pulse. This is the adequate stimulus for the hair cells to be activated.This optoacoustic effect might also be used for electromechanical stimulation that was described above .In contrast to optoacoustic stimulation, that does not directly activate auditory neurons, the optogenetic stimulation uses the sensitization of the neuronal tissue by initially applied pigments. They can permanently be generated via genetic manipulation so that the treated spiral ganglia cells are sensitized for optical stimuli .Advances in battery and microphone technology allowed fully implantable hearing systems. The energy supply is performed by transcutaneously charged batteries. The sound reception occurs transcutaneously. If needed, an external speech processor can be coupled. Currently battery lifetimes of about 10–15 years are considered as being realistic. The patient gains further freedom of action and loses the stigma of disability. One disadvantage is the restriction to software updates to participate in the technological progress outside the re-implantation period.By including the cochlear implant in a superordinate communication system, the possibilities of audiotechnology and telecommunication can be fully used for the cochlear implant. The control is performed for example via Bluetooth.Rapid technological progress of cochlear implants makes the development of the bionic ear more and more realistic. Improved hearing results will lead to the fact that more patients benefit from this technology. Currently there are about 1 million candidates for cochlear implantation only in Germany, actually about 50,000 are implanted. To reach all patients, according advances in the fields of technology and biology are necessary. Those technical developments will allow performing surgery in a minimally invasive way, if possible under local anesthesia, during a short-term intervention. Suitable automated procedures will allow for an adequate fitting and thus create the preconditions for hearing that is as natural as possible.The author declares that he has no competing interests.


CIDP), exclusion of secondary axonal degeneration is challenging with conventional methods such as nerve conduction study (NCS), needle electromyography, and nerve biopsy. Increased echo intensity (EI) and decreased muscle thickness (MT) identified on muscle ultrasound (MUS) examination represent muscle denervation due to axonal degeneration in neurogenic disorders, suggesting MUS as a new tool to detect secondary axonal degeneration in patients with CIDP.In chronic inflammatory demyelinating polyneuropathy .EI and low MT, suggesting denervation following secondary axonal degeneration. These six muscles belonged to two patients with long disease history, unresponsiveness to treatment, and long interval from onset to initial therapy. There were no significant differences in EI and MT  between the CIDP and NC groups, although NCS results revealed obvious demyelinating abnormalities in all CIDP patients, suggesting the fact that muscle structures will be preserved, and EI and MT will not change unless secondary axonal degeneration occurs in CIDP.Six of 45 muscles showed abnormally high MUS is a promising tool for evaluating secondary axonal degeneration in patients with CIDP. The NCSs on median and ulnar nerves and the MUS on APB, ADM, and FDI were performed on the clinically more affected side determined by the same neurologist (MS). Both NCSs and MUS were conducted on the same day or with an interval of less than 1 week. All MUS procedures were performed by the same examiner (KH), whereas NCSs were performed by several trained doctors or technicians other than KH. The MUS and NCS results were withheld from the examiner of the other test. In addition, detailed medical history including disease duration, severity of weakness, and medications was also blinded, although the diagnosis of the patient was known to every examiner. In NCs, MUS was conducted only on the dominant hand.2.3Motor NCSs were performed on median and ulnar nerves in all subjects via standard methods of our laboratory using an MEB‐2200 EMG device . The CMAPs of the APB were recorded in median NCS with stimulations at the wrist and elbow. The CMAPs of the ADM and FDI were recorded simultaneously in ulnar NCS with stimulations at