Proactive interference (PI) is the capacity to resist interference to the acquisition of new memories from information stored in the long-term memory. Previous research has shown that PI correlates significantly with the speech-in-noise recognition scores of younger adults with normal hearing. In this study, we report the results of an experiment designed to investigate the extent to which tests of visual PI relate to the speech-in-noise recognition scores of older adults with hearing loss, in aided and unaided conditions. The results suggest that measures of PI correlate significantly with speech-in-noise recognition only in the unaided condition. Furthermore the relation between PI and speech-in-noise recognition differs to that observed in younger listeners without hearing loss. The findings suggest that the relation between PI tests and the speech-in-noise recognition scores of older adults with hearing loss relates to capability of the test to index cognitive flexibility. Proactive interference (PI) refers to an effect whereby the acquisition of new memories is disrupted by interference from similar information that has been learned previously. PI is a robust phenomenon, having been observed in a variety of contexts including memory for odors  and the Effects of PI have been demonstrated in both long-term and short-term memories . HoweverAnother complex task, known to be predicted by WM span scores is the perception of distorted speech see . This beThe aim of the present study is therefore to investigate whether the speech-in-noise recognition scores of listeners with an age-related hearing loss is significantly related to performance in a visual PI test. Whether this relation differs depending on whether the speech-in-noise task is completed in an aided or unaided condition will also be investigated, along with the degree to which performance in the PI test relates to aided benefit to speech-in-noise perception.A sample of 23 participants 16 male) aged between 65 and 77 years old (mean age = 70 years) were recruited via the audiology clinic at Linköping University Hospital to take part in the study. Listeners were required to be native speakers of Swedish and have a moderate—to—severe  symmetrical sensorineural hearing loss and at least 1 year of hearing aid experience, which was binaural for all participants except one. Participants’ better-ear audiograms are displayed in Figure 6 male agAll testing was completed in one session, lasting approximately 1.5 h. Upon arrival, participants completed a questionnaire about their hearing loss and a pure-tone audiogram was obtained (at frequencies between 125 and 8000 Hz). Participants then completed the PI test and finally, the speech-in-noise recognition test. The order in which these tests were completed was not counterbalanced, as it was expected that fatigue could affect performance in either of these tests, thus we wished to keep the order the same for all participants. In order to reduce the potential effects of fatigue, participants were encouraged to take breaks in between the tests.Six blocks of 10 sentences from the Swedish HINT corpus  were preThe PI test consisted of three blocks of trials. Each block consisted of four lists of seven words, the first three lists belonging to one semantic category  and the final list belonging to a second semantic category . Words were presented orthographically on a computer screen. After the presentation of each list, participants completed a distractor task for 16 s to prevent rehearsal. The distractor task involved participants being presented  with a letter-number sequence  and being asked to continue the sequence . After the distractor task, participants were given 20 s to recall as many words as possible from the list. Participants gave their answers verbally and their responses were noted down by the experimenter. Two outcome measures were then calculated: Resistance to PI , where a lower score indicates greater resistance to PI and Release from PI , where a higher score indicates greater release from PI. See Figure t-tests were used. Correlational analyses were then conducted to investigate the relation between the measures of speech recognition and those of PI. Partial correlations, with the effect of high frequency pure tone average  removed were also conducted to examine the extent to which the relation between the measures of PI and speech recognition was influenced by degree of hearing loss. Reported p-values are based on 1-tailed hypotheses.Prior to analysis, the normality of the data was confirmed, thus parametric tests were conducted. In order to determine whether there was evidence of an effect PI in the data, t-tests confirm significant effects of both resistance to PI  and release from PI  thus replicating the expected effects using this task.The mean number of items in each list correctly recalled in the PI task is depicted in Figure r = 0.47, p = 0.015), with the relation between unaided SIN performance and resistance to PI failing to reach significance .The relation between unaided performance in the SIN test and both resistance to (panel A) and release from PI (panel B) can be seen in Figure r = 0.46, p = 0.015) showing a significant correlation and the relation between unaided SIN and resistance to PI failing to reach significance .Partial correlational analyses, with the effect of HFPTA removed, revealed the same pattern of results with the relation between unaided SIN and release from PI , with the relation between aided SIN performance and resistance to PI failing to reach significance .The relation between aided performance in the SIN test and both resistance to (panel A) and release from PI (panel B) can be seen in Figure r = 0.30, p = n.s.) nor the relation between aided SIN performance and resistance to PI  were significant.Once the effect of HFPTA had been removed, the results of the partial correlational analyses indicated that neither the relation between aided SIN performance and release from PI  nor the relation between aided benefit in the SIN test and resistance to PI  were significant.The relation between aided benefit in the SIN test and both resistance to (panel A) and release from PI (panel B) can be seen in Figure r = –0.25, p = n.s.) nor the relation between aided benefit in the SIN test and resistance to PI  reaching significance.Partial correlational analyses, with the effect of HFPTA removed, revealed the same pattern of results with neither the relation between aided benefit in the SIN test and release from PI (The results of the study provide clear evidence of resistance to and release from PI on a semantically-based word recall task, based on the modified Brown–Peterson paradigm . The finThe results of the study provide evidence of significant effects of both resistance to and release from PI. The magnitude of this effect was greater than that observed in our earlier study on younger listeners with normal hearing . This diIt may also be that, despite the fact that the test of PI used in this case contained no auditory information, that listeners with a hearing loss were disadvantaged anyway, due to the association between hearing loss and cognitive decline (see for example,Due to differences in the methodologies employed, it is difficult to draw direct comparisons between the magnitude of the effects of PI observed in the present study and the results reported in previous studies. However, the only methodological difference between this study and that reported by The results of the study indicate that, in the case of older listeners with hearing loss, release from PI correlates significantly with both aided and unaided speech in noise. This pattern of results differs to that observed in younger adults without hearing loss, for whom resistance to, rather than release from, PI was significantly related to speech-in-noise recognition. Furthermore, the magnitude of the observed effects of both resistance to, and release from, PI were greater in the present study than in our earlier study on young adults with normal hearing .One possible explanation for these results may relate to the fact that older adults are known to have a greater bias to respond in a context-congruent manner and be less able than younger adults to constrain responses to a given category . These tThe fact that that release from PI correlates with speech in noise perception in the unaided condition only is consistent with the ELU model , 2013 ifThe results seem to indicate both that PI is involved in speech perception and that hearing aids facilitate a decreased reliance on cognitive function. The findings seem to be inconsistent with the suggestion that release from PI is an automatic process and unrelated to WM capacity see . In the RE and JR contributed equally to designing the study, interpreting the data and preparing the manuscript. RE collected the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hearing loss is a common feature in human aging. It has been argued that dysfunctions in central processing are important contributing factors to hearing loss during older age. Aging also has well documented consequences for neural structure and function, but it is not clear how these effects interact with those that arise as a consequence of hearing loss. This paper reviews the effects of aging and adult-onset hearing loss in the structure and function of cortical auditory regions. The evidence reviewed suggests that aging and hearing loss result in atrophy of cortical auditory regions and stronger engagement of networks involved in the detection of salient events, adaptive control and re-allocation of attention. These cortical mechanisms are engaged during listening in effortful conditions in normal hearing individuals. Therefore, as a consequence of aging and hearing loss, all listening becomes effortful and cognitive load is constantly high, reducing the amount of available cognitive resources. This constant effortful listening and reduced cognitive spare capacity could be what accelerates cognitive decline in older adults with hearing loss. Normal aging in humans is often accompanied by hearing loss  and normal hearing (N = 75) older adults in a baseline scan and a follow-up scan (on average) 6.4 years later. They found no significant differences between the groups at the baseline measure, but after the follow-up scan those with hearing impairment had an accelerated volume decline in the whole brain, and particularly in the right temporal lobe. In short, the use of more specific, hypothesis-driven definitions of regions of interest, in combination with longitudinal approaches, could aid some light on the mixed effects found when measuring gray matter changes as a consequence of hearing loss.In trying to understand discrepancies in the observed effects of hearing loss and aging on brain morphometry, there are three salient factors: (1) Lack of specificity when defining regions of interest, as explained above ; (2) Measuring the effects of high frequency hearing thresholds vs. average pure tone thresholds. Hearing loss tends to be greater for higher-frequency sounds, and as discussed by Eckert et al. , using tAnother non-invasive brain imaging technique to study brain structure is diffusion MRI (dMRI). This technique measures microstructural parameters, including fractional anisotropy (FA) and mean diffusivity (MD), which reflect properties such as the degree of density and orientation dispersion of neuronal fiber bundles Jones, . It alsoN = 12–15 per experimental group; Profant et al., Microstructural changes associated with hearing loss have been found in subcortical components of the auditory pathway [reduction in fractional anisotropy (FA) and increase in radial diffusivity], such as the lateral lemniscus and the inferior colliculus . By detecting changes in blood flow that occur as a consequence of neural activity, this technique allows indirect measurement of brain function in a non-invasive manner and with a spatial resolution of millimeters , and it is not clear whether it is mediated by age itself or by age-related hearing loss  , aids in adaptive control during word recognition  . In adults, the most prominent peaks are N1 (~100 ms post-stimulus onset) and P2 (~175–200 ms). Smaller peaks P1 and N2, preceding N1 and succeeding P2 respectively, are also often described , resulting in elevated thresholds and reduced amplitudes , Action on Hearing Loss (Project 598), and the Economic and Social Research Council of Great Britain (Grant RES-620-28-0002).The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the absence of a national newborn hearing screening program in Italy, parent associations have been working with the Italian Paediatric Federation (FIMP), the Society of Neonatology (SIN) and members of the Italian Society of Audiology and Phoniatrics (SIAF) to promote guidelines, best practice and training courses in early hearing detection intervention that incorporate sensitivity training for professionals working with families of deaf children. The establishment of the Italian Paediatric Federation’s Audiology Network is the result of an international collaboration between parents and medical professionals designed to promote an effective model in developing Early Hearing Detection Intervention Programs (EHDI) that recognize the role of parents as partners in the process. Among other factors, one important component frequently underestimated in most early intervention programs, both in the USA and in other countries, involves the role of parental involvement within the EHDI process.  From scrAfter five years of working region by region in Italy, the network recently held its first National Pediatric Course of Audiology where the parental voice was fundamental in providing sensitivity training and in recruiting the participation of pediatricians with patients diagnosed with hearing loss.In the absence of a Regional Protocol for Newborn Hearing Screening in Sicily, the Association Io Sento offers assistance to families of children with hearing loss. The Association collaborates with health structures and agencies to offer resources that inform, educate and support families regarding diagnosis, school services, the cochlear implant, speech habilitation by using a network created by information technology.


