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.

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.

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.

Evaluation of hearing and auditory nerve function by combining ABR, DPOAE and eABR tests into a single recording session.

In this article, we describe an efficient method for testing both auditory receptor and auditory nerve function in a single recording session. Auditory receptor function is tested in response to pure tone, tone burst and click acoustic stimuli (i.e. distortion products of otoacoustic emissions, DPOAE; and auditory-evoked brainstem responses, ABR). The function of the auditory neurons and nerve is measured in response to direct electric current stimulation (i.e. electrically evoked auditory brainstem responses, eABR). All measurements were obtained from anesthetized laboratory rats during single recording sessions using hardware and software stimulation and analysis programs developed by Intelligent Hearing Systems, Miami, FL.