Single-sided deafness (SSD) and asymmetric hearing loss (AHL) have recently been proposed as a new indication for cochlear implantation. There is still no recommended treatment for these hearing deficits, and most options considered rely on the transfer of sound from the poor ear to the better ear, using Contralateral Routing of the Signal (CROS) hearing aids or bone conduction (BC) devices. In contrast, cochlear implantation allows the poor ear to be stimulated and binaural hearing abilities to be partially restored. Indeed, most recently published studies have reported an improvement in the spatial localisation of an incoming sound and better speech recognition in noisy environments after cochlear implantation in SSD/AHL subjects. It also provides consistent relief of tinnitus when associated. These encouraging hearing outcomes raise the question of the cost-utility of this expensive treatment in an extended indication.The final endpoint of this national multicentre study is to determine the incremental cost-utility ratio (ICUR) of cochlear implantation in comparison to the current standard of care in France through simple observation, using a randomised controlled trial. Firstly, the study comprises a prospective and descriptive part, where 150 SSD/AHL subjects try CROS hearing aids and a BC device for three weeks each. Secondly, the choice is made between CROS hearing aids, BC implanted device and cochlear implantation. Hearing outcomes and quality of life measurements are described after 6 months for the subjects who chose CROS, BC or declined any option. The subjects who opt for cochlear implantation are randomised between one group where the cochlear implant is inserted without delay and one group of simple initial observation. Hearing outcomes and quality of life measurements are compared after 6 months.The present study was designed to assess the efficiency of cochlear implantation in SSD/AHL. A favourable cost-utility ratio in this extended indication would strengthen the promising clinical results and justify a reimbursement by the health insurance. The efficiency of other options  will also be described.ClinicalTrials.gov , the 29th July 2014 under the n°NCT02204618.This research has been registered in  Treatment of single-sided deafness (SSD) and asymmetric hearing loss (AHL) is a recurrent focus of interest in contemporary otorhinolaryngology because their consequences were globally underestimated until the late 1970’s, by both the general public and professionals of auditory disorders, assuming that the good ear would compensate the deficit of the poor ear. Although the level of handicap generated varies, SSD and AHL are actually disabling conditions in both children and adults. In the paediatric population, several case-control studies have demonstrated the impact of such hearing deficits on cognitive functions  and schoFrom a physiological point of view, the deficit produced by SSD and AHL is first related to the disruption of binaural hearing and its binaural advantages. Indeed, in normal hearing subjects, an incoming sound arrives with differences between the two ears in terms of timing  and amplitude , depending on its spatial position and spectral content  hearing aids system are considered to be the standard care in several countries, French authorities have not yet recommended any official guideline. Clinical experience shows that some patients adapt to this condition or simply use a conventional hearing aid for the better ear in cases of AHL , but recAlthough cochlear implantation is traditionally used to treat bilateral profound deafness, it has recently been proposed as a new treatment option in SSD/AHL, specifically because it ensures the stimulation of the poor ear. Bilateral auditory input can therefore be restored in those patients, which is a major difference to CROS hearing aids and BC devices and is the theoretical prerequisite for an improvement of binaural hearing. Furthermore, the beneficial effect of cochlear implant on tinnitus has been consistently demonstrated in numerous studies on bilateral profound deafness  and can In a prospective pilot study, Arndt et al.  showed tAll these differences between the possible treatments of SSD/AHL probably make cochlear implantation an option that is set apart from the others. Therefore in the recent consensus papers, it is not considered as an alternative among different options, but as a second-line treatment, in the event CROS or BAHA systems fail.The present article describes a prospective multicentre randomised controlled trial initiated in France, which allows the subjects to choose their treatment option . The decision was made to randomise CI versus observation after a consecutive trial of CROS hearing aids and BAHA on a headband (Baha® or Ponto®) see Fig. . EventuaThe main objective of this study is to assess the efficiency of CI in SSD/AHL, compared to the current standard of care in France (observation), after failure of CROS and BC trials, using a cost-utility analysis.assess the changes in quality of life and binaural hearing performance for each successive intervention ;search for associations between changes in hearing performance scores and changes in quality of life for each intervention ;assess the evolution of tinnitus intensity and the level of annoyance for each intervention .This study also aims to:The cost-utility ratio of other treatment options  will be determined. Hearing outcomes of each option  will be described using tests for speech recognition in noise and for horizontal localisation.The affordability to the French Health Insurance of the widespread implementation of CI in SSD/AHL will be assessed.This prospective multicentre national study combines two major steps:- a prospective, descriptive observational cohort study, with a 6-month follow-up for SSD/AHL adult subjects treated by CROS, SSD/AHL subjects treated by an implanted bone conduction device (Baha or Ponto), and adult patients who decline all the options.- and an open-label randomised controlled clinical trial for adults with SSD or AHL after failure of both CROS and BC systems, in two parallel groups: Observation for 6 months versus Cochlear implantation.Participants are recruited in 7 tertiary referral centres in France. Once the inclusion/criteria are checked and the consent from the subject is obtained by one of the investigators, a reference evaluation is made for quality of life, binaural hearing performances  and tinnitus severity. Then, CROS hearing aids and a BC device on a headband are successively tried by the subjects for three weeks each. The evaluation of quality of life, binaural hearing abilities, and tinnitus severity is repeated after the first trial (CROS) and the second trial (BC). At this point, the subject may choose the treatment he/she is willing to continue. If one of the trials (CROS or BC) is considered to be successful and the subject is willing to continue with the corresponding device, it is then provided and a new evaluation is performed 6 months later. If none of the trials is considered to be successful and the subject does not want any other treatment, then only a new evaluation is performed 6 months later. If none of the trials is considered to be successful and the subject wants treatment by cochlear implantation, the second major step of the study begins with the randomisation procedure.Subjects who chose a cochlear implant are thus randomised between two arms: in the first one (“CI” arm), a cochlear implantation is performed within a month and a new evaluation on quality of life, binaural hearing performances, and tinnitus severity is planned 6 months after CI activation. Subjects of the second arm (“observation” arm) simply undergo the new evaluation 6 months after randomisation, without any intercurrent treatment for SSD/AHL. For obvious ethical reasons, subjects of the second arm benefit from a cochlear implantation after this 6-month evaluation.Data from all the subjects included will be anonymised, collected and analysed. If a serious adverse event does not allow the end of the protocol to be reached and/or if a subject withdraws his/her consent, the data which were collected until this event will also be taken into account in the final analysis. If a serious adverse event occurs, it will be reported to the coordinating centre  and to the “Agence Nationale de Sécurité du Médicament” .Age eighteen years or older.French speaking.French social security affiliation.Ability and willingness to participate in all the assessments, with written consent to the protocol.Poorer ear: severe-to-profound hearing loss measured using pure tone audiometry with pure-tone average  ≥ 70 dB, confirmed by auditory brainstem responses, with minimal benefit of a conventional hearing aid. In order to account for the heterogeneity of this population, no selection was applied on the duration of deafness or the severity of the tinnitus.Better ear: normal hearing thresholds (≤20 dB HL) to moderate hearing loss (PTA ≤ 60 dB HL).Retrocochlear pathology .Major cochlear ossification or malformation which could prevent the full insertion of a cochlear implant, controlled on CT scan and MRI of temporal bones.Subjects under juridical protections or tutelage measure.Neurological or psychiatric disorder.Previous treatment with CROS, BC or CI.Medical condition that contraindicates CI surgery.Randomisation of CI versus observation was based on a 1:1 ratio and was stratified per centre. The allocation sequence was randomly generated by a computer , providing a sequence of treatments randomly permuted in blocks of varying size , 6. TherThe sample size was calculated based on the expected number of failures of CROS hearing aids, BC trials and on the expected improvement of quality of life in cochlear implanted subjects with SSD/AHL. In a pilot study performed in our centre (unpublished), 40 to 50% of SSD/AHL subjects declined both CROS and BC devices after a three-week trial. This rate is consistent with previous studies performed on BC implantation after a headband trial . An impThe purpose of this study is to assess the cost-utility ratio of CI in subjects with SSD/AHL after failure of more conventional treatments. Therefore, every subject will successively try CROS hearing aids and a BC device placed on a headband, for three weeks each. The baseline assessment of each trial relies on auditory measures  and quality of life questionnaires. Once each trial has been performed, the subject will decide which treatment he/she opts for. This choice is mainly based on the subjective feelings of the subject after the two trials, but also guided by the auditory outcomes obtained with each device, and the counselling of the physician. The programme provides all costs directly related to the treatment, i.e. the cost of the medical device and the following fitting sessions.CI may restore binaural hearing to some extent but with a significant inter-individual variability;its effect is more consistent on ipsilateral tinnitus when associated with unilateral profound deafness;The impact of CI in this indication has mainly been studied and demonstrated in subjects where the duration of profound deafness was less than 10 years.The information regarding the results which may be expected from a cochlear implant is critical. A CI is the only device which cannot be herein tried before the decision is made and its choice intrinsically relies on hedging a bet. Across the seven centres, this information is standardised through several elements indicated to the subjects willing to try a CI, based on the literature on cochlear implantation outcomes in