Background: Bone conductive implants (BCI) have been reported to provide greater beneficial effects for the auditory and perceptual functions of the contralateral ear in patients presenting with asymmetric hearing loss (AHL) compared to those with single-sided deafness (SSD). The aim of the study was to assess the effects of wearing a conventional hearing aid in the contralateral ear on BCI in terms of an improved overall auditory performance. Methods: eleven AHL subjects wearing a BCI in their worse hearing ear underwent an auditory evaluation by pure tone and speech audiometry in free field. This study group was obtained by adding to the AHL patients those SSD subjects that, during the follow-up, showed deterioration of the hearing threshold of the contralateral ear, thus presenting with the features of AHL. Four different conditions were tested and compared: unaided, with BCI only, with contralateral hearing aid (CHA) only and with BCI combined with CHA. Results: all of the prosthetic conditions caused a significant improvement with respect to the unaided condition. When a CHA was adopted, its combination with the BCI showed significantly better auditory performances than those achieved with the BCI only. Conclusions: the present study suggests the beneficial role of a CHA in BCI-implanted AHL subjects in terms of overall auditory performance. Single-sided deafness (SSD) and asymmetric hearing loss (AHL) represent two distinctive audiological conditions that differ on the basis of having the contralateral, better-hearing ear either normal or impaired by hearing loss to different degrees. More specifically, AHL presents a pure tone average (PTA) of 70 dB or more in the worse ear and a PTA from 30 to 55 dB in the better ear, with an interaural threshold difference of least 15 dB ,3. The aContrary to previous reports , it was It is a common observation in clinical practice that some subjects, first identified as SSD cases, show a deterioration of the hearing threshold in the better ear at a later stage, in relation to aging or to causing pathology or other. The temporal progression of the auditory function, in this case, could transform the features of SSD into those that characterize AHL, suggesting the adoption of a conventional hearing aid in the better, yet abnormal, hearing ear. By regularly monitoring SSD and AHL patients implanted with BCI in their worse ear, we observed that some of the SSD BCI-implanted subjects were undergoing an auditory deterioration in the contralateral ear that was consequently rehabilitated by a conventional hearing aid (CHA). The aim of the present study was to assess the efficacy of CHA in AHL BCI-implanted subjects, while comparing their audiological performances with those of unaided, BCI-only-aided and CHA-only-aided conditions.In total, 11 patients, 8 males and 3 females aged between 50 and 80 years old mean 67.2), were included in the study for having previously received a BCI for SSD or AHL, according to the standardized classification [, were inp < 0.05.The t-paired test was used to verify the hypothesis of difference among the groups, with a significant difference of The mean values of the air and bone conduction thresholds at the time of the implant (pre) and at the last follow-up (post) confirmed the hearing threshold deterioration in the better ear . The meaThe mean values of PTA, WRS maximum and dB HL related to WRS max in free field in the four different auditory conditions  are reported in p = 0.0014); BAHA + CHA vs. unaided 26 dB in favor of BAHA + CHA (p = 0.0002); BAHA vs. unaided 6 dB in favor of BAHA (p = 0.03); BAHA vs. CHA 15 dB in favor of CHA (p = 0.0086); BAHA vs. CHA + BAHA 20 dB in favor to CHA + BAHA (p = 0.0008); CHA vs. CHA + BAHA 5 dB in favor of CHA + BAHA (p > 0.05) (Unaided 61 dB HL (SD 16.5); CHA 41 dBHL (SD 7.4); BAHA 55 dBHL (SD 14.4); BAHA + CHA 37 dBHL (SD 7.6). The comparison of PTA under the different situations showed the following PTA gains: CHA vs. unaided 21 dB in favor of CHA ( > 0.05) .Unaided: WRS 70% 74 dB; CHA: WRS 93% 60 dB; BAHA WRS 83% 65 dB; BAHA + CHA WRS 95% 56 dB.p = 0.0008); a significant improvement of 22% in WRS (<0.05) and of 9 dB HL in BAHA vs. unaided (p < 0.05); a significant improvement of 28% in WRS and of 18 dB HL in CHA + BAHA vs. unaided (p < 0.05); a non-significant improvement (p > 0.05) of 4% in WRS and of 5 dB HL in CHA vs. BAHA; a non-significant improvement (p > 0.05) of 2% in WRS and 4 dB HL in BAHA + CHA vs. CHA; a non-significant improvement of 6% in WRS and of 9 dB HL (p > 0.05) in BAHA + CHA vs. BAHA  and of 14 dB HL in CHA vs. unaided , provided some interesting results that are worth discussing. The “unaided” PTA and speech audiometry in quiet achieved similar results in the ear contralateral to the implant, whilst the comparison between the “unaided” condition and the other differently aided solutions resulted in significantly better scores for the latter. Considering the possibility of adopting only one solution, either a CHA or a BCI, it was found that CHA provided a better PTA threshold without similar significant advantages in WRS and loudness values. The possibility of implementing the auditory rehabilitation with more than one tool, as was tested in this study, shows that the combination of BAHA with CHA offers significant advantages in terms of PTA improvement compared to either BAHA or CHA only for PTA, WRS and loudness, but this latter finding was without statistical significance. These findings confirm the beneficial effects of a CHA in addition to a BCI for AHL patients, therefore verifying the study hypothesis of the positive role played by the CHA. The present study supports the findings of our previous study on the possibility of achieving, in AHL, the improvement of the auditory and perceptual functions of the contralateral better ear when the worse ear is implanted with a BCI. Moreover, it has allowed us to assess the effect of using a BCI to improve the outcome of wearing a CHA in the ear contralateral to the implant itself. It would therefore be advisable to perform a follow-up of both SSD and AHL patients rehabilitated with a BCI on the worse ear in order to monitor the auditory function of the better ear and eventually propose bimodal rehabilitation with a BCI and a CHA.


Background: The aim of this study was to compare, in users of bimodal cochlear implants, the performance obtained using their own hearing aids (adjusted with the standard NAL-NL1 fitting formula) with the performance using the Phonak Naìda Link Ultra Power hearing aid adjusted with both NAL-NL1 and a new bimodal system ) developed by Advanced Bionics and Phonak Corporations. Methods: Eleven bimodal users  were enrolled in our study. The users’ own hearing aids were replaced with the Phonak Naìda Link Ultra Power and fitted following the new formula. Speech intelligibility was assessed in quiet and noisy conditions, and comparisons were made with the results obtained with the users’ previous hearing aids and with the Naída Link hearing aids fitted with the NAL-NL1 generic prescription formula. Results: Using Phonak Naìda Link Ultra Power hearing aids with the Adaptive Phonak Digital Bimodal fitting formula, performance was significantly better than that with the users’ own rehabilitation systems, especially in challenging hearing situations for all analyzed subjects. Conclusions: Speech intelligibility tests in quiet settings did not reveal a significant difference in performance between the new fitting formula and NAL-NL1 fittings (using the Naída Link hearing aids), whereas the performance difference between the two fittings was very significant in noisy test conditions. Cochlear implants (CIs) are able to successfully restore a sense of hearing in patients with severe-to-profound hearing loss. In recent years, indication criteria for CI candidacy have become less stringent , allowinBimodal hearing has consistently been shown to outperform listening with the CI alone. Benefits have been demonstrated regarding speech intelligibility, sound localization, sound quality, listening effort, and subjective benefit ,3,4,5,6.To further improve performance in bimodal listeners, Phonak and Advanced Bionics have developed a dedicated bimodal system. The Naída Link Has produced by the Phonak company based in Stafa, Switzerland, and specifically the accompanying bimodal fitting formula  ), have bThe goal of traditional HA fitting is to optimize speech intelligibility with the HA; frequencies crucial to speech intelligibility (1–4 kHz) are amplified to maximize audibility. In bimodal CI users, especially those with limited contralateral residual hearing, often the CI ear dominates speech intelligibility , with thPrevious research has demonstrated this alignment between CI and HA to be beneficial. Matching the AGC characteristics between the HA and the CI led to improved speech intelligibility in noise tasks using a single competing talker, and the matched AGC was preferred to a standard HA AGC in questionnaires and in a subjective preference test .In this study, speech intelligibility in bimodal CI users upgraded to the Naída Link HA was evaluated in quiet and in noise and compared to results obtained using the previously used HA model as well as the Naída Link HA fitted with the generic NAL-NL1 prescription. The study sought to evaluate not the effect of directional microphones in the noisy environment, but rather, the validity of the APDB formula compared to the NAL, and for this reason, the operating mode of the microphones was set to omnidirectional, and all automated functions were disabled in both the HA and CI.Eleven unilateral CI users with aidable hearing in the contralateral ear participated in this study. They included 6 adults and 5 children, with a mean age of 23 ± 20 years. Four subjects were female. At the time of testing, subjects had on average 5 ± 3 years of experience with their CI and stable maps. All subjects used the Naída CI Q70 sound processor. All subjects had a severe-to-profound hearing loss in the contralateral ear, clinically aided with a GN Resound Enzo or Phonak Naída Sky UP Q70 hearing aid. Detailed subject information is provided in The Adaptive Phonak Digital Bimodal (APDB) fitting formula aims at optimally complementing hearing with the Naída CI by aligning the behavior of the contralateral HA to the Naída CI regarding frequency response, loudness growth functions, and automatic gain control (AGC) characteristics.The frequency response is aligned by optimizing low-frequency gain and bandwidth. Low-frequency gain optimization uses the model of effective audibility  to ensurLoudness growth is aligned by implementing the input-output function of the cochlear implant in the hearing aid ;The dynamic compression behavior is aligned by porting the Naída CI dual loop AGC into the hearing aid.In contrast to traditional HA fitting approaches, the APDB fitting formula provides 3 fundamental characteristic points:Exclusively available