We have designed a whole-animal screen that tests for rescue of both up- and down-regulated gene expression in mutant embryos, together with analysis of weak and strong alleles. From a screen of 3120 structurally diverse compounds, we have identified 68 that reduce versican b expression in the adgrg6 mutant ear, 41 of which also restore myelin basic protein gene expression in Schwann cells of mutant embryos. Nineteen compounds unable to rescue a strong adgrg6 allele provide candidates for molecules that may interact directly with the Adgrg6 receptor. Our pipeline provides a powerful approach for identifying compounds that modulate GPCR activity, with potential impact for future drug design.Adgrg6 (Gpr126) is an adhesion class G protein-coupled receptor with a conserved role in myelination of the peripheral nervous system. In the zebrafish, mutation of  Targeteethality . In humaongenita . Peripheongenita . Human Acoliosis   components and ECM-modifying enzymes (adgrg6 mutants (Adgrg6 (Gpr126) mRNA is known to be expressed in the mouse ear , leading to elevated cAMP levels and activated protein kinase A (PKA) to induce transcription of downstream target genes, such as egr2 and oct6 , a seven-pass transmembrane domain (7TM), and a short intracellular domain  or Tg(mbp:eGFP) fluorescent transgenic lines to screen for small molecules that increase progenitor or myelinating oligodendrocyte cell number. While elegant in design, and successful in identifying hit compounds, these screens required the use of sophisticated and costly high-resolution imaging platforms and relied on detailed quantitative assays for cell number, techniques that are not available to all labs and are potentially limiting in scalability and throughput.To date, over one hundred drug screens using different zebrafish disease models have been conducted, some identifying lead compounds that have subsequently been tested in mammalian model systems or entered clinical trials  reviewe. Two scrversican b (vcanb) as an easily-scored qualitative readout to identify compounds that can reduce vcanb overexpression back to normal levels in a hypomorphic mutant allele for adgrg6, tb233c. We used expression of mbp in the posterior lateral line ganglion of tb233cadgrg6 mutants as a secondary screening assay, with the aim of identifying chemical classes capable of rescuing the expression of both genes, which may thus represent agonists of the Adgrg6 signalling pathway. To identify ligands that potentially bind directly to Adgrg6, we then tested hit compounds for their ability to rescue a strong loss-of-function adgrg6 allele, fr24, which predicts a severely truncated protein. Several compounds were unable to rescue fr24adgrg6 mutants, including a group with similar structures from the gedunin family of compounds. Compounds able to rescue both alleles include colforsin, a known activator of adenylyl cyclase, demonstrating proof-of-principle that our screen can identify compounds that restore GPCR pathway activity downstream of the receptor. These alternative assays for both down-regulation and up-regulation of gene expression, combined with a comparison of rescue in both weak and strong alleles, have facilitated selection of a strong cohort of hit compounds that can be differentiated by the different screens used. Our approach is scalable and can be used to screen additional compound collections. In parallel, chemoinformatics analysis of the compound libraries and identified hits has enabled classification and prioritisation of selected hit compounds.In this study, we have developed an in vivo drug screening assay based on semi-automated in situ hybridisation (ISH) to identify modulators of the Adgrg6 pathway. We have used the otic expression of vcanb expression in the adgrg6 mutant ear for our primary screen. vcanb has a number of advantages for screening, including highly localised expression in the otic vesicle, very strong and reproducible staining intensity in adgrg6 mutant embryos, and a clear difference between staining in mutant and wild-type embryos at the stage chosen, making it ideal for manual scoring , which has the advantage of being a simple, reproducible assay that can be semi-automated . We sele scoring . We thertb233c) and strong (fr24) alleles for the adgrg6 mutant  in the fourth transmembrane domain of the receptor, whereas the fr24 allele is a nonsense mutation (L463X), predicting a severely truncated protein lacking the hormone-binding, GAIN, 7TM and C-terminal domains , but is then strongly down-regulated after fusion; by 4 days post fertilisation (dpf), very little expression is detectable in the ear .vcanb expression in the ear, the zebrafish adgrg6 mutant also shows a reduction or loss of expression of the myelin basic protein (mbp) gene in the PNS  associated with the posterior lateral line ganglion (PLLg)  is not affected in either allele, obscuring any reduction of mbp staining in the PNS without performing a detailed analysis. This made mbp expression unsuitable for a primary screen, but useful for a secondary screen of hit compounds identified from the vcanb screen.In addition to an upregulation of  the PNS . This adutations . Expressn (PLLg)   . The fr2l nerves   and strong (fr24) alleles of adgrg6 mutants are homozygous viable, enabling large batches of 100% mutant embryos to be generated for each assay. We decided to use the hypomorphic allele (tb233c) in our primary screen, for four main reasons: (1) adult fish homozygous for the tb233c allele produce a larger number of healthy embryos than adults homozygous for the fr24 allele; (2) a lower concentration of our positive control compound IBMX was sufficient to rescue the phenotype in tb233c mutants compared with fr24 mutants  vcanb expression, although not as dramatically affected as in fr24, is still robustly over-expressed in the tb233c allele, and (4) we predicted that any small molecules that interact with the active site of the receptor or act as allosteric modulators would be missed in a screen using fr24 mutants, which should only be able to identify compounds acting on targets downstream of the receptor. By using tb233c, we should be able to identify modulators of the pathway acting both downstream and at the level of the receptor itself.Our strategy for the screening protocol and analysis pipeline is outlined in  mutants , suggestvcanb expression and a rescue of projection fusion in the ears of tb233cadgrg6 mutants (nacre (-/-mitfa) strain, which has reduced pigmentation, facilitating visualisation of ISH staining patterns, was used as an untreated wild-type control. Three embryos per well were treated with compounds at 25 µM in E3 medium from 60 to 90 hpf, after which they were fixed and analysed for expression of vcanb by whole-mount ISH. At the embryonic stage assayed by ISH (90 hpf), expression of vcanb in untreated mutant embryos is very specific to the ear, making it clearly visible as two dark spots in the head of each embryo within the well. All controls gave results as expected in all assay plates tested: DMSO-treated mutant embryos showed strong otic staining for vcanb, untreated wild-type embryos showed very little staining in the ear, and IBMX-treated mutant embryos showed rescued (down-regulated) otic vcanb expression  as a positive control. We have previously shown that addition of 100 μM IBMX at 60 hpf is optimal for both down-regulation of  mutants . At this mutants . Compounpression .In order to test a wide range of compounds, we chose to screen two commercially available small molecule libraries. The Tocriscreen Total library (‘Tocris’) consists of 1120 compounds representing known bioactive compounds with diverse structures. The Spectrum Collection  comprises 2000 compounds, including FDA-approved drugs for repurposing, bioactive compounds and natural products. Scaffold analysis of the two libraries highlights the structural diversity present . Based ovcanb mRNA levels, we used a scoring system from 0 to 3  compounds from the Tocris library and 205 (10%) from the Spectrum library that scored in categories A–C (vcanb expression (category D). The largest category , as expected, represented compounds that had no rescuing or other effect at the concentration used (25 µM). To visualise the complete set of screening results and to identify any clusters of hit compounds with similar structures, compounds were displayed as individual data points on a polar scatterplot . Compound position along the circumference of the plot for each library is based on position on the respective similarity dendrogram . Specifically, 83 out of the 92 possible hit compounds from the Tocris library and 145 of the 205 possible hits from the Spectrum library were retested twice, again with three embryos per well. By increasing the number of embryos screened to a total of nine per compound, we aimed to eliminate any false-positive hits that had an increased average score over these two retests or did not show a clear rescue (score >7) in any individual test. In total, 91 compounds from the combined list of hits  that showed consistent rescue of y assays .adgrg6 ear phenotype  inhibitor, is present in the Spectrum library and was identified as a hit in the primary screen . The moshenotype , but forvcanb score average from the two individual screens. 39 (25%) of the 155 duplicate compounds yielded a vcanb score average that differed by 1–2 units between the two libraries; 12 (8%) of the compounds yielded a vcanb score average that differed by 3–6 units, whereas only 4 (3%) compounds had a score average that differed by 7–9 units. In summary, 90% (139/155) of the compounds common to both libraries showed similar scores for the vcanb assay from each library (scores differing by ≤2 units), whereas 10% (16/155) of the compounds resulted in differing levels of vcanb down-regulation between the two different libraries. After retesting, the difference between the two vcanb score averages for nine of these compounds was reduced; however, for seven compounds, the scores between the two libraries remained significantly different. These discrepancies could be either due to differences in compound purity between the two suppliers, or could be due to experimental error . In cases where the same compound was scored as toxic in one assay and not in another, the health condition of the embryos in a particular well could be the underlying reason. Duplicated compounds have been included in the data for each library in the polar scatter plots  had exactly the same er plots  and 4.vcanb assays were combined to give a complete list of 89 unique compounds, with baicalein and gedunin present in both libraries. The list covers a wide spectrum of naturally-derived and synthetic molecules, with known and unknown functions  that scored A–C in all three unctions . The hitvcanb expression significantly reduced the possibility of false-positive results due to experimental error (e.g. in the ISH protocol), but the list of hits could still contain false-positive compounds that may generally inhibit transcription. In order to eliminate such compounds, we exploited the expression of mbp as a secondary screening assay, scoring for rescue of expression around the posterior lateral line ganglion (PLLg)  were subjected to this secondary assay for mbp expression. We used the same assay format and treatment time window as for vcanb, as we had previously found that treatment with IBMX between 60 and 90 hpf was also able to rescue mbp expression in adgrg6 mutants (not shown).The two retests for n = 6 fish tested per drug), compounds were categorised into groups based on their average mbp score. These included groups of compounds that showed rescue of the mutant phenotype ; no rescue , and those that resulted in a decrease in mbp expression, as shown in mbp expression and thus represent possible modulators of Adgrg6 pathway , it is