SSD/AHL , 34, 35:Once this information has been delivered, each subject makes a final choice among four options: abstention, CROS hearing aids, Bone-Anchored hearing aid (Baha or Ponto), or CI. Subjects who chose CI are randomised between two arms (“CI” arm versus “observation” arm) and re-assessed and compared after 6 months their choice has been implemented, and the results of the two arms are compared. Subjects who chose abstention, CROS or BAHA are re-assessed 6 months after their choice has been implemented, and the results of each group are described.Subjects in this group who do not experience any benefit from the CROS hearing aid trial or from the BAHA trial, and choose no specific treatment for SSD/AHL.In CROS systems, a remote microphone which is placed on the poorer ear receives the surrounding incoming signal and transmits it to the second hearing aid on the better ear, using a wireless link. The second hearing aid delivers the signal using air conduction and can also apply an additional amplification in case of hearing loss in the better ear.A Phonak CROS system will be used for the trials and in case the subject eventually opts for this type of rehabilitation . The hearing aids will be fitted by an experienced audiologist, using the fitting software provided by the company (Phonak Target™ 3.0).Bone-anchored hearing aids transmit the auditory signal received by the processor placed behind the poor ear to the better ear through bone conduction of the skull. The programme does not provide any additional hearing aid in the better ear if the subject has AHL.Baha BP 110® (Cochlear Ltd) and Ponto® (Oticon) will be used on a headband for the trial period and placed on the corresponding abutment after a surgical procedure if the subject chooses this option. The surgery consists of the placement of the implant which aims at being osteo-integrated, on which an abutment is screwed. The processor may be used for 3 to 6 weeks following the surgery according to wound healing. It is then fitted by an experienced audiologist following the company’s guidelines.A surgical procedure is performed to allow the placement of the receptor under the temporal muscle and the introduction of the electrode array into the scala tympani via the round window or a cochleostomy. According to the company, the electrode array supports 12 to 22 intra-cochlear electrodes which aim at stimulating the first neurons of the auditory nerve. Four cochlear implant companies are involved in this study: Advanced Bionics, Cochlear, MedEl and Oticon. The choice of the brand will depend on the physician in charge of the patient. Approximately four weeks after surgery, the external processor can be activated and the patient will therefore receive electric auditory stimulation of the implanted ear.The primary outcome measure will be applied to the two randomised arms to determine the incremental cost-utility ratio (ICUR) of cochlear implantation versus initial observation at 6 months. The utility is defined in this study as survival weighted by the quality of life of patients at 6 months (QALY). Quality of life will be measured using a generic questionnaire, the EuroQol-5D-3 L scale. This instrument is recommended by the national French agency (Haute Autorité de Santé) for cost-utility studies .The EQ-5D-3 L is a self-administered, generic and multidimensional questionnaire . It has Healthcare costs will be assessed from the French health insurance perspective , 40. DirConsumption of healthcare resources will be retrospectively gathered from the French Social Health Insurance databases, using a bottom-up approach. Administrative data corresponding to the name, surname, date of birth, place of living and gender were recorded for patients included in the study.Inpatient stays will be valued using the French Disease Related Groups (DRGs). Outpatient care, which includes visits, medical and paramedical acts, will be valued using the tariffs reimbursed by the French health insurance. Visits and paramedical acts will be valued using the French General Nomenclature of Professional Acts. Medical acts will be valued using the French Common Classification of Medical Acts, except for laboratory tests for which valuation will be based on the Nomenclature of Biological Acts. For all these fees, we will apply the corresponding reimbursement rate and we will subtract, if necessary, the medical deductible that is due by the patient and not reimbursed by the French Social Health Insurance (FSHI).A cost-utility analysis will be performed. This study will establish a link between costs and medical consequences, expressed in QALY gained, at 6 months between the two care management strategies. The ICUR between SSD/AHL patients treated by CI and those that are only followed-up, will be calculated as follows :Where ΔC and ΔU were increments of costs and utilities, respectively.A Budget Impact Analysis (BIA) will be implemented to measure the net costs to the social health insurance of the care management of SSD/AHL patients, taking into account all positive and negative variations, the use of health resources that could result from the CI .The BIA, which will be conducted using international and French guidelines, will include the predictable variation of context elements  , 42. TheThe analysis will include three phases:- An inventory of the care management of SSD/AHL patients,- A care management model of these patients after the introduction of the new strategy (i.e. CI) to their care,- The estimated cost for health insurance for these situations and the difference between the two strategies, to estimate the financial impact of the introduction of the new strategy in the treatment of SSD/AHL patients.The introduction of the strategy based on the CI can change their own cost, but also have an impact on the size of the population (target and reached population), patterns of care performed prior to and after, the unit costs of resources mobilised in the context of strategies. The BIA should take these factors into account.All participants are asked to complete several quality of life questionnaires and hearing tests at inclusion and 6 months after their choice of the treatment . Pre/post treatment analyses will be carried out in each of these groups. Comparisons will be made in the group of subjects who chose a CI, randomised between immediate CI and initial observation.As described above, a generic evaluation of quality of life is performed using the EuroQol-5D-3 L scale. Hearing-specific quality of life questionnaires will also be administered.The Nijmegen Cochlear implant questionnaire (NCIQ) contains 60 questions exploring 6 areas concerned with the quality of auditory perception  . Indeed,An evaluation of the discomfort related to the possible tinnitus associated with the deafness will rely on a visual analogue scale (VAS) ranging from 0 to 10. This scale is presented as a 17 cm plastic ruler with a vertical arrow on one side and a graduated scale on the other side (0 to 10 cm). The subject first indicates the level of annoyance generated by the tinnitus on the vertical arrow using a cursor and the corresponding numeric value is reported by the evaluator. Then, the intensity of tinnitus is assessed using another ruler with the same dimensions.www.hoertech.de) and presented via loudspeakers and amplifier . Speech signal is presented at a fixed level of 65 dB SPL and the level of competing noise is adjusted using the adaptive procedure described by Jansen et al. to obtain the signal-to-noise ratio in dB for 50% correct word recognition (SNR50).Speech recognition in competing noise is measured using the French Matrix test  in soundSNR50 is obtained in three different spatial configurations: one with speech and noise presented from a single loudspeaker in front of the subject at 0° and two conditions with speech and noise presented from separate loudspeakers at 60° to the left and right of the subject. The choice of a spatial configuration  was selected based on previous results  showing Horizontal localisation ability is assessed using an array of 7 horizontal loudspeakers and an amplifier  located at intervals of 30 degrees from – 90 degrees to 90 degrees in a frontal semicircle diameter of 1.2 m at the subject’s head level. The stimulus comprises two 150 msec white gaussian noises from 20 Hz to 20 kHz with a 0.05 msec ramp. A silence of 150 msec is inserted between the two noises for total stimulus duration of 450 msec. This stimulus is similar to the one used by Slattery and Middlebrooks  and is pOnce the validity of the database is double-checked, descriptive analyses will be carried out for each treatment option . Quantitative variables will be described as means, standard deviations, and percentile distribution. The number and proportion of subjects will be used to describe categorical data.Regarding the randomised controlled trial phase comparing CI versus observation, the analysis will be conducted on all subjects who were randomised on an intention-to-treat basis. For each outcome , the value measured at 6 months will be compared between the two groups using bivariate analyses (Student’s T test or Wilcoxon rank sum tests) and linear regressions adjusted for the baseline value.For each intervention, we will assess the associations between changes in hearing performances and changes in quality of life. Data will be presented according to the Consolidated Standards of Reporting Trials (CONSORT) Statement , 48.In order to test the robustness of the ICUR, deterministic and probabilistic sensitivity analyses will be conducted.As part of the sensitivity analysis, we will determine the robustness of the results by testing the impact on the outcome of the variation of different cost and utility parameters . ProbabiThe detrimental consequences of SSD and AHL have been demonstrated for auditory performances as well as for the quality of life experienced by the patients , 51. HowThe present protocol attempts to give account for this heterogeneity through the wide inclusion criteria which have been chosen. The choice of treatment and the outcomes of different subgroups  will therefore be specifically described. The theoretical advantage of cochlear implantation over CROS hearing aids and BC implants is to restore an auditory stimulation of the poor ear. This represents the mandatory condition to restore, at least partially, the perception of interaural differences. Further, alleviation of tinnitus is an established side-effect of CI, and subjects with incapacitating tinnitus associated with SSD/AHL might benefit more from this treatment than other SSD/AHL subpopulations.The current study falls within the framework of validation of SSD/AHL as a new indication of cochlear implantation in France. In this line, the NCIQ was chosen as specific subjective evaluation of hearing before and after treatment, to refer to previous studies on benefits of CI in bilateral severe-to-profound deafness , 55–58. The calculation of an intervention utility relies on a generic evaluation of quality of life. Among the different instruments which may be used, the Health Utility Index Mark 3 has been identified as sensitive to detect hearing changes after a treatment , 60. NevThe present study is the first randomised controlled trial on CI in SSD/AHL with the determination of the cost-utility ratio as a primary endpoint. It also provides an assessment of hearing modifications which may be expected from most of treatments for SSD/AHL, such as CROS systems and BAHA. Eventually, it will help delineating the national new criteria for CI indication.Inclusions completed. Data controls ongoing.


