Refolding is known as the bottleneck in inclusion body (IB) downstream processing in the pharmaceutical industry: high dilutions leading to large operating volumes, slow refolding kinetics and low refolding yields are only a few of the problems that impede industrial application. Solubilization prior to refolding is often carried out empirically and the effects of the solubilizate on the subsequent refolding step are rarely investigated. The results obtained in this study, however, indicate that the quality of the IB solubilizate has a severe effect on subsequent refolding. As the solubilizate contains chaotropic reagents in high molarities, it is commonly analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE, however, suffers from a long analysis time, making at-line analytical implementation difficult. In this study, we established an at-line reversed phase liquid chromatography method to investigate the time-dependent quality of the solubilizate. To verify the necessity of at-line solubilization monitoring, we varied the essential solubilization conditions for horseradish peroxidase IBs. The solubilization time was found to have a major influence on subsequent refolding, underlining the high need for an at-line analysis of solubilization. Furthermore, we used the developed reversed phase liquid chromatography method for an in-process control (IPC). In conclusion, the presented reversed phase liquid chromatography method allows a proper control of IB solubilization applicable for tailored refolding. Escherichia coli, present a dominant fraction of the microbial production segment [E. coli can be carried out at very low costs in short fermentation run-times and high target protein concentrations at a high purity can be achieved [To date, approximately 20–30% of all approved biopharmaceuticals are produced in microbial hosts ,2. Insolachieved ,5,6. Earachieved ,8. In paachieved ,10. Protachieved . The addachieved . Refoldiachieved  comparedachieved .Singh et al. reported that mild solubilization boosts the refolding yield . The redUnfortunately, this gel-based method is not suitable for in-process control (IPC) as the sample treatment, method running time and staining protocols are very time-consuming. However, according to quality by design (QbD) principles in pharmaceutical manufacturing, an IPC must be applied  and, forSeveral liquid chromatography (LC) separation principles can tolerate the high molarities of the chaotropic agents required for the sample dissolution ,28. LC iIn this study, we implemented an RPLC method applicable for an at-line solubilizate analysis. In this context, at-line defines a measurement that is performed in close proximity to the process stream as specified by regulatory agencies . To demoE. coli BL21 (DE3)  with details stated in previous publications [HRP was produced in ications ,38. Afteications ,39,40, tCell disruption was carried out at 1200 bar for 3 passages using a high-pressure homogenizer . After centrifugation at 10,000 rpm, 4 °C and 20 min , the soluble fraction was discarded. The resulting IB pellet was washed two times with a buffer . The IB pellets were then resuspended in water, aliquoted to a defined wet IB weight, centrifuged  and stored at −20 °C until further use.2; 7% v/v glycerol) containing either 0 mM, 1.27 mM or 2.54 mM GSSG (glutathione disulfide) [The aliquoted IBs were thawed and resuspended in an HRP solubilization buffer . After the resuspension, DTT (1 M DTT stock) was added to a final concentration of 0 mM, 7.11 mM and 14.22 mM, respectively. The solubilization was performed at RT and a slight agitation and the samples were drawn after 0.5 h, 2 h, 4 h, 6 h, 8 h and 21 h. The samples were centrifuged  and the supernatant was used for refolding, the RPLC analysis and diluted 1:2 in 2x Laemmli buffer for the SDS-PAGE analysis. For refolding, the solubilizate was diluted 1:40 in a pre-cooled HRP refolding buffer  during the induction was varied. Batch 1 was conducted at a qs of 0.25 g/g/h during the induction whereas Batch 2 was performed at a qs of 0.35 g/g/h during the induction. The harvest, cell disruption, IB wash, aliquoting and storage were done identically for both batches.In order to demonstrate the applicability of the at-line RPLC method for the IPC, we produced two different IB batches. Fermentations for both batches were conducted as described here  only the2; 7% v/v glycerol; 1.27 mM GSSG). For IB Batch 2, two different refolding approaches were performed: the solubilizate was diluted 1:40 (= “fixed dilution”) and the dilution was adapted based on the RPLC results in order to achieve the same monomeric HRP concentration as in Batch 1. In this case, the dilution was reduced to 1:17 due to a lower concentration of the target protein HRP in IB Batch 2.The inclusion bodies from both batches were solubilized at a concentration of 100 g wet IB/L solubilization mix. The solubilization buffer consisted of 50 mM glycine pH 10; 6 M Urea. DTT was added to a final concentration of 7.11 mM (1 M DTT stock). The solubilization was performed for 0.5 h at RT on a rocker-shaker. After centrifugation , the supernatant was analyzed using the described RPLC method. Batch 1 solubilizate was diluted 1:40 in a refolding buffer  trifluoroacetic acid. Ultrapure water was acquired from a Milli-Q system from Merck Millipore . Acetonitrile (HPLC-grade) and trifluoroacetic acid  were obtained from Carl Roth . For the analysis, a recently published RPLC method, which had been developed and validated according to QbD principles, was modified in terms of gradient, column temperature and flow rate [The RPLC measurements were performed using a Dionex UltiMate 3000 system with a quaternary solvent delivery pump, an auto-sampler with a sample thermostat and a UV detector . The wavelength for the UV detection was set to 280 nm in order to monitor the protein absorption allowing the quantification of the target protein and its impurities. The instrument control and data acquisition were carried out via Chromeleon 7.2 software (Thermo Fisher). Prior to the RPLC measurement, all samples were centrifuged  to separate the aggregates from the soluble fraction. We used a BioResolve RP mAb Polyphenyl column   connected to a pre-column  of the same stationary phase. The mobile phase was composed of ultrapure water  and acetonitrile (eluent B) both supplemented with 0.1%  standard calibration ranging from 0.0625 g/L to 1.0000 g/L. The BSA was used for the quantification as no non-glycosylated standard was available for HRP.For the SDS-PAGE analysis, the samples were mixed with 2× concentrated Laemmli buffer to achieve a 1× concentration of Laemmli buffer in the final dilution. The used buffer solution did not contain β-mercaptoethanol (non-reducing conditions) in order to analyze the solubilization quality in regard to disulfide bridge formation. The samples were heated to 95 °C for 10 min. A total of 5 µL of each sample was loaded onto pre-cast SDS gels . The gels were run in a Mini-PROTEAN Tetra System (Bio-Rad) for 30 min at 180 V and stained with Coomassie Blue. The protein bands were evaluated densitometrically using ImageLab software (Bio-Rad).v/v glycerol) depending on their volumetric activity. A total of 170 µL of ABTS solution  was mixed with 10 µL of the respective diluted sample in the well. The reaction was started by adding 20 µL hydrogen peroxide . Immediately after the start of the reaction, the change in the absorbance at 420 nm was recorded at 30 °C for 2 min. The volumetric enzyme activity was calculated using Equation (1):The HRP enzyme activity was measured with a Tecan Infinite M200 PRO  using flat-bottom polystyrene 96-well plates, as described previously . The samtotalV—total well volume (μL).A/min—change in absorption (∆Abs 420 nm/min).∆Dilution—dilution of the sample.sampleV—volume of the sample (µL).d—length of the beam path through the well (d = 0.58 cm).420 = 36 mM−1 cm−1).ε—extinction coefficient  and the model predictability (Q2).An analysis of the used design of experiments (DoEs) was done using a multivariate data assessment program  with the model being based on a multiple linear regression. The results were analyzed for the statistical relevance of the model by the measure of fit  was defined as the concentration of monomeric HRP. We hypothesized that the enzymatic activity after refolding would directly correlate with the concentration of monomeric HRP in the solubilizate. The aggregated HRP was believed to result in a structure not applicable for refolding whereas monomeric HRP was defined as completely reduced and denatured during solubilization. Therefore, a monomeric HRP titer was chosen as the target response for the DoE. To test whether both RPLC and SDS-PAGE could predict the targeted solubilization for tailored refolding, all solubilizates were refolded and the volumetric activity (U/mL) after refolding was recorded.For the SDS-PAGE analysis, the HRP target band was found at 34 kDa . We hypo2 and Q2) are summarized in A multivariate data approach was applied in order to quantify the effects of the varied DTT concentrations and solubilization times on the monomeric HRP concentration during solubilization. The contour plots  of the multivariate data analysis for SDS-PAGE and RPLC in solubilization and the enzymatic activity after refolding are shown in The results obtained for the model responses varied between the SDS-PAGE and the RPLC analysis for the conducted DoE A,B. SurpThe effects on the product purity during solubilization can be found in To assess which analytical method was better suited for refolding yield prediction, the volumetric activity (U/mL) after refolding was determined C. The GSTherefore, the presented RPLC method met the criteria required for an at-line monitoring tool for the solubilization of HRP IBs. In comparison with the commonly used SDS-PAGE, the models based on the RPLC data were able to predict the influence of DTT concentration and time during solubilization on the subsequent refolding step and the refolding yield correctly. Furthermore, due to the versatility and short analysis time , RPLC coThe refolding yield highly depends on the protein concentration during refolding with lower protein concentrations favoring higher refolding yields ,9. The pSolubilizates were diluted at a fixed ratio of 1:40, as previously determined ;Solubilizates were diluted according to the product quantity assessed by RPLC .In order to evaluate if the presented RPLC method was suited for the IPC, we conducted solubilization and refolding experiments comparing two IB batches. Early DSP until the refolding step were kept constant for both IB batches. The refolding was performed by two different approaches:The corrected dilution in refolding led to a variation in the redox system because a higher DTT carry-over occurred at lower dilutions. This shift of the redox system potentially influences the refolding yield .For the demonstration purpose of the IPC via RPLC, drastic deviations from the USP were targeted (i.e. 54% of titer deviation). However, the protein concentration adjustment in refolding via at-line RPLC from solubilization might be even less error-prone for smaller deviations.Furthermore, standard deviations resulting from the RPLC measurements and enzymatic assay could explain further deviations .As shown in In the case of HRP IBs, the presented RPLC method was applied successfully as an IPC tool. Based on the rapid analysis time of the developed RPLC method, the deviations caused by the USP could be monitored at-line and the dilution was adapted to minimize the deviations during the refolding step. In addition, the received IB fingerprint  obtained via RPLC could provide valuable information using a reference impurity pattern for industrial applications.The solubilization of IBs prior to refolding is essential to obtain the desired protein conformation and protein concentrations in refolding. In this study, we developed an at-line RPLC method to monitor the target protein concentration during the solubilization unit operation of IB processing. DTT concentration and the solubilization time were varied for HRP IBs and the monomeric HRP concentration was recorded using SDS-PAGE and RPLC. The short analysis time (8.1 min), facile sample preparation and the high accuracy of RPLC (as demonstrated for the generic method ) allowedMoreover, we demonstrated the IPC making use of the developed RPLC method, determining USP alterations. As downstream operations are performed sequentially in industry, subsequent unit operations are influenced by initial deviations. In this study, the protein concentration in refolding could be adapted by adjusting the dilution factor based on the at-line RPLC analysis. This allowed for a more robust refolding process against the deviations contrived from the USP and a reduction of the refolding buffer compared with empirical dilutions thus facilitating a more economic process.Concluding, the developed RPLC method can be applied to accelerate process development in IB solubilization and for in-process monitoring therefore allowing IPC, which facilitates tailored IB refolding.