possible that some or all of these compounds could rescue mbp expression at a higher concentration . The 28 members of this group are structurally and functionally diverse . Groups with five or more compounds included the pyridines  modulator  confirmed that the pyridines cilnidipine, nifedipine and tracazolate hydrochloride were able to decrease otic vcanb expression and increase mbp expression around the PLLg in mutant embryos for the tb233c allele, whereas FPL 64176 was able to reduce vcanb expression but was unable to restore mbp expression to wild-type levels -Bay K 8644, which had originally scored as a non-hit in the primary screen (25 µM). We found that this compound rescued otic periment . Interesperiment . All comperiment .tb233cadgrg6 embryos to concentrations ranging from 0.3 μM to 222.2 μM between 60–110 hpf. Nine embryos were tested for each concentration, and a 1.5-fold dilution series of each drug was used. ISH analysis of the 110-hpf embryos revealed a robust, dose-dependent down-regulation of vcanb mRNA expression in response to treatment with all four drugs  optics at 110 hpf . Consistent with the vcanb scores for the number of projections stained, live DIC images of the inner ear revealed a dose-dependent rescue of projection fusion and pillar formation, which was greater at higher doses , nine compounds were inactive and four were toxic. The majority of the gedunin-related compounds that passed both rounds of retesting were later found also to rescue mbp expression . The shared structural characteristics of the gedunin group may give useful clues for candidate structures of agonistic ligands for Adgrg6. In summary, our study demonstrates a novel screening approach which, when combined with chemoinformatics analysis, is able to delineate both expected downstream rescuers of the Adgrg6 pathway and several candidates for drugs that may interact directly with the Adgrg6 receptor.Interestingly, four of the 19 compounds in the last group are in the cluster of gedunin derivatives identified in ibraries  and 8. Ivcanb expression in the inner ear of adgrg6 mutants, exploiting an easily identifiable phenotype that could be scored manually. Following our primary screen of 3120 small molecules, we tested 89 hit compounds in a counter screen for rescue of the myelination defect in adgrg6 mutant embryos. We identified 41 compounds that can both rescue vcanb expression in the inner ear and mbp expression in Schwann cells of adgrg6 hypomorphic mutants, suggesting these are Adgrg6 pathway-specific modulators. Further analysis of a strong adgrg6 allele, fr24, identified a subset of 19 compounds that are potential direct interactors of the Adgrg6 receptor. This analysis, combined with chemoinformatics analysis of the identified hit compounds, has identified clusters of compounds acting at different levels of the Adgrg6 pathway.Adhesion GPCRs are critical regulators of development and disease, driving cell-cell and cell-ECM communications to elicit internal responses to extrinsic cues. This study set out to identify positive modulators of the Adgrg6 signalling pathway, a key regulator of myelination and inner ear development in the zebrafish embryo. Use of a whole-animal phenotypic (mutant rescue) screen gave the potential to identify compounds affecting the entire Adgrg6 pathway in the correct cellular context. We have used a simple in situ hybridisation approach to assay vcanb expression in the ear of adgrg6 mutant fish compared with the low expression in a small dorsal region of the wild-type ear at four dpf to produce a robust scoring system for our phenotype rescue.An optimal drug screening assay design identifies the maximum number of hit compounds with the minimum number of false positives and false negatives. Chemical screening assays using zebrafish range from simple morphology screens  through vcanb, enabled us to bypass the difficulties of scoring and quantification of mbp staining on a large scale; instead, mbp expression was used as a counter screen on a limited number of cherry-picked hits. Contrary to the primary assays, which screened for down-regulation of vcanb expression, the counter screen assayed for up-regulation of mbp expression, enabling the identification of 21 false-positive compounds that down-regulate the expression of both genes (presumably by inhibiting transcription).Relatively few zebrafish screens have been undertaken to identify compounds that can increase myelination  or restovcanb in the ear and negative for mbp, it is likely that tissue-specific functions of Adgrg6 are mediated through different downstream pathways or are stimulated by different ligands.Determining the false-negative rate for any screen is difficult. In our assay we used only one concentration of compound (25 µM), so it is likely that some of the compounds that were toxic or showed no effect at 25 µM—and thus eliminated from our screen—would be effective at lower or higher concentrations, respectively. One possibility would be to run a parallel screen at a lower or higher concentration or use an alternative protocol with shorter incubation times, an approach that has recently proved successful at identifying different compounds influencing segmentation in zebrafish . Here, tfr24 allele. Both these observations highlight the robustness of the assay and the consistency of the scoring process. In total, the final number of hit compounds identified was similar in both the compound libraries screened, with 42 compounds identified from the Spectrum library (2.1%) and 27 compounds from the Tocris library (2.4%). These hit rates are comparable to those found in other similar screens  belong to the chemical group of dihydropyridines (cluster 1), and initial investigations into compounds with similar structures identified four additional compounds in this class. Several dihydropyridines are known to have neuroprotective effects in mammalian models. Nimodipine, for example, has been shown to trigger remyelination in a mouse model of multiple sclerosis and to improve repair in peripheral nerve crush injuries in rats  and nonsense (fr24) mutations. In total, we found 19 hits that could rescue vcanb expression and mbp expression in the tb233c allele, but were unable to rescue the fr24 allele. We hypothesise that the fr24 allele is unable to produce the full-length Adgrg6 protein including the CTF, and therefore any compounds that interact directly with the receptor would not be able to rescue any CTF-dependent function in this strong allele. Further analysis will be needed to determine whether any of these compounds can bind directly to the Adgrg6 receptor, for example by assessing stimulation of cAMP and direct binding in in vitro assays. However, this approach of using a combination of null and hypomorphic alleles in zebrafish whole-organism screening with the aim of identifying target-specific compounds is particularly exciting and one that the advent of CRISPR/Cas9 technology is placed to take full advantage of, since it is now possible to generate designer mutations in the zebrafish through homology-directed repair . One of these compounds, deoxygedunin, has previously been identified as a TrkB agonist that has neuroprotective properties  and the tetranortriterpenoid (gedunin-derived) compounds (cluster 2), there are also clusters of steroid hormones  and flavonoid compounds . The flavonoids are a group of molecules with wide ranging activities, including anti-cancer  and neurvcanb expression, but did not rescue mbp expression, which may provide useful tools to manipulate semicircular canal formation in vivo. Versican and other chondroitin sulphate proteoglycans (CSPGs) are associated with a number of human pathologies; Versican overexpression has been shown to be strongly involved in inflammation, cancer progression and the development of lung disorders  strain (ZDB-GENO-990423–18), which lack melanophores . adgrg6 mutant alleles used were tb233clau (formerly tb233cbge) and fr24lau (ZDB-GENE-070117–2161)  , and wer17–2161) . Prior t17–2161)  at 28.5°nacre) and homozygous tb233cadgrg6 mutant embryos were raised to 50 hpf at 28.5°C in E3 medium, dechorionated manually with forceps, and then incubated at 20°C overnight to slow down development and facilitate timing of experimental treatments. This regime reduced ear swelling, but did not reduce otic vcanb levels, in mutant embryos. Embryos at the 60 hpf stage were aliquoted at three embryos per well into MultiScreen-Mesh mesh-bottomed plates (Millipore) and transferred to the drug plate . Assay plates were incubated at 28.5°C for 28 hr and the embryos were then transferred to 4% paraformaldehyde and stored at 4°C overnight. Embryos were bleached according to the standard protocol  and The Spectrum Collection  were arrayed in MultiScreen-Mesh 96-well culture receiver trays (Millipore) in columns 2–11 and diluted to 25 μM in E3 medium for drug screening. Control wells contained either IBMX , DMSO  or E3, in columns 1 and 12 (see diagram of the plate layout in protocol  and storvcanb (mbp (mbpa) (Digoxigenin-labelled RNA probes for vcanb  and mbp p (mbpa)  were prep (mbpa) , modifiep (mbpa) . Stainedvcanb mRNA levels, a scoring system from 0 to 3 was used, with 0 being the score for a very efficient drug (a ‘hit’) that can suppress vcanb expression back to almost wild-type levels, and 3 the score for a drug that did not have any effect on vcanb mRNA levels expressed in the adgrg6 mutant ear. Scores 1 and 2 were given to drugs that showed an ability to down-regulate vcanb expression to some extent, with 1 given for a stronger down-regulation than 2  and a score of 1 in cases where the mbp expression was identical to the one seen in untreated tb233cadgrg6 mutants (i.e. lacking mbp expression around the PLLg). The fact that mbp expression is not missing altogether from other areas of the PNS in tb233cadgrg6 mutants allowed us to use a score of 0 in cases where mbp expression levels were lower than those typically seen in tb233cadgrg6 mutants.For the r screen , a scorevcanb expression, respectively. Drugs from category F caused severe developmental abnormalities, heart oedema, brain oedema or death at the end of the treatment and therefore were characterised as toxic. Category G represented drugs that were potentially corrosive, as no fish were found in these wells at the end of the treatment, although this could also have resulted from death of the embryos followed by digestion by microorganisms, or through experimental error. Drugs that fell into any of the categories D–G and most from category C (59) were eliminated from the assay and were not followed further. In addition, 10 compounds from category A and B were unable to be tested further due to compound availability issues.Drugs categorised as A or B were considered successful and were cherry-picked into new drug assay plates for further testing. Drugs categorised as C were potentially interesting and a few were used to complete a 96-well cherry-pick plate (37/96 compounds). Drugs categorised as D and E were considered to show incomplete or no inhibition of Compounds taken forward for secondary assays were chosen by two criteria: 1. A final average score for three replicates  with a category A–C; 2. No individual score >7. In total, 91 compounds were picked for the counter screen, including two that were present in both libraries, resulting in 89 individual compounds. The screening pipeline is shown in tb233cadgrg6 mutant embryos, the ear-to-ear width was measured from photographs of live embryos mounted dorsally, and normalised for the size of the head, using CELLB software , using 16 LWT wild-type embryos  per concentration. To avoid cross-contamination from dead embryos, each wild-type (LWT) embryo was kept in a separate well of a 96-well plate. At the end of each treatment, the number of dead embryos (no heartbeat for 