It is anticipated that demands on ambulatory HIV services will increase in coming years as a consequence of the increased life expectancy of HIV patients on highly active anti-retroviral therapy (HAART). Accurate cost data are needed to enable evidence based policy decisions be made about new models of service delivery, new technologies and new medications.A micro-costing study was carried out in an HIV outpatient clinic in a single regional centre in the south of Ireland. The costs of individual appointment types were estimated based on staff grade and time. Hospital resources used by HIV patients who attended the ambulatory care service in 2012 were identified and extracted from existing hospital systems. Associations between patient characteristics and costs per patient month, in 2012 euros, were examined using univariate and multivariate analyses.The average cost of providing ambulatory HIV care was found to be €973  per patient month in 2012. Sensitivity analysis, varying the base-case staff time estimates by 20% and diagnostic testing costs by 60%, estimated the average cost to vary from a low of €927 per patient month to a high of €1019 per patient month. The vast majority of costs were due to the cost of HAART. Women were found to have significantly higher HAART costs per patient month while patients over 50 years of age had significantly lower HAART costs using multivariate analysis.This study provides the estimated cost of ambulatory care in a regional HIV centre in Ireland. These data are valuable for planning services at a local level, and the identification of patient factors, such as age and gender, associated with resource use is of interest both nationally and internationally for the long-term planning of HIV care provision. Highly active anti-retroviral therapy (HAART), available since the mid-1990s, has transformed HIV infection into a chronic disease ,3. HowevSince HIV testing began in the early 1980s over 6,600 people have been diagnosed with HIV in Ireland . Of thesData on the use and cost of services are needed to estimate the potential impact the changing HIV epidemiology will have on the health service in Ireland, as well as to support evidence based decisions about new models of service delivery, technologies and medications . It is nper diem costs. Top-down costing studies tend to be relatively quick and easy to carry out, however they are also less precise and cannot provide information on individual factors driving the costs [There are two general approaches to costing healthcare: top-down and bottom up. A top-down approach estimates the cost of an individual service on average, usually using routinely available data e. g. average he costs . A bottohe costs . Studieshe costs -15.Existing data on the cost of outpatient care in Ireland are limited. The national average cost of an outpatient visit in Ireland was estimated to be €130 in 2011 using a top-down methodology , however no information is available on how this cost may vary across specialties. A previous study looking specifically at the pharmacoeconomics of HIV in Ireland carried out in the early HAART era reported an average outpatient cost of IR£493 per active patient month in 1999 which would be equivalent to €1,184 in 2012  ,17.Ireland is currently in the process of restructuring its healthcare funding system from one where hospitals are funded based on historical levels of funding adjusted for activity and patient mix to a prospective case based payment system (“Money Follows the Patient”) . While iThe aim of this study was to estimate the total cost of providing an outpatient HIV service in a single centre in 2012 from the health service provider perspective, and to identify associated patient factors. We also compare our results with other available sources of cost data.Ethical approval for this study was obtained from the Clinical Research Ethics Committee of Cork University Hospital.Cork University hospital (CUH) is one of the largest university teaching hospitals in Ireland, and provides HIV care for the counties of Cork, Kerry, Waterford, and South Tipperary (covering approximately 20% of the Irish population). The infectious disease (ID) department holds two routine outpatient clinics specifically for HIV patients: the main HIV clinic (“HIV clinic”) held once a week and a joint ID-maternity clinic  held once a month in the adjoining maternity hospital. Patients attending the antenatal-HIV clinic attend both obstetric and ID services during their appointment.Local clinical guidelines, based on international best-practice -21, recoAll patients who attended for HIV care in 2012 were identified from a pre-existing clinical database which contains the results of all viral load tests performed in CUH. The database also contains basic demographic and risk factor information on the patients who have attended the public HIV clinic in CUH. Patients who attended the public HIV clinic at least once in 2012 were eligible to be included in the study. Patients who attended for private HIV outpatient care, who did not attend any outpatient appointment in 2012, or who routinely attended another HIV centre for their HIV care were excluded.For new patients to the clinic in 2012 (newly diagnosed or transferred from another clinic) the date of their first appointment attended or admission was taken as the start date of their care, for all other patients the start date of care was 01/01/2012. Patients were considered to be in care until 31/12/2012 if they used any hospital service (outpatient/in-patient/emergency department) at least once between 01/07/2012 and 31/12/2012. The files of patients who did not use any hospital service in the last 6 months of 2012 were manually checked, and these patients were censored at the date of their last scheduled appointment where appropriate. Patients who had either a CD4 count < 350 cells/μl or an AIDS defining illness at the time of diagnosis recorded in their medical notes were categorised as late diagnoses.Information on the number and dates of all outpatient appointments and diagnostic tests  were extracted from the hospital information system. Data on the total number of months of each HAART regimen dispensed, as well as any relevant start and/or stop dates, were extracted from pharmacy patient files. The medical records of patients with no pharmacy files were cross-checked to confirm the patient did not receive HAART in 2012. In addition, information on the number of doses of prophylactic antibiotics  dispensed to HIV patients was provided by the hospital pharmacy. Diagnostic tests were linked with patient appointments on the basis of matching test and appointment dates. Radiology and cardiology tests, which may not occur on the date of the outpatient appointment where the test was ordered, were linked with the nearest previous outpatient appointment attended.A micro-costing study was carried out to estimate the cost of typical HIV outpatient appointments from the service-provider perspective in 2012. Staff routinely involved in providing or supporting the HIV clinic  were interviewed to assess the HIV-clinic related tasks performed by the staff member and the estimated patient-facing and non patient-facing time involved. Doctors and nurses self-recorded their patient-facing times at six consecutive HIV clinics in March and April 2013 (127/153 appointments timed). Pharmacy staff self-recorded their HIV related patient-facing and non patient-facing time for the month of April, 2013. The accuracy of self-timed doctor patient-facing times was confirmed by independent observation of approximately 50% of appointments. Average costs per appointment were then calculated based on estimated time and salary of relevant staff using recommended national guidelines .Eight basic types of appointment were identified: 1) did not attend scheduled outpatient appointment (DNA), patient was not on HAART at the time ; 2) DNA, patient was on HAART at the time ; 3) attended the HIV-clinic, patient was not on HAART at the time ; 4) attended the HIV-clinic, patient was on HAART at the time ; 5) attended the antenatal HIV-clinic, patient was not on HAART at the time ; 6) attended the antenatal-HIV clinic, patient was on HAART at the time ; 7) first attended appointment with the ID service for HIV care, patient was not on HAART at the time ; and 8) first attended appointment with the ID service for HIV care, patient was on HAART at the time . Appointments were categorised as DNA if the patient did not attend their scheduled appointment without cancelling or rescheduling the appointment prior to the start of the clinic they were due to attend. The cost of a DNA appointment was estimated based on the same non patient-facing administrative and pharmacy staff time (where applicable) as attended appointments as well as the time incurred rescheduling an appointment. In general, a patient is considered lost to follow-up if they DNA three consecutive appointments. For the antenatal-HIV clinic appointments only the cost of the care delivered by the ID team was included in the micro-costing.The unit costs of diagnostic tests were obtained from the finance department of the hospital and the National Virus Reference Laboratory. The monthly costs of anti-retroviral medications and prophylactic antibiotics were provided by the hospital pharmacy. In Ireland, the price of all prescribed drugs are set at a national level ,24, althThe hospital finance department also provided outpatient costs per patient for comparison purposes from a newly implemented activity based costing system. These data were broken down into direct and indirect costs by category , but contained little information on individual patient characteristics, appointment types or diagnostic tests. Drug costs (with the exception of a few very high cost drugs) were not assigned at a patient level in the hospital activity based costing system in 2012.All information was entered into a Microsoft Access ® database. Data were analysed using Microsoft Excel® and STATA 12 . Weighted average costs per patient month (ppm) were calculated to account for differences in patient lengths of follow-up. Chi-squared tests were used to test for differences in proportions. Logistic regression was used to estimate the odds ratios of being treated with PI-based regimens compared to NNRTI-based regimens in patients receiving a single type of regimen. Generalized linear models were used to model costs ppm on HAART as they are suitable for modelling cost data, which tend to be skewed, without the need for transformation . The mosThe unit costs of individual appointment types, common diagnostic tests and regimen types are shown in Table To reflect the uncertainty in the estimates of staff time and laboratory costs staff times were varied by 20% and unit costs of diagnostic testing by 60% in a sensitivity analysis. The 20% variation in staff time was based on the approximate width of the confidence intervals of the average doctor and nurse patient-facing times, while the choice of 60% variation for diagnostic testing costs was based on the difference in the weighted average test costs calculated using alternative unit costs which were available for a limited number of the tests.In total 326 patients (3659 patient months) were included in this study. The characteristics of the patient population can be seen in Table Three patients died in 2012, two of these were considered HIV-related deaths both of which were of people who were newly diagnosed with advanced illness (CD4 < 50 cells/μl) at the time of diagnosis.There were a total of 1315 HIV outpatient appointments scheduled during 2012, with an average of 