Weight loss usually decreases energy expenditure (EE) because of changes in body composition (BC). The reduction in EE may contribute, in part, to long-term weight regain. Patients undergoing bariatric surgery might experience a decrease in EE, mainly due to reduced resting metabolic rate (RMR), explained by a decreased lean body mass (LBM), similarly to what occurs to patients after diet-induced weight loss.To assess the effects of Roux-en-Y Gastric Bypass (RYGB) on RMR and BC in severe obese patients after RYGB.This is a prospective cohort study with 28 patients who have undergone RYGB. RMR was assessed prior to surgery and 6 months postoperative by indirect calorimetry (IC). BC was measured at these same time-points using dual-energy X-ray. RMR was adjusted for changes in body weight (BW), i.e., kilocalories per kilogram, and in free fat mass (FFM).2 and a mean BW of 128±19 Kg, half of which composed by fat mass (FM) (50±5%). The mean RMR was 2218±595 Kcal/day. Baseline RMR correlated with FFM   of 49±9 Kg/mWeight loss following RYGB Results in FM as well as LBM reduction, which lead to decrease RMR. Such decrease in RMR may limit weight loss over time and even favor weight regain.


A single non-invasive gene expression profiling (GEP) test (AlloMap®) is often used to discriminate if a heart transplant recipient is at a low risk of acute cellular rejection at time of testing. In a randomized trial, use of the test (a GEP score from 0–40) has been shown to be non-inferior to a routine endomyocardial biopsy for surveillance after heart transplantation in selected low-risk patients with respect to clinical outcomes. Recently, it was suggested that the within-patient variability of consecutive GEP scores may be used to independently predict future clinical events; however, future studies were recommended. Here we performed an analysis of an independent patient population to determine the prognostic utility of within-patient variability of GEP scores in predicting future clinical events.We defined the GEP score variability as the standard deviation of four GEP scores collected ≥315 days post-transplantation. Of the 737 patients from the Cardiac Allograft Rejection Gene Expression Observational (CARGO) II trial, 36 were assigned to the composite event group  and 55 were assigned to the control group (non-event patients). In this case-controlled study, the performance of GEP score variability to predict future events was evaluated by the area under the receiver operator characteristics curve (AUC ROC). The negative predictive values (NPV) and positive predictive values (PPV) including 95 % confidence intervals (CI) of GEP score variability were calculated.The estimated prevalence of events was 17 %. Events occurred at a median of 391 (inter-quartile range 376) days after the final GEP test. The GEP variability AUC ROC for the prediction of a composite event was 0.72 (95 % CI 0.6-0.8). The NPV for GEP score variability of 0.6 was 97 % (95 % CI 91.4-100.0); the PPV for GEP score variability of 1.5 was 35.4 % (95 % CI 13.5-75.8).In heart transplant recipients, a GEP score variability may be used to predict the probability that a composite event will occur within 3 years after the last GEP score.NCT00761787Clinicaltrials.gov identifier The online version of this article (doi:10.1186/s12872-015-0106-1) contains supplementary material, which is available to authorized users. In a landmark article published more than 40 years ago, Dr. Philip Caves and colleagues described the percutaneous transvenous endomyocardial biopsy (EMB) and reported their experience with the 67 cardiac biopsies performed in 17 cardiac transplant recipients . DespiteA number of alternative tests have been developed but have either failed to deliver adequate performance or their implementation proved complex . In contIn addition to the diagnostic utility of the single GEP score, Deng and colleagues noted an association between low variability of serial GEP scores and the clinical stability in patients . These iTherefore, in the current study, we expanded on the earlier findings by conducting an analysis of an independent patient population from the Cardiac Allograft Rejection Gene Expression Observational (CARGO) II study to determine: (1) the prognostic utility of within-patient variability of GEP scores in predicting future significant clinical events; (2) the negative predictive value (NPV) and the positive predictive value (PPV) of GEP score variability in predicting future significant clinical events.The CARGO II study was approved by local institutional review boards or ethics committees of 12 participating institutions (Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), SERGAS. Universidade da Coruña, Spain; University Hospital Muenster, Muenster, Germany; Medical University of Vienna, Vienna, Austria; Hannover Medical School, Hannover, Germany; Toronto General Hospital, Toronto, Canada; Papworth Hospital, Papworth Everard, Cambridge, United Kingdom; Silesian Center for Heart Disease, Zabrze, Poland; Ospedali Riuniti di Bergamo, Bergamo, Italy; Innsbruck Medical University, Innsbruck, Austria; Groupe Hospitalier Pitié-Salpêtrière, Paris, France; University Hospital of Leuven, Leuven, Belgium; Deutsches Herzzentrum Berlin, Berlin, Germany). The study was appropriately registered  and was conducted in accordance with the declaration of Helsinki and Good Clinical Practices . PatientThe CARGO II, a prospective observational multi-center trial, was carried out at 17 academic centers (13 from Europe and 4 from North America). Between May 2005 and February 2009, the study enrolled 737 new and existing heart transplant patients who were ≥18 years old and received post-transplant care at the participating centers. Patients who were enrolled in a double blind trial investigating immunosuppressive drugs were excluded from the study. During the study, data on clinical status, EMB grades, echocardiography results and blood for GEP testing were collected at routine surveillance visits. After the last study visit, all participating centers were asked to collect further outcome data on all previously enrolled study subjects. This follow-up case report form included information on the patient’s vital status and causes of death , re-transplantation and events of graft failure see .For this report, we utilized a case cohort study design, a powerful choice for follow-up studies of multiple event types of interest, particularly in the field of human genomics . We selenot experiencing ≥2R acute cellular rejection [The heart transplant recipients enrolled in the study underwent long-term rejection surveillance, with or without EMB. With regards to GEP testing, prepared, frozen blood samples were shipped in standardized packages to the Clinical Laboratory Improvement Amendments (CLIA) certified laboratory  where the GEP testing was performed .The event cohort consisted of all patients who had an event ≥315 days post-transplant. Previous studies have reported that a mean of 3.5  or 4.4  tests/paOf the 737 patients enrolled in the CARGO II study, 333 were excluded (236 from 5 centers not providing follow-up events data and 97 with only one GEP score). Of the 404 remaining patients with longitudinal GEP scores and at least a 3 year follow-up, 288 patients did not have events and were suitable to be controls, but as these were in abundance, we randomly chose 55 patients to be controls and excluded 233. The final cohort of 91 patients thus included all 36 patients who experienced at least one of the predefined clinical events and were included in the event group and 55 patients without events serving as the control group  was defined as the standard deviation of the four consecutive scores collected ≥315 days post-transplantation. For this calculation, the individual scores used were the direct output of the GEP linear discriminate algorithm (LDA) prior to the step that transforms the LDA score to the non-linear GEP score that fits within the 0–40 scale used for the AlloMap report , according to the study-group assignment. The area under the receiver operating characteristics curve (AUC ROC) was used to evaluate the prognostic utility of GEP score variability in discriminating between low- and high-risk patients with respect to the occurrence of future events. Sensitivity and specificity were determined for all GEP score variability values between 0.1 and 2.1. Using observed event rates from the CARGO II study, estimates and confidence intervals for PPV and NPV were computed. Confidence intervals (CI) for sensitivity and specificity were calculated using the Clopper-Pearson exact method. Confidence intervals for AUC ROC, NPV and PPV were calculated using re-sampling methods. Demographic variables were compared using the two-sample t-test (age) and two-tailed Fisher’s exact test . The comparisons of days between visits were done using the Wilcoxon test and were reported in terms of medians and inter-quartile range (IQR).All statistical analyses were performed using R softwareThe baseline characteristics of patients were well matched between the two study groups Table . The medP = 0.351. In the event group and the control group, the last test was performed at a median of 1489 (IQR 2005) and 1119 (IQR 1262) days post-transplant respectively, P = 0.399. There was no statistical difference between the groups in number of days between the first and the last GEP test; in the event group, the median was 336 days (IQR 147) and in the control group, the median was 315 (IQR 108) days, P = 0.858.In the event group, the median interval between transplant and the first GEP test eligible for the GEP variability computation was 1298 (IQR 1975) days; in the control group the median interval was 794 (IQR 1147) days, Of all patients, 63 % (57/91) had a GEP score variability ≤1.0; 16 % (15/91) of patients had a score variability ≤0.5. The majority of patients had GEP score variability ranging from 0.5 to 1.5 . The distribution of the GEP scores in both study groups is shown in Fig. The estimated prevalence of events in our data set was 17 %. The AUC ROC for the GEP score variability to predict a composite, clinically significant event was 0.72, 95 % CI from 0.61 to 0.81 Fig. .Fig. 4ThThe GEP score variability NPV and PPV performance characteristics are shown in Table In the event group 58 % (21/36) of patients died, 31 % (11/36) experienced graft failure and 11 % (4/36) underwent cardiac re-transplantation.The most important finding of this study is that the GEP score variability may be useful in estimating the probability of future events of death, re-transplantation or graft failure in heart transplant recipients undergoing surveillance with GEP testing ≥315 days post-transplantation. A low variability of sequential GEP scores (≤0.6), which was found in 24.2 % of the study population, rendered NPVs of ≥97.0 %  using a proprietary algorithm . In the future, we would aim to collect larger numbers and/or more specific subtypes of endpoints with sufficient power to be separately analyzed (e.g. deaths from infections or cancers due to long term sequelae of over-immunosuppression). Additionally, the expression levels of the informative genes used to compute GEP scores in some cases may reflect a feature of the status of the recipient’s immune system that is not directly associated with the allograft . PreviouIn our study, the performance of the GEP score variability in predicting outcomes for patients who were monitored with GEP testing ≥315 days post-transplantation  is similar to the performance of the established single GEP score in ruling out acute cellular rejection for patient samples ≥2-6 month post-transplant . The limited PPV of the GEP score variability (Table The GEP score variability may be used in estimating the likelihood of events of death, re-transplantation or graft dysfunction occurring in patients beyond 315 days post-transplant and thus may aid physicians in identifying heart transplant recipients who may need more or less intensive further surveillance. Prospective studies of GEP score variability including a larger number of patients and events from the Outcome AlloMap Registry are desirable in the future.


