Combining a mobile application-based vestibular diary called the DizzyQuest and an iPad-based hearing test enables evaluation of the relationship between experienced neuro-otological symptoms and hearing thresholds in daily life setting. The aim was to investigate the relationship between self-reported hearing symptoms and hearing thresholds in patients with Meniere’s disease (MD), using the DizzyQuest and the iPad-based hearing test simultaneously.The DizzyQuest was administered for 3 weeks in 21 patients. Using the experience-sampling-method (ESM), it assessed hearing loss and tinnitus severity for both ears separately. Each day after the DizzyQuest, an iPad-based hearing test was used to measure hearing thresholds. A mixed model regression analysis was performed to investigate relationships between hearing thresholds and self-reported hearing loss and tinnitus severity.p < 0.001). Self-reported tinnitus severity significantly increased with self-reported hearing loss severity in affected (p = 0.011) and unaffected ears (p < 0.001).Fifteen patients were included. Overall, pure-tone averages (PTAs) were not correlated with self-reported hearing loss severity and tinnitus. Individual differences in PTA results between both ears did not significantly influence the difference in self-reported hearing loss severity between both ears. Self-reported hearing loss and tinnitus scores were significantly higher in ears that corresponded with audiometric criteria of MD (Combining the DizzyQuest and iPad-based hearing test, facilitated assessment of self-reported hearing loss and tinnitus severity and their relationship with hearing thresholds, in a daily life setting. This study illustrated the importance of investigating neuro-otological symptoms at an individual level, using multiple measurements. ESM strategies like the DizzyQuest should therefore be considered in neuro-otological research.The online version contains supplementary material available at 10.1007/s00415-021-10909-8. Meniere’s disease (MD) is an inner ear disease that results in attacks of vertigo, combined with fluctuating ear symptoms like tinnitus, aural fullness and sensorineural hearing loss. The prevalence is estimated around 34–190 in 100,000 people  and it cFurthermore, a new app-based vestibular diary was recently introduced by the Dizzynet network , 7: the The objective of this study was to investigate the relationship between self-reported hearing symptoms and hearing thresholds in MD patients, using the DizzyQuest and the iPad-based hearing test simultaneously. It was hypothesized that a significant relationship would exist between individual hearing threshold differences and corresponding differences in self-reported hearing symptoms. Overall, a weaker correlation was expected between hearing thresholds and self-reported hearing symptoms due to interpersonal differences in symptom experience and coping mechanisms –12.MD patients with audiovestibular symptoms including fluctuating sensorineural hearing loss, were recruited by the Department of Otorhinolaryngology and Head & Neck Surgery of Maastricht University Medical Center+  and Haga hospital . Inclusion criteria comprised: aged 18 years or older, proficient in Dutch language and basic understanding of English texts, diagnosed with MD by an otorhinolaryngologist according to the Barany Society criteria  and in pwww.dizzyquest.com). It runs from the UM ESM (v2.04) application. This application is an experimental version of the PsyMate™ app (www.psymate.eu) and can be downloaded on a smartphone or tablet. It acts as a platform for medical research that enables the administration of multiple questionnaires and sampling schemes. The DizzyQuest consists of four different questionnaires, of which only the evening questionnaire was used for this study. The evening questionnaire reflects on the past day using questions about vestibular symptoms and their psychosocial context . A seven-point Likert scale is used to grade the symptoms from 1  to 7 (very much). The other DizzyQuest questionnaires  were not suited for this study due their nature, timing and frequency at which they were administered [The DizzyQuest is an app-based diary for vestibular patients  automated audiometer on iPad  was used to conduct air conduction audiometry. The following settings were used: noise alert mode off, masked air testing on, no Maximum Permissible Ambient Noise Level protocol, minimum volume 0 dB, and maximum volume 90 dB. RadioEar  DD450 headphones were used for testing air conduction thresholds ..Air conduction hearing thresholds were measured for tonal stimuli at 250, 500, 1000, 2000, 3000, 4000 and 8000 Hz. The right ear was always tested first. A play task format was used for this hearing test . AudiogrPatients were instructed to fill in the Dizziness Handicap Inventory (DHI)  and HospThe HADS is a questionnaire to screen for states of anxiety and depression in outpatients. It consists of 14 multiple choice questions of which 7 focus on anxiety and 7 on depression. Depending on the answer, 0, 1, 2 or 3 points can be scored per question. A higher score indicates a greater risk of anxiety or depression. Total scores are summed for anxiety and depression separately. The following cut-off scores were used: 0–7 for none, 8–10 for possible and > 11 for probable anxiety or depression .Patients were invited to participate in a 3-week trial. At baseline, the DHI and HADS questionnaires were completed. Then, patients were asked to complete the DizzyQuest and perform the iPad-based hearing test each day consecutively during the trial period. Before the trial, patients were contacted by two of the authors  and received an instructional video on how to use the DizzyQuest. Next to this, other authors  instructed them on how to use the iPad-based hearing test, and a written instruction for future reference was provided. The necessity of a quiet testing environment was emphasized.Each day during the 3-week trial, the evening questionnaire became available in the DizzyQuest at 7:30 PM. Patients were alerted by a notification on their phone at 8 PM. The evening questionnaire remained available until 4:30 AM the next morning. Immediately after completing the evening questionnaire, the iPad-based hearing test had to be performed. This order was chosen to prevent anchoring bias (audiometric results were shown directly after completion of the test). In case a patient did not complete the hearing test, this was considered as missing data since average PTAs could not be calculated for that given day.Five questions from the DizzyQuest evening questionnaire were selected: (1) Today I suffered from hearing loss: left; (2) Today I suffered from hearing loss: right; (3) Today I suffered from tinnitus: left; (4) Today I suffered from tinnitus: right; (5) Today I suffered from sensitivity to sounds”. This selection was made in consensus between two authors  and was based on their theme: aural symptoms. The first four questions regarding hearing loss and tinnitus were included as main outcome parameters. The fifth question “I suffered from sensitivity to sound” was included to analyse a possible confounding effect of hyperacusis, since severe self-reported handicap of tinnitus is closely associated with severity of hyperacusis .In case it was very clear that the left and right side of the headphones were accidentally switched by the patient , hearing thresholds were corrected for their side. Suspected hearing test artefacts  were excluded from analysis.It was checked for each patient whether they audiometrically fulfilled the diagnostic criteria for MD during the study period. For this, the audiometric criteria for MD were applied to the obtained iPad-based hearing test results of each ear . For eacPatients were selected for analysis if they had at least 10 “valid testing days”. A valid testing day was considered when the hearing test was completed to such extent that the main outcome parameters could be analysed. This involved obtainment of all hearing thresholds on the octave frequencies of 500–4000 Hz to calculate the Pure Tone Average (PTA) on both ears.Data were analysed with IBM SPSS Statistics 25 . For eveTo visually explore the different relationships, scatterplots per patient were made. Since every patient was tested multiple times, the assumption of independence was violated. Therefore, mixed effects regression analysis was used to analyse the data. The most appropriate model was selected using likelihood ratio testing. To correct (adjust) for a possible confounding effect of hyperacusis  and time (number of testing days) separate analyses were performed including these variables in addition to the variable of interest. A significance level of 0.05 was applied.Finally, hearing tests obtained with the iPad-based at home, were compared to previously obtained hearing tests from an audiological center which were part of routine clinical care. For each patient, hearing thresholds per octave frequency (500–1000–2000–4000 Hz) of a hearing test obtained at an audiological center, were compared to the average hearing thresholds per octave frequency of the iPad-based hearing test. The time difference between the hearing test in the audiological center and the first day of the iPad-bed hearing test, was calculated in days. Data analysis was performed by calculating the Correlation Coefficient (ICC) using a single-measure, two-way mixed-effects model (absolute agreement) . Mean ICApproval for this study was given by the research ethics board of Maastricht University Medical Center (MUMC+) (protocol: METC 2018-0809). Informed consent was obtained from all patients through the DizzyQuest application.Twenty-one patients were enrolled in this study. Three patients were lost to follow-up  and three patients were excluded since they completed less than 10 valid testing days. Therefore, data obtained from 15 patients was suited for analysis. Tables B = 0.023, t (8.823) = 1.489, p = 0.171). Results were similar after adjusting for hyperacusis and number of testing days. However, self-reported hearing loss was significantly lower in the non-affected ears, compared to the affected ears: on average 2.4 points on the Likert scale  = − 4.713, p < 0.001). This effect was also comparable after adjusting for hyperacusis and number of testing days. When comparing both ears of individual patients, the PTA difference between both ears did not significantly influence the difference in self-reported hearing loss severity  = 1.418, p = 0.182).Figure B = 0.020, t (9.540) = 1.524, p = 0.160). Results were similar after adjusting