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Real-time PCR with intercalating dyes can only be performed once. The expensive fluorescent hydrolysis probes are target specific and are suitable to detect multiplex targets. Uracil-DNA N-glycosylase (UNG), which specifically hydrolyzes and degrades any uracil-containing PCR products, is often applied before PCR to reduce carryover contamination. We developed an optimized protocol for recovering DNA from PCR products and revaluating by real-time PCR with intercalating dye using UNG processing, which is particularly useful when the sample volume is very small and insufficient for multiple assays of real-time PCR. • A real-time PCR master mix with dUTP instead of dTTP was used. • UNG at 1 % and 10 % concentrations of PCR product volumes were used for the first and second processing. • The second real-time PCR was performed with different primer pairs than the first real-time PCR.

Lead (Pb) can damage organs and also have undesirable effects on neural development. To explore the effects of Pb on olfactory cells, we investigated Pb‐induced cell toxicity in the DBC1.2 olfactory cell line, with a focus on endoplasmic reticulum (ER) stress, apoptosis, and necroptosis. Representative markers of ER stress, apoptosis, and necroptosis were analyzed by quantitative PCR. The mRNA expression levels of GRP94, GRP78, spliced XBP1, PERK, and ATF6 increased significantly after Pb exposure in a dose‐dependent manner. The expression of Caspase 3 and Caspase 12 did not increase after Pb exposure, which suggested that apoptosis‐induced cell death was not activated after Pb exposure. However, the mRNA of RIPK3 and MLKL showed increases in expression, which indicated that necroptosis‐induced cell death was activated after Pb exposure. These results indicate that Pb exposure induced dose‐dependent cytotoxicity through ER stress and necroptosis pathways in DBC1.2 cells, whereas the apoptosis pathway was not significantly stimulated. HEPES buffer showed a partial protective effect in terms of ER stress, apoptosis, and necroptosis. In summary, the necroptosis pathway plays a crucial rule in Pb exposure‐induced cytotoxicity in olfactory cells.

Abstract Objective To investigate the effects of self‐management vestibular rehabilitation (VR) on the subjective symptoms of dizziness and postural stability in persistent postural‐perceptual dizziness (PPPD). Study design Retrospective case review. Methods The medical records of 30 patients newly diagnosed with PPPD based on the Bárány Society's diagnostic criteria were reviewed. Nineteen patients (4 males and 15 females; age range 27–84 years, mean age ± standard deviation 57.4 ± 14.2 years) who was newly instructed to self‐management VR were included and instructed to perform self‐management VR for 2 months. Results One patient did not visit the outpatient clinic again, and in the remaining 18 patients, 4 (22%) discontinued VR at their own discretion. In the 12 patients who completed 2 months of VR (67%), there was a significant improvement in Niigata PPPD Questionnaire (NPQ) and Dizziness Handicap Inventory (DHI) scores after VR compared to those before VR (p .05). Conclusions For PPPD, self‐management VR improved subjective symptoms of dizziness, but not stability of standing posture. It is necessary to improve patients' adherence to the treatment. Level of evidence 4.

Abstract We investigated whether the oxidoreductase cofactor pyrroloquinoline quinone (PQQ) prevents noise-induced and age-related hearing loss (NIHL and ARHL) in mice. To assess NIHL, 8 week-old mice with and without PQQ administration were exposed to noise for 4 h. PQQ was orally administered for one week before and after noise exposure and subcutaneously once before noise exposure. For ARHL evaluation, mice were given drinking water with or without PQQ starting at 2 months of age. In the NIHL model, PQQ-treated mice had auditory brainstem response (ABR) thresholds of significantly reduced elevation at 8 kHz, a significantly increased number of hair cells at the basal turn, and significantly better maintained synapses beneath the inner hair cells compared to controls. In the ARHL model, PQQ significantly attenuated the age-related increase in ABR thresholds at 8 and 32 kHz at 10 months of age compared to controls. In addition, the hair cells, spiral ganglion cells, ribbon synapses, stria vascularis and nerve fibers were all significantly better maintained in PQQ-treated animals compared to controls at 10 months of age. These physiological and histological results demonstrate that PQQ protects the auditory system from NIHL and ARHL in mice.