the wrist, below and above the elbow, the upper arm, and the Erb's point. Peak‐to‐peak CMAP amplitude, distal latency, distal CMAP duration, and nerve conduction velocity were evaluated. Abnormal findings were determined using normal data obtained at our laboratory. In the ulnar NCS with FDI recordings, only CMAP amplitudes were evaluated as normal values for other parameters were not available. Skin temperature was measured on the palm and was maintained at 33°C or higher throughout the examination.2.4MUS was performed in APB, ADM, and FDI. All MUS examinations were conducted using a Nemio MX ultrasound device and a 7.5–11 MHz linear array probe with a peak frequency at 11 MHz . Equipment settings were maintained as follows during the entire examination: gain, 84 dB; compression, 55; time‐gain compensation in neutral position; and three focal points at 1.5, 2.0, and 2.5 cm. All subjects laid in the supine position. The probe was placed along the line connecting the midpoint of the volar aspect of the first metacarpophalangeal joint and the volar prominence of the scaphoid bone for APB, along the line connecting the midpoint of the fifth metacarpophalangeal joint and the pisiform bone for ADM, and on the dorsal surface of the hand perpendicular to the axis of the second metacarpal bone at the level of the first metacarpophalangeal joint for FDI . Region of interest (ROI) was set to cover the entire muscle cross‐section in the ultrasound screen, and the mean gray‐scale value of the ROI was measured and expressed as a value between 0 (black) and 255 (white). The measurement was repeated three times and the mean value was adopted. MT was measured using an electronic caliper equipped with an ultrasound device. The thickest location of the muscle belly was measured in each muscle. All quantitative assessments were performed by the same examiner (KH).2.6z‐scores for standardization using values from the corresponding NC group. Normal ranges for EI and MT values were determined according to the z‐scores of the NC group. The z‐scores of both groups were tested using the Mann–Whitney U test to determine significant differences between the CIDP and NC groups.To evaluate the test–retest reliability of consecutive quantitative MUS assessments performed by the same examiner (KH), intraclass correlation coefficient (ICC)  for EI and MT was assessed in 10 randomly selected subjects in the NC group. The same examiner (KH) performed three separate tests for each subject. The first and second tests were performed on the same day with an interval of at least 3 hours, and the third test was performed 1–3 days after the last test. The ICC  was calculated from the three examinations. Stepwise multiple linear regression analysis was performed in the NC group to evaluate whether gender and age were independent predictors of EI and MT, as previously reported  were enrolled. Disease duration was 27.5 ± 33.2 months . The MRC scales of APB, ADM, and FDI were 3.3 ± 1.1, 3.0 ± 1.0, and 3.2 ± 1.0, respectively. Eight patients were newly diagnosed with CIDP. Of the eight patients with recurrent CIDP, seven patients had previously received intravenous immunoglobulin therapy (IVIg). However, the last course had been administered more than 5 months prior to entry into this study. Three patients were treated with corticosteroids and/or immunosuppressants as maintenance therapy. One patient had not received any medications previously  Table . Data ob3.3The ICCs  of EI and MT displayed substantial agreement among the muscles. The ICCs  of EI and MT in the APB, ADM, and FDI were 0.78, 0.74, and 0.80 and 0.85, 0.90, and 0.89, respectively. These results determined that the reproducibility of the quantitative assessment was guaranteed and further experiments were warranted.In total, 45  and 180  muscles from the CIDP and NC groups, respectively, were included in the final analysis. Stepwise multiple linear regression analysis in the NC group revealed that gender significantly affected the EI of the ADM and FDI as well as the MT of every muscle. Furthermore, within the NC group, age significantly affected the EI of every muscle and the MT of the FDI Table .3.4z‐scores as +2.5 for EI and −2.5 for MT, as all values of the NC group fell within this range. In the CIDP group, although