for hyperacusis and number of testing days. In contrast, non-affected ears showed a significantly lower Likert score (on average 2.8 points) than affected ears  = − 6.335, p < 0.001). This effect was also comparable after adjusting for hyperacusis and number of testing days. No significant relationship was found between PTA difference and the difference in self-reported tinnitus severity between ears of the same patient  = 1.881, p = 0.090).Figure B = 0.270, t (13.033) = 2.963, p = 0.011 and B = 0.642, t  = 6.356, p < 0.001, respectively). Results were similar after adjusting for hyperacusis and number of testing days in affected and unaffected ears.Figure The time difference between the test at an audiological center and the start of the iPad-hearing test ranged from 5 days to 56 months (SD 558.8 days). The average hearing thresholds obtained with the iPad-based hearing test at home, showed a good correlation for both the right  and left ears  with the hearing thresholds obtained at audiological centers.The aim of this study was to investigate the relationship between self-reported hearing symptoms and hearing thresholds in patients with Meniere’s disease (MD), using the DizzyQuest and the iPad-based hearing test simultaneously. This enabled the opportunity to perform multiple measurements in the same individuals, in daily life setting. It was shown that overall, PTAs were not correlated with self-reported severity of hearing loss and tinnitus. Furthermore, no significant relationship was found between individual PTA difference and corresponding difference in self-reported hearing loss or tinnitus severity between the two ears of the same subjects. However, self-reported hearing loss and tinnitus severity were on average reported to be higher in ears that (during this study period) corresponded with the audiometric criteria of MD, than ears that did not comply with the criteria. Additionally, self-reported tinnitus severity and self-reported hearing loss were significantly related: self-reported tinnitus severity increased with self-reported hearing loss severity. This study illustrates one of the positive aspects of using the ESM method: the same individuals are measured multiple times. Multiple measurements facilitate more reliable assessment of symptom severity on an individual level than when using only a single momentary assessment .It was demonstrated that (relatively) objective measurements like PTAs do not correlate well with self-reported hearing loss or tinnitus, but that self-reported items between themselves, can correlate to a certain extent. The discrepancy between the (relatively) objective hearing tests and self-reported hearing loss is congruent with previous literature . HoweverFurthermore, no significant effect was found when comparing individual differences in hearing threshold with corresponding differences in experienced hearing loss or tinnitus between both ears of the same patient. However, this does not imply that no effect can be present.Main challenge of this study was the fact that PTAs within the same ears did not show much variability during the study period. This was against expectations, since patients indicated to experience fluctuations in hearing loss at time of study inclusion. This finding demonstrates one of the strengths of prospective repeated measurements: recall bias is lowered. After all, it would be unlikely that patients suddenly stopped having fluctuations in hearing loss at time of inclusion. Most likely due to recall bias, episodes of fluctuations in hearing loss were overreported at time of inclusion. This is also congruent with a previous study with the DizzyQuest, in which the reported frequency of vertigo attacks was much higher in a retrospective end of day questionnaire than in the questionnaires which needed to be completed right after a vertigo attack . The lacEars that corresponded with the audiometric criteria of MD (affected ears) showed on average higher self-reported scores with respect to hearing loss and tinnitus severity. This was expected, since the inclusion criteria for MD include hearing loss and tinnitus. Therefore, a selection bias was most likely present. Nevertheless, this shows that the DizzyQuest is able to measure these aspects with significant differences compared to unaffected ears, demonstrating its validity in this context.Finally, the good correlation between the iPad-based hearing test results and results from “conventional” audiometry obtained in audiological centers, strengthens the use of portable hearing tests in future research , 27–29Taking all these aspects into account, this study demonstrates that technological advances provide the opportunity to perform multiple measurements in daily life setting, including assessment of neuro-otological symptoms using the DizzyQuest, and assessment of objective hearing thresholds using home-based hearing tests. Using these strategies most likely improves reliability (due to increase in data points) and it increases ecological validity of the findings, since results more closely reflect real-life setting. They also highly facilitate assessment of findings on an individual level, improving precision medicine . It is tTwo main limitations were present in this study. Firstly, during home-based audiometry using the iPad-based hearing test, the noise alert mode was off. Therefore it was not known whether background noise was present when patients performed the hearing test, which might have influenced the results of hearing thresholds. Nevertheless, all patients were explicitly instructed  to perform the test in a quiet room.Secondly, some patients already started with the DizzyQuest and iPad-based hearing test before the intended starting date, which might have affected the study results. However, most of the patients achieved a high number of valid testing days during the study period . This shThe combination of the DizzyQuest and iPad-based hearing test, facilitated assessment of self-reported hearing loss and tinnitus severity and their relationship with hearing thresholds (PTA 500–4000 Hz), in a daily life setting. Overall, PTAs were not correlated with self-reported severity of hearing loss and tinnitus. On an individual level, no significant relationship was found between individual PTA difference and corresponding difference in self-reported hearing loss or tinnitus severity between the two ears of the same subjects. However, self-reported hearing loss and tinnitus severity scores were significantly higher in ears that corresponded with audiometric criteria of MD and self-reported hearing loss and tinnitus were significantly related. This study illustrated that it is important to investigate neuro-otological symptoms on an individual level, using multiple measurements. ESM strategies like the DizzyQuest should therefore be considered in future neuro-otological research.Supplementary file1 (PDF 209 KB)Supplementary file2 (PDF 384 KB)Below is the link to the electronic supplementary material.


The writing performance of the easy-cone magnetic tunnel junction (MTJ) and perpendicularly magnetized MTJ (pMTJ) under various temperatures was investigated based on the macrospin model. When the temperature is changed from 273 K to 373 K, the switching current density of the pMTJ changes by 56%, whereas this value is only 8% in the easy-cone MTJ. Similarly, the temperature-induced variation of the switching delay is more significant in the pMTJ. This indicates that the easy-cone MTJ has a more stable writing performance under temperature variations, resulting in a wider operating temperature range. In addition, these two types of MTJs exhibit opposite temperature dependence in the current overdrive and write error rate. In the easy cone MTJ, these two performance metrics will reduce as temperature is increased. The results shown in this work demonstrate that the easy-cone MTJ is more suitable to work at high temperatures compared with the pMTJ. Our work provides a guidance for the design of STT-MRAM that is required to operate at high temperatures. FL and mPL, can only be parallel or antiparallel. According to the tunneling magnetoresistance effect, the electrical resistance of MTJ is low (high) when mFL and mPL are parallel , which can be used to represent the binary states  is determined by the spin polarization factor and polar angle of FL magnetization.The current driven magnetization dynamics is studied by solving the Landau–Lifshitz–Gilbert–Slonczewski (LLGS) equation d × σSTT)  with theHthermal, which arises from the thermal fluctuations. To investigate the intrinsic writing performance, we firstly exclude the Hthermal. To determine the equilibrium state for the easy-cone MTJ, θc at various temperatures is calculated using Equation (3), which is shown in θc at T = 273 K to T = 373 K using the numerical simulation. The results of the calculation and simulation fit well. It can be seen that the misalignment between the FL and PL will become more pronounced at a higher T. The maximum θc in our simulation reaches 19° at T = 373 K. It has been demonstrated that a larger θc will lead to lower intrinsic switching current density (Jsw0) and switching delay (tsw). Jsw0 is defined as the critical current density above which STT can overcome the damping and magnetization switching will be initialized. A small Jsw0 should be promised to ensure a low-power consumption. For the easy-cone MTJ and pMTJ, Jsw0 is given by Equations (8) and (9), respectively , which is determined by the combined effect of θ and Jsw0 [T, benefiting from the stronger thermal fluctuations and reduced Ku1,eff, θ will be enlarged, leading to a smaller overdrive. However, this will be compensated by the reduced Jsw0 at higher T (see T is increased, while that of the easy-cone MTJ decreases at elevated T. This is attributed to the larger variations of Jsw0 compared with the change of θ in the pMTJ. Therefore, Jsw0 dominates the variations of the overdrive. However, for the easy-cone MTJ, since Jsw0 barely changes as T is varied, θ dominates the variations of the current overdrive. As mentioned above, θ will be enlarged at higher T. Therefore, the overdrive will decrease as T is raised. In both devices, the variations of the overdrive mainly arise from the temperature dependence of the physical parameters. In contrast, the thermal fluctuations are less important in the fast precession regime. However, when τ is 5 ns, the overdrive of the easy-cone MTJ is almost unchanged as T is varied. It is confirmed that the easy-cone MTJ enters dynamic reversal regime, which is a transitory stage of