Heavy metals are often found in soil and can contaminate drinking water, posing a serious threat to human health. Molecular pathways and curation therapies for mitigating heavy metal toxicity have been studied for a long time. Recent studies on oxidative stress and aging have shown that the molecular foundation of cellular damage caused by heavy metals, namely, apoptosis, endoplasmic reticulum stress, and mitochondrial stress, share the same pathways as those involved in cellular senescence and aging. In recent aging studies, many types of heavy metal exposures have been used in both cellular and animal aging models. Chelation therapy is a traditional treatment for heavy metal toxicity. However, recently, various antioxidants have been found to be effective in treating heavy metal-induced damage, shifting the research focus to investigating the interplay between antioxidants and heavy metals. In this review, we introduce the molecular basis of heavy metal-induced cellular damage and its relationship with aging, summarize its clinical implications, and discuss antioxidants and other agents with protective effects against heavy metal damage.

Abstract Objective Time restricted feeding (TRF), which is an intermittent fasting protocol, has been reported to decrease the toxicity and mortality rate associated with systemic gentamicin (GM) administration. The aim of this study is to evaluate the effect of TRF on GM‐induced vestibular and auditory function in mice. Methods Japan Central Laboratory for Experimental Animals:Institute of Cancer Research (Jcl:ICR) mice were housed in a light–dark (LD) cycle (12:12) and were divided into three groups: (1) GM treatment at a dose of 220 mg/kg with TRF (feeding time: 8 h [9:00–17:00] during the light phase [7:00–19:00]) (GM + TRF group), (2) GM treatment at a dose of 220 mg/kg without TRF (GM group), and (3) saline injection with TRF (NS + TRF group). GM or saline was injected subcutaneously for 18 days (three courses of 5 days' injection + 2 days' rest, and an additional 3 days' injection). The auditory brainstem response (ABR) and vestibular evoked potential (VsEP) were tested after the treatments. The number of sensory hair cells in the cochlear organs and the vestibular organs were quantified using microscopic images. Results All animals survived until the end of the experiment. One day after the last injection, GM + TRF mice showed significantly lower ABR thresholds at 4 kHz compared to GM mice, and there was no significant difference between the GM + TRF and NS + TRF groups. There was a significant difference of VsEP between GM and GM + TRF mice only in symmetric parabolic waves with linear acceleration and ramps waveform stimulation. GM + TRF mice showed significantly less outer and inner hair cell loss compared to GM mice. GM + TRF mice showed significantly less type II hair cell loss in the utricle and the ampulla compared to GM mice. Conclusion TRF with daytime feeding reduced GM cytotoxicity in the cochlea and vestibular organs of ICR mice. Level of Evidence NA

The circadian system, which is essential for the alignment of sleep/wake cycles, modulates adult neurogenesis. The olfactory epithelium (OE) has the ability to generate new neurons throughout life. Loss of olfactory sensory neurons (OSNs) as a result of injury to the OE triggers the generation of new OSNs, which are incorporated into olfactory circuits to restore olfactory sensory perception. This regenerative potential means that it is likely that the OE is substantially affected by sleep deprivation (SD), although how this may occur remains unclear. The aim of this study is to address how SD affects the process of OSN regeneration following OE injury. Mice were subjected to SD for 2 weeks, which induced changes in circadian activity. This condition resulted in decreased activity during the night-time and increased activity during the daytime, and induced no histological changes in the OE. However, when subjected to SD during the regeneration process after OE injury, a significant decrease in the number of mature OSNs in the dorsomedial area of the OE, which is the only area containing neurons expressing NQO1 (quinone dehydrogenase 1), was observed compared to the NQO1-negative OE. Furthermore, a significant decrease in proliferating basal cells was observed in the NQO1-positive OE compared to the NQO1-negative OE, but no increase in apoptotic OSNs was observed. These results indicate that SD accompanied by disturbed circadian activity could induce structurally negative effects on OSN regeneration, preferentially in the dorsomedial area of the OE, and that this area-specific regeneration delay might involve the biological activity of NQO1.

