Design of AsLOV2 domain as a carrier of light-induced dissociable FMN photosensitizer.

Flavin mononucleotide (FMN) is a highly efficient photosensitizer (PS) yielding singlet oxygen (1 O2 ). However, its 1 O2 production efficiency significantly decreases upon isoalloxazine ring encapsulation into the protein matrix in genetically encoded photosensitizers (GEPS). Reducing isoalloxazine ring interactions with surrounding amino acids by protein engineering may increase 1 O2 production efficiency GEPS, but at the same time weakened native FMN-protein interactions may cause undesirable FMN dissociation. Here, in contrast, we intentionally induce the FMN release by light-triggered sulfur oxidation of strategically placed cysteines (oxidation-prone amino acids) in the isoalloxazine-binding site due to significantly increased volume of the cysteinyl side residue(s). As a proof of concept, in three variants of the LOV2 domain of Avena sativa (AsLOV2), namely V416C, T418C, and V416C/T418C, the effective 1 O2 production strongly correlated with the efficiency of irradiation-induced FMN dissociation (wild type (WT) < V416C < T418C < V416C/T418C). This alternative approach enables us: (i) to overcome the low 1 O2 production efficiency of flavin-based GEPSs without affecting native isoalloxazine ring-protein interactions and (ii) to utilize AsLOV2, due to its inherent binding propensity to FMN, as a PS vehicle, which is released at a target by light irradiation.

Isotopic Depletion Increases the Spatial Resolution of FPOP Top-Down Mass Spectrometry Analysis.

Protein radical labeling, like fast photochemical oxidation of proteins (FPOP), coupled to a top-down mass spectrometry (MS) analysis offers an alternative analytical method for probing protein structure or protein interaction with other biomolecules, for instance, proteins and DNA. However, with the increasing mass of studied analytes, the MS/MS spectra become complex and exhibit a low signal-to-noise ratio. Nevertheless, these difficulties may be overcome by protein isotope depletion. Thus, we aimed to use protein isotope depletion to analyze FPOP-oxidized samples by top-down MS analysis. For this purpose, we prepared isotopically natural (IN) and depleted (ID) forms of the FOXO4 DNA binding domain (FOXO4-DBD) and studied the protein-DNA interaction interface with double-stranded DNA, the insulin response element (IRE), after exposing the complex to hydroxyl radicals. As shown by comparing tandem mass spectra of natural and depleted proteins, the ID form increased the signal-to-noise ratio of useful fragment ions, thereby enhancing the sequence coverage by more than 19%. This improvement in the detection of fragment ions enabled us to detect 22 more oxidized residues in the ID samples than in the IN sample. Moreover, less common modifications were detected in the ID sample, including the formation of ketones and lysine carbonylation. Given the higher quality of ID top-down MSMS data set, these results provide more detailed information on the complex formation between transcription factors and DNA-response elements. Therefore, our study highlights the benefits of isotopic depletion for quantitative top-down proteomics. Data are available via ProteomeXchange with the identifier PXD044447.

Top-Down Proteoform Analysis by 2D MS with Quadrupolar Detection.

Two-dimensional mass spectrometry (2D MS) is a multiplexed tandem mass spectrometry method that does not rely on ion isolation to correlate the precursor and fragment ions. On a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS), 2D MS instead uses the modulation of precursor ion radii inside the ICR cell before fragmentation and yields 2D mass spectra that show the fragmentation patterns of all the analytes. In this study, we perform 2D MS for the first time with quadrupolar detection in a dynamically harmonized ICR cell. We discuss the advantages of quadrupolar detection in 2D MS and how we adapted existing data processing techniques for accurate frequency-to-mass conversion. We apply 2D MS with quadrupolar detection to the top-down analysis of covalently labeled ubiquitin with ECD fragmentation, and we develop a workflow for label-free relative quantification of biomolecule isoforms in 2D MS.

Systematic Literature Review of Hearing Preservation Rates in Cochlear Implantation Associated With Medium- and Longer-Length Flexible Lateral Wall Electrode Arrays.