the fast precession and thermally activated regimes. In this regime, restricted to the short τ, the reduction in overdrive is more gradual. Only when τ is further increased, and the magnetization switching enters thermally activated regime, the overdrive can be further decreased and becomes less than 1 [In the fast precession regime, the required current density for the magnetization switching is several times of er T see b. It cans than 1 . In concJ normalized by Jsw0 at different τ. An opposite temperature dependence of WER can be observed for the pMTJ and easy-cone MTJ. For the pMTJ, it requires a larger J/Jsw0 to ensure an appropriate write error rate as T is raised, resulting in the right shift of the write error rate curves. In contrast, for the easy-cone MTJ, the J/Jsw0 required to achieve the same write error rate becomes smaller at elevated T. For instance, in −3 in the easy-cone MTJ, the required J/Jsw0 is 2.67 at T = 373 K, and this value is increased to 3.18 at a lower T = 273 K. However, in the pMTJ, the corresponding J/Jsw0 is 6.31 at T = 373 K and decreased to 3.62 at T = 273 K. It can be observed in T changes from 273 K to 373 K, the required J for a write error rate of 10−3 at τ = 2 ns will decrease by 22.9% and 23.4% in the easy-cone and pMTJ, respectively. However, as shown in Jsw0 of the pMTJ changes by 56% and this value in the easy-cone MTJ is only 8%. This indicates that although Jsw0 of the pMTJ has been reduced a lot at elevated T, the required J for an appropriate write error rate cannot benefit from it. In contrast, J in the easy-cone MTJ becomes much smaller when T is increased. This demonstrates that the easy-cone MTJ exhibits a better performance at high temperatures. In addition, it can be observed that the slope of the write error rate curves in both devices is almost unchanged as T is varied. This is attributed to the opposite impact of Msat and P on the write error rate slope. When T is raised, both Msat and P are reduced, and it has been shown that a smaller Msat leads to a larger slope, whereas a reduced P makes the curve more gradual [T is manifested in the shift of the curves, which is much larger in the pMTJ due to the large variations of J/Jsw0 required for a specific write error rate. In contrast, there is negligible shifts in the easy-cone MTJ, which is beneficial when the device is required to work stably in a wide temperature range.The write error rate is the probability of non-switching cases with current applied. In order to ensure the data writing function, the write error rate should be low enough.  gradual . Since tJsw0 and tsw of both devices were decreased at high temperatures, and for the pMTJ, they fell even faster. With the effective thermal field introduced, we then performed the stochastic simulations. In the fast precession regime, the easy-cone MTJ and pMTJ had an opposite temperature dependence in the current overdrive Jsw/Jsw0. At higher temperatures, easy-cone MTJ had a smaller Jsw/Jsw0. Meanwhile, the write error rate curve shifted in opposite directions with increased temperatures for the two devices. It was also demonstrated that the required current density for an appropriate write error rate is smaller in the easy-cone MTJ. Our work reveals the potential of the easy-cone MTJ-based STT-MRAM in commercial electronic devices. Benefiting from its outstanding performance at high temperatures, the easy-cone MTJ is also fully capable of working in the automotive IC.In conclusion, we used the macrospin model to investigate the temperature dependence of the writing performance of the easy-cone MTJ and pMTJ. Consistent with the previous studies, at the same temperature, easy-one MTJ has a better writing performance ,17,24. I


Fusarium and Beauveria bassiana, respectively. Both compounds are cytotoxic and ionophoric. In the present study, the mechanism of cell death induced by these compounds was investigated. Epidermal carcinoma-derived cell line KB-3-1 cells were treated with different concentrations of these compounds. The extracellular secretion of cathepsin B increased in a concentration-dependent manner, and the lysosomal staining by lysotracker red was reduced upon the treatment with any of the compounds. However, the extracellular secretion of cathepsin L and cathepsin D were not affected. Inhibition of cathepsin B with specific inhibitor CA074 significantly reduced the cytotoxic effect of both compounds, while inhibition of cathepsin D or cathepsin L did not influence the cytotoxic activities of both compounds. In vitro labelling of lysosomal cysteine cathepsins with Ethyl -epoxysuccinate-Leu-Tyr-Acp-Lys (Biotin)-NH2 (DCG04) was not affected in case of cathepsin L upon the treatment with both compounds, while it was significantly reduced in case of cathepsin B. In conclusion, ENN B and BEA increase lysosomal Ph, which inhibits delivery of cathepsin B from Golgi to lysosomes, thereby inducing cathepsin B release in cytosol, which activates caspases and hence the apoptotic pathway.Enniatin B (ENN B) and Beauvericin (BEA) are cyclohexadepsipeptides that can be isolated from  Fusarium, while BEA was isolated from Beauveria bassiana [ENN B and BEA belong to a class of peptides named cyclohexadepsipeptides, which contain six monomers . These cbassiana . These cbassiana . Moreovebassiana .Previous studies were carried out to assess the risk of inhalation of both compounds, which confirmed no acute affects . DiffereEscherichia coli, Listeria monocytogenes, Eubacterium biforme, Enterococcus faecium, Clostridium perfringens, Bifidobacterium adolescentis, Bacillus cereus, and B. pumilis [Hemiberlesia rapax, Calliphora erythrocephala, and Aedes aegypti. BEA exhibits antibacterial activities against both pathogenic Gram-positive and Gram-negative bacteria and shows an effective capacity to inhibit human immunodeficiency virus type-1 integrase [Several investigations reported the antibacterial, antiviral and pesticidal properties of both compounds. ENN B for instance showed antibacterial effects against different bacteria species, in particular,  pumilis . Anotherntegrase .ENN B increases the lysosomal permeability through lysosomal membrane protein LAMP-2 destabilization . Other sLysosomal cathepsins are classified into three classes according to their active sites, cysteine cathepsins that include cathepsins B, H, V, W, K, L, X, C, S, F, and O , serine 4Cl was found to interfere with M6P receptors’ function by increasing the lysosomal Ph, leading to the inhibition of ligand/lysosomal hydrolases dissociation and release of newly synthesized lysosomal hydrolases from Golgi to cytosol instead of transporting them to endosomal/lysosomal compartments [Several investigations proved the importance of acidic lysosomal environment in lysosomal cathepsins sorting ,26,27. Tartments .Fusarium mycotoxin (ENN B) and Beauveria bassiana mycotoxin (BEA) and the role of some lysosomal peptidases in the apoptotic pathway of both compounds.As both Enniatin B and Beauvericin exert cytotoxic activities by inducing mitochondrial modifications and cell cycle disruption, finally resulting in apoptotic cell death, recent studies reported a potential anticancer activity ,31. The This study intends to fill the gap of the possible cytotoxicity mechanisms of the two mycotoxins (ENN B and BEA), potentially classified as anticancer agents ,33. PrevIt was hypothesized that both ENN B and BEA could interrupt lysosomal compartments’ Ph environment to interfere with M6P-tagged peptidases transfer from Golgi to lysosomes, which leads to peptidase release into cytosol and hence extracellular secretion induction rather than lysosomal translocation. In order to confirm this hypothesis, KB-3-1 cells were treated with ascending concentrations of ENN B or BEA prior to incubation with acidic compartment-specific dye lysotracker red, which is considered to be an excellent reagent to detect lysosomal Ph shifts ,36. The According to these results, lower concentration of ENN B 0.652 μM inhibited the lysosomal compartment staining to 36%, while the same concentration of BEA inhibited the lysosomal staining to 44%. The higher the concentrations of either ENN B or BEA, the more inhibition of the lysosomal compartment staining.The approachability of the active sites of cysteine cathepsins to the activity-based probe DCG04 was investigated. DCG04 was initially intended to be a selective activity-based probe of cysteine peptidases . In thisTwo specific bands were clearly appeared in DCG04 labelled cells—the higher band was approximately 30 KDa and the lower band was approximately 26 KDa. These findings showed that the cellular contents of the upper band were clearly reduced by ENN B treatment, while they were not affected by ENN B treatment in the lower band .In order to identify both bands, a pull-down experiment using streptavidin–Sepharose beads was performed, the proteins that conjugated to streptavidin–Sepharose beads were subjected to protein SDS-PAGE and Western blotting with antibodies against both cathepsin B and cathepsin L .The results of pull-down experiments supported the previous results of in vitro labelling of cysteine cathepsins with DCG04, and the cellular contents of cathepsins B were reduced to 57% of ENN B non-treated cells, and this range is similar to results of in vitro labelling experiments.On the other side, the cellular level of cathepsin L was not affected by ENN B treatment .Similar results were observed with BEA—concisely, the cellular contents of cathepsins B were reduced in BEA treated cells in comparison to BEA non-treated cells, while the cellular content of cathepsin L did not show any remarkable change upon treatment with BEA .In order to identify these two bands, pull-down experiments were carried out to cells treated with only DCG04 or BEA and DCG04, and protein samples were subjected to electrophoresis and immunoblotting using antibodies specific to cathepsin B and cathepsin L .From those experiments, we can assume that both ENN B and BEA behave similarly, either by inhibiting the proteolytic activity of cathepsin B or by interfering with biosynthetic pathway and targeting cathepsin B from Golgi to the lysosomal compartments.In vitro labelling studies of cysteine cathepsins have brought two hypotheses, that cathepsin B could be inhibited by ENN B or BEA or the extracellular secretion of cathepsin B was just increased upon treatment with one of those two compounds. Extracellular secretion studies were needed to answer this question. KB-3-1 cells were treated for 24 h with different concentrations of ENN B or BEA. Media of different