Mycoplasma hyopneumoniae, the primary pathogenic bacterium causing enzootic pneumonia, significantly affects worldwide swine production. The infection is usually persistent and bacterial identification and isolation of M. hyopneumoniae in clinical samples are challenging due to the fastidious requirements for its growth. Hence, new practical surveillance tools that improve or complement existing diagnostics on M. hyopneumoniae are desirable, especially in early infection. The objective of this study was to identify potential metabolite markers of early M. hyopneumoniae infection in pigs through metabolomics analysis. Samples obtained from pigs in a previous M. hyopneumoniae experimental infection were used in this study. Briefly, two pigs served as mock inoculated controls and ten pigs were intra-tracheally inoculated with M. hyopneumoniae. Sera, laryngeal swabs (LS), and tracheo-bronchial lavage fluid (TBLF) were collected from all pigs at 0, 2, 5, 9, 14, 21 and 28 days post-inoculation (dpi). Bronchial swabs (BS) were collected post-mortem at 28 dpi. Mycoplasma hyopneumoniae infection was confirmed by PCR in LS, TBLF and BS. Serum metabolites were profiled using high-resolution liquid chromatography–mass spectrometry (LC–MS) analysis. Metabolite markers were identified by structural analysis following multivariate analysis of LC–MS data. The results showed that M. hyopneumoniae infection time-dependently altered the serum levels of selective amino acids and fatty acids. α-Aminobutyric acid and long-chain fatty acids were markedly increased at 14 and 21 dpi in inoculated pigs (p < 0.05). These results indicated that M. hyopneumoniae infection caused systemic changes in host metabolism, warranting further studies to determine underlying biochemical and physiological mechanisms responsible for the observed changes. Mycoplasma hyopneumoniae (M. hyopneumoniae) is a major health risk in the porcine industry, causing enzootic pneumonia (EP), a chronic respiratory disease in growing pigs. Enzootic pneumoniae is often chronic, with a pattern of slow spreading and progression over the course of months, which usually results in herd morbidities that can reach up to 70–100% + = 556.2771 m/z) in real time. Mass chromatograms and mass spectral data were acquired and processed by MassLynx software (Waters Corporation) in centroided format. Additional structural information was obtained by tandem MS (MS/MS) fragmentation with collision energies ranging from 15 to 40 eV [A 5 μL aliquot of each sample was injected into an ultraperformance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC–QTOFMS) system  and separated by a BEH C18 column (Waters Corporation) with a gradient of mobile phase ranging from water to 95% aqueous ACN containing 0.1% formic acid over a 10-min run. Capillary voltage and cone voltage for electrospray ionization were maintained at 3 kV and 30 V for positive mode detection, whereas source temperature and desolvation temperature were set at 120 and 350 °C, respectively. Nitrogen was used as both cone gas (50 L/h) and desolvation gas (600 L/h), and argon was used as collision gas. For accurate mass measurement, the mass spectrometer was calibrated with sodium formate solution . MarkerLynx provided a multivariate data matrix containing information on sample identity, ion identity (retention time and m/z), and ion abundance. The abundance of each ion was calculated by normalizing the single ion counts to the total ion counts in the entire chromatogram, and the data matrix was then exported into SIMCA-P+ software . Principal components analysis (PCA) was used to model the data for the mock inoculated controls and inoculated groups. Metabolite markers were identified by analyzing ions contributing to sample separation in PCA models. QuanLynx software (Waters Corporation) was used for the quantification of metabolites, and standard curves were generated by incorporating amino acid and fatty acid standard molecules in the LC–MS run. The concentrations or relative abundances of identified metabolite markers in the samples and correlations among these metabolite markers were evaluated using GraphPad and R program .t tests for paired data within a time point. Friedman test, the non-parametric alternative to the one-way ANOVA with repeated measures, was used to test for differences among metabolites between mock inoculated controls and inoculated pigs over time. Hierarchical cluster analysis (HCA) was performed to identify metabolite clusters contributing to sample separation. Pearson analysis was performed to evaluate the correlation between metabolite markers and detection of M. hyopneumoniae in serum samples. Differences between the mock inoculated controls and inoculated pigs were considered significant if p < 0.05 and were considered a trend when between 0.05 and 0.10.Statistical analysis of metabolomic parameters was performed as a two-tailed Student’s M. hyopneumoniae throughout the study. Among inoculated pigs, M. hyopneumoniae DNA was detected in LS and TBLF of all pigs (100%) starting at 9 dpi  and individual FFA, including myristic acid, palmitoleic acid, oleic acid, and linoleic acid in serum samples were significantly elevated at 14 and 21 dpi in inoculated pigs , a non-proteinogenic amino acid, was identified at 14 and 21 dpi in inoculated pigs (M. hyopneumoniae detection in TBLF (p < 0.05). A negative correlation was indicated based on low Ct values (high bacterial burden and greater correlation with the increased concentration of AABA; Figure p < 0.05; Figures A significant correlation was observed between the concentration of AABA detected in serum of inoculated pigs and Ct values for F p < 0.0. A negatF p < 0.0. A negatMycoplasma hyopneumoniae has been recognized as an endemic pathogen globally. The typical lesions of EP have been reported to occur in 30 to 80% of slaughter pigs in different countries [M. hyopneumoniae in a survey conducted by the National Animal Health Monitoring System [M. hyopneumoniae limits current efforts to control and manage the clinical disease in the field. Furthermore, the mechanisms of immune modulation and the immunopathology associated with EP are not fully understood [M. hyopneumoniae infections were identified using state-of-art metabolomics analysis.ountries . In the g System . The lacM. hyopneumoniae challenge were observed as early as 5 dpi. It has been shown that the attachment of bacterial cells to tracheal cilia is one of the events preceding M. hyopneumoniae infection and it may occur 5 days after experimental challenge [The serum metabolomics profile identified both, infection-dependent and independent changes in experimentally infected pigs. The host metabolic responses to hallenge , when mohallenge , 29, 30.A significant increase in the levels of AABA was identified at 14 and 21 dpi in this study. AABA is a non-proteogenic amino acid and often considered to be a catabolic product of other amino acids. As shown in humans, AABA is biosynthesized by transamination of α-ketobutyric acid, a metabolite in branched chain amino acids biosynthesis . AlternaSeveral studies have also reported AABA as a metabolic indicator of disease severity in human patients with sepsis, as well as multiple organ failure syndrome , 36. IncM. hyopneumoniae is a surface lipoprotein which has recently been identified to exhibit lipolytic activity with affinity to a wide range of fatty acid esters. Schmidt et al. speculated that the lipolytic function of p65 could be the reason of the reduced function of surfactants in pneumonic pig lungs [M. hyopneumoniae [The levels of FFA in serum samples were elevated at 14 and 21 dpi in inoculated pigs. This observation is consistent with the reported lipolytic activity of mycoplasmas . The p65ig lungs . Pulmonaig lungs , 39. Amoig lungs . Genome eumoniae .Mycoplasma organisms for fatty acids is well known since their discovery. The fastidious nature of mycoplasmas in vitro is defined by the requirement of a exogenous supply of amino acids and fatty acids in their growth medium [Mycoplasma species including M. hyopneumoniae, lack the genes involved in fatty acid synthesis and therefore, the building units for bacterial membrane lipids are obtained from long chain saturated and unsaturated fatty acids present in the host [M. hyopneumoniae membrane lipid composition, of which oleic, as well as palmitic acids are the major fatty acids [M. hyopneumoniae both in vitro and in vivo [The nutritional requirement of h medium . A majorthe host . Fatty aty acids . Recentl in vivo .M. hyopneumoniae infection. The requirement of fatty acids by the pathogen was justified by the observed increased abundance of long chain fatty acids in the serum. The increased levels of AABA along with altered kinetics of amino acids may indicate protein hyper-catabolism in hosts. It is also important to note that the host metabolic signature in response to M. hyopneumoniae infection reflected the active metabolic pattern of the pathogen, rather than that of the defense mechanisms. Thus, through characterizing the metabolic signature of M. hyopneumoniae infection in pigs, the study examined the metabolic responses to early disease which was sparsely reported before. The information procured would aid to improve the capabilities to diagnose, control, and eradicate this bacterium from swine herds. Nevertheless, the changes observed in the experimental inoculation need to be further validated in the natural course of infection. Moreover, the molecular mechanisms involved in host responses to M. hyopneumoniae infection need to be deciphered.The present study identified significant changes in the host amino acid and fatty acid profiles during early stages of


Drosophila – has a very different structure and morphology to that of the hearing organs of vertebrates. Nevertheless, it is becoming clear that vertebrate and invertebrate auditory organs share many physiological, molecular and genetic similarities. Here, we compare the molecular and cellular features of hearing organs in Drosophila with those of vertebrates, and discuss recent evidence concerning the functional conservation of Usher proteins between flies and mammals. Mutations in Usher genes cause Usher syndrome, the leading cause of human deafness and blindness. In Drosophila, some Usher syndrome proteins appear to physically interact in protein complexes that are similar to those described in mammals. This functional conservation highlights a rational role for Drosophila as a model for studying hearing, and for investigating the evolution of auditory organs, with the aim of advancing our understanding of the genes that regulate human hearing and the pathogenic mechanisms that lead to deafness.Johnston's organ – the hearing organ of  Summary: This Review discusses the similarities and differences between hearing in mammals and the fruit fly Drosophila, and describes how recent technological developments allow Drosophila to be used as a model to understand the function of human deafness genes. Drosophila, might also provide convenient and more rapid ways to assay the function of candidate deafness genes.Hearing loss is one of the most common neurological disorders in humans, affecting 2 to 3 of every 1000 children in the USA . ApproxiDrosophila also have a hearing organ – Johnston's organ , a2 (or pedicelus), which contains Johnston's organ and responds to sound, gravity and wind flow, and a3 (or funiculus), the most distal segment, which is an olfactory organ.Arista: a delicate, feather-like structure projecting from the third  antennal segment of Drosophila that is involved in sound detection.Chordotonal organs: these organs are ciliated stretch receptors that detect motion in joints of the insect body. They are made