Background: The last two decades have demonstrated that preoperative functional acoustic hearing (residual hearing) can be preserved during cochlear implant (CI) surgery. However, the relationship between the electrode array length and postoperative hearing preservation (HP) with lateral wall flexible electrode variants is still under debate. Aims/Objectives: This is a systematic literature review that aims to analyze the HP rates of patients with residual hearing for medium-length and longer-length lateral wall electrodes. Method: A systematic literature review methodology was applied following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) recommendations to evaluate the HP rates of medium-length and longer-length lateral wall electrodes from one CI manufacturer (medium length FLEX 24, longer length FLEX 28 and FLEX SOFT, MED-EL, Innsbruck, Austria). A search using search engine PubMed (https://www.ncbi.nlm.nih.gov/pubmed/) was performed using the search terms "hearing preservation" or "residual hearing" and "cochlear implant" in "All fields." Articles published only in English between January 01, 2009 and December 31, 2020 were included in the search. Results: The HP rate was similar between medium-length (93.4%-93.5%) and longer (92.1%-86.8%) electrodes at 4 months (p = 0.689) and 12 months (p = 0.219). In the medium-length electrode group, patients under the age of 45 years had better HP than patients above the age of 45 years. Conclusions: Both medium-length and longer electrode arrays showed high hearing preservation rates. Considering the hearing deterioration over time, implanting a longer electrode at primary surgery should be considered, thus preventing the need for future reimplantation.

Considerations for Fitting Cochlear Implants Bimodally and to the Single-Sided Deaf.

When listening with a cochlear implant through one ear and acoustically through the other, binaural benefits and spatial hearing abilities are generally poorer than in other bilaterally stimulated configurations. With the working hypothesis that binaural neurons require interaurally matched inputs, we review causes for mismatch, their perceptual consequences, and experimental methods for mismatch measurements. The focus is on the three primary interaural dimensions of latency, frequency, and level. Often, the mismatch is not constant, but rather highly stimulus-dependent. We report on mismatch compensation strategies, taking into consideration the specific needs of the respective patient groups. Practical challenges typically faced by audiologists in the proposed fitting procedure are discussed. While improvement in certain areas (e.g., speaker localization) is definitely achievable, a more comprehensive mismatch compensation is a very ambitious endeavor. Even in the hypothetical ideal fitting case, performance is not expected to exceed that of a good bilateral cochlear implant user.

Multi-Frequency Intraoperative Monitoring of Hearing Preservation during Cochlear Implantation.

(1) Background: Current indications for cochlear implants (CIs) have expanded to include patients with appreciable low-frequency hearing. However, longitudinal results indicate that only one-third of these recipients retain full hearing preservation. In another words, the remaining two-thirds lose this facility either partially or fully. This points to the need to better understand the impact of cochlear implantation on cochlear integrity. Intracochlear electrocochleography (ECochG) involves the recording of electrical potentials generated in the inner ear in response to acoustic stimuli, and previous studies have shown that these potentials give an indication of residual inner ear function. Aim of the research: The aim is to monitor intracochlear ECochG during CI surgery and gain a better understanding of how the implant impacted inner ear function. A newly developed SPL Chirp was used for stimulation. (2) Methods: Intracochlear ECochG signals were measured in a subject with residual preoperative low-frequency hearing, while an electrode array was introduced into the cochlea and was continued until the round window was sealed. Afterwards, surgical events were reviewed with the surgeon; preoperative and postoperative radiological data and hearing thresholds were also evaluated. (3) Conclusions: Real-time intraoperative monitoring, with multifrequency evaluation and video recording, has the potential to allow surgeons and audiologists to continuously assess cochlear function. ECochG monitoring may be a useful tool during cochlear implantation to gain frequency-specific information on the status of the patient's hearing, assisting surgeons to lower hearing trauma during the operation.

In Vivo Basilar Membrane Time Delays in Humans.