treatments were collected, and cell lysates were prepared from cells. Both of cell lysates and media were subjected to SDS-PAGE and Western blotting using antibodies against cathepsin B, cathepsin D, and cathepsin L. Interestingly, the cellular contents of cathepsin B were significantly decreased upon treatment with ENN B or BEA. Moreover, the situation was opposite in media. However, the cellular contents or media of cathepsin D and cathepsin L were not changed upon the treatment with any of both compounds .In addition, the extracellular secretion of cathepsin B was correlated with concentrations of ENN B or BEA. According to those results, the cellular content of cathepsin B was decreased to 38% after treatment with 2.5 µM ENN B, and the expression of the same protein was not detectable after treatment with 5 µM ENN B. Examination of this secretion level of cathepsin B in medium was increased upon treatment with BEA. Therefore, the secretion levels were concentration-dependent. In summary, the extracellular secretion increased to 1.8-fold after treatment with 2.5 µM ENN B and almost two-fold after treatment with 5 µM BEA.The same experiments were carried out with BEA, and similar results to ENN B were obtained . HoweverThe target of these experiments was to study if the change in cathepsin B cellular contents was due to the change in cathepsin B expression and extracellular secretion. KB-3-1 cells were treated for 24, 48, and 72 h by different concentrations of ENN B or BEA. Cellular extracts were prepared, and media were collected. Cathepsin B enzymatic assays were performed using Fluorogenic Peptide Substrate Z-R-R-AMC. Cathepsin B activities were found to be decreased in cellular extracts after treatment by ENN B or BEA, and the inhibition was correlated with time and concentrations of both compounds . TreatmeInterestingly, reduction of cathepsin B activities in cellular extracts were correlated with increasing of cathepsin B activities in media .These data indicate that both ENN B and BEA enhancing cathepsin B extracellular secretion. Furthermore, they do not have inhibition activity on cathepsin B since the reduction of cathepsin B activities in cells was correlated with increasing of enzymatic activities in media. However, measurement of cathepsin L activities in both cellular and media using Fluorogenic Peptide Substrate Z-F-R-AMC did not show significant changes in cathepsin L activities extracts . In the In this experiment, KB-3-1 cells were treated with 330 nM sortilin inhibitor, AF38469, and no significant extracellular secretion pattern changes of cathepsin B were observed, while cathepsin D and cathepsin L secretion was increased more than 50% , which iTo study if the lysosomal cathepsins are implicated in cytotoxic effect of ENN B and BEA, KB-3-1 cells were treated with different concentrations of ENN B or BEA in the presence or absence of 20 mM ammonium chloride at different time span. The effect was not strongly obvious at 12 h, while it was marked after 24- and 48-h treatment. An amount of 85% of cells survived upon treatment with 1.25 µM at 24 h treatment with ENN B, while it reached 61% in the presence of ammonium chloride. The effect was greater after treatment with 2.5 µM ENN B at the same time span, while about 60% survived in the absence of ammonium chloride, and the survival range reached about 50% in the presence of ammonium chloride. About 33% of cells were survived after treatment with 5 µM ENN B in the absence of ammonium chloride, and 13% of cells survived in the presence of ammonium chloride. The effect was greater and more marked when the treatment was for 48 h A. SimilaThese results indicate the lysosomal cathepsins are implicated in the cytotoxicity of both ENN B and BEA.To determine which of lysosomal cathepsins are involved in ENN B or BEA cytotoxicity, KB-3-1, cells were treated with different concentrations of ENN B or BEA at different time in the presence or absence of 10 µM of cathepsin B inhibitor CA074, cathepsin D inhibitor pepstatin A, or the specific cathepsin L inhibitor Z-FY (tBu)DMK A. CathepIn order to provide a direct evidence whether cathepsin B mediates the apoptotic pathway induced by ENN B or BEA, KB-3-1, cells were treated for 24 h with different concentrations of ENN B or BEA in the presence and absence of 10 µM of cathepsin D inhibitor (pepstatin A) or the same concentration of cathepsin L inhibitor (Z-FY(tBu)DMK) or cathepsin B inhibitor (CA074). Cells were harvested, and cell lysates were prepared from cells. The lysates were then subjected to SDS-PAGE and Western blotting using antibodies against the cleaved forms of some cathepsin activation dependent apoptotic proteins .The expression of these proteins was obviously elevated upon treatment with different doses of ENN B or BEA. The effect of these compounds on the expression of these proteins was significantly reduced upon treatment with the specific cathepsin B inhibitor (CA074) . InteresPrevious investigations showed that ENN B has ionophoric properties to induce lysosomal membrane disruption, which increases lysosomal permeability and hence the lysosomal hydrolases release and extracellular secretion. ENN B was found to destabilize the lysosomal membrane protein LAMP-2 . This stIt has been reported that ENN B can induce extracellular secretion of many lysosomal cathepsins as a consequence of lysosomal membrane destabilization . In thisAs a consequence of ENN B or BEA treatments, the protein expression in media was significantly increased for cathepsin B, but not for cathepsin L or cathepsin D . The celThe lysosomal cathepsins have a unique pathway pattern of N-glycosylation, hence their N-glycan is mannose 6-phosphrylated, which makes them suitable for interaction with one of the two mannose 6-phsphate specific receptors, MPR46 and M6P/IGF2R. These receptors transport the lysosomal hydrolases from Golgi to the lysosomal compartments. In the lysosomal compartments, the receptors dissociate from the ligands due to the low pH of the lysosomal compartments, and the dissociated receptors return to Golgi to bring the newly synthesized lysosomal hydrolases ,38. For Sortilin is not specific for M6p tagged proteins. Therefore, it is not pH-dependent. Interestingly, it was observed that sortilin can transport some cathepsins, such as cathepsin L, cathepsin H, and cathepsin D, but not cathepsin B . In thisCathepsin B inhibition, but not cathepsin D or cathepsin L, significantly reduced the cytotoxic effects of ENN B or BEA . MoreoveWe expect that this fluctuation was due to the presence of both cathepsin D and cathepsin L in the cytosol and their physiological roles in proteins processing and turning over. Cytosolic cathepsin D and L, for instance, play an important role in activation of some proteins that are involved in apoptosis.In addition, cytosolic cathepsin D and L are implicated in Bid degradation and activation . CathepsIn summary, both ENN B and BEA have different mechanisms for apoptosis. The lysosomal pathway of apoptosis is involved in these mechanisms. Both compounds can induce cytosolic release and extracellular secretion of cathepsin B, but not cathepsin L or cathepsin D, to induce apoptosis. Only cathepsin B secretion was affected upon treatment with one of both compounds due to different sorting mechanisms of lysosomal hydrolases, and cathepsin B sorting has a unique M6P-dependent pathway, while cathepsin D and cathepsin L can be sorted with M6P-dependent pathway and non M6P-dependent pathway via the sortilin receptor.In conclusion, ENN B and BEA have the ability to induce lysosomal permeabilization ,14, and,Taking together our findings and other studies to future investigations, for instance, drug delivery-based nanoparticles studies on both ENN B and BEA to target certain tumor types in mice models might be developed. Therefore, we assume that this study can contribute to further therapeutic cancer research insights.2 atmosphere.Epidermal carcinoma-derived cell line KB-3-1  was used in the study. The cell line was propagated in Dulbecco’s Eagle’s medium , supplemented with 4 mM glutamine. Antibiotics 100 units/mL penicillin and 100 ug/mL streptomycin  were added, and cells were incubated at 37 °C in a 5% COCells were seeded in 96 well plates at a density of 2000 cells/well in 100 μL/well medium and incubated for 24 h. After that, cells were incubated separately with ENN B and BEA , and concentrations ranged from 1.25 to 5 μM. The percentage of viable cells was determined at 24, 48, and 72 h post ENN B and BEA treatment by 3--2 and -5 diphenyltetrazolium bromide (MTT)-based viability assay . An amount of 20 μL of EZ4U solution was added to each well. After incubation for 2 h at 37 °C, the absorbance was measured by a microplate reader  at 450 nm with 620 nm as reference to the unspecific background values. All experimental work was repeated three times in triplicate.7) were treated for 24, 48, and 72 h at 37 °C in a complete medium containing 10 μM inhibitor of E64-d , CA074 , leupeptin , and pepstatin A . Dimethylsulphoxide solvent  was used as a control.Cell lines  before being treated with 600 nM LysoTracker dye and DAPI staining  for 10 min at room temperature (RT).These experiments were performed according to . Cells wImages were captured using an Olympus BX51 upright microscope with a 60× 1.4NA objective and Picture framer software .The lysosomal area was calculated using Image J software.w/v) triton X-100). The samples were incubated for 5 min at 37 °C before the addition of 10 μM Z-F-R-AMC (Z-Phe-Arg-7-amido-4-methycoumarin)  as substrate, then cathepsin B activity was measured using specific substrate that is hardly cleaved by another cathepsin [The cysteine cathepsin activity of cellular extract and media was determined spectrofluorimetrically according to . Brieflyathepsin , Z-R-R-ACysteine cathepsins can be labelled in cultured cells by using the activity-based probe DCG04 , which is a biotinylated form of general cysteine peptidase inhibitor E-64. DCG04 is a selective activity-based probe of cysteine peptidases. It is a biotinylated form of the general cysteine peptidase inhibitor E64 (which line number). It has the ability to bind to active cysteine peptidases in protein mixture . Cells wg for five min, and supernatant was incubated overnight with 40 μL settled streptavidin beads at 4 °C. Beads were precipitated by centrifugation for 5 min at 3000 rpm, then they were washed five times with 20 mM sodium acetate (pH 5.5), 150 mM sodium chloride, and 0.1% triton X-100 and twice with 10 mM Tris-HCL, pH 6.8 [In order to pull down the in vitro