ILDR1) gene encodes angulin-2/ILDR1, a recently discovered tight junction protein, which forms tricellular tight junction (tTJ) structures with tricellulin and lipolysis-stimulated lipoprotein receptor (LSR) at tricellular contacts (TCs) in the inner ear. Previously reported recessive mutations within ILDR1 have been shown to cause severe to profound nonsyndromic sensorineural hearing loss (SNHL), DFNB42. Whole-exome sequencing of a Korean multiplex family segregating partial deafness identified a novel homozygous ILDR1 variant (p.P69H) within the Ig-like domain. To address the pathogenicity of p.P69H, the angulin-2/ILDR1 p.P69H variant protein, along with the previously reported pathogenic ILDR1 mutations, was expressed in angulin-1/LSR knockdown epithelial cells. Interestingly, partial mislocalization of the p.P69H variant protein and tricellulin at TCs was observed, in contrast to a severe mislocalization and complete failure of tricellulin recruitment of the other reported ILDR1 mutations. Additionally, three-dimensional protein modeling revealed that angulin-2/ILDR1 contributed to tTJ by forming a homo-trimer structure through its Ig-like domain, and the p.P69H variant was predicted to disturb homo-trimer formation. In this study, we propose a possible role of angulin-2/ILDR1 in tTJ formation in the inner ear and a wider audiologic phenotypic spectrum of DFNB42 caused by mutations within ILDR1.The immunoglobulin (Ig)-like domain containing receptor 1 ( Tight junctions (TJs) are specialized structures within the barrier of epithelial cells. TJs prevent the passage of some solutes through the paracellular pathway of adjacent epithelial cells ,2. TJs cTRIC (tricellulin) and ILDR1 (angulin-2/ILDR1) have been reported to cause autosomal recessive nonsyndromic deafness DFNB49 (MIM 610153) [To date, tricellulin, angulin-1/lipolysis-stimulated lipoprotein receptor (LSR), angulin-2/immunoglobulin-like domain containing receptor 1 (ILDR1) and angulin-3/immunoglobulin-like domain containing receptor 2 (ILDR2) have been sequentially identified as protein components of tTJs ,8,9. AmoB49 MIM 6153 [10–1 610153)  and DFNB 610153) ,14, resp 610153) . Interes 610153) .ILDR1 (p.P69H) located in the immunoglobulin-like (Ig-like) domain through whole-exome sequencing (WES) that segregates as an autosomal recessive nonsyndromic sensorineural hearing loss (SNHL) allele in a Korean family. This novel ILDR1 mutant allele is associated with weakened localization of angulin-2/ILDR1 at TCs, as well as a milder auditory phenotype compared with the originally reported DFNB42 profound deafness phenotype. Using protein modeling, we predict that angulin-2/ILDR1 contributes to tTJs by forming a trimer structure through its Ig-like domain, and its variant p.P69H localizes in the interface of the trimer complex to disrupt the stability of the structure.In the present study, we identified a novel mutant allele of This study was approved by the Institutional Review Boards (IRBs) of Seoul National University Hospital (IRBYH-0905–041–281) and Seoul National University Bundang Hospital (IRB-B-1007–105–402). Written informed consent for participation in the study was obtained from participants or from a parent/guardian in the case of child participants (SH23–52). A three-generation pedigree was established for the family (SNUH23) . Among tBoth pure tone audiometry (PTA) and physical examinations were performed on four members . PTA, wiWES was performed on two siblings, SH23–98 and SH23–52, at the Samsung Genome Institute. Genomic DNA was captured using the SureSelect Human All Exon Kit V3  and sequenced using a HiSeq2000 system  with 101 base-paired end reads. Sequence reads were aligned with the human genome reference sequence (hg19) using BWA-v0.6.2 with the default settings. Sam tools v0.1.18, GATK v2.4–9 and Picard v1.88 were used to process SAM/BAM files, perform local realignment, and mark duplicates. Base recalibration and variant calling were performed using the GATK unified genotyper . Variants were also recalibrated using GATK based on dbSNP137, Mills indels, Hap Map, and Omni. Perl script, offered by ANNOVAR, was used to annotate the variants. Only exonic and splicing variants including non-synonymous variants and small indels were filtered. Variants with an allele frequency greater than 1% were discarded based on NHLBI-ESP 6500, 1000 Genomes Project, and an in-house database consisting of 44 Korean individuals. Of the variants identified in both siblings, only the variants that have not been reported in dbSNP137 were included. Low quality reads (< 20) and genotyping (<30) or variant/total depth < 0.2 were ruled out subsequently.The rat anti-tricellulin antibody (Ab) used in this study has been described previously . Mouse aMouse mammary epithelial EpH4 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS). The knockdown of angulin-1/LSR in EpH4 cells and the cDNA sequence encoding human angulin-2/ILDR1 have been described previously . The p.PFor immunofluorescent staining, each clone was cultured on coverslips, fixed with 1% formaldehyde in phosphate-buffered saline (PBS), blocked with 2% bovine serum albumin (BSA) in PBS, and incubated with the above-described primary and secondary antibodies. After washing with PBS, samples were embedded in FluorSave reagents  and observed under a fluorescence microscope  equipped with a cooled charge-coupled device camera  controlled by Power Macintosh G5 and IPLab V3.9.5 software  at room temperature.http://blast.ncbi.nlm.nih.gov/Blast.cgi) and HHsearch (http://toolkit.tuebingen.mpg.de/hhpred). Among these structures, 1XT5 , which demonstrated the highest alignment score, was chosen as the template structure. The three-dimensional (3D) structural model for a single Ig-like domain of angulin-2/ILDR1was then constructed using Modeller  (https://www.rosettacommons.org/). To assess the physiochemical impact of the p.P69H mutation, we built a complex structural model of the Ig-like domains of angulin-2/ILDR1, assuming that the complex of Ig-like domains of angulin-2/ILDR1 formed a homo-trimeric structure based on their colocalization at the extracellular region of three neighboring epithelial cells [https://www.rosettacommons.org/). These structures were then ranked by their interface energy score. After choosing the upper 500 (1%) structures, the most plausible trimeric structure was then selected as the final trimer model. The criteria used were as follows: (i) the putative membrane location for each chain was plausible , (ii) the interface energy score, and (iii) the number of polar interactions between interface residues capable of forming hydrogen bonds. The mutant (p.P69H) trimer model was then constructed from the wild-type trimer model using Fold X (http://foldx.crg.es/) (http://www.gromacs.org/) to compare the dynamic stability of wild-type and mutant trimer models to ultimately assess the impact of the p.P69H mutation.To obtain structural insight regarding the effects of the p.P69H mutation on tricellular localization of the angulin-2/ILDR1 protein, a structural model of the Ig-like domain of angulin-2/ILDR1 was constructed. Since the Ig-like domain is located within the extracellular domain at the site where the mutation occurs, only the Ig-like domain was modeled rather than the entire protein. First, we identified feasible template structures for the Ig-like domain of angulin-2/ILDR1 from its homologs using BLAST (deller/) .. In addal cells . First, crg.es/) . A molecTMPRSS3, the gene encoding transmembrane protease serine 3, is the most frequently detected gene in subjects with ski-slope type hearing loss in the Korean population [TMPRSS3 sequence present in SH23–98 and SH23–52 to identify any mutation(s) located within the exons and/or splice junctions; however, we failed to identify any mutations within this gene.PTA was performed on five family members, three of whom  exhibited a post-lingual onset and partial deafness . SH23–98pulation , we examWe have also performed a rigorous systemic physical examination to detect, if any, subtle abnormalities relevant to the recently proposed role of angulin-2/ILDR1 , howeverMUC12 and CHD3) were excluded by Sanger sequencing. An additional four variants from two genes (BRCA1 and ABCA10) did not co-segregate with the auditory phenotype among the remaining family members. Four homozygous variants from ZSCAN26, NR1H2, VSIG10L, and PLK1S1 were also excluded, since they were recently reported as polymorphisms  in dbSNP138. Finally, MICA and ATXN3 variants were detected in three and four unrelated Korean normal hearing controls, respectively, of 276 chromosomes, suggesting that these variants are Korean-specific polymorphisms. Finally, only one novel homozygous missense variant, c.C206A in ILDR1, resulting in the substitution of p.P69H, remained and was validated by Sanger sequencing. This variant was found in a partially deafened SH23–99 (The whole exomes of two siblings (SH23–98 and SH23–52) with hearing loss were sequenced; the mean depth of the target regions was greater than 80× coverage, with nearly 90% with at least 10× coverage . To iden SH23–99 . The nov SH23–99 . This vap = 1.14 x 10–60) (p = 7.34 x 10–65)  . Tricellx 10–65) . p.R97Q x 10–65) . These rR497X/R497X mice lacking tricellulin at TCs, indicating that tricellulin does not drive angulin-2/ILDR1 localization [The localization of angulin-2/ILDR1 at TCs was reported recently . In the lization . We furtlization  were botTo further evaluate the stability of the model and to evaluate the mutational effect in greater detail, we performed molecular dynamics (MD) simulations. MD simulations were executed a total of eight times . We found that the root-mean-square deviation (RMSD) of residue 69 converged to ~5 Å in the wild-type trimer model, but the RMSD in the p.P69H mutant trimer model did not converge, varying between 3~7 Å .. This rILDR1 most likely accounts for the partial deafness in this family. Less severe, but significant mislocalization of the p.P69H substitution of angulin-2/ILDR1 was noted in angulin-1/LSR knockdown EpH4 cells, further supporting its pathogenic potential. Recently, WES contributed significantly to the detection of a series of new deafness genes [Systematic exclusion of the variants either in the homozygous or compound heterozygous state from 10 autosomal genes based upon a segregation study among family members of SNUH23 indicated that the p.P69H allele of ss genes –27. AlthTMPRSS3 gene [ILDR1 can also manifest as discrete, audiologic phenotypes depending upon the degree of localization of angulin-2/ILDR1 at the TC.A ski-slope type sensorineural hearing loss, now referred to as ‘partial deafness’, characterized by an abrupt downsloping from mild to severe/profound hearing loss across high frequencies, is a challenge for conventional auditory rehabilitation methods, such as hearing aids or cochlear implants. Based upon the progression of hearing loss and the degree of residual low-frequency hearing, appropriate auditory rehabilitation varies from patient to patient. However, the molecular etiology underlying this special configuration of hearing loss has not been elucidated, with the exception of the previously identified recessive mutations within the SS3 gene ,29. DepeSS3 gene ,30, two ILDR1 mutant in this study led to the incomplete recruitment of tricellulin to TCs in LSR knockdown epithelial cells. This incomplete recruitment of tricellulin due to partial mislocalization of angulin-2/ILDR1 could be a direct cause of progressive partial deafness in the patients described in this study since mislocalization of tricellulin causes deafness in humans and mice regardless of angulin-2/ILDR1 [Angulin-2/ILDR1 protein, a component of tTJs, provides a strong barrier property to the epithelium in cultured epithelial cells. The majority of DFNB42-associated mutations within the angulin-2/ILDR1 protein did not localize at TCs in the angulin-1/LSR knockdown epithelial cells . Higashi-2/ILDR1 ,16. The in vivo. Recently, Morozko et al. proposed that angulin-2/ILDR1 was not required for initial recruitment of tricellulin to TCs in vivo. Their data indicated that failure in recruitment of tricellulin itself does not account for the early- onset severe hearing loss in DFNB49 and in angulin-2/ILDR1 deficient in mice. They observed gradual mislocalization of tricellulin at TCs after the first postnatal week in angulin-2/ILDR1 deficient mice [in vivo results contradict our in vitro findings showing that tricellulin localization was tightly correlated with that of angulin-2/ILDR1 in angulin-1/LSR knockdown EpH4 cells. The presence of other tight junction proteins in vivo, such as angulin-1/LSR and occludin could contribute to this discrepancy; LSR is co-expressed with angulin-2/ILDR1 in the inner ear [in vitro system may be able to recruit tricellulin to the TCs in the inner ear in the absence of angulin-2/ILDR1 expression. Given these in vivo findings, it is also possible that the mutation within ILDR1 may lead to deafness via a different mechanism unrelated to failed tricellulin recruitment at TCs, or through a combination of these mechanisms. Rather, we proposed a direct effect of ILDR1 alterations upon the tTJ structure irrespective of the status of tricellulin recruitment, based upon our protein modeling of the Ig-like domain. In fact, the position of the p.P69H mutation within the Ig-like domain and the instability of the predicted homo-trimer complex of angulin-2/ILDR1 caused by this mutation suggest that p.P69H may exert pathogenicity irrespective of the failure to recruit tricellulin at TCs. Specifically, the Ig-like domain of angulin-2/ILDR1 shares the same motif as other cell adhesion molecules (CAM) [ILDR1 knockout or knock-in mice of a specific ILDR1 mutation, as well as further biostructural studies, will be needed to address this issue. Indeed, freeze-fracture electron microscopy has revealed alteration of the ultrastructure of inner ear tTJ from angulin-2/ILDR1 deficient mice [However, there may be another mechanism for hearing loss due to alteration of angulin-2/ILDR1 ent mice . These inner ear , and occnner ear . Therefoes (CAM) , indicates (CAM) . It is des (CAM) . If we aent mice .ILDR1 variants  [ILDR1 variants reported thus far, including the three variants described herein, are associated with severe or a more severe degree of flat-type hearing loss [R497X/R497X mice, which lack tricellulin at TCs at P16, revealed that cochlear hair cell degeneration was more pronounced in the mid and basal turns compared with the apical turn [Results from our study are intriguing, in that a milder auditory phenotype characterized by a ‘partial deafness’ was observed in all three siblings from this family. A higher degree of hearing loss with a flat or slightly downsloping configuration was the main audiological feature of the DFNB42 family . What acerfsX15) , albeit ing loss ,13,14. IILDR1 could result in the auditory phenotype that is more pronounced in mid and high frequencies. Disruption of the tTJ, due to abnormal angulin-2/ILDR1, may damage the organ of Corti preferentially in the mid and basal turn of the cochlea. To date, only three ILDR1 variants, including p.P69H, have been reported to reside in this domain. The p.Asn109_Pro111dup mutation, reported in a consanguineous Saudi family, was associated with a non-progressive downsloping audiogram from severe to profound across higher frequencies. The residual low-frequency hearing in this Saudi family was not as significant as that in our study [Alternatively, mutations within the Ig-like domain of ur study . With reur study , the detILDR1 in this study was compatible with aggravated hearing thresholds in a mouse model lacking tricellulin (Tricp.R497X/p.R497X mice) [ILDR1 mutations suggest that the pathogenic mechanism of p.P69H may differ from those of other variants. It may be that the p.P69H mutation in the Ig-like domain affects TJ structure in the inner ear to the extent that the inner ear sensory epithelium develops normally and can be maintained for a while, but subsequently begins to degenerate due to a toxic microenvironment in the extracellular space of the hair cells especially from the basal turns. In addition, this progression should be considered for appropriate auditory rehabilitation of partial deafness.It should also be noted that significant progression of hearing loss was observed in homozygous carriers of p.P69H in this study. This is in contrast to a previous description in which DFNB42 deafness was characterized by bilateral non-progressive moderate to profound sensorineural hearing impairment . This wo7X mice) . It has 7X mice) . SignifiILDR1 gene associated with partial deafness, and identified partial mislocalization of the mutant angulin-2/ILDR1 protein at TCs. More importantly, we have proposed a new pathogenic mechanism of ILDR1 mutations residing in the Ig-like domain, based upon the calculated effect of our novel ILDR1 mutation on the stability of a predicted trimer model.In this study, we reported a novel missense mutation within the S1 FigThe red and orange residues are the 69th and 97th residue, respectively. Those two residues are the sites for the mutation p.P69H and p.R97Q, respectively.(TIF)Click here for additional data file.S2 FigThe MD simulations executed 8 times; 4 times for a wild type model (wt1~wt4) and 4 times for a mutant model (m1~m4). The RMSD values tend to converge on around 5 Å for the wild type model, while they tend to widely vary between 3~7 Å for the mutant model.(TIF)Click here for additional data file.S1 TableStatistical analysis was performed using Picard with the module ‘CalculateHsMetrics’. ‘UNIQUE READS’ represent the number of reads not marked as duplicates. ‘UQ READS ALIGNED’ represent the number of unique reads that align with a mapping score > 0 to the reference. ‘ON+NEAR BAIT’ represents the percentage of on + near bait bases against bases aligned, and ‘OFF BAIT’ represents the percentage of aligned bases that mapped neither on nor near a bait. ‘ZERO CVG TARGETS PCT’ represents the number of targets that did not reach coverage = 2 over any base. PCT: percent, UQ: unique, CVG: coverage.(DOCX)Click here for additional data file.