for Phonak Naída Link HAs, the APDB fitting formula was used in this study to program a Phonak Naída Link Ultra Power (UP) HA without using any adaptive parameters . The patients were wearing their own earmolds, and the output was analyzed by a probe mic.Speech intelligibility was tested using phonetically balanced lists of 20 bisyllabic Italian words  in quietSpeech was presented from the front (0°) in all test conditions. Noise was presented from 90° and 270°.Subjects were invited to 2study appointments. At the first appointment, speech intelligibility with their own HAs was measured in quiet and in noise. Subsequently, a Naída Link UP HA was fitted according to each subject’s hearing loss using the APDB fitting formula. At the second appointment following a 7-day acclimatization period to the new HA prescription, speech intelligibility in quiet and in noise was measured with the Naída Link UP HA using the APDB fitting formula and the formula (NAL-NL1) used in their previous HA.All subjects tried the APDB formula last because the old HAs were already programmed with the NAL-NL1 system.Statistical analyses were performed using Statistica 12  with the level of significance set at 0.05. Main effects of noise condition and fitting approach as well as the interaction between them were analyzed using a repeated measures analysis of variance (ANOVA). Post-hoc analyses were performed using Wilcoxon-signed rank tests. Bonferroni corrections for multiple comparisons were applied.Median percent correct speech intelligibility is presented in p < 0.001), a statistically significant main effect of the fitting condition  = 57.110, p < 0.001), as well as a statistically significant interaction between the two  = 6.9471, p < 0.001).Repeated measures ANOVA revealed a statistically significant main effect of the noise condition  = 386.20, p = 0.035), at 0 dB SNR , and at -5 dB SNR . Performance with the Naída Link HA was significantly better using the APDB fitting formula that using NAL-NL1 at 0 dB SNR  as well as at -5 dB SNR .Post-hoc analysis using Wilcoxon signed rank tests with Bonferroni corrections for multiple comparisons revealed statistically significant differences between the different fitting conditions within each noise condition. The Naída Link HA with APDB yielded significantly better speech intelligibility than the subjects’ own HAs in quiet  while the performance difference between the two fittings was significant in both noisy test conditions. In quiet, the sensitivity of the test material used was limited by the ceiling effect: using the APDB fitting, the median speech intelligibility score in quiet was 100% with six out of eleven subjects scoring 100%. This may have obscured a benefit of the APDB fitting compared to the NAL-NL1 fitting, which can be seen in the noisy test conditions. More difficult speech material or testing at a lower level could have decreased subject performance to avoid the ceiling effect and therefore might have revealed a significant difference also in the quiet test condition.Similarly, performance at the most unfavorable SNR of <5 dB was limited by floor effects: five out of eleven subjects scored 0% when using the Naída Link HA with the NAL-NL1 fitting and when using their own HA. However, neither the tests in quiet nor the tests at the more favorable SNR condition of 0 dB revealed a statistically significant difference between the Naída Link NAL-NL1 fitting and their own HA; therefore, the ceiling effect likely did not obscure a significant difference at <5 dB SNR, either.Previous studies investigating speech intelligibility of bimodal CI listeners revealed equal performance between the dedicated bimodal APDB fitting and the generic HA fitting formulae NAL-NL2 and DSLv5 ,12 when An additional analysis of the signal audibility, based on the group-average hearing loss, revealed a difference in audibility between the APDB and NAL-NL1 fittings of 8 dB for frequencies below 1 kHz, offering a plausible explanation for the superior performance of the APDB fitting in this subject group. In previous studies ,12, the Compared to each subject’s own HA fitted with a generic fitting formula, the Naída Link HA fitted with the APDB formula also resulted in superior speech intelligibility performance. Acclimatization to each HA would in this case favor the subject’s own, clinically used HA with a long period of acclimatization before testing over the Naída Link HA with only 7 days of acclimatization before testing. However, the Naída Link UP provided higher output levels than the clinically used HAs (GN Resound Enzo or Phonak Naída Sky UP Q70), potentially explaining the superior speech intelligibility outcomes. Additionally, the Naída Link HA was fitted for each subject within the study protocol, based on current audiograms, whereas the fitting of their own HA was based on less recent audiogram data and therefore may not have been adequate anymore.Overall, the superior performance with the APDB fitting may also have been affected by the test order: APDB was always tested last within the test session. If any training effects occurred, they were in favor of the APDB fitting.After the study, 10 out of 11 subjects continued using the APDB fitting formula as their new clinical provision. The remaining subject, despite better performance with APDB, preferred to return to the previous fitting formula as a result of a more pleasing sound, in his opinion, whereas no one else preferred to resume using their previous hearing aid. The number of patients participating in the study did not allow us to break down the results according to age; in general, it did not appear that the results depended on the age of the subject.The dedicated bimodal fitting formula APDB provided statistically significant speech intelligibility benefits over the generic NAL-NL1 fitting formula, especially in challenging listening situations. Most of the subjects involved in the study preferred to continue using the APDB for the fitting of hearing aids.


Retinopathy of prematurity (ROP) is an ocular vascular disease affecting premature infants, characterized by pathological retinal neovascularization (RNV), dilated and tortuous retinal blood vessels, and retinal or vitreous hemorrhages that may lead to retinal detachment, vision impairment and blindness. Compared with other neovascular diseases, ROP is unique because of ongoing and concurrent physiological and pathological angiogenesis in the developing retina. While the disease is currently treated by laser or cryotherapy, anti-vascular endothelial growth factor (VEGF) agents have been extensively investigated but are not approved in the U.S. because of safety concerns that they negatively interfere with physiological angiogenesis of the developing retina. An ideal therapeutic strategy would selectively inhibit pathological but not physiological angiogenesis. Our group recently described a novel strategy that selectively and safely alleviates pathological RNV in animal models of ROP by targeting secretogranin III (Scg3), a disease-restricted angiogenic factor. The preclinical profile of anti-Scg3 therapy presents a high potential for next-generation disease-targeted anti-angiogenic therapy for the ROP indication. This review focuses on retinal vessel development in neonates, the pathogenesis of ROP and its underlying molecular mechanisms, including different animal models, and provides a summary of current and emerging therapies. Retinopathy of prematurity (ROP) with pathological retinal neovascularization (RNV) is the most common cause of blindness in children, primarily afflicting preterm infants . During ROP first appeared as a clinical disorder in the 1940s, coincident with the liberal use of supplemental oxygen to improve the survival of preterm infants with respiratory distress syndrome (RDS) . The disROP is currently treated by laser therapy or cryotherapy that provide only limited efficacy and significant risk of adverse side effects. Anti-vascular endothelial growth factor (VEGF) drugs have been used off-label in the U.S. but are not FDA approved because of the potential for adverse side effects caused by indiscriminate inhibition of all forms of angiogenesis in the developing retina. The recent discovery by our group that secretogranin III (Scg3) is a disease-selective angiogenic factor presents a unique opportunity to selectively target and alleviate ROP safely without affecting physiological angiogenesis and normal retinal development.The retina is supplied by two vascular networks: the retinal vasculature supplies the inner retina while the choroidal vasculature supports the retinal pigment epithelium and photoreceptors  6]. Abn. Abn6]. The human retina has two temporal vascular networks that develop sequentially in a precisely coordinated manner: the transient hyaloid vasculature supplies the retina and lens only in the early stage of gestation while the lifelong retinal vasculature supports only the retina. The hyaloid vasculature originates from the central hyaloid artery that enters the embryonic optic fissure at about 6 weeks of GA, progresses through the vitreous, surrounds the lens and then begins to regress about 13 weeks of GA .Concomitantly, the human retinal vasculature begins to develop at about 16 weeks of GA in utero and is fully mature within 40 weeks of GA, normally before birth. Vascularization starts from the optic nerve head, extends toward the peripheral edge of the retina and forms the superficial retinal vascular plexus at about 36 weeks of GA. This vasculature further penetrates into the retina to form the intermediate and deep vascular plexuses. Together, these three layers of vessels form the retinal vascular network in the upper half of the retina, whereas the bottom half of the retina, including photoreceptors, remains avascular for the entire lifespan . The vesMice and rats share similar timelines of retinal vascularization, which are markedly different from humans. Mice are born with a hyaloid vasculature, and the retinal arteries sprout out of the optic nerve head into the retina on postnatal day 1 (P1), followed by outward branching and extension toward the peripheral retina . Mouse sDespite difference in timelines, humans and rodents share similar sequential patterns of hyaloid and retinal vasculature development. Additionally, both species have a network of astrocytes that spread outward from the optic nerve head before physiological RNV, and retinal vessels follow the network of astrocytes ,11. As oSupplemental oxygen triggers ROP in extremely low-birth weight preterm infants through two distinct phases A.Phase 1: Vaso-obliteration is initiated at birth and subsides on termination of supplemental oxygen treatment, usually at approximately 32–34 weeks of GA. At birth, premature infants lose placental and maternal growth factors and are exposed to extrauterine high oxygen tension. Hyperoxia suppresses the expression of angiogenic factors that support physiological