Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation. Endosomes are central organelles in orchestrating cell interactions with the extracellular environment, whether by regulating the composition of signaling molecules at the plasma membrane or by facilitating uptake and digestion of certain nutrients or degrading toxic or no longer needed material. Their wide range of functions is accomplished through a sequential and highly regulated process known as endosome maturation . Early e2 in late endosomes P, SNX1 and Rab11 recruitment, cargo recycling, and acidification likewise occurred. Finally, matured late endosomes fused with lysosomes, resulting in functional endolysosomes. Our minimally invasive assay provides an inexpensive and robust way to evaluate relative kinetics of key mediators of endosome maturation at individual endosome level. This assay does not require any specialized equipment, and maturation is detected with the ease of a conventional widefield microscope. We used this assay to investigate whether Rab conversion is coordinated with the recruitment of Rab11 recycling compartments and endosomal acidification. We found that interactions between the Rab11 compartment and the maturing endosome did not strongly correlate with Rab conversion. Fusing GalT to ratiometric pHlemon  P levels on endosomes during maturation.Driving early endosome identity, Rab5 recruits the PI(3)P kinase VPS34 and forms a positive feedback loop with PI(3)P . CoincidA major property of endosomes is their ability to undergo extensive sorting, to recycle components back to the cell surface and to the Golgi and to internalise membrane cargo destined for ILV-mediated degradation. Our electron microscopy data provides evidence that the nigericin-induced enlarged endosomes are capable of ILV formation and internalisation of the GalT marker . AdditioTo corroborate the apparent lack of strict coordination between Rab5 removal and Snx1 persistence at the endosomes, we co-expressed Snx1-GFP and mApple-Rab7 . ConsistTo further support our hypothesis that sorting can occur continuously from early to late endosomes, we examined the behaviour of a key component of the recycling pathway to the plasma membrane, Rab11. If our hypothesis were correct, we would expect Rab11 to show a similar behaviour as Snx1. Therefore, we investigated the dynamics of GFP-Rab11 in relation to Rab5 or Rab7-positive endosomes. Rab11 docks on the tubular part of maturing/sorting endosomes and promotes the recycling of cargo to the plasma membrane . SurprisThe ability of the enlarged endosomes to recruit mediators of recycling, Snx1 and Rab11 vesicles, suggests that our assay can recapitulate recycling pathways. To further probe this possibility, we monitored the fate of three cargoes: TfR-GFP for recycling to the plasma membrane, cation-dependent mannose-6-phosphate receptor (GFP-CDMPR) for recycling to the TGN and GalT-GFP, which we have shown above to remain in the endosome to be transported to the lysosome. All three cargoes were observed on Rab5-positive endosomes . While tBoth, Rab conversion and acidification are essential for endosome maturation but how the two are coordinated is poorly understood. To follow endosomal acidification at individual endosome level, we have tested several available endosomal pH sensors, such as mApple-Lamp1-pHluorin and NHE6-pHluorin2 ; howeverEquipped with a sensor locating to endosomes and responding to endosomal pH changes, we investigated the kinetics of endosomal acidification relative to Rab5 and Rab7 recruitment, using cells transiently transfected with mApple-Rab5 and GalT-pHlemon, as well as cells stably expressing mApple-Rab7 and transiently transfected with GalT-pHlemon. The nigericin-induced enlarged endosomes showed a dramatic decrease in YFP signal, which always coincided with Rab5-positive stages of endosome maturation  and withIf endosomal acidification is dependent on the progression of endosome maturation, then blocking endosome maturation by impairing Rab conversion should undermine acidification. To block Rab conversion, we knocked out the Ccz1, a subunit of the Rab7GEF, which has been shown to promote Rab conversion . Ccz1 deTo test our hypothesis that impaired Rab conversion compromises endosomal acidification, we expressed mApple-Rab5 and GalT-pHlemon in Ccz1 KO and control cells, and monitored the YFP/CFP ratio kinetics of the pH sensor on Rab5-positive endosomes . While iC. elegans Rab7GEF recruitment to PI(3)P-rich endosomes  supplemented with 10% fetal calf serum , 2 mM L-Glutamine, 1 mM Sodium Pyruvate, and 1 x Penicillin and Streptomycin . Cos-1 cells were grown in low-glucose DMEM supplemented as above. All cell lines were a kind gift of Dr Martin Spiess, with their identities authenticated by STR analysis by Microsynth AG , except Cos-1, the identity of which was validated by morphology analysis. All cell lines were confirmed to be mycoplasma-negative by PCR.HeLa CCL2, HeLa Kyoto-α, HEK293 and Neuro2A cells were grown at 37°C and 5% COFor transient cell transfections, cells were plated into 6-well plates to reach 70 % confluency the following day and transfected with 0.5 µg plasmid DNA complexed with Helix-IN transfection reagent (OZ Biosciences). Cells were used in imaging assays at 48 hr post transfection. For the Ccz1 rescue experiments, given the larger size of the plasmid, 2 µg plasmid DNA was used, and cells were transfected for 72 hr.For cell growth assays, cells were plated into 12-well plates at 10,000 cells per well. The following day, a sample was taken for counting (0 hr time point), and the remaining cells were incubated in complete medium with or without nigericin for 20 min, washed, incubated for specified time periods and collected for counting. Both conditions were reaching 90 % confluency by 72 hr. Three wells were measured per condition. Doubling time was calculating using the formulawhere c1 and c2 are cell counts for two consecutive time points t1 and t2.DNA constructs mApple-Rab5a, mApple-Rab7a, GalT-mCherry, mNeptune2-C1 and mApple-Lamp1-pHluorin plasmids were a gift from Michael Davidson . GFP-Rab11a was a gift from Richard Pagano (Addgene # 12674) . EGFP-2xFor stable GalT-GFP cell line generation, a sequence encoding GalT-EGFP  was generously provided by Jennifer Lippincott-Schwartz . GalT-EGFP was amplified using primers with restriction site overhangs for NotI and PacI, and subcloned into the Retro-X Q vector pQCXIP (Takara Bio). The GalT-EGFP plasmid for transient transfections was generated from GalT-mCherry plasmid and the EGFP insert from LAMP1-EGFP, using NEBuilder HiFi Assembly cloning kit  with the primers designed by the NEBuilder Assembly Tool  followinFor Ccz1 rescue experiments, the myc tag was removed from the Ccz1-myc plasmid using NEB site-directed mutagenesis (SDM) kit (NEB) following manufacturer’s instructions and primers selected using NEBaseChanger tool . NuclearFor the pH sensor constructs, we used pHlemon, which consists of mTurquoise2 and EYFP in tandem with a 21 bp linker in between . Separat2.To generate GalT-EGFP stable cell lines, Phoenix Ampho packaging cells  were grown in complete medium supplemented with 1 mM sodium pyruvate and transfected with pQCXIP-GalT-EGFP using FuGENE HD (Promega) to produce viral particles. The viral supernatant was harvested after 48–72 hr, passed through a 0.45 µm filter, supplemented with 15 µg/ml polybrene, and added to target HeLa-α cells. The next day, complete medium with 1.5 µg/ml puromycin was added. Following selection, cells were subjected to cell sorting on a FACSAria III (BD Biosciences) to obtain a cell pool with homogenous expression levels. HeLa-α-GalT-EGFP were maintained in complete medium supplemented with 1.5 µg/ml puromycin at 37 °C in 5 % COStable expression of mApple-Rab5 in HeLa cells was achieved by transfection of HeLa CCL2 cells with the mApple-Rab5a plasmid and three rounds of bulk-sorting by FACS at 15, 33 and 61 days post transfection. The cell line stably expressing mApple-Rab7a were generated as previously described . BrieflyCcz1 has a homolog, Ccz1b, which differs by four nucleotides and is identical in amino acid sequence. Guide RNAs (gRNA) were designed to target both genes simultaneously. The CRISPR strategy was described previously . Briefly2, 2.7 mM KCl, 0.5 mM MgCl2 in PBS supplemented with 10 % FCS and Penicillin and Streptomycin). Where specified, cells were treated with 10 µM Nigericin  or 5 µM Monensin  or 10 mM ammonium chloride prior or during imaging. Where specified, 100 nM Lysotracker Green  or 10 nM Lysotracker Red in imaging buffer was added 20 min prior to imaging. For live cell imaging of HEK293, Neuro-2A and Cos-1 cells, imaging chambers were pre-coated with poly-L-lysine to enhance attachment of these three cell types.Cells were plated into 8-well imaging chambers (Miltenyi) the day before imaging to reach 50–70% confluency on the day of imaging. Just prior to imaging, cell medium was replaced with pre-warmed imaging buffer  with a Plan Apochromat N 63×/1.40 oil DIC M27 objective and a Photometrics Prime 95B camera. Filters with standard specifications for CFP, GFP, YFP, dsRed and APC were used to image mTurquoise2, GFP, YFP, mApple and mNeptune2, respectively. To minimise overexpression artifacts, cells were selected for imaging that expressed minimal amount of each fluorescent marker that was sufficient to produce good quality signal. For time-lapse experiments, to monitor endosome maturation kinetics, cells were treated with nigericin for 20 min, washed four times in imaging buffer and imaging chamber mounted onto an automated microscope stage. Several fields of view (FOV) were selected for imaging per condition. The microscope was programmed to image all FOVs sequentially and repeat the imaging at 1–2 min intervals. The obtained images corresponded to the recovery time between 40 min and 190 min post nigericin treatment. For experiments comparing Ccz1 WT to KO clones, with and without rescue, all four conditions were imaged in a single time-lapse experiment. All experiments were performed three times on different days.For co-localisation studies of GalT-GFP or GalT-pHlemon with mApple-Rab5 or Lysotracker Red, imaging buffer was replaced with cold PBS to slow down endosome movements, and cells were imaged immediately. Z-stack images were deconvolved using Huygens deconvolution online software tool.For endolysosomal labelling, 0.5 mg/mL Dextran-AF488  was pulsed into cells for 4 hr and chased for 1.5 hr into lysosomes. For endosomal dextran labelling, 2.5 mg/mL Dextran-AF488 or 1 mg/mL Dextran-AF647 was pulsed into cells for 20 min post nigericin treatment  and subsequently washed away for imaging within the following 10 min and at 1 min intervals thereafter.To visualize endocytosis and trafficking of surface transferrin receptor, TfR-EGFP-expressing cells were treated with mCherry-tagged nanobodies against GFP to tag the plasma membrane TfR-EGFP. Nanobodies were produced as previously reported . BrieflyUsing the time-lapse images, endosomes were selected for analysis with the following criteria. For mApple-Rab5 expressing cells, endosomes initially devoid of Rab5 and later acquiring and