4.1  scheduled appointments per patient and 3.6  attended appointments per patient. The frequencies of each type of appointment are shown in Table In total 304 patients were on HAART for 3272 patient-months, with 3322 months of HAART dispensed in total. The majority of regimens dispensed consisted of a nucleoside reverse-transcriptase inhibitor (NRTI) backbone combined with either a protease inhibitor (PI) or a non-nucleoside reverse-transcriptase inhibitor (NNRTI). NNRTI-based regimens were the most frequently dispensed  followed by PI-based regimens , with a small number of patients being dispensed other regimens types . Fifteen percent (n = 45) of patients had more than one regimen prescribed during the study period, most of which were within the same class, although a small number of patients were prescribed regimens from different classes during the study period (n = 15).The overall average cost of providing HIV outpatient care was estimated to be €973 ppm in 2012 . As the cost of HAART accounted for the majority (88%) of total costs, varying the staff time estimates and unit costs of diagnostic testing did not result in substantial changes to the average costs ppm in the sensitivity analysis, with total costs ppm only varying by about 5% , there are many other factors apart from cost which influence regimen choice, including patient , clinical  and health system factors . Significantly lower proportions of new patients and newly diagnosed patients received HAART in 2012 compared to existing and previously diagnosed patients . While a significantly higher proportion of those diagnosed late (i.e. with a CD4 count <350 cells/μl or an AIDS defining illness at diagnosis) received HAART . On multivariate logistic regression of these three factors late diagnosis was the only factor that remained significant with an odds ratio (OR) of 15.5 (95% CI 2–120). Logistic regression of regimen type  found women were significantly more likely to be prescribed a PI-based regimen , while those over 50 years were less likely to be prescribed a PI-based regimen  when adjusted for risk factor, patient type, years since diagnosis, CD4 < 350 cells/μl and late diagnosis.The results of multivariate analysis of non-HAART, HAART and total costs ppm on HAART are shown in Table Patient level costs for the HIV patients included in the micro-costing study were provided by the hospital finance department. The data are activity based costing data, the cost components of which are generated using a top-down methodology. The total number of HIV outpatient appointments in the finance dataset was very similar to the total scheduled number of appointments from the utilisation study (1310 vs 1315) which would be expected given that the finance costs are based on information recorded in the same hospital information system as the utilisation data were extracted from. As the costs of most drugs were not assigned at a patient level in this system in 2012, it was not appropriate to compare drug costs, however the estimated non-drug cost ppm based on the hospital activity-based cost data was €63 ppm (95% CI €59-€67) compared to €117 (95% CI €111-€124) in the micro-costing study. Use of the national average outpatient appointment cost (€130.56 in 2012) would have resulted in an even lower estimate of €47 ppm (95% CI €44-€49 ppm). While our estimate is greater than the estimates generated using routine data, the estimated total cost (€973 ppm) is about 20% lower than the IR£493 per active patient month in 1999 reported by a previous micro-costing study . Although that study was carried out in the early HAART era and the patient population was quite different to the patient population in this study .This study reports the results of a bottom-up costing study of routine HIV outpatient care performed in a regional referral centre in the south of Ireland. The total estimated cost of the service in 2012 was €973 ppm, nearly 90% of which was due to the cost of HAART. While it is difficult to compare costs across countries, HAART is reported to account for 60%-80% of the total cost of HIV care (including inpatient care) in many studies in developed countries -33.Despite its cost, HAART is considered a very cost-effective treatment ,34, and The most important factor on multivariate analysis which influenced total costs ppm on HAART was gender, with women being 15% more expensive than men. It was also interesting to note that patients over 50 years of age had significantly reduced costs ppm on HAART. This was due to a combination of older patients tending to be on less expensive NNRTI-based regimens, as well as having fewer scheduled appointments . The lower appointment rate in older patients contrasts with previous studies which have found older patients have higher utilisation rates , and mayThere are several limitations to this study. The data collection was restricted to one year, so changes over time cannot be examined. The usage and cost of other services  were not included in this analysis. While the majority of HIV-related care is now provided on an out-patient basis by the ID team, a small minority of patients use a substantial amount of other services . Newly dAccurate cost data are essential for ensuring HIV services are effective, efficient, and equitable and cost information should be used to guide policy, planning and implementation . This is


Small molecules are being increasingly used for inducing the targeted differentiation of stem cells to different cell types. However, until now no systematic method for selecting suitable small molecules for this purpose has been presented. In this work, we propose an integrated and general bioinformatics- and cheminformatics-based approach for selecting small molecules which direct cellular differentiation in the desired way. The approach was successfully experimentally validated for differentiating stem cells into cardiomyocytes. All predicted compounds enhanced expression of cardiac progenitor  and mature cardiac markers  significantly during and post-cardiac progenitor formation. The best-performing compound, Famotidine, increased the percentage of Myh6-positive cells from 33 to 56%, and enhanced the expression of Nkx2.5 and Tnnt2 cardiac progenitor and cardiac markers in protein level. The approach employed in the study is applicable to all other stem cell differentiation settings where gene expression data are available. Transcription factors have been used for inducing the differentiation of embryonic stem cells in a step-wise manner to various cells of interest such as dopamine neurons, retinal pigment epithelium, floor plate cells, hematopoietic cells, endothelial cells, pancreatic cells and cardiomyocytes.11 while gene expression data for 1309 compound treatments has been employed as provided in the Connectivity Map (CMap)12 database. The CMap database has been previously used particularly in drug repositioning and also predicting the mode-of-action of drugs,14 however, its use in the context of cellular differentiation is novel.Hence, in this work we present a systematic approach for the selection of potent small molecules for inducing the differentiation of pluripotent stem cells to the tissue of interest. We have applied and experimentally validated this approach, which is based both on bioinformatics and cheminformatics components, by selecting small molecules to promote the differentiation of stem cells to cardiomyocytes. The approach presented here employs publicly available gene expression data for the transition from stem cells to cardiomyocytes from the cellular side, and gene expression data that are the result of compound treatment from the other side. On the basis of matching changes in gene expression in both spaces  our algorithm predicts candidate compounds to induce the differentiation of stem cells to cardiomyocytes . The gen15 Experimental evidence supporting this hypothesis has been presented in several studies, usually also based on the CMap database,14 such as the repurposing of the antiulcer drug Cimetidine for lung cancer15 and antiepileptic Topiramate for inflammatory bowel disease.15 On the basis of the same hypothesis, the authors have recently developed transcriptional approaches for repurposing drugs for various diseases with prospective successful validation for leukemia and breast cancer.16In the area of drug repositioning it is hypothesized that if the gene expression signature of compound treatment is strongly anticorrelated to a given disease signature , then that compound is potentially capable of treating this disease.17 The GSEA approach yields in connectivity scores that represents how much the genes targeted by each compound is related to genes differentially expressed in the heart signature where correlated, anticorrelated and not correlated gene signatures (between compound and heart signature) results in positive connectivity, negative connectivity and zero connectivity scores.In this work, we have been inspired by the idea of matching gene signatures of a disease to drugs and applied it into novel application area of stem cell differentiation. Although in drug repositioning disease and healthy samples are compared with constitute the gene expression profile of a disease, in the case of selecting small molecules for stem cell differentiation adult heart samples are compared with embryonic stem cells to constitute the heart gene expression profile. Subsequently, the gene expression profile of this tissue is matched with compound signatures using Gene Set Enrichment Analysis (GSEA).18 that was previously developed was extended  and all the putative protein targets of the compounds contained in CMap were predicted. In addition, for the purpose of the study here all the predicted protein targets in cardiovascular diseases using the Comparative Toxicogenomics Database (CTD)19 were determined. The aim of this step was to enhance the compound selection process by giving insights to the potential targets of the compounds and thereby providing a clear mechanistic hypothesis for the mode-of-action of the compounds selected for differentiation.In addition to this gene expression-based ranking of compounds for a particular purpose we also took its computationally predicted bioactivity spectra (its predicted activities against a set of 1080 protein targets) into account. For this purpose a cheminformatics target prediction algorithmSubsequently, the combined bioinformatics and cheminformatics approach presented here was employed in the novel area of systematic selection of small molecules for promoting differentiation of stem cell to cardiomyocytes. It was observed that administration of four novel-predicted compounds significantly enhance expansion rate and expression of cardiac precursor and cardiac markers in gene and protein level. This method is generally applicable in cases where gene expression data of the target tissue are available.