BackgroundThe head impulse test is a valuable clinical test that can help identify peripheral vestibular dysfunction by observing corrective saccades that return the eyes to the target of interest. Corrective saccades have been classified as covert if the onset occurs before the end of the head impulse and as overt if they occur afterwards. However, the mechanism that trigger these saccades remain unclear.ObjectiveThe objective of this study was to examine the role of neck input in generating overt as well as covert saccades.MethodsSixteen patients (9 males and 7 females: age 35-80 years, average 62.7 years old) who showed corrective saccades during the head impulse test were included. Twelve patients had unilateral vestibular dysfunction, and 4 patients had bilateral vestibular dysfunction. Patients underwent both the head impulse test (HIT) and the body impulse test (BIT) in a randomized order. While the head is rotated horizontally in HIT, the body is rotated horizontally in BIT. During BIT, the neck is fixed by a cervical collar (neck lock extrication collar) to reduce somatosensory input from the neck. The head movements and eye movements were recorded and analyzed by the video HIT recording system.ResultsIn all 16 patients, corrective saccades were observed in HIT as well as in BIT. While there were no significant differences in peak head velocities between HIT and BIT (p = 0.33, paired t-test), the VOR gain in BIT was significantly smaller than that in HIT (p = 0.011, paired t-test). The number of overt saccades per trial in BIT was significantly decreased compared to that in HIT (p < 0.001, paired t-test) whereas there were no significant differences in the number of covert saccades between the two tests. The proportion of overt saccades among all corrective saccades in BIT was significantly lower than the proportion in HIT (p < 0.001, paired t-test).ConclusionsSomatosensory input from the neck contributes to the generation of overt saccades and reinforces the vestibulo-ocular reflex complementing the retinal slip during high frequency head movements.

Abstract Oxidative stress causes tissue damage, affecting age-related pathologies. Protein restriction (PR) provides a powerful intervention strategy for reducing oxidative stress, which may have a positive effect on individual organs. However, it is unknown whether PR intervention influences the olfactory system. Here, we investigated how 10 months of PR could affect the cell dynamics of the olfactory epithelium (OE) in mice. We found that PR reduced age-related loss of outer hair cells in the cochlea, providing preventive effects against age-related hearing loss. In contrast, PR resulted in reduced mature olfactory sensory neurons (OSNs), increased proliferative basal cells, and increased apoptotic OSNs in zone 1 (the only area containing neurons expressing NQO1 [quinone dehydrogenase 1]) of the OE in comparison with animals given a control diet. Substantial oxidative stress occurred in NQO1-positive cells and induced apoptotic OSNs in zone 1. These results indicate that in contrast to the positive effect on the auditory system, PR induces oxidative stress and structurally and functionally negative effects on OSNs in zone 1, which is probably involved in the bioactivation of NQO1.

Abstract This case report presents bilateral hearing loss after vestibular‐evoked myogenic potential (VEMP) testing. The loud acoustic stimulus used in VEMP testing can cause noise‐induced hearing loss in some cases with high sensitivity to noise exposure or by exceeding individual cochlear safety thresholds.