subsequently losing Rab5 were identified. For mApple-Rab7 expressing cells, endosomes initially devoid of Rab7 and subsequently acquiring Rab7 and stabilising its expression were identified. This ensured that the entire Rab conversion event was captured in the kinetic.To quantify the recruitment of markers to the endosome, an oval selection tool in Fiji was used to draw a circular region-of-interest (ROI) closely following the rim of the endosome in the channel for the most visible marker or by predicting the location of the rim if cases where the enlarged endosome was negative for both markers and appeared as a dark circle . For lesEndosomal recruitment marker measurements were background-subtracted and adjusted for the minimum and maximum values of the entire measured kinetic, to represent a range from 0 to 1. The pHlemon measurements were kept as background-adjusted YFP/CFP ratios. For averaging, kinetics were aligned for Rab5 peak or for Rab7 at 50 % of its final maximum value, representing the point of Rab conversion . At leas2, 2 mM MgCl2, 10 mM Glucose, 10 mM HEPES (for pH 5.5–9.0) or 10 mM MES (for pH 4.0–5.0), and pH was adjusted with NaOH or HCl. Images were taken at 15 min after adding the buffers to the cells. Golgi ribbon structures were selected with a segmented line tool in Fiji, and MFI was calculated in both channels and subtracted for background measured in a field devoid of cells. The equation for the line of best fit was generated in GraphPad Prism based on log(dose) response curve with variable slope, where log(dose) is pH and response is YFP/CFP values  to disrupt intracellular proton gradients. The buffers consisted of 138 mM NaCl, 5 mM KCl, 2 mM CaCl2O for 1 hr at 4 °C. Following a wash with water, cells were further stained for proteins and nucleic acids with 2 % uranyl acetate in H2O overnight at 4 °C. Samples were subsequently dehydrated in acetone/H2O in stepwise increases in acetone concentration of 20%, 50%, 70%, 90 % and 3 × 100 %. The samples were infiltrated with 50 % epon embedding medium in acetone for 1 hr at room temperature, and subsequently with 100 % epon resin overnight. Next day, fresh epon resin was added and samples were polymerised for 24 hr at 60 °C. Sections of 60–70 nm were collected on carbon-coated Formvar-Ni-grids and were viewed with a Phillips CM100 electron microscope.HeLa CCL2 cells were grown in 10 cm dishes, treated for 20 min with nigericin and left to recover. At specified times, cells were fixed in DMEM containing 2.5 % glutaraldehyde and 3 % formaldehyde for 2 hr at room temperature. Cells were washed with PBS and cell membranes stained with 2 % osmium tetroxide and 1 % potassium hexacyanoferrate in H2O, and subsequently stained for 10 min with 2 % uracyl acetate and 2 min Reynold’s solution.To prepare cells for immunolabeling, HeLa cells stably expressing GalT-GFP were prepared as previously described . SectionHeLa cells were plated onto coverslips 24 hr prior to 20 min nigericin treatment and recovery. At specified times post nigericin treatment, cells were fixed in 2 % paraformaldehyde, permeabilised with 0.1 % Triton X-100, blocked in PBS containing 2 % BSA and 5 % goat serum, and stained with anti-GalT  followed by AF594-conjugated goat anti-rabbit and DAPI. HeLa cells stably expressing mApple-Rab5 were plated onto coverslips 24 hr prior to treatment. Cells were left untreated, treated for 16 hr with bafilomycin , or treated with nigericin for 20 min followed by a 60 min washout. Following treatment, cells were fixed for 10 min with methanol at –20 °C, blocked in PBS containing 2 % BSA and 5 % goat serum, and stained with anti-LC3b  followed by AF488-conjugated goat anti-rabbit. Coverslips were mounted onto glass slides with Fluoromount G (Southern Biotech) and sealed with nail polish. Following z-stack image acquisition, images were deconvolved using Huygens deconvolution online software tool.ACATTTAGCCCATCAAAACCTGC, CCGAACAACCATGACCATCC). Ccz1 expression was normalized for β-actin expression.RNA was extracted and purified from cells using RNeasy kit or using the Trizol-Chloroform extraction and isopropanol precipitation, following manufacturer’s instructions. cDNA was reverse-transcribed using GoScript reverse transcriptase primed with a mix of Oligo(dT)s and random hexamers (Promega). qRT-PCR was performed using GoTaq qPCR master mix (Promega) and primers specific for spanning the intron junction between exons 3 and 4 of ccz1  and denatured in Laemmli buffer at 65 °C for 5 min. Samples were resolved by SDS-PAGE and transferred onto nitrocellulose membrane. Samples were blocked in milk, stained with anti-myc  and HRP-conjugated anti-mouse, and revealed with WesternBright ECL HRP substrate (Advansta). The manuscript describes and validates a method for observing endosome dynamics in living cells. The approach may open the door to mechanistic studies of endosome maturation. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Christopher G Burd as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Vivek Malhotra as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Benjamin S Glick (Reviewer #3).Thank you for submitting your article "A novel live cell imaging assay reveals regulation of endosome maturation" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The authors describe a technical innovation that swells endosomes and the terminal compartments of the Golgi of cultured human cells, allowing them to observe the maturation of the dramatically enlarged compartments by fluorescence microscopy. Ionophore treatment is a significant perturbation, but a variety of findings validate that the enlarged endosomes undergo Rab5-to-Rab7 conversion over a time course that is roughly similar to the rate of Rab endosome conversion in unperturbed cells. The results further establish that endosome recruitment of a recycling/retrograde cargo sorting factor, Snx1, is not correlated with the Rab5-to-Rab7 early-to-late conversion process. Endosomes decorated with another regulator of recycling, Rab11, are reported to make transient contact with Rab-negative and Rab5/7-positive endosomes, leading the authors conclude that cargo recycling/retrograde sorting pathways are unaffected by nigericin-induced swelling. In contrast, acidification of the endosome is reported to be linked to Rab5-to-Rab7 conversion. It is further reported that the terminal compartments of the Golgi apparatus, identified by the presence of GalT, are swelled by nigericin and they are observed to fuse with a Rab5-negative endosome which subsequently acquires Rab5, initiating to Rab5-to-Rab7 conversion. Overall, this is a high-quality study that presents a useful tool for observing endosome dynamics in live cells. The approach was used to elucidate several unappreciated aspects of endosome maturation and Golgi-endosome interactions that are intriguing though largely preliminary in significance.1. A critical concern throughout the manuscript regards extrapolations of endosome protein recruitment as a direct indication of cargo transport to or out of an endosome. No cargo transport assays are included in the study, and the reviewers concluded that important points, described below, are not firmly substantiated.A) It is asserted that the Rab conversion process under investigation indicates physiological trafficking of endocytosed cargo to the lysosome via endosome maturation. However, cargo trafficking and/or turnover was not examined in the study. One dextran uptake experiment suggested that some dextran-containing compartments become decorated with tagged Rab5, but the prevalence of this could not be ascertained from the presentation and transport to the lysosome was not tested. This could be accomplished by examining endocytosis and subsequent trafficking using a fluorescent cargo .B) It is asserted that recruitment of tagged Snx1 or Rab11 to the endosome indicates recycling of endocytosed cargo to the plasma membrane. However, trafficking of a recycling cargo protein was not examined. Testing if recycling cargo is exported from the endosome via Snx1-coated tubules or under Rab11 regulation is necessary to support the conclusions regarding export of cargo from the endosome. This is especially important because many of the reporter proteins are tagged with a fluorescent protein and expressed transiently at high level in cells that also express the native proteins. It is also important because presumed changes in the tension of the plasma membrane of rapidly swelled endosomes may affect the formation of carriers of recycling pathways, such as documented for phagosomes during their maturation .It is noted that accurate descriptions of findings are included in the text , however, equating endosome recruitment of sorting factors with cargo trafficking is the norm throughout the manuscript, including in the Abstract.2. The conclusion that Rab11+ compartments directly contact Rab5-negative and Rab5/7-positive endosomes is not convincingly supported by the data. The Rab11 fluorescence signals are very complex, with so many puncta present throughout the crowded cytoplasm that specific  contacts between Rab11-decorated endosomes and early endosomes were not (perhaps cannot) distinguished by the approach used. The observations may also be influenced by the expression level of GFP-Rab11. The reviewers considered these conclusions to be preliminary in significance.3. The conclusion that entire late Golgi compartments are directed into the endosome maturation pathway is poorly supported by the data. This aspect of the study is provocative, yet the reviewers consider this aspect of the study to be preliminary in significance. This finding was not even addressed in the Discussion.(A) The critical observations are determinations of the localization of GalT-fluorescent fusion proteins in nigericin-treated cells. Residents of trans/TGN Golgi compartments cycle between the Golgi and the endosome. Golgi residents that fail to be sorted into the retrograde pathway continue to the lysosome and are degraded (via the MVB pathway). While the authors' observations might indicate the convergence of the secretory and endosome maturation pathways, other possibilities were not addressed. It may be that the retrograde pathway fails to some degree when late Golgi compartments are swollen , resulting in 'leakage' of GalT fusion proteins to the lysosome. This is especially concerning because most of the experiments employed GalT fusion proteins where the increased expression may saturate retrograde pathways.