Phoca vitulina) revealed bilateral loss of inner hair cells  while the outer hair cells (sensory cells responsible for sound amplification and frequency selectivity and sensitivity) were intact. The selective inner hair cell loss (up to 84.6% of loss) was more severe in the basal turn, where the high frequencies are encoded. Potential causes and consequences are discussed. This is the first report of a case of selective inner hair cell loss in a marine mammal neonate, likely congenital.Congenital hearing loss  is recognized in humans and other terrestrial species, but there is a lack of information on congenital malformations and associated hearing loss in pinnipeds . Baseline knowledge on marine mammal inner ear malformations is essential to differentiate between congenital and acquired abnormalities, which may be caused by infectious agents, age, or anthropogenic interactions, such as noise exposure. Analysis of the cochlea of a neonate harbor seal (Phoca vitulina) by scanning electron microscopy revealed bilateral loss of inner hair cells with intact outer hair cells. The selective inner hair cell loss was more severe in the basal turn, where high-frequency sounds are encoded. The loss of inner hair cells started around 40% away from the apex or tip of the spiral, reaching a maximum loss of 84.6% of hair cells at 80–85% of the length from the apex. Potential etiologies and consequences are discussed. This is believed to be the first case report of selective inner hair cell loss in a marine mammal neonate, likely congenital.Congenital hearing loss is recognized in humans and other terrestrial species. However, there is a lack of information on its prevalence or pathophysiology in pinnipeds. It is important to have baseline knowledge on marine mammal malformations in the inner ear, to differentiate between congenital and acquired abnormalities, which may be caused by infectious pathogens, age, or anthropogenic interactions, such as noise exposure. Ultrastructural evaluation of the cochlea of a neonate harbor seal ( Toxoplasma gondii, rubella, cytomegalovirus, herpes, and syphilis infections. Within these infectious agents, congenital cytomegalovirus is the most common cause of non-hereditary sensorineural hearing loss in childhood , Ceftriaxone Sodium , ceftiofur crystalline free acid Excede  and benzylpenicillin procaine and benzylpenicillin benzathine suspension Duplocillin LA . Due to a poor prognosis, the pup was humanely euthanized and presented for necropsy. The animal was approximately 1 month of age when it died, with a weight of 9.0 kg and a total length of 58 cm.An extensive post-mortem examination was conducted following international protocols ,19 at thThe head was removed, and the inner ears were collected at the University of British Columbia (UBC) within 4.25 h post-mortem. The skull was opened with a hand saw to extract the brain, and the occipital bone was removed with a chisel T-Shape (Virchow skull breaker) post-mortem from the occipitomastoid suture a. The eaThe periotic bones (surrounding the cochlea) were decalcified with 14% Ethylenediaminetetraacetic acid (EDTA) tetrasodium salt, changing the media every 7–15 days for 47 and 192 days . Both cochleae were dissected to remove the bone, vestibular wall, Reissner and tectorial membranes, and dehydrated with increasing concentrations of ethanol.® 2021.The right cochlea was critical point dried , coated with platinum/palladium, and imaged with a Hitachi S-4700 SEM at the UBC Bioimaging Facility, Canada. The left cochlea was critical point dried , coated with gold, and imaged with a Zeiss Crossbeam 340 FIB-SEM at the University Medical Center Hamburg-Eppendorf, Germany. The brightness and contrast of images were adjusted in Adobe  Photoshop® software (https://imagej.nih.gov/ij/index.html accessed on 19 April 2021) from SEM micrographs at the level of the limit between the first row of OHCs and the inner pillar cells. A total of 33 micrographs were used for the calculation of the cochlear spiral from the left ear. Specific points on each image were identified to achieve an accurate consecutive delineation of length. Contiguous measurements avoided overlapping or gaps in the calculation of the structure (see Girdlestone and colleagues [The cochlear length was measured with ImageJlleagues ).Once the cochlear length was measured, it was possible to identify equidistant locations every 5% along the cochlear spiral. Due to damage in the end of the base or hook region, the locations were evaluated up to 88.6% from the apex. Counting of IHCs was performed at 5% increments to determine the number of IHCs present and absent. In addition, to confirm the number of absent IHCs at each location, the length of the cuticular plate of the IHCs was measured and averaged every 10% of the cochlear spiral. To illustrate the results, location 5% represents the counting of IHCs from 0 to 5%, location 10% from 5.01 to 10%, onwards.Pseudomonas aeruginosa was cultured from the spleen, with moderate mixed growth of Staphylococcus sp., Corynebacterium sp., Psychrobacter sp., Escherichia coli, and Enterococcus sp. isolated from a lymph node. Based on the nature of the bacterial isolates and histopathology (vasculitis and pneumonia), the Pseudomonas aeruginosa was considered significant. The lack of more significant growth from the lung was attributed to antemortem antimicrobial administration. Molecular studies of pooled tissues  [T. gondii.The animal presented for necropsy in moderate body and good post mortem condition. Morphologic diagnoses included marked bronchopneumonia, with transmural vasculitis and atelectasis, multifocal necrotizing adrenocortical adenitis with intralesional inclusions consistent with phocid herpesvirus infection, hepatocellular hemosiderosis, splenic extramedullary hematopoiesis, and renal congestion. No bacteria were recovered from the lung, but light d liver)  did not Ultrastructural evaluation of the organ of Corti revealed selective IHC loss throughout the cochlear spiral . While tThe left cochlea was better dissected and exposed than the right. The cochlear length was 27.19 mm. In the left ear, because the basilar membrane was artefactually folded, the sensory cells of the organ of Corti from the hook region (88.6% to 99% from the apex) could not be assessed. However, the rest of the cochlea was well preserved, with some signs of post-mortem decomposition due to delay between the death of the individual and the fixation of the inner ear. The number of IHCs present and absent were counted every 5% of the cochlear length. There was little loss of IHCs in the apical region, up to 35% from the apex, and an increasing trend of IHC loss towards the base of up to 84.6% loss of IHCs at 80 to 85% of the apex . The expThe right cochlea was well preserved, especially in the region of the apical and middle turns. However, there was a dissection and processing artifact that hampered the ultrastructural evaluation of the reticular lamina of the sensory epithelium in the majority of the basal turn. In those locations where the organ of Corti was visible, there was a comparable distribution of IHCs loss as in the left cochlea, while the OHCs appeared morphologically intact. However, as the regions of the base where the sensory cells were visible were limited, there was insufficient exposure of the right ear to confirm comparable severity in the bilateral loss of IHCs.Cochlear ultrastructural analysis of a neonate harbor seal showed an extremely rare pattern of selective loss of IHCs, which was particularly severe in the basal turn . Since tIn most cases of sensorineural hearing loss in terrestrial mammals, either due to noise exposure , ototoxiConversely, systemic administration of the anti-neoplastic drug carboplatin in the chinchilla, damages IHCs, leaving the OHCs morphologically intact . In otheHerpesviridae. Since this seal pup had lesions consistent with phocid herpesvirus infection, we considered the possibility that the ultrastructural features found in the inner ear of this individual were correlated with herpesvirus infection. However, selective IHC loss was not previously described with a congenital viral infection, making a viral etiology unlikely.Many cases of congenital hearing loss are due to viral infections during different stages of fetal development (see review by Karimi-Boroujeni and colleagues ). AlthouSelective IHC loss was reported in the Bronx waltzer mutant mice (bv/bv), where the IHCs were either absent or abnormally haired, but the OHCs appeared normal . SelectiConnexin 26 mutation (encoded by the GJB2 gene) is considered the most common cause for non-syndromic hereditary deafness ,42. In tAuditory neuropathy spectrum disorder refers to several hearing dysfunctions characterized by compromised signal processing along the auditory nerve or by deficient transmission of this signal to the auditory nerve by the presynaptic IHCs with normal function of OHCs (see review by De Siati and colleagues ). There On initial presentation to the MMR, this harbor seal neonate was not deemed premature. In addition, since the dissection of the cochlea prepared for SEM was optimized to image the sensory cells of the organ of Corti, it was not possible to evaluate if there was a degeneration of type I afferent neurons, precluding evaluation for potential auditory neuropathy spectrum disorder.In summary, based on the review of human and laboratory studies, there are no apparent precedents that may account for the pattern of IHC loss observed in the harbor seal of our study. However, it is possible that harbor seals have unique hereditary diseases, distinct to humans and reported rodent models.Since the disposition of the first sensory cells in the extreme hook in mammals can be variable , the finLosses of up to 70% of IHCs and 50% of cochlear neurons were associated with a moderate elevation of hearing threshold (by an average of 20 dB SPL) in Bronx waltzer mutant mice . As a reThis study highlights the importance to have baseline knowledge on “natural” congenital malformation of the hearing apparatus of marine mammals to be able to further differentiate from potential damage caused by exposure to factors (including noise) that the individuals might encounter during their lifetime.This is the first study to report a case of selective IHC loss in a neonate marine mammal, likely congenital.