10 s) was recorded.Still images of live embryos were taken using an Olympus BX51 microscope, C3030ZOOM camera and CELLB software, and assembled with Adobe Photoshop. All micrographs are lateral views with anterior towards the left and dorsal towards the top, unless otherwise stated. For archiving, fixed and stained embryos were imaged in MultiScreen-Mesh plates containing 50% glycerol, using a Nikon AZ100 microscope with an automated stage (Prior Scientific). A compressed in-focus image was generated using the NIS-Elements Extended Depth of Focus software (Nikon).fr24adgrg6 homozygous mutant embryos in a transgenic background  were mounted at 60 hpf in 0.7% agarose with anaesthetic  and 0.003% PTU (to prevent pigment formation). Images were taken of a dorsal view of the ear every 5 min . A control time-lapse of a wild-type sibling embryo  was taken on a separate day. Images were cropped and a subset of z-slices through the anterior (fr24adgrg6) and posterior  projections were used to make Maximum Intensity Projection videos of projection fusion in the wild-type sibling and the swollen projections in fr24adgrg6 mutant embryo. The two videos do not correspond exactly to the same developmental stage.Time-lapse imaging of live embryos was performed on a Zeiss Z.1 light-sheet microscope. http://www.rdkit.org/, accessed 06 Nov. 2018) in Python . Morgan fingerprints of radius 2 . The polar scatterplot was created using the matplotlib library (printed version)  were coversion)  and plotversion)  was compversion) .www.graphpad.com. The Strictly Standardised Mean Difference    for the  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.adgrg6 (gpr126) mutant" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: David W Raible (Reviewer #3).Thank you for submitting your article "Identification of compounds that rescue otic and myelination defects in the zebrafish The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:adgrg6 (gpr126) mutant," Diamantopoulou, Baxendale et al., present the results of a set of screening and validation protocols that identify classes of compounds that rescue phenotypes associated with disruption to the adhesion gpcr adgrg6, aka gpr126. Disruption to gpr126 in zebrafish leads to defects in inner ear morphogenesis and peripheral nerve myelination by Schwann cells. The availability of gpr126 mutant alleles (missense and nonsense mutations) has allowed construction of a clever primary screen to identify regulators of mutant-related vcanb expression in the ear), and a secondary screen to identify compounds that do/don't also affect myelination. Extensive follow-up validation and categorisation has been carried out, culminating in the identification of compounds that may represent direct agonists of the aGPCR itself, which would be of great interest. In general the data are of a very high standard, the manuscript is well written, and all major claims are well substantiated by observations.In "Identification of compounds that rescue otic and myelination defects in the zebrafish The reviewers and handling editor agreed that the major strength of this current work is as an excellent exemplar of how to execute and follow-up chemical genetic screens in zebrafish, rather than in the provision of specific insights into mechanisms of ear/glial cell biology. As such, we would encourage a revision in the Tools and Resources format. All reviewers and the handling editor agree that essential revisions outlined below should strengthen the manuscript and be achievable within the standard revision period.Essential revisions:Additional analyses are required related to the screen and follow-up analyses1) The network analysis is a nice way to categorize and display the different compounds and reveal possible commonalities. A good test of whether these commonalities are related to function would be to test additional related compounds not in the original panels. This is the principal request for new experimental data.2) The identification of 90 compounds out of 3000 (3%) seems rather high, suggesting that either the assays have a potentially high rate of false positive hits or there is the potential for nonspecific action revealed by the assays. Could the authors please comment on this?mbp staining assay. These data are in Figure 5A, but the results appear more variable than the vcanb assay. Are there possible false-positives here?Similarly, it would be nice if the authors provided a summary of the reproducibility of the 3) The dose-response functions provide a good measure of concentration dependent activity. It would have been nice if the controls were repeated with each dose-response function , as these would give a better representation of the reliability of the assay. It would be good if the z-prime was calculated for this assay.4) The authors describe the similarities in results between 155 compounds found between the two libraries. It would be useful to report the statistical correlation between the two sets of scores as a measure of reliability.A little more clarity in presentation/ writing is required in a few places throughout the manuscript.5) Following how compounds advanced through the testing funnel is a little hard to follow. It would be good if there were a way to easily represent this such as a flow chart or other graphic. For example it is not clear how the 91 compounds were identified by the secondary screen . Why were only some compounds advanced for retesting (227/297 compounds)? What criteria advanced them into secondary screening? I can't see an easy way to get 91 compounds from categories in Figure 4A (I get 88 compounds from retest categories A and B).6) The flow of paragraphs (subsection “Compounds that can rescue both inner ear and myelination defects”) is not quite right in that the first paragraph talks about selecting two groups for further analysis and then the next paragraph also mentions compounds chosen for further study. This second announcement sounds independent of the first one. Perhaps in the second paragraph the authors could state, "An additional selection…" or a 'third group'.fr24 allele encodes a highly truncated protein, but I am not sure I see any evidence of this, or in the previous cited paper. One might expect, instead, that this allele would lead to nonsense mediated mRNA decay, but even if this did not occur, it is rather hard to see how the predicted protein/ peptide fragment could be presented in a physiological manner. The authors should either remove, qualify, or amend this statement.7) The authors mention that the 8) It is not clear why the one screening concentration was selected? Can the authors explain, and discuss further in the context of screen design and execution.9) In Figure 8B the use of gray lines makes it difficult to visualize the gray circles.10) What is the significance of the yellow colors in the table shown in Figure 4A?11) For dose-response curves, were measurements done blinded?12) It is not clear what the two sets of graphs are in Figure 7A – are these replicates? Essential revisions:Additional analyses are required related to the screen and follow-up analyses1) The network analysis is a nice way to categorize and display the different compounds and reveal possible commonalities. A good test of whether these commonalities are related to function would be to test additional related compounds not in the original panels. This is the principal request for new experimental data.adgrg6 rescue assays; encouragingly, all four can also rescue the down-regulation of otic vcanb expression in tb233cadgrg6 mutant embryos.We thank the reviewers for their suggestion and agree the network analysis is useful for the display of structural commonalities and as a basis for determining structure-function relationships. To test additional related compounds, we have taken the dihydropyridine network analysed in Figure 6 as a starting point and have selected four additional dihydropyridines that are structurally related to nifedipine, but which were not represented in the Spectrum or Tocris libraries. The new compounds tested are nilvadipine, nemadipine A, felodipine and lercanidipine, which are all drugs of the type used to treat hypertension and are available from Sigma. We have tested these additional compounds in vcanb expression and a strong rescue of mbp expression around the posterior lateral line ganglion . We also demonstrated a dose-dependent rescue of otic vcanb expression with higher concentrations of nilvadipine, with a reduction of expression to wild-type levels after treatment with 50.6 µM .Nilvaldipine has a high structural similarity to nifedipine (Tanimoto similarity coefficient >0.5), and we have modified the network in Figure 6B to show this (green circle). Nilvadipine was initially tested at 22.5 µM and showed a partial rescue of otic vcanb expression, felodipine partially rescued and lercanidipine failed to rescue vcanb expression . We next tested if a higher drug concentration was effective for the latter two compounds. At 40 µM, felodipine still showed partial rescue of vcanb , but lercanidipine was able to rescue vcanb expression at 50 µM .The additional three new compounds showed a range of effects at 25 µM. Nemadipine A fully rescued otic vcanb expression . The testing of (±)-Bay K 8644 highlights how the network analysis can aid in prioritising compounds to test at alternative doses. These data are also relevant for our response to point 8 (see below).We also tested the Tocris compound (±)-Bay K 8644, which is included in the same network as nifedipine but did not score as a hit in the primary screen (25 µM). At a higher concentration (40 µM), we found that (±)-Bay K 8644 was now able to rescue otic We have described these new data in the Results  and have also modified the Discussion (fourth and sixth paragraphs). See also modified Figure 6, and new Figure 7—figure supplement 1. Information on the source for the additional compounds is included in the Materials and methods .2) The identification of 90 compounds out of 3000 (3%) seems rather high, suggesting that either the assays have a potentially high rate of false positive hits or there is the potential for nonspecific action revealed by the assays. Could the authors please comment on this?This point also relates to point 5 asking for further explanation of the screening funnel for how compounds were selected. To address this, we have included a new graphical summary in Figure 4G, which we hope will clarify the screening results and outline our strategy to remove false positives and those compounds with non-specific action.mbp expression also identified compounds that reduced transcription of both vcanb and mbp—potential false positives—and this reduced the number of verified hits with vcanb to 68 (2.3% of 2965). This counter screen also used two independent phenotypes (ear and myelination) to identify any hits that were not specific for the Adgrg6 pathway, and this reduced the number of hit compounds that could rescue both vcanb and mbp expression to 41 (1.4%). We further tested whether compounds were unable to rescue the strong allele as potential interactors with the Adgrg6 protein and identified 19 compounds (0.6%) in this final assay.To summarise, the list of 91 hit compounds included two compounds that were present in both the Spectrum and the Tocris libraries, and therefore the total number of unique compounds was 89 out of a total of 3120 compounds . The counter screen for up-regulation of We therefore feel that the overall hit rate for Adgrg6 modulators of (1.4%) is not higher than we would expect in comparison to published phenotypic screens. Wiley, Redfield and Zon (2017) reviewed the percentage of hits identified from a variety of drug screens in zebrafish and our hit rate is well within the range seen in other phenotypic screens . We have now cited this study in the Discussion (fifth paragraph). It should also be taken into account that the Tocris and Spectrum libraries include known bioactives and FDA-approved drugs, which are more likely to have activity in an in vivo screen than a completely novel synthetic compound library, where the hit rate would be expected to be lower. We expect the list of hits will include compounds, like colforsin and our control compound IBMX, acting downstream of Adgrg6 signalling by increasing cAMP levels, and we would expect there to be a number of compounds with similar activity in these two libraries.We therefore prefer to retain the higher rate of false positives after the primary screen and then use the retests and counter screens to validate hits. The screening design could be made more robust by increasing the number of embryos screened or performing multiple repeats; however, in our experience, the time and financial cost of screening in duplicate or triplicate at the primary screen stage in order to identify fewer primary hits is not cost effective and would reduce the number of compounds screened overall. This is already discussed in the text .Similarly, it would be nice if the authors provided a summary of the reproducibility of the mbp staining assay. These data are in Figure 5A, but the results appear more variable than the vcanb assay. Are there possible false-positives here?mbp staining between wild-type and mutant embryos is highly reproducible, even when using the hypomorphic allele. Although expression in the PNS as a whole in the tb233cadgrg6 mutant is variable, expression around the posterior lateral line ganglion is one of the few regions that is consistently absent, as discussed in the Results . To quantify this, and to illustrate the variability of mbp staining intensity around the posterior ganglion between individual wild-type embryos and adgrg6 mutants, we have mounted individual embryos for imaging. In dorsally mounted embryos, it is possible to distinguish the peripheral mbp staining (decreased in mutants) from the CNS staining (unaffected in mutants). We have taken 10 tb233cadgrg6 mutant embryos and 10 siblings from the same in situ hybridisation experiment and analysed the left and right posterior lateral line ganglion region from each embryo. After thresholding for colour using the HSB settings in Fiji we have plotted the percentage area of mbp staining. The results show a statistically significant reduction in staining area in mutant embryos. We have included these data in a new supplementary figure .The difference in mbp staining results show variability between individual embryos treated with a given concentration of drug , but as we are looking for up-regulation of gene expression in this assay, we would expect to have more false-negatives than false-positives at this stage. Note also that the individual mounting required for the quantitative analysis described above is not practicable for a large-scale primary screen.In the screen, some of the 3) The dose-response functions provide a good measure of concentration dependent activity. It would have been nice if the controls were repeated with each dose-response function , as these would give a better representation of the reliability of the assay.To clarify, two different dose response assays were performed in separate 96-well plates and each plate included a set of controls. One plate screened increasing doses of FPL 64176 using an optimised assay window of 60-90 hpf and the second plate had increasing doses of nifedipine, tracazolate and cilnidipine using the time window of 60-110 hpf. There are therefore two sets of controls, one for the 60-90 hpf assay and another set for the 60-110 hpf assay. The experiment was repeated for both assay plates, and so the controls contain data points from two replicates. To make this information clearer we have rearranged this figure and legend so that the FPL 64176 data including controls is on one graph  and the other three compounds, with their controls, are on a second graph .It would be good if the z-prime was calculated for this assay.n) is low. In order to have a significant z-prime score it is necessary to have well-separated distributions  between the positive and negative control.In general, z-prime scores are not suitable for in vivo phenotypic screens, such as this one, where the results are initially qualitative and then assigned to an arbitrary numerical scoring system, or where the sample size  for assessing assay quality. This method has been used in RNAi screens . We used the data from Figure 7D (ear width measurements) and the SSMD formula for two independent groups,β=μ1-μ2σ12+σ221 is the mean ear width distance of the tb233cadgrg6 mutants treated with DMSO (negative control) and µ2 is the mean ear width distance of the tb233cadgrg6 mutants treated with IBMX (positive control), σ12is the variance of the negative control and σ22 is the variance of the positive control. This generated an SSMD score of 2.811 for the controls on the plate with nifedipine, cilnidipine and tracazolate, which demonstrates that this assay has a strong threshold for hit detection. Scores for the hit compounds, compared to the negative control, were also strong at the higher concentrations . The controls for the FPL 64176 plate have a slightly lower score of 2.03 after a 24-hour treatment, compared with the 48-hour treatment on the other plate. FPL 64176 had a similar score of 2.04 at 6.7 μM. We have included all the scores in a modified Figure 7—figure supplement 2 and included the method in the statistics section of the Materials and methods  .where µ4) The authors describe the similarities in results between 155 compounds found between the two libraries. It would be useful to report the statistical correlation between the two sets of scores as a measure of reliability.As mentioned in response to point 3, our primary assay scores are not suited to statistical correlation as they are not quantitative. We had analysed the duplicated compounds in detail and had already discussed the few differences we found in the text .A little more clarity in presentation/ writing is required in a few places throughout the manuscript.5) Following how compounds advanced through the testing funnel is a little hard to follow. It would be good if there were a way to easily represent this such as a flow chart or other graphic. For example it is not clear how the 91 compounds were identified by the secondary screen . Why were only some compounds advanced for retesting (227/297 compounds)? What criteria advanced them into secondary screening? I can't see an easy way to get 91 compounds from categories in Figure 4A (I get 88 compounds from retest categories A and B).As discussed in response to point 2, we have now added further clarification of our screening funnel in the Materials and methods section and also made a new panel summarising the hit selection steps for Figure 4G. Briefly, from the primary screen, the list of hits included those in categories A–C. While we were interested in those that had the strongest rescue  we also wanted to determine how many compounds in category C would come through the retests as a hit. We therefore made up the cherry-pick ‘hit’ plates with extra compounds from the C category, although not all of category C compounds were retested. In addition, 10 compounds from the B category were not followed up due to a lack of compound availability in the cherry-pick plates—this is mentioned in the Materials and methods (subsection “Hit selection”). The table in the original Figure 4A confirmed that the majority of compounds from category C retested as category D (non-hit). However, we have taken the reviewers’ comments on board and have removed this table and replaced it with a schematic of the hit selection process, which we hope will be clearer for the reader. We have moved some of the details of the screening process from the Results section to the Materials and methods section. The revised text can be found in the Results  and Materials and methods (subsection “Hit selection”). See also the new Figure 4G.6) The flow of paragraphs (subsection “Compounds that can rescue both inner ear and myelination defects”) is not quite right in that the first paragraph talks about selecting two groups for further analysis and then the next paragraph also mentions compounds chosen for further study. This second announcement sounds independent of the first one. Perhaps in the second paragraph the authors could state, "An additional selection…" or a 'third group'.mbp expression, and identification of false positives”, last paragraph). We have also clarified which of the selected compounds were from the two groups that correspond to the clusters in Figure 4D and Figure 5B and which were chosen separately and hopefully this is now clearer (subsection “Compounds that can rescue both inner ear and myelination defects”).We have now amended this section and moved the discussion of the heatmap to the previous section (subsection “Secondary screen for rescue of 7) The authors mention that the fr24 allele encodes a highly truncated protein, but I am not sure I see any evidence of this, or in the previous cited paper. One might expect, instead, that this allele would lead to nonsense mediated mRNA decay, but even if this did not occur, it is rather hard to see how the predicted protein/ peptide fragment could be presented in a physiological manner. The authors should either remove, qualify, or amend this statement.fr24 allele. We have been careful to explain this in the text  and have checked all other references in the manuscript to fr24 to remove any ambiguity. As shown in Figure 1 and in our previous study , the fr24 allele predicts a stop codon L463X which is within the extracellular domain of the protein, N-terminal to the hormone-binding domain and GAIN domain.The reviewers are correct that we do not have any experimental evidence for the presence or absence of a truncated protein product in the adgrg6 transcript in fr24adgrg6 mutants by in situ hybridisation. In some regions expression is weaker, but expression in the swollen projections in the ear is strong . Given that some transcript is produced, it is possible that it is translated into protein. If the truncated N-terminal fragment (amino acids 1–462) is expressed as protein it would contain the signal peptide needed to export the peptide and also the CUB and PTX domains. We note that another truncating adgrg6 allele, st49, is known to produce a slightly larger peptide, which retains the hormone binding domain and is biologically active in vivo, being able to restore radial sorting of axons in adgrg6 mutants, so there is precedent for the production of truncated Adgrg6 peptides . This is now discussed in the aforementioned paragraph.Although nonsense-mediated mRNA decay is a possibility, we have previously tested the presence of the fr24 allele in the current study is to distinguish between compounds that can activate downstream signalling of the Adgrg6 pathway (which are active whether the Adgrg6 protein is present or not) and those that potentially interact with the Adgrg6 protein . In the latter case, even if an N-terminal peptide is produced and is active in fr24 mutants, the mutation predicts that the transmembrane domain necessary for G protein coupling to activate downstream signalling will be missing.However, the purpose of using the 8) It is not clear why the one screening concentration was selected? Can the authors explain, and discuss further in the context of screen design and execution.The choice of compound concentration for screening is always a compromise between efficacy and toxicity. The concentration 25 μM was chosen empirically after analysing results from previous screens performed at both 10 μM  and 25 μM . These data suggested that the number of toxic compounds did not increase significantly at the higher concentration. 