The aims of this study were to (1) develop a series of short interactive videos (or reusable learning objects [RLOs]) covering a broad range of practical and psychosocial issues relevant to the auditory rehabilitation for first-time hearing aid users; (2) establish the accessibility, take-up, acceptability and adherence of the RLOs; and (3) assess the benefits and cost-effectiveness of the RLOs.The study was a single-center, prospective, randomized controlled trial with two arms. The intervention group  received the RLOs plus standard clinical service including hearing aid(s) and counseling, and the waitlist control group  received standard clinical service only. The effectiveness of the RLOs was assessed 6-weeks posthearing aid fitting. Seven RLOs  were developed using a participatory, community of practice approach involving hearing aid users and audiologists. RLOs included video clips, illustrations, animations, photos, sounds and testimonials, and all were subtitled. RLOs were delivered through DVD for TV (50.6%) and PC (15.2%), or via the internet (32.9%).RLO take-up was 78%. Adherence overall was at least 67%, and 97% in those who attended the 6-week follow-up. Half the participants watched the RLOs two or more times, suggesting self-management of their hearing loss, hearing aids, and communication. The RLOs were rated as highly useful and the majority of participants agreed the RLOs were enjoyable, improved their confidence and were preferable to written information. Postfitting, there was no significant between-group difference in the primary outcome measure, overall hearing aid use. However, there was significantly greater hearing aid use in the RLO+ group for suboptimal users. Furthermore, the RLO+ group had significantly better knowledge of practical and psychosocial issues, and significantly better practical hearing aid skills than the RLO− group.The RLOs were shown to be beneficial to first-time hearing aid users across a range of quantitative and qualitative measures. This study provides evidence to suggest that the RLOs may provide valuable learning and educational support for first-time hearing aid users and could be used to supplement clinical rehabilitation practice. A series of seven multimedia interactive videos (or reusable learning objects [RLOs]) covering a range of practical and psychosocial information were developed for first-time hearing aid users. A prospective randomized controlled trial of 203 patients showed that the intervention group that received the RLOs had better knowledge of hearing-related issues, better practical hearing aid skills, and greater hearing aid use in suboptimal hearing aid users, compared with the control group at 6 weeks post-fitting. RLOs were rated as highly useful, and user feedback on the RLOs was very positive. Take-up was high and adherence was at least 67%. RLOs provide valuable educational support.Supplemental Digital Content is available in the text. Hearing loss results in speech perception and communication difficulties, which in adults can lead to social withdrawal, depression, employment problems, an increased risk of dementia, and reduced quality of life . ProvisiDespite evidence that hearing aids are effective in providing hearing-specific benefits and improved quality of life , a signiProvision of high-quality information by audiologists to hearing aid users, particularly those using them for the first time, may help to address many of the above issues. Typically, much of the information offered in a clinical setting is delivered verbally, with the result that many patients forget the information given to them. As follow-up appointments are not routine in the UK National Health Service , the impKnowledge of hearing-related matters in the general public is generally poor , and eveRemote delivery of a more cost-effective individual home-communication program delivered by videotapes showed improvement in the use of communication strategies and improved quality of life and satisfaction at a 6-month follow-up . SimilarIn other fields, educational and psychological research provides evidence that visual approaches can enhance learning and motivation by providing concrete depictive representations of subjects to be learned . In the Internet delivery of educational programs, along with other teleaudiology applications, has the added potential of being accessible to many people, including hard-to-reach populations, provided they have access to the internet . While tDevelopment of an intervention should be based on an identified evidence base and an underlying theory. Feasibility of the intervention informs recruitment and retention of participants, delivery, accessibility and take-up of the intervention, in addition to acceptability and adherence of participants with the intervention. The evaluation phase assesses the effectiveness of the intervention, ideally by an RCT to prevent selection bias, where selection of appropriate outcome measures is crucial. The final stage, implementation, ensures that the intervention translates into clinical practice and assessment of long-term outcomes, to show whether the intervention can generalize to wide-scale implementation and effectiveness.We aimed to develop and evaluate a series of RLOs for first-time hearing aid users, based on guidance on the development, evaluation, and implementation of complex interventions provided by the The specific aims of this research were to:develop a series of short interactive RLOs to cover a broad range of auditory rehabilitation content, both practical and psychosocial, which could be accessible to hearing aid users and their family and friends;establish accessibility, take-up, acceptability, and adherence of the RLOs;assess the benefits and cost-effectiveness of the RLOs in first-time hearing aid users.This study is reported in accordance with the CONSORT statement  that offAdult first-time hearing aid users were recruited via the Nottingham Audiology Service at the Nottingham University Hospitals NHS Trust who had been referred for hearing assessment by their family doctor. Of the 553 patients who attended the assessment appointment and had not previously worn hearing aids, 203 (36.7%) were willing to take part and met the inclusion criteria. These were (1) adults ages ≥18 years, (2) first-time hearing aid users, and (3) spoken English as first language or good understanding of English. Exclusion criteria were those who were unable to (1) access DVD, PC or internet, or (2) complete questionnaires due to age-related problems.The design was a prospective, registered clinical RCT with two arms. The intervention group  received the RLOs plus standard clinical service including hearing aid(s) and counseling, and the waitlist control group  received standard clinical service only. Patients who met the eligibility criteria at the hearing assessment appointment were asked by the clinical audiologist if they were interested in participating in the study. Details of the study, including randomization and offer of the RLOs to the control group postevaluation, were explained both verbally and in the participant information sheet. Informed, written consent was obtained by the audiologist at the hearing aid fitting appointment approximately 4 weeks later to meet the ethical requirement of at least a 24-hr consideration period. Eight experienced clinical audiologists received training in the consent procedure, as well as how to use and demonstrate the RLOs as part of the study protocol by MF and MB.Postconsent, participants were allocated to one of two groups. Allocation was based on a computer-generated pseudo-random code using random arrangement of blocks of randomly varying size, created by the Nottingham Clinical Trials Unit to provide a robust randomization method. Allocation to groups was in the ratio of 1:1, stratified by age , and made by the recruiting clinical audiologist who accessed a remote-controlled randomization system managed by the Nottingham Clinical Trials Unit. Allocations were revealed to the research team on completion of the study.Those who were allocated to the RLO+ group chose their preferred RLO delivery method from one of four options: (1) interactive DVD for television, (2) interactive DVD for PC, (3) interactive RLOs via the internet, and (4) autoplay DVD for television with no interactivity. The DVD was given at the hearing aid fitting appointment, and access to the internet was given within 1 to 2 days of receiving the hearing aid. Participants were requested to watch all the RLOs, preferably no more than two per day to minimize information overload.Participants attended an evaluation session approximately 5 to 8 weeks postfitting . The research audiologists (n = 2) were blinded as to whether the participants had received the RLOs or not. Before attending the evaluation session, the participants were requested not to reveal their group until the end of that session, to minimize researcher bias in outcome measurement. On completion of the evaluation session, the RLO− group was offered the RLOs although there was no further evaluation. The primary outcome measure was hearing aid use measured by the Glasgow Hearing Aid Benefit Profile GHABP, . On the The study was approved by the Nottingham Research Ethics Committee and Nottingham University Hospitals NHS Trust Research and Development department. Participants were paid a nominal inconvenience fee and travel expenses to attend the evaluation session.RLOs were developed using a participatory, community of practice approach, and were based on pedagogical design principles  describehttp://links.lww.com/EANDH/A236). The RLOs were based on pedagogical principles, introducing specific learning outcomes at the outset, reinforcement of good behaviors, and explaining the consequences of poor behaviors, with an interactive multiple-choice quiz at the end of each RLO so the user could see what they had learned. The RLOs included video clips, illustrations, animations, photos, sounds and testimonials, and all were subtitled. The importance of psychosocial aspects of hearing loss was evident from the Delphi review and workshops. Therefore, aspects such as emotions, confidence, and involvement of friends and family members were included in the Expectation, Adaptation and Communication RLOs.There were seven RLOs , with duration between 4 and 11 min . There was also a short introduction (2.8 min), and altogether the total duration of the RLOs was 58.7 min. Sample RLO clips can be found in the videos in Supplemental Digital Content 1 (The interactive nature of the RLOs included (1) choice of ear mold ; (2) choice of RLOs from a user-friendly interface; (3) the option to rewind, fast-forward, and pause; and (4) the interaction with a two- or three-question multiple-choice quiz, based on a three-option, three-alternate forced-choice paradigm, after each RLO via the TV remote control handset or PC mouse. After selecting the answer, the participant was shown whether this was correct or not, along with some additional advice, before moving onto the next question. Participants were able to replay the quiz as many times as they wished. An autoplay version was developed for those unable to use a TV remote control handset. Using a platform for DVD delivery meant that the participants’ interactivity with RLOs was limited compared with that for RLOs developed specifically for internet delivery. However, given the low PC and internet use in the typical first-time hearing aid user group, we decided the compromise between lower interactivity and higher accessibility for typical first-time hearing aid users was acceptable.Hearing aids  were fitted, programmed using the NAL-NL1 algorithm and verified by real-ear measurement in accordance with local protocols and national guidelines . HearingPure-tone air conduction thresholds were measured at octave frequencies (0.25 to 8 kHz) for each ear, and bone-conduction thresholds as required (0.5 to 4 kHz), following the procedure recommended by the All questionnaires were completed by interview at the evaluation session unless specified otherwise.GHABP  that has been previously validated  using data logging information integral to the hearing aid was obtained for each participant for the period between the fitting and evaluation appointments.Video Diary recorded how often and when  a participant viewed each RLO, and how useful each RLO was . Participants were instructed to watch all the RLOs, preferably across several days, and to concurrently note their