(B) The time required acquisition of Rab5 on GalT-positive compartments is long – on the order of 1-2 hours. It is unclear if the observations of swollen late Golgi compartments reveal a physiologically meaningful transition.4. The conclusion that endosome acidification is linked to Rab5-to-Rab7 conversion is not convincingly supported by the data. The graphical presentations of the data shown in figures 10 C, E, F indicate that acidification was initiated on Rab5-positive endosomes and that the rates of acidification did not change once Rab7 was acquired. On the other hand, the data presented in figures 10B and 11B are suggestive of a temporal correlation between acidification and the acquisition of Rab7. This discrepancy needs to be resolved.Reviewer #1 (Recommendations for the authors):I have a few concerns regarding some other points made using the approach.1. The analyses of rab11, Snx1, and endosome acidification are interesting, though the accuracy of such analyses is not rigorously validated at this stage due to the small size of native endosomes. Hence, it is unclear if the conclusions apply to native endosomes. Related, a recent paper from Sergio Grinstein and colleagues  showed that ion transport-driven changes in membrane tension during phagosome maturation (membrane "crenation") are necessary to trigger Snx1, Snx2 and Snx5 recruitment to the phagosome. This may not apply to native endosomes, but the physical principal  is applicable to any membrane compartment, so there is good reason to be concerned about confounding effects due to the swelling of these compartments.2. I am not convinced that it has been shown that swelled trans/TGN compartments provides the membrane of rab5 and rab7 positive endosomes. Figure 2 shows that a compartment, probably the TGN, is swelled by nigericin and it is argued (lines ~155-162) that the presence of overexpressed (and tagged) GalT-GFP in ILVs of MVBs probably indicates that rab5 and rab7 positive endosomes are derived from the swollen trans Golgi/TGN. Proteins considered to be residents of the trans/TGN compartments cycle between the endosome and the Golgi and Golgi residents that fail to be sorted into the retrograde pathway continue to the lysosome and are degraded (via the MVB pathway). It may be that the retrograde pathway fails to some degree when all of these compartments are swollen (point 1). In my opinion, the paper would be improved by removing this material.Reviewer #2 (Recommendations for the authors):(1) The most serious problem is with the data and conclusions related to Rab11 interaction with early endosomes in Figure 8. Unlike the Snx1 data, where association with the early endosomes is fairly convincing, there are simply too many Rab11 puncta in the cell to tell if they are meaningfully interacting with the early endosomes, or if the data simply reflects random collisions of small recycling endosomes with the enlarged early endosomes. To determine if these collisions are functionally significant the authors would need to show data on the transfer of recycling cargo from Rab5 endosomes to Rab11 endosomes during such interactions. Without this I would recommend removing this data from the paper. Along these lines, while the Snx1 data was more convincing, it was overinterpreted as "recycling" or "sorting" even though no cargo was followed. The authors should more carefully word the text, especially the abstract and discussion, to avoid such gross overinterpretation of the Snx1 data.(2) To really establish this endosome maturation model the authors should establish if the enlarged endosomes contain endocytosed cargo, as opposed to Golgi-derived cargo, and determine how long it takes to acquire such cargo. This could be accomplished using Tf, EGF, or perhaps dextran at early timepoints after nigericin washout.(3) Figure 7 – It was not convincing that data in panels F and G are different from each other.(4) Figure 11 – I find the interpretation of panel D confusing. How can we interpret this as connected to Rab conversion when even the labeled compartments at the earliest time point in the czz1 knockout have abnormally high pH, and during the time-course even the last timepoint for czz1 KO is higher than that of the earliest timepoint for WT?(5) Figure 12 – The criteria used to determine which GalT structures are Golgi or lysosomes seems flawed. Morphology alone is not sufficient to identify the compartments with high accuracy, especially after perturbation. Also, to what extent does GalT-CFP label lysosomes without nigericin treatment?Reviewer #3 (Recommendations for the authors):– What is the status of recycling endosomes? These structures must have some relationship to maturing endosomes but the literature is confusing. Rab11 is the traditional marker for recycling endosomes, and the authors describe Rab11 vesicles that "contact" maturing endosomes. How is this pathway envisioned to occur? Does internalized material ever accumulate in Rab11-positive recycling endosomes? It would be useful to bring the earlier work on the FERARI complex into a model that incorporates the current data.– Do late endosomes fuse completely with lysosomes, or do they typically undergo kiss-and-run fusion as has been described in both mammalian and yeast cells? Some of the images here seem to argue against complete fusion. What are the implications? Specifically, if late endosomes are constantly being generated by maturation but are not consumed by fusion with lysosomes, why doesn't the cell fill up with late endosomes?– The most striking result is that TGN-derived compartments go on to become Rab5-labeled endosomes, yet this topic is not even mentioned in the Discussion. Do the authors infer that there is a close relationship between the TGN and early endosomes, as has been observed in plant and yeast cells? Or is it more likely that existing Rab5-positive early endosomes become more prominent due to fusion with enlarged TGN-derived compartments?In Figure 1D, homotypic fusion is readily apparent but I don't see the Rab conversion described in the legend.The videos are hard to match to the figures. For example, the "Figure 1C supplement 1" video seems to have nothing to do with Figure 1C, and I couldn't find a description of this video.Figure 5: PI(3)P is presumably generated, not recruited. Essential revisions:The authors describe a technical innovation that swells endosomes and the terminal compartments of the Golgi of cultured human cells, allowing them to observe the maturation of the dramatically enlarged compartments by fluorescence microscopy. Ionophore treatment is a significant perturbation, but a variety of findings validate that the enlarged endosomes undergo Rab5-to-Rab7 conversion over a time course that is roughly similar to the rate of Rab endosome conversion in unperturbed cells. The results further establish that endosome recruitment of a recycling/retrograde cargo sorting factor, Snx1, is not correlated with the Rab5-to-Rab7 early-to-late conversion process. Endosomes decorated with another regulator of recycling, Rab11, are reported to make transient contact with Rab-negative and Rab5/7-positive endosomes, leading the authors conclude that cargo recycling/retrograde sorting pathways are unaffected by nigericin-induced swelling. In contrast, acidification of the endosome is reported to be linked to Rab5-to-Rab7 conversion. It is further reported that the terminal compartments of the Golgi apparatus, identified by the presence of GalT, are swelled by nigericin and they are observed to fuse with a Rab5-negative endosome which subsequently acquires Rab5, initiating to Rab5-to-Rab7 conversion. Overall, this is a high-quality study that presents a useful tool for observing endosome dynamics in live cells. The approach was used to elucidate several unappreciated aspects of endosome maturation and Golgi-endosome interactions that are intriguing though largely preliminary in significance.1. A critical concern throughout the manuscript regards extrapolations of endosome protein recruitment as a direct indication of cargo transport to or out of an endosome. No cargo transport assays are included in the study, and the reviewers concluded that important points, described below, are not firmly substantiated.A) It is asserted that the Rab conversion process under investigation indicates physiological trafficking of endocytosed cargo to the lysosome via endosome maturation. However, cargo trafficking and/or turnover was not examined in the study. One dextran uptake experiment suggested that some dextran-containing compartments become decorated with tagged Rab5, but the prevalence of this could not be ascertained from the presentation and transport to the lysosome was not tested. This could be accomplished by examining endocytosis and subsequent trafficking using a fluorescent cargo .To further examine cargo trafficking, we performed additional dextran-Alexa647 uptake assays in mApple-Rab5 and GFP-Rab7 expressing cells. The experiments revealed that dextran-Alexa647 is present in Rab5-positive endocytic vesicles that go on to fuse with Rab5-negative enlarged structures, which subsequently become Rab5 positive. Those Rab5 enlarged endosomes undergo Rab conversion, turn Rab7 positive and form endolysosomes, which mature to lysosomes, still containing the dextran. These data are now included, replacing the shorter dextran-Alexa488/mApple-Rab5 panel .As for the prevalence of endocytic Dextran-containing compartments, we now include a higher-quality image set, showing Rab5-positive enlarged endosomes, both with and without detectable Dextran-AF488, as well as Dextran-containing Rab5-negative (presumably Rab-converted) endosomes. We cannot really measure prevalence because Dextran endocytosis is too fast to capture its early and highly-synchronized fusion events with the asynchronously-generated and highly-transient Rab5-positive enlarged compartments. .As a second piece of evidence, we transfected cells with BFP-Rab5 and transferrin receptor tagged to GFP (TfR-GFP). We then added a GFP-nanobody, which was tagged with mCherry, resulting in illumination of surface TfR and its subsequent trafficking. Already within a few minutes after addition of the nanobody, we were able to detect the nanobody together with Rab5 in close proximity of the enlarged endosomes; the presence of the nanobody on the limiting membrane became more obvious after about 15-20 min of the addition. Thus, plasma membrane derived cargo appears to reach the enlarged endosome in a reasonable time frame. Proper quantification over many endosomes as we did for the other experiments proved to be difficult because BFP-Rab5 seems to be less well tolerated by cells compared to the GFP or mApple versions, and only a few cells with a weakly detectable Rab5 expression could be imaged. Further difficulties stem from the short window of opportunity to trace the synchronous uptake of nanobody-tagged surface TfR and its trafficking to the highly-asynchronous and transient Rab5-positive enlarged endosomes. Moreover, the signal for the mCherry-tagged GFP-nanobody was rather weak, limiting the number of endosomes with detectable TfR. Nevertheless, we observed multiple events and two examples are now shown in the manuscript in supplementary information . Additionally, we show quantification for TfR-GFP acquisition to and removal from the enlarged endosomes relative to Rab5 recruitment . These data show concomitant arrival of Rab5 and TfR to the endosome and prompt removal of TfR as the endosome undergoes Rab conversion.Taken together, we provide more evidence that cargo from the plasma membrane reaches the enlarged early endosomal structures and that we can recapitulate the lifetime of an endosome from early-to-late to endolysosome to lysosome.B) It is asserted that recruitment of tagged Snx1 or Rab11 to the endosome indicates