angiogenesis. Supplemental oxygen to treat RDS further downregulates angiogenic factors in the retina, leading to vaso-obliteration.Phase 2: Vaso-proliferation begins at the time of withdrawal of supplemental oxygen. As preterm infants mature, the developing retina becomes metabolically active and relatively hypoxic because of delayed physiological RNV, causing upregulation of angiogenic factors, including VEGF and IGF-1 that trigger pathological RNV. Unlike the stepwise developmental program of physiological angiogenesis that is regulated by precise gradients of angiogenic factors diffusing from the astrocyte network, the burst of angiogenic factors during Phase 2 lacks such regulated temporal and spatial gradients, thereby triggering pathological RNV with a disorganized retinal vasculature (culature B. Excess2) of preterm babies during the first few weeks of life also contribute to the risk of ROP, and a high incidence of intermittent hypoxia is associated with severe ROP ,4645,46]Initial efforts to develop OIR in rats by exposing neonates to high oxygen for 5–10 days failed to reproduce ROP-like vascular manifestations . Penn etCats with a normal gestation of 63–67 days share a similar pattern of physiological RNV to humans and rodents but with an intermediate timeline. In kittens, the retinal artery emerges from the optical disc between the 35th and 45th days of GA but does not reach the periphery until 3 weeks after birth .The first kitten model of OIR was established in 1954 by varying the onset, duration and concentrations of oxygen exposure . Severe Compared with rodent models, the canine retina is more vascularized at birth . A dog mOver the past 70 years, numerous clinical trials have been implemented to investigate the optimal range of supplemental oxygen administered during Phase 1 and 2 to reduce the risk of ROP. These finding are summarized below. Detailed discussions on the clinical management of oxygen therapy are covered by other excellent reviews ,55.Phase 1: A clinical survey found that the risk and severity of ROP is associated with high SpO2 [2 of >98% in the first two weeks of birth vs. 3% for those with maximal SpO2 of ≤98%. After 2 weeks of age, the rate of severe ROP was 3.3% with maximal SpO2 of >92% vs. 1.3% with SpO2 of ≤92%.igh SpO2 . The rat2 (91–95%), infants with low SpO2 (85–89%) sustained a marked reduction in severe ROP, but again, the benefit was accompanied by significantly increased mortality [Five large multicenter, randomized, masked, controlled trials, collectively known as The Neonatal Oxygen Prospective Meta-Analysis or NeOProM Collaboration, enrolled approximately 5000 premature infants <28 weeks, including the SUPPORT trial in the U.S., BOOST II trials in Australia, New Zealand and United Kingdom, and the COT trial in Canada. Compared with premature infants exposed to high SpOortality ,57.Phase 2: Based on the two-phase theory of ROP pathogenesis, it was postulated that increased oxygen supplementation in Phase 2  would mitigate the disease. A multicenter STOP-ROP clinical trial found that high oxygenation (SpO2 96–99%) neither caused additional progression of pre-threshold ROP nor significantly reduced the number of infants requiring peripheral ablative surgery [2 range of either 91–94% or 95–98% at 32 weeks of GA [ surgery . This fiks of GA . Again, 2 is within the range of 90–95% and should be personalized within this range as indicated for individual cases [2 during the first few weeks of life are associated with increased risk and severity of ROP. Therefore, strict management to avoid fluctuation in SpO2 is important. Additional clinical trials are warranted to optimize oxygen therapy and reduce ROP.In summary, numerous large multicenter clinical trials implemented over the previous 70 years were unable to identify an optimal oxygen saturation range that favorably balanced the risks between mortality and ROP in Phase 1, and no clinical benefit was demonstrated for high oxygen treatment in Phase 2. Despite the setbacks, valuable knowledge generated from these studies has improved clinical management of ROP. Firstly, exposure of premature infants to excessively high levels of supplemental oxygen confers increased risk of ROP. Optimal SpOal cases . Second,Cryotherapy and laser photocoagulation are ablative surgeries to destroy non-neovascularized area in the peripheral retina and convert them into non-functional scar tissues for preserving the central vision and are reviewed in detail elsewhere ,61. In bGiven the critical role of VEGF in pathological RNV of ROP and the clinical successes of anti-VEGF agents for other neovascular indications, anti-VEGF has generated interests as a therapeutic option for ROP. However, safety is a major concern for anti-VEGF therapy of ROP. VEGF is an essential growth factor for physiological angiogenesis that supports the development of the retina and other organs during embryonic and neonatal stages. Indeed, mice with homozygous deletion of either VEGFR1 or 2 die in utero ,64. SimiCurrently, there are five approved anti-VEGF drugs for wet AMD and diabetic retinopathy, including ranibizumab, pegaptanib, aflibercept, brolucizumab and conbercept (approved in China). Additionally, bevacizumab approved for cancer therapy is often used off-label for ocular diseases .Bevacizumab was the first anti-VEGF drug to be reported for ROP therapy . BEAT-ROThe randomized, multicenter RAINBOW trial recently found that intravitreal ranibizumab (0.2 mg/eye) was superior to laser therapy with fewer unfavorable structural outcomes and an acceptable 24-week safety profile . Based oIn ROP infants, intravitreal injection of bevacizumab or ranibizumab resulted in significantly decreased serum VEGF up to 12 weeks . Given tWe recently reported that intravitreal aflibercept inhibited vessel development in the retina, leading to reduced retinal vascularization and decreased b-wave amplitude of electroretinography (ERG) . SystemiOver the past 70 years, numerous clinical trials have failed to identify effective and safe therapeutic strategies for ROP ,84. SuchOral propranolol, a non-selective β-blocker that has been extensively used in newborn infants with indications for other disorders, effectively reduced severe ROP but with serious adverse side effects ,91. ClinBy applying a new technology called comparative ligandomics, our group recently discovered that Scg3 is a novel disease-restricted pro-angiogenic factor . Of thouThese findings suggest that an as-yet-unknown Scg3 receptor (Scg3R) is markedly upregulated on the surface of diabetic endothelial cells (by >1700-fold) to account for the enhanced binding with only moderately increased ligand Scg3. We found only a 1.38-fold increase of Scg3 in diabetic vitreous fluid versus controls . This is−/− mice have a normal gross phenotype attest to the predicted safety of anti-Scg3 therapy in OIR mice and possibly human ROP by extension [−/+ mice are embryonic lethal with severe defects in vasculogenesis [To investigate the novel concept of ligand-guided disease-targeted anti-angiogenic therapy, we developed Scg3-neutralizing monoclonal antibodies (mAbs) and confirmed their high efficacy to alleviate retinal vascular leakage in diabetic mice . The resxtension . In contogenesis . A caveaogenesis  but is yDue to the prerequisite to selectively target pathological angiogenesis while sparing physiological angiogenesis, ROP represents a unique clinical challenge . This coLigand-guided disease-targeted anti-Scg3 therapy offers a new hope to selectively suppress pathological angiogenesis with minimal inhibition of physiological angiogenesis and a high safety profile. This is in marked contrast with anti-VEGF-based therapies that lack the obligatory vascular selectivity to treat human ROP. Despite our reports of safety and efficacy in a mouse OIR model , the the


HULC, PVT1, MEG3, SPRY4-IT1, LINC-ROR and DSCAM-AS1 lncRNAs to appraise their transcript levels in the circulation of CIDP and GBS cases versus controls. Expression of HULC was higher in CIDP patients compared with healthy persons (Ratio of mean expression (RME) = 7.62, SE = 0.72, P < 0.001). While expression of this lncRNA was not different between female CIDP cases and female controls, its expression was higher in male CIDP cases compared with male controls . Similarly, expression of HULC was higher in total GBS cases compared with healthy persons  and in male cases compared with male controls . Similar pattern of expression was detected between total cases and total controls. PVT1 was up-regulated in CIDP cases compared with controls  and in both male and female CIDP cases compared with sex-matched controls. Similarly, PVT1 was up-regulated in GBS cases compared with controls  and in total patients compared with total controls . Expression levels of DSCAM-AS1 and SPRY4-IT1 were higher in CIDP and GBS cases compared with healthy subjects and in both sexes compared with gender-matched healthy persons. Although LINC-ROR was up-regulated in total CIDP and total GBS cases compared with controls, in sex-based comparisons, it was only up-regulated in male CIDP cases compared with male controls . Finally, expression of MEG3 was up-regulated in all subgroups of patients versus controls except for male GBS controls. SPRY4-IT could differentiate CIDP cases from controls with AUC = 0.84, sensitivity = 0.63 and specificity = 0.97. AUC values of DSCAM-AS1, MEG3, HULC, PVT1 and LINC-ROR were 0.80, 0.75, 0.74, 0.73 and 0.72, respectively. In differentiation between GBS cases and controls, SPRY4-IT and DSCAM-AS1 has the AUC value of 0.8. None of lncRNAs could appropriately differentiate between CIDP and GBS cases. Combination of all lncRNAs could not significantly enhance the diagnostic power. Taken together, these lncRNAs might be involved in the development of CIDP or GBS.Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and Guillain-Barré syndrome (GBS) are inflammatory neuropathies with different clinical courses but similar underlying mechanisms. Long non-coding RNAs (lncRNAs) might affect pathogenesis of these conditions. In the current project, we have selected  Both conditions are associated with dysregulation of immune response4. In GBS, such responses are believed to be triggered by infectious conditions in the respiratory or gastrointestinal tract leading to a functional failure in the blood–nerve barrier and damage of myelin sheaths and/or nerve fibers5. Almost all aspects of immune function including humoral responses, complement, T cells and macrophages participate in the pathogenesis of these immune-mediated neuropathies6. However, the underlying cause of such extensive immune dysregulation is not thoroughly identified3. Long non-coding RNAs (lncRNAs) have central influences on the activity of immune system8. Contribution of a number of these transcripts in the pathogenesis of CIDP and GBS has been recently verified by our group9. However, the role of several members of lncRNAs in autoimmune neuropathies needs to be elucidated. In the current project, we have selected HULC, PVT1, MEG3, SPRY4-IT1, LINC-ROR and DSCAM-AS1 lncRNAs to appraise their transcript levels in the circulation of CIDP and GBS cases versus controls. The reason for selection of these lncRNAs was their roles in modulation of immune responses. HULC has been identified as one of important factors in induction of pro-inflammatory responses in the course of liposaccharide-associated sepsis in endothelial cells10. Pvt1 has been shown to modulate the immunosuppression function of granulocytic myeloid-derived suppressor cells in animal models11. MEG3 has been reported to induce imbalance between regulatory T cells and Th17 cells12. SPRY4-IT1 interacts with ERRα13, a nuclear receptor which regulates innate immunity14. LINC-ROR has functional interaction with TGF-β to regulated hypoxia-induced cellular cascades15. Finally, DSCAM-AS1 has been shown to regulate several genes which are implicated in inflammatory responses16. These lncRNAs regulate immune reactions via different routes.Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and Guillain-Barré syndrome (GBS) are inflammatory neuropathies with different clinical courses. While CIDP has a slowly progressive onset17 and National Institute of Neurological Disorders and Stroke18. In addition, electrophysiological criteria were used for diagnosis of GBS19. Blood samples were obtained when patients entered the remission phase and were not on any treatment. All were responsive to corticosteroids or IVIg treatment. No concomitant treatment was used for these patients. None of them had any comorbid condition. Persons recruited as controls had no recent or chronic infection, malignant condition, or any systemic diseases. The study protocol was approved by the ethical committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.MSP.REC.1399.575) and the study protocol is performed in accordance with the relevant guidelines. Informed consent forms were signed by all recruited persons.A total of 32 CIDP patients with typical type , 25 GBS patients , and 58 healthy individuals  participated in the current investigation. CIDP cases had symmetric muscle weakness which affected both proximal and distal muscles. The course of disorder was compliant with a motor-predominant neuropathy. Patients were assessed using the guidelines stated by American Academy of NeurologyHULC, PVT1, MEG3, SPRY4-IT1, LINC-ROR and DSCAM-AS1.Three milliliters of the peripheral blood of all recruited people were obtained for RNA extraction. This phase was performed using the GeneAll kit . The retrieved RNA was then transformed to cDNA using the kit prepared by the Thermo Fisher Scientific Company . Expression levels of mentioned lncRNAs were measured in GBS and CIDP cases versus healthy persons using the Ampliqon master mix . Reactions were executed in the Step One Plus Real-Time PCR system . Table 20. Transcript magnitudes of these lncRNAs in comparison with the levels of B2M gene were measured from Ct and efficiency values. The obtained figures were log2 transformed. The significance of difference in mean values of transcript intensities of lncRNAs was judged using the t-test. Correlations between expression quantities were appraised using Spearman correlation test. Receiver operating characteristic (ROC) curves were plotted to quantify the diagnostic values of expression levels of lncRNAs. Youden's J statistic was used to determine the optimum threshold. Area under curve (AUC) values were quantified.Expression of selected lncRNAs were analyzed in the R V.34 softwareAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent forms were obtained from all study participants. The study protocol was approved by the ethical committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.MSP.REC.1399.575). All methods were performed in accordance with the relevant guidelines and regulations.Table HULC was higher in CIDP patients compared with controls (Ratio of mean expression (RME) = 7.62, SE = 0.72, P < 0.001). While expression of this lncRNA was similar between female CIDP cases and female controls, its expression was up-regulated in male CIDP cases compared with male controls . Similarly, expression of HULC was higher in total GBS cases compared with controls  and in male cases compared with male controls . Similar pattern of expression was detected between total cases and total controls. PVT1 was up-regulated in CIDP cases compared with controls  and in both male and female CIDP cases compared with sex-matched healthy persons. Similarly, PVT1 was up-regulated in GBS cases compared with controls  and in total patients compared with total controls . Expression levels of DSCAM-AS1 and SPRY4-IT1 were higher in CIDP and GBS cases compared with controls and in both sexes compared with gender-matched healthy subjects. Although LINC-ROR was up-regulated in total CIDP and total GBS cases compared with controls, in sex-based comparisons, it was only up-regulated in male CIDP cases compared with male controls . Finally, expression of MEG3 was up-regulated in all subgroups of patients versus controls except for male GBS controls   Fig. .Figure 2HULC/DSCAM-AS1 and HULC/LINC-ROR pairs (r = 0.84 for both pairs)  Fig. .Figure 3Finally, diagnostic power of lncRNAs for distinguishing patients from healthy subjects was assessed Fig. .Figure 4SPRY4-IT could differentiate CIDP cases from controls with AUC = 0.84, sensitivity = 0.63 and specificity = 0.97. AUC values of DSCAM-AS1, MEG3, HULC, PVT1 and LINC-ROR were 0.80, 0.75, 0.74, 0.73 and 0.72, respectively. In differentiation between GBS cases and controls, SPRY4-IT and DSCAM-AS1 has the AUC value of 0.8. None of lncRNAs could appropriately differentiate between CIDP and GBS cases. Combination of all lncRNAs could not significantly enhance the diagnostic power  compared with exosomes retrieved from normal blood samples21. The same study has reported up-regulation of mentioned lncRNAs in addition to H19 antisense, HAR1B and GAS5 in peripheral blood mononuclear cells of these patients21. ENST00000483588 is another lncRNA which has been shown to be up-regulated in fibroblast-like synoviocytes of patients with RA22. A number of selected lncRNAs in the current project have been previously shown to be up-regulated in immune-mediated conditions. For instance, PVT1 has been reported to be up-regulated in fibroblast-like synoviocytes of RA models parallel with down-regulation of sirt6, a putative target for this lncRNA. PVT1 silencing or sirt6 over-expression could suppress cell proliferation and inflammation, while inducing cell apoptosis23. MEG3 has been demonstrated to regulate RA pathogenesis through targeting NLRC524. LINC-ROR, MEG3, SPRY4-IT1 and UCA1 have been among lncRNA with higher expression in patients with schizophrenia compared with normal subjects25.LncRNAs have been shown to take part in the pathogenesis of immune-related conditions. Up-regulation of lncRNAs has been reported in a number of these conditions. For instance, expression levels of HULC was higher in CIDP patients compared with controls. While expression of this lncRNA was not different between female CIDP cases and female controls, its expression was higher in male CIDP cases compared with male controls. Similarly, expression of HULC was higher in total GBS cases compared with controls and in male cases compared with male controls. Similar pattern of expression was detected between total cases and total controls. HULC has been shown to regulate immune responses through miR-128-3p/RAC1 axis26. In line with our observations, miR-128-3p has been shown to be down-regulated in cerebrospinal fluid of animal models of GBS27. RAC1 regulates a number of inflammatory pathways such as STAT3 and NF-κB28. NF-κB pathway has a documented effect in the pathogenesis of immune-related neuropathies29. Therefore, HULC/miR-128-3p/RAC1 axis might also been involved in the pathogenesis of CIDP and GBS.CIDP and GBS disorders are two immune-mediated conditions in which lncRNAs might contribute. We measured expression of amounts of six immune-related lncRNAs in the circulation of these patients versus healthy controls. Expression of PVT1 was up-regulated in CIDP cases compared with controls and in both male and female CIDP cases compared with sex-matched controls. Similarly, PVT1 was up-regulated in GBS cases compared with controls and in total patients compared with total controls. Contrary to this finding, we have previously reported down-regulation of PVT1 in the peripheral blood of patients with multiple sclerosis30. Therefore, this lncRNA might have distinctive effects in these two inflammatory conditions.DSCAM-AS1 and SPRY4-IT1 were higher in CIDP and GBS cases compared with controls and in both sexes compared with sex-matched controls. Therefore, these lncRNAs have a consistent pattern of expression among CIDP and GBs patients potentiating them as biomarkers for these conditions.Expression levels of LINC-ROR was up-regulated in total CIDP and total GBS cases compared with controls, in sex-based comparisons, it was only up-regulated in male CIDP cases compared with male controls indicating the possible interactions between this lncRNA and sex-related parameters, since there was no gender-based difference in phenotype of the patients in terms of severity of illness.Although MEG3 was up-regulated in all subgroups of patients versus controls except for male GBS controls. Expression of MEG3 has been shown to be elevated in CD4 + T cells of patients with immune thrombocytopenic purpura. Expression of this lncRNA has been reduced in CD4 + T cells cultured with dexamethasone12. Functionally, MEG3 inhibits Foxp3 expression and increases RORγt expression, thus inducing imbalance between regulatory T cells and Th17 cells12. The imbalance between these subsets of T cells might participate in the pathogenesis of GBS or CIDP as previous studies have shown the therapeutic effects of regulatory T cells in animal models of GBS31.Finally, expression of SPRY4-IT and DSCAM-AS1 could differentiate CIDP cases from controls with appropriate diagnostic power values. Similarly, these lncRNAs had high power in differentiation between GBS cases and controls. Since expression levels of lncRNAs were almost similar between CIDP cases and GBS cases, none of lncRNAs could appropriately differentiate between CIDP and GBS cases. Combination of all lncRNAs could not significantly enhance the diagnostic power. Taken together, these lncRNAs might be involved in the development of CIDP or GBS. These transcripts might be regarded as marker for these immune-related conditions as well. Future studies should appraise expression of these transcripts in other immune-related conditions to evaluate their suitability as diagnostic markers for GBS/CIDP.The correlations between expression levels of mentioned lncRNAs were not meaningfully different between patients and controls based on the measured correlation coefficients.