up of repeating multicellular units called scolopidia (see below).Cochlea: the coiled, snail shell-shaped part of the mammalian inner ear, the innermost portion of which, the cochlear duct, contains the organ of Corti (see below), which detects sound.CRISPR-mediated homology-directed repair: the CRISPR-Cas9 system can be used to generate targeted double-stranded DNA breaks (DSBs) when coupled with a sequence-specific guide RNA (gRNA). When Cas9 and a gRNA are introduced into a cell with a piece of DNA flanked by homology arms, the DSB can be repaired by homologous recombination, inserting the new piece of DNA into the locus of interest.Forward genetic screens: a genetic screen, typically of induced mutations, which identifies genes responsible for a particular phenotype, for example, hearing defects.GAL4-UAS system: a two-component genetic system that activates gene expression in Drosophila. It uses the yeast GAL4 transcription factor to activate transcription of a transgene adjacent to an upstream activation sequence (UAS).Johnston's organ: the largest chordotonal organ in Drosophila, containing ∼200 scolopidia. It is located in the second antennal segment and detects auditory signals (courtship songs), as well as wind flow and gravity.Lethal mosaic screen: lethal homozygous mutations cannot be studied beyond the age they cause lethality. To circumvent this, clones of cells carrying two lethal mutant alleles can be generated in an otherwise heterozygous organism using a variety of recombination techniques. This approach allows researchers to assess the phenotypes caused by lethal mutations.Mechanotransduction channels: ion channels that are gated by mechanical force.Organ of Corti: an epithelial specialization of the cochlear duct, which detects sound through its mechanosensitive hair cells. The organ of Corti sits on a thin, flexible basilar membrane, which oscillates at different frequencies corresponding to its position along the cochlea.Recombination-mediated cassette exchange (RMCE): regions of genomic DNA can be flanked by DNA sequences that act as targets for site-specific integrases. For example, the attB and attP DNA motifs are targets for the phiC31 integrase. If a piece of DNA bearing attB sites (a recombination cassette) is introduced into a cell that carries a DNA sequence flanked by attP sites in the presence of the phiC31 integrase, the intervening sequence will be replaced by the recombination cassette.Scolopidium: a sensory unit of chordotonal organs, each containing 1-3 mechanosensitive sensory neurons, the cilia of which are surrounded by a scolopale cell. The scolopidium is anchored to the insect cuticle at its proximal and distal ends by a ligament cell and a cap cell, respectively. When flexional or rotational force is applied to the insect's cuticle, it is transferred to the sensory neurons in each scolopidium.Stereocilia: elongated, actin-rich microvilli that protrude from the apical surface of sensory hair cells in vertebrates. Their displacement by force opens mechanosensitive ion channels, with their distal ends acting as likely sites of active mechanotransduction in hair cells. Rows of stereocilia of increasing length form a staircase-like bundle structure in hair cells, and the coupling of adjacent stereocilia by tip links transfers force to the entire bundle.Drosophila hearing organ have mammalian counterparts that function in the differentiated cells of the cochlea and are required for hearing in mammals. In particular, we focus on recent findings that Usher syndrome proteins in Drosophila appear to physically interact in protein complexes that are similar to those described in mammals. We also describe recent work that indicates that some Usher syndrome proteins interact physically with Myosin II, the variants of which can cause MYH9-related diseases, including deafness. Various approaches, including forward genetic screens in Drosophila  basic helix-loop-helix (bHLH) transcription factor was discovered in Drosophila as a proneural gene required for the formation of photoreceptors and of some mechanoreceptors /Gfi1, which can be regulated by ato/Atoh1, is required for the development of the chordotonal organ in Drosophila embryos and for the survival of hair cells in the mouse , appears to function in neurite outgrowth rather than in neuronal specification  and cousin of atonal (cato)  . Althougce versa , Neurog1t degree , 2015, sDrosophila, studies of the fluid that bathes a variety of insect and crustacean mechanoreceptors have reported that this fluid has a positive potential of about +70 mV relative to the hemolymph , a homeodomain transcription factor, is specifically expressed by scolopale cells in Johnston's organ  domain glycoprotein that is produced and secreted by scolopale cells. It forms acellular filaments that connect the mechanosensory neuronal dendrites to the third antennal cuticle  #276900, #276904, #601067, #276901, #605472, #611383] that is characterized by varying degrees of deafness and retinitis pigmentosa-induced vision loss, and is the leading cause of deaf-blindness in humans . Based o2+-mediated signaling ] is required for hearing: loss of ck abolishes a fly's auditory responses and causes the scolopidia to detach from the cuticle of the a2/a3 antennal joint in Johnston's organ , Cib2 (cib2), whirlin (dyschronic) and clarin 1 (CG1103) , do not appear to have any obvious homologs in Drosophila  . Althougosophila . Moreoveosophila , and theyosin II , the mamMYH9-related diseases are caused by mutations in MYH9, which encodes nonmuscle myosin IIA. These diseases are characterized by large platelets and thrombocytopenia from birth, and are associated with a variable onset of progressive sensorineural hearing loss, presenile cataracts, elevated liver enzymes and renal disease, which initially manifests as a glomerular nephropathy , NMIIB (encoded by MYH10) and NMIIC (encoded by MYH14). Mice that are homozygous for a Myh9 null mutation die during embryogenesis by embryonic day (E) 7.5  7.5 . Mice heresholds . This su anomaly . Althougair cell , NMIIA iDrosophila homolog of NMIIA is encoded by the myosin II gene, also known as zipper. This gene is expressed in Johnston's organ, and Myosin II is enriched at the apical tips of scolopidia, adjacent to Myosin VIIA (spaghetti squash (sqh), genetically interacts with myosin VIIA in Johnston's organ and affects hearing  lines are available , as shown by their ability to rescue null alleles  of interest under the control of a UAS promoter from a separate locus in the Drosophila genome  seeks to find possible genetic etiologies for rare human diseases (www.mousephenotype.org), with the US effort coordinated through an NIH-funded Knockout Mouse Project (www.komp.org). Because auditory testing is part of the standard IMPC phenotyping pipeline, it is likely that many new mouse genes will be identified that can cause hearing loss in homozygous or heterozygous mutants, and this might support the identification of human variants that also cause hearing loss. Mutagenesis-based screens in mice have also revealed new genes responsible for hearing loss and age-related hearing loss -funded Undiagnosed Diseases Network  or proteins acting downstream of the affected proteins, and slow the aggregation of pathogenic protein variants. Once again, we suggest that


The JNDs and interquartile ranges for 0.25, 1, and 4 kHz octave-band increments were 6.3 [4.0–7.8], 6.7 [4.6–9.1], and 9.6 [7.3–12.4] dB, respectively. The JNDs and interquartile ranges for low-, mid-, and high-frequency dual-octave-band increments were 3.7 [2.5–4.6], 3.8 [2.9–4.7], and 6.8 [4.7–9.1] dB, respectively. The JND for broadband increments was 2.0 [1.5–2.7] dB. High-frequency dual-octave-band JNDs were positively correlated with high-frequency pure-tone thresholds and sensation levels, suggesting an effect of audibility for this condition. All other JNDs were independent of pure-tone threshold and sensation level. JNDs were independent of age and hearing-aid experience. These results suggest using large initial adjustments when using short sentences in a hearing-aid fitting to ensure patient focus, followed by smaller subsequent adjustments, if necessary, to ensure audibility, comfort, and stability.During a hearing-aid fitting, the gain applied across frequencies is often adjusted from an initial prescription in order to meet individual needs and preferences. These gain adjustments in one or more frequency bands are commonly verified using speech in quiet . Such adjustments may be unreliable and inefficient if they are not discriminable. To examine what adjustments are discriminable when made to speech, this study measured the just-noticeable differences (JNDs) for gain increments in male, single-talker sentences. Sentences were presented with prescribed gains to the better ears of 41 hearing-impaired listeners. JNDs were measured at  The adjustment of frequency-dependent gain is common in hearing-aid fittings, whether in real-ear verification or in fine-tuning to patient feedback. While fine-tuning to patient feedback is routine , evidencThe authors previously reported the just-noticeable differences (JNDs) for gain increments in frequency-specific speech–spectrum-shaped noise regions: approximately 3 dB for octave bands from 500  to 4000 Hz . Those dIncreasingly popular self-fitting technology allows patients to adjust their own devices to real-world stimuli in their own acoustical environments  rather tPrevious research only provides evidence on noticeable broadband gain adjustments. In examining gain preferences with speech in noise, This study directly measured the JNDs for gain increments in speech. Participants listened to sentences that were processed with prescribed National Acoustics Laboratories-Revised (NAL-R) gains and discriminated level increments in a fixed-level (method of constant stimuli), same-different task. Octave-band gain adjustments are often used in real-ear measurements for meeting prescription targets, while larger, multiple octave-band gain adjustments are often used for fine-tuning and automated fittings . TherefoForty-one participants  were recruited from local audiology clinics. The median age of participants was 69 years (range: 51–75 years). All participants had some degree of hearing loss in at least one ear. All participants had experience wearing hearing aids. The median hearing-aid experience was 10 years (range: 2–35 years), and the median proportion of life wearing hearing aids was 0.14 (range: 0.03–0.53 of life). Unmasked pure-tone thresholds were manually measured for both ears just prior to the experiment . For theThe experiment was ethically approved by the University of Glasgow research ethics system committee (application number 200160138). All participants provided written informed consent prior to the experiment.Stimuli were sentences taken from the Bamford–Kowal–Bench (BKB) corpus , spoken For each trial, a single sentence was randomly selected from the corpus of 336 to be in both intervals . Gains were calculated for each participant’s BE4FA ear using the NAL-R formula . The NALStandard signals were sentences plus prescribed gains. Target signals were sentences plus prescribed gains , plus a band-specific level increment of 5, 10, 15, or 20 dB see . There wStandard stimuli were calibrated  so that with a gain of 0 dB in each band, the long-term average root mean square (RMS) presentation level across all sentences was 60 dB(A). Target stimuli were also calibrated to confirm the frequency-band and broadband increments. Audibility of the sentences was checked with the participant following their practice trials by one of the authors.The two stimuli in each trial were separated by a silent interstimulus interval