In this study, we determined the aetiologies of profound bilateral sensorineural hearing loss (pbSNHL) with a view to provide the relevant data needed for preventive and therapeutic intervention among children in Ekiti State, South Western Nigeria.medical records of children with pbSNHL seen over a ten-year period were analysed.in all, records of 142 children with pbSNHL were analysed. The results showed spectrum of aetiologies that were similar to those reported decades ago with the 'unknown' assuming a recurring decimal. Of the known (acquired) cases, measles takes up the largest 'chunk' with a prevalence of 45.8%. Twenty-eight (32.2%) of those with febrile illnesses had treated their fever with ototoxic antibiotics. Late diagnosis was characteristic of the pbSNHL.this study shows that there hasn't been any significant shift in the common causes of pbSNHL. Of great concern is the persistence of the 'unknown' causes which assumes a recurring decimal in this and previous studies. Also worrisome is the high prevalence of measles-induced pbSNHL despite the availability of anti-measles vaccine. We therefore suggest effective immunization against measles and other vaccine-preventable causes of pbSNHL. The need for intensive research on the unknown causes of pbSNHL is hereby stressed. Also recommended is routine hearing assessment for survivors of febrile conditions so as to detect, if any, hearing loss promptly. Ibekwe noted that up to 500,000 children in our environment are profoundly deaf at the time of their report [Profound bilateral sensorineural hearing loss (pbSNHL) in children is an otologic tragedy. Apart from being a hidden disability which makes early diagnosis difficult, the condition is largely irreversible and without rehabilitation, the victim risks being deaf and dumb. In Nigeria, the affected children often suffer untold hardship, negligence and isolation. Sometimes, the grown-up victims become destitutes and resort to street begging. Even those who are institutionalized in the school for the deaf are not fully integrated with members of the society. Citing Holborow r report . Smith ar report . In Eastr report .et al. found: unknown 34.8%; febrile illness 18.3%; measles 13.9%; meningitis 8.7%; mumps 6.9% and severe birth asphyxia 4.3% as the main differential diagnosis of profound bilateral sensorineural hearing loss [In his own report, James Coplan said, 1 child per 1000 is born deaf and additional 2 are deafened during childhood  while Naet al. the cause of hearing loss is often understood from medical history [The aetiologies of sensorineural hearing loss could be identified prenatally. According to Glenn Isaacson, advances in the field of antenatal diagnosis have made possible the detection of profound sensorineural hearing loss prior to birth . Also, t history . Also no history . Thus, hA major challenge to accurate diagnosis of pbSNHL in our own setting however, is failure to recall all the relevant diagnostic information. This, often, is due to late presentation which has been attributed to failure of parents to seek early otolaryngologic help for their deaf children . Also, aStudy setting: this study was conducted at the otolaryngological and paediatric clinics of two tertiary hospitals in South Western Nigeria between January 2009 to December 2018. The hospitals provide specialist care for the host community and neighbouring states. Study design: children diagnosed with profound bilateral sensorineural hearing loss in otolaryngologic and paediatric clinics were retrospectively studied for the causes of their hearing loss. The tools of investigations were records of their illnesses retrieved from the central and peripheral medical information units of our hospitals. Information retrieved were socio-demographic data of parents/guardians, immunization status of the children, age of speech acquisition, onset of hearing loss, onset of speech loss, past medical history, pregnancy and birth history, records of otoscopic examinations, audiological assessment , otoacoustic emission (OAE), auditory brainstem response (ABR) and treatments previously received.The degree of hearing loss was classified based on the World Health Organsation as mild (26 - 40db), moderate (41 - 60db), severe (61 - 80db) and profound (>80db). Exclusion criteria: excluded were cases of isolated conductive hearing loss, unilateral hearing loss, mild or moderate hearing loss. Ethical considerations: the study was approved by the institution's ethics and research committee. Data analysis: the data generated were entered into personal computer and simple descriptive statistics was performed using SPSS version 14.In all, records of 142 children were analysed. They were aged 6 to 15 years. There were 73 males and 69 females , giving Profound bilateral sensorineural hearing loss (pbSNHL) is an otologic tragedy. The condition constitutes huge educational, social and economic liability for individual, families and the society at large. The present study focused on the aetiologies and distribution of pbSNHL among children in Ekiti State, South Western Nigeria. The results showed a spectrum of aetiologies similar to those reported by other scholars decades ago in the West Africa subregion ,11-13. TRanking highest among the known causes of pbSNHL in this study was measles with a prevalence of 45.8%. This was recorded in some other studies . HoweverNext in ranking among the acquired causes of pbSNHL in this study is meningitis. Our records showed that meningitis was responsible for 17 (12.0%) of the cases in this study. This is proportionally similar to, though numerically different from the 33 (11%) of the 298 profoundly deaf children recorded by Ijaduola in Lagos, Nigeria. Further analysis of our own series showed that: in 23.5% of the meningitic cases, chronic suppurative otitis media (CSOM) was the focus of infection. Although, there was no active ear disease at the time of review, this finding underscores the need to pay close attention to CSOM in children as studies have shown that it could be due to parental negligence. As noted by Okafor, chronic suppurative otitis media is a complication, often due to negligence, of the acute variety. As he put it, acute otitis media are often delayed while looking around for a local remedy . Also, tet al. malaria infection may cause deafness either by local action with microvascular changes in the end arteries of the cochlea or in a general way by lowering resistance to disease and thus enhancing the adverse effects of other infections. Although, high fever which is characteristics of most malaria infection in children, may damage the sensitive hair cells of the inner ear, the clogging of inner ear micro vessels similar to what is suggested in sickle-cell induced SNHL seems to be more relevant. This is supported by finding pbSNH in 6.3% of patients with sickle cell disease in this and bilateral profound sudden sensorineural hearing loss in a child with sickle cell anaemia in Mace et al. [Surprisingly, malaria, a very common cause of febrile illness in Nigeria was responsible for only 14 (9.9%) of the cases in this study. This finding is at variance with the 26 (41.3%) reported by Ibekwe [e et al.  studies.e et al. -24. Furte et al. . Overalle et al. , hence bMumps was recorded in 11 (7.7%) of the cases in this series. The most common manifestation was bilateral parotitis. This might blur a co-existing sensorineural hearing loss. A high index of suspicion of otologic involvement is therefore important to clinch a diagnosis. Birth asphyxia (BA) and severe neonatal jaundice (SNNJ) were non-infectious disorders recorded as causes of pbSNHL in this study. They contributed 2.1% and 1.4% prevalence rate respectively. They were diagnosed based on data from pregnancy and birth records. Although, advances in the field of antenatal diagnosis have made possible the detection of profound sensorineural hearing loss prior to birth , such waAnother major finding in this study is the abuse of ototoxic antibiotics by patients with infection-induced pbSNHL. Some patients with measles, meningitis and other febrile illnesses were noted to have used and abused ototoxic antibiotics without doctors` prescription. In such circumstances, it becomes a diagnostic conundrum to differentiate between ototoxicity and the febrile causes of the auditory shutdown. Ignorance, poverty and the ease with which antibiotics can be bought over the counter without appropriate prescription have been identified as the main cause of antibiotic abuse in our own setting. Health education and improved socio-economic wellbeing are therefore important to prevent pbSNHL caused by the abuse of ototoxic antibiotics. Periodic serum assay of ototoxic antibiotics used in those on hospital admission is equally important to keep the drugs within the safety margin. Also, there is the need for the government agency to enforce the rules guiding the sales of on the counter antibiotics among the general public.Important to note in this study is the late diagnosis of pbSNHL. As observed, the patients were seen between 2-7 years after the onset of the pbSNHL. Although, this could be attributed to poor access to otolaryngologists, the failure to seek early otolaryngologic help is a major contributing factor. According to Ijaduola, one reason for late diagnosis is because mothers delay seeking help; they wait until their children have failed to acquire speech and even when speech has failed to develop, they delay further with various excuses ranging from having late starters in their family or the child having “only tongue tie” . Given tThis study shows that there hasn't been any significant shift in the common causes of pbSNHL. Of great concern is the persistence of the 'unknown' causes which assumes a recurring decimal in this and previous studies. Also worrisome is the high prevalence of measles-induced pbSNHL despite the availability of anti-measles vaccine. We therefore suggest effective immunization against measles and other vaccine-preventable causes of pbSNHL. The need for intensive research on the unknown causes of pbSNHL is hereby stressed. Also recommended is routine hearing assessment for survivors of febrile conditions so as to detect, if any, hearing loss promptly.Aetiologies of profound bilateral sensorineural hearing loss could be congenital or acquired;Some of the acquired varieties can be prevented by immunization.Chronic suppurative otitis media is a risk factor in profound bilateral sensorineural hearing loss;Need for routine hearing assessment of survivors of febrile condition.