most of the EI and MT values fell within this range, there were four muscles with abnormally high EI and abnormally low MT, in addition to two muscles with abnormally high EI. These six muscles belonged to patients 9 and 15 . There were fair correlations between the dCMAP/LLN ratio and the EI and MT values in patients with CIDP  amplitude values measured in three different muscles, we determined the ratio of the measured values to the lower limit of values obtained from normal subjects (LLN) utilized in our laboratory. There was no significant difference in the dCMAP/LLN ratio between the six muscles with abnormally high EI and/or low MT values and the remaining muscles with normal MUS findings in patients with CIDP (44.1Several recent studies reported that the determination of EI and MT by quantitative MUS was highly accurate and reproducible in detecting hand muscle denervation (Kim, Seok, & Kim, In this study, muscle denervation was determined only by quantitative MUS. Needle EMG was not performed in this study with an academic focus due to its invasiveness on small hand muscles. Pillen et al. insisted that MUS was equally or slightly less capable of detecting focal neuropathies than EMG, with a sensitivity estimated at 70% (Pillen et al., The two patients with abnormally high EI and/or low MT by MUS exhibited several characteristics that could have contributed to the development of secondary axonal degeneration such as the history of a long disease course, unresponsiveness to medication, and the long interval from onset to initiation of therapy (Hahn, Bolton, Zochodne, & Feasby, 4.2Although the NCS findings of all CIDP patients in this study showed definite demyelinating abnormalities, no significant differences in EI and MT were noted between the CIDP and NC groups. This finding might reflect the fact that the main pathology during the early phase of CIDP is segmental demyelination, unless secondary axonal degeneration occurs (Boukhris et al., Reinnervation is another possible mechanism that might be preventing structural changes in muscles. However, the mechanism by which reinnervation affects MUS findings remains controversial. Specifically, Arts et al. revealed that some patients with amyotrophic lateral sclerosis exhibited increased MT and decreased EI in 6‐month sequential studies (Arts, Overeem, Pillen, Schelhaas, & Zwarts,In recurrent cases with normal MUS findings, immune therapies such as IVIg, CS, and IS may have contributed to the prevention of secondary axonal degeneration and the promotion of reinnervation. Further studies are required to determine the mechanisms by which therapies may affect MUS findings.4.3In this study, there was no significant difference in the dCMAP/LLN ratio between the six muscles with abnormal MUS findings suggesting axonal degeneration and the remaining muscles with normal MUS findings. This reflects the challenge in distinguishing secondary axonal degeneration from segmental demyelination in patients with CIDP by measuring dCMAP amplitudes in conventional NCSs as both pathologies could result in decreased dCMAP amplitudes (Harbo et al., We found fair correlations of dCMAP amplitude with both EI and MT in the CIDP group. Specifically, the dCMAP amplitude was negatively correlated with EI and positively correlated with MT Figure . These f4.4Our study has several limitations. First, the number of enrolled subjects was small. Evidently, future studies with larger sample sizes will be necessary to increase the statistical power. However, this study is the largest study examining MUS in patients with CIDP. Second, several variables, such as handedness and body mass index, which can affect MUS findings and in turn the study findings were not considered in our analyses (Arts, Pillen, Schelhaas, Overeem, & Zwarts, 5Quantitative MUS is a promising tool for detecting secondary axonal degeneration by determination of denervation in patients with CIDP. This approach can potentially be utilized to predict prognosis and responsiveness to treatment in patients with CIDP. Further studies with large populations and long‐period monitoring using objective clinical scales, such as the INCAT disability scale, are required to clarify the impact of MUS findings on the clinical course of patients with CIDP.None declared.