α-Estradiol (rank 20). Meglumine is capable of differentiating bone marrow mesenchymal stem cells to cardiomyocytes,20 whereas Troglitazone induces heart cell proliferation.21 Estradiol demonstrates cardioprotective effect against cardiac injury22 and induces proliferation, but not differentiation, of undifferentiated myoblast cells. On the other hand Resveratrol (rank 2) and Pyrvinium (rank 29) show strong negative connectivity, and it has been established before that Resveratrol-modified cardiac stem cells regenerate infarcted myocardium.23 Pyrvinium, on the other hand, promotes wound repair and post-myocardial infarct cardiac remodeling.24 It also ameliorates myocardial contractile dysfunction in a mouse model of myocardial infarction.25 Overall significant literature support for the claimed effects could hence be identified in the first step of this study.The expression profile of undifferentiated human embryonic stem cells was compared with the expression profile of adult ventricular cardiac tissue using database GSE50704 in GEO , Prilocaine (rank 4), Famotidine (rank 12) and Sodium Phenylbutyrate  have been selected for prospective experimental validation owing to their high rank and novelty in the given context. The effect of treatment of these compounds on stem cell differentiation was investigated as presented in the next section.To investigate the effect of Butyrate, Famotidine, Prilocaine and Bethanechol on cardiogenesis from mouse embryonic stem cells (mESCs) embryonic bodies (EBs) were treated with each individual compound from day 2 to 7  and cardiomyocyte differentiation was assessed both qualitatively and quantitatively .Qualitative assessment was performed by comparing morphological features of the beating cells to controls. It was found that the treatment of EBs with all selected compounds, particularly Famotidine, increased their expansion rate of plated or beating EBs, as well as the degree of contraction  higher than for control. This marker was increased at the protein level in the presence of both Famotidine and Butyrate  and Famotidine . On day 7, the expression of of Gata4  was incrButyrate . SignifiActc1 and Myh6 as cardiomyocytes-specific markers and Tagln as smooth muscle marker were examined also by RT-qPCR. The specific expression of the Actc1 (α-actin) gene represents the start of differentiation of cardiac cells29 and it is expressed in adult heart.30Myh6 on the other hand is expressed in healthy, but not failing, human heart and has a major role in cardiac contractility.31Tagln is expressed in the embryonic heart32 but its expression diminishes in adult heart.33 Treatment with Butyrate, Bethanechol, Prilocaine and Famotidine significantly enhanced mRNA levels of Actc1  and Myh6  on day 15 versus their respective controls, while levels of Tagln remained unchanged , whereas Tagln (smooth muscle marker) was overexpressed (1.7 and 1.8 folds) by Butyrate and Bethanechol treatment, respectively. Western bloting confirmed the expression of Tnnt2 as a mature cardiac marker for all of the compounds, particularly Prilocaine and Butyrate, at the protein level  signature and the set of the 50 most upregulated genes of Famotidine instance in CMap . These genes were found to be GRPR, RHBG, FSCN1, FGFR3, KIAA1609, WIPI2, ST8SIA3, GLP1R, ING1, SLC24A1, CHRNA3 and DNAJC16. Among those genes particularly FGFR3 and GRPR are of interest here, as fibroblast growth factors including FGFR3 regulate epicardial signaling and myocardial growth and differentiation.37GRPR on the other hand has a role in smooth muscle contraction.38 On the other hand the most enriched downregulated genes of the Famotidine instance, that are also downregulated in the ESCvsHRT signature, are SIPA1L3, ROM1, HABP4, SLC25A23, SCRN3, CCL14, PLEKHO1, CTDSPL, SOS2, ZNF639, FSTL3, ARHGEF9 and LRP6. The Famotidine instance downregulated an essential Wnt co-receptor, LRP6, from the ‘Canonical Wnt signaling’ pathway which was also downregulated in the ESCvsHRT. Modulating the Wnt signaling pathway is known to have an important role in differentiation of stem cells to cardiomyocytes.39 The compound also downregulates Notch2 from the ‘Notch Signaling Pathway’, which was upregulated in the ESCvsHRT signature. Perturbation of the Notch Signaling Pathway regulates cardiac differentiation during development in mammals and also has an important role in recovery from adult myocardial damage.40When considering the gene expression changes induced by Famotidine, the GSEA algorithm was used to identify the most enriched upregulated genes in common between the embryonic stem cells To summarize, the positive effect of Famotidine in ischemia seems to go far beyond its activity on the Histamine receptor alone, and by combining predicted protein targets and differentially expressed genes after compound application provides a far more unified picture of the mode-of-action of Famotidine in this context.7 Famotidine is predicted to act by inhibiting of Complement C1s and thrombin at the protein level, and by upregulating GRPR and FGFR3, as well as downregulating LRP6 and Notch2 on the gene level, which gives a much more unified picture of compounds action both from the ligand-target interaction level and the genetic level than previous studies have found.In this work, a general method for selecting small molecules for directed stem cell differentiation has been presented. To this end, large scale gene expression profiles of compound treatments (1309 compounds in CMap) were utilized, which were then matched with gene expression profiles of the particular target cell line of interest, compared with the cell line of origin . This approach has then experimentally been validated by predicting compounds for promoting differentiation of stem cells to cardiomyocytes. Four compounds  were experimentally validated in this study for their ability to stimulate gene and protein levels of cardiac precursor and cardiac markers. Each of the four compounds stimulated the markers in agreement with the hypothesis. However, Famotidine and Butyrate had more significant effect on the cardiac and cardiac progenitor markers and hence had superior effect on the differentiated cardiomyocytes. Famotidine and Butyrate increased the percentage of Myh6-positive cells from 33% and 39%, respectively, to 56% and 58%, respectively, compared with control groups . To put those numbers into context, a recently discovered small molecule for cardiomyocyte differentiation, Zebularine, increased the percentage of Myh7-protein-positive cells  from 27 to 35% (an 1.29-fold increase), so at considerably lower levels.in silico target prediction, can be used for the selection of small molecules to promote the targeted differentiation of embryonic stem cells to cardiomyocytes, as well as suggesting possible mechanism of actions of the small molecule. Given the general nature of the approach it is expected that this method can be extended to the selection of small molecules for promotion of stem cell differentiation to any tissue of interest.Overall, the computational approach proposed here shows that gene expression data, in combination with 17 was employed in this work. This method checks whether a query gene signature is occurring at the extremes (top or bottom) of a rank-ordered list of genes, or whether it shows closer to random distribution . An enrichment score was calculated by descending the rank-ordered list of genes and incrementing a variable when encountering a gene in the given query signature. The magnitude of increment depends on the position of the gene in the list, which is chosen corresponding to a weighted Kolmogorov–Smirnov statistic. The enrichment score can hence range between −1 and 1, where −1 shows strong negative connectivity, 1 identifies strong positive connectivity and 0 represents zero connectivity. In our particular case, the signature of each compound from CMap was used to query rank-ordered list of genes of a disease and both positive and negative connectivities were analyzed subsequently.For identifying compounds that are anticorrelated with the heart signature GSEA as implemented by the Broad Instituteversus drug the two signatures were used to query the disease profile, hence giving rise to two scores, the ‘up’ score and the ‘down’ score. The following formula was used to combine the two scores into one score:up and scoredown are the scores calculated for the compound given its most x up- and downregulated genes, respectively. In the GSEA results a negative score means that the query signature was found at the bottom of the list and a positive score means that the query signature was found at the top of the list. The combined score was used to sort the compounds and come up with a rank-ordered list.The input of the GSEA algorithm is a query gene signature, a gene expression profile (gct), a phenotype annotation file (cls), and a probeset to gene symbol translation file (chip). A further parameter is the number of top and bottom genes of the rank-ordered list of compound gene expression profile to be selected, where in the current study this was selected to be 50. In this list the ranking of genes was based on log2 ratio of the treatment of compound to vehicle. Generally, how many genes to select is based as much on intuition as on rational criteria, and in our experience the best number of genes to be selected generally varied between those numbers. This left us with two query signatures, namely one of the most upregulated genes, and one of the most downregulated genes. For each disease 19 has been used to identify genes that are associated with cardiovascular diseases, where disease–gene links were downloaded separately as provided on the CTD website. The CTD database provides an Inference score for each target, which indicates the level of relevance of each target with cardiovascular diseases, based on text mining approaches of a large set of scientific publications. The gene–protein links were retrieved from ChEMBL41 to map gene identifiers to proteins implicated in diseases.Although gene expression data are able to map compound and disease spaces with the parameters chosen, we also employed computational tools to hypothesize protein target-based bioactivity spectra for selected compounds, and to evaluate the mode-of-action of those , based on additional database information we integrated. For this purpose, the Comparative Toxicogenomics Database (CTD)18 has been utilized to predict protein targets of compounds in the CMap databases using the Naïve Bayes approach. This algorithm predicts a score for each protein target included in the training set, which represents the probability of the compound to bind to this target . The extraction of compound-target pairs was identical to the benchmarking data set query introduced in the previous work18 (which included targets with binding affinity <10 μM and confidence level of 9 or 10) except that it was applied on ChEMBL41 v.17 and hence left us with a larger training database of 385 126 compound-protein pairs, 1643 distinct proteins and 226 791 unique compounds. Compounds were standardized and ECFP4 fingerprints were generated using the JChem package of ChemAxon.42 The standardization options were Aromatize, RemoveExplicitH, Clean 2D, Clean 3D, RemoveFragment and Neutralize. The Laplacian modified Naïve Bayes version of the algorithm provided in the previous publication18 was then trained on the extracted.A target prediction algorithm as established beforeThe protein target predictions for all compounds to be analyzed  was incorporated with the gene expression-based results and the whole information is then displayed to the user, enabling the selection of compounds based on connectivity derived from gene expression profiles, predicted bioactivity spectra (modes-of-action), as well as relevance of each target for the disease. ChEMBL Therapeutic Flags of compounds have been used to check whether the drug is approved.