labelled cysteine cathepsins, 250 μL (about 400 μg) of protein concentrations of cells extracts were measured according to Bradford 1976 ), and ce, pH 6.8 . Settled, pH 6.8 ,48,49,50, pH 6.8 .Different general cathepsin inhibitors were used to assess the effect of the lysosomal cathepsins on ENN B and BEA cytotoxicity. Around or CA074 ,52. For or CA074 ,54. For 2 incubator in DMEM medium supplemented with 5% fetal bovine serum. Medium was then aspirated, and cells were washed twice with PBS. Cells were scrapped in lysis buffer  and transferred to 1.5 μL centrifuge tube. The cells were then homogenized on ice by ultrasonication, and then 0.1% Triton X-100 was added. The homogenized cells were then extracted on ice for 30 min. Samples were then cleared by centrifugation 15,000× g for 10 min. The proteins were separated under reducing conditions by 12.5% SDS-PAGE. Proteins were then transferred to nitrocellulose membrane  by semi-dry blotting at 25 V for 30 min. Unspecific sites were then blocked for 3 h by blocking solution (3% BSA in PBS). Membrane was then incubated for 90 min with the primary antibodies, cathepsins B, D, or L  (1:2000), cleaved Bid, Bcl-2, caspase 3  (1:1000), and β-actin  (1:3000), and then it was washed five times with PBST and incubated another 90 min with the corresponding secondary antibodies and washed three times by PBST and one time by PBS. Enhanced chemiluminescence  was used for visualization. Membranes were exposed to X-ray films . Experimental films were scanned and quantified by ImageJ .Experiments were conducted as previously described ,34,35,364Cl for 24, 48, and 72 h. Media were then collected, and cells were harvested and subjected to protein extraction prior to Western blot analysis [In order to induce lysosomal hydrolases secretion, 80% confluent cells were treated with 20 mM NHanalysis ,56,57. Ft-test to compare between two groups, and, if there were more than two groups, one-way ANOVA or two-way ANOVA were used according to the number of independent variables. When an analysis of variance (ANOVA) F test is significant, it does not report which pairs of means are different. Post hoc tests are used to uncover specific differences between three or more group means. Dunnett’s can be used after the ANOVA has been run to identify the pairs with significant differences. Dunnett’s test (Dunnett’s multiple comparison) compares means from several experimental groups against one fixed control group mean to see if there is any difference. The Bonferroni test is a statistical test used to reduce the instance of false positive, if needed. Bonferroni designed an adjustment to prevent data from incorrectly appearing to be statistically significant. All statistical analyses were performed with GraphPad Prism (GraphPad Software), San Diego, CA, USA, as well as Microsoft Excel 365. The probability level of p < 0.05 was considered statistically significant. Data were presented as mean ± SE. Detailed statistical parameters for specific experiments were described in the appropriate section or T legends.Statistical analysis was performed using a non-parametric


Despite its high prevalence in the aged, hearing loss has been poorly investigated. Audiometry is the gold standard for evaluation of hearing loss, but large-scale use of the procedure involves operational difficulties. Thus, self-report may be an alternative.To determine if a single global question is valid for use in epidemiologic research.Systematic review.Study Selection and Data Extraction: The articles that compared the results obtained with self-report to a single global question with those obtained by pure tone audiometry were selected. Data about the prevalence of hearing loss, and sensitivity, specificity and predictive values were extracted. Data Synthesis: Ten longitudinal studies were included. A single global question seems to be an acceptable indicator of hearing loss, sensitive and reasonably specific, mainly if the hearing loss is identified as the tone average that includes frequencies up to 2 or 4 kHz, at 40 dBHL level, in the best ear.A search of the medical literature from 1990 to 2004 was performed using MEDLINE and LILACS. The references of the articles identified in the electronic search were also reviewed. A single global question shows good performance in identifying older persons with hearing loss and can be recommended for an epidemiologic study if audiometric measurements cannot be performed. Considering the age of 60 years as the dividing line between elderly and non-elderly, we observed based on IBGE data (Brazilian Institute of Geography and Statistics) that there has been percentage increase in number of elderly people (60 years and over) in Brazil, from 5.07% in 1970 to 8.56% in 2000Among pathologies whose frequency increases with aging, we can include those related with inner ear. Hearing loss is the third most prevalent chronic condition among American elderly patients, third to hypertension and arthritisDespite the high prevalence among elderly patients, hearing loss is one of the problems not investigated during routine medical examination in this age rangeConsidering that audiometry is the gold standard for hearing loss detection and requires trained team, soundproof booth and equipment, hindering its execution in large scale, our final goal was to determine whether one single global question would be valid to be used in epidemiological research studies. This paper aimed at reviewing studies whose purposes were to compare results obtained with use of isolated questions and results obtained through pure tone audiometry in assessment of hearing loss in the elderly.hearing loss, hearing impairment, deafness, presbycusis, questionnaires, self-report, question, validity, audiometry, isolated or in combination and accepted texts in all languages. We also analyzed articles cited in the references of other articles identified in the electronic search. We selected 10 articles from MEDLINE and none from LILACS. We excluded one article because it did not provide isolated data on validity of the use of one single global question on hearing loss assessment compared to pure tone audiometryWe conducted a literature review on databases MEDLINE and LILACS, in the period between 1990 and 2004 to retrieve articles comparing results obtained in the assessment of hearing loss in the elderly through self-report using one single global question and pure tone audiometry. In the electronic search we used the terms We extracted data on prevalence of hearing loss observed by audiometry, estimated loss by the question and difference between prevalence of observed and estimated hearing loss. Sensitivity, specificity and predictive values of hearing loss assessment through one single global question in comparison to audiometry were also included. One study presented estimate of odds ratio (OR) of the question concerning hearing loss measured by audiometry and another one assessed the association between the question and the mean thresholds in different frequencies.In Pure tone audiometry criteria varied according to frequency, intensity and assessed ears. Seven studies assessed pure tone mean (MT), with variation of frequencies used in the calculation between 0.5, 1, 2, 3 and 4 kHzThe questions used were similar one to the other, and five studies used questions with yes-no questionsObserved prevalence by pure tone audiometry ranged from 2% . To both men and women, prevalence of self-reported hearing impairment increased with each 5-year increase in age group . Pure tone mean in the best ear in 683 patients that reported an auditory problem was significantly worse than in 979 cases that did not report hearing problems. Pure tone mean in the best ear was significantly higher among men  than among women . As a result of increased age, there is generalized worsening of thresholds in all frequencies, especially in higher ones. Authors detected a significant difference  between proportion of men (32.5%) and women (26.7%) that were classified as having hearing loss with MT 0.5-2 kHz.Gates et al. (1990)Reuben et al. (1998)Nondahl et al. (1998)Sindhusake et al. (2001)Uchida et al. (2003)Hashimoto et al. (2004)The present study aimed at comparing the prevalence estimates of hearing loss obtained using two methods: self-report and audiometry. According to the analysis of the studied literature, the single global question seems to be an acceptable indicator of hearing loss which is sensitive and reasonably specific, especially when the loss is identified as being pure tone mean up to frequencies 2 or 4 kHz, at 40 dBHL on the best ear.The comparison of hearing loss prevalence among the studies is hindered by the differences in investigated populations and in audiometric criteria used for its definitionDefinitions of hearing loss assessed by pure tone mean in the literature range according to the ear used to classify the subject  and to frequencies included in the pure tone mean to determine the best and the worst earAs to self-report of hearing loss comparing to pure tone audiometry classification criteria, the use of the best ear is justified by the fact that the worst ear tends to be compensated by the function of the side that has the best subjective perceptionAs to questions used, multiple-choice questionsVoeks et al. (1993)According to Hashimoto, Nomura and Yano (2004)Screening approaches try to increase the likelihood a person with a specific dysfunction has to be identified (sensitivity), excluding those without the dysfunction (specificity). In practice, not all cases are identified , and some people without dysfunction will be misdiagnosed As to results of the studies based on gender and age, Gates et al. (1990)As to both studies that did not present results of sensitivity, specificity and predictive values, Reuben et al. (1998)Gates et al. (1990)We should also add that we did not find any Brazilian studies that compared one single question questionnaire to pure tone audiometry, and we do not have estimates that can be generalized to the Brazilian population.One single global question has good performance to identify elderly people with hearing loss and it may be recommended for epidemiological studies that cannot apply audiometric measurements. Considering that we do not have estimates that can be generalized to the Brazilian population, it would be recommendable to conduct a validation study of one single global question compared to audiometry, allowing the use of this useful tool in Brazilian epidemiological studies, with the advantage of being able to study a large number of subjects.