Endothelial cell dysfunction is an essential pathophysiological feature of preeclampsia (PE). It has been reported that cathepsin C is upregulated in the maternal vascular endothelium of PE patients. The excessive activation of p38 MAPK leads to various diseases, including PE. NF-κB pathway can promote uteroplacental dysfunction, endothelial stress and development of PE. Moreover, it has been verified that cathepsin C can activate p38 MAPK/NF-κB pathway. In the present work, hypoxia/reoxygenation (H/R) injury model of HUVECs was established to discuss the biological functions of cathepsin C in endothelial cell dysfunction and to elucidate the underlying molecular mechanism. The correlation between cathepsin C and p38 MAPK/NF-κB pathway in H/R-stimulated HUVECs as well as the effects of cathepsin C and p38 MAPK/NF-κB pathway on viability, apoptosis, invasion, in vitro angiogenesis of HUVECs and oxidative stress were assessed. The results revealed that H/R injury elevated cathepsin C expression and activated p38 MAPK/NF-κB pathway in HUVECs and cathepsin C knockdown inhibited the activity of p38 MAPK/NF-κB pathway in H/R-stimulated HUVECs. Downregulation of cathepsin C improved viability, inhibited apoptosis and enhanced invasion of H/R-stimulated HUVECs. In addition, downregulation of cathepsin C alleviated oxidative stress and induced stronger HUVEC angiogenesis in vitro. Furthermore, the protective effects of cathepsin C knockdown against endothelial cell dysfunction were reversed by p38 MAPK activator anisomycin. In other words, downregulation of cathepsin C could improve HUVEC viability and enhance anti-apoptotic capacity, anti-oxidative capability, invasive ability, as well as angiogenic potential of H/R-stimulated HUVECs by repressing p38 MAPK/NF-κB pathway. Preeclampsia (PE) is a multisystem progressive disease unique to pregnancy . It is cCathepsin C (CatC), also known as dipeptidyl peptidase I (DPPI), is one of the cathepsins with lysosomal exo-cysteine protease activity . Cathepsp38 mitogen activated protein kinase (p38 MAPK) is a highly conserved member of the serine/threonine MAPK family whose primary function is to transmit extracellular signals to the cells . p38 MAPNuclear factor-kappaB (NF-κB), a common nuclear transcription factor, is widely present in the cytoplasm, and acts as a signaling pathway when cells are stimulated . As a clTo sum up, the correlation between cathepsin C and p38 MAPK/NF-κB pathway in H/R-stimulated HUVECs as well as the effects of cathepsin C and p38 MAPK/NF-κB pathway on viability, apoptosis, invasion, in vitro angiogenesis of HUVECs and oxidative stress were assessed, aiming to discuss the biological functions of cathepsin C in endothelial cell dysfunction and to elucidate the underlying molecular mechanism.2.Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection  and maintained in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum  and 1% penicillin/streptomycin in a humidified incubator at 37°C with 5% COCathepsin C siRNA and the control siRNA were obtained from GenePharma . For in vitro transfection, HUVECs were transfected with si-cathepsin C or si-NC using Lipofectamine 3000  for 48 h following the manufacturer’s instructions.To investigate the specific effects of p38 MAPK pathway, HUVECs were pre-incubated with or without p38 MAPK activator anisomycin  for 30 min.2 and 95% N2 in a humidified incubator for 8 h and then replaced with a fresh medium. Afterward, HUVECs were cultured under normal growth conditions for 16 h of reoxygenation [To establish H/R injury model of HUVECs, HUVECs were cultured in preconditioned hypoxic medium under 5% COgenation .4 cells/well) for 24 h. Then, CCK-8 solution  was added into each well, and incubated for another 4 h. The absorbance was measured at 450 nm with a microplate reader .The viability of the HUVECs was measured by CCK-8 assay. In short, HUVECs were cultured in 96-well plates (1 × 106) were harvested and then washed twice with ice-cold PBS. Then, HUVECs were resuspended in 400 μl of 1× binding buffer, and cell suspension was incubated with Annexin V and PI in the dark for 15 min at room temperature. Next, HUVECs were assessed by a FACS flow cytometer .The proportions of apoptotic cells were determined using FITC Annexin V Apoptosis Detection Kit . Briefly, HUVECs , and cell supernatant of lysates was collected by centrifugation. Levels of MDA and SOD were analyzed using MDA assay kit and SOD assay kit .HUVECs were resuspended in a serum-free medium and seeded onto the upper chamber of transwell chambers  precoated Matrigel . Next, FBS-containing DMEM was placed to the lower chamber. After 24 h incubation, the invaded cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet  for 30 min. The penetrated cells were photographed and counted under an inverted microscope .4 cells/well) into 96-well plates precoated with 50 μl Matrigel . After 24 h of incubation, tube formation was observed and photographed under an inverted microscope . Quantitative analysis of tubules was performed using AngioSys 2.0 image analysis software .HUVECs were trypsinized, resuspended with a serum-free medium and seeded  following the manufacturer’s instructions. 1 µg RNA was reversely transcribed into cDNA using a reverse transcription kit . Next, PCR reactions were performed on ABI 7500 quantitative PCR instrument using SYBR Master Mixture . The PCR conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. The primers were as following: cathepsin C, forward: 5′- CCAACTGCACCTATCTTGACC −3′ and reverse: 5′- AAGGCAAACCACTTGTAGTCATT −3′; GAPDH, forward: 5′- GAAGGTGAAGGTCGGAGTC −3′ and reverse: 5′- GAAGATGGTGATGGGATTTC −3′. The relative expression of mRNA was normalized to GAPDH and calculated using 2HUVECs were lysed in ice-cold RIPA lysis buffer , and protein concentration was measured by BCA Protein Assay Kit . Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Next, membranes were blocked with 5% BSA for 1 h at room temperature and then incubated overnight 4°C with the primary antibodies as follows: cathepsin C , p-p38 MAPK , p38 MAPK , p-NF-κB p65 , NF-κB p65 , p-IκBα , IκBα , Bcl-2 , Bax , Birc5 , Cleaved PARP , PARP  and GAPDH . On the second day, the membranes were incubated with the corresponding secondary antibody  for 1.5 h at room temperature. Protein blots were visualized using electrochemiluminescence  method and analyzed by a Bio-Rad imaging system .Each experiment is performed in triplicate. Experimental data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test using GraphPad Prism 7.00 and expressed as mean ± standard deviation. Differences in statistical significance were set at p < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001The biological roles of cathepsin C and p38 MAPK/NF-κB pathway in H/R injury model of HUVECs were evaluated. H/R injury enhanced cathepsin C expression in HUVECs ). BesideTo elucidate the correlation between cathepsin C and p38 MAPK/NF-κB pathway, H/R-stimulated HUVECs were transfected with si-cathepsin C-1 or si-cathepsin C-2, and then the activity of p38 MAPK/NF-κB pathway was assessed. Transfection with si-cathepsin C-1 or si-cathepsin C-2 distinctly downregulated cathepsin C expression ). Due toH/R injury led to decreased HUVEC viability and increased apoptosis of HUVECs. Downregulation of cathepsin C improved the viability of H/R-stimulated HUVECs, which was partially abrogated by treatment with p38 MAPK activator anisomycin ). MoreovH/R injury increased the levels of MDA, OXSR1 and Nitrotyrosine as well as decreased the levels of SOD and p-eNOS in HUVECs. Downregulation of cathepsin C reduced the levels of MDA, OXSR1 and Nitrotyrosine as well as elevated the levels of SOD and p-eNOS in H/R-stimulated HUVECs. Furthermore, anisomycin treatment reversed the regulating effects of cathepsin C knockdown on the levels of oxidative stress associated markers ). In sumAttenuated invasion of HUVECs was observed in H/R-stimulated HUVECs. Downregulation of cathepsin C enhanced invasion of H/R-stimulated HUVECs and the promoting effect of cathepsin C knockdown on invasion of HUVECs was partly abolished by anisomycin treatment ).FigureResults of tube formation assay revealed that H/R injury resulted in a week in vitro angiogenesis in HUVECs. Downregulation of cathepsin C strengthened in vitro angiogenesis in H/R-stimulated HUVECs. The enhancing effect of cathepsin C knockdown on HUVEC angiogenesis in vitro was partially abrogated by anisomycin treatment ).FigureIt has been demonstrated that the main causes of PE include abnormal remodeling of spiral arteries in placenta, placental defects, oxidative stress at maternal–fetal interface, inflammation in maternal circulation and disturbance of angiogenesis. These events result in a systemic and diffuse endothelial cell dysfunction, an essential pathophysiological feature of PE ,7.Cathepsin C is a lysosomal cysteine protease belonging to the papain superfamily . Catheps2O2 treatment could promote p38 MAPK phosphorylation in HUVECs, while p38 MAPK inhibitor could restrain H2O2-induced apoptosis and alleviate PE-related endothelial cell injury. Kim et al. [Previous reports indicate that p38 MAPK/NF-κB signaling pathway plays a vital role in PE progression. Zhao et al.  discoverm et al.  report tm et al. ,33. In tTo conclude, downregulation of cathepsin C improved HUVEC viability and enhanced anti-apoptotic capacity, anti-oxidative capability, invasive ability, as well as angiogenic potential of H/R-stimulated HUVECs by repressing p38 MAPK/NF-κB pathway. The study on the protective mechanism of cathepsin C knockdown against endothelial cell dysfunction may suggest a novel promising approach for PE therapies.