β-mercaptoethanol  and 1000 U/ml leukemia inhibitory factor , 1 μm PD0325901(Sigma-Aldrich) and 10 μm SB431542 (Sigma-Aldrich), 1% N2 and 2% B27 (3).Cell culture chemicals were supplied by Life Technologies  unless indicated otherwise. Mouse embryonic stem cells strain RB20 from C57BL/6 strain was used as reported. Twenty cells cultured in serum-free media, Dulbecco’s modified eagle medium F12 (DMEM F12) and Neurobasal media with 0.5% BSA, 2 mM glutamine, 0.1 mM nonessential amino acids, 1% penicillin–streptomycin , 0.1 mM L-glutamine, 0.1 mM beta-mercaptoethanol, 1% penicillin–streptomycin and ascorbic acid. For cardiac differentiation, EBs were formed by adding 800 ESCs per hanging drop over two consecutive days (day 2). Following collection of EBs, they were cultured in suspension for the next five days (day 7). On the seventh day, the EBs were plated on gelatin coated 12-well plates  for further 6 to 8 days (day13–15).43The media used for cardiac differentiation consisted of K-DMEM, supplemented with 15% ES-FCS, 0.1 mM nonessential amino acids, 2 mM μM, respectively. 200 μM of Butyrate was also used where indicated in results.To obtain the optimal concentration of each predicated compound, the concentration predicated based on pervious literature was taken as the bases and lower and higher concentration was added during EB formation and the concentrations which reduced the EB size were taken as toxic concentration and the lower concentrations which had no toxic effect was selected for further experiments. Therefore, the selected concentrations for Butyrate, Famotidine, Prilocaine and Bethanechol were 50, 10, 15 and 20 To evaluate the effect of predicted compounds on cardiac precursor formation, the compounds were added during the first 5 days of EB formation (day 2–7), whereas for cardiac differentiation, the compounds were added during plating (day 7–15). All the compounds were dissolved in water, except for Famotidine, which was dissolved in dimethyl sulfoxide (DMSO). Therefore, equal amount of the DMSO was also added to the control of this group. To evaluate the effect of these compounds on cardiac precursor formation and cardiac differentiation, the cells were evaluated microscopically for morphology, by RT-qPCR for gene expression, by western blot for protein expression and by flow cytometry for efficiency of cardiac differentiation.μg of each RNA sample, and random hexamer primers according to the manufacturer’s protocol. Reverse transcription quantitative real-time PCR (RT-qPCR) was implemented by gene specific primers and SYBR Green . Applied Biosystems step one plus Real-Time PCR system  was used for Real time PCR analyses. For each reaction in a final volume of 10 μl, the PCR mixture contained 5 μl Rotor-Gene SYBR Green PCR Master Mix, 3 pmole of each primer, and 25 ng of cDNA. The relative mRNA concentrations were calculated using the software provided by the manufacturer. In order to normalize the data, Gapdh expression level was measured in the same sample. All measurements were carried out in triplicates, and the data was assessed and reported according to 2−ΔΔCt method. Specific primer pairs were designed by the Beacon designer  and Perl-primer ordered through metabion company .RNA was extracted using RNeasy Mini Kit  and cDNA was made using Moloney murine leukemia virus reverse transcriptase, 1 43 The primary antibodies were anti-mouse antibodies against Myosin Heavy Chain . The secondary antibody was, fluorescein isothiocyanate (FITC)-goat anti-mouse IgG . Meanwhile, the nuclei were counterstained with DAPI. The stained cells were analyzed with a fluorescent microscope  and images were acquired with an Olympus DP70 camera .An indirect immunofluorescence light microscopy was applied to analyze the cells as previously described.μg of solubilized protein fraction of each sample was subjected to SDS-PAGE electrophoresis and transferred to Polyvinylidene fluoride (PVDF) membrane. The samples were blocked on the membrane by adding 5% skim milk and the respective bands were labeled with mouse anti Tnnt2 antibody , rabbit anti NKX2.5 antibody  and mouse anti GAPDH antibody . The secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG  and HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology). HRP-conjugated IgG bound to each protein band was visualized by an Amersham ECL Advance Western Blotting Detection kit .Cells were lysed with TRI reagent (Sigma-Aldrich) according to the manufacturer’s protocol. Thirty In order to estimate the content of Myh6 in cardiac cells, cells were fixed with 4% paraformaldehyde, and permeabilized by treatment with Triton X-100 at a final concentration of 0.4% (V/V). Treated cells were incubated with Myh6 antibody for 1h at 37 °C, and subsequently were labeled with goat fluorescence isothiocyanate (FITC) anti-mouse as a secondary antibody for 40 min at 37 °C. The fluorescence intensity of cells, which represented Myh6 expression levels were analyzed by a Becton Dickinson FACSCalibur flow cytometer . For each sample, 104 events were recorded in the forward light scatter/side light scatter (FSC/SSC) dot plot. Both green fluorescence FITC for Myh6 were detected in a fluorescence detector 1 (FL-1) with a 530/30 nm band pass filter. Data were analyzed by comparison of the mouse IgG1 negative isotype control .P<0.05 (*) was used to indicate levels of statistical significance.SPSS  was used to present data as means±SEM obtained from three independent treatments of the replicated observations. One-way analysis of variance (ANOVA) was applied to identify the statistical differences between the three or more treatments. Also independent t-test analysis was carried out to identify statistical differences between the two treatments. The threshold of


MK2 inhibition in the absence of synergistic chemotherapy had not been investigated for its inherent cytotoxicity, and Munoz published a study aimed to fill this gap in our knowledge of MK2.4 For their MK2 cytotoxicity study in glioblastoma cells, Munoz et al. combined the use of siRNA and chemical probes. Three MK2 inhibitors were chosen for their distinct structures and mechanism of action (Ki 330 nM).5 CMPD1 is not simply a non-selective p38 inhibitor as it shows no inhibition of the phosphorylation of two other p38 substrates, ATF2 and MBP. MK2i is a classical ATP-competitive kinase inhibitor, which inhibits MK2 (IC50 126 nM).2 PF-3644022 is a more potent ATP-competitive MK2 inhibitor (Ki 5 nM).6 Although MK2i and PF-3644022 are both ATP-competitive MK2 inhibitors, they differ significantly in potency and are structurally orthogonal.7There has been much interest in developing inhibitors of the checkpoint kinases Chk1/2 to augment the effects of DNA-damaging agents for chemotherapy.f action . CMPD1 iKi of MK2 phosphorylation . A decrease in Bcl-Xpoptosis .In vitro fluorescent detection of tubulin polymerization showed that CMPD1 was indeed a potent inhibitor of this process. Munoz et al. completed their study by showing that CMPD1 is less cytotoxic to normal astrocytes over glioblastoma cells.The final clue to how CMPD1 was exerting its cytotoxic effects came from examining U87-cell morphology following compound treatment . The celThe study by Munoz is an example of deciphering the target of a small-molecule inhibitor using thorough cell biology techniques. This work is more significant due to the reported role of CMPD1 as an MK2 inhibitor, which is irrelevant to its antiproliferative effects in glioblastoma.8 In addition to kinase off-targets, there have been a number of kinase inhibitors that have recently been disclosed to have other pharmacology beyond kinases. For example, the c-Met inhibitor tivantinib is also reported to have potent anti-tubulin activity much like CMPD1.9 The off-target activity of kinase inhibitors has also strayed into bromodomains.10In early kinase drug discovery, it was common to discover additional kinase targets of putative selective inhibitors. In response to this incomplete characterization of inhibitors, the number of kinases available for selectivity screening increased dramatically to cover the majority of the kinome. It is increasingly common to have the complete kinome selectivity of an inhibitor disclosed.11 CETSA potentially extends the biological annotation of a chemical probe to the entire proteome, but in practice is limited to proteins sensitive enough to stabilization by ligand binding.12 Future approaches to chemical probe characterization will likely include pharmacological finger-print matching via transcriptomics and high-content imaging.13As chemical probes are used more in target discovery to link a phenotype to a target via small-molecule inhibition just as Munoz was attempting to do with CMPD1, it is imperative that we understand the pharmacology of the tools used. Technologies such as kinobeads and ActiveX attempt to do this by interrogating the binding of a molecule to the entire active kinome in a cell lysate, but are still limited to one protein family.9 of the 22 commercial suppliers of tivantinib listed in eMolecules and ChemSpider, only Cayman Chemical mentions the anti-tubulin activity in the product description (eMolecules (www.emolecules.com) and ChemSpider (www.chemspider.com) was searched on 20 September 2015 for ‘tivantinib.' The catalog of each of the 27 suppliers listed in either search were subsequently searched for ‘tivantinib' or ‘ARQ-197' and it was found in 22 supplier catalogs. Nine suppliers have no description for tivantinib beyond the structure and molecular weight. Twelve list it as a selective c-Met inhibitor. Only Cayman Chemical describes tivantinib's additional anti-tubulin activity). The remaining suppliers' catalogs either lack biological annotation for tivantinib or erroneously describe it as a selective c-Met inhibitor.A final, low-cost way to increase the utility of chemical probes is via better sharing of pharmacology annotation. Munoz put considerable effort into deciphering the true target of CMPD1 in preventing proliferation. The anti-tubulin effects of CMPD1 should be immediately known to the next researcher who purchases this compound as an MK2 inhibitor. Although commercial vendors have been essential for making chemical probes available for target discovery, they can be slow in responding to further characterization of probes in the literature. Although tivantinib was first described as a tubulin inhibitor in February 2013,et al,14 who advocated the creation of an easily accessible information source for researchers to add their own references and findings for chemical probes. Since then, the website www.chemicalprobes.org has been set up, which aims to create a first point-of-call for those interested in finding chemical probes with the latest annotation and using them in their research. Initiatives like this should ensure that the hard work done by Munoz et al. characterizing the true pharmacology of CMPD1 is not missed.The lack of consistent descriptions of an individual chemical probe's strengths and weaknesses, including poly-pharmacology, was the subject of a recent letter from Arrowsmith