recycling of endocytosed cargo to the plasma membrane. However, trafficking of a recycling cargo protein was not examined. Testing if recycling cargo is exported from the endosome via Snx1-coated tubules or under Rab11 regulation is necessary to support the conclusions regarding export of cargo from the endosome. This is especially important because many of the reporter proteins are tagged with a fluorescent protein and expressed transiently at high level in cells that also express the native proteins. It is also important because presumed changes in the tension of the plasma membrane of rapidly swelled endosomes may affect the formation of carriers of recycling pathways, such as documented for phagosomes during their maturation .We agree with the reviewers that we did not show any recycling cargo in the original manuscript. We now used TfR-GFP and mApple-Rab5 to identify the enlarged endosomes, from which TfR would recycle to the plasma membrane. We now include data in the manuscript showing that TfR-GFP is present in enlarged Rab5 positive endosomes and that the TfR-GFP signal gets incorporated into vesicles and goes down over time in the enlarged endosomes, consistent with recycling . Likewise, we used CDMPR-GFP, which recycles to the Golgi and we observed again a reduction of the CDMPR signal in Rab5 positive enlarged endosomes over time and the formation of CDMPR positive vesicles . Importantly, we did not observe the same reduction of the signal (or its packaging into vesicles) for GalT-GFP, which go down the route to the lysosome . Therefore, the reduction in the GFP signal cannot be explained by bleaching and thus we assume that we observe recycling of TfR and CDMPR to the plasma membrane and the Golgi, respectively. Regardless of the recycling pathway or the destination of the recycled cargo, these data indicate that sorting and recycling appear to be functional in the enlarged endosomes. We have not investigated whether TfR or CDMPR is exported away from the endosome via Snx1-coated tubules or Rab11-mediated transport carriers, as this would require triple transfection with BFP-Rab5, which cells do not tolerate well.To provide evidence for the role of Snx1 or Rab11 in mediating recycling in the enlarged endosomes, we used CRISPR-Cas9 SNX1 and RAB11a KO cell lines. We did not see a strong effect on recycling of TfR-GFP, which might be due to redundancy issues. SNX4 can compensate for SNX1 and presumably Rab11b for Rab11a, and over time the cell lines potentially adapt to the loss of SNX1 and RAB11a, respectively. To have a more acute readout, we knocked down Rab11a and b with siRNAs. Unfortunately, we obtained mixed results as we observed a delay in TfR recycling upon Rab11a+b knockdown in some experiments but not in others. Of course, this might be related to the knockdown efficiency in individual experiments and in the experiments where we observed an effect the efficiency might have been higher. Even though we sometimes observed an effect, we do not feel comfortable putting these data into the manuscript. Since the direct correlation between Rab11/Snx1 and cargo recycling has not been shown, we therefore adjusted the manuscript text accordingly.As with regards to potential changes in membrane tension and its effect on endosome recycling function, while we cannot exclude that these changes may occur because we did not measure them in our system, we now include data in the manuscript showing that uptake from the plasma membrane works well and that cargo is recycled away from the endosome (TfR and CDMPR data). The crenation, as described in Freeman et al., paper, is driven by changes in osmolarity, which the cells could promptly (within minutes) readjust after experimental intervention. As we allow recovery after the acute nigericin treatment, we predict there to be no big perturbations in osmolarity over the subsequent hours during which we can image cells and observe traffic events. Our assumptions here are supported by the stable endosome size during endosome maturation and the new evidence we provide on the cargo recycling.It is noted that accurate descriptions of findings are included in the text , however, equating endosome recruitment of sorting factors with cargo trafficking is the norm throughout the manuscript, including in the Abstract.We assume that the reviewers mean lines 78-79. We changed the text to reflect that we determine the interaction of Rab11 vesicles with enlarged endosomes as recruitment. Even though we do not mention recycling to the plasma membrane explicitly in the abstract, we changed the wording in the abstract and are more careful throughout the manuscript not to mention any specific cargo recycling, unless warranted.2. The conclusion that Rab11+ compartments directly contact Rab5-negative and Rab5/7-positive endosomes is not convincingly supported by the data. The Rab11 fluorescence signals are very complex, with so many puncta present throughout the crowded cytoplasm that specific  contacts between Rab11-decorated endosomes and early endosomes were not (perhaps cannot) distinguished by the approach used. The observations may also be influenced by the expression level of GFP-Rab11. The reviewers considered these conclusions to be preliminary in significance.To address the reviewers’ concern, we took videos in which images were taken in much shorter, 2-sec intervals. These videos show that Rab11 contact directly enlarged Rab5 negative and positive compartments and stay on there for various times, indicating that this interaction is not incidental. These data are now included in the manuscript. .3. The conclusion that entire late Golgi compartments are directed into the endosome maturation pathway is poorly supported by the data. This aspect of the study is provocative, yet the reviewers consider this aspect of the study to be preliminary in significance. This finding was not even addressed in the Discussion.off and Vassalli (J. Exp Med. 1977) reported the TGN swelling/dispersal after ionophore treatment, including nigericin. There were a few studies in the 1980’s and 1990’s , which report the swelling of the outer Golgi leaflet with additional cisternae getting affected with longer treatment, suggesting that our acute treatment would partially preserve the Golgi compartment. Indeed, this partial disruption of the Golgi agrees well with our data that recycling of CDMPR remains functional. Since the Golgi dispersal was well documented in the literature, we did not include this into the discussion. However, upon the reviewers’ suggestion, we now discuss our finding that Golgi compartments can fuel the endocytic pathway.We apologize for the confusion. We agree with the reviewers that we cannot conclude that the entire late Golgi compartments are directed into the endosome maturation pathway as we can only have a qualitative but not quantitative measure. We changed the text to make this clearer and we now discuss the swelling of the TGN in the discussion. We are actually not the first ones to notice this. Already Tartak(A) The critical observations are determinations of the localization of GalT-fluorescent fusion proteins in nigericin-treated cells. Residents of trans/TGN Golgi compartments cycle between the Golgi and the endosome. Golgi residents that fail to be sorted into the retrograde pathway continue to the lysosome and are degraded (via the MVB pathway). While the authors' observations might indicate the convergence of the secretory and endosome maturation pathways, other possibilities were not addressed. It may be that the retrograde pathway fails to some degree when late Golgi compartments are swollen , resulting in 'leakage' of GalT fusion proteins to the lysosome. This is especially concerning because most of the experiments employed GalT fusion proteins where the increased expression may saturate retrograde pathways.Indeed, we did not address the functionality of the retrograde transport to the TGN after nigericin washout in the original manuscript. The new experiments with the CDMPR described under point 1 would argue that the retrograde transport is functional at least at the level of cargo removal from the enlarged endosome. We can show that CDMPR positive structures bud off from the Rab5 positive enlarged endosomes and that the levels of CDMPR at the enlarged endosome drop over time. Moreover, these endosomes seem to be capable of also accepting CDMPR-containing vesicles. We take this as a strong indication that the Golgi-endosome shuttle is intact after the nigericin washout .If overexpression of proteins involved in the Golgi-endosome shuttle resulted in a saturated retrograde pathway, we would see a compromised removal of the overexpressed CDMPR-GFP from the endosome. However, this is not the case. In contrast to GalT constructs, all of the observed endosomes promptly removed CDMPR during or shortly before Rab5 recruitment. Thus, retention at the endosome and failure to recycle back to the Golgi is limited to the GalT fusion proteins.Although endogenous GalT is rarely observed outside of the Golgi ribbon , the constructs appear in punctate structures in addition to the Golgi ribbon, which occasionally colocalized with Rab5 and lysosomes . This suggests that GalT construct trafficking to the endosome is a property of the construct and not nigericin-mediated perturbation of retrograde trafficking. The combination of intact retrograde trafficking and the retention of GalT constructs in endosomes make GalT-pHlemon an excellent tool to study intralumenal pH of endosomes with minimum disruption to endosome function.