This study was aimed at examining the effects of an adaptive non-linear frequency compression algorithm implemented in hearing aids  at different parameter settings and auditory acclimatization on speech and sound-quality perception in native Mandarin-speaking adult listeners with sensorineural hearing loss.Data consisted of participants’ unaided and aided hearing thresholds, Mandarin consonant and vowel recognition in quiet, and sentence recognition in noise, as well as sound-quality ratings through five sessions in a 12-week period with three SR2 settings .Twenty-nine native Mandarin-speaking adults aged 37–76 years old with symmetric sloping moderate-to-profound sensorineural hearing loss were recruited. They were all fitted bilaterally with Phonak Naida V90-SP BTE hearing aids with hard ear-molds.The participants demonstrated a significant improvement of aided hearing in detecting high frequency sounds at 8 kHz. For consonant recognition and overall sound-quality rating, the participants performed significantly better with the SR2 default setting than the other two settings. No significant differences were found in vowel and sentence recognition among the three SR2 settings. Test session was a significant factor that contributed to the participants’ performance in all speech and sound-quality perception tests. Specifically, the participants benefited from a longer duration of hearing aid use.Findings from this study suggested possible perceptual benefit from the adaptive non-linear frequency compression algorithm for native Mandarin-speaking adults with moderate-to-profound hearing loss. Periods of acclimatization should be taken for better performance in novel technologies in hearing aids. High-frequency components of acoustic signals convey useful information in speech and music. They play an important role in sound-quality perception, sound localization, speech perception in noise, and language development in children . Many paFor people with severe-to-profound hearing loss, more aggressive settings with a lower CT and a higher CR are required because the patients have a narrower audible frequency bandwidth and the inaudible frequencies start at a lower frequency point in comparison to people with mild or moderate hearing loss. While the use of a lower CT ensures that a wider range of high frequencies can be shifted down so that they become audible to hearing aid users, it may also introduce unwanted detrimental effects to consonant and vowel perception  and sounSo far, there has been a number of studies examining the efficacy of NLFC on various aspects of speech perception including phoneme and word recognition, sentence perception, and sound-quality perception . While mAs adaptive NLFC is a newly developed algorithm, only a few studies tested the use of this algorithm in hearing-impaired listeners e.g., . In thesM = 66.7, SD = 8.8). All participants were diagnosed with sloping moderate-to-profound sensorineural hearing loss. The average pure-tone audiometric thresholds between 500 and 4,000 Hz for both ears were between 40 and 90 dB HL. The individual and group average pure-tone thresholds are shown in The participants included 29 Mandarin-speaking adults  aged between 37 and 76 years old  programmed in Phonak’s Target fitting software (v. 5.1). To ensure proper amplification of sound across the entire speech spectrum and to limit acoustic feedback, occluding hard ear-molds made of acrylic materials were used with different vent sizes based on the recommendation of the fitting software. After the feedback and real-ear test using the estimated RECD  and recommended vents, the APDT  gain algorithm was chosen as the prescriptive target. The output gain level was initially set to 100%, decreasing in a 10% step size in case the participant reported that the hearing aids were too loud. Three SR2 settings were tested in the study: SR2 off, SR2 default, and SR2 strong . As a group, the parameters CT1 and CT2 changed from 3.77 ± 1.35 (mean ± SD) and 5.12 ± 1.21 kHz in SR2 default to 2.17 ± 0.55 and 3.78 ± 0.67 kHz in SR2 strong. The group average of the parameter CR remained unchanged in SR2 default and SR2 strong (1.22 ± 0.10 vs. 1.22 ± 0.06). Note that the use of lower CT1 and CT2 in SR2 strong could potentially move more high-frequency energy to the audible range but create greater disruption of low-frequency information. A schematic diagram of the signal processing for SR2 is available in our previous acoustic study of non-linear frequency compression See . The othThe perceptual performance of each participant was evaluated through speech perception tests and sound-quality rating tasks. The speech perception tests included Mandarin-Chinese consonant, vowel, and sentence recognition tests./, sh //, and h /x/) and six Mandarin affricates  embedded in a /Ca/ syllable in tone 1. The 11 words containing the target consonants are  (fā),  (sā),  (zā),  (cā),  (xiā),  (jiā),  (qiā),  (shā),  (zhā),  (chā), and  (hā). The tokens were recorded from 6 adult Mandarin speakers . Thus, the consonant recognition test comprised 66 tokens (11 words × 6 speakers) that were randomly presented to the participants. The intensity of the stimuli was set at 65 dB SPL.The consonant recognition test included five Mandarin fricatives  embedded in a /dV/ syllable structure in tone 1. The 12 monosyllabic words are  (dā),  (dāi),  (dāo),  (dē),  (dī),  (diāo),  (diē),  (diū),  (dōu),  (dū),  (duī), and  (duô). The tokens were recorded from the same 6 adult Mandarin speakers. In total, there were 72 tokens for the vowel recognition test (12 words × 6 speakers). The intensity of the stimuli was also set at 65 dB SPL.The Mandarin vowel list included 12 Mandarin vowels  . MHINT cAll participants were asked to rate the loudness, clarity, naturalness, and overall sound quality of different types of sounds including own voice, male voice, female voice, bird chirp, and music. Own voice was referred to as the participant’s natural and spontaneous vocal production in daily life after wearing the experimental hearing aids. The male voice and female voice were reading text composed of 127 Chinese characters by one male and one female talker. The lengths of the recordings were 34 and 36 s for the male and female voices, respectively. They were presented at 65 dB(A) in quiet. The bird chirps were provided by MATLAB software . The original 8 chirps  were repThe five types of stimuli were presented in a fixed order as above with no repetition being allowed. After listening to each stimulus, participants were asked to rate four aspects of sound quality including loudness, clarity, naturalness, and overall quality using a continuous bar with two ends being 0 (extremely poor) and 10 (perfect). No practice was provided for this task.All participants were tested at five different sessions separated by 3 weeks between each two consecutive sessions. In the first session, the participants were fitted with bilateral hearing aids (Phonak Nadia V90-SP BTE) with individualized hard ear-molds. The sound-field aided thresholds with SR2 off, SR2 default, and SR2 strong settings were measured using warble tones at 250, 500, 1,000, 2,000, 3,000, 4,000, 6,000, and 8,000 Hz. Perceptual tests including speech perception  and sound-quality rating with SR2 off and SR2 default or SR2 strong settings were then conducted. After the first session, the participants were sent home with the hearing aids on either SR2 default or SR2 strong. To count-balance the order of SR2 settings, 14 of the 29 participants wore SR2 default for the first 6 weeks (Group A) and the remaining 15 of the 29 participants wore SR2 strong for the first 6 weeks (Group B). After 6 weeks, the SR2 settings for both groups were switched. Statistical analyses were conducted using R software (version 3.63). The percent-correct data of the recognition tests were treated as binomial data . A generF-values ranging from 8.87 to 77.92, all p < 0.001) except 250 Hz . Tukey-adjusted pairwise comparisons revealed no significant differences in aided hearing thresholds among SR2 off, SR2 default, and SR2 strong settings from 250 Hz to 6 kHz . At 8 kHz, the hearing thresholds for SR2 off setting and unaided condition were comparable (adjusted p = 0.34), and those for SR2 default and SR2 strong settings were not significantly different (adjusted p = 0.29). However, the hearing threshold was significantly higher at for unaided or SR2 off setting than for SR2 default setting  and SR2 strong setting .The group mean unaided and aided hearing thresholds under the three SR2 settings  were anat-test comparing the recognition performance between Groups A and B revealed no order effect . Thus, in the following presentations of results, data from Groups A and B were pooled together and were not treated separately.Individual and group average performance of consonant, vowel, and sentence recognition with the three SR2 settings in five sessions is shown in Hz250–4,000 )  with exception of one condition . In addition, we used the difference scores in speech recognition between SR2 default and SR2 off settings or those between SR2 strong and SR2 off settings as potential NLFC benefit scores. However, Pearson correlation analyses revealed no significant correlation of the latter and the participants’ aided hearing thresholds at high frequencies or PTAHz250–4,000 . We also found that the participants’ age was not correlated with any of the speech recognition performance nor the potential NLFC benefit scores.Large individual variability in speech recognition, especially in consonant recognition in quiet and sentence recognition in noise (+5 dB SNR), was evident . Pearson2 = 21.90, p < 0. 0001; sessions: χ2 = 68.95, p < 0.0001]. Post-hoc multiple comparisons revealed a modestly better performance for SR2 default setting than for SR2 off setting (adjusted p = 0.001) or SR2 strong setting (adjusted p < 0.001). No significant differences between SR2 off and SR2 strong settings were observed (adjusted p = 0.41). The participants also showed improved performance from Session 1 to Session 5. Multiple comparisons revealed that the participants performed similarly at Sessions 4 and 5 (adjusted p = 0.98) which were both significantly better than at Sessions 1, 2, and 3 . The interaction between test session and SR2 setting was not significant .2 = 61.73, p < 0.00]. Specifically, the vowel recognition at Session 5 was significantly better than that at Sessions 1, 2, and 3 . Between Sessions 4 and 5, however, no significant differences were observed (adjusted p = 0.006). This was also true for the pairwise comparisons among Sessions 1, 2, and 3 .For vowel recognition, unlike consonant recognition, only test session contributed to the improved performance  and no interaction was found between the two factors . The sentence-recognition performance for SR2 default setting was the highest as compared to SR2 off setting (adjusted p = 0.001) or SR2 default setting (adjusted p = 0.003). No difference between SR2 off and strong was found (adjusted p = 0.99).For sentence recognition in noise, similar to consonant perception, both test sessions and SR2 settings contributed to the improved performance  and type of stimulus . SR2 settings  as well as the interaction between test session and SR2 setting  were not significant. The participants’ satisfaction with loudness improved progressively from Session 1 to Session 5 . Among the five different types of stimuli, the rating score for own voice was significantly lower than all the other four types of stimuli . In addition, the overall quality rating for the female voice was found to be higher than for the male voice (adjusted p = 0.028). No other significant differences were observed .F = 4.10, p = 0.016; SR2 setting: F = 71.16, p < 0.0001; type of stimulus: F = 18.59, p < 0.0001] but there was no interaction between test session and SR2 setting . The participants rated clarity significantly higher with SR2 default setting than with SR2 strong setting (adjusted p = 0.018), while no difference was detected between SR2 off and SR2 default settings (adjusted p = 0.17) or between SR2 off and SR2 strong settings (adjusted p = 0.041). The clarity rating at Sessions 4 and 5 was the highest compared to that at Sessions 1, 2, and 3 , yet no significant difference was observed at Session 5 compared with Session 4 (adjusted p = 0.59). Finally, own voice clarity scores were found to be the lowest among all types of stimuli . The female voice, on the other hand, received rating scores significantly higher than the male voice (adjusted p < 0.001) or music (adjusted p = 0.001) but similar scores to the chirp (adjusted p = 0.39). All other pairwise comparisons were not significant otherwise .In terms of clarity rating, all the three main factors were significant  but not the interaction terms . Both naturalness and overall ratings improved from Session 1 to 4  but not from Session 4 to 5 . Both ratings were found to be better for SR2 default setting than for SR2 strong setting (both adjusted p = 0.002), but no difference was found for the other comparisons among SR2 settings. Among different types of stimuli, own voice was rated with the lowest scores for both naturalness  and overall quality scores . In addition, the female voice was rated with significantly higher naturalness than music (adjusted p = 0.009) and with higher overall quality scores than the other types of stimuli .For the naturalness and overall rating, all the three main factors  played significant roles in the outcome measures [Naturalness: In the present study, we evaluated the efficacy of the new adaptive NLFC scheme on Mandarin speech perception and sound-quality ratings in adult hearing aid users. A series of tests were conducted over a 3 month period to evaluate aided hearing thresholds, consonant and vowel recognition in quiet, sentence recognition in noise, and subjective sound-quality ratings in 29 participants with severe-to-profound hearing impairment./ (“x”), especially with the stronger SR2 settings.For sound detection, our results demonstrated an improvement of more than 30 dB in detecting high-frequency sounds at 8 kHz with the application of adaptive NLFC scheme . A similFor vowel recognition, most of the participants had recognition scores above 90% correct . The adaUnlike vowel recognition, the use of adaptive NLFC significantly improved consonant recognition performance. , t, th/  concentrates at a lower frequency region that was less affected by frequency compression. By contrast, the alveolar sounds /s, ts, tsh/  have spectral energy distributed at a higher-frequency region. Frequency compression shifted the spectral energy of alveolar sounds to a lower-frequency region similar to retroflex sounds. The more aggressive compression setting caused greater distortion of spectral energy distribution and thus greater confusion. While both alveolar and alveolopalatal sounds are high-frequency sounds that present accessibility challenges in people with severe hearing impairments, the higher accuracy and less confusion of alveolopalatal sounds were likely associated with the phonotactic constraints in Mandarin Chinese. Mandarin /, t, th/  is always followed by /i/, /y/, or vowels starting with these two. In the current study, the alveolopalatal sounds were followed by /ia/ which differed from other tested consonants that were followed by /a/. The distinct vowel environment likely assisted in the recognition by the hearing aid users.Our consonant confusion analyses  showed tAlthough adaptive NLFC technology helped improve patients’ consonant recognition and audibility of high-frequency information in quiet, it did not provide significant perceptual benefits to sentence recognition in noise. Previous research revealed mixed findings on sentence recognition in noise with the NLFC technology . Chen etSound quality is another core criterion in evaluating patients’ willingness to accept technology and degree of comfort after using it. Some researchers reported no obvious changes in sound-quality ratings with or without NLFC . Some reper se. This finding was consistent with the previous SR study (Researchers have found that a period of acclimatization is necessary for the human brain to gradually learn and obtain benefits from any newly applied technologies including the NLFC scheme. SR study . The preIn summary, the adaptive NLFC technology implemented the hearing aids provided benefit to high-frequency sound detection, as well as Mandarin consonant recognition, and sound-quality ratings in listeners with moderate-to-profound sensorineural hearing loss. As expected, vowel recognition performance with or without the adaptive NLFC algorithm was consistently good, indicating no deterioration of vowel perception using the adaptive NLFC. However, for sentence recognition in noise, the adaptive NLFC algorithm showed limited effects. Among different settings, SR2 default provided more benefits than SR2 strong setting in our group of participants with moderate to profound hearing loss. Furthermore, participants showed continuous improvement of recognition performance with increased length of SR2 use, which indicated a potential effect of auditory acclimatization.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Review Boards of Ohio University and Beijing Tongren Hospital. The patients/participants provided their written informed consent to participate in this study.XC, JR, VK, and LX conceived and designed the study. SQ, XT, HH, and JG collected the data. XW performed the statistical analyses. SQ, JY, and LX wrote the manuscript. All authors discussed the results and implications and commented on the article at all stages.XT, HH, and JG were employed by the company Sonova China, Shanghai. JR and VK were employed by the company Sonova AG Switzerland. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mixed model analyses revealed that both reactive aggression and proactive aggression decreased with age, whereas shame and guilt peaked in early adolescence. Adolescents with hearing loss reported higher levels of proactive aggression, lower levels of shame and guilt, and showed protracted development for guilt compared to their hearing peers. In both groups, shame contributed to an increase in reactive aggression, whereas guilt contributed to a decrease in proactive aggression. These longitudinal associations highlight the unique role that shame and guilt play in the development of adolescent aggression.This longitudinal study examined how shame and guilt contribute to the development of reactive and proactive aggression in adolescents with and without hearing loss. Adolescents between 9 and 16 years old (adolescents with hearing loss ( Aggression is any form of behaviour that has the goal of harming or injuring someone else , explain behaviour based on mental states, and actively stimulate children’s perspective taking abilities . Thus, communication about the social world around the child is crucial to the development of shame and guilt. But many children with hearing loss cannot access this kind of full communication. Not surprisingly, cross-sectional studies have indicated lower levels of shame and guilt in children with hearing loss  or negative emotions  following imagined moral transgressions is an important predictor of aggression  in the development of reactive and proactive aggression. Adolescents with hearing loss have less access to the social world solely due to limitations on auditory input. It is therefore plausible that differences between both groups are the result of less access to the social world. Group differences in this study are therefore viewed as a proxy for social access.The first aim of this study was to compare the levels and development of proactive and reactive aggression and, shame and guilt between adolescents with and without hearing loss. We expected higher levels of reactive and proactive aggression, and lower levels of shame and guilt in adolescents with hearing loss compared to their hearing peers  an unaided hearing loss of at least 40 dB in the better ear. An individual with a loss of 40 dB has difficulties hearing normal speech even at close distances. 2) The hearing loss had to be detected before the age of four, meaning that the hearing loss occurred before or during language acquisition. 3) All adolescents were born to hearing parents not proficient in sign language. Adolescents without hearing loss were recruited from primary and secondary schools in the Netherlands. Inclusion criteria for both adolescents with and without hearing loss were 1) age between 9 and 16 years at Time 1 (T1), 2) average intellectual functioning, 3) no diagnosed developmental disabilities or learning difficulties, and 4) living in the Netherlands or the Dutch speaking part of Belgium. Parents indicated on the informed consent form whether their child was currently or previously diagnosed with any psychiatric condition or learning difficulty. Intellectual functioning was assessed by using two nonverbal subtests of the Wechsler Intelligence Scale for Children – Third Edition  Self-Report . Questionnaires were administered individually in a quiet room at the participant’s school or home. Participants were seated in front of a computer, and questions were presented one by one. For adolescents with hearing loss, all instructions and questions were accompanied by a video providing a translation in Dutch Sign Language.We administered self-report questionnaires to participants at all three time points with intervals of approximately 9 months . This analytic technique is also appropriate for datasets with missing data  + Shame (baseline & change) + Guilt (baseline & change).Proactive aggression ~ Age + Group + Gender + Proactive aggression (baseline & change) + Shame (baseline & change) + Guilt (baseline & change).Second, linear mixed models were used to assess whether shame and guilt contributed to the linear development of reactive and proactive aggression. In the first step, we used baseline levels and change levels. Baseline levels represent the score of the participant at the first time point. . Change scores represent the difference relative to the baseline, meaning that scores of the second time point and third time point are subtracted by the score of the first time point . We also included age, group and gender  in the analyses. This resulted in the following models:In the second step, interactions with group were added . Again, we made a comparison between nested models by comparing AIC and BIC values . All analyses were performed in SPSS version 24.0. Graphs were made in R version 3.4.3 using the Ggplot2 function.Intraclass correlations (ICC) were calculated to test nesting of observations within individuals across the three time points. We used a two-way mixed effects model with a measure of absolute agreement and interpreted average measures. ICC were good with values of 0.76 for reactive aggression, 0.73 for proactive aggression, 0.77 for shame, and 0.79 for guilt. Pearson correlations between the averages of all study variables  are displayed in the Supplementary Appendix Table .The outcomes for the best fitting model of the multilevel analyses are displayed in Table b = 0.97, p = 0.084), but adolescents with hearing loss displayed higher levels of proactive aggression  compared to hearing adolescents  and lower levels of guilt .As shown in Table For proactive aggression, the baseline level and change in guilt contributed to a decrease in proactive aggression, controlling for reactive aggression Below is the link to the electronic supplementary material.