COL2A1) and Aggrecan (ACAN) at the gene level. Furthermore, AGEs inhibited the expression of SRY-related high mobility group-box gene 9 (SOX-9), promoting the expression of COL2A1 and ACAN. COL2A1, ACAN, and SOX-9 in chondrocytes were significantly elevated by treatment with Etomidate alone. Consistently, Etomidate ameliorated AGEs-induced downregulation of COL2A1, ACAN, and SOX-9 in a dose-dependent manner. Importantly, we found that knockdown of SOX-9 eliminated the beneficial effects of Etomidate against AGEs-induced decrease in COL2A1 and ACAN genes. Based on these findings, we demonstrated that Etomidate could ameliorate AGEs-induced reduction of extracellular matrix gene expression in chondrocytes by upregulating SOX-9.Osteoarthritis (OA) is a rheumatic disease common in the elderly. AGEs are the end products of glycation reactions and play an important role in the development of OA. Etomidate is a general anesthesia-inducing agent recently reported to exert significant anti-inflammatory effects. The present study aims to explore the protective effect of Etomidate against advanced glycation end-products (AGEs)-induced reduction of extracellular matrix gene expression in chondrocytes. In the present study, we found that AGEs significantly reduced the expression of Collagen II (  Osteoarthritis (OA) is a rheumatic disease with high morbidity in people over 60 years of age . It is aCOL2A1) [As the main cell type of cartilage tissues, chondrocytes are responsible for the synthesis of extracellular matrix (ECM) and provide sufficient mechanical support for the shear force and pressure produced from articular movements ,8. ECM iCOL2A1) . It is rAdvanced glycation end-products (AGEs) are the end products of glycation reactions. It has been reported that increased accumulation of AGEs in cartilage plays an important role in the pathogenesis of OA . The intSOX-9 and COL2A1 [COL2A1 in cartilage tissues was directly regulated by SOX-9 through binding with the promoter of COL2A1 [Aggrecan is another main component of ECM and the decomposition of aggrecan induced by elevated expressions of MMPs and ADAMTS, is involved in the pathogenesis of OA. SOX-9 is an important transcriptional factor involved in the regulation of developing cartilage , reported COL2A1 . Bel claf COL2A1 , which if COL2A1 . Hence, f COL2A1  and apprf COL2A1 . Etomida2 [SW1353 chondrocytes were obtained from ATCC  and cultured in DMEM supplemented with 5% FBS at 37°C and 5% CO2 . A speci2 . EtomidaHuman SW1353 chondrocytes were stimulated with Etomidate alone at 0.25, 0.5, 1.25, 2.5, 5, 25, and 50 μM for 24 hours. After treatment, media was discarded from cell cultures. 50 µL of serum-free media and 50 µL of 3--dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) solution were added into each well. After incubation for 37°C for 3 hours, 150 µL DMSO was added to dissolve the MTT formazan by wrapping the plate in foil and shaking it on an orbital shaker for 15 minutes. Absorbance was read at 590 nM to calculate cell viability.−ΔΔCt method was applied to calculate the expression of target genes, with GAPDH as a normalization gene [After isolating the total RNA from the treated human SW1353 chondrocytes using the TRIzol reagent , the RNAs were quantified and transcribed into cDNA utilizing the Revert Aid First-strand cDNA Synthesis Kit  based on the manufacturers’ instruction. Then, the SYBR system  was used to perform the real-time PCR in a 20 μL PCR reaction system. Lastly, the 2ion gene . The priAfter lysing the treated human SW1353 chondrocytes with the cell lysis buffer , the total proteins were extracted for quantification using a BCA kit  and were further loaded and run on the SDS-PAGE. Subsequently, the proteins were transferred onto the PVDF membrane, followed by being blocked with 5% nonfat milk. After washing 3 times, the membrane was incubated with primary antibodies against SOX-9  and GAPDH  at 4°C overnight, followed by being incubation with secondary antibody. Lastly, the blots were incubated with ECL solution and exposed to the Tonan exposure . The blots were further quantified using the Image J software .After washing the human SW1353 chondrocytes with PBS buffer, cells were fixed using 4% paraformaldehyde  and permeabilized with 0.1% Triton X-100 , followed by being incubation with the primary antibody against SOX-9  at room temperature for 2 hours. Subsequently, the appropriate secondary antibodies conjugated with TRITC and DAPI (a nuclear marker and the color is blue) were used to incubate with the collected cells. Lastly, the fluorescence density of each group was observed under a fluorescence microscope  .All experiments were repeated at least three times. All data were expressed as the mean ± SD and analyzed by ANOVA, followed by Turkey’s post-hoc test, using Graph Pad Prism 6.0. P less than 0.05 was considered significant .in vitro SW1353 chondrocytes model stimulated with AGEs, we examined the function of Etomidate in the expression of matrix biomarkers. We found that Etomidate mitigated AGEs-induced reduction of COL2A1 and ACAN by increasing the expression of the transcriptional factor SOX-9.Using an To screen the optimized concentrations of Etomidate for cell culture, human SW1353 chondrocytes were stimulated with Etomidate alone at 0.25, 0.5, 1.25, 2.5, 5, 25, and 50 μM, cell viability was measured using the MTT assay. Interestingly, we found that when the concentration of Etomidate was lower than 5 μM, it did not affect cell viability. However, exposure to 25, and 50 μM Etomidate resulted in lower cell viability of human SW1353 chondrocytes . TherefoCOL2A1 and ACAN were significantly elevated by the presence of Etomidate  with Etomidate. Compared to the control group, COL2A1 and ACAN were reduced by AGEs, which were upregulated by treatment with Etomidate, suggesting a beneficial effect of Etomidate on ECM degradation induced by AGEs.To evaluate the protective effect of Etomidate on ECM degradation induced by AGEs, cells were stimulated with AGEs  for 24 hours. As shown in COL2A1 and ACAN, human C28/I2 chondrocytes cell lines were used. We found that exposure to AGEs reduced the expression of both COL2A1 and ACAN in human C28/I2 chondrocytes, which were rescued by Etomidate in a dose-dependent manner .To further confirm the beneficial effects of Etomidate on the expression of We firstly evaluated the effects of Etomidate on SOX-9 in chondrocytes. The gene and protein expression levels of SOX-9 were significantly suppressed by the stimulation with AGEs . In the COL2A1 and ACAN were remarkably reduced in AGEs-challenged cells and pronouncedly elevated by treatment with Etomidate. However, the knockdown of SOX-9 abolished the effects of Etomidate against AGEs-induced reduction of COL2A1 and ACAN. These data indicate that Etomidate might protect the reduction of ECM genes through upregulating SOX-9.To further confirm that Etomidate protected the degradation of ECM through upregulating SOX-9, cells were transfected with SOX-9 siRNA. The expression of SOX-9 was significantly downregulated in the siRNA transfected cells, indicating that the SOX-9 silenced chondrocytes were successfully established . COL2A1 The development of OA is closely related to factors such as aging, heredity, development, metabolism, and trauma, with aging being one of the most important variables. Excessive development and buildup of advanced glycation end products (AGEs) are commonly generated as people get older ,26.in vitro injury model on chondrocytes, which was verified by the downregulation of COL2A1 and ACAN, indicating that the ECM degradation might be induced by the stimulation with AGEs.AGEs are the stable covalent compounds derived from the non-enzymatic glycation between proteins or lipids and glucose or other reductive monosaccharides, which is an inescapable and irreversible process in our body. It is reported that compared to other tissues, AGEs are highly expressed on cartilage tissues and are closely associated with the pathogenesis of OA . The colCOL2A1 and ACAN were dramatically elevated, showing that Etomidate could help to restore the degraded ECM caused by AGEs. In our future work, more elements will be taken into consideration to better understand the protective effect of Etomidate against ECM degradation, including measuring the expression of MMPs and detecting the production of inflammatory factors, which are reported to be involved in the pathological changes in chondrocytes induced by AGEs [After Etomidate treatment,  by AGEs . FurtherCOL2Α1 by targeting specific binding sites located in the intron of COL2Α1 [SOX-9 gene into the chondrocytes [SOX-9 is widely expressed in precursor cells of cartilage and mature chondrocytes. It is reported that it directly regulates the transcription process of f COL2Α1 . SOX-9 nf COL2Α1  claimed drocytes . In addidrocytes ,36. In tAlthough the pathological mechanism of OA is still complex, it is well known that the inflammatory response plays a critical role in the progression of OA . Pro-infin vitro cell culture studies. It should be noted that the pathological mechanism of OA is complicated and various risk factors have been involved [There are several limitations of the current study. Firstly, the major findings here are based on involved . In vivoinvolved .COL2A1 and ACAN to prevent reduction of extracellular matrix genes in chondrocytes by upregulating SOX-9.In conclusion, our data revealed that Etomidate might rescue the AGEs-induced downregulation of Click here for additional data file.