FAM65B mutations cause hearing loss by an unknown mechanism. Using biochemistry and stochastic optical reconstruction microscopy (STORM), we show here that Fam65b oligomers form a circumferential ring near the basal taper of the mechanically sensitive stereocilia of murine hair cells. Taperin, a second protein near the taper, forms a dense-core-like structure that is disrupted in the absence of Fam65b. Stereocilia of Fam65b-deficient murine hair cells start to develop, but mechanotransduction is affected and stereocilia deteriorate. Yeast-two-hybrid screens identify RhoC as a Fam65b binding partner. RhoC co-localizes with Fam65b in stereocilia and regulates Fam65b oligomerization. Binding to RhoC and oligomerization are critical for Fam65b function. Our findings thus reveal a highly organized compartment near the base of stereocilia that is critical for hair cell function and affected in disease.Cochlear hair cells convert sound-induced vibration into electrical signals. DOI:http://dx.doi.org/10.7554/eLife.14222.001 Hair cells of the mammalian cochlea have a specialized morphology that is optimized for their role as mechanical sensors for sound. Each hair cell contains at the apical surface a bundle of stereocilia that are organized in rows of decreasing height . StereocFAM65B causes hearing loss in humans  and generated Fam65b cause hearing impairment, we created a Fam65b-deficient mouse line. In the mutant mice, the coding region of the Fam65b gene was replaced with a LacZ transgene. We will refer to the modified allele as LacZFam65b (LacZ/LacZFam65bmice. Measurements of the auditory brain stem response (ABR) to broadband click stimuli in 4-week old animals revealed that homozygous mutant mice were deaf  are presented. Outer hair cells (OHCs) amplify the distortions and they are propagated back through the middle ear and ear canal and can be measured in sound pressure waveforms , which are mechanical distortions generated in the inner ear when two primary tones   . The Famear duct  with strear duct . During d retina .10.7554/LacZ/LacZ Fam65bmice . In whole mounts of the cochlea at P5, we observed expression in OHCs and IHCs where Fam65b immunoreactivity outlined the shape of the stereociliary bundle . AdditioacZ mice . Higher acZ mice . To confacZ mice  to expreacZ mice . When weacZ mice , the proacZ mice .Previous studies have shown that taperin is localized near the basal taper of stereocilia . To furtLacZ/LacZFam65b mice in whole mount cochlea using phalloidin staining combined with fluorescence deconvolution microscopy. Whole mount staining at P2 revealed that the sensory epithelium in mutant mice was patterned properly into three rows of OHCs and one row of IHCs  against displacement. The resulting curve in the mutant animals was slightly shifted to the right and broadened . These findings provide evidence that while both Fam65b and taperin are present near the taper of stereocilia, they show a distinct distribution pattern. Taperin is more widely expressed throughout the core of the stereocilium and Fam65b is present in a circumferential ring that might partially overlap with the distribution of taperin. Taperin is no longer concentrated near the taper region in Fam65b-deficient hair cells , while the taperin structure had a diameter of 228 ± 9 nm . Thus, the two structures appear to overlap, although this is difficult to ascertain because the dimension of the primary and secondary antibody affects the measured dimension. As with deconvolution microscopy, both proteins could no longer be detected in the taper region in ir cells , which cWe next analyzed whether Fam65b might directly bind to taperin, or to CLIC5 and radixin, which are thought to form a protein complex with taperin . We alsoThe ring-like distribution of Fam65b near the taper of stereocilia suggested that Fam65b might form oligomeric structures. To determine the extent to which Fam65b oligomerizes we cloned full-length Fam65b as well as N- and C-terminal fragments . First, To determine the extent to which oligomerization of Fam65b is important for its function, we wanted to disrupt interactions between Fam65b molecules in mechanosensory hair cells. We hypothesized that overexpression of a Fam65b fragment containing one Fam65b binding site might disrupt homophilic interactions between Fam65b molecules thus acting as a dominant negative construct. To test this model, we first determined the extent to which interactions between full-length Fam65b-GFP with Myc-N1 was disrupted by co-expression of N2-GFP in heterologous HEK293 cells. Co-immunoprecipitation experiments demonstrated that N2-GFP significantly reduced interactions between Fam65b-GFP and Myc-N1 . Next weOur co-immunoprecipitation data demonstrated that Fam65b does not interact with taperin or with other proteins that have been localized near the base of stereocilia such as CLIC5, radixin, and whirlin . We therTo determine the extent to which RhoC and Fam65b might functionally interact in vivo, we wanted to determine the subcellular distribution of RhoC in mechanosensory hair cells. Unfortunately, available antibodies for RhoC were not of sufficient quality for immunolocalization studies. We therefore expressed by injectoporation HA-tagged RhoC and Fam65b-GFP in mechanosensory hair cells at P2 and evaluated their localization two days later. Stereocilia were visualized by staining with Alexa Fluor 647 phalloidin. Remarkably, RhoC was localized near the base of stereocilia and its distribution largely overlapped with the distribution of Fam65b . We thusPrevious studies in immune cells have suggested that Fam65b might regulate RhoA activity . Fam65b Next, we expressed full-length Fam65b-GFP with N-terminal (Myc-N1) and C-terminal (Myc-C1) fragments of Fam65b either alone or together with RhoC. Interactions between Fam65b-GFP and Myc-N1 but not Myc-C1 were strongly enhanced by RhoC , suggestRL/AA) that are thought to be critical for Fam65b function  at 37°C. Sections were washed 3 times for 20 min in PBS and post-fixed overnight at 4°C in 4% PFA. Sections were washed in distilled water, dehydrated and mounted.LacZ staining was performed as described . In brieCochlear whole mount staining was carried out as described . Whole mPrimary antibodies were as follows: α-Fam65b ; α-HA ; α-Myc ; α-taperin ; α-espin ; anti-acetylated-α-tubulin . Additional reagents were: Alexa Fluor 488-phalloidin, Alexa Fluor 647-phalloidin , Alexa Fluor 488 and Alexa Fluor 647 conjugated goat anti-rabbit secondary antibodies.2; 5 mM MgCl2; 0.9% NaCl). Sample was fixed for 2 hr at RT, and dissected in washing buffer . The stria vascularis, Reissner’s membrane and tectorial membrane were removed. Samples were dehydrated and processed to critical drying point in an Autosamdri-815A (Tousimis). Cochlea were mounted with carbon tape and coated with iridium . Samples were imaged with a Hitachi S-4800-ll Field Emission Scanning Electron.The experiment was performed as described . In brieDNA constructs are described in Supplementary Information. Expression of the constructs, immunoprecipitations, and western blots were carried out as described . Immunop2+ imaging, we used G-CaMP3 (Addgene 22692). Hair cells were imaged on an upright Olympus BX51WI microscope mounted with a 60x water-immersion objective and Qimaging ROLERA-QX camera, controlled by Micro-Manager 1.3 software  was pulled with a P-97 pipette puller (Sutter), and polished with MF-830 microforge (Narishige) to resistances of 3–5 MΩ. Hair bundles were deflected with a glass pipette mounted on a P-885 piezoelectric stack actuator (Physik Instrument). The tip of the pipette was fire-polished to ~4 μm diameter to fit the shape of OHC bundles. The actuator was driven with voltage steps that were low-pass filtered at 10 KHz with a 900CT eight-pole Bessel filter (Frequency Devices). The output driving voltage to the actuator stack was monitored by an oscilloscope to ensure a rise time <50 μs. The tip of the probe was cleaned in chromic acid to allow adherence to hair bundles. Whole cell recordings were carried out and currents were sampled at 100 KHz with an EPC 10 USB patch-clamp amplifier operated by Patchmaster 2.35 software (HEKA). To record macroscopic currents, the patch pipette was filled with intracellular solution . Cells were clamped at -70 mV.The experiment was performed as described . In brie0)/F0, where F0 is the averaged fluorescence baseline at the beginning. Transduction current-displacement curves (I(X)) were fitted with a three-state Boltzmann model  and Igor pro 6 . Calcium signal (△F/F) was calculated with the equation:  and Gary Westbrook as the Senior Editor. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Thank you for submitting your work entitled "Super-resolution microscopy resolves protein organization in stereocilia critical for mechanosensory hair cell function" for consideration by Summary:FAM65B in sensory hair cells of the inner ear. Mutation in the FAM65B gene has previously been linked to deafness in humans. The current manuscript addresses the localisation and function of the FAM65B protein in sensory inner hair cells in the mouse. As you will see, all three reviewers have commended your work, but have also raised a number of concerns. These should be addressed as follows:This paper uses biochemical, imaging and genetic studies to characterise the protein Essential revisions:FAM65B and taperin, and FAM65B and RhoB) should be cautious unless further analysis can strengthen the evidence for these interactions.1) A thorough, objective and quantitative analysis of the STORM imaging data should be provided, together with appropriate statistical analysis where required, as requested by Reviewer 1. Inferences about direct interactions (between 2) Likewise, the interpretation of the co-IP results in HEK-293 cells should be discussed with appropriate caution, as requested by Reviewer 2. If additional co-IP results from a different cell line are available, these should be included.3) A full structural analysis of the stereociliary bundle phenotype, including cohesion, growth, presence of tapers and stiffness should be provided, as requested by Reviewer 3.A) It was agreed that further electrophysiological analyses of the transduction currents  would be beyond the scope of this manuscript, and so are not required. However, the authors should acknowledge and discuss any limitations of the methods they have used.4) A comment on the presence or absence of the outer hair cells and of hair cell polarity defects in the mutant should be included.5) Any discrepancies between the findings and those of Diaz-Horta et al. should be discussed, as requested by Reviewer 3.6) The title should be revised, as requested by Reviewer 2, to reflect the findings of the manuscript more generally.FAM65B will help with cross-reference to existing literature.7) The name 'basilin' should be removed from the manuscript. There appears to be no strong reason to re-name the protein, and indeed, several arguments (as raised by Reviewer 3) the name 'basilin' is unsuitable. Retaining the name The full reviews are appended below for your information.Reviewer #1:FAM65B, which is renamed as basilin, in structural integrity of stereocilia in hair cells. Most of the conclusions, based on genetic, biochemical and imaging data, are quite convincing. Nevertheless, the reviewer has several major concerns regarding the super-resolution data:This manuscript reveals the functional role of 1) 2) 3) Reviewer #2:FAM65B (renamed "Basilin"), a protein previously shown to be linked to inherited forms of deafness but whose molecular and cellular function has remained unknown. The major findings are: 1) basilin is expressed at the taper end of the sensory inner hair cell stereocilia and hair bundles are disorganized in basilin deficient mice; 2) basilin forms head to head and tail to tail oligomers assembled in a ring like pattern at the base of each stereocilia; 3) oligomer formation depends on RhoC (a Rho kinase) binding which also regulates basilin activity. Interestingly, while basilin has been shown to interact with RhoA in neutrophils , the current work reveals only weak interaction between basilin and RhoA but highly efficient interaction with RhoC. Somewhat surprising, co-IP does not show any interaction between basilin and Cdc42 and Rac1, other Rho family members expressed in hair cells and demonstrated to play a role in cellular patterning, polarization and planar polarity . A major part of this report is based on Co-IPs (12 panels), which assess interaction between basilin and basilin constructs  with taperin, CLIC5, radixin, whirlin, RhoA, RhoC, Rac1 and Cdc42. Super-resolution