(B) The time required acquisition of Rab5 on GalT-positive compartments is long – on the order of 1-2 hours. It is unclear if the observations of swollen late Golgi compartments reveal a physiologically meaningful transition.We can detect this also at earlier timepoints. We wanted the cells to reestablish the proper pH, and setting up the microscope (we record multiple cells simultaneously) takes some time. To be consistent, we usually start imaging 40 min after washout. We now include a time-lapse image series, in which Rab5 is recruited to an enlarged endosome, which then converts into a Rab7-positive endosome, within 20 min after nigericin washout . Thus, these events happen already very early after nigericin washout and continue for hours. There is no synchronous wave after washout, rather we observe asynchronous events. Additionally, as the Golgi swell up, the early swollen compartments are easy to miss as they are frequently in different focal planes to smaller endosomes. As the cell adjusts to its new physiology, the enlarged compartments rearrange to sit in a single focal plane and are more practical to image en masse. We clarify this in the text.4. The conclusion that endosome acidification is linked to Rab5-to-Rab7 conversion is not convincingly supported by the data. The graphical presentations of the data shown in figures 10 C, E, F indicate that acidification was initiated on Rab5-positive endosomes and that the rates of acidification did not change once Rab7 was acquired. On the other hand, the data presented in figures 10B and 11B are suggestive of a temporal correlation between acidification and the acquisition of Rab7. This discrepancy needs to be resolved.We think that the issue might be related to the way the experiments were performed. In Figure 10 , we used HeLa cells stably expressing either mApple-Rab5 or mApple-Rab7, while in Figure 11  Rab5 and Rab7 were transiently expressed in the ccz1 KO cell lines. To be consistent we repeated the experiments shown in Figure 10  with transient expression. We generated a new data set, which shows stabilization of pH upon Rab conversion, and now all data are consistent.Reviewer #1 (Recommendations for the authors):I have a few concerns regarding some other points made using the approach.1. The analyses of rab11, Snx1, and endosome acidification are interesting, though the accuracy of such analyses is not rigorously validated at this stage due to the small size of native endosomes. Hence, it is unclear if the conclusions apply to native endosomes. Related, a recent paper from Sergio Grinstein and colleagues  showed that ion transport-driven changes in membrane tension during phagosome maturation (membrane "crenation") are necessary to trigger Snx1, Snx2 and Snx5 recruitment to the phagosome. This may not apply to native endosomes, but the physical principal  is applicable to any membrane compartment, so there is good reason to be concerned about confounding effects due to the swelling of these compartments.Indeed, the Freeman et al., 2020 paper  explore the idea of osmotic/ionic pressure (and the associated membrane tension) changes playing a role in sorting and recycling, which could be of relevance to our system where the swollen Golgi likely arise from osmotic/ionic imbalances during nigericin treatment. The swelling in our system is most likely driven by interference to the proton gradient, since the protonophore FCCP inhibited monensin-induced swelling , and we observed a similar effect with V-ATPase inhibitor bafilomycin abolishing nigericin-induced swelling (data not shown). However, unlike the above-mentioned studies, where treatments were continuous, the nigericin treatment in our set up is highly transient and we allow our cells to recover and re-establish pH, thereby minimizing the possibility of any ionic imbalances during the observation phase. The macropinosomes in the Freeman et al., study respond to external ionic/osmotic stimuli within minutes of their addition, strongly suggesting that any ionic imbalances within our system would also be resolved within minutes of nigericin removal. Consistent with this notion, the enlarged endosomes do not rapidly shrink with the recruitment of Snx1, and rather remain stable in size until the endolysosomal stages. The mechanism driving the enlarged endosome shrinkage during endolysosomal stages will be subject of future studies. Although we do not show the functionality of Snx1 directly, we now include the CDMPR and TfR data to show that cargo recycling is functional at the enlarged endosomes. .2. I am not convinced that it has been shown that swelled trans/TGN compartments provides the membrane of rab5 and rab7 positive endosomes. Figure 2 shows that a compartment, probably the TGN, is swelled by nigericin and it is argued (lines ~155-162) that the presence of overexpressed (and tagged) GalT-GFP in ILVs of MVBs probably indicates that rab5 and rab7 positive endosomes are derived from the swollen trans Golgi/TGN. Proteins considered to be residents of the trans/TGN compartments cycle between the endosome and the Golgi and Golgi residents that fail to be sorted into the retrograde pathway continue to the lysosome and are degraded (via the MVB pathway). It may be that the retrograde pathway fails to some degree when all of these compartments are swollen (point 1). In my opinion, the paper would be improved by removing this material.Indeed, Figure 2 alone is not sufficient to argue that the enlarged endosomes derive from TGN-derived compartments. This is why we back up our hypothesis with Figure 3A, which shows a vesiculated GalT-GFP positive compartment acquire Rab5. To rule out any potential failure of retrograde transport, we now include CDMPR data, which shows prompt removal of CDMPR from the enlarged early endosome . We do consider it important to leave these data in the manuscript because they provide also the basis for the use of GalT-pH lemon with which explore endosomal acidification.Reviewer #2 (Recommendations for the authors):(1) The most serious problem is with the data and conclusions related to Rab11 interaction with early endosomes in Figure 8. Unlike the Snx1 data, where association with the early endosomes is fairly convincing, there are simply too many Rab11 puncta in the cell to tell if they are meaningfully interacting with the early endosomes, or if the data simply reflects random collisions of small recycling endosomes with the enlarged early endosomes. To determine if these collisions are functionally significant the authors would need to show data on the transfer of recycling cargo from Rab5 endosomes to Rab11 endosomes during such interactions. Without this I would recommend removing this data from the paper. Along these lines, while the Snx1 data was more convincing, it was overinterpreted as "recycling" or "sorting" even though no cargo was followed. The authors should more carefully word the text, especially the abstract and discussion, to avoid such gross overinterpretation of the Snx1 data.We provide now more evidence for the interaction of Rab11 vesicles with the enlarged endosomes. We made videos with shorter intervals between the individual frames . These data clearly show that this is not an accidental bumping into an endosome but that Rab11 vesicles can circle around endosomes and stay for several minutes.In addition, we imaged TfR-GFP together with mApple-Rab5. These data show that TfR-GFP positive vesicles bud off from mApple-Rab5 positive endosomes and that the GFP fluorescence intensity goes down over time in enlarged endosomes . These data are consistent with recycling of TfR to the plasma membrane.Moreover, CDMPR-GFP, which cycles between the TGN and endosomes was found to be present on Rab5 negative enlarged structure, which then turned Rab5 positive, and subsequently lost the CDMPR signal . Importantly those endosomes could regain CDMPR, which we interpret as acquisition from the TGN . These data may indicate that the TGN-endosome shuttle is intact after nigericin washout.That the TfR and CDMPR are really transported out of the enlarged endosome is also supported by our finding that GalT-GFP stayed in the enlarged endosome and the signal intensity did not significantly drop .The dependency of cargo trafficking on Rab11 and Snx1 could not be shown most likely because of the redundancy of these recycling pathways, with alternative pathways compensating for any deficiency in Rab11a or Snx1 . Therefore, we followed the advice of the Reviewer 2 to be more careful with the wording in order to avoid overinterpretation of Snx1 and Rab11 data.Regardless of the recycling pathway or the destination of the recycled cargo, these data show that sorting and recycling appear to be functional in the enlarged endosomes. We have not investigated whether TfR or CDMPR is exported away from the endosome via Snx1-coated tubules or Rab11-mediated transport carriers, as this would require triple transfection with BFP-Rab5, which cells do not tolerate well.(2) To really establish this endosome maturation model the authors should establish if the enlarged endosomes contain endocytosed cargo, as opposed to Golgi-derived cargo, and determine how long it takes to acquire such cargo. This could be accomplished using Tf, EGF, or perhaps dextran at early timepoints after nigericin washout.As pointed out above, we show now that TfR-GFP is present in enlarged endosomes and is lost from these endosomes over time . Moreover, we performed experiments with dextran-Alexa647 and mCherry-nanobodies directed against GFP to show that endocytosed material from the plasma membrane indeed reached the enlarged endosomes . TfR-GFP recruitment to the Rab5-positive endosome coincided with the recruitment of mCherry-tagged anti-GFP nanobody-labeled surface transferrin, suggesting that most of TfR-GFP at the enlarged endosome did indeed come from the plasma membrane. In contrast, CDMPR-GFP is present at the enlarged compartments prior to Rab5 recruitment and is promptly removed during or shortly before Rab5 recruitment . These observations suggest that the maturing endosomes readily accept endocytosed cargo and actively remove Golgi-derived cargo. We observed that endocytosed material reached the enlarged endosomes as they became Rab5 positive .(3) Figure 7 – It was not convincing that data in panels F and G are different from each other.We agree with the reviewer that the difference between the data presented in panel F and G is not very big. These panels represent the average of many endosomes and with the averaging the differences from the individual traces get cancelled out. The process is asynchronous and thus in this case the individual traces are more telling than the averaged traces. Nevertheless, we decided to keep the average traces in the manuscript because the highlight the asynchronous nature of the process. We modified the text to make this point clear.(4) Figure 11 – I find the interpretation of panel D confusing. How can we interpret this as connected to Rab conversion when even the labeled compartments at the earliest time point in the czz1 knockout have abnormally high pH, and during the time-course even the last timepoint for czz1 KO is higher than that of the earliest timepoint for WT?C. elegans and Mon1a/b knockdown in mammalian cells . Acidification eventually will occur, but it is massively slowed down; the molecular basis of which is still under investigation in our lab. Even in ccz1 KO cells endosomal traffic down the degradation pathway is not completely blocked, similarly to what we observed for sand-1 (-/-) C. elegans and Mon1a/b knockdown in mammalian cells . Acidification eventually will occur, but it is massively slowed down; the molecular basis of which is still under investigation in our lab.We agree that the ccz1 KO cells display higher endosomal pH than WT cells throughout the time-course. However, the cells in which we express the rescue plasmid of Ccz1 also have apparently less acidified endosomes, even though Ccz1 can still drive Rab conversion, and the pH dropped at an intermediate rate, when comparing rescued cells to control and ccz1 KO cells. Even in ccz1 KO cells endosomal traffic down the degradation pathway is not completely blocked, similarly to what we observed for sand-1 (-/-) (5) Figure 12 – The criteria used to determine which GalT structures are Golgi or lysosomes seems flawed. Morphology alone is not sufficient to identify the compartments with high accuracy, especially after perturbation. Also, to what extent does GalT-CFP label lysosomes without nigericin treatment?To address these issues, we co-labelled cells with lysotracker. GalT-CFP (pHlemon) and lysotracker showed a very high degree of co-localization. These data are included in the manuscript .Reviewer #3 (Recommendations for the authors):– What is the status of recycling endosomes? These structures must have some relationship to maturing endosomes but the literature is confusing. Rab11 is the traditional marker for recycling endosomes, and the authors describe Rab11 vesicles that "contact" maturing endosomes. How is this pathway envisioned to occur? Does internalized material ever accumulate in Rab11-positive recycling endosomes? It would be useful to bring the earlier work on the FERARI complex into a model that incorporates the current data.We would predict that the Rab11 vesicles would undergo kiss-and-run on the enlarged endosomes, similarly to what we have described previously. We added a sentence to this effect in the discussion. In fact, we compared Rab11 in control and FERARI KO cells, and it seems as if we indeed see a difference in residence time. However, we consider these experiments and the additional work necessary to make a strong case would be way beyond the scope of this manuscript. Therefore, we included wording in the discussion to integrate our FERARI findings.