comments.The RLO Feedback Questionnaire was adapted from a questionnaire from the RLO Center for Excellence in Teaching and Learning toolkit  were led by two authors (PL and MF), and included participants from the RCT (n = 20) and their communication partners (n = 5). The aims were to obtain feedback on the RLOs in terms of content, how they were used and how they affected participants’ hearing and communication. In addition, topic ideas and feedback for future RLO development was sought. The focus groups were recorded, transcribed, and analyzed using thematic analysis according to t test or Mann–Whitney test. Group means (or median) and the 95% confidence intervals were presented as planned. For significant results, an adjusted analysis using multiple linear regression, adjusting for age, gender, and hearing loss (better-ear average [BEA] for 0.25 to 4 kHz) was performed. Bonferroni correction to account for multiple comparisons was applied where necessary. For outcome measures obtained at both fitting and evaluation (HADS and PAM), an analysis of variance was performed with “group” and “visit” as factors. Mode of RLO delivery was not a covariate. Effect size (Cohen’s d) was categorized as small (0.2), moderate (0.5), and large for (0.8). Significance was set to p ≤ 0.05.Planned analysis initially examined the difference between groups for the primary outcome measure (GHABP hearing aid use) and the secondary outcome measures obtained at evaluation, using either an independent For the cost-effectiveness analysis, responses from the EQ-5D were transformed into a quality-adjusted life year (QALY) score using the scoring metric as described by p < 0.001) for age, gender, and hearing loss between those who participated in the RCT (P+) and those who had a hearing test but did not participate (P−). Age was lower in the P+ group , there were fewer females in the P+ group  and the BEA was better  than in the P− group.Demographic and clinical characteristics of those who 1) were initially assessed, (2) were eligible and consented at the fitting appointment, and (3) participated in the evaluation session are shown in Table  were iniOf the 553 patients who attended the hearing assessment appointment who had never worn hearing aids, 370 (66.9%) met all the eligibility criteria, and of those 203 (54.9%) consented to participate in the study. The numbers recorded who did not meet the eligibility criteria were poor understanding of English, n = 32 (5.8%), unable to access a DVD, PC, or the internet, n = 116 (20.9%), unable to complete the questionnaires, n = 53 (9.8%), or fully understand the study requirements, n = 40 . Of the four delivery formats used, DVD for TV was the most commonly used (50.6%), followed by internet delivery (32.9%), DVD for PC (15.2%), and DVD autoplay (1.3%).Of those who were eligible to participate at the hearing assessment, 290 (78.4%) expressed an interest in participating in the study at the hearing assessment. At the fitting appointment, one person who was eligible declined to consent because they were not interested in watching the RLOs. Adherence in those who attended the evaluation session and watched the RLOs was very high , with only two participants failing to watch any of the RLOs. Video diaries were completed by 71 (89.9%) participants, and of those, 67 (94.3%) participants watched all seven RLOs . Of the four participants who did not watch them all, two participants watched five RLOs and two participants watched three RLOs, one of whom had difficulties accessing the RLOs online as he used the Linux operating system. The average number of views per participant was 13.0 . On average, around half (49.2%) the participants watched the RLOs at least twice, and around a fifth (22.1%) watched the RLOs at least three times, with some participants watching the RLOs as many as seven times (Table p = 0.02). Reasons given for nonattendance were health problems (n = 8), personal not study related (n = 5), poor mobility (n = 2), husband ill/died (n = 2), could not take time off work (n = 2), no longer wishes to take part (n = 2), hearing better, no longer wears aids (n = 1), no reason obtained .Attrition between the fitting and evaluation sessions was slightly higher than anticipated at 17.8% (n = 39), and slightly more for the RLO+ group (20.3%) than the RLO− group (15.0%). There was no significant difference in age and hearing loss between those who attended and did not attend the evaluation session . There was, however, a significant difference in gender, with more women (28.4%) dropping out at evaluation session than men . Similarly, there was no group difference for hearing aid use measured by data logging .There were significant differences between the two groups for overall practical hearing aid handling skills , and overall knowledge of hearing aids and communication issues . These remained significant after Bonferroni correction. None of the other outcome measures showed a significant between-group difference.Table t(37) = −2.3; p = 0.03], where the RLO+ group used their hearing aids on average 15.2% more than the RLO− group , with a large effect size , these results were no longer significant after applying Bonferroni corrections. There were 5 (5.6%) hearing aid nonusers in the RLO− group compared with none in the RLO+ group.Secondary analysis assessed for hearing aid use (GHABP) in suboptimal hearing aid users. There is no consensus in the literature on what constitutes suboptimal use, so we chose GHABP use <70%, which was approximately the mean use from a large sample of hearing aid users who had hearing loss in the better ear at 30 to 39 dB HL . There wZ = −3.4; p = 0.001) and Cleaning Ear mold domains . These remained significant after Bonferroni correction. There was no significant difference between the groups for hearing aid insertion/removal  and battery door/change . A quantile regression analysis of PHAST overall score, accounting for age, gender, and BEA, showed there remained a significant effect of group (p = 0.002) and there was a significant effect of age (p = 0.04), where older people had poorer practical skills, but no effect of gender (p = 0.10) or BEA (p = 0.11).Table t(139) = 5.5, p < 0.001, d = 0.94], and both the practical  and psychosocial questions  nor visit  nor any significant interaction. For the PAM, there was no significant main effect of group  nor visit  nor any significant interaction.There was no significant effect of group for the remaining outcome measures obtained at the evaluation session Table . The HADcontent, the vast majority (>92%) agreed the illustrations and videos aided their understanding and the RLOs held their interest. For activity and engagement, 91% agreed the interactive quiz gave a clear message, and 88% would refer back to RLOs if they had a problem. For self-assessment, there was high agreement (>88%) that the quiz was valuable and gave clear messages as to what was right and wrong.Table In addition, the top five key words chosen by each person from a list of 60 words to describe the RLOs are shown in the word cloud in Figure Three focus groups, with between 7 and 10 participants, were held comprising (1) 20 people who had participated in the RCT, and were representative of the RCT sample , and (2) five communication partners . The key themes from the thematic analysis of the postevaluation focus groups were that the RLOs were aligned to people’s experiences, and the content was supported by the vast majority.I did find it very helpful especially in areas when they talked about sounds and how loud things were because that quite shocked me at first, but then watching the video I found that was a normal reaction…I think the information they provided was fantastic. I got everything I wanted.…I have watched it and I found it very useful. The first time was little tips on how to put hearing aids in, that was good. One interesting thing was how your brain learns what is loud and what isn’t loud and adjusts accordingly.Although not everyone was positive about the RLOs.I don’t think there was so much need to prolong everything so much and to talk as though we still couldn’t hear.My daughter said it was very long…you could have put what they said in three minutes, not six minutes.Other themes included:1. repeated watching of RLOs… I found that I had missed something. So I went back and looked at it again,2. sharing of videos with others, such as family, friends, and neighborsI have passed my DVD on to an old couple who both have hearing aids … I kept telling her, Play that DVD and you will know why, because you have got to get used to it, haven’t you?3. the RLOs provided reassurance, helped people to remember things and improved awareness and confidenceIt [the DVD] explained how we have to learn to rehear things. That is not an aspect that I [was aware of], to re-educate the brain to interpret what you hear.4. involvement of family members and friendsWell, I went through them, right the way through. I begged my wife to watch them as well which I thought was important.I sat my husband down. I said, I would like you to watch this. Well he said, What do I need to watch this for? I am not deaf, but I said, It will help you to understand me. So he did…. yes, I did find it very, very useful for that.p = 0.91) and 6-week follow-up QALYs (p = 0.25) nor for incremental QALYs (p = 0.68). Using costs of the service from the healthcare purchaser’s perspective, there was a significant difference in total costs between both the control and intervention groups of −£0.90 (p < 0.0001). The mean ICER was −£479.41 (95% CI = −£20784 to £16155), indicating the DVD intervention was dominant over usual care (as it was more effective and cheaper). At a willingness to pay of £20,000 per QALY  for first-time hearing aid users, assess the feasibility of delivering the RLOs to those attending hearing aid fitting appointments in an audiology clinic, and then evaluate the effectiveness of the home-delivered RLOs using an RCT, based on a framework for developing and evaluating complex interventions .RLO content was developed using pedagogical principles from learning theory by taking a participatory approach that involved both hearing aid users and audiologists using a Delphi review, focus groups and workshops and iterative feedback during development . The parRecruitment of the participants in the present study differed from other studies that have evaluated remotely delivered educational programs. These generally recruited existing users by advertising through the media thereby recruiting later in the patient journey as well as running the risk of sampling biases, such as self-selection and being better educated  of 65- to 74-year-olds was 26.2% . Seven people did not have the means to play the DVD, three did not want the DVD, and one could not understand English.Acceptability of the RLOs was high, as demonstrated by take-up, ratings of usefulness, usability and desirability, the feedback questionnaire and postevaluation focus groups. The highly useful RLO rating (8.9/10) was consistent with the usability descriptors where “useful” was listed in the top three choices. There was high agreement on the feedback questionnaire for all three RLO components . Practically, the RLOs were not difficult to use and were pitched at the right level. In terms of impact on their everyday lives, participants agreed they would be more likely to contact Audiology, and the RLOs gave them more confidence to discuss hearing aids and communicate with others. This suggests the RLOs were more than simply a tool to enhance information and knowledge in that they also had a positive impact on other activities and actions in their lives. Improved confidence has been suggested to be a critical element in the success of an intervention , only 67% watched all the RLOs. One of the interesting findings was that RLO reuse was considerable with at least half watching the RLOs two or more times, and some using them up to six to seven times. This reusability, which was also evident in the postevaluation focus groups, suggests that the participants were using the RLOs as a means to self-manage their hearing loss, hearing aids, and communication. This is an important finding as it is recognized in other health domains that patients who are motivated and actively participate in their care are more likely to adopt health behaviors that then lead to better patient outcomes , although there were no nonhearing aid users in the RLO+ compared with the RLO− group (n = 5). However, for