CDKL5 deficiency disorder (CDD) is a rare neurodevelopmental condition, primarily affecting girls for which no cure currently exists. Neuronal morphogenesis and plasticity impairments as well as metabolic dysfunctions occur in CDD patients. The present study explored the potential therapeutic value for CDD of FRAX486, a brain‐penetrant molecule that was reported to selectively inhibit group I p21‐activated kinases (PAKs), serine/threonine kinases critically involved in the regulation of neuronal morphology and glucose homeostasis.Cdkl5‐knockout male mice (Cdkl5‐KO) and in vivo  on Cdkl5‐KO heterozygous females (Cdkl5‐Het).The effects of treatment with FRAX486 on CDD‐related alterations were assessed in vitro (100 nM for 48 h) on primary hippocampal cultures from Cdkl5‐KO mouse neurons. In vivo, FRAX486 normalized the general health status, the hyperactive profile and the fear learning defects of fully symptomatic Cdkl5‐Het mice. Systemically, FRAX486 treatment normalized the levels of reactive oxidizing species in the whole blood and the fasting‐induced hypoglycemia displayed by Cdkl5‐Het mice. In the hippocampus of Cdkl5‐Het mice, treatment with FRAX486 rescued spine maturation and PSD95 expression and restored the abnormal PAKs phosphorylation at sites which are critical for their activation (P‐PAK‐Ser144/141/139) or for the control cytoskeleton remodeling (P‐PAK1‐Thr212).The in vitro treatment with FRAX486 completely rescued the abnormal neuronal maturation and the number of PSD95‐positive puncta in Present results provide evidence that PAKs may represent innovative therapeutic targets for CDD. CDKL5 deficiency disorder (CDD) is a severe neurological disease for which no cure exists. A systemic 5‐day long treatment with FRAX486 rescued both the neurobehavioral alterations and hippocampal spine imbalance in a female mouse model of CDD at a clearly symptomatic stage. In mouse blood, FRAX486 rescued the increased oxidative stress status and the fasting‐induced hypoglycemia. Importantly, the treatment also restored the normal activity of PAKs kinase family. Available data point to a crucial role of CDKL5 in fundamental neurodevelopmental processes such as activity‐dependent regulation of neuronal morphogenesis and plasticity.Cdkl5‐KO) and in induced‐pluripotent stem cells (iPSCs)‐derived neurons from CDD patients.CDKL5 deficiency disorder is a X‐linked dominant disorder caused by de novo mutations in the Cdkl5‐KO brains was also found, which was accompanied by an impairment in mitochondrial ATP production rate and reduced ATP whole brain levels.A role for CDKL5 in the regulation of energy metabolism is also emerging. Recent findings in fact provide evidence that mitochondrial dysfunction and oxidative stress occur in CDD.Since loss of CDKL5 affects neuronal morphology and a role for CDKL5 in the regulation of cytoskeleton dynamics has been highlighted, in the present study we focused our attention on group I p21‐activated kinases (PAKs), a family of serine/threonine kinases (PAK1/2/3) that are critically involved in the regulation of microtubule and actin‐cytoskeleton remodeling.Interestingly, PAKs are also emerging as regulators of glucose homeostasis in peripheral tissues as well as in neurons.Cdkl5‐KO mouse model. Preliminary in vitro evaluation was performed to verify whether treatment with FRAX486 was able to rescue neuronal alterations in the complete absence of Cdkl5. Based on the promising results obtained, we next assessed FRAX486 therapeutic efficacy in vivo on heterozygous Cdkl5‐KO female (Cdkl5‐Het) mice, the model that better reproduces the genetic and hormonal milieu of CDD patients.The present study tested the possibility that pharmacological treatment with the brain penetrant FRAX486,To verify the activation status of PAKs in CDD mouse brain and FRAX486 effects thereon, phosphorylation levels at sites which are considered indices of PAKs activation  were also assessed. Given its emerging crucial role in the proper regulation of the cytoskeleton during neurite outgrowth,2Mouse colony. Experimental subjects were derived from the Cdkl5‐KO strain developed byCdkl5‐Het female mice and wild‐type (wt) or hemizygous male mice. The day of birth was designated as postnatal day (P) 0 and animals with 24 h of age were considered as P1 old animals. After weaning at P25‐30, mice were housed according to sex in groups of 2–5 per cage on a 12 h light/dark cycle in a temperature‐controlled environment (21 ± 1°C and relative humidity at 60 ± 10%) with a complete pellet diet  and tap water provided ad libitum. All experimental procedures were conducted in conformity with the European Communities Council Directive (10/63/EU) as well as the Italian law (26/2014) and approved by the Italian Ministry for Health.Genotyping. DNA was prepared as previously described.Drug. FRAX486 was supplied by DBA  and stored at −20°C, protected from light. FRAX486 was dissolved in either 20% (weight/volume) hydroxypropyl‐β‐cyclodextrin  in saline or DMSO  for in vivo and in vitro experiments, respectively.2.1Cdkl5‐KO mice (N = 3–4 per group), as previously described.50,Primary hippocampal neurons were prepared from P1 old wt and Immunostaining was performed using rabbit polyclonal anti‐MAP2  and mouse monoclonal anti‐PSD95  antibodies. Detection was performed using rabbit FITC‐conjugated  and mouse Cy3‐conjugated  secondary antibodies. Fluorescent images were acquired using a Nikon Eclipse TE600 microscope equipped with a Nikon Digital Camera DXM1200 ATI System . Neurite outgrowth was analyzed using the image analysis system Image‐Pro Plus as previously described.N = 3–4 samples and 8–10 neurons per sample.Acquired images of MAP2 staining, specifically identifying dendrites, were analyzed thanks to the “Sholl Analysis” plugin of the FiJi software (htpp://Fiji.sc).2.22.2.12.2.1.1Cdkl5‐Het mice were treated once daily for 5 consecutive days via subcutaneous (s.c.) injections of either 20 mg/Kg of FRAX486 or ctrl (N = 12 per group) starting at 8:00 am, as previously described.N = 15). Mice were tested at an advanced stage of the disease  production and hippocampi were dissected and immediately frozen for subsequent analyses.e Figure b 6 h aft2.2.1.2Cdkl5‐Het and wt mice at the time of hippocampus collection . The oxidation of the spin probe 1‐hydroxy‐3‐carboxypyrrolidine  to the correspondent 3‐carboxy‐proxyl radical (CP•)• is not specific to a singular oxidant but is suitable for screening the totality of ROS  produced in biological samples. If the intensity of CP• is significantly increased, the presence of a pro‐oxidant status is suggested. Briefly, CPH  was added to 100 μl of whole blood of wt or Cdkl5‐Het mice and the intensity of CP• was measured after 20 min at 37°C. Samples were drawn up into a gas‐permeable Teflon tube with 0.81 mm internal diameter and 0.05 mm wall thickness . The Teflon tube was folded four times, inserted into a quartz tube, and fixed to the cavity (4108 TMH) of a Bruker ECS 106 EPR spectrometer equipped with a variable temperature unit (ER4111VT). Spectrometer conditions were: modulation frequency, 100 kHz; microwave frequency, 9.4 GHz; microwave power, 20 mW; gain, 1 × 104; modulation amplitude, 1G; conversion time, 20.5 ms; time constant, 82 ms; sweep time, 21 s; and number of scans, 1.Trunk blood was collected into heparinized tubes on the 5t Figure b, 6 h fr2.2.1.5N = 4–11 per group). The hippocampal formation was homogenized in ice‐cold RIPA buffer supplemented with 1 mM PMSF , and with 1% protease  and phosphatase inhibitor cocktail . Protein concentration was determined using the Lowry method.WB analysis was conducted on hippocampi collected 6 h from the last s.c. injection (2.2.22.2.2.1Cdkl5‐Het mice and wt littermates (N = 3–4 per group) that had not undergone behavioral assessment was treated once daily for 5 consecutive days via s.c. injections of either 20 mg/Kg of FRAX486 or ctrl, to evaluate FRAX486 treatment effects on spine density/maturation . Dendritic spine density was measured by manually counting the number of dendritic spines on apical dendrites of CA1‐pyramidal neurons. In each mouse, 10–15 dendritic segments  were analyzed and the linear spine density was calculated by dividing the total number of counted spines by the length of the sampled segment. Based on their morphology, dendritic spines can be divided into five different classes, which also reflect their state of maturation . The total number of spines was expressed per 10 μm, and the number of spines belonging to each class was counted and expressed as a percentage. All steps of sectioning, imaging, and data analysis were conducted blindly.2.2.32.2.3.1Cdkl5‐Het mice show abnormal blood glucose levels (glycemia) at a fully symptomatic stage, an independent cohort of symptomatic