– Do late endosomes fuse completely with lysosomes, or do they typically undergo kiss-and-run fusion as has been described in both mammalian and yeast cells? Some of the images here seem to argue against complete fusion. What are the implications? Specifically, if late endosomes are constantly being generated by maturation but are not consumed by fusion with lysosomes, why doesn't the cell fill up with late endosomes?This is really difficult to distinguish at this point. We think that we observe both events. On one hand we see lysosomes contacting and ‘dancing around’ the enlarged endosomes. On the other hand, we also see them disappearing after contacting endosomes, consistent with a fusion event. At any rate, the enlarged endosomes acidify further and become lysotracker positive. At which point, they will lose their round and becomes smaller over time. In a way the endolysosome matures into a lysosome. We surmise that also membrane recycling will occur, but this is a subject for future studies. We do not observe an accumulation of late endosomes because they turn into endolysosomes and then mature to lysosomes, which then can go on and fuse with late endosomes again. Our data in new Figure 10 figure supplement 1 supports this model.– The most striking result is that TGN-derived compartments go on to become Rab5-labeled endosomes, yet this topic is not even mentioned in the Discussion. Do the authors infer that there is a close relationship between the TGN and early endosomes, as has been observed in plant and yeast cells? Or is it more likely that existing Rab5-positive early endosomes become more prominent due to fusion with enlarged TGN-derived compartments?We now included a discussion about TGN-derived compartments becoming Rab5-positive endosomes. We speculate that the Rab5 identity is mostly brought about through fusion with Rab5 positive endocytic structures, as we frequently observe such events. However, we cannot exclude other possible mechanisms such as direct recruitment of Rab5 from the cytoplasm. These alternative or additional mechanisms may just be much harder to recognize and to interpret, especially if occurring in combination.In Figure 1D, homotypic fusion is readily apparent but I don't see the Rab conversion described in the legend.We agree with the reviewer that in the particular example Rab conversion is more difficult to detect as the Rab7 signal at the endosome is very weak. However, in Figure 1C this is easy to see. Therefore, we do not refer to panel D anymore when describing Rab conversion. In addition we provide more examples of Rab conversion throughout the manuscript.The videos are hard to match to the figures. For example, the "Figure 1C supplement 1" video seems to have nothing to do with Figure 1C, and I couldn't find a description of this video.We apologize for the mistake. This was apparently a mistake introduced by the renaming of all files. We provide now the correct file names. We further integrate the links to all the videos in the figure legends, as the descriptions are identical to the time lapse image series provided in the figures.Figure 5: PI(3)P is presumably generated, not recruited.Of course, the reviewer is right. We changed the text accordingly.


To access the awareness of dental practitioners of Nepal towards COVID-19 transmission through aerosols.The study involved 384 dentists from all over Nepal and was conducted for a period of 3 months. A self-reported online questionnaire was developed using Google forms and the link was shared. It emphasized the awareness related to the aerosol and ventilation system in their daily practices was prepared. The data were analyzed in Statistical Package for Social Sciences version 20.0 software.n = 203) and within the age groups of <30 years 57% (n = 219). Participants from Bagmati Province were 60.4% (n = 232), with least from Sudurpaschim Province 0.5% (n = 2). 60% of participants provided only emergency services during the COVID-19 pandemic and few (7%) provided consultations via telephone. The current ventilation system used was a well-ventilated room with open windows 65.4% (n = 251). However, 52.8% (n = 203) preferred specialized operatory incorporating high-efficiency particulate air (HEPA) filters and ultraviolet (UV) light. More than 60% of respondents were unaware of the particle size of the aerosol.The majority of participants were female 52.9% (The obtained results signify the need for the proper ventilation system with appropriate air filtration systems in dental clinical setups. Its infections may lead to the common cold, to more serious manifestations.2 The incubation period for COVID-19 is reported to be 2–14 days during which patients show symptoms within 11.5 days.3 The main symptoms of COVID-19 include fever, cough, fatigue, and patients may also show sputum production, headache, hemoptysis, diarrhea, dyspnea, and lymphopenia. COVID-19 is characterized by rapid transmission and can occur by close contact with an infected person or through respiratory droplets and contact routes.7 The knowledge regarding the disease is evolving and the other ways of transmission may be highlighted by the scientific evidence in the future.Coronavirus disease 2019 (COVID-19), an emerging respiratory infection, was first discovered in late December 2019, in Wuhan city, Hubei Province, China.8 During the window period of the disease, patients may present to the dentist. The infected patient may be asymptomatic but may have an active state of infection and can also be a source of cross transmission. As procedures in a dental clinic generally involve close contact between patients and dentists, the risk of acquiring respiratory infection in this setting can be high.9Centers for Disease Control and Prevention (CDC) Guidelines for Infection Control in Dental Health-Care Settings—2003 notes postponing any non-emergency or elective dental procedures until a patient is no longer contagious with diseases that can be transmitted through the airborne, droplet, or contact transmission.It is, therefore, critical that adequate information should be conveyed to dental healthcare professionals in the time of this global emergency. Studies assessing the awareness of aerosols in the context of COVID-19 have not been carried out in our country. In this regard, this study aims to access the awareness of dental practitioners towards COVID-19 transmission through aerosols.A cross-sectional study was conducted among the dental practitioners of Nepal, from June 2020 to August 2020. Ethical approval was obtained from the Institutional Review Committee (IRC), Kantipur Dental College and Teaching Hospital (KDCH). The data were collected online, via a self-reported questionnaire, using Google forms. A link to the survey was distributed personally or in groups, via e-mails, and various social media applications.Z = 1.96 at confidence level = 95%; p = 0.5 (50%)10 and margin of error (e) = 0.05 (5%)The sample size of 384 was calculated by employing the following formula for infinite population:13 The questions were prepared to emphasize the awareness related to the aerosol and ventilation system in their practice. Suggestions for improvement in the questionnaires were incorporated from subject experts and modified accordingly. The respondents were clearly informed about the background and objectives of the study on the first page of the online questionnaire. They were also informed that they were free to withdraw at any time, without giving a reason, and the study maintained their privacy and confidentiality of the collected information. Dental practitioners of Nepal who wish to participate in the study were instructed to complete the questionnaire. Online informed consent was obtained before proceeding with the questionnaire. This questionnaire consisted of two sections: the first consisted of the sociodemographic and professional aspects and the second section consisted of questions related to knowledge, attitude, and practice regarding the aerosol related to COVID-19 transmission. After the completion of the questionnaire, the study participants were also provided with resources regarding the subject matter. After the desired sample size was obtained on removing the double entries by the participants, the form was set for not accepting responses. The data from Google forms were entered in Microsoft Excel and analyzed using descriptive statistics and presented as frequency and percentages in Statistical Package for Social Sciences (SPSS) version 20.0 software.The self-reported online questionnaire was developed by the authors after reviewing the pertinent literature.n = 203) and within the age groups of <30 years 57% (n = 219). More than 60% (n = 232) of the respondents were from Bagmati province with only 0.5% (n = 2) participants from Sudurpaschim Province. An almost equal distribution of respondents with their years of dental practice experience participated in this study. A total of 148 (38.5%) had 2–5 years of dental practice experience and 12.8% (n = 49) of the participants had >10 years of experience. The various characteristics of the respondents are mentioned in Table Altogether 400 responses were received. On removing the duplications, the desired sample size of 384 was obtained and included for further analysis. The majority of the respondents were female 52.9%  than Province 5. It was also observed that the mean attitude score of 31–40 years was significantly higher  than <31 years [Table The mean knowledge scores were observed to be significantly different among the subgroups based on residence with ANOVA test. On further analysis, Post-hoc (Tukey test) showed the mean knowledge score of Province 1 was significantly higher  have the potential to penetrate and lodge in the smaller passages of the lungs which are thought to carry the greatest potential for transmitting infections. Bio-aerosols on the other hand are aerosols that contain particles of any kind of organism.15 In the context of the COVID-19 pandemic, the awareness of aerosol-related transmission among dentists becomes more relevant which is the basis for this study.The