25 μM is within the range frequently used in zebrafish assays . This is discussed in the fourth paragraph of the Discussion.As highlighted in our response to point 1, the new Figure 7—figure supplement 1 now includes a panel of dihydropyridine compounds that were only partially active in the assay at 25 μM, but were active at higher concentrations of 40 or 50 μM. Lowering the concentration in the primary screen to 10 μM would have resulted in more hit compounds being missed, including nifedipine, based on our dose-response data in Figure 7B. Screening the compounds in duplicate with two different concentrations would have reduced the overall number of compounds we could screen, taking in to account the number of embryos available and the cost of other reagents. One potential compromise would be to take all the compounds that proved to be toxic at 25 μM and re-screen these cherry-picked compounds at a lower concentration.9) In Figure 8B the use of gray lines makes it difficult to visualize the gray circles.The colour of the lines has been modified to make the network easier to visualise in Figures 6, 8 and Figure 7—figure supplement 1. The entire network is also available as an interactive version, where individual networks can be enlarged to aid visualisation.10) What is the significance of the yellow colors in the table shown in Figure 4A?The yellow colours in the previous Figure 4A highlighted the categories with the highest numbers of hit compounds after retesting. However, after consideration of the reviewers’ points 2 and 5 we have decided to remove this table and have replaced it with a schematic of the hit selection process that also still includes the numbers of compounds in each category at each stage . See also our response to point 5 above.11) For dose-response curves, were measurements done blinded?vcanb staining were still available; we went back to the original plates and re-ordered them to do a retrospective blinded score. Here, we found the same overall trends as shown in Figure 7, with only a marginal difference between the intermediate dose scores, while the scores for the ‘rescued’ and ‘not rescued’ were the same in both cases. We have not amended the data in Figure 7B but have mentioned our method of scoring in the Materials and methods .The ear measurements from the ear swelling dose response assays were not done blinded. In general, all plates were scored twice and by two different scorers. For the dose-response assay the reviewer refers to, the in situ hybridisation results for the 12) It is not clear what the two sets of graphs are in Figure 7A – are these replicates?vcanb expression). Figure 7B shows the overall score of the vcanb staining as defined in Figure 3A. We have modified the figure legend to make this clearer and explanation in the text is found in the first paragraph of the subsection “Nifedipine, cilnidipine, tracazolate hydrochloride and FPL 64176 rescue otic defects in tb233cadgrg6mutants in a dose-dependent manner”.The two sets of graphs record different measurements, but were scored on the same set of embryos. We have now made these two panels separate to make this clearer. The first panel  is scored for the number of projections that have fused to form a pillar (i.e. morphological rescue of the inner ear defect, not just reduced


Loss of proteostasis and cellular senescence are key hallmarks of aging, but direct cause-effect relationships are not well understood. We show that most yeast cells arrest in G1 before death with low nuclear levels of Cln3, a key G1 cyclin extremely sensitive to chaperone status. Chaperone availability is seriously compromised in aged cells, and the G1 arrest coincides with massive aggregation of a metastable chaperone-activity reporter. Moreover, G1-cyclin overexpression increases lifespan in a chaperone-dependent manner. As a key prediction of a model integrating autocatalytic protein aggregation and a minimal Start network, enforced protein aggregation causes a severe reduction in lifespan, an effect that is greatly alleviated by increased expression of specific chaperones or cyclin Cln3. Overall, our data show that proteostasis breakdown, by compromising chaperone activity and G1-cyclin function, causes an irreversible arrest in G1, configuring a molecular pathway postulating proteostasis decay as a key contributing effector of cell senescence. Like most other cell types, individual yeast cells display a finite lifespan as they undergo subsequent replication cycles and, due to their relative simplicity, have become a very fruitful model to study the causal interactions among the different hallmarks of cell aging. Since yeast daughter cells are rejuvenated during most of the mother cell lifespan, it is generally accepted that aging is the result of asymmetric segregation of factors such as extrachromosomal rDNA circles (ERCs), dysfunctional mitochondrial and vacuolar compartments, or resilient protein aggregates . Proteossir2 mutants  that can be detected by fluorescence microscopy with no gross effects on cell cycle progression   as an orThe intersection value of autocorrelation functions obtained by RICS depends on a second parameter related to the number of fluorescent molecules in the moving particles, termed brightness (B). Interestingly, aged cells displayed lower B values for both Ssa1-GFP and Ydj1-GFP compared to young cells , which wFirefly luciferase (FFL) refolding and enzymatic activity recovery has been widely used to assay chaperone activity in vitro  and in vAs previously mentioned, overall Hsp104-mCh levels increased much faster after the last budding event . Howeverinsets). Simulated interdivision time in consecutive cycles remained rather constant, but progressively increased during the last generations before the final G1 arrest  and 48 hr (25–30 generations), respectively. Overexpressing CLN3 in pre-aged cells did not affect their budding size  that cooperate in ER-release and proper Cdk-cyclin complex formation was duplicated  were not arrested in G1 at death and showed a slightly shorter lifespan, which agrees with published findings . PFD-ovesir2 mutants  mutants . On the aperones , and we RIM15, which is strongly upregulated by nutrient starvation but not by heat stress, is also upregulated in aged cells, indicating that G0 and aged cells might activate common transcriptional programs other than the ESR.Aged cells display increased levels of expression of many genes of the environmental stress response (ESR) , which sProteostasis defects have been associated with cell aging in many different model organisms , and the11A fusion protein contains a hypoactive and hyperstable cyclin with 11 amino acid substitutions  that allows its detection by fluorescence microscopy with no gross effects on cell cycle progression  as described , and see2) was repetitively photobleached at full laser power while the cell was imaged at low intensity every 0.5 s to record fluorescence loss. After background subtraction, fluorescence data from an unbleached cell region were made relative to the initial time point, and a mobility index was calculated as the inverse of the fluorescence half-life obtained by fitting an exponential function. We noticed a clear dependency of this mobility index on cell size. Using a dataset of cells with very wide size range and expressing free GFP, we obtained an expression to correct the mobility index for cell size with c = MI .r1.49MI where MI is the raw mobility index, r is the cell radius and cMI is the corrected mobility index (We used fluorescence loss in photobleaching (FLIP) to analyze chaperone mobility in a Zeiss LSM780 confocal microscope with a 40X/1.2NA water-immersion objective at room temperature. FLIP was used as a qualitative assay to determine Ssa1-GFP and Ydj1-GFP mobility in the whole cell. A small circular region of the cytoplasm , for each cell was obtained and averaged for each group of cells to determine the diffusion coefficient (D) and the number of molecules in the focus (N) by fitting the data to a simple diffusion model. In order to assess the uncertainty of the predicted D values from pooled RICS data by non-linear regression methods we used a Monte Carlo approximation.Raster Image Correlation Spectroscopy (RICS) analysis was performed in a Zeiss LSM780 confocal microscope with a 63X/1.3NA water-immersion objective; specifically, we used a 35 nm pixel size and 12.6μs dwell time, tacking a stack of 100 frames (2 s/frame) in photon-counting detection mode, at room temperature. To obtain the coefficient of diffusion, 64 × 64 pixel stacks were used to remove the immobile fraction with a five frame moving average and analyzed using a set of plugins written by Jay Unruh (Stowers Institute) for ImageJ. The resulting autocorrelation function (ACF) in the scanning direction, that is GCoincidence analysis with RICS data was carried out with CoinRICSJ . Briefly4 cfu/cm2 onto 35 mm 2% agar plates containing SC medium with 2% glucose or 2% galactose and 1 µM β-estradiol. Once the plates were dry, they were incubated at 30°C for 4 days. Finally, the microcolonies were imaged using a Leica AF7000 microscope with a 20X/0.5NA dry objective. As a proof of concept for the method, we measured the microcolony size produced by cells pre-aged in liquid media with 1 µM β-estradiol for increasing amounts of time, and we observed a progressive decrease in microcolony size as a function of pre-aging time in liquid media before plating  or YDJ1 (SIS1), genetic ablation of these genes was simulated by applying different degrees of reduction in the concentrations of the respective proteins .Sample size is always indicated in the figure legend and, unless stated otherwise, median and quartile (Q) values are shown in all plots with single-cell data. Pairwise comparisons were performed with a Mann-Whitney U test; and the resulting p values are shown in the corresponding figure panels. For percentages, 95% confidence limits (CL) are always shown. Time-lapse data recorded from single cells in the CLiC microfluidics chamber are represented as the mean value of the population along time , while the shadowed area represent the 95% confidence limits of the mean. Logistic regression analysis was performed with the aid of a Java applet developed by J.C. Pezzullo .Thank you for submitting your article "Proteostasis collapse halts G1 progression and delimits replicative lifespan" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.SummaryIn this study, Moreno et al. establish that old yeast mother cells show a strong increase in proteostasis defects in their last divisions, as measured by chaperone mobility, levels and localisation. In parallel, these cells fail to properly fold the G1 cyclin Cln3, as reported by its failure to enter the nucleus, and the majority of the cells arrest their last cell cycle in the G1 phase of the cell cycle before dying. Cells induced to accumulate an aggregating protein show a similar increase in proteostasis defects, defect in Cln3 localisation to the nucleus and cell cycle arrest in G1. Based on a quantitative in silico model of this process, model that is is largely corroborated by the data, the authors propose that the collapse of proteostasis titrates the chaperones that normally ensure the proper activity and localisation of Cln3 and thereby lead to cell cycle arrest. Supporting this model, CLN3 over-expression extends the life-span of these cells. All three reviewers found that this manuscript is very well written, the experimental design is generally appropriate for testing the authors' hypotheses, and the experiments are well executed and convincing.Essential revisionsThe fact that CLN3 over-expression extends the longevity of the cells whereas overexpression of the chaperones does not is actually confusing. This seems at odds with the statement