microscopy demonstrates distinct localization between taperin and basilin  had been shown to be responsible for deafness , the role of this protein in hair cells has been unclear. Here the authors combine biochemical (mostly Co-IP), super-resolution microscopy and functional studies to provide insight into  basilin  and lineThe title of the manuscript is not justified. While STORM microscopy resolves localization of basilin and taperin  in stereCo-IP results in HEK-293 cells, in particular the lack of interactions between some of the players may not reflect the situation in native hair cells. Since a large portion of the work is based on co-IP, it is important to discuss these findings critically. Co-Ip using a different cell line such as PK1-CL4 cells or pull down assay might reveal different results. In particular, the lack of interaction with CLIC5, Radixin and whirlin is puzzling in part because Taperin has been shown to interact with CLIC5 . Have the authors also tested interactions with PTPRQ or Myosin VI also present in the tapering region of the stereocilia?Since Rho family members have been shown to play a role in planar polarity, is there evidence of planar polarity defects (kinocilium position or bundle orientation) in the basilin deficient mouse?Reviewer #3:FAM65B, thought by some to be a PX-BAR domain protein, (i) forms oligomeric ring-like structures in a Rho-C dependent at the base of hair-cell stereocilia and (ii) is required for the normal development and maintenance of hair-bundle structure in the mouse cochlea. The paper considerably extends upon the recent paper from Dias-Horta and colleagues showing that a deletion in FAM65B causes deafness in a human family and is required for the normal function and survival of hair cells in the zebrafish. Whilst the extent to which the paper reveals 'insights into the mechanisms by which proteins regulate hair cell function' remains debatable, this is an impressive body of work that certainly provides considerable insight into the ever increasing molecular complexity of the sensory hair bundle.An interesting and exciting paper that utilizes an impressive suite of methodologies to provide evidence that Additional comments:Introduction: 'Mutation in nearly all of the genes…'. Are there mutations in genes that encode proteins that localise to the base of stereocilia that do not cause deafness?FAM65B appears to have quite a widespread distribution. It may be worth mentioning here that FAM65B may be a PX-BAR domain protein that could interact with lipids and regulate membrane curvature.The value of giving a protein yet another name is questionable. Aside from confusion causing by renaming, is basilin the best of names? It was originally used for a protein that was found in the basilar membrane but then dropped as the protein proved to be EMILIN-2. Many things are basally located in many structures, and then there are basal bodies, basal laminae…Introduction: 'Basilin does not bind to taperin or any of its known binding partners'. Slightly ambiguous; rephrase.FAM65B?Results first paragraph, first line: Is the ko mouse really a model for a human disease in which there is an in frame deletion in Results, end of second paragraph: Are the OHCs even present, let alone functional? They disappear in the zebrafish MO knockdown so do they have hair bundles or survive to 4 weeks of age?In the third paragraph of the subsection “Basilin expression in hair cells of the inner ear”: Is the commercially available antibody from Sigma any better than the two used by Dias -Horta et al.? Do the latter give staining in the Fam65b ko? Are these among the many antibodies that not of sufficient quality for immunolocalisation studies and/or STORM? Is there some explanation as to why Dias-Horta found localisation to the apical membrane and the membrane of stereocilia and this study does not? The 'validation' image in In the first paragraph of the subsection “Basilin deficiency affects hair bundle morphogenesis and stereociliary growth”. Indicate in figure with arrows which stereocilia are abnormally thick and large. Are the stereocilia fusing?FAM65B affect hair bundle cohesion and growth? The TEM is not of very good quality. At best one can see the base of the tapered region in one stereocilium in the ko. Does the taper not form in the ko? This really needs to be addressed. Is taperin displaced up the stereocilia in the Fam65b ko? The apical protrusions are interesting; is this due to a loss of membrane cytoskeletal interactions at the base of the stereocilium? Are the perturbations seen really due to 'defects in bundle cohesion and growth'?In the second and third paragraphs of the subsection “Basilin deficiency affects hair bundle morphogenesis and stereociliary growth” and 2+ was reduced to 0.02 mM? Also, were reverse polarity currents measured (as indicated in methods?). Considering the localisation data and the theoretical contribution of the ankle region to hair-bundle properties it is surprising that the authors have not addressed whether the stiffness or resilience of the hair bundle is altered in any way in the ko. Displacement , Tanya Whitfield (Reviewing editor), and three reviewers. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Thank you for resubmitting your work entitled "Fam65B forms ring-like structures at the base of stereocilia critical for mechanosensory hair cell function" for further consideration at 1) There now appear to be three different versions of the revised title – one on the cover page, one on the first page of the manuscript and one in the rebuttal letter. Please clarify which of these you propose to use.2) For the new measurements provided for the Fam65B rings, please state what the confidence bounds represent .3) Reviewer 1 recommends that the HEK images  should b4) There was no mention of TRIOBP in the text, as stated in the rebuttal.Further detail is provided in the assessments from Reviewers 1 and 3 below, for your information. Reviewer 2 was satisfied with your response and did not have any further comments.Reviewer #1:The revised manuscript has satisfactorily addressed previous concerns regarding For the HEK cell STORM images in Reviewer #3:FAM65B. Whilst the quantitation of the diameters of the FAM65b and taperin staining patterns provide support for the structural model, these numbers are presumably derived from a subset of the FAM65b clusters seen at the base of the stereocilia; the standard errors (?) are small and judging from The authors have addressed most of the points raised by the reviewers well. The additional SEM data is fascinating, although it is hard to immediately understand why the thin, tapered region of the stereocilium should get longer in the absence of  Essential revisions:1) A thorough, objective and quantitative analysis of the STORM imaging data should be provided, together with appropriate statistical analysis where required, as requested by Reviewer 1. Inferences about direct interactions  should be cautious unless further analysis can strengthen the evidence for these interactions.We have quantified the diameter of the Fam65b circumferential ring and the taperin dense-core-like structure at the base of stereocilia. The diameter of Fam65b structure is 229 ± 7 nm (n=41) and taperin structure is 228 ± 9 nm (n=56). While the data indicate a potential overlap between the two structures , we provide substantial new data supporting the view that Fam65b and taperin do not interact directly. We performed Co-IP experiments in HEK293 cells  and in response to the reviewers' comments now also in CL4 cells, which have been used especially by the Bartles and Berryman laboratories to study interactions between proteins important for hair cell function. We provide positive controls demonstrating that we can reveal known interactions (e.g. taperin with CLIC5). In addition, we have carried out yeast-two-hybrid assays to evaluate interactions between Fam65b and taperin but cannot detect any interactions but we identify new interactions (e.g. Fam65b with RhoC). Taken together, the findings suggest that Fam65b does not bind directly to taperin. However, the two proteins might interact via an intermediate protein. We have revised the text to explain our data more clearly.2) Likewise, the interpretation of the co-IP results in HEK-293 cells should be discussed with appropriate caution, as requested by Reviewer 2. If additional co-IP results from a different cell line are available, these should be included.As outlined above, we have performed additional Co-IP experiments in CL4 cells and carried out additional yeast-two-hybrid experiments. We include appropriate positive controls. Our data confirm the published interaction of taperin with CLIC5 but do not reveal any interaction between taperin and Fam65b. The data are included in the revised manuscript.3) A full structural analysis of the stereociliary bundle phenotype, including cohesion, growth, presence of tapers and stiffness should be provided, as requested by Reviewer 3.We have substantially expanded our analysis of the morphological phenotype of hair cells using SEM and include an additional figure  as well Given the strong structural defects and variability in morphology, we did not include measurements of hair bundle stiffness. Most biophysical measures on hair-bundle stiffness that have been published were collected from non-mammalian species that have substantially longer stereocilia than mice. These measurements are extremely difficult to quantify in murine hair cells and while we attempted measurements we could not determine reliable parameters that could be applied to the morphologically abnormal hair bundles in our mutant mice.A) It was agreed that further electrophysiological analyses of the transduction currents  would be beyond the scope of this manuscript, and so are not required. However, the authors should acknowledge and discuss any limitations of the methods they have used.We have revised the text to outline the limitations of our approach. The experiments are very interesting but we confront a similar problem as for stiffness measurements. It is very difficult to assign primary versus secondary effects due to heterogeneous degenerative changes in hair cells.4) A comment on the presence or absence of the outer hair cells and of hair cell polarity defects in the mutant should be included.We now include a quantification of hair cell survival at 2 and 4 weeks of age . There i5) Any discrepancies between the findings and those of Diaz-Horta et al. should be discussed, as requested by Reviewer 3.Unlike Diaz-Horta in studies with zebrafish, we did not observe significant hair cell loss at 2 and 4 weeks in mice, which suggests that the hearing phenotype in our rodent model is not caused by death of hair cells. There could be a species difference in Fam65b function and/or hair cells in zebrafish might die more readily when their hair bundles show morphological abnormalities. We discuss this in the revised manuscript.Diaz-Horta et al. reported that Fam65b is localized in murine hair cells along the length of stereocilia. However, judging from the images in the manuscript, they show high expression at the base of stereocilia and less along their lengths. We purchased the two commercial antibodies used by Diaz-Horta and found that we could not detect convincing staining for Fam65b in hair cells. This might perhaps be explained by differences in antibody batches provided by the manufacturer at different times. We would also like to point out that the two commercial antibodies used by Diaz-Horta were raised against the N-terminus of Fam65b, which shows 70% similarity with Fam65a and