term “aerosol” in the dental environment was first used by Micik and colleagues.16 The majority of the participants of this study knew about the dental procedures generating aerosol and 13% of them even believed that simple extraction and hand scaling generate aerosol. The highly contagious nature of the COVID-19 might have led the dentists to be skeptical for each dental procedure as an aerosol-generating procedure. A similar finding was observed by Teja et al.,13 where they reported that 60% of the participants felt that restorative procedures and any procedures producing aerosols have higher chances to transmit COVID-19 and 35% of them felt that surgical procedures have higher chances of the transmission of infection.13Sources of bio-aerosols in dental setups are ultrasonic scalers, high-speed handpieces, air turbines, three in one syringe, and air-water syringes.8 In a study, done among Turkish dentists 49.95% avoided performing aerosol-forming procedures as much as possible.17 CDC states that emergency dental procedures like emergency access opening of acute pulpitis case with a dental handpiece can be the source of virus transmission to operatory and dental professionals with aerosol generation.8 A recent systematic review even recommends lower power settings be considered to reduce the amount and spread of contamination during the operative procedures.18 This highlights the importance of taking precautions to avoid any airborne contamination in a dental operatory.A total of 230 (60%) respondents were practicing only emergency dental services during the pandemic which is in accordance with CDC guidelines which states that non-emergency dental procedures should be postponed.12 They observed that the source of information regarding COVID-19 was primarily the internet (37.7%). This shows the increased use of social media during the lockdown period.Majority of the participants, 43.3% of this study utilized social media to get updated with the information related to COVID-19 which is higher as compared to the study done by Kamate et al.8A total of 296 (77%) participants in this study were aware that particles may enter through the route between mask/eyewear and facial skin. This awareness can be the result of following recent CDC guidelines by the dentist of Nepal which states that protective eyewear  with gaps between glasses and the face likely do not protect eyes from all splashes and sprays.19 Dental patients and dental health-care professionals (DHCPs) and other persons not directly involved in patient care but potentially exposed to infectious agents like administrative, clerical, housekeeping, maintenance, or volunteer personnel can be exposed to pathogenic microorganisms.11More than 82% of the respondents of the current study agreed that high-efficiency particulate air (HEPA) filter along with high-volume evacuator should be used to reduce the airborne contamination. SARS CoV-2 can remain viable in aerosol and survive up to 3 days on inanimate surfaces at room temperature, with a greater preference for humid conditions.20 The majority of the respondents (82%) of the current study agreed that N90/ N-95 mask should be routinely worn in dental practice due to the current outbreak. In a study conducted among Indian dentists, revealed that almost 50% of them selected Surgical N95  as a first option.21 An interesting finding has been reported by Gambarini et al.,22 where 70% of the dentists consider dental settings more dangerous for the diffusion of COVID-19 than other social behaviors .Particulate respirators  are usually recommended for a routine dental practice.23 assuming that the professors would be older than the lecturer.The present study revealed that the mean attitude score for the participants within the age group of 31–40 years was found to be statistically significantly higher than <31 years. Almost similar findings were noted in a cross-sectional study which showed that Professors/Associate professors were equipped with better knowledge and attitude regarding COVID-19 disease than lecturers24Regarding the current ventilation system of the operatory, a total of 35 participants were practicing in an air-conditioned room with a closed chamber and 28 of them were not aware of their practice ventilation system of their practice. Swedish government recommends that dental practices can only provide aerosol-generating procedures (AGPs) in practice if they have surgical space with external ventilation. The clearance of infectious particles after an AGP is dependent on the ventilation and rate of air change within the room and treatment rooms must be decontaminated after completion of an AGP. The ‘rule of thumb’ below should be followed until further definitive advice is available: (a) For a treatment room with more than 10 air changes per hour (ACH) and which can be evidenced to the National Health Service (NHS) Board, a minimum of 20 min ‘fallow time’  before cleaning is recommended. (b) For a treatment room with external ventilation  with less than 10 ACH or with no data on the number of air changes per hour available, the fallow time would be 60 min. (c) For a treatment room with no external ventilation , the absence of air changes means that AGPs should not be undertaken.25, airborne Infection Isolation Rooms (AIIRs) should be reserved for patients. Air from these rooms should be extracted directly to the outside or be filtered through a HEPA filter.8Although, isolation and high-volume suction are effective to reduce ultrafine dental aerosol particles26 stated that the use of a high-volume evacuator (HVE) may reduce bacterial contamination in aerosols less than one foot (~30 cm) from a patient’s mouth but not at longer distances. The participants of the present study who preferred HEPA filter along with UV light and high-volume evacuator in their operatory for the management of participants during this pandemic were 53%. But, none of them reported having installed this system during the study period. This emphasizes the need to modify dental operatory for preventing airborne contamination.The findings from a systematic review conducted by Kumbargere et al.,On following dental setups of our colleagues, certain changes in the operatory like installation of high-volume evacuator, modifying the closed air-conditioned operatory to cross-ventilated one, etc. were made in few setups. However, the majority of the dentists were practicing in the existing operatory without any modifications. This may reflect the financial constraint faced by the Nepalese dentist despite being aware of the aerosol-related cross-contamination in the dental clinics which needs serious consideration.Despite having certain limitations, this study has put forth the concern regarding the transmission of COVID-19 and also have made the dental professionals think about the risk that their patients and they themselves may be at high risk for infection as well as following standard guidelines. The use of only internet tools to acquire data might have excluded those not using it and a short period of time for data collection might not have included a greater number of participants.


This study aimed to evaluate the effect of oral dexamethasone in reducing kidney scars in infants with a first febrile urinary tract infection (UTI).Children aged between 2 and 24 months with their first presumed UTI, at high risk for kidney scarring based on procalcitonin levels (≥1 ng/mL), were randomly assigned to receive dexamethasone in addition to routine care or routine care only. Kidney scars were identified by kidney scan at 6 months after initial UTI. Projections of enrollment and follow-up completion showed that the intended sample size could not be reached before funding and time to complete the study ran out. An amendment to the protocol was approved to conduct a Bayesian analysis.We randomized 48 children, of whom 42 had a UTI and 18 had outcome kidney scans (instead of 128 planned). Kidney scars were found in 0/7 and 2/11 patients in the treatment and control groups respectively. The probability that dexamethasone could prevent kidney scarring was 99% in the setting of an informative prior probability distribution  and 98% in the low-informative scenario (which discounted the prior literature information by 50%). The probabilities that dexamethasone could reduce kidney scar formation by up to 20% were 61% and 53% in the informative and low-informative scenario, respectively.Dexamethasone is highly likely to reduce kidney scarring, with a more than 50% probability to reduce kidney scars by up to 20%.EudraCT number: 2013-000388-10; registered in 2013 (prospectively registered)The online version contains supplementary material available at 10.1007/s00467-021-05117-5. Kidney scarring can occur in 10 to 40% of children with a urinary tract infection (UTI) despite appropriate antibiotic treatment . InfrequWhile risk factors are still largely unknown, the inflammatory process, rather than the bacterial component, seems responsible for the permanent tissue damage of the kidney , 7. On t99m-Tc-dimercaptosuccinic acid (DMSA) scan found that adjuvant oral methylprednisolone was associated with a lower kidney scarring rate compared with the control group (33.3% versus 60%). [In 2011, a randomized controlled trial (RCT) including children with pyelonephritis confirmed on acute us 60%). . HoweverProcalcitonin (PCT), a blood biomarker of infection, has shown good correlation to both acute pyelonephritis and kidney scarring in children , 13–16, Based on these premises, we conducted a multicenter RCT to determine the effectiveness of adjuvant steroids in reducing kidney scar formation in young children with a first febrile UTI deemed at higher risk for kidney scarring based on their PCT values. Projections of enrollment and completion of study follow-up were calculated halfway through the study because of unanticipated low recruitment and high attrition rate. As projections showed that the intended sample size could not be reached before funding and time to complete the study ran out, an amendment to the analysis plan was approved to use Bayesian analysis. In Bayesian analyses, the probability of treatment effect (posterior probability) is estimated considering the trial data and incorporating the prior probability distribution. The prior distribution includes information on treatment effect provided by previous relevant studies , when available .In this paper, we describe the original study plan and the study results using a Bayesian analysis approach.This multicenter RCT was conducted at five Italian hospitals. Three centers , were part of the study since its inception, in May 2014, while two centers  joined the study in May 2016. The trial was approved by the Ethics Committee of the respective institutions and registered with the European Clinical Trial Database, EudraCT number: 2013-000388-10. We obtained written informed consent from parents or guardians.Children aged 2 to 24 months with the first episode of presumed febrile UTI at high risk of kidney scarring based on PCT levels ≥ 1 ng/mL were eligible for enrolment. A presumed febrile UTI was defined as axillary temperature > 37.5°C and