of 375 ms. Presentation level was roved from trial to trial by a randomized amount selected from a flat distribution of ± 3 dB to minimize the potential learning of a fixed-level standard. There were four possible stimulus combinations for each trial: two same (standard–standard or target–target) and two different (standard–target or target–standard). These combinations were counterbalanced and presented randomly.The experiment was conducted in a single, one-and-a-half-hour session for each participant. A screening questionnaire on hearing-aid usage was completed and unmasked pure-tone thresholds were measured. Participants were seated in an audiometric booth for the experiment. Stimuli were presented monaurally over circumaural headphones  after being converted from a 44.1 kHz, 16-bit digital signal to analog via an external soundcard . Participants completed two blocks of trials with a break between, with each block lasting approximately 20 to 25 minutes. Twenty practice trials were embedded into the start of each block.A fixed-level (method of constant stimuli), same-different task was used. Participants were asked to listen to each presentation and decide “did the sentences sound the same or different?” Participants responded by choosing the appropriate button (“same” or “different”) on a touch-screen monitor. Participants were not informed of a dimension on which to base their judgments, and therefore discrimination could be based on multiple dimensions . Visual feedback (“correct” or “incorrect”) was provided following each response.Twenty-eight adjustments were presented: four-level increments (5–20 dB) for each of the six frequency-band conditions  and four-level increments (1–4 dB) for the broadband  condition. Coupled with four stimulus combinations (two same and two different) repeated twice, there were 224 trials  presented in a randomized order per block. Each participant completed two blocks, totaling 64 trials for each of the seven conditions.Three types of JNDs were measured: (a) increment JNDs in three octave-bands, (b) increment JNDs in three dual bands, and (c) broadband increment JNDs.d’  task and 76% correct by an unbiased observer in a two-alternative forced-choice task , dual-band JNDs , and broadband JNDs . Therefore, median JNDs are reported, and nonparametric analyses are employed. To avoid the influence of outliers in the JND data, Spearman rank-order correlation coefficients (ρ) are reported . However, 0.25 and 1 kHz JNDs were significantly smaller (better) than 4 kHz JNDs . A similar effect of center frequency was found for the dual-band JNDs. While LF and MF JNDs were not significantly different , they were smaller than the HF JNDs . Of note are the large individual differences, reflected in the interquartile ranges , 1 kHz JNDs were greater than MF JNDs , and 4 kHz JNDs were greater than HF JNDs . Octave-band JNDs were also greater than broadband JNDs , as were dual-band JNDs .Octave-band JNDs were greater (poorer) than dual-band JNDs: 0.25 kHz JNDs were greater than LF JNDs (p < .05). For dual-band discrimination, LF and MF JNDs were not significantly correlated with corresponding average pure-tone thresholds (both p > .05), while participants’ averages of pure-tone threshold at 4 and 6 kHz were positively correlated with their HF JNDs . To examine audibility more directly, JNDs were also compared with sensation level, which was approximated from individual thresholds and long-term presentation level as determined by individual prescription. The same relationship was found: only dual-band HF sensation levels were correlated with HF JNDs . There were no significant correlations between JNDs and age or hearing-aid experience when adjusting for multiple comparisons with the Holm-Bonferonni correction .Correlations between participants’ frequency-specific JNDs and their corresponding pure-tone thresholds were examined. Correlations between individual 0.25, 1, or 4-kHz JNDs and their respective 0.25, 1, or 4-kHz pure-tone thresholds were insignificant  than HF dual-band JNDs, indicating discrimination ability is more dependent on the energy within a band than bandwidth. With the scale of these octave- and dual-band JNDs, it was possible that participants were judging changes in overall loudness rather than spectral shape. For most octave-band and dual-band adjustments, however, a gain increment of one JND did not exceed the broadband JND , which was driven by participants with the most severe 6 kHz pure-tone thresholds. These findings suggest that patients will require even larger adjustments than the median JNDs to be noticeable if they are not provided with adequate sensation levels. As in There was a positive correlation between HF JNDs and averaged pure-tone thresholds at 4 and 6 kHz (ρ = 0.34). Further analyses revealed that the two participants with HF JNDs of 18 dB or greater had averaged 4 and 6 kHz pure-tone thresholds of at least 75 dB HL. There was also a negative correlation between HF JNDs and sensation level at 4 and 6 kHz (ρ = –0.50). These correlations suggest an effect of audibility for this condition. The sensation levels in these higher frequency bands were not sufficient for all participants to make accurate judgments on lesser increments, resulting in elevated JNDs. The 2.0 dB broadband JND measured in this study is commensurate with the unaided and aided broadband JNDs for sentences in Measuring intelligibility and sound quality ratings with hearing aids, This study presented recorded male, single-talker sentences from the BKB corpus. This material is standardized, providing a starting point for examining the discriminability of speech adjustments. However, there is some variation in spectral content between talkers: these JNDs may be over or underestimates depending on speech characteristics such as language or gender . Accent Given the sparseness of information in any frequency band of speech at any time in these short sentences (mean duration 1.6 s), it is possible that longer duration speech—such as an ongoing monologue—may improve JNDs cf. . A more Judgments were made on a short-term, trial-by-trial basis, similar to those made in a hearing-aid fitting. However, it is not known whether the JNDs change with time. It may be that sensitivity improves with listening experience; that a small adjustment previously unnoticeable in the clinic becomes noticeable and appreciable after a period of real-world use. In this case, these results may still be of use to a clinician to inform a patient in an appointment that while instant benefits from fine-tuning are unlikely, they may be appreciable with time. Alternatively, it may be that sensitivity worsens with time. This study was conducted in favorable listening conditions, and it is reasonable to expect that noisier environments would degrade sensitivity. Further research on the longitudinal, real-world effects of JND-sized adjustments on subjective and objective outcomes is required. Earlier studies have disagreed on whether JND-sized deviations from prescriptions would be detrimental  or not . Recent Frequency-dependent gain is routinely adjusted to patient feedback in hearing-aid fittings. Speech is a common test signal for these adjustments, particularly in fine-tuning and troubleshooting practices. The JNDs reported here see  provide


Haemaphysalis longicornis exhibits two separate reproductive populations: bisexual and parthenogenetic, which have diploid and triploid karyotypes, respectively. The parthenogenetic population can undergo engorgement without copulation and produce viable female-only offspring with a longer incubation period than the bisexual population. Three enzymes, cathepsin B, cathepsin D and acid phosphatase, were found to be involved in vitellin degradation during the embryonic development of bisexual H. longicornis. However, the expression and activity profiles of these enzymes during the embryonic development of parthenogenetic ticks remain unknown. In the present study, the transcriptional expression profile, enzyme activity and roles in embryogenesis of the three enzymes during the embryonic development of parthenogenetic H. longicornis were investigated.The tick Quantitative real-time polymerase chain reaction (qPCR) and fluorescence detection were used to analyze the dynamic changes in the three enzymes during embryogenesis. The roles of the three enzymes during embryogenesis were also explored using RNA interference (RNAi).H. longicornis. The expression of cathepsin B was highest on day 15, whereas that of cathepsin D was highest on day 3 and the peak of acid phosphatase expression occurred on day 9. The activity of cathepsin B was highest on day 3 and lowest on day 5, then gradually increased and remained stable. Cathepsin D activity was highest on day 1 and showed a gradually decreasing trend, whereas acid phosphatase showed the opposite trend and reached a peak on day 23. RNA interference experiments in engorged female ticks revealed that there was no significant difference in the number of eggs laid, but the hatching rate of the eggs was significantly decreased.The three enzymes were all expressed during embryonic development in parthenogenetic H. longicornis, but the expression patterns and changes in the activity of the enzymes in the bisexual and parthenogenetic populations are different. The results will help a better understanding of the similarities and differences underlying embryonic development in the bisexual and parthenogenetic populations and contribute to the future exploration of the development of the parthenogenetic population of H. longicornis.The three enzymes all play important roles in embryonic development of  Ticks are obligate blood-sucking ectoparasites and over 900 tick species have been reported around the world . Ticks aHaemaphysalis longicornis is a three-host tick that is widely distributed in Australia, New Zealand, Korea, Japan and China [Anaplasma, Babesia, Borrelia, Ehrlichia and Rickettsia bacteria [Haemaphysalis longicornis exhibits two reproductive populations: bisexual and obligate parthenogenetic populations [H. longicornis are diploid and triploid, respectively. Parthenogenetic H. longicornis can complete their life-cycle without fertilization, without any males being observed throughout the life-cycle and they show strict reproductive isolation from the bisexual population [nd China . Recentlnd China . It is abacteria . Haemaphulations . Compariulations , 13. In pulation , 12. Becpulation , 12.Bombyx mori [Rhodnius prolixus [Periplaneta americana [Blattella germanica [Culex quinquefasciatus [Musca domestica [Anticarsia gemmatalis [Dipetalogaster maxima [Spodoptera exigua [Helicoverpa armigera [Ornithodoros moubata [Rhipicephalus microplus [H. longicornis [H. longicornis [H. longicornis were investigated. The results will contribute to a better understanding of the similarities and differences underlying embryonic development between the bisexual and parthenogenetic populations and to the future exploration of the development of the parthenogenetic population of H. longicornis.Embryonic development is an important physiological process in reproduction and development. Many proteolytic enzymes accumulate in oocytes together with vitellin and regulate vitellin degradation to provide nutrients during embryonic development . This phbyx mori –17, Rhodprolixus , 19, Permericana , Blattelermanica , Culex qasciatus , Musca domestica , Anticarmmatalis , Dipetalr maxima , Spodopta exigua  and Heliarmigera . However moubata , Rhipiceicroplus , 29–33 agicornis . Our pregicornis . HoweverH. longicornis ticks were collected by flag dragging from Cangxi County  in Sichuan Province, China. They were maintained for several generations in our laboratory