Fam65c. We used an antibody to the C-terminus of Fam65b, which has very low homology with Fam65a/c. Perhaps hair cells express Fam65a, b, c and Diaz-Horta observed with their batch of antibody not only Fam65b. The antibody that we used is clearly specific as it does not stain hair cells from Fam65b mutant mice. We have revised the text to discuss the discrepancy between the studies and possible explanations for the difference.6) The title should be revised, as requested by Reviewer 2, to reflect the findings of the manuscript more generally.We have revised the title: "Fam65b and RhoC cooperate to form ring-like structures at the base of stereocilia critical for mechanosensory hair cell function"7) The name 'basilin' should be removed from the manuscript. There appears to be no strong reason to re-name the protein, and indeed, several arguments (as raised by Reviewer 3) the name 'basilin' is unsuitable. Retaining the name FAM65B will help with cross-reference to existing literature.We now refer throughout to Fam65b.The full reviews are appended below for your information.Reviewer #1:This manuscript reveals the functional role of FAM65B, which is renamed as basilin, in structural integrity of stereocilia in hair cells. Most of the conclusions, based on genetic, biochemical and imaging data, are quite convincing. Nevertheless, the reviewer has several major concerns regarding the super-resolution data:1) We have revised the manuscript to address this point. As the reviewer correctly points out, we had to image regions at the edge of cells where label-density is low. The choice was deliberate because it is impossible to reach conclusions from areas with very dense labeling because there are just too many clustered dots, which cannot be assigned to different substructures. Also, we did not mean to imply that the HEK images show that Fam65b forms ring-like structures, just beads-on-a-string like arrangements that would be consistent with oligomerization . The arrangements of the dots is not random, reproducibly seen in many cells and does not resemble any cellular structure that we have seen for any protein in HEK cells. It is difficult to quantify the data in an unbiased manner because we cannot resolve the structures in more densely labeled areas of a cell and are thus selective in the images that we show.We have left the images in the revised manuscript and describe them much more carefully, also pointing out the limitations associated with the figure and finding. If the reviewer considers it is the better option, we are happy to remove the image from the manuscript. Not showing the data does not affect the central conclusion of our manuscript.2) We entirely agree with the reviewer that the STORM images cannot be used to rule in/out direct interactions between Fam65b and taperin. We formulated our text poorly. Our biochemical data (now extended), but not the imaging data, support that Fam65b and taperin do not interact directly.Following the reviewer’s suggestion, we have now quantified the STORM images. The diameter of the Fam65b ring is 229 ± 7 nm (n=41) and of the taperin structure 228 ± 9 nm (n=56). While the data indicate a potential overlap between the two structures , we provide substantial new data supporting the view that Fam65b and taperin do not interact directly . However, the similarity in diameter of the structures labeled by the two antibodies suggests that the two structures are potentially connected, perhaps by a linker protein. We discuss this now more carefully.The antibodies we used for STORM were raised in rabbits. We tried several different antibodies and dyes, but we could not find a good combination that allowed us to carry out double-labeling for Fam65b and taperin. We are in the process of generating additional antibodies including monoclonals, but this will take substantial time.3) We agree that co-localization does not mean that two proteins directly interact and we did not want to use the data as support for a direct interaction. To show that Fam65b and RhoC interact, we carried out yeast-two-hybrid analysis and Co-IP experiments. However, having identified an interaction between the two proteins in these heterologous systems, we wanted to confirm that they are present in hair cells in a similar distribution as a first step towards a validation that the biochemical data may be physiologically relevant. Clearly, the proteins are appropriately localized in hair cells to act in a common subcellular compartment. We attempted to further solidify the data and carry out STORM microscopy with RhoC antibodies but did not identify antibodies that worked with the technique. We have carefully modified the text to explain the localization data better. However, we still find that we provide significant evidence for a physical and functional interaction between Fam65b and RhoC: (i) Co-IP and yeast-two-hybrid to demonstrate direct interaction; (ii) demonstrating in cell lines that RhoC promotes Fam65b oligomerization; (iii) demonstrating in hair cells that Fam65b oligomerization and RhoC binding are critical for hair bundle morphogenesis.Reviewer #2:[…] The title of the manuscript is not justified. While STORM microscopy resolves localization of basilin and taperin  in stereWe have revised the title: "Fam65b and RhoC cooperate to form ring-like structures at the base of stereocilia critical for mechanosensory hair cell function".Co-IP results in HEK-293 cells, in particular the lack of interactions between some of the players may not reflect the situation in native hair cells. Since a large portion of the work is based on co-IP, it is important to discuss these findings critically. Co-Ip using a different cell line such as PK1-CL4 cells or pull down assay might reveal different results. In particular, the lack of interaction with CLIC5, Radixin and whirlin is puzzling in part because Taperin has been shown to interact with CLIC5 . Have the authors also tested interactions with PTPRQ or Myosin VI also present in the tapering region of the stereocilia?We have carried out additional experiments to strengthen the biochemical data. We have carried out immunoprecipitations in 293 and CL4 cells and we provide new yeast-two-hybrid data. The results from all experiments agree, revealing no interaction of Fam65b with taperin. As a positive control, we show that taperin interacts with CLIC5 as published. We also performed Co-IPs of the cytoplasmic domain of PTPRQ with Fam65b but did not observe an interaction. Finally, we carried out more yeast-two-hybrid screens but never identified interactions of Fam65b with proteins of the taperin/CLIC5/Whirlin complex, or with PTPRQ and MyoVI. We do not consider this a concern. The data suggest that there are several distinct protein complexes at the base, which is in agreement with the STORM images for Fam65b and taperin – clearly the proteins do not show the same distribution in hair cells. We anticipate that there might be a yet-to-be identified protein that links Fam65b to the taperin/CLIC5/Whirlin complex and we are trying to identify this protein. We also like to point out that there are other proteins such as TRIOBP that are abundant near the base of stereocilia and that form independent structures with no known biochemical link to the taperin/CLIC5/Whirlin complex.We have revised the text and carefully address the limitations of our biochemical analysis.Since Rho family members have been shown to play a role in planar polarity, is there evidence of planar polarity defects (kinocilium position or bundle orientation) in the basilin deficient mouse?Yes, we have analyzed this. We now include data on hair-bundle polarity and observe clear polarity defects in hair cells at P2 .Reviewer #3:[…] Introduction: 'Mutation in nearly all of the genes…'. Are there mutations in genes that encode proteins that localise to the base of stereocilia that do not cause deafness?We have corrected the text. So far all genes that have been localized to the base seem to be linked to deafness.The value of giving a protein yet another name is questionable. FAM65B appears to have quite a widespread distribution. It may be worth mentioning here that FAM65B may be a PX-BAR domain protein that could interact with lipids and regulate membrane curvature.We refer now to Fam65b throughout. We now discuss extensively that Fam65b might be a PX-BAR domain protein.Aside from confusion causing by renaming, is basilin the best of names? It was originally used for a protein that was found in the basilar membrane but then dropped as the protein proved to be EMILIN-2. Many things are basally located in many structures, and then there are basal bodies, basal laminae… We refer throughout to Fam65b.Introduction: 'Basilin does not bind to taperin or any of its known binding partners'. Slightly ambiguous; rephrase.Sorry, that was not a good formulation. We have modified the text.Results first paragraph, first line: Is the ko mouse really a model for a human disease in which there is an in frame deletion in FAM65B?We have modified the text to be more accurate. We do not mimic the precise mutation linked to disease in humans and clearly describe this now in the text.Results, end of second paragraph: Are the OHCs even present, let alone functional? They disappear in the zebrafish MO knockdown so do they have hair bundles or survive to 4 weeks of age?We did not observe significant hair-cell loss at 2 and 4 weeks, which suggests that the hearing phenotype in our mice is not caused by death of hair cells. We have included the data in the revised manuscript. Diaz-Horta et al. studied zebrafish, and there could be a species difference in Fam65b function or hair cells; zebrafish hair cells might die more readily when their hair bundles show morphological abnormalities. We discuss the differences between the two studies in the revised manuscript. We also show data for mechanotransduction recordings, revealing functional defects in the hair cells.In the third paragraph of the subsection “Basilin expression in hair cells of the inner ear”: Is the commercially available antibody from Sigma any better than the two used by Dias -Horta et al.? Do the latter give staining in the Fam65b ko? Are these among the many antibodies that not of sufficient quality for immunolocalisation studies and/or STORM? Is there some explanation as to why Dias-Horta found localisation to the apical membrane and the membrane of stereocilia and this study does not?Diaz-Horta et al. reported that Fam65b is localized in murine hair cells along the length of stereocilia. However, judging from the images in the manuscript, they show very high expression at the base of stereocilia and less along their lengths. We purchased the two commercial antibodies used by Diaz-Horta and found that we could not detect convincing staining for Fam65b in hair cells. This might have to do with the fact that we likely obtained different batches of antibody from the manufacturer. We would also like to point out that the two commercial antibodies used by Diaz-Horta were raised against the N-terminus of Fam65b, which shows 70% similarity with Fam65a and Fam65c. We used an antibody to the C-terminus of Fam65b, which has very low homology with Fam65a/c. Perhaps hair cells express Fam65a, b, c and Diaz-Horta observed with their batch of antibody not only Fam65b. We have revised the text to discuss the discrepancy between the studies and possible explanations for the difference.In our hands, the Sigma antibody works very well and does not stain hair cells from Fam65b mutant animals. We have purchased several batches and obtained reproducible results.The 'validation' image in We apologize for