Contrary to the increasing incidence of gastroenteropancreatic neuroendocrine tumors (GEP-NETs), GEP-NETs of the accessory papilla of the duodenum are extremely rare. Furthermore, there have been no recommendations regarding the treatment strategy for GEP-NETs of the accessory papilla of the duodenum. We present a case of GEP-NET of the accessory papilla of the duodenum successfully treated with robotic pancreatoduodenectomy.A case of a 70-year-old complaining of no symptoms was diagnosed with GEP-NET of the accessory papilla of the duodenum. A 8-mm tumor was located at the submucosal layer with a biopsy demonstrating a neuroendocrine tumor grade 1. The patient underwent robotic pancreatoduodenectomy as curative resection for the tumor. The total operative time was 406 min with an estimated blood loss of 150 mL. The histological examination revealed a well-differentiated neuroendocrine tumor with low Ki-67 index (< 1%). In the posterior areas of the pancreas, the lymph node metastases were detected. The patient was followed up for 6 months with no recurrence postoperatively.Considering the potential risks of the lymph node metastases, the standard treatment strategy for GEP-NETs of the accessory papilla of the duodenum should be radical resection with pancreatoduodenectomy. Minimally invasive approach can be the alternative to the conventional open surgery. Although the incidence of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) has increased, GEP-NETs of the duodenum are rare , 2. FurtA 70-year-old man was referred to our hospital for further examination of a tumor at the duodenum. Esophagogastroduodenoscopy identified a submucosal tumor at the accessory papilla in the second part of the duodenum Fig. a, b. EndAlthough there have been no recommendations regarding the treatment strategy for GEP-NETs of the accessory papilla of the duodenum, radical resection with pancreatoduodenectomy has been recommended as the first-line treatment for GEP-NETs of the ampulla of Vater due to relatively poor prognosis . FurtherOur standardized protocol for robotic pancreatoduodenectomy was previously reported . FollowiThe total operative time was 406 min, and the estimated blood loss was 150 mL. Uneventful postoperative course including no complications was observed with the patient being discharged on postoperative day 8. The final histological diagnosis was neuroendocrine tumor G1 based on the findings of synaptophysin positive, chromogranin A positive, and Ki-67 index (< 1%) Fig. . The preThe patient was followed up for 6 months without recurrence postoperatively.To our knowledge, this is the first description on GEP-NETs of the accessory papilla of the duodenum with lymph node metastases successfully treated with robotic pancreatoduodenectomy. The present case indicates that a well-differentiated neuroendocrine tumor G1 with less than 1 cm at the accessory papilla of the duodenum could have the potential risks of the lymph node metastases. Therefore we suggest that radical surgical resection with pancreatoduodenectomy instead of local excision should be the first-line treatment option for GEP-NETs of the accessory papilla of the duodenum.n = 4) or endoscopically (n = 1). Although the margin of resected tumor was confirmed to be positive in a case with endoscopic intervention, no recurrences during the follow-up were observed in five cases undergoing local excision. In contrast, five cases underwent radical resection with pancreatoduodenectomy. The results of case series suggested the efficacy of radical resection for the tumors. Surprisingly, lymph node metastases at the resected regional areas were identified in four cases out of five. Therefore, it should be recognized that GEP-NETs of the accessory papilla of the duodenum could metastasize to the lymph nodes regardless of size of the tumor.Contrary to the increasing number of GEP-NETs, GEP-NETs of the accessory papilla of the duodenum are very rare. The characteristics in reported cases of GEP-NETs of the accessory papilla of the duodenum are demonstrated in Table With respect to treatment strategy, it suggests that pancreatoduodenectomy should be the first-line treatment for GEP-NETs of the accessory papilla of the duodenum. In the light of malignant potential, radical resection should be considered even for small primary tumors. Minimally invasive pancreatoduodenectomy can be optional given the safety and oncologic efficacy of minimally invasive surgery . In contIn conclusion, we present an extremely rare case of GEP-NET of the accessory papilla of the duodenum accompanied by lymph node metastases. Although carcinogenic mechanism of this tumor remains uncertain because of the limited reports, radical resection with pancreatoduodenectomy should be performed due to the potential risks of the lymph node metastases. Minimally invasive approach should be considered as the alternative to the conventional open surgery.


Following cardiac stress/damage, membrane-associated cavin-4 recruits the signaling molecule ERK to caveolae, which activates key cardio-protective responses. Evaluation of ERK signaling revealed a profound inhibition, below physiological baseline, in the 5cvmdx mouse heart. Expression of μDys in 5cvmdx mice prevented the development of cardiac histopathology but did not rescue membrane localization of cavins nor did it normalize ERK signaling. Our study provides the first comparative analysis of purified protein complexes assembled in vivo by full-length dystrophin and a therapeutic micro-dystrophin construct. This has revealed disruptions in cavins and ERK signaling that may contribute to DMD cardiomyopathy. This new knowledge is important for ongoing efforts to prevent and treat heart disease in DMD patients.ΔR4-R23/ΔCT micro-dystrophin (μDys) is a miniaturized version of dystrophin currently evaluated in a Duchenne muscular dystrophy (DMD) gene therapy trial to treat skeletal and cardiac muscle disease. In pre-clinical studies, μDys efficiently rescues cardiac histopathology, but only partially normalizes cardiac function. To gain insights into factors that may impact the cardiac therapeutic efficacy of μDys, we compared by mass spectrometry the composition of purified dystrophin and μDys protein complexes in the mouse heart. We report that compared to dystrophin, μDys has altered associations with α1- and β2-syntrophins, as well as cavins, a group of caveolae-associated signaling proteins. In particular, we found that membrane localization of cavin-1 and cavin-4 in cardiomyocytes requires dystrophin and is profoundly disrupted in the heart of  Patients with Duchenne muscular dystrophy (DMD) lack sufficient expression of a functional dystrophin protein in all striated muscles, leading to loss of ambulation by 13 years of age, progressive respiratory insufficiency and dilated cardiomyopathy . Althougmdx mouse model of DMD  promoter (MMRRC Stock No: 41194-JAX). For biochemical analyses, we transferred the μDys transgene to the 5cvmdx mouse model  because it has lower residual dystrophin expression compared to mdx mice (To study the μDys-associated protein complex (μDAPC) in the heart, we used transgenic mdx mice , renderi5cvmdx mice (μDYS-5cvmdx mice), we performed immunohistochemistry and western blot analyses with the MANEX1011B antibody that recognizes an epitope shared by dystrophin and μDys located close to Hinge 1 of the rod domain  is expressed on average 3.9-fold above wild-type levels in μDYS-5cvmdx heart total protein lysates, with inter-individual differences ranging from 2 to 6-fold , while expression of α1-syntrophin and α-dystrobrevins was normalized to wild-type levels  and signaling  functions. We first analyzed transcript levels by quantitative RT-PCR and found that loss of dystrophin expression in ype mice . These iv hearts . Therefoype mice . In μDYSe levels . By immurobrevin . Additiomyocytes . In μDYSectively  in agreeectively . Howevermyocytes  in contrmyocytes . Overall5cvmdx mouse model shows late onset cardiac fibrosis and cardiac dysfunction similar to mdx mice  in heart sections of 5cvmdx mice but not μDYS-5cvmdx mice between 6 and 12 months of age  at 6 months of age to 8.7% ± 1.2 (n = 5) at 1 year of age  evaluation at 6 months of age, revealed significant abnormalities in 5cvmdx mice compared to wild type  that were normalized by μDys  using the MANEX1011B monoclonal antibody as previously described  Fig. 3AFig. 3A. and μDys . FurtherN = 3) and μDys IPs from μDYS-5cvmdx hearts (N = 3). The μDys sequence was manually added to the peptide search database for accurate peptide matching and was given the ID P11531-A. No peptides matching to domains of dystrophin lacking in μDys were identified in μDYS-5cvmdx IPs. A total of 121 proteins were identified by MS (5cvmdx mice (N = 3) or in a control IP on wild-type lysates with an isotype-matched antibody . Utrophin, a homologue of dystrophin, was detected in MANEX1011B IPs indicating some antibody cross-reactivity with utrophin  was performed on dystrophin IPs from wild-type hearts  or to the amount of dystrophin/μDys within each sample (emPAIDMD). Regardless of the normalization method used, α1-syntrophin, cavin-1, cavin-2 and cavin-4 were found to be significantly decreased or absent in μDys IPs compared to dystrophin IPs (P = 0.12), it was not detected in any μDys IP suggesting that its association with μDys might be impaired similar to cavins-1, -2 and -4  in protein expression detected in protein lysates by western blot, translate into more modest increases in membrane fluorescence. Furthermore, in the case of cavins, similar protein expression levels were detected by western blot between the genotypes studied, yet membrane localization was profoundly different. Therefore, analyses on total protein lysates on their own are not a good proxy for DAPC protein expression at the membrane or for identification of dystrophin-associated proteins. Overall, our study shows that MS, immuno-precipitation and quantification of protein membrane fluorescence provide important additional information when evaluating DAPC rescue by micro-dystrophin constructs developed for gene therapy. While the approach we used is ideal to rapidly identify and validate differential protein associations, additional complementary biochemical approaches should be considered to explore further the differences we found between the cardiac DAPC and μDAPC. Among these, approaches previously used to study the assembly, composition and stoichiometry of the DAPC  of dystrophin required for association with cavins could help guide the design of future micro-dystrophins with improved cardiac protection, and to inform future exon skipping or gene editing strategies aimed at the heart.Detailed methods are provided in the supplemental material.Animal breeding and experimental procedures followed approved protocols by the Institutional Animal Care and Use Committees at Nationwide Children’s Hospital and University of Missouri.PAI-1, where PAI (Protein Abundance Index) denotes the ratio of observed to observable peptides for a given protein. For quantitative analyses, the emPAI was normalized either to the protein amount in each sample using the normalization function in Scaffold, or divided by the emPAI of dystrophin/μDys within the same sample.Purification and MS analyses of complexes assembled by dystrophin (DAPC) and μDys (μDAPC) were performed as previously described  using tht-test or a one-way ANOVA followed by a Bonferroni test for pair-wise comparisons with a P value set at 0.05 and with correction for multiple comparisons. Non-normally distributed data were analyzed using the non-parametric Kruskal-Wallis analysis followed by the Dunn’s test for pair-wise comparisons. A two-way repeated measure ANOVA was used for superplots involving nested measurements of membrane fluorescence intensity for n = 3 mice per group.The Shapiro–Wilk normality test was performed. Normally distributed data were analyzed using a two-tailed Student’s Wang_et_al_Suppl_Figures_revised_ddab133Click here for additional data file.Wang_et_al_Supplemental_Tables_ddab133Click here for additional data file.SUPPLEMENTAL_MATERIAL_Revised_Final_ddab133Click here for additional data file.