KCNQ4 gene is a common genetic factor of ADNSHL. Few studies have investigated the association between hearing impairment and the variant c.546C>G of KCNQ4. Here, we investigated the phenotype and clinical manifestations of the KCNQ4 variant. Study subjects were selected from the participants of the Taiwan Precision Medicine Initiative. In total, we enrolled 12 individuals with KCNQ4 c.546C>G carriers and 107 non-carriers, and performed pure tone audiometry (PTA) test and phenome-wide association (PheWAS) analysis for the patients. We found that c.546C>G variant was related to an increased risk of hearing loss. All patients with c.546C>G variant were aged >65 years and had sensorineural and high frequency hearing loss. Of these patients, a third (66.7%) showed moderate and progressive hearing loss, 41.7% complained of tinnitus and 16.7% complained of vertigo. Additionally, we found a significant association between KCNQ4 c.546C>G variant, aortic aneurysm, fracture of lower limb and polyneuropathy in diabetes. KCNQ4 c.546C>G is likely a potentially pathogenic variant of ADNSHL in the elderly population. Genetic counseling, annual audiogram and early assistive listening device intervention are highly recommended to prevent profound hearing impairment in this patient group.Clinical presentation is heterogeneous for autosomal dominant nonsyndromic hearing loss (ADNSHL). Variants of  Hearing loss is a common disability worldwide, involving inherited sensory defects. The global prevalence of hearing impairment in 2008 was 1.4% for children aged 5–14 years, 12.2% for males >15 years of age, and 9.8% for females >15 years of age ,4, and tWFS1, KCNQ4, COCH, and GJB2 are more common as causes of ADNSHL [KCNQ4 accounts for 9% of ADNSHL cases [KCNQ4 gene starts in the second decade and its audio profile presents high-frequency and progressive hearing loss [Current reports show that 123 genes are pathogenically related to non-syndromic HHI . HHI is f ADNSHL . Among ting loss .KCNQ4 is a voltage-activated potassium channel. Potassium recycling in the inner ear is an important process for maintaining auditory sensitivity. In the labyrinth, this process modulates how hair cells, such as the vestibular type I cell and cochlear outer hair cell, handle their responses to sensory stimulation, by exuding potassium back to the endolymph [ndolymph ,12. Spirndolymph .KCNQ4, 30 of them are inherited via missense mutation [KCNQ4; these variants are likely related to late-onset hearing loss [KCNQ4  from three carriers. The carrier for c.546C>G exhibits prelingual and profound deafness [Currently, of the 38 identified variants of mutation . There amutation . The maimutation ,17. In amutation . Anothering loss . One studeafness ,20. HoweKCNQ4 gene to post-lingual non-syndromic HHIs in a hospital-based population in Taiwan.Here, we aimed to explore the possible contribution of the c.546C>G variant of the This case-control study was conducted using data from the Taiwan Precision Medicine Initiative (TPMI), which gathered information and specimens from a convenience sample of Taiwanese volunteer participants in Taichung Veterans General Hospital (TCVGH) during the period from June 2019 to June 2020. Between June 2019 to June 2020, patients aged >18 years who visited 28 medical and surgical outpatient clinics in Taichung Veterans General Hospital (TCVGH) were invited to participate in the study. Our study cohort consisted of 32,728 patients  with genotyping information, demographics, and medical history, as well as biochemical reports, and all of the participants provided informed consent. The study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the Institutional Review Board of TCVGH (SF19153A). This case-control study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines.KCNQ4 c.546C>G variant and with audiograms were assigned to the control group. We further analyzed the association between the KCNQ4 c.546C>G variant and non-syndromic hearing loss. In the SNHL/MHL group, 12 patients had the KCNQ4 c.546C>G variant and were defined as KCNQ4 c.546C>G variant carriers, and the remaining 224 patients who did not have KCNQ4 c.546C>G variant were defined as KCNQ4 c.546C>G variant non-carriers.As illustrated in Audiometric tests were conducted with a pure tone audiometer . Pure tone bone and air conduction hearing thresholds were assessed according to standard procedures at frequencies of 250, 500, 1000, 2000, 4000, and 8000 Hz. Pure tone averages (PTA) were ciphered from frequencies of 500, 1000, 2000, and 4000 Hz. The level of the individual subject’s hearing loss was graded based on disparity levels against PTA, as normal hearing (<20 dB), mild hearing loss (21–40 dB), moderate hearing loss (41–70 dB), severe hearing loss (71–95 dB), or profound hearing loss (>95 dB). Hearing loss was typed as SNHL, mixed hearing loss, or conductive type hearing loss according to the patterns in pure-tone air- and bone-conduction thresholds. Sensorineural and mixed types of hearing loss were defined as hearing loss in this study. High-frequency hearing loss was defined as hearing loss in the range between 2000 and 8000 Hz.p values of individual phenotypes. Blue and red horizontal lines mark p values at 0.05 and 0.001. A Quantile–Quantile (Q–Q) plot was used to evaluate whether the observed distribution was different from the expected distribution under the null hypothesis .Clinical diagnoses of c.546C>G carriers and non-carriers were extracted from TCVGH electronic health records according to the diagnostic codes of the International Classification of Diseases, Ninth Revision (ICD-9). Phenotypes were detected by guiding ICD-9 codes and separated disease entities. Patients were defined to have a certain phenotype if they had the matching ICD diagnosis on one or more dates, while phenotypic controls consisted of those participants who had never matched the ICD code. The PheWAS Manhattan plot was used to correlate phenotype with genotype. Such plots revealed any significant association between different phenotypes of the variant c.546C>G. On the x-axis, we display different disease groups in different colors. The y-axis shows the Taiwan Biobank version 2 (TWBv2)  array adopted the whole-genome sequence (WGS) data from Taiwan Biobank (TWB) participants to select single nucleotide polymorphisms (SNPs) best for imputation in Han Chinese samples in Taiwan, enclosed 114,000 risk variants in 2831 unusual disease genes chosen from the ClinVar, ACMG, HGMD, GWAS Catalog, locus-specific databases and published literature . We genoKCNQ4 c.546C>G variant for hearing loss. Statistical analyses were evaluated with Windows SPSS Statistics , version 22. p values < 0.05 were considered statistically significant.The basic characteristics of hearing loss-associated variants in the TPMI were described in number and minor allele frequencies. Fisher’s exact and chi-square tests were applied to compare categorical variables between case and control groups. Univariable logistic regression was used to evaluate the odds ratio (OR) of the KCNQ4 c.546C>G was 0.61% at TCVGH, which was higher than that of the East Asian population in the GnomAD database. The MAFs of the SLC26A4 c.919-2A>G, GJB2 c.299_300del, and MT-RNR1 rs28358569 were 0.78%, 0.1%, and 4.49%, respectively, and were similarly higher than that of the East Asian population. On the other hand, the MAFs of SLC26A4 c.2168A>G and GJB2 c.235del were 0.09% and 0.59%, respectively, and were low compared with those of the East Asian population. No compound heterozygotes were observed in our cohort. These results suggested that KCNQ4 c.546C>G was the most common variant among those affected by ADNSHL in the Taiwanese population.Between June 2019 and June 2020, the TPMI identified 32,728 patients  from TCVGH with genotyping microarrays. They all had 11 identified risk variants in seven rare diseases related to hearing loss based on the TWBv2 array . The minKCNQ4 c.546C>G variant, and hypertension were all significantly higher in the case group.In this study, we identified 348 patients with available audiograms, 236 of whom had SNHL/MHLand were designated as thecase group. The 107 participants without SNHL/MHL wereassigned to thecontrol group . The demKCNQ4 c.546C>G variant  heterozygous variant  in patients aged over 70.The KCNQ4 c.546C>G and clinical diagnosis identified in the electronic health records. We found an association between KCNQ4 and aortic aneurysm , fracture of lower limb  and polyneuropathy in diabetes . The Q-Q plot of the observed versus expected p-values that showed clear enrichment of p-values in KCNQ4 c.546C>G, and we had sufficient power to detect modest association signals for diseases.p value (logistic regression) for each PheWAS signal. False discovery rate (FDR) corrected significant threshold (p < 0.001) is represented by a red line. The blue horizontal line is displayed as the significance level of p = 0.05.Refer to table for number of cases and controls per genotype, OR (95% CI), and KCNQ4 c.546C>G variant was associated with a four-fold increased risk for SNHL and high-frequency hearing loss. We showed that genetic factors likely contributed to age-related hearing loss. We also explored the associations between a hearing loss-related genetic variant and clinical phenotypes of different disease entities. Our results suggested that annual hearing check-ups could be beneficial for KCNQ4 c.546C>G carriers. Genetic evaluations might provide additional information for patients with age-related hearing loss to guide early intervention with assistive