To date, objective measurements and psychophysical experiments have been used to measure frequency dependent basilar membrane (BM) delays in humans; however, in vivo measurements have not been made. This study aimed to measure BM delays by performing intracochlear electrocochleography in cochlear implant recipients. Sixteen subjects with various degrees of hearing abilities were selected. Postoperative Computer Tomography was performed to determine electrode locations. Electrical potentials in response to acoustic tone pips at 0.25, 0.5, 1, 2, and 4 kHz and clicks were recorded with electrodes at the frequency specific region. The electrode array was inserted up to the characteristic cochlear frequency region of 250 Hz for 6 subjects. Furthermore, the array was inserted in the region of 500 Hz for 15 subjects, and 1, 2, and 4 kHz were reached in all subjects. Intracochlear electrocochleography for each frequency-specific tone pip and clicks showed detectable responses in all subjects. The latencies differed among the cochlear location and the cochlear microphonic (CM) onset latency increased with decreasing frequency and were consistent with click derived band technique. Accordingly, BM delays in humans could be derived. The BM delays increased systematically along the cochlea from basal to apical end and were in accordance with Ruggero and Temchin, 2007.

Utilization of Fast Photochemical Oxidation of Proteins and Both Bottom-up and Top-down Mass Spectrometry for Structural Characterization of a Transcription Factor-dsDNA Complex.

A combination of covalent labeling techniques and mass spectrometry (MS) is currently a progressive approach for deriving insights related to the mapping of protein surfaces or protein-ligand interactions. In this study, we mapped an interaction interface between the DNA binding domain (DBD) of FOXO4 protein and the DNA binding element (DAF16) using fast photochemical oxidation of proteins (FPOP). Residues involved in protein-DNA interaction were identified using the bottom-up approach. To confirm the findings and avoid a misinterpretation of the obtained data, caused by possible multiple radical oxidations leading to the protein surface alteration and oxidation of deeply buried amino acid residues, a top-down approach was employed for the first time in FPOP analysis. An isolation of singly oxidized ions enabled their gas-phase separation from multiply oxidized species followed by CID and ECD fragmentation. Application of both fragmentation techniques allowed generation of complementary fragment sets, out of which the regions shielded in the presence of DNA were deduced. The findings obtained by bottom-up and top-down approaches were highly consistent. Finally, FPOP results were compared with those of the HDX study of the FOXO4-DBD·DAF16 complex. No contradictions were found between the methods. Moreover, their combination provides complementary information related to the structure and dynamics of the protein-DNA complex. Data are available via ProteomeXchange with identifier PXD027624.

Comparative Analysis of Cortical Auditory Evoked Potential in Cochlear Implant Users.

OBJECTIVES: The primary goal of the study was to investigate electrical cortical auditory evoked potentials (eCAEPs) at maximum comfortable level (MCL) and 50% MCL on three cochlear implant (CI) electrodes and compare them with the acoustic CAEP (aCAEPs), in terms of the amplitude and latency of the P1-N1-P2 complex. This was achieved by comparing the eCAEP obtained with the method described and stimulating single electrodes, via the fitting software spanning the cochlear array and the aCAEP obtained using the HEARLab system at four speech tokens. DESIGN: Twenty MED-EL (MED-EL Medical Electronics, Innsbruck, Austria) CI adult users were tested. CAEP recording with HEARLab System was performed with speech tokens /m/, /g/, /t/, and /s/ in free field, presented at 55 dB SPL. eCAEPs were recorded with an Evoked Potential device triggered from the MAX Programming Interface (MED-EL Medical Devices) with 70 msec electrical burst at 0.9 Hz at the apical (1), middle (6), and basal (10 or 11) CI electrode at their MCL and 50% MCL. RESULTS: CAEP responses were recorded in 100% of the test subjects for the speech token /t/, 95% for the speech tokens /g/ and /s/, and 90% for the speech token /m/. For eCAEP recordings, in all subjects, it was possible to identify N1 and P2 peaks when stimulating the apical and middle electrodes. This incidence of detection decreased to an 85% chance of stimulation at 50% MCL on the same electrodes. A P1 peak was less evident for all electrodes. There was an overall increase in latency for stimulation at 50% MCL compared with MCL. There was a significant difference in the amplitude of adjacent peaks (P1-N1 and N1-P2) for 50% MCL compared with MCL. The mean of the maximum cross-correlation values were in the range of 0.63 to 0.68 for the four speech tokens. The distribution of the calculated time shift, where the maximum of the cross-correlation was found, was distributed between the speech tokens. The speech token /g/ had the highest number of valid cross-correlations, while the speech token /s/ had the lowest number. CONCLUSIONS: This study successfully compared aCAEP and eCAEP in CI users. Both acoustic and electrical P1-N1-P2 recordings obtained were clear and reliable, with good correlation. Latency increased with decreasing stimulation level, while amplitude decreased. eCAEP is potentially a better option to verify speech detection at the cortical level because it (1) uses direct stimulation and therefore creates less interference and delay of the sound processor and (2) creates more flexibility with the recording setup and stimulation setting. As such, eCAEP is an alternative method for CI optimization.