suboptimal users (GHABP hearing aid use <70%), there was significantly greater use in the intervention (RLO+) group compared with the control (RLO−) group. It could be argued that improvements in hearing aid use were shown where it mattered, which is when hearing aid use is lower than it could be. This suggests that if clinical resources are limited, RLOs are best targeted at those who are less likely to use their hearing aids optimally. Difficulties in noisy situations and in background noise are a major reason for nonuse of hearing aids  and althThere were benefits for the RLO+ compared with the RLO− group in terms of significantly better practical hearing aid skills and better knowledge on hearing aids and communication. The effect sizes were generally large, which is important when considering implementing an intervention into clinical practice as the clinical significance of the intervention is as important, if not moreso, than the statistical significance .For practical hearing aid skills, there was no group difference for hearing aid insertion/removal and battery functions, where both groups performed at ceiling. This probably reflects the importance audiologists place on ensuring that new users can manage the basics of hearing aid and battery insertion before leaving the clinic as an inability to do either renders the hearing aid virtually useless. Despite this, other studies have shown that difficulties with ear mold insertion and batteries are problematic in between 9 and 17% . InteresKnowledge of psychosocial issues  was significantly poorer than practical issues  in both groups. This suggests that either psychosocial issues receive less attention in hearing aid appointments or retention of knowledge of the more complex psychosocial issues is poorer. Either way those who received the RLOs had significantly better knowledge of hearing aids and communication 6-weeks postfitting, with large effect sizes, indicating that the RLOs facilitated improved knowledge and awareness of hearing-related issues. Furthermore, There were no between-group differences in the remaining hearing aid outcome measures . The results of the IOI-HA were similar to those reported in other studies of educational programs . HoweverThere were no between-group differences in the remaining hearing aid outcome measures . The results of the IOI-HA were similar to those reported in other studies of educational programs . HoweverThe RLOs were shown to be cost-effective in just over two-thirds of users. Such a finding with the EQ-5D is not common in hearing research as the EQ-5D is usually insensitive to hearing-related interventions . The relThe choice of outcome measures to assess the benefits of interventions is a major issue in adult rehabilitation research. There is no “gold standard” measure for “patient benefit” or consensus on which outcome measure(s) are optimal, and many studies use multiple measures to tap into different domains . HoweverThe long-term vision is to have educational resources, such as these RLOs, available to all hearing aid users in the UK. To this end, we have partnered with industrial and third sector partners and the RLOs, now branded as “C2Hear,” have been available to the UK audiology centers (NHS and independent sector) since end of 2014. An online version (C2Hear Online) is now freely available on YouTube. Further research needs to done to identify real-world benefits in a late phase clinical trial.There were a number of limitations in this research. First of all, there was no active control group, so the participants could not be blinded with respect to the intervention. Similarly, although a number of precautions were taken to blind the research audiologists to the intervention and control groups , the explicit request for participants not to reveal their group was not always successful. A double-blind trial with an active control group  should be considered in future studies of this type. There was no long-term follow-up, which has been identified as major problem in hearing rehabilitation research . This waWith the increasing functionality available of online and smartphone technology, there are a number of potential future developments. Individualized tailoring of resources would provide a more user-centered intervention to meet individuals’ needs rather than the current “one-size-fits-all” approach taken in this research. This would allow the more technologically able to explore new technologies and reduce the basics, whereas for the less able, the converse would be more relevant. New technological developments in multilingual mobile translation  could alThis study developed an educational program for first-time hearing aid users based on the concept of RLOs. This concept has been trialed for the first time in audiology in one of the largest RCTs of an educational intervention for individualized use in adult auditory rehabilitation to date. The results of this interactive, multimedia educational intervention showed a range of benefits, suggesting that it may be a valuable supplement to the clinical management of first-time hearing aid users.http://www.hearing.nihr.ac.uk/, or view the C2Hear Online RLOs via YouTube.The authors thank Holly Thomas for collecting the data and Victoria Owen and Matthew Jones for analyzing some of the data. Special thanks to the media developers James Henderson and Michael Taylor, the public patient involvement panel , and our willing video stars  for their valuable input into developing the RLOs. The authors gratefully acknowledge the audiologists at Nottingham Audiology Services who recruited the participants , and special thanks to Helen Bastow. The authors also thank Ashana Tittle and Sandra Smith for their help with the data and Martin Morrison for setting up the internet portal. To view C2Hear Online RLOs, please go to the NIHR Nottingham Hearing Biomedical Research Unit website


Finally, DDAH1+/− and DDAH2−/− deficient mice were studied in the oxygen-induced retinopathy (OIR) model, a model used to emulate retinal ischemia and neovascularization, and VEGF and ADMA levels were quantified by ELISA and liquid chromatography tandem mass spectrometry. In the OIR model, DDAH1+/− exhibited a similar phenotype compared to wildtype controls. DDAH2 deficiency, in contrast, resulted in elevated retinal ADMA which was associated with attenuated aberrant angiogenesis and improved vascular regeneration in a VEGF independent manner. Taken together this study suggests, that in retinal ischemia, DDAH2 deficiency elevates ADMA, promotes vascular regeneration and protects against aberrant angiogenesis. Therapeutic inhibition of DDAH2 may therefore offer a potential therapeutic strategy to protect sight by promoting retinal vascular regeneration and preventing pathological angiogenesis.Ischemia-induced angiogenesis is critical for tissue repair, but aberrant neovascularization in the retina causes severe sight impairment. Nitric oxide (NO) has been implicated in neovascular eye disease because of its pro-angiogenic properties in the retina. Nitric oxide production is inhibited endogenously by asymmetric dimethylarginines (ADMA and L-NMMA) which are metabolized by dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2. The aim of this study was to determine the roles of DDAH1, DDAH2, ADMA and L-NMMA in retinal ischemia-induced angiogenesis. First, DDAH1, DDAH2, ADMA and L-NMMA levels were determined in adult C57BL/6J mice. The results obtained revealed that DDAH1 was twofold increased in the retina compared to the brain and the choroid. DDAH2 expression was approximately 150 fold greater in retinal and 70 fold greater in choroidal tissue compared to brain tissue suggesting an important tissue-specific role for DDAH2 in the retina and choroid. ADMA and L-NMMA levels were similar in the retina and choroid under physiological conditions. Next, characterization of DDAH1 •Nitric oxide has been implicated in neovascular eye disease.•Key inhibitor of NO production is ADMA, which is metabolized by DDAH.•DDAH2 deficiency results in elevated ADMA and reduced neovascularization in mice.•Therapeutic inhibition of ADMA or DDAH2 may offer a potential therapeutic strategy. In the mature retina, however, ischemia-induced angiogenesis is typically misdirected into the vitreous gel where it not only fails to redress retinal ischemia but also exacerbates impairment of sight with haemorrhage into the vitreous gel and tractional retinal detachment . Patholol-arginine residues, and are metabolized to citrulline and dimethylamine by dimethylarginine dimethylaminohydrolases-1 (DDAH1) and -2 (DDAH2)  promotes vascular dilatation by cGMP-induced smooth muscle relaxation . In the  (DDAH2)  (Leiper  (DDAH2) . These t (DDAH2)  suggesti (DDAH2) .Here we show that in retinal ischemia, DDAH2 deficiency elevates ADMA, promotes retinal vascular regeneration and protects against aberrant neovascularization.22.1DDAH1+/− knockout mice were generated as previously described  and bred to generate homozygous DDAH2−/−, heterozygous DDAH2+/− and wildtype DDAH2+/+ mice. All animals were managed in accordance with the guidelines of the Association for Research in Vision and Ophthalmology. In all experiments, weight-matched wildtype littermates were compared to circumvent inter-litter variability.escribed . Since g+/− mice . Heteroz2.22 in an oxygen supply chamber from postnatal day (p) 7 to p12, returned to room air on p12 and culled at p12 or p17 as described elsewhere  as previously described  controls. At p17 however, heterozygous DDAH2+/− knockout mice developed greater revascularization of the area of retinal vascular ablation, resulting in less extensive ischemia, and less extensive aberrant pre-retinal neovascularization. Having identified an effect of DDAH2 haploinsufficiency we then determined that in DDAH2 null (DDAH2−/−) mice the magnitude of this response to OIR was greater still , a model of retinal ischemia-induced neovascularization. In OIR, exposure of young mice to hyperoxia  from postnatal day 7 (p7) results in ablation of immature retinal vasculature. On return to room air at p12 the ischemic retina becomes hypoxic, leading to upregulation of adaptive angiogenic processes. Neovascularization, however, fails to revascularize ischemic retina appropriately and instead is misdirected into the vitreous, in a pattern that recapitulates key features of proliferative diabetic retinopathy. Heterozygous controls G and H. er still A–I. Thes3.4DDAH2-deficient mice during OIR. The concentration of VEGF protein was significantly raised in the retina during the hypoxic phase of OIR. However, the concentration of VEGF was unaffected by DDAH2 deficiency , which is well recognized for its pro-angiogenic role in OIR, we next investigated retinal VEGF protein levels in ficiency J indicat3.5DDAH2−/− mice , a feature of age-related macular degeneration, we measured the extent of CNV induced by laser-rupture of Bruch’s membrane in heterozygous controls  suggesti4Therapeutic strategies that promote new vessel growth into the ischemic retina and away from the vitreous body would be extremely beneficial for patients with ischemic retinopathy, such as proliferative diabetic retinopathy and retinal vein occlusion. In this study we aimed to explore the role of ADMA and L-NMMA and its catabolizing enzyme DDAH1 and DDAH2, which are potent regulators of NO synthesis, on vascular regeneration and pathological neovascularization. To do this we investigated the expression of ADMA and L-NMMA in the ischemic murine retina and characterized DDAH1 and DDAH2 knockout mice in health and in an established model for retinal ischemia and neovascularization.We found that DDAH1 and DDAH2 are expressed to a similar extent in the murine retina while DDAH2 is the predominant isoform found in the choroid and RPE. Interestingly, DDAH2 expression is approximately 1000fold greater in the retina than in the brain indicating an important tissue-specific role for DDAH2 in the retina.+/− and DDAH2−/− deficient mice in two established models of ocular neovascularization. We could previously show that DDAH1+/− mice exhibit reduced DDAH1 protein expression and elevated circulating and tissue ADMA concentrations  in cultured endothelial cell ; CL holds a grant from the Dr. Jackstädt Stiftung, The Wellcome Trust Seeding Drug Discovery Initiative and MRC Intramural Funding to JL.This work was supported by funding from the The authors declare no conflict of interests.The authors declare no competing financial interests.