Cdkl5‐Het and wt littermates . Glycemia was measured at 0 (baseline), 30, 60, 120, and 180 min after i.p. injection.After a 15‐h overnight fasting (from 7:30 pm to 10:30 am) mice were intra‐peritoneally (i.p.) loaded with 2 g/kg body weight of One week later, mice were starved for 5 h (from 9:00 am to 2:00 pm), and glycemia was measured immediately before  and 15 min after an i.p. injection with 0.4 U/kg body weight of human recombinant insulin .2.2.3.2Cdkl5‐Het and wt mice  was tested to confirm the hypoglycemic profile of Cdkl5‐Het mice in fasting conditions and to verify whether FRAX486 treatment may rescue the impaired glucose metabolism in CDD  on the 5th day of treatment. The following week, Cdkl5‐Het mice received once daily, for 5 consecutive days, s.c. injections of 20 mg/kg of FRAX486, while wt mice received ctrl solution, starting at 8:00 am. Glycemia was measured as in the first week.A separate cohort of symptomatic D Figure d. In the2.32.3.12 at 37°C. Cell medium was replaced every 3 days and the cells were sub‐cultured once they reached 90% confluence. Cells were transfected with pCMV14‐3xFLAG‐hCDKL5 carrying the CDKL5_1 isoform that generates a protein of 960 amino acids (107 kDa), pCMV14‐3xFLAG‐hCDKL5 carrying the CDKL5_1 kinase dead isoform K42R and pCMV14‐3xFLAG empty backbone as a control  using Metafectene Easy Plus .293 T cell line was maintained in Dulbecco Modified Eagle Medium  supplemented with 10% heat‐inactivated FBS, 2 mM of glutamine, and antibiotics , in a humidified atmosphere of 5% of CO2.3.2N = 3 per group). Total proteins from 293 T cells transfected with wt CDKL5 or empty vector were lysed in ice‐cold RIPA buffer  supplemented with 1 mM PMSF , and with 1% protease  and phosphatase inhibitor cocktail . Protein concentration was determined using the Bradford method.Twenty‐four hours after transfection cells were processed for WB , with Dunnett's test for post hoc comparisons, setting Cdkl5‐KO, ctrl or Cdkl5‐Het, ctrl as control group. Repeated measures ANOVA  followed by Tukey's post hoc test was also used. Animals identified as outliers by the use of Grubb's test were excluded from the analyses.p < 0.05 was considered to be statistically significant. Results are presented as mean ± standard error of the mean  or as 10th, 25th, 50th (median), 75th, and 90th percentiles (box plots). Statistical analyses results are reported in Tables Statistical analysis was performed with IBM SPSS Statistics Version 26.0. Normality and homoscedasticity were evaluated using Shapiro–Wilk's and Levene's tests, respectively. Datasets with normal distribution were analyzed for significance using Student's 33.1Cdkl5‐KO mice, a reliable in vitro disease‐related model that recapitulates many of the most striking neuronal CDD phenotypes.Cdkl5‐KO mice showed reduced neurite outgrowth  ± SEM: basal: wt = 145.400 ± 4.681; Cdkl5‐Het = 137.857 ± 4.611; fasted: wt = 140.800 ± 5.183; Cdkl5‐Het = 119.286 ± 4.069; data not shown]. These findings provide innovative evidence that aberrant CDKL5 functioning may disrupt mechanisms that regulate glucose homeostasis. To test whether alterations in PAKs activation may be involved in the impaired glucose metabolism of Cdkl5‐Het mice, we next assessed whether a 5‐day long treatment with FRAX486 may restore the abnormal blood glucose levels in fasted Cdkl5‐Het mice. We found that a 5‐day long treatment with FRAX486 completely rescued the fasting‐induced hypoglycemia of Cdkl5‐Het mice Cdkl5‐Het mouse hippocampus compared with wt controls (p < 0.01 for Cdkl5‐Het, ctrl vs. wt, ctrl; Figure Cdkl5‐Het hippocampal homogenates, thus restoring wt‐like levels (p < 0.05 for Cdkl5‐Het, ctrl vs. Cdkl5‐Het, FRAX486; Figure We found that the levels of P‐PAKs/PAKs ratio, which provides an index of the net functionality of the kinase, was shifted toward decreased phosphorylation for Cdkl5‐Het mice also showed increased levels of P‐PAK1 Thr212 in comparison with wt mice (p < 0.01 for Cdkl5‐Het, ctrl vs. wt, ctrl; Figure Cdkl5‐Het mice (p < 0.001 for Cdkl5‐Het, ctrl vs. Cdkl5‐Het, FRAX486; Figure WB analyses on hippocampal extracts from p < 0.05 relative to control; Figure Given the fundamental role played by this phosphorylation site in the regulation of cytoskeleton dynamics4The present study demonstrates that systemic treatment with FRAX486 exerts compelling beneficial effects on neurobehavioral alterations in a female mouse model of CDD and rescues the increased levels of ROS and fasting‐induced hypoglycemia in CDD mouse blood. Interestingly, these effects were accompanied by a normalization of PAKs hypoactivation in CDD mouse hippocampus. The first evidence that CDKL5 dysfunction affects the phosphorylation of Thr‐212 PAK1, a site that has emerged as crucial for the proper regulation of neuronal morphology,Cdkl5‐Het female mouse brain, the model which more closely resembles the genetic and hormonal milieu of CDD patients.The evidence demonstrating that CDKL5 plays a crucial role in brain development and function is overwhelming. However, the mechanisms leading to the neurological symptoms associated with CDD have not been completely clarified.Cdkl5‐KO male mice in the same brain area,Surprisingly, however, we found that systemic treatment with FRAX486 normalized PAKs hypoactivation in CDD mouse hippocampus. Given the reported role of the molecule as a PAKs inhibitor,While the mechanisms underlying PAKs hypoactivation in CDD mouse brain and FRAX486 effects thereon have to be further explored, it is reliable to speculate that the beneficial effects of the treatment with FRAX486 on CDD‐related dysfunction in mouse neurons may be attributed to a rescue of altered PAKs‐dependent cytoskeleton dynamics. PAKs are in fact critical regulators of cytoskeleton dynamics underlying neuronal morphological changes and plasticity and, in particular, the Thr‐212 phosphorylation site of PAK1 has been proposed to affect the cross‐talk between microtubules and microfilaments during the forward movement and turning of neuronal growth cones,Importantly, the normalization of the altered neuronal phenotype was accompanied by the rescue of CDD‐related behavioral alterations in the CDD mouse model, suggesting the potential therapeutic value of this treatment strategy for CDD. In particular, FRAX486 treatment restored fear memory defects in CDD female mice, confirming a number of evidence suggesting a tight link between PAKs alterations and cognitive dysfunction.Another major finding of the present study regards the demonstration that CDD female mice at an advanced stage of disease display alterations in blood glucose levels that are rescued by systemic treatment with FRAX486. In fact, despite recent studies have suggested an important role for CDKL5 in the regulation of energy metabolism,Cdkl5‐Het mice. Interestingly, numerous evidence have highlighted a tight link between metabolic dysfunction and the increased generation of ROS,We also provide evidence that the aberrant pro‐oxidant status occurring in CDD mouse blood can be substantially improved by systemic treatment with FRAX486. This result is consistent with previous studies demonstrating an increase in oxidative stress markers in CDD patients‐derived fibroblasts and plasma.In conclusion, the present study identifies PAKs as promising therapeutic targets for CDD, a highly debilitating disorder for which no cure exists. Although the surprising effect of the treatment with FRAX486 on PAKs activation status in CDD mouse brain challenges the translational value of this molecule, present data provide innovative evidence that PAKs activating drugs may represent promising therapeutic candidates for CDD. The emerging role of PAKs as crucial regulators of glucose homeostasis is also confirmed, further stressing the need for PAKs expression levels to be finely regulated both in the brain and in the periphery. Present results also shed some new light on CDD metabolic alterations, by demonstrating that glucose homeostasis is compromised in a female mouse model of the disorder. Furthermore, increased levels of reactive oxidizing species in whole blood of CDD mice were first highlighted, that could be exploited as markers of treatment efficacy.CF, MCT, and BDF designed experiments. MCT and LC performed behavioral analyses. CF and GM performed in vitro studies and western blots. NM performed in vitro image analysis and quantification. MCQ and DP performed EPR analysis. LC and CU performed data analysis. DP, EC, AF, and BDF provided resources and supervised data collection and analysis. BDF, LC, and CU wrote the article. BDF conceived the study, acquired funding, and administered the project. All the authors reviewed the article.The authors declare no conflict of interest.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.