Background: Vestibular dysfunction is a complication of cochlear implantation (CI). Reports on the evaluation of vestibular function before and after CI are limited, especially in children. We investigated the effect of CI on vestibular function in pediatric patients.Patients and Methods: We routinely evaluated vestibular function before but not immediately after CI. Therefore, patients who underwent sequential bilateral CI were enrolled in this study. Seventy-three children who underwent sequential CI from 2003 to 2020 at our hospital were included. Since the vestibular function of the first implanted ear was evaluated before the second surgery for the contralateral ear, post-CI evaluation timing differed among the cases. The evaluation included a caloric test, a cervical vestibular-evoked myogenic potential (cVEMP) test, and a damped rotation test. The objective variables included the results of these tests, and the explanatory variables included the age at surgery, cause of hearing loss, electrode type, and surgical approach used. The associations of these tests were analyzed.Results: cVEMP was the most affected after CI (36.1%), followed by the caloric test (23.6%), and damped rotation test (7.8%). Cochleostomy was significantly more harmful than a round window (RW) approach or an extended RW approach based on the results of the caloric test (p = 0.035) and damped rotation test (p = 0.029). Perimodiolar electrodes affected the caloric test results greater than straight electrodes (p = 0.041). There were no significant associations among these tests' results.Conclusions: Minimally invasive surgery in children using a round window approach or an extended round window approach with straight electrodes is desirable to preserve vestibular function after CI.

Objective: To evaluate various machine learning algorithms in predicting peripheral vestibular dysfunction using the dataset of the center of pressure (COP) sway during foam posturography measured from patients with dizziness.Study Design: Retrospective study.Setting: Tertiary referral center.Patients: Seventy-five patients with vestibular dysfunction and 163 healthy controls were retrospectively recruited. The dataset included the velocity, the envelopment area, the power spectrum of the COP for three frequency ranges and the presence of peripheral vestibular dysfunction evaluated by caloric testing in 75 patients with vestibular dysfunction and 163 healthy controls.Main Outcome Measures: Various forms of machine learning algorithms including the Gradient Boosting Decision Tree, Bagging Classifier, and Logistic Regression were trained. Validation and comparison were performed using the area under the curve (AUC) of the receiver operating characteristic curve (ROC) and the recall of each algorithm using K-fold cross-validation.Results: The AUC (0.90 ± 0.06) and the recall (0.84 ± 0.07) of the Gradient Boosting Decision Tree were the highest among the algorithms tested, and both of them were significantly higher than those of the logistic regression (AUC: 0.85 ± 0.08, recall: 0.78 ± 0.07). The recall of the Bagging Classifier (0.82 ± 0.07) was also significantly higher than that of logistic regression.Conclusion: Machine learning algorithms can be successfully used to predict vestibular dysfunction as identified using caloric testing with the dataset of the COP sway during posturography. The multiple algorithms should be evaluated in each clinical dataset since specific algorithm does not always fit to any dataset. Optimization of the hyperparameters in each algorithm are necessary to obtain the highest accuracy.

A 39-year-old man presented with chief complaints of epistaxis and pain on the right cheek that lasted for approximately 2 months. A very hard calcified black mass with a foreign body was found in the right inferior nasal meatus. The patient underwent endoscopic endonasal surgery and antimicrobial therapy, which included intravenous injection of Penicillin G 4 million units 6 times/day for 1 week and oral amoxicillin 1500 mg/day for 6 months. Actinomyces species was identified from the calcified foreign body that measured 5 mm in size. There was no recurrence for 1 year after the surgery. Surgical removal and long-term high-dose penicillin for 6 months or longer are necessary because death caused by poor compliance with antibiotics have been reported in previous studies. Keywords: Rhinolithiasis, Actinomyces

Auditory system senescence: Mitochondrial dynamics and respiration The mitochondrial morphology and physiology could influence the process of age-related hearing loss. Prof. Tatsuya Yamasoba’s research group at the University of Tokyo has examined the functional changes of mitochondria in terms of its respiratory function, membrane potential and morphology under premature senescence induced by oxidative stress in an auditory cell line. The morphological and functional mitochondrial damage were observed as the respiratory capacity deficiency and the fluctuation of the fusion/fission balance. Their results provide evidence of the fundamental interdependence between mitochondrial metabolic activity and its network structure in premature senescence process of auditory cells. This is a pioneer study to indicate the influence of mitochondrial dynamics and respiratory system on the premature senescence process of auditory cells. Further studies into inter cellular communication including cytoskeleton and nucleus can help us understand the etiology underlying age-related hearing loss.