The database is freely available and can be easily scaled and updated to keep pace with the rapid rise in genomic information.The HGTree database provides putative genome-wide horizontal gene transfer (HGT) information for 2472 completely sequenced prokaryotic genomes. This task is accomplished by reconstructing approximate maximum likelihood phylogenetic trees for each orthologous gene and corresponding 16S rRNA reference species sets and then reconciling the two trees under parsimony framework. The tree reconciliation method is generally considered to be a reliable way to detect HGT events but its practical use has remained limited because the method is computationally intensive and conceptually challenging. In this regard, HGTree ( Vertical inheritance refers to the transfer of genetic information from parents to offspring. Vertically inherited genes typically show higher degree of similarity between species that are closely related than those that are distantly related. This aids in reliable recognition of species and understanding their classification and evolution. For example, ribosomal RNA (rRNA) genes have been historically used to determine the taxonomic structure of cellular life . HoweverHGT allows gain of novel molecular functions and can provide selective evolutionary advantages to species. For example, transfer of antibiotic resistance and virulence factor genes between bacterial species poses significant challenges to human health . SimilarAccurate detection of HGT however remains a computational and conceptual challenge. Existing databases such as HGT-DB  and Darkhttp://hgtree.snu.ac.kr) that provides putative genome-wide HGT information for 2472 completely sequenced prokaryotic genomes. HGTree defines HGT by comparing the gene tree for each orthologous gene set to the reference species tree. Conflict between gene and species trees is taken as indication of non-vertical evolution. Specifically, different hypotheses regarding the evolution of gene sets are evaluated and only those corresponding to HGT are kept and stored in the database. Results are displayed graphically for quick understanding. The friendly user-interface allows quick retrieval of already processed results for HGT analysis. Currently, three major services are provided: (i) HGT browser to display the molecular functions, gene family and phylogenetic relationships of HGT-acquired genes for all the genomes in the database, (ii) HGT analysis between and within (user selected) genomes and (iii) HGT analysis of user submitted gene and genome sequences. For each service, donor and recipient genomes are also graphically labeled for quick understanding. The database is freely available, does not require registration or login credentials and can be easily scaled and updated to keep pace with the continuous rise in genomic information. Importantly, HGTree represents the most complete existing resource for HGT-related information built on an explicit evolutionary model of tree reconciliation.Here, we introduce HGTree   , informah, 2015) . Out of h, 2015)    database  using HM < 10−3) . RNammer < 10−3)  was usedE-value cutoff of 10−6. Second, a sparse graph was built that described relationships between genes based on BLAST results. Third, homologous clusters were generated using hcluster_sg  were removed, as they do not provide enough information for reliable estimation of phylogenetic relationships. FastTree (ver. 2.0) was used to reconstruct phylogenetic trees for each orthologous gene set and the corresponding species tree (a posteriori using the Newick Utility (ver. 1.6)  of orthologous gene sets was generated using CLUSTAL Omega (ver.1.2.1)  under deies tree . FastTreies tree . It is mies tree  and RAxMies tree  and is oies tree . Specieser. 1.6) . The reler. 1.6)  similar er. 1.6)  was useder. 1.6) . All HGTE-value threshold of < 10−6 and enabling soft-masking (E-mail.User submitted sequences are processed in the following manner: (i) Prodigal ver. 2.6) is used to detect protein-coding genes; (ii) orthologous groups are assigned to predicted genes by searching against already constructed orthologous gene sets using reciprocal-BLAST search Figure . Several-masking ; (iii) o.6 is useFisher's exact test was performed to test the significance of the null hypothesis stating that HGT events of a particular phylum were not greater compared to other phyla. For this purpose, 2 × 2 contingency tables for each phylum were analyzed. Specifically, the counts of HGT-related and total genes for each phylum were compared with the counts of HGT-related and total genes in all other phyla. The odds ratio greater than one favored the alternate hypothesis stating that HGT events of a particular phylum were significantly greater than the HGT events of all other phyla.http://mariadb.org/) management system. The database consists of four tables with more than 13 million records. The web-based user interface was written in HTML5, PHP, CSS and JavaScript. User interface widgets were implemented using jQWidgets (ver.3.8.1) (http://www.jqwidgets.com) and jQuery (ver.1.11) (http://jquery.com). Circular phylogenetic trees were generated by jsPhyloSVG-1.55 (The database server was developed using MariaDB (ver.10.0.13)  HGT Browser, (ii) HGT Analysis within Selected Genomes, (iii) Between-group HGT Analysis, (iv) HGT Analysis of User Query and (v) Gene or Keyword Search. Each of the sub-menus is described below. In addition, users can download FASTA formatted protein and 16S rRNA sequences, general description files for each genome, and pre-computed alignments and phylogenetic trees corresponding to all genes and species sets from the Downloads menu. To facilitate easy navigation and understanding, step-by-step tutorials are also available from the Tutorial menu.The interface of HGTree consists of six main menus: HGT Browser gives complete information related to all genomes and HGT events stored in the database. The current version of HGTree contains a total of 660 894 HGT events detected in 2472 microbial genomes (Supplementary Table S2). A search box allows users to search for their genome of interest. Alternatively, users may navigate from the classification window provided on the left under ‘Taxonomic Tree’  using both NJ and ML. In turn, it took 57 and 88 minutes to process the largest genome  under NJ and ML environments respectively  using the Gene or Keyword Search utility provided by the Search menu.While HGT Browser can be used to find genome-wide HGT events of a genome against all other genomes, s Figure . For this Figure , as we hs Figure  because s Figure . For exaly Table . TherefoHodgkinia and Mycoplasma haemofelis) to 0.59 (Borrelia garinii) (Supplementary Table S2). While most microbial genomes showed linear relationship between the total number of genes and total number of horizontally transferred genes, some outlier genomes with significantly (P < 0.05) lower or higher HGT-index were also detected. Specifically, we focused on the 5% upper (HGT-index < 0.16) and lower (HGT-index > 0.42) percentiles of HGT-index as shown in the red-dotted line in Figure Chlamydia, Rickettsia and Mycobacterium genera belonged to the upper percentile, while Mycoplasma to the lower. Interestingly, these organisms are notable parasites of other species suggesting that symbiosis and parasitism lead to significant increase/decrease in horizontal exchange  enriched by HGT. In turn, Chlorobi (9 members) and the unclassified archaeon . HoweveTo ensure high speed and optimal accuracy in tree reconstruction, we implemented FastTree program to infer approximate ML phylogenies for each orthologous gene set and its corresponding species tree . FastTreTo accurately define orthologs, we incorporated Mestortho, which is an orthology detection algorithm based on minimum evolution . To imprHGTree is a non-commercial public database developed to support various fields of research. It has a user-friendly interface allowing easy access to large amount of HGT information. To our knowledge, it is the most comprehensive available resource of HGT-related information generated by large-scale phylogenetic analyses. However, not all transfers can be detected by tree reconciliation. For example, transfers that occur between sister taxa do not yield topological incongruence. Similarly, an HGT-acquired gene in the common ancestor of two (or more) species may later be lost in only one (or more) of the descendants. Here, favoring either transfer or loss can be conceptually challenging because the gene has experienced both events. The decision to record the event either as HGT or to ignore (i.e. treat as loss) is based on the most parsimonious embedding of gene tree inside the species tree. In our opinion, recording such events as transfers is more appropriate because HGT followed by loss nullifies the first gain and restores the original state. Technically, such events could still be recorded as losses if they yield the most parsimonious reconciliation and hence will be excluded from the HGTree repository. In other words, some true positives have likely been missed and false-positives included thanks to the biological complexity of the HGT-detection problem. We expect to quantify these rates in a future version. Because tree reconciliation method is susceptible to topologies of both species and gene trees, short branch lengths of species tree may also sometimes lead to incorrect estimation of HGT. However, no method for HGT detection can be 100% accurate. Therefore, genes output by HGTree should be taken as putative HGT-genes to aid further downstream analysis. Moreover, results are dependent upon the choice and accuracy of existing programs for tree reconstruction and reconciliation and will no doubt improve in precision with the availability of better alternatives in future.The precision and use of HGTree can be improved with additional upgrades. In the present version, we removed orthologous gene sets containing >50% of total microbial genomes. While some widely distributed gene families may also be subject to HGT (e.g. aminoacyl-tRNA synthetase), their accurate detection via phylogenetic inferences can be more challenging. In turn, these transfers can perhaps be better detected via comparative genomics approaches, as shown in . Moreove