and it was verified that no males were observed throughout the entire life-cycle to ensure that the ticks were obligate parthenogenetic. These ticks were reared on the ears of rabbits in the laboratory as described previously [Parthenogenetic eviously . After fH. longicornis, the sequences of several ticks were aligned and compared to search for conservative sequences upon which to design corresponding primers. For cathepsin B, sequences from H. longicornis (GenBank: AB255051), Ixodes ricinus (GenBank: EF428206) and Ixodes scapularis (GenBank: XM_002435418) were used. For cathepsin D, sequences from H. longicornis (GenBank: EU019715), I. ricinus (GenBank: HQ615697) and R. microplus (GenBank: FJ655904) were used. For acid phosphatase, sequences from H. longicornis (GenBank: HM150759), I. scapularis (GenBank: XM_002410276) and Anoplophora glabripennis (GenBank: XM_018710438) were used. The primers for the β-actin gene were designed using the sequence from H. longicornis (GenBank: AY254898). All primers were designed with Primer Premier 5 (Premier Biosoft) and had lengths of 18–22 bp and were free of dimers and hairpins . The purified products were inserted into the pEASY®-T1 Simple Cloning Vector (TransGen), which was then transformed into Escherichia coli DH5α cells (Invitrogen). The transformed cells were submitted to Sangon (Sangon Biotech) and the resulting sequences were analyzed by BLASTn searches in the NCBI database to ensure that the primers were correct.Primers for quantitative analysis were designed using sequence information from the National Center for Biotechnology Information (NCBI). Because of the paucity of sequences from parthenogenetic 2O, 1 μl of cDNA template, 0.4 μl of Passive Reference Dye (TransGen Biotech) and 0.4 μl of each primer at 10 μM  using an AxyPrep™ Multisource Total RNA Miniprep Kit (Axygen). Then, total RNA from each sample was reverse transcribed to cDNA using TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech) following the manufacturer’s protocol. qPCR was performed in an Mx3005P system (Agilent Technologies) using TransStart® Top Green qPCR SuperMix (TransGen Biotech) following the manufacturer’s instructions. The reaction system was as follows: 10 μl of 2× TransStart® Top Green qPCR SuperMix, 7.8 μl of Hq method .rpm for 10 min at 4 °C to obtain the supernatants. The bicinchoninic acid (BCA) method was used to adjust the protein concentration for consistency [Total protein was extracted from eggs at different developmental stages (1.0 g each) by using 50 mM sodium acetate buffer (pH 5.0). Then, the homogenates were centrifuged at 12,000 × sistency . CathepsGFP) from Tetraselmis subcordiformis (GenBank: KJ668651) were also used. The PCR products were gel purified to synthesize RNA by using the T7 RiboMAXTM Express RNAi System  according to the manufacturer’s protocol. Double-stranded RNA (dsRNA) injection was performed as described previously [Primers for RNAi were designed in the same way as the qPCR primers. The TAA TAC GAC TCA CTA TAG G (T7) promoter sequence was added to the 5ʹ end of the primers Table . Primer eviously . Engorgeeviously . A portiP ≤ 0.05. Experimental values were obtained from three independent assays and expressed as the mean ± standard error.All experiments were repeated three times and the data were analyzed using ANOVA with SPSS 12.0 software (IBM). The t-test statistical analysis was performed using the SAS JMP statistical program 13.2 (SAS Institute Inc). The differences were considered statistically significant when cathepsin B, cathepsin D and acid phosphatase genes were 133 bp, 88 bp and 180 bp, respectively  = 129.77, P ≤ 0.0001). However, the expression of cathepsin B decreased to a low level after day 15.To illustrate the functions of cathepsin B during embryogenesis, the transcriptional expression profile of ent Fig. a. The exF = 14.98, P ≤ 0.0001), but it decreased to the lowest level on day 5 and increased to a stable level thereafter.The analysis in which the fluorescent substrate was used to detect the activity of cathepsin B showed that its activity was high throughout embryonic development Fig. b. On daycathepsin D was detectable in the eggs from different stages of embryonic development  = 33.59, P ≤ 0.0001). Thereafter, the expression level gradually declined. The activity of cathepsin D was highest on day 1  = 21.15, P ≤ 0.0001) and decreased on days 3 and 5  = 50.14, P ≤ 0.0001). Thereafter, its expression dropped to a low level, where it remained until the end of embryogenesis. The activity trend of acid phosphatase showed a gradual increase until reaching the highest level on day 23  = 145.08, P ≤ 0.0001).On the first five days, the ion Fig. a. After g. F11, 2 = 50.14,cathepsin B, cathepsin D and acid phosphatase were 430 bp, 497 bp and 539 bp, respectively  = 11.13, P = 0.0032), cathepsin D  = 7.57, P = 0.0101) and acid phosphatase  = 126.43, P ≤ 0.0001)resulted in a decrease in their relative levels compared to GFP  = 0.129, P = 0.940). However, the hatching rates of these groups were 49.33%, 52.17%, 66.40% and 84.87%, respectively  = 22.127, P ≤ 0.0001).The engorged ticks of the experimental and control groups were maintained in the incubator to lay eggs. The oviposition of the ticks and the hatching rate of the eggs were recorded. The weights of the eggs laid by the ticks in the ely Fig. a. There The main biochemical event in the embryonic development of arthropods is the utilization of yolk components . As the cathepsin B was low on days 1–11 and rapidly increased to reach the highest level on day 15, then decreased sharply in the late development stage. However, in bisexual H. longicornis, cathepsin B showed a rapid rising trend on days 1–5, followed by a slow decline on later days [cathepsin B presents a high expression level at different developmental stages [H. longicornis, cathepsin B is more likely to be highly expressed in late embryonic development. The activity of cathepsin B fluctuated and was maintained at a high level throughout embryonic development in the parthenogenetic population, but in the bisexual population, it gradually decreased. Combined with the results obtained for cathepsin B expression, it can be concluded that cathepsin B played a vital role in late embryonic development in the parthenogenetic population, while in the bisexual population, cathepsin B tended to play a role in the early stage.Cathepsin B is a cysteine protease found in the cytolysosomes that plays a vital role in the degradation of yolk during embryogenesis. In our study, the expression of ter days . The expter days . In othel stages , 43; thecathepsin D rapidly reached the highest level on day 3 and then gradually decreased to a low level in the later development stage. The activity of cathepsin D was also highest in the early stage (on day 1) and was maintained at a high level during the later stage, except on day 23. However, in bisexual H. longicornis, the expression and activity of cathepsin D were highest on days 11 and 13, respectively [H. longicornis, cathepsin D tended to play a role in the early stage, while in the bisexual population, cathepsin D was more likely to regulate embryogenesis in the late stage. Although both cathepsin B and D were indicated to regulate embryogenesis, their dynamic changes were not the same in the parthenogenetic and bisexual populations. In our previous work, we found that the vitellin of the parthenogenetic population exhibits 9 subunits, ranging from 31 kDa to 203 kDa; however, in the bisexual population, vitellin exhibits only 8 subunits, ranging from 52 kDa to 112 kDa [Cathepsin D is the major lysosomal aspartic protease and is widely distributed in the cells, where it regulates programmed cell death, autophagy and the degradation of yolk protein , 25. Ourectively . In cont 112 kDa , 45. ThiIn contrast to cathepsins B and cathepsins D, acid phosphatase is a classical lysosomal enzyme that catalyzes the hydrolysis of orthophosphoric monoesters . The expH. longicornis [Radopholus similis [Schistosoma mansoni [Schistosoma japonicum [Schmidtea mediterranea [cathepsin B gene affects the development of the eggs and the hatching rate and even slows development and results in a shorter body length. In B. mori, cathepsin B participates in programmed cell death during metamorphosis and RNAi knockdown of cathepsin B leads to the stagnation of the larval-pupal metamorphosis [S. exigua has also been reported and RNAi knockdown of cathepsin D reduces the survival rate in the fifth-instar [B. mori, in which deficiency of cathepsin D affects the pupation of larvae [cathepsin D in S. mansoni affects its growth and reproduction of polypides in mice, which indicates the important role of cathepsin D in S. mansoni development [Trypanosoma brucei, researchers screened acid phosphatase and the functional analysis of this enzyme through RNAi showed that it was involved in growth and development [After RNAi treatment, the expression of the three targeted genes in the eggs was found to be significantly suppressed and the hatching rate was significantly reduced compared with that in the GFP group. However, the oviposition of the ticks showed no significant change. Similar results were previously found in bisexual gicornis , 47. In  similis , Schisto mansoni , 50, Schaponicum  and Schmterranea , knockinorphosis . The rolh-instar . A similf larvae . RNAi knelopment . Throughelopment . These rP. americana and M. domestica and the hydrolysis of vitellin is increased when both enzymes are present [R. prolixus, acid phosphatase can hydrolyze polyphosphate (polyP) to abolish the inhibitory effect of polyP on cathepsin D, which could facilitate the degradation of vitellin by cathepsin D [D. maxima [The dynamic changes and functions of these three genes in embryonic development have been studied, but the enzyme-enzyme and enzyme-vitellin interactions are still not clear. The interaction of cathepsin B and acid phosphatase has been found in  present , 23. In hepsin D , 19. The. maxima . In futuH. longicornis have been identified. The three enzymes all play an important role in embryonic development, but the expression patterns and changes in the activity of the enzymes in the bisexual and parthenogenetic populations are different. After the knockdown of the genes encoding the enzymes, oviposition was not affected, whereas the hatching rate of eggs was significantly decreased. These above results will help a better understanding of the similarities and differences in embryonic development between the bisexual and parthenogenetic populations and will contribute to the future exploration of the development of the parthenogenetic population of H. longicornis.The dynamic changes in cathepsin B, cathepsin D and acid phosphatase during the embryonic development of parthenogenetic