Triclosan (TCS) is a widely used antimicrobial agent and TCS resistance is considered to have evolved in diverse organisms with extensive use of TCS, but distribution of TCS resistance has not been well characterized. Functional screening of the soil metagenome in this study has revealed that a variety of target enoyl acyl carrier protein reductases (ENR) homologues are responsible for the majority of TCS resistance. Diverse ENRs similar to 7-α-hydroxysteroid dehydrogenase (7-α-HSDH), FabG, or the unusual YX7K-type ENR conferred extreme tolerance to TCS. The TCS-refractory 7-α HSDH-like ENR and the TCS-resistant YX7K-type ENR seem to be prevalent in human pathogenic bacteria, suggesting that a selective enrichment occurred in pathogenic bacteria in soil. Additionally, resistance to multiple antibiotics was found to be mediated by antibiotic resistance genes that co-localize with TCS resistance determinants. Further comparative analysis of ENRs from 13 different environments has revealed a huge diversity of both prototypic and metagenomic TCS-resistant ENRs, in addition to a selective enrichment of TCS-resistant specific ENRs in presumably TCS-contaminated environments with reduced ENR diversity. Our results suggest that long-term extensive use of TCS can lead to the selective emergence of TCS-resistant bacterial pathogens, possibly with additional resistance to multiple antibiotics, in natural environments. The emergence of bacterial resistance to antimicrobials and the growing number of multidrug-resistant bacteria have become a global public health concern3Various environments, including waste water treatment plants (WWTPs), sediment, surface water, sewage, sludge and soil, have been known to serve as potential reservoirs of antibiotic resistance genes (ARGs)The widely used biocide triclosan  has a broad-spectrum capacity to kill many microorganisms and is a constituent in a variety of personal care productsIn addition to being targeted by TCS, ENRs are also targeted by a variety of other clinically important antibiotics and antimicrobials, such as Isoniazid131114TCS-resistant bacteria are abundant in nature. Several mechanisms that confer resistance to this biocide are known and include: (i) overexpression of ENR18An enormous amount of TCS has been released into the environmentEscherichia coli K-12 genomes) from alluvial soil (AS) and industrially contaminated soil (ICS)  , were scil (ICS)  on the bfabI or fabG mutants of E. coli, respectively -based taxonomic assignment of metagenomic clones revealed that the majority belonged to the phylum proteobacteria . The mette clade . Both thte clade E. coli ENR FabI and to the Mycobacterium ENR InhA include amino acid substitutions such as G93-V, G93-S, M159-T, F203-L and F203-C. However, the metagenomic FabI ENRs carried unique patterns of substitutions  in the FabI ENRs of these organisms. Among these substitutions, the frequency of G93-A replacement was higher (50%) than F203-A (7%) in ENRs from these organisms. Few of the FabI ENR homologues carried substitutions, which were different from both previously known and metagenome-derived substitutions. Additionally, the metagenome-derived FabI ENRs, with different patterns of substitutions, not only conferred parental levels of resistance to TCS but also complemented ENR activity  of this biocide by >700-fold. These metagenomic 7-α-HSDH homologues also shared structural similarity with some of the previously known prototypic ENRs .Seven of the completely TCS-refractory metagenomic clones, pBF1-5, pL-2, pY-4, pR-2-3, pQ-2-3, pQ2N-2 and pBC1-1 , Table 1pic ENRs  and inde species . These mosteroni . HoweverEpsilonproteobacteria – Campylobacteraceae, Helicobacteraceae and Nautiliaceae – the first two of which include many human and other animal pathogens  of the metagenomic FabG-like ENR candidates differed from the functional FabG of E. coli by the replacement of Tyr151 with a nonpolar valine. While metagenomic FabG-like ENRs did not carry the highly conserved Asn113, a characteristic of the FabL ENRs, these two homologues complemented the growth of a FabI-defective E. coli JP1111 mutant  , suggesting that TCS is not being removed or inactivated by those seven clones, nor is ENR activity complemented in those clones.Nine of the TCS-resistant metagenome-derived clones did not carry any previously known ENR homologues   were alsomologue . The remomologue , althougacrB gene  obtained from MG-RAST, a public-repository web site  reservoir in the natural environment at the metagenomic level. The level of TCS detected in our samples suggested potential TCS contamination of these sites, and is in concordance with various other studiesStaphylococcus aureus isolate was mediated by an unusual additional sh-fabI allele originated from and 100% identical to that