Electrode estimation in the acoustic region of the human Cochlea.

Background: In this study, a method to estimate number of electrodes in the acoustic region of Electric Acoustic Stimulation (EAS) subjects was proposed. Aims/Objectives: To develop and validate an anatomy-based method for EAS subjects to estimate the number of electrodes within the acoustic region.Material and methods: The postoperative CTs of adults with various degree of hearing implanted with lateral wall electrodes with mean insertion depth of 23.9 mm (18.0-28.2 mm) and mean insertion angle of 505° (355-695°) were evaluated.Results: The difference between the estimated and measured angle varied between -18 and 25°, with a mean of 0.9°. For the insertion angle of 230° and higher, the maximum difference was 24°. Taking this uncertainty into account, all electrodes in the acoustic region were predicted correctly.Conclusions and significance: The method decides on non-overlapping acoustic and electric stimulation in terms of place in the cochlea. With the accuracy of 0.84 mm for the electrode arrays inserted for more than 230°, the method was sufficient to estimate the exact number of electrodes in the acoustic region of cochlear implantees. The benefit of this method may be in fitting of EAS subjects with some portion of the electrode array in the acoustic region.

Cochlear Microphonics in Hearing Preservation Cochlear Implantees.

OBJECTIVES: The intracochlear electrocochleography (ECoG) could be recorded directly from the cochlear implant (CI) electrode in CI recipients with residual hearing. The primary objective of this study is to identify the most sensitive frequency to record cochlear microphonics (CM) in CI users with a wide degree of hearing abilities and deep electrode insertion. The secondary objective is to identify the optimum location within the cochlea to record intracochlear potentials. MATERIALS AND METHODS: CMs were recorded from the CI electrodes in eight females and eight males implanted with CIs Pulsar, Concerto, or Sonata, Med-El Corp. RESULTS: Among the tone pips of various frequencies, 1k or 500 Hz were the most sensitive for CI users. The most sensitive place in the cochlea to record the CM potentials depended on the tone frequency used. The deeper into the cochlea the mean maximum CM peak-to-peak amplitude was measured, the lower the stimulating tone frequency was. CONCLUSION: The most optimal recording parameters identified for intracochlear CM recording can be useful for intraoperative and postoperative monitoring of cochlear health in CI users with residual hearing.

Improving the Sequence Coverage of Integral Membrane Proteins during Hydrogen/Deuterium Exchange Mass Spectrometry Experiments.