confusing images. We now provide images with matched exposure.There is considerable staining of the microvilli on the apical surface of all the supporting cells, and no images are shown of the apical surface of Hensens's cells. Do the microvilli on these cells have basal tapers, and are there any subtle defects in the structure or function of these microvilli? Is any lacz staining seen in Dieters' and pillar cells if the X-gal staining time is increased? Or is the microvillar staining seen on the surfaces of all supporting cells except Hensen's cells 'non-specific'?Our analysis of hair bundle morphology suggests that Fam65b is not essential to form tapers but that stereocilia degenerate during their maturation. Thus, Fam65b could be present on other cell types even in microvilli without tapers.Regarding the b-gal staining results and the immunohistochemistry, we have substantially revised the text and provide new data. The b-gal data show particular prominent expression in hair cells and Hensen's cell. However, b-gal is not a very sensitive read-out and we cannot exclude that there is weaker expression in other support cells. Using antibody staining, we see prominent labeling of hair cells but also clear signal in Hensen's cells and in some of the microvilli on the top of support cells. The staining is not seen in mutant mice, indicating that the signal is specific. We thus conclude that Fam65b is expressed in hair cells but also likely in support cells including Hensen's cells. New images for Hensen's cells are now included and we discuss the limitations of the b-gal staining data and our results more clearly.By SEM, we have not been able to observe structural defect in microvilli of support cells and mention this now in the text.In the first paragraph of the subsection “Basilin deficiency affects hair bundle morphogenesis and stereociliary growth”. Indicate in figure with arrows which stereocilia are abnormally thick and large. Are the stereocilia fusing?We describe now in revised In the second and third paragraphs of the subsection “Basilin deficiency affects hair bundle morphogenesis and stereociliary growth” and We have substantially expanded our analysis of the morphological phenotype of hair cells using SEM. We found that the morphological phenotype in hair cells is very heterogeneous with some cells showing defects in polarity, cohesion and/or growth already during early stages of hair cell development. In addition, the taper region of stereocilia appears abnormal and we observed unusual membranous protrusions from the apical cell surface. The defects at the taper are consistent with the localization of Fam65b to this region. We provide a quantitative analysis of the morphological phenotypes by grouping the hair cells according to different parameters into four morphological classes. We also provide a quantification of hair bundle polarity defects. Overall, the findings suggest that stereocilia grow and start to form a bundle with a staircase but tapers are abnormally shaped and bundles start to deteriorate before they reach maturity.We acknowledge that we do not fully understand the cellular mechanisms by which mutation in Fam65b causes defects in hair bundles but our data suggest that it has a critical structural role at the base of stereocilia. We discuss this carefully in the revised manuscript and try to avoid over-interpretation. However, even without a detailed mechanism, we still find that our findings are a significant step forward in our understanding of Fam65b function outlining subcellular distribution, physical and functional interactions with RhoC and a clear link between Fam65b and the morphogenesis of hair bundles.2+ was reduced to 0.02 mM? Also, were reverse polarity currents measured (as indicated in methods?). Considering the localisation data and the theoretical contribution of the ankle region to hair-bundle properties it is surprising that the authors have not addressed whether the stiffness or resilience of the hair bundle is altered in any way in the ko. Displacement  and of the taperin structure 228 ± 9 nm (n=56). The data indicate a potential overlap between the two structures . We discuss this now more carefully.We tried extensively to use in STORM microscopy several different labels together with antibodies to Fam65b and taperin. We attempted to label membranes  as well as F-actin. However, we were unable to visualize double labeling with our instrumentation. For membrane staining, Vybrant DiI, Vybrant DiO, and Vybrant CM-DiI were readily taken up by supporting cells in the cochlea, but not very efficiently by the hair cells themselves; not just the stereocilia bundles, but also the apical membranes in hair cells did not stain significantly. Background was too high with biotin-based membrane staining methods. We were also looking for a good fluorophores coupled to phalloidin that could bleach with our laser illumination set-up , but using commercially available fluorophore-conjugated phalloidin we could not achieve good F-actin staining using STORM. We have more recently approached researchers at other institutions for collaborations to explore other technologies  but do not yet have access to these instruments. Even without these data, we find that our studies make a substantial contribution and reveal clear differences in the distribution of taperin and Fam65b at the base of stereocilia. The diameter of the structures that we image is in good agreement with the diameter of stereocilia, suggesting localization of Fam65b near the membrane, as depicted in the diagram. Please note that we do not claim that our data show affinity of Fam65b for membranes or lipids (others have done that in the published literature). We merely depict a plausible model and have revised the text carefully to point this out.We only excluded TRIOBP because it is more widely localized along rootlets and the figure is already crowded. However, we now mention TRIOBP in the text. It would indeed be very interesting to explore a connection between TRIOBP and Fam65b but we have not carried out experiments along these lines.In the subsection “Basilin oligomerization is important for its function” and Expression in some of the displayed cells was low and did not display well in the provided image. We provide now a more suitable and representative images. We also show one cell that expresses very low levels of the protein and, as expected, no morphological disruptionIn the last paragraph of the subsection “RhoC binding to basilin is critical for basilin function in hair cells” and We specify this now in the text. The morphological defects of cultured Fam65b hair cells are more severe than in vivo. Most of them assume a round-shape or have strong defects in cohesion. This indicates that the bundles are likely fragile and that morphological defects are exaggerated by the dissection procedure. Following rescue by gene expression, the hair bundles show a clear organized morphology with a characteristic V-shape.Discussion, fourth paragraph: Are the ring like structures seen when FAM65B is expressed in heterologous cells associated with any structures? Surely physiological studies will be also be required to reveal if FAM65B oligomers form spring-link structures at the base of the stereocilia?As pointed out in response to the comments of reviewer 2, we have modified our section on the structures in heterologous cells – the data are qualitative in nature and if the reviewers consider them inappropriate for inclusion we can remove them without affecting the conclusions of the manuscript.Regarding the "spring-like structures", we entirely agree that we do not have data to claim that the molecules form springs. We wanted to bring this up as an interesting discussion point. We have now further modified the text to express this explicitly: "One interesting possibility would be that basilin oligomers form spring-like structures near the membrane at the base of stereocilia that affect the mechanical properties of the bundle. While there is currently no experimental evidence for this model, we are carrying out structural studies to address this possibility."Methods: Minimal details are provided for STORM analysis.We now provide a detailed description of the method:“Whole mount staining and super-resolution imagingCochlear whole mount staining was carried out as described . Whole mount images were captured by fluorescent deconvolution microscopy (Deltavision). […] Additional reagents were: Alexa Fluor 488-phalloidin, Alexa Fluor 647-phalloidin , Alexa Fluor 488 and Alexa Fluor 647 conjugated goat anti-rabbit secondary antibodies.”Thank you for resubmitting your work entitled "Fam65B forms ring-like structures at the base of stereocilia critical for mechanosensory hair cell function" for further consideration at eLife. Your revised article has been favorably evaluated by Gary Westbrook (Senior editor), a Reviewing editor (Tanya Whitfield), and three reviewers. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:1) There now appear to be three different versions of the revised title – one on the cover page, one on the first page of the manuscript and one in the rebuttal letter. Please clarify which of these you propose to use.We apologize for the confusion. We propose the following title: “Murine Fam65B forms ring-like structures at the base of stereocilia critical for mechanosensory hair cell function.” We have modified the text of the manuscript accordingly.2) For the new measurements provided for the Fam65B rings, please state what the confidence bounds represent .The result is represented as Mean ± SEM. This is now stated in the first paragraph of the subsection “Analysis of Fam65b and taperin distribution in hair cells” .3) Reviewer 1 recommends that the HEK images  should bThe images have been removed and the text and figure legends have been adjusted.4) There was no mention of TRIOBP in the text, as stated in the rebuttal.We now cite TRIOBP: “In addition, TRIOPB forms rootlets that extend from stereocilia into the cuticular plate and traverse the taper region .”Further detail is provided in the assessments from Reviewers 1 and 3 below, for your information. Reviewer 2 was satisfied with your response and did not have any further comments.Reviewer #1:The revised manuscript has satisfactorily addressed previous concerns regarding We have now revised the text accordingly: “We therefore carried out measurements to determine the diameter of Fam65b ring-like structures and taperin circles under in the tallest row of stereocilia of P7 inner hair cells using NIS-Elements AR analysis software. The ring-like Fam65b structure had a diameter of 229 ± 7 nm , while the taperin structure had a diameter of 228 ± 9 nm .”Reviewer #3:The authors have addressed most of the points raised by the reviewers well. The additional SEM data is fascinating, although it is hard to immediately understand why the thin, tapered region of the stereocilium should get longer in the absence of FAM65B. Whilst the quantitation of the diameters of the FAM65b and taperin staining patterns provide support for the structural model, these numbers are presumably derived from a subset of the FAM65b clusters seen at the base of the stereocilia; the standard errors (?) are small and judging from We have now revised the text accordingly: “We therefore carried out measurements to determine the diameter of Fam65b ring-like structures and taperin circles under in the tallest row of stereocilia of P7 inner hair cells using NIS-Elements AR analysis software. The ring-like Fam65b structure had a diameter of 229 ± 7 nm , while the taperin structure had a diameter of 228 ± 9 nm .”