of S. haemolyticusS. aureus and S. epidermidis identical to sh-fabI homologues were located on plasmids, which suggested high mobility potential of these ENR homologuesCampylobacter lari and Helicobacter bilis, indicate that 7-α-HSDH is the only ENR in these organisms. FabG-like and completely TCS-refractory ENRs were predominant in Streptomyces spp., which are known for having various polyketide synthesis pathways and might therefore have different types of ENRs for fatty acid biosynthesis. The slight structural similarities and differences in TCS resistance levels between metagenomic FabG and prototypic FabL ENRs suggest the possibility that enzymes of intermediate structures may have, over evolutionary history, left different versions of these enzymes throughout a diverse range of microorganisms. For instance, FabL and FabI are structurally very similar, with similar catalytic domains, but both have different cofactor requirements and different affinities for TCS. Additionally, phylogenetic analysis suggests that the metagenomic 7-α-HSDH, FabG and YX7K-like ENRs may have potentially diverged from FabL or FabI-like ENRs or from either 7-α-HSDH or FabG. Consequently, these different types of ENRs may be difficult to distinguish by sequence comparison or sequence annotation alone.Intriguingly, our results revealed for the first time that a similar YX7K-type ENR is shared by both eukaryotic and prokaryotic intracellular pathogenic microorganisms, suggesting either a potential evolutionary link in the ENRs among these intracellular pathogens or a potential horizontal gene transfer event among these intracellular pathogenic organisms. Potential ENR dissemination via horizontal gene transfer have previously been assessed, where TCS resistance in a The unusual characteristics of two of the metagenomic prototypic FabV and FabK-like ENR homologues might be attributed to the absence of the conserved FAD-binding domainacrB gene homologue revealed the presence of different versions of this protein or different codon usageIt has been proposed that additional cellular targets of TCS might exist in addition to the previously known targetThe co/cross-resistance to other antibiotics observed in the metagenomic clones may be associated with the colocalized genes encoding efflux pumps, antibiotic-modifying enzymes, ARG clusters and other genes. The ARGs that colocalized with TCS resistance determinants indicate that TCS may selectively enrich the complex patterns of multiple resistance to other antibiotics in the environment. TCS-mediated overexpression of the AcrB efflux system2231Metagenomic comparisons from different environments suggest that TCS may act as a driving force to influence the total ENR diversity in TCSREs, which are the most likely sites for TCS resistance developmentWe conclude that different versions of ENRs and novel TCS resistant genes (TRGs) are abundant in nature. The vast diversity of ARG pools has made it possible for only some of the ARGs to be selectively transferred to many bacteria8Helicobacter pylori HPKTCC B0100 was obtained from the Korean Type Culture Collection (KTCC), Korea National Research Resource Bank (KNRRC). The genomic DNA from Campylobacter jejuni NCTC11168 was kindly provided by Dr. Jong-Hyun Kim at the Centers for Disease Control and Prevention. E. coli strains/mutants DH5α, EPI-300, JP1111  and CL37 (kindly provided by Dr. John Cronan at the University of Illinois at Urbana-Champaign) were routinely grown at 37 °C in Luria-Bertani (LB) broth or LB agar supplemented with appropriate antibiotics. The antibiotic concentrations used were as follows; ampicillin, 100 μg/ml; kanamycin, 50 μg/ml; chloramphenicol, 30 μg/ml and TCS, 0.1–650 μg/ml. Fosmid pCC1FOS  was used to construct genomic libraries, whereas pUC119 and pGEM®-T Easy were used for further subcloning experiments.The bacterial strains and plasmids used in this study are described in E. coli EPI-300 with pCC1FOS and DH5α with pUC119 were first grown to an OD600 of 1.0, and these bacterial suspensions were serially diluted to 1 × 105 colony-forming units (CFU)/ml. The cell suspension (1 × 105 CFU/ml) was spread onto LB agar medium containing antibiotics and TCS in a range of 0.1–5 μg/ml. The LB plates were incubated at 37 °C for three days, and bacterial colony formation was examined at regular 24 h intervals. The lowest concentration of TCS (0.9 μg/ml) that prevented bacterial growth of E. coli EPI-300 was considered the MIC for TCS. To determine the MICs for the clones carrying various ENR homologues, the procedure was followed as above, but the negative control E. coli EPI-300 carried an additional chromosomal fabI gene from wild-type E. coli K12 in the pCC1FOS fosmid vector to prevent ENR overexpression effect. This experiment was performed in triplicate for