Aged residential care (ARC) admission needs are increasing beyond the available capacity in many countries, including New Zealand. Therefore, identifying modifiable factors which may prevent or delay ARC admissions is of international importance. Hearing impairment is common among older adults and thought to be an important predictor, although the current evidence-base is equivocal. Using the largest national database to date, competing-risk regression analysis was undertaken on 34,277 older adults having standardised home care assessments between 1 July 2012 and 31 May 2014, aged ≥65 years, and still living in the community 30 days after that assessment. Minimal hearing difficulty was reported by 10,125 (29.5%) participants, moderate difficulty by 5,046 (14.7%), severe difficulty/no hearing by 1,334 (3.9%), while 17,769 (51.8%) participants reported adequate hearing. By 30 June 2014, the study end-point, 6,389 (18.6%) participants had an ARC admission while 6,082 (17.7%) had died. In unadjusted competing-risk regression analyses, treating death as a competing event, hearing ability was significantly associated with ARC admission (p < 0.001). However, in adjusted analyses, this relationship was completely confounded by other variables (p = 0.67). This finding implies that screening for hearing loss among community-living older adults is unlikely to impact on ARC admission rates. This demographic shift poses serious challenges across multiple sectors, including health and aged residential care (ARC) services, through rapidly increasing age-related chronic disease and condition numbers. So much so that the current approach to health and disability services provision is considered unsustainable in New Zealand3, as it has been elsewhere4. Integrated health systems centred on enabling people to stay in their own homes longer, with an interconnected structure supporting them to live well and take greater responsibility for their own health, has been mooted as one solution6. Among the principal envisioned downstream impacts of this strategy is a reduction in ARC entry and hospital admission demand7. At the heart of this integrated strategy is an emphasis on prevention, efficaciously targeting and ameliorating modifiable risk factors. One potentially modifiable risk factor, hearing impairment, is common among older people and may benefit from such an approach.Globally, and within New Zealand, an uncharted demographic phenomenon is occurring; the accelerating ageing of populations through declining fertility and increasing life expectancies8. In a 2018 New Zealand hearing loss prevalence study, conducted on 16,080 nationally representative participants, 23.5% of those aged 65–74 years and 39.4% of those aged ≥75 years reported impairment9. Similarly, 19.5% and 36.7% of adults aged 65–74 years and ≥75 years, respectively, reported hearing impairment across the United Kingdom, Germany and France in 201810. Of the 50 million Americans aged >65 years, approximately 35% have hearing loss which is sufficient to make them hearing-aid candidates11. Despite hearing loss being ranked as the fifth leading cause of years lived with disability12, unmet treatment need remains high – even in developed countries such as the USA13. Yet some forms of hearing loss are preventable14, and many other forms are readily treatable11.Hearing loss can have profound effects on individuals, not only on interpersonal communication, but also on health, independence, wellbeing, and quality-of-life15. In a longitudinal study investigating predictors of institutionalization among adults age ≥75 years, those having mild hearing loss had increased odds of institutionalization estimated to be 2.2  compared to those without hearing loss, while people with severe/profound hearing loss had estimated odds of 9.4 16. However, these findings have not been replicated elsewhere. In an unadjusted analysis, Evans and colleagues identified a statistically significant relationship between hearing impairment and ARC admission, which disappeared after adjustment17. Another study failed to find a statistically significant unadjusted relationship in their non-disabled, community-living sample of adults aged ≥70 years18. These equivocal findings are likely due to important study design and analytical differences19. Also, the competing risk of death has not been explicitly considered before – a factor which is germane to older populations where the likelihood of death is non-ignorable21.Hearing impairment is more frequent among people within ARC facilities than their age-standardised community counterparts, and opined to be an important antecedent disability which contributes to admission2. However, there are insufficient financial returns to support building new capacity and replacing ageing stock2. To mitigate this economic and resource burden, mutable factors contributing to ARC admission need to be identified and attenuated. Although many forms of hearing impairment are readily treatable11, it has received limited research attention with respect to ARC admission; meta-analyses and reviews often fail to identify or consider hearing loss within their scope22. Using the largest ever population dataset to date, this study seeks to determine whether hearing loss is an independent predictor of ARC admission for those aged ≥65 years, after controlling for a suite of confounders and employing apposite statistical methods that adjust for the competing event of death.Using current service delivery models projections, the required sector bed numbers within New Zealand ARC facilities needs to increase by 78% to 110% to accommodate the increase in extra residents and replacement of ageing facilities by 2026A time-to-event study following a national cohort.Home-based people age ≥65 years in New Zealand having an interRAI-HC assessment undertaken between 1 July 2012 and 31 May 2014, inclusive, and who consented to their data being used for planning and research purposes. Those who were admitted to ARC or died within 30 days of their assessment were excluded.23. The interRAI-HC assessment form version 9.1 , modified with permission for New Zealand, is used under license to the Ministry of Health (www.interrai.co.nz). It is comprised of 236 questions over multiple health and social domains, which form 27 standardized instruments, and yields internationally valid and reliable scales24. Hearing was elicited by asking respondents about their ability to hear , with response options: adequate – no difficulty in normal conversation, social interaction, listening to TV; minimal difficulty – difficulty in some environments ; moderate difficulty – problem hearing normal conversation, requires quiet setting to hear well; severe difficulty – difficulty in all situations ; and, no hearing. InterRAI-HC information is stored electronically and is National Health Index (NHI) number-linked, using encryption for data security23. The NHI is a unique identifier that is assigned to every person who uses health and disability support services in New Zealand.The interRAI-HC instrument is used for all community care assessments on older people needing publicly funded long-term community services or ARC in New Zealand25. At the time of data release, the CPSS database was complete and finalised for ARC information collected on or before the 30 June 2014, so this was assigned as the study end date. For those with an interRAI-HC assessment and a subsequent ARC admission captured within the CPSS database, ARC status was indicated and study time was equated to the difference between the ARC admission and interRAI-HC assessment dates.ARC entry status and date of entry data were obtained from the Ministry of Health’s Contracted Care Payment System (CPSS) database. The CPSS contains all people who are publicly funded for such care . However, CPSS also contains information on approximately 50% of those who are self-funding, as this is voluntarily entered by providers. Hence the CPSS database contains data on approximately 90% of those entering ARC26. A cut-off date of 30 June 2014 was also applied, to ensure complete capture of registered deaths and consistency with the CPSS dataset. For those with an interRAI-HC assessment whose death was subsequently captured within the National Mortality Collection Register and who had not admitted to an ARC, death was indicated and study time was equated to the difference between death and interRAI-HC assessment dates. Note, death after an ARC admission was not relevant for this analysis – as it occurred after the event of interest. Eligible participants without an ARC admission and who survived beyond the study end date were defined as being censored, with study times equal to 30 June 2014 minus their interRAI-HC assessment dates.Survival status and date of death data were extracted from the National Mortality Collection Register, also held by the Ministry of HealthA suite of demographics and potentially confounding variables have been associated with ARC admission. Consistent with previous published studies and clinical experience, the following variables were utilized: age; sex; ethnicity; marital status; living arrangements; body mass index (BMI); activities of daily living (ADL); instrumental ADL (IADL), excluding phone use; alcohol consumption; smoking status; vision status; depression; delirium; history of falls; recent hospitalisation; diagnoses of stroke/cerebrovascular accident (CVA), chronic obstructive pulmonary disease (COPD), cancer, congestive heart failure (CHF), and coronary heart disease (CHD); fatigue; timed 4 m walk; urinary incontinence; and, self-rated health. All these measures arose from the interRAI-HC assessment, and the variable definition and classification details appear in the supplementary materials.23. In brief, the standardised interRAI-HC instrument is used for the conduct of all community care assessments on older people needing publicly funded long-term community services or ARC. Individuals are referred by a health practitioner to have their needs assessed by one of the more than 1,800 trained interRAI-HC assessors. Assessors visit clients in their own home to produce individualized care-plans according to a standardized protocol. Participants are explicitly asked if they consent to their de-identified interRAI-HC information being used for planning and research purposes; approximately 93% of participants provide this consent23. Where an individual had more than one interRAI-HC assessment between 1 July 2012 and 31 May 2014, only the first assessment was utilized.A detailed account of the interRAI-HC assessment instrument and procedure within New Zealand has been described previouslyThree separate national databases were interrogated and research datasets created, each containing encrypted NHI numbers for their respective members; encryption was undertaken by the Ministry of Health as an additional layer of identity protection. These databases included the: (i) interRAI-HC; (ii) CPSS; and, (iii) National Mortality Collection Register. The interRAI-HC research dataset contained information on all people who had at least one interRAI-HC assessment between 1 July 2012 and 31 May 2014 and consented to their data being used for planning and research purposes. Importantly here, the CPSS research dataset contained the date of entry for those admitted to ARC facilities, while the National Mortality Collection Register research dataset contained the date of death for those who had died. These latter two databases were known to have complete information on or before the 30 June 2014, thus this was assigned as the study end date.23, exclusion criteria applied, and then subjected to pursuant statistical analyses.All data were extracted by the Ministry of Health, using the interRAI-HC dataset membership as the index. This was done by extracting NHI numbers for all consenting members between 1 July 2012 and 31 May 2014 from this database, and then separately deterministically linking them to the CPSS and National Mortality Collection Register databases. When a NHI match was found, then this populated its respective research dataset, together with the date of admission or death whichever applicable. People changing ARC facilities have multiple admission dates; in these instances the first date was used. The interRAI-HC, CPSS and National Mortality Collection Register research datasets were securely released to the research team, deterministically matched by participants’ encrypted NHI numbers27. Analyses employed competing-risk regression models, using the Fine and Grey method28, and treated ARC admission as the primary event of interest and death as the competing event. Both unadjusted and