Insight into the structure-function relationship of membrane proteins is important to understand basic cell function and inform drug development, as these are common targets for drugs. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is an established technique for the study of protein conformational dynamics and has shown compatibility with membrane proteins. However, the digestion and mass analysis of peptides from membrane proteins can be challenging, severely limiting the HDX-MS experiment. Here we compare the digestion of four integral membrane proteins-Cl-/H+ exchange transporter (ClC-ec1), leucine transporter (LeuT), dopamine transporter (DAT), and serotonin transporter (SERT)-by the use of porcine pepsin and three alternative aspartic proteases either in-solution or immobilized on-column in an optimized HDX-MS-compatible workflow. Pepsin was the most favorable for the digestion of ClC-ec1 and LeuT, providing coverage of 82.2 and 33.2% of the respective protein sequence; however, the alternative proteases surpassed pepsin for the digestion of DAT and SERT. By also screening quench solution additives, we observe that the denaturant urea was beneficial, resulting in improved sequence coverage of all membrane proteins, in contrast to guanidine hydrochloride. Furthermore, significant improvements in sequence coverage were achieved by tailoring the chromatography to handle hydrophobic peptides. Overall, we demonstrate that the susceptibility of membrane proteins to proteolytic digestion during HDX-MS is highly protein-specific. Our results highlight the importance of having multiple proteases and different quench buffer additives in the HDX-MS toolbox and the need to carefully screen a range of digestion conditions to successfully optimize the HDX-MS analysis of integral membrane proteins.

Usefulness of Electrical Auditory Brainstem Responses to Assess the Functionality of the Cochlear Nerve Using an Intracochlear Test Electrode.

OBJECTIVE: To use an intracochlear test electrode to assess the integrity and the functionality of the auditory nerve in cochlear implant (CI) recipients and to compare electrical auditory brainstem responses (eABR) via the test electrode with the eABR responses with the CI. SETTING: Otolaryngology department, tertiary referral hospital. PATIENTS: Ten subjects (age at implantation 55 yr, range, 19-72) were subsequently implanted with a MED-EL CONCERTO CI on the side without any useful residual hearing. INTERVENTIONS: Following identification of the round window (RW), the test electrode was inserted in the cochlea previous to cochlear implantation. MAIN OUTCOME MEASURES: To assess the quality of an eABR waveform, scoring criteria from Walton et al. (2008) were chosen. The waveforms in each session were classified by detecting waves III and V by the algorithm and visual assessment of the waveform. Speech performance was evaluated with monosyllables, disyllables, and sentence recognition tests. RESULTS: It was possible to evoke electrical stimulation responses along with both the test electrode and the CI in all subjects. No significant differences in latencies or amplitudes after stimulation were found between the test electrode and the CI. All subjects obtained useful hearing with their CI and use their implants daily. CONCLUSIONS: The intracochlear test electrode may be suitable to test the integrity of the auditory nerve by recording eABR signals. This allows for further research on the status of the auditory nerve after tumor removal and correlation with auditory performance.

Cochlear implantation with hearing preservation yields significant benefit for speech recognition in complex listening environments.

OBJECTIVE: The aim of this study was to assess the benefit of having preserved acoustic hearing in the implanted ear for speech recognition in complex listening environments. DESIGN: The present study included a within-subjects, repeated-measures design including 21 English-speaking and 17 Polish-speaking cochlear implant (CI) recipients with preserved acoustic hearing in the implanted ear. The patients were implanted with electrodes that varied in insertion depth from 10 to 31 mm. Mean preoperative low-frequency thresholds (average of 125, 250, and 500 Hz) in the implanted ear were 39.3 and 23.4 dB HL for the English- and Polish-speaking participants, respectively. In one condition, speech perception was assessed in an eight-loudspeaker environment in which the speech signals were presented from one loudspeaker and restaurant noise was presented from all loudspeakers. In another condition, the signals were presented in a simulation of a reverberant environment with a reverberation time of 0.6 sec. The response measures included speech reception thresholds (SRTs) and percent correct sentence understanding for two test conditions: CI plus low-frequency hearing in the contralateral ear (bimodal condition) and CI plus low-frequency hearing in both ears (best-aided condition). A subset of six English-speaking listeners were also assessed on measures of interaural time difference thresholds for a 250-Hz signal. RESULTS: Small, but significant, improvements in performance (1.7-2.1 dB and 6-10 percentage points) were found for the best-aided condition versus the bimodal condition. Postoperative thresholds in the implanted ear were correlated with the degree of electric and acoustic stimulation (EAS) benefit for speech recognition in diffuse noise. There was no reliable relationship among measures of audiometric threshold in the implanted ear nor elevation in threshold after surgery and improvement in speech understanding in reverberation. There was a significant correlation between interaural time difference threshold at 250 Hz and EAS-related benefit for the adaptive speech reception threshold. CONCLUSIONS: The findings of this study suggest that (1) preserved low-frequency hearing improves speech understanding for CI recipients, (2) testing in complex listening environments, in which binaural timing cues differ for signal and noise, may best demonstrate the value of having two ears with low-frequency acoustic hearing, and (3) preservation of binaural timing cues, although poorer than observed for individuals with normal hearing, is possible after unilateral cochlear implantation with hearing preservation and is associated with EAS benefit. The results of this study demonstrate significant communicative benefit for hearing preservation in the implanted ear and provide support for the expansion of CI criteria to include individuals with low-frequency thresholds in even the normal to near-normal range.