that chaperones are the limiting factor – does overproduction of Cln3 bypass the need for chaperones or what is going on? Does CLN3 over-expression promote expression of chaperones? If this is not the case, then the model needs to be amended at this time as it might not be as straightforward, and linear, a relationship as the authors suggest. Indeed, this observation could support two independent conclusions: Either proteostasis defects are accumulating more rapidly than any chaperone increase could control (and then it would be interesting to understand why), or one or several other processes limit the lifespan of the cell together with proteostasis and are not mitigated by chaperone over-expression.Depending on the results of the additional experiments requested, the title of the paper might need to be amended.eLife. Your revised article has been favorably evaluated by David Ron (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for resubmitting your work entitled "Proteostasis collapse halts G1 progression and delimits replicative lifespan" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:While there is clear enthusiasm about your study and its findings, the reviewers were still unanimously concerned about whether the overall conclusion, as claimed in the title is truly supported by the data. The fact that Cln3 over-expression does prolongate the lifespan of wild type cells is a very interesting and important finding. However, the fact that chaperone over-expression does not show such effect remains a strong concern, and this is for two reasons.1) It raises the question of whether the proteostasis collapse is THE process that delimits the replicative lifespan of yeast cells, i.e., is the ultimate cause of the death of the cells. Indeed, proteostasis defects could also be a symptom of another defect, which could be the actual lethal event. You very clearly show that proteostasis collapse can be lethal, as many other events, but you also clearly show that in that case chaperon over-expression does restore longevity. This strongly undermines the arguments made on the basis of in silico modelling.2) It raises issues about the interpretation of how Cln3 over-expression promotes longevity. Indeed, if proteostasis collapse is the lethal event, how would cell cycle progression allow cells to escape it, in the context of your model of an exponential accumulation of unfolded proteins?Therefore, the reviewers decided to insist on their request that you investigate whether CLN3 over-expression promotes chaperon expression. If this were not to be the case, the reviewers would find it more appropriate to discuss the issue more thoroughly and tone down the main conclusion of the paper. Essential revisionsThe fact that CLN3 over-expression extends the longevity of the cells whereas overexpression of the chaperones does not is actually confusing. This seems at odds with the statement that chaperones are the limiting factor – does overproduction of Cln3 bypass the need for chaperones or what is going on? Does CLN3 over-expression promote expression of chaperones? If this is not the case, then the model needs to be amended at this time as it might not be as straightforward, and linear, a relationship as the authors suggest. Indeed, this observation could support two independent conclusions: Either proteostasis defects are accumulating more rapidly than any chaperone increase could control (and then it would be interesting to understand why), or one or several other processes limit the lifespan of the cell together with proteostasis and are not mitigated by chaperone over-expression.As also suggested by the reviewers in their last minor point, we have added to main Figure 5 our data and model simulations corresponding to chaperone overexpression in aging cells. We have modified the text (subsection “Cln3 overexpression increases replicative lifespan in a chaperone-dependent manner” and Discussion paragraph five) to describe these data as follows: While Ydj1-deficient cells displayed a reduced lifespan as previously observed , concurrent overexpression of Ssa1 and Ydj1 did not increase lifespan significantly . Albeit surprisingly, our model predicted that chaperone overexpression would have a very limited effect on lifespan . By analyzing in detail the kinetics of Ydj1 levels and the appearance of protein aggregates in stochastic simulations  we observed that, due the positive feedback loop inherent to autocatalytic aggregation, once the first protein aggregates appear they rapidly overcome Ydj1 levels by several orders of magnitude, thus making ineffective the relatively small (ca. 50%) increase in Ydj1 levels attained by GAL1p-driven overexpression .In addition, in the Discussion section (paragraph five) we mention that, similarly to our results with Hsp70/40, overexpression of Hsp104 restores proteasome activity in aging cells  and suppresses lifespan defects of sir2 mutants , but does not increase lifespan significantly in wild-type cells .Finally, as we discuss in Discussion paragraph two, the accumulation of ERCs in aged cells  may play a more direct role repressing CLN1,2 expression . This would explain why, regarding lifespan extension, CLN3 overexpression is much more efficient than increasing chaperone expression levels.Depending on the results of the additional experiments requested, the title of the paper might need to be amended.We believe that, considering also our data above, the title reflects the main findings of our work, particularly in view of the important effects caused by precocious PD-protein aggregation in lifespan, and their suppression by either G1-cyclin or chaperone overexpression.While there is clear enthusiasm about your study and its findings, the reviewers were still unanimously concerned about whether the overall conclusion, as claimed in the title is truly supported by the data. The fact that Cln3 over-expression does prolongate the lifespan of wild type cells is a very interesting and important finding. However, the fact that chaperone over-expression does not show such effect remains a strong concern, and this for two reasons.1) It raises the question of whether the proteostasis collapse is THE process that delimits the replicative lifespan of yeast cells, i.e., is the ultimate cause of the death of the cells. Indeed, proteostasis defects could also be a symptom of another defect, which could be the actual lethal event. You very clearly show that proteostasis collapse can be lethal, as many other events, but you also clearly show that in that case chaperon over-expression does restore longevity. This strongly undermines the arguments made on the basis of in silico modelling.We agree with your first point that, although we find very strong temporal correlations, our data do not demonstrate that proteostasis collapse is THE reason why cell proliferation declines and cells arrest in G1 before death. We also accept that the initially proposed title was a bit ambiguous in this respect and we propose a new title as follows: “Proteostasis collapse, a hallmark of aging, hinders the chaperone-Start network and arrests cells in G1”. In line with this, the end of the Abstract has been modified to clarify the idea of a “molecular pathway postulating proteostasis decay as a key contributing effector of cell senescence.” In addition, although we had already addressed this point in our paper, the Discussion section has also been modified to emphasize the existence of other mechanisms as follows: “[…], the inability of chaperone overexpression to extend lifespan would also underscore the existence of chaperone-independent mechanisms affecting execution of Start in aged cells as above mentioned, such as ERC-mediated downregulation of G1/S genes .”2) It raises issues about the interpretation of how Cln3 over-expression promotes longevity. Indeed, if proteostasis collapse is the lethal event, how would cell cycle progression allow cells to escape it, in the context of your model of an exponential accumulation of unfolded proteins?Therefore, the reviewers decided to insist on their request that you investigate whether CLN3 over-expression promotes chaperon expression. If this were not to be the case, the reviewers would find it more appropriate to discuss the issue more thoroughly and tone down the main conclusion of the paper.CLN3 overexpression fully suppresses the requirement of the Ydj1 chaperone for cell cycle entry in young cells, with no effects on the expression of related chaperones . We had interpreted this result as if retention of Cln3 at the ER was not a very efficient mechanism and could not cope with very high levels of translated Cln3. In any event, the above suppression would explain why, even if proteostasis is seriously affecting chaperone availability in very old cells, high levels of Cln3 would suffice to prolong replicative lifespan. We have added this comment to the Discussion section (paragraph five).Regarding your second point, we would like to point out that


This hybrid structure consisted of CCHH and RGO , in which RGO uniformly dispersed in CCHH, being used as the gas sensing film. The resistivity of the hybrid structure was highly sensitive to the changes on NH3 concentration. CCHH in the hybrid structure was the sensing material and RGO was the conductive channel material. The hybrid structure could improve signal-to-noise ratio (SNR) and the sensitivity by obtaining the optimal mass proportion of RGO, since the proportion of RGO was directly related to sensitivity. The gas sensor with 0.4 wt% RGO showed the highest gas sensing response reach to 9% to 1 ppm NH3. Compared to a conventional gas sensor, the proposed sensor not only showed high gas sensing response at room temperature but also was easy to achieve large-scale production due to the good stability and simple synthesis process.A novel hybrid structure sensor based on cobalt carbonate hydroxide hydrate (CCHH) and reduced graphene oxide (RGO) was designed for room temperature NH As an irritating odor and highly toxic gas, ammonia is widely found in industrial exhaust emissions, which is gradually threatening the health of humans and animals. Therefore, it is very crucial to develop highly sensitivity ammonia sensors and detection equipment.2O3 nanowires to prepare gas sensors with a great gas sensing properties to NO2 [2 nanowire had good gas sensing properties for hydrogen [Semiconductor gas sensors, all using metal oxide semiconductors as sensing materials, are widely applied to detect various gases owing to the good repeatability and low cost ,2,3. Xu s to NO2 . The SnOhydrogen . Wagh ethydrogen . In ordehydrogen ,8. The ohydrogen ,10. Cons2 microspheres was fabricated by a hydrothermal method and showed excellent sensitivity and good selectivity to ammonia gas [2 composite sensor by mixing the RGO obtained from thermal reduction with the commercial SnO2 nanoparticles. Although the sensor had good gas sensing properties at room temperature, its noise was very high [In recent years, many works on gas sensors based on composite structures of graphene and sensing materials have been published ,15,16,17ery high .Combining with the previous research, a new research idea has been gradually produced recently, which is to synthesize the ideas proposed above so as to further improve the gas sensing properties. ZnO nanoparticles loaded onto 3D RGO with hydrothermal method was very sensitive to CO . Liu et 3)0.5OH•0.11H2O), referred as CCHH has been usually utilized as the precursor to obtain Co3O4 by annealing process at 300–500 °C [Cobalt carbonate hydroxide hydrate  was dispersed in deionized water (60 mL), vibrated and sonicated for 4 h. After GO dispersed evenly in the solution, cobalt chloride hexahydrate  and urea  were added into the dispersed solution, stirred and sonicated for 3 min. When the two chemicals were completely