fully adjusted models were undertaken. Rather than using the bivariable analyses to screen risk factors or potential confounders, in the spirit of Sun and colleagues29, all candidate variables were included in the multivariable model regardless of their statistical significance. Sub-hazard ratios (SHRs) and associated 95% CIs were reported, and Wald’s type III χ2 statistic used to determine a variable’s significance.Reporting of analyses were informed by the RECORD guidelines30, and the Wald’s type III χ2 statistic associated with the hearing ability variable calculated. To establish whether there was a likely causal relationship, rather than simply associative, stratified interaction terms were assessed and reported31. Spearman’s ρ was employed to measure the correlation between ordinal variables.To gauge the extent of confounding explained between hearing ability and ARC admission, a forward stepwise selection process was undertaken with the hearing ability status variable forced into the model. At each step, the variable combination which minimized the Bayes information criterion (BIC) was selectedc-statistic was employed32. Models are typically considered reasonable when the c-statistic >0.7 and strong when >0.833. To derive the c-statistic, the sample was randomly partitioned into two datasets of equal size; the first used as a training dataset to fit the model, and the second used as a test dataset to make prediction assessments32. All analyses and graphics were performed using Stata SE version 15.1 , and α = 0.05 defined statistical significance.To assess the contribution of the hearing ability status variable in the adjusted model, Harrell’s Clearance for this study and its protocol was approved by the Ministry of Health’s Health and Disability Ethics Committee (14/STH/140). All methods were performed in accordance with that Ethics Committee’s relevant guidelines and regulations. The study only included those participants who provided written and informed consent to their data being used for planning and research purposes. The Ministry of Health does not release data to researchers for those who do not provide this consent. As this investigation is a secondary analysis of routine collected de-identified data, written and informed consent was not obtained for this specific study nor was it deemed necessary in the Ethics Committee’s approval. The research databases released by the Ministry of Health contained participants’ encrypted NHI numbers, needed for matching, but has all personally identifying information removed. The encryption code is held by the Ministry of Health and no other.Overall, 47,257 individuals had an interRAI-HC assessment between 1 July 2012 and 30 June 2014. Of these, 2,797 were aged <65 years, 1,907 had their assessment after 31 May 2014, and 8,276 were admitted to ARC or had died within 30 days of their assessment, and so were excluded from the study. This left an eligible sample of 34,277 people, with an average age of 82.1 years . Their demographic profiles are presented in Table 1, Q3) follow-up times were 4.6 months , 6.2 months , and 9.4  for each outcome, respectively. In total, this equated to 25,168.5 person-years participants were at risk and under observation. Of the 6,389 participants admitted to ARC, 2,545 (39.8%) subsequently died prior to the study end-point (30 June 2014). The demographic breakdown of participants by the study outcomes is also contained within Table By 30 June 2014, 6,389 (18.6%) participants had been admitted to ARC, 6,082 (17.7%) had died without entering such care, and 21,806 (63.6%) remained at home. The median  participants were classified as having no hearing, and thus were combined with those having severe difficulty hearing. The distribution of hearing ability is presented in Table % particiApplying competing-risk regression models, treating death as a competing event, hearing ability was significantly associated with ARC admission p < 0.001), and the likelihood of admission appeared to be dependent on hearing ability status; see Table , and theTables p = 0.17), as was the main effect term associated with hearing impairment (p = 0.12). Within each IADL stratum, all the SHR estimates for hearing impairment yielded 95% CIs which included the null value, whereas the pattern of SHR estimates increased similarly and monotonically for IADL categories between the hearing impairment strata – and had overlapping 95% CIs ; not importantly different from that when hearing ability status was excluded with 0.6810 . This represents better than chance prediction but less than the threshold (of 0.7) for a model demonstrating ‘reasonable’ predictive power.Finally, the predictive power of the full multivariable model, fitted on a training dataset and calculated on the test dataset, yielded a Harrell’s 17, a strong and significant relationship between hearing impairment and ARC admission was initially identified in this study. For instance, those with severe hearing difficult or no hearing had odds of ARC admission 1.42  that of their counterparts with adequate hearing. However, like that identified by Evans and colleagues17, this relationship was fully explained by other variables – so that those with severe hearing difficult or no hearing had odds of ARC admission 1.00  that of their counterparts with adequate hearing in the adjusted analyses. In predicting ARC admission, the inclusion of age and IADL into the statistical model rendered hearing impairment as being non-significant (p = 0.81) in its contribution, completely confounding the significant unadjusted relationship. Here, IADL excluded phone use, as it might be considered a direct marker for hearing loss, but included: meal preparation; ordinary housework; managing finances; managing medications; stairs use; shopping; and, transportation . By design, IADLs measure basic functional necessities, no matter where one lives, and typically represent the first areas requiring outside support. This functional assessment tool allows for the monitoring of older adults across the health continuum, from relative independence through to episodes of care34.Consistent with previous literature35 and ARC admission36, and it might also be argued that it lies on the causal pathway between hearing impairment and ARC admission. Informed by the recommendations of Knol and VanderWeele31, stratum-specific SHRs were presented. It was evident from these analyses that hearing impairment was not differentially affected by IADL level, and that its non-significant relationship with ARC admission within each IADL group was consistent across all the IADL quartiles. In contrast, the patterns observed between IADL groups and ARC admission within the hearing impairment groups were similar. If hearing impairment was causally related to ARC admission, a different non-significant pattern would be expected within at least one IADL stratum. Finally, it might be argued that the hearing ability and IADL were largely explaining a similar health domain. However, the weak correlation between these variables makes it difficult to support this notion.It has been recognised that IADL is associated with both hearing impairmentc-statistic < 0.7. This implies that other important unmeasured variables exist in predicting ARC admission beyond a particular individual’s interRAI-HC profile, such as family, community, geographic, economic, and political driving forces2. Notwithstanding, in the final adjusted model, several other potentially mutable factors appear and deserve further investigation, including vision impairment and depression.Predictively, it is noteworthy that the final adjusted model remained relatively weak, with Harrell’s 23. The large number of person-years, high uptake of participants consenting to the use of their data, ability to deterministically link national datasets through encrypted NHIs, and the use of apposite biostatistical methods in competing-risk regression analyses and prediction all add to the study’s strength and robustness20. However, the study is not without limitations. Arguably, the primary weakness centres on the variable of interest; hearing impairment was not determined through comprehensive audiological assessment. Nonetheless, clear differential patterns emerged in the unadjusted analyses and the question itself has strong face validity – which adds strength to this measure. Moreover, this hearing measure has been successfully employed elsewhere38. Another potential weakness is that hearing impairment is likely to be time-varying, yet only a baseline assessment was used here. However, if the purpose is to identify modifiable factors for intervention, then intrinsically this is not problematic but serves to reduce the predictive capacity of the final adjusted model. Another limitation is that the CPSS database only contains approximately 50% of participants who privately fund their ARC admission; which accounts for around 10% of all older adults entering care25. This 10% is likely to have different demographic and socio-economic profiles compared to the 90% contained within the CPSS database, but the extent and consequence of these differences is unknown. Moreover, the 30-days exclusion criterion applied within this study was designed to identify and remove older adults with acute needs or terminal illnesses, and the interRAI-HC assessment was simply a vehicle to expedite ARC admission. Although employed elsewhere25, this period remains arbitrary and the potential for misclassification exists.This large, national, contemporary study derived from a standardised instruments with broad suite of variables and few missing values, administered by a trained workforce has a number of strengths13. Future research establishing the validity of the interRAI-HC hearing impairment variable against audiologically assessed standardised measures would strengthen the robustness of this evidence, as would the replication of this study’s findings in other geographically dispersed populations. Furthermore, future research aimed at developing models which improve ARC admission prediction power, through identification of additional confounders and reduction of measurement variability, is also encouraged. Particularly if these additional confounders are potentially modifiable, and amenable to change. Given the high unmet treatment need, yet detrimental sequelae of hearing loss, it would benefit from routine screening in primary health care for older adults39. Those suffering hearing loss should have treatment options discussed, managed, and be repeatedly assessed. Ultimately, this is likely to both enhance older adults’ health and wellbeing, and significantly reduce the deleterious effects of hearing loss among their families and society at large.Although not found to be statistically related to ARC admission, hearing loss among older adults is common and unmet treatment remains high40. Especially when the current evidence-base is of mixed quality and producing equivocal findings. As hearing loss does not appear to be an independent predictive antecedent to ARC admission, screening for hearing is unlikely to impact on admission rates.While hearing impairment is often underserved in the older population, and there are compelling reasons why some have suggested that hearing loss may be an antecedent to ARC admission, no evidence was found here to support this supposition. The increased likelihood of ARC admission for those with hearing impairments was completely explained by other factors. While a null finding, it has been argued that replication studies are valuable and, indeed, keeps science on trackSupplementary materials for: Hearing ability is not a risk factor for admission to aged residential care of older persons in New Zealand