listening devices.In this hospital-based real-world study of an elderly Taiwanese population, we found that the KCNQ4 c.546C>G variant is a missense mutation (F182L), which is located in the S3 domain of KCNQ4 and is highly conserved in KCNQ4 among various species. KCNQ4 encodes a protein that constitutes a potassium channel. The potassium channel has a P-loop region forming the channel pore and six transmembrane domains. The glycine–tyrosine–glycine sequence within the P-loop encompasses the selectivity filter distinguishing potassium ions for transport needs [KCNQ4 channels in Xenopus oocytes, and measurements were performed using a two-electrode voltage clamp. The KCNQ4 outward current induced by the calcium salt, ionomycin, was significantly increased (about 1.7-fold) from the control current amplitude of +60 mV. Additionally, mutation F182L of the KCNQ4 gene was investigated in the subcellular localization of HeLa cells, which resulted in proteins with impaired trafficking, localized in perinuclear vesicles, but not localized to the membrane [The rt needs . The aniutation F2L, whichrt needs  investigutation F2L, whichmembrane . KCNQ4 c.648C>T and KCNQ4 c.1503C>A both cause protein changes  and the onset of symptoms during childhood. Su et al. [KCNQ4 and the function of the K+ channel. On the contrary, the T501T mutant results in a protein that is localized to the membrane, similar to a normal KCNQ4 protein. Therefore, the mutation T501T of the KCNQ4 gene did not affect the synthesis of KCNQ4 proteins, nor would their transportation to the cell membrane to form functional potassium channels. The role of the T501T mutant in hereditary hearing loss requires further investigation to evaluate its function in the DFNA2/KCNQ4 gene.In Taiwan, two silent mutations of uncertain clinical significance were reported. Variants such as u et al.  discoverKCNQ4 c.546C>G variant, CADD scores were taken from the “CADD” project website (version v1.5) [KCNQ4 c.546C>G variant is more likely to be stimulated and have deleterious effects based on different genomic features. A CADD Phred-scaled score of 20.9 means that the variant is potentially pathogenic, with a cutoff value of 15. According to the ACMG guidelines and the Annovar database, the KCNQ4 c.546C>G variant is classified as 2PM (pathogenic moderate) and 1PP (pathogenic supporting) evidence [KCNQ4 c.546C>G variant. Although the risk did not reach statistical significance, we did observe SNHL in multiple unrelated patients with the c.546C>G variant. Therefore, our study provided further evidence of moderate pathogenicity. Taken together, we judge the KCNQ4 c.546C>G variant as 3PM and 1PP, i.e., likely pathogenic, by ACMG guidelines.To judge the pathogenicity of the on v1.5) . The valevidence . MeanwhiKCNQ4 pathogenic variants are missense alterations, and the phenotype reflects the effect of faulty proteins in the inner ear [KCNQ4 are missense variants, affecting the P-loop protein domain, and the onset of symptoms appears during childhood [Most nner ear . Some panner ear . Currenthildhood . Howeverhildhood . We are KCNQ4, results in a higher risk of noise-induced hearing loss for noise-exposed Chinese workers [KCNQ4 gene is likely related to the complex interactions between age, environmental noise, and hearing loss. Collectively, our findings suggest that carriers of all variants of the KCNQ4 gene should avoid noise exposure to prevent further damage to the inner ear.In the elderly, age-related hearing loss is the most common sensory loss. Noise exposure, metabolic stress, and genetic propensities are causes of age-related hearing loss . Both he workers . Our finKCNQ4 variant with aortic aneurysm and diabetic polyneuropathy. An animal study in rats reported that the KCNQ4-encoded voltage-dependent potassium (Kv7.4) channel contributes to the β-adrenoceptor-mediated vasodilatation of smooth muscles in renal arteries [In the present study, the PheWAS data implied an association of the arteries . Down-rearteries . Since carteries ,30,31,32KCNQ4 variant and the fracture of lower limbs. In a prior family study, no vestibular problem was observed in families with pathogenic variants of KCNQ4 [KCNQ4 [KCNQ4 c.546C>G carriers are needed to confirm our hypothesis.In addition, this study demonstrated the correlation between the of KCNQ4 . Our res4 [KCNQ4 . Hearing4 [KCNQ4 . With a 4 [KCNQ4 . We hypoKCNQ4 c.546C>G carriers in a hospital-based population. An association of KCNQ4 c.546C>G with hearing loss in an elderly population was established. Lastly, only patients with blood tests participated in the TPMI study. The presence of selection bias is likely, underestimating minor allele frequencies.Our study has several limitations, despite being based on real-world data from patients with hearing loss. Firstly, we used the SNP array for genetic profiling. Not every variant of hearing loss was included in the probe design . TherefoKCNQ4 gene is associated with postlingual hearing loss in an elderly population in Taiwan. Genetic factors might be involved in age-related hearing loss. Our data suggested that genetic tests for the KCNQ4 c.546C>G variant are necessary for patients with age-related hearing loss or indecipherable postlingual hearing loss. Early family genetic counseling, annual audiograms, and early assistive listening device interventions are crucial to prevent profound hearing impairments in this group of patients.In summary, this hospital-based study demonstrated that the c.546C>G variant of the


Hearing loss is a major health challenge that can have severe physical, social, cognitive, economic, and emotional consequences on people’s quality of life. Currently, the modifiable factors linked to socioeconomic inequalities in hearing health are poorly understood. Therefore, an online database search  was conducted to identify literature that relates hearing loss to health inequalities as a determinant or health outcome. A total of 53 studies were selected to thematically summarize the existing literature, using a critical interpretive synthesis method, where the subjectivity of the researcher is intimately involved in providing new insights with explanatory power. The evidence provided by the literature can be summarized under four key themes: (a) There might be a vicious cycle between hearing loss and socioeconomic inequalities and lifestyle factors, (b) socioeconomic position may interact with less healthy lifestyles, which are harmful to hearing ability, (c) increasing health literacy could improve the diagnosis and prognosis of hearing loss and prevent the adverse consequences of hearing loss on people’s health, and (d) people with hearing loss might be vulnerable to receiving low-quality and less safe health care. This study uses elements from theoretical models of health inequalities to formulate a highly interpretive conceptual model for examining hearing health inequalities. This model depicts the specific mechanisms of hearing health and their evolution over time. There are many modifiable determinants of hearing loss, in several stages across an individual’s life span; tackling socioeconomic inequalities throughout the life-course could improve the population’s health, maximizing the opportunity for healthy aging.  Hearing loss involves the partial or total inability to hear sounds from one or both ears. It can be categorized as mild, moderate, severe, or profound, according to its severity. Approximately 15% of the global adult population suffers from some degree of hearing loss . Approxiour understanding of age-related hearing loss is very limited .Hearing loss is far beyond a sensory disorder, as it is associated with negative physical, social, cognitive, economic, and emotional consequences. In high-income countries, hearing loss is the third most common chronic health condition among older adults, following high blood pressure and arthritis . Neverthage-related hearing loss has similar characteristics to sensorineural hearing loss that can occur at any age. Based on that, as the knowledge on the causes of hearing loss in patients with older age increases, the need for expressions of hearing problems without specific etiology on older age, through terms such as “presbyacusis,” is likely to be diminished  provide an interpretive synthesis of the existing literature and give insight into the socioeconomic disparities in hearing health and (b) formulate a conceptual model for hearing health inequalities, which depicts the specific mechanisms for hearing health and their evolution over time.This review’s scope is broader than testing a specific research question, which could be achieved through a systematic review or scoping review. This article aims to integrate diverse forms of research evidence. To do so, the methodology of critical interpretive synthesis (CIS) is adopted . The CISsynthetic constructs or themes. These themes are then linked and supported by the relevant evidence, which is placed within its context to build a highly interpretive conceptual model , which included participants with hearing loss (with no age restrictions) and presented associations between hearing loss and health inequalities either as a determinant factor or as a health outcome. English-written articles from any country and setting were eligible for inclusion in this study. A two-stage screening process was applied: First, titles and abstracts were screened against the inclusion criteria; second, a detailed review of the potentially eligible full-texts was completed. Two reviewers were involved in the data screening process . Disagreements were