Glucocorticoid-induced osteoporosis (GIOP) is a major cause of secondary osteoporosis, and the pathogenic mechanisms of GIOP remain to be elucidated. Here, we show a rapid dexamethasone-induced osteoporosis animal model using zebrafish scales. Intraperitoneal injection of dexamethasone over a 5-day period suppressed the regeneration of scales. Furthermore, the circularity of the newly formed regenerated scales was also slightly reduced compared to that of the control group on day 5. The changes in bone-related enzymes, such as cathepsin K, tartrate-resistant acid phosphatase (TRAP) for bone resorption, and alkaline phosphatase (ALP) for bone formation, provide insight into the progression of bone diseases; therefore, we further developed a method to measure the activities of cathepsin K, TRAP, and ALP using zebrafish scales. We found that a lysis buffer with detergent at neutral pH under sonication efficiently helped extract these three enzymes with high activity levels. Interestingly, treatment with a dexamethasone injection produced considerably higher levels of cathepsin K activity and a lower Ca/P ratio than those in the control group, suggesting that dexamethasone increased osteoclast activity, with no significant changes in the activities of TRAP and ALP. Our GIOP model and enzyme assay method could help to design better treatments for GIOP. Osteoporosis is an age-related bone disease that increases the risk of bone fragility by decreasing the bone strength and density, especially in postmenopausal women and elderly individuals . Aging aSeveral animal models for GIOP have been reported, including in mice and rats. However, mammalian GIOP models are expensive and time-consuming to generate. Zebrafish have become a popular biomedical model in a wide variety of studies ,12. In cTherefore, we designed the adult zebrafish scale GIOP model by investigating the activity of bone biomarkers, including the cathepsin K, TRAP, and ALP enzymes . CathepsIn this study, we aimed to establish a dexamethasone-induced GIOP zebrafish model in a short period (5 days) using zebrafish scales and evaluated cathepsin K, TRAP, and ALP activity in the same sample by our developed method.To evaluate zebrafish scales as an in vivo model for dexamethasone-induced osteoporosis, we examined the size and shape of both the ontogenetic and regenerated scales from fish with or without dexamethasone treatment on day 0 and day 5. The term ‘ontogenetic scales’ was used to describe the original scale that grows during ontogeny, while the ‘regenerated scales’ referred to the newly formed scales after regeneration. The surface area of the ontogenetic scales collected on day 5 was not significantly different from that of the original scale at day 0, regardless of dexamethasone treatment A. The avTo evaluate the enzymatic activities of the bone-related enzymes cathepsin K, TRAP, and ALP in scales, we designed and developed a method to improve enzyme extractability by the use of a nonionic detergent (1% NP-40) and a sonication process in a basal lysis buffer . The scale samples lysed in the buffer containing 1% NP-40 showed stronger cathepsin K activity than did those lysed in the buffer without detergent, while the cathepsin K activity obtained by sonication was comparable to that obtained without sonication A. IntereWe then evaluated the inhibitor specificity in lysate samples extracted from our method. The cathepsin K activities from samples lysed in buffer containing 1% NP-40 were comparable to those from 1% NP-40 and sonication A. The spAdditionally, TRAP and ALP analyses used the same lysate and substrate, and the inhibitor study was further performed using levamisole, which is a well-known endogenous ALP inhibitor. The samples lysed under 1% NP-40 and sonication conditions were pretreated with levamisole before incubation in the individual reaction buffers. The relative TRAP activity of the control group was comparable to that of the levamisole-treated group B. ConverWe further used scale lysates to investigate the effectiveness of our method. When comparing our method with previously reported methods using raw scales ,36,43, tTo better understand the mechanisms underlying the dexamethasone-induced reduction in scale regeneration, we further used our developed assay to evaluate the activity of enzymes involved in bone remodeling from the same sample. In total, five zebrafish scales were pooled and incubated with lysis buffer. After centrifugation, the solution was separated into two parts, Lysate 1 (L1) and Lysate 2 (L2), as shown in We next examined the effects of dexamethasone on cathepsin K, TRAP, and ALP activity after a 5-day period, as described previously. The cathepsin K activity of ontogenic scales in the dexamethasone-treated groups on day 5 tended to be higher than that of ontogenetic scales in the control group, although the difference was not statistically significant A. In conTo further evaluate the effect of dexamethasone on scales, the calcium and phosphorus contents were measured in the original scales and regenerated scales . The calIn this study, dexamethasone treatment increased osteoclast activity  but not osteoblast activity . This observation is supported by similar findings in scales treated with prednisolone, in which the expression of osteoblast markers, including ALP, was not increased . FurtherTypically, a ratio of calcium phosphate of approximately 1.67 indicates pure hydroxyapatite in the bone . After tAccordingly, our dexamethasone-induced model recapitulated the early GIOP phenotype  but did not reduce osteoblast cell activity well. Our experiment used dexamethasone, which is a more potent glucocorticoid than prednisolone, to induce early-stage osteoporotic characteristics in zebrafish ,47. AlthOur study used cathepsin K and TRAP as bone-degradation markers and ALP as a bone-formation marker. However, TRAP and cathepsin K are not confined to osteoclasts ,50, simiIn our study, we established a method to extract cathepsin K, as well as ALP and TRAP, from zebrafish scales using a Tris-based buffer containing detergent (1% NP-40). Compared with previously reported methods, our method exhibits a beneficial capability to determine the activities of three bone-related enzymes using the same samples. It allows simple and reproducible analyses with reduced variation originating from the individual scales. Previous experiments used raw scales to analyze the activities of TRAP and ALP ,59. To mArtemia cysts  and kept for one week before starting experiments.Adult zebrafish were purchased from Masuko Co., Ltd. (Japan). The fish were separated and kept in a 1.3 L tank (Meito System) under a normal light/dark cycle of 14 h/10 h at 28 °C. The fish were fed twice daily with Otohime B2  and freshly hatched w/v sodium chloride (NaCl) solution was used as a vehicle for dissolving dexamethasone and for treatment in the control group. On the first day of the experiment (day 0), the original scales were removed. Dexamethasone (9.68 mM) and NaCl solution were injected through the peritoneum of the fish in the treated group and control group, respectively. The injections were given on days 0, 2, and 4 in both groups. At the end of the experiment (day 5), the regenerated scales and ontogenetic scales were harvested and collected for further experiments.Dexamethasone 21-phosphate was purchased from Wako Inc. . The concentration of dexamethasone solution used was 30 μg per g fish body weight . The metP is the perimeter of the scale).Scale images were captured using AxioCam MRc  on a Leica microscope, and the surface area and perimeter were measured by the ImageJ program . The scaw/v tricaine methanesulfonate  solution [2HPO4, 1.8 mM KH2PO4, pH 7.3) in a 1.5 mL microcentrifuge tube. When starting an experiment, the PBS solution was removed, and 100 μL of lysis buffer  was added to lyse the scales. The scales were incubated in lysis buffer for 40 min on ice and then centrifuged at 17,400× g for 15 min at 4 °C. The first half of the lysate was collected for cathepsin K activity measurement. The remaining lysate was sonicated for a total time of 12.5 min. Then, this solution was used to measure TRAP and ALP activity.Prior to scale harvesting, the zebrafish were anesthetized using 0.016% solution . Scales A method for determining cathepsin K activity was adapted from Bossard et al. . Ten micTo determine the cathepsin K activity of each assay, the following formula was used:The method for TRAP activity measurement was performed according to a previously described protocol . Ten mic2 and 0.1 mM ZnCl2). The total enzymatic reaction was activated by adding 20 mM pNPP, and the mixture was then incubated for 1 h at 28 °C. Subsequently, the reaction was stopped by adding 50 μL of 3 M sodium hydroxide (NaOH). The molecular absorbance was determined using a FilterMax F5  at a wavelength of 405 nm.The method for measuring ALP activity followed a previous protocol with some modifications . Ten micTRAP and ALP activities were indirectly calculated from the hydrolysis of pNPP to p-nitrophenol (pNP), which is a colored compound. The concentration of pNP was used as a standard curve, and the following formula was used:For the inhibitor specificity of cathepsin K, 10 μL of lysate extracted in buffer containing 1% NP-40 was incubated with 10 μL of 10 μM Z-Leu-Leu-Leu-H , which is a cathepsin K inhibitor, and the assay buffer was incubated for 40 min on ice before adding the cathepsin K substrate, followed by the method for cathepsin K measurement described above.For the TRAP and ALP assays, the scale lysates extracted under 1% NP-40 sonication conditions were pretreated with 1 mM levamisole for 40 min at 28 °C. The samples were then incubated with an individual reaction buffer containing pNPP solution for 1 h at 28 °C. The molecular absorbance was determined, followed by TRAP and ALP assays, as mentioned earlier.3) on a hot plate at 230 °C for 5 h. Fifty microliters of the sample was diluted in 5 mL of 1% diluted HNO3 and kept at 4 °C until the determination mentioned below. Mineral contents such as calcium and phosphorus elements were determined using ICP-MS . Then, the Ca/P molar ratios were calculated. This method was adapted from previous protocols [Ten ontogenetic and regenerated scales each were collected and lysed with 100 μL 61% nitric acid .All statistical analyses were performed using GraphPad Prism Version 6 for Windows . One-way and two-way ANOVA with Tukey’s multiple comparisons test were used to determine the interaction between groups in several experiments. Comparisons between two groups were performed by Student’s The need for high-throughput screening has arisen from the demands of research activities. Our study provides a straightforward bone assessment method using adult zebrafish scales to measure the activities of cathepsin K, TRAP, and ALP. Furthermore, an increase in osteoclastic activity was observed on day 5 of regeneration after dexamethasone exposure, as indicated by the high levels of cathepsin K and lower Ca/P ratio. To summarize, our zebrafish scale GIOP model and the scale lysis method offer useful tools for bone research, especially in medium-throughput drug screening.