dissolved and evenly distributed in the solution, the solution was transferred into a reaction kettle to conduct hydrothermal reactions for 3 h under 130 °C. The solution fabricated by hydrothermal reaction was centrifuged and washed with deionized water several times. The obtained material was dried in oven under 60 °C for 12 h and the final product was obtained. The fabrication process of CCHH with RGO hybrid structure was shown in The previously cleaned target solution mainly being CCHH with RGO hybrid material, was transferred onto the fresh interdigital electrode (IDE) and heated in oven under 50 °C for 10 min and then the gas sensor named as CCHH-RGO was obtained.In order to obtain the highest sensing response, the effect of the proportion of RGO in the hybrid structure on gas sensing response has been studied. According to the different RGO proportion, several gas sensors with different RGO mass proportion were fabricated at the same time and listed in The structure and morphology of CCHH-RGO were investigated with scanning electron microscopy . X-ray photoelectron spectroscopy  and X-ray diffraction  were used to characterize the elemental composition and state of the composites, respectively.The whole ammonia gas sensing system was based on the homemade testing chamber shown in The structure and morphology of the synthesized CCHH-RGO was characterized by various instruments. Considering the influence of different RGO proportion on the structure and gas sensing properties, CCHH-RGO-0.4 with the optimized gas sensitivity was systematically characterized as a typical one.3O4 were detected in the sample pattern, meaning that high purity CCHH was well synthesized in the RGO dispersed solution with the hydrothermal method.2+ in the sample. Data from XPS and XRD showed that CCHH-RGO-0.4 was mainly composed of Co2+. The existence of sub-peaks in the spectra further revealed that Co2+ was not oxidized to the Co3O4, which well matched the research results by Varghese [32− at 284.9, 286.5, 287.1 and 289.7 eV, separately. Combined with the C 1s peaks of the sample and GO, we concluded that GO was completely reduced to RGO due to the drastic reduction of oxygen-containing functional groups such as C-OH and C-O-C. Meanwhile, the new peak appearing at 289.7 represented presence of CO32−, which was mainly derived from the formation of CCHH.For further characterization, we performed XPS analysis of CCHH-RGO-0.4 to further investigate the chemical composition of the sample. The Co 2p spectrum displayed two major peaks with the Binding energy (BE) values at 781.2 and 797.1 eV in the Varghese . The C 1In order to study the gas sensing properties of CCHH and RGO hybrid sensors, a series of gas sensing tests were implemented in the proposed self-made test system at six different concentrations of ammonia at room temperature. The resistance values of several sensors changed with the injection and discharge of ammonia. The gas sensing response can be obtained as follow equation.ammonia and Rair are the electrical resistance values with and without ammonia gas, respectively. The gas sensing response is defined as a ratio expressed in percentage.RAs the ammonia gas in, the response curves of sensors began to increase rapidly and then slowly stabilized at a fixed maximum. Next, the response values decreased rapidly and returned to near the initial value after ammonia gas off. The black, red, blue and green curves correspond to the responses of four sensors  to six concentrations of ammonia gas  in To study the gas sensing mechanism of the hybrid structure sensor, we performed a gas sensing test on the 10 ppm ammonia gas for four sensors with a gas on (360 s)/gas off (1200 s) cycle of 1560 s at the same time. Similar to metal oxide, there were a large number of lattice defects, surface discontinuities and a certain concentration of electron donor and acceptor energy levels on the surface of CCHH, which could exchange electrons with oxygen molecules in the air to form oxygen anions (n region .When ammonia gas reacted with the oxygen anion on the surface, ammonia molecules would release an electron to form ammonium ions. While the electron could be smoothly transferred to the surface of CCHH and then further diverted to RGO due to coupling effect between CCHH and RGO, which could give rise to the resistivity increase of the sensor owing to the prepared RGO being p-type material. Once the ammonia gas was removed, the ammonium ions would be converted to ammonia molecules and the resistivity of the sensor would decrease. Besides, CCHH prepared in this experiment was a sisal-like structure with a high specific surface area and the structure was conducive to the gas sensing response increase because the sisal-like structure was suitable for gas circulation.The introduction of RGO greatly reduced the resistivity of the sensor due to the high conductivity of RGO, which not only facilitated the electronic conduction of the sensor after adsorbing gases but also increased the signal current to improve SNR and weaken the effect of noise. CCHH covered with RGO could sense tiny charge changes from adsorbed gases and provide a basis for realizing room temperature gas sensing tests. However, the proportion of RGO was not the more the better. The experimental results showed that with the increase of RGO proportion, the response of the sensor decreased gradually. The main reason may be that excessive RGO covered the surface of CCHH, which prevented the gas molecules from reacting with the CCHH, thus preventing the change of conductivity.The exposure of the CCHH-RGO-0.4 to four continuous cycles of 10 ppm ammonia was shown in 2 as the sensing materials, could obtain a good gas sensing response at a high operating temperature. Although the remaining four sensing materials could be used for gas sensors at room temperature, their gas sensing responses were relatively low. Compared with these sensors, the proposed sensor in this paper with the simple fabrication process, not only could achieve gas sensing test at room temperature but also had excellent gas sensing performances.There were several performance comparisons of different sensors reported by other journals. The sensing material, operating temperature, gas concentration and response of those sensors were listed in In summary, the paper propose a novel CCHH-RGO gas sensor fabricated by a by one-step hydrothermal method. The simple synthesis not only simplified the experimental process but also facilitates mass production. The prepared sisal-like CCHH with high specific surface area could provide abundant gas adsorption interface and a suitable pathway for gas molecules. Meanwhile, the synthesized RGO uniformly dispersed on CCHH surface significantly improved the signal current and gas sensitivity caused by gas molecular adsorption. When the mass proportion of RGO was 0.4%, CCHH-RGO hybrid structure had the optimized gas sensing response, reach to 9% to 1 ppm ammonia. In addition, it could be found that CCHH-RGO hybrid structure had a good selectivity to ammonia by comparing several different gases as acetone, isopropanol, ethanol and formaldehyde. We believe that the novel hybrid sensor has the potential to be used in mass-produced gas sensors and is valuable for the future research on gas sensors.


International guidelines recommend using basal insulin in patients with type-2 diabetes mellitus if glycaemic target cannot be attained on non-insulin anti-diabetic drugs. Available choices of basal insulin include intermediate-acting neutral protamine Hagedorn (NPH) insulin and long-acting insulin analogues like insulin glargine U100. Despite clear advantages of glargine U100, the existing practice in Hong Kong still favours NPH insulin due to lower immediate drug costs.The objective of this study was to assess the cost-effectiveness of insulin glargine U100 compared to NPH insulin in patients with type-2 diabetes uncontrolled with non-insulin anti-diabetic agents alone in Hong Kong.The IQVIA™ Core Diabetes Model (CDM) v9.0 was used to conduct the cost-effectiveness analysis of glargine U100 versus NPH. Baseline characteristics were collected from the Hong Kong Diabetes Registry. Efficacy rates were extracted from a published study comparing glargine U100 and NPH in Asia, utilities from published literature, and costs constructed using the Hong Kong Hospital Authority (HA) Gazette . The primary outcome was an incremental cost-effectiveness ratio (ICER).Insulin glargine U100 resulted in an ICER of HKD 98,663 per Quality Adjusted Life Year (QALY) gained. The incremental gains in QALY and costs were 0.217 years and HKD 21,360 respectively. Results from scenario and probabilistic sensitivity analyses were consistent with that from base case analysis.Insulin glargine U100 is a cost-effective treatment for patients with type 2 diabetes compared to NPH insulin in setting in Hong Kong. This was mainly driven by the significantly lower rates of hypoglycaemia of insulin glargine U100 than NPH insulin.The online version of this article (10.1186/s12962-019-0180-9) contains supplementary material, which is available to authorized users. Type 2 diabetes mellitus is a chronic medical condition characterised by inadequate insulin production and action resulting in hyperglycaemia. People with diabetes are at risk of developing micro-/macrovascular complications of serious consequences particularly if glycaemia and other metabolic risk factors are inadequately managed –3. MaintThe objective of the current study was to assess the cost-effectiveness of insulin glargine U100 compared to NPH insulin in patients with type-2 diabetes uncontrolled with non-insulin anti-diabetic agents alone in Hong Kong. We took the societal perspective in Hong Kong for this cost-effectiveness analysis. The analysis was conducted using the internet-based computer simulation IQVIA CORE Diabetes Model (CDM) which will be discussed in further details below.The IQVIA™ Core Diabetes Model (CDM) v9.0 was used to predict the lifelong costs and outcomes of using insulin Glargine U100 and NPH insulin in patients uncontrolled on non-oral anti-diabetic drugs. A detailed description of the CDM and its operational features have been published elsewhere , 12. TheCDM is often used as a policy analysis tool because it is a non-product specific model. It comprises of a series of 15 sub-models, where each sub-model is a combination of semi-Markov model structures and Monte Carlo simulations, which simulate the major complications of diabetes including, but not limited to, congestive heart failure, myocardial infarction, stroke, end-stage renal disease, lower extremity amputation, foot ulcer and hypoglycaemia. The model uses time, state, and diabetes type-dependent probabilities derived from published sources, in addition to utilizing tracker variables to overcome the “memory-less” properties of standard Markov models. This allows the interconnectivity and interaction between individual complications’ sub-models and hence allows the patient cohort to develop multiple complications within each model cycle. The CDM projects the outcomes for the population based on the following non-exhaustive list: the cohort’s baseline characteristics, past history of complications, concomitant medications, and changes in physiological variables over time. From there, the model can calculate the incidence of complications, life expectancy, quality-adjusted life expectancy, and total costs within the population. The results are expressed in terms of quality-adjusted life years gained and incremental cost-effectiveness ratio (ICER). An ICER threshold of 343,312 Hong Kong Dollars (HKD) in Hong Kong (2016) was used in this analysis based on the guidance by the World Health Organization (WHO) which recommends an ICER threshold that is equal to the Gross Domestic Product (GDP) per