resolved through discussion until a consensus was reached. If the two authors could not reach a consensus, the team of four coauthors would discuss until a consensus was reached.Quality appraisal: We assessed the methodological quality of the included studies, using criteria provided in the guidance on quality assessment components and ratings ;Data collection methods: use of psychoacoustic hearing assessment tools, which are valid and reliable.This article relies on CIS reviews’ described guidelines to ensure that the reporting is transparent and coherent . The folData extraction: A data extraction table was developed, including the following elements of the selected studies: names of authors, publication year, country, and key point(s) made by the authors and in which synthetic constructs they were applied. Four recurring concepts/themes were identified from the studies, and the literature was placed within its context, to inform the emerging research themes  is used instead of the term socioeconomic status, to refer specifically to the components of economic and social well-being; this is in line with the suggestion of SEP is linked to both childhood and adult social class positions. It includes both resource-based  and prestige-related characteristics, which refer to the individual’s rank or status in a social hierarchy. We decided to include education, occupation, income, and wealth as the selected indicators of SEP; according to the list of SEP indicators proposed by The term hearing loss instead of the term hearing impairment, which looks beyond pathology, addressing issues that interact to affect the individual’s ability to maintain as high a level of health and well-being as possible and function within society: According to the Sociopolitical Model of Disability, hearing disability is being approached through the lens of the loss or limitation of opportunities, rooted in societal barriers  may account for the higher prevalence of hearing loss among males . MoreoveAn increasing number of studies attest the fact that people who are less likely to adopt beneficial health behaviors have low health literacy. The concept of health literacy refers to one’s ability to make judgments and decisions concerning health care, disease prevention, and health promotion in their everyday lives . PreviouHealth literacy is a multidimensional construct that also refers to one’s ability to navigate the healthcare system and work out the best care for them and their ability to decide which providers they need to see . For thiMoreover, after the onset of hearing loss, individuals may face substantial disparities in accessing and using hearing health care . As a reHowever, in addition to costs, other factors, such as a low level of education and disability, also contribute to the lower uptake of hearing aids among lower socioeconomic groups . The exiAn explanation to the aforementioned paradox is that the perception of hearing ability acts as a strong predictor of hearing aid acquisition, even when financial factors are mitigated . Ng and Historically, hearing loss has primarily been conceptualized as impairment within a biomedical model and managed clinically within an isolated care model, with little consideration of comorbidities . HoweverPeople with hearing loss also face significant challenges in their communication with healthcare professionals . The comPoor communication between providers and patients can result in a variety of adverse outcomes. It affects patients’ awareness of healthy behaviors, appropriate use of health services, understanding the importance of specific management and treatment approaches and the effective transfer of health knowledge . It may fundamental causes and the wider environmental influences  that emerged from the CIS  and incoThe HHI model proposes that children born to parents from lower SEP tend to experience more health-related issues. The antibiotics used to treat a bacterial infection, especially in sick babies with a genetic predisposition, may affect their hearing health. Thus, many individuals with disabling hearing impairment are disadvantaged children who have been exposed to several risk factors during their prenatal, perinatal, or neonatal period of development or have experienced inequalities in access to screening tests . Parent The exact mechanisms behind the link between parental SEP and hearing health inequality are still unclear. Previous analyses that study this connection  suggest The consequences of hearing loss in childhood may include impairment in language skills and lower educational achievement compared with children with normal hearing . Having Having a lower educational status is also related to lower health literacy , which hHearing health inequalities in middle adulthood can then affect older adults’ retirement status and income by impacting their ability to continue working or to advance occupationally . Lastly,Also, hearing health inequalities may accumulate: The higher a person’s socioeconomic status, the better their hearing health can be throughout their life span. On the other hand, those who are persistently exposed to inadequate socioeconomic resources during their childhood and adulthood face a disproportionately higher chance of suffering from hearing loss. It is now clear how the low SEP and hearing loss form a vicious cycle, as hearing loss can be a consequence and a causal contributor to socioeconomic disparity.the cognitive and social skills that determine individuals' motivation and ability to gain access to, understand and use information in ways that promote and maintain good health  a vicious cycle between hearing loss and socioeconomic inequalities and lifestyle factors exists, (b) SEP prompts healthy or unhealthy lifestyles which affect people’s hearing ability, (c) people with hearing loss are more at risk of receiving low quality and less safe health care, and (d) increasing health literacy could improve the diagnosis and prognosis of hearing loss and prevent the adverse consequences of hearing loss on people’s health. The HHI model identifies determinants of hearing loss using a life-course approach, which aims to shine new light on the current hearing health research debates. This model can be used as a tool for preventing, identifying, and managing hearing health inequalities and for policy formulation to reduce hearing loss risks.There are significant limitations in terms of the type and quality of existing published literature on the topic. The field of hearing health inequalities is an emerging research field, and most of the evidence cited in the article stems from cross-sectional studies which demonstrate associations. However, although the CIS approach demands attention to study design, it also allows for the inclusion of less methodologically robust papers as long as they are essential in their theoretical contribution . Thus, iauthorial voice in examining a network of synthetic constructs (themes) and the relationships between them and places a great deal of emphasis on the researcher’s interpretation e needed .preventing involves acknowledging those who have limited access to hearing health aid and whose working and living conditions put them more at risk of suffering from poor hearing health. Several primary prevention activities—such as improved prenatal, perinatal, or neonatal care; universal vaccination programs; and antibiotic stewardship practices—can be implemented to reduce the incidence of hearing loss from preconception to adulthood. In addition, secondary and tertiary prevention activities—such as prompt intervention, fitting of hearing devices , and training in sign language and special or inclusive education—are needed and should be actively encouraged  socioeconomic inequalities and hearing loss, (b) hearing loss and comorbidity, and (c) hearing impairment and related socioeconomic disparities, this review is the first to examine the mechanisms and explain the relationship between socioeconomic inequalities and hearing health in a life-course perspective, synthesizing the existing evidence.Apart from the physiological and pathological aging of sense organs, the HHI model provides a visual representation of several modifiable determinants of hearing loss in distinct life stages, supporting the argument that a substantial proportion of hearing loss in older adulthood is preventable, treatable, and even postponable. Understanding that hearing deterioration occurs over a prolonged period of time is an essential step in addressing the burden of hearing loss not within an isolated model of care which focuses on the acquired hearing loss among older adults, but as a lifelong process.Although reducing hearing health inequalities is a complex ambition, the life-course approach can lead to the development of appropriate interventions and public health strategies that can have significant health policy and practice implications. The management of hearing loss must involve integrated care, which entails considering an individual’s entire health profile and providing ongoing support for each person’s adaptation and self-management. In that way, we will ensure that a more substantial proportion of the population receives high-quality health care and maximizes the opportunity for healthy aging.Click here for additional data file.Supplemental material, sj-pdf-1-tia-10.1177_23312165211002963 for Conceptual Model of Hearing Health Inequalities (HHI Model): A Critical Interpretive Synthesis by Dialechti Tsimpida, Evangelos Kontopantelis, Darren M. Ashcroft and Maria Panagioti in Trends in Hearing