Conservation of hearing and protection of hair cells in cochlear implant patients' with residual hearing.

This review covers the molecular mechanisms involved in hair cell and hearing losses which can result from trauma generated during the process of cochlear implantation and the contributions of both the intrinsic and extrinsic cell death signaling pathways in producing these trauma/inflammation induced losses. Application of soft surgical techniques to conserve hearing and protect auditory sensory cells during the process of cochlear implantation surgery and insertion of the electrode array during the process of cochlear implantation are reviewed and discussed. The role of drug therapy and mode of drug delivery for the conservation of a cochlear implant patient's residual hearing is presented and discussed.

Evoked stapedius reflex and compound action potential thresholds versus most comfortable loudness level: assessment of their relation for charge-based fitting strategies in implant users.

AIM: The main goal of the present study was to assess the feasibility of using evoked stapedius reflex (eSR) and evoked compound action potential (eCAP) thresholds to create speech processor programs for children using Med-El Maestro software. The secondary goals were (1) to compare the eSR and eCAP thresholds recorded using charge units in experienced adults fitted with Med-El Pulsar CI100 cochlear implants with most comfortable loudness levels (MCLs) obtained for the apical, medial and basal electrodes, and (2) to compare eSR and eCAP thresholds for the apical, medial and basal electrodes between adults and children. METHODS: Fourteen children and 16 adults participated in the study. eSR and eCAP thresholds were measured in both groups using the auditory nerve response telemetry algorithm, with MCL being behaviourally measured only in the adult group. RESULTS: In the adult population, the correlation between eSR threshold and MCL was better for apical, medial and basal electrodes than that between eCAP threshold and MCL. There was no significant difference in the means obtained for eCAP and eSR thresholds in children and adults for any of the electrodes tested. This finding suggests that in children, the correlations between eCAP thresholds and MCL values, and those between eSR thresholds and MCL values are not lower than those generally found in adults. CONCLUSIONS: Although the eSR threshold is a better predictor of MCL values, both eSR and eCAP thresholds can be useful tools for assisting with map creation for children.

Recording of electrically evoked auditory brainstem responses after electrical stimulation with biphasic, triphasic and precision triphasic pulses.

Biphasic electrical pulses are the standard stimulation pulses in current cochlear implants. In auditory brainstem recordings biphasic pulses generate a significant artifact that disrupts brainstem responses, which are magnitudes smaller. Triphasic pulses may minimize artifacts by restoring the neural membrane to its resting potential faster than biphasic pulses and make auditory brainstem responses detection easier. We compared biphasic pulses with triphasic and precision triphasic pulses to evoke brainstem responses in human subjects. For this purpose, electrically evoked brainstem response audiometry was performed in 10 (11 ears) cochlear implant patients. Artifacts and brainstem responses evoked by bi- and triphasic stimulation were analyzed. Artifact amplitude and decay time were related to pulse pattern shape, but application of averaging and alternation reduced the deterioration of electrically evoked brainstem responses independent of pulse pattern shape. Contrary to our expectations, biphasic pulses showed a higher detectability in comparison to triphasic pulse stimulation at the same stimulation amplitude.