capita , 19.The baseline characteristics for the base case analysis were collected from the HKDR after applying the inclusion criteria of the LEAD study Table . Also, aWhen certain characteristics’ values were required in the CDM but were not captured within the registry or the LEAD study, the default values in the CDM were used, which are based on published literature –27. ThesIn the current analysis, we compared the intermediate-acting neutral protamine Hagedorn (NPH) insulin (comparator) versus the long-acting insulin analogue insulin glargine U100 (intervention) in patients with T2DM uncontrolled with non-insulin non-diabetic agents alone.The current analysis compared insulin glargine U100 versus NPH Insulin and the efficacy data of each treatment was taken from the results reported in the LEAD study. In the intention-to-treat analysis, reductions in HbA1c for glargine U100 and NPH were 1.10% and 0.92% respectively and the difference between adjusted mean changes in the two treatment groups was 0.22 (p = 0.0319). After the first year, HbA1c was set to increase following the natural progression as defined by the Hong Kong Diabetes Registry risk equation. The rates for non-severe hypoglycaemia used as input were 671.67 and 990 per 100 patient-years for glargine U100 and NPH respectively (p < 0.004)  was incorporated into the model through using health utilities. Since there are no QoL data specific to the Chinese population, the research team relied on published literature –37 (TablDrug acquisition costs for insulin Glargine U100 and NPH were based on the purchase prices paid by the HA to the supplier (payer perspective) in 2018. The current cost of insulin glargine U100 was HKD 0.40 per unit, and the cost of insulin glargine U100 was fourfold that of NPH insulin. During the first year, a Drug Daily Dose (DDD) of 32.1 units (glargine U100) and 32.8 units (NPH) was applied which were the doses used in the LEAD trial. The dose for each treatment was then up-titrated in the second year by 10% and remained stable afterwards. It was assumed that there would be no adjustment to non-insulin and anti-diabetic medications throughout the simulation.Costs of treating diabetes-related complications in 2018 were constructed from the HA Gazette  Table 44 which sTable Within this analysis, we also considered indirect healthcare costs, specifically absenteeism costs. This means that for patients who are absent from work due to diabetes complications, we quantify the economic value of these absent days Table . A diabeIndirect costs are captured based on the human capital approach, which takes into account the value of lost production resulting from morbidity and mortality associated with the disease for patients of working age.Costs per day absent from work are calculated separately for males and females based on the average annual salary  and the number of working days per year. Each complication is associated with days absent from work and this is assigned to each patient in each year of the simulation.Table A lifetime horizon of 50 years was deemed appropriate and used for this analysis with a 3% discount rate for both costs and outcomes as recommended by the Chinese Center for Health Economics Research .2 versus 25.36 ± 4.04 kg/m2) in the LEAD study cohort compared with base case. In scenario 2, the proportion of severe hypoglycaemia requiring medical assistance was adjusted to 50% instead of 11.8% as used in the base case. This enabled examination of the magnitude of impact that medical assistance in severe hypoglycaemic episodes would have on the costs. We also repeated the analysis assuming that the rates of severe hypoglycaemia were the upper bound of 95% confidence interval (CI) of glargine U100 treatment and the lower bound of 95% CI of NPH treatment (scenario 3). However, it should be noted that probabilistic sensitivity analysis in CDM v9.0 excludes variation in the variable (hypoglycaemia rates). The next updated version of CDM will include variation on hypoglycaemia rates. Although the 95% CI was not reported by Pan et al. a poisson distribution was assumed for the number of hypoglycaemic events and in turn a 95% CI was calculated and used. The scenario evaluated the robustness of the results produced by glargine U100 even under extreme unfavorable rates of hypoglycaemic events. Two further scenarios (scenario 4 and 5) were also simulated where an alternative set of risk equations were used, namely the UKPDS and PROcam risk equations. The UKPDS 82 risk equations are used globally in health economic analyses and were therefore applied here. The PROcam risk equation was proven to be a good predictor of cardiovascular outcomes in Asia despite of being developed for Germany, Austria, and Switzerland [Scenario analyses were conducted to test the consistency of results to changes in various input variables Table . Scenaritzerland . The priIn version 9.0 of the CDM, the cohort baseline values , the treatment effects on physiological parameter levels and transition probabilities for cardiovascular events were subject to random sampling based on their standard error (SE). Direct- and indirect costs are also included in the PSA based on a defined variation of 20%. Utilities and disutilities were reported without SE and as such not considered in the PSA. Finally, please note that version 9.0 of the model does not allow inclusion of hypoglycaemia rates in the PSA. Results of the PSA are the ICER cloud scatterplot and the complementary cost-effectiveness acceptability curve (CEAC).In the base case analysis, 1000 patients were treated with insulin glargine U100 or NPH insulin for a time horizon of 50 years (lifetime) and incurring costs in the public setting. Total costs of treating diabetes which included costs of insulin, costs related to diabetes complications and indirect costs amounted to HKD 762,136 for a patient receiving glargine U100 and HKD 740,776 for a patient using NPH Table . The breUnder scenario 1, the analysis was repeated using the baseline characteristics reported in the LEAD study. This resulted in slightly larger incremental gains in favor of glargine U100 compared to the base case (QALY: 0.224 vs 0.217 years) Table . The ICEIn scenario 2, the proportion of severe hypoglycaemia that required medical assistance was equal to that not requiring medical assistance (i.e. 50% versus 50%). The realized ICER under this scenario was HKD 10,583 per QALY gained which was significantly lower in comparison to the base case and all the other scenarios Table . This drThe analysis for scenario 3 assumed that the rates of severe hypoglycaemia were at the upper bound of the 95% CI of glargine U100 treatment and the lower bound of 95% CI of NPH treatment which effectively attenuated the difference in the rates of hypoglycaemia between the two treatments. The ICER under this scenario at HKD 253,115 was higher than that of the base case  is deemed highly cost-effective and is mainly driven by the reduced rates of hypoglycaemic events experienced with glargine U100. The results from the PSA further supported the robustness of the calculated ICER and glargine U100 is expected to be cost-effective in real-life if it would be reimbursed in Hong Kong.The analysis was based on clinical data extracted from the LEAD study which is the only published trial to date that compares insulin glargine U100 with NPH insulin in Asia including Hong Kong. In turn, the current cost-effectiveness analysis is the first to utilize Asian-specific clinical data to compare glargine U100 and NPH using the validated Core Diabetes Model. All the scenarios that were defined and run consistently showed that glargine U100 is cost-effective with ICERs well below the local WTP threshold.From a clinical perspective, insulin glargine U100 has been demonstrated to produce greater reduction in HbA1c levels than NPH insulin . ImportaIt is worth noting that the additional glucose lowering effect of glargine U100 compared with NPH did not lead to a significant reduction in the rates of vascular complications from diabetes or improvement in life expectancy in the present analysis. This is not unexpected since the between-group difference in attained HbA1c was too small to have a sustained impact. As seen in other trials, long-term vascular and mortality benefits from intensive glycaemic control were observed only in younger patients with shorter disease duration and not in older adults with long-standing diabetes and multiple co-morbidities , 45. TheThe results from this cost-effectiveness analysis concur with previous analyses comparing insulin glargine U100 with NPH insulin. Brandle et al.  used theThe study has a number of limitations that need to be acknowledged. Firstly, the efficacy rates of insulin glargine U100 compared with NPH insulin were based on the results of a single clinical trial. For reasons related but not limited to patient selection, treatment compliance, and overall medical care delivered, results from clinical trials are often not reproducible in real world clinical practice. For instance, it is possible that the frequency of severe hypoglycaemia in our local setting differ from that reported in the LEAD trial which could affect the outcome of the analysis. On the other hand, the LEAD study was conducted in Asia with inclusion of patients from Hong Kong. In this regard, clinical profile and responses to treatment should approximate that of patients in Hong Kong. Secondly, some of the baseline characteristics and management settings were not available in the LEAD study or the HKDR and were filled in with default values of the CDM which might not be specific to the local disease population. However, such a limitation is considered common among cost-effectiveness studies and is expected to have only minimal effect on the results. Thirdly, the unit costs of some medical procedures that were considered complex were based on inputs from medical experts in Hong Kong and could vary slightly compared to the realistic procedures used. Again, it is believed that these slight variations would not affect the overall results of the analyses since the medical resources used were based on the rates of the occurrence of adverse events which in turn were based on published literature. Fourthly, we assumed that the dose of insulin was fixed after the second year. In actual practice, insulin regimen would be adjusted, for instance, an increase of basal insulin dose, a change to pre-mixed insulin, or addition of prandial insulin. Although the exact impact of this manoeuvre cannot be determined, it is reasonable to deduce that insulin adjustment pertains to both glargine U100 and NPH groups equally and should not greatly alter the conclusion of the study. Lastly, the Asian population is different from the Western population with respect to risks of complications characterised by more strokes and fewer myocardial infarctions among Asians. We corrected for this by applying Hong Kong-specific risk equations, although testing with the UKPDS 82 equation did not alter the results strongly.Insulin glargine U100 is a cost-effective treatment for patients with type 2 diabetes when compared with NPH insulin in the Asian setting in Hong Kong. The major driver was the significantly lower rates of hypoglycaemia of glargine U100 than NPH. All the scenarios conducted under the current analysis proved glargine U100 being cost-effective even when the rates of hypoglycaemia were increased for glargine U100 and lowered for NPH. To conclude, these results support the use of insulin glargine U100 in Hong Kong even when the upfront drug acquisition costs are deemed higher than NPH insulin.Additional file 1: Table S1. Base Case Breakdown of direct costs. Table S2. Results of hypoglycaemia adverse events (per patient).Additional file 2: Figure S1. Scatterplots and CEACs of the different scenarios.