each antibiotic concentration.To determine the MIC of TCS, http://blast.ncbi.nlm.nih.gov). Multiple alignment analysis was performed using BioEdit software in combination with GeneDoc.Standard recombinant DNA techniques were followed as previously describedThe metagenomic library from alluvial soil (AS) was previously constructed352PO4, 0.3 g; Na2HPO4, 0.6 g; MgSO4, 0.2 g; gelatin, 0.1 g), and fosmid clones from the metagenomic library were spread on LB agar containing 30 μg/ml chloramphenicol and 5 μg/ml TCS. E. coli colonies that grew on LB with TCS were picked and further tested at higher concentrations of TCS until they were unable to grow or were found refractory to TCS. Pure cultures of TCS-resistant clones were processed for fosmid isolation followed by BamHI restriction digestion, and the unique clones were finally selected based on BamHI restriction profiles.To select TCS-resistant clones from the fosmid library, the library pool stocks were diluted in a buffer  activity of two of the metagenomic clones, complementation studies were performed in a similar manner as described for ENR complementation but using a E. coli DH5α with various metagenomic clones carrying TCS resistance showed any differential responses to a variety of antibiotics, we first grew these cells to an OD600 of 1.0 in the presence or absence of 0.1 μg/ml TCS, a sub-lethal concentration (to observe cross-resistance). These bacterial cultures were then serially diluted to 1 × 105 CFU/ml, and 3 μl of each cell suspension (1 × 105 CFU/ml) was spotted onto LB agar medium containing the antibiotics to be tested for co/cross-resistance. LB plates were incubated at 37 °C for three days, and bacterial colony formation was examined at regular intervals of 24 h. The lowest concentration of antibiotic that prevented bacterial growth was considered the MIC for that antibiotic. This experiment was performed in triplicate for each concentration of each antibiotic. Prior to testing for co/cross-resistance, two independent experiments were performed in which a set of TCS-resistant clones was cultured in LB broth supplemented with 0.1 μg/ml TCS (for cross-resistance studies) with an identical set not exposed to TCS (to observe co-resistance). This experiment was performed in triplicate for each concentration of each antibiotic. MIC of E. coli carrying TCS resistance genes was determined to 19 different antibiotics of 8 different classes . Detailed information about how metagenomic data sets were selected and about the TRG reference database can be found in To investigate the abundance and diversity of genes encoding ENR homologues in presumably TCS-contaminated and TCS-free environments, 49 metagenome datasets were selected from 13 different environments. The presumably TCS-rich environments (TCSREs) included 3 samples from WWTPs  and one sample from the ocean sediment in China. The presumably TCS-free or low-TCS environments included 9 samples: 2 human oral samples , 4 fresh water samples , 2 forest soil samples (a Mediterranean forest soil from Spain and a Temperate forest soil from Finland) and one glacier sample (from Austria). The metagenomic data sets for these samples were downloaded from the public repository MG-RAST web site (−10. The annotated read numbers (hits) from individual metagenomic datasets were normalized as previously describedA TRG reference database was constructed. The TRG database contained the deduced amino acid sequences of well-known prototypic and metagenome-derived ENRs identified in this study . TRG seqNormalization of the data for each dataset was performed as described using the following equationNTRG homologous sequence is the number of annotated TRG-like sequences, annotated as one specific TRG reference sequence; LTRG reference sequence is the sequence length of the corresponding specific ENR reference sequence in the TRG database; N16S sequence is the number of 16S rRNA gene sequences identified in the metagenomic data; L16S sequence is the average length of the 16S rRNA gene sequence found in the Greengenes database ; n is the number of mapped TRG reference sequences; and Lmetagenomic data read length is the sequence length for the corresponding sequence technology used, such as Illumina HiSeq, Sanger sequencing, or 454 pyrosequencing (refer to where refer to .http://www.r-project.org/) using VeganAll comparisons of the relative abundance of TRG homologues between metagenomes were performed using the normalized abundance reads for each ENR divided by the total number of normalized ENR reads in the dataset . To compHow to cite this article: Khan, R. et al. Triclosan Resistome from Metagenome Reveals Diverse Enoyl Acyl Carrier Protein Reductases and Selective Enrichment of Triclosan Resistance Genes. Sci. Rep.6, 32322; doi: 10.1038/srep32322 (2016).