This study aimed to explore the relationship between body mass index (BMI) and hearing loss. We analyzed data from the Korean National Health Insurance Service Health Screening Cohort 2009–2019 . Both patient groups were subsequently divided into four groups according to BMI: <18.5 (underweight), 18.5–24.9 , 25–29.9 (obese I), and ≥30 (obese II). To evaluate the relationship between BMI and hearing loss, multivariate logistic regression analysis was used, adjusting for age, sex, smoking, alcohol consumption, blood pressure, triglycerides, total cholesterol, low-density lipoprotein, proteinuria, serum creatinine, aspartate aminotransferase, alanine aminotransferase, and fasting glucose levels. The adjusted odds ratio (OR) of the underweight group for hearing loss was 1.21 (95% CI = 1.19–1.24) compared to the normal BMI group, whereas the adjusted ORs of obese I and obese II groups for hearing loss were 0.95 and 0.87, respectively. Being underweight was generally associated with an increased prevalence of hearing loss in the Korean adult population. In the last few years, over 5% of the world’s population—an estimated 1.57 billion people—has been reported to have hearing loss. Of those, the population of the Western Pacific region accounted for the largest proportion 540.2 million), followed by Southeast Asian population (400 million)  million,,7,8. Pop2 had 1.59-fold increased odds of sudden sensorineural hearing loss than those with a BMI < 23.5 kg/m2 [2, respectively, compared to individuals with BMI < 25.0 kg/m2 [Obesity constitutes an important public health concern as its prevalence has continued to increase worldwide in the past few decades . Several.5 kg/m2 . In addi.0 kg/m2 . Factors.0 kg/m2 .2 compared to the normal range of 19–24.9 kg/m2) was not significantly associated with hearing loss [2) or decreased (OR = 1.312 for BMI < 18.5 kg/m2) along with BMI. However, the conclusions were limited to severe cases as the study excluded mild to moderate degrees of hearing loss [However, other studies have reported no relationship between obesity and hearing loss prevalence. A prospective cohort study of male patients aged 40–74 years in the US showed that a history of hypertension , diabetes mellitus , or obesity  is a cohort of individuals who participated in health screening programs provided by the NHIS in the Republic of Korea. We used data based on the NHIS-HEALS database from 2009 to 2019. The Ethics Committee of CHA Bundang Medical Center (2022-04-009) approved the use of these data. This study was exempted from the need for written informed consent by the Institutional Review Board.The total number of participants was 9,535,376. Of these, 2,688,340 were excluded because of missing data on hearing loss in one or both ears, smoking, alcohol consumption, height and weight beyond established thresholds, abnormal blood pressure, proteinuria, and abnormal blood levels of creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, triglycerides, low-density lipoprotein (LDL), and fasting glucose. We detected outliers by determining an interval spanning over the mean plus/minus three standard deviations, as a result, 466,589 were excluded. Consequently, 316,623 participants with hearing loss and 6,530,413 control participants were included in the final analysis .Of a total of 9,535,376 participants, 316,623 hearing loss patients and 6,530,413 control participants were enrolled.2) was categorized as <18.5 (underweight), 18.5–24.9 , 25–29.9 (obese I), and ≥30 (obese II) [Independent variable. BMI (kg/mbese II) .Dependent variable. Hearing loss was defined as an HT > 40 dB HL at 1 kHz using pure-tone audiometry.Covariates. Age groups were categorized using five-year intervals . Proteinuria was determined using the dipstick method, which shows a certain degree of color change toward green within 60 s after dipping a test paper into urine. The degree of color change was indicative of protein levels in urine. Proteinuria status was expressed as negative (−), weakly positive (±), 30 mg/dL (+1), 100 mg/dL (+2), 300 mg/dL (+3), and 1000 mg/dL (+4), and categorized into two groups: normal  and abnormal . Creatinine was measured in serum and categorized as <1.4  and ≥1.4 . Smoking status was assessed using a questionnaire and categorized into three groups: current smokers, past smokers, and non-smokers. Alcohol consumption status was categorized into two groups: consumers and non-consumers. Blood pressure was categorized into two groups: systolic blood pressure < 120 mmHg and diastolic blood pressure < 80 mmHg , and systolic blood pressure ≥ 120 mmHg or diastolic blood pressure ≥ 80 (hypertension). AST and ALT blood levels were categorized as ≤40  and >40 . Total cholesterol levels were categorized as <200  and ≥200 . Triglyceride levels were categorized as <200  and ≥200 . LDL levels were categorized as <130  and ≥130 . Fasting glucose levels were categorized as <100  and ≥100 .A chi-square test was performed to determine the differences in categorical variables between hearing loss and control groups. Multiple logistic regression was used to analyze the crude and adjusted OR for hearing loss. In this analysis, crude (simple) and adjusted models were used. Adjustments were made for age, sex, smoking, alcohol consumption, blood pressure, triglycerides, total cholesterol, LDL, proteinuria, serum creatinine, AST, ALT, and fasting glucose levels.p < 0.05. The 95% CIs were calculated. The results were statistically analyzed using SPSS statistical software platform .For subgroup analyses, we subdivided the participants according to age  and sex. The P value was set at p < 0.001, p < 0.001, The following analyzed variables—age, sex, smoking, alcohol consumption, BMI, blood pressure, total cholesterol, proteinuria, creatinine, AST, ALT, and fasting glucose levels—were different between the hearing loss and control groups  and sex. Consistent with the results for all age groups , men demp < 0.001 for BMI < 18.5 and 40–59 years old; adjusted OR = 1.11, 95% CI = 1.07–1.14, p < 0.001 for 25 ≤ BMI < 29.9 and 40–59 years old; adjusted OR = 1.06, 95% CI = 1.01–1.11, p < 0.001 for BMI < 18.5 and 60–79 years old; adjusted OR = 1.11, 95% CI = 1.08–1.13, p < 0.001 for for 25 ≤ BMI < 29.9 and 60–79 years old; adjusted OR = 1.31, 95% CI = 1.24–1.38, p < 0.001 for for BMI < 18.5 and over 80 years old; In women, the association of hearing loss with BMI differed according to age , lower BMI (<18.5 kg/m2) was associated with an increased risk of hearing loss even after adjusting for age, sex, smoking, alcohol consumption, and past medical histories . In the BMI 25–29.9 kg/m2 group, although the odds of hearing loss were high in the crude model, they were equivalent to that in the normal BMI group after adjusting for the described variables, indicating that obesity was not related to an increased risk of hearing loss. Our results are partly consistent with those of previous studies in which being underweight was associated with an elevated risk of hearing loss [The association between BMI and hearing loss in the Korean population was analyzed in this study. Compared to normal BMI , which is characterized by normal levels of triglycerides, blood pressure, and fasting glucose, and reduced high-density lipoprotein cholesterol [Combining the above research, our conclusion that low body weight is associated with an increased risk of hearing loss can be explained by introducing the concept of sarcopenic obesity, which describes a condition in which individuals lose their lean mass or skeletal muscle instead of fat . From thlesterol . Both saBased on the result of subgroup analyses according to age and sex, being underweight contributed to an increased risk of hearing loss in men of all age group. In the elderly population, being underweight can be partially attributed to malnutrition, which has been suggested to induce high morbidity and mortality ,35.This study has several strengths, including a large-scale population-based study with a national health screening cohort. While many studies have previously analyzed the association between obesity and hearing loss, we enrolled patients who were adjusted for many confounders, including age, sex, smoking, alcohol consumption, blood pressure, dyslipidemia, proteinuria, serum creatinine, liver function, and diabetes. However, this study has several limitations. Causality between hearing loss and underweight or obesity cannot be concluded in this cross-sectional study. Despite our efforts to include many confounding factors, there may be residual confounders, such as socioeconomic status, education level, depressive mood, noise exposure, drug use, underlying cardiovascular disease, and congenital hearing loss . In addiBeing underweight was associated with an increased prevalence of hearing loss. These results were more consistent in men than in women. Since Asians have a lower prevalence of obesity than Western populations, proper weight control, including optimal nutrient intake, is needed from the perspective of hearing loss.