positive dipstick (≥ 1+ leukocyte esterase and/or nitrites) on urine samples collected by urine catheterization, in children with unexplained fever and no other signs of infection.We excluded children who received antibiotics in 48 h before evaluation; who had known underlying kidney diseases or urinary tract abnormalities, a history of previous UTI and UTI recurrence before DMSA scan at 6 months for detection of kidney scars, a history of prematurity , known immunodeficit, and contraindication to steroid therapy; or whose urine culture eventually resulted negative. Patients who were hospitalized could be approached within 48 h after starting antibiotic therapy.Study participants were randomly allocated to receive dexamethasone (0.15 mg/kg per dose every 12 h for 4 days) in addition to routine care or routine care only. Dexamethasone could be administered up to 48 h after starting antibiotic therapy in case hospitalized patients could not be approached in the emergency department. Routine care consisted of oral amoxicillin-clavulanate for a total of 10 days for well-appearing children. In case of allergy or previous adverse reactions to amoxicillin-clavulanate, cefixime was administered. Routine care for ill or toxic-appearing children was intravenous ceftriaxone followed by oral antibiotic therapy after the fever had resolved for at least 48 h, for a total 10-day duration of treatment. Antibiotics could be changed according to antibiogram results on positive urine cultures or ceased if urine cultures resulted negative. The antipyretic of choice was acetaminophen at all centers, with ibuprofen recommended only if acetaminophen was ineffective in relieving the fever-related patient discomfort.We used a computer-generated randomization list accessed through a web-based system, which was password-protected. The computerized system attributed study allocation for each consecutively enrolled patient (independently of the recruiting center). An allocation ratio of 1:1, with random block sizes of 12, was used.Quantitative measurements of PCT levels were performed in blood samples drawn at the time of initial assessment.Children randomized to the dexamethasone arm received the oral drop formulation with detailed instructions on the weight-based dose and times of administration. Patients who were discharged received a first follow-up call as soon as the result of the urine culture was available. A diagnosis of UTI was confirmed in the presence of a positive urine culture defined as the growth of only one micro-organism ≥ 50,000 CFU/mL. In children with a confirmed diagnosis of UTI, a kidney and bladder ultrasound was recommended as the standard of care.A telephone follow-up was also conducted at 10–15 days after the diagnosis of UTI to survey the compliance with prescribed treatment and the occurrence of any possible adverse events.At 6 months after the diagnosis of UTI, a clinical follow-up visit was performed in conjunction with a DMSA scan to detect kidney scarring. The DMSA scan was performed according to the current European guidelines of the European Association of Nuclear Medicine . Kidney The primary outcome was the presence of kidney scars on the DMSA scan performed at the 6-month follow-up. Outcome assessors were two nuclear medicine physicians, blinded to study allocation, and unaware of the patient’s clinical data, who interpreted the scans independently. Discrepancies were resolved by consensus if necessary.Secondary outcomes were the presence of kidney scarring in the subgroup of children with higher PCT values and the acceptability of adjuvant steroid treatment in terms of the rate of discontinuation of treatment and the reported side effects.p-value of 0.05 as statistically significant.We based the original sample size calculation on the hypothesis that dexamethasone would determine a kidney scar reduction from 40%  to 20%.Projections of enrollment and completion of study follow-up showed that the intended sample size could not be reached before funding and time to complete the study ran out. For this reason, an amendment to the study protocol was approved by the participating site Ethics Committes, as well as by the Italian drugs regulatory authority (AIFA), to conduct a Bayesian analysis. The Bayesian method allows the incorporation of the available knowledge on treatment effect (translated into prior probability distribution), combining it with the trial data, such as to reduce uncertainty rather than provide a definitive response to the study hypothesis. The prior probability distribution is based on biological plausibility and on the results of other previous relevant studies, or on clinical experience , 19.Πtreat ∼ Beta and Πcontrol ∼ Beta. The ALC achieved sample size consisted of 18 patients (9+9).The sample size estimation was carried out considering a Bayesian procedure based on a Beta Binomial model for a difference in proportion outcome . An averA Beta Binomial model was used to analyze the primary outcome, namely, the difference in scar proportions between the treatment and control group , 22. TheInformative, which fully incorporates in the final inference the information on the treatment effect provided by previous relevant published studies [ studies , 12Low-informative, which discounts the prior literature information weight on the final inference by 50%Uninformative, which assumes an absolute lack of a priori knowledge on the treatment effect estimateAs the inference was expected to be seriously conditioned by the prior probability distribution  given that only a few data points from the study were available to estimate the likelihood of treatment effect, a sensitivity analysis was performed to assess the robustness of the inferential conclusion concerning different prior distributions. In this regard, the prior probability distributions (representing the a priori knowledge on the treatment effect) considered for the analysis were as follows:The final results were analyzed evaluating the alternative hypothesis that the treatment effect estimate (the difference in the proportions of kidney scar events in treatment versus control) is less than the hypothesized absolute difference of 20% in scar frequency between the groups (assuming that steroids would determine 20% fewer scars compared with the control group): margin of –0.2. The final results were also evaluated considering no difference between the groups (absolute difference of 0%): margin of 0. The analyses were conducted using R 3.6.2 .Online Resource.Additional details on the Bayesian analysis for this study have been recently published  and are -Online ResourceE. Coli. Nearly 60% of patients initially received parenteral antibiotic treatment, and the overall median duration of treatment was 9.5 days. A total of 10 patients (21%) underwent a voiding cystourethrography, and vesicoureteral reflux was found in three. One of these patients had bilateral reflux and presented recurrent UTIs in the first 6 months after enrollment, which determined exclusion from the study, as per study protocol.Children were recruited between May 2014 and June 2017 and follow-up completed by December 2017. A total of 437 children were assessed for eligibility, of whom 225 (51.5%) either did not meet inclusion criteria or met exclusion criteria. Of the remaining 212 patients, 131 (61.8%) did not complete the study procedures, namely, determination of serum PCT and/or urine collection through catheterization, to assess eligibility and were considered “potentially eligible.” Of the 81 eligible patients, 12 (14.8%) could not be approached by research staff and 21 (25.9%) declined consent; thus 48 (59.3%) patients underwent randomization, and 18 completed the 6-month follow-up for the primary outcome assessment and were included in the analysis  patients allocated to the dexamethasone group discontinued the treatment. There was only one reported side effect of transient behavioral change with reported increased irritability in the treatment arm.Our study, unfortunately, failed to assess the effectiveness of adjuvant steroid treatment in reducing kidney scarring in children with acute pyelonephritis, as originally designed. Due to unanticipated significant challenges with patient recruitment and high attrition rate, we eventually used a Bayesian analysis approach to estimate the probability of the treatment effect. Bayesian analysis has been previously used for trials where difficult recruitment was expected . The BayThe Taiwanese RCT by Huang et al.  enrolledE. Coli are at high risk for the development of kidney scarring [Successful recruitment and retention of patients in clinical trials are known to be some of the greatest challenges in conducting RCTs. In our trial, we similarly found many barriers to both recruitment and retention. First, adherence to study procedures by clinicians was a significant challenge to patient recruitment in our study. Failure to perform blood tests for PCT determination prevented the assessment of eligibility for the study, with PCT ≥ 1 ng/mL being one of the inclusion criteria. Clinicians often felt that determination of blood tests, although part of local management protocols, was time-consuming and was of limited added value in well-appearing previously healthy children with a first uncomplicated UTI episode. The role and yield of blood markers in the management of first febrile UTIs episodes are debatable and are not routinely recommended by the Italian guidelines, the National Institute of Care Excellence (NICE), and the American Academy of Pediatrics (AAP) guidelines , 26–29. scarring . Howeverscarring , a DMSA scarring  DMSA scascarring , “the stAs reported above, our study suffered from many limitations that prevented its completion as per original design. In addition, some patients may have received one or more doses of ibuprofen for the treatment of their fever, which might have had an influence on the study outcome. However, the study steering committee made a pragmatic choice not to deviate from the standard of care on fever management at participating sites to avoid parental confusion with respect to antipyretic administration for possible future febrile illnesses. In making this decision, the study steering committee considered that randomization would equally distribute known and unknown confounding factors between the study arms. Furthermore, the study sample size was achieved with respect to the Bayesian design; however, the allocation in the two treatment groups appears to be unbalanced (11 versus 7). Despite all these limitations, our Bayesian analysis could provide probabilities of treatment effect that could be used in clinical practice.Conducting a trial to assess the effectiveness of adjuvant steroid treatment in reducing kidney scar development in children with acute pyelonephritis has proven challenging using a frequentistic approach. A Bayesian analysis approach showed that adjuvant steroids are very likely to reduce kidney scarring, with a more than 50% probability to reduce kidney scar formation by up to 20%, in the setting of an informative or low-informative prior probability distribution.ESM